AIDS IN IDENTIFYING CANDIDATES FOR HER2-TARGETED THERAPY

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1 AIDS IN IDENTIFYING CANDIDATES FOR HER2-TARGETED THERAPY

2 THE HERMARK BREAST CANCER ASSAY HERmark is based on our proprietary VeraTag technology that precisely quantifies HER2 proteins and protein complexes in formalin-fixed, paraffin-embedded (FFPE) tissue specimens. 1 Produces direct quantitative measurement of HER2 protein, the target of current anti-her2 drug therapies. 2 Provides a continuous, rather than semiquantitative, measurement of HER2 total protein over a 3-log dynamic range. 2 Demonstrates a 7-to-10 fold improvement in sensitivity over IHC, with detection of HER2 levels down to ~2,500 receptors per cell. 2 Clinical Significance of HERmark HERmark Provides Accurate HER2 Assessment and Prognostic Value Accurate assessment of HER2 status in breast cancer is critical in identifying patients who may benefit from HER2-targeted therapy. Studies have shown that there still is a relatively high discordance level (18-27%) between central and local HER2 testing. 3,4 Representing real-world HER2 testing, a recent retrospective, multicenter Collaborative Biomarker Study (CBS) of breast cancer patients (not treated with trastuzumab) showed that the HERmark assay offers accurate quantification of HER2 protein expression that correlated best with central laboratory HER2 IHC re-testing, and less so with local laboratory HER2 IHC and FISH results: 5 HERmark reclassified 15% of local HER2 status negative results as HERmark positives. HERmark offers a more accurate assessment of HER2 status and may change therapy selection in approximately 20% of patients. HERmark has shown to be a better prognostic factor in overall survival in patients for whom HER2 test results were discordant between HERmark and routine HER2 testing. The difference in overall survival with the discordant groups did not appear to be due to clinicopathologic factors but more likely due to local HER2 misclassifications. A Quantitative H2T Levels A. Discordant High 9% A. Discordant High 9% 10% 10% B Overall Survival (%) A. Discordant High Concordant Nega ve Concordant Nega ve A. Concordant Discordant Posi ve High Concordant Posi ve Local HER2 negative Local HER2 positive Time Since Diagnosis (months) Adapted from Yardley et al., Breast Cancer Research 2015; 17:41 5 Figure A. HER2 status as stratified by local HER2 status and HERmark levels. HERmark may offer a more accurate assessment of HER2 status when compared to local HER2 measurements, and may change therapy selection in approximately 20% of patients (A. and B.). Figure B. Kaplan-Meier overall survival analyses for corresponding HERmark concordant and discordant groups. HERmark High (A. Discordant High) and HERmark Low () had similar overall survival curves compared to concordant HERmark/local HER2 positive and concordant HERmark/local HER2 negative, respectively.

3 Better Correlation with Clinical Outcomes on Trastuzumab 6 Our HERmark clinical study showed HERmark outperformed FISH as a predictor of time to progression (TTP) in patients with metastatic breast cancer treated with trastuzumab (n=102). 2.5 EXAMINATION OF HERMARK/FISH DISCORDANCE 100 TIME TO PROGRESSION (TTP) 2.0 CONCORDANT POSITIVE FISH(+), HER2 Total high HERmark HER2 Total high, FISH(+): Median TTP = 11.3 months Hazard Ratio (HR) compared to: HERmark HER2 Total low, FISH(+) = 0.43, P = 0.01 HERmark HER2 Total low, FISH( ) = 0.42, P < Log 10 HER2 Total Negative H2T CUTOFF LOG(H2T) = 1.14 CONCORDANT NEGATIVE FISH( ), HER2 Total low FISH DISCORDANT FISH(+), HER2 Total low Positive Percent Progression Free HERmark HER2 Total low, FISH(+): Median TTP = 3.7 months HERmark HER2 Total low, FISH( ): Median TTP = 4.5 months Time (Months) Patients with HER2 FISH-positive (HER2/CEP17 ratio 2.2) samples that were reclassified by HERmark as HER2-low (H2T low) had similar TTP as patients with FISH-negative results (3.7 months and 4.5 months, respectively) (HR, 1.0; p=0.99). HERmark Provides Predictive Value HERmark measurements were performed in tumors of patients (n=324) enrolled in the NeoALTTO trial. The NeoALTTO trial evaluated neoadjuvant treatment of lapatinib with trastuzumab vs. either treatment alone for HER2-positive early stage breast cancer. The results of this study include: HERmark results were predictive of pathologic complete response (pcr) when patients received trastuzumab or trastuzumab in combination with lapatinib. 7 HERmark positive patients had better outcomes and achieved a pcr rate of 39% vs. 11% for HERmark negative (plus equivocal). 8 Higher HERmark values achieved better outcomes in the combination arm (lapatinib and trastuzumab) compared to trastuzumab alone. 7 Higher HERmark value=increased benefit from addition of lapatinib to trastuzumab Lower HERmark (but still HER2 positive by IHC/FISH)=reduced benefit from combination treatment Predicted probability of pcr HERmark-negative Eq. HERmark-positive HER2 levels (all patients) Addition of lapatinib to tastuzumab Large benefit Reduced benefit

4 Better Correlation with Time to Brain Metastasis 9 HERmark HER2 protein levels correlated with risk of brain metastases in HER2-positive metastatic breast cancer patients receiving trastuzumab therapy. Time to brain metastases by A.) HERmark (H2T) median cutoff or B.) FISH/CEP17 ratio median cutoff within the HER2 FISH-positive group of patients A B HR, 2.4; p=0.005 HR, 1.3; p=0.4 Comparison with Other HER2 Testing Methods In validation studies involving more than 1,000 clinical tumor samples, HERmark demonstrated a high level of concordance* with other HER2 testing methods performed in central reference laboratories. Central IHC 10,11 n=808 Central FISH 12 n=116 Central IHC and CISH 10 n=218 HERmark Assay 96-98% 93% 97% *Note: Concordance values exclude equivocal results 13 HERmark results are reported as Patient HER2 Status (positive, negative, or equivocal) as well as the patient s precise quantitative HER2 level. The HER2 level is compared to a database with established HERmark reference ranges (1,090 breast cancer patient samples previously defined as HER2-positive or negative according to central reference lab IHC and FISH/CISH). When to Use HERmark HERmark offers an alternative HER2 testing method in order to identify candidates for HER2-targeted therapy. Consider HERmark: When a fully quantitative and validated HER2 protein measurement is desired. When HER2 status is inconclusive: Cases with discordant HER2 testing results in IHC and/or FISH (CISH) Cases with HER2 status equivocal by IHC and/or FISH (CISH) Cases that demonstrate significant heterogeneity in the expression of HER2 protein or gene amplification status of HER2 In cases with HER2 status negative by IHC and/or ISH (FISH/CISH), but the patient s clinical picture (by physician s assessment and experience) suggests an aggressive (possibly HER2-positive) disease.

5 HERmark Assay Methodology 1 The HERmark assay specifically quantifies the full range of HER2 protein expression (H2T) levels in FFPE breast cancer tumor samples. The VeraTag methodology uses two primary HER2-specific monoclonal antibodies and proximity-based conjugated fluorescent VeraTag reporters that are quantified using capillary electrophoresis. The amount of released VeraTag reporters is proportional to the amount of HER2 protein in the invasive tumor sample. HERmark breast cancer assay based on VeraTag technology Novel proximitybased method Direct, quantitative measure of the drug target, HER2 protein HERmark breast cancer assay based FISH, CISH, on SISH, VeraTag and microarrays technology are indirect assays of protein expression Direct, Dual antibody quantitative format measure enhances specificity, of the drug target, HER2 protein minimizes background, and FISH, results CISH, in SISH, greater and signal/noise microarrays ratio are indirect assays of protein expression Dual antibody format enhances specificity, minimizes background, and results in greater signal/noise ratio 2 References 1. Shi, Y, Huang, W, Tan, Y, et al. A Novel Proximity Assay for the Detection of Proteins and Protein Complexes: Quantitation of HER1 and HER2 Total Protein Expression and Homodimerization in Formalin-fixed, Paraffin-Embedded Cell Lines and Breast Cancer Tissue. Diagn Mol Pathol 2009; 18(1): Larson, JS, Goodman, LJ, Tan, Y, et al., Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark ) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens. Patholog Res Int. 2010; 2010: Paik, S et al., Real-World Performance of HER2 Testing National Surgical Adjuvant Breast and Bowel Project Experience. J Natl Cancer Inst 2002; 94: Denkert, C et al., HER2 and ESR1 mrna expression levels and response to neoadjuvant trastuzumab plus chemotherapy in patients with primary breast cancer. Breast Cancer Research 2013; 15:R Yardley, DA et al., Quantitative measurement of HER2 expression in breast cancers: comparison with real-world routine HER2 testing in a multicenter Collaborative Biomarker Study and correlation with overall survival. Breast Cancer Research 2015; 17: Lipton, A, Köstler, W, Leitzel, K, et al., Quantitative HER2 protein levels predict outcome in fluorescence in situ hybridization-positive patients with metastatic breast cancer treated with trastuzumab. Cancer 2010;116:(22): Scaltriti, M et al., High HER2 Expression Correlates with Response to the Combination of Lapatinib and Trastuzumab. Clin Cancer Res 2015; 2193): Scaltriti, M et al., High HER2 Expression Correlates with Response to Trastuzumab and the Combination of Trastuzumab and Lapatinib in the NeoALTTO Phase III Trial. Cancer Res 2013; 73(24 Suppl):Abstract nr P Duchnowska, R, Biernat, W, Szostakiewicz, B, et al., Correlation Between Quantitative HER-2 Protein Expression and Risk for Brain Metastases in HER-2+ Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy. The Oncologist 2012; 17(1): Joensuu, H, Weidler, J, Lie, Y, et al., Quantitative measurements of HER2 expression and HER2 homodimers using a novel proximity based assay: comparison with HER2 status by immunohistochemistry and chromogenic in situ hybridization in the FinHer study. San Antonio Breast Cancer Symposium, Abstract # Huang, W, Reinholz, M, Weidler J, et al., Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity based assay. Am J Clin Pathol 2010;134: Duchnowska, R, Szostakiewicz, B, Jankowski, T, et al., Correlation between quantitative HER2 protein level and the risk of brain metastasis (BM) in metastatic breast cancer (MBC) patients (pts) treated with trastuzumab-containing therapy. ASCO Annual Meeting J Clin Oncol 28:15s, 2010 (suppl; abstr 1030). 13. Wolff, AC, Hammond, ME, Schwartz, JN, 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 2006; 25:

6 Specimen Requirement Options Unstained slides 5-μm sections on positively-charged glass slides, 1 section per slide. A total of 5 unstained slides per patient is required (sections should contain 10 mm 2 invasive tumor). Freshly cut sections, stored at 4 C, and sent within 1 week. OR 1 paraffin-embedded tissue block (requires formalin-fixed tissue) If multiple blocks are available, select the tissue block with the highest amount of viable invasive tumor submit only 1 block. Note: Invasive carcinoma of the breast is required; cases with in situ disease only (e.g., DCIS or LCIS) are not acceptable. Fine needle aspiration (FNA) specimens are not acceptable. Excisional biopsy specimens are preferred; large core biopsies are also acceptable. Reimbursement Assistance Gateway Services can assist your office and your patients with obtaining coverage and reimbursement for HERmark. Gateway makes reimbursement assistance as easy as 1-2-3: Call Gateway at prior to ordering a HERmark assay. The appropriate application forms will be sent to you via fax or . Complete the application forms and return them to Gateway via fax, Receive notification from Gateway of patient eligibility, typically within 24 to 48 hours. In the event that verification/coverage cannot be established, Gateway can assist with the patient s case and/or research alternative coverage Laboratory Corporation of America Holdings. All rights reserved. onc-488-v L