1. Kathleen D. Danenberg Response Genetics, Inc. Predictive and Prognostic Markers for Gastric Cancer

2. ERCC1

3. Why would a predictive test for platinum efficacy be desirable?

4. Dank et al. Randomized phase III study comparing irinotecan combined with 5-fluorouracil and folinic acid to cisplatin combined with 5-fluorouracil in chemotherapy naive patients with advanced adenocarcinoma of the stomach or esophagogastric junction. Annals of Oncology. 2008; 19:1450-1457. IF (n=170): irinotecan, folinic acid 5-fluorouracil; CF (n=163): cisplatin and 5-fluorouracil Median TTP IF: 5.0 months CF: 4.2 months Median OS: IF: 9.0 months CF: 8.7 months Non-platin and platin therapy have similar outcomes

5. …but non-platin treatment is less toxic: Comparative toxicity profiles of CF and IF Dank et al. Annals of Oncology. 2008; 19:1450-1457.

6. “… Irinotecan/5-fluorouracil (IF) is a platinum-free regimen that has similar efficacy to cisplatin/5- fluorouracil (CF) but with improved tolerance. As such, IF could represent a potential platinum-free alternative backbone to be combined with new targeted agents to be explored for the treatment of metastatic gastric cancer. Dank et al. Annals of Oncology. 2008; 19:1450-1457. Conclusions

7. The platins react with DNA to form inter- and intra-strand crosslinks

8. ERCC1 is part of the nucleotide excision repair complex that repairs platin crosslinks in DNA

9. Pre-clinical studies show ERCC1 to be a direct determinant of cisplatin efficacy ERCC1 small interfering RNA expression reduces ERCC1 expression and sensitizes the cells to platinum-containing chemotherapeutic agents. Youn et al. Oncogenic H-Ras Up-Regulates Expression of ERCC1 to Protect Cells from Platinum-Based Anticancer Agents Cancer Res 2004:64, 4849-4857.

10. ERCC1 gene expression in gastric cancer cells negatively correlates with sensitivity to cisplatin. ERCC1 mRNA expression levels and sensitivity to cisplatin in cells from malignant effusions collected from untreated gastric cancer patients (P= 0.014, r = 0.685). Wang et al. ERCC1 and BRCA1 mRNA expression levels in metastatic malignant effusions is associated with chemosensitivity to cisplatin and/or docetaxel. BMC Cancer 2008;8:97.

11. Clinical Study ERCC1 Threshold for Platin Sensitivity: Response Genetics Scale Percent Patients with Low ERCC1BenefitRef NSCLC: GILT (Platin Doublets)ERCC1<1.753RR=53%Cobo et al JCO 2007 NSCLC: MADeIT (Platin Doublets)ERCC1<1.4450 RR=44%, Increased SurvivalSim et al JCO 2007 CRC: FOLFOXERCC1<1.780 Increased Survival and ResponseShirota et al JCO 2001 CRC: FOLFOX ValidationERCC1<1.780 Increased SurvivalLenz et al ASCO 2008 Gastric: 5-FU/CisERCC1<1.4650 Increased SurvivalMetzger et al JCO 1998 Gastric: FOLFOXERCC1<1.7964 Increased SurvivalJ Wei et al ASCO 2007 Gastric: FOLFOXERCC1<2.280 Increased Survival J Wei et al British J of Cancer 2008 Gastric: Platin (S-1/Oxaliplatin)ERCC1<1.8567 Increased RR and Survival Matsubara et al British J of Cancer 2008 ERCC1 thresholds and benefit of low ERCC1 from platin therapy

12. ERCC1 mRNA levels and response in gastric cancer patients receiving FP Metzger R, et al. J Clin Oncol. 1998;16:309-316. p=0.004 by Kruskal- Wallis test. ERCC1 Expression 20 16 12 8 4 0 ResponseNo Response

13. ERCC1 mRNA levels and survival of advanced gastric cancer patients treated with a FOLFOX regimen Conclusion: “In patients with high mRNA levels of ERCC1, alternative chemotherapy regimens should be considered.” p<0.0001 Wei J et al. Br J Cancer. 2008;98:1398-402.

14. Effect of ERCC1 protein expression on survival in FOLFOX chemotherapy of advanced gastric cancer Overall survival curve according to ERCC1 expression measured by IHC (P = 0.0396). ERCC1 was the only significant independent prognostic factor impacted on OS (hazard ratio 1.91, P = 0.037). Kwon et al. Ann Oncol. 2007;18:504-9.

15. Low ercc1High ercc1 P valueHazard Adjuvant therapy 47 mos.7 mos.0.012.77 Surgery only 12 mos.33 mos.0.038 Low ercc1High ercc1 P valueHazard Adjuvant therapy undefined13 mos.0.0072.68 Surgery only 21 mos.43 mos.0.0040.18 Median RFS Median OS Yiu et al. ASCO 2010 abstract 29 Prediction of survival by ERCC1 expression in gastric cancer treated with surgery followed by FOLFOX or receiving surgery alone.

16. PELF = cisplatin (P), epirubicin (E), leucovorin (L), 5-fluorouracil (F) Conclusion: “IHC studies for ERCC1 might be useful to predict the clinical outcome in MGC patients treated with PELF regimen.” Natoli et al. J Clin Oncol 28, 2010 (suppl; abstr e14603) ERCC1 expression and activity of PELF regimen as first-line treatment of metastatic gastric cancer.

17. Br J Cancer. 2008; 98: 832–839.Matsubara et al. ERCC1expression and outcomes of advanced gastric cancer patients treated with cisplatin and S-1.

18. 0.0 0.2 0.4 0.6 0.8 1.0 0 12 24 36 48 60 72 84 96 log-rank P <.001 Months since start of 1st-line chemotherapy Probability of survival Low ERCC1 and low DPD expression: median survival time, 15.5 months Any high expression: median survival time, 10.2 months Impact of low ERCC1 and DPD on the outcomes of advanced gastric cancer. Br J Cancer. 2008; 98: 832–839.Matsubara et al.

19. ASSIGNMENTASSIGNMENT RANDOMRANDOM n=200, Endpoints: feasibility and increase of PFS Genotypic Arm: ERCC1 Selection High ERCC1: CPT11/docetaxel ASSIGNMENTASSIGNMENT RANDOMRANDOM High ERCC1: FOLFOX SWOG proposed prospective trial using ERCC1 to select CPT11/docetaxel or FOLFOX

20. EGFR expression

21. Chemotherapy with EGFR-targeted agents: KRAS and EGFR mutations are rare in gastric adenocarcinoma Mammano E etal. Anticancer Res. 2006;26:3547-50. –in 49 gastric adenocarcinomas, no specific EGFR gene mutations were detected. S.W. Han et al. Br J Cancer 2009;100:298-304. –In 38 gastric patients, no EGFR amplification or K-ras mutations were observed. These findings suggest a priori that: a)due to lack of EGFR mutations, gastric tumors will not be very sensitive to EGFR-directed TKI’s (e.g., gefitinib and erlotinib) b) however, due to the lack of KRAS mutations, they may be sensitive to EGFR-directed antibodies

22. EGFR Tyrosine Kinase Inhibitors: Phase II, Adenocarcinoma GastricNumber Patients% Response Dragovich (Erlotinib) 250% Doi (Gefitinib)751% GE Junction Ferry (Gefitinib)2711% Janmaat (Gefitinib)260% Tew (Erlotinib)170% Dragovich (Erlotinib) 43 Total: 7/113 9% 6% Doi 1036 Proc ASCO 22, 2003; Ferry Clin Can Res 132:5869; 2007 Janmaat JCO 24: 1612; 2006;Tew GI ASCO 2005; Dragovich JCO 24: 4922; 2006

23. Bouche O et al. J Clin Oncol 2004;22:4319-4328. PFS and OS with “classical chemotherapy” SurvivalLV5FU2 (n=45) LV5FU2- cisplatin (n=44) LV5FU2- irinotecan (n=45) OS, months6.89.511.3 1-yr OS, %3143 PFS, months3.24.96.9 Abbreviations: LV5FU2), leucovorin-5-FU; OS, overall survival; PFS, progression-free survival

24. Moehler et al.. Ann Oncol. 2010 Nov 30.: Patients with a complete response (CR) or partial response (PR) had significantly longer OS times and PFS times than patients with SD or PD. Moehler et al.. Ann Oncol. 2010 Nov 30. PFS and OS are increased by addition of cetuximab Bouche et al.. J Clin Oncol 2004;22:4319-4328

25. Predicting cetuximab activity Since KRAS mutations and EGFR mutations are rare in gastric tumors, can EGFR expression levels predict response to cetuximab?

26. -For EGFR (+) patients, both TTP (median 7.2 vs 5.0 months, P=0.020) and OS (not reached vs 7.6 months, P=0.013) were significantly longer after adjusting for clinical factors. S.W. Han et al Br J Cancer. 2009;100:298-304. Phase II study and biomarker analysis of cetuximab combined with modified FOLFOX6 in advanced gastric cancer. Response Overall (n=38)50% EGFR(+), low serum ligands (n=11) 100% Remainder (n=27) 37%

27. -but tumor EGFR expression did not correlate with PFS (log-rank P = 0.567) or OS (log-rank P = 0.663). Moehler et al. Ann Oncol. 2010 Nov 30. Cetuximab with irinotecan, folinic acid and 5-FU as first-line treatment in advanced gastroesophageal cancer: a prospective multi-center biomarker-oriented phase II study.

28. -EGFR expression did not significantly correlate with ORR Pinto et al. Phase II study of cetuximab in combination with FOLFIRI in patients with untreated advanced gastric or gastroesophageal junction adenocarcinoma (FOLCETUX study). Ann Oncol 2007;18:510-517. Phase II study of cetuximab plus FOLFIRI in patients with untreated advanced gastric or GE junction adenocarcinoma (FOLCETUX study).

29. Discrepant results for EGFR expression level as a predictive factor for cetuximab therapy

30. HER2 status

31. HER2 inhibitors trastuzumab and lapatinib in gastric cancer ASCO 2008, Abstr 4526, Bang, et al. –Analysis of 2484 gastric cancer samples from the Ph III ToGA trial –21.9% HER2 positivity ASCO 2009, Abstr LBA 4509, ToGA Trial –Rand Ph III, HER2+ gastric cancer –5-FU/capecitabine + cisplatin +/- trastuzumab –RR 47.3 vs. 34.5%, OS 13.5 vs. 11.1 mo (p = 0.0048) –HR 0.74 (0.60-0.91) –Practice changing!!! LOGIC Trial –Rand Ph III, HER 2+ gastric cancer –Capecitabine + oxaliplatin +/- lapatinib

32. The ToGA trial: Primary end point- OS Time (months) 294 290 246 223 209 185 173 143 147 117 113 90 64 71 47 56 32 43 24 30 16 21 14 13 7 12 6 6565 4040 1010 0000 No. at risk 11.113.8 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0246810 12 14 16 182022242628303234 36 Event FC + T FC Events 167 182 HR 0.74 95% CI 0.60, 0.91 p value 0.0046 Median OS 13.8 11.1 T, trastuzumab

33. Rüschoff et al. Virchows Arch. 2010 457:299-307. HER2 assay by IHC for gastric cancer required a different set of guidelines than for breast

34. Sources of HER2 Testing Variation with IHC Pre-analytic Time to fixation Method of tissue processing Time of fixation Type of fixation Analytic Assay validation Equipment calibration Use of standardized laboratory procedures Training and competency assessment of staff Type of antigen retrieval Test reagents Use of standardized control materials Use of automated laboratory methods Post-analytic Interpretation criteria Use of image analysis Reporting elements Quality assurance procedures Laboratory accreditation Proficiency testing Pathologist competency assessment 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;25:118-45.

35. Press et al. HER-2 Gene Amplification, HER-2 and Epidermal Growth Factor Receptor mRNA and Protein Expression, and Lapatinib Efficacy in Women with Metastatic Breast Cancer. Clin Cancer Res 2008; 14: 7861 HER-2 mRNA expression by PCR correlates with HER-2 FISH (r=0.83) and IHC (r=0.72)

36. Comparison of HER2 expression and amplification in primary breast tumors (T) and corresponding lymph node metastases (N) determined with IHC, FISH, and quantitative RT-PCR Vinatzer et al. Clin Cancer Res 2005;11:8348-8357 IHC HER2 expression scored as 0 and 1+ (=negative) or 2+ and 3+ (=positive) is indicated. FISH analysis: red, HER2 signals; green, centromere 17 signals. +, specimens harboring a HER2 amplification; −, nonamplified specimens. Quantitative RT-PCR: red line, the cutoff between high (scored as HER2 positive) and low relative expressions of HER2.

37. PCR quantitation of HER2 expression gives the same clinical information as IHC and FISH Vinatzer et al. Clin Cancer Res 2005;11:8348-8357 Quantitative RT-PCR: -simple, cost-effective, -rapidly produces quantitative, numerical, and reproducible results. -easily amenable to standardization, insensitive to inter-observer variability -results are a number, which can be either above or below a predetermined threshold. IHC -interpretation of IHC results is inherently difficult and time-consuming, requires experienced pathologists -is influenced by use of different antibodies, fixatives, staining protocols, and inter-observer variability. FISH -is quantitative and reproducible but results are more difficult to interpret than those of quantitative RT-PCR. -time-consuming, and requires specialized expertise and equipment.

38. Her2 gene expression associated with OS in patients with metastatic gastric cancer treated with lapatinib Chang H et al,Journal of Clinical Oncology, 2007 ASCO Annual Meeting Proceedings Part I. Vol 25, No. 18S (June 20 Supplement), 2007: 4647

39. Summary and conclusions ERCC1 mRNA expression appears to be a viable predictive marker for platin therapy. The jury is still out on EGFR expression as a predictive marker for cetuximab therapy. The IHC and FISH-based assay of HER2 has many issues so PCR should be investigated as an additional tool or as an alternative.