Early HER2 dysregulation in gastric and oesophagealcarcinogenesis

Matteo Fassan,1,2,* Luca Mastracci,3,* Federica Grillo,3 Vittorina Zagonel,4 Sara Bruno,3

Giorgio Battaglia,4 Francesca Pitto,3 Donato Nitti,2 Tiziana Celiento,3 Giovanni Zaninotto,5

Roberto Fiocca3 & Massimo Rugge1,4

1Surgical Pathology & Cytopathology Unit, Department of Medicine (DIMED), University of Padua, Padua,2General Oncology & Surgery Unit, Department of Surgical Oncology and Gastroenterology Sciences (DiSCOG), University

of Padua, Padua, 3Department of Anatomical Pathology, University of Genoa and S. Martino University Hospital, Genoa,4Istituto Oncologico Veneto, IOV-IRCCS, Padua, and 5Gastroenterology Unit, Department of Surgical Oncology and

Gastroenterology Sciences (DiSCOG), University of Padua, Padua, Italy

Date of submission 15 December 2011Accepted for publication 12 March 2012

Fassan M, Mastracci L, Grillo F, Zagonel V, Bruno S, Battaglia G, Pitto F, Nitti D, Celiento T, Zaninotto G, Fiocca R &

Rugge M

(2012) Histopathology

Early HER2 dysregulation in gastric and oesophageal carcinogenesis

Aims: To explore human epidermal growth factorreceptor 2 (HER2) status in the histological phenotypes[metaplasia, intraepithelial neoplasia (IEN, i.e. dyspla-sia), and adenocarcinoma] involved in the morpho-genesis of both intestinal-type gastric cancer (GC) andBarrett’s adenocarcinoma (BAc).Methods and results: A consecutive series of 275samples of stomach and oesophagus tissue (representingthe whole spectrum of the phenotypic changes involvedin gastric and Barrett’s carcinogenesis) was studied.HER2 status was assessed by applying two immunohis-tochemistry (IHC) protocols, using the antibodies 4B5and CB11. Dual-colour silver chromogenic in-situhybridization (SISH) was also performed on the sametissue samples. In both oesophageal and gastric samples,the rate of HER2 overexpression rose significantlyfrom low-grade to high-grade IEN to adenocarcinoma

(P < 0.001), with the two IHC protocols showingconsistent staining (consistency 95%; k = 0.78;P < 0.001). Intratumour heterogeneity was docu-mented in both GC and BAc (using both IHC protocols).The rate of HER2 amplification (using SISH) increasedsignificantly along with IEN dedifferentiation (P <0.001). Neither native nor metaplastic mucosa samples(obtained from either stomach or oesophagus) evershowed HER2 amplification. There was excellentagreement between HER2 amplification and proteinoverexpression (both IHC protocols: SISH ⁄ 4B5––consis-tency 97.8%, k = 0.89, P < 0.001; SISH ⁄ CB11—consistency 97.8%, k = 0.91, P < 0.001).Conclusions: There is early involvement of HER2dysregulation (amplification and protein overexpres-sion) in both gastric (intestinal-type) and Barrett’soncogenesis.

Keywords: carcinogenesis, dysplasia, HER2, immunohistochemistry, SISH

Abbreviations: BAc, Barrett’s adenocarcinoma; CEP, chromosome 17 enumeration probe; FISH, fluorescencein-situ hybridization; G ⁄ HG-IEN, gastric high-grade intraepithelial neoplasia; G ⁄ IM, gastric intestinal metaplasia;G ⁄ LG-IEN, gastric low-grade intraepithelial neoplasia; GC, gastric cancer; HER2, human epithelial growth factorreceptor 2; HG-IEN, high-grade intraepithelial neoplasia; IEN, intraepithelial neoplasia ⁄ dysplasia; IHC, immuno-histochemistry; IM, intestinal metaplasia; ISH, in-situ hybridization; LG-IEN, low-grade intraepithelial neoplasia;

Address for correspondence: M Rugge, Surgical Pathology & Cytopathology Unit, Department of Medicine (DIMED), University of Padua, Istituto

Oncologico Veneto-IRCCS, 61 Via Aristide Gabelli, 35121 Padua, Italy. e-mail: massimo.rugge@unipd.it

*These authors contributed equally to this work.

� 2012 Blackwell Publishing Limited.

Histopathology 2012 DOI: 10.1111/j.1365-2559.2012.04272.x

O ⁄ GM, oesophageal gastric cardiac-type metaplasia; O ⁄ HG-IEN, oesophageal high-grade intraepithelialneoplasia; O ⁄ IM, oesophageal intestinal metaplasia; O ⁄ LG-IEN, gastric low-grade intraepithelial neoplasia;SISH, silver in-situ hybridization

Introduction

Intestinal-type gastric cancer (GC) and Barrett’sadenocarcinoma (BAc) both result from the progressiveaccumulation of genetic dysregulations combined withphenotype dedifferentiation. Both conditions are alsotriggered primarily by longstanding inflammation,resulting in the native mucosa being replaced bymetaplastic epithelia (gastric intestinalization andcolumnar Barrett’s metaplasia).1,2 Intestinal metapla-sia (IM), in particular, has been consistently establishedas the ‘carcinogenic field’ in which intraepithelialneoplasia [IEN, formerly named dysplasia; be it low-grade (LG-IEN) or high-grade (HG-IEN)] can develop,possibly progressing to invasive adenocarcinoma.3–9

Although the ‘cascade’ of phenotypic changesleading from native epithelia to full-blown cancer hasbeen thoroughly described, the molecular eventsunderlying the neoplastic transformation(s) have yetto be clarified in detail.10,11

The human epidermal growth factor receptor 2proto-oncogene (HER2; c-erbB2) encodes a tyrosinekinase receptor, which is consistently overexpressed inseveral cancers (including GC and BAc).12–25 It is onthese grounds that a molecularly targeted therapyagainst HER2 has been proposed. The recent Trast-uzumab for Gastric Adenocarcinoma (ToGA) trialsuccessfully applied anti-HER2 therapy (i.e. trast-uzumab) in advanced gastro-oesophageal cancers,and this strategy has become the standard option foradvanced, HER2-positive gastric or gastro-oesophagealadenocarcinomas.12 Only fragmentary information isavailable, however, on HER2 gene amplificationand ⁄ or protein overexpression in each of the histolog-ical lesions involved in the spectrum of gastric andoesophageal carcinogenesis.14–16,19,25

On the basis of the clinicobiological similaritiesbetween gastric and Barrett’s adenocarcinomas, thisstudy systematically assessed HER2 status in each of thephenotypic alterations involved in (intestinal-type) gas-tric and (Barrett’s) oesophageal carcinogenesis. Withthis aim, two different immunohistochemistry (IHC)protocols and silver chromogenic in-situ hybridization(SISH) were applied to a consecutive series of 275 biopsysamples representing the whole spectrum of the pheno-typic changes encountered in both (intestinal-type)gastric and (Barrett’s) oesophageal carcinogenesis.

Materials and methods

tissue samples

The cases considered in this study were retrospectivelycollected from the files of the Surgical Pathology andCytopathology Unit at the University of Padova andfrom the Veneto Region’s multicentre Barrett’s Esoph-agus Registry (EBRA, Padova Unit; http://www.esofagodibarrett.it).7 The institutes’ ethical regulations onresearch conducted on human tissues were followed.

The study was conducted on a total of 275 endoscopicbiopsy samples (obtained from different biopsy sets). Forgastric oncogenesis, the tissue samples (all obtained fromthe distal gastric mucosa, i.e. antrum and ⁄ or incisuraangularis) included: (i) 25 biopsies of extensive intestinalmetaplasia of the distal gastric mucosa [(G ⁄ IM), obtainedfrom cases of atrophic gastritis in Operative Link onGastritis Assessment (OLGA) stages III–IV];1,5,26 (ii) 25cases of gastric low-grade IEN (i.e. low-grade dysplasia;G ⁄ LG-IEN); (iii) 25 cases of gastric high-grade IEN (i.e.high-grade dysplasia; G ⁄ HG-IEN); and (iv) 25 cases ofwell-differentiated or moderately differentiated intesti-nal-type distal adenocarcinoma (GC; all pT1 ⁄ T2 cases)(Figure 1). 27 For Barrett’s oncogenesis, representativetissue samples were all obtained from long-segmentBarrett’s oesophagus, and included: (i) 25 cases of non-intestinal columnar metaplasia (gastric cardiac-type;O ⁄ GM); (ii) 25 cases of oesophageal intestinal metaplasia(O ⁄ IM); (iii) 25 cases of low-grade IEN (O ⁄ LG-IEN); (iv)25 cases of high-grade IEN (O ⁄ HG-IEN); and (v) 25 casesof well-differentiated or moderately differentiated BAc (allpT1 ⁄ pT2 cases) (Figure 1).27

Biopsy samples of normal gastric antral mucosa (25cases) and normal squamous oesophageal mucosa (25cases) obtained from dyspeptic patients were used asnormal controls.

immunohistochemistry

In each case, IHC was performed by applying twocommercial immunostaining protocols, according tothe manufacturers’ recommendations:28,29 (i) PATH-WAY HER-2 ⁄ neu (4B5) rabbit monoclonal antibody(Ventana Medical Systems, Milan, Italy), using theautomated Benchmark XT platform (Ventana MedicalSystems); and (ii) the ORACLE HER2 Bond IHC system(CB11) mouse monoclonal antibody (Menarini Diag-

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� 2012 Blackwell Publishing Ltd, Histopathology

nostics, Florence, Italy), using the automated LeicaMicrosystems Bondmax (Leica, Wetzlar, Germany).

her2 dual-colour silver-enhanced in-s itu

hybridization assay

HER2 amplification status was tested using SISH, asdescribed elsewhere.18,30 Briefly, HER2 and the chro-mosome 17 enumeration probe (CEP) were tested on thesame slide, using bright-field automated in-situ hybrid-ization (ISH) and the Ventana HER2 dual-colour ISHassay (Ventana Medical Systems). Automated stainingwas performed using the Benchmark XT platform(Ventana Medical Systems). The assay protocol con-sisted of an extended pretreatment with the citrate bufferCell Conditioning 2 (Ventana Medical Systems), pH 6.0,followed by protein digestion with ISH-protease 3 for12 min, and then incubation with specific dinitrophe-nyl-labelled DNA probes for 6 h. The ultraView SISHDetection kit and accessory reagents (Ventana MedicalSystems) were used; nuclear silver precipitation in

single copies of HER2 results in single black dots; singlecopies of chromosome 17 CEP are featured as red dots.The slides were counterstained with haematoxylin IIand a bluing reagent. Samples with known HER2amplification (one breast cancer and one oesophagealadenocarcinoma) were used as positive controls.

ihc and sish scoring

Both IHC tests were assessed jointly by three pathologists(M.F., L.M., and M.R.) blinded to any clinicopathologicalinformation. HER2 expression was scored according tothe four-tiered Herceptest, as modified for gastric ade-nocarcinoma as follows:13,18 score 0 (negative), absenceof any stain or membrane staining; score 1+ (negative),tumour cell cluster with faint or barely perceptiblemembrane reactivity irrespective of percentage oftumour cells stained; score 2+ (equivocal), tumour cellcluster with weak to moderate (complete, lateral, orbasolateral) reactivity irrespective of percentage oftumour cells stained; score 3+ (positive), tumour cell

A B C

D E F

Figure 1. Preneoplastic lesions of the gastric and oesophageal mucosa: gastric intestinal metaplasia (A), gastric low-grade intraepithelial

neoplasia (IEN) (B), gastric high-grade IEN (C), Barrett’s mucosa (D), oesophageal low-grade IEN (E), and oesophageal high-grade IEN (F).

HER2 status in gastro-oesophageal carcinogenesis 3

� 2012 Blackwell Publishing Ltd, Histopathology

cluster with moderate to strong (complete, lateral, orbasolateral) reactivity irrespective of percentage oftumour cells stained. For scoring purposes, any (nuclearor cytoplasmic) background staining was disregarded.

The SISH results were assessed on a Cyres system(Zeiss, Jena, Germany) consisting of a conventionaloptical microscope connected to a monitor via a 3CCDcolour camera (JVC, Tokyo, Japan). The serial slidesstained with haematoxylin and eosin and 4B5 antibodywere re-examined before ISH, and areas containing thehighest HER2 counts were preferentially assessed,counting HER2 and chromosome 17 CEP in at least 40nuclei. Only nuclei with a distinct nuclear border wereconsidered; overlapping nuclei were always excluded.The ratio of HER2 to CEP was calculated, and HER2 wasconsidered to be amplified when the ratio of gene-specificHER2 to CEP signals was ‡2.0, or when there wasevidence of HER2 signal clusters.13,18,19

statistical analysis

Differences and correlations between groups were testedwith the modified Kruskal–Wallis non-parametric testfor trend. The k-coefficient was used to check the level ofagreement between samples for HER2 staining obtained

with 4B5 versus CB11, and HER2 status established byIHC or SISH. The k-values, ranging from 0.61 to 0.80,were assumed to indicate very good agreement. P-values<0.05 were considered to be statistically significant.Statistical analysis was performed with stata software(Stata Corporation, College Station, TX, USA).

Results

immunohistochemistry

Excellent agreement was observed in the 275 samplesanalysed between the staining obtained using 4B5 andthat using CB11 (agreement 95.3%; k = 0.78;P < 0.001), with only 13 ⁄ 275 discrepancies (Table 1).A non-membranous staining pattern was occasionallyobserved with both antibodies (in normal and non-dysplastic surrounding tissues too), but was disre-garded for scoring purposes.

HER2 overexpression (2+ and 3+) was also apparent(to a lesser degree than in adenocarcinomas) in LG-IENand HG-IEN in both gastric and oesophageal tissuesamples (Table 1; Figures 2 and 3). The rate of thisoverexpression increased significantly with increasingdedifferentiation of the preneoplastic lesions (from LG-

Table 1. HER2 status tested by immunohistochemistry (using two different antibodies) and dual-colour silver in-situhybridization (SISH) in gastro-oesophageal carcinogenesis

Model Samples

PATHWAYHER-2 ⁄ neu (4B5), no. (%) Oracle HER2 Bond (CB11), no. (%)SISH-positive (%)0 1+ 2+ 3+ 0 1+ 2+ 3+

Gastriccarcinogenesis(intestinal type)

G ⁄ N 25 (100) 0 (0) 0 (0) 0 (0) 25 (100) 0 (0) 0 (0) 0 (0) 0 (0)

G ⁄ IM 22 (88) 3 (12) 0 (0) 0 (0) 24 (96) 1 (4) 0 (0) 0 (0) 0 (0)

G ⁄ LG-IEN 20 (80) 3 (12) 1 (4) 1 (4) 21 (84) 2 (8) 1 (4) 1 (4) 1 (4)

G ⁄ HG-IEN 16 (64) 4 (16) 2 (8) 3 (12) 17 (68) 4 (16) 1 (4) 3 (12) 4 (16)

GC 11 (44) 6 (24) 3 (12) 5 (20) 12 (48) 5 (20) 3 (12) 5 (20) 8 (32)

Barrett’scarcinogenesis

O ⁄ N 25 (100) 0 (0) 0 (0) 0 (0) 25 (100) 0 (0) 0 (0) 0 (0) 0 (0)

O ⁄ GM 25 (100) 0 (0) 0 (0) 0 (0) 25 (100) 0 (0) 0 (0) 0 (0) 0 (0)

O ⁄ IM 23 (92) 2 (8) 0 (0) 0 (0) 25 (100) 0 (0) 0 (0) 0 (0) 0 (0)

O ⁄ LG-IEN 18 (72) 3 (12) 3 (12) 1 (4) 19 (76) 3 (12) 2 (8) 1 (4) 2 (8)

O ⁄ HG-IEN 15 (60) 3 (12) 3 (12) 4 (16) 17 (68) 1 (4) 3 (12) 4 (16) 5 (20)

BAc 13 (52) 4 (16) 2 (8) 6 (24) 14 (56) 4 (16) 2 (8) 5 (20) 7 (28)

BAc, Barrett’s adenocarcinoma; GC, gastric adenocarcinoma; G ⁄ HG-IEN, gastric high-grade intraepithelial neoplasia; G ⁄ IM,gastric intestinal metaplasia; G ⁄ LG-IEN, gastric low-grade intraepithelial neoplasia; G ⁄ N, normal gastric antral mucosa; O ⁄ GM,oesophageal gastric cardiac-type metaplasia; O ⁄ HG-IEN, oesophageal high-grade intraepithelial neoplasia; O ⁄ IM, oesophagealintestinal metaplasia; O ⁄ LG-IEN, oesophageal low-grade intraepithelial neoplasia; O ⁄ N, normal squamous oesophageal mucosa.

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IEN to HG-IEN to adenocarcinoma; Kruskal–Wallis,P < 0.001 for both antibodies, and for the models ofboth gastric and oesophageal carcinogenesis).

As observed previously for both gastric and oesoph-ageal adenocarcinomas,18 there was marked intratu-mour heterogeneity in five cases of HG-IEN, whicheverantibody was used (Figure 3C).

her2 A M P L I F I C A T I O N

HER2 amplification as determined by dual-colour SISHwas observed in one case of G ⁄ LG-IEN, four cases ofG ⁄ HG-IEN, eight cases of GC, two cases of O ⁄ LG-IEN,five cases of O ⁄ HG-IEN, and seven cases of BAc(Table 1; Figure 4). No HER2 amplification was appar-ent in the native gastric and oesophageal mucosasamples, or in the metaplastic lesions (G ⁄ IM, O ⁄ GM,and O ⁄ IM). The HER2 amplification rates rose signif-icantly from LG-IEN to HG-IEN to adenocarcinoma(Kruskal–Wallis, P < 0.001 for both gastric andoesophageal carcinogenesis). There was an excellentagreement between the SISH results and HER2 proteinoverexpression (2+ and 3+) assessed using both 4B5(agreement 97.8%; k = 0.89; P < 0.001) and CB11(agreement 98.5%; k = 0.91; P < 0.001). It is worthnoting that all cases scoring 3+ with both antibodiesalso featured HER2 amplification.

Discussion

Overexpression of HER2 and amplification of HER2 ⁄c-erbB2 are hallmarks of human cancer, includinggastric and oesophageal adenocarcinomas, amongwhich HER2 is dysregulated in 7–34% of primarytumours.13–25 It is on these grounds that anti-HER2therapy (i.e. trastuzumab in combination with tradi-

tional chemotherapy) is currently recommended as abiologically targeted therapy for gastric and gastro-oesophageal cancers overexpressing HER2.12

Few studies have reported on the up-regulationof HER2 in gastro-oesophageal dysplasticlesions,14–16,19,25 and none has systematically explored(on paired biopsy samples) protein expression and ⁄ orgene amplification in the whole spectrum of phenotypicchanges occurring in the carcinogenic cascades leadingto intestinal-type GC and BAc. This study assessed HER2status by using both IHC (two different anti-HER2antibodies: 4B5 and CB11) and SISH in a large series ofbiopsy samples covering all of the phenotypic stepsinvolved in gastric and oesophageal carcinogenesis.

HER2 overexpression (2+ and 3+) was seen in LG-IENand HG-IEN (both gastric and oesophageal), andincreased significantly from LG-IEN to HG-IEN, and toadenocarcinoma (with no significant differences inprotein expression between the two applied antibodies).The results obtained provide unequivocal evidence forthe early involvement of HER2 dysregulation in theneoplastic transformation of both gastric and oesopha-geal metaplastic mucosa. Villanacci14–16 and Lee25

found a similar picture in high-grade dysplastic lesions(in the oesophageal and gastric settings, respectively). Noprospective studies are currently available that havetested the timing of HER2 dysregulation in the ‘cascade’of the phenotypic alterations included in the single-cancer natural history (from intraepithelial to invasiveneoplasia). Such information could theoretically con-tribute to clarifying the biological background of theintratumour variability affecting both oesophageal andgastric adenocarcinoma. The information obtainedpostpones HER2 dysregulation to the ‘stage’ ofmetaplastic transformation (in both oesophageal andgastric settings); moreover, the heterogeneous HER2

B CA

Figure 2. HER2 is overexpressed in gastric and oesophageal low-grade dysplasia. A,B, HER2 overexpression (3+) in a case of oesophageal

low-grade dysplasia. Note the concordance between the two different antibodies. C, A case of HER2 3+ in low-grade dysplasia within

the gastric mucosa. All cases showed HER2 amplification on silver in-situ hybridization analysis. A,C, Stained with 4B5. B, Stained with CB11.

HER2 status in gastro-oesophageal carcinogenesis 5

� 2012 Blackwell Publishing Ltd, Histopathology

status demonstrated in IEN suggests that the moleculardysregulation may (clonally) affect only a subset ofintraepithelial neoplastic cell populations. Tumourheterogeneity represents the biological rationale forassessing HER2 status in multiple tissue samples (theItalian Division of the International Academy of Pathol-ogy recommends the evaluation of at least six tumourbiopsies).

The results obtained further support the theory thata non-negligible subset of both oesophageal and gastricHG-IENs feature HER2 overexpression and HER2amplification (2+ and 3+ = from 15% to 20%): this

should be considered in the assessment of HER2 statusin those endoscopy samples in which intraepithelialand invasive neoplasia coexist (Figure 3D).

The reliability of the assessment method (togetherwith sample-dependent heterogeneity) is a key issue inselecting patients eligible for anti-HER2 therapy.18

Bearing this in mind, the use of SISH (with itsundoubted benefit of bright-light assessment) insteadof dark field fluorescence in-situ hybridization (FISH)offers significant advantages in morphologically char-acterizing which cell population (neoplastic non-inva-sive versus invasive) exhibits HER2 dysregulation.

A B

C D

Figure 3. HER2 is overexpressed in gastric and oesophageal high-grade dysplasia ⁄ adenocarcinoma. A–C, Three representative cases of

oesophageal (A) and gastric (B,C) high-grade dysplasia samples. In (C), note the intralesional heterogeneity (3+ pattern coexisting with 1+

staining). D, An HER2 1+ oesophageal adenocarcinoma enclosed by HER2 3+ dysplastic epithelia. All 3+ cases showed HER2 amplification

on silver in-situ hybridization analysis. A, Stained with 4B5. B–D, Stained with CB11.

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Moreover, the diagnostic efficiency of SISH (particu-larly the dual-colour technique) could be usefullyexploited in the routine assessment of small biopsysamples to save irreplaceable material for any furtherdiagnostic and ⁄ or research testing. Further studiesshould test SISH reliability in the selection of patientsfor HER2-targeted therapy, and SISH results in com-parison with the different applied antibodies.

In conclusion, the present study provides unequiv-ocal evidence of an early role for HER2 in thegastric ⁄ oesophageal mucosa’s acquisition of the neo-plastic phenotype. From a technical viewpoint (whichis not a minor issue), only trivial discrepancies havebeen shown with the application of different IHC

protocols; additionally, substantial consistency wasshown between IHC and ISH procedures. In view ofthe (possible) HER2 dysregulation in IEN (frequentlycoexisting with invasive cancer in biopsy specimens),SISH bright light (instead of FISH) provides morereliable information on HER2 status in the invasivecancer cell population targeted by biological therapy.

Acknowledgements

We wish to acknowledge the continuous support of the‘G. Berlucchi’ and the ‘G. B. Morgagni’ Foundationsand of the EBRA registry. This work was partlysupported by a grant from the Italian Association for

A B

C D

Figure 4. HER2 is amplified in gastric and oesophageal low-grade and high-grade dysplasia. A, Normal HER2 copy number in a case of low-

grade dysplasia of the oesophagus. B–D, Silver in-situ hybridization analysis showed HER2 amplification in the HER2 3+ areas of low-grade

gastric dysplasia (B), and high-grade oesophageal (C) and gastric (D) dysplasia.

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Cancer research (AIRC Regional grant 2008). We alsothank Dr Mariangela Balistreri, Dr Vincenza Guzzardo,Dr Marco Pizzi, Dr Cristiano Lanza and Dr VanniLazzarin for their excellent technical support. Dr Fassanis a paid consultant for Menarini Diagnostics s.r.l. onmatters of diagnostic testing.

Author contributions

All authors of this research paper participated directlyin the planning and execution of the study, and in theanalysis of the results.

Conflict of interest

The authors have no competing interests to declare.

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