Highlights in the Management of Breast Cancer Roma, 10 Maggio 2013 Molecular tools for decision making in adjuvant therapy Enrico Ricevuto & Valentina Cocciolone Oncologia Medica Ospedale San Salvatore Università degli Studi di L’Aquila

2 Adjuvant Breast Cancer Treatment Key issues Patients selection according to biomarkers Selection of appropriate treatment

3 Adjuvant Breast Cancer Treatment Molecular Tools for decision-making Biomarkers  ER/PR  HER2 Topoisomerase II  BRCA1/BRCA2  KI67 Multi-Genes expression profiles (GEPs) Circulating Tumoral Cells (CTCs)

4 BC Prevalence according to ER/PR and HER2 ER/PR HER2 Total+++Negative Positive Total

5 Hormone receptor status: prognostic potential Patients with ER-positive/PgR-positive and ER-positive/PgR- negative BC had significantly better prognoses than patients with ER-negative/PgR-negative disease. Patients with ER-positive/PgR-negative tumors tended to have slightly worse disease-free and overall survival than patients with ER-positive/PgR-positive tumors, but the differences did not achieve statistical significance (P.05) Bordou et al. J Clin Oncol 2003; 21:

6 Hormone receptor status: predictive potential multivariate analyses confirmed that both ER and PgR are independent significant predictors of DFS and OS among patients who received adjuvant endocrine therapy; the reduction in RR of recurrence was 53% for ER-positive/PgR-positive patients and 25% for ER-positive/PgR- negative patients (P.0001); patients whose tumors are positive for both receptors have the greatest reduction of RR of death compared with patients whose tumors are ER-negative and PgR-negative. Bordou et al. J Clin Oncol 2003; 21:

7 Her2 status: prognostic potential Pritchard et al. N Engl J Med 2006;354:

8 Her2 status: predictive potential Pritchard et al. N Engl J Med 2006;354:

9 BC Prevalence according to ER/PR and HER2 ER/PR HER2 Total+++Negative Positive Total

10 Topoisomerase II and responsiveness to adjuvant Anthracyclines O’Malley et al. J Natl Cancer Inst 2009;101: CEF CMF CEF CMF amplified or deleted TOPO2A normal TOPO2A

11 Topoisomerase II and responsiveness to adjuvant Anthracyclines amplified or deleted TOPO2A normal TOPO2A O’Malley et al. J Natl Cancer Inst 2009;101: CEF CMF

12 TOPO2A HR 0.53 for RFS (p 0.09) HR 0.38 for OS (p 0.02) Topoisomerase II and responsiveness to adjuvant Anthracyclines HER2 HR 0.40 for RFS (p 0.008) HR 0.44 for OS (p 0.02) Adjusted test for interaction: O’Malley et al. J Natl Cancer Inst 2009;101:

13 Topoisomerase II and responsiveness to adjuvant Anthracyclines Slamon et al. N Engl J Med 2011;

14 BRCA1-ness in TNBC ER/PR HER2 Total+++Negative BRCA1+ 11% “BRCA1-ness”>50% Positive Total

15 BRCA1, BRCA2 predisposition carriers Breast Cancer prognosis BRCA1 + BRCA2 + Non carriers Pts Risk recurrence (CI)1.19 ( )1.63 ( ) p Risk death (CI) 1.43 ( ) 1.81 ( ) p Goodwin et al, JCO’12: 30; 19-26

16 Ki67: prognostic role in EBC de Azambuja et al. Br J Cancer 2007;96: Despite some limitations, this meta-analysis supports the prognostic role of Ki-67 in early BC, by showing a significant association between its expression and the risk of recurrence and death in all populations considered and for both outcomes, DFS and OS.

17 Two different gene sets: first, a set of 476 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Breast Cancer Genomics and Clinical Classification Sørlie et al. Proc Natl Acad Sci USA 2001;98:

18 Gene expression profiling predicts clinical outcome of breast cancer Van de Vijver et al. N Engl J Med 2002;347: Panel A shows the pattern of expression of the 70 marker genes in a series of 295 consecutive patients with breast carcinomas. Each row represents the prognostic profile of the 70 marker genes for one tumor, and each column represents the relative level of expression of one gene. The tumors are numbered from 1 to 295 on the y axis, and the genes are numbered from 1 to 70 on the x axis. Red indicates a high level of expression of messenger RNA (mRNA) in the tumor, as compared with the reference level of mRNA, and green indicates a low level of expression. The dotted line is the determined threshold between a good-prognosis signature and a poor-prognosis signature. Panel B shows the time in years to distant metastases as a first event for those in whom this occurred, and the total duration of follow-up for all other patients. Panel C shows the lymph-node status (blue marks indicate lymph- node–positive disease, and white lymph-node–negative disease), the number of patients with distant metastases as a first event (blue marks), and the number of patients who died (blue marks).

19 Gene-expression signature is a predictor of survival in breast cancer ASSOCIATION BETWEEN CLINICAL CHARACTERISTICS AND THE PROGNOSIS SIGNATURE: the prognosis profile was significantly associated with: - the histologic grade of the tumor (P<0.001); - the estrogen-receptor status (P<0.001); -age (P<0.001) but not with: - the diameter of the tumor; - the extent of vascular invasion; - the number of positive lymph nodes - treatment Van de Vijver et al. N Engl J Med 2002;347:

20 Gene-expression signature is a predictor of DFS and OS in breast cancer Van de Vijver et al. N Engl J Med 2002;347: Among the overall population: HR for distant metastases as a first event was 5.1 (95% confidence interval, 2.9 to 9.0; P<0.001); HR for overall survival was 8.6 (95 %confidence interval, 4 to 19; P<0.001).

21 The St. Gallen and NIH criteria classify patients as at low risk or high risk on the basis of various histologic and clinical characteristics. This comparison shows that the prognosis profile assigned many more patients with lymph-node–negative disease to the low-risk (good-prognosis signature) group than did the traditional methods (40 percent, as compared with 15 percent according to the St. Gallen criteria and 7 percent according to the NIH criteria). Comparison with St. Gallen criteria and NIH Consensus Criteria Van de Vijver et al. N Engl J Med 2002;347:

22 Histologic grade Simpson et al. J Clin Oncol 2000; 18:

23 Molecular basis of histologic grade Sotiriou et al. J Natl Cancer Inst 2006; 98: Most genes are overexpressed in grade 3 tumors (high expression is RED) and have functions that have been previously associated with cell cycle progression and proliferation.

24 Molecular basis of histologic grade Sotiriou et al. J Natl Cancer Inst 2006; 98:

25 Oncotype DX 21 Gene Recurrence Score (RS) Assay For ER-positive patients CategoryRS (0 – 100) Low riskRS < 18 Intermediate risk RS ≥ 18 and < 31 High riskRS ≥ 31 N Events Paik S, et al. Breast Cancer Res Treat 88 (S1):A24, 2004

26 Validation in tamoxifen-treated patients with node- negative, ER–positive breast cancer (NSABP-B14) Paik et al. N Engl J Med 2004;351:

27 Validation in tamoxifen-treated patients with node-negative, ER–positive breast cancer Paik et al. N Engl J Med 2004;351:

28 Percentage of patients on tamoxifen with larger N0 tumors free of recurrence at 10 years varies by Recurrence Score in NSABP B14 Percentage of patients on tamoxifen with moderately/poorly differentiated tumors free of recurrence at 10 years varies by Recurrence Score in NSABP B14 Validation in tamoxifen-treated patients with node-negative, ER–positive breast cancer Paik et al. N Engl J Med 2004;351:

29 Prognostic disease-free survival and overall survival analyses by Recurrence Score group in patients treated with TAMOXIFEN ALONE Validation in tamoxifen-treated patients with node-positive, ER–positive breast cancer Albain et al. Lancet Oncol 2010; 11:55–65

30 Dowsett et al. J Clin Oncol 2010; 28:

31 Many patients live normal life expectancy free of breast cancer recurrence after surgical treatment alone 1.3% of recurrences occurred after 20 years (3.7% of the 20-year survivors) Albain KS. Presented at SABCS 2012

32 10-Year Survival Rate by axillary node status for patients treated with radical mastectomy Study1-3+ Nodes4+ Nodes Valagussa, %24% Haagensen, %27% Fisher B, %13% Ferguson, %27% Albain KS. Presented at SABCS 2012

33 Prediction of recurrence in NSABP-B20 (Tam vs Tam + CMF) Paik et al. J Clin Oncol 2006; 24:

34 NSABP-B20 Paik et al. J Clin Oncol 2006; 24:

35 Prediction of anthracycline-based chemotherapy benefit by RS: DFS Albain et al. Lancet Oncol 2010; 11:55–65 The RS was a strong predictive factor of CAF benefit for DFS, but degree of CAF benefit depended on the RS: NO apparent benefit for scores <18 (p=0.97; HR=1.02) or 18– 30 (p=0.48; HR=0.72); SIGNIFICANT advantage for CAF-T compared to tamoxifen alone for patients with RS ≥31 (p=0.033; HR=0.59)

36 Impact on clinical practice (where available) Albain et al. The Breast 2009;18: S141–S145

37 Available tests and prospective ongoing clinical trials Goncalves and Bose. J Natl Compr Canc Netw 2013;11:

38 Theoretical spectrum of sensitivity to adjuvant systemic therapy Hayes D. J Clin Oncol 2012; 30:

39 TAILORx trial Goncalves and Bose. J Natl Compr Canc Netw 2013;11:

40 RxPONDER trial Goncalves and Bose. J Natl Compr Canc Netw 2013;11:

41 MINDACT trial Goncalves and Bose. J Natl Compr Canc Netw 2013;11:

42 Adjuvant Breast Cancer Treatment Key questions Patients selection according to biomarkers Selection of appropriate treatment  no adjuvant treatment  Chemotherapy  Hormonal therapy

43 Circulating Tumoral Cells in Adjuvant Therapy Three typical patterns of response observed: Decrease >10x Marginal Change Increase >10x Pachmann et al. J Clin Oncol 2008; 26: Peripherally circulating tumor cells are influenced by systemic CT An increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells

44 Circulating Tumoral Cells in Adjuvant Therapy Tamoxifene Trastuzumab

45 Metastatic Tumors Evolution of medical treatment Markers None“One fit (unfit) all” Clinical “One fit some” (>10%) Monogene“One fit few”(<10%) Multigenes“One fit one”(<1%)

46 Early Breast carcinoma Evolution of medical treatment Markers None“One fit (unfit) all” Clinical “One fit some” (>10%) Monogene“One fit few”(<10%) Multigenes“More fit many”(>30%)

47

48 Renal cell carcinoma Evolution of medical treatment Parameters None“One fit (unfit) all” Bio-Clinical “One fit some” (>10%)  Patientfitness (age, comorbidities)  Tumorprognostic risk  Drugsprediction (safety/toxicity, efficacy)

49 Renal cell carcinoma Evolution of medical treatment Markers None“One fit (unfit) all” Clinical “One fit some” (>10%) Monogene“One fit few”(1-10%)  Other genetic alterations  Heterogeneity (tumor/metastasis)

50 Tailor therapy for individual patients Unanswered questions Are CTCs detected in the peripheral blood released from existing micrometastases or are they the source of distant metastases after “seeding” organ sites? Are there stem cells capable of “seeding”new tumor sites in the CTC? Could theCTCs be used to assess sensitivity of resistant tumor cells to alternative agents?

51 Adjuvant Breast Cancer Treatment Key questions Selection of appropriate treatment  Chemotherapy anthracycline-based taxane-based +Trastuzumab+Hormonetherapy A/T-based  Hormonetherapy SERMs Aromatase Inhibitors

52 Multi-Gene Predictors in Breast Cancer (1) Classification Perou Grading Sotiriou OncotypeDx Genomic Health MammaPrint Agendia Number of Genes427/33, IndicationTumor SubtypesMolecular GradingPrognosis Tamox/CMF Prognosis Guide to Specific Therapy No Yes CMF ER/HER2 No PlatformStanford/ AffymetrixAffymetrixRT-PCRGE/Agilent Formalin Paraffin In DevelopmentNoYesNo FDA ApprovalNo ? PendingYes Commercial Status None On the Market

53 Breast Cancer Prevalence according to ER and HER2 status Total+++Negative Positive Total HER 2 ER/PR

54 Breast Cancer Prevalence according to ER and HER2 status Total+++Negative Positive Total HER 2 ER/PR

55 CTCs Identification and Detection Semiquantitative determination Prognostic/Predictive factor Molecular Characterization

56 CTCs Detection Methods Immunofluorescent staining (CellSearch System, Veridex): FDA approved  Immunomagnetic separation  Fluorescent staining Cristofanilli M et al, NEJM’04 Harris et al, JCO’07: 25; 5287 RT-PCR Xenidis et al, JCO’06

57 CTCs Cell Search System (Veridex) Cut-off >5 CTCs/7.5 ml bloodMetastatic BC >1 CTC/7.5 ml bloodEarly BC

58

59 Pharmaco-genetics Modulations of activity dependent from genetic alterations (DNA)  structural mutations (point- or rearrangements)  constitutive  somatic Allelic variants (constitutive)  Functional (methylation) Pharmaco-genomics Modulations of activity dependent from or inducing alterations of gene expression (RNA, protein) Pharmaco-Genetics and -Genomics

60 Breast Cancer Genomics and Clinical Classification Gene Expression ProfileBreast Cancer “Basal-like”“Triple-negative” (ER/PR/HER2-negative) HER2-positiveHER2-postive (IHC +++ /FISH ampl ) Luminal A/B/CER and/or PR-positive Sørlie et al. Proc Natl Acad Sci USA 2001;98:

61 Ki67: predictive role in EBC There are not robust evidence that Ki67 can serve as a tool to identify patients who will benefit from a specific chemotherap y or endocrine treatment. Yerushalmi et al. Lancet Oncol 2010; 11:174–83

62 Gene expression profiling Unfixed samples of tumor tissue obtained during surgery are the starting material for gene-expression profiling. The expression levels of a set of prognostically relevant genes are determined by DNA-microarray analysis. The resulting molecular signatures allow the patients to be classified into groups with a poor prognosis or a good prognosis, thus facilitating therapeutic decision making. Van’t Veer et al. J Clin Oncol 2005; 23:

63 Red= upregulation Green =Down-regul Black = constiutive exp Gene expression profiling

64 Thus, are there tumors with favorable prognosis which don’t benefit from chemotherapy?

65 Of 89 assessable patients, 11 (12%) had a pCR; 86 genes correlated with pCR (unadjusted P 0.05); pCR was more likely with higher expression of proliferation-related genes and immune-related genes, and with lower expression of ER–related genes; The Recurrence Score was positively associated with the likelihood of pCR (P 0.005), suggesting that the patients who are at greatest recurrence risk are more likely to have chemotherapy benefit Gianni et al. J Clin Oncol 2005; 23:

66 Chang et al. Breast Cancer Res Treat 2008; 108:

67 Single gene/protein predictors of treatment tailoring Bedard et al. The Breast 2009; 18:S25–S30

68 Topoisomerase II and responsiveness to adjuvant Anthracyclines

69

70 Cancer Genetics and Genomics Genetics→ genetic alterations (DNA)  structural mutations (point- or rearrangements)  constitutive  somatic Allelic variants (constitutive)  Functional (methylation) Genomics → alterations of gene expression (RNA, protein)

71 Genomics Identification of biomarkers  Response Prediction  Target identification  Between patients  Within patientsduring treatment between primary and mets

72 Breast cancer Prognostic/predictive factors “Simple markers”  HR  NG  Tumor size  N status GEP CTCs

73 Candidate targets and pathways in TNBC Berrada et al. Ann Oncol 2010; 21(S7): vii30-vii35

74 TNBC paradox Tang et al. J Translational Medicine 2012, 10(Suppl 1):S4

75 EGFR and pCR in TNBC Tang et al. J Transl Med 2012, 10(Suppl 1):S4

76 Cancer Genetics and Genomics Genetics→ genetic alterations (DNA)  structural mutations (point- or rearrangements)  constitutive  somatic Allelic variants (constitutive)  Functional (methylation) Genomics → alterations of gene expression (RNA, protein)

77 Microtubule-Associated Protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in ER-positive BC Andrè et al. Clin Cancer Res 2007;13: No adjuvant therapy Neoadjuvant T/FAC Adjuvant tamoxifen

78 Prognostic and predictive value of tau-mRNA expression Higher tau-mRNA expression showed borderline non significant association with better prognosis in the absence of systemic adjuvant therapy Higher tau-mRNA expression was significantly associated with no recurrence (at 5 and10 years, P =0.005 and P = 0.05, respectively) in patients treated with tamoxifen, indicating a predictive value for endocrine therapy Tau expression was significantly lower in patients who achieved pCR to paclitaxel/FAC chemotherapy (P < 0.001) Andrè et al. Clin Cancer Res 2007;13:

79 C-Myc alterations and association with benefit from adjuvant trastuzumab DFS (median follow-up, 4.0 y): MYC:CEP8 ratio ≤ 2.2 ↓ HR 0.46 (p 0.001) MYC:CEP8 ratio > 2.2 ↓ HR 0.67 (p 0.33) interaction p 0.38 MYC copies/nucleus ≤ 5.0 ↓ HR 0.52 (p 0.002) MYC copies/nucleus > 5.0 ↓ HR 0.48 (p 0.02) interaction p 0.94 Perez et al. J Clin Oncol 2011; 29:

80 C-Myc alterations and association with benefit from adjuvant trastuzumab MYC:CEP8 ratio < 1.3 with normal chromosome 8 copy number ↓ HR 0.66 (p 0.28) MYC:CEP8 ratio ≥ 1.3 or < 1.3 with polysomy 8 ↓ HR 0.44 (p 0.001) interaction p 0.23 Perez et al. J Clin Oncol 2011; 29: MYC copies/nucleus < 2.5 ↓ HR 1.07 (p 0.87) MYC copies/nucleus ≥ 2.5 ↓ HR 0.42 (p 0.001) interaction p 0.05

81 Fumagalli, J Natl Cancer Inst 2011; How to improve the development of predictive tools

82 Gene-expression signature is a predictor of DFS and OS in breast cancer Van de Vijver et al. N Engl J Med 2002;347:

83 Molecular basis of histologic grade Sotiriou et al. J Natl Cancer Inst 2006; 98:

84 Prognostic utility of Recurrence Score compared to clinicopathologic features The 21-gene assay was a more accurate predictor of relapse than standard clinical features for individual patients with HR- positive operable breast cancer treated with chemohormonal therapy and provides information that is complementary to features typically used in anatomic staging, such as tumor size and lymph node involvement. Goldstein et al. J Clin Oncol 2008; 26:

85 Paik et al. J Clin Oncol 2006; 24: NSABP-B20

86 Prediction of anthra-based CT benefit by RS: OS Albain et al. Lancet Oncol 2010; 11:55–65 There was no statistically significant benefit to CAF for the low (p=0.63) or intermediate (p=0.85) RS groups. However, there was a significant CAF benefit in the high RS group (p=0.0271), which did not vary by age.

87 Prediction over time Albain et al. Lancet Oncol 2010; 11:55–65 Increasing involvement of axillary lymph nodes was prognostic. At 10 years, the treatments start diverging at approximately RS=10, though any clinically significant CAF benefit is not evident until much higher RS. RS has better short- than long-term prediction: the treatments are equivalent up to approximately RS=20, but diverge at higher RS values