Fatima Cardoso, MD Jules Bordet Institute & TRANSBIG 2006 European Breast Cancer Meeting Stockholm, Sweden 20–21 May 2006 USING PROGNOSTIC & PREDICTIVE FACTORS IN BREAST CANCER

PROGNOSTIC FACTOR % Treat. A Treat. B + -

PREDICTIVE FACTOR Case 1 Case 2 % % Treat. B Treat. A Treat. B Treat. A

PROGNOSTIC FACTORS Who needs a treatment? PREDICTIVE FACTORS Which treatment is best? THERAPEUTIC CHOICES AVOID UNDER AND OVER TREATMENT INDIVIDUALIZE TREATMENT WHY DO WE NEED PROGNOSTIC AND PREDICTIVE FACTORS

ER-ER+ RFS Basal-like1 HER-2-like Luminal1 Luminal2Luminal3 Basal-like2 Adapted from Sotiriou et al, PNAS, 2003 BC GENE EXPRESSION PATTERNS and OUTCOME Molecular (re-)classification of BC

PROGRESS IN ADJUVANT CHEMOTHERAPY FOR BREAST CANCER L-PAM, MF CMF x 6 AC x 4 FAC  FEC x 6 A(E)  CMF AC x 4  Paclitaxel x 4 TAC x 6 FEC  docetaxel AC  paclitaxel dose-dense ± ± + Average treatment effect Financial toxicity 1970’s1980’s 1990’s 2000’s Successive generations of adjuvant CT regimens Adapted with permission from G. Hortobagyi d)  $ c)  $ b)  $ a)  800 $ +++ ADJUVANT TRASTUZUMAB +++

New prognostic factors accepted: HER-2, vascular invasion Node+ 1-3: in average risk group, if HER-2– and no vascular invasion St Gallen 2005 Consensus: What’s new? Beyond St Gallen 2005 … uPA, PAI-1 Cyclin E Genomic signatures Oncotype DX* (predictive & Px) Oncotype DX* (predictive & Px) Topo-II-  *Genomic Health

uPA-PAI-1

CLINICAL RELEVANCE OF uPA & PAI-1 IN PRIMARY BREAST CANCER uPA and PAI-1: first novel tumor biological factors in breast cancer with clinical relevance validated at highest level of evidence (LOE I)  Standardized quality assured ELISA tests: Sweep et al, Br J Cancer 78: , 1998  Prospective multi-center therapy trial („Chemo N0“): Jänicke et al, JNCI 93: , 2001  EORTC RBG meta analysis (n=8,377): Look et al, JNCI 94:116-28, 2002  Recommended for clinical risk assessment: AGO Therapy Guidelines „breast cancer“ (since 2002): N. Harbeck – used with permission

uPA AND PAI-1 FIRST NOVEL TUMOR BIOLOGICAL FACTORS IN BC WITH LEVEL 1 OF EVIDENCE WHY ARE THEY NOT WIDELY USED? 1. ELISA not commonly used in pathological practice a.Biochemistry lab required b.Further personnel training required c.€€££$$ required 2. Frozen tumor specimen required 3. Large quantity (100 µg) required Target population = small tumors – feasible ? 4. Population used in validation studies: Interaction with ER status not well defined (?)

HOW CAN THEY BECOME WIDELY USED? 1.Refining ELISA test – less tissue 2.Alternative techniques – other protein assays – gene expression 3.Further validation according to ER status ALL ONGOING uPA AND PAI-1 FIRST NOVEL TUMOR BIOLOGICAL FACTORS IN BC WITH LEVEL 1 OF EVIDENCE

GENOMIC SIGNATURES

BIG-TRANSBIG Secretariat– Used with permission IMPROVED RISK ASSESSMENT OF EARLY BREAST CANCER THROUGH GENE EXPRESSION PROFILING microarray Gene-expression profile Good signature Poor signature N Engl J Med, Vol 347 (25), Dec ~4% die of breast cancer ~96% survive breast cancer ~50% die of breast cancer ~50% survive breast cancer

TRANSLATING MOLECULAR KNOWLEDGE INTO EARLY BREAST CANCER MANAGEMENT

BIG-TRANSBIG Secretariat– Used with permission Audited clinical data INDEPENDENT VALIDATION : DESIGN RNA Achieved n = 307 Target n = 400 Amsterdam Gene expression profiling Agilent platform Agilent platform 70-gene prognostic 70-gene prognostic custom designed custom designed chip chip High or low gene signature risk Clinical data « Local » pathological data Brussels Comparison of clinical vs gene signature assessment of prognostic risk Endpoints 1. TDM 2. OS 3. DMFS, DFS Tissue samples  UK (Guy’s, Oxford) : 1984 => 1996  France (IGR, CRH) : 1978 => 1998  Sweden (Karolinska) : 1980 => 1990 Node negative, untreated Node negative, untreated < 60 years old < 60 years old > 5 years follow-up > 5 years follow-up T1, T2 T1, T2 Tumor cell % > 50% Tumor cell % > 50% Centrally reviewed path data (Milan)

BIG-TRANSBIG Secretariat– Used with permission OVERALL SURVIVAL by GENE SIGNATURE RISK Amsterdam/Agendia Signature 10-year OS 89% (81%-94%) 10-year OS 70% (62%-76%) Average Survival HR 2.66 Average Survival HR  2.66 M. Buyse et al. JNCI In press

BIG-TRANSBIG Secretariat– Used with permission TRANSBIG INDEPENDENT VALIDATION The best signature? Amsterdam’s Signature 70 genes Rotterdam’s Signature 76 genes TEST ALL IN VALIDATION SERIES & DECIDE Only few genes in common … But similar biological pathways Brussels’ GGI signature

BIG-TRANSBIG Secretariat– Used with permission OVERALL SURVIVAL by GENE SIGNATURE RISK Rotterdam/Veridex Signature 5-year survival: low risk group: 0.98 ( ) high risk group: 0.84 ( ) 10 year survival: low risk group: 0.87 ( ) high risk group: 0.72 ( ) C. Desmedt et al. Presentated at: EBCC 2006

CONCLUSIONS VALIDATION PHASE The Amsterdam 70-gene signature has been independently validated The Amsterdam 70-gene signature has been independently validated The Rotterdam 76-gene & Genomic Grade signatures have been independently validated using the same TRANSBIG validation series The Rotterdam 76-gene & Genomic Grade signatures have been independently validated using the same TRANSBIG validation series The performances of the signatures are similar The performances of the signatures are similar There is a strong time dependency of all signatures (better predictors of EARLY RELAPSE), which was not seen for the clinical risk There is a strong time dependency of all signatures (better predictors of EARLY RELAPSE), which was not seen for the clinical risk The Amsterdam 70-gene test is robust (laboratory reproducibility) and available for patient diagnostic testing The Amsterdam 70-gene test is robust (laboratory reproducibility) and available for patient diagnostic testing GREEN LIGHT FOR MINDACT TRIAL! GREEN LIGHT FOR MINDACT TRIAL!

Evaluate Clinical-Pathological risk and 70-gene signature risk Clinical-pathological and 70-gene both HIGH risk Discordant cases Clin-Path HIGH 70-gene LOW Clin-Path LOW 70-gene HIGH Clinical-pathological and 70-gene both LOW risk Use Clin-Path risk to decide Chemo or not Use 70-gene risk to decide Chemo or not 55% 32%13% R1 Chemotherapy N=3300 N=780 Endocrine therapy EORTC-BIG MINDACT TRIAL DESIGN 6,000 Node negative women N=1920 Potential CT sparing in 10-15% pts

GENOMIC GRADE

Histologic Grade G1 G2 G3 Genomic Grade GG1 GG2 GG3 Sotiriou et al., ASCO 2005 Poor inter observer reproducibility G2: difficult treatment decision making, under- or over treatment likely Findings consistent across multiple data sets and microarray platforms More objective assessment Easier treatment decision-making High proportion of genes involved in cell proliferation ! C. Sotiriou – used with permission

HistologicalGrade 3 Histological Grade 3 HG3 GENOMIC GRADE IN EACH OF THE HISTOLOGIC GRADE SUBGROUPS Genomic Grade 1 Genomic Grade 3 Histological Grade 2 HG2 Histological Grade 1 HG1 C. Sotiriou – used with permission C. Sotiriou et al. JNCI 2006

Oncotype DX NSABP & Genomic Health

MULTI GENE RT-PCR ASSAY FOR PREDICTING RECURRENCE IN NODE NEGATIVE BC PATIENTS 250 candidate genes Tested using RT-PCR Three studies 21 GENE PREDICTOR Recurrence score lowintermediatehigh

PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 ESTROGEN ER PGR Bcl2 SCUBE2 INVASION Stromolysin 3 Cathepsin L2 HER2 GRB7 HER2 GSTM1 REFERENCEBeta-actinGAPDHRPLPOGUSTFRC Best RT-PCR performance and most robust predictors CD68 BAG1 Paik et al, N Engl J Med 2004 THREE BREAST CANCER STUDIES USED TO SELECT CANDIDATE GENES FOR A RECURRENCE SCORE UNDER TAMOXIFEN TREATMENT Recurrence score for TAM-treated pts established and subsequently validated

338 pts 149 pts 181 pts B14-RESULTS DRFS—Low, Intermediate, High RS Groups Paik et al, N Engl J Med 2004

PREDICTIVE MARKERS

HER-2 neu 95% Negative predictive value <5% chances of responding to TRASTUZUMAB (HER-2) or to HT (ER) 30-70% Positive predictive value Accepted Predictive Markers In Breast Cancer ER/PgR OxfordOverview2000 St Gallen ConsensusPanel2003 NIHConsensusPanel2000 ASCOGuidelines %-70% chances of responding to HT (ER) & 40%-50% of responding to TRASTUZUMAB (HER-2)

PREDICTIVE MARKERS FOR CHEMOTHERAPY

FISH IHC ADJUVANT SETTING CMF vs. ANTHRA-BASED TOPO II  RESULTS Di Leo A et al, Clin Cancer Res, 2002 All pts with HER-2 amplification Di Leo A et al, Ann Oncol 2001

4 x AC 60/600 mg/m 2 4 x Docetaxel 100 mg/m 2 6 x Docetaxel and Carboplatin 75 mg/m 2 AUC 6 1 Year Trastuzumab N=3,222 1 Year Trastuzumab AC  T AC  TH TCH Her2+ (Central FISH) N+ or high risk N- 4 x AC 60/600 mg/m 2 4 x Docetaxel 100 mg/m 2 Slamon D., SABCS 2005 BCIRG 006 Stratified by Nodes and Hormonal Receptor Status

Disease Free Survival % Disease Free Year from randomization 77% 86% 80% 73% 84% 80%86% 93% 91% Patients Events AC->T AC->TH TCH HR (AC->TH vs AC->T) = 0.49 [0.37;0.65] P< HR (TCH vs AC->T) = 0.61 [0.47;0.79] P= Slamon D., SABCS 2005 AC->TH AC->T TCH

DFS CO-AMPLIFIED TOPO II BY ARM % Disease Free Months Patients EventsTreatment AC->T 26513AC->TH 25221TCH Logrank P= 0.24 TCH AC->TH AC->T Slamon D., SABCS 2005

DFS NON CO-AMPLIFIED TOPO II BY ARM % Disease Free Months Patients EventsTreatment 45892AC->T 47245AC->TH 44654TCH Logrank P= <0.001 TCH AC->TH AC->T Slamon D., SABCS 2005

HER-2 AND TOPOISOMERASE-II  PROMISING POTENTIAL PREDICTIVE MARKERS OF ANTHRACYCLINE EFFICACY HOW TO OBTAIN LEVEL 1 EVIDENCE LARGE PROSPECTIVE TRIALS META-ANALYSIS

HER-2 AND TOPOISOMERASE-II  AS POTENTIAL PREDICTIVE MARKERS OF ANTHRACYCLINE EFFICACY: A META-ANALYSIS DANISH TRIAL FEC vs CMF UK TRIAL E  CMF vs CMF NCIC-CTG TRIAL CEF vs CMF BELGIAN TRIAL EC vs CMF Tampere University Laboratory Central evaluation of HER-2/TOPO II by FISH Central evaluation of HER-2/TOPO II gene amplification by FISH Correlation with outcome of CMF or anthracycline-based therapy with  4,500 tumor samples

TOP TRIAL OR « TRIAL OF PRINCIPLE » Operable tumors, > 2 cm ER-negative EPIRUBICIN 100 mg/m² x 4 SURGERY Docetaxel x 4 Radiotherapy ± HT Hypothesis :  pCr in HER-2 / Topo2 co-amplified tumors  pCr in HER-2 - / basal-like 1 tumors Incisional biopsy Snap frozen sample HER2/Topo2 FISH analysis (Vysis probe) Genomic signature of response to anthracyclines Inflammatory or LABC ER-negative EPIRUBICIN 100 mg/m² x 6 dose dense / 2w + G-CSF Gene expression analysis

EORTC-BIG-p53 TRANSLATIONAL RESEARCH TRIAL: STUDY DESIGN Target accrual= 1300 (872 p53 -, 436 p53 + ) Hypothesis: ↑ DFS at 3 y by 5% in p53 - and by 20% in p53 + RANDRAND Non Taxane arm FEC 100 or Canadian FEC Taxane arm T-T-T-ET-ET-ET Sample 1: standard fixation Incisional biopsy Sample 2: snap frozen. Loc. adv.. Infl.. Large Operable Local ± TAM therapy Local ± TAM therapy P53 pathway P53 analysis

FRAGRANCE trial months Letrozole 15 days Microarray Analysis Microarray Analysis Genomic signature of de novo AI resistance Microarray Analysis Postmenopausal patients (no age limits) Non-candidates for CT T  2 cm Stages I, II & III ER and/or PgR+

INTEGRATING TRANSLATIONAL RESEARCH IN CLINICAL RESEARCH & PRACTICE Multidisciplinarity Multidisciplinarity Collaboration (between specialties, between centers…) Collaboration (between specialties, between centers…) Bench-to-bedside-to-bench Bench-to-bedside-to-bench Biological material collection (unethical not to do it!) Biological material collection (unethical not to do it!) Patient selection & treatment tailored to the individual Patient selection & treatment tailored to the individual New technologies, new statistical methods… New technologies, new statistical methods… Costs ?? Costs ?? INDISPENSABLE and already ongoing

Total expected costs:  €35, 000,000 EU funding €7,000,000 OTHER: National Funding Pharmaceutical Industry Biotechnology companies (Agendia) Other grants NATIONAL FUNDING FOR NATIONAL PATIENTS (indispensible) MINDACT & TRANSBIG FUNDING - 1

ACKNOWLEDGEMENTS BIG-TRANSBIG TeamBordet Fellows Translational Research Team M. Piccart