1. Predictors of HER2 FISH amplification in immunohistochemistry score 2+ infiltrating breast cancer: a single institution analysis Maria Vittoria Dieci 1, Elena Barbieri 1, Stefania Bettelli², Federico Piacentini 1, Guido Ficarra², Sara Balduzzi 1, Massimo Dominici 1, PierFranco Conte 1, Valentina Guarneri 1 1 Department of Oncology, Hematology and Respiratory Diseases, and ²Department of Pathology, University Hospital, Modena, Italy

2. Background Herbst. Int J Radiat Oncol Biol Phys. 2004;59(suppl):21; Roskoski. Biochem Biophys Res Commun. 2004;319:1; Rowinsky. Annu Rev Med. 2004;55:433

3. Background (cont’d)  HER2 amplification is the primary mechanism for overexpression  HER2 is amplified in 18-20% of infiltrating breast cancers  HER2 overexpression is associated with higher risk of recurrence and death, relative resistance to endocrine therapy, apparent lesser benefit from certain chemotherapeutic regimens  The MoAb antiHER2 Trastuzumab was FDA-approved in 1998 for treatment of metastatic disease  Adjuvant trastuzumab reduces the risk of recurrence and death by 1/2 and 1/3, respectively, in patients with early stage, high-risk HER2 positive breast cancers  One year trastuzumab costs: $70,000-$110,000

4. The Need For Accurate Testing  Various strategies to determine or confirm HER2 expression have been used in clinical trials in  Correct HER2 assessment is critical for patients and clinicians since anti-HER2 therapies are effective in HER2+ disease only

5. IHC Images by Kornstein, MD, Medical College of Virginia Abnormal 2+Abnormal 3+Normal 0 Normal Normal 1+ Normal Abnormal low amplification Abnormal high amplification HER2 expression

6. From FDA to ASCO/CAP IHC  For IHC: the cut-off for 3+ score was raised from 10% to 30% of invasive tumor cells with uniform and intense membrane staining FISH amplification  For FISH amplification: a HER2/CEP17 ratio greater than 2.2, rather than equal or greater than 2.0  Recent studies have shown that the concordance between FISH and IHC is higher when the cut-off level to define HER2 3+ score is > 30% Mass. Proc Am Soc Clin Oncol. 2000; Shah, Hum Pathol 2010 As a consequence:  a higher proportion of breast cancer specimens are scored IHC 2+  but only 24% of the IHC 2+ tumors have gene amplification when tested by FISH  the request for FISH assay is increasing

7. Endpoint To evaluate if routinely assessed pathologic parameters such as tumor grade, hormone receptor status and Ki67 are able to predict FISH results in patients with an equivocal IHC HER2 score 2+

8. Tissue specimens 480 infiltrating breast cancer with IHC 2+ and evaluable HER2/CEP17 ratio ANTIBODY: Novocastra Laboratories, clone CB11 until June 2008 and Ventana, clone 4B5 from July 2008 PROBE: PathVysion HER-2 DNA Probe Kit (Vysis Inc., Downers Grove, IL) 96% were primary surgical samples or core biopsy for patients undergoing neoadjuvant therapy 4% were samples from bilateral breast cancer or metastases biopsy

9. Tumor characteristics Samples 480 Hormone Receptor expression Negative (ER and PgR) Positive (ER and/or PgR) Not evaluable 44 433 3 9.2% 90,2% 0.6% Estrogen Receptor expression Negative (<10%) Positive (>10%) Not evaluable 56 420 4 11.7% 87.5% 0.8% Progesteron Receptor expression Negative (<10%) Positive (>10%) Not evaluable 133 345 2 27.7% 71.9% 0.4% Ki67 expression Low proliferation (<15%) High proliferation (>15%) Not evaluable Median Ki67 expression (range) 156 311 13 23.5% (0-98) 32.5% 64.8% 2.7% Histologic Grade G1-2 G3 Not evaluable 153 268 59 31.9% 55.8% 12.3%

10. Distribution of FISH results Negative (<1.8) Borderline (1.8-2.2) Positive (>2.2) ASCO/CAP 332 (69.2%) 48 (10.0%) 100 (20.8%) Negative (<2.0) Positive (>2.0) FDA 348 (72.5%) 132 (27.5%)

11. Distribution of FISH results by G, HR and Ki67 (ASCO/CAP criteria) NNegative(<1.8) Borderline (1.8-2.2) Positive (>2.2) p value G1-2153 126 (82%) 13 (9%) 14 (9%) 0.000 G3268 166 (62%) 30 (11%) 72 (27%) HR -ve 44 33 (75%) 3 (6.8%) 8 (18%) 0.638 HR +ve 433 297 (68.6%) 45 (10.4%) 91 (21%) Low Ki67 156 117 (75%) 20 (13%) 19 (12%) 0.003 High Ki67 311 206 (66%) 26 (8%) 79 (25%)

12. Distribution of FISH results by G, HR and Ki67 (FDA criteria) NNegative(<2.0) Positive (>2.0) p value G1-2153 126 (82%) 27 (18%) 0.000 G3268 178 (66%) 90 (34%) HR –ve 44 35 (79.5%) 9 (20.5%) 0.274 HR +ve 433 311 (72%) 122 (28%) Low Ki67 156 123 (79%) 33 (21%) 0.022 High Ki67 311 214 (69%) 97 (31%)

13. OR for FISH positive test ASCO/CAP and FDA criteria ASCO/CAP OR for FISH positivity 95% CI p value G1-2 Ref. 1.97-6.720.000 G3 3.64 Low Ki67 Ref. 1.42-4.220.001 High Ki67 2.45FDA OR for FISH positivity 95% CI p value G1-2 Ref. 1.45-3.830.001 G3 2.35 Low Ki67 Ref. 1.07-2.650.023 High Ki67 1.68

14. Discussion & Conclusions  While the association between HER2 status and pathological and molecular characteristics has been largely documented in literature, the possibility to predict HER2 amplification in HER2 equivocal (IHC 2+) cases has been less extensively studied  This is the largest single-institution series of HER2 IHC equivocal breast cancer specimens tested by FISH and analyzed to identify potential predictors for FISH amplification  This is the first report on the role of Ki67 index in such contest  On a series of 480 HER2 equivocal samples, both tumor grade and proliferation were found to be significantly associated with HER2 FISH amplification, according to the FDA and ASCO/CAP guidelines