Upload
others
View
3
Download
0
Embed Size (px)
Citation preview
UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
The serrated neoplasia pathway: investigating the role of serrated polyps in colorectal cancerdevelopment
Boparai, K.S.
Link to publication
Citation for published version (APA):Boparai, K. S. (2011). The serrated neoplasia pathway: investigating the role of serrated polyps in colorectalcancer development.
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
Download date: 18 May 2020
1
The serrated neoplasia pathway
Investigating the role of serrated
polyps in colorectal cancer development
Karam Singh Boparai
l
2
The Serrated Neoplasia Pathway Thesis, University of Amsterdam, the Netherlands ISBN: Cover: Forest in Corbett National Park, India Printed by: Ipskamp Drukkers BV, Amsterdam Copyright © K.S. Boparai, Amsterdam, the Netherlands The copyrights of the articles that have been accepted for publication published has been transferred to the respective journals The research described in this thesis was performed at the Departments of Gastroenterology & Hepatology and Pathology, AMC, Amsterdam The printing of this thesis was financially supported by AMC, Eurotec, Tramedico, Ferring, Olympus, Merck Sharp & Dohme B.V., US Endoscopy, Nederlandse Vereniging voor
Gastroenterologie,
l
3
The serrated neoplasia pathway
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. D.C.van den Boom ten overstaan van een door het college voor promoties ingestelde
commissie in het openbaar te verdedigen in de Agnietenkapel op vrijdag 9 december 2011, te 12:00
door
Karam Singh Boparai
geboren te Purmerend
l
4
Promotiecommissie Promotores: Prof. dr. P. Fockens
Prof. dr. C.J.M. van Noesel Co-promotores: Dr. E. Dekker
Prof. dr. E.M.H. Mathus-Vliegen Overige leden: Prof. dr. G.A. Meijer Dr. F.M. Nagengast Prof dr. W.A. Bemelman Prof dr. G.J.A. Offerhaus Prof dr. F. Baas
Dr. J.E. East Faculteit der Geneeskunde
l
5
l
6
TABLE OF CONTENTS Chapter 1: General introduction and outline of 9
the thesis (submitted)
Part I: Clinical studies – Colorectal cancer in patients
and relatives Chapter 2: Multicentre cohort analysis: 41
colorectal cancer risk (Gut 2009)
Chapter 3: Increased CRC risk 1st degree 65
relatives in HPS (Gut 2010)
Part II: Molecular analyses – Etiology and colorectal
cancer pathways Chapter 4: Serrated polyps in MYH-associated 83
polyposis (Gastroenterology 2008)
Chapter 5: Serrated polyps in Lynch syndrome 101
(submitted) Chapter 6: Pathways leading to CRC in HPS 123
(American Journal of Pathology 2011) Part III: Endoscopic imaging – Detection and
differentiation of polyps Chapter 7: Increased polyp detection using NBI 153
(Endoscopy 2011) Chapter 8: Differentiation of polyps using ETMI in HPS 177
(Gastrointestinal Endoscopy 2009)
l
7
Summary and future perspectives 201 Samenvatting en toekomstperspectief 209 Acknowledgements 219 Curriculum Vitae 227
8
l
l
9
General introduction and outline of the thesis Submitted
Ch
ap
ter
10
l
General introduction and outline of the thesis
11
GENERAL INTRODUCTION Colorectal cancer causes
Colorectal cancer (CRC) ranks as the second most common
cause of cancer-related death in the western world.1 In general, CRC
can be subdivided into sporadic and familial cases. The overall
majority of individuals with CRC (~80%) are sporadic cases, lacking
any indication of an underlying inherited disorder. Approximately 20%
of cases are attributable to a familial (hereditary) disorder. However, in
only 5% of these patients the causative genetic defect is identified
such as in familial adenomatous polyposis syndrome (FAP), Lynch
syndrome or other rare CRC syndromes such as MUTYH-associated
polyposis (MAP), Peutz-Jeghers and juvenile polyposis (figure 1).
Recently a relatively new condition, called hyperplastic polyposis
syndrome (HPS), has been identified which is characterised by the
presence of multiple colorectal serrated polyps. This presumed
hereditary condition, has been shown to harbour a significantly
increased CRC risk.2-6
Although rare, the above mentioned hereditary disorders have
proven to be important models to study colorectal carcinogenesis in
general and based on these models major advances have occurred
regarding our understanding of the molecular events leading to CRC
and which potential pathways involving different polyp types are
implicated.
Chapte
r 1
Chapter I
12
Figure 1. Causes of colorectal cancer
Histological polyp types
Traditionally, colorectal polyps were divided in to two separate groups,
the adenoma which can be divided into tubular, tubulovillous and
villous subtypes7, and the hyperplastic polyp (HP), which displays
serration in the upper one-half to one-third of the crypt.8 Adenomas
have long been known to represent neoplastic precursor lesions of
most CRCs, especially those adenomas harbouring a villous
component. HPs on the other hand, which constitute the most common
occurring serrated polyps, were regarded until recently as innocuous
lesions lacking any premalignant potential. Untill very recently, clinical
guidelines did not recommend surveillance colonoscopies for
individuals with HPs because the associated CRC risk was not
believed to be higher than the risk in individuals without polyps.9
General introduction and outline of the thesis
13
Recent morphological reappraisals of polyps revealed however that
serrated polyps can be further subdivided into different histological
entities with possibly different premalignant potential.
Hyperplastic polyps (HPs)
Colorectal HPs are typically diminutive polyps (<5mm in diameter)
which have a predilection for the sigmoid colon and rectum although
larger sized HPs with a proximal localization have also been
described.10 Besides being generally small these lesions are often flat
or sessile in shape, unremarkable in colour and often covered with
mucus.11, 12 Serration, which is caused by infolding of the crypt
epithelium leading to a saw-toothed appearance in longitudinal section
and a star-shaped appearance on cross-section, is limited to the upper
half to one-third of the crypt. Crypts are generally narrow and straight,
with a normal distribution of the proliferative zone in the base of the
crypts.8 HPs can be further subdivided into microvesicular, goblet cell-
rich and mucin-poor subtypes on the basis of mucin content of the
epithelial cells although the reproducibility hereof among pathologists
is doubtful and the clinical relevance questionable.
Sessile serrated lesion (SSL)
Torlakovic and Snover identified a new hyperplastic polyp variant,
formerly known as the sessile serrated adenoma.8, 13 These polyps are
currently referred to as sessile serrated lesions.109 These authors
showed that a subset of lesions that were originally diagnosed as HPs
in patients with Hyperplastic polyposis syndrome demonstrated a
different morphology with abnormal proliferation and concluded that
these SSLs were distinct lesions which should be differentiated from
HPs. SSLs are defined by predominantly architectural distortion with
Chapte
r 1
Chapter I
14
irregular dilated crypts, including dilatation of the base of the crypts
that often have a boot, L or inverted T shape. Serration can also be
identified at the base of the crypts as well as abnormal proliferation
and maturation with mature goblet or foveolar cells.14 Cytological
dysplasia is not usually demonstrated. However, if an area of dysplasia
is present, this should be specified. In contrast, HPs have narrow crypt
bases and serration is restricted to the upper half of the crypt.
Previous studies have shown that SSLs and HPs are
histologically hard to distinguish, leading to confusion with regard to
categorization of these serrated polyps. Indeed, it has recently been
shown that at re-evaluation the interobserver agreement for the
differentiation of serrated polyps remains only moderate.13, 15-17
Molecular analysis for the differentiation of these serrated polyps has
thus far shown that right-sided SSLs have increased MLH-1 and p16
methylation compared to left-sided SSLs and HPs.18 Some studies
have also shown that SSLs are more often BRAF mutated than HPs.11,
19 Nevertheless, unambiguous molecular differentiation between these
two lesions has as yet not been achieved.
Traditional serrated adenoma (TSA)
Longacre & Fenoglio Preiser, in a large polyp survey, re-evaluated a
group of polyps displaying mixed histological features of both
hyperplastic and adenomatous polyps. Rather than representing mixed
tumors, these lesions were considered to be adenomas with a serrated
configuration similar to HPs and were thus coined serrated adenomas
(currently named traditional serrated adenomas).20 Criteria for TSAs
include a pedunculated polyp shape with elongated villiform
projections uniformly lined with atypical cells having elongated nuclei
and eosinophilic cytoplasm.8, 21, 22 In the mentioned survey, TSAs
General introduction and outline of the thesis
15
represented less than 1% of all colorectal polyps. Before re-evaluation,
approximately one third of these TSAs were originally diagnosed as
HP, one third as conventional adenoma and one third as intermediate
lesions. This variability underlines the difficulty in diagnosing these rare
lesions. TSAs are distinguishable from SSLs by their uniform
population of abnormal epithelial cells as well as their pedunculated
shape rather than sessile.
Colorectal carcinogenesis
CRC pathways
Notwithstanding this re-classification of serrated polyps,
conventional adenomas are considered to be the main lesions with an
undisputable premalignant potential. Clear histological evidence that
CRCs develop from adenomas was first established in 1975 by Muto
et al. which was further genetically described by Vogelstein et al. who
demonstrated in FAP patients that the adenoma-carcinoma sequence
is caused by aberrant activation of the Wnt signalling pathway.23, 24
This multi-step process of carcinogenesis is characterised by an initial,
bi-allelic inactivation of the adenomatous polyposis coli gene (APC)
followed by accumulation of genetic mutations in key oncogenes and
tumor-suppressor genes including KRAS, DCC and p53. These
events, in conjunction with associated chromosomal instability (CIN),
result in adenoma initiation and progression to CRC.
Subsequently, an alternative molecular pathway to CRC, the
microsatellite instability pathway, was discovered in patients with
Lynch syndrome.25 This pathway is initiated by deletion or inactivation
of one of the mismatch repair genes (MLH-1, MSH-2, MSH-6 or PMS-
2). This causes defective repair of replication errors in repetitive DNA
sequences which are most clearly demonstrated in so called
Chapte
r 1
Chapter I
16
microsatellite regions throughout the genome.26 Microsatellite regions
are abundant in non-encoding regions but are also present in encoding
regions of growth regulatory genes like tumor suppressor genes such
as TGFßRII, IGF2R and BAX. Alterations in such regions may then
cause inactivation of these genes and subsequent CRC formation with
microsatellite instability (MSI).27-29 MSI-CRCs can be the result of an
inherited germline defect of a mismatch-repair gene, as a part of Lynch
syndrome in 3% of all CRC cases, but can also be caused by a
somatic deletion or inactivation of a mismatch repair gene in up to 15%
of the sporadic CRCs (figure 1).30, 31
Alternative pathway to MSI CRC?
Both CRCs with CIN and CRCs with MSI from patients with Lynch
syndrome have been shown to harbour predominantly APC, KRAS
and P53 mutations.25, 32-36 In contrast, sporadic MSI CRCs infrequently
harbour these mutations which are typically found in conventional
adenomas.37-39 Instead, sporadic MSI CRCs are shown to have high
levels of mutations in the BRAF oncogene and extensive CPG- island
methylation of multiple genes (CIMP) including MLH-1, 40-44 which are
rare findings in sporadic adenomas34, 41, 43, 45, 46 but are commonly seen
in serrated polyps.11, 41, 45, 47 The association between serrated polyps
and specifically sporadic MSI-H CRCs is further emphasised by the
infrequent presence of BRAF mutations and CIMP in Lynch syndrome
MSI-H CRCs.42, 48 These findings suggested that serrated polyps,
instead of conventional adenomas, could represent precursor lesions
of sporadic MSI CRCs.
In parallel, clinicohistological evidence arose that serrated
polyps may also play a role in colorectal carcinogenesis. These reports
involve both sporadic HPs and HPs in the context of hyperplastic
General introduction and outline of the thesis
17
polyposis syndrome and comprise CRCs in close vicinity of large
hyperplastic polyps49, 50; CRCs identified in mixed hyperplastic and
adenomatous polyps51; increased incidence of HPs and serrated
adenomas in patients with sporadic microsatellite-unstable CRCs52-54;
and a high prevalence of CRC in HPS patients13, 50, 55-57. More recent
cross-sectional association studies also showed a strong and
independent association between large (>1cm) sporadic serrated
polyps, i.e. HPs, SSLs and/or TSAs and synchronous advanced
CRC.58, 59 In another study, 5.8% of CRCs were found to have an
adjacent “serrated adenoma” which were more commonly MSI (38%)
than in CRCs without an adjacent serrated adenoma (11%).53
Currently however, proof of the existence of a serrated CRC pathway
data on the natural history of serrated polyps are lacking. Therefore,
the appropriate management of serrated polyps is unclear.
Serrated neoplasia pathway: proposed sequence of molecular events
While a single activating point mutation in BRAF (V600E) is an
oncogenetic hit in the mitogen-activated protein kinase (MAPK)
pathway resulting in augmented cell proliferation, survival and
inhibition of apoptosis, epigenetic alterations like CPG-island
methylation, in which the DNA sequence remains unaltered, can cause
inactivation of genes. CPG-islands are seen in approximately 50% of
human genes and consist of dense clusters of cytosine-guanosine
dinucleotides (CpG) that are susceptible to methylation of the cytosine
base resulting in gene silencing. Within the context of CRC, CPG-
island methylation can cause transcriptional repression of tumor
suppressor genes, like MLH1 in sporadic MSI CRCs.60-63 Despite their
different mechanisms of tumor development, BRAF mutations and
CIMP have been shown to be strongly associated in CRC, with an
Chapte
r 1
Chapter I
18
odds ratio of 203.64 This strong association between BRAF and CIMP
might be explained by the fact that mutations in the BRAF oncogene
alone induce an initial burst of proliferation followed by cell
senescence.64 Cell senescence is regulated by genes such as p16 and
p53 which can restrain the initially induced cell proliferation.65, 66
Further development to CRC may then be dependent on silencing of
these regulatory genes so that a stable senescent state is overcome. It
has thus been proposed that BRAF-mutant HPs may only progress to
CRC, possibly via SSLs, after a second molecular change such as
methylation-induced silencing of p16.67, 68 Interestingly, in a previous
study, analyzing the molecular differences between HPs and SSLs, it
was shown that p16 was significantly more methylated in SSLs than in
HPs, supporting the supposition that SSLs represent more advanced
lesions.18 Similarly, IGFBP7, a mediator of P53-induced senescence
has been shown to be methylated and silenced in BRAF mutated
CRCs.69, 70 Thus, it seems that the serrated pathway involves a
necessary multi-step process of genetic and epigenetic changes to
CRC.
Polyposis syndromes such as FAP and MAP are valuable
models for analyzing the molecular mechanisms of the adenoma-
carcinoma sequence due to the multitude of conventional adenomas
and coincident CRC in these patients. Similarly, HPS patients who
harbour multiple serrated polyps and have been shown to have
concurrent CRC in up to 50% of cases at clinical presentation3, 5, 6, 26, 51,
71-74, may provide a “scientific workbench” for further analysis of the
genetic sequence of events in the serrated neoplasia pathway.
General introduction and outline of the thesis
19
Hyperplastic polyposis syndrome (HPS)
HPS is characterized by the presence of multiple serrated polyps
spread throughout the colorectum and has been defined by the World
Health Organization as the presence of at least five histologically
diagnosed HPs proximal to the sigmoid colon, of which 2 larger than
10mm in diameter, or more than 30 HPs distributed throughout the
colon.2 However, besides multiple HPs, SSLs and traditional
adenomas are common findings in this condition as well. HPs and
SSLs, both displaying a serrated phenotype, have been shown to be
histologically very similar and difficult to differentiate microscopically
with only moderate concordance.16-18, 75 Consequently, originally
diagnosed HPs may represent SSLs and vice versa. It was therefore
recently decided to rename the condition ‘serrated polyposis
syndrome’ or SPS, with inclusion of all serrated polyp subtypes.76
From a practical perspective, considering that most articles in this
thesis were published before this new definition was implemented, the
term hyperplastic polyposis syndrome (HPS) will be used throughout
this thesis.
From a clinical viewpoint, a further reclassification of HPS may
be beneficial to identify subtypes that have a higher risk of CRC
development. For example, it is assumable that HPS patients with a
higher number of polyps or larger polyps also have a higher CRC risk.
Also from an etiological viewpoint reclassification of HPS seems
prudent. The heterogeneous phenotype of HPS such as (i) the
presence of different polyp histologies and (ii) difference in polyp
localization and (iii) number of polyps, suggests that HPS represents
separate genetic conditions. Also molecular differences are found
between HPS phenotypes, such as more CPG-island methylation in
Chapte
r 1
Chapter I
20
right-sided serrated polyps as compared to left-sided serrated polyps,
however these differences are not distinctive.
Concerning the prevalence of HPS in the general population, a
previous screening sigmoidoscopy study in asymptomatic people aged
55 to 64 years estimated the prevalence to be 1:3000.77 However, in
this screening study only the rectosigmoid was investigated without
visualization of the remaining proximal colon. In addition, considering
that HPs are often diminutive in size, flat in shape and unremarkable in
colour, it is also possible that polyps were left undetected. This
suggests that the actual prevalence of HPS is probably higher than
currently estimated and in any case more frequent in occurrence than
other polyposis syndromes such as FAP (1:13.000).78 Concordantly, a
recent large prospective colonoscopy screening study involving 50.148
asymptomatic volunteers in Poland revealed 28 HPS patients leading
to an estimated incidence of 1:1800.79
HPS and CRC risk
HPS is associated with an increased CRC-risk. Previously published
case series report CRC at clinical presentation in up to 50% of HPS
patients.3, 5, 6, 26, 51, 71-74 However, because these findings could also be
interpreted as two-co-existent unrelated abnormalities, a causal
relationship between HPS and the development of CRC can not by
default be made but does seem presumable. Ideally, a large cohort of
asymptomatic HPS patients, derived from a population based
colonoscopy screening study, that is subsequently surveyed for
multiple years would be useful to adequately determine the risk of
CRC in these patients. Until such a study is performed, clinical
evidence for an increased CRC risk after HPS diagnosis is thus far
based on cohort studies with associated ascertainment bias. One such
General introduction and outline of the thesis
21
study describes a case series of 13 HPS patients who were
prospectively followed and who received regular endoscopies. Three
of 13 (23%) patients developed CRC during follow-up, suggesting that
patients with HPS have an increased risk of malignant progression of
serrated polyps.5 Molecular supporting evidence include studies
showing a higher number of BRAF mutations and CIMP in serrated
polyps of HPS patients compared to sporadic serrated polyps.41, 80, 81
Moreover, HPS patients have far more serrated polyps than people in
the general population.
Nevertheless, considering that conventional adenomas, which
are traditionally considered premalignant polyps, are also common in
HPS, these polyps and the associated Wnt-pathway can not be
disregarded. Thus, based on the mixture of different precursor lesion
types, HPS patients represent a unique model to evaluate which CRC
pathways play a role in HPS and consequently which polyps are
clinically relevant and to obtain new evidence supporting the existence
of a serrated CRC pathway in humans.
Endoscopic diagnostics of HPS
Considering the presumed increased risk of malignant progression of
polyps in HPS, detection and removal of polyps seems necessary to
prevent CRC development in these patients. In HPS patients however,
serrated polyps, which are the overall majority, are generally small, flat
in shape and unremarkable in colour.82-84 These features are
associated with polyp miss-rates of up to 26% using standard
colonoscopy.85-87 Improved detection of these polyps seems therefore
desirable which can be achieved by improving bowel preparation;
clearing of fluid and debris; implementing a minimally accepted
withdrawal time of 6 minutes; visualizing the proximal sides of folds,
Chapte
r 1
Chapter I
22
flexures, and valves; and adequate colon distension.88-92 In addition,
chromoendoscopy has been shown to improve the detection of small
and flat lesions, specifically HPs, in patients undergoing surveillance
colonoscopy.86, 87, 93-95 Novel advanced endoscopic techniques, such
as narrow-band imaging (NBI) may also be an attractive alternative to
improve the detection of polyps in HPS. In contrast with
chromoendoscopy, which is a labour-intensive and time-consuming
technique, NBI (‘electronic chromoendoscopy’) is an easier push-of-a-
button technique that enhances mucosal and vascular detail without
the use of dyes. It has proven to be superior to high-resolution
endoscopy (HRE) for the detection of sporadic HPs but has not been
evaluated in HPS.96, 97
In addition, although removal of all larger polyps seems
necessary in any case, accurate differentiation of diminutive polyps
may aid the endoscopist in only removing advanced lesions (e.g.
conventional adenomas, SSLs and TSAs) and leaving ‘innocuous’
HPs, which display relatively lower levels of BRAF mutations and
CIMP, in situ.11, 16, 18, 19 Since HPS patients have many diminutive
polyps, this may save time and considerably prevent complications
from unnecessary polypectomies of HPs.3 Whereas chromoendoscopy
and NBI may also aid in the differentiation of HPs and adenomas, it is
unknown whether differentiation between HPs and SSLs is possible as
well.3, 98-101 Finally, autofluorescence imaging (AFI), which facilitates
differentiation of adenomas from non-adenomatous polyps based on
different fluorescence emission spectra102-104, may also be of value for
the differentiation of polyps in HPS.
General introduction and outline of the thesis
23
Treatment of HPS
Due to this risk of malignant transformation of polyps, HPS patients are
either advised to undergo endoscopic surveillance with removal of
polyps or a surgical colonic resection. Recent expert opinions
recommend surveillance intervals ranging from one to three years.
Concerning the removal of polyps, advice varies from removal of only
proximally located polyps to complete removal of all polyps >5mm.3, 105
As a consequence, lack of clarity exists regarding the recommended
surveillance interval and which polyps should be removed. Therefore it
seems possible that a proportion of HPS patients may be insufficiently
treated and consequently be at risk of developing CRC under
surveillance (interval CRC). Conversely, over-treatment with
unnecessary removal of possibly harmless lesions which may result in
complications should also be avoided.
Familial risk
As opposed to FAP which has an autosomal dominant inheritance
pattern, in HPS the mode of inheritance is yet unknown. However,
although no genetic substrate has yet been identified, families have
previously been reported from which both an autosomal recessive and
autosomal dominant inheritance could be considered.3, 73, 106, 107 For
this reason, screening colonoscopies of first-degree relatives is
currently advised by some authorities.3, 108
The possible increased risk of developing CRC for first-degree
relatives is however unclear. In first-degree relatives CRC has been
reported, ranging in frequency from 0 to 50%.3, 4 In one retrospective
case series the incidence of polyps and CRC in first degree relatives
was studied.107 Of 17 first degree relatives who underwent screening
colonoscopy, 10/17 (59%) had polyps. In three patients HPs were
Chapte
r 1
Chapter I
24
detected, predominantly in the rectum and one patient (6%) was
diagnosed with HPS. This patient had multiple concomitant adenomas
as well. In the other 6 patients adenomas were discovered proximal to
the sigmoid colon. Of these patients, seven were under the age of 45
and the youngest was 32 years of age. No carcinoma was found.
However, these data were not prospectively acquired and instead of
performing a screening colonoscopy on all first-degree relatives of
patients with HPS, screening was primarily performed because of CRC
occurrence in the index-patient or because of an established family
history of CRC. To make an adequate estimate of the incidence of
polyps and HPS in first-degree relatives and the CRC-risk in this group
due to HPS, prospective screening of larger series of first-degree
relatives should be performed.
General introduction and outline of the thesis
25
OUTLINE OF THE THESIS The foundation of this research project was laid at the endoscopy unit and out-patient clinic of the Academic Medical Center where an increasing number of patients with hyperplastic polyposis syndrome (HPS) have been diagnosed. HPS is characterized by the presence of multiple colorectal hyperplastic polyps which are traditionally considered to be benign lesions. The surprisingly common occurrence of colorectal cancer (CRC) in these patients prompted further research in to the clinical and molecular characteristics of HPS patients and the associated endoscopic management. Part I: Clinical analyses – Colorectal cancer risk The first part of the thesis focuses on the clinicopathological features of HPS patients and their first-degree relatives. Previously published case series report CRC at clinical presentation in up to 50% of HPS patients. Chapter 2 evaluates the risk of developing CRC after HPS diagnosis and which variables are associated with CRC in a large multi-centre cohort of HPS patients undergoing endoscopic surveillance. In chapter 3 the prevalence of polyps and CRC in first-degree relatives is described and compared with the background prevalence in the general population. Part II: Molecular analyses – Etiology and colorectal cancer pathways Although an underlying genetic disorder seems likely, thus far no genetic causes of HPS have been identified. However, previous case series of patients with a known genetic disorder have reported individuals with multiple serrated polyps. In chapter 4 and 5 we aimed to molecularly analyze whether serrated polyps identified in the setting of MYH-associated polyposis and Lynch syndrome are causally related to these respective disorders. Besides hyperplastic polyps (HPs), sessile serrated lesions (SSLs) and conventional adenomas are also common findings in HPS. Due to this heterogeneity, it is unknown which polyps eventually lead to CRC in HPS and thus are clinically relevant. In chapter 6 we aimed to analyze which polyps lead to CRC in HPS patients by performing combined immunohistological and molecular analyses.
Chapte
r 1
Chapter I
26
Part III: Endoscopic imaging – Detection and differentiation of polyps Considering the presumed increased risk of malignant progression of polyps in HPS, detection and removal of polyps seems necessary to prevent CRC development in these patients. Besides following general quality guidelines of colonoscopy, novel advanced endoscopic techniques, such as narrow-band imaging (NBI) may improve the detection of polyps in HPS. Chapter 7 involves a randomized cross-over study comparing NBI and high-resolution endoscopy with regard to their miss-rates of polyps. In addition to improved detection of polyps in HPS, accurate differentiation of HPs and SSLs, which appear endoscopically very similar, may aid the endoscopist in only removing SSLs and leaving HPs, which display comparatively lower levels of genetic mutations, in situ. In chapter 8 we evaluated high resolution white-light endoscopy, NBI and autofluorescence imaging for the endoscopic differentiation of polyps in HPS.
General introduction and outline of the thesis
27
REFERENCES
1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,
Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK. Annual Report to the Nation on the Status of Cancer, 1975-2005, Featuring Trends in Lung Cancer, Tobacco Use, and Tobacco Control. J Natl Cancer Inst 2008.
2. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.
3. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
4. Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004;99:2012-2018.
5. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
6. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
7. Hamilton SR, Vogelstein B, Kudo S, et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000:104-119.
8. Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81.
9. Winawer SJ, Zauber AG, Fletcher RH, Stillman JS, O'Brien MJ, Levin B, Smith RA, Lieberman DA, Burt RW, Levin TR, Bond JH, Brooks D, Byers T, Hyman N, Kirk L, Thorson A, Simmang C, Johnson D, Rex DK. Guidelines for colonoscopy surveillance after polypectomy: a consensus update by the US Multi-Society Task Force on Colorectal Cancer and the American Cancer Society. CA Cancer J Clin 2006;56:143-159.
Chapte
r 1
Chapter I
28
10. DiSario JA, Foutch PG, Mai HD, Pardy K, Manne RK. Prevalence and malignant potential of colorectal polyps in asymptomatic, average-risk men. Am J Gastroenterol 1991;86:941-945.
11. Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T, Simms LA, Young J, James M, Montgomery GW, Appleyard M, Hewett D, Togashi K, Jass JR, Leggett BA. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400-1407.
12. Yano T, Sano Y, Iwasaki J, Fu KI, Yoshino T, Kato S, Mera K, Ochiai A, Fujii T, Yoshida S. Distribution and prevalence of colorectal hyperplastic polyps using magnifying pan-mucosal chromoendoscopy and its relationship with synchronous colorectal cancer: prospective study. J Gastroenterol Hepatol 2005;20:1572-1577.
13. Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996;110:748-755.
14. Torlakovic EE, Gomez JD, Driman DK, Parfitt JR, Wang C, Benerjee T, Snover DC. Sessile Serrated Adenoma (SSA) vs. Traditional Serrated Adenoma (TSA). Am J Surg Pathol 2008;32:21-29.
15. Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY, Mino-Kenudson M. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008;32:30-35.
16. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.
17. Wong NA, Hunt LP, Novelli MR, Shepherd NA, Warren BF. Observer agreement in the diagnosis of serrated polyps of the large bowel. Histopathology 2009;55:63-66.
18. Sandmeier D, Benhattar J, Martin P, Bouzourene H. Serrated polyps of the large intestine: a molecular study comparing sessile serrated adenomas and hyperplastic polyps. Histopathology 2009;55:206-213.
General introduction and outline of the thesis
29
19. Carr NJ, Mahajan H, Tan KL, Hawkins NJ, Ward RL. Serrated and non-serrated polyps of the colorectum: their prevalence in an unselected case series and correlation of BRAF mutation analysis with the diagnosis of sessile serrated adenoma. J Clin Pathol 2009;62:516-518.
20. Longacre TA, Fenoglio-Preiser CM. Mixed hyperplastic adenomatous polyps/serrated adenomas. A distinct form of colorectal neoplasia. Am J Surg Pathol 1990;14:524-537.
21. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.
22. Snover DC. Serrated polyps of the large intestine. Semin Diagn Pathol 2005;22:301-308.
23. Muto T, Bussey HJ, Morson BC. The evolution of cancer of the colon and rectum. Cancer 1975;36:2251-2270.
24. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532.
25. Aaltonen LA, Peltomaki P, Leach FS, Sistonen P, Pylkkanen L, Mecklin JP, Jarvinen H, Powell SM, Jen J, Hamilton SR, . Clues to the pathogenesis of familial colorectal cancer. Science 1993;260:812-816.
26. Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y, Watanabe H. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52:5-9.
27. Souza RF, Appel R, Yin J, Wang S, Smolinski KN, Abraham JM, Zou TT, Shi YQ, Lei J, Cottrell J, Cymes K, Biden K, Simms L, Leggett B, Lynch PM, Frazier M, Powell SM, Harpaz N, Sugimura H, Young J, Meltzer SJ. Microsatellite instability in the insulin-like growth factor II receptor gene in gastrointestinal tumours. Nat Genet 1996;14:255-257.
28. Rampino N, Yamamoto H, Ionov Y, Li Y, Sawai H, Reed JC, Perucho M. Somatic frameshift mutations in the BAX gene in colon cancers of the microsatellite mutator phenotype. Science 1997;275:967-969.
Chapte
r 1
Chapter I
30
29. Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Vogelstein B, . Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science 1995;268:1336-1338.
30. Ionov Y, Peinado MA, Malkhosyan S, Shibata D, Perucho M. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 1993;363:558-561.
31. Thibodeau SN, Bren G, Schaid D. Microsatellite instability in cancer of the proximal colon. Science 1993;260:816-819.
32. Fujiwara T, Stolker JM, Watanabe T, Rashid A, Longo P, Eshleman JR, Booker S, Lynch HT, Jass JR, Green JS, Kim H, Jen J, Vogelstein B, Hamilton SR. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol 1998;153:1063-1078.
33. Huang J, Papadopoulos N, McKinley AJ, Farrington SM, Curtis LJ, Wyllie AH, Zheng S, Willson JK, Markowitz SD, Morin P, Kinzler KW, Vogelstein B, Dunlop MG. APC mutations in colorectal tumors with mismatch repair deficiency. Proc Natl Acad Sci U S A 1996;93:9049-9054.
34. Konishi M, Kikuchi-Yanoshita R, Tanaka K, Muraoka M, Onda A, Okumura Y, Kishi N, Iwama T, Mori T, Koike M, Ushio K, Chiba M, Nomizu S, Konishi F, Utsunomiya J, Miyaki M. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111:307-317.
35. Losi L, Ponz de LM, Jiricny J, di GC, Benatti P, Percesepe A, Fante R, Roncucci L, Pedroni M, Benhattar J. K-ras and p53 mutations in hereditary non-polyposis colorectal cancers. Int J Cancer 1997;74:94-96.
36. Tomlinson I, Ilyas M, Johnson V, Davies A, Clark G, Talbot I, Bodmer W. A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 1998;184:148-152.
General introduction and outline of the thesis
31
37. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, Meltzer SJ, Wright C, Searle J, Young J, Leggett BA. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52:455-460.
38. Salahshor S, Kressner U, Pahlman L, Glimelius B, Lindmark G, Lindblom A. Colorectal cancer with and without microsatellite instability involves different genes. Genes Chromosomes Cancer 1999;26:247-252.
39. Jass JR, Barker M, Fraser L, Walsh MD, Whitehall VL, Gabrielli B, Young J, Leggett BA. APC mutation and tumour budding in colorectal cancer. J Clin Pathol 2003;56:69-73.
40. Jass JR. Serrated adenoma of the colorectum and the DNA-methylator phenotype. Nat Clin Pract Oncol 2005;2:398-405.
41. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.
42. McGivern A, Wynter CV, Whitehall VL, Kambara T, Spring KJ, Walsh MD, Barker MA, Arnold S, Simms LA, Leggett BA, Young J, Jass JR. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3:101-107.
43. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.
44. Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA, Borten MM, Stitz R, Searle J, McKeone D, Fraser L, Purdie DR, Podger K, Price R, Buttenshaw R, Walsh MD, Barker M, Leggett BA, Jass JR. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 2001;159:2107-2116.
45. Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63:4878-4881.
Chapte
r 1
Chapter I
32
46. Ikehara N, Semba S, Sakashita M, Aoyama N, Kasuga M, Yokozaki H. BRAF mutation associated with dysregulation of apoptosis in human colorectal neoplasms. Int J Cancer 2005;115:943-950.
47. Yang S, Farraye FA, Mack C, Posnik O, O'Brien MJ. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am J Surg Pathol 2004;28:1452-1459.
48. Wang L, Cunningham JM, Winters JL, Guenther JC, French AJ, Boardman LA, Burgart LJ, McDonnell SK, Schaid DJ, Thibodeau SN. BRAF mutations in colon cancer are not likely attributable to defective DNA mismatch repair. Cancer Res 2003;63:5209-5212.
49. Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000;43:1309-1313.
50. Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994;89:123-125.
51. Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984;8:551-556.
52. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93:1307-1313.
53. Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193:286-294.
54. Goldstein NS, Bhanot P, Odish E, Hunter S. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119:778-796.
55. Jeevaratnam P, Cottier DS, Browett PJ, Van de Water NS, Pokos V, Jass JR. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179:20-25.
General introduction and outline of the thesis
33
56. Kusunoki M, Fujita S, Sakanoue Y, Shoji Y, Yanagi H, Yamamura T, Utsunomiya J. Disappearance of hyperplastic polyposis after resection of rectal cancer. Report of two cases. Dis Colon Rectum 1991;34:829-832.
57. Shepherd NA. Inverted hyperplastic polyposis of the colon. J Clin Pathol 1993;46:56-60.
58. Li D, Jin C, McCulloch C, Kakar S, Berger BM, Imperiale TF, Terdiman JP. Association of large serrated polyps with synchronous advanced colorectal neoplasia. Am J Gastroenterol 2009;104:695-702.
59. Lazarus R, Junttila OE, Karttunen TJ, Makinen MJ. The risk of metachronous neoplasia in patients with serrated adenoma. Am J Clin Pathol 2005;123:349-359.
60. Ahuja N, Mohan AL, Li Q, Stolker JM, Herman JG, Hamilton SR, Baylin SB, Issa JP. Association between CpG island methylation and microsatellite instability in colorectal cancer. Cancer Res 1997;57:3370-3374.
61. Cunningham JM, Christensen ER, Tester DJ, Kim CY, Roche PC, Burgart LJ, Thibodeau SN. Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability. Cancer Res 1998;58:3455-3460.
62. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999;59:793-797.
63. Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner R. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res 1997;57:808-811.
64. Weisenberger DJ, Siegmund KD, Campan M, Young J, Long TI, Faasse MA, Kang GH, Widschwendter M, Weener D, Buchanan D, Koh H, Simms L, Barker M, Leggett B, Levine J, Kim M, French AJ, Thibodeau SN, Jass J, Haile R, Laird PW. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat Genet 2006;38:787-793.
Chapte
r 1
Chapter I
34
65. Collado M, Serrano M. The power and the promise of oncogene-induced senescence markers. Nat Rev Cancer 2006;6:472-476.
66. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997;88:593-602.
67. Minoo P, Jass JR. Senescence and serration: a new twist to an old tale. J Pathol 2006;210:137-140.
68. Leggett B, Whitehall V. Role of the serrated pathway in colorectal cancer pathogenesis. Gastroenterology 2010;138:2088-2100.
69. Wajapeyee N, Serra RW, Zhu X, Mahalingam M, Green MR. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell 2008;132:363-374.
70. Suzuki H, Igarashi S, Nojima M, Maruyama R, Yamamoto E, Kai M, Akashi H, Watanabe Y, Yamamoto H, Sasaki Y, Itoh F, Imai K, Sugai T, Shen L, Issa JP, Shinomura Y, Tokino T, Toyota M. IGFBP7 is a p53-responsive gene specifically silenced in colorectal cancer with CpG island methylator phenotype. Carcinogenesis 2010;31:342-349.
71. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.
72. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, Cohn D, Furlong KL, Walsh MD, Palazzo J, Edmonston TB, Fishel R, Young J, Leggett BA. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47:43-49.
73. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
74. Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y, Oda J, Mizutani M, Takayanagi S, Kishi D, Shinohara T, Yamada K, Matumoto J, Imamura K. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008.
General introduction and outline of the thesis
35
75. Khalid O, Radaideh S, Cummings OW, O'Brien MJ, Goldblum JR, Rex DK. Reinterpretation of histology of proximal colon polyps called hyperplastic in 2001. World J Gastroenterol 2009;15:3767-3770.
76. Snover DC, Ahnen D, Burt R, Odze R.D. Serrated Polyps of the Colon and Rectum and Serrated Polyposis. WHO Classification of Tumours of the Digestive System. 4th edition ed. Lyon, France: IARC, 2010.
77. Lockett MJ, Atkin W.S. Hyperplastic polyposis: prevalence and cancer risk. 48 ed. 2001:A4.
78. Bisgaard ML, Fenger K, Bulow S, Niebuhr E, Mohr J. Familial adenomatous polyposis (FAP): frequency, penetrance, and mutation rate. Hum Mutat 1994;3:121-125.
79. Orlowska J, Kiedrowski M, Kaminski F.M., Regula J, Butruk E. Hyperplastic polyposis syndrome in asymptomatic patients: the results from the colorectal-cancer screening program. Virchows Arch 2009;Abstract: OP 13.8.
80. Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S, Broaddus RR, Issa JP, Hamilton SR, Rashid A. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005;166:1069-1075.
81. Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002;160:529-536.
82. Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997;29:1-3.
83. Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999;49:736-742.
84. Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996;87:305-309.
85. van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006;101:343-350.
Chapte
r 1
Chapter I
36
86. Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS, Cardwell CR, Williams CB. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002;56:333-338.
87. Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004;53:376-380.
88. Barclay RL, Vicari JJ, Doughty AS, Johanson JF, Greenlaw RL. Colonoscopic withdrawal times and adenoma detection during screening colonoscopy. N Engl J Med 2006;355:2533-2541.
89. Froehlich F, Wietlisbach V, Gonvers JJ, Burnand B, Vader JP. Impact of colonic cleansing on quality and diagnostic yield of colonoscopy: the European Panel of Appropriateness of Gastrointestinal Endoscopy European multicenter study. Gastrointest Endosc 2005;61:378-384.
90. Harewood GC, Sharma VK, de GP. Impact of colonoscopy preparation quality on detection of suspected colonic neoplasia. Gastrointest Endosc 2003;58:76-79.
91. Rex DK. Colonoscopic withdrawal technique is associated with adenoma miss rates. Gastrointest Endosc 2000;51:33-36.
92. Rex DK. Maximizing detection of adenomas and cancers during colonoscopy. Am J Gastroenterol 2006;101:2866-2877.
93. Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006;38:444-448.
94. Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A, Sautereau D, Chaussade S, Galmiche JP. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006;4:349-354.
95. Park SY, Lee SK, Kim BC, Han J, Kim JH, Cheon JH, Kim TI, Kim WH. Efficacy of chromoendoscopy with indigocarmine for the detection of ascending colon and cecum lesions. Scand J Gastroenterol 2008;43:878-885.
General introduction and outline of the thesis
37
96. Adler A, Pohl H, Papanikolaou IS, bou-Rebyeh H, Schachschal G, Veltzke-Schlieker W, Khalifa AC, Setka E, Koch M, Wiedenmann B, Rosch T. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008;57:59-64.
97. Aschenbeck J, Adler A, Yenerim T, Mayr M, Aminalai A, Drossel R, Schroder A, Scheel M, Wiedenmann B, Rosch T. Narrow-Band Versus White-Light HDTV Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008.
98. Su MY, Hsu CM, Ho YP, Chen PC, Lin CJ, Chiu CT. Comparative study of conventional colonoscopy, chromoendoscopy, and narrow-band imaging systems in differential diagnosis of neoplastic and nonneoplastic colonic polyps. Am J Gastroenterol 2006;101:2711-2716.
99. Chiu HM, Chang CY, Chen CC, Lee YC, Wu MS, Lin JT, Shun CT, Wang HP. A prospective comparative study of narrow-band imaging, chromoendoscopy, and conventional colonoscopy in the diagnosis of colorectal neoplasia. Gut 2007;56:373-379.
100. East JE, Suzuki N, Bassett P, Stavrinidis M, Thomas HJ, Guenther T, Tekkis PP, Saunders BP. Narrow band imaging with magnification for the characterization of small and diminutive colonic polyps: pit pattern and vascular pattern intensity. Endoscopy 2008;40:811-817.
101. van den Broek FJ, Reitsma JB, Curvers WL, Fockens P, Dekker E. Systematic review of narrow-band imaging for the detection and differentiation of neoplastic and nonneoplastic lesions in the colon (with videos). Gastrointest Endosc 2009;69:124-135.
102. DaCosta RS, Andersson H, Cirocco M, Marcon NE, Wilson BC. Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa. J Clin Pathol 2005;58:766-774.
103. Haringsma J, Tytgat GN, Yano H, Iishi H, Tatsuta M, Ogihara T, Watanabe H, Sato N, Marcon N, Wilson BC, Cline RW. Autofluorescence endoscopy: feasibility of detection of GI neoplasms unapparent to white light endoscopy with an evolving technology. Gastrointest Endosc 2001;53:642-650.
Chapte
r 1
Chapter I
38
104. Wang TD, Van DJ, Crawford JM, Preisinger EA, Wang Y, Feld MS. Fluorescence endoscopic imaging of human colonic adenomas. Gastroenterology 1996;111:1182-1191.
105. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplastic polyps and serrated adenomas of the colon: classification, molecular genetics, natural history, and clinical management. Gastroenterol Clin North Am 2008;37:25-46, v.
106. Jeevaratnam P, Cottier DS, Browett PJ, Van de Water NS, Pokos V, Jass JR. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179:20-25.
107. Lage P, Cravo M, Sousa R, Chaves P, Salazar M, Fonseca R, Claro I, Suspiro A, Rodrigues P, Raposo H, Fidalgo P, Nobre-Leitao C. Management of Portuguese patients with hyperplastic polyposis and screening of at-risk first-degree relatives: a contribution for future guidelines based on a clinical study. Am J Gastroenterol 2004;99:1779-1784.
108. Young J, Jenkins M, Parry S, Young B, Nancarrow D, English D, Giles G, Jass J. Serrated pathway colorectal cancer in the population: genetic consideration. Gut 2007;56:1453-1459
109 Vieth M, Quirke P, Lambert R, von KL, Risio M. Annex to Quirke et al.
Quality assurance in pathology in colorectal cancer screening and diagnosis: annotations of colorectal lesions. Virchows Arch 2011;458:21-30.
CRC cancer risk in HPS patients during follow up
39
Clinical analyses – Colorectal cancer risk
P A
R T
Chapter 2
40
CRC cancer risk in HPS patients during follow up
41
Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study
K.S. Boparai E.M.H. Mathus-Vliegen
J.J. Koornstra F.M. Nagengast M. van Leerdam
C.J.M. van Noesel
M. Houben
A. Cats
L.P. van Hest
P. Fockens
E. Dekker
Gut. 2010 Aug;59(8):1094-100
Ch
ap
ter
Chapter 2
42
ABSTRACT
Background and aims: Patients with hyperplastic polyposis syndrome
(HPS) receive endoscopic surveillance to prevent malignant
progression of polyps. However, the optimal treatment and
surveillance protocol for these patients is unknown. The aim of this
study was to describe the clinical and pathological features of a large
HPS cohort during multiple years of endoscopic surveillance. Methods:
Databases were searched for HPS patients who were retrospectively
analysed. Endoscopy reports and histopathology reports were
collected to evaluate frequency of endoscopic surveillance and to
obtain information regarding polyp and CRC presence. Results: In 77
HPS patients, 1984 polyps were identified during a mean follow-up
period of 5.6 years (range:0.5-26.6). In 27(35%) patients CRC was
detected of which 22(28.5%) at initial endoscopy. CRC was detected
during surveillance in five patients (cumulative incidence: 6.5%) after a
median follow-up time of 1.3 years and a median interval of 11 months.
Of these interval CRCs, 4/5 were detected in diminutive serrated
polyps (range: 4-16mm). The cumulative risk of CRC under
surveillance was 7% at five years. At multivariate logistic regression,
an increasing number of hyperplastic polyps (odds ratio:1.05, p=0.013)
and serrated adenomas (odds ratio:1.09, p=0.048) was significantly
associated with CRC presence. Conclusions: HPS patients undergoing
endoscopic surveillance have an increased CRC risk. The number of
serrated polyps is positively correlated with the presence of CRC in
HPS, thus supporting a ‘serrated pathway’ to CRC. To prevent
malignant progression, adequate detection and removal of all polyps
seems advisable. If this is not feasible, surgical resection should be
considered.
CRC cancer risk in HPS patients during follow up
43
INTRODUCTION
Colorectal cancer (CRC) ranks as the third most common
cause of cancer related death in the western world.1 A well known
mechanism describing CRC development is the adenoma-carcinoma
sequence which in the majority of cases is initiated by activation of the
Wnt signalling pathway.2, 3 Much information regarding this pathway
has been derived from polyposis syndromes such as familial
adenomatous polyposis (FAP) and MYH-associated polyposis (MAP).
In addition to this classical adenoma-carcinoma sequence, a proposed
“serrated neoplasia pathway”, describes the progression of serrated
polyps (i.e. hyperplastic polyps, sessile serrated adenomas and
traditional serrated adenomas) to CRC.4 It is proposed that this
possible alternative pathway is also associated with hyperplastic
polyposis syndrome (HPS). There are strong indications that mixed
pathways also exist in which both conventional adenomas and
serrated polyps are involved.5
Clinically, the condition HPS is characterized by the presence
of multiple hyperplastic polyps (HPs) spread throughout the colorectum
and is associated with an increased CRC-risk. Indeed, numerous
patients with CRC and concurrent HPS have been reported.6-12 While
previously the indicated management of HPS patients was unknown,
experts presently believe that these patients should undergo regular
endoscopic surveillance to prevent malignant progression of polyps.7, 13
However, the optimal treatment and surveillance protocol for HPS
patients is largely speculative. Therefore it seems possible that a
proportion of HPS patients may be insufficiently treated and
consequently be at risk of developing CRC under surveillance (interval
CRC).
Chapte
r 2
Chapter 2
44
The aim of this study was to describe the clinical and
pathological features of a large HPS cohort (n=77) during multiple
years of endoscopic surveillance. Furthermore, we assessed the
cumulative incidence and incidence rate of CRC during surveillance
and its association with the interval and frequency of surveillance
endoscopies. Finally, we analysed possible predictive variables that
may be associated with the occurrence of CRC in HPS.
PATIENTS AND METHODS
Study population
Databases of 7 medical centres in the Netherlands were searched for
patients satisfying the diagnostic criteria of HPS (i.e. at least five
histologically diagnosed HPs proximal to the sigmoid colon, of which 2
greater than 10mm in diameter, or more than 20 HPs distributed
throughout the colon) and undergoing endoscopic surveillance.8, 11
Owing to the common presence of both HPs and (sessile) serrated
adenomas (SAs) in HPS and the difficult histological differentiation
between these two groups, both HPs and SAs were used to fulfil the
criteria. 14-17 Of these patients, clinical data from May 1982 to June
2008 were retrospectively analysed. Adherence to the described
criteria was assessed by analysing endoscopy reports with
corresponding histopathology reports as well as histopathology reports
of colonic surgical resection specimens. This study was conducted in
accordance with the research code of our institutional medical ethical
committee on human experimentation, as well as in agreement with
the Helsinki Declaration of 1975, as revised in 1983. Patients with a
known germline APC mutation or bi-allelic MYH mutation were
excluded from the study.
CRC cancer risk in HPS patients during follow up
45
Clinical characteristics
Demographic data of patients concerning age, sex and history
of CRC were ascertained. Endoscopy reports with corresponding
histopathology reports during follow-up were collected to evaluate the
duration, interval and frequency of endoscopic surveillance and to
derive information regarding the, number, size, distribution and
histology of polyps. If applicable, histopathology reports of surgical
colonic resection specimens were also used to obtain the above
mentioned polyp characteristics. Also, if genetic mutation analysis was
performed, these data were retrieved.
Polyps were classified as HP, serrated adenoma (SA), mixed
polyp (MP) or conventional adenoma. Because the distinction between
sessile serrated adenoma and traditional serrated adenoma had not
been made throughout the study period by each medical center and
because they are both considered to be precursor lesions in the
‘serrated pathway’, the category SAs comprised both types of
lesions.16 Regarding the number of polyps detected in this study, all
polyps were tallied once i.e. when a detected polyp was not removed
during endoscopy this polyp was not re-tallied at subsequent
endoscopies.
Information concerning the nature and reason of performed
colorectal surgery was obtained if applicable. Detailed information
regarding co-existent CRC and CRC incidence during surveillance was
examined by evaluating histopathology reports of colectomy resection
specimens and/or endoscopy reports. An interval CRC was defined as
a CRC detected after HPS diagnosis after at least two previous
endoscopies.
Chapte
r 2
Chapter 2
46
Statistical analysis
Statistical analyses were performed by using a statistical
software package (Statistical Package for the Social Sciences 15.0.1;
SPSS Inc, Chicago, Ill). The cumulative risk of developing CRC during
follow-up was analyzed by Kaplan-Meier survival analysis. Observation
time was measured from date of HPS diagnosis to the incidence of
carcinoma or end of the study period. Univariate binary logistic
regression was performed for chosen variables that may be associated
with the presence of CRC. For multivariate regression analyses, only
variables which showed an association (p<0.1) on univariate analysis
were used in a final multivariate model.
RESULTS
Patients
Data of 77 HPS patients from the period 1982-2008 were
retrospectively analysed in this study. Clinical characteristics of
patients are shown in table 1. The median age at diagnosis of HPS
was 56 years (range: 40-74). There were 42 males and 35 females.
Fifty-nine of 77 patients (77%) had >5 proximal HPs (of which 2 were
larger than 10mm) or >20 HPs spread throughout the colon. The other
17 patients had >5 proximal HPs/SAs (of which 2 were larger than
10mm) or >20 HPs/SAs spread throughout the colon. In 52/77 (68%)
patients, germline APC and MYH-mutation analysis was performed. In
all cases mutation analysis was negative except for one patient with a
mono-allelic MYH-mutation (Y165C) who had ≥ 15 adenomas. In all
patients harbouring ≥ 15 adenomas (n=5) mutation analysis was
performed. Main reasons for initial presentation were: colorectal polyps
detected elsewhere (n=23), a positive family history for CRC or
colorectal polyps (n=16), bloody stools/positive faecal occult blood test/
CRC cancer risk in HPS patients during follow up
47
iron-deficiency anaemia (n=15), altered defecation pattern (n=8),
abdominal pain with or without altered bowel habits (n=5), polyps
detected at sigmoidoscopy screening programme (n=4) and personal
history of CRC (n=3).
Cumulatively, a median number of 15 HPs per patient were
found in this cohort. In 47 (61%) patients HPs ≥ 10mm were detected.
SAs and adenomas were identified in 52% and 69% of patients,
respectively. CRC was diagnosed in 27 (35%) patients: 22 (28.5%) at
initial endoscopy and 5 (6.5%) during follow-up.
A surgical colonic resection was performed in 33/77 (43%)
patients: 2 total colectomies, 13 subtotal colectomies, 8 hemi-
colectomies (6 left-sided) and 10 (recto)sigmoidal resections. Seven
patients underwent a surgical resection because of extensive
polyposis or due to difficult advancement of the endoscope during
examination resulting in incomplete visualization of the colon. The
other 26 patients underwent a colonic resection due to CRC diagnosis.
Consequently, during (a part of) follow-up 44 patients received
endoscopic surveillance of the intact colon; 17 patients of the
remaining segment proximal to the rectosigmoid colon; 15 patients of
the remaining distal colon segment and 2 patients did not receive
endoscopic surveillance after undergoing a proctocolectomy.
Chapte
r 2
Chapter 2
Nu
mb
er o
f ad
eno
ma
s
Nu
mb
er o
f MP
s
Nu
mb
er o
f SA
s
Nu
mb
er o
f HP
s
Tota
l poly
ps
- inte
rval C
RC
- at in
itial e
ndoscopy
Patie
nts
with
CR
C
Media
n n
um
ber o
f ad
en
om
as
in p
atie
nts
(rang
e)
Patie
nts
with
≥1
aden
om
a
Media
n n
um
ber o
f SA
s in
patie
nts
(ra
ng
e)
Patie
nts
with
≥1
SA
N
um
ber o
f pa
tients
with
an H
P ≥
10m
m
Media
n n
um
ber o
f pro
x. H
Ps
in p
atie
nts
(rang
e)
Media
n n
um
ber o
f HP
s
in p
atie
nts
(rang
e)
Media
n in
terv
al e
nd
osco
pie
s
in m
onth
s (ra
ng
e)
Mea
n F
U-tim
e a
fter H
PS
dia
gnosis
in
yrs
(rang
e)
Mea
n F
U-tim
e in
yrs
(ran
ge)
Media
n a
ge a
t dia
gnosis
(ran
ge
)
273
(14%
)
2 (0
.1%
)
302
(15%
)
140
7 (7
2%
)
198
4
5 (6
.5%
)
22 (2
8.5
%)
27 (3
5%
)
2 (0
-26)
53 (6
9%
)
1 (0
-24)
40 (5
2%
)
47 (6
1%
)
8 (1
-45)
15 (2
-53
)
11 (1
-96
)
4.0
(0.4
-21.0
)
5.6
(0.5
-26,6
)
56 (4
0-7
4)
All c
entre
s
(n=
77)
160
(14%
)
2 (0
.2%
)
259
(23%
)
705
(63%
)
112
4
4 (2
7%
)
11 (7
3%
)
15 (3
5%
)
2 (0
-26)
31 (7
2%
)
5 (0
-24)
32 (7
4%
)
25 (5
8%
)
7 (1
-30)
14 (2
-53
)
9 (1
-96)
3.2
(1.3
-6.1
)
4.4
(1.3
-9.3
)
55 (4
0-7
4)
Centre
1
(n=
43)
31 (1
1%
)
0
6 (2
%)
258
(87%
)
295
0
6 (1
00%
)
6 (7
5%
)
2 (0
-16)
6 (7
5%
)
0 (0
-6)
1 (1
3%
)
5 (6
3%
)
29 (5
-45
)
29 (1
1-7
3)
23 (5
-26
)
8.1
(1.1
-20.8
)
10.6
(2.9
-26.4
)
51 (4
2-6
9)
Centre
2
(n=
8)
46 (2
2%
)
0
9 (4
%)
161
(74%
)
216
1 (3
3%
)
2 (6
7%
)
3 (4
3%
)
6 (0
-14)
6 (8
6%
)
0 (0
-8)
2 (2
9%
)
5 (7
1%
)
10 (2
-21
)
25 (6
-45
)
7 (3
-63)
5.8
(1.1
- 11
.7)
6.4
(1.1
-12.3
)
56 (4
6-6
5)
Centre
3
(n=
7)
12 (1
4%
)
0
1 (1
%)
74 (8
5%
)
87
0
1 (1
00%
)
1 (1
7%
)
1 (0
-9)
3 (5
0%
)
0 (0
-1)
1 (1
7%
)
3 (5
0%
)
7 (2
-12)
10 (6
-27
)
6 (1
-17)
2.1
(1.2
-4-4
)
2.6
(1.2
-5.1
)
58 (5
6-6
7)
Centre
4
(n=
6)
Table
1. C
hara
cte
ristic
s o
f HP
S p
atie
nts
div
ided p
er c
entre
.
9 (8
%)
0
23 (2
5%
)
61 (6
7%
)
93
0
0
0
2 (0
-3)
4 (8
0%
)
1 (0
-20)
3 (6
0%
)
4 (8
0%
)
6 (2
-12)
12 (2
-22
)
8 (1
-24)
3.3
(1.9
-4.7
)
4.3
(3.0
-5.7
)
51 (4
3-7
2)
Centre
5
(n=
5)
8 (7
%)
0
0
109
(93%
)
117
0
0
0
1 (0
-5)
3 (6
0%
)
0
0
4 (8
0%
)
10 (4
-30
)
21 (9
-41
)
11 (3
-53
)
2.2
(1.5
-4.1
)
2.2
(1.5
-4.1
)
52 (4
5-6
1)
Centre
6
(n=
5)
7 (1
4%
)
0
4 (8
%)
39 (7
8%
)
50
0
2 (1
00%
)
2 (6
7%
)
0 (0
-7)
0 (0
-1)
0 (0
-1)
1 (3
3%
)
1 (3
3%
)
5 (5
-13)
16 (5
-18
)
13 (1
0-2
5)
3.2
(1.3
-6.1
)
4.4
(1.3
-9.3
)
57 (4
8-6
9)
Centre
7
(n=
3)
Clin
ical c
hara
cte
ristic
s o
f HP
S p
atie
nts
from
multip
le c
entre
s in
the N
eth
erla
nds
CRC cancer risk in HPS patients during follow up
The mean follow-up period of patients was 5.6 years (range:
0.5 – 26.6) and from the point HPS was diagnosed this was 4.0 years
(range: 0.4 -21.0). During follow-up, the number of surveillance
endoscopies varied among patients. In the observed time period, 207
surveillance endoscopies were performed (median 3, range 0-11). One
patient was diagnosed with HPS based on the surgical resection
specimen and had not yet undergone surveillance endoscopies. The
median interval between surveillance endoscopies was 10 months
(range: 1-96).
Polyps
Polyp characteristics are outlined in table 1. In this study, 847/1407
(60%) HPs, 197/302 (65%) SSAs and 165/273 (60%) adenomas were
detected proximal to the sigmoid colon. The maximum size of HPs and
SAs was 30mm which were located in the ascending and transverse
colon, respectively. The largest adenoma detected in this cohort was
75mm, which was located in the ascending colon. Polyps were
detected during endoscopy with standard or high-resolution white-light
endoscopy. Narrow-band imaging was used in 22/294 (7%)
endoscopies in 22 patients.
CRC
Of the 77 HPS patients included in this study, 27 (35%) patients had
CRC (median age 56 years; range 36-75). In 14/27 (52%) of these
patients, CRC was located proximal to the sigmoid colon. One patient
had two separate synchronous CRCs, one proximally and one distally
located.
Chapte
r 2
Chapter 2
50
While CRC was diagnosed at initial colonoscopy in the majority
(22/27) of HPS patients, in five patients (cumulative incidence: 6.5%)
with a median age of 58 years (range 49-68) CRC was detected during
surveillance after the diagnosis HPS was made (mean follow-up time
5.6 yrs) without any prior history of CRC. The median follow-up time in
this group was 1.3 years (range: 0.4-6.7) with a median of 3
endoscopies (range: 2-4). Clinical and histological data of these
patients are summarized in table 2. During a total of 294.6 person
years of follow-up, this corresponds with a CRC- incidence rate during
surveillance of 17 per 1000 person-years. In four of the five (80%)
patients, CRC was detected during a planned endoscopy and was
located within a HP (3/4) or a SA (1/4) without causing clinical
symptoms. The median size of these polyps was 10mm (range: 4-
16mm). One patient (patient 2) presented with weight loss and fatigue
after a surveillance interval of more than 3 years. At endoscopy a large
CRC was detected. In four of five patients, CRC was located
proximally to the sigmoid colon. The median interval between
surveillance endoscopies in patients with an interval CRC was 11
months (range: 3-43) compared to 10 months (range: 1-96) in HPS
patients without an interval CRC (ns). The median interval between the
last surveillance endoscopy and CRC detection in patients with interval
carcinomas was also 11 months (range: 4-43). The calculated
cumulative risk of CRC in HPS during surveillance was 7% at five
years (figure 1). When analysing the cumulative risk separately for
patients with an intact colon and for patients with a surgical colonic
resection, the 5-year cumulative risk was 6% and 4% respectively.
CRC cancer risk in HPS patients during follow up
5
4
3
2
1
Patie
nt
48
48
58
67
49
Age
F
M
M
F
M
Sex
3
2
4
3
2
Num
ber o
f end
oscopie
s
untill C
RC
Desce
ndin
g c
olo
n
Tra
nsvers
e c
olo
n
Rectu
m
Ascen
din
g
colo
n
Ascen
din
g
colo
n
Locatio
n
CR
C
not s
tate
d
(Tis
N0M
O )
10m
m
(T1N
0M
0)
4m
m
(Tis
N0M
0)
95m
m
(T3N
0M
0)
16m
m
(T3N
0M
0)
Siz
e C
RC
(T
NM
)
HP
HP
HP
non
e
SA
Imm
edia
te
adja
cen
t poly
ps
15.6
4.3
36.4
80.4
11.4
Tim
e (m
onth
s)
betw
ee
n H
PS
dia
gnosis
and
CR
C d
ete
c
10.1
4.3
11.6
44.2
7.7
Tim
e (m
onth
s)
betw
ee
n la
st
end
oscopy a
nd
dia
gnosis
CR
C
Caecum
Caecum
Sig
moid
(sub
tota
l cole
cto
my)
Tra
nsvers
e c
olo
n
Caecum
Most p
roxim
al
intu
batio
n p
oin
t la
st e
nd
oscopy
Ta
ble
2: C
hara
cte
ristic
s o
f HP
S p
atie
nts
in w
hic
h a
n in
terv
al c
arc
inom
a w
as d
ete
cte
d.
Multip
le p
oly
ps th
roug
ho
ut th
e
colo
rectu
m., w
hic
h w
ere
only
dia
gnostic
ally
bio
psie
d
Multip
le p
oly
ps d
ete
cte
d, o
f whic
h
11 p
oly
ps w
ere
rem
ove
d. M
ultip
le
poly
ps re
main
ed in
situ
All v
isib
le re
cto
sig
moid
al p
oly
ps
rem
oved.
Multip
le p
oly
ps th
roug
ho
ut th
e
colo
rectu
m. P
roce
dure
com
plic
ate
d
by p
erfo
ratio
n (a
borte
d). M
ultip
le
poly
ps re
main
ed in
situ
. Pa
tien
t re
turn
ed w
ith s
ym
pto
ms a
fter 4
4.2
m
on
ths.
>20 p
oly
ps w
hic
h w
ere
only
dia
gnostic
ally
bio
psie
d.
Abno
rmalitie
s a
nd tre
atm
en
t durin
g la
st e
ndoscop
y
befo
re d
iagn
osis
CR
C
Chapte
r 2
Chapter 2
52
To analyse an association with CRC in HPS, univariate logistic
regression was performed for 8 independent variables: age; sex;
number of HPs; number of SAs; number of adenomas; largest HP;
largest SA and largest adenoma (table 3). At univariate logistic
regression, the number of HPs and the number of SAs were
associated with CRC (p<0.1). At subsequent multivariate logistic
regression the number of HPs (p=0.013) and the number of SAs
(p=0.048) were significantly associated with CRC with corresponding
odds ratios of 1.05 (95%-CI: 1.01-1.10) and 1.09 (95%-CI: 1.00-1.19)
respectively.
Prognostic variables (univariate analysis)
Odds ratio 95%-CI p-value
Age (per year) 1.04 0.98-1.11 0.162
Male sex 0.67 0.26-1.72 0.408
Number of HPs (per polyp) 1.05 1.01-1.09 0.018*
Number of SAs ( per polyp) 1.08 0.99-1.17 0.076*
Number of adenomas (per polyp) 1.01 0.92-1.11 0.870
Largest HP (per mm) 0.98 0.91-1.06 0.611
Largest SA (per mm) 1.03 0.97-1.10 0.302
Largest adenoma (per mm) 0.99 0.96-1.04 0.887
Prognostic variables (multivariate analysis)
Odds ratio 95%-CI p-value
Number of HPs (per polyp) 1.05 1.01-1.10 0.013*
Number of SAs ( per polyp) 1.09 1.00-1.19 0.048*
Table 3. Results of univariate and multivariate logistic regression analysis: independent prognostic variables and corresponding odds ratios for the presence of colorectal cancer in hyperplastic polyposis syndrome. * Statistically significant p-value for univariate analysis: p< 0.1 and for multivariate analysis: p< 0.05
CRC cancer risk in HPS patients during follow up
53
Figure 1. Cumulative proportion of HPS patients with colorectal cancer during surveillance
DISCUSSION
This multicentre cohort study showed that in a total of 27/77 (35%)
HPS patients, CRC was detected. Interestingly, CRC was detected in
5/77 (6.5%) patients during surveillance of which four CRCs within a
diminutive serrated polyp (HP or SA) resulting in a cumulative risk of
CRC under endoscopic surveillance of 7% in 5 years. This is
substantial considering that the lifetime risk of developing CRC in the
general population is estimated to be 6%.18 Of these patients with
interval CRCs, two CRCs were detected within a year (table 2: 11.4
and 4.3 months) after the diagnosis HPS was made and after two
previous endoscopies. The high frequency of endoscopies in a short
time period suggest that these patients were probably still in an
Chapte
r 2
Chapter 2
54
orientating treatment phase when CRC was detected. If surveillance
was defined as endoscopies performed after HPS diagnosis after at
least one year follow-up, the cumulative risk under surveillance would
be 4% at five years.
Although different management protocols have recently been
advised, thus far no uniform and adequately substantiated
management protocol exists for the endoscopic management of HPS
patients. Consequently, lack of clarity exists regarding the
recommended surveillance interval and which polyps to remove.
Recent studies recommend surveillance intervals ranging from one to
three years and concerning the removal of polyps, advice varies from
removal of only proximally located polyps to complete removal of all
polyps >5mm.7, 13
This absence of a standardized treatment protocol may also be
associated with the incidence of interval CRCs in this retrospective
multicentre study dating back to 1982. Possible explanations for the
incidence of interval CRCs could be that previously an association
between HPS and CRC was not made or that only proximal and/or
larger lesions were considered clinically significant, resulting in
incomplete removal of polyps. Also when considering the relatively
short median interval between endoscopies (median interval:
11months), it is likely that the interval CRCs were also present at prior
surveillance endoscopies but were not removed. This was indeed the
case for two of five of these patients who underwent incomplete
removal of all detected polyps during the last surveillance endoscopy
before CRC diagnosis (table 2: patient 2 and 4), underlining the
importance of comprehensive polyp removal during surveillance
Conversely, in three of five patients (patients 1, 3 and 5) all
detected polyps were biopsied or removed at previous endoscopy. A
CRC cancer risk in HPS patients during follow up
55
possible explanation for this contrary finding could be that these CRCs
originating in serrated polyps were simply missed. This could possibly
be due to the multiplicity of polyps seen in HPS patients resulting in a
sub-optimal overview of all colorectal polyps. Alternatively, typical HPs
and (sessile) serrated adenomas seldom exceed 10mm in size,
suggesting that most polyps in HPS are diminutive.19-25 It has been
shown that the miss-rate of polyps <10mm can be as high as 23%.26
This could explain why these relatively small CRCs originating in
diminutive serrated polyps were not detected at previous endoscopy.
Nevertheless, considering their small size and the unknown
progression rate in HPS, it can not be excluded that these CRCs,
originating in serrated polyps (4/5) developed since the last
endoscopy. A previous retrospective polyp study of consecutive
patients with an average risk for CRC showed that the estimated
growth rates of HPs and SAs (both sessile- and tradional) compared to
conventional adenomas were similar or significantly higher.27
Moreover, a recent case report described the progression of a sessile
serrated adenoma to carcinoma within 8 months.28 In this respect, it is
conceivable that in HPS a subset of serrated polyps have an increased
progression rate leading to CRC. This is an interesting point
considering that the risk of high grade dysplasia or even invasive
cancer in diminutive lesions (<10mm) has been shown to be <2%. 29-31
The finding of CRC within a small serrated polyp in 4/5 (80%) interval
carcinomas suggests that in HPS small polyps have a greater
malignant potential than in the general population.
When considering the management of HPS patients, this study
suggests that the absence of a clear treatment protocol plays a role in
the presence of CRCs during surveillance. Considering that CRCs
detected in this study were as small as 4mm (detected in a HP: patient
Chapte
r 2
Chapter 2
56
3), removal of all polyps seems advisable but needs to be
prospectively assessed. Although these recommendations seem of
importance in preventing malignant progression in HPS, practical
difficulties may present when trying to comply with these guidelines in
a clinical setting. Firstly, besides being small, detection of HPs and
SAs is also complicated by their predominantly flat shape,
unremarkable colour and mucus coating which possibly increases
polyp miss rates.19, 32 Secondly, removal of all detected polyps during
endoscopic surveillance sessions in HPS patients with a large quantity
of polyps can be time-consuming and unfeasible, especially when
endoscopic mucosal resection (EMR) is indicated for predominantly
flat shaped serrated polyps.
With regard to polyp detection, previous randomized controlled
trials demonstrated that chromoendoscopy and narrow-band imaging
(NBI) increased the detection of HPs.33-38 Although not formally
investigated, these techniques could in this respect also be of value for
the detection of serrated polyps in HPS. Concerning polyp removal,
the multiplicity of polyps and the use of EMR can indeed lead to
increased duration of endoscopic procedures in HPS patients. In this
respect, it is important that these endoscopies are performed by
endoscopists experienced in EMR for complete, prompt and safe polyp
removal and that allowances are made for sufficient procedure time.
Annual surveillance by an experienced endoscopist specialized in
HPS, having advanced imaging techniques available such as
chromoendoscopy and NBI seems therefore advisable. Alternatively,
when complete endoscopic removal of all polyps is not feasible,
surgical colonic resection should seriously be considered since these
patients have an increased risk of malignant progression of polyps.
CRC cancer risk in HPS patients during follow up
57
In this study, at multivariate logistic regression, an increasing
number of HPs and SAs was significantly associated with CRC
presence (OR of 1.05 and 1.09 respectively per polyp). In other words,
the CRC risk will increase by 5% and 9% respectively with each
additional HP or SA. Concordantly, results from previous literature
reports strongly suggest that SAs in particular play a role in a ‘serrated
pathway’ leading to CRC in HPS.19, 39-41 A possible explanation for the
significant association between HPs and CRC in this study could be
that HPs and SAs (primarily sessile serrated adenomas) are
histologically hard to distinguish, leading to incorrect differentiation and
misdiagnosis of these serrated polyps. Indeed, it has recently been
shown that even at re-evaluation the interobserver agreement for the
differentiation of serrated polyps remains only moderate (к=0.55).14, 15,
42 Nevertheless, the significant association between serrated polyps
and CRC in this study supports the hypothesis of a ‘serrated pathway’
leading to CRC in HPS (figure 2).
Figure 2. Endoscopic image of a serrated adenoma (10mm, ascending colon) detected in a patient with hyperplastic polyposis syndrome (A). At microscopy a focus of adenocarcinoma was seen within the serrated adenoma (B and C).
In conclusion, HPS is associated with an increased personal
CRC risk, even under endoscopic surveillance. Considering that these
advanced lesions were detected in polyps as small as 4mm (median:
Chapte
r 2
Chapter 2
58
10mm), which were not recognized as such, all polyps in HPS seem at
risk of representing advanced lesions warranting removal of all polyps.
However, the miss rate of polyps <10mm (which represents the
majority of polyps in HPS) has been shown to be as high as 23% with
standard white-light endoscopy suggesting that a considerable number
of polyps in HPS are missed. 26 Advanced endoscopic imaging
techniques such as chromoendoscopy and NBI may in this respect be
of additional value for the detection of polyps in HPS. Alternatively, if
endoscopically unfeasible, preventive colonic resection should be
considered. An increasing number of serrated polyps are associated
with the presence of CRC in HPS, supporting the theory of a ‘serrated
pathway’ leading to CRC. Future prospective data from large HPS
cohorts, undergoing a standardized treatment protocol are required to
further enhance our knowledge with regard to the rate of polyp
progression in these patients and to determine the optimal treatment
and surveillance protocol for these patients.
CRC cancer risk in HPS patients during follow up
59
REFERENCES 1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,
Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK. Annual Report to the Nation on the Status of Cancer, 1975-2005, Featuring Trends in Lung Cancer, Tobacco Use, and Tobacco Control. J Natl Cancer Inst 2008.
2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767.
3. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532.
4. Makinen MJ. Colorectal serrated adenocarcinoma. Histopathology 2007;50:131-150.
5. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.
6. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.
7. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
8. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
9. Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y, Watanabe H. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52:5-9.
Chapte
r 2
Chapter 2
60
10. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, Cohn D, Furlong KL, Walsh MD, Palazzo J, Edmonston TB, Fishel R, Young J, Leggett BA. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47:43-49.
11. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
12. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
13. East JE, Saunders BP, Jass JR. Sporadic and syndromic hyperplastic polyps and serrated adenomas of the colon: classification, molecular genetics, natural history, and clinical management. Gastroenterol Clin North Am 2008;37:25-46, v.
14. Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY, Mino-Kenudson M. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008;32:30-35.
15. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.
16. Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81.
17. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.
18. Winawer S, Fletcher R, Rex D, Bond J, Burt R, Ferrucci J, Ganiats T, Levin T, Woolf S, Johnson D, Kirk L, Litin S, Simmang C. Colorectal cancer screening and surveillance: clinical guidelines and rationale-Update based on new evidence. Gastroenterology 2003;124:544-560.
19. Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T, Simms LA, Young J, James M, Montgomery GW, Appleyard M, Hewett D, Togashi K, Jass JR, Leggett BA. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400-1407.
CRC cancer risk in HPS patients during follow up
61
20. DiSario JA, Foutch PG, Mai HD, Pardy K, Manne RK. Prevalence and malignant potential of colorectal polyps in asymptomatic, average-risk men. Am J Gastroenterol 1991;86:941-945.
21. Estrada RG, Spjut HJ. Hyperplastic polyps of the large bowel. Am J Surg Pathol 1980;4:127-133.
22. Hayashi T, Yatani R, Apostol J, Stemmermann GN. Pathogenesis of hyperplastic polyps of the colon: a hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 1974;66:347-356.
23. Imperiale TF, Wagner DR, Lin CY, Larkin GN, Rogge JD, Ransohoff DF. Results of screening colonoscopy among persons 40 to 49 years of age. N Engl J Med 2002;346:1781-1785.
24. Johnson DA, Gurney MS, Volpe RJ, Jones DM, VanNess MM, Chobanian SJ, Avalos JC, Buck JL, Kooyman G, Cattau EL, Jr. A prospective study of the prevalence of colonic neoplasms in asymptomatic patients with an age-related risk. Am J Gastroenterol 1990;85:969-974.
25. Lieberman DA, Prindiville S, Weiss DG, Willett W. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-2967.
26. van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006;101:343-350.
27. Lazarus R, Junttila OE, Karttunen TJ, Makinen MJ. The risk of metachronous neoplasia in patients with serrated adenoma. Am J Clin Pathol 2005;123:349-359.
28. Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y, Oda J, Mizutani M, Takayanagi S, Kishi D, Shinohara T, Yamada K, Matumoto J, Imamura K. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008.
29. Butterly LF, Chase MP, Pohl H, Fiarman GS. Prevalence of clinically important histology in small adenomas. Clin Gastroenterol Hepatol 2006;4:343-348.
30. East JE, Suzuki N, Saunders BP. Comparison of magnified pit pattern interpretation with narrow band imaging versus chromoendoscopy for diminutive colonic polyps: a pilot study. Gastrointest Endosc 2007;66:310-316.
Chapte
r 2
Chapter 2
62
31. Sano Y, Ikematsu H, Fu KI, Emura F, Katagiri A, Horimatsu T, Kaneko K, Soetikno R, Yoshida S. Meshed capillary vessels by use of narrow-band imaging for differential diagnosis of small colorectal polyps. Gastrointest Endosc 2008.
32. Yano T, Sano Y, Iwasaki J, Fu KI, Yoshino T, Kato S, Mera K, Ochiai A, Fujii T, Yoshida S. Distribution and prevalence of colorectal hyperplastic polyps using magnifying pan-mucosal chromoendoscopy and its relationship with synchronous colorectal cancer: prospective study. J Gastroenterol Hepatol 2005;20:1572-1577.
33. Kiesslich R, von BM, Hahn M, Hermann G, Jung M. Chromoendoscopy with indigocarmine improves the detection of adenomatous and nonadenomatous lesions in the colon. Endoscopy 2001;33:1001-1006.
34. Lecomte T, Cellier C, Meatchi T, Barbier JP, Cugnenc PH, Jian R, Laurent-Puig P, Landi B. Chromoendoscopic colonoscopy for detecting preneoplastic lesions in hereditary nonpolyposis colorectal cancer syndrome. Clin Gastroenterol Hepatol 2005;3:897-902.
35. Lee JH, Kim JW, Cho YK, Sohn CI, Jeon WK, Kim BI, Cho EY. Detection of colorectal adenomas by routine chromoendoscopy with indigocarmine. Am J Gastroenterol 2003;98:1284-1288.
36. Ratiu N, Gelbmann C, Rath HR, Herfarth H, Kullmann F, Scholmerich J, Messmann H. Chromoendoscopy with indigo carmine in flexible sigmoidoscopy screening: does it improve the detection of adenomas in the distal colon and rectum? J Gastrointestin Liver Dis 2007;16:153-156.
37. Adler A, Aschenbeck J, Yenerim T, Mayr M, Aminalai A, Drossel R, Schroder A, Scheel M, Wiedenmann B, Rosch T. Narrow-Band Versus White-Light High Definition Television Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008.
38. Kaltenbach T, Friedland S, Soetikno R. A randomised tandem colonoscopy trial of narrow band imaging versus white light examination to compare neoplasia miss rates. Gut 2008;57:1406-1412.
39. Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D, Young J. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49:121-131.
CRC cancer risk in HPS patients during follow up
63
40. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.
41. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J, Jass JR. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467-1474.
42. Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996;110:748-755.
Chapte
r 2
Chapter 2
64
Increased colorectal cancer risk in first degree relatives of patients with hyperplastic polyposis syndrome
K.S. Boparai J.B. Reitsma
V. Lemmens
T.A.M. van Os E.M.H. Mathus-Vliegen
J.J. Koornstra
F.M. Nagengast
L.P. van Hest
J.J. Keller
E. Dekker
Gut. 2010 Sep;59(9):1222-5
Ch
ap
ter
Chapter 3
66
ABSTRACT Introduction Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal hyperplastic polyps and is associated with an increased colorectal cancer (CRC) risk. For first-degree relatives of HPS patients (FDRs) this has not been adequately quantified. Reliable evidence concerning the magnitude of a possible excess risk is necessary to determine whether preventive measures, like screening colonoscopies, in FDRs are justified. Aims and methods We analyzed the incidence rate of CRC in FDRs and compared this with the general population through person-year analysis after adjustment for demographic characteristics. Population-based incidence data from the Eindhoven Cancer Registry during the period 1970-2006 were used to compare observed numbers of CRC cases in FDRs with expected numbers based on the incidence in the general population. Results A total of 347 FDRs (41% male) from 57 pedigrees were included, contributing 11.053 person-years of follow-up. During the study period, a total of 27 CRC cases occurred among FDRs compared to 5 expected CRC cases (p<0.001). The relative risk of CRC in FDRs compared to the general population was 5.4 (95%-CI: 3.7-7.8). Four FDRs satisfied the criteria for HPS. Based on the estimated HPS prevalence of 1:3000 in the general population the projected relative risk of HPS in FDRs was 39 (95%-CI: 13-121). Conclusions FDRs of HPS patients have an increased risk for both CRC and HPS compared to the general population. Hence, as long as no genetic substrate has been identified, screening colonoscopies for FDRs seem justified but this needs to be prospectively evaluated.
CRC cancer risk in first-degree relatives of HPS patients
Introduction
Hyperplastic polyposis syndrome (HPS) is a condition characterized by
the presence of multiple hyperplastic polyps (HPs) spread throughout
the colon and is associated with an increased colorectal cancer risk. 1-5
Because of this increased risk of malignant progression, HPS patients
undergo endoscopic surveillance with removal of polyps or surgical
colonic resection. Although an increased CRC risk for these patients
has been established, it is uncertain whether an increased risk of CRC
and/or HPS for first-degree relatives (FDRs) exists and consequently
whether preventive measures, like screening colonoscopies, should be
performed in this group.
Although HPS was initially considered to be non-familial,
previously published case series report that up to 50% of HPS patients
have a FDR with CRC. 2, 6 In addition, HPS presence has been
described in multiple family members.2, 3, 7-9 Based on these reports, a
yet unidentified underlying genetic defect seems to play a role in at
least some HPS cases.
Concordantly, previous studies have shown that HPS is caused in a
small proportion of patients by a germline mutation in the MUTYH
gene resulting in a phenotype of multiple conventional adenomas and
serrated polyps.2, 10 However, in the overall majority it is unclear what
proportion of FDRs are at risk of developing CRC and/or HPS.
Furthermore, the possible mode of inheritance is unknown. Families
have been reported previously from which both an autosomal
recessive and autosomal dominant inheritance could be considered.
2, 6, 8, 9 Thus, to adequately inform FDRs regarding their risk of CRC
and/or HPS, reliable evidence concerning the magnitude of this excess
Chapte
r 3
Chapter 3
68
risk is necessary. The aim of this study was to estimate the incidence
rate of CRC and HPS in FDRs of HPS patients and to compare this
with the general population so as to determine whether preventive
measures such as screening colonoscopies in this group are justified.
Methods
Besides HPs, HPS patients often have multiple sessile serrated
adenomas (SSAs), traditional serrated adenomas and conventional
adenomas. HPs and SSAs have been shown to be histologically very
similar and difficult to differentiate microscopically with only moderate
concordance.11-14 For this reason HPS was defined as at least five
histologically diagnosed HPs and/or SSAs proximal to the sigmoid
colon, of which 2 greater than 10mm in diameter, or more than 20 HPs
and/or SSAs distributed throughout the colon. HPS patients (probands)
from 4 medical centres in the Netherlands were included when
satisfying the above mentioned diagnostic criteria for HPS. Of these
patients, family history data were obtained through face to face- and
telephone interviews or by examining data from the departments of
Clinical Genetics. Patients with a known germline APC mutation or bi-
allelic MUTYH mutation were excluded from the study. This study was
conducted in accordance with the research code of our institutional
medical ethical committee on human experimentation, as well as in
agreement with the Helsinki Declaration of 1975, as revised in 1983.
Risk assessment for CRC was performed by including person-
years at risk for CRC from 1 January 1970 until 1 January 2009
(censory date). FDRs were considered at risk from birth until date of
CRC diagnosis, date of death or the censory date. It was presumed
that from the time the proband developed CRC FDRs would have a
CRC cancer risk in first-degree relatives of HPS patients
69
higher chance of receiving endoscopic screening for CRC which could
cause a bias when comparing with the general population. For this
reason, person-years at risk also ended at the date of CRC diagnosis
in the proband if applicable.
Person-years at risk were stratified according to 5-year age
group, gender and calendar year using SAS software, version 9.1
(SAS Institute Inc, Cary, NC). Similarly stratified population-based data
from the Eindhoven Cancer Registry were used to compare the
incidence of CRC in FDRs with that of the general population.15 The
expected CRC incidence in FDRs was calculated by applying the age-,
gender- and calendar-specific incidence rates of the general
population to the composition and follow-up years of the FDR cohort.
The observed versus expected number of CRC cases were formally
compared by calculating a relative risk and 95% confidence interval
(CI). This relative risk is calculated by taking the ratio of observed to
expected number of cases and its confidence interval by assuming a
Poisson distribution of cases. Two-sided P-values <0.05 were
considered statistically significant.
RESULTS
First-degree relatives (FDRs)
In this study a total of 347 FDRs (142 male) from 57 pedigrees were
included, contributing 11.053 person-years of follow-up (figure 1). The
median age at end of follow-up was 60 years (interquartile range: 44-
71). These FDRs consisted of 165 (48%) siblings, 100 (35%) parents
and 82 (17%) children. In total, 27 FDRs, consisting of 14 parents, 12
siblings and 1 child were excluded because (i) they died before 1
January 1970 (n=17) or (ii) information regarding that individual was
unknown (n=10).
Chapte
r 3
Chapter 3
70
Figure 1. Flow diagram
Colorectal cancer
During the study period, a total of 27 (8%) CRC cases occurred among
FDRs. The median age of CRC occurrence was 62 years (interquartile
range: 57-78) and 15 (56%) were of the male gender (table 1). The
relative risk of CRC in FDRs compared to the general population was
5.4 (95%-CI: 3.7-7.8). One additional CRC case occurred outside the
study period (before 1970) and was therefore not included. There were
no excluded CRC cases in FDRs which developed after CRC
diagnosis in the proband. Of the 27 FDRs with CRC, 4 probands
developed CRC at a later stage. In total, 16/57 (28%) probands were
diagnosed with CRC during the study period.
CRC cancer risk in first-degree relatives of HPS patients
71
Table 1. Risk analysis of colorectal cancer (CRC) in first-degree relatives
of HPS patients compared with population-based data from the Eindhoven Cancer
Registry
Group Person-
years
Observed
CRCs
Expected
CRCs
RR
(observed/e
xpected)
95%-CI
Males 4736 15 2.3 6.5 3.9 -10.8
Females 6317 12 2.6 4.6 2.6 - 8.0
Combined 11053 27 5.0 5.4 3.7 -7.8
The difference in CRC risk between men and women within the FDR
group was not statistically different (RR: 1.9, 95%-CI: 0.9-4.1, Pearson
Chi-Square test: p=0.089). Also when comparing this between siblings
(brothers and sisters) and non-siblings (parents and children) within
the FDR group, no significant difference was seen (RR: 1.0, 95%-CI:
0.4-2.5, Pearson Chi-Square test: p=0.96). Other malignancies
recorded in the study period included breast cancer (n=10), gastric
cancer (n=2), ovarian cancer (n=2) and others (n=12).
Polyps
Endoscopies were performed in 65/347 (19%) of FDRs. Reasons for
endoscopy were not recorded. In this group, 35/65 (54%) individuals
had colorectal lesions from whom 24 the histology was known. In 7
FDRs multiple histologically confirmed HPs (≥ 5) were identified at a
median age of 58 years (range: 50-75) of which 6 had HPs proximal to
the rectosigmoid colon (table 2). Four of these FDRs from 4 different
pedigrees satisfied the criteria for HPS. Based on the estimated HPS
prevalence of 1:3000 in the general population, the projected relative
risk of HPS in FDRs would be 39 (95%-CI: 13-121). In FDRs with
CRC, 3/27 (11%) of cases had multiple HPs (two had HPS) compared
Chapte
r 3
Chapter 3
72
to 4/320 (1%) non-CRC cases (Fisher’s Exact Test: p=0.012). In 3
other FDRs, multiple polyps were detected of which the histology was
unknown. The difference in risk of having multiple HPs between men
and women within the FDR group was not statistically different (RR:
1.6, 95%-CI: 0.4-7.4, Fisher’s exact test: p=0.71). Also when
comparing this between siblings and non-siblings within the FDR group
no significant difference was seen (RR: 3.1, 95%-CI: 0.6-16.1, Fisher’s
exact test: p=0.252).
Table 2. First degree relatives (FDR) of HPS patients with multiple (≥ 5)
hyperplastic polyps (HPs).
* Patients satisfying the criteria for HPS.
FDR Relation Age at
endoscopy
Number of
HPs
Location Colorectal
cancer
(location)
1 mother 75 6 Distal colon No
2 sister 59 6 Pancolonic Yes (right)
3 brother 50 9 Rectosigmoid No
4 father 66 >10* Pancolonic Yes (right)
5 sister 56 >40* Pancolonic Yes (right)
6 brother 58 >10* Pancolonic No
7 brother 58 23* Distal colon No
CRC cancer risk in first-degree relatives of HPS patients
73
Discussion
This retrospective study describes the largest series of FDRs of HPS
patients in which the presence of CRC and polyps was assessed and
is, to our knowledge, the first to quantify the relative risk of CRC and/or
HPS in FDRs. Our results showed that FDRs of HPS patients have an
increased risk for both CRC and HPS compared to the general
population warranting screening colonoscopies for this group.
A limitation of this study is that study data were collected in a
retrospective manner and based on medical charts and self-reported
information about family history. An important question therefore is
whether our approach could have lead to an over- or underestimation
of the incidence of CRC in FDRs. An overestimation of CRC risk may
cause stress16, unneeded referrals for genetic counseling and possible
unnecessary endoscopic procedures or surgery.17, 18 However,
previous studies evaluating the accuracy of patient reporting of familial
CRC by comparing patient family history reports with cancer registries,
showed that the specificity (i.e. the proportion of CRC-negatives which
are correctly identified as not having CRC) was 92-99%. These
findings imply that patients seldom incorrectly report CRC-negative
FDRs as having CRC, which would lead to an overestimation of CRC-
incidence in FDRs. Moreover, the sensitivity (i.e. the proportion of
CRC-positives which are correctly identified as having CRC) was 53-
86%. In other words, patients tend to under-report the incidence of
CRC in FDRs, leading to underestimation of CRC-incidence in
FDRs.19-21 Furthermore, the majority of probands included in this study
were symptomatic patients and thus represent a selected patient
population. These patients may have a more aggressive form of HPS
and also a higher risk of (familial) CRC than other unidentified
asymptomatic HPS cases. Therefore, our findings concerning their
Chapte
r 3
Chapter 3
74
FDRs can not by default be extrapolated to all (including unidentified)
HPS cases. However, considering that the aim of our study was to
analyze the risk of CRC and polyps in FDRs of all identified HPS
patients, we believe our findings are relevant for the management of
HPS patients and their relatives in a clinical setting. Finally, in this
study, 19% of FDRs received an endoscopy during follow-up for which
the reason was unknown. It was unknown at what rate endoscopies
were performed in the general population during the study period.
However, considering that FDR follow-up time ended at the date of
CRC diagnosis (if applicable) in the proband, we believe that the
amount of performed screening endoscopies for familial CRC in FDRs
will be comparable to the general population. Similarly, screening
endoscopies for HPS in FDRs have only recently been proposed by
some authorities and is to this date not standard practice of care.2, 6, 22
For this reason it is also unlikely that FDRs in this study received more
screening endoscopies for HPS than the general population.
In probands (i.e. HPS patients) a higher CRC incidence was
observed (16/57: 28%) during the study period compared to FDRs
(27/347: 8%). This finding was expected considering that not all FDRs
also had HPS. However, of the 4 FDRs with HPS, 2 (50%) had CRC
suggesting that the presence of multiple serrated polyps is associated
with CRC. These findings are concordant with a previous large HPS
cohort study which showed that the number of serrated polyps was
positively correlated with the risk of CRC.23
In a previous flexible sigmoidoscopy screening study performed
in a large cohort of asymptomatic individuals (n=40.673), the true
prevalence of HPS was estimated to be 1:3000 after subsequent
colonoscopies.24 Colorectal polyps, particularly HPs, do not
necessarily cause symptoms and thus could be left undiagnosed in
CRC cancer risk in first-degree relatives of HPS patients
75
individuals if an endoscopy is not performed. In this study only 19% of
FDRs received an endoscopy. Consequently, although our study
concluded that FDRs have an increased risk of having HPS, an
underestimation of the true prevalence of HPS in FDRs seems likely.
In addition, of the FDRs with colorectal polyps the histology was
unknown in 11/35 cases. Of these individuals five were reported to
have multiple polyps of unknown histology. It seems possible that
these individuals could potentially have HPS too but this could not be
verified.
Alternatively, because the HPS prevalence in the general population
was based on a previous screening sigmoidoscopy study, a degree of
uncertainty exists regarding this estimation. This uncertainty decreases
the validity of our projected relative risk of HPS in FDRs.
With regard to the mode of inheritance, our study did not show
a significant difference in CRC incidence between siblings and non-
siblings (RR: 1.0, 95%-CI: 0.4-2.5, Pearson Chi-Square test: p=0.96).
These findings make it difficult to postulate a preference for either a
vertical or a horizontal transmission. Also concerning polyp incidence
in FDRs, although 3/4 (75%) of FDRs with HPS were siblings, these
numbers are too few to make any conclusions about the mode of
inheritance. These results are in concordance with previously reported
FDRs with HPS (table 3: 6 siblings vs 7 non-siblings). Alternatively, the
presence of FDRs with an intermediate phenotype of <10 HPs,
suggest that a co-dominant mode of inheritance, involvement of
several low penetrance genes, high risk genes with reduced
penetrance or even environmental factors may play a role.
Chapte
r 3
Chapter 3
76
Author
Family
member
(age yrs)
Histology
(detected/biopsied) Location HPS Comments
Chow et al.2
(2 families) Sister
1 (78)
HPs (70/33)
Adenomas (8/8) Prox. colon Yes <10mm
Son
1
(not stated)
Adenomas (4/4)
HP (1/1) Pancolonic No
Son2 (53) HPs (21/21) Dist. colon Yes <10mm
Son2 (51)
HPs (50-100/30)
Adenoma (1/1) Pancolonic Yes <10mm
Lage et al.6
(3 families) Brother
1 (67) HPs/SAs (19/19) Dist. colon Yes 3-25mm, CRC
Brother1 (69) Adenomas (12/4) Prox. colon No 3-7mm
Daughter2 (26) HPs (63/63) Distal colon Yes 2-5mm
Son3 (42)
HPs (50/4)
Adenoma (1/1) Distal colon Yes 2-3mm
Rashid et
al.9
(I family)
Brother1 (67) HPs (>20/>20) Not stated Yes
Son1 (42)
HPs (>20/>20)
Adenoma (1/1) Not stated Yes
Jeevaratnam
et al. 8 (1
family)
Sister1 (73)
HPs (multiple/3)
SAs (multiple/5) Pancolonic Yes
Largest 40mm,
CRC
Brother1 (55) HPs (4/4) Not stated No
3 HPs > 1cm,
CRC
Son1 (43)
HPs (9/9)
Mixed polyp (1/1) Pancolonic No <10mm
Table 3. Previous literature data concerning first-degree relatives with multiple colorectal polyps of known histology (>3 polyps). HP: hyperplastic polyp. SA: serrated adenoma. HPS: hyperplastic polyposis syndrome. CRC: colorectal cancer.
Regarding the management of FDRs, as long as no underlying
genetic cause has been identified, screening colonoscopies seem
justified for this group. Interestingly, of the 27 FDRs with CRC, only 4
probands had CRC, suggesting that screening colonoscopies should
CRC cancer risk in first-degree relatives of HPS patients
77
be performed in all FDRs, independent of CRC presence in the
proband. The appropriate age at which screening colonoscopies
should be commenced is somewhat speculative. We advise
commencement at the age of 35 years or 5 years younger than the
lowest incidence age of HPS reported in the family. Subsequent
surveillance colonoscopies are advised at 6 year intervals with shorter
intervals when polyps are detected. However, future large prospective
screening studies in FDRs are required to further evaluate the
incidence of CRC and HPS and the optimal screening programme in
this group.
Chapte
r 3
Chapter 3
78
REFERENCES 1. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen
LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.
2. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
3. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
4. Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25:177-184.
5. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
6. Lage P, Cravo M, Sousa R, Chaves P, Salazar M, Fonseca R, Claro I, Suspiro A, Rodrigues P, Raposo H, Fidalgo P, Nobre-Leitao C. Management of Portuguese patients with hyperplastic polyposis and screening of at-risk first-degree relatives: a contribution for future guidelines based on a clinical study. Am J Gastroenterol 2004;99:1779-1784.
7. Jass JR, Cottier DS, Pokos V, Parry S, Winship IM. Mixed epithelial polyps in association with hereditary non-polyposis colorectal cancer providing an alternative pathway of cancer histogenesis. Pathology 1997;29:28-33.
8. Jeevaratnam P, Cottier DS, Browett PJ, Van de Water NS, Pokos V, Jass JR. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179:20-25.
9. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
10. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile
CRC cancer risk in first-degree relatives of HPS patients
79
serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.
11. Khalid O, Radaideh S, Cummings OW, O'Brien MJ, Goldblum JR, Rex DK. Reinterpretation of histology of proximal colon polyps called hyperplastic in 2001. World J Gastroenterol 2009;15:3767-3770.
12. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.
13. Sandmeier D, Benhattar J, Martin P, Bouzourene H. Serrated polyps of the large intestine: a molecular study comparing sessile serrated adenomas and hyperplastic polyps. Histopathology 2009;55:206-213.
14. Wong NA, Hunt LP, Novelli MR, Shepherd NA, Warren BF. Observer agreement in the diagnosis of serrated polyps of the large bowel. Histopathology 2009;55:63-66.
15. van Steenbergen LN, Lemmens VE, Louwman MJ, Straathof JW, Coebergh JW. Increasing incidence and decreasing mortality of colorectal cancer due to marked cohort effects in southern Netherlands. Eur J Cancer Prev 2009;18:145-152.
16. Douglas FS, O'Dair LC, Robinson M, Evans DG, Lynch SA. The accuracy of diagnoses as reported in families with cancer: a retrospective study. J Med Genet 1999;36:309-312.
17. Fry A, Campbell H, Gudmunsdottir H, Rush R, Porteous M, Gorman D, Cull A. GPs' views on their role in cancer genetics services and current practice. Fam Pract 1999;16:468-474.
18. Sweet KM, Bradley TL, Westman JA. Identification and referral of families at high risk for cancer susceptibility. J Clin Oncol 2002;20:528-537.
19. Aitken J, Bain C, Ward M, Siskind V, MacLennan R. How accurate is self-reported family history of colorectal cancer? Am J Epidemiol 1995;141:863-871.
20. Kerber RA, Slattery ML. Comparison of self-reported and database-linked family history of cancer data in a case-control study. Am J Epidemiol 1997;146:244-248.
Chapte
r 3
Chapter 3
80
21. Mitchell RJ, Brewster D, Campbell H, Porteous ME, Wyllie AH, Bird CC, Dunlop MG. Accuracy of reporting of family history of colorectal cancer. Gut 2004;53:291-295.
22. Young J, Jenkins M, Parry S, Young B, Nancarrow D, English D, Giles G, Jass J. Serrated pathway colorectal cancer in the population: genetic consideration. Gut 2007;56:1453-1459.
23. Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ, Houben M, Cats A, van Hest LP, Fockens P, Dekker E. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009.
24. Lockett MJ, Atkin W.S. Hyperplastic polyposis: prevalence and cancer risk. 48 ed. 2001.
Association between serrated polyps and MYH-associated polyposis
81
Molecular analyses – Etiology and colorectal cancer pathways
P A
R T
Chapter 4
82
Association between serrated polyps and MYH-associated polyposis
83
Hyperplastic polyps and sessile serrated adenomas as phenotypic expression of MYH-associated polyposis (MAP)
K.S. Boparai E. Dekker
S. van Eeden
M.M. Polak
J.F.W.M. Bartelsman
E.M.H. Mathus –Vliegen
J.J. Keller
C.J.M. van Noesel
Gastroenterology. 2008 Dec;135(6):2014-8
Ch
ap
ter
Chapter 4
84
ABSTRACT
Background and aims: MYH-associated polyposis (MAP) is a disorder
caused by a bi-allelic germline MYH mutation, characterised by
multiple colorectal adenomas. These adenomas typically harbour
G:C→T:A transversions in the APC and K-ras genes caused by MYH
deficiency. Occasional hyperplastic polyps (HPs) have been described
in MAP patients but a causal relationship has never been investigated.
We examined the presence of HPs and sessile serrated adenomas
(SSAs) in 17 MAP patients and studied the occurrence of G:C→T:A
transversions in the APC and K-ras gene in these polyps. Methods:
MAP patients were analysed for the presence of HPs/SSAs. APC-
mutation cluster region and K-ras codon 12 mutation analysis was
performed in adenomas(n=22), HPs(n=63) and SSAs(n=10) from
these patients and from a control group of sporadic adenomas(n=17),
HPs(n=24) and SSAs(n=17). Results: HPs/SSAs were detected in
8/17(47%) of MAP patients of which 3(18%) met the criteria for
hyperplastic polyposis syndrome (HPS). APC mutations were only
detected in adenomas and comprised exclusively G:C→T:A
transversions. K-ras mutations were detected in 51/73(70%)
HPs/SSAs in MAP patients, compared to 7/41(17%) in sporadic
HPs/SSAs in the control group (p<0.0001). In HPs/SSAs, 48/51(94%)
of K-ras mutations exhibited G:C→T:A transversions, compared to
2/7(29%) of sporadic HPs/SSAs in the control group (p<0.0001).
Conclusions: HPs and SSAs are a common finding in MAP patients.
The detection of almost exclusively G:C→T:A transversions in the K-
ras gene of HPs/SSAs strongly suggests that these polyps are
causally related to MYH deficiency. This implies that distinct pathways,
i.e. APC-gene related in adenomas and non-related in HPS/SSAs,
appear to be operational in MAP.
Association between serrated polyps and MYH-associated polyposis
85
Background and aims
Familial adenomatous polyposis (FAP) is an autosomal dominant
disorder characterised by the development of hundreds of colorectal
adenomas and eventually colorectal cancer (CRC) at young age. FAP
is caused by a germline mutation in the APC tumor suppressor gene.
A milder form of FAP, known as attenuated FAP (AFAP), is caused by
mutations in the extreme distal or proximal portion of the APC gene
resulting in fewer adenomas and an older age of onset. 1, 2
Recently, an autosomal recessive polyposis syndrome caused
by bi-allelic germline mutations in the mutY human homologue (MYH)
gene has been identified, known as MYH associated polyposis (MAP).
MYH is a DNA glycosylase that plays a key role in base excision repair
(BER) of 8-oxoG:A mismatches caused by reactive oxygen species.3
Deficiency of this pathway results in somatic G:C→T:A transversions
that are indeed found in the APC and K-ras genes in adenomas of
these patients.4-6 Clinically, MAP resembles AFAP with an average age
of onset around the mid 50’s and often fewer than 100 adenomas,
predominantly in the proximal colon. Polyps have been reported to be
mainly tubular adenomas, some tubulo-villous adenomas and
occasional hyperplastic polyps (HPs). 6-9
Chow et al reported one patient with a bi-allelic MYH mutation
and multiple HPs in addition to adenomatous polyposis.10 Here we
describe 17 MAP patients of which 8 (47%) harboured HPs and sessile
serrated adenomas (SSAs) in addition to conventional adenomas. We
provide evidence that the HPs and SSAs in MAP patients are not rare
and are causally associated with the MYH deficiency, reflected by
G:C→ T:A transversions in K-ras mutated genes of these polyps.
Chapte
r 4
Chapter 4
86
Materials and Methods
Patients and specimens
For this study, our cohort of 17 patients with a bi-allelic MYH mutation,
receiving treatment at the endoscopy department of the Academic
Medical Center (Amsterdam) from 21-7-1988 to
12-3-2008, was analysed for the presence of HPs or SSAs. MAP
patients with HPs/SSAs were arbitrarily classified in to two groups:
patients with multiple HPs and SSAs (≥10); or patients with occasional
HPs and SSAs (<10).
Polyp characteristics were recorded retrospectively from
previous colonoscopy reports or the gross description of the resection
specimens. All polyps were blindly re-evaluated and diagnosed
separately by two experienced pathologists as HP, SSA or adenoma.
SSA was defined by irregular crypts with architectural distortion,
serration and dilation extending to the base of the crypts that often
display boot- or T-shaped branching, and abnormal proliferation and
maturation with mature goblet or foveolar cells at the base of the
crypts.11 In case of disagreement, consensus was reached during a
multi-headed microscope session. This study was conducted in
accordance with the research code of our institutional medical ethical
committee on human experimentation, as well as in agreement with
the Helsinki Declaration of 1975, as revised in 1983.
Somatic mutation analysis
Epithelial cells from HPs (n=63), SSAs (n=10) and adenomas (n=22)
were microdissected and DNA was isolated as described previously.12
In addition, DNA was isolated from sporadic HPs (n=24) and SSAs
(n=17) of similar average size (median 3mm, range: 2-12mm), from a
randomly selected group of patients without a polyposis syndrome
Association between serrated polyps and MYH-associated polyposis
87
(control group). Using previously described primers and assays, DNA
was analysed for mutations in the APC-mutation cluster region (codon
1250-1550), K-ras codon 12.13, 14 Detected mutations were confirmed
in a second experiment.
Statistics
Statistical analyses were performed by using a statistical software
package (Statistical Package for the Social Sciences 12.0.2; SPSS
Inc, Chicago, Ill). Somatic K-ras codon 12 and APC-mutation cluster
region (APC-MCR) configurations were compared with those of a
control panel using a two-sided Fisher exact test. A p-value of < 0.05
was considered statistically significant.
Results
From 17 patients with MAP, 8 (47%) unrelated patients were identified
having at least 1 HP and/or SSA. Of these 8 patients, 3 had >10 HPs
and/or SSAs (table 1). The median age was 50 (range 34-67) years.
Besides multiple adenomas (median 25, range: 3-39), a total of 145
HPs and 19 SSAs were identified from biopsies and polypectomy
specimens (figure 1). The median size of detected HPs and SSAs was
3mm (range: 2-9 mm).
Chapte
r 4
Chapter 4
88
Table 1. Histologically confirmed hyperplastic polyps and sessile serrated adenomas in 8 patients with a bi-allelic MYH mutation. HP: hyperplastic polyp; SSA: sessile serrated adenoma; TC: total colectomy; STC: subtotal colectomy; RC: right colectomy 1Biopsied polyps
A (hemi)colectomy was performed in 6/8 patients, due to concerns that
their polyps were too numerous to guarantee adequate colonoscopic
surveillance, resulting in the detection of most polyps in the remaining
distal colon or rectum. In all 3 patients with multiple HPs and/or SSAs,
more polyps (>30) were seen than biopsied (figure 2).
Patient Age
(yrs) Sex Resection
HPs1
analyzed/total
SSAs1
analyzed/total
Median size HPs/SSAs
(range mm)
MAP patients with multiple (≥10) hyperplastic polyps/sessile serrated
adenomas
1 67 F TC 24/106 8/17 4(2-6)/4(2-6)
2 46 M None 13/13 1/1 2 (2-3)/3
3 67 F RC 14/14 0/0 2(2-3)
MAP patients with occasional (<10) hyperplastic polyps/sessile serrated
adenomas
4 50 M None 2/2 1/1 4(2-5)/4
5 49 F STC 3/3 0/0 2(2-8)
6 47 M STC 4/4 0/0 5(2-9)
7 79 F STC 1/1 0/0 3
8 34 F TC 2/2 0/0 2
Total 50 63/145 10/19 3 (2-9)
Association between serrated polyps and MYH-associated polyposis
89
Somatic APC and K-ras mutations
From the patients with MAP, 22 adenomas, 63 HPs and 10 SSAs were
analysed for APC-MCR and K-ras codon 12 mutations. In adenomas,
APC mutations were found in 9/22 (41%) of polyps, and comprised
exclusively G:C →T:A transversions, whereas no G:C →T:A
transversions were found in APC mutated adenomas in the control
group (p<0.0001). K-ras mutations were found in 5/22 (23%) of
adenomas, also comprising exclusively G:C →T:A transversions.
In HPs and SSAs of MAP patients and of the control group, no
APC mutations were detected (table 2). K-ras mutations were detected
in 51/73 (70%) of HPs/SSAs in MAP patients, which was significantly
more than the frequency in sporadic HPs/SSAs (7/41:17%) in the
control group (p<0.0001). Furthermore, in K-ras mutated HPs/SSAs
from our patients with MAP 48/51(94%) of mutations comprised a G:
C→T: A transversion (figure 3), compared to 2/7 (29%) of mutations in
sporadic HPs/SSAs in the control group (p<0.0001).
Chapte
r 4
Chapter 4
90
Figure 1. Examples of the spectrum of lesions found in the colon: hyperplastic polyp (top panel), sessile serrated adenoma (middle panel) and tubular adenoma (bottom panel).
Association between serrated polyps and MYH-associated polyposis
91
Table 2. Detected APC-mutation cluster region and K-ras codon 12 mutations in hyperplastic polyps (HP), sessile serrated adenomas (SSA) and adenomas (AD) and distribution of G:C→T:A transversions compared to a control panel. *Statistically significant p-value for adenomas in patients with MYH-associated polyposis (MAP) compared to adenomas in the control group **Statistically significant p-value for HPs/SSAs in patients with MAP compared to HPs/SSAs in the control group.
Discussion
MAP is an autosomal recessive polyposis syndrome, caused by a bi-
allelic germline MYH gene mutation. Similar to FAP, MAP is
characterised by the presence of multiple adenomas in the colorectum
and a high cancer risk.4, 5 Here, we show that HPs and SSAs can also
be considered a phenotypic expression of MAP, reflected by the
detection of HPs/SSAs in 8/17 (47%) of patients of which 3 (18%) also
met the criteria for hyperplastic polyposis syndrome (HPS).6, 9
Interestingly, in previous large series of patients with MAP, only
‘occasional’ HPs and no SSAs were described. The presence of
HPs/SSAs in combination with adenomas may thus be more common
than previously thought. A possible cause for this discrepancy may lie
Somatic
mutation
spectrum
Patients with MYH-associated
polyposis (n=8)
Control group P-value
Polyps AD
(n=22)
HP
(n=63)
SSA
(n=10)
AD
(n=17)
HP
(n=24)
SSA
(n=17)
APC mutation 9 0 0 7 0 0
G:C→T:A 9(100%) 0 0 0 0 0 <0.0001*
K-ras
mutation 5 45 6 4 5 2 <0.0001**
G:C→T:A 5(100%) 42(93%) 6(100%) 3(75%) 1 (20%) 1 (50%) <0.0001** Chapte
r 4
Chapter 4
92
in the fact that HPs/SSAs are more difficult to detect endoscopically
due to their often diminutive size and flat or sessile shape.
Furthermore, in patients with multiple adenomatous polyps like in
MAP, the finding of small hyperplastic-looking polyps at colonoscopy
may seem of little clinical interest and are consequentially not biopsied,
preventing documentation of HPs/SSAs as a phenotypic feature of
MAP.
Figure 2. Endoscopic picture of multiple hyperplastic polyps in a patient with MYH-associated polyposis.
HPS is a recently recognised condition, frequently linked with
CRC and defined as at least five HPs proximal to the sigmoid colon, of
which two are greater than 10mm in diameter, or more than 20 HPs
distributed throughout the colon.15-18 Besides multiple HPs, the
presence of SSAs, traditional serrated adenomas and conventional
adenomas are common findings in this condition.10, 19 The combination
Association between serrated polyps and MYH-associated polyposis
93
of BRAF mutations and CPG-island methylation (CIMP), evolving in
SSAs, are considered to be the key mechanism in the ‘serrated
neoplasia pathway’ leading to CRC in these patients.20-23 In our cohort
of MAP patients, 3 unrelated patients also met the criteria for HPS.
Two of these bi-allelic MYH mutations carriers were compound
heterozygote (Y176C and P402L; Y165C and G382D) and one was
monozygote (G382D). Most HPs/SSAs from these patients were
detected in the distal colon and rectum and not proximally. This could
partly be due to the fact that 2/3 of patients had received a
(hemi)colectomy so that only the distal colon and rectum remained. It
has been suggested that proximal HPs/SSAs have more BRAF
mutations than distal HPs/SSAs and are more likely to develop CRC
and thus more clinically relevant than distal HPs/SSAs.17, 20 Other
studies however, have shown that distal HPs/SSAs have a similar
frequency of BRAF mutations as proximal HPs/SSAs.19, 22 In addition,
in the largest case series describing HPS patients, 6/10 of patients had
a left-sided CRC, indicating that distal HPs/SSAs are also of clinical
significance in HPS.10
Chapte
r 4
Chapter 4
94
Figure 3. Somatic G:C →T:A transversion in codon 12 of K-ras in a hyperplastic polyp of a patient with MAP (above) compared to a hyperplastic polyp in the control group (below).
Including our 3 cases, 4 patients with MAP also meeting the
criteria for HPS have now been described.10 In these patients,
coincident multiple adenomas (median number 37, range: 25-40) were
also present. However, bi-allelic MYH gene mutation with HPs in the
absence of adenomas has never been reported. Alternatively, reported
analysis on the germline MYH gene in HPS patients (n=38) revealed a
bi-allelic MYH mutation in only one (3%) patient with 40 adenomas
compared to a median adenoma count of only 2 (range 0-22) in the
other HPS patients.10 These collective data suggest that in HPS
patients MYH mutation analysis should only be considered when
multiple (for example: ≥25) adenomas are also present. Additional
studies are however required to further understand to what extent
MYH testing should be performed in patients with HPS.
The adenomas in our series of MAP patients contained APC-
MCR and K-ras codon 12 mutations. In HPs/SSAs of MAP patients, no
Association between serrated polyps and MYH-associated polyposis
95
APC mutations were detected. However, in these patients we found
significantly higher rates of K-ras mutations in HPs (70%) and SSAs
(60%) as compared to those found in sporadic HPs (21%) and SSAs
(12%) of the control group as well as to reported K-ras mutation
frequencies in sporadic HPs (4-47%) 19, 23-31 and SSAs (0-8%). 20, 22, 23,
26
MYH deficiency characteristically results in somatic G:C→T:A
transversions in the APC and K-ras genes of adenomas.4-6 Indeed, all
APC-MCR mutations detected in adenomas of our MAP patients were
G:C→ T:A transversions, compared to none in the control group.
Accordingly, in K-ras codon 12 mutated HPs/SSAs of these patients
with MYH deficiency, significantly more G:C→T:A transversions (94%)
were found compared to only 29% in control group polyps. This latter
percentage is concordant with previous reports of K-ras mutations in
HPs from patients without a polyposis syndrome showing G:C→T:A
transversions in only 0-33% of polyps.24, 30, 32 Similarly, in previous
large case series of pancreatic carcinomas, having no known
association with MYH deficiency, G:C→T:A transversions were
observed in 6-29% of K-ras mutated carcinomas.33-35 This frequency
range seems to reflect a random statistical chance, considering that
there are 9 different single mutations possible in codon 12 of K-ras
leading to amino acid replacement, of which 2 (22%) result in a
G:C→T:A transversion (i.e. GGT→TGT or GTT) .
In conclusion, our findings suggest that HPs and SSAs are
causally related to bi-allelic MYH mutations. Distinct colonic polyposis
pathways thus seem to prevail in at least a proportion of patients with
MYH deficiency, i.e. a pathway leading to conventional adenomas with
APC and/or K-ras mutations and a separate, non APC-route leading to
HPs/SSAs with K-ras mutations.
Chapte
r 4
Chapter 4
96
REFERENCES 1. Fearnhead NS, Britton MP, Bodmer WF. The ABC of APC. Hum Mol
Genet 2001;10:721-733.
2. Lamlum H, Al TN, Jaeger E, Frayling I, Sieber O, Reza FB, Eckert M, Rowan A, Barclay E, Atkin W, Williams C, Gilbert J, Cheadle J, Bell J, Houlston R, Bodmer W, Sampson J, Tomlinson I. Germline APC variants in patients with multiple colorectal adenomas, with evidence for the particular importance of E1317Q. Hum Mol Genet 2000;9:2215-2221.
3. Slupska MM, Baikalov C, Luther WM, Chiang JH, Wei YF, Miller JH. Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage. J Bacteriol 1996;178:3885-3892.
4. Al-Tassan N, Chmiel NH, Maynard J, Fleming N, Livingston AL, Williams GT, Hodges AK, Davies DR, David SS, Sampson JR, Cheadle JP. Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors. Nat Genet 2002;30:227-232.
5. Jones S, Emmerson P, Maynard J, Best JM, Jordan S, Williams GT, Sampson JR, Cheadle JP. Biallelic germline mutations in MYH predispose to multiple colorectal adenoma and somatic G:C-->T:A mutations. Hum Mol Genet 2002;11:2961-2967.
6. Lipton L, Halford SE, Johnson V, Novelli MR, Jones A, Cummings C, Barclay E, Sieber O, Sadat A, Bisgaard ML, Hodgson SV, Aaltonen LA, Thomas HJ, Tomlinson IP. Carcinogenesis in MYH-associated polyposis follows a distinct genetic pathway. Cancer Res 2003;63:7595-7599.
7. Gismondi V, Meta M, Bonelli L, Radice P, Sala P, Bertario L, Viel A, Fornasarig M, Arrigoni A, Gentile M, Ponz de LM, Anselmi L, Mareni C, Bruzzi P, Varesco L. Prevalence of the Y165C, G382D and 1395delGGA germline mutations of the MYH gene in Italian patients with adenomatous polyposis coli and colorectal adenomas. Int J Cancer 2004;109:680-684.
8. Sampson JR, Dolwani S, Jones S, Eccles D, Ellis A, Evans DG, Frayling I, Jordan S, Maher ER, Mak T, Maynard J, Pigatto F, Shaw J, Cheadle JP. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet 2003;362:39-41.
Association between serrated polyps and MYH-associated polyposis
97
9. Sieber OM, Lipton L, Crabtree M, Heinimann K, Fidalgo P, Phillips RK, Bisgaard ML, Orntoft TF, Aaltonen LA, Hodgson SV, Thomas HJ, Tomlinson IP. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med 2003;348:791-799.
10. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
11. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.
12. Nielsen M, Franken PF, Reinards TH, Weiss MM, Wagner A, van der KH, Kloosterman S, Houwing-Duistermaat JJ, Aalfs CM, Ausems MG, Brocker-Vriends AH, Gomez Garcia EB, Hoogerbrugge N, Menko FH, Sijmons RH, Verhoef S, Kuipers EJ, Morreau H, Breuning MH, Tops CM, Wijnen JT, Vasen HF, Fodde R, Hes FJ. Multiplicity in polyp count and extracolonic manifestations in 40 Dutch patients with MYH associated polyposis coli (MAP). J Med Genet 2005;42:e54.
13. de Leng WW, Keller JJ, Luiten S, Musler AR, Jansen M, Baas AF, de Rooij FW, Gille JJ, Menko FH, Offerhaus GJ, Weterman MA. STRAD in Peutz-Jeghers syndrome and sporadic cancers. J Clin Pathol 2005;58:1091-1095.
14. de Leng WW, Westerman AM, Weterman MA, de Rooij FW, Dekken HH, De Goeij AF, Gruber SB, Wilson JH, Offerhaus GJ, Giardiello FM, Keller JJ. Cyclooxygenase 2 expression and molecular alterations in Peutz-Jeghers hamartomas and carcinomas. Clin Cancer Res 2003;9:3065-3072.
15. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.
16. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
17. Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25:177-184.
Chapte
r 4
Chapter 4
98
18. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
19. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.
20. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.
21. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J, Jass JR. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467-1474.
22. Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T, Simms LA, Young J, James M, Montgomery GW, Appleyard M, Hewett D, Togashi K, Jass JR, Leggett BA. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400-1407.
23. Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D, Young J. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49:121-131.
24. Zauber P, Sabbath-Solitare M, Marotta S, Zauber A, Bishop T. Comparative molecular pathology of sporadic hyperplastic polyps and neoplastic lesions from the same individual. J Clin Pathol 2004;57:1084-1088.
25. Fogt F, Brien T, Brown CA, Hartmann CJ, Zimmerman RL, Odze RD. Genetic alterations in serrated adenomas: comparison to conventional adenomas and hyperplastic polyps. Hum Pathol 2002;33:87-91.
26. O'Brien MJ, Yang S, Mack C, Xu H, Huang CS, Mulcahy E, Amorosino M, Farraye FA. Comparison of microsatellite instability, CpG island methylation phenotype, BRAF and KRAS status in serrated polyps and traditional adenomas indicates separate pathways to distinct colorectal carcinoma end points. Am J Surg Pathol 2006;30:1491-1501.
Association between serrated polyps and MYH-associated polyposis
99
27. Yang S, Farraye FA, Mack C, Posnik O, O'Brien MJ. BRAF and KRAS Mutations in hyperplastic polyps and serrated adenomas of the colorectum: relationship to histology and CpG island methylation status. Am J Surg Pathol 2004;28:1452-1459.
28. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994;54:5523-5526.
29. Otori K, Oda Y, Sugiyama K, Hasebe T, Mukai K, Fujii T, Tajiri H, Yoshida S, Fukushima S, Esumi H. High frequency of KRAS mutations in human colorectal hyperplastic polyps. Gut 1997;40:660-663.
30. Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63:4878-4881.
31. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
32. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW. Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994;54:5523-5526.
33. Motojima K, Urano T, Nagata Y, Shiku H, Tsurifune T, Kanematsu T. Detection of point mutations in the Kirsten-ras oncogene provides evidence for the multicentricity of pancreatic carcinoma. Ann Surg 1993;217:138-143.
34. Rozenblum E, Schutte M, Goggins M, Hahn SA, Panzer S, Zahurak M, Goodman SN, Sohn TA, Hruban RH, Yeo CJ, Kern SE. Tumor-suppressive pathways in pancreatic carcinoma. Cancer Res 1997;57:1731-1734.
35. Song MM, Nio Y, Dong M, Tamura K, Furuse K, Tian YL, He SG, Shen K. Comparison of K-ras point mutations at codon 12 and p21 expression in pancreatic cancer between Japanese and Chinese patients. J Surg Oncol 2000;75:176-185.
Chapte
r 4
Association between serrated polyps and MYH-associated polyposis
101
Serrated polyps and Lynch syndrome: are they associated?
K.S. Boparai
E. Kurpershoek M.M. Polak A.R. Musler
E.Dekker C.J.M. van Noesel
Submitted
Ch
ap
ter
Chapter 5
102
ABSTRACT
Background and aims: Adenomas and colorectal cancers (CRCs) of
Lynch syndrome patients display defective mismatch repair (MMR)
associated with microsatellite instability (MSI). Serrated polyps have
also been described, but previous studies investigating a causal
relationship with Lynch syndrome by MMR testing have not been
conclusive. Presence of a BRAF mutation is an alternative method to
distinguish Lynch-associated CRCs from sporadic CRCs and may thus
be of value to investigate Lynch association in serrated polyps. We
studied the occurrence of defective MMR and BRAF mutations in
serrated polyps occurring in 101 Lynch patients. Methods: Lynch
patients with >1 serrated polyp were included. In all polyps of these
patients, MLH1, MSH2, MSH6 and PMS2 protein expression was
evaluated. In addition, APC-mutation cluster region, KRAS (exon 2)
and BRAF (exon 15) mutation analysis was performed in adenomas
and serrated polyps from these patients and from a control group of
sporadic adenomas (n=17) and serrated polyps (n=42). Results: We
identified 10/101 Lynch patients with >1 serrated polyp of whom 2/10
had multiple (≥10) and 8/10 occasional (<10) serrated polyps. A total of
69 polyps were available for analysis: 32 conventional adenomas and
37 serrated polyps. In 13/32 (40%) conventional adenomas and 0/37
serrated polyps, loss of MMR protein expression was observed.
Overall, BRAF mutations were identified in 16/37 (43%) serrated
polyps in Lynch compared to 20/42 (48%) serrated polyps in the
control group (NS). In Lynch patients with occasional serrated polyps,
BRAF mutations were significantly lower (23%) than control group
serrated polyps (49%: p=0.04) Conclusions: Overall, serrated polyps
do not seem to be a product of the MSI pathway in Lynch syndrome
reflected by the absence of defective MMR in these polyps and a
similarly high frequency of BRAF mutations as in sporadic serrated
polyps. For occasional serrated polyps a causal relationship with
Lynch syndrome can not be excluded.
Association between serrated polyps and MYH-associated polyposis
103
INTRODUCTION
It is generally accepted that conventional adenomas of the
colorectum are the main lesions with an undisputable malignant
potential. The classical model describing colorectal cancer (CRC)
development is the adenoma-carcinoma sequence associated with
activation of the Wnt signalling pathway and chromosomal
instability.(1;2) An alternative route to CRC, the microsatellite instability
(MSI) pathway, was discovered in patients with Lynch syndrome.(3)
Lynch syndrome is an autosomal dominant disorder associated with an
increased risk (25-70%) of developing CRC.(4-6) Precursors of CRC in
these patients are also conventional adenomas which are assumed to
progress more rapidly to CRC than their sporadic counterparts.(7) The
MSI pathway in these patients is initiated by a bi-allelic deletion or
inactivation of one of the mismatch repair genes (MLH1, MSH2, MSH6
or PMS2). The hallmark of Lynch-associated tumors is microsatellite
instability (MSI) and loss of immunostaining of the affected mismatch
repair protein. MSI is found in more than 95% of Lynch associated
CRCs as opposed to only 15% of sporadic CRCs.6, 7 CRCs from Lynch
patients harbour predominantly APC, KRAS and P53 mutations and
virtually never a BRAF mutation.(3;8-13) BRAF testing has therefore
been shown to be an ideal method to exclude Lynch associated
tumors from sporadic tumors.(14;15) Recently, a newly proposed
‘serrated neoplasia pathway’ has been described involving the
progression of serrated polyps to CRC through accumulation of
genetic mutations, namely BRAF mutations and CPG-island
methylation. Supporting clinicohistological and molecular evidence for
such a pathway has been derived primarily from patients with serrated
polyposis syndrome (SPS).(13;16-21)
Chapte
r 5
Chapter 5
104
In Lynch syndrome, serrated polyps (i.e. hyperplastic polyps,
HPs; sessile serrated adenomas, SSAs; and traditional serrated
adenomas, TSAs) have also been described.(7;22-27) In a number of
previous studies, an attempt was made to verify whether these
serrated polyps are causally related to Lynch syndrome, reflected by
the presence of defective mismatch repair (MSI) and/or loss of
immunostaining. These studies have thus far been inconclusive. Both
conventional adenomas (24-93%)(23;28-37) and HPs/hyperplastic
abberant crypt foci (0-100%)(26;29;35;38;39) express defective
mismatch repair in highly variable frequencies. To analyze whether
serrated polyps are causally related to Lynch syndrome, BRAF
mutation analysis may be a valuable adjunct to mismatch repair gene
testing. Sporadic serrated polyps have been shown to display high (70-
78%) levels of BRAF mutations.33 It seems presumable that, like CRCs
in Lynch, serrated polyps when causally related to Lynch syndrome,
will display BRAF mutations at a far lower frequency than their
sporadic counterparts.
In this study, we describe the frequency of serrated polyps
(HPs/SSAs/TSAs) in 101 patients with a genetically confirmed
diagnosis of Lynch syndrome. We show that, overall, serrated polyps
in Lynch syndrome lack defective mismatch repair (MSI and/or loss of
immunostaining) and harbour a similarly high frequency of BRAF
mutations as their sporadic counterparts, strongly suggesting that they
are not associated with the MSI-pathway. At sub-analysis, we show
that occasional serrated polyps in Lynch patients have significantly
lower levels of BRAF mutations than their sporadic counterparts.
Therefore, a causal relationship with the MSI-pathway can not be
excluded in these polyps.
Association between serrated polyps and MYH-associated polyposis
105
MATERIALS AND METHODS
Patients and specimens
For this study, 102 patients known at our centre with Lynch syndrome
and an identified germline mutation in one of the MMR genes was
analysed for the presence of serrated polyps. Patients were included if
>1 serrated polyp was identified. These patients were then arbitrarily
classified in to two groups: patients with multiple serrated polyps (≥10);
or patients with occasional serrated polyps (2-10).
Polyp characteristics were recorded retrospectively from
previous colonoscopy reports or the gross description of the resection
specimens at histopathology. All polyps were blindly re-evaluated and
diagnosed by a single experienced pathologist (CvN) as HP, SSA,
TSA or adenoma based on the histological features on H&E
staining.(40-42) Lesions proximal from the caecum, ascending colon,
transverse colon and descending colon were regarded as proximal and
polyps from the sigmoid colon and rectum were regarded as distal. For
the purpose of comparison, a previously selected control group
consisting of sporadic HPs (n=24), SSAs (n=18) and conventional
adenomas (n=17) from patients without a suspicion for Lynch
syndrome was used to compare somatic mutations.This study was
conducted in accordance with the research code of our institutional
medical ethical committee on human experimentation, as well as in
agreement with the Helsinki Declaration of 1975, as revised in 1983.
Molecular analysis
Mutation analysis
Using previously described primers and assays, DNA was analysed for
mutations in the APC-mutation cluster region (APC-MCR), KRAS
(exon 2) and BRAF (exon 15) of all polyps.37, 38 Previously performed
Chapte
r 5
Chapter 5
106
mutation analysis of these genes in a randomly selected sporadic
polyps served as a control group. Detected mutations were confirmed
in a second experiment.
Immunohistochemistry
Immunohistochemistry was performed on all polyps of the included
Lynch patients. Unstained 5-µm sections were cut from paraffin blocks
and the slides were deparaffinized. Primary monoclonal antibodies
used were specific for MLH1(1:50 BD Pharmingen, San Diego, USA);
MSH2(1:100 Oncogene Research Prod., San Diego, USA);
MSH6(1:200 BD Transduction Lab., San Jose, USA); PMS2(1:250 BD
Transduction Lab., San Jose, USA). Slides were immersed in 0.3%
hydrogen peroxide in methanol for 20 minutes. Subsequently, antigen
retrieval was carried out by 10 minutes of boiling in 10mM Tris/1mM
EDTA (pH 9) followed by incubation with above mentioned diluted
primary antibodies during 1 hour at room temperature. Post-antibody
block (Immunologic) in PBS was performed followed by
implementation of an antipolyvalent HRP detection system
(Immunologic) to visualize antibody binding sites with 3.3’-
diaminobenzidine as a chromogen. Sections were counterstained with
haematoxylin. Stains for MLH1, MSH2, MSH6 and PMS2 were
considered negative when there was complete absence of nuclear
expression in all neoplastic cells. Negative staining in a part of a lesion
or in a single crypt was registered separately.
Microsatellite analysis
In a subset of Lynch patients, harbouring many serrated polyps and
conventional adenomas (>20), MSI analysis was performed.
Microsatellite status was determined using an international standard
Association between serrated polyps and MYH-associated polyposis
107
panel of 5 microsatellite markers (D17S250, D2S123, D5S346, BAT25
and BAT26) using standard techniques. A high degree of microsatellite
instability (MSI-high) was defined as two (40%) or more unstable
markers, MSI-Low as one unstable marker, and microsatellite stable
(MSS) as no unstable markers.
Statistics
Statistical analyses were performed by using a statistical software
package (Statistical Package for the Social Sciences 12.0.2; SPSS
Inc, Chicago, Ill). Somatic mutations in polyps of Lynch patients were
compared with those of the control group using a two-sided Fisher
exact test. A p-value of < 0.05 was considered statistically significant.
RESULTS
From 101 Lynch syndrome patients with a known germline mutation,
10 (10%) patients were identified with >1 serrated polyp. Of these
patients, 2 had ≥10 serrated polyps (table 1). The median age was 62
years (range: 44-75) with a male: female ratio of 3:7. In 10 patients, a
total of 89 polyps were identified of which 69 were available for
analysis: 32 conventional adenomas (21 tubular adenomas and 11
tubulovillous adenomas) and 37 serrated polyps (32 HPs and 5 SSAs).
The median size of serrated polyps was 2mm (range: 1-10mm) and
4mm (range: 2-30mm) for conventional adenomas. Of 34/37 serrated
polyps location could be ascertained. Nineteen of 34 (51%) serrated
polyps were located proximal to the sigmoid colon. In 8/10 (80%)
patients, serrated polyps were identified proximal to the sigmoid colon.
In 1/11 tubulovillous adenomas (20mm) an adenocarcinoma was
identified (at surgery: T2N0M0). The other 10 tubulovillous adenomas
displayed low-grade dysplasia. In 6/22 tubular adenomas, features of
Chapte
r 5
Chapter 5
108
high-grade dysplasia were seen. In 1/10 patients a subtotal colectomy
had been performed previously because of an adenocarcinoma
(T3N0M0, tissue not available for analysis). Previously performed
germline mutation analysis in patient 2 due to the high number of
adenomas showed a mono-allelic mutation of the MUTYH gene.
Table 1: Number of histologically verified polyps and total number of detected polyps in Lynch patients.
Patient
Age
(yrs)
Germline
mutation
Sex
Adenomas
Analyzed/total
HPs
Analyzed/total
SSAs
analyzed/total
Location
serrated
polyps
Lynch patients with multiple (≥10) serrated polyps (=SPS)
1 44 MSH6 F 2/2 12/>30 0/0 Pancolonic
2 69 MSH6 M 14/14 1/10 2/2 Pancolonic
Lynch patients with occasional (<10) serrated polyps (≠SPS)
3 60 MLH1 M 5/5 2/2 0/0 Pancolonic
4 62 MSH6 M 1/1 2/2 1/1 Pancolonic
5 50 PMS2 F 0/0 2/2 0/0 Rectosigmoid
6 68 MLH1 F 5/5 4/4 0/0 Pancolonic
7 62 MLH1 F 1/1 2/2 1/1 Right-side
8 62 MSH2 M 2/2 3/3 0/0 Rectosigmoid
9 75 MSH2 F 1/1 3/3 0/0 Left-side
10 65 MSH2 F 1/1 1/1 2/2 Right-side
Total 12 32/32 32/>59 5/5
Association between serrated polyps and MYH-associated polyposis
109
Somatic APC, KRAS and BRAF mutation analysis
A total of 32 adenomas, 32 HPs and 5 SSAs were analysed for
APC-MCR, KRAS exon 2 and BRAF exon 15 mutations. In adenomas,
APC mutations were found in 6/32 (19%) of polyps. No KRAS
mutations or BRAF mutations were detected in adenomas. No
significant differences were seen compared to the control group
regarding APC and BRAF mutations (table 2). More KRAS mutations
were identified in sporadic adenomas (4/17) of the control group
compared to Lynch adenomas (0/32: p=0.01). The adenocarcinoma
identified in a tubulovillous adenoma displayed an APC mutation. In
serrated polyps, no APC mutations were identified. KRAS mutations
were identified 6/37 (16%) serrated polyps vs 7/42 (17%) in the control
group. BRAF mutations were detected in 16/37 (43%) serrated polyps
in Lynch patients compared to 20/42 (48%) in the control group (n.s.).
There was no significant difference between proximal and distal
serrated polyp location regarding BRAF mutation status.
When comparing patients with multiple serrated polyps and
satisfying the criteria for SPS (n=2) to patients with occasional serrated
polyps (n=8), we identified significantly (p=0.002) more BRAF
mutations in SPS serrated polyps (11/15 =73%) than in occasional
serrated polyps (5/22=23%). After subdivision, comparison of BRAF
mutations showed that occasional serrated polyps in Lynch patients
harboured a significantly lower BRAF frequency (23%) than the control
group (49%: p=0.04). Comparison of SPS serrated polyps with control
group serrated polyps showed no significant differences regarding
mutations.
Chapte
r 5
Chapter 5
110
Table 2. Detected APC, KRAS and BRAF mutations in serrated polyps (HPs, SSAs) and adenomas (AD) compared to a control group. * significantly more KRAS mutations in sporadic adenomas than Lynch adenomas
Immunohistochemistry and microsatellite analysis
Loss of expression of one of the MMR proteins was observed in 13/33
(39%) conventional adenomas in Lynch patients which correlated in all
cases with the germline MMR mutation. There was no correlation
between histology (10 tubular adenomas and 3 tubulovillous
adenomas) and loss of MMR expression (p=0.259). Also when grade
of dyspasia was analyzed, no association was observed with MMR
expression (p=0.672). The adenocarcinoma identified within a
tubulovillous adenoma in a Lynch patient did not demonstrate loss of
MMR expression. None of the serrated polyps showed loss of
expression of one of the MMR proteins. MMR protein expression in the
crypts of the serrated polyps was comparable to that of crypts in
normal epithelium of the colorectum. Expression of all four MMR
proteins was observed in most nuclei in the base of the crypts whereas
few to none of the luminal nuclei expressed any of the MMR proteins.
Somatic mutation Patients with Lynch Control group P-value
Serrated polyps
(n=37)
AD
(n=32)
Serrated polyps
(n=42)
AD
(n=17)
APC mutation 0 6
(19%) 0
7
(41%) ns
BRAF mutation 16 (43%) 0 20 (49%) 0 ns
KRAS mutation 6 (16%) 0 7 (17%) 4
(24%) 0.01*
Association between serrated polyps and MYH-associated polyposis
111
Microsatellite analysis was performed in 30 polyps of Lynch
patients 1 and 2, both with a germline mutation in MSH6 and ≥10
serrated polyps, also satisfying the clinical diagnosis of SPS. Fifteen
adenomas, 13 HPs and 2 SSAs were analyzed. In patient 1, both
adenomas (2/2) were MSI-high and showed corresponding loss of the
MSH6 protein at immunohistochemistry. All serrated polyps were MSS
in this patient. In patient 2, 0/14 adenomas (including the
adenocarcinoma) was MSI-H and none showed immunohistochemical
loss of the MSH-6 protein. All serrated polyps were microsatellite
stable in this patient.
DISCUSSION
In this comprehensive cohort study of Lynch syndrome patients with a
proven germline MMR gene mutation, we demonstrated that a small
proportion (2%) of patients harbor multiple serrated polyps in addition
to conventional adenomas, two of whom also satifying the criteria for
SPS. Using conventional immunohistochemistry and MSI analysis,
previous studies have been performed to analyze whether serrated
polyps are causally related to Lynch syndrome. Results from these
studies have however been inconclusive. Based on the principle that
BRAF mutation testing is a highly sensitive method to distinguish
sporadic CRCs (high frequency of mtuations) from Lynch-associated
CRCs (low frequency of mutations)(14;15;43), we demonstrated that
serrated polyps most likely are not associated with Lynch syndrome,
reflected by both a similarly high frequency of BRAF mutations as
sporadic serrated polyps and an absence of MMR deficiency in all of
these polyps.
Our study is the first to utilize an alternative molecular approach
involving mutation analysis (APC, KRAS and BRAF) in adjunct to
Chapte
r 5
Chapter 5
112
defective MMR testing to evaluate whether serrated polyps are
causally related to Lynch syndrome. In addition, our study
encompasses the largest analysis of serrated polyps (including SSAs)
from Lynch patients who have been unambiguously defined by a
germline mutation thus representing true Lynch syndrome patients.
Prior reports evaluating solely defective MMR in serrated polyps
defined Lynch syndrome in a broader sense: fulfilment of either genetic
or clinical (Amsterdam) criteria. However, correlation between the
Amsterdam criteria and a germline mutation are imperfect. Indeed, it
has been shown that families satisfying the Amsterdam criteria may
carry an alternative diagnosis such as Syndrome X or serrated
polyposis syndrome (SPS).(19;44)
In Lynch syndrome, carcinogenesis is traditionally considered
to proceed through an accelerated adenoma-carcinoma sequence
driven by MMR deficiency. However, in the literature not all
conventional adenomas in these patients have been shown to express
defective MMR (24-93%). We also found loss of expression of one of
the MMR proteins in only 13/33 (39%) conventional adenomas. It is
conceivable that the remaining adenomas without loss of expression of
one of the MMR proteins represent co-existing sporadic conventional
adenomas. Concordantly, sporadic conventional adenomas show
defective mismatch repair in far lower percentages: 0-3% of
adenomas.(30;45) Nevertheless, independent of Lynch-associaton,
removal of all conventional adenomas reduces the incidence of CRC
and is therefore standard practice of care in these patients.(46;47)
Serrated polyps, which were traditionally considered to be
benign lesions, have recently also been considered to be lesions with
malignant potential through a serrated CRC pathway. The early
genetic events of this route, as yet identified, are predominantly BRAF
Association between serrated polyps and MYH-associated polyposis
113
mutations and a generally enhanced CPG-island methylation status of
multiple genes.(20;29;48-53) Additional combined clinicohistological
and molecular evidence supporting a serrated CRC pathway include
right-sided carcinomas identified within serrated polyps harboring
identical BRAF mutations as the serrated polyp component(17) and
increased incidence of serrated polyps in patients with sporadic
microsatellite-unstable CRCs.(20;48;54) Removal of these lesions, in
particular large, right-sided serrated polyps, is therefore clinically
important.(13;17) However, serrated polyps associated with the
accelerated MSI-pathway may represent present precursor lesions of
MSI-CRC which evolve even faster than in the serrated CRC pathway
alone. Investigation of an association between serrated polyps and
Lynch is therefore clinically relevant. Three previous studies reported
defective MMR in serrated polyps. In two studies, MSI was observed in
hyperplastic aberrant crypt foci (4/4:100%)(39) and HPs
(2/17:11%)(23). Finally, in one case report a Lynch patient was
described with a mixed HP/adenoma in contiguity with a CRC.(55) We
found no defective MMR in serrated polyps which is in concordance
with a number of prior studies also showing an absence of defective
MMR in serrated polyps.(22;26;38;56) In addition, the similarly high
frequency of BRAF mutations in serrated polyps as their sporadic
counterparts in this study further supports the notion that serrated
polyps are not causally related to the MSI carcinogenesis pathway in
Lynch syndrome.
It is possible that two oncogenic pathways, a MSI-pathway and
a serrated CRC pathway, co-exist within Lynch syndrome patients with
serrated polyps. However, considering that a large proportion of
serrated polyps in our study were BRAF-mutated and thus far no
CRCs with a BRAF mutation have been identified in Lynch patients, it
Chapte
r 5
Chapter 5
114
seems that the MSI pathway predominates over the serrated CRC
pathway because of a relatively higher frequency of conventional
adenomas in Lynch patients, favouring a stochastic process of
carcinogenesis. Alternatively, the serrated CRC pathway may
represent a slower route of carcinogenesis than the MSI pathway.
BRAF mutations are more commonly associated with serrated polyps
and the serrated CRC pathway than KRAS mutations. Nevertheless, a
serrated CRC pathway, skewed towards initial KRAS mutations in a
minority of serrated polyps can not be excluded.
Interestingly, after subdividing Lynch patients in SPS Lynch
patients with multiple serrated polyps and non-SPS Lynch patients with
occasional serrated polyps, we found that BRAF mutations were more
common in serrated polyps of Lynch-SPS patients: 11/15 (73%)
compared to 5/22 (23%) in non-SPS patients (p=0.002). Based on
these findings we conclude that serrated polyps in SPS Lynch patients
in particular are an expression of a serrated-CRC pathway.
Alternatively, only 23% of serrated polyps in non-SPS patients were
BRAF mutated. This proportion is significantly lower than control group
serrated polyps (49%: p=0.04). Considering this low frequency of
BRAF mutations in serrated polyps of non-SPS Lynch patients, a
causal relationship with the MSI-pathway can not be excluded despite
lack of defective MMR. Thus, these occasional serrated polyps, lacking
BRAF mutations, may follow an accelerated route to CRC via the MSI-
pathway.
SPS is a condition characterized by the presence of multiple
serrated polyps spread throughout the colon and is associated with an
increased CRC risk.(57-60) However, the heterogeneous phenotype of
SPS such as the presence of different polyp histologies, differences in
polyp localization and number of polyps, suggest that SPS can be
Association between serrated polyps and MYH-associated polyposis
115
subdivided into separate (genetic) conditions. Two Lynch patients
(patients 1 and 2) harboured ≥10 colorectal serrated polyps. While
patient 1 satisfied the classical criteria for SPS with few classical
adenomas(16), patient 2 had less serrated polyps and more
conventional adenomas thus representing an intermediate SPS with a
mixed phenotype. MSI analysis in adenomas of patient 1 showed high
degree of MSI in all (2/2) adenomas as well as concordant lack of
MSH-6 expression, indicating that these adenomas are a manifestation
of Lynch syndrome. Interestingly, 0/14 adenomas of patient 2,
including the adenocarcinoma, were MSI-H. The lack of MSI in all
adenomas of this Lynch patient with a mixed SPS phenotype suggests
that these adenomas are not associated with Lynch syndrome. To
substantiate this, we selected a control group of conventional
adenomas (n=15) which were matched for size, location, histology and
grade of dysplasia from Lynch patients without serrated polyps to
compare MSI status and found MSI-H in 9/15 adenomas (p=0.016,
data not shown). Although less common, it is possible that the
adenomas in patient 2 are an expression of mono-allelic MUTYH
deficiency.(61) However, only 2/6 APC-mutated adenomas displayed
G:C→T:A transversions, which are typical for MUTYH-deficiency. No
KRAS mutations were identified in these adenomas. In the literature, a
subset of serrated polyps has also been described to be causally
related to bi-allelic MUTYH-deficiency.(62) This seems unlikely in
patient 2 considering that of the mutated serrated polyps, 2 were
BRAF mutated and only one KRAS mutated (although it did have a
G:C→ T:A conversion). Another explanation could be that these
adenomas are in fact part of a mixed subtype of SPS. It remains
however difficult to verify this hypothesis considering that no hallmark
genetic characteristics of SPS have been identified. Alternatively,
Chapte
r 5
Chapter 5
116
these adenomas could simply be sporadic adenomas. However,
considering the multitude of adenomas, this seems unlikely.
Based on our observations, we conclude that, overall, serrated
polyps play an insignificant role in the MSI carcinogenesis pathway in
Lynch syndrome. A causal relationship between occasional serrated
polyps and the MSI-pathway can not be excluded. BRAF-mutated
serrated polyps may represent components of a co-existent serrated
CRC pathway. The MSI pathway of carcinogenesis seems however to
predominate over the serrated CRC pathway in these patients
reflected by the described absence of BRAF mutations in Lynch-
associated CRCs.
Association between serrated polyps and MYH-associated polyposis
117
REFERENCES (1) Fearon ER, Vogelstein B. A genetic model for colorectal
tumorigenesis. Cell 1990;61(5):759-67.
(2) Vogelstein B, Fearon ER, Hamilton SR et al. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319(9):525-32.
(3) Aaltonen LA, Peltomaki P, Leach FS et al. Clues to the pathogenesis of familial colorectal cancer. Science 1993;260(5109):812-6.
(4) Hendriks YM, Wagner A, Morreau H et al. Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance. Gastroenterology 2004;127(1):17-25.
(5) Quehenberger F, Vasen HF, van Houwelingen HC. Risk of colorectal and endometrial cancer for carriers of mutations of the hMLH1 and hMSH2 gene: correction for ascertainment. J Med Genet 2005;42(6):491-6.
(6) Plaschke J, Engel C, Kruger S et al. Lower incidence of colorectal cancer and later age of disease onset in 27 families with pathogenic MSH6 germline mutations compared with families with MLH1 or MSH2 mutations: the German Hereditary Nonpolyposis Colorectal Cancer Consortium. J Clin Oncol 2004;22(22):4486-94.
(7) Lindgren G, Liljegren A, Jaramillo E et al. Adenoma prevalence and cancer risk in familial non-polyposis colorectal cancer. Gut 2002;50(2):228-34.
(8) Fujiwara T, Stolker JM, Watanabe T et al. Accumulated clonal genetic alterations in familial and sporadic colorectal carcinomas with widespread instability in microsatellite sequences. Am J Pathol 1998;153(4):1063-78.
(9) Huang J, Papadopoulos N, McKinley AJ et al. APC mutations in colorectal tumors with mismatch repair deficiency. Proc Natl Acad Sci U S A 1996;93(17):9049-54.
(10) Konishi M, Kikuchi-Yanoshita R, Tanaka K et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111(2):307-17.
Chapte
r 5
Chapter 5
118
(11) Losi L, Ponz de LM, Jiricny J et al. K-ras and p53 mutations in hereditary non-polyposis colorectal cancers. Int J Cancer 1997;74(1):94-6.
(12) Tomlinson I, Ilyas M, Johnson V et al. A comparison of the genetic pathways involved in the pathogenesis of three types of colorectal cancer. J Pathol 1998;184(2):148-52.
(13) Kambara T, Simms LA, Whitehall VL et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53(8):1137-44.
(14) McGivern A, Wynter CV, Whitehall VL et al. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3(2):101-7.
(15) Domingo E, Laiho P, Ollikainen M et al. BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet 2004;41(9):664-8.
(16) Boparai KS, Mathus-Vliegen EM, Koornstra JJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2010;59(8):1094-100.
(17) Boparai KS, Dekker E, Polak MM et al. A serrated colorectal cancer pathway predominates over the classic WNT pathway in patients with hyperplastic polyposis syndrome. Am J Pathol 2011;178(6):2700-7.
(18) Chan TL, Zhao W, Leung SY et al. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63(16):4878-81.
(19) Jeevaratnam P, Cottier DS, Browett PJ et al. Familial giant hyperplastic polyposis predisposing to colorectal cancer: a new hereditary bowel cancer syndrome. J Pathol 1996;179(1):20-5.
(20) Makinen MJ, George SM, Jernvall P et al. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193(3):286-94.
(21) Nosho K, Kure S, Irahara N et al. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 2009;137(5):1609-20.
Association between serrated polyps and MYH-associated polyposis
119
(22) Andersen SH, Lykke E, Folker MB et al. Sessile serrated polyps of the colorectum are rare in patients with Lynch syndrome and in familial colorectal cancer families. Fam Cancer 2008;7(2):157-62.
(23) Iino H, Simms L, Young J et al. DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer. Gut 2000;47(1):37-42.
(24) Jarrar AM, Church JM, Fay S et al. Is the phenotype mixed or mistaken? Hereditary nonpolyposis colorectal cancer and hyperplastic polyposis syndrome. Dis Colon Rectum 2009;52(12):1949-55.
(25) Liljegren A, Lindblom A, Rotstein S et al. Prevalence and incidence of hyperplastic polyps and adenomas in familial colorectal cancer: correlation between the two types of colon polyps. Gut 2003;52(8):1140-7.
(26) Rijcken FE, van der Sluis T, Hollema H et al. Hyperplastic polyps in hereditary nonpolyposis colorectal cancer. Am J Gastroenterol 2003;98(10):2306-11.
(27) Walsh MD, Buchanan DD, Walters R et al. Analysis of families with Lynch syndrome complicated by advanced serrated neoplasia: the importance of pathology review and pedigree analysis. Fam Cancer 2009;8(4):313-23.
(28) Rijcken FE, Hollema H, Kleibeuker JH. Proximal adenomas in hereditary non-polyposis colorectal cancer are prone to rapid malignant transformation. Gut 2002;50(3):382-6.
(29) Iino H, Jass JR, Simms LA et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52(1):5-9.
(30) Aaltonen LA, Peltomaki P, Mecklin JP et al. Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients. Cancer Res 1994;54(7):1645-8.
(31) Jacoby RF, Marshall DJ, Kailas S et al. Genetic instability associated with adenoma to carcinoma progression in hereditary nonpolyposis colon cancer. Gastroenterology 1995;109(1):73-82.
(32) Konishi M, Kikuchi-Yanoshita R, Tanaka K et al. Molecular nature of colon tumors in hereditary nonpolyposis colon cancer, familial polyposis, and sporadic colon cancer. Gastroenterology 1996;111(2):307-17.
Chapte
r 5
Chapter 5
120
(33) Akiyama Y, Iwanaga R, Saitoh K et al. Transforming growth factor beta type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer. Gastroenterology 1997;112(1):33-9.
(34) Sasaki S, Masaki T, Umetani N et al. Microsatellite instability is associated with the macroscopic configuration of neoplasms in patients with multiple colorectal adenomas. Jpn J Clin Oncol 1998;28(7):427-30.
(35) Pino MS, Mino-Kenudson M, Wildemore BM et al. Deficient DNA mismatch repair is common in Lynch syndrome-associated colorectal adenomas. J Mol Diagn 2009;11(3):238-47.
(36) Muller A, Beckmann C, Westphal G et al. Prevalence of the mismatch-repair-deficient phenotype in colonic adenomas arising in HNPCC patients: results of a 5-year follow-up study. Int J Colorectal Dis 2006;21(7):632-41.
(37) Giuffre G, Muller A, Brodegger T et al. Microsatellite analysis of hereditary nonpolyposis colorectal cancer-associated colorectal adenomas by laser-assisted microdissection: correlation with mismatch repair protein expression provides new insights in early steps of tumorigenesis. J Mol Diagn 2005;7(2):160-70.
(38) Halvarsson B, Lindblom A, Johansson L et al. Loss of mismatch repair protein immunostaining in colorectal adenomas from patients with hereditary nonpolyposis colorectal cancer. Mod Pathol 2005;18(8):1095-101.
(39) Pedroni M, Sala E, Scarselli A et al. Microsatellite instability and mismatch-repair protein expression in hereditary and sporadic colorectal carcinogenesis. Cancer Res 2001;61(3):896-9.
(40) Hamilton SR, Vogelstein B, Kudo S et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000: 104-19.
(41) Jass JR, Baker K, Zlobec I et al. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49(2):121-31.
(42) Snover DC, Jass JR, Fenoglio-Preiser C et al. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124(3):380-91.
Association between serrated polyps and MYH-associated polyposis
121
(43) Loughrey MB, Waring PM, Tan A et al. Incorporation of somatic BRAF mutation testing into an algorithm for the investigation of hereditary non-polyposis colorectal cancer. Fam Cancer 2007;6(3):301-10.
(44) Lindor NM, Rabe K, Petersen GM et al. Lower cancer incidence in Amsterdam-I criteria families without mismatch repair deficiency: familial colorectal cancer type X. JAMA 2005;293(16):1979-85.
(45) Young J, Leggett B, Gustafson C et al. Genomic instability occurs in colorectal carcinomas but not in adenomas. Hum Mutat 1993;2(5):351-4.
(46) Mecklin JP, Aarnio M, Laara E et al. Development of colorectal tumors in colonoscopic surveillance in Lynch syndrome. Gastroenterology 2007;133(4):1093-8.
(47) Winawer SJ, Zauber AG, Ho MN et al. Prevention of colorectal cancer by colonoscopic polypectomy. The National Polyp Study Workgroup. N Engl J Med 1993;329(27):1977-81.
(48) Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93(17):1307-13.
(49) Jass JR, Biden KG, Cummings MC et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52(6):455-60.
(50) Jass JR, Iino H, Ruszkiewicz A et al. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47(1):43-9.
(51) Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000;37(4):295-301.
(52) Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001;193(3):283-5.
(53) Yao T, Nishiyama K, Oya M et al. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000;190(4):444-9.
Chapte
r 5
Chapter 5
122
(54) Goldstein NS, Bhanot P, Odish E et al. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119(6):778-96.
(55) Jass JR, Cottier DS, Pokos V et al. Mixed epithelial polyps in association with hereditary non-polyposis colorectal cancer providing an alternative pathway of cancer histogenesis. Pathology 1997;29(1):28-33.
(56) Meijer TW, Hoogerbrugge N, Nagengast FM et al. In Lynch syndrome adenomas, loss of mismatch repair proteins is related to an enhanced lymphocytic response. Histopathology 2009;55(4):414-22.
(57) Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47(12):2101-4.
(58) Leggett BA, Devereaux B, Biden K et al. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25(2):177-84.
(59) Rubio CA, Stemme S, Jaramillo E et al. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38(3):266-70.
(60) Rashid A, Houlihan PS, Booker S et al. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119(2):323-32.
(61) Sieber OM, Lipton L, Crabtree M et al. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med 2003;348(9):791-9.
(62) Boparai KS, Dekker E, van ES et al. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135(6):2014-8.
CRC pathways in patients with HPS
123
A serrated colorectal cancer pathway predominates over the classical WNT-pathway in patients with hyperplastic polyposis syndrome
Karam S. Boparai
Evelien Dekker
Mirjam M. Polak
Alex R. Musler
Susanne van Eeden
Carel J.M. van Noesel
American Journal of Pathology 2011 Jun;178(6):2700-7
Ch
ap
ter
Chapter 6
124
ABSTRACT
Background and aims: Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC-pathway in humans. Methods: To establish which CRC-pathways play a role in HPS and particularly to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps(n=84) and CRCs(n=19) in 17 HPS patients as compared to control groups of various sporadic polyps(n=59) and sporadic, microsatellite-stable CRCs(n=16). Results: In both HPS and sporadic polyps, APC mutations were exclusively identified in adenomas whereas BRAF mutations were confined to serrated polyps. Six of 19(32%) HPS CRCs were identified within a serrated polyp. Mutation analysis performed in both the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located within an adenoma, both components harboring an identical APC mutation. Overall, 10/19(53%) HPS CRCs carried a BRAF mutation, compared to none in control group CRCs (p=0.001). Six (60%) of the BRAF-mutated HPS CRCs were microsatellite-unstable (MSI-high) due to MLH1 methylation. Conclusion: Our findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating both microsatellite-stable and microsatellite-instable CRCs.
CRC pathways in patients with HPS
125
INTRODUCTION
Colorectal cancer (CRC) ranks as the second most common
cause of cancer-related death in the western world.1 The classical
model which describes CRC development is the adenoma-carcinoma
sequence associated with activation of the WNT signalling pathway.2, 3
This pathway is characterised by an initial, bi-allelic inactivation of the
adenomatous polyposis coli gene (APC) followed by mutations in key
oncogenes and tumor-suppressor genes, including KRAS, DCC and
TP53, resulting in adenoma initiation and progression to CRC. This
multi-step process of carcinogenesis has been elaborately studied and
much information has been derived from the familial adenomatous
polyposis (FAP) and MUTYH-associated polyposis (MAP) syndromes.
In addition to the adenoma-carcinoma sequence, an
alternative, microsatellite instability (MSI) pathway exists which is
characterized by deletion or inactivation of mismatch repair (MMR)
genes. Loss of one of the MMR genes occurs in 10-15 % of the
sporadic CRC, whereas 1-2% of CRCs with MMR gene loss is due to a
hereditary predisposition, i.e. in Lynch syndrome patients carrying a
mono-allelic MMR gene defect in the germline.
Recently a, “serrated neoplasia pathway”, has been proposed
which involves the progression of serrated polyps, i.e. hyperplastic
polyps (HPs), sessile serrated adenomas (SSAs) and/or traditional
serrated adenomas (TSAs), to CRC. The early genetic events of this
route, as yet identified, are BRAF or KRAS mutations and an
enhanced CPG-island methylation status of multiple genes.4-11 There is
evidence to suggest that a proportion of sporadic MSI CRCs originate
from serrated polyps as both these lesions commonly harbour
hypermethylated MLH1 in combination with BRAF mutations.12-16 In
addition, clinicohistological reports supporting a serrated CRC pathway
Chapte
r 6
Chapter 6
126
include CRCs in close vicinity of large hyperplastic polyps17, 18; CRCs
identified in mixed hyperplastic and adenomatous polyps19; and
increased incidence of serrated polyps in patients with sporadic
microsatellite-unstable CRCs.4, 10, 20 Currently however, proof of the
existence of a serrated CRC pathway, demonstrated by the combined
histological finding of a serrated polyp directly adjacent to a CRC and
concurrent molecular evidence for a sequential relationship has not
been delivered.
Hyperplastic Polyposis Syndrome (HPS) is a condition
characterized by the presence of multiple colorectal serrated polyps.
The genetic cause(s) of HPS is/are largely unknown. We recently
demonstrated that HPS can occur in the context of MAP21, but MUTYH
mutations seem to occur in only a small proportion of HPS patients.22
HPS is associated with an increased CRC-risk.5, 7, 19, 22-27 Previously
published case series report CRC at clinical presentation in up to 50%
of HPS patients and interval carcinomas, i.e. carcinomas occurring
after HPS diagnosis and during endoscopic surveillance, in up to 25%
of patients.24, 28 However, since HPS is a heterogeneous condition,
comprising serrated polyps of different categories i.e. HPs and SSAs
but also co-existent conventional adenomas22, 23, 28-30, it is uncertain
which polyps eventually lead to CRC in these patients and thus are
clinically relevant. In the case that the CRCs do originate from the
serrated polyps, HPS may prove to be a valuable model for studying
the serrated CRC pathway.
Within a cohort of 56 HPS patients, we identified 17 patients
with CRC. By combined histopathological and molecular analyses of
the polyps and CRCs in these patients, we obtained novel evidence for
a serrated CRC pathway in HPS which predominates over the
classical WNT pathway of carcinogenesis.
CRC pathways in patients with HPS
127
MATERIALS AND METHODS
Subjects
From a cohort of 56 HPS patients undergoing endoscopic
treatment/surveillance at the Academic Medical Centre in the
Netherlands, 21 HPS patients had CRC. From 17 of these patients
tissue was available (n=19) which was included in this study. HPS was
defined as at least five histologically diagnosed HPs and/or SSAs
proximal to the sigmoid colon, of which 2 greater than 10mm in
diameter, or more than 20 HPs and/or SSAs distributed throughout the
colon.28 Because both HPs and SSAs are common findings in HPS
and have been shown to be difficult to differentiate microscopically, all
serrated polyps were included in our criteria. 31-34 Patients with a
known germline APC mutation or a bi-allelic MUTYH mutation were
excluded from the study. The study was conducted in accordance with
the research code of our institutional medical ethical committee on
human experimentation, as well as in agreement with the Helsinki
Declaration of 1975, as revised in 1983.
Specimens
All retrieved CRCs (n=19) were formalin-fixed and paraffin-embedded.
H&E stained tissue sections were re-evaluated by two pathologists
(CvN, SvE). In addition, all CRCs were re-evaluated for the presence
of an adjacent serrated component and for features of a serrated
adenocarcinoma as claimed by others.10 A control group was selected
consisting of sporadic, microsatellite stable (MSS) CRCs (n=14) from
non-polyposis patients, matched for age, gender and CRC-location.
These CRCs were re-evaluated as described above.
In the case of a mutation identified in a CRC, ≥ 5 polyps in the closest
proximity of the CRC were selected and reviewed by a single
Chapte
r 6
Chapter 6
128
pathologist (CvN) who was blinded for patient characteristics and
original histological diagnosis. Polyps were classified as HP, SSA,
TSA, mixed polyp or conventional adenoma based on the histological
features on H&E staining.35-37 Polyps with a serrated morphology i.e.
HPs, SSAs, TSAs and mixed polyps were collectively designated as
‘serrated polyps’. A polyp control group was also selected consisting of
sporadic HPs (n=24), SSAs (n=18) and conventional adenomas (n=17)
from non-polyposis patients. For the purpose of analysis, lesions from
the caecum, ascending colon, transverse colon and descending colon
were regarded as proximal and those from the sigmoid colon and
rectum were regarded as distal.
Somatic mutation analysis
Epithelial cells from polyps and CRCs were microdissected and DNA
was isolated as described previously.38, 39 Using previously described
primers and assays, DNA was analysed for mutations in the APC-
mutation cluster region (APC-MCR), KRAS (exon 2), BRAF (exon 15)
and NRAS (exons 1 and 2).38, 39 In the case of a CRC in a polyp,
mutation analysis of TP53 (exons 4-10) was performed in an attempt
to assess whether these two components were clonally related. In
case of an identified genetic mutation in a CRC, mutation analysis of
surrounding polyps (≥5) was performed as described above. Detected
mutations were confirmed in a second independent experiment.
Microsatellite instability analysis
Microsatellite status of the CRCs of HPS patients was determined
using an international standard panel of 5 microsatellite markers
(D17S250, D2S123, D5S346, BAT25 and BAT26) using standard
techniques. A high degree of microsatellite instability (MSI-high) was
CRC pathways in patients with HPS
129
defined as two (40%) or more unstable markers, MSI-Low as one
unstable marker, and microsatellite stable (MSS) as no unstable
markers.
Immunohistochemistry
Immunohistochemistry was performed on CRCs and polyps of the
HPS patients. Unstained 5-µm sections were cut from paraffin blocks
and the slides were deparaffinized. Primary monoclonal antibodies
used were specific for MLH1(1:50 BD Pharmingen, San Diego, USA);
MSH2(1:100 Oncogene Research Prod., San Diego, USA);
MSH6(1:200 BD Transduction Lab., San Jose, USA); PMS2(1:250 BD
Transduction Lab., San Jose, USA); SMAD4(1:200 Santa Cruz, USA);
CTNNB1(1:10.000 BD Biosciences, San Diego, USA) and
TP53(1:2000 Neomarkers, Fremont, USA). Slides were immersed in
0.3% hydrogen peroxide in methanol for 20 minutes. Subsequently,
antigen retrieval was carried out by 10 minutes of boiling in 10mM
Tris/1mM EDTA (pH 9) followed by incubation with above mentioned
diluted primary antibodies during 1 hour at room temperature. Post-
antibody block (Immunologic) in PBS was performed followed by
implementation of an antipolyvalent HRP detection system
(Immunologic) to visualize antibody binding sites with 3.3’-
diaminobenzidine as a chromogen. Sections were counterstained with
haematoxylin.
Immunoreactivity for CTNNB1 (β-catenin) was regarded as
positive when strong nuclear staining was observed in >25% of the
cells. Stains for TP53 were regarded to be indicative of TP53
dysfunction or deletion when >75% of the lesional nuclei were
strongly positive or completely negative (absent staining). Stains for
Chapte
r 6
Chapter 6
130
SMAD4, MLH1, MSH2, MSH6 and PMS2 were considered negative
when there was complete absence of nuclear expression in all lesional
cells. Negative staining in a part of a lesion or in a single crypt was
registered separately.
Statistics
Statistical analyses were performed by using a statistical software
package (Statistical Package for the Social Sciences 12.0.2; SPSS
Inc, Chicago, Ill). Somatic mutations in CRCs and polyps of HPS
patients were compared with those of a control panel using a two-
sided Fisher exact test. A p-value of < 0.05 was considered statistically
significant.
RESULTS
Patients
The clinico-pathological features of the HPS patients are summarized
in table 1. The median age of this cohort of 17 HPS patients at CRC
diagnosis was 58 years (range: 41-75) with a male: female ratio of 8:9.
In all patients, germline APC and MUTYH-mutation analyses were
previously performed and found negative. A surgical colonic resection
was performed in 14/17 (82%) patients: 6 subtotal colectomies, 4
hemi-colectomies (2 right-sided) and 4 (recto)sigmoidal resections.
Histological evaluation of biopsies and surgical resection specimens of
these patients revealed a median of 16 HPs, 7 SSAs and 2
conventional adenomas per patient. All patients satisfied the criteria for
HPS defined by the World Health Organization (WHO).30
CRC pathways in patients with HPS
131
Mutation analysis in HPS polyps and control-group polyps
A total of 84 polyps, originating from 17 HPS patients with CRC, were
analysed for pathogenic mutations in the APC-mutation cluster region
(APC-MCR), KRAS (codons 12 and 13), BRAF (codon 600) and NRAS
(exons 1 and 2). The 84 HPS polyps consisted of 21 HPs, 38 SSAs, 3
TSAs, 2 mixed polyps (64 serrated polyps) and 20 conventional
adenomas. The control group of sporadic lesions (n=59) consisted of
24 HPs, 18 SSAs (42 serrated polyps) and 17 conventional adenomas
(table 2).
Molecular analysis, of both the HPS polyps and control group
polyps, showed APC-MCR mutations exclusively in the conventional
adenomas and BRAF mutations exclusively in the serrated polyps:
BRAF mutations were detected in 48/64 (75%) HPS serrated polyps,
whereas 20/42 (48%) of the control group serrated polyps harbored a
BRAF mutation (p=0.007). Also when evaluating HPS patients
individually, each patient harbored predominantly BRAF mutations in
their serrated polyps. In four patients, beside BRAF mutations a single
KRAS mutation was identified in a distal serrated polyp. No significant
difference in frequency of KRAS mutations in serrated polyps was
seen between groups (6% vs 17%; p=0.1), but in conventional
adenomas of HPS patients no KRAS mutations were detected,
compared to 4/17 (24%) conventional adenomas in the control group
(p=0.029). In none of the polyps KRAS and BRAF mutations were
found together.
Chapte
r 6
Chapter 6
132
Table 1. Clinico-pathological features of HPS patients with colorectal cancer G= gender, Location=location of carcinoma, AC=ascending colon, TC= transverse colon DC=descending colon
When location in the colon was analyzed, no association was
seen between the presence of a BRAF mutation and polyp location in
both HPS and in the control group (table 3). BRAF mutations were
observed in 36/46 (82%) proximal HPS serrated polyps and in 12/18
(67%) distal HPS serrated polyps. In HPS, KRAS mutated serrated
Case no. Age G CRC Location CRC Size (TNM) Adjacent polyps
1 59 F Caecum 56 mm (T3N0M0) -
2 41 F Rectosigmoid 25 mm (T2N0M0) -
3 75 M AC 50 mm (T3N0M0) -
4 58 M Rectosigmoid 4 mm (TisN0M0) HP
5 59 M AC < 20 mm (T1N0M0) SSA
6 68 F AC 95mm (T3N0M0) -
7 43 F DC 40mm (T4N1M0) -
8 49 F DC Not stated(TisN0M0) HP
9 54 M DC 50mm (T3N0M0) -
9 Rectosigmoid 20mm (T1N0M0) -
10 54 F AC 16mm (T1N0M0) SSA
11 56 F Rectosigmoid 65mm (T2N0M0) Adenoma
12 61 F Rectosigmoid
20mm (T1N0M0) -
13 52 M Rectosigmoid
40mm (T3N1M0) -
14 66 M AC 8 mm (T1N0M0) SSA
15 63 M AC 6 mm (T1N0M0) SSA
16 69 M TC 120mm (T3N1M1) -
17 68 F AC 12mm (TisNoM0) -
17 68 F Rectosigmoid 29mm (T2N0M0) -
CRC pathways in patients with HPS
133
polyps (4/15: 27%) were exclusively detected in the distal colon
(p=0.005).
Immunohistochemistry in polyps
In none of the 84 HPS polyps, loss of expression of the mismatch
repair genes (MLH1, MSH2, MSH6 and PMS2) or SMAD4 was
observed (not shown). In addition, none of these polyps showed
abnormal TP53 staining (either strong nuclear TP53 staining or
complete TP53 loss, not shown). In 9 conventional HPS adenomas,
we detected strong nuclear CTNNB1 staining (i.e. in >25% of lesional
cells, not shown). In 5 of these adenomas, an APC-MCR mutation was
identified. In four other APC-MCR mutated adenomas no abnormal
nuclear β-catenin staining was detected. Nuclear CTNNB1 was not
found in any of the serrated polyps of either group.
Table 2. Detected APC, KRAS and BRAF mutations in serrated polyps (HPs, SSAs, TSAs and mixed polyps) and adenomas (AD) compared to a control panel. *Statistically significant p-value for BRAF mutation frequency in serrated polyps of HPS patients compared to serrated polyps in the control group **Statistically significant p-value KRAS mutation frequency in adenomas of HPS patients compared to adenomas in the control group.
Somatic mutation Patients with HPS Control group P-value
Serrated polyps
(n=64) AD
(n=20) Serrated polyps
(n=42) AD
(n=17)
APC mutation 0 9
(45%) 0 7 (41%) Ns
BRAF mutation 48 (75%) 0 20 (48%) 0 0.007*
KRAS mutation 4 (6%) 0 7 (17%) 4 (24%) 0.029**
Chapte
r 6
Chapter 6
134
Histological characteristics of the colorectal carcinomas
Upon histological re-evaluation of CRCs, 6/19 (32%) HPS CRCs were
identified within/directly adjacent to a serrated polyp. In 1/19 (5%)
cases, the HPS CRC was identified within a conventional adenoma
(Fig.1). One patient had two synchronous CRCs. Although 6 CRCs
were identified within serrated polyps, no HPS CRCs displayed a
distinguishing morphology justifying the diagnosis serrated
adenocarcinoma.10
Mutation: BRAF mutation KRAS mutation
Group: HPS Control group HPS Control group
Location: Proximal Distal Proximal Distal Proximal Distal Proximal Distal
HP 8/13 (62) 5/8 (63) 1/2 (50) 9/22 (41) 0/13 (0) 3/8 (38) 0/2 (0) 5/22 (23)
SSA 25/30 (83) 5/8 (63) 10/18 (56) 0/1 (0) 0/30 (0) 1/8 (13) 2/17 (12) 0/1 (0)
TSA 1/1 (100) 2/2 (100) 0/0 0/0 0/1 (0) 0/2 (0) 0/0 0/0
MP 2/2 (100) 0/0 0/0 0/0 0/2 0/0 0/0 0/0
Table 3. Serrated polyp location and BRAF and KRAS mutation. MP=mixed polyp NOTE. All values are expressed as n (%).
CRC pathways in patients with HPS
135
Figure 1. Two colorectal carcinomas, one located within a serrated polyp (A and B, magnification 50x) and one within a villous adenoma (C, magnification 50x) of two HPS patients.
Mutation analysis in HPS CRCs and control-group CRCs
BRAF mutations were detected in 10/19 (53%) HPS CRCs, whereas
no BRAF mutations were detected in the CRCs of the control group
(p=0.001, table 4 and figure 2). All BRAF mutations involved the same
thymine to adenine transversion at nucleotide 1796, resulting in a
valine to glutamine substitution at codon 600 (V600E). We found no
Chapte
r 6
Chapter 6
136
NRAS mutations in the CRCs of either group. Of the 6 CRCs identified
within a serrated polyp, 5/6 (83%) carried the same BRAF mutation
(GTG → GAG) in both the polypous and tumor components (Table 5).
However, considering that these are hotspot mutations, these findings
do not prove a clonal relationship between these lesions. TP53 (exons
4-10) sequence analysis, performed on these 6 CRCs in order to
further explore the clonal relationship between the carcinomas and
precursor lesions, yielded no mutations.
Mutation
HPS carcinomas (n=19)
Carcinomas control group (n=14)
P-value
APC 2 (11%) 4 (29%) Ns
KRAS 1 (5%) 5 (36%) Ns
BRAF 10 (53%) 0 0.001*
NRAS 0 0 Ns
MSS 12 (63%) 14 NA
MSI-L 1 (5%) 0 NA
MSI-H 6 (32%) 0 NA
Table 4. Mutation spectrum of colorectal cancers in HPS patients compared to control-group CRCs. ns: not significant; NA: not applicable. *statistically significant p-value compared to control group CRC
CRC pathways in patients with HPS
137
Figure 2. Mutation profiles of HPS polyps/CRCs compared to sporadic control group polyps/CRCs. SPs = serrated polyps, Ads = conventional adenomas, CRCs = colorectal cancers
APC-MCR mutations were identified in 2/19 (11%) HPS CRCs
compared to 4/14 (29%) in the control group CRCs (not significant)
and 1/19 (5%) KRAS mutations were detected in HPS CRCs
compared to 5 (36%) in the control group (not significant). In one HPS
CRC (patient 6), both a BRAF mutation and an APC-MCR mutation
were detected. Interestingly, one of the HPS CRCs (patient 11),
harbouring an APC-MCR mutation (insertion 1364), was the single
HPS CRC found within a villous adenoma (Fig.1C). Subsequent APC-
MCR sequence analysis of the adenoma revealed the same mutation
(insertion 1364), establishing a clonal relationship between these
Chapte
r 6
Chapter 6
138
lesions. Accordingly, both the adenomatous and malignant
components displayed evident nuclear CTNNB1 staining (Fig.3).
When location in the colon was analysed, all BRAF-mutated
HPS CRCs were exclusively detected in the proximal colon (p=0.007).
No association was seen between location and KRAS mutations in the
control group or APC-mutations in both groups. The entire list of
molecular analyses on a per-carcinoma basis can be found as a
supplementary table online (http://ajp.amjpathol.org).
Table 5. Mutations in CRCs and adjacent polyps.
Carcinoma
Adjacent polyp
Mutation in CRC
Mutation in adjacent polyp
4 HP None None
5 SSA BRAF (GTG→GAG)
BRAF (GTG→GAG)
8 HP BRAF (GTG→GAG) BRAF (GTG→GAG)
10 SSA BRAF (GTG→GAG) BRAF (GTG→GAG)
11 Adenoma APC (insertion G 1354) APC (insertion G 1354)
15 SSA BRAF (GTG→GAG) BRAF (GTG→GAG)
16 SSA BRAF (GTG→GAG) BRAF (GTG→GAG)
CRC pathways in patients with HPS
139
Figure 3. Nuclear CTNNB1 expression in the adenocarcinoma and the adjacent adenoma of patient 11, both harbouring the same APC gene mutation (magnification 200x).
Genetic events detected immunohistochemically and by
microsatellite analysis in CRCs
In 6/19 (32%) HPS CRCs, loss of expression was seen of at least 1
mismatch repair (MMR) protein. In 6 CRCs, there was complete loss of
expression of both MLH1 and PMS2. All these 6 HPS CRCs were
microsatellite-unstable (MSI-high), were proximally located and
harboured a BRAF mutation. Two of the 6 MSI-high CRCs involved
combined serrated polyp-CRC lesions. In these lesions, the serrated
polyp components were microsatellite stable (MSS).
Strong nuclear β-catenin staining (>25%) was observed in 5/19
(26%) HPS CRCs. In one of these CRCs, an APC-MCR mutation was
identified and none of these was BRAF mutated. In all 5 of these HPS
CRCs, abnormal TP53 staining was observed (>75% nuclear staining
or complete absence), indicative of loss of functional TP53. Three
additional HPS CRCs displayed a perturbed TP53 status. In 2/19
(11%) CRCs loss of SMAD4, either focal or complete, was detected.
Chapte
r 6
Chapter 6
140
DISCUSSION
In this first comprehensive cohort study of HPS patients with
both CRCs and polyps, we demonstrated that the serrated polyps and
conventional adenomas of HPS patients not only morphologically
resemble their respective sporadic counterparts but also have similar
molecular profiles (table 3, figure 2). Interestingly, we identified a high
number of combined serrated polyp-CRC lesions which showed
identical BRAF mutations in both components, supporting the
existence of a serrated CRC pathway. Overall, we demonstrated that
both microsatellite-stable and –unstable CRCs in HPS predominantly
originate from the serrated polyps thus confirming that HPS patients
provide a valuable model to analyze the molecular characteristics of
the serrated CRC pathway.
In accordance with previous reports 13, 40, 41, the HPS serrated
polyps harboured significantly more BRAF mutations than those of the
control group (80 vs. 48%: p=0.001). The slight difference in the KRAS
mutation frequencies between both groups (7 vs. 17%) was not
statistically significant. Thus in HPS patients, BRAF mutations
correlate even stronger with serrated polyps than in non-HPS patients.
This contrasts with serrated polyps in MAP patients with a germline
MUTYH gene mutation, which contain significantly more KRAS
mutations (70%) than BRAF mutations (4%).21 Both BRAF and KRAS
have been identified as early or instigating events in the serrated
pathway. It has to be determined however, whether serrated lesions
with these mutually exclusive mutations are biologically equivalent and
have the same risk of developing to CRC.
The mutation profiles of the HPS CRCs were clearly more
similar to those found in the serrated polyps than in the conventional
adenomas (table 3 and figure 2). The profiles of the control group MSS
CRC pathways in patients with HPS
141
CRCs, as expected, were comparable to the profiles of the
conventional adenomas. As many as 10 of the included 19 HPS CRCs
were BRAF-mutated compared to none of the 14 control group CRCs
(p=0.001). Interestingly, all these BRAF-mutated CRCs were
proximally located. Previous molecular studies in HPS CRCs
encompassed small non-controlled series of selected cases.23, 42-44 and
a comparison between our results and these studies is not
straightforward owing to variation in patient selection criteria and
analysis of different or single genes. Overall however, BRAF mutations
were identified in 6/15 (40%) HPS-related CRC-cases described in the
assembled literature until now. We identified no BRAF mutations in the
control group of age-and sex-matched, MSS CRCs from non-polyposis
patients. Concordantly, BRAF mutations have been reported in only 5-
10% of sporadic MSS CRCs of non-polyposis patients (n>100).45-48
Considering that in the literature serrated polyps are suggested to be
precursor lesions of particularly MSI CRCs, we chose MSS
carcinomas in our control group in order to exclude MSI-CRCs from
potentially unrecognized HPS patients. However, this strategy is
arbitrary and eliminates sporadic MSI CRCs from non-HPS patients.
Hence, our statistically significant difference in BRAF mutation
frequency between HPS CRCs and control group CRCs is higher
compared to an unselected cohort of CRCs. We detected only one
KRAS mutation in a distally located HPS CRC, which corroborates two
previously published studies together analyzing a total of 15 HPS
CRCs.40, 41 To our knowledge, only one other study has described the
presence of a KRAS mutation in a single distally located HPS CRC,
supporting the notion that KRAS plays a minor role in the
carcinogenesis of HPS and is confined to the distal colon.23
Chapte
r 6
Chapter 6
142
In our study, 2 patients had synchronous CRCs. Theoretically,
a field effect may account for the development of synchronous
polyps/CRCs.49 In one patient (patient 9), no mutations were identified
in both CRCs. The other patient (patient 17) harboured one KRAS
mutation and one BRAF mutation respectively in each of the CRCs.
Hence, although a field effect associated with a serrated CRC pathway
may cause simultaneous CRCs with identical mutations, we were not
able to demonstrate this in patient 9 due to the lack of any mutations
and seems excluded in patient 17. Of note, clonal markers are
necessary to be able to demonstrate a true field effect. Such clonal
markers associated with the serrated pathway are currently not
available.
The histological characteristics of the HPS CRCs as a group,
either or not BRAF-mutated, were inconspicuous and not obviously
different from the control group CRCs. In particular, a serrated growth
pattern, as has been reported, 50, 51 was not apparent in our series of
HPS CRCs. We found a remarkable number of HPS CRCs (6/19:
32%) to be located within or directly adjacent to a serrated polyp (Fig.
1). In 5/6 of these combined lesions an identical hotspot mutation at
codon 600 (GTG→GAG) of BRAF was detected in both components.
These combined histological and molecular data are strongly
suggestive for a sequential relationship between serrated polyps and
CRC. A clonal relationship between the carcinomas and their assumed
precursor lesions could not be further substantiated due to lack of
appropriate clonal markers.
In the literature it has been shown that 90% of sporadic MSI-H
CRCs are caused by loss of MLH1 function as a result of methylation
of this mismatch repair gene. In these lesions, BRAF mutations, which
are associated with serrated polyps, are also a common finding
CRC pathways in patients with HPS
143
suggesting that the serrated pathway can generate MSI-H CRCs.13, 15
Interestingly, 6/19 HPS CRCs were MSI-H due to loss of MLH1 and
PMS2 and all 6 carried BRAF mutations. Additional analysis in these
CRCs also showed hypermethylation of MLH1 in all cases (data not
shown). These findings suggest a causal relationship between this
novel carcinogenic route and microsatellite-unstable CRCs. On the
other hand, our findings demonstrate that the serrated pathway, at
least in HPS, also generates MSS-CRCs.
Previous large cohort studies (>30 patients) have reported the
presence of at least one conventional adenoma in 69- 85% of HPS
patients and >5 conventional adenomas in 21-32% of cases.22, 23, 28-30
Concordantly, in our study APC-MCR mutations were detected in two
(13%) HPS CRCs. The identification of an HPS CRC (patient 11)
located within a conventional adenoma of the same clonal origin,
reflected by the identical APC-MCR mutation in both components, is
significant since this proves that the classical adenoma-carcinoma
pathway52 is also operational in HPS patients.
The apparent dominance of the serrated over the non-serrated
CRC pathway in HPS may either be due to a greater intrinsic risk of
tumour progression of the serrated polyps, or be simply a reflection of
the numerical prevalence of serrated polyps over adenomas. To our
knowledge, the only study that has addressed this issue in 2 HPS CRC
cases, reported no APC mutations.23 In that study however, patients
with >10 conventional adenomas were excluded. In our study, all
cases satisfied the WHO criteria for HPS in which the presence or
number of conventional adenomas is not an issue. Interestingly, in
HPS patient 11 with the classical CRC, a relative abundance of
conventional adenomas was observed (12 adenomas versus 17
serrated polyps), suggesting a stochastic rather than an intrinsically
Chapte
r 6
Chapter 6
144
biased process of carcinogenesis. Interestingly, in the other APC-
MCR-mutated HPS CRC (patient 6) also a BRAF mutation was
identified. Assuming that this CRC is of monoclonal origin, it may be
the outcome of an elusive ‘fusion’ pathway which combines
mechanisms associated with both conventional adenomas and
serrated polyps, as proposed previously.36
We observed 4 HPS CRCs with nuclear CTNNB1 staining,
associated with the WNT-pathway. Although in these CRCs, no APC-
MCR mutations or CTNNB1 exon 4-10 hotspot mutations were
identified, alternative mechanisms may be operational to activate the
WNT -pathway, e.g. methylation of the APC promotor regions and
MSI-related frameshift mutations in WNT pathway regulators like
AXIN2.53 In these HPS patients, a median of only 1 adenoma (range:
0-5) was identified, suggesting that the WNT-pathway indeed may be
involved at some stage of carcinogenesis via the serrated pathway.
Considering that no BRAF mutations or directly adjacent serrated
polyps were found, it seems unlikely that these CRCs are the outcome
of a proposed fusion pathway, but formally this can not be excluded.
Based on these observations, we conclude that distinct, APC-
and non-APC mediated CRC pathways are functional in HPS. A
serrated pathway, skewed towards initial BRAF mutations and
proximal localization, however seems to predominate, most likely due
to the numerical prevalence of serrated polyps in these patients. From
this it is inferred that all polyp types in HPS, should be considered
clinically relevant and be removed.
CRC pathways in patients with HPS
145
REFERENCES 1. Jemal A, Thun MJ, Ries LA, Howe HL, Weir HK, Center MM, Ward E,
Wu XC, Eheman C, Anderson R, Ajani UA, Kohler B, Edwards BK. Annual Report to the Nation on the Status of Cancer, 1975-2005, Featuring Trends in Lung Cancer, Tobacco Use, and Tobacco Control. J Natl Cancer Inst 2008.
2. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759-767.
3. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532.
4. Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93:1307-1313.
5. Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y, Watanabe H. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52:5-9.
6. Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E, Meltzer SJ, Wright C, Searle J, Young J, Leggett BA. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999;52:455-460.
7. Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ, Cohn D, Furlong KL, Walsh MD, Palazzo J, Edmonston TB, Fishel R, Young J, Leggett BA. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000;47:43-49.
8. Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000;37:295-301.
9. Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001;193:283-285.
Chapte
r 6
Chapter 6
146
10. Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001;193:286-294.
11. Yao T, Nishiyama K, Oya M, Kouzuki T, Kajiwara M, Tsuneyoshi M. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000;190:444-449.
12. Jass JR. Serrated adenoma of the colorectum and the DNA-methylator phenotype. Nat Clin Pract Oncol 2005;2:398-405.
13. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.
14. McGivern A, Wynter CV, Whitehall VL, Kambara T, Spring KJ, Walsh MD, Barker MA, Arnold S, Simms LA, Leggett BA, Young J, Jass JR. Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer. Fam Cancer 2004;3:101-107.
15. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B, Velculescu VE. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934.
16. Young J, Simms LA, Biden KG, Wynter C, Whitehall V, Karamatic R, George J, Goldblatt J, Walpole I, Robin SA, Borten MM, Stitz R, Searle J, McKeone D, Fraser L, Purdie DR, Podger K, Price R, Buttenshaw R, Walsh MD, Barker M, Leggett BA, Jass JR. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 2001;159:2107-2116.
17. Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000;43:1309-1313.
18. Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994;89:123-125.
CRC pathways in patients with HPS
147
19. Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984;8:551-556.
20. Goldstein NS, Bhanot P, Odish E, Hunter S. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119:778-796.
21. Boparai KS, Dekker E, van ES, Polak MM, Bartelsman JF, Mathus-Vliegen EM, Keller JJ, van Noesel CJ. Hyperplastic polyps and sessile serrated adenomas as a phenotypic expression of MYH-associated polyposis. Gastroenterology 2008;135:2014-2018.
22. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
23. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.
24. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
25. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
26. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
27. Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y, Oda J, Mizutani M, Takayanagi S, Kishi D, Shinohara T, Yamada K, Matumoto J, Imamura K. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008.
28. Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ, Houben M, Cats A, van Hest LP, Fockens P, Dekker E. Increased colorectal cancer risk during follow-up in patients
Chapte
r 6
Chapter 6
148
with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009.
29. Buchanan DD, Sweet K, Drini M, Jenkins MA, Win AK, Gattas M, Walsh MD, Clendenning M, McKeone D, Walters R, Roberts A, Young A, Hampel H, Hopper JL, Goldblatt J, George J, Suthers GK, Phillips K, Young GP, Chow E, Parry S, Woodall S, Tucker K, Muir A, Field M, Greening S, Gallinger S, Green J, Woods MO, Spaetgens R, de la Chapelle A, Macrae F, Walker NI, Jass JR, Young JP. Phenotypic diversity in patients with multiple serrated polyps: a genetics clinic study. Int J Colorectal Dis 2010;25:703-712.
30. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.
31. Khalid O, Radaideh S, Cummings OW, O'Brien MJ, Goldblum JR, Rex DK. Reinterpretation of histology of proximal colon polyps called hyperplastic in 2001. World J Gastroenterol 2009;15:3767-3770.
32. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.
33. Sandmeier D, Benhattar J, Martin P, Bouzourene H. Serrated polyps of the large intestine: a molecular study comparing sessile serrated adenomas and hyperplastic polyps. Histopathology 2009;55:206-213.
34. Wong NA, Hunt LP, Novelli MR, Shepherd NA, Warren BF. Observer agreement in the diagnosis of serrated polyps of the large bowel. Histopathology 2009;55:63-66.
35. Hamilton SR, Vogelstein B, Kudo S, et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000:104-119.
36. Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D, Young J. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49:121-131.
37. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.
CRC pathways in patients with HPS
149
38. de Leng WW, Keller JJ, Luiten S, Musler AR, Jansen M, Baas AF, de Rooij FW, Gille JJ, Menko FH, Offerhaus GJ, Weterman MA. STRAD in Peutz-Jeghers syndrome and sporadic cancers. J Clin Pathol 2005;58:1091-1095.
39. de Leng WW, Westerman AM, Weterman MA, de Rooij FW, Dekken HH, De Goeij AF, Gruber SB, Wilson JH, Offerhaus GJ, Giardiello FM, Keller JJ. Cyclooxygenase 2 expression and molecular alterations in Peutz-Jeghers hamartomas and carcinomas. Clin Cancer Res 2003;9:3065-3072.
40. Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S, Broaddus RR, Issa JP, Hamilton SR, Rashid A. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005;166:1069-1075.
41. Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002;160:529-536.
42. Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S, Broaddus RR, Issa JP, Hamilton SR, Rashid A. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005;166:1069-1075.
43. Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003;63:4878-4881.
44. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J, Jass JR. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467-1474.
45. Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML. Poor survival associated with the BRAF V600E mutation in microsatellite-stable colon cancers. Cancer Res 2005;65:6063-6069.
46. Barault L, Charon-Barra C, Jooste V, de la Vega MF, Martin L, Roignot P, Rat P, Bouvier AM, Laurent-Puig P, Faivre J, Chapusot C, Piard F. Hypermethylator phenotype in sporadic colon cancer: study on a population-based series of 582 cases. Cancer Res 2008;68:8541-8546.
Chapte
r 6
Chapter 6
150
47. Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Meyerhardt JA, Loda M, Giovannucci EL, Fuchs CS. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 2009;58:90-96.
48. de VS, Weijenberg MP, Herman JG, Wouters KA, de Goeij AF, van den Brandt PA, de Bruine AP, van EM. MGMT and MLH1 promoter methylation versus APC, KRAS and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events. Ann Oncol 2009;20:1216-1222.
49. Nosho K, Kure S, Irahara N, Shima K, Baba Y, Spiegelman D, Meyerhardt JA, Giovannucci EL, Fuchs CS, Ogino S. A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 2009;137:1609-1620.
50. Makinen JM, Makinen MJ, Karttunen TJ. Serrated adenocarcinoma of the rectum associated with perianal Paget's disease: a case report. Histopathology 2002;41:177-179.
51. Makinen MJ. Colorectal serrated adenocarcinoma. Histopathology 2007;50:131-150.
52. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM, Bos JL. Genetic alterations during colorectal-tumor development. N Engl J Med 1988;319:525-532.
53. Thorstensen L, Lind GE, Lovig T, Diep CB, Meling GI, Rognum TO, Lothe RA. Genetic and epigenetic changes of components affecting the WNT pathway in colorectal carcinomas stratified by microsatellite instability. Neoplasia 2005;7:99-108.
NBI increases polyp detection in HPS
151
Endoscopic imaging – Detection and differentiation of polyps
P A
R T
Chapter 7
152
NBI increases polyp detection in HPS
153
Increased polyp detection using narrow-band imaging compared to high-resolution endoscopy in patients with hyperplastic polyposis syndrome
Karam S. Boparai
Frank J.C. van den Broek
Susanne van Eeden
Paul Fockens
Evelien Dekker
Endoscopy. 2011 Aug;43(8):676-82
Ch
ap
ter
Chapter 7
154
SUMMARY
Background and study aims: Hyperplastic polyposis syndrome
(HPS) is associated with colorectal cancer and is characterized by
multiple hyperplastic polyps (HPs), sessile serrated adenomas (SSAs)
and adenomas. Narrow-band imaging (NBI) may improve the detection
of polyps in HPS. We aimed to compare polyp miss-rates of NBI
compared to high-resolution endoscopy (HRE).
Patients and Methods: This was a single center, randomized cross-
over study in which consecutive HPS patients underwent tandem
colonoscopy with HRE and NBI, in randomized order with removal of
all detected polyps.
Results: In 22 patients with HPS, 209 polyps were detected: 27
normal histology, 116 HPs, 42 SSAs and 24 adenomas. Within
patients assigned to HRE first (n=11) a total of 78 polyps was
detected; subsequent NBI added 44 polyps. In patients examined with
NBI first, 78 polyps were detected and subsequent HRE added 9.
Polyp miss-rates of HRE and NBI were 36% and 10% (OR 0.21; 0.09-
0.45). Flat polyp shape was independently associated with increased
miss-rate.
Conclusion: NBI significantly reduces polyp miss-rates in HPS
patients. We recommend using either NBI or chromoendoscopy for
colonoscopic surveillance of HPS patients with removal of all detected
polyps.
NBI increases polyp detection in HPS
155
INTRODUCTION
Hyperplastic polyposis syndrome (HPS) is characterized by the
presence of multiple hyperplastic polyps (HPs) spread throughout the
colon and is associated with an increased colorectal cancer (CRC)
risk.[1-4] Besides HPs, sessile serrated adenomas (SSAs) and
conventional adenomas are common findings in this condition as well.
The presence of SSAs is even considered typical for HPS.[5-7]
Whereas sporadic HPs are traditionally considered to be low-
risk lesions, a novel serrated neoplasia pathway has been suggested
which describes the progression of serrated polyps (i.e. HPs, SSAs
and traditional serrated adenomas) to CRC through accumulation of
genetic mutations.[8-15] Molecular research strongly suggest that
serrated polyps are lesions which may lead to CRC with BRAF and
CPG-island methylator phenotype (CIMP).[16-20] In addition,
clinicohistological reports supporting a serrated neoplasia pathway
include CRCs in close vicinity of large hyperplastic polyps [21,22] and
CRCs identified in serrated polyps [23-25]. Therefore, serrated polyps
seem to represent direct premalignant lesions. SSAs have even been
recommended to be managed as conventional adenomas.[26]
Serrated polyps in HPS however, have been shown to have even
higher numbers of BRAF mutations and CIMP compared to sporadic
serrated polyps.[20,27] Moreover, HPS patients have far more
serrated polyps than people in the general population. This may (in
part) explain the increased risk of CRC in HPS patients. Numerous
HPS patients with CRC arising in a serrated polyp have been
reported.[23] Thus, detection and removal of serrated polyps seems
necessary to prevent CRC in patients with HPS.
Chapte
r 7
Chapter 7
156
In HPS patients however, SSAs and HPs are generally small
and flat.[28-30] These features are associated with polyp miss-rates of
up to 26% using standard colonoscopy.[31-33] Improved detection of
these polyps by using advanced endoscopic techniques seems
therefore desirable. Chromoendoscopy has previously been shown to
improve the detection of small and flat lesions, specifically HPs, in
patients undergoing surveillance colonoscopy.[32-36] However,
chromoendoscopy is a labour-intensive and time-consuming
technique. Narrow-band imaging (NBI) is an easier push-on-a-button
technique that enhances mucosal and vascular detail without the use
of dyes, and has proven to be superior to high-resolution endoscopy
(HRE) for the detection of sporadic HPs.[37,38] The aim of this
randomized trial was to compare NBI and HRE for the detection of
polyps in patients with HPS.
PATIENTS AND METHODS
Patients
Between October 2007 and October 2008, consecutive HPS
patients were recruited for this study at the Academic Medical Center
in Amsterdam. A diagnosis of HPS is based on the following criteria: 1)
≥20 HPs found during previous colonoscopies; 2) ≥5 HPs proximal to
the sigmoid colon of which 2 were larger than 1cm; or 3) any HP
occurring proximal to sigmoid colon in an individual who has a first-
degree relative with HPS.[1,4] However, owing to the common
presence of both HPs and SSAs in HPS and the difficult histological
differentiation between these two groups, both HPs and SSAs were
used to fulfil the criteria. [26,39-41] Patients were excluded in case of
inflammatory bowel disease, severe coagulopathy, <18 years of age,
NBI increases polyp detection in HPS
157
insufficient bowel preparation (<90% of colonic mucosa visible) and a
known germline APC mutation or bi-allelic MYH mutation. From
included patients informed consent was obtained and the study was
approved by our institutional review board.
Endoscopic equipment
For this study the Evis Lucera system (CV-260, Olympus Inc.,
Tokyo, Japan) and a high-resolution video colonoscope (CF-H260Z)
were used integrating HRE, NBI and optical magnification (100×). The
endoscopist could easily switch between the imaging modes by
pressing a button on the shaft of the endoscope. As only high-
resolution monitors were used, the high-definition signal of the system
was not utilized.
Study design and randomization
We used a cross-over study design with randomized order.
Consecutive patients underwent tandem colonoscopy with HRE and
NBI by the same endoscopist and the order of these techniques was
randomized (figure 1). After informed consent, block randomization
was performed by a single investigator by opening sealed opaque
envelopes (containing notes with ‘HRE’ or ‘NBI’ in a 1:1 ratio) once the
cecum was reached.
Chapte
r 7
Chapter 7
158
Figure 1. Flow diagram
Colonoscopic procedure
Patients were prepared with 4 liters polyethylene glycol solution
(Kleanprep, Norgine Inc., Amsterdam, Netherlands) and underwent
colonoscopy under conscious sedation with midazolam and fentanyl.
All procedures were performed by the same endoscopist (ED) who
was highly trained in NBI (>500 NBI colonoscopies).
The colonoscope was advanced to the cecum in the HRE mode
of the endoscope. No attention was paid to polyps during the insertion
phase. Cecal intubation was confirmed by identification of the
appendiceal orifice and ileocecal valve. After extensive rinsing and
suctioning of remaining stools, the level of bowel preparation was
determined as excellent (100% of colonic mucosa visible), good (90-
NBI increases polyp detection in HPS
159
99%) or poor (<90%). Patients with poor bowel preparation were
excluded from this study.
Hereafter, each colonic segment (ascending, transverse,
descending and recto sigmoid colon) was examined twice, once with
HRE and once with NBI. The order of the two techniques was
determined by randomization. During withdrawal with the first
technique, each segment of the colon was meticulously inspected for
the presence of polyps. Of all detected polyps the size (estimated by
an opened biopsy forceps), location (colonic segment and distance
from the anus) and Paris classification were noted.[42] Hereafter, each
polyp was immediately removed by endoscopic mucosal resection or
biopsy removal (if <5mm) and sent for pathology in separate jars.
After first inspection and polyp clearance of each colonic
segment, the colonoscope was advanced again to the beginning of the
segment. The other imaging technique was then used for the second
inspection of the same segment. In case of indistinctive hepatic or
splenic flexures, a random biopsy was taken for reference of each
colonic segment. If additional polyps were detected during the second
examination, their size, location and Paris classification were noted
before removal.
Withdrawal times during the HRE and NBI examinations were
measured by using a stopwatch. Time for performing polypectomy was
not included in these withdrawal times. A maximum colonoscopy time
of 2 hours was set for the entire endoscopic procedure; otherwise the
procedure was too long and inconvenient for the patient. It was not
possible to blind the endoscopist to the imaging intervention, and,
logistically, it was not possible to have a different endoscopist perform
the second examination.
Chapte
r 7
Chapter 7
160
Histopathology
Resection specimens were evaluated by an expert GI
pathologist (SvE) who was blinded for endoscopic technique. Lesions
were classified as normal mucosa, HP, SSA, traditional serrated
adenoma, mixed polyp or conventional adenoma based on the
morphological features on H&E staining.[19,26,43] As previously
described by Torlakovic et al, SSAs were defined by architectural
distortion with irregular dilated crypts, including dilatation of the base of
the crypts that often have a boot, L or inverted T shape, serration
including at the base of the crypts and abnormal proliferation and
maturation with mature goblet or foveolar cells at the base of the
crypts.[44]
Outcome measures
Primary outcome measure was the polyp miss-rate of each technique,
defined as the number of polyps detected during the second inspection
divided by the total number of polyps detected during both
examinations. Additional exploratory sub-analyses were performed
regarding polyp location, -size and -shape.
Statistical analysis and sample size
Polyp miss-rates of NBI and HRE were compared by Chi-square
testing. Logistic regression analysis was used to evaluate associations
between polyp characteristics and polyp miss-rate (i.e. dependent
variable), using odds ratios (OR) plus 95%-conficence interval to
represent the strength of the association. Histopathology of each polyp
served as reference standard. The STARD statements were used for
reporting diagnostic test accuracy.[45] In addition the CONSORT
statements were used for reporting this randomized controlled trial.[46]
NBI increases polyp detection in HPS
161
Previous research comparing NBI and HRE for adenoma
detection showed a 3.3-fold increase in detection of HPs with NBI.[47]
As the general polyp miss-rate is 22%, we hypothesized a 3.3-fold
decrease in miss-rate with NBI resulting in a polyp miss-rate of
6.7%.[31] To detect this difference in polyp miss-rate with a power of
80% and significance level of 5%, a total of 188 polyps were required.
In a previous analysis of HPS patients undergoing surveillance
endoscopies at our department we found a mean number of 9 polyps
per HPS patient, resulting in (188/9=) 22 patients for inclusion in this
trial.[48]
RESULTS
A total of 22 patients with HPS were randomized to tandem
colonoscopy with either HRE first (n=11) or NBI first (n=11). Patient
characteristics are demonstrated in table 1 and were comparable
between the randomization groups.
Chapte
r 7
Chapter 7
162
Demographics Randomization
HRE (n=11) NBI (n=11) P
Mean age, yrs (range) 58 (33-73) 62 (43-76) .331
Male 8 (73%) 4 (36%) .198
Personal history of high-grade neoplasia or CRC 6 (55%) 4 (36%) .670
Partial colectomy 5 (45%) 3 (27%) .659
Number of previous polyps (mean number per patient)
Hyperplastic polyps 18.1 11.3 .121
Sessile serrated adenomas 4.4 7.6 .183
Adenomas 2.9 4.2 .508
Bowel preparation
Excellent 10 (91%) 6 (55%) .149
Good 1 (9%) 5 (45%)
Examination time (minutes), mean (±SD)
First inspection 15.0 (3.7) 13.9 (3.8) .485
Second inspection 11.2 (3.6) 8.9 (2.3) .115
Table 1: demographics of patients randomized to HRE and NBI as first inspection
technique
Polyp miss-rates
High-resolution endoscopy: During HRE as first examination
technique, a total number of 78 polyps (mean size 6.0mm; range 2-15)
were detected. Histology demonstrated normal tissue in 8 polyps, HP
in 58, SSA in 5 and conventional adenoma in 7. Subsequent
inspection with NBI added 44 polyps (mean 6.1mm; 2-20) of which 4
with normal histology, 29 HPs, 8 SSAs and 3 conventional adenomas.
The corresponding overall polyp miss-rate of HRE hence was 36%
(95%-CI: 28-45).
NBI increases polyp detection in HPS
163
Narrow-band imaging: During NBI as first examination
technique, a total number of 78 polyps (mean size 5.4mm; range 2-20)
were found (13 normal, 26 HP, 25 SSA, 14 adenomas). Subsequent
inspection with HRE added 9 polyps (mean 4.7mm; 2-10) of which 2
had normal histology, 3 were HP and 4 SSA. The corresponding
overall polyp miss-rate of NBI was 10% (95%-CI: 5.5-19).
The overall polyp miss-rate for NBI was significantly lower than
for HRE (OR 0.21; 95%-CI: 0.094-0.45; p<0.001). Table 2
demonstrates the polyp miss-rates for HPs, SSAs and adenomas
separately. Table 3 shows the polyp miss-rates for flat (Paris 0-IIa, 0-
IIb, 0-IIa+c) and protruded (Paris 0-Is, 0-Ip) lesions and for proximal
and distal colonic locations of polyps.
On multivariable logistic regression analysis, the use of NBI
was independently associated with a reduction in polyp miss-rate (OR
0.17; 95%-CI: 0.08-0.39), whereas flat macroscopic appearance was
associated with an increased miss-rate (OR 3.73; 1.72-8.05). Polyp
size, colonic location and histology were not associated with the miss-
rate.
Chapte
r 7
Chapter 7
164
HRE NBI P
Overall polyps
First inspection, n 78 78 .741
Second inspection, n 44 9 -
Miss-rate, % (95%-CI) 36 (28-45) 10 (5.5-19) <.001
Hyperplastic polyps
First inspection, n 58 26 .181
Second inspection, n 29 3 -
Miss-rate, % (95%-CI) 33 (24-44) 10 (3.6-26) .017
Sessile serrated adenomas
First inspection, n 5 25 .081
Second inspection, n 8 4 -
Miss-rate, % (95%-CI) 62 (36-82) 14 (5.5-31) .003
Adenomas
First inspection, n 7 14 .271
Second inspection, n 3 0 -
Miss-rate, % (95%-CI) 30 (11-60) 0 (0-22) .059
Table 2: polyp detection during the first and second inspection and polyp miss-rates among patients randomized to either HRE or NBI as first inspection technique, subdivided for histopathological outcome of polyps.
NBI increases polyp detection in HPS
165
HRE NBI P
Flat polyps
First inspection, n 37 50 .665
Second inspection, n 35 7 -
Miss-rate, % (95%-CI) 49 (37-60) 12 (6.1-23) <.001
Protruded polyps
First inspection, n 41 28 .595
Second inspection, n 9 2 -
Miss-rate, % (95%-CI) 18 (9.8-31) 6.7 (1.9-21) .195
Proximal location
First inspection, n 31 45 .349
Second inspection, n 19 5 -
Miss-rate, % (95%-CI) 38 (26-52) 10 (4.4-21) .001
Distal location
First inspection, n 47 33 .302
Second inspection, n 25 4 -
Miss-rate, % (95%-CI) 35 (25-46) 11 (4.3-25) .011
Table 3: polyp detection during the first and second inspection and polyp miss-rates among patients randomized to either HRE or NBI as first inspection technique, subdivided for macroscopic appearance and colonic location of polyps.
DISCUSSION
Previous large prospective randomized trials comparing NBI with HRE
for adenoma detection showed that NBI was associated with an
increased sporadic HP detection rate, although adenoma detection
rates were equal.[47,49] Table 4 lists all randomized clinical trials
comparing NBI with standard white-light endoscopy for the detection of
HPs and non-adenomatous polyps. These studies showed that NBI
was particularly of value for the detection of HPs.
Chapte
r 7
Chapter 7
166
Hyperplastic polyp detection
Author Study design N WLE NBI p-value
Adler 2008[47] RCT: NBI vs WLE 401 Detected: 23 Detected: 56 <0.001
Adler 2009[49] RCT: NBI vs WLE 1256 Detected: 116 Detected: 146 0.03
Non-adenomatous polyp detection
Author Study design N WLE NBI p-value
Inoue 2008[50] RCT: NBI vs WLE 109 Detected: 12 Detected: 24 n.a.
Kaltenbach
2009[51]
Tandem design:
WLE-NBI 276
Miss rate:
12.6%
Miss rate:
10.1% n.s.
Table 4: Randomized clinical trials comparing the detection of hyperplastic polyps between narrow-band imaging (NBI) and white light endoscopy (WLE). N, number of patients; RCT, Randomized controlled trial; n.a., not analyzed; n.s., not significant.
This study demonstrated that NBI had a significantly lower
polyp miss-rate than HRE (10% versus 36%; OR 0.21; p<0.001) in
HPS patients harboring multiple serrated polyps. As in previous
studies, NBI did not prove of additional value for the detection of
adenomas. These findings could be regarded as disappointing.
However, whereas serrated polyps traditionally are considered to be
harmless lesions, especially when they are small, recent molecular
research in serrated polyps in HPS suggests these are high-risk
lesions leading to CRC. [11,16,52,53] Furthermore, a previous large
cohort study showed that 5/77 (7%) HPS patients developed CRC
despite endoscopic surveillance of which 4/5 were detected within
relatively small serrated polyps (range: 4-16mm).[54] This supports our
opinion that the increased detection of serrated polyps with NBI in HPS
is of clinical relevance.
Our study additionally showed that NBI is of particular value for the
detection of serrated polyps which are flat in shape (HRE miss-rate
49% vs. NBI miss-rate 12%; p<0.001). Previous studies demonstrated
NBI increases polyp detection in HPS
167
that NBI did not detect more flat adenomas than HRE.[47,49,51,55] A
possible reason for this incongruence could be the fact that flat
adenomas are generally red in colour and therefore easier visible than
flat HPs and SSAs, which have the same colour as their surroundings
and are often covered by a layer of mucus. During NBI, serrated
polyps appear whiter in colour, thereby increasing the contrast
between the polyps and surrounding colonic tissue (figure 2).
Particularly these features may explain the higher miss-rate of serrated
polyps by HRE.
Figure 2. Examples of detected flat sessile serrated adenomas imaged with high-resolution endoscopy (A + C) and corresponding images with narrow-band imaging (B+D)
Several remarks regarding this study must be made before
making any firm recommendations based on our results. Adler et al
Chapte
r 7
Chapter 7
168
previously postulated that the level of experience with NBI may induce
a learning effect for improved recognition of polyps with HRE as well,
causing the difference in polyp detection between NBI and HRE to be
larger at the beginning of the learning curve.[56] However, in our study
the endoscopist had already performed more than 500 colonoscopies
with NBI, making a learning effect with regard to HRE unlikely.
Secondly, there was an unequal total number of detected polyps within
the randomization groups (a total of 122 polyps for HRE randomization
vs. 87 for NBI). These numbers approximate the true (detected and
undetected) number of baseline polyps in each group. This difference
was difficult to overcome considering the fact that both a patient with
>5 proximal HPs as well as a patient with more than 30 HPs satisfied
the criteria for HPS. The large variance of the number of polyps
between HPS patients will easily lead to unequal numbers of polyps
after randomization of only 22 patients. Due to this expected unequal
distribution of baseline polyp numbers, we chose a cross-over study
design which compares the percentage of missed polyps (i.e. polyp
miss rates) with each modality independent of the total number of
baseline polyps in each group instead of comparing the total number of
detected polyps (i.e. detection rates). Contrary to polyp miss-rates,
polyp detection rates are also dependent on the total number of
baseline polyps present in each group. Detection rate analysis is
therefore only possible when baseline polyp numbers are equal
between groups. For this reason detection rate analysis in the setting
of our study would have been unsuitable. If we would have compared
polyp detection rates at first inspection, one would expect a bias in
favour of the group with more baseline polyps. In our study that would
have resulted in a bias in favour of HRE (n=122) and against NBI
(n=87). This bias also explains the equal number of detected polyps at
NBI increases polyp detection in HPS
169
first inspection with HRE and NBI while after second inspection polyp
miss-rates with HRE were higher. Finally, this pilot study was
performed by a single experienced endoscopist who was unblinded to
the imaging intervention. Although comparison of investigation times
between randomization groups (both >6 minutes per inspection)
showed no significant differences at first and second inspection, a bias
by the endoscopist in favour of NBI can not be excluded. Nevertheless,
the significantly large difference in miss-rates between NBI and HRE
for the detection of polyps in HPS warrants confirmation in a
subsequent multi-centre study involving more patients and different
endoscopists.
With regard to the management of HPS patients, considering
that in these patients CRCs as small as 4mm have been described,
removal of all polyps ≥3mm seems indicated in any case, but this
surveillance strategy needs to be prospectively assessed. Concerning
polyps <3mm, a previous study analyzing the differentiation of polyps
in HPS showed that 20/35 polyps <3mm were high-risk sessile
serrated adenomas (9/35) and conventional adenomas (11/35).[48]
Differentiating these diminutive premalignant polyps from HPs with NBI
by means of polyp colour differentiation (lighter than the surrounding
mucosa is unsuspicious; darker than or the same as the surrounding
mucosa is suspicious) rendered a sensitivity of 95% for sessile
serrated adenomas and conventional adenomas (diagnostic accuracy:
78%). For this reason removal of polyps darker than or the same as
the surrounding mucosa may be sufficient for polyps of this size.
Previous randomized studies have shown that
chromoendoscopy increases the detection of sporadic HPs in non-
HPS patients compared to standard white-light endoscopy.[57-60] Our
Chapte
r 7
Chapter 7
170
study with NBI (“electronic chromoendoscopy”), which allows the
endoscopist to switch between modalities at a switch of the button,
showed similar results for the detection of serrated polyps in HPS
patients. Although the value of chromoendoscopy in HPS has not
formally been investigated, this cheap and readily available technique
seems to be a valid alternative for the endoscopic surveillance of HPS
patients.
In summary, this pilot study demonstrated that NBI is
associated with a reduced polyp miss-rate when compared to HRE in
patients with HPS. These findings suggest that all polyps in patients
with HPS need to be resected during colonoscopic surveillance, which
should be done using either NBI or chromoendoscopy.
NBI increases polyp detection in HPS
171
REFERENCES 1 Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the risk
of colorectal cancer. Dis Colon Rectum 2004; 47: 2101-2104
2 Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001; 25: 177-184
3 Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006; 38: 266-270
4 Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000; 119: 323-332
5 Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L et al. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006; 131: 30-39
6 Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004; 99: 2012-2018
7 Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996; 110: 748-755
8 Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001; 93: 1307-1313
9 Iino H, Jass JR, Simms LA, Young J, Leggett B, Ajioka Y et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999; 52: 5-9
10 Jass JR, Biden KG, Cummings MC, Simms LA, Walsh M, Schoch E et al. Characterisation of a subtype of colorectal cancer combining features of the suppressor and mild mutator pathways. J Clin Pathol 1999; 52: 455-460
11 Jass JR, Iino H, Ruszkiewicz A, Painter D, Solomon MJ, Koorey DJ et al. Neoplastic progression occurs through mutator pathways in hyperplastic polyposis of the colorectum. Gut 2000; 47: 43-49
Chapte
r 7
Chapter 7
172
12 Jass JR, Young J, Leggett BA. Hyperplastic polyps and DNA microsatellite unstable cancers of the colorectum. Histopathology 2000; 37: 295-301
13 Jass JR. Serrated route to colorectal cancer: back street or super highway? J Pathol 2001; 193: 283-285
14 Makinen MJ, George SM, Jernvall P, Makela J, Vihko P, Karttunen TJ. Colorectal carcinoma associated with serrated adenoma--prevalence, histological features, and prognosis. J Pathol 2001; 193: 286-294
15 Yao T, Nishiyama K, Oya M, Kouzuki T, Kajiwara M, Tsuneyoshi M. Multiple 'serrated adenocarcinomas' of the colon with a cell lineage common to metaplastic polyp and serrated adenoma. Case report of a new subtype of colonic adenocarcinoma with gastric differentiation. J Pathol 2000; 190: 444-449
16 Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD et al. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004; 53: 1137-1144
17 Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR et al. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006; 55: 1467-1474
18 Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T et al. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006; 131: 1400-1407
19 Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D et al. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006; 49: 121-131
20 Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S et al. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005; 166: 1069-1075
21 Azimuddin K, Stasik JJ, Khubchandani IT, Rosen L, Riether RD, Scarlatto M. Hyperplastic polyps: "more than meets the eye"? Report of sixteen cases. Dis Colon Rectum 2000; 43: 1309-1313
22 Warner AS, Glick ME, Fogt F. Multiple large hyperplastic polyps of the colon coincident with adenocarcinoma. Am J Gastroenterol 1994; 89: 123-125
NBI increases polyp detection in HPS
173
23 Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2010; 59: 1094-1100
24 Oono Y, Fu K, Nakamura H, Iriguchi Y, Yamamura A, Tomino Y et al. Progression of a Sessile Serrated Adenoma to an Early Invasive Cancer Within 8 Months. Dig Dis Sci 2008;
25 Urbanski SJ, Kossakowska AE, Marcon N, Bruce WR. Mixed hyperplastic adenomatous polyps--an underdiagnosed entity. Report of a case of adenocarcinoma arising within a mixed hyperplastic adenomatous polyp. Am J Surg Pathol 1984; 8: 551-556
26 Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005; 124: 380-391
27 Chan TL, Zhao W, Leung SY, Yuen ST. BRAF and KRAS mutations in colorectal hyperplastic polyps and serrated adenomas. Cancer Res 2003; 63: 4878-4881
28 Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997; 29: 1-3
29 Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999; 49: 736-742
30 Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996; 87: 305-309
31 van Rijn JC, Reitsma JB, Stoker J, Bossuyt PM, van Deventer SJ, Dekker E. Polyp miss rate determined by tandem colonoscopy: a systematic review. Am J Gastroenterol 2006; 101: 343-350
32 Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS et al. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002; 56: 333-338
33 Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004; 53: 376-380
Chapte
r 7
Chapter 7
174
34 Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006; 38: 444-448
35 Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A et al. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006; 4: 349-354
36 Park SY, Lee SK, Kim BC, Han J, Kim JH, Cheon JH et al. Efficacy of chromoendoscopy with indigocarmine for the detection of ascending colon and cecum lesions. Scand J Gastroenterol 2008; 43: 878-885
37 Adler A, Pohl H, Papanikolaou IS, bou-Rebyeh H, Schachschal G, Veltzke-Schlieker W et al. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008; 57: 59-64
38 Aschenbeck J, Adler A, Yenerim T, Mayr M, Aminalai A, Drossel R et al. Narrow-Band Versus White-Light HDTV Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008;
39 Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY et al. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008; 32: 30-35
40 Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007; 450: 613-618
41 Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003; 27: 65-81
42 Endoscopic Classification Review Group. Update on the paris classification of superficial neoplastic lesions in the digestive tract. Endoscopy 2005; 37: 570-578
43 Hamilton SR, Vogelstein B, Kudo S et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System.Lyon, France: IARC Press, 2000: 104-119
NBI increases polyp detection in HPS
175
44 Torlakovic EE, Gomez JD, Driman DK, Parfitt JR, Wang C, Benerjee T et al. Sessile Serrated Adenoma (SSA) vs. Traditional Serrated Adenoma (TSA). Am J Surg Pathol 2008; 32: 21-29
45 Simel DL, Rennie D, Bossuyt PM. The STARD statement for reporting diagnostic accuracy studies: application to the history and physical examination. J Gen Intern Med 2008; 23: 768-774
46 Schulz KF, Altman DG, Moher D. CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials. PLoS Med 2010; 7: e1000251
47 Adler A, Pohl H, Papanikolaou IS, Abou-Rebyeh H, Schachschal G, Veltzke-Schlieker W et al. A prospective randomised study on narrow-band imaging versus conventional colonoscopy for adenoma detection: does narrow-band imaging induce a learning effect? Gut 2008; 57: 59-64
48 Boparai KS, van den Broek FJ, van ES, Fockens P, Dekker E. Hyperplastic polyposis syndrome: a pilot study for the differentiation of polyps using high resolution endoscopy, autofluorescence imaging and narrow-band imaging. Gastrointest Endosc 2009;
49 Adler A, Aschenbeck J, Yenerim T, Mayr M, Aminalai A, Drossel R et al. Narrow-Band Versus White-Light High Definition Television Endoscopic Imaging for Screening Colonoscopy: A Prospective Randomized Trial. Gastroenterology 2008;
50 Inoue T, Murano M, Murano N, Kuramoto T, Kawakami K, Abe Y et al. Comparative study of conventional colonoscopy and pan-colonic narrow-band imaging system in the detection of neoplastic colonic polyps: a randomized, controlled trial. J Gastroenterol 2008; 43: 45-50
51 Kaltenbach T, Friedland S, Soetikno R. A randomised tandem colonoscopy trial of narrow band imaging versus white light examination to compare neoplasia miss rates. Gut 2008; 57: 1406-1412
52 Beach R, Chan AO, Wu TT, White JA, Morris JS, Lunagomez S et al. BRAF mutations in aberrant crypt foci and hyperplastic polyposis. Am J Pathol 2005; 166: 1069-1075
53 Chan AO, Issa JP, Morris JS, Hamilton SR, Rashid A. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002; 160: 529-536
Chapte
r 7
Chapter 7
176
54 Boparai KS, Mathus-Vliegen EM, Koornstra JJ, Nagengast FM, van LM, van Noesel CJ et al. Increased colorectal cancer risk during follow-up in patients with hyperplastic polyposis syndrome: a multicentre cohort study. Gut 2009;
55 Paggi S, Radaelli F, Amato A, Meucci G, Mandelli G, Imperiali G et al. The impact of narrow band imaging in screening colonoscopy: a randomized controlled trial. Clin Gastroenterol Hepatol 2009; 7: 1049-1054
56 Adler A. Narrow Band Imaging (NBI) Influences the Learning Curve for Conventional Endoscopy - Final Results of a Prospective Randomized Study in the Detection of Colorectal Adenomas [abstract]. Gastrointest Endosc 2007; 65: AB116
57 Brooker JC, Saunders BP, Shah SG, Thapar CJ, Thomas HJ, Atkin WS et al. Total colonic dye-spray increases the detection of diminutive adenomas during routine colonoscopy: a randomized controlled trial. Gastrointest Endosc 2002; 56: 333-338
58 Hurlstone DP, Cross SS, Slater R, Sanders DS, Brown S. Detecting diminutive colorectal lesions at colonoscopy: a randomised controlled trial of pan-colonic versus targeted chromoscopy. Gut 2004; 53: 376-380
59 Lapalus MG, Helbert T, Napoleon B, Rey JF, Houcke P, Ponchon T. Does chromoendoscopy with structure enhancement improve the colonoscopic adenoma detection rate? Endoscopy 2006; 38: 444-448
60 Le RM, Coron E, Parlier D, Nguyen JM, Canard JM, Alamdari A et al. High resolution colonoscopy with chromoscopy versus standard colonoscopy for the detection of colonic neoplasia: a randomized study. Clin Gastroenterol Hepatol 2006; 4: 349-354
Hyperplastic polyposis syndrome: a pilot study for the differentiation of polyps using high resolution endoscopy, autofluorescence imaging and narrow-band imaging
K.S. Boparai
F.J.C. van den Broek
S. van Eeden
P. Fockens
E. Dekker
Gastrointestinal Endoscopy 2009 Nov;70(5):947-55
Ch
ap
ter
Chapter 8
ABSTRACT
Background: Endoscopic differentiation and removal of potentially
premalignant sessile serrated adenomas (SSAs) may be an important
step in preventing colorectal cancer (CRC) development in
hyperplastic polyposis syndrome (HPS).
Objective: To assess the value of high resolution endoscopy (HRE),
autofluorescence imaging (AFI) and narrow-band imaging (NBI) for
differentiating polyps in HPS.
Design: A prospective polyp series.
Setting: Single tertiary referral center.
Patients and Interventions: 7 patients with HPS underwent
colonoscopy using endoscopic trimodal imaging (ETMI), which
incorporates HRE, AFI and NBI in one system. All detected polyps
were analysed with AFI for colour and with NBI for Kudo pit pattern
and vascular pattern intensity (VPI).
Main outcome measurements: The accuracy, sensitivity and
specificity of AFI and NBI in differentiating detected polyps were
determined by using histology as a gold standard.
Results: A total of 19 hyperplastic polyps (HPs), 32 SSAs and 15
adenomas were detected. For differentiating SSAs from HPs, AFI-
colour, Kudo pit pattern and VPI resulted in a diagnostic accuracy of
55%, 55% and 52% respectively. For differentiating adenomas from
HPs, this was 65%, 94% and 90% respectively. Macroscopically, the
combination of size ≥3mm and proximal location resulted in the highest
accuracy (76%) for differentiating SSAs from HPs.
Limitations: Small sample size.
Conclusion: Endoscopic differentiation between HPs and SSAs using
ETMI proved unsatisfactory. Differentiation of adenomas from HPs
was well possible with NBI but not with AFI.
NBI increases polyp detection in HPS
179
INTRODUCTION
Hyperplastic polyposis syndrome (HPS) is a recently
recognised condition characterised by the presence of multiple (>30)
hyperplastic polyps (HPs) spread throughout the colon and has
frequently been linked with colorectal cancer (CRC).1-4 Besides
multiple HPs, serrated adenomas are frequently seen in HPS as well.
1-8 In fact, the co-existence of sessile serrated adenomas (SSAs) has
been considered by some as a characterizing feature of this condition.9
Molecular research in SSAs strongly suggests that these
polyps are precursor lesions which may lead to CRC.10-13 Accordingly,
authorities recommend that SSAs should be endoscopically managed
like conventional adenomas.14 In this respect, endoscopic
differentiation of SSAs from HPs and removal of SSAs may be an
important step in preventing cancer development in HPS. However,
HPs and SSAs, being both often small in size and sessile/flat in shape,
are similar in appearance and therefore difficult to distinguish from
each other when using standard endoscopy (figure 1).15-18
Novel endoscopic imaging techniques may aid in the
differentiation of conventional adenomas and HPs with high
accuracy.19-22 Autofluorescence imaging (AFI) facilitates differentiation
of adenomas from non-adenomatous polyps based on different
fluorescence emission spectra.23-25 The use of AFI in differentiating
polyps in HPS patients has not been described before. Narrow band
imaging (NBI) utilizes short wavelength visible light to provide
improved details of the mucosal pit pattern and microvasculature. Pit
pattern analysis by applying the Kudo classification has shown to be a
reliable approach for distinguishing adenomas from non-adenomatous
polyps and has also been used to describe serrated adenomas.26-31 Chapte
r 8
Chapter 8
180
Figure 1: Similar endoscopic appearance of a hyperplastic polyp (left) and a sessile serrated adenoma (right) using conventional white light endoscopy with corresponding haemotoxylin and eosin stains below
However, at the times of these studies the diagnosis SSA was
not yet in practice and Kudo pit patterns for serrated adenomas varied
considerably from II to as high as IV.27, 28 In addition, the assessment
of microvasculature and vascular pattern intensity (VPI) with NBI is
believed to be a relatively easy method for differentiating adenomas
from non-adenomatous polyps, but has not been evaluated in HPS.20,
29, 30, 32-34
The aim of this study was to assess the diagnostic accuracy of
high resolution endoscopy (HRE), AFI and NBI for the differentiation of
polyps in patients with HPS.
NBI increases polyp detection in HPS
181
PATIENTS AND METHODS
Study population
This study was conducted at the Academic Medical Center
Amsterdam and was approved by the local medical ethics committee.
Consecutive patients with HPS were invited to participate when
fulfilling the criteria for HPS in accordance with the World Health
Organisation (WHO): (1) at least five histologically confirmed HPs
proximal to the sigmoid colon, of which two are greater than 10mm in
diameter, or (2) more than 30 HPs distributed throughout the colon.35
Patients <18 years or patients with severe coagulopathy or insufficient
bowel cleansing were excluded from this study.
Endoscopic equipment
All procedures were performed with the endoscopic tri-modal
imaging (ETMI) system, which integrates HRE, AFI and NBI into one
unit (XCV-260 HP, Olympus Inc., Tokyo, Japan). The endoscope
(XCF-H240FZL) is equipped with a movable lens for optical
magnification (up to 100×) and two high-quality charge coupled
devices: one for HRE/NBI and one for AFI. The light source used in
this system (XCLV-260HP) was of the type “sequential RGB-
illumination”. The ETMI specifications have previously been described
in detail.36, 37 During colonoscopy, the endoscopist could easily switch
between the three imaging modalities by pressing a button on the shaft
of the endoscope. A high resolution monitor was used for all
procedures.
Chapte
r 8
Chapter 8
182
Colonoscopy procedure
Patients were prepared with 4-6 L polyethylene glycol solution
(Kleanprep; Norgine GmbH, Marburg, Germany) and underwent
colonoscopy under conscious sedation with midazolam and/or
fentanyl. The colonoscope was advanced until cecal intubation was
confirmed by identification of the appendiceal orifice and ileocecal
valve. Upon reaching the cecum, the level of bowel preparation was
determined as good (100% of the mucosa visible), moderate (90-
100%) or poor (<90%) after rinsing and suctioning.
During withdrawal of the endoscope, HRE was used to detect
colonic polyps. All detected polyps were assessed for size (open
biopsy forceps: 8mm), shape (Paris classification) and location.38
Subsequently, each polyp was assessed with AFI for polyp colour:
green, ambiguous or purple. Purple and ambiguous colours were
considered suspicious for adenoma and green was considered non-
suspicious for adenoma. Hereafter, the Kudo pit pattern (I-V) was
assessed with NBI.31 Kudo pit pattern III-V were considered suspicious
for adenoma, while pit pattern I-II were non-suspicious. Still images
(BMP-format) with all modalities were acquired after which the polyp
was resected and harvested for histopathology. All procedures and
instant assessments with AFI and NBI were performed by one
experienced endoscopist (ED), who has performed >2,500
colonoscopies and >50 ETMI colonoscopies.
In addition, representative NBI images of all detected lesions
were later assessed for vascular pattern intensity (VPI) as described
by East et al.20 For this purpose, all sharp high-quality images were
displayed in a random order to the same endoscopist who was blinded
for final histopathology. Images were directly displayed on a personal
NBI increases polyp detection in HPS
183
computer in standard format (3.2 × 2.4 inch; 200 pixels/inch) without
any post-processing. The VPI was scored as lighter (weak), the same
(normal) or darker than (strong) the surrounding mucosa. Strong VPI
was considered suspicious for adenoma, whereas weak/normal VPI
was considered non-suspicious.
Reference standard
All polyp specimens were blindly evaluated by a gastrointestinal
pathologist (SvE). Lesions were classified as HP, SSA, traditional
serrated adenoma (TSA), mixed polyp or conventional adenoma based
on the morphological features on H&E staining which was used as
reference standard.13, 14, 39
Statistical analysis
SSAs were regarded as adenomatous polyps, owing to their
premalignant potential.10-14 Consequently, it was examined whether
these polyps were suspicious on AFI (ambiguous/purple) and/or NBI-
VPI (strong). In addition, NBI pit patterns of SSAs were expected to be
comparable to HPs (Kudo II) as these polyps are described to be
microscopically difficult to distinguish from each other.14, 18, 40, 40 TSAs,
mixed polyps and conventional adenomas were expected to be
suspicious on AFI and NBI as these polyps harbour neoplastic
changes of the epithelium.
The sensitivity, specificity, and diagnostic accuracy plus 95%-
confidence interval (95%-CI, using the Wilson procedure without
correction for continuity) for differentiating SSAs, TSAs, mixed polyps
and conventional adenomas (i.e. adenomatous group) from HPs were
determined for each modality by comparing the endoscopic diagnosis Chapte
r 8
Chapter 8
184
to final histopathology, which served as reference standard. Lesions
histologically diagnosed as normal mucosa were excluded from the
analysis. The size, location and shape of all lesions were summarized
and compared between the different polyps using the Chi-Square test,
Fisher’s exact test, Kruskal-Wallis test or Mann-Whitney U test when
appropriate. To make statistical comparisons and to calculate the 95%-
CI, it is necessary to assume that results for individual polyps
constitute statistically independent observations, even when there may
have been more than one polyp assessed in individual patients. A p-
value less than 0.05 from a single test was considered statistically
significant, but it is recognized that there was multiple testing of
outcome data arising from individual polyps. Examining the nominal p-
values in light of correction for multiple testing using the method of
correction of Bonferroni, it is suggested that only those nominal p-
values less than 0.01 will retain significance after correction. The
uncorrected p-values are presented with the warning that p-values
between 0.01 and 0.05 should be considered as provisional. For
reporting the results of this study, the STARD guidelines were used.41
Results
From January 2005 to July 2006, 7 patients (5 male) who met
the criteria for HPS, underwent colonoscopy with ETMI. The median
age of all patients was 55.8 (range 54-71) years. At colonoscopy all
patients had good to moderate bowel preparation. A total of 66 polyps
(19 HPs, 32 SSAs and 15 tubular adenomas) were detected as well as
10 additional lesions displaying normal mucosa on histology (excluded
from the analysis). Macroscopic polyp characteristics are summarized
in table 1. Overall, SSAs were larger than HPs (p<0.001, Mann-
NBI increases polyp detection in HPS
185
Whitney U test) and adenomas (p<0.001, Mann-Whitney U test). There
was no significant difference in size between HPs and adenomas.
Concerning the differentiation of SSAs from HPs, the odds ratio
for predicting a polyp to be SSA was 2.3 (95%-CI: 1.2-4.4) for size (per
mm increase), 4.9 (1.3-18.0) for flat shape and 3.9 (1.1-14.7) for
proximal location. The combination of size ≥3mm and proximal location
yielded the largest differential value (p<0.0001) between SSAs (21/32:
66%) and HPs (1/19: 5%) with a corresponding odds ratio of 34.4 (4.0-
292). The sensitivity and specificity of this combination for
differentiating SSAs from HPs would be 66% (95%-CI: 48-80%) and
95% (75-99%) respectively. The overall diagnostic accuracy would be
76% (62-87%).
Autofluorescence imaging
With AFI, 10/19 (53%) HPs displayed a green colour versus
14/32 (44%) SSAs and 3/15 (20%) adenomas (p=0.142). The
sensitivity, specificity and diagnostic accuracy of AFI for discriminating
SSAs from HPs based on colour were 56% (95%-CI: 39-72%), 53%
(32-73%) and 55% (41-68%) respectively. Differentiation with AFI
between adenomas and HPs had a sensitivity, specificity and accuracy
of 80% (55-93%), 53% (32-73%) and 65% (48-79%).
Chapte
r 8
Chapter 8
186
Polyp
characteristics
All polyps
(n=66)
HP
(n=19)
SSA
(n=32)
Adenoma
(n=15)
P-
value
Median size mm
(interquartile range) 2 (2-5) 2 (1-2) 3 (2-8) 2 (1-3) 0.001*
Location 0.013**
Proximal colon 51 (77%) 11 (58%) 27 (84%) 13 (87%)
Distal colon 6 (9%) 1 (5%) 3 (9%) 2 (13%)
Rectum 9 (14%) 7 (37%) 2 (7%) 0 (0%)
Shape 0.048**
0-Ip 0 0 0 0
0-Is 18 (38%) 9 (47%) 5 (16%) 4 (27%)
0-II 48 (62%) 10 (53%) 27 (84%) 11 (73%)
Table 1: Clinicopathological characteristics of all detected polyps in 7 patients with hyperplastic polyposis syndrome. Proximal colon = cecum, ascending and transverse colon; distal colon = descending colon and sigmoid colon. *Kruskal-Wallis test (only SSAs differed significantly from HPs and adenomas on additional testing with the Mann-Whitney U test). **Pearson Chi-Square test. Note that Bonferroni correction for multiple testing removes statistical significance except where p<0.01 in this table.
Narrow band imaging
The sensitivity, specificity and diagnostic accuracy of the Kudo
classification with NBI for differentiation of SSAs from HPs were 28%
(95%-CI: 16-33%), 100% (83-100%) and 55% (41-68%). For
differentiating adenomas from HPs, the obtained sensitivity, specificity
and diagnostic accuracy were 87% (81-98%), 100% (83-100%) and
94% (62-96%) (table 2). During the subsequent NBI image evaluation,
the VPI was assessed for 14 HPs, 28 SSAs and 15 adenomas. Images
of the other detected polyps could not be included for analysis
because VPI assessment was not possible (e.g. blurry images). The
sensitivity, specificity and diagnostic accuracy of VPI for differentiating
SSAs from HPs, were 36% (21-54%), 86% (6-96%) and 52% (38-67%)
respectively. When adenomas were compared with HPs the sensitivity,
NBI increases polyp detection in HPS
187
specificity and diagnostic accuracy were 93% (74-96%), 86% (60-96%)
and 90% (70-98%).
NBI HP (n=19) SSA (n=32) Adenoma (n=15) P-value
Pit pattern <0.0001*
Kudo I-II 19 (100%) 23 (72%) 2 (13%)
Kudo III-V 0 9 (28%) 13 (87%)
NBI HP (n=14) SSA (n=28) Adenoma (n=15) P-value
VPI <0.0001*
Weak/normal 12 (86%) 18(64%) 1 (7%)
Strong 2 (14%) 10 (36%) 14 (93%)
Table 2: Kudo pit pattern classification and vascular pattern intensity (VPI) assessment of all detected polyps with NBI. *p-value for adenomas compared to HPs and SSAs.
Table 3 lists the sensitivities, specificities and diagnostic accuracies of
the different endoscopic modalities for the grouped differentiation of
both high-risk SSAs and conventional adenomas from low-risk HPs.
Table 3: Sensitivity, specificity and diagnostic accuracy of all endoscopic modalities for differentiating sessile serrated adenomas and conventional adenomas (high risk) from hyperplastic polyps (low risk) in patients with hyperplastic polyposis syndrome.
SSA/adenoma vs. HP
Modality AFI NBI-pit pattern NBI-VPI
Sensitivity
(95%-CI)
30/47: 64%
(50-76%)
22/47: 47%
(33-61%)
24/43: 56%
(41-70%)
Specificity
(95%-CI)
10/19: 53%
(32-73%)
19/19: 100%
(83-100%)
12/14: 86%
(60-96%)
Diagnostic accuracy
(95%-CI)
40/66: 61%
(49-72%)
41/66:62%
(50-73%)
36/57: 63%
(50-74%)
Chapte
r 8
Chapter 8
188
DISCUSSION
In the present study, SSAs were significantly larger and more
often proximally located than HPs but the latter finding has its nominal
significance removed by Bonferroni correction for multiple testing of
data. This is in concordance with recent comparative studies, in which
sporadic SSAs were also found to be larger than sporadic HPs and
preferentially located in the right colon whereas HPs were more often
found distally.12, 18, 42 Interestingly, in our study the combination of size
≥3mm (median size of SSAs) and proximal location, showed a highly
significant difference between SSAs and HPs but with a corresponding
diagnostic accuracy of only 76%.
This pilot study demonstrated that the diagnostic accuracy of
AFI was unsatisfactory for differentiating SSAs from HPs (accuracy
55%). The presence of epithelial dysplasia and hypervascularisation
within adenomatous polyps are considered to be responsible for their
different fluorescence emission spectra when compared to non-
adenomatous polyps and hence lead to a different colour on AFI.24, 43
Although epithelial dysplasia has occasionally been described in
advanced SSAs progressing to carcinomas 44, SSAs generally lack
these histopathological features as confirmed by the present study
(figure 2).
NBI increases polyp detection in HPS
189
Figure 2 (left column). Green (A), ambiguous (B) and purple (C) coloured sessile serrated adenomas using autofluorescence imaging (AFI). Figure 3 (right column). Variation of pit-pattern characteristics in sessile serrated adenomas using narrow-band imaging (NBI): Kudo I (A), Kudo II (B), Kudo IIIL (C)
Therefore, as expected a-priori, AFI appeared insufficient to
distinguish HPs from SSAs. Furthermore, the diagnostic accuracy of
AFI for differentiating adenomas from HPs was also insufficient (65%).
Chapte
r 8
Chapter 8
190
This is in accordance with a previous study evaluating AFI for
differentiation of neoplastic and non-neoplastic lesions in the colon, in
which a similar diagnostic accuracy (68%) was obtained.45
Kudo pit pattern analysis with NBI demonstrated an equally
insufficient diagnostic accuracy for differentiation of SSAs from HPs as
AFI (55%). SSAs are microscopically defined as polyps with irregular
crypts displaying dilatation, branching and exaggerated serration,
especially at the base of the crypts. However, despite these defined
crypt characteristics, microscopic differentiation of SSAs from HPs
remains difficult.14, 18, 40, 42 NBI pit pattern analysis, which describes the
orifices of crypts, showed similar (Kudo II) phenotypes in HPs and
SSAs, confirming that crypt characteristics of these polyps are not only
microscopically but also endoscopically difficult to use for
differentiation purposes (figure 3).
As HPs and SSAs mainly differ in histological anatomy at the
base of the crypts, it was therefore expected a-priori that the pit pattern
of these polyps, which is assessed at the luminal site of the crypts,
was comparable. Differentiation of adenomas from HPs however was
very well possible with NBI (accuracy 94%). This corresponds with
previous studies in which the diagnostic accuracy of NBI for
differentiation of adenomas from non-neoplastic polyps ranged from
77-99%.19-21, 29, 30, 32, 33, 46
Previous use of VPI in sporadic polyps suggested that this
method has a comparable diagnostic accuracy in differentiating
adenomas from non-adenomatous polyps as pit pattern analysis.20, 29,
46 Based on the principle of increased vascularization, adenomatous
polyps would have a stronger VPI and thus have a darker colour when
viewed with NBI. 19, 20, 29, 30, 32-34, 46 Owing to the presumed premalignant
potential of SSAs, we examined whether a darker colour due to
NBI increases polyp detection in HPS
191
hypervascularization could be observed in these polyps. Overall only
10/28 (36%) of SSAs displayed a darker colour than the surrounding
mucosa, resulting consequently in a low diagnostic accuracy and
insufficient differential value (figure 4).
Figure 4: Weak (A), normal (B) and strong (C) vascular pattern intensity (VPI) displayed in three different sessile serrated adenomas using narrow-band imaging (NBI).
NBI-VPI analysis did however show a similarly high diagnostic
accuracy as NBI-pit pattern for differentiating adenomas from HPs.
These high diagnostic accuracies observed with NBI (pit-pattern and
Chapte
r 8
Chapter 8
192
VPI) are interesting considering that the median size of all polyps in
this study was only 2mm. This corresponds with results from previous
studies evaluating NBI for differentiating diminutive (<10mm) polyps. 20,
47-49
This study was performed in HPS patients in a tertiary referral
center by a single endoscopist specialized in HPS. This could explain
the diminutive size of polyps detected in these patients as they
undergo annual surveillance endoscopies with removal of most polyps,
leaving only small ones in situ. However, typical HPs seldom exceed
5mm in size and in a recent prospective study of unselected
consecutive patients undergoing colonoscopy, 83% of detected SSAs
were ≤10mm and 36% were ≤5mm.12, 50-55 These findings suggest that
the predominant polyps in HPS, i.e. HPs and SSAs, are typically small.
Nevertheless, HPS is associated with a significantly increased risk of
developing CRC.5 Also in our personal (unpublished) experience of
annual HPS surveillance, intra-mucosal carcinomas have been
detected in serrated polyps as small as 4mm. Therefore, unlike in the
general population, diminutive polyps should be considered clinically
relevant in HPS.
A possible limitation of this study is that only HPS patients were
selected for endoscopic differentiation of polyps using ETMI. Thus, our
results for differentiating polyps can not by default be extrapolated to
polyps in non-HPS patients, as these are not necessarily identical.
Furthermore, one may question the generalizability of this pilot study
since the sample size of SSAs was relatively small. However, as the
accuracies of AFI, NBI-pit pattern and NBI-VPI were all far from
acceptable (52-55%) in differentiating SSAs from HPs, and the upper
limit of the 95%-confidence interval of these accuracies was 68% at
best, a larger sample size is unlikely to alter the conclusion of this pilot
NBI increases polyp detection in HPS
193
study. Moreover, during real-time polyp assessment, AFI was used
first after which an assessment with NBI was performed. This may
cause AFI-colour to influence the subsequent NBI assessment and
thus cause bias. However, when differentiating adenomas from HPs,
NBI results were markedly better than prior AFI results, suggesting that
bias due to the order of assessment was minimal. Finally, in this study
diminutive lesions displaying normal mucosa on histology (n=10) were
excluded from analysis. When these lesions were grouped with HPs at
additional analysis, diagnostic accuracies for the different endoscopic
modalities remained largely unchanged with a maximum difference of
5% (range: 1-5%).
In summary, the diagnostic accuracy of AFI, NBI-pit pattern and
NBI-VPI proved unsatisfactory for differentiating SSAs from HPs in
patients with HPS. Differentiation of conventional adenomas from HPs
was well possible using both NBI-pit pattern and NBI-VPI. Proximal
colonic location combined with a size ≥ 3mm proved to be the most
valuable for differentiating SSAs from HPs. Nevertheless, the
diagnostic accuracy of this combination appears still too low for clinical
use (76%).
Chapte
r 8
Chapter 8
194
REFERENCES 1. Hyman NH, Anderson P, Blasyk H. Hyperplastic polyposis and the
risk of colorectal cancer. Dis Colon Rectum 2004;47:2101-2104.
2. Leggett BA, Devereaux B, Biden K, Searle J, Young J, Jass J. Hyperplastic polyposis: association with colorectal cancer. Am J Surg Pathol 2001;25:177-184.
3. Rubio CA, Stemme S, Jaramillo E, Lindblom A. Hyperplastic polyposis coli syndrome and colorectal carcinoma. Endoscopy 2006;38:266-270.
4. Rashid A, Houlihan PS, Booker S, Petersen GM, Giardiello FM, Hamilton SR. Phenotypic and molecular characteristics of hyperplastic polyposis. Gastroenterology 2000;119:323-332.
5. Carvajal-Carmona L, Howarth K, Lockett M, Polanco-Echeverry G, Volikos E, Gorman M, Barclay E, Martin L, Jones A, Saunders B, Guenther T, Donaldson A, Paterson J, Frayling I, Novelli M, Phillips R, Thomas H, Silver A, Atkin W, Tomlinson I. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2007;212:378-385.
6. Chow E, Lipton L, Lynch E, D'Souza R, Aragona C, Hodgkin L, Brown G, Winship I, Barker M, Buchanan D, Cowie S, Nasioulas S, du SD, Young J, Leggett B, Jass J, Macrae F. Hyperplastic polyposis syndrome: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30-39.
7. Ferrandez A, Samowitz W, DiSario JA, Burt RW. Phenotypic characteristics and risk of cancer development in hyperplastic polyposis: case series and literature review. Am J Gastroenterol 2004;99:2012-2018.
8. Lage P, Cravo M, Sousa R, Chaves P, Salazar M, Fonseca R, Claro I, Suspiro A, Rodrigues P, Raposo H, Fidalgo P, Nobre-Leitao C. Management of Portuguese patients with hyperplastic polyposis and screening of at-risk first-degree relatives: a contribution for future guidelines based on a clinical study. Am J Gastroenterol 2004;99:1779-1784.
9. Torlakovic E, Snover DC. Serrated adenomatous polyposis in humans. Gastroenterology 1996;110:748-755.
NBI increases polyp detection in HPS
195
10. Kambara T, Simms LA, Whitehall VL, Spring KJ, Wynter CV, Walsh MD, Barker MA, Arnold S, McGivern A, Matsubara N, Tanaka N, Higuchi T, Young J, Jass JR, Leggett BA. BRAF mutation is associated with DNA methylation in serrated polyps and cancers of the colorectum. Gut 2004;53:1137-1144.
11. Minoo P, Baker K, Goswami R, Chong G, Foulkes WD, Ruszkiewicz AR, Barker M, Buchanan D, Young J, Jass JR. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467-1474.
12. Spring KJ, Zhao ZZ, Karamatic R, Walsh MD, Whitehall VL, Pike T, Simms LA, Young J, James M, Montgomery GW, Appleyard M, Hewett D, Togashi K, Jass JR, Leggett BA. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400-1407.
13. Jass JR, Baker K, Zlobec I, Higuchi T, Barker M, Buchanan D, Young J. Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a 'fusion' pathway to colorectal cancer. Histopathology 2006;49:121-131.
14. Snover DC, Jass JR, Fenoglio-Preiser C, Batts KP. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124:380-391.
15. Jaramillo E, Watanabe M, Rubio C, Slezak P. Small colorectal serrated adenomas: endoscopic findings. Endoscopy 1997;29:1-3.
16. Matsumoto T, Mizuno M, Shimizu M, Manabe T, Iida M, Fujishima M. Serrated adenoma of the colorectum: colonoscopic and histologic features. Gastrointest Endosc 1999;49:736-742.
17. Rubio CA, Jaramillo E. Flat serrated adenomas of the colorectal mucosa. Jpn J Cancer Res 1996;87:305-309.
18. Sandmeier D, Seelentag W, Bouzourene H. Serrated polyps of the colorectum: is sessile serrated adenoma distinguishable from hyperplastic polyp in a daily practice? Virchows Arch 2007;450:613-618.
19. Chiu HM, Chang CY, Chen CC, Lee YC, Wu MS, Lin JT, Shun CT, Wang HP. A prospective comparative study of narrow-band imaging, chromoendoscopy, and conventional colonoscopy in the diagnosis of colorectal neoplasia. Gut 2007;56:373-379. C
hapte
r 8
Chapter 8
196
20. East JE, Suzuki N, Saunders BP. Comparison of magnified pit pattern interpretation with narrow band imaging versus chromoendoscopy for diminutive colonic polyps: a pilot study. Gastrointest Endosc 2007;66:310-316.
21. Hirata M, Tanaka S, Oka S, Kaneko I, Yoshida S, Yoshihara M, Chayama K. Magnifying endoscopy with narrow band imaging for diagnosis of colorectal tumors. Gastrointest Endosc 2007;65:988-995.
22. McCallum AL, Jenkins JT, Gillen D, Molloy RG. Evaluation of autofluorescence colonoscopy for the detection and diagnosis of colonic polyps. Gastrointest Endosc 2008;68:283-290.
23. DaCosta RS, Andersson H, Cirocco M, Marcon NE, Wilson BC. Autofluorescence characterisation of isolated whole crypts and primary cultured human epithelial cells from normal, hyperplastic, and adenomatous colonic mucosa. J Clin Pathol 2005;58:766-774.
24. Haringsma J, Tytgat GN, Yano H, Iishi H, Tatsuta M, Ogihara T, Watanabe H, Sato N, Marcon N, Wilson BC, Cline RW. Autofluorescence endoscopy: feasibility of detection of GI neoplasms unapparent to white light endoscopy with an evolving technology. Gastrointest Endosc 2001;53:642-650.
25. Wang TD, Van DJ, Crawford JM, Preisinger EA, Wang Y, Feld MS. Fluorescence endoscopic imaging of human colonic adenomas. Gastroenterology 1996;111:1182-1191.
26. Kudo S, Rubio CA, Teixeira CR, Kashida H, Kogure E. Pit pattern in colorectal neoplasia: endoscopic magnifying view. Endoscopy 2001;33:367-373.
27. Morita T, Tamura S, Miyazaki J, Higashidani Y, Onishi S. Evaluation of endoscopic and histopathological features of serrated adenoma of the colon. Endoscopy 2001;33:761-765.
28. Oka S, Tanaka S, Hiyama T, Ito M, Kitadai Y, Yoshihara M, Haruma K, Chayama K. Clinicopathologic and endoscopic features of colorectal serrated adenoma: differences between polypoid and superficial types. Gastrointest Endosc 2004;59:213-219.
29. Tischendorf JJ, Wasmuth HE, Koch A, Hecker H, Trautwein C, Winograd R. Value of magnifying chromoendoscopy and narrow band imaging (NBI) in classifying colorectal polyps: a prospective controlled study. Endoscopy 2007;39:1092-1096.
NBI increases polyp detection in HPS
197
30. Machida H, Sano Y, Hamamoto Y, Muto M, Kozu T, Tajiri H, Yoshida S. Narrow-band imaging in the diagnosis of colorectal mucosal lesions: a pilot study. Endoscopy 2004;36:1094-1098.
31. Kudo S, Rubio CA, Teixeira CR, Kashida H, Kogure E. Pit pattern in colorectal neoplasia: endoscopic magnifying view. Endoscopy 2001;33:367-373.
32. Hirata M, Tanaka S, Oka S, Kaneko I, Yoshida S, Yoshihara M, Chayama K. Evaluation of microvessels in colorectal tumors by narrow band imaging magnification. Gastrointest Endosc 2007;66:945-952.
33. Rastogi A, Bansal A, Wani S, Callahan P, McGregor DH, Cherian R, Sharma P. Narrow-band imaging colonoscopy-a pilot feasibility study for the detection of polyps and correlation of surface patterns with polyp histologic diagnosis. Gastrointest Endosc 2007.
34. Konerding MA, Fait E, Gaumann A. 3D microvascular architecture of pre-cancerous lesions and invasive carcinomas of the colon. Br J Cancer 2001;84:1354-1362.
35. Burt RW, Jass J. Hyperplastic polyposis. In: Hamilton SR and Aaltonen LA, eds. World Health Organisation Classification of Tumours Pathology and Genetics. Berlin: Springer-Verlag, 2000:135-136.
36. Curvers WL, Singh R, Song LM, Wolfsen HC, Ragunath K, Wang K, Wallace MB, Fockens P, Bergman JJ. Endoscopic tri-modal imaging for detection of early neoplasia in Barrett's oesophagus: a multi-centre feasibility study using high-resolution endoscopy, autofluorescence imaging and narrow band imaging incorporated in one endoscopy system. Gut 2008;57:167-172.
37. van den Broek FJ, Fockens P, van ES, Reitsma JB, Hardwick JC, Stokkers PC, Dekker E. Endoscopic tri-modal imaging for surveillance in ulcerative colitis: randomised comparison of high-resolution endoscopy and autofluorescence imaging for neoplasia detection; and evaluation of narrow-band imaging for classification of lesions. Gut 2008;57:1083-1089.
38. Endoscopic Classification Review Group. Update on the paris classification of superficial neoplastic lesions in the digestive tract. Endoscopy 2005;37:570-578.
Chapte
r 8
Chapter 8
198
39. Hamilton SR, Vogelstein B, Kudo S, et al. World Health Organization Classification of Tumours, Pathology and Genetics of Tumours of the Digestive System. Lyon, France: IARC Press, 2000:104-119.
40. Farris AB, Misdraji J, Srivastava A, Muzikansky A, Deshpande V, Lauwers GY, Mino-Kenudson M. Sessile serrated adenoma: challenging discrimination from other serrated colonic polyps. Am J Surg Pathol 2008;32:30-35.
41. Simel DL, Rennie D, Bossuyt PM. The STARD statement for reporting diagnostic accuracy studies: application to the history and physical examination. J Gen Intern Med 2008;23:768-774.
42. Torlakovic E, Skovlund E, Snover DC, Torlakovic G, Nesland JM. Morphologic reappraisal of serrated colorectal polyps. Am J Surg Pathol 2003;27:65-81.
43. Georgakoudi I, Jacobson BC, Van DJ, Backman V, Wallace MB, Muller MG, Zhang Q, Badizadegan K, Sun D, Thomas GA, Perelman LT, Feld MS. Fluorescence, reflectance, and light-scattering spectroscopy for evaluating dysplasia in patients with Barrett's esophagus. Gastroenterology 2001;120:1620-1629.
44. Goldstein NS. Small colonic microsatellite unstable adenocarcinomas and high-grade epithelial dysplasias in sessile serrated adenoma polypectomy specimens: a study of eight cases. Am J Clin Pathol 2006;125:132-145.
45. F.J.C.van den Broek, Curvers WL, Hardwick JC, Fockens P, Dekker E. Interobserver variability and accuracy of colonic pit patterns by narrow band imaging and the additional value of autofluorescence characteristics. 65 ed. Gastrointestinal Endoscopy: 2007:AB349.
46. Su MY, Hsu CM, Ho YP, Chen PC, Lin CJ, Chiu CT. Comparative study of conventional colonoscopy, chromoendoscopy, and narrow-band imaging systems in differential diagnosis of neoplastic and nonneoplastic colonic polyps. Am J Gastroenterol 2006;101:2711-2716
47. East JE, Suzuki N, Bassett P, Stavrinidis M, Thomas HJ, Guenther T, Tekkis PP, Saunders BP. Narrow band imaging with magnification for the characterization of small and diminutive colonic polyps: pit pattern and vascular pattern intensity. Endoscopy 2008;40:811-817.
48. Rogart JN, Jain D, Siddiqui UD, Oren T, Lim J, Jamidar P, Aslanian H. Narrow-band imaging without high magnification to differentiate
NBI increases polyp detection in HPS
199
polyps during real-time colonoscopy: improvement with experience. Gastrointest Endosc 2008;68:1136-1145.
49. Sano Y, Ikematsu H, Fu KI, Emura F, Katagiri A, Horimatsu T, Kaneko K, Soetikno R, Yoshida S. Meshed capillary vessels by use of narrow-band imaging for differential diagnosis of small colorectal polyps. Gastrointest Endosc 2008.
50. DiSario JA, Foutch PG, Mai HD, Pardy K, Manne RK. Prevalence and malignant potential of colorectal polyps in asymptomatic, average-risk men. Am J Gastroenterol 1991;86:941-945.
51. Estrada RG, Spjut HJ. Hyperplastic polyps of the large bowel. Am J Surg Pathol 1980;4:127-133.
52. Hayashi T, Yatani R, Apostol J, Stemmermann GN. Pathogenesis of hyperplastic polyps of the colon: a hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 1974;66:347-356.
53. Imperiale TF, Wagner DR, Lin CY, Larkin GN, Rogge JD, Ransohoff DF. Results of screening colonoscopy among persons 40 to 49 years of age. N Engl J Med 2002;346:1781-1785.
54. Johnson DA, Gurney MS, Volpe RJ, Jones DM, VanNess MM, Chobanian SJ, Avalos JC, Buck JL, Kooyman G, Cattau EL, Jr. A prospective study of the prevalence of colonic neoplasms in asymptomatic patients with an age-related risk. Am J Gastroenterol 1990;85:969-974.
55. Lieberman DA, Prindiville S, Weiss DG, Willett W. Risk factors for advanced colonic neoplasia and hyperplastic polyps in asymptomatic individuals. JAMA 2003;290:2959-2967.
Chapte
r 8
NBI increases polyp detection in HPS
201
Summary & Future Perspectives
202
Summary and future perspectives
203
SUMMARY
Colorectal cancer (CRC) is a condition associated with a high mortality
rate. Previous molecular research performed in patients with FAP and
Lynch syndrome has revealed that conventional adenomas are pre-
malignant lesions that can progress to CRC. It has been shown that
cancer development in these adenomas can follow different molecular
pathways involving seperate oncogenes and tumor-suppressor genes.
Recently, a novel CRC pathway has been suggested involving
histologically different polyps namely serrated polyps. In these serrated
polyps, alternative molecular processes are suggested to lead to CRC.
In this thesis we investigated a proposed ‘serrated neoplasia pathway’.
To this aim, patients with hyperplastic polyposis syndrome (HPS)
harboring multiple serrated polyps but also conventional adenomas
represented a unique demographic opportunity for clinical and
molecular analysis of serrated polyps and their association with CRC.
Clinical analyses – Colorectal cancer risk
In chapter 2 of this thesis we describe the clinical and pathological
features of a large multi-centre HPS cohort during multiple years of
unprotocollized endoscopic surveillance. We showed that one third of
HPS patients presented with co-existent CRC and in an additional 7%
of patients CRCs were identified despite endoscopic surveillance
(interval-CRCs). These findings are substantial considering that the
lifetime risk of developing CRC in the general population is estimated
to be only 6%. Interestingly, at multivariate logistic regression, serrated
polyps (i.e. HPs and SSLs) and not conventional adenomas were
significantly associated with CRC presence thus supporting the
existence of a predominant ‘serrated pathway’ to CRC in these
Sum
mary
& f
utu
re p
ers
pectives
204
patients. Considering that CRCs were detected in polyps as small as
4mm during endoscopic surveillance, all polyps in HPS seem at risk of
representing advanced lesions warranting removal of all polyps (at
least polyps ≥3mm) at annual surveillance endoscopies. If this is not
feasible, surgical resection should be considered.
Although an increased CRC risk for HPS patients has been
established based on previous cohort studies, it was yet unknown
whether first-degree relatives (FDRs) have an increased risk of CRC
and/or HPS. Consequently, the need for preventive measures, like
screening colonoscopies, in this group was doubtful. In chapter 3 we
performed a retrospective study involving, at the time, the largest
described cohort of FDRs in which we analyzed the incidence rate of
CRC and HPS. This incidence we subsequently compared with the
general population through person-year analysis. This way we were
able to quantify the suggested increased CRC incidence in the
literature by means of a relative risk. Our results showed that FDRs of
HPS patients have an increased risk for both CRC (RR: 3.7-7.8) and
HPS (RR: 13-121) compared to the general population warranting
screening colonoscopies for this whole group as long as no genetic
substrate is identified which can help us indentify FDRs with a high
risk.
Molecular analyses – Etiology and colorectal cancer pathways
Although an underlying genetic disorder seems likely, thus far no
genetic causes of HPS have been identified. However, previous case
series of patients with a known genetic disorder have reported
individuals with multiple serrated polyps. In chapter 4 we investigated
the presence of serrated polyps in MYH-associated polyposis (MAP)
and analysed whether these polyps were causally related to MAP.
Summary and future perspectives
205
Phenotypically, MAP polyps encompass conventional adenomas and
very rarely serrated polyps. Conversely, at re-evaluation of all polyps in
our cohort of MAP patients we found that almost half of all patients had
at least one serrated polyp and 18% had ≥ 10 serrated polyps. At
subsequent molecular analysis, we found serrated polyps to be
causally related to MYH-deficiency, reflected by the presence of
almost exclusively G:C→T:A transversions in the KRAS gene of these
lesions. This implies that distinct pathways, i.e. APC-gene related in
adenomas and non-related in serrated polyps, appear to be
operational in MAP.
Similar to patients with MAP, detected polyps in patients with
Lynch syndrome, who have a germline defect in one of the MMR
genes, invariably represent conventional adenomas. However, recent
studies have also described serrated polyps in Lynch patients.
Subsequent molecular studies evaluating whether these polyps are
causally related to Lynch syndrome by means of
immunohistochemistry have however been inconclusive. In chapter 5
the presence of serrated polyps in a large cohort of genetically proven
Lynch patients was analysed. To examine whether serrated polyps are
causally related to Lynch we utilized an alternative molecular approach
involving mutation analysis (APC, KRAS and BRAF) in adjunct to
defective MMR testing. The possible finding of an association between
Lynch and serrated polyps could imply that these lesions follow an
accelerated route to CRC. Based on the principle that Lynch CRCs are
not BRAF mutated, BRAF analysis is method to distinguish Lynch-
associated CRCs from sporadic CRCs. We hypothesised that a high
frequency of BRAF mutations in serrated polyps, comparable to
sporadic control group serrated polyps, would indicate that these
polyps are not Lynch-associated. In this study, we demonstrated that
Sum
mary
& f
utu
re p
ers
pectives
206
serrated polyps in Lynch-HPS patients (>10 serrated polyps) are not
associated with Lynch due to the high frequency of BRAF mutations in
these polyps. Alternatively, in Lynch patients with occasional serrated
polyps (<10), serrated polyps had significantly lower levels of BRAF
mutations than control group polyps. It can be surmised that
considering this low frequency of BRAF mutations in serrated polyps of
non-HPS Lynch patients, a causal relationship with the MSI-pathway
can not be excluded despite lack of defective MMR. These polyps
associated with the accelerated MSI-pathway may represent precursor
lesions of MSI-CRC which evolve even faster than in the serrated CRC
pathway alone.
Because of the unique mixture of different precursor lesion
types in HPS, it was yet unknown which polyps lead to CRC in HPS
and thus which polyps are clinically relevant. In chapter 6 we
evaluated which pathways are operational in HPS by studying the
histological and molecular characteristics of all available CRCs in a
large cohort of HPS patients. We found in this comprehensive cohort
study of HPS patients a high number of combined serrated polyp-CRC
lesions which showed identical BRAF mutations in both components,
supporting the existence of a serrated CRC pathway. Overall, we
demonstrated that both microsatellite-stable and –unstable CRCs in
HPS predominantly originate from serrated polyps, reflected by a high
percentage of BRAF mutations in these CRCs, but also from
conventional adenomas. The predominance of a serrated CRC
pathway over the classical Wnt-pathway seems due to the numerical
prevalence of serrated polyps in HPS patients. From this it is inferred
that all polyp types in HPS, should be considered clinically relevant.
Furthermore, we showed at molecular analysis of lesions in the same
cohort that CRCs evolving through the serrated CRC pathway were
Summary and future perspectives
207
predominantly right-sided. Considering that CRC in HPS can be as
small as 4mm, it seems advisable to remove at least all polyps ≥3mm
and especially proximally located serrated polyps.
Endoscopic imaging – Detection and differentiation of polyps
Due to the increased risk of malignant progression of HPS polyps,
optimal endoscopic detection, differentiation and removal of in
particular high-risk polyps is necessary to prevent CRC development in
these patients. Chapter 7 showed that NBI significantly reduces polyp
miss-rates in HPS. To evaluate for which polyps NBI was particularly
of value, we compared miss-rates for different polyp histologies and
shapes. NBI was especially of value for the detection of flat serrated
polyps as opposed to conventional adenomas. Considering the
predominance of a serrated CRC pathway in HPS, our findings are
clinically relevant and support the implementation of NBI for the
endoscopic surveillance of HPS patients.
Chapter 8 evaluates the value of ETMI (high-resolution
endoscopy, NBI and autofluorescence imaging) for the real-time
endoscopic differentiation of polyps in HPS. None of the three
modalities rendered a sufficient diagnostic accuracy for the
differentiation of SSLs from HPs. Differentiation of adenomas from
HPs however was well possible with NBI but not with AFI. Based on
these findings, we conclude that ETMI offers insufficient diagnostic
tools to differentiate between high-risk SSLs from relatively ‘innocuous’
HPs but is of value for the differentiation of conventional adenomas
from HPs.
Sum
mary
& f
utu
re p
ers
pectives
208
FUTURE PERSPECTIVES
Research in this thesis provides additional supporting evidence for a
serrated CRC pathway in humans. This pathway is particularly
predominant in patients with HPS who have a high-risk of malignant
progression, even under unprotocollized endoscopic surveillance.
Currently, no uniform and adequately substantiated management
protocol exists for these patients. Prospective management studies,
based on previous clinical and molecular research, will hopefully
provide us with data with which we can construct a safe and effective
management protocol for these patients. In this light, adequate
endoscopic detection as well as real-time differentiation and removal of
only high-risk polyps in HPS would strongly increase management
efficiency. Regarding polyp detection, future multi-centre studies need
to be performed to evaluate whether NBI indeed increases polyp
detection in HPS. Concerning real-time differentiation, results from
recent studies analyzing polyp differentiation using NBI, AFI and even
endomicroscopy have been disappointing, especially regarding SSAs.
Future endoscopic techniques with which molecular characteristics
such as mutation- and methylation status (“molecular imaging”) can be
evaluated real-time during endoscopy may be of value in this respect.
Thus far, HPS patients have been mostly identified when
symptomatic (the reason for the endoscopy), and a large proportion of
them have a co-incident CRC at the time of diagnosis. With the recent
development of whole-genome sequencing, genome-wide association
studies are currently being performed in order to unravel the genetic
make-up and inheritance pattern of HPS patients. Based on these
findings a reclassification of HPS may be possible so as to identify
subtypes that have a higher risk of CRC development (e.g. patients
with a higher number of polyps or larger polyps) or a more dominant
Summary and future perspectives
209
inheritance pattern. Based on these findings we will hopefully be able
to recognize which individuals need to receive (more frequent)
screening colonoscopies. In addition, causative target genes may be
identified which in turn can be treated with specific gene-inhibitors.
Until then, prospective screening studies should be performed in first-
degree relatives of HPS patients to adequately assess the risk of HPS
in these individuals.
Sum
mary
& f
utu
re p
ers
pectives
211
Samenvatting
& Toekomstperspectief
212
Samenvatting & toekomstperspectief
213
Colorectaal carcinoom (CRC) is een aandoening die geassocieerd is
met een hoge mortaliteit. Voorgaande moleculaire studies bij patiënten
met FAP en Lynch syndroom toonden aan dat conventionele
adenomen pre-maligne lesies zijn die zich kunnen ontwikkelen tot
CRC. Progressie tot kanker in deze adenomen kan volgens
verschillende moleculaire routes verlopen waarbij verschillende
oncogenen en tumor-suppresor genen een rol spelen. Recentelijk is
een nieuwe CRC route voorgesteld waarbij niet adenomen maar
serrated poliepen een rol spelen. In deze serrated poliepen treden
alternatieve moleculaire veranderingen plaats welke leiden tot CRC. In
dit proefschrift onderzochten wij de voorgestelde “serrated neoplasia
pathway”. Hiervoor maakten wij gebruik van patiënten met
hyperplastische polyposis syndroom (HPS). HPS patiënten hebben
multipele serrated poliepen maar ook conventionele adenomen en
vormen hierom een unieke groep voor klinische en moleculaire
analyse van serrated poliepen en hun relatie met CRC.
Klinisch onderzoek – Colorectaal kanker risico
In hoofdstuk 2 van dit proefschrift beschreven wij de klinische en
pathologische eigenschappen van een groot multi-center HPS cohort
na meerdere jaren ongeprotocoliseerd endoscopische surveillance. Wij
toonden aan dat een derde van de HPS patiënten zich al presenteerde
met gelijktijdig CRC. Daarnaast werd er bij nog 7% van de HPS
patiënten CRC ontdekt, ondanks endoscopische surveillance (interval-
CRCs). Deze bevindingen zijn substantieel aangezien het geschatte
levensrisico op CRC in de algehele bevolking slechts 6% is. Bij
multivariaat logistische regressie bleken serrated poliepen en niet
adenomen significant geassocieerd te zijn met de aanwezigheid van
Sam
envattin
g &
toekom
stp
ers
pectief
214
CRC. Deze bevindingen ondersteunen de aanwezigheid van een
overheersend ‘serrated CRC pathway’ in HPS patiënten. Aangezien
carcinomen tijdens endoscopische surveillance ook werden ontdekt in
poliepen van slechts 4mm, lijken alle poliepen pre-maligne lesies te
kunnen zijn waardoor verwijdering van deze poliepen (in ieder geval
poliepen ≥3mm) tijdens jaarlijke endoscopische surveillance
geïndiceerd lijkt. Indien dit niet mogelijk is, dient chirurgische resectie
overwogen te worden.
In hoofdstuk 3 hebben wij een retrospectieve studie verricht,
betreffende het grootste cohort eerste-graads familieleden van HPS
patiënten waarbij we de incidentie van CRC en HPS onderzochten.
Deze incidenties werden vervolgens vergeleken met de algemene
bevolking door middel van persoonsjaar analyse. Zo konden wij het
gesuggereerde verhoogd familiair risico kwantificeren met een relatief
risico. Onze resultaten toonden een verhoogd relatief risico voor zowel
CRC (RR: 3.7-7.8) als HPS (RR: 13-121) in vergelijking met de
algemene bevolking. Hierom zijn screening coloscopieën voor deze
hele groep geïndiceerd zolang er geen genetische markers bekend
zijn die ons kunnen helpen hoog-risico individuen te identificeren.
Moleculaire analyses – etiologie en colorectaal kanker routes
Hoewel een onderliggend genetische aandoening aannemelijk is, is er
tot nog toe geen genetische oorzaak voor HPS ontdekt. Er zijn echter
studies beschreven waarbij patiënten met een bekende genetische
aandoening multipele serrated poliepen hadden.
In hoofdstuk 4 onderzochten wij de aanwezigheid van serrated
poliepen in MYH-associated polyposis (MAP) en of deze poliepen
causaal gerelateerd zijn met MAP. Poliepen in MAP bestaan
voornamelijk uit conventionele adenomen en zeer zelden uit serrated
Samenvatting & toekomstperspectief
215
poliepen. Bij herevaluatie van alle poliepen in ons cohort MAP
patiënten vonden wij echter dat bijna de helft van de patiënten
minstens 1 serrated poliep had en dat 18% ≥ 10 serrated poliepen
had. Bij moleculair onderzoek zagen wij dat serrated poliepen causaal
gerelateerd waren met MYH-deficiëntie doordat gevonden mutaties in
deze poliepen bijna exclusief uit specifieke G:C→T:A transversies in
het KRAS gen bestonden. Dit suggereert dat verschillende pathways
operatief zijn in MAP, namelijk APC-gen gerelateerd in adenomen en
niet-APC gerelateerd in serrated poliepen.
Vergelijkbaar met MAP bestaan poliepen in patiënten met
Lynch syndroom die een kiembaan mutatie hebben in een van deze
MMR genen voornamelijk uit conventionele adenomen. In voorgaande
studies zijn er echter ook serrated poliepen beschreven bij deze
patiënten. Hierop volgende moleculaire studies, die met behulp van
immunohistochemie onderzochten of serrated poliepen causaal
gerelateerd zijn met Lynch syndroom, waren niet conclusief.
In hoofdstuk 5 werd de aanwezigheid van serrated poliepen in het
grootste cohort genetisch bewezen Lynch syndroom patiënten
beschreven. Om een causaal verband te onderzoeken maakten wij
naast immunohistochemie gebruik van mutatie analyse (APC, KRAS
en BRAF). Een associatie tussen Lynch en serrated poliepen zou
kunnen wijzen op een versnelde CRC route in deze poliepen.
Aangezien Lynch-geassocieerde CRCs zelden BRAF gemuteerd zijn,
is BRAF analyse een manier om onderscheid te maken tussen Lynch-
geassocieerde CRCs en sporadische CRCs. Wij veronderstelden dat
een hoge frequentie BRAF mutaties in serrated poliepen, vergelijkbaar
met die van een controle groep sporadische serrated poliepen, erop
zou wijzen dat deze poliepen niet geassocieerd zijn met Lynch. Wij
toonden aan dat serrated poliepen in patiënten met Lynch-HPS (≥10
Sam
envattin
g &
toekom
stp
ers
pectief
216
serrated poliepen) niet geassocieerd zijn met Lynch vanwege de hoge
frequentie BRAF mutaties in deze poliepen. In tegenstelling, serrated
poliepen van Lynch patiënten met <10 serrated poliepen hadden
signicant lagere frequenties BRAF mutaties dan in de controle groep.
Gezien deze lage frequentie BRAF mutaties in patiënten met weinig
serrated poliepen, kan een causaal verband tussen deze poliepen en
de MSI-pathway niet worden uitgesloten. MSI-pathway geassocieerde
serrated poliepen zouden precursor lesies kunnen zijn die zich nog
sneller ontwikkelen tot CRC dan serrated poliepen die alleen
geassocieerd zijn met de serrated CRC pathway. Vanwege de unieke
diversiteit aan precursor lesies in HPS was het tot nog toe onduidelijk
welke poliepen zich tot CRC ontwikkelen in HPS en welke poliepen
derhalve klinisch significant zijn. In hoofdstuk 6 onderzochten wij
welke pathways operationeel zijn in HPS. Hiervoor onderzochten wij
de histologische en moleculaire eigenschappen van alle beschikbare
CRCs in een groot cohort HPS patiënten. Wij leverden nieuw
ondersteunend bewijs voor een serrated CRC pathway door in
combinatie (serrated poliep–CRC) lesies, identieke BRAF mutaties
aan te tonen in beide componenten. Tevens toonden wij aan dat
microsatteliet stabiele en –instabiele CRCs in HPS zich voornamelijk
ontwikkelen via een serrated CRC pathway en in mindere mate via de
Wnt-pathway aangezien de meerderheid van de CRCs een BRAF
mutatie hadden. De overheersing van een serrated CRC pathway ten
opzichte van de klassieke Wnt-pathway lijkt te verklaren door een
numerieke meerderheid aan serrated poliepen in HPS. Hierom lijken
alle histologische subtypes klinisch relevant te zijn. CRCs die zich
ontwikkelden via de serrated CRC pathway waren voornamelijk
rechtszijdig. Aangezien CRCs in HPS tot 4mm klein zijn beschreven
Samenvatting & toekomstperspectief
217
lijkt resectie van alle poliepen ≥3mm en voornamelijk proximaal
gelocaliseerde serrated poliepen geïndiceerd.
Endoscopische imaging – Detectie en differentiatie van poliepen
Gezien het verhoogde risico op maligne progressie van HPS poliepen
is endoscopische detectie, differentiatie en resectie van in het
bijzonder hoog-risico poliepen noodzakelijk om CRC ontwikkeling te
voorkomen. Hoofdstuk 7 demonstreerde dat NBI het percentage
gemiste poliepen significant vermindert in HPS in vergelijking met
high-resolution endoscopie (wit-licht). Om te evalueren welke poliepen
minder gemist werden met NBI, vergeleken wij miss-rates voor
verschillende histologieën en vormen. NBI was vooral van
toegevoegde waarde voor de detectie van vlakke serrated poliepen en
niet voor de detectie van adenomen. Aangezien de serrated CRC
pathway domineert in HPS patiënten, zijn deze bevindingen van
klinisch belang en ondersteunen zij het gebruik van NBI voor de
endscopische surveillance van HPS patiënten. In hoofdstuk 8
beschreven we het gebruik van ETMI (high-resolution endoscopie, NBI
en autofluorescentie imaging) voor ‘real-time’ endoscopische
differentiatie van poliepen in HPS. Geen van de modaliteiten toonden
voldoende diagnostische accuratesse om hoog risico sessile serrated
lesions van relatief ‘onschuldige’ HPs te onderscheiden. NBI (en niet
AFI) was echter wel waardevol voor de differentiatie tussen
hyperplastische poliepen en adenomen. S
am
envattin
g &
toekom
stp
ers
pectief
218
Acknowledgements
219
Acknowledgements
220
Acknowledgements
221
ACKNOWLEDGEMENTS
Het laatste hoofdstuk van dit proefschrift, maar misschien ook wel het
belangrijkste hoofdstuk van dit proefschrift. Niet omdat het voor
sommigen van u ook meteen het enige is dat u zult lezen, (uiteraard
neem ik u hiervoor absoluut niet kwalijk) maar meer omdat ik hier de
kans krijg om alle mensen persoonlijk te bedanken die hebben
bijgedragen aan de totstandkoming van dit boekje.
Allereerst mijn twee directe begeleiders die van het begin tot het eind
mij hebben bijgestaan: Prof. dr. C.J.M. van Noesel en Dr. E. Dekker.
Where molecular meets clinical. Beste Carel, jij hebt me bijgebracht
wat moleculaire wetenschap is. Jouw open en rustige houding als ook
je positiviteit zijn enorm inspirerend geweest. Een ‘walk of despair’
richting jouw kamer transformeerde vaak tot een ‘walk of triumph’ de
kamer uit. Jij bent het voorbeeld van een academicus die hoogstaande
wetenschap met plezier bedrijft. Bedankt voor je onvoorwaardelijke
steun, laagdrempeligheid en de vrijheid die je me hebt gegeven. Beste
Evelien, jouw visie, enthousiasme en vrolijkheid zijn de fundering van
dit proefschrift. Hoewel het volgens mij onmogelijk is voor elk gewoon
mens om jou bij te benen, weet ik dat je dit ons allen niet kwalijk neemt
en ons indien nodig een zetje (of twee) in de rug zal geven als we te
ver achterblijven. Bedankt voor de kans die je me hebt gegeven om
met jou aan dit onderzoeksproject te werken. Ik hoop dat we in de
toekomst nog veel samen kunnen brainstormen en mooi onderzoek
kunnen verrichten.
222
Prof. dr. E.M.H. Mathus-Vliegen, beste Lisbeth, als gevestigd MDL-arts
met zeer veel kennis en ervaring omtrent erfelijke polyposis
syndromen, zijn jouw inhoudelijke adviezen en commentaar van groot
belang geweest bij dit onderzoeksproject. Bedankt voor de altijd
plezierige samenwerking.
Prof. dr. P. Fockens, beste Paul, in het begin was je nog wat plagerig
als het om serrated poliepen ging. Jouw suggestie om mijn eerste
artikel naar de “Journal of serrated polyps of zo..” te sturen zal ik dan
ook zeker niet vergeten (het blad bestaat nog niet overigens)! Bedankt
voor je belangrijke bijdrage aan dit boekje en met name bij de imaging
studies.
Dr. J.B. Reitsma, beste Hans, statistiek is ingewikkeld. Maar daarmee
is de kous niet af. Statistiek is verwarrend. Statistiek is frustrerend.
Maar statitistiek is ook van essentieel belang om de waarde van
onderzoeksresultaten te kwantificeren. Veel dank voor al je hulp met
de analyses, in het bijzonder hoofdstuk 3.
Beste mede-auteurs die nog niet werden vernoemd in dit dankwoord.
Hartelijk dank voor jullie bijdrage en kritische verbeteringen aan de
stukken van dit proefschrift: J.F.W.M. Bartelsman, A. Cats, S. van
Eeden, L.P. van Hest, M. Houben, J.J. Keller, J.J. Koornstra, E.
Kurpershoek, M. van Leerdam, V. Lemmens, T.A.M. van Os.
Geachte leden van leescommissie: Prof. dr. F. Baas, Prof. dr. W.A.
Bemelman, Prof. dr. G.A. Meijer, Dr. F.M. Nagengast en Prof. dr.
G.J.A. Offerhaus. Veel dank voor uw bereidheid dit proefschrift op zijn
wetenschappelijke waarde te beoordelen. Dear Dr. J.E. East, it is an
Acknowledgements
223
honour for me that you were willing to critically review my thesis and
that you will be present during my defence.
Moleculair analysten van het MD-Lab: Mirjam, Alex, Mireille en Jitske.
Zonder al jullie hulp was dit boekje nooit tot stand gekomen. Mirjam, jij
bent mijn lab guru geweest gedurende mijn tijd bij jullie. Veel dank
voor alle tijd die je in mij hebt gestoken en al je geduld bij de vele
mislukte proefjes maar vooral voor je opgewektheid, je energie en je
humor. Alex, bedankt voor al je hulp bij de immuno’s en het flinterdun
snijden van piepkleine poliepjes. Jij bent niet alleen de koning op de
woensdag! Mireille, bedankt voor je hulp bij de MSI en methylatie
analyses zonder jou was het een hopeloze onderneming geweest.
Jitske, jij bent een allrounder waar ik vaak met vragen terecht kon –
bedankt hiervoor en de mooie woordenwisselingen..;) !!
Mijn dank aan mijn collega-artsonderzoekers van de Tytgatsuite
waarmee ik het geluk had om een kamer te delen: Anne, Simone en
Rogier jullie zijn pionier-Tytgatters en zullen dat altijd blijven. Latere
versterkingen: Denny, bedankt voor het mooie up-to-date
dierennieuws; Koen en Thomas, jullie hebben het concept
kantoorgrappen tot een hoger niveau getild; Boney, veel succes met je
eind-examen (na de school-onderzoeken natuurlijk). Jullie zijn allen
dierbare collega’s geworden en ik weet zeker dat we ook in de
toekomst met veel plezier vele jaren samen zullen werken. Alle andere
collega onderzoekers: Wouter, Bart, Renée, Wout (dude), David, Joep,
Annikki, Charlotte, Noor, Tessa, Willemijn, Susan, Esmerij, Wietske,
Sjoerd, Claire, Babette, Olivia, Femme en Emma, bedankt voor de
gezellige tijd samen!
224
Collega’s van B1-245. Het was zoals jullie weten de eerste dagen
behoorlijk wennen op de kamer. De stilte was oorverdovend en een
speld had geen kans om onomgemerkt op de zacht maar oud ex-
bestuurstapijt te landen. Maar gelukkig kon uiteindelijk hard getik
afgewisseld worden met overleg, goede (en slechte) verhalen, koffie
pauzes en een sporadisch youtube filmpje. Bedankt voor een
fantastische tijd samen roomies. Persoonlijk bericht voor jullie:
Frans van der Pants, bedankt voor ons vele overleg, gezelligheid en
hulp bij hoofdstuk 7 en 8. Ik weet zeker dat je een koningschirurg zal
worden! Roos Poe, ontzettende tofferd met je Apple laptop én je
‘gewone’ PC. Ik ben benieuwd welk boek je nu weer aan het lezen
bent. Freddepet, nog steeds als laatste naar huis? Lorenzio, bedankt
voor je mooie verhalen over het mooie leven van Spaanse stieren en
hoe ze uiteindelijk Spaanse worst worden. Nadine, veel succes met je
promotie! Teaco en Yark, met jullie was er gelukkig wel iets frequenter
een kort intermezzo in te lassen.
Yark, jij bent de nieuwe HPS/SPS koning. Ik vond het erg gezellig met
je samen te werken en wens je veel succes met de Eclipse, de
PROTECT en alle andere lopende en toekomstige studies. Hopelijk
kunnen we geregeld brainstormen over nieuwe studies en lopende
zaken onder het genot van een biertje.
Christine Cohen, jij hebt het overzicht, jij kent de patiënten en jij weet
wie aan welke studies deelneemt. Bedankt voor al je hulp, positiviteit
en enthousiasme de afgelopen jaren!
Lisbeth Mathus-Vliegen, Ellen de la Mar en Monique Dijselhof. Om de
nodige onderzoekscentjes te verdienen heb ik het eerste half jaar met
Acknowledgements
225
veel plezier met jullie samengewerkt aan een fase-2 studie betreffende
een afslankmiddel bij obese patienten. Hoewel het inhoudelijk vrij
weinig met poliepen te maken had, was deze tijd met jullie erg
bijzonder waarbij we veel plezier hebben gehad samen. Om een paar
episodes te noemen: spookrijden in Genève, de altijd verloren bril van
Ellen etc. Laten we vaak blijven afspreken.
Mijn paranimfen: Laurien, jij hebt al vanaf mijn eerste dag als
onderzoeker gezeurd dat je mijn paranimf wilde zijn. Daar kon ik niet
om heen en stiekem zou ik dat ook niet willen. Je bent mij erg
dierbaar! Patrick, huisarts living the good life, bedankt voor je
onvoorwaardelijke vriendschap en we gaan zeker binnenkort de
Himalaya trek doen samen.
Al mijn vrienden waarvan sommigen nog steeds niet weten waar ik
onderzoek naar heb gedaan. Bedankt voor de goede tijden samen, de
afleiding, ontspanning en jullie vriendschap. Biram old man, you
deserve a special mention. Thank you for being my best friend. You
like to explore different paths of life but I am confident that you will
eventually make your own path. Don’t stop making lemonade!
My family, extended family and close friends in India and dispersed
around the globe: although we are far away from each other, I luckily
get to see you regularly. Despite the distances we will always remain
close!
226
The Boparai family:
My parents, Ma and Pa, without you I wouldn’t be where I am today.
There are so many things to thank you for but most importantly I thank
you for being the best parents I could ever wish for.
Neelu, you snaggle tooth-vulture, thank you for being an ever caring
elder sister. I am glad you have Ed, Kiran and little Tej to take care of
you a little too.
Mohini (monu), little-big sister, world-traveller and carnivore of the
highest order, I hope we will still be laughing at the same jokes when
we are old wrinkled prunes.
Acknowledgements
227
CURRICULUM VITAE
Karam Boparai was born in Purmerend, the Netherlands, on the 31st
January 1981. Here he attended high school at the Jan van Egmond
College. After graduating in 1999 he ventured to Delft to study
Mechanical Engineering. After realizing that this was not his calling, he
promptly decided to study Medicine at the University of Amsterdam
(UVA). During his studies he grew increasingly interested in the field of
Gastroenterology and Hepatology. This interest led him to Dr. Evelien
Dekker and Prof. dr. Carel van Noesel at the Academic Medical Center
in Amsterdam with whom he performed a research project regarding
serrated polyps and their association with MYH-associated polyposis.
After graduating from Medical school in March 2007 he joined the
department of Gastroenterology and Hepatology to continue both
clinical and molecular research concerning the Serrated Neoplasia
Pathway under supervision of Dr. Evelien Dekker, Prof. dr. Carel van
Noesel, Prof. dr. E.M.H. Mathus-Vliegen and Prof. dr. P. Fockens
which led to this dissertation. He was (co)applicant of the successful
grant proposal to the KWF which will further focus on the clinical
management of patients with serrated polyposis syndrome. Since
October 2010, Karam is doing his Internal Medicine residency at the
Onze Lieve Vrouwe Gasthuis in Amsterdam. He will commence his
training as Gastroenterologist at the Onze Lieve Vrouwe Gasthuis and
the Academic Medical Center from October 2012.
Curriculum Vitae
228