HER2 and adjuvant trastuzumab Soonmyung Paik, MD NSABP Foundation

Target = Degree of Benefit

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

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

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

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

ER*tam interaction: p-value= 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

Disease-Free Survival B-31 N9831 Years AC->T+H AC->T AC->T AC->T+H N Events HR=0.45, 2P=1x10 -9 HR=0.55, 2P= AC->T+H AC->T

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

CP 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)

CP Adjuvant Trastuzumab May Benefit Pts with Normal HER2 Breast Tumors (n=103) p = 0.12 p = 0.26 p = 0.14

CP Hazard Ratio of Benefit to Trastuzumab by HER2 FISH Ratio (328) < 2.0 (156) (253) (515) (473) ≥ 15.0 (70) p = 0.12 p = 0.05 p = 0.03 p = p = 0.96 p = Ratio (N) p-value Hazard ratio

CP DFS Based on Chromosome 17 Status and Treatment Arm p = 0.04 All had HER2 amplification

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

B-31 FISH neg without polysomy 17 Years After Surgery % Disease-free Trt N Events ACT ACTH 69 7 RR=0.38 p=

FISH negative with Polysomy 17 Years After Surgery % Disease-free Trt N Events ACT 34 4 ACTH 24 1 RR=0.32 p=

 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

 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

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

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

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

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

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

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

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

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

Adjusted P=0 HER2 mRNA (210930_s_at) HER2 negHER2 pos

Adjusted P= e-32 C17orf37 mRNA HER2 negHER2 pos

SKBR3 (10) MCF7 (10) B30 HER neg (33) B30 HER2 pos (10) both negative (154) FISH+ IHC- or FISH- IHC+ (121) both positive (299) HER2 mRNA Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

SKBR3 (10) MCF7 (10) B30 HER neg (33) B30 HER2 pos (10) both negative (154) FISH+ IHC- or FISH- IHC+ (121) both positive (299) HER2 mRNA Central HER2 assay negative cases have similar HER2 mRNA levels as in HER2 negative cases from B-30 trial

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

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.

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.

VeraTag HER2 Total protein assay in FFPE

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

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

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.

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