GENES, CHROMOSOMES & CANCER 50:82–94 (2011)

Common Pathogenetic Mechanism Involving HumanChromosome 18 in Familial and Sporadic IlealCarcinoid Tumors

Janet L. Cunningham,1 Teresita Dıaz de Stahl,2 Tobias Sjoblom,2 Gunnar Westin,3

Jan P. Dumanski,2 and Eva T. Janson1*

1Departmentof Medical Sciences,Section of Endocrine Oncology,Uppsala University,Uppsala,Sweden2Departmentof Genetics and Pathology,Uppsala University,Uppsala,Sweden3Departmentof Surgical Sciences,Section of Endocrine Surgery,Uppsala University,Uppsala,Sweden

Serotonin producing endocrine carcinoma of small intestine (ileal carcinoid) is a clinically distinct endocrine tumor. It is

generally considered as a sporadic disease and its molecular etiology is poorly understood. We report comprehensive clin-

ical and molecular studies of 55 sporadic and familial patients diagnosed with this condition. Nine pedigrees encompassing

23 affected subjects were established, consistent with autosomal dominant mode of inheritance. Familial and sporadic

patients demonstrated indistinguishable clinical pictures. Molecular analyses of 61 tumors from 45 individuals, including

eight familial and 37 sporadic patients, aimed at determination of global copy number aberrations using BAC and Illumina

SNP arrays and gene expression profiling by Affymetrix chips. Chromosome 18 aberrations were identified in both spo-

radic and in familial tumors; 100% vs. 38%, respectively. Other, less frequent aberrations were also common for both

groups. Global expression profiles revealed no differentially expressed genes. Frequent gain of chromosome 7 was exclu-

sively observed in metastases, when patient matched primary tumors and metastases were compared. Notably, the latter

aberration correlated with solid growth pattern morphology (P < 0.01), a histopathological feature that has previously

been related to worse prognosis. The clinical and molecular similarities identified between sporadic and familial cases sug-

gest a common pathogenetic mechanism involved in tumor initiation. The familial variant of ileal carcinoid represents a pre-

viously unrecognized autosomal dominant inherited tumor disease, which we propose to call Familial Ileal Endocrine

Carcinoma (FIEC). Our findings indicate the location of a FIEC tumor suppressor gene near the telomere of 18q, involved

in development of inherited and sporadic tumors. VVC 2010 Wiley-Liss, Inc.

INTRODUCTION

Well differentiated serotonin producing endo-

crine carcinomas that originate in the small intes-

tine and proximal colon (the midgut) are

commonly denoted ileal carcinoids (Modlin et al.,

2008). They are malignant and most patients

present with metastatic disease. The median age

at diagnosis is 61 years and the incidence is 1-2/

100 000 inhabitants per year. The tumor is usu-

ally slow growing and the Ki67 proliferation index

is below 1% in the majority of cases (Cunning-

ham et al., 2007). Many patients present with ab-

dominal pain due to intestinal obstruction caused

by tumor growth. Hormones produced by the tu-

mor cells give rise to the carcinoid syndrome,

including flush, diarrhea, the carcinoid heart dis-

ease and bronchial constriction. The only curative

treatment is surgery, but this can be accom-

plished only in a minority of cases. First-line

medical treatment includes alpha-interferon and

somatostatin analogs of which the latter are

potent in reducing hormone secretion and thus

symptoms of the carcinoid syndrome (Modlin

et al., 2008).

Ileal carcinoid is generally considered a spo-

radic tumor. However, during the past five deca-

des some case reports of inherited variants have

been published describing families with 2 or 3

affected members (Eschbach and Rinaldo, 1962;

Moertel and Dockerty, 1973; Wale et al., 1983;

Kinova et al., 2001; Pal et al., 2001; Jarhult et al.,

2010). In contrast to patients with ileal carcinoid,

Additional Supporting Information may be found in the onlineversion of his article.

Supported by: The Swedish Research Council, Lions founda-tion for Cancer research at Uppsala University Hospital, SelandersFoundation, the Swedish Children’s Cancer Foundation and theSwedish Cancer Society.

The first two authors contributed equally to this work.

*Correspondence to: Eva T. Janson, Department of MedicalSciences, Uppsala University, University Hospital entrance 40, 5thfloor, 751 85 Uppsala, Sweden.E-mail: Eva.Tiensuu_Janson@medsci.uu.se

Received 12 July 2010; Accepted 11 October 2010

DOI 10.1002/gcc.20834

Published online 22 November 2010 inWiley Online Library (wileyonlinelibrary.com).

VVC 2010 Wiley-Liss, Inc.

subjects affected with multiple endocrine neo-

plasia type 1 (MEN1) develop lesions in the

pituitary, parathyroid and pancreas. Well differen-

tiated endocrine carcinomas originating in the

lung, thymic, gastric and duodenal area, earlier

denoted foregut carcinoids, are also associated

with MEN1. Furthermore, a large epidemiologi-

cal study failed to demonstrate an association

between ileal carcinoids and endocrine tumors

typically associated with the MEN1 syndrome

(Hemminki and Li, 2001). Recent genomic

(Kytola et al., 2001; Lollgen et al., 2001; D’Adda

et al., 2002) and expression studies (Duerr et al.,

2006) indicate that malignant ileal carcinoids

have a different etiology than other neuroendo-

crine tumors and genetic screening for mutation

in the MEN1 gene in two of the previously

described ileal carcinoid families was negative

(Kinova et al., 2001; Jarhult et al., 2010).

The genetic events leading to development of

an ileal carcinoid tumor are largely unknown. Pre-

vious studies have shown that deletion of 18q21-

qter is a common event (Lollgen et al., 2001). This

was confirmed by another group, which narrowed

the region to 18q22qter (Kytola et al., 2001). Other

alterations described include loss on 11q22-23, loss

on 16q21 and gain on 4p14. In two recent studies

using genomic profiling of ileal carcinoids, loss of

chromosome 18 (61% and 74%, respectively) was

confirmed ( Kulke et al., 2008; Andersson et al.,

2009). Other common aberrations were loss of 9p,

11q and 16q, as well as gain of chromosome 14.

The latter abnormality was associated with poor

survival in one of the studies (Andersson et al.,

2009). These reports provided information about

the genetic changes involved in the sporadic dis-

ease. There is, however, no available data on

genetic aberrations in familial cases.

In this study, we have characterized nine fami-

lies with a history of ileal carcinoid. To evaluate

the degree to which molecular events in familial

tumors resembles that of sporadic cases, we per-

formed high-resolution genomic and gene expres-

sion profiling in a comprehensive series of tumors

derived from familial and sporadic patients.

MATERIALS AND METHODS

Patient Samples

The Department of Endocrine Oncology at Upp-

sala University Hospital, Sweden has been a national

referral center for neuroendocrine tumors since the

1980’s. For this study we have collected clinical

records for 55 patients with ileal carcinoid, diagnosed

at the Laboratory of Pathology and Cytology and

treated at our department. The diagnosis was based

on international recommendations for the classifica-

tion of endocrine tumors (Solcia et al., 2000; Kloppel

et al., 2009). For nine of these patients, diagnosed

between 1988 and 2005, a family history of carcinoid

disease was uncovered (our index patients). During

the past 2 decades, 9 further patients were diagnosed

at our clinic within these families. Five additional

patients belonging to these families were identified

at other hospitals as having a confirmed ileal carci-

noid, but they were not subject to clinical or molecu-

lar studies. For these additional patients pathology

reports were reviewed and tissue specimens re-eval-

uated when necessary to establish the diagnosis.

When an index patient was discovered the patient

was interviewed and a family pedigree established.

A trained research nurse contacted living family

members to collect relevant data.

Plasma chromogranin A (CgA), urinary 5-

hydroxyindole acetic acid (U-5HIAA) and radiol-

ogy was performed at diagnosis. Data were ana-

lyzed using the statistical program package SPSS.

The end point for survival analyses was event-

free survival time calculated from diagnosis to

carcinoid related death or, for censored observa-

tions, until last follow-up. A possible correlation

between number of genetic aberrations and tu-

mor morphology was tested using a Spearman

Rank test. Independent effects of specific chro-

mosome aberrations on tumor growth pattern

were tested using stepwise logistic regression

analysis. All patients developed metastatic dis-

ease; 4 cases having WHO stage IIIb and the

remaining 51 with stage IV.

Sixty-one tumor samples derived from 45 of

these patients (8 familial and 37 sporadic cases)

were subjected to genetic analysis. Twenty-seven

samples represented primary tumors and 34 me-

tastases (30 mesenterial and 4 hepatic). For 14

patients, patient-matched primary and metastatic

samples were profiled. Tumor material was

sampled during operation and stored at �80�C.Tumor tissue for DNA and RNA extraction was

carefully selected to obtain >70% tumor cells

content. Sections from frozen tissue were taken

before and after material collection, stained for

CgA and examined to ensure high tumor content.

Evaluation of tumor morphology was performed

as previously described (Cunningham et al.,

2007). The study was approved by the Uppsala

University Hospital ethics committees and all

patients gave informed consent.

FAMILIAL AND SPORADIC ILEAL CARCINOIDS 83

Genes, Chromosomes & Cancer DOI 10.1002/gcc

Array-Based Comparative Genomic

Hybridization: 32K Array

The 32K array was previously established (de

Stahl et al., 2008). DNA labeling, hybridization,

washing and scanning of arrays were performed

as earlier described (Buckley et al., 2002; de Stahl

et al., 2008). Blood DNA from a healthy female

(F1) was used as reference in all experiments.

The raw data were uploaded to a laboratory infor-

mation management system database (LCB;

http://base.lcb.uu.se) and filters were applied to

remove oversaturated spots (>5%), spots with

low signal-to-noise-ratio (<3) and spots flagged as

bad or absent (de Stahl et al., 2008). The data

was normalized using print-tip locally weighted

scatter-plot smoothing (Otsuka et al., 2008). Log2

ratio was exported into Nexus Copy Number 4.0

copy number analysis program (BioDiscovery,

Inc., El Segundo, CA). BioDiscovery’s Rank seg-

mentation algorithm was used for calling of copy

number alterations (CNAs). Copy number data

were used for statistical analysis using Fisher’s

Exact Test.

High-Density SNP Genotyping Arrays:

Illumina 610Q Chips

Profiling with SNP arrays was done using the

Illumina InfiniumII assay (Steemers et al., 2006)

on Illumina Human610Q beadchips (Illumina,

San Diego, CA). Normalization and calling was

done in BeadStudio v3.3 (Illumina). The median

call rate was 94.38% (range: 90.62-95.10%). Log

R ratio (LRR) and B-allele frequency (BAF)

were then exported into Nexus Copy Number

4.0 copy number analysis program (BioDiscovery,

Inc.). CNAs were called using BioDiscovery’s

SNPRank segmentation algorithm. The raw data

from 32K BAC array and Illumina beadchips are

being submitted to Gene Expression Omnibus

database.

Microarray Expression Analysis

Total RNA was prepared by RNeasy Mini Kit

(Qiagen, Hilden, Germany). RNA concentration

and quality was assessed as previously described

(Nord et al., 2010). 100 ng of total RNA were

used to prepare biotinylated fragmented cRNA

using a two-cycle amplification step, according to

the GeneChipVR

Expression Analysis Technical

Manual (Rev. 5, Affymetrix Inc., Santa Clara,

CA). Affymetrix GeneChipVR

expression arrays

(GeneChipVR

Human Genome U133 Plus 2.0

Array) were hybridized, washed and stained and

data analyzed as described in Nord et al. (2010).

To search for differentially expressed genes an

empirical Bayes moderated t-test was then

applied (Smyth, 2004), using the ‘limma’ package

(Smyth, 2005) and P-values were adjusted accord-

ing to Benjamini and Hochberg (Benjamini et al.,

2001). Hierarchical clustering of the samples was

made in Genesis (Sturn et al., 2002) using all

genes, Euclidian distance and average linkage.

Sequencing of Candidate Genes

RefSeq gene and transcript coordinates, human

genome sequence, and single nucleotide poly-

morphisms were obtained from the UCSC data-

base (http://genome.ucsc.edu). For each gene,

protein-encoding exons as well as flanking

intronic sequences and 5’ UTR and 3’ UTR

sequences were extracted. Primers used for PCR

amplification and sequencing have previously

been published (Sjoblom et al., 2006; Wood

et al., 2007). A sequencing primer (M13 forward,

5’-GTAAAACGACGGCCAGT-3’) was appended

to the 5’ end of one of the primers in each primer

pair. Whole genome amplification (WGA) was

used to increase quantities of DNA from tumor

and normal samples, according to published pro-

tocol (Sjoblom et al., 2006). For each sample, a

minimum of 5 independent WGA reactions were

pooled to reduce the effects of any allelic or locus

bias that may have occurred during amplification.

PCR amplification, dye terminator sequencing

and somatic mutation analysis was performed as

previously described (Sjoblom et al., 2006).

RESULTS

Familial and Sporadic Ileal Carcinoids Have

Similar Clinical Characteristics

Twenty-three patients were identified with a

family history of ileal carcinoids (Fig. 1) and com-

plete clinical data were available for 18 of them.

In two families, ileal carcinoids were seen in two

branches linked by an unaffected individual sug-

gesting an incomplete penetrance of the disease.

The clinical details of familial cases compared

with the sporadic ones are summarized in Table

1. No significant differences in age at diagnosis,

hormone levels or proliferation index were

observed between the two groups. A higher fre-

quency of other non-malignant endocrine condi-

tions (hyperparathyroidism and thyrotoxicosis)

was found in patients with a family history of

84 CUNNINGHAM ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

ileal carcinoids, when compared with sporadic

cases. One family stands out (family E, Fig. 1)

where five out of six siblings as well as their

mother presented with thyrotoxicosis and three of

the siblings also developed ileal carcinoid tumors.

Similar Genomic Profiles of Familial and

Sporadic Ileal Carcinoids

Genomic aberrations in 61 tumor samples were

evaluated using a 32K BAC array and confirmed

with Illumina 610Q beadchips. Overall, 252

CNAs were identified in the 27 primary tumors

and 34 metastases (Table 2). The frequency of

copy number changes was calculated for all tumor

samples as well as for familial and sporadic

groups separately (Fig. 2A). Aberration of chro-

mosome 18 was the dominating finding and was

observed in 88.8% of tumors, including all 37

sporadic and three (out of eight) familial cases.

Monosomy 18 was often found in combination

with other changes, but was also observed as the

only detectable CNA in the tumors from 12

patients (Table 2, Figs. 2 and 3 and Supporting

Information Fig. 1). The two affected individuals

in family G displayed different CNAs on chromo-

some 18, one presented with monosomy 18 while

the other patient showed copy number neutral

loss of heterozygozity (LOH) of the whole chro-

mosome, without copy number reduction (ID6

and ID7, respectively, Fig. 3 and Table 2). The

data from tumor ID7 are consistent with loss of

one copy of chromosome 18 and reduplication of

the remaining one. The results from this family

show that two different second hits affected the

putative tumor suppressor gene located on 18q.

The comparison of genomic profiles between all

familial and all sporadic tumors revealed that

most of the genetic changes were in common

between the groups. As for differences, we

observed a tendency for higher number of chro-

mosomes affected by CNAs in familial vs. spo-

radic cases, average 5.6 vs. 3.4 /sample, (t-testP-value: 0.028). The identification of more aber-

rations in individuals with familial disease may,

however, be a consequence of over-representation

of liver metastases in this group (three liver

metastases out of eight familial cases vs. one out

of 37 in sporadic cases). Interestingly and in con-

tradiction to the above, one familial case (ID8)

with a thoroughly verified tumor content (>70%,

displayed no CNAs in the samples taken from

both primary tumor and metastasis.

Another intriguing aberration of chromosome

18 was noted in one sporadic tumor ID11 (Sup-

porting Information Fig. 1 and Table 2). In this

sample, the Log R ratio (LRR) values reflecting

fluorescent intensity and B-allele frequency

Figure 1. Pedigrees of nine families (A–I) showing patients affectedwith ileal carcinoid are in agreement with autosomal dominant inheri-tance pattern and incomplete penetrance. Individuals with ileal carci-noids are represented as filled symbols. Patients studied molecularlyare denoted by Arabic numerals, which correspond to patient ID inTable 2. Generations are shown using Roman numerals on the leftand diagonal lines indicate deceased patients. Other diagnoses are:thyrotoxicosis (TT), hyperparathyroidism (HP), hypernephroma (HN)and colon adenocarcinoma (CA).

TABLE 1. Clinical Data From Patients with Familiar andSporadic Ileal Carcinoids

ParameterFamilial ilealcarcinoid

Sporadic ilealcarcinoid

Number of patients 18 37Age at diagnosis (years) 62 [24–73] 61 [37–85]Sex (M:F) 7:11 19:18Survival (months) 79 [27–310] 75 [7–399]Status (DWD:AWD)a 11:7 8:29Ki67 (%) <1 [<1–10] <1 [<1–4]U-5HIAA (lmol/24 h) 141 [28–2720] 300 [16–2112]CgA (nmol/L) 46 [4–1280] 22 [4–382]Other malignancy 1b 5c

Other endocrine disease 6d 2e

M, Male; F, Female; DWD, dead with disease; AWD, alive with

disease; Survival is calculated as months from diagnosis to death;

U-5HIAA, Urinary 5-hydroxyindoleacetic acid (serotonin metabolite)

normal reference is 50 lmol/24 h; CgA, Chromogranin A, normal

reference is <4 nmol/L; Ki67 (%), proliferation index in tumor areas

with highest proliferation.aA higher number of patients died in the familial group than among

sporadic cases. This is likely due to the longer duration of follow up

in familial group of patients as there is no difference in duration of

survival between the two groups.bColon adenocarcinoma (1).cBreast cancer (1), colon adenocarcinoma (1), folicular thyroid cancer

(1), uterus adenocarcinoma (1), prostate cancer (1).dThyrotoxicosis (4), hyperparathyroidism (2).eHypothyroidism (1), thyrotoxicosis (1).

FAMILIAL AND SPORADIC ILEAL CARCINOIDS 85

Genes, Chromosomes & Cancer DOI 10.1002/gcc

TABLE2.SummaryofFindings

forFamilialandSporadicIlealCarcinoid

Tumors

Pat

IDa

Typeb

Site

c

Age

d

(years)

Follow-upe

(months)

Gains

Deletions

ChrwithCNAf

Array

g

1A

FP

45

250

4,5,14,20

432K,610Q,U133þ2

2B

FL

62

180DW

D4,20

232K,610Q

3B

FP

43

68DW

D4,5,6q25.3-q26,10,12,14,

19q13.33-q13.41,20

11p11.2

932K,610Q

4F

FM

66

33

1p33-p32.3,3p21.31-p11.1,12q24.32-q24.33,

17p13.3-q11.1,17q12-q21.2,17q21.33-q22,

18

5610Q

5F

FL

38

310DW

D1q23.3-q44,2p12,2p11.2,2q24.3-q31.1,

2q36.2-q37.3,3p26.3-p26.1,3p24.2-p24.1,

3p23-p22.3,3p22.1-p21.31,3p14.3-p14.2,

3p14.1-p11.1,3q11.2-q13.31,3q22.1-

q26.31,3q26.33,5q33.1,5q34,6q22.1,

11q13.5-q25

632K,610Q

6G

FL

68

77DW

D3p22.1-p21.31,4q22.1-q24,5q14.3-q21.3,

6p25.3-p24.1,6p12.3-p12.1,10q24.1-

q24.31,14q11.2-q21.1,17q21.31-q24.2

3p24.3-p24.2,3p21.1-p14.1,3p13-p12.3,

12p13.2-p12.3,12p12.2-p11.23,12q12-

q13.11,12q13.13,12q13.3-q15,14q24.1-

q24.3,14q31.3-q32.11,18

932K,610Q

7G

FM

50

145

2,4,5,7,10,11,14,15,20

17p13.3-q11.1,CNN-LOH

18h

10

32K,610Q,U133þ2

8H

FP

52

35

032K,610Q

8H

FM

032K,610Q,U133þ2

9S

P68

152DW

D5q31.2-q35.3

11q14.3-q24.2,16q13-q24.3,18

432K,610Q

10

SP

40

156DW

D10p12.33,18

232K,610Q,U133þ2

10

SM

10p12.33,18

232K

11

SP

52

159

18

132K

11

SM

11q14.1-q23.3,18i

232K,610Q

12

SP

55

137

18

132K,U133þ2

13

SM

41

194DW

D4p16.1-p15.1,5,14,20

9p24.3-p11.2,18,22q13.1-q13.33

732K

14

SP

50

142

11q14.1-q23.3,18

232K

14

SM

4,5,7,14,20

9q33.3-q34.3,13,16q12.1-q24.3,18

932K

15

SP

70

87DW

D11q14.1-q24.2,18

232K

16

SM

61

103DW

D2p25.3-p22.3,4,5,20

9,11q22.1-q25,12p13.31-p12.3,12q21.33-

q22,18

732K

17

SM

37

102

14q13.2-q22.3,18

232K

18

SM

64

82

9,11q22.1-q24.1,13,18

432K

19

SM

51

94

4,5,7,10,14,20,21q21.2-q22.3

18

832K

20

SP

40

399

9q33.2-q34.3,11q12.2-q13.3,16q23.3-q24.3,

18

432K

21

SP

55

106

13q21.33-q34

16q12.1-q24.3,18

332K

21

SM

13q21.33-q34

16q12.1-q24.3,18

332K

22

SP

67

99

18

132K

22

SM

18

132K

23

SP

52

62

18,22

232K

24

SP

71

82

418

232K

25

SM

71

57

4,5,7,10,12,14,20

2q24.3-q32.1,18

932K,610Q (Continued)

86 CUNNINGHAM ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

TABLE2.SummaryofFindings

forFamilialandSporadicIlealCarcinoid

Tumors

(Continued)

Pat

IDa

Typeb

Site

c

Age

d

(years)

Follow-upe

(months)

Gains

Deletions

ChrwithCNAf

Array

g

26

SM

85

25DW

D12p13.33-p13.32,17q21.32-q24.1

16q22.1-q24.3,18

432K

27

SP

52

142

18

132K

27

SM

16q12.2-q24.1,18

232K

28

SL

45

44

4,5,8,10,12,14,17,20,21

9p24.3-p11.2,17q11.2,18,22

13

32K

29

SM

67

34DW

D18

132K,610Q,U133þ2

30

SM1

70

48

18q22.1-q22.2

132K,610Q

30

SM2

18

132K

31

SP

78

41

16q12.2-q24.1,18

232K

31

SM

4,20q11.23-q13.33

16q12.2-q24.3,18

432K

32

SP

74

7DW

D9,18

232K

33

SM

47

133

18

132K,610Q,U133þ2

34

SP

51

37

18

132K

34

SM

18

132K

35

SP

65

35

18

132K

35

SM

18

132K

36

SP

76

39

16q12.2-q24.3,18

232K

37

SP

71

34

4,5q11.2-q35.3,14,20

2q36.1-q37.3,9p24.3-p12,18q21.1-q21.31

732K,610Q,U133þ2

37

SM

4,5q11.2-q35.3,14,20

2q36.1-q37.3,9p24.3-p12,18q21.1-q21.31

732K

38

SM1

40

75

18

132K

38

SM2

14

9p24.3-p12,18

332K

39

SM

68

32

2p25.3-p25.1,17p13.3-p11.2,18

332K

40

SP

71

34

10q26.13-q26.3,20q13.13-q13.33

12p13.31-p12.3,18q21.1-q21.32,18q22.3-

qter

432K,610Q,U133þ2

41

SP

41

37

18

132K

41

SM

18

132K

42

SP

66

30

18

132K

42

SM

2p25.3-p25.1,2p21-p16.3,2q24.1-q24.3,

4p16.2-p15.33,7p21.1-p15.1,

8q21.3,8q24.12-q24.22,14q32.2-q32.33,

17p12-q11.1,20q13.33

3p21.31,11q22.3-q25,12q24.31-q24.33,18

11

32K,610Q

43

SP

43

130

4,10,14,15,20

16q13-q24.3,18,22

832K

44

SP

65

67

6q23.3,13q12.3-q13.2,13q13.3-q21.2,18

332K

44

SM

5,7,10,14,20

18

632K

45

SP

57

72

4,5,20

18

432K

Copy

numberalterations(C

NAs)

identifiedin

61tumorsamplesderivedfrom

45patients

withfamilialandsporadicilealcarcinoid.

aThefamily

towhichindividualsbelongisindicatedbyacapitalsuperscriptletter(A,B,F,G

orH),seeFig.1.

bS,

Sporadic;F,Familialilealcarcinoid.

cP,Primarytumor;

M,Mesenterial

metastasis;L,Livermetastasis.Fo

r14patients

matchedprimaryandmetastatic

tumorwere

profiled.In

6ofthematchedpairs

(ID:11,14,27,31,42,and44),additional

alterationswere

observedin

metastasis.Fo

rID:30and38tw

ometastasesderivedfrom

thesamepatientwere

profiled(M

1andM2),whichrevealedtumorheterogeneity.

dAge

atdiagnosis.

ePatients

DW

D,dead

withdiseaseareindicated.

f NumberofchromosomesaffectedbyCNA.

gThearrayusedto

profile

thetumorsamplesisindicated:32K,Illumina610Q

orAffym

etrix

U133þ2

chips.

hCNN-LOH

18,copy

numberneutralLOH

ofchromosome18.

i Loss

ofchromosome18withoutallelic

imbalance,seetextfordetails.

FAMILIAL AND SPORADIC ILEAL CARCINOIDS 87

Genes, Chromosomes & Cancer DOI 10.1002/gcc

Figure 2. Summary of findings in ileal carcinoids. (A) The frequencyof copy number changes was calculated for all measurement points inthe arrays and plotted relative to the position along the chromosomefor all tumors and separately for familial as well as for sporadicgroups. Green bars above the horizontal line indicate the frequency inpercent of copy number gains and red bars below the horizontal lineindicate the frequency of copy number losses. For patient wheremore than one lesion was profiled only one tumor was included inthese plots. When both primary tumor and metastasis was studied,metastasis data are presented. Whenever results from BAC array andIllumina beadchip were available for the same sample, Illumina data areshown (Table 2). The statistical significance of the differences betweenfamilial and sporadic cases is shown below. Regions altered betweengroups are denoted by red and green bars for deletions and gains,respectively (relative frequency difference >25%, significance P <0.05). (B) Histopathological sections from sporadic ileal carcinoid usedfor DNA isolation display strong chromogranin A immunoreactivityand a high tumor cell content (magnification 100�). Tumors with solidmorphology (bottom) have increased number of genomic aberrationscompared to those with organoid morphology (top) (P ¼ 0.01) and anindependent correlation to gain of chromosome 7 (P < 0.01). Differ-

ences in vascularisation are demonstrated in inset showing CD31 andCD34 immunoreactivity. (C) Chromosome 18 ideogram indicating theposition of minimal regions of deletion determined in previous reports(Kytola et al., 2001; Lollgen et al., 2001; Kulke et al., 2008; Anderssonet al., 2009) and our study. Lollgen et al. identified a minimal regionthrough LOH screening with microsatellite markers. LOH wasobserved for D18S541 and D18S844, while D18S858, the next in-formative marker was retained. Our samples ID30, 37 and 40 definedthree regions (R1, R2 and R3), highlighted in grey. The summary ofresults from comparative analysis of global gene expression for tran-scripts from chromosome 18 in sporadic vs. familial tumor samples isshown below. Fold change (log2) for chromosome 18 transcript levels,established with Affymetrix array, is shown. Probes representing can-didate genes selected for mutational studies are indicated as red dots.Hierarchical clustering for samples profiled with expression arrayswas performed in Genesis (Sturn et al., 2002) using all genes. Familial(1A, 7G and 8H) and sporadic cases did not cluster into separategroups. (D) Frequency plot of copy number changes for 14 patient-matched primary and metastatic samples, revealing additional aberra-tions in metastases. The layout of this panel is the same as describedabove for panel A.

(BAF) data showing allelic status demonstrated

inconsistent results for chromosome 18. LRR val-

ues indicate monosomy 18 but BAF values did

not show the expected allelic imbalance. It

should be stressed that this tumor also showed in-

terstitial deletion of chromosome 11 with the

expected appearance of both LRR and BAF val-

ues. Several explanations can be proposed to

these unexpected results. For instance, this tumor

sample might be bi-clonal with two tumor cell

populations co-existing at a similar relative fre-

quency and each cell population having lost

either the maternal or paternal chromosome.

Another possible explanation is the presence of

tetraploid cancer cells in this tumor, exhibiting

loss of two copies (one maternal and one pater-

nal) of chromosome 18.

The high resolution of the applied analysis

platforms and a considerable number of studied

tumors permitted us to identify four partial dele-

tions on 18q in sporadic cases, which defined

three candidate intervals: 18q21.1-q21.31 (45.80-

54.36Mb), 18q22.1-q22.2 (62.68-65.99Mb) and

18q22.3-q23 (68.38-76.12Mb) indicated as R1, R2

and R3 (Fig. 2C and Table 2). These regions

were 8.56, 3.31 and 7.73 Mb in size and encom-

passed 30, 6, and 29 genes, respectively. A sec-

ond major region affected by deletions in six

sporadic and one familial tumors mapped to

11q22.3-23.3 (Fig. 2). Genes mapped to this locus

include several members of the cysteine-aspartic

acid protease (caspase) family: CASP4, CASP1and CASP5 as well as MLL and SDHD. Restrictedregions of deletion were also observed at 3p12.3-

p14.3, in 3 familial tumors, two of these being

liver metastasis (Fig. 2 and Table 2). The pres-

ence of deletions at 11q22.3-23.3 and 3p12.3-

p14.3 and loss of chromosome 18 were not mutu-

ally exclusive indicating that these deletions may

have a synergistic effect on tumor progression.

Copy number gains frequently encompassed

whole chromosomes 4, 5, 14 and 20 and were

observed in more than 20% of cases (Fig. 2). The

above mentioned array results are best compatible

with trisomy for these chromosomes in the tumor

cells and these aberrations were often present con-

comitantly. Since gain of chromosome 14 has been

suggested to be a predictor of poor outcome in ileal

carcinoids (Andersson et al., 2009), survival was com-

pared in patients with and without this alteration. In

this study, 14 patients displayed partial or complete

gain. The shortest observation time within the group

with chromosome 14 gain was 34 months and the

shortest survival time was 68 months (Table 3).

Kaplan-Meier survival analysis showed no difference

in survival between patients with and without gain of

chromosome 14 (not shown). Eleven cases (10 spo-

radic and 1 familial), revealed gain of chromosome 14

together with loss of chromosome 18, another familial

case displayed gain of chromosome 14 along with

copy number neutral LOH of chromosome 18, while

2 familial cases displayed gain of chromosome 14

without deletions on chromosome 18. Segmental

gains on chromosome 14 were observed in 2 patients,

at position chr14:22,093,320-37,596,612 in case ID6

and at position chr14:97,434,551-106,368,585 in case

ID4 (Table 1). Interestingly, a locus on chromosome

14 at position chr14:19,704,670-22,817,404 showed

the highest copy number gain consistent with four

copies, which was present on top of trisomy 14 (case

ID3, band 14q11.2). The overlap between the seg-

mental gain in case ID6 and the high copy gain in

case ID3 defines a candidate region at chr14:

22,093,320-22,817,404.

Familial and Sporadic Ileal Carcinoids Reveal

Similar Expression Profiles

To identify gene expression differences leading

to the familial or sporadic variants of ileal carcinoids,

global gene expression profiles were established

using U133Plus2.0 arrays with RNA extracted from

9 tumors (6 sporadic and 3 familial, Table 2). We

first searched for differentially expressed genes

along chromosome 18. Mean expression levels for

transcripts along the whole chromosome were com-

parable in both groups of samples. Only four probes

representing the SYT4, SERPINB5, DOK6 and

CBLN2 genes presented with the absolute mean

fold change in log2 ratio > 1 (Fig. 2C). However,

none of these transcripts were significantly differen-

tially expressed (P-value > 0.7) between the familial

and sporadic tumors. At the genome wide level, only

one probe (212143_s_at), representing IGFBP3 (in-

sulin-like growth factor binding protein 3), was

found differentially expressed between the groups

(P-value ¼ 0.040, log2 fold change �1.93, sporadic

vs. familial). However, the second probe represent-

ing this gene (210095_s_at) was not expressed differ-

entially (P-value ¼ 0.631, log2 fold change �1.70).

Clustering analysis showed that familial and sporadic

cases did not cluster into separate groups (Fig. 2C).

Sequencing-Based Mutation Analysis of

Candidate Genes

In an attempt to identify a gene involved in

the development of familial and sporadic ileal

FAMILIAL AND SPORADIC ILEAL CARCINOIDS 89

Genes, Chromosomes & Cancer DOI 10.1002/gcc

carcinoids, we performed mutation analysis of 18

genes from three candidate regions (Fig. 2C).

The protein encoding sequences of SMAD7,ACAA2, ST8SIA3, ONECUT2, FECH, NARS,ATP8B1, NEDD4L, ALPK2, CDH7, CDH19,DSEL, TXNDC10, CCDC102B, DOK6, CD226,SOCS6, and CBLN2 were studied in 23 sporadic

tumors as well as in patient-matched blood DNA.

The sequencing encompassed � 37 kb target

sequence (175 exons including at least four flak-

ing bases in the splice donor- and acceptor sites),

generating a total of 888 kb of Sanger sequence.

The sequence coverage for the studied genes is

shown in Supporting Information Table 1. Exons

with putative mutations were re-sequenced in tu-

mor and matched normal tissue. However, no tu-

mor-specific mutations were confirmed in these

genes.

Comparison of Patient-Matched Primary Tumors

and Metastases

Sixteen pairs of tumor samples, derived from

the same individual, were profiled. For 14

patients, DNA was isolated from matched pri-

mary and metastatic tumor, and for the remaining

two from two different metastases. Loss of chro-

mosome 18 was seen in all primary tumors and

Figure 3. Familial ileal carcinoid tumors derived from two individ-uals in family G presented with different second hit events. Wholegenome results from samples ID6 and ID7, profiled with Illumina610Q chips are shown. Red arrows indicate chromosome 18. Panels

A and C show Log R ratio (LRR) data from the tumors, while panelsB and D represent B-allele frequency (BAF) values. Sample ID6 dis-played monosomy 18, while ID7 presented with copy number neutralLOH for the entire chromosome 18, without copy number change.

90 CUNNINGHAM ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

matched metastases. A metastasis from case ID30

displayed a regional loss at 18q22.1-q22.2, which

defines a new region on chromosome 18, while

another metastasis from the same patient dis-

played total loss of chromosome 18. The remain-

ing eight pairs showed indistinguishable profiles

in primary and metastatic tumors. In four cases,

both primary tumors and metastases displayed

loss of chromosome 18, as the only detectable

change, further confirming that this aberration is

an early event in the pathogenesis of ileal

carcinoids.

Aberrations additional to those identified in pri-

mary tumors were observed in the metastases of

eight matched pairs. These included mostly gains

of entire chromosomes 4, 5, 7, 14 and/or 20,

which could indicate a tendency towards increas-

ing genomic instability in the metastatic tumors

(Fig. 2D). Interestingly, gain of chromosome 7

was exclusively observed in metastases of the

matched samples. Furthermore, an examination

of the whole tumor series shows that five out of

totally 32 metastases, but none of the 27 primary

tumors, presented with gain of entire chromo-

some 7 (Table 2).

Gain of Chromosome 7 was Related to a Distinct

Morphological Feature

Thirty-two molecularly studied tumors were

morphologically categorized as organoid (small tu-

mor nests surrounded by a highly vascularised

stroma), 11 as solid (large tumor areas with pene-

trating vascular network) and 18 as mixed, with

both patterns present (Fig. 2B). Tumors with

solid morphology presented with higher number

of genomic aberrations. This was found in both

primary tumors (rs 0.6, P < 0.01) and metastases

(rs ¼ 0.8, P < 0.01). Gain of chromosome 7 was

the only aberration with an independent associa-

tion to ‘‘solid’’ tumor morphology (P<0.01).

DISCUSSION

We have investigated a comprehensive series of

sporadic and familial well differentiated serotonin

producing endocrine carcinomas originating in the

small intestine and proximal colon, commonly

known as ileal carcinoids. Nine families were char-

acterized encompassing 23 patients with an inher-

ited disease, making this the largest collection of

hereditary cases described to date. The histopatho-

logical characteristics such as immunoreactivity for

relevant markers and proliferation index are similar

for the familial and sporadic tumors. Furthermore,

the clinical features of familial cases, including age

at disease onset, symptoms, hormone production

and survival are indistinguishable from sporadic

patients. The pattern of inheritance suggests an

autosomal dominant two-hit inherited susceptibility

to ileal carcinoid, first elucidated for retinoblastoma

and further shown as operative for numerous other

inherited tumor diseases (Vogelstein and Kinzler,

2002). For familial ileal carcinoids, the disease pen-

etrance is difficult to evaluate as the disease usu-

ally is diagnosed late in life and the tumors are

seldom recognized before symptoms due to meta-

static disease occur. It is further very likely that

some individuals in the families are carrying clini-

cally silent tumors, as suggested by autopsy studies

(Berge and Linell, 1976). There are previous

reports of families with two or more members

affected with ileal carcinoid tumors (Eschbach and

Rinaldo, 1962; Moertel and Dockerty, 1973; Wale

et al., 1983; Kinova et al., 2001; Pal et al., 2001;

Jarhult et al., 2010). The most recent one describes

a three generation family from Sweden which is

not included in our report (Jarhult et al., 2010).

There are also epidemiological studies supporting

the notion of a familial variant of this disease

(Babovic-Vuksanovic et al., 1999; Hemminki and

Li, 2001). Based on these results and on the results

presented here, we propose that the Familial IlealEndocrine Carcinoma (FIEC) represent a so far

unrecognized inherited tumor disease.

The patients included in this study represent a

carefully selected cohort. All met the WHO crite-

ria for diagnosis and patients with other gastroin-

testinal endocrine tumors (i.e. appendix

carcinoids) were excluded, making this a homoge-

nous group. We have included tumor tissue from

both sporadic and familial cases in all assays; the

results show that there is no obvious difference

in genetic aberrations or gene expression between

the two groups. Chromosome 18 aberrations

detected in both sporadic and familial forms of

TABLE 3. Median Time from Diagnosis to Death or LatestFollow-Up for Patients with and Without Gain of

Chromosome 14

Statusa

No gain ofchromosome 14

Gain ofchromosome 14

NMedian survival

(range in months) NMedian survival

(range in months)

AWD 22 65 (30–159) 11 94 (34–250)DWD 9 103 (7–310) 3 77 (68–194)

N, number of patients; AWD, alive with disease; DWD, dead with

disease.

FAMILIAL AND SPORADIC ILEAL CARCINOIDS 91

Genes, Chromosomes & Cancer DOI 10.1002/gcc

ileal carcinoids (100% vs. 38%, respectively) indi-

cates that these tumor variants share a common

mechanism and strengthens the notion of a gene

involved in tumor development located to this

chromosome. The frequency of chromosome 18

aberration in sporadic tumors was considerably

higher in our study compared with previous

reports (43%-88%) (Kytola et al., 2001; Lollgen

et al., 2001; 2002; Zikusoka et al., 2005; Kulke

et al., 2008). This might be due to strict patient

inclusion criteria and careful dissection of the tis-

sue. To ensure high tumor cell content, we

examined CgA stained 4 lm tissue sections,

taken both before and after dissection of the tu-

mor tissue.

The data regarding chromosome 18 reinforce the

notion that this chromosome contains the primary

ileal carcinoid gene. Our results identified, how-

ever, three minimal overlapping regions of dele-

tions in sporadic cases, mapping to 18q21.1-q21.31,

18q22.1-q22.2 and 18q22.3-q23 (Fig. 2C, Table 2).

Two of these loci are novel and the third one,

mapping to the telomeric end of the q-arm, overlap

with that previously reported by Lollgen et al.

(2001) and Andersson et al. (2009). The fact that

these regions are not overlapping is intriguing and

renders identification of a suppressor gene involved

in ileal carcinoid more difficult. It also points to

the possible involvement of more than one 18q-

located gene in the tumorigenesis. We performed a

comprehensive somatic mutation analysis of 18

genes from three candidate regions but were

unable to observe any tumor-specific sequence var-

iants upon comparison with the patient-matched

blood DNA, which suggest that these 18 genes are

unlikely tumor suppressor candidates. It is note-

worthy in this context that 18q deletions have

been proposed as a molecular predictor of hepatic

metastasis in e.g. colorectal cancer (Tanaka et al.,

2009), which is a very common feature of ileal car-

cinoids. It is, therefore, plausible that chromosome

18 contains more than one gene involved in the

course of the disease of ileal carcinoids; allowing

for an initiating tumor suppressor and a metastasis-

related gene(s). The MEN1 gene on 11q13.1 maps

outside the minimal candidate interval and only

one sporadic tumor in our series (sample ID25)

presented with a deletion encompassing MEN1.This is in agreement with previous studies showing

that MEN1 alterations are rare in ileal carcinoids

(Debelenko et al., 1997; Lollgen et al., 2001).

We have applied two platforms (BAC array and

Illumina SNP beadchips) for characterization of tu-

mor-specific genomic aberrations. This permits an

important validation step of results obtained from

one platform by another methodologically distinct

approach. Furthermore, Illumina beadchips allow

two levels of analysis. In addition to the LRR val-

ues representing fluorescent intensity of probes on

the array, the BAF data displays allelic status of

genotyped SNPs. The BAF-derived results allowed

us to uncover copy number neutral LOH, of an

affected chromosomal segment. This type of aber-

ration is consistent with loss of one parental allele

followed by reduplication of the remaining one.

The findings from family G are particularly impor-

tant as two family members displayed two differ-

ent second hits affecting the putative tumor

suppressor on 18q, one of them being a copy num-

ber neutral LOH. This result is consistent with

segregation of a ‘‘first hit’’ inherited mutation in

the FIEC predisposition gene.

A recent publication suggested that gain of chro-

mosome 14 was associated with short survival for

ileal carcinoid patients; 7 of 8 patients died within

20 months from diagnosis (Andersson et al., 2009).

Since the survival of ileal carcinoid patients has

been reported as considerably longer [median sur-

vival 92 months (Janson et al., 1997)], the gain of

chromosome 14 (Andersson et al., 2009) was sug-

gested as an indicator of poor prognosis and FISH

analysis was proposed to be performed in all ileal

carcinoid tumors. We were, however, not able to

confirm this result. We observed only three death

events in patients with tumor-specific gain of chro-

mosome 14, after 68, 77 and 194 months (Table 2).

The median duration of follow-up in our patients

with chromosome 14 gain was 92 months (range

34-250). Furthermore, we could not demonstrate

difference in overall survival time between patients

with and without chromosome 14 gain. The differ-

ence between the two studies regarding the possi-

ble effect of chromosome 14 gain on survival

merits further investigation. However, we currently

see no indication to incorporate FISH analysis in

the general work-up of ileal carcinoid patients.

In a previous study of ileal carcinoid tumor

morphology a defined morphologic feature, the

solid growth pattern, was shown to be an inde-

pendent risk factor for shorter survival (Cunning-

ham et al., 2007). In the present study this

morphologic feature is correlated to genomic

instability and demonstrates an independent cor-

relation to trisomy 7, which was only present in

metastases. ‘‘Solid’’ morphology is distinguished

from ‘‘organiod’’ by larger tumor cell aggrega-

tions, intratumoral vascular structures and the ab-

sence of palisade cell arrangement. Future

92 CUNNINGHAM ETAL.

Genes, Chromosomes & Cancer DOI 10.1002/gcc

understanding of the role of trisomy 7 in the reg-

ulation of tumor growth pattern may yield impor-

tant insights of prognostic and therapeutic

significance.

In conclusion, similarities between sporadic

and familial cases suggest that FIEC represent a

new autosomal dominant inherited tumor disease.

One major point of our report is raising the

awareness about the existence of a familial vari-

ant of ileal carcinoid disease. Loss of chromosome

18 is, without doubt, a very early event in the de-

velopment of both familial and sporadic tumors

and the presented evidence suggests a shared

pathogenetic mechanism, while gain of chromo-

some 7 seems to correlate to a change in growth

pattern. However, the actual mutated gene(s)

that initiates tumor development still remains to

be identified. Based on our clinical findings we

propose that patients presenting with abdominal

pain or hormone-related symptoms and a positive

family history of ileal carcinoid should be investi-

gated on a wide indication for biochemical and

radiological signs of such tumors.

ACKNOWLEDGMENTS

The authors thank Dr. Carl Bruder and Dr.

Johanna Sandgren for producing the BAC arrays

and Monica Lindman and Daniel Moreno Bergg-

ren for performing the sequence analysis. The

Illumina genotyping was performed at the SNP

Technology Platform, Uppsala, Sweden (www.ge-

notyping.se), supported by Uppsala University

and the Knut and Alice Wallenberg foundation.

The Affymetrix expression analysis was per-

formed at the Uppsala Array Platform

(www.medsci.uu.se/klinfarm/arrayplatform/).

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Genes, Chromosomes & Cancer DOI 10.1002/gcc