Since the approval of trastuzumab

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1 Diagnostics HER2 Diagnostics in Gastric Cancer Oliver Stoss, PhD, Martina Schmitt, PhD, Dirk Zielinski, PhD, Thomas Henkel, PhD Iris Nagelmeier, MD Josef Rüschoff, MD Targos Molecular Pathology Germaniastrasse 7 D Kassel, Germany Pathologie Nordhessen Germaniastrasse 7 D Kassel, Germany Since the approval of trastuzumab (Herceptin Genentech/Roche) a humanized monoclonal antibody directed against the HER2 receptor as the first targeted therapy in advanced gastric cancer in February 2010, the analysis of HER2 status in gastric cancer has evolved to be a very important task. The data of the ToGA trial showed that patients with HER2 protein over-expression benefit from this type of anti-her2 therapy (1). An ongoing phase III clinical trial with lapatinib in gastric cancer (LOGiC) is also dependent on accurate HER2 based patient stratification. Importance of the HER2 Receptor in Gastric Cancer The human epidermal growth factor receptor (EGFR) family consists of four receptors (ErbB1-4) that can be activated by ligand induced homo- or heterodimerization. Up to now, no ligand has been identified for ErbB2 (HER2). However, from breast cancer biology, it is known that the HER2 receptor plays an important role due to the fact that this receptor is constitutively active as a result of its special conformation. Upon over-expression, the ErbB2 receptor is a preferred binding partner for the other family members (2, 3) and triggers signal transduction which affects cell growth, apoptosis, metastasis and angiogenesis in breast cancer. Although much less is known about the molecular pathways induced by the HER2 receptor in gastric cancer compared to breast cancer, the current publication status suggests that the HER2 receptor plays a similar role in gastric cancer (Fig. 1). There is evidence that HER2 is a prognostic factor for gastric cancer. Similar to breast cancer, HER2 over-expression correlates with a shorter overall survival (4-7). A systematic analysis of ErbB receptor family members and their ligands along the horizontal and vertical gradient in the gastrointestinal tract has not yet been performed. In the ToGA trial, the rate of HER2 gene amplification and/or membrane over-expression was 22.1%, which is in a similar range to breast cancer. Nevertheless, there is a broad variation of HER2-positivity ranging from 6.8% to 34.0% for IHC and 7.1% to 42.6% for FISH reported in the literature (8). Apart from the different Based on the histological divergence of gastric cancer, in situ hybridization techniques assessable with bright field microscopy such as CISH are an attractive alternative to FISH. scoring systems that have been applied, the discrepancy is thought to be caused by additional parameters that are unique for gastric cancer. First, the underlying biology for the development of HER2-positive gastric cancer appears to be different in terms of expression pattern. In gastric cancer, the HER2 expressing cells are distributed more heterogeneously, pointing towards differences in the role of HER2 within the carcinogenesis between both diseases (9). ErbB-1, = epidermal growth factor receptor (EGFR) : ErbB-2, = HER2 in humans and neu in rodents: ErbB-3 = HER3 and ErbB-4, = HER4 (Courtesy: Wikipedia) HER2 is a gene (also called HER2/neu) 34 Connection 2010

2 Basolateral Compartment Apical Compartment Ligand-dependent Receptor Activation HER4 HER3 HER2 EGF-R Proliferation Anti-Apoptosis Migration Angiogenesis cbl Ub Receptor Ub Internatlisation and Degradation Ion Channel Sodium Influx Na+ Chloride Efflux CI- Receptor Transactivation and Signaling Crosstalk TACE Protease VEGF-R VEGF-R TNF-α-R Proteolytic Release of Ligands TGF-α 7TM Receptors GPCR Figure 1. Signaling events associated with the EGF receptor family. Whereas most of the interactions have been reported for the EGF receptor, HER2 is the preferred heterodimerization partner and thus important for activation of downstream signaling events. The constitutively active HER2 itself may trigger the described pathways completely by ligand-independent means of homodimerization. Activation of the EGF receptor may occur either ligand-dependent or ligand-independent via HER2 and leads to proliferation and induction of anti-apoptotic pathways as well as stimulation of migration and angiogenesis. All these processes may be induced by signaling crosstalk of a variety of other receptor tyrosine kinases, such as VEGF-R, IGF-R or TNF-α-R. G-protein coupled receptors may transactivate the EGF-R as well and trigger release of EGF-R ligands. Intracellular signaling of the EGF-R is regulated by ubiquitin-mediated receptor internalization and subsequent degradation. Activation of the EGF-R has impact on ion influx and efflux and thus maintenance of the normal gastric milieu. Growth factor receptors are exclusively located to the basolateral compartment of gastric cancer cells. Abbreviations: EGF-R: epidermal growth factor receptor; HER: human epidermal growth factor receptor; cbl: E3 ubiquitin ligase; Ub: ubiquitin; VEGF-R: vascular endothelial growth factor receptor; IGF-R: insulin-like growth factor receptor; TNF-α-R: tumor necrosis factor alpha receptor; TGF-α: transforming growth factor alpha; TACE: tumor necrosis factor alpha converting enzyme; 7TM Receptors: seven tans-membrane-domain receptor; GPCR: G-protein coupled receptor Connection

3 Second, HER2 over-expression/amplification has been observed predominantly in intestinal type gastric cancer and much less in diffuse gastric cancer specimens (1, 4). Finally, the rate of HER2 over-expression or amplification also depends on the exact tumor location. HER2 expression is higher in the cancer of the gastroesophageal junction compared to the stomach and may even be higher in esophageal cancer (1). Since the pathological effect of HER2 over-expression also depends on its binding partners, the next few paragraphs summarize the current knowledge about the other family members in gastric cancer. EGFR expression is observed in up to 63% of gastric cancer cases and is in 15% of the cases co-expressed with HER2. EGFR over-expression has been linked to worsen the prognosis of gastric cancer (10). Transforming growth factor TGF-α is thought to be the major EGFR ligand in the gastrointestinal tract (11). TGF-α suppresses gastric acidity, stimulates gastric epithelial cell proliferation and induces gastric mucus production. Increased EGFR expression is common in squamous cell carcinoma of the esophagus and has been associated with poor outcomes (12, 13). In addition, mutations in EGFR have been detected in 11.7% of esophageal adenocarcinomas and 14.2% of cases of Barrett s esophagus (14). ErbB2/ErbB3 heterodimers have been reported to play a central role in breast cancer progression. Recently, Zhang, et al. reported that they did not observe a coexpression of HER2 and HER3 in gastric cancer, with HER3 being predominantly expressed in 26.2% of tumors of the diffuse type (15). Therefore, HER2/HER3 dimers may play a minor role in gastric cancer compared to breast cancer. Further analysis of HER3 in gastric cancer are necessary due to conflicting data in this research area (16). HER3 may play a role in the progression to more dedifferentiated gastric cancers (17). Only very limited knowledge about ErbB4/ HER4 is currently available. In contrast to HER2, ErbB4 IHC staining appeared to be most intense in signet ring cancers (18). Although this section is focused on ErbB receptors, one should be aware that intensive cross talks between GPCRs, VEGF, TNF-α and IGF-Receptors exist (19). In addition, important cell surface signaling events include the regulation of ErbB receptor ligand release by proteolytic cleavage which also holds true for gastric cancer cells (20). Status of HER2 Testing: Immunohistochemistry and In situ hybridization By the use of the determination of the HER2 status in gastric cancer, the pathologist now decides about the use of high budget therapies of about 30,000 to 50,000 EUR per year (21). In addition, targeted therapeutics such as trastuzumab do have side effects and it is the ethical goal, not only to select HER2-positive patients for a therapy, but also to efficiently exclude HER2-negative patients from a HER2 targeted therapy. Different techniques are currently available to assess the HER2 status from tissue sections, among them; fluorescence in situ hybridization (FISH), silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), immunohistochemistry (IHC) or RT-PCR based methods. In addition, HER2 extracellular domain (ECD) can also be measured from serum samples using ELISA. The most commonly used techniques currently applied in routine testing are FISH and IHC. The advantage of IHC is that the method is inexpensive in terms of reagents and equipment. However, discussions about the accuracy of the test results are still ongoing due to the fact that IHC produces inconsistent results in breast cancer. HER2 IHC results depend on a large variety of external factors (Table 1). Therefore, large efforts have been undertaken to standardize HER2 IHC testing in breast cancer (22). The improvement can be measured by interlab discordance rates that were at about 27% in the early 2000s and are now in the range of about 14% (23). IHC results can typically be controlled by the use of batch controls and/or external on-slide controls. These types of controls verify if the staining reaction per se, was within the expected range. However, still one of the major disadvantages for IHC remains the absence of adequate internal controls within the tissue of interest to exclude sample related problems. Up to 8 of the 14 major factors influencing HER2 IHC can be standardized by the use of validated, commercially available HER2 testing kits (points 5-12 in Table 1). Nevertheless, different validated HER2 testing Predominant External Factors Influencing HER2 IHC Results: 1. Fixative 2. Fixation duration 3. Tissue slide thickness 4. Time between section preparation and staining 5. Antibody retrieval method 6. Antibody type 7. Antibody dilution 8. Incubation temperature 9. Incubation time 10. Antibody detection system 11. Use of autostainer 12. Scoring system 13. Training of pathologist 14. Use of HER2 IHC analysis software Table 1. Predominant external factors influencing HER2 IHC results. 36 Connection 2010

4 kits are available on the marked showing scan, which is unfortunatly time consuming different results especially in the equivocal and expensive. Table 2 shows an overview scoring range. In addition, a lot of efforts about major external factors influencing the and quality control programs exist in order to HER2 FISH results. standardize the evaluation of HER2 IHC results by pathologists in breast cancer. In gastric What are the implications for HER2 IHC and cancer, adequate training of the pathologist FISH in gastric cancer? In the ToGA trial, will even be of much higher importance due both HER2 IHC and FISH were used for the to the increased complexity of the scoring determination of HER2 status. The correlation system itself and due to the heterogeneous of the results obtained by both methods is staining pattern in gastric cancer. significant in gastric cancer, but appears to be less evident than in breast cancer. 7.5% of The problems of standardization of HER2 IHC 0 or 1+ cases are FISH positive suggesting IHC are the motivation for some institutions that, although gene amplification still appears to use FISH as the standard testing to be the major mechanism for HER2 receptor method for HER2. Indeed, according to our over-expression, other mechanisms for the experience, the use of different commercially regulation of HER2 expression also play a role available FISH kits is not the primary source in gastric cancer. Since the amount of HER2 of discrepancy between different labs. IHC positive, but FISH negative cases is very However, apart from the commonly referred small, it remains difficult to decide which of disadvantages of FISH, such as higher assay both methods has the higher predictive value costs and special equipment, one should for trastuzumab response. not forget that the selection of the area of interest is still subjective and may lead to Since we are at the beginning of HER2 testing, discrepancies between two readers. Second, there is currently no single method of choice FISH typically shows a higher sample related in gastric cancer. FISH reading appears to be failure rate since FISH is very sensitive to the more challenging in gastric cancer due to the fixative and the duration of fixation. Finally, large amount of cases with dispersed single documentation of FISH remains a challenge amplified cells, as well as the danger to oversee since the fluorescence signals diminish on the small amplified foci in large tissue sections slides. Also for proper image documentation, with heterogeneous HER2 amplification. one should be able to perform a full slide In addition, macrophages appear to show Predominant External Factors Influencing HER2 FISH Results: 1. Fixative 2. Fixation duration 3. Tissue slide thickness 4. Time between probe hybridization and analysis (due to signal bleaching) 5. Scoring system (including the amount of counted cells) 6. Training of pathologist / use of technician for counting 7. Use of HER2 FISH analysis software Table 2. Predominant external factors influencing HER2 FISH results. unspecific signals with currently available commercial FISH probes. It has also to be expected that, due to the subjective selection of tissue areas in cases with heterogeneous amplification patterns, the inter-lab variation will be higher in gastric cancer. Additional challenges for HER2 immunohistochemistry in gastric cancer include the unspecific staining of gastric mucosal metaplasia, as well as the heterogeneity of the intensity and the distribution of stained tumor cells. Finally, one type of discrepancy between IHC and FISH is simply caused by the official guidelines. Whereas for FISH, 20 tumor cells are generally sufficient to be counted, 10% of cells (usually several thousand cells) need to be stained by IHC according to the existing guidelines. This is especially a problem in case of focal HER2 expression which is more prominent in gastric cancer than in breast cancer. Based on the histological divergence of gastric cancer, in situ hybridization techniques assessable with bright field microscopy such as CISH are an attractive alternative to FISH. In breast cancer, these assays have been shown to deliver comparable results and comparative studies are required in gastric cancer. Testing Guidelines for HER2 in Gastric Cancer The origin for the development of HER2 IHC and FISH testing guidelines for gastric cancer is based on the experience with the Dako HercepTest and FISH evaluation criteria valid for breast cancer. For HER2 evaluation in breast cancer, a minimum cut-off for membrane-stained tumor cells exists. Below this cut-off value, the IHC result should be scored 0. Since at the start of the ToGA trial, the 10% cut-off for stained tumor cells was the standard for breast cancer HER2 IHC evaluation, this cut-off was also kept for gastric cancer surgical specimens. However, a separate gastric cancer validation Connection

5 The origin for the development of HER2 IHC and FISH testing guidelines for gastric cancer is based on the experience with the Dako HercepTest study initiated in 2005, detected necessary changes that need to be applied for HER2 IHC assessment in gastric cancer. Due to the heterogeneity of HER2 staining in gastric cancer, a consensus meeting of international pathologists agreed to skip the 10% cut-off rule for biopsies (8, 24). Since HER2 IHC negative and positive gastric cancer tumor regions may be adjacently located, biopsy sampling errors are much more likely in gastric than in breast cancer. Another adaption of the HER2 IHC assessment was necessary due to the histo-morphological nature of gastric adenocarcinoma cells. A complete HER2 membrane staining may not be achieved since the receptors are predominantly expressed at the basolateral surface. The pathophysiological significance of apical HER2 receptors, if any, is unknown. Therefore, HER2 membrane staining was also defined to be acceptable if it occurs either basolateral or lateral. The FISH cut-off ratio (HER2/CEP17) was kept at 2.0 (also standard for breast cancer in 2005) for the definition of HER2 gene amplification (assessed on 20 cells). Re-assessment of FISH results with 40 cells in the range of 1.8 to 2.2 is recommended. Based on the data of the ToGA trial, a HER2 analysis algorithm accepted by the European Medicines Agency (EMEA) is the primary use of IHC. If the specimen shows IHC 0 or IHC 1+ results, a patient will not receive trastuzumab. If the specimen is scored 3+, the patient is eligible for trastuzumab therapy. A score of 2+ is considered as equivocal and should be subject to ISH analysis. Quality Assurance Quality assurance programs for HER2 IHC testing in gastric cancer have now been initiated. HER2 staining performance and assessment are now included into the Quality Initiative in Pathology (QuIP) study, and other training programs will follow. Training courses for HER2 evaluation in gastric cancer are organized by different national pathology organizations as well as by commercially available international training courses (25). A French-German ring study was conducted recently in order to identify the pitfalls for HER2 testing in gastric cancer (26). In addition, attempts for standardization of HER2 testing and the recommendation of quality controls for breast cancer are also applicable for gastric cancer (22). Finally, plausibility checks can further reduce the failure rate. For instance, although HER2-positive results for diffuse gastric cancer are possible, they are relatively seldom and should be rechecked. Open Questions Open questions now arise in HER2 gastric cancer testing: Do patients with small HER2-positive foci benefit from a HER2 targeted therapy in the same manner than those with a high percentage of HER2-positive cells in the tumor? No data is available now that would address this question. Do patients with heterogeneously stained tumors benefit from a HER2 targeted therapy in the same manner than those with homogenously stained tumors? It is tempting to speculate that the underlying biology of a tumor showing dispersed amplified tumor cells is different from tumors showing homogenous amplification patterns. The question is, if this also holds true on the therapy level. Do HER2 IHC positive, but HER2 FISH negative patients benefit from a HER2 targeted therapy? Since the number of cases with this combination is very small, we will probably not receive an answer in the next few years. Do HER2 FISH positive but HER2 IHC negative patients benefit from a HER2 targeted therapy? The ToGA data suggest that the response to trastuzumab is less efficient for this group compared to the HER2 FISH positive and HER2 IHC positive group, but the number of cases is still small. Is the FISH cut-off value to distinguish positive from negative cases adequate? Currently, the HER2/CEN-17 cut-off is 2.0 whereas for breast cancer, the cut-off is 2.2. It remains to be verified if an increase of the cut-off in gastric cancer would increase the predictive value of the test. Is the IHC cut-off value for stained cells (currently 10% for gastric cancer) correct? For breast cancer, the cut-off has been increased from 10% to 30% (22). Since the staining pattern in gastric cancer is different, it remains questionable if the same cut-off values can be applied for in gastric and breast cancer. 38 Connection 2010

6 What is the difference between HER2 expression of primary and metastatic specimens? From breast cancer, we know that the concordance of the HER2 status between primary tumor and metastases is very high; although this may be dependent on the nature of the clinical trial (metastatic breast cancer, adjuvant or neo-adjuvant breast cancer). Data for gastric cancer are currently not available to our knowledge. How well does HER2 testing results of biopsies and matched surgical specimens correlate? Due to the heterogeneous HER2 expression in gastric cancer, one may assume that discrepancies in the HER2 results derived from biopsies compared to surgical specimens may be more likely compared to breast cancer. In addition, biopsies and surgical specimens need different fixation times that may influence the HER2 result. Studies looking at this aspect are currently ongoing. What about polysomy in gastric cancer? Polysomy defined as more than two centromeric signals of chromosome 17 per cell in average is a major cause of false negative FISH results. In case polysomy plays a major role in gastric cancer, it may be important to know if polysomic cases respond to HER2 targeted therapies. Conclusion The aim of future HER2 testing in gastric cancer will be the standardization of IHC and FISH testing. Since there are a number of pathologists world wide who are familiar with HER2 testing in breast cancer, but not in gastric cancer, there is a danger that pathologists may use breast cancer scoring criteria on gastric cancer samples that may lead to an underestimation of positive cases (27). From the technical perspective, the application of HER2 testing methods and guidelines in gastric cancer will benefit from the experiences in breast cancer. Due to the complexity of FISH evaluation in gastric cancer, it is tempting to speculate that ISH technologies based on bright field microscopy will replace FISH in the future since the parallel assessment of histology and HER2 amplification is highly appreciated. In addition, bright field ISH techniques will provide the possibility for easier long term slide storage at ambient temperature as well as for faster digitalization with less complex equipment. References 1. Van Cutsem, et al. Efficacy results from the ToGA trial: A phase III study of trastruzumab added to standard chemotherapy (CT) in first-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC). 2010; ASCO Abstract LBA Graus-Porta, et al. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 1997; 16: Waterman, et al. A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor. Mol Cell Biol 1996; 16: Tanner, et al. Amplification of HER2 in gastric carcinoma: association with topoisomerase II alpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab. Ann Oncol 2005; 16: Nakajima, et al. The prognostic significance of amplification and over-expression of c-met and c-erb B-2 in human gastric carcinomas. Cancer 1999; 85: Park, et al. HER2(neu amplification is an independent prognostic factor in gastric cancer. Dig Dis Sci 2006; 51: Yonemura, et al. Expression of c-erbb-2 protein is an independent indicator of poor short-term prognosis in patients with gastric carcinoma. Cancer 1991; 67: Hofmann, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology 2008; 52: HER2 amplification is highly homogenous in gastric cancer. Hum Pathol 2010; 41: ; author reply Tokunaga, et al. Clinical significance of epidermal growth factor (EGF), EGF receptor, and c-erb-2 in human gastric cancer. Cancer 1995; 75: Cartlidge, et al. Transforming growth factor alpha and epidermal growth factor levels in normal human gastrointestinal mucosa. Br J Cancer 1989; 60: Gibault, et al. Diffuse EGFR staining is associated with reduced overall survival in locally advanced oesophageal squamous cell cancer. Br J Cancer 2005; 93: Yano, et al. Immunohistologic detection of the epidermal growth factor receptor in human esophageal squamous cell carcinoma. Cancer 1991; 67: Kwak, et al. Epidermal growth factor receptor kinase domain mutations in esophageal and pancreatic adenocarcinomas. Clin Cancer Res 2006; 12: Zhang, et al. Comparative Study on Over-expression of HER2/neu and HER3 in Gastric Cancer. World J Surg 2009; 33: Sanidas, et al. Expression of the c-erbb-3 gene product in gastric cancer. Int J Cancer 1993; 54: Kobayashi, et al. Activation of ErbB3-PI3-kinase pathway is correlated with malignant phenotypes of adenocarcinomas. Oncogene 2003; 22: Kataoka, et al. Expression of mrna for heregulin and its receptor, ErbB-3 and ErbB4, in human upper gastrointestinal mucosa. Life Sci 1998; 63: Rodland, et al. Multiple mechanisms are responsible for transactivation of the epidermal growth factor receptor in mammary epithelial cells. J Biol Chem 2008; 283: Shida, et al. Lysophospholipids transactivate HER2/neu (erbb-2) in human gastric cancer cells. Biochem Biophys Res Commun 2005; 327: Neyt, et al. An economic evaluation of Herceptin in adjuvant setting: The Breast Cancer International Research Group 006 trial. Ann Oncol 2006; 17: Wolff, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 2007; 25: Roche, et al. Concordance between local and central laboratory HER2 testing in the breast intergroup trial N9831. J Natl Cancer Inst 2002; 94: Rüschoff, et al. HER2 testing in castric cancer: What is different in comparison to breast cancer? Pathologe 2010; 31: International HER2 training courses: Rüschoff, et al. HER2 Testing in Gastric Cancer: Guideline Validation and Development. Virchows Archiv; submitted. 27. Barros-Silva, et al. Association of ERBB2 gene status with histopathological parameters and disease-specific survival in gastric carcinoma patients. Br J Cancer 2009; 100: Connection