Determination of the HER2 status

Transcription

1 Diagnostics Developing Pharmacodiagnostics * for HER2-Positive Gastric Cancer Jan Trøst Jørgensen, MScPharm, PhD Director, Dx-Rx Institute Baunevaenget 76 DK-3480 Fredensborg, Denmark External Lecturer, Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences University of Copenhagen, Denmark Determination of the HER2 status serves as an important predictive test for treatment with HER2 inhibitors in breast cancer, such as the monoclonal antibody trastuzumab (Herceptin, Genentech/Roche) and the tyrosine kinase inhibitor lapatinib (Tykerb, GSK) (1, 2). The relationship between HER2-positivity and a positive outcome, when treating with HER2 inhibitors, has been demonstrated in a number of studies (3, 4, 5). The HER2 protein is a 185-kDa transmembrane tyrosine kinase receptor and a member of the epidermal growth factor receptor (EGFR) family. Over-expression of HER2 has been found to promote tumorigenesis and to be involved in the pathogenesis of several human cancers (6). The gene that codes for the HER2 protein is located on chromosome 17 and has been found to be amplified with an increased copy number in about 20-25% of women with breast cancer (1). Over-expression of HER2 and amplification of the HER2 gene in breast cancer has been associated with a poor prognosis in this disease (1). A number of slide-based assays are available for the detection of both over-expression of the HER2 protein, measured by immunohistochemistry (IHC), and amplification of the HER2 gene, measured by fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization(cish) (1, 2). Amplifications and/or over-expression of HER2 have been reported in malignancies other than breast cancer, such as ovarian, prostate, colorectal, pancreatic and gastric cancers (7-11). Gastric cancer, especially, has attracted attention recently due to the encouraging results from the ToGA (trastuzumab with chemotherapy in HER2-positive advanced Gastric Cancer) trial. In this multicenter phase III study HER2-positive patients with advanced gastroesophageal and gastric adenocarcinoma were randomized to receive chemotherapy, or chemotherapy plus trastuzumab (12). Gastric cancer is a major global health problem. Recent data indicates that 1.4 million new cases of gastroesophageal and gastric cancer are diagnosed annually, and 1.1 million deaths are attributed to the diseases (13). There is a marked geographic variation in the incidence of the disease, which to some extent has shown to be higher in the developing countries (14). Gastric cancer is found more often in men than women with a distribution rate of approximately 2 to 1. Despite some advances in the prevention and treatment of the disease, the 5-year survival in most parts of the world still remains around 20%. Although the incidence of gastric cancer is declining, the prognosis for the disease remains poor (15). The poor survival rate is mainly explained by the advanced stage of the disease at the time of diagnosis. If screening for gastric cancer was performed as is the standard practice in Japan, the tumors could be detected at an earlier stage and thus surgical resection performed. Surgical resection has shown to increase the 5-year survival to above 50% (15). So far, treatment of advanced gastric cancer has been palliative, and no real consensuses exist in relation to the modalities used. Various chemotherapy combination have been tried, and it seems that the combination of 5-FU/capecitabine (Xeloda, Roche) and cisplatin or oxaliplatin, either *The word pharmacodiagnostic is a registered trademark of Dako. The word has been used to describe a pre-treatment measurement performed to aid in the assessment of whether a patient is likely to respond to a given therapy. 40 Connection 2010

2 Figure 1. Adenocarcinoma of the stomach stained with HercepTest (Dako), 3+ staining (20 ). Figure 2. Adenocarcinoma of the stomach stained with HER2 FISH pharmdx Kit (Dako), HER2 gene amplification (HER2/CEN ) (40 ). Connection

3 Reference Type of Assay N HER2-Positive Rate ToGA Trial (22) IHC/FISH 3, % Review publication (21) IHC 6, % Review publication (21) FISH/CISH % Table 1. HER2-Positive Rate in Gastric Cancer. alone or in combination with a third drug such as epirubicin (Pharmorubicin, Pfizer) or docetaxel (Taxotere, Sanofi-Aventis), are the most effective combinations. In a recent phase III study 457 patients with advanced gastric cancer were randomized to receive docetaxel, cisplatin, and 5-FU or cisplatin and 5-FU, and the median overall survival were 9.2 months and 8.6 months, respectively (14). Similarly in another study, 507 patients with advanced esophageal or gastric cancer were randomized to receive epirubicin, cisplatin, and 5-FU or epirubicin, oxaliplatin, and capecitabine. Here the overall survival was marginally higher, namely 9.6 months and 11.2 months, respectively (17). With a median overall survival of 8-11 months, the medical needs for new effective treatment modalities in advanced gastric cancer are obviously high. The use of the principles of stratified medicine where predictive biomarkers are combined with targeted drugs is an approach that will probably lead to a more effective treatment of patients with advanced gastric cancer as has been demonstrated in breast cancer (2, 18). In this brief review the background for, and the clinical development of, pharmacodiagnostics/ companiondiagnostics for HER2-positive gastroesophageal and gastric cancer will be discussed. The HER2 Status in Patients with Gastric Cancer Amplification of the HER2 gene and over-expression of the HER2 protein in gastric cancer were first described in 1986 (19, 21), and since then a number of studies have confirmed these findings. In a recent review publication, data on over-expression of the HER2 protein from 6,542 patients comprising 24 independent studies were listed (21). A rather large variation between the individual studies regarding the percentage of patients with HER2-positive tumors was seen (range %). The explanation of this is presumably multifactorial, such as the differences in the populations studied, but the most important aspects are probably the use of non-standardized assays and the application of different scoring criteria for the stained slides. When a weighted mean for the 24 studies was calculated, the percentage of patients with HER2-positive tumors was 19.0%. A number of these studies also reported that HER2 protein over-expression was associated with a poor prognosis of the disease (21). A somewhat smaller number of studies have investigated the amplification of the HER2 gene in patients with gastric cancer measured either by FISH or CISH, and these studies comprise data from a total of 869 patients (21). Some variation with regard to the percentage of patients with amplification of the HER2 gene ( %) is seen, though the magnitude is much smaller than seen for IHC. This may be explained by the fact that the FISH and CISH assays are quantitative methods compared to IHC, which use a semi-quantitative scoring system. When a weighted mean for the 5 studies was calculated, the percentage of patients with amplification was 19.4% (21). Examples of adenocarcinoma of the stomach with over-expression of the HER2 protein (IHC 3+) and amplification of the HER2 gene (HER2/CEN ) are shown in Figures 1 and 2, respectively. From the recent ToGA trial tumor samples from 3,667 gastric cancer patients were successfully screened with respect to HER2 status, which is by far the largest population published until now. In this study both HER2 gene amplification by FISH (HER2 FISH pharmdx, Dako) and HER2 protein over-expression by IHC (HercepTest, Dako) were measured in all patients during the screening phase (22). The results from the screening part of the study showed that 22.1% of the patients with advanced gastric cancer were HER2-positive either by FISH or IHC (22, 23). When looking at the European and the Asian patient populations no difference could be detected with regard to the HER2-positivity rate. As previously observed, the number of HER2-positive patients were shown to be higher when the cancer was localized to the gastroesophageal junction compared to when the cancer was localized to the stomach (24). When the tumors were classified according to the Laurén classification the intestinal type showed a higher HER2-positive rate than the diffuse or mixed type (23). The HER2 results from the ToGA trial should be regarded as reliable due to the fact that the analysis were performed at one single central laboratory 42 Connection 2010

4 using standardized HER2 IHC and FISH assays (25). Table 1 compares the findings of IHC and FISH for the HER2-positive rate of gastric cancer from the ToGA trial using the data from a recent review article (21). For approximately half of the studies reported in the literature on the HER2 status, either over-expression or amplification provides prognostic information (21). This is also in line with what is known from breast cancer. A recent meta-analysis, based on data from 39,730 patients, showed an overall HER2-positive rate of 22.2% and a mean relative risk for overall survival (OS) of 2.74 (1). A few studies have investigated the relationship between HER2 amplification and HER2 over-expression in gastric cancer patients using FISH and IHC, and from these studies the concordance between the two assays seems to be relatively high, ranging from 86.9% to 96.4% (25, 26, 27). The results from these studies seem to indicate that an amplification of the HER2 gene results in an over-expression of the HER2 protein as is known from breast cancer (28, 29, 30). Most of the studies that have investigated the relationship between HER2 amplification and HER2 over-expression in gastric cancer are relatively small, at least compared to the screening population from the ToGA trial that consists of 3,667 successfully tested patients. If we look at the results available so far, the relationship between the gene and the protein does not seem to be so unambiguous as the results from the studies described above. In the abstract from the ASCO 2009 meeting it was stated that the concordance between IHC and FISH was 87.5% (23), which is in line with what has been reported from other studies. However, in the abstract it has also been mentioned that In breast cancer most IHC 0/1+ samples are FISH negative, but in ToGA, the frequency of IHC 0/1+ samples testing FISH positive was almost as high as IHC 2+/FISH positive samples (23% vs. 26%) (23). As indicated in the abstract this is different from what has been observed in breast cancer, where the best assays show a concordance around 95% (30). The population with IHC 0/1+ and FISH+ tumors comprised 131 patients, which corresponded to approximately 23% of the 584 patients enrolled in the ToGA trial (12). At the European Cancer Organization (ECCO 15)/ European Society for Medical Oncology (ESMO 34) meeting in Berlin in September 2009 an abstract/poster about the HER2 testing in the ToGA trial was presented (22). This poster provided a more detailed explanation of how the high concordance was reached despite the relative large patient population with IHC 0/1+ and FISH+ tumors in the ToGA trial. It was here reported that the concordance between IHC and FISH was calculated based on the screening population and not the study population. From the poster it also appeared that 190 out of the 3,667 successfully tested patients had tumors that were IHC 0/1+ and FISH+ (22). Based on this recent data it may be concluded that the concordance between IHC and FISH, for the patients screened for the ToGA trial, is in line with what has previously been reported from other studies in gastric cancer (25, 26, 27), and what is known from breast cancer (28, 29, 30). Preclinical Studies Based on the observations of over-expression and amplification of HER2 in gastric cancer tumors and the experiences from breast cancer studies, it seems rational to test the hypothesis of an antitumor effect of HER2 inhibition in preclinical tumor models. The first in vitro and in vivo studies were conducted in 1992 using a combination of two monoclonal anti-her2-specific antibodies. A growth HER2 has shown its importance as target in the treatment of cancer and paves the way for a new treatment modality in patients with unresectable gastric cancer. inhibitory effect of the HER2 inhibition was demonstrated in vitro on the cultured human gastric tumor cell line NCI-N87, and in vivo when the same cell line was growing as xenografts in nude mice (31). Subsequently, these studies have been repeated with both trastuzumab and lapatinib using the NCI-N87 cell line as well as other HER2-positive gastric tumor cell lines, such as SMU-216, 4-1ST, and MKN-45P (24, 32-35). In these different preclinical studies trastuzumab and lapatinib have shown to be effective both as single agents as well as in combination with the chemotherapeutic agents that are widely used for the treatment of gastric cancer, such as 5-FU, capecitabine (Xeloda, Roche), irinotecan (Camptosar, Pfizer), cisplatin, paclitaxel (Taxol, Bristol Myers Squibb), etc. In one of the in vivo studies the three-drug combination of capecitabine, cisplatin and trastuzumab showed a remarkable tumor growth inhibitory effect in the NCI-N87 tumor xenograft model (34). In fact, this drug combination is the one that was used later in the ToGA trial, and proved effective in patients with advanced gastric cancer (12). Based on these results from epidemiological studies with regard to the frequency of HER2-positivity and the preclinical in vitro and in vivo studies, the scientific rationale for initiating clinical trials with trastuzumab in HER2-positive gastric cancer seem to have been well substantiated. Connection

5 The ToGA Trial Except for the data from the ToGA trial, clinical documentation for the use of trastuzumab in gastric cancer patients is relatively modest. A few case studies have been published in 2005 and 2006 where trastuzumab in combination with chemotherapy (5-FU/capecitabine and docetaxel) was successfully given to a few patients (36, 37). Further, abstracts on ongoing pilot/phase II studies have been presented where trastuzumab is given either as monotherapy or in combination chemotherapy (cisplatin, docetaxel), but no final results from these studies have been reported yet (38-40). The ToGA trial is, so far, the only well-controlled published study with a HER2 inhibitor in advanced gastric cancer. The study was conducted in 24 countries, which included the following parts of the world: Asia, Australia, Europe, Central and South America, and South Africa (12). The trial was designed as an open labeled, randomized multicenter phase III study in HER2-positive patients with advanced gastroesophageal and gastric adenocarcinoma. HER2-positivity was defined as being either IHC positive (3+) or positive by HER2 FISH (HER2/CEN ). The IHC testing was performed using the HercepTest (Dako) with slightly modified scoring criteria as described in the publication by Hofmann, et al. (25). The FISH testing was performed using the HER2 FISH pharmdx Kit (Dako). After enrollment in the study the patients were randomized to receive chemotherapy (5-FU or capecitabine and cisplatin) or chemotherapy plus trastuzumab. In total, 584 patients were randomized. Trastuzumab was given as a loading dose of 8 mg/kg followed by 6 mg/kg every third week until progression of the disease. The primary endpoint in the study was overall survival (OS), and the secondary endpoints included, among others, overall response rate (ORR) and progression free survival (PFS) (12). The aim of the ToGA trial served two purposes; firstly, it showed the safety and efficacy of trastuzumab combined with standard chemotherapy, and secondly, it demonstrated the predictive characteristics of the HER2 IHC and FISH tests used in the study. For the primary endpoint the combination of chemotherapy plus trastuzumab showed to be statistically superior to chemotherapy alone. The median OS increased from 11.1 to 13.8 months (p=0.0046) with a hazard ratio of 0.74 (95% CI: ). The secondary endpoints, both ORR and PFS, also showed superiority in favor of the combined treatment with chemotherapy and trastuzumab (12). A subgroup analysis performed looking at the effect of the combined treatment of trastuzumab plus chemotherapy, with respect OS (Months) IHC0/FISH+ (n=61) IHC1+/FISH+ (n=70) to HER2 status showed that the survival benefit provided by trastuzumab seems to be dependent on the level of HER2 protein over-expression. The single group of patients with the greatest survival benefit was the one with a HER2 test result of IHC 3+. Here the median OS increased to 17.9 months for the group treated with the combination of trastuzumab and chemotherapy. The hazard ratio for this group of patients was 0.58 (95% CI: ) (12). The results of the subgroup analysis for the different IHC scores are shown in Figure 3. Based on the information from the subgroup analysis, an explorative analysis was performed on a sub-population of the originally included patients. This population IHC2+/FISH+ (n=159) IHC3+/FISH+ (n=256) C C+T Figure 3. Median Overall Survival (OS) in months for the four individual HER2 IHC scores for two treatment groups; namely the chemotherapy group (C) and the chemotherapy plus trastuzumab group (C+T) (12). 44 Connection 2010

6 comprised the patients who were IHC 3+ positive or IHC 2+ positive and FISH positive. A total of 446 patients fulfilled these criteria, and the median OS for the group of patients who had received chemotherapy plus trastuzumab increased to 16.0 months compared to 11.8 months for the patients on chemotherapy alone. The hazard ratio for this analysis was 0.65 (95% CI: ). The median follow-up for all the patients in the ToGA trial was reported to be 17.1 months (12). Concerning the predictive properties of the two HER2 assays, both the subgroup and the explorative analysis showed that the IHC test should be used as the primary test for selection of patients for treatment with trastuzumab. As shown in Figure 3, the effect of trastuzumab seems to be dependent on the degree of over-expression of the HER2 protein, with the best median OS in the group of patients that were IHC 3+. Further, when looking at the HER2 test results from the ToGA trial only, but not the screening population, the agreement between over-expression of the HER2 protein and amplification of the gene is found to be somewhat lower in gastric cancer than normally observed in breast cancer. A relatively high number of HER2 FISH positive cases were found among the IHC 0 and IHC 1+ tumors (22, 23). In December 2009, the European Medicines Agency s (EMEA) Committee on Human Medicinal Products (CHMP) gave a positive opinion for extending the indication of Herceptin, Genentech/ Roche to include treatment of patients with HER2-positive metastatic adenocarcinoma of the stomach or gastro-esophageal junction based on the results from the ToGA trial. With respect to the treatment selection, the CHMP stated in the approval letter that Herceptin should only be used in patients with metastatic gastric cancer whose tumors have HER2 over-expression as defined by IHC 2+ and a confirmatory FISH+ result, or IHC 3+ as determined by an accurate and validated assay (41). This again emphasizes the fact that IHC should be regarded as the primary test for the selection of gastric cancer patients who may be considered potential candidates for treatment with trastuzumab. Conclusion The data from the ToGA trial should be considered encouraging and a major step forward in the treatment of advanced gastric cancer. Again, HER2 has shown its importance as target in the treatment of cancer and paves the way for a new treatment modality in patients with unresectable gastric cancer. Hopefully, this will open up for a development parallel to what has been seen in breast cancer treatment where the principles of stratified medicine have been practiced for years (42). The key driver in a more individualized and effective cancer treatment, including gastric cancer, is molecular diagnostics, and we will hopefully see an increasing number of drug-diagnostic combinations being introduced in the years to come (18, 43). Based on the encouraging results from the ToGA trial, it is now expected that routine HER2 testing of all patients with advanced gastric cancer will be performed worldwide. Glossary Lauren Classification: A histological classification of gastric carcinoma into the intestinal and diffuse/mixed types based on the criteria proposed by Pekka Lauren in 1965 (44). Hazard Ratio: An estimate of relative risk, which is frequently used in the statistical analysis of binary outcomes data, e.g., in clinical trials. In the ToGA trial it was used to compare the risk of death, in the group of patients who received trastuzumab plus chemotherapy versus the group of patients who received chemotherapy alone. CI: Confidence Interval. References 1. Ross JS, Slodkowska EA, Symmans WF, et al. The HER2 receptor and breast cancer: ten years of targeted anti-her2 therapy and personalized medicine. Oncologist 2009; 14: Jørgensen JT, Nielsen KV, Ejlertsen B. Pharmacodiagnostics and Targeted Therapies A rational approach for individualizing medical anti-cancer therapy in breast cancer. Oncologist 2007; 2: Slamon DJ, Leyland-Jones B, Shak S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that over-expresses HER2. N Engl J Med 2001; 344: Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005; 353: Piccart-Gebhart MJ, Procter M, Leyland-Jones B, et al. Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 2005; 353: Moasser MM. The oncogene HER2: Its signaling and transforming functions and its role in human cancer pathogenesis. Oncogene 2007; 26: Slamon DJ, Godolphin E, Jones LA, et al. Studies of the HER2/neu proto-oncogene in human breast and ovarian cancer. Science 1989; 244: Morris MJ, Reuter VE, Kelly, WK, et al. HER2 Prolining and Targeting in Prostate Carcinoma. Cancer 2002; 94: Schuell B, Gruenberger T, Scheithauer W, et al. HER2/neu protein expression in colorectal cancer. BMC Cancer 2006; 123: Safran H, Steinhoff M, Mangray S, et al. Over-expression of the HER2/neu oncogene in pancreatic adenocarcinoma. Am J Clin Oncol 2001; 24: Gravalos C, Jimeno A. HER2 in gastric cancer: a new prognostic factor and a novel therapeutic target. Ann Oncol 2008; 19: Van Cutsem E, Kang Y, Chung H, et al. Efficacy results from the ToGA trial: A phase III study of trastuzumab added to standard chemotherapy in first-line human epidermal growth factor receptor 2 positive advanced gastric cancer. J Clin Oncol 2009; 27:18s, (suppl; abstr LBA2409). ( 13. Comella P, Franco L, Casaretti R, et al. Emerging Role of Capecitabine in Gastric Cancer. Pharmacotherapy 2009; 29: Catalano V, Labianca R, Beretta GD, et al. Gastric Cancer. Crit Rev Oncol Hematol 2009; 71: Kamangar F, Dores GM, Anderson WF. Patterns of Cancer Incidence, Mortality, and Prevalence across Five Continents: Defining Priorities to Reduce Cancer Disparities in Different Geographic Regions of the World. J Clin Oncol 2006; 24: Connection

7 16. Van Cutsem, E, Moiseyenko VM, Tjulandin S, et al. Phase III Study of Docetaxel and Cisplatin Plus Fluorouracil Compared With Cisplatin and Fluorouracil As First-Line Therapy for Advanced Gastric Cancer: A Report of the V325 Study Group. J Clin Oncol 2006; 24: Cunningham D, Starling N, Rao S, et al. Capecitabine and Oxaliplatin for Advanced Esophagogastric Cancer. N Engl J Med 2008; 358: Jørgensen JT. From blockbuster medicine to personalized medicine. Per Med 2008; 5: Fukushige S, Matsubara K, Yoshida M, et al. Localization of a Novel c-erbb-related Gene, c-erbb-2. On Human Chromosome 17 and its Amplification in a Gastric Cancer Cell Line. Mol Cell Biol 1986; 6: Sakai K, Mori S, Kawamoto T, et al. Expression of epidermal growth factor receptors on normal epithelia and gastric carcinomas. J Natl Cancer Inst 1986; 77: Jørgensen JT. Targeted HER2 Treatment in Advanced Gastric Cancer. Oncology 2010; 78: Chung H, Bang YJ, Xu JM, et al. Human epidermal growth factor receptor 2 (HER2) in gastric cancer (GC): results of the ToGA trial screening programme and recommendations for HER2 testing. Eur J Cancer 2009; 7s:364 (Abstract 6511, ECCO 15/ESMO 34). 23. Bang Y, Chung H, Xu J, et al. Pathological features of advanced Gastric Cancer (GC): Relationship to human epidermal growth factor receptor 2 (HER2) positivity in the global screening programme of the ToGA trial. J Clin Oncol 2009; 27:15s (suppl; abstr 4556). 24. Tanner M, Hollmén M, Junttila TT, 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: 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: Yano T, Doi T, Ohtsu A, et al. Comparison of HER2 gene amplification assessed by fluorescence in situ hybridization and HER2 protein expression assessed by immunohistochemistry in gastric cancer. Oncol Rep 2006; 5: Marx AH, Tharun L, Muth J, et al. HER2 amplification is highly homogenous in gastric cancer. Hum Pathol 2009; 40: Lottner C, Schwarz S, Diermeier S, et al. Simultaneous detection of HER2/neu gene amplification and protein over-expression in paraffin-embedded breast cancer. J Pathol 2005; 205: O Malley PF, Thomson T, Julian J, et al. HER2 Testing in a Population-Based Study of patients with Metastatic Breast Cancer Treated with Trastuzumab. Arch Pathol Lab Med 2008; 132: Jørgensen JT. A Model for Drug-Diagnostic Co-Development - Trastuzumab (Herceptin ) and HercepTest. Abstract presented at the 4th Danish Conference on Biotechnology and Molecular Biology May 28-29, Vejle, Denmark. 31. Kasprzyk PG, Song SU, Di Fiore PP, et al. Therapy of an animal model of human gastric cancer using a combination of anti-erbb-2 monoclonal antibodies. Cancer Res 1992; 52: Kim SY, Kim HP, Kim YJ, et al. Trastuzumab inhibits the growth of human gastric cancer cell lines with HER2 amplification synergistically with cisplatin. Int J Oncol 2008; 32: Matsui Y, Inomata M, Tojigamori M, et al. Suppression of tumor growth in human gastric cancer with HER2 over-expression by an anti-her2 antibody in a murine model. Int J Oncol 2005; 27: Fujimoto-Ouchi K, Sekiguchi F, Yasuno H, et al. Antitumor activity of trastuzumab in combination with chemotherapy in human gastric cancer xenograft models. Cancer Chemother Pharmacol 2007; 59: Kim JW, Kim HP, Im SA, et al. The growth inhibitory effect of lapatinib, a dual inhibitor of EGFR and HER2 tyrosine kinase, in gastric cancer cell lines. Cancer Lett 2008; 272: Inui T, Asakawa A, Morita Y, et al. HER2 over-expression and targeted treatment by trastuzumab in a very old patient with gastric cancer. J Intern Med 2006; 260: Rebischung C, Barnoud R, Stéfani L, et al. The effectiveness of trastuzumab (Herceptin ) combined with chemotherapy for gastric carcinoma with over-expression of the c-erbb-2 protein. Gastric Cancer 2005; 8: Rech J, Arnold D, Folprecht G. et al. A pilot study of Trastuzumab mono therapy in patients who progressed while on chemotherapy for metastatic or locally advanced HER2-postive gastric cancer. Ann Oncol 2006; 17 (Suppl 9):314 (Abstr. 1096P). 39. Nicholas G, Cripps C, Heather-Jane A, et al. Early results of a trial of Trastuzumab, Cisplatin, and Docetaxel (TCD) for the treatment of metastatic gastric cancer over-expressing HER2. Ann Oncol 2006; 17 (Suppl 9): 316 (Abstr. 1105P). 40. Cortes-Funes H, Rivera F, Ales I, et al. Phase II of trastuzumab and cisplatin in patients with advanced gastric cancer with HER2/neu over-expression/ amplification. J Clin Oncol 2007; 15; 18S (Abstr. 4613). 41. Committee for Medical Products for Human use Post-Authorisation Summary of Positive Opinion for Herceptin. EMEA-CHMP, London, 17 December, Jørgensen JT, Winther H. The new era of personalized medicine: 10 years later. Per Med 2009; 6: Jørgensen JT. Are we approaching the post-blockbuster era? Pharmacodiagnostics and rational drug development. Expert Rev Mol Diagn 2008; 8: Lauren P. The Two histological main types of gastric carcinoma: Diffuse and so-called Intestinal-type carcinoma. Acta Path et Mircobiol Scandinav 1965; 64: Connection 2010