Dinsdag 14 en woensdag 15 juni e Bossche Mamma Congres De Ruwenberg, Sint-Michielsgestel. Changing therapy?

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1 Dinsdag 14 en woensdag 15 juni e Bossche Mamma Congres De Ruwenberg, Sint-Michielsgestel Changing therapy?

2 No financial disclosures (including Agendia). Disclosures Emiel Rutgers

3 No, it will not. Changing therapy?

4 Will it change indication for chemotherapy? Yes, it will certainly do

5 Adjuvant chemotherapy? Two steps 1. Prognosis 2. Prediction

6 Two steps 1. Prognosis: is prognosis so good that chemotherapy does not add to breast cancer related outcome Individual decision: balanced information

7 Two steps Prediction: does chemo really works? The chemoprediction tools: - Her-2, BRCA-ness/HRD, - further one size fits all

8 Primary analysis of the EORTC 10041/ BIG 3-04 MINDACT study: A prospective, randomized study evaluating the clinical utility of the 70-gene signature (MammaPrint ) combined with common clinical-pathological criteria for selection of patients for adjuvant chemotherapy in breast cancer with 0 to 3 positive nodes Martine Piccart, Emiel Rutgers, Laura van t Veer, Leen Slaets, Suzette Delaloge, Giuseppe Viale, Jean Yves Pierga, Peter Vuylsteke, Etienne Brain, Suzan Vrijaldenhoven, Peter Neijenhuis, Bruno Coudert, Tineke Smilde, Miguel Gil, Alastair Thompson, Isabel Rubio, Rodolfo Passalaqua, Erika Matos, Urlike Nitz, Mauro Delorenzi, Geraldine Thomas, Theodora Goulioti, Carolyn Straehle, Konstantinos Tryfonidis, Jan Bogaerts & Fatima Cardoso On behalf of TRANSBIG consortium and MINDACT investigators Presented at AACR, April 18,

9 Let s start with a question Lady, 54, screen detected abnormality - Left, palpable, UOQ, mm - Core biopsy: IDC Gr 2, ER 90%, PgR 0%, Her2, - US axilla: one node cortex 3 mm, FNAC negative - Wide local excision and SN. Histology: IDC 18 mm, compleet margins, IHC idem, Gr 2, Ki 67 15%, SN macro met 5 mm, second SN.

10 Adjuvant therapy: your advice? 1. 3 rd generation ChT (TAC or equivalent) > 7 yrs anti estrogen Rx 2. Only 7 yrs anti estrogen Rx 3. Order multi-gene test and advice patient accordingly y/n chemotherapy

11 First presentation by Martine Piccart AACR 18 April 2016 This presentation is a MINDACT team effort

12 ST. GALLEN DEFINITIONS OF RISK Low G1 T 2 Node HER2 LVI absent Only 20% of patients! R I S K Intermediate High AGE < 35 G2-3 T>2 Node, HER2+ or LVI present Node + (1-3) and HER2 - Node + (1-3) and HER2 + Node + 4 Most difficult group for CT decision! R I S K Other similar guidelines exist : NCCN, ESMO,

13 Adjuvant! Online for breast cancer (Updated version) used for a standardized approach to Clinical Risk P. Ravdin

14 IMPROVED RISK ASSESSMENT OF EARLY BREAST CANCER L. Van t Veer THROUGH GENE EXPRESSION PROFILING R. Bernards 78 untreated N primary tumors 295 partially treated N / N + tumors 44 w/o relapse at 8 y follow-up 34 with a relapse within 5 y microarray Gene-expression profile 5000 genes 231 genes 70 genes Poor prognosis signature Van t Veer L., Nature 2002; 415 (31) : Van de Vijver MJ, N Engl J Med 2002; 347 (24):

15 The development pathway of MINDACT Two important milestones before the launch of the trial MINDACT recruitment [ ] TRANSBIG Independent validation of the 70- gene (MammaPrint ) signature Clinical validity proven N= 302 pts (no CT) Median fup: 13y / 92 events O.S. H.R. High vs Low Mammaprint risk = 2.66 (19% absolute survival difference at 10 years) Buyse, JNCI, 2006 TRANSBIG Interlaboratory reproducibility of Mammaprint MINDACT protocol developed 3 laboratories Same protocol Excellent reproducibility Ach, BMC Genomics, 2007 Analytical validity proven

16 Why a 92% threshold for 10y OS w/o adjuvant chemotherapy using Adjuvant! Online? Overall Survival Predicted OS without chemotherapy 92% 92% 4% 88% Adj. Endocrine Therapy Predicted benefit versus harm of adjuvant CT in this population Adj. CTX + 2% gain 2% risk of CHF leukemia ER - ER + Consensus reached within the TRANSBIG consortium, including EuropaDonna patient advocates Gain in survival counterbalanced by treatment associated risks

17 The MINDACT study design Diagnosis of breast cancer Screening informed consent Surgery Local pathology (T1-3, 0 to 3 positive nodes, ER status, HER2 status) Agendia (frozen tumor sample shipment, RNA extraction, microarray analysis) Clinical risk (c) Adjuvant Online! Enrollment Genomic risk (g) 70-gene signature or MammaPrint c-low/g-low Discordant c-high/g-high c-low/g-high c-high/g-low R-T No Chemotherapy If HR+ R-E If HR+ Chemotherapy R-C

18 The development pathway of MINDACT Three important milestones after the launch of the trial S. Mook E. Rutgers G. Viale Mammaprint is strongly prognostic in 241 pts with 1-3 N+ (Med Fup 8y; 53% had CTX) HR for distant mets 4.13 ( ) 76% vs 91% at 10y DMFS Study logistically feasible Discordant risk populations : 34% > 92% compliance with randomisation High concordance between locally and centrally assessed ER 98 % PgR 95 % HER2 97 % Breast Canc Res Treat, 2009 MINDACT OPENED TO women with 1-3 N+ EJC, 2011 Breast Canc Res Treat, 2015 MINDACT in line with expectations MINDACT «clinical» strategy based on local pathology is a solid clinical trial arm

19 MINDACT recruitment 9 European countries 111 centers EORTC (Sponsor) and 6 additional Cooperative Groups -BOOG -GOIRC -NCRI-UK -SOLTI -UNICANCER -WSG

20 Key eligibility criteria Enrollment & Randomization Women with histologically proven operable invasive breast cancer (clinical Max. 56 days Mandatory-Shipment tumor tissue (in liquid nitrogen) to Central Lab for 70- gene signature assay (Amsterdam) and central pathology evaluation (Milan) stage T1, T2 or operable T3) & 0-3 positive lymph nodes and of any subtype (postsurgery) A frozen tumor sample should have been taken from the excised primary tumor (a core punch biopsy) And 10 ml blood sample plus 1 paraffin block Confirmation of 70- gene signature performance & clinical risk assessment by Adjuvant! Online (8.0 including HER2 status)

21 The primary analysis population Enrolled N = Clinical risk (c) Adjuvant Online! Genomic risk (g) 70-gene signature or MammaPrint N = 644 c-low/g-low N=2745 No Chemotherapy N=592 Discordant N=1550 c-low/g-high c-high/g-low R-T Chemotherapy c-high/g-high N=1806

22 Primary test Analysis population for the primary test (PT population) The set of patients who have a low risk gene prognosis signature and high risk clinical-pathological criteria at enrollment, and who were randomized (R-T) to use the 70-gene signature risk and thus receive no chemotherapy, and did not deviate from this: no change in risk status post enrollment and no adjuvant chemotherapy received.

23 Primary test Primary endpoint: Distant metastasis free survival (DMFS) at 5 years Null hypothesis: 5-year DMFS rate in PT population = 92% Alpha: 2.5% (1-sided) Power: 80% when true 5-year DMFS rate=95% Primary test: 95% 2-sided Confidence interval (CI) for the 5-year DMFS rate will be compared to 92% significant if 92% is excluded from the CI

24 Primary test Distant metastasis free survival (DMFS) Events: distant metastatic recurrences and deaths (for any cause) Timing of primary analysis when two conditions are met: o o The standard error of DMFS rate at 5 years is 0.01 or less At least one third of the patients in the above dataset have been followed for five years.

25 RESULTS

26 The MINDACT study: Patient demographics N = 6,693 Mean age = 55y Node - 80% Node + 20% T1 tumours 58% Grade 2 49% HR positive 88% HER2+ 10% N=2745 clinical Low/ genomic Low Discordant N=1806 clinical High/ genomic High N=592 clinical Low/ genomic High N=1550 clinical High/ genomic Low

27 MINDACT: patient demographics and compliance with assigned therapy Risk group clinical Low / genomic Low Risk group clinical High / genomic High N = 2745 med. age=55y N = 1806 med. age = 53y T size < 2cm 96% Grade 1 or 2 99% Node negative 94% Luminal 96% HER2+ 4% T size > 2cm 48% Grade 3 76% Node positive 26% Luminal 50% Triple 31% HER2+ 19% Assigned: NO CHEMOTHERAPY Compliance = 99% Received Endocrine therapy: 79% Assigned: CHEMOTHERAPY Compliance = 96% Received Endocrine therapy: 59% Received trastuzumab: 15%

28 MINDACT: patient demographics and compliance with assigned therapy Risk group clinical Low / genomic High N = 592 med. age = 55y Risk group clinical High / genomic Low N = 1550 med. age = 55 y T size < 2cm 98% Node negative 98% T size > 2cm 58% Node positive 48% Grade 1 or 2 85% Luminal 79% HER2+ 12% [triple 9%] Grade 3 29% Luminal 91% HER2+ 8% triple - 1% Randomization Randomization No chemotherapy Chemotherapy Compliance = 80% Compliance = 88% Received E.T.: 82% Received Trastuzumab: 7% No chemotherapy Compliance = 89% Chemotherapy Compliance = 85% Received E.T.: 94% Received Trastuzumab: 5%

29 EFFICACY RESULTS N = 6,693 patients randomized

30 Outcome results per corrected risks Randomization outcome : per intent-to-treat T Y P E O F E V E N T S Events across the entire MINDACT population Median follow-up = 5 years DMFS Distant relapses Deaths (all causes) N = 362 Relapses 74% Deaths 26% DFS Distant relapses Deaths Local relapses Contralateral BC Secondary cancers N = 672 Distant relapses 36% Locoreg relapses 16% 2 nd prim. 42% Deaths 6% OS Deaths (all causes) N = 208

31 Clinical outcome of the MINDACT population at 5y median follow-up A) CONCORDANT RISK GROUPS (using corrected risk) DMFS DFS OS curve curve % at 5y (95% CI) % at 5y % at 5y cl/gl 97.6 ( ) 92.8 ( ) 98.4 ( ) ch/gh 90.6 ( ) 85.3 ( ) 94.7 ( )

32 The MINDACT population: CT assignment according to a Clinical vs a Genomic strategy Whole population N = 6,693 N=2745 clinical Low/ genomic Low Discordant N=1806 clinical High/ genomic High N=592 clinical Low/ genomic High N=1550 clinical High/ genomic Low «Clinical» strategy CT to = 3,356 pts = 50 % 14% reduction «Genomic» strategy CT to = 2,398 pts = 36 %

33 Clinical outcome of the MINDACT population at 5y median follow-up B) DISCORDANT RISK GROUPS: PRIMARY TEST The primary analysis population c-low /g-high Discordant risks RANDOMIZATION c-high/g-low The primary statistical test (DMFS at 5Y) No chemotherapy N = 748 CT No change in risk post enrollement and no CT received N = 644 Null Ho set à 92% Observed 5Y DMFS = 94.7% 95% CI % excludes 92%!!!

34 Clinical outcome of the MINDACT population at 5y median follow-up Discordant groups: outcome in relation with adjuvant chemotherapy Yes or NO?

35 Efficacy: ACT vs no ACT in discordant risk groups Intent-to-treat analysis Allocated Treatment strategy % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) ACT 95.9 (94.0, 97.2) 1.00 no ACT 94.4 (92.3, 95.9) 1.52 (0.86, 2.71) p-value (adjusted logrank) Allocated Treatment strategy % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) ACT 95.8 (92.9, 97.6) 1.00 no ACT 95.0 (91.8, 97.0) 0.92 (0.46, 1.86) p-value (adjusted logrank) Allocated to: Allocated to:

36 cl/gh: CT versus no CT per protocol cl/gh ACT vs no ACT per protocol population Treatment received Patients Observed Events % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) DMFS DFS OS ACT (92.4, 98.1) 0.90 (0.40,2.01) no ACT (89.6, 96.5) 1.00 ACT (87.9, 95.7) 0.74 (0.40,1.39 no ACT (85.7, 93.8) 1.00 ACT (94.9, 99.3) 0.72 (0.23,2.24) no ACT (93.8, 98.6)

37 ch/gl: CT versus no CT per protocol ch/gl ACT vs no ACT per protocol population Treatment received Patients Observed Events % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) DMFS DFS OS ACT (94.7, 98.0) 0.65 (0.38,1.10) no ACT (92.6, 96.3) 1.00 ACT (90.7, 95.2) 0.64 (0.43,0.95) no ACT (87.6, 92.4) 1.00 ACT (97.4, 99.5) 0.63 (0.29,1.37) no ACT (95.6, 98.4)

38 Future clinical use of Mammaprint in the clinical high risk population Enrolled N = Clinical risk (c) Adjuvant Online! Genomic risk (g) 70-gene signature or MammaPrint c-low/g-low Discordant c-high/g-high c-low/g-high c-high/g-low R-T N=1550 N=1806 Following the «genomic» strategy means no longer prescribing CT to 1550/3356 women = 46%, who would have been otherwise considered CT candidates based on «clinical» risk

39 The MINDACT Trial Conclusions (1) has played a major educational role in Europe, mobilized hundreds of professionals and popularized the concept of biology-driven treatment demonstrated that genomics can provide important information in order to treat patients with early breast cancer implemented the logistics to collect and freeze tumour materials in a quality-controlled fashion and built an invaluable biobank for future research in B.C (including full gene- array bank).

40 Conclusions (2) Mindact results provide prospective evidence of the clinical utility of Mammaprint for assessing the lack of a clinically relevant chemotherapy benefit in the clinically high risk (c-high) population. C-High/g-Low patients had a 5-year DMFS rate in excess of 94%, whether they were randomized to adjuvant CT or no CT. (These results are similar in Node negative and Node positive patients and in the population of «Luminal» breast cancers, eg : hormone receptor positive/her2 negative/ data in the manuscript) The clinical use of Mamaprint among the c-high patients is associated with a 46% reduction in chemotherapy prescription.

41 Acknowledgements ALL THE MANY MINDACT PATIENTS! We are most grateful to you for your significant contribution to our research endeavour and for your faith in our work!

42 Acknowledgements Other Principal Investigators MINDACT s leading scientist Fatima Cardoso Martine Piccart Laura Van t Veer Leading pathologist EORTC HQ Statistical & Medical Team Giuseppe Viale Jan Bogaerts Leen Slaets Kostas Tryfonidis

43 Acknowledgements All National Teams and Participating Cooperative Groups Country Enrolled pts Netherlands (NKI) 2092 France (UCBG) 2065 Germany (WSG) 835 Belgium (EORTC) 828 Spain (SOLTI) 546 IOL Italy (GOIRC) 199 UK (NCRI-BCG ) 66 Slovenia (IOL) 37 Switzerland (EORTC) 25 Total 6693 L-R, from top: Emiel Rutgers (NL), Suzette Delaloge (FR), Mahasti Saghatchian (FR), Ulrike Nitz (DE), Isabel T. Rubio (ES), Rodolfo Passalacqua (IT), Alastair Thompson (UK), Erika Matos (SL), Khalil Zaman (CH)

44 Acknowledgements - Funding Research Grants European Commission Framework Program VI (FP6-LSHC-CT ) Novartis Sanofi-Aventis Veridex LLC the Breast Cancer Research Foundation Susan G. Komen for the Cure Fondation Contre le Cancer / Stichting tegen Kanker (Belgian Cancer Society) F. Hoffmann-La Roche Eli Lilly Agendia EBCC-Breast Cancer Working Group asbl Jacqueline Seroussi Memorial Foundation Cancer Research UK KWF Kankerbestrijding (Dutch Cancer Society) Association Le cancer du sein, parlons-en! Deutsche Krebshilfe (German Cancer Aid) Grant Simpson Trust Prix Mois du Cancer du Sein the (U.S.) National Cancer Institute NIF Trust EORTC Charitable Trust * Agendia s investment is in services provided and not direct funds Brussels Breast Cancer Walk-Run & American Women s Club of Brussels

45 Acknowledgements And the great many others who contributed to MINDACT: The MINDACT Steering and Executive Commmittee members EUROPA Donna the European Breast Cancer Coalition, and ECCO, the European CanCer Organization BIG-TRANSBIG-MINDACT fellows: Kim Aalders (NL), Jacques Bines (BR), Philippe Bedard (CA), Sofia Braga (PT), Ivana Bozovic (RS), Carlos Castaneda (PE), Aleksandar Celebic (RS), Camelia Colichi (RO), Carmen Criscitiello (IT), Lissandra Dal Lago (BR/BE), Gaston Demonty (AR/BE), Fei Fei (CN), Michela Lia (IT), Sherene Loi (AU), Stella Mook (NL), Camilo Moulin (BR), Roman Sreseli (GE), Gustavo Werutsky (BR) The headquarters teams from the EORTC and BIG The many academic medico-scientific institutions / individual scientists involved over the life of TRANSBIG and MINDACT The entities providing scientific / logistical support: Adjuvant!Online, Agendia, Fundazione IEO, IBBL, IDDI, Novartis, Roche, Sanofi, SIB

46 Behandelstrategieën in adjuvante setting bij ER+/HER2- tumoren bij vrouwen jaar Tot 2cm, SN-ve, graad 1-2: geen MammaPrint, geen chemotherapie, HT > 2cm graad1-2, > 1cm graad 3, (S)N 1-3 +ve: MammaPrint + consequenties Wat als echo oksel ve & SN macrometa? OKD of MP? Kans op N4+ is <8 % (Amaros): keus voor MP is te verdedigen

47 ALND results Amaros Median number of all nodes removed (Q1-Q3) ALND (744 pts) 15 (12-20) Number of additional positive nodes (besides SN) % 25.0 % 7.8 %

48 Behandelstrategieën in neo-adjuvante setting bij ER+/HER2- tumoren bij vrouwen jaar De MRI is leidend bij het bepalen van het ct stadium. Bij een tumorgrootte < 5 cm (indien N0) en < 3 cm (indien N+) komt patiente in aanmerking voor een Mammaprint om te bepalen of (neo-)adjuvante chemotherapie achterwege gelaten kan worden De N-status wordt bepaald met echo en met PET. Indien er > 1 PET-avide klier is (in aanwezigheid van een FDG-avide primaire tumor) wordt (neo-) adjuvante chemotherapie geadviseerd, onafhankelijk van de mammaprint Bij een bulky klier in de axilla waarbij twijfel bestaat of het meerdere klieren zou kunnen betreffen, wordt (neo-) adjuvante chemotherapie geadviseerd, onafhankelijk van de mammaprint ct1cn0 en graad I /II en highly endocrine-sensitive (ER en PR >50%) wordt geen chemotherapie (en geen Mammaprint) geadviseerd indien sprake is van N+ ziekte (max 1 axillaire klier op PET) en een low risk Mammaprint, kan de klier met een jodiumzaadje worden gelokaliseerd (Localization of Axillary node with Radioactive Iodine LARI). Na het verwijderen van deze klier bij de operatie wordt axillaire bestraling geadviseerd

49 BACK-UP

50 The MINDACT study: Patient screening and enrollment Diagnosis of BC Informed consent for screening 70- gene array N = Local pathology evaluation Surgery Tissue sample not appropriate for Genomic analysis: Patient/investigator decision: Higher nr. of nodes positive: Inadequate sample: Ineligible: Other: 26% 20% 17% 17% 10% 11% Agendia microarray analysis (frozen sample) [ 40% «drop out» rate ] Enrolled N = 6.693

51 Patients «as enrolled» and subsequently randomized (if discordant) MINDACT Presentation of results Patients with a corrected risk postenrollment slightly less discordant patients N = 6,693 N = 6,693 Discordant N = 2,187 Discordant N = 2,142 Concordant Discordant 1.7% Discordant Concordant 2.3% = Intention-to-treat population = Corrected risk (per protocol) populations

52 Two important milestones along the MINDACT pathway 2011 (EJC) 2015 (Breast Canc Res Treat) E. Rutgers First 800 patients enrolled Study logistically feasible Discordant risk populations : 34% > 92% compliance with randomisation G. Viale All patients enrolled High concordance between locally and centrally assessed ER 98 % PgR 95 % HER2 97 %

53 MINDACT: Confronting the «genomic» versus the «clinical» strategy in predicting patient outcome 1. Distant metastasis free survival 2. Disease free survival 3. Overall survival

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57 Independent validation of the 70-gene signature: Design RNA N = 307 Tissue samples UK (Guy s, Oxford) France (IGR, CRH) Sweden (Karolinska) Node negative, untreated < 60 years old > 5 years follow-up T1, T2 Tumor cell % > 50% Amsterdam Gene expression profiling Agilent platform 70-gene prognostic custom designed chip Clinical data «Local» pathological data Audited clinical data Centrally reviewed path data (Milan) High or low gene signature risk Brussels Comparison of clinical vs gene signature assessment of prognostic risk Endpoints 1. OS 2. DMFS, DFS Buyse et al, JNCI 98: , 2006

58 Adjuvant! Online for breast cancer (Updated version) used for a standardized approach to Clinical Risk Clinical low risk, in agreement with EuropaDonna patient advocates, defined as predicted 10-year breast cancer survival probability (without adjuvant therapy): 92% for ER- patients 88% for ER+ patients (will equal 92% with adjuvant endocrine therapy)

59 N = 302 pts 92 events Med fup 13.6y Average Survival HR 2.66 OVERALL SURVIVAL by 70-GENE SIGNATURE RISK Results of the TRANSBIG validation study Probability Patients Events Risk group Genetic low risk Genetic high risk 10-year OS 70% (62%-76%) 10-year OS 89% (81%-94%) Green light for! Year CLR CHR Number at risk Buyse et al, JNCI 2006; 98: ,

60 ROBUST INTERLABORATORY REPRODUCIBILITY OF THE 70-GENE SIGNATURE MEASUREMENT Milestone of the TRANSBIG PROJECT Collaboration between: Agilent Technologies Robert Ach Institute Gustave Roussy Vladimir Lazar Agendia Arno Floore Using 4 samples from the validation series CONCLUSIONS Adherence to standard protocol crucial High significant reproducibility R.A. Ach et al., BMC Genomics 2007; 8:148

61 Greater trust in the 70-gene profile for patients with 1-3 positive nodes Distant metastases as first event 91% Good profile (40%) Poor profile (60%) Breast cancer specific survival 96% 73% 71% P<0.001 HR 3.8 ( ; p<0.001) HR adjusted 2.8 ( ; p=0.009) P<0.001 HR 6.1 ( ; p<0.001) HR adjusted 5.4 ( ; p=0.001) Of note: 53% had adjuvant CT / Med Fup= 7.8y Mook et al, Breast Cancer Res Treat 2009; 116:

62 The development pathway of MINDACT: 12 years from conception to first results study TRANSBIG Independent validation of the 70- gene (MammaPrint ) signature Clinical validity proven Analytical validity proven Fine-tuning of MINDACT DESIGN MINDACT protocol developed MINDACT recruitment

63 Two additional optional randomizations supported by 3 pharmaceutical companies (Novartis / Sanofi Aventis / Hoffman La Roche) Chemotherapy Endocrine therapy

64 Risk corrections post enrollment C-risk/G-risk at enrollment cl/g? (N=1) cl/gl (N=2744) Corrected C-risk/G-risk cl/gh (N=592) ch/gl (N=1550) ch/gh (N=1806) Total (N=6693) N (%) N (%) N (%) N (%) N (%) N (%) cl/gl 1 (100.0) 2600 (94.8) 3 (0.5) 30 (1.9) 0 (0.0) 2634 (39.3) cl/gh 0 (0.0) 95 (3.5) 580 (98.0) 0 (0.0) 15 (0.8) 690 (10.3) ch/gl 0 (0.0) 44 (1.6) 0 (0.0) 1450 (93.5) 3 (0.2) 1497 (22.4) ch/gh 0 (0.0) 5 (0.2) 9 (1.5) 70 (4.5) 1788 (99.0) 1872 (28.0)

65 Reason for clinical risk change Reason for clinical risk assessment change Total (N=103) N (%) change in tumor size 21 (20.2) change in tumor grade 19 (18.3) change in ER status 4 (3.8) change in HER2 status 12 (11.5) change in Nodal status 24 (23.1) clinical risk of LN2/3+ was miscalculated as LN 4/9+ 6 (5.8) clinical risk of 12% was miscalculated as low risk 6 (5.8) other/combination 11 (10.6)

66 Reason for genomic risk change Reason for clinical risk assessment change Total (N=177) N (%) Change in RNA extraction solution 153 (86.4) Sample swap 5 (2.8) other/combination 19 (10.7)

67 Sensitivity analysis excluding the period of shift in risk due the change in RNA extraction solution (1)

68 Allocated Treatment strategy Patients (N) Events % at 5 Year(s) (95% CI) (adjusted Cox model) (O) (95% CI) Sensitivity analysis excluding the period of shift in risk due the DFS DMFS OS ACT (90.3, 95.4) 0.57 (0.37,0.87) change in RNA extraction solution (2) no ACT (85.7, 91.3) 1.00 ACT (94.1, 97.9) 0.60 (0.34,1.06) no ACT (91.4, 95.8) 1.00 ACT (97.1, 99.5) 0.54 (0.23,1.26) no ACT (94.9, 98.2) 1.00 PPS population (excluding G-risk shift period): clgh p-value (adjusted logrank) Allocated Treatment strategy Patients (N) Observed Events (O) % at 5 Year(s) (95% CI) Hazard Ratio p-value (adjusted Cox model) (adjusted logrank) (95% CI) DFS DMFS OS ACT (86.8, 96.0) 0.69 (0.34,1.39) no ACT (84.6, 93.9) 1.00 ACT (91.8, 98.3) 0.86 (0.35,2.14) no ACT (89.2, 96.9) 1.00 ACT (94.7, 99.7) 0.45 (0.11,1.85) no ACT (92.7, 98.5)

69 Subgroup analysis of the HR+/HER2-/N negative patients (analyses in the subgroups with discordant risk) DMFS - ITT1 population: chgl subgroup Allocated Treatment strategy Patients (N) Observed Events (O) % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) HR+/HER2-/N- CT (92.5, 97.3) 0.80 (0.44,1.45) no CT (90.6, 96.1) 1.00 DMFS - ITT1 population: clgh subgroup Allocated Treatment strategy Patients (N) Observed Events (O) % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) HR+/HER2-/N- CT (91.5, 97.2) 1.45 (0.68,3.08) no CT (91.6, 97.6)

70 Multivariate analysis Factor Levels Final model for DMFS (N=6643) (After backward selection) N Interaction terms with chemo Hazard Ratio (95% CI) P-value C-risk low high (1.05, 2.13) G-risk low <0.001 high (1.79, 3.26) LN status LN LN (0.99,1.67) T status 1 cm <0.001 CT and HER2 status interaction 1-2 cm (0.78, 1.79) 2-5 cm (1.20, 3.06) > 5cm (0.04, 2.39) HER2 negative HER2 positive No CT CT 0.56 (0.40, 0.78) 176 No CT CT 0.24 (0.13,0.49) CT effect: <0.001 HER2 effect: Interaction effect between HER2 and CT: HR status negative positive (0.63, 1.05) grade (1.04, 2.13) (1.08, 2.63) surgery Mastectomy Breast conserving surgery (0.58, 0.94)

71 Distant Metastasis Free Interval ITT1 population: chgl Allocated Treatment strategy Patients (N) Observed Events (O) % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) DMFI CT (94.8, 97.8) 0.76 (0.47,1.22) no CT (93.4, 96.6) 1.00 ITT1 population: clgh Allocated Treatment strategy Patients (N) Observed Events (O) % at 5 Year(s) (95% CI) Hazard Ratio (adjusted Cox model) (95% CI) p-value (adjusted logrank) DMFI CT (95.7, 99.1) 0.63 (0.27,1.47) no CT (92.5, 97.5)