1. HER2 and adjuvant trastuzumab Soonmyung Paik, MD NSABP Foundation

2. Target = Degree of Benefit

3. NSABP B-14 N-, ER+ (over 10 fmole) Placebo Tamoxifen When positive, more ER the better response to tamoxifen?

4. Median risk High risk Low risk Amount of target (ER) Expected prognosis of tamoxifen treated patients

5. ER by IHC is not a linear prognosticator for tam pts ER percent staining HR

6. ER by QRT-PCR is linear HR ER by QRT-PCR

7. ER*tam interaction: p-value=0.0006 Degree of benefit ER Expression by RT-PCR (relative to ref genes; log2) Association of Quantitative ER Expression by QRT-PCR and Tamoxifen Benefit Cut-off based on 10 fmole LBA

8. Disease-Free Survival B-31 N9831 Years AC->T+H 864 83 AC->T 872 171 AC->T 807 90 AC->T+H 808 51 N Events HR=0.45, 2P=1x10 -9 HR=0.55, 2P=0.0005 AC->T+H AC->T

9. Interaction p=0.60 for FISH Interaction p=0.26 for IHC Note: RR adjusted for ER and nodal status

10. CP1270832-10 Clinical Significance of Polysomy 17 in the HER2+ NCCTG N9831 Intergroup Adjuvant Trastuzumab Trial Reinholz MM, Jenkins RB, Hillman D, Lingle WL, Davidson N, Martino P, Kaufman P, Kutteh L, and Perez EA. NCCTG, ECOG, SWOG, CALGB Reinholz et al: SABCS 2007 (abstract #36)

11. CP1270832-11 Adjuvant Trastuzumab May Benefit Pts with Normal HER2 Breast Tumors (n=103) p = 0.12 p = 0.26 p = 0.14

12. CP1270832-12 Hazard Ratio of Benefit to Trastuzumab by HER2 FISH Ratio 0.00 0.50 1.00 1.502.00 2.503.00 3.504.00 11.0-15.0 (328) < 2.0 (156) 2.0-5.0 (253) 5.0-8.0 (515) 8.0-11.0 (473) ≥ 15.0 (70) p = 0.12 p = 0.05 p = 0.03 p = 0. 04 p = 0.96 p = 0.004 Ratio (N) p-value Hazard ratio

13. CP1270832-13 DFS Based on Chromosome 17 Status and Treatment Arm p = 0.04 All had HER2 amplification

14. DFS for HER2 Normal (by IHC and FISH) Patients by Chromosome 17 Status and Treatment Arm

15. B-31 FISH neg without polysomy 17 Years After Surgery % Disease-free 012345 0 20 40 60 80 100 Trt N Events ACT 80 19 ACTH 69 7 RR=0.38 p=0.02667

16. FISH negative with Polysomy 17 Years After Surgery % Disease-free 012345 0 20 40 60 80 100 Trt N Events ACT 34 4 ACTH 24 1 RR=0.32 p=0.30719

17.  Disease-free-Survival According to Local Immunohistochemistry for HER2 and Central FISH for Patients Treated with Adjuvant Chemotherapy with/without Trastuzumab in the HERA Trial _____________________ McCaskill-Stevens W, Procter M, Azambuja E, Dafni U, Leyland-Jones B, Ruschoff J, Dowsett M, Jordan B, Dolci S, Abramovitz M, Stoss O, Viale G, Gelber RD, Piccart-Gebhart M, for the HERA Study Team

18.  Prospective testing for eligibility before randomization Local IHC 3+ → central IHC 3+ Local IHC 2+ → central FISH+ Local FISH + → central FISH+ HER2 status testing central FISH+ = FISH Ratio ≥ 2.0 Central FISH results are available for: 1131 pts. prospectively (eligibility screening) 940 pts. retrospectively (assay banked specimens) 2071 (61%) total out of the 3401 patients

19. 

20.  p-value for interaction = 0.29 (FISH ratio ≥ 2 only)

21.  p-value for interaction = 0.38 (HER2 copy ≥ 4 only)

22. Interaction p=0.60 for FISH Interaction p=0.26 for IHC Interaction p=0.60 for FISH Interaction p=0.26 for IHC

23. Questions Testing issue?? –Non-linearity of current tests

24. Questions Testing issue?? –Non-linearity of current tests Artifact of subset analyses??

25. Questions Testing issue?? –Non-linearity of current tests Artifact of subset analyses?? Differences in mechanism of action of trastuzumab in metastatic versus adjuvant setting??

26. Chromosome 17q HER2 locus is not amplified in HER2 negative tumors in B-31 P-value cut off at 0.0001 for blue color Height indicates fold difference between HER2 pos and negative Chr 17 Chr 1

27. Chromosome 17q HER2 locus is not amplified in HER2 negative tumors in B-31 P-value cut off at 0.0001 for blue color Height indicates fold difference between HER2 pos and negative Chr 17 Chr 1

28. Adjusted P=0 HER2 mRNA (210930_s_at) 4 8 16 32 64 128 256 512 1024 HER2 negHER2 pos

29. Adjusted P= 4.62507e-32 C17orf37 mRNA 8 16 32 64 128 256 512 1024 2048 HER2 negHER2 pos

30. SKBR3 (10) MCF7 (10) B30 HER neg (33) B30 HER2 pos (10) both negative (154) FISH+ IHC- or FISH- IHC+ (121) both positive (299) 1 2 4 8 16 32 64 128 256 512 HER2 mRNA Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

31. SKBR3 (10) MCF7 (10) B30 HER neg (33) B30 HER2 pos (10) both negative (154) FISH+ IHC- or FISH- IHC+ (121) both positive (299) 1 2 4 8 16 32 64 128 256 512 HER2 mRNA Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

32. STEPP Analysis Lower 1/3 Mid 1/3 Upper 1/3A gene on 8q is a predictor of degree of benefit from adjuvant trastuzumab

33. HER-2 total (H2T) HER-2 homodimerization (H22D) The Principle of VeraTag Technology - A Novel Proximity-based Assay A monoclonal antibody specific for a unique epitope of HER2 is conjugated to a fluorescein VeraTag reporter (Pro11) or a molecular scissors (S) by means of a cleavable tether. The molecular scissors liberates singlet O2 upon irradiation with red light.

34. HER-2 total (H2T) HER-2 homodimerization (H22D) The Principle of VeraTag Technology - A Novel Proximity-based Assay The free radicals cleave all thioether bonds within approximately 200 nM, releasing the “VeraTag reporter.” The signal (Pro11) can then be collected and analyzed on a capillary electrophoresis array.

35. VeraTag HER2 Total protein assay in FFPE

36. VeraTag H2T Assay Performance Characteristics The H2T and H22D assays quantitate HER2 expression and HER2:HER2 dimers over a 3-log dynamic range. Accuracy, precision, sensitivity, and reproducibility. CLIA-validated and CAP-certified. Similar results for H22D. Accuracy H2T Sensitivity H2T Precision H2T Reproducibility H2T ELISA: MDA MB 435: ~2 ng/mg Replicates: 15 each (105 pairwise comparisons) 100% within 1.7-fold >95% within 1.45-fold 55 replicates 96% within 2-fold FFPE breast tumors FFPE cell lines

37. IHCFISH Comparison of HER2 assessments by VeraTag with IHC (N=170) and FISH (N=64) in FFPE breast tumors

38. Figure 3: Univariate Kaplan-Meier (KM) analyses examining the impact of Trastuzumab treatment on time-to-progression (TTP) and overall survival (OS) in patients with high vs. low HER2 expression (H2T, left) and HER2 homodimer (H22D, right) levels.

40. Summary Three large adjuvant studies failed to demonstrate association between the amount of HER2 gene copy or protein expression and degree of benefit from trastuzumab added to chemotherapy HER2 testing method is evolving to more quantitative tests with improved linearity of prediction of trastuzumab response