Diseases of the Esophagus (2015) 28, 380–385 DOI: 10.1111/dote.12204
Original article
Human epidermal growth factor receptor 2 overexpression and amplification in endoscopic biopsies and resection specimens in esophageal and junctional adenocarcinoma P. van Hagen,1 K. Biermann,2 J. E. Boers,3 O. Stoss,4 H. F. Sleddens,2 J. J. B. van Lanschot,1 W. N. M. Dinjens,2 J. Rueschoff,4,5 B. P. L. Wijnhoven1 Department of Surgery, 2Department of Pathology, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, 3Department of Pathology, Isala Clinics, Zwolle, The Netherlands; and 4TARGOS Molecular Pathology GmbH, and 5Institute of Pathology, Klinikum Kassel, Kassel, Germany 1
SUMMARY. Human epidermal growth factor receptor 2 (HER2) is overexpressed in a subset of esophageal adenocarcinomas. Frequently, biopsy material is used for evaluation of HER2 status. The aim of the study was to determine if HER2 expression in preoperative endoscopic biopsies is representative for the entire tumor. Preoperative endoscopic biopsies and matched resection specimens were collected from 75 patients who underwent esophagectomy for esophageal adenocarcinoma. Immunohistochemical staining (IHC) on HER2 and dual-color in situ hybridization (ISH) were performed. HER2 status was determined by following a clinical algorithm, first determining HER2 overexpression on immunohistochemistry and, when equivocal (2+), determining HER2 amplification on ISH. Seventy-one of 75 (95%) biopsies and 69/75 (92%) resection specimens could be analyzed due to technical failure. HER2 positivity was seen in 18/71 (25%) biopsies and in 15/69 (22%) resection specimens. Overall, HER2 status in the biopsy was concordant with HER2 status in the resection specimen in 94% of cases. Interobserver agreement on IHC scoring for all three observers was 83% in biopsies and 85% in resection specimens. HER2 positivity was detected in 22% of esophageal adenocarcinomas. Although interobserver agreement was moderate, HER2 status of a primary tumor can be reliably determined based on the endoscopically obtained pretreatment biopsy. KEY WORDS: esophageal adenocarcinoma, HER2, protein expression.
INTRODUCTION Cancer of the esophagus is the sixth cause of cancer death worldwide. It is a highly lethal disease in which only one third of patients have a resectable tumor at the time of presentation. Surgery with neoadjuvant chemo(radio)therapy offers the best chances for long-term survival.1 However, still half of patients suffer from recurrence of disease after multimodality therapy.2–4 Clearly, new treatments are needed to improve the outcome of patients with esophageal cancer. Address correspondence to: Dr Bas P.L. Wijnhoven, MD, PhD, Erasmus MC University Medical Centre Rotterdam, PO Box 2040, Rotterdam 3000 CA, The Netherlands. Email: [email protected] Disclosure: This study was financially supported by an unrestricted grant from Roche b.v., Woerden, the Netherlands. 380
The human epidermal growth factor receptor 2 (HER2) regulates cell growth and proliferation. Overexpression of HER2 has been identified in a wide variety of carcinomas, including esophageal cancer. HER2 overexpression correlates with advanced tumor stage and poor survival.5–9 Trastuzumab, a monoclonal antibody against the HER2 receptor, inhibits the receptor activity and oncogenic downstream targets.10 In HER2-positive breast cancer, trastuzumab improves overall and disease-free survival in early and metastatic disease.11 Overexpression of HER2 is not uncommon in esophageal cancer with up to 43% of the cancers being HER2-positive.10,12–17 Hence, trastuzumab could well be an effective drug in the multimodal treatment of esophageal cancer.18 In metastatic gastric cancer, the addition of trastuzumab to standard palliative chemotherapy has proved to be of benefit.19 © 2014 International Society for Diseases of the Esophagus
HER2 expression in esophageal cancer
Trastuzumab is only effective in cancer cells overexpressing HER2 and/or cells that show HER2 gene amplification.6 The HER2 status is evaluated by immunohistochemical staining and HER2 gene amplification by in situ hybridization (ISH).20 Abundant tumor tissue is available for analysis from the resection specimen. However, preoperative biopsies from esophageal cancer are limited in size. Moreover, expression of oncogenes and tumor suppressor genes in adenocarcinomas of the upper gastrointestinal tract are well known to be heterogeneous that questions the representativeness of a small tissue fragment taken at endoscopy.21 This might lead to undertreatment of patients in whom HER2 overexpression/ amplification was falsely scored negative. On the other hand, false-positive biopsies may lead to an increase of toxicity and costs, and moreover, inadequate prognostication with a potential negative impact on clinical decision-making. The aims of the present study were to determine if the HER2 status of an esophageal adenocarcinoma can be reliably determined based on endoscopic biopsies and to assess the interobserver agreement in determining HER status.
METHODS Patients and samples Patients who had undergone an esophagectomy for adenocarcinoma of the esophagus or OGJ between January 2000 to January 2010 in the Erasmus MC, Rotterdam, the Netherlands were identified from the institutional database. Seventy-five patients with a cT2-cT3 tumor were selected. Excluded were cT1 and cT4 tumors, and patients who underwent any form of neoadjuvant or adjuvant chemo(radio)therapy. cT1 tumors were excluded because of the limited additional effect of neoadjuvant therapy targeted at Her2 that can be expected after resection in this group of tumors that already has a relatively high survival rate after resection. cT4 tumors were excluded because in our center, cT4 tumors are not resected but are treated with definitive chemoradiotherapy, after which no resection specimen is available to compare with the pretreatment biopsies. Also, patients with incomplete or inadequate archived tumor tissue (either in biopsies or the surgical resection specimen) were excluded. Patients were selected consecutively whenever possible. Tissue blocks of the endoscopic biopsies, routinely taken from multiple areas of the tumor, as well as the resection specimen for each patient were retrieved. All tissue samples had been fixed using 4% buffered formalin and embedded in paraffin after dehydration. All biopsies and a representative area of invasive tumor of the resection specimen that was identified using a hematoxylin and eosin (H&E) stain. Subse© 2014 International Society for Diseases of the Esophagus
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quently, 4-μm sections were cut from the tissue blocks and were used for immunohistochemistry (IHC) and ISH. Several patient and tumor characteristics were identified including patients’ age (at the time of diagnosis), sex and tumor stage according to the Union for International Cancer Control TNM, 6th edition (as determined by endoultrasonography and computed tomography scanning of the chest and abdomen, or from pathology report when clinical investigations were not conclusive).22 The study was reviewed and approved by the local medical ethics committee, and the requirement of obtaining patients’ informed consent was waived. HER2 IHC and dual-color ISH The 4-μm sections were mounted on starfrost slides (Knittel Gläser, Braunschweig, Germany) and dried. The immunohistochemical staining was performed using a BenchMark ULTRA automated slide staining instrument (Ventana Medical Systems, Tucson, AZ, USA) and using the PATHWAY HER2 4B5 antibody, according to the manufacturer’s instructions. ISH staining was also performed fully automated with the BenchMark ULTRA, with the INFORM HER2 DNA probe and the Red ISH V probe (both Ventana Medical Systems). Evaluation of HER2 status HER2 expression and amplification was independently assessed by three experienced pathologists (KB, JEB and JR) from three institutions (Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The Netherlands; Isala Clinics Zwolle, The Netherlands; and TARGOS Molecular Pathology, Kassel, Germany). The pathologists were blinded for the patients. Biopsies and resection specimens were rotating among the centers in three batches making sure that matching biopsies and resection specimens were never present at the same location simultaneously. The H&E staining, IHC, and ISH results of the tissues from a single patient’s biopsy or resection specimens were at the disposal of the pathologist. For determining the HER2 status, an algorithm was used (Fig 1). In case of no or weak HER2 expression (1+), the tumor is scored as HER2-negative, and in case of strong expression (3+), the tumor is scored as HER2-positive. In case of moderate expression (2+), the results of ISH are evaluated, and the HER2 status is determined based on the absence (negative) or presence (positive) of HER2 amplification (Fig 2). Evaluation of the IHC for HER2 was done according to the scoring system for gastric and OGJ cancer.23 In brief, HER2 expression in the resection specimen was scored as 2+ when ≥10% of tumor cells showed a weak to moderate complete or basolateral
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Diseases of the Esophagus Table 1 Patient and tumor characteristics of 75 patients with esophageal or esophagogastric junction adenocarcinoma Characteristics
Fig. 1 Algorithm for the assessment of human epidermal growth factor receptor 2 (HER2) status. Intensity of reactivity: 0–1+ = absent–faint; 2+ = moderate; 3+ = strong. HER2 : CEP17, HER2 and centromeric probe 17 ratio; IHC, immunohistochemistry; ISH: dual color-silver in situ hybridization.
Gender (n, %) Male Female Age (years) Mean (SD) Tumor differentiation (n, %) Good Moderate Poor Missing Clinical T-stage (n, %)† cT2 cT3 No endoscopic pass Clinical N-stage (n, %)‡ N0 N1 Missing
— 56 (75%) 19 (25%) — 63 (11) — 3 ( 4%) 29 (39%) 42 (56%) 1 (1%) — 20 (27%) 48 (64%) 7 (9%) — 23 (31%) 45 (60%) 7 (9%)
†Clinical T-stage: T-stage as defined by endoultrasonography and/or CT scanning according to UICC TNM Cancer Staging, 6th edition. ‡Clinical N-stage: clinical N-stage as defined by endoultrasonography and/or CT scanning and/or FDG-PET according to UICC TNM Cancer Staging, 6th edition. CT, computed tomography; FDG-PET, fluorodeoxyglucose-positron emission tomography; SD, standard deviation; UICC, Union for International Cancer Control.
membranous reactivity. For preoperative biopsies, the presence of cohesive IHC 3+ and/or ISH+ clones resulted in a positive HER2 status irrespective of size.23 For ISH evaluation, the HER2 : centromeric probe 17 ratio was determined. A sample was considered positive for HER2 amplification when this ratio was >2.2. Statistical analysis HER2 status was defined as positive or negative.23 The statistical analysis was performed using Statistical Package for Social Sciences version 17.0 (SPSS, Inc., Chicago, IL, USA).
RESULTS Patients A total of 75 patients were selected who met the inclusion criteria. The male : female ratio was 3 : 1, and the mean age was 63 years. Forty-eight patients (64%) had a cT3 tumor, and 45 (60%) patients were staged as node positive (Table 1). Fig. 2 Hematoxylin and eosin (A, B), immunohistochemistry (Aa, Ba) and in situ hybridization (ISH) (Ab, Bb) in two different invasive esophageal adenocarcinomas. In (A), the adenocarcinoma is human epidermal growth factor receptor 2 (HER2)-negative in IHC, and correspondingly, there is no amplification of HER2 gene in ISH. In contrast, adenocarcinoma in (B) shows moderate HER2 expression (2+), and the corresponding ISH reveals HER2 amplification in some but not all tumor cells.
Biopsies A mean of five biopsies (standard deviation ±2) per patient were collected. The biopsies of four (5%) patients could not be evaluated for HER2 expression by IHC. One biopsy was identified as squamous cell carcinoma, and in the three remaining biopsies, the IHC © 2014 International Society for Diseases of the Esophagus
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Table 2 Conventional immunohistochemistry scoring and results with the application of a clinical algorithm† of 71 evaluable biopsies and 69 evaluable resection specimens of esophageal adenocarcinoma IHC
Biopsies (n = 71) Resection specimens (n = 69)
After following algorithm
0–1+
2+
3+
Neg
Pos
NE
52 (73%) 53 (78%)
3 (4%) 3 (4%)
16 (23%) 13 (19%)
53 (75%) 53 (77%)
18 (25%) 15 (22%)
0 (0%) 1 (1%)
†Clinical algorithm: IHC 0–1+: HER2-negative; IHC 2 + → HER2/centromere17 ratio on ISH 2.2: HER2-positive; IHC3+: HER2-positive. Intensity of reactivity, 0–1+: absent–faint; 2+: moderate; 3+: strong. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; NE, not evaluable; Neg, negative; Pos, positive.
failed. In 16/71 (23%) patients, expression of HER2 by IHC was 3+; in 3/71 (4%) patients, this was 2+ . Two of these biopsies showed HER amplification. Hence, 18/71 (25%) biopsies were HER2-positive. (Table 2). Resection specimens Six (8%) patients were excluded; there was one squamous cell carcinoma, two samples contained no representative tumor cells, and in three, IHC staining failed. Thirteen patients (19%) were 3+, while 3/69 (4%) were 2+. Two of these showed HER2 gene amplification, resulting in a total of 15/69 (22%) HER2-positive resection specimens. In the third 2+ specimen, the ISH failed, resulting in one specimen that could not be classified as positive or negative after using the clinical algorithm (Table 2). Concordance, sensitivity, and specificity Concordance of HER2 positivity between the biopsies and matching resection specimens was found in 14/18 (78%) patients. In three (16%) patients, the matching resection specimens were HER2-negative, and in one patient, the resection specimen could not be evaluated due to technical failure. For the positive resection specimens, 14/15 (93%) biopsies were concordant with the positive status. One of the 53 (2%) HER2-negative biopsies was collected from a tumor that showed HER2 overexpression in the resection specimen (Table 3). SensiTable 3 Concordance in HER2 status (based on clinical algorithm† for HER2 status) in 75 matched pairs of biopsies and resection specimen of esophageal adenocarcinoma Resection specimen
Biopsy
Negative Positive NE Total
Negative
Positive
NE
Total
47 3 3 53
1 14 0 15
5 1 1 7
53 18 4 75
†Clinical algorithm: IHC 0–1+: HER2-negative; IHC 2+ → HER2/centromeric probe 17 ratio by ISH 2.2: HER2-positive; IHC3+: HER2-positive. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; NE, not evaluable. © 2014 International Society for Diseases of the Esophagus
tivity for determining HER2 status of a tumor in an endoscopic biopsy was 93%; specificity was 94%. The positive predictive value of HER2 testing in biopsies was 82%; the negative predictive value was 98%.
Interobserver agreement There was agreement among the pathologists for the assessment of HER2 expression in 83% of biopsies and in 85% of the resection specimens. Agreement on HER2 gene amplification was found in 55% of the biopsies, and 47% of the resection specimens.
DISCUSSION The treatment of esophageal cancer with monoclonal antibodies targeted against the HER2 receptor is only indicated when HER2 is overexpressed.6 Hence, careful selection of HER2-positive tumors is essential to prevent unnecessary (neo-)adjuvant treatment of patients with HER2-negative tumors, who are not likely to benefit. This study found that 22% of the esophageal adenocarcinomas are HER2-positive. Most importantly, we showed that HER2 expression can be evaluated in small diagnostic tissue fragments taken at the time of upper gastrointestinal endoscopy, and HER2 status in these biopsies matches the HER2 status of the resection specimen available after surgery in 94% of the cases. The findings of this study are of importance for clinical practice. In many patients, only a formalinfixed, paraffin-embedded endoscopic biopsy is available in which this HER2 status can be determined. Hence, the indication for cancer treatment with trastuzumab relies on accurate determination of HER2 expression in these small tissue fragments. Nowadays, neoadjuvant therapy is given to most patients with esophageal cancer that induces complete tumor regression in 25% of patients, and no vital tumor cells are present for HER evaluation. Also, genetic modifications may take place during chemotherapy and radiotherapy that can make determination of the HER2 status unreliable or impossible.24
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It has been questioned that small pretreatment biopsies are not representative for the genetic profile of the primary tumor. Great heterogeneity was observed in HER2 expression throughout esophageal adenocarcinomas.25 Several other studies were performed using tissue arrays for determining HER2 status, risking false-positive or false-negative results because of sampling error in heterogeneous tumors.26,27 By evaluating all biopsies taken during the first endoscopy from multiple areas of the tumor and representative sections of the resection specimens, we feel that this possible sampling error was reduced to a minimum in the present study. However, because we still encountered three false-positive biopsies, sampling error might still be an issue, even when using multiple biopsies. Problems were encountered with the quality of the ISH. Because IHC already provided sufficient information to determine HER2 status according to the algorithm, it was only necessary to repeat ISH in two patients that had been scored as HER2+ by IHC. Also, the use of archival material has not led to a deviating proportion of HER-positive tumors. In fact, 22% positivity for HER2 is well within the range of other studies.12,13,16,17 A concordance rate of 89–98% for HER2 expression between core biopsies and excision specimens has been demonstrated for breast cancer.28–30 This is comparable with the 94% concordance rate in the present study. However, it is also known that after neoadjuvant treatment, the HER2 status of a tumor can differ from the pretreatment status, which might make it more difficult to show concordance in patients who were neoadjuvantly treated.12 When considering both neoadjuvant and adjuvant treatment, the concordance in HER2 status between the primary tumor and metastases (both regional and distant) is of importance. A study by Reichelt et al. reported 100% concordance in HER2 status between the primary tumor and lymph-node metastases as well as distant metastatic deposits of esophageal cancer when analyzed using a fluorescence ISH technique.26 A more recent study by Bozetti et al. also found high levels of concordance between primary gastric tumors and metastasis.31 Fassan et al. found a concordance between primary esophagogastric junction tumors and lymph-node metastases of more than 98%, using a chromogenic in situ hybridization technique.32 These results suggest that both neoadjuvant and adjuvant treatment targeted at HER2 in patients with tumors of the upper gastrointestinal tract will affect the primary tumor as well as possible metastases in the majority of cases. The IHC scoring algorithm complicates a comparison in HER2 expression between biopsies and resection specimens. Discrepant HER2 expression between biopsies and resection specimens could be explained by different cut-off criteria for HER2 positivity or
be true differences based on biology. However, the different cut-off criteria would only lead to a falsepositive result in biopsies (because of the 10% rule in resection specimens), and this also only holds true for IHC 1+ or IHC 0 resection specimens with a focus of less than 10% of strong membrane staining. These cases are rare. In the present study, the interobserver agreement was moderate with respect to the assessment of ISH. There are several possible explanations for this finding. First, experience in evaluating ISH results differed between the observers. Second, the equipment used for the analyses was likely not the same in the three centers influencing the quality of the staining. However, the disagreement among the pathologists is likely reduced to a minimum when using the algorithm, not affecting the finally determined HER2 status of a specimen. The developments in HER2 targeted therapy for cancers of the upper gastrointestinal tract raise questions about the accuracy of determining the HER2 status of the tumor in small tissue fragments. The present study shows that this can be reliably performed by following an algorithm, when using multiple endoscopically obtained pretreatment biopsies.
Acknowledgment The authors are indebted to Mrs C. M. VollebregtUiterwijk for her dedicated collection of the patient data.
References 1 van Hagen P, Hulshof M C, van Lanschot J J et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med 2012; 366: 2074–84. 2 Burmeister B H, Smithers B M, Gebski V et al. Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial. Lancet Oncol 2005; 6: 659–68. 3 Tepper J, Krasna M J, Niedzwiecki D et al. Phase III trial of trimodality therapy with cisplatin, fluorouracil, radiotherapy, and surgery compared with surgery alone for esophageal cancer: CALGB 9781. J Clin Oncol 2008; 26: 1086–92. 4 Urba S G, Orringer M B, Turrisi A, Iannettoni M, Forastiere A, Strawderman M. Randomized trial of preoperative chemoradiation versus surgery alone in patients with locoregional esophageal carcinoma. J Clin Oncol 2001; 19: 305–13. 5 Dreilich M, Wanders A, Brattstrom D et al. HER-2 overexpression (3+) in patients with squamous cell esophageal carcinoma correlates with poorer survival. Dis Esophagus 2006; 19: 224–31. 6 Hudis C A. Trastuzumab – mechanism of action and use in clinical practice. N Engl J Med 2007 Jul 5; 357: 39–51. 7 Polkowski W, van Sandick J W, Offerhaus G J et al. Prognostic value of Lauren classification and c-erbB-2 oncogene overexpression in adenocarcinoma of the esophagus and gastroesophageal junction. Ann Surg Oncol 1999; 6: 290–7. 8 Sato-Kuwabara Y, Neves J I, Fregnani J H, Sallum R A, Soares F A. Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using fluorescence in situ hybridization (FISH) and immunohistochemistry. BMC Cancer 2009; 9: 6. © 2014 International Society for Diseases of the Esophagus
HER2 expression in esophageal cancer 9 Tanner M, Hollmen M, Junttila T T et al. Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Ann Oncol 2005; 16: 273–8. 10 al-Kasspooles M, Moore J H, Orringer M B, Beer D G. Amplification and over-expression of the EGFR and erbB-2 genes in human esophageal adenocarcinomas. Int J Cancer 1993; 54: 213–9. 11 Slamon D J, Godolphin W, Jones L A et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244: 707–12. 12 Schoppmann S F, Jesch B, Zacherl J et al. HER-2 status in primary oesophageal cancer, lymph nodes and distant metastases. Br J Surg 2011; 98: 1408–13. 13 Radu O M, Foxwell T, Cieply K et al. HER2 amplification in gastroesophageal adenocarcinoma: correlation of two antibodies using gastric cancer scoring criteria, H score, and digital image analysis with fluorescence in situ hybridization. Am J Clin Pathol 2012; 137: 583–94. 14 Jankowski J, Coghill G, Hopwood D, Wormsley K G. Oncogenes and onco-suppressor gene in adenocarcinoma of the oesophagus. Gut 1992; 33: 1033–8. 15 Hardwick R H, Shepherd N A, Moorghen M, Newcomb P V, Alderson D. c-erbB-2 overexpression in the dysplasia/ carcinoma sequence of Barrett’s oesophagus. J Clin Pathol 1995; 48: 129–32. 16 Schoppmann S F, Jesch B, Friedrich J et al. Expression of Her-2 in carcinomas of the esophagus. Am J Surg Pathol 2010; 34: 1868–73. 17 Yoon H H, Shi Q, Sukov W R et al. Association of HER2/ ErbB2 expression and gene amplification with pathologic features and prognosis in esophageal adenocarcinomas. Clin Cancer Res 2012; 18: 546–54. 18 Safran H, DiPetrillo T, Akerman P et al. Phase I/II study of trastuzumab, paclitaxel, cisplatin and radiation for locally advanced, HER2 overexpressing, esophageal adenocarcinoma. Int J Radiat Oncol Biol Phys 2007; 67: 405–9. 19 Bang Y J, Van Cutsem E, Feyereislova A et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastrooesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010; 376: 687–97. 20 Schneider S, Uchida K, Brabender J et al. Downregulation of TS, DPD, ERCC1, GST-Pi, EGFR, and HER2 gene expression after neoadjuvant three-modality treatment in patients with esophageal cancer. J Am Coll Surg 2005; 200: 336–44.
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21 Grabsch H, Sivakumar S, Gray S, Gabbert H E, Muller W. HER2 expression in gastric cancer: rare, heterogeneous and of no prognostic value – conclusions from 924 cases of two independent series. Cell Oncol 2010; 32: 57–65. 22 Sobin L H, Wittekind C. International Union against Cancer. TNM: Classification of Malignant Tumours, 6th edn. New York: Wiley-Liss, 2002. 23 Hofmann M, Stoss O, Shi D et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008; 52: 797–805. 24 Adams A L, Eltoum I, Krontiras H, Wang W, Chhieng D C. The effect of neoadjuvant chemotherapy on histologic grade, hormone receptor status, and HER2/neu status in breast carcinoma. Breast J 2008; 14: 141–6. 25 Boers J E, Meeuwissen H. Methorst N. HER2 status in gastrooesophageal adenocarcinomas assessed by two rabbit monoclonal antibodies (SP3 and 4B5) and two in situ hybridization methods (FISH and SISH). Histopathology 2011; 58: 383–94. 26 Reichelt U, Duesedau P, Tsourlakis M et al. Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus. Mod Pathol 2007; 20: 120–9. 27 Thompson S K, Sullivan T R, Davies R, Ruszkiewicz A R. HER-2/neu gene amplification in esophageal adenocarcinoma and its influence on survival. Ann Surg Oncol 2011; 18: 2010– 7. 28 Shousha S, Peston D, Amo-Takyi B, Morgan M, Jasani B. Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens. Histopathology 2009; 54: 248–53. 29 Sutela A, Vanninen R, Sudah M et al. Surgical specimen can be replaced by core samples in assessment of ER, PR and HER-2 for invasive breast cancer. Acta Oncol 2008; 47: 38–46. 30 Apple S K, Lowe A C, Rao P N, Shintaku I P, Moatamed N A. Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol 2009; 22: 1151–9. 31 Bozzetti C, Negri F V, Lagrasta C A et al. Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma. Br J Cancer 2011; 104: 1372–6. 32 Fassan M, Ludwig K, Pizzi M et al. Human epithelial growth factor receptor 2 (HER2) status in primary and metastatic esophagogastric junction adenocarcinomas. Hum Pathol 2012; 43: 1206–12.
Original article
Human epidermal growth factor receptor 2 overexpression and amplification in endoscopic biopsies and resection specimens in esophageal and junctional adenocarcinoma P. van Hagen,1 K. Biermann,2 J. E. Boers,3 O. Stoss,4 H. F. Sleddens,2 J. J. B. van Lanschot,1 W. N. M. Dinjens,2 J. Rueschoff,4,5 B. P. L. Wijnhoven1 Department of Surgery, 2Department of Pathology, Erasmus MC, University Medical Centre Rotterdam, Rotterdam, 3Department of Pathology, Isala Clinics, Zwolle, The Netherlands; and 4TARGOS Molecular Pathology GmbH, and 5Institute of Pathology, Klinikum Kassel, Kassel, Germany 1
SUMMARY. Human epidermal growth factor receptor 2 (HER2) is overexpressed in a subset of esophageal adenocarcinomas. Frequently, biopsy material is used for evaluation of HER2 status. The aim of the study was to determine if HER2 expression in preoperative endoscopic biopsies is representative for the entire tumor. Preoperative endoscopic biopsies and matched resection specimens were collected from 75 patients who underwent esophagectomy for esophageal adenocarcinoma. Immunohistochemical staining (IHC) on HER2 and dual-color in situ hybridization (ISH) were performed. HER2 status was determined by following a clinical algorithm, first determining HER2 overexpression on immunohistochemistry and, when equivocal (2+), determining HER2 amplification on ISH. Seventy-one of 75 (95%) biopsies and 69/75 (92%) resection specimens could be analyzed due to technical failure. HER2 positivity was seen in 18/71 (25%) biopsies and in 15/69 (22%) resection specimens. Overall, HER2 status in the biopsy was concordant with HER2 status in the resection specimen in 94% of cases. Interobserver agreement on IHC scoring for all three observers was 83% in biopsies and 85% in resection specimens. HER2 positivity was detected in 22% of esophageal adenocarcinomas. Although interobserver agreement was moderate, HER2 status of a primary tumor can be reliably determined based on the endoscopically obtained pretreatment biopsy. KEY WORDS: esophageal adenocarcinoma, HER2, protein expression.
INTRODUCTION Cancer of the esophagus is the sixth cause of cancer death worldwide. It is a highly lethal disease in which only one third of patients have a resectable tumor at the time of presentation. Surgery with neoadjuvant chemo(radio)therapy offers the best chances for long-term survival.1 However, still half of patients suffer from recurrence of disease after multimodality therapy.2–4 Clearly, new treatments are needed to improve the outcome of patients with esophageal cancer. Address correspondence to: Dr Bas P.L. Wijnhoven, MD, PhD, Erasmus MC University Medical Centre Rotterdam, PO Box 2040, Rotterdam 3000 CA, The Netherlands. Email: [email protected] Disclosure: This study was financially supported by an unrestricted grant from Roche b.v., Woerden, the Netherlands. 380
The human epidermal growth factor receptor 2 (HER2) regulates cell growth and proliferation. Overexpression of HER2 has been identified in a wide variety of carcinomas, including esophageal cancer. HER2 overexpression correlates with advanced tumor stage and poor survival.5–9 Trastuzumab, a monoclonal antibody against the HER2 receptor, inhibits the receptor activity and oncogenic downstream targets.10 In HER2-positive breast cancer, trastuzumab improves overall and disease-free survival in early and metastatic disease.11 Overexpression of HER2 is not uncommon in esophageal cancer with up to 43% of the cancers being HER2-positive.10,12–17 Hence, trastuzumab could well be an effective drug in the multimodal treatment of esophageal cancer.18 In metastatic gastric cancer, the addition of trastuzumab to standard palliative chemotherapy has proved to be of benefit.19 © 2014 International Society for Diseases of the Esophagus
HER2 expression in esophageal cancer
Trastuzumab is only effective in cancer cells overexpressing HER2 and/or cells that show HER2 gene amplification.6 The HER2 status is evaluated by immunohistochemical staining and HER2 gene amplification by in situ hybridization (ISH).20 Abundant tumor tissue is available for analysis from the resection specimen. However, preoperative biopsies from esophageal cancer are limited in size. Moreover, expression of oncogenes and tumor suppressor genes in adenocarcinomas of the upper gastrointestinal tract are well known to be heterogeneous that questions the representativeness of a small tissue fragment taken at endoscopy.21 This might lead to undertreatment of patients in whom HER2 overexpression/ amplification was falsely scored negative. On the other hand, false-positive biopsies may lead to an increase of toxicity and costs, and moreover, inadequate prognostication with a potential negative impact on clinical decision-making. The aims of the present study were to determine if the HER2 status of an esophageal adenocarcinoma can be reliably determined based on endoscopic biopsies and to assess the interobserver agreement in determining HER status.
METHODS Patients and samples Patients who had undergone an esophagectomy for adenocarcinoma of the esophagus or OGJ between January 2000 to January 2010 in the Erasmus MC, Rotterdam, the Netherlands were identified from the institutional database. Seventy-five patients with a cT2-cT3 tumor were selected. Excluded were cT1 and cT4 tumors, and patients who underwent any form of neoadjuvant or adjuvant chemo(radio)therapy. cT1 tumors were excluded because of the limited additional effect of neoadjuvant therapy targeted at Her2 that can be expected after resection in this group of tumors that already has a relatively high survival rate after resection. cT4 tumors were excluded because in our center, cT4 tumors are not resected but are treated with definitive chemoradiotherapy, after which no resection specimen is available to compare with the pretreatment biopsies. Also, patients with incomplete or inadequate archived tumor tissue (either in biopsies or the surgical resection specimen) were excluded. Patients were selected consecutively whenever possible. Tissue blocks of the endoscopic biopsies, routinely taken from multiple areas of the tumor, as well as the resection specimen for each patient were retrieved. All tissue samples had been fixed using 4% buffered formalin and embedded in paraffin after dehydration. All biopsies and a representative area of invasive tumor of the resection specimen that was identified using a hematoxylin and eosin (H&E) stain. Subse© 2014 International Society for Diseases of the Esophagus
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quently, 4-μm sections were cut from the tissue blocks and were used for immunohistochemistry (IHC) and ISH. Several patient and tumor characteristics were identified including patients’ age (at the time of diagnosis), sex and tumor stage according to the Union for International Cancer Control TNM, 6th edition (as determined by endoultrasonography and computed tomography scanning of the chest and abdomen, or from pathology report when clinical investigations were not conclusive).22 The study was reviewed and approved by the local medical ethics committee, and the requirement of obtaining patients’ informed consent was waived. HER2 IHC and dual-color ISH The 4-μm sections were mounted on starfrost slides (Knittel Gläser, Braunschweig, Germany) and dried. The immunohistochemical staining was performed using a BenchMark ULTRA automated slide staining instrument (Ventana Medical Systems, Tucson, AZ, USA) and using the PATHWAY HER2 4B5 antibody, according to the manufacturer’s instructions. ISH staining was also performed fully automated with the BenchMark ULTRA, with the INFORM HER2 DNA probe and the Red ISH V probe (both Ventana Medical Systems). Evaluation of HER2 status HER2 expression and amplification was independently assessed by three experienced pathologists (KB, JEB and JR) from three institutions (Erasmus MC, University Medical Centre Rotterdam, Rotterdam, The Netherlands; Isala Clinics Zwolle, The Netherlands; and TARGOS Molecular Pathology, Kassel, Germany). The pathologists were blinded for the patients. Biopsies and resection specimens were rotating among the centers in three batches making sure that matching biopsies and resection specimens were never present at the same location simultaneously. The H&E staining, IHC, and ISH results of the tissues from a single patient’s biopsy or resection specimens were at the disposal of the pathologist. For determining the HER2 status, an algorithm was used (Fig 1). In case of no or weak HER2 expression (1+), the tumor is scored as HER2-negative, and in case of strong expression (3+), the tumor is scored as HER2-positive. In case of moderate expression (2+), the results of ISH are evaluated, and the HER2 status is determined based on the absence (negative) or presence (positive) of HER2 amplification (Fig 2). Evaluation of the IHC for HER2 was done according to the scoring system for gastric and OGJ cancer.23 In brief, HER2 expression in the resection specimen was scored as 2+ when ≥10% of tumor cells showed a weak to moderate complete or basolateral
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Diseases of the Esophagus Table 1 Patient and tumor characteristics of 75 patients with esophageal or esophagogastric junction adenocarcinoma Characteristics
Fig. 1 Algorithm for the assessment of human epidermal growth factor receptor 2 (HER2) status. Intensity of reactivity: 0–1+ = absent–faint; 2+ = moderate; 3+ = strong. HER2 : CEP17, HER2 and centromeric probe 17 ratio; IHC, immunohistochemistry; ISH: dual color-silver in situ hybridization.
Gender (n, %) Male Female Age (years) Mean (SD) Tumor differentiation (n, %) Good Moderate Poor Missing Clinical T-stage (n, %)† cT2 cT3 No endoscopic pass Clinical N-stage (n, %)‡ N0 N1 Missing
— 56 (75%) 19 (25%) — 63 (11) — 3 ( 4%) 29 (39%) 42 (56%) 1 (1%) — 20 (27%) 48 (64%) 7 (9%) — 23 (31%) 45 (60%) 7 (9%)
†Clinical T-stage: T-stage as defined by endoultrasonography and/or CT scanning according to UICC TNM Cancer Staging, 6th edition. ‡Clinical N-stage: clinical N-stage as defined by endoultrasonography and/or CT scanning and/or FDG-PET according to UICC TNM Cancer Staging, 6th edition. CT, computed tomography; FDG-PET, fluorodeoxyglucose-positron emission tomography; SD, standard deviation; UICC, Union for International Cancer Control.
membranous reactivity. For preoperative biopsies, the presence of cohesive IHC 3+ and/or ISH+ clones resulted in a positive HER2 status irrespective of size.23 For ISH evaluation, the HER2 : centromeric probe 17 ratio was determined. A sample was considered positive for HER2 amplification when this ratio was >2.2. Statistical analysis HER2 status was defined as positive or negative.23 The statistical analysis was performed using Statistical Package for Social Sciences version 17.0 (SPSS, Inc., Chicago, IL, USA).
RESULTS Patients A total of 75 patients were selected who met the inclusion criteria. The male : female ratio was 3 : 1, and the mean age was 63 years. Forty-eight patients (64%) had a cT3 tumor, and 45 (60%) patients were staged as node positive (Table 1). Fig. 2 Hematoxylin and eosin (A, B), immunohistochemistry (Aa, Ba) and in situ hybridization (ISH) (Ab, Bb) in two different invasive esophageal adenocarcinomas. In (A), the adenocarcinoma is human epidermal growth factor receptor 2 (HER2)-negative in IHC, and correspondingly, there is no amplification of HER2 gene in ISH. In contrast, adenocarcinoma in (B) shows moderate HER2 expression (2+), and the corresponding ISH reveals HER2 amplification in some but not all tumor cells.
Biopsies A mean of five biopsies (standard deviation ±2) per patient were collected. The biopsies of four (5%) patients could not be evaluated for HER2 expression by IHC. One biopsy was identified as squamous cell carcinoma, and in the three remaining biopsies, the IHC © 2014 International Society for Diseases of the Esophagus
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Table 2 Conventional immunohistochemistry scoring and results with the application of a clinical algorithm† of 71 evaluable biopsies and 69 evaluable resection specimens of esophageal adenocarcinoma IHC
Biopsies (n = 71) Resection specimens (n = 69)
After following algorithm
0–1+
2+
3+
Neg
Pos
NE
52 (73%) 53 (78%)
3 (4%) 3 (4%)
16 (23%) 13 (19%)
53 (75%) 53 (77%)
18 (25%) 15 (22%)
0 (0%) 1 (1%)
†Clinical algorithm: IHC 0–1+: HER2-negative; IHC 2 + → HER2/centromere17 ratio on ISH 2.2: HER2-positive; IHC3+: HER2-positive. Intensity of reactivity, 0–1+: absent–faint; 2+: moderate; 3+: strong. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; NE, not evaluable; Neg, negative; Pos, positive.
failed. In 16/71 (23%) patients, expression of HER2 by IHC was 3+; in 3/71 (4%) patients, this was 2+ . Two of these biopsies showed HER amplification. Hence, 18/71 (25%) biopsies were HER2-positive. (Table 2). Resection specimens Six (8%) patients were excluded; there was one squamous cell carcinoma, two samples contained no representative tumor cells, and in three, IHC staining failed. Thirteen patients (19%) were 3+, while 3/69 (4%) were 2+. Two of these showed HER2 gene amplification, resulting in a total of 15/69 (22%) HER2-positive resection specimens. In the third 2+ specimen, the ISH failed, resulting in one specimen that could not be classified as positive or negative after using the clinical algorithm (Table 2). Concordance, sensitivity, and specificity Concordance of HER2 positivity between the biopsies and matching resection specimens was found in 14/18 (78%) patients. In three (16%) patients, the matching resection specimens were HER2-negative, and in one patient, the resection specimen could not be evaluated due to technical failure. For the positive resection specimens, 14/15 (93%) biopsies were concordant with the positive status. One of the 53 (2%) HER2-negative biopsies was collected from a tumor that showed HER2 overexpression in the resection specimen (Table 3). SensiTable 3 Concordance in HER2 status (based on clinical algorithm† for HER2 status) in 75 matched pairs of biopsies and resection specimen of esophageal adenocarcinoma Resection specimen
Biopsy
Negative Positive NE Total
Negative
Positive
NE
Total
47 3 3 53
1 14 0 15
5 1 1 7
53 18 4 75
†Clinical algorithm: IHC 0–1+: HER2-negative; IHC 2+ → HER2/centromeric probe 17 ratio by ISH 2.2: HER2-positive; IHC3+: HER2-positive. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; ISH, in situ hybridization; NE, not evaluable. © 2014 International Society for Diseases of the Esophagus
tivity for determining HER2 status of a tumor in an endoscopic biopsy was 93%; specificity was 94%. The positive predictive value of HER2 testing in biopsies was 82%; the negative predictive value was 98%.
Interobserver agreement There was agreement among the pathologists for the assessment of HER2 expression in 83% of biopsies and in 85% of the resection specimens. Agreement on HER2 gene amplification was found in 55% of the biopsies, and 47% of the resection specimens.
DISCUSSION The treatment of esophageal cancer with monoclonal antibodies targeted against the HER2 receptor is only indicated when HER2 is overexpressed.6 Hence, careful selection of HER2-positive tumors is essential to prevent unnecessary (neo-)adjuvant treatment of patients with HER2-negative tumors, who are not likely to benefit. This study found that 22% of the esophageal adenocarcinomas are HER2-positive. Most importantly, we showed that HER2 expression can be evaluated in small diagnostic tissue fragments taken at the time of upper gastrointestinal endoscopy, and HER2 status in these biopsies matches the HER2 status of the resection specimen available after surgery in 94% of the cases. The findings of this study are of importance for clinical practice. In many patients, only a formalinfixed, paraffin-embedded endoscopic biopsy is available in which this HER2 status can be determined. Hence, the indication for cancer treatment with trastuzumab relies on accurate determination of HER2 expression in these small tissue fragments. Nowadays, neoadjuvant therapy is given to most patients with esophageal cancer that induces complete tumor regression in 25% of patients, and no vital tumor cells are present for HER evaluation. Also, genetic modifications may take place during chemotherapy and radiotherapy that can make determination of the HER2 status unreliable or impossible.24
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It has been questioned that small pretreatment biopsies are not representative for the genetic profile of the primary tumor. Great heterogeneity was observed in HER2 expression throughout esophageal adenocarcinomas.25 Several other studies were performed using tissue arrays for determining HER2 status, risking false-positive or false-negative results because of sampling error in heterogeneous tumors.26,27 By evaluating all biopsies taken during the first endoscopy from multiple areas of the tumor and representative sections of the resection specimens, we feel that this possible sampling error was reduced to a minimum in the present study. However, because we still encountered three false-positive biopsies, sampling error might still be an issue, even when using multiple biopsies. Problems were encountered with the quality of the ISH. Because IHC already provided sufficient information to determine HER2 status according to the algorithm, it was only necessary to repeat ISH in two patients that had been scored as HER2+ by IHC. Also, the use of archival material has not led to a deviating proportion of HER-positive tumors. In fact, 22% positivity for HER2 is well within the range of other studies.12,13,16,17 A concordance rate of 89–98% for HER2 expression between core biopsies and excision specimens has been demonstrated for breast cancer.28–30 This is comparable with the 94% concordance rate in the present study. However, it is also known that after neoadjuvant treatment, the HER2 status of a tumor can differ from the pretreatment status, which might make it more difficult to show concordance in patients who were neoadjuvantly treated.12 When considering both neoadjuvant and adjuvant treatment, the concordance in HER2 status between the primary tumor and metastases (both regional and distant) is of importance. A study by Reichelt et al. reported 100% concordance in HER2 status between the primary tumor and lymph-node metastases as well as distant metastatic deposits of esophageal cancer when analyzed using a fluorescence ISH technique.26 A more recent study by Bozetti et al. also found high levels of concordance between primary gastric tumors and metastasis.31 Fassan et al. found a concordance between primary esophagogastric junction tumors and lymph-node metastases of more than 98%, using a chromogenic in situ hybridization technique.32 These results suggest that both neoadjuvant and adjuvant treatment targeted at HER2 in patients with tumors of the upper gastrointestinal tract will affect the primary tumor as well as possible metastases in the majority of cases. The IHC scoring algorithm complicates a comparison in HER2 expression between biopsies and resection specimens. Discrepant HER2 expression between biopsies and resection specimens could be explained by different cut-off criteria for HER2 positivity or
be true differences based on biology. However, the different cut-off criteria would only lead to a falsepositive result in biopsies (because of the 10% rule in resection specimens), and this also only holds true for IHC 1+ or IHC 0 resection specimens with a focus of less than 10% of strong membrane staining. These cases are rare. In the present study, the interobserver agreement was moderate with respect to the assessment of ISH. There are several possible explanations for this finding. First, experience in evaluating ISH results differed between the observers. Second, the equipment used for the analyses was likely not the same in the three centers influencing the quality of the staining. However, the disagreement among the pathologists is likely reduced to a minimum when using the algorithm, not affecting the finally determined HER2 status of a specimen. The developments in HER2 targeted therapy for cancers of the upper gastrointestinal tract raise questions about the accuracy of determining the HER2 status of the tumor in small tissue fragments. The present study shows that this can be reliably performed by following an algorithm, when using multiple endoscopically obtained pretreatment biopsies.
Acknowledgment The authors are indebted to Mrs C. M. VollebregtUiterwijk for her dedicated collection of the patient data.
References 1 van Hagen P, Hulshof M C, van Lanschot J J et al. Preoperative chemoradiotherapy for esophageal or junctional cancer. N Engl J Med 2012; 366: 2074–84. 2 Burmeister B H, Smithers B M, Gebski V et al. Surgery alone versus chemoradiotherapy followed by surgery for resectable cancer of the oesophagus: a randomised controlled phase III trial. Lancet Oncol 2005; 6: 659–68. 3 Tepper J, Krasna M J, Niedzwiecki D et al. Phase III trial of trimodality therapy with cisplatin, fluorouracil, radiotherapy, and surgery compared with surgery alone for esophageal cancer: CALGB 9781. J Clin Oncol 2008; 26: 1086–92. 4 Urba S G, Orringer M B, Turrisi A, Iannettoni M, Forastiere A, Strawderman M. Randomized trial of preoperative chemoradiation versus surgery alone in patients with locoregional esophageal carcinoma. J Clin Oncol 2001; 19: 305–13. 5 Dreilich M, Wanders A, Brattstrom D et al. HER-2 overexpression (3+) in patients with squamous cell esophageal carcinoma correlates with poorer survival. Dis Esophagus 2006; 19: 224–31. 6 Hudis C A. Trastuzumab – mechanism of action and use in clinical practice. N Engl J Med 2007 Jul 5; 357: 39–51. 7 Polkowski W, van Sandick J W, Offerhaus G J et al. Prognostic value of Lauren classification and c-erbB-2 oncogene overexpression in adenocarcinoma of the esophagus and gastroesophageal junction. Ann Surg Oncol 1999; 6: 290–7. 8 Sato-Kuwabara Y, Neves J I, Fregnani J H, Sallum R A, Soares F A. Evaluation of gene amplification and protein expression of HER-2/neu in esophageal squamous cell carcinoma using fluorescence in situ hybridization (FISH) and immunohistochemistry. BMC Cancer 2009; 9: 6. © 2014 International Society for Diseases of the Esophagus
HER2 expression in esophageal cancer 9 Tanner M, Hollmen M, Junttila T T et al. Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Ann Oncol 2005; 16: 273–8. 10 al-Kasspooles M, Moore J H, Orringer M B, Beer D G. Amplification and over-expression of the EGFR and erbB-2 genes in human esophageal adenocarcinomas. Int J Cancer 1993; 54: 213–9. 11 Slamon D J, Godolphin W, Jones L A et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science. 1989;244: 707–12. 12 Schoppmann S F, Jesch B, Zacherl J et al. HER-2 status in primary oesophageal cancer, lymph nodes and distant metastases. Br J Surg 2011; 98: 1408–13. 13 Radu O M, Foxwell T, Cieply K et al. HER2 amplification in gastroesophageal adenocarcinoma: correlation of two antibodies using gastric cancer scoring criteria, H score, and digital image analysis with fluorescence in situ hybridization. Am J Clin Pathol 2012; 137: 583–94. 14 Jankowski J, Coghill G, Hopwood D, Wormsley K G. Oncogenes and onco-suppressor gene in adenocarcinoma of the oesophagus. Gut 1992; 33: 1033–8. 15 Hardwick R H, Shepherd N A, Moorghen M, Newcomb P V, Alderson D. c-erbB-2 overexpression in the dysplasia/ carcinoma sequence of Barrett’s oesophagus. J Clin Pathol 1995; 48: 129–32. 16 Schoppmann S F, Jesch B, Friedrich J et al. Expression of Her-2 in carcinomas of the esophagus. Am J Surg Pathol 2010; 34: 1868–73. 17 Yoon H H, Shi Q, Sukov W R et al. Association of HER2/ ErbB2 expression and gene amplification with pathologic features and prognosis in esophageal adenocarcinomas. Clin Cancer Res 2012; 18: 546–54. 18 Safran H, DiPetrillo T, Akerman P et al. Phase I/II study of trastuzumab, paclitaxel, cisplatin and radiation for locally advanced, HER2 overexpressing, esophageal adenocarcinoma. Int J Radiat Oncol Biol Phys 2007; 67: 405–9. 19 Bang Y J, Van Cutsem E, Feyereislova A et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastrooesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010; 376: 687–97. 20 Schneider S, Uchida K, Brabender J et al. Downregulation of TS, DPD, ERCC1, GST-Pi, EGFR, and HER2 gene expression after neoadjuvant three-modality treatment in patients with esophageal cancer. J Am Coll Surg 2005; 200: 336–44.
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21 Grabsch H, Sivakumar S, Gray S, Gabbert H E, Muller W. HER2 expression in gastric cancer: rare, heterogeneous and of no prognostic value – conclusions from 924 cases of two independent series. Cell Oncol 2010; 32: 57–65. 22 Sobin L H, Wittekind C. International Union against Cancer. TNM: Classification of Malignant Tumours, 6th edn. New York: Wiley-Liss, 2002. 23 Hofmann M, Stoss O, Shi D et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008; 52: 797–805. 24 Adams A L, Eltoum I, Krontiras H, Wang W, Chhieng D C. The effect of neoadjuvant chemotherapy on histologic grade, hormone receptor status, and HER2/neu status in breast carcinoma. Breast J 2008; 14: 141–6. 25 Boers J E, Meeuwissen H. Methorst N. HER2 status in gastrooesophageal adenocarcinomas assessed by two rabbit monoclonal antibodies (SP3 and 4B5) and two in situ hybridization methods (FISH and SISH). Histopathology 2011; 58: 383–94. 26 Reichelt U, Duesedau P, Tsourlakis M et al. Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus. Mod Pathol 2007; 20: 120–9. 27 Thompson S K, Sullivan T R, Davies R, Ruszkiewicz A R. HER-2/neu gene amplification in esophageal adenocarcinoma and its influence on survival. Ann Surg Oncol 2011; 18: 2010– 7. 28 Shousha S, Peston D, Amo-Takyi B, Morgan M, Jasani B. Evaluation of automated silver-enhanced in situ hybridization (SISH) for detection of HER2 gene amplification in breast carcinoma excision and core biopsy specimens. Histopathology 2009; 54: 248–53. 29 Sutela A, Vanninen R, Sudah M et al. Surgical specimen can be replaced by core samples in assessment of ER, PR and HER-2 for invasive breast cancer. Acta Oncol 2008; 47: 38–46. 30 Apple S K, Lowe A C, Rao P N, Shintaku I P, Moatamed N A. Comparison of fluorescent in situ hybridization HER-2/neu results on core needle biopsy and excisional biopsy in primary breast cancer. Mod Pathol 2009; 22: 1151–9. 31 Bozzetti C, Negri F V, Lagrasta C A et al. Comparison of HER2 status in primary and paired metastatic sites of gastric carcinoma. Br J Cancer 2011; 104: 1372–6. 32 Fassan M, Ludwig K, Pizzi M et al. Human epithelial growth factor receptor 2 (HER2) status in primary and metastatic esophagogastric junction adenocarcinomas. Hum Pathol 2012; 43: 1206–12.
