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Journal of Clinical Oncology, Vol 22, No 23 (December 1), 2004: pp. 4737-4745 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.05.142 Copy Number of Chromosome 17 but Not HER2 Amplification Predicts Clinical Outcome of Patients With Pancreatic Ductal AdenocarcinomaFrom the Chirurgische Klinik, and Institut für Pathologie, Universitätsklinikum Hamburg-Eppendorf, Hamburg; Institut für Immunologie, Ludwig-Maximilians-Universität München, München; and Koordinierungszentrum für klinische Studien, Universitätsklinikum Düsseldorf, Düsseldorf, Germany Address reprint requests to Nikolas H. Stoecklein, MD, Klinik für Allgemein-und Viszeralchirugie, Universitätsklinikum Düsseldorf, Moorenstr. 5, D-40225 Düsseldorf, Germany; e-mail: Nikolas.Stoecklein{at}uni-duesseldorf.de
PURPOSE: To determine the frequency and the potential clinical use of HER2 (17q21) gene amplification and chromosome 17 aneuploidy in pancreatic ductal adenocarcinoma (PDAC). MATERIALS AND METHODS: Serial tissue sections of 50 resected PDACs were analyzed with chromogenic in situ hybridization using locus-specific HER2 probes and centromeric probes for chromosome 17. Centromeric probes for chromosome 7 and 8 were hybridized to confirm ploidy levels. Expression of HER2 protein was assessed by immunohistochemistry. Correlations of experimental findings with clinical and follow-up data were tested. RESULTS: The HER2 gene locus was frequently (24%) amplified in PDAC and the rate of overexpression (2+ and 3+) was 10%, but no prognostic significance was found. Copy number analysis of chromosomes 7, 8, and 17 revealed disomic (40%), trisomic (36%), and hypertetrasomic (24%) tumors. Compared with patients with disomic tumors, patients with hypertetrasomic tumors exhibited a significantly decreased relapse-free and overall survival (5.0 v 13.0 months, P = .0144 and 7.0 v 20.0 months, P = .0099, respectively). Multivariate analysis confirmed the independent prognostic significance of hypertetrasomy. CONCLUSION: Tumor ploidy levels correlate with prognosis of PDAC patients, indicating characteristic biologic properties of PDAC with high chromosomal instability. In contrast, no prognostic influence on patient outcome was found for the amplification of the HER2 oncogene or p185HER2 overexpression. Therefore, evaluation of ploidy levels offers new opportunities for patient stratification in clinical trials and enables novel approaches to study the well-known aggressiveness of PDAC.
Pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic malignancy,1 is a highly aggressive cancer and major cause of cancer-related death in the western world.1-3 Eighty percent of the PDAC patients present with inoperable advanced disease and will die within 4 to 6 months after diagnosis. Of the remaining patients who are selected for potentially curative surgery, only 20% to 30% survive longer than 5 years.4-6 Because efficient standard chemotherapeutic protocols are still lacking,7 new therapeutic targets and better prognostic markers for patient stratification are desperately needed. Recently, a humanized monoclonal antibody (trastuzumab) has shown significant antitumor activity in breast cancer patients,8 whose tumors display increased expression of the receptor tyrosine kinase p185HER2 that transmits growth signals into the cell. Interestingly, only increased expression of p185HER2 arising from gene amplification correlated significantly with advanced disease, metastasis, and poor clinical outcome9 in several human cancers, whereas overexpression without gene amplification did not.10 These data suggest that only cancers that depend on amplified HER2 as the tumor-driving oncogene may be successfully treated by HER2-based therapies. In PDAC the incidence of HER2 amplification and hence its prognostic impact and suitability as therapy target is unknown. However, staining of p185HER2 in pancreatic cancer tissues has been repeatedly, albeit not consistently, observed.11-19 This prompted us to study the genomic basis of an increased expression of p185HER2 in pancreatic cancer patients using DNA probes directed against the HER2 locus on chromosome 17q12. However, it has recently become evident that solid cancers can be differentiated by the distributions of the number of their cytogenetic imbalances, which can be monotonically decreasing (eg, breast cancer, colon) and bimodal (eg, pancreatic cancer, lung cancer).20 Given that the bimodal distribution seen in pancreatic cancers is likely due to an increased level of chromosomal instability, we also tested for genome-wide chromosomal abnormalities. In doing so, we intended to control for HER2-dependent and -independent effects on clinical outcome.
Patients All patients (N = 50) in this study underwent partial pancreatoduodenectomy and radical lymphadenectomy with curative intent at the University Hospital of Hamburg (Hamburg, Germany). All patient cases had pancreatic ductal carcinomas identified by histopathology. Clinical data and pathology data for all patients were acquired with approval from the ethics committee of the chamber of physicians of Hamburg, Germany. Formalin-fixed and paraffin-embedded samples were used for this study and were reviewed by a pathologist (A.E.). The clinical data for all investigated patient cases are summarized in Table 1. The resection margins of 49 samples, including the retroperitoneal margins and the resection margins of the biliary tree, were free of tumor in the final histopathologic assessment. Survival analysis was calculated from 41 patients who received no neoadjuvant or adjuvant chemoradiotherapy therapy. Nine patients were excluded from survival analysis: one patient received adjuvant chemoradiotherapy therapy, two patients died during the hospital stay, and no complete follow-up could be obtained for at least 3 months for six patients. The median clinical observation period was 13 months (range, 3 to 99 months).
Fluorescence In Situ Hybridization The PathVision (Vysis Inc, Downers Grove, IL) dual-color fluorescence in situ hybridization (FISH) kit for simultaneous hybridization of differently labeled probes was used for probes of chromosome 17 and HER2. The slides underwent pretreatment, proteolytic digestion, fixation, DNA denaturation, hybridization, and posthybridization washes according to instructions provided by the manufacturer. The hybridization results were evaluated with a Leica DMRA (Leica, Bensheim, Germany) microscope, equipped with appropriate filters. Sixty intact tumor interphase nuclei were enumerated for FISH signals. First we determined the chromosomal index (CI) for chromosome 17 and the copy number index (CNI) of HER2. The CI is defined as the mean number of chromosome copies per nucleus in the sample of enumerated nuclei and in analogy, the CNI is defined as the mean gene copy number in the sample of the assessed nuclei. For both the CI and the CNI, a value of 2.0 is expected for normal diploid cells, but in tissue sections this value is smaller. The HER2 amplification was determined by calculating the ratio of HER2 signals and chromosome 17 centromere signal counts. Ratios smaller than 2.0 were considered as nonamplified, those greater than 2.0 were considered as amplified, and those greater than 5.0 were scored as high-level amplification.
Chromogenic In Situ Hybridization To determine the chromosome 17 copy number, we followed the recommendations of Mendelin et al.21 Briefly, tumors were classified as monosomic for chromosome 17 if more than 50% of the nuclei showed one or no ISH signal. Tumors were classified as disomic, trisomic, or tetrasomic if at least 20% of the enumerated nuclei showed two, three, or four signals, respectively. Hypertetrasomy (aneuploidy) of chromosome 17 was interpreted when we counted more than four signals in at least 10% of the nuclei of the tumor.
Immunohistochemistry
Statistical Analysis
Comparison of FISH and CISH Usually, HER2 gene amplification is assessed in tissue sections by dual-color FISH with differently labeled probes for HER2 and chromosome 17 to distinguish between polysomy and HER2 gene amplification.24 However, histologic assessment of most PDACs is difficult, making the discrimination between nuclei of benign and malignant pancreatic ducts often impossible when fluorescence staining is used. Therefore, we applied chromogen-based hybridization signal detection (CISH), which enables the correct assessment of the histologic hallmarks of PDAC and of DNA copy number changes. Given that CISH signals can only be developed with one color per sample, two consecutive sections were hybridized with the two probes. Because of this difference compared with the standard procedure, we confirmed the reliability of CISH by FISH on samples with unambiguous cancer morphology. The HER2 and chromosome 17 ratios were determined by CISH and FISH on consecutive sections of six different malignant and nonmalignant tissue samples. All nonamplified, amplified, and high-level amplified (Fig 1A) samples found by CISH were confirmed with dual-color FISH (Table 2). No statistically significant differences between the FISH and CISH results were detected (P = .486).
Chromosome 17 Copy Number in PDAC Using the CISH method, we first counted the hybridization signals for chromosome 17 in 200 cells of nonpathologic exocrine pancreatic acini that served as normal controls. On average, we observed no hybridization signal in 11.4% ± 4.09, one signal in 33.5% ± 3.91, two signals in 51.9% ± 4.45, three signals in 2.8% ± 1.86, four signals in 0.4% ± 0.68, and never more than four signals in the nuclei of the normal controls, respectively (Fig 1B). Therefore, all nonpathologic acini were judged as disomic. In contrast, of the 50 tumors, 19 tumors were disomic (38%), 19 were trisomic (38%), and 12 were found to be hypertetrasomic (24%) for chromosome 17. None of the tumors were found to be monosomic or tetrasomic for chromosome 17 (Fig 1D). The CI for chromosome 17 in nonpathologic acini was 1.48 ± 0.10 (range, 1.29 to 1.68), whereas the mean CI for chromosome 17 of all investigated PDAC was significantly higher, with a value of 2.54 ± 0.62 (range, 1.65 to 4.26; P < .001; Fig 2A). The degree of aneuploidy did not correlate with T stage, N stage, or the grade of tumor differentiation.
Confirmation of Tumor Ploidy by Additional Centromeric Probes To determine whether the increased copy number of chromosome 17 is a specific mutational event in some of our PDAC tumors or whether it reflects tumor cell ploidy in general, we applied additional centromeric probes for chromosomes 7 and 8 on consecutive tissue sections. Chromosomes 7 and 8 are among the most frequently altered chromosomes in PDAC,25,26 and hence tumors disomic for chromosome 17 should be truly diploid if they were judged to be disomic for these two chromosomes as well. We found that all but two tumors disomic for chromosome 17 were also disomic for chromosomes 7 and 8 (Fig 3A). The mean CI of chromosomes 7 and chromosome 8 was 1.69 ± 0.29 and 1.80 ± 0.34, respectively, and thus was similar to the CI of chromosome 17. When we tested five unselected samples of the trisomic group and five samples of the hypertetrasomic group with the additional probes, we could also confirm the aneuploidy of the tumors (Fig 3B). The CI and the distribution of the hybridization signals of all tested chromosomal probes defined similar groups of ploidy among PDAC tumors.
HER2 Amplification in PDAC Next we hybridized specific probes binding to the HER2 region on chromosome 17q12 (Fig 1C) and compared the signal counts between PDAC and normal cells (Fig 1E). The mean HER2 CNI for the tumor samples was significantly higher than that of nonpathologic pancreatic acini (4.11 ± 3.57; range, 2.04 to 18.8 v 1.39 ± 0.91; range, 1.21 to 1.55; P < .001; Fig 2A). Gene amplification is defined by the excess of a gene-specific hybridization signal over the chromosome-specific signal, and for HER2 is crucial for the prognostic impact in several human cancers. Therefore, we formed the ratio of the HER2 signals and the chromosome 17 signals. Twelve of 50 tumors (24%) displayed HER2 gene amplification, including two tumors with intrachromosomal high-level amplification (Fig 1C, arrowhead). Interestingly, HER2 amplifications were independent from the copy number of chromosome 17 and no significant correlation between the two was found (Fig 2B). In addition, HER2 amplification was not associated with primary tumor stage or grade of tumor differentiation (Table 1).
p185HER2 Expression in PDAC
Effect of Chromosome 17 Aneuploidy, HER2 Amplification, and Overexpression of p185HER2 on Survival The prognostic impact of HER2 amplification, p185HER2, and tumor ploidy, as measured by copy number of chromosome 17, was tested by Kaplan-Meier analysis. Although amplification of HER2 and overexpression of p185HER2 had no influence on the survival of PDAC patients (Figs 4A and B), we found significant differences for relapse-free and overall survival of patients with disomic, trisomic, and hypertetrasomic tumors. Patients with tumors hypertetrasomic for chromosome 17 had a significantly decreased relapse-free survival (median, 5.0 months) versus patients with trisomic tumors (median, 10.0 months) and patients with disomic tumors (median survival, 13.0 months; P = .0144; Figs 5A and B). The same effect of tumor cell ploidy was seen for the median cumulative overall survival (7, 12, and 20 months for hypertetrasomic, trisomic, and disomic tumors, respectively; P = .0099; Fig 5C). For Cox regression analysis, we used the AIC for model selection and included chromosome 17 aneuploidy, sex, age, pN and pT status, and grading in the first model. The starting AIC for the first model for relapse-free survival was 198.01 and the AIC for the final model was 194.94. The result showed a borderline independent prognostic influence of chromosome 17 hypertetrasomy for relapse-free survival (Table 4). The AIC of the starting model for overall survival was 172.61 and the final model with an AIC of 167.88 revealed an independent prognostic significance of chromosome 17 aneuploidy (Table 4).
Our study indicates that tumor ploidy levels correlate with disease-free and overall survival of operable PDAC patients. In contrast, no influence on patient outcome was found for the amplification of the HER2 oncogene or p185HER2 overexpression. We obtained the data from a relatively large cohort of PDAC patients applying an enzymatic DNA in situ hybridization method (CISH) that enabled us to probe chromosomal- and gene-specific loci by DNA hybridization and simultaneously assess the morphologic dignity of hematoxylin-stained tissue. We could define three distinct groups of tumors by their predominant chromosome 17 copy number: disomic, trisomic, and hypertetrasomic tumors. The three groups exhibited a significantly divergent biologic behavior. PDACs with predominantly disomic tumor cell populations were the least aggressive, reflected by the best postoperative survival among our patients. In contrast, patients with predominantly hypertetrasomic tumor cells suffered from an aggressive course of disease with extremely early metastatic relapse and early tumor-related death. The significant prognostic impact of tumor ploidy was independent from other established prognostic markers. When we hybridized additional centromeric probes to chromosomes 7 and 8, we were able to demonstrate that the chromosome 17 signals correctly reflected the ploidy level of the tumors and can therefore be used to investigate ploidy in PDAC. Interestingly, a bimodal distribution of the number of cytogenetic abnormalities has been observed in pancreatic cancers.20 It seems that at least two groups of tumors with differing biologic and clinical behaviors underlie this bimodal distribution. Thus, our subset of diploid PDAC with good prognosis may resemble cancers with a monotonically decreasing distribution of cytogenetic aberrations, such as breast and colon cancers that often show relatively long survival rates.20 In contrast, hypertetrasomic cancers comprising the second type within the bimodal distribution apparently form a group of aggressive cancers with unfavorable prognosis. It will be of clinical interest to investigate whether such subgroups also exist in other bimodal tumor entities such as head and neck, lung, and ovarian cancer. Surprisingly, neither oncogene amplification of HER2 nor protein overexpression conferred similar prognostic information as tumor ploidy levels, although we found an unexpectedly high frequency of HER2 gene locus amplifications (24%) in primary PDAC tumors.27 Of these, only a subgroup showed intrachromosomal high-level amplifications (homogeneously stained regions),28 which were associated with strong p185HER2 overexpression. In breast cancer, homogeneously stained regions represent the most frequent type of HER2 amplification and induce p185HER2 overexpression in 95% of the tumors.28 Apparently, high-level amplification engenders p185HER2 overexpression also in PDAC, albeit at a lower frequency when compared with breast cancer. Because it was shown recently that an antibody against p185HER2 (trastuzumab) inhibits growth of positive pancreatic carcinoma cell lines in vitro and in vivo,29 immunotherapy might be beneficial in PDAC patients with high-level HER2 amplification and strong p185HER2 overexpression. However, the majority of tumors with amplification of the HER2 locus did not overexpress p185HER2. In these tumors we observed clear single hybridization signals in excess of chromosome 17 signal numbers. It remains unclear whether this reflects amplification of the locus 17q12 in the form of double minutes, another type of amplification, or whether it reflects a gain of DNA from chromosome 17q. The high frequency of 17q gain that has been observed previously25,26 might indicate the existence of additional genes with relevance for pancreatic cancer progression. In conclusion, despite frequent amplification of the HER2 gene locus 17q12 and rare overexpression of p185HER2, no prognostic significance of HER2 was seen in PDAC. In contrast, patients with diploid and chromosomal-stable PDAC tumors had a favorable course of disease when compared with patients whose tumors displayed extensive chromosomal instability as measured by chromosome 17 CISH. Given that the latter group is at high risk for metastatic relapse, chromosome 17 CISH should be useful for patient stratification in therapeutic trials.
The authors indicated no potential conflicts of interest.
We thank Oleg Schmidt-Kittler, Thomas Blankenstein, and Jürgen Kraus for critical review of the manuscript.
Supported in part by a grant from the Hamburger Krebsgesellschaft, a grant from the Deutsche Forschungsgemeinschaft STO 464/1-1, and by a grant from the Werner Otto Stiftung. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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8. Slamon DJ, Leyland-Jones B, Shak S, et al: Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783-792, 2001 9. Yarden Y, Sliwkowski MX: Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2:127-137, 2001[CrossRef][Medline] 10. Sauer T, Wiedswang G, Boudjema G, et al: Assessment of HER-2/neu overexpression and/or gene amplification in breast carcinomas: Should in situ hybridization be the method of choice? Apmis 111:444-450, 2003[CrossRef][Medline] 11. Battifora H: HER2/neu immunostaining in pancreatic carcinoma. Hum Pathol 25:549-550, 1994[Medline] 12. Yamanaka Y, Friess H, Kobrin MS, et al: Overexpression of HER2/neu oncogene in human pancreatic carcinoma. Hum Pathol 24:1127-1134, 1993[CrossRef][Medline] 13. Okada N, Ohshio G, Yamaki K, et al: Elevated serum c-erbB-2 protein levels in patients with pancreatic cancer: Correlation to metastasis and shorter survival. Oncology 52:392-396, 1995[Medline] 14. Day JD, Digiuseppe JA, Yeo C, et al: Immunohistochemical evaluation of HER-2/neu expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasms. Hum Pathol 27:119-124, 1996[CrossRef][Medline] 15. Dugan MC, Dergham ST, Kucway R, et al: HER-2/neu expression in pancreatic adenocarcinoma: Relation to tumor differentiation and survival. Pancreas 14:229-236, 1997[Medline] 16. Koeppen HK, Wright BD, Burt AD, et al: Overexpression of HER2/neu in solid tumours: An immunohistochemical survey. Histopathology 38:96-104, 2001[CrossRef][Medline] 17. Novotny J, Petruzelka L, Vedralova J, et al: Prognostic significance of c-erbB-2 gene expression in pancreatic cancer patients. Neoplasma 48:188-191, 2001[Medline] 18. Safran H, Steinhoff M, Mangray S, et al: Overexpression of the HER-2/neu oncogene in pancreatic adenocarcinoma. Am J Clin Oncol 24:496-499, 2001[CrossRef][Medline] 19. Koka V, Potti A, Koch M, et al: Role of immunohistochemical identification of Her-2/neu and detection of variability in overexpression in pancreatic carcinoma. Anticancer Res 22:1593-1597, 2002[Medline] 20. Hoglund M, Gisselsson D, Mandahl N, et al: Multivariate analyses of genomic imbalances in solid tumors reveal distinct and converging pathways of karyotypic evolution. Genes Chromosomes Cancer 31:156-171, 2001[CrossRef][Medline] 21. Mendelin J, Grayson M, Wallis T, et al: Analysis of chromosome aneuploidy in breast carcinoma progression by using fluorescence in situ hybridization. Lab Invest 79:387-393, 1999[Medline] 22. Burnham KP, Anderson DR: Model Selection and Multimodel Interference (ed 2). New York, Springer, 2002
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
