1. Real world HER2 testing - are they reliable? Result of the planned analysis of the initial 104 cases enrolled by IHC in NSABP B-31 Soonmyung Paik, MD Division of Pathology National Surgical Adjuvant Breast and Bowel Project

2. NSABP B-31 AC x 4 Taxol x 4 TAM x 5 yrs.* AC x 4 Taxol x 4 + Herceptin weekly for 1 year TAM x 5 yrs.* Operable Breast Cancer HER-2 Positive Tumor Path Positive Axillary Nodes Randomization * ER+ or PgR+, optional for > 50 yrs. whose tumors are ER– and PgR–

3. Eligibility determination for B-31 trial for adjuvant Herceptin Herceptest 3+ by any laboratory other antibodies: more than 33% of cells show strong membrane staining gene amplification by FISH

4. Planned central review of the initial 100 cases in B-31 Objective: To make sure that we are capturing the real HER2 positive patients for B-31 If more than 20% of the cases are negative by both Herceptest and FISH, consider changing the eligibility criteria

5. Method Central Herceptest and PathVysion FISH test performed by a designated third party central laboratory (Laboratory Corporation of America) Results are available for both assays from 104 cases Results were reconfirmed by NSABP Pathology lab using tissue array of 81 cases

6. Central Herceptest versus FISH (n=104) * 94% agreement

7. B-31 Tissue Microarray

8. FISH versus FISH (N=81) * 95% agreement

9. B-31 central review of the first 104 cases Small volume lab: process no more than 99 cases per month Larger volume lab: process at least 100 cases per month

10. B-31 central review of the first 104 cases

11. One publication showed CISH is a reliable alternative to FISH

12. FISH versus CISH (B-31) * 92% agreement

13. CISH vs. FISH (NSABP B15, n=123) 92% agreement with FISH

14. Central review of cases entered by FISH (N=27) * 26/27 cases by PathVysion, 1/27 case by Inform test

15. Conclusions from central review of B-31 Herceptest provided by larger volume reference labs was reliable in determining 3+ cases (about 95% agreement with FISH) –According to survey, all larger volume labs perform both IHC and FISH, this may contribute to better QC IHC performed by small volume labs tends to be less reliable PathVysion FISH test is reliable CISH in the current format is not a good alternative to FISH

16. Eligibility change for B-31 If FISH positive (amplification - as determined by PathVysion or HER2 Inform test) - eligible regardless of IHC result If 3+ IHC by an approved lab - eligible If 3+ IHC by a non-approved lab - needs to be confirmed by an approved lab

17. Lab Approval for B-31 Automatic approval if the lab process more than 100 cases per month with Herceptest If a lab process less than 100 cases with Herceptest or using different antibody method, we will determine the quality of the lab on individual evaluation (based on concordance rate with FISH, track record) Automatic approval if using FISH

18. Plan Reliability of real world FISH remains an open question (can pathology labs that cannot do IHC do FISH well??) We will conduct another 100 cases review after implementation of the protocol amendment All cases are being arrayed in triplicate before return of the blocks