Breast Cancer Heterogeneity

Breast Cancer Heterogeneity Session 2: 8:15 AM 9:00 AM Molecular Subsets and Molecular Diagnostics in Breast Cancer ER + subtypes ER-negative subtypes Lisa A. Carey, MD University of North Carolina Lineberger Comprehensive Cancer Center Courtesy Chuck Perou 2 Genome-wide studies markedly enhance our understanding of breast cancer biology and behavior Genomic Profiling Assays in Breast Cancer: The Good, The Bad, The Un-interpretable Genomic Profiling Analyses Good: Expand existing knowledge Identify relevant biology Not reliant on single markers Bad: Too much information Gene protein Un-interpretable: What population / indication? Prognostic? Predictive? Validation requires prospective or exceptional datasets Unsupervised are samples different? Good for identifying biologically distinct groups Not good for predicting anything Example: Intrinsic subtypes Supervised can gene sets predict clinical event? Generally good for predicting what they were designed to predict. Tricky validating is hard, need homogeneous population and Rx. Genes may be part of pathways, not themselves relevant Example: 70-Gene/Mammaprint, Recurrence Score 3 4 Unsupervised Analysis Gives Us Breast Cancer Intrinsic Subtypes Breast Cancer Subtypes and Prognosis Heterogeneity in ER+ prognosis ER + subtypes ER-negative subtypes Relative uniformity of ER-, at least until HER2 targeting 5 Courtesy Chuck Perou N= 311 6 Courtesy C. Perou p < 0.0000001 1

Hormone Receptor-Driven Subtypes: Luminal Subtypes HER2+/ER- Subtype Luminal B Luminal A Majority of tumors high expression hormone receptorrelated gene cluster. HER2 Basal 15-20% of tumors High HER2 cluster expression Low ER (and related genes) cluster expression HER2 + or - Luminal ER+ is a different subtype Can be proliferative or not Very proliferative 7 Sørlie T, et al. Proc Natl Acad Sci U S A. 2001;98(19):10869-10874. Most heterogeneous group 8 Proliferation Basal-like (Also Known as Triple Negative ) Subtype Potential Targets Can Be Found Within Subtypes Luminal Basal About 15% of tumors Low HER2 cluster expression High basal cluster (CK 5, 17, EGFR, αb crystallin, c-kit etc) expression Low ER (and related genes) cluster expression Luminal Basal The basal cluster includes CK 5, 17, EGFR, αb crystallin, c-kit etc) expression. Molecular target? 54%+ by IHC on TMA Cell lines EGFR dependent Proliferation Very proliferative ~50% are p53 mutant Proliferation 9 10 TBCRC 001: EGFR Inhibition With Cetuximab (C) Added to Carboplatin (P) in Triple-Negative Insight into Treatment Effect from Serial Biopsies Rand. Phase II Stage IV Triple Neg Largely pretreated Cetuximab PD Cetuximab+carboplatin Cetuximab+carboplatin C alone C and P N 31 71 CR - 1% PR 6% 15% SD 4% 23% CB 10% 31% RR 6% 17% EGFR pathway EGFR activated Pre-therapy: EGFR expressed One week cetuximab + carboplatin Post-therapy: Clinical responder! EGFR inactivated by therapy 11 Carey LA, et al. ASCO 08 12 Courtesy of: Charles M. Perou, PhD. 2

Targeting Heterogeneous Tumors: Lessons from the Cetuximab / Carboplatin Study The Truth About Targeted Therapy EGFR expressed Pre-therapy Post 1 wk cetux+carbo Rx 13 EGFR activated 16 tumors with serial biopsies from target lesion 12 EGFR expressed and activated Treatment effect on EGFR activation clusters: 1 inactivated (PR), 3 decreased activation (1SD, 2PD) 8 no significant change (all PD) other mechanisms at work?! BUT no change with Rx 14 Citri A, et al. Nat Rev Mol Cell Biol. 2006;7(7):505-516. Human cellular pathways are both modular and redundant especially in cancer Using profiles to identify drug targets is reasonable, but single agents likely to fail in most reliant on single markers again We need more tissue-based studies! Unsupervised vs Supervised Analyses Basal-like HER- Luminal A 2+/ER- Luminal B Normal Good prognosis signature Genomics and Prognostication Poor prognosis signature 15 Sørlie T, et al. Proc Natl Acad Sci U S A. 2001;98(19):10869-10874. Van de Vijver MJ, et al. N Engl J Med. 2002;347(25):1999-2009 16 Audience Response Question? Top-Down Assay Development: The Mammaprint 70-gene Prognosticator A 45-year-old woman was diagnosed with a T1c N0 ERpositive, PR-negative, HER2-nonoverexpressing right breast cancer. She comes for an initial visit one week postoperatively to discuss her risk of recurrence and adjuvant options. Which of the following test results would be the most valuable in determining the additional value of chemotherapy added to endocrine therapy? 1. Mammaprint 70-gene prognostic profile 2. 76-gene Rotterdam prognostic profile 3. Oncotype Dx Recurrence Score 4. Two-gene ratio 5. Molecular profiling for intrinsic (luminal, basal, HER2) subtype Tumors Genes Gene discovery from 2 cohorts relapsed early and not Apply to dataset with known outcome Outcome by Gene expression signature N Engl J Med, Vol 347 (25), Dec. 2002 17 18 Van t Veer, Nature 2002 3

70-Gene Prognosticator Validation 76-Gene Prognosticator Validation N=326, node-negative, <61 years old, no adjuvant Rx. Overall HR ~2.5 (not as good as initial series) Independent of clinical risk Gene signature HR adjusted for clinical risk N=180 node-negative, no adjuvant Rx Assay developed to represent ER+ and ER- gene sets Validation study very few ER- tumors (16) N=198 node-negative, <61yo, no adjuvant Rx Proportion in high clinical risk group 19 High clinical risk = Probability of 10y Buyse M, JNCI 2006 OS less than 20 It works.. Mostly to predict early relapse Foekens J et al, JCO 06; Desmedt C et al, Clin Cancer Res 07 Bottom-Up Development Using Tailored Gene Lists: The Recurrence Score The Recurrence Score Differentiates Luminal A versus B 250 candidate genes (literature) RT-PCR assay on fixed specimens 3 datasets Tailored to 16 genes Validation subsets from prospective NSABP datasets N0, ER+ Tamoxifen-treated HER2 and GRB7 21 + 1.04 + 0.47-0.34 PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 INVASION Stromolysin 3 Cathepsin L2 HER2 GRB7 HER2 GSTM1 CD68 BAG1 ESTROGEN ER PGR Bcl2 SCUBE2 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC Paik et al, SABCS 2003 Paik, NEJM 2004 22 Courtesy C. Perou ER, SCUBE2, BCL2 Ki-67, STK6, Survivin, Cyclin B1 and MYBL2 Relapse in NSABP B14 by Recurrence Score (Node-Negative, HR+, Tamoxifen-Treated) Too Many Prognostic Profiles? 23 100% Low RS 7% distant mets 90% 80% Int RS 14% distant mets 70% High RS 31% distant mets 60% 50% NCCN St. Gallen s RS 40% Risk % DRFS @10y % pts DRFS % pts DRFS pts @10y @10y 30% Low 8 93% 8 95% 51 93% 20% Intermed - - 33 91% 22 86% 10% High 92 85% 59 81% 27 69% 0% 0 2 4 6 8 10 12 14 16 Years RS far less useful in HER2+ Paik S et al, NEJM 04, SABCS 04 (mostly high) NSABP B-14 DRFS Amsterdam 70-gene Signature Van de Vijver NEJM 2002 Oncotype Recurrence Score 24 Paik, NEJM 2004 Rotterdam 76-gene signature Wang, Lancet 2005 Especially in 70 gene poor Wound Response Signature In ER high for age Chang, PNAS 2005 Proliferation Signature Dai, Ca Res 2005 Independent of wound response Stem Cell IGS signature Liu, NEJM 2007 4

Levels of Biomarker Evidence Node-Negative Prognostic Profiles Level Type Name Validation population Outcome HR I II III IV V Prospective, high power, designed to test marker (does not need to be randomized) OR Meta-analysis of Level II/III Prospective clinical trial not designed to test marker. Good specimen ascertainment. Large but retrospective studies OR Variable proportion of specimens obtained Small retrospective studies, case-control studies Pilot studies Mammaprint (70-gene) 1 76-gene 2 Recurrence Score 3 302 LNno Rx, > 10y 180 LN- Any Rx, >5y 668 ER+ LN- Rx tam, >10y Time to death 2.6 Needs fresh/frozen tissue Distant met-free survival Needs frozen tissue 11.3 Distant mets 2.8 Validated in population-based sets Reliance on standard gene sets? 3 genes overlap 25 26 1.Buyse M, et al. J Natl Cancer Inst. 2006;98(17):1183-1192. 2. Wang Y, et al. Lancet. 2005;365(9460):671-679. 3. Paik S, et al. N Engl J Med. 2004;351(27):2817-2826. 0 LN Recurrence Score Low RS Int. RS High RS 1-3 LN Low RS Int. RS High RS Low RS 4+ LN Int. RS High RS P=NS 0 0.1 0.2 0.3 0.4 Prognostic Profiles in Node-Positive Mammaprint / 70-gene HR 4.1 (1.7-10.0) 241 N+ Confounded by heterogeneous pts and Rx (for example almost all ER- had poor signature) ECOG 2197 substudy 465 HR+, 0-3LN All treated with chemoendocrine Rx Goldstein L, et al. Presented at: 30th San Antonio Breast Cancer Symposium (SABCS); Dec 15, 2007; 27 Abstract 63.; Mook et al, BCRT 08 28 Why Don t the Gene Sets Overlap? A Selection of the 76 Genes.... clone MGC:13188 hypothetical protein FLJ23468 (FLJ23468) Consensus includes gb:aa772093 /neuralised (Drosophila)-like chromatin-specific transcription elongation factor, 140 kda subunit (FACTP140) Consensus includes gb:u07802 /DEF=Human Tis11d gene Rho GDP dissociation inhibitor (GDI) β (ARHGDIB) proteasome (prosome, macropain) 26S subunit, ATPase, 2 (PSMC2) hypothetical protein DKFZp434E2220 (DKFZp434E2220) Consensus includes gb:r39094 /KIAA1085 protein Similar to CD44 antigen (homing function and Indian blood group system) Consensus includes gb:al117652.1 /DEF=Homo sapiens mrna solute carrier family 35 (CMP-sialic acid transporter), member 1 (SLC35A1) cyclin E2 (CCNE2) Consensus includes gb:bf055474 / putative zinc finger protein NY-REN-34 Ag It s about pathways, not individual genes Allows us to be ignorant for now Molecular Profiles Are Often Concordant Subtype N Recurrence Score 70-gene Wound healing 2-gene Basal-like 53 Adding 100% them 100% together 94% didn t 79% prognosticate any better than each alone HER2+/ER- 35 they 100% are not measuring 91% 100% independent 80% processes Luminal B 55 91% 84% 93% 45% Genomics and Adjuvant Therapy Luminal A 123 29% 29% 63% 36% 29 Fan C, et al. N Engl J Med. 2006;355(6):560-569. 30 5

Audience Response question? Chemotherapy Advances Benefit ER-Negative More A 60-year-old woman recently underwent breast conserving surgery for DCIS with micro-invasion noted on final pathology. The invasive component was a high grade infiltrating ductal carcinoma that was hormone receptor-positive and HER2-positive. Staging sentinel lymph nodes were negative. She is referred to your office to discuss adjuvant systemic therapy. Physical examination is WNL. Her surgical incisions are well healed, and her cardiac examination is normal. Which of the below is the best adjuvant systemic therapy recommendation for her? 1. AC-TH followed by endocrine therapy 2. TCH followed by endocrine therapy 3. Oncotype Dx testing with chemotherapy and trastuzumab added to endocrine therapy if the Recurrence Score is high. 4. Endocrine therapy only. Improvement in DFS 70 63% 60 50 36% 40 32% 30 25% 23% 20 14% 12% 10% 10 0 CALGB Trial 8541 9344 9741 Overall Optimizing Adding Optimizing anthracycline taxane taxane ER- ER+ 31 32 Berry DA, et al. JAMA. 2006;295(14):1658-1667. Breast Cancer Relapse is Heterogeneous Chemotherapy Affects Early Relapse ER- ER+ CALGB 8541: Optimizing anthracycline Higher risk of early relapse CALGB 9344: Adding taxane Constant risk of relapse CALGB 9741: Optimizing taxane 33 Anderson WF, et al. Breast Cancer Res Treat. 2006;100(1):121-126. 34 Berry DA, et al. JAMA. 2006;295(14):1658-1667. Response to the Same Chemotherapy Differs by Intrinsic Subtype Survival by pcr in Triple Negative and Non-Triple Negative T-FAC (N=82)* AC-T (n=107)* Luminal A/B 7% 7% Normal-like 0 NA HER2+/ER- 45% 36% Basal-like 45% 26% * P<0.01 ER-negative subtypes are more sensitive to chemotherapy Those with pathologic complete response (pcr) do well (>90% 5y DDFS) Residual disease only The reason ER-negative group has overall worse prognosis is because there is higher risk among residual disease pcr do well, regardless of subtype Non-pCR do not do well, especially if triple negative 35 Rouzier R, et al. Clin Cancer Res. 2005;11(16):5678-5685; Carey LA, et al. Clin Cancer Res. 2007;13(8)2329-2334. 36 Liedtke C, et al. J Clin Oncol. 2008;26(8):1275-1281. 6

Chemotherapy Benefit in ER-positive: Role of the Recurrence Score Recurrence Score Validation Studies 37 Several of the genes reflect markers already associated with chemosensitivity -0.34 + 1.04 + 0.47 PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 INVASION Stromolysin 3 Cathepsin L2 Paik S, et al. J Clin Oncol. 2006;24(23):3726-3734. HER2 GRB7 HER2 GSTM1 CD68 BAG1 ESTROGEN ER PGR Bcl2 SCUBE2 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC 38 NSABP B-14 N0, ER+ NSABP B-20 N0, ER+ 1. Paik S, et al. N Engl J Med. 2004;351(27):2817-2826. 2. Paik S, et al. J Clin Oncol. 2006;24(23):3726-3734. Placebo Tamoxifen First report Paik1, NEJM 04 Risk of distant relapse despite tamoxifen Tamoxifen Tamoxifen + MF or CMF Benefit of chemo added to tamoxifen Second report Paik2, JCO 062 CMF/MF Chemotherapy Benefit in Node-Negative: NSABP B-20 Low RS <18 Absolute benefit of MF or CMF added to tamoxifen n = 353 CAF Benefit in Node-Positive HR+ Postmenopausal: SWOG 8814 Low RS (<18) SWOG 8814: Postmenop. N+, ER+ Tamoxifen Tamoxifen + CAF Int RS 18-30 n = 134 P=0.97 at 10 yrs As in node-negative, the benefit of adding CAF to tam was mostly in the high RS group Intermed. RS High RS ( 31 ) High RS 31 n = 164 0 10% 20% 30% 40% % Increase in DRFS at 10 Yrs (mean ± SE) 39 Paik et al. J Clin Oncol. 2006. 40 Paik et al. J Clin Oncol. 2006. P=0.48 at 10 yrs P=0.033 at 10 yrs Albain K, et al. Presented at: 30th San Antonio Breast Cancer Symposium; Dec 13, 2007; Abstract 10. Recurrence Score and AT Chemotherapy Recurrence Score Marks General Chemosensitivity Probability of pcr 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% Pathologic complete response to neoadjuvant AT No pcr pcr Predicts benefit of CMF/MF in node-negative Predicts pcr to AT in locally advanced Predicts benefit of CAF in node-positive It does NOT tell you which regimen to use. 41 0% 0 10 20 30 40 50 60 70 80 90 100 Recurrence Score Gianni L et al, JCO 2005 42 7

Adjuvant Endocrine Therapy? Caveat: You Still Need Your Pathologist Several gene sets developed to predict hormonal sensitivity, none yet adequately validated Response to tamoxifen in advanced disease (n=69): Biology does not trump anatomy Tumor size matters: B-14 High RS only distant mets 1 < 1cm ~ 80% 1-4 cm ~ 70% 4+ cm ~ 45% High RS only Number of nodes matter: RS and relapse in S8814 2 Similar findings in E2197 3 Risk of relapse or death 43 Kok M et al, BCRT 08 44 1.Paik S, et al. N Engl J Med. 2004;351(27):2817-2826. 2. Albain K, et al. Presented at: 30th San Antonio Breast Cancer Symposium (SABCS); Dec 13, 2007; Abstract 10. 3. Goldstein L, et al. SABCS; Dec 15, 2007; Abstract 63. Optimizing Use of All Factors TAILORx Study Design Anatomic Factors? Integration into clinical practice Pre-REGISTER ONCOTYPE DX ASSAY REGISTER Specimen Banking Biologic Factors Secondary Study Group 1 RS < 11 ~29% of Population Primary Study Group RS 11-25 ~44% of Population Secondary Study Group 2 RS > 25 ~27% of Population 45 Prospective Clinical Trials TailoRx MINDACT 46 ARM A Hormonal Therapy Alone RANDOMIZE Stratification Factors: Tumor Size, Menopausal Status, Planned Chemo, Planned Radiation ARM B Hormonal Therapy Alone ARM C Chemotherapy Plus Hormonal Therapy ARM D Chemotherapy Plus Hormonal Therapy EORTC-BIG MINDACT TRIAL N= 6000 node negative women Evaluate Clinical-Pathological risk and 70-gene signature risk Clinical & 70-gene HIGH / HIGH 55% 32% 13% Discordant Clin-Path HIGH 70-gene LOW 70% N=1344 Clinical and 70-gene LOW / LOW Clin-Path LOW 70-gene HIGH 30% N=576 R1 N=1920 Genomics and Specific Chemotherapy Decision-making Use Clin-Path risk to decide Chemo or not Use 70-gene risk to decide Chemo or not 47 Potentially spares chemotherapy in 10-15% pts 48 8

Phases of Biomarker Development Guilt by Association: Basal-like Breast Cancer and BRCA1 Intrinsic gene list applied to Van t Veer dataset (Nature 2002) Basal-like 49 TET/FEC predictor (Bonnefoi Lancet Oncol 2007) Adapted from Andre F, et al. Nat Clin Pract Oncol. 2006;3(11):621-632. In the absence of specific chemotherapy profiles, can subtype help you choose agent? = BRCA1+ = BRCA2+ Sorlie T et al. PNAS 03 Most cancers in BRCA1 mutation carriers are basal-like But Most basal-like breast cancers are not in BRCA1 carriers Is BRCA1 function different in them? Why should you care? Hereditary and Sporadic Basal-like Breast Cancers Share Certain Characteristics BRCA1 Expression Is Abnormal in Triple Negative Breast Cancer Xiso XIST Loss Hereditary Basal-like BRCA1 Triple Neg Cancer Genomic Instability Sporadic Basal-like like Xiso XIST Loss BRCA1?? Triple Neg Cancer Genomic Instability Nuclear expression 1940 invasive breast cancers Multiple markers on TMA BRCA1 nuclear expression: loss 15% Reduced 39% Aberrant expression common in basal-like: ER-/PR-/HER2-/CK5 or EGFR+ (77%) triple negative (80%) Courtesy J. Garber Loss of expression Rakha et al, Hum Pathol 2008 BRCA1 functions: cell cycle arrest for DNA damage repair apoptosis in response to microtubule damage Implications of BRCA1 Function in Chemosensitivity? BRCA1 Loss or Malfunction Neoadjuvant Cisplatin (CDDP) in Triple-Negative Breast Cancer A Little Clinical Data N = 28, > 2-cm stage II/III triple negative Single-agent cisplatin 75 mg/m 2 q3w x 4 cycles Within selected group only predictor of pcr was age (p<0.04) Pathologic CR 6 (22%) Clinical CR 4 (14%) Clinical PR 10 (36%) Stable Disease 5 (17%) Not bad for a single agent These are NOT ENOUGH DATA to change routine clinical practice. Definitive guidance awaits clinical trials. Garber et al Abs 3074, SABCS 2006 Kennedy RD et al. J Natl Cancer Inst 2004 Sensitivity Resistance N = 30, stage II/III triple negative, treated with neoadjuvant cisplatin/epirubicin/5fu paclitaxel 43% in-breast pcr rate Torrisi et al, Cancer Chemother Pharmacol, 2007 9

Intergroup/CALGB 40603: Triple Negative Neoadjuvant Trial (Sikov, PI) Summary Paclitaxel N=400 ER/PR/HER2- Stage II-IIIB Paclitaxel Carboplatin No carboplatin Carboplatin No carboplatin Dose-dense AC S U R G E R Y RT prn Genomics opens the window further on the heterogeneity of breast cancer. Intrinsic subtypes allow biology-driven segregation Several prognostic profiles are validated for specific uses (Recurrence Score, 70- and 76-gene profiles) Both anatomy and biology are important. Breast imaging Blood MUGA Tumor Biopsy* Bevacizumab Breast imaging Blood Breast imaging Blood MUGA Clinical utility of prognostic profiles will be answered by prospective clinical trials Keep supporting them! Predictive profiles are still in their infancy but are promising. 56 Molecular Subsets and Diagnostics in Breast Cancer Questions & Answers? Thank you for attending Master Class for Oncologists 57 Audience Response Question? Audience Response Question? You have been treating a 37-year-old mother of two elementary age children who was diagnosed 3 months ago with a clinical stage III (T3N1) ERnegative, PR-negative, HER2-negative infiltrating ductal carcinoma of the left breast. After two cycles of neoadjuvant TAC, her clinical tumor size remains at 5.5 cm, and the lymph node remains palpable. Your best course of action is: 1. Continue TAC 2. Change to gemcitabine/carboplatin 3. Continue TAC, add bevacizumab 4. Change to vinorelbine/capecitabine A 60-year-old woman recently underwent breast conserving surgery for DCIS with microinvasion noted on final pathology. The invasive component was a high grade infiltrating ductal carcinoma that was hormone receptor-positive and HER2-positive. Staging sentinel lymph nodes were negative. She is referred to your office to discuss adjuvant systemic therapy. Physical examination is WNL. Her surgical incisions are well healed, and her cardiac examination is normal. Which of the below is the best adjuvant systemic therapy recommendation for her? 1. AC-TH followed by endocrine therapy 2. TCH followed by endocrine therapy 3. Oncotype Dx testing with chemotherapy and trastuzumab added to endocrine therapy if the Recurrence Score is high. 4. Endocrine therapy only. 59 60 10

Audience Response Question? Audience Response Question? A 45-year-old woman was diagnosed with a T1c N0 ERpositive, PR-negative, HER2-nonoverexpressing right breast cancer. She comes for an initial visit one week postoperatively to discuss her risk of recurrence and adjuvant options. Which of the following test results would be the most valuable in determining the additional value of chemotherapy added to endocrine therapy? 1. Mammaprint 70-gene prognostic profile 2. 76-gene Rotterdam prognostic profile 3. Oncotype Dx Recurrence Score 4. Two-gene ratio 5. Molecular profiling for intrinsic (luminal, basal, HER2) subtype Which of the following SSRIs is the safest to give with tamoxifen? 1. Paroxetine (Paxil) 2. Venlafaxine (Effexor) 3. Fluoxetine (Prozac) 4. Sertraline (Zoloft) 61 62 Audience Response Question? You have been treating a 37-year-old mother of two elementary age children who was diagnosed 3 months ago with a clinical stage III (T3N1) ER-negative, PR-negative, HER2-negative infiltrating ductal carcinoma of the left breast. After two cycles of neoadjuvant TAC, her clinical tumor size remains at 5.5 cm, and the lymph node remains palpable. Your best course of action is: 1. Continue TAC 2. Change to gemcitabine/carboplatin 3. Continue TAC, add bevacizumab 4. Change to vinorelbine/capecitabine 63 11