Tratamiento neoadyuvante: Enfermedad residual como marcador de resistencia Carlos L. Arteaga, MD Vanderbilt-Ingram Cancer Center Vanderbilt

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1 Tratamiento neoadyuvante: Enfermedad residual como marcador de resistencia Carlos L. Arteaga, MD Vanderbilt-Ingram Cancer Center Vanderbilt University

2 Neoadjuvant (preoperative) therapy Surgery Systemic therapy Adjuvant Neoadjuvant Systemic therapy Surgery NSABP B-18: Preoperative versus postoperative AC N=1450 clinical T1-3, N0-1 DFS DDFS OS AC OR OR AC Fisher B et al, JCO 1998

3 NOAH (NeOAdjuvant Herceptin) study HER2-positive LABC (IHC 3+ or FISH+) HER2-negative LABC (IHC 0/1+) (n=115) H + AT q3w x 3 cycles H + T q3w x 4 cycles H q3w x 4 cycles + CMF q4w x 3 cycles Surgery followed by radiotherapy a H continued q3w to week 52 (n=113) AT q3w x 3 cycles T q3w x 4 cycles CMF q4w x 3 cycles Surgery followed by radiotherapy a 19 crossed over to H (n=99) AT q3w x 3 cycles T q3w x 4 cycles CMF q4w x 3 cycles Surgery followed by radiotherapy a IHC, immunohistochemistry; FISH, fluorescence in situ hybridisation; H, trastuzumab (8 mg/kg loading dose then 6 mg/kg); AT, doxorubicin (60 mg/m 2 ), paclitaxel (150 mg/m 2 ); q3w, every 3 weeks; T, paclitaxel (175 mg/m 2 ); q4w, every 4 weeks a Hormone receptor-positive patients will receive adjuvant tamoxifen Gianni et al. Lancet 375:377, 2010

4 Pathological CR rate in NOAH: Intent-to-treat population Patients (%) % p=0.002 p= % 16% 0 With H Without H HER2 negative HER2 positive pcr Gianni et al. Lancet 375:377, 2010

5 Event-free survival in HER2+ patients Probability, EFS H + CT CT Patients Events Months HR a 0.59 p a Median follow-up is 3 years a Unadjusted for stratification variables: adjusted HR=0.58, p= HR, hazard ratio; CI, confidence interval; CT, chemotherapy

6 Can we use neoadjuvant trials to simplify large adjuvant trials (Neo-ALTTO ALTTO)

7 If we knew the results of Neo-ALTTO before ALTTO, had a lapatinib arm been necessary in ALTTO?

8 . after accelerated approval, demonstration of an improvement in disease-free or overall survival would be required NEJM 366:2438, 2012

9 Triple negative breast cancer and neoadjuvant chemotherapy Neoadjuvant chemotherapy (NAC) is used for breast conservation A pathologic complete response (path CR; no evidence of disease at surgery) after NAC is achieved in 20-30% of patients and provides prognostic information In patients with br ca who do not achieve a path CR, a high tumor cell proliferation (by Ki67) in the residual cancer after NAC predicts poor outcome Thus, the molecular profile of the tumor cells remaining after selection with NAC may serve as a proxy for the alterations present in clinically-silent, drug-resistant micro-metastases destined to recur Liedtke et al. JCO 26:1275, 2008 Jones et al. Breast Cancer Res. Treat, 2009

10 Change in HER2 status in HER2+ breast cancers treated with neoadjuvant chemotherapy + trastuzumab correlate with patient outcome + to + + to negative (FISH) Speculation: In patients whose tumors switched from HER2+ to HER2-negative, micrometastases were also HER2-negative and, thus, did not benefit from adjuvant trastuzumab (?) Mittendorf et al. Clin Cancer Res. 15:7381, 2009

11 The CD44+/CD24 /low-ms gene signature is enriched in breast tumors after treatment with antiestrogens or chemotherapy Creighton C J et al. PNAS 2009;106: by National Academy of Sciences

12 JNCI 99:167, 2007 Baseline Ki67 p= week post-anastrozole Ki67 but not the baseline Ki67 predicted long-term outcome 2-week Ki67 p=0.008

13 a-estrogensensitive a-estrogenresistant Presurgical clinical trial of letrozole in ER+/HER2 operable breast cancer (Vanderbilt SPORE) Letrozole Post-letrozole Ki67 25% <1%

14 PIK3CA mutations correlate with a higher Ki67 following estrogen suppression with letrozole

15 TCGA data on ~10,000 tumors

16 PI3K pathway mutations

17 Combined inhibition of ER and PI3K induces complete regressions of ER+/PIK3CA-mutant br ca xenografts Miller et al. Cancer Discovery 1:338, 2011

18 Other mutations in the 4 patients with the highest post-letrozole Ki67 (kinome deep SEQ, 612 genes) Gene Tumor Symbol % Variant in Tumor Amino Acid Change Method Identified Domain A TEC 9% V->I Kinome-SEQ Kinase A CSNK1G3 9% T->A Kinome-SEQ Kinase A TSSK4 3% L->V Kinome-SEQ Kinase A TP53 41% R->G Kinome-SEQ DNA binding A PIK3CA 16% H->R (H1047R) Kinome-SEQ/OncoMap Catalytic B EPHA7 10% Y->H Kinome-SEQ Ligand binding B PIK3CA 10% Q->E (Q546E) Kinome-SEQ Helical C LYN 8% D->Y Kinome-SEQ SH2 C ATM 10% D->H Kinome-SEQ None C DYRK2 18% R->W Kinome-SEQ Kinase C MYLK3 7% Q->E Kinome-SEQ None C PIK3CA 14% T->A (T1025A) Kinome-SEQ/OncoMap Catalytic D PTK2 1% A->V Kinome-SEQ Unknown D WNK2 10% G->R Kinome-SEQ None D DYRK2 3% M->I Kinome-SEQ Kinase D DYRK1A 4% R->H Kinome-SEQ None D PIK3CA 2% H->R (H1047R) Kinome-SEQ Catalytic Fox E, Balko J,.. Arteaga CL, Unpublished

19 RNAi kinome screen identifies LYN is required for hormone-independent ER+ breast cancer growth MCF-7/LTED LYN

20 Copy number increases in LYN occur in ~10% of breast cancers Analysis of 550 breast cancers in the TCGA identified four additional LYN mutations: E159K (SH2), K188N (SH2), G418R (Kinase), and A503D (Kinase).

21 D189Y LYN confers resistance to estrogen deprivation, fulvestrant and PI3K inhibitors in ER+/PIK3CA mutant breast cancer cells

22 Inhibition of the SFKs with dasatinib enhances the effect of BKM120 and fulvestrant against ER+/PIK3CA mutant tumors log 2 (Tumor Volume) 4096 Vehicle (n=9) Dasatinib (n=9) 2048 BKM + Fulv (n=10) 1024 BKM + Fulv + Das (n=10) * # Time of Treatment (wks)

23 Implications ER+ tumors that maintain high proliferation upon shortterm estrogen deprivation will harbor multiple molecular lesions potentially associated with antiestrogen resistance These alterations can be interrogated in the surgical specimen after short term therapy or after neoadjuvant therapy

24 A second cohort of triple negative breast cancers after neoadjuvant chemotherapy (NAC) Immunohistochemistr y Ki67, ER, PR, HER2, AR 112/ clinically-defined TNBC patients with RD after NAC Next generation sequencing 182 oncogenes and tumor suppressors 81/114 Nanostring digital expression analysis 450 genes 89/114 Median Min Max Age N % Stage IIa 3 3% IIb 5 5% IIIa 13 12% IIIb 77 69% IIIc 10 9% NA 3 3% Taxane Yes 55 50% No 53 48% NA 3 3% Menopaus e Pre 55 50% Post 53 48% NA 3 3% Node status Pos 70 63% Neg 37 33% NA 4 4%

25 Ki67 in TNBC after neoadjuvant chemotherapy does not predict survival Ki67 was scored by IHC in the residual tumor after NAC At least 500 tumor cells were counted in each case A cutoff of 15% was used based on previously published literature % Samples Basal-like Her2-enriched Luminal A Luminal B Normal-like Ki67 score after NAC (%) Basal-like Her2-enriched ANOVA P= Luminal A Luminal B Normal-like Percent relapse-free P=0.42 Ki67 > 15% Ki67 < 15% Time (years) Percent surviving P=0.84 Ki67 > 15% Ki67 < 15% Time (years)

26 Better survival in non-basal-like vs. basal-like post-chemotherapy cancers P=0.1 P=0.05

27 Randomized trial of post-operative cisplatin vs. observation in pts with TNBC who do not achieve a path CR after NAC and have basal-like gene expression in the residual tumor pcr Not eligible TNBC Post-NAC N ~ Residual disease (> 0.5 cm) at surgery Tissue collection PAM50 Large Scale Profiling Not basal-like Basal-like N = 221 (2:1) N = 147 Cisplatin 75 mg/m2 x 4 doses Observation N = 74 We expect ~ 80% of the residual post-nac tumors to fall within the basal-like TNBC subtype Mayer, VICC

28 Next generation sequencing of post-nac TNBC (n=81) 182 oncogenes and tumor suppressors in a CLIA certified lab (Foundation Medicine, Cambridge MA) Data were evaluable for 81 tumors with a sufficient coverage to determine CNAs in 72/81 Mean depth of coverage was 635 (range: ) HER2 amplification was identified by NGS in 7/81 samples and these samples were excluded from survival analyses % of samples TP53 MCL1 N=81 MYC PIK3CA PTEN RB1 BRCA1 JAK2 CDKN2A Amplifications/deletions or mutations of known or implied functional significance Gene NF1 KRAS CCND1 AKT3 CCND2 CCND3 IGF1R CDK6 CCNE1 Novel mutations of unknown significance could not be assessed due to lack of normal controls

29 MCL1 amplification occurs at high rates in TNBC and co-occurs with MYC amplification Amplification of the anti-apoptosis gene MCL1 was identified in 56% of tumors. MCL1 IHC p=0.001 MYC amplification was identified in 33% of tumors. MYC % of cases WT Amplified MYC status MCL1 status WT Amplified MCL1 Oncogene induced apoptosis/ senescence

30 MYC and MCL1 cooperate on transformation of mammary epithelial cells

31 JAK2 amplification in TNBC correlates with IL6 overexpression Amplifications in the JAK2 locus were identified in 8/72 (11%) of patients IL6 IL6ST JAK2 STAT3 IL6 mrna IL6 mrna expression p=0.008 WT Amplified JAK2 status

32 Clinically targetable pathways in TNBC Number of samples with aberrations AKT3 PTEN PIK3CA TSC1 AKT2 AKT1 RAPTOR RICTOR PIK3R1 BRCA2 BRCA1 ~90% of all patients had an alteration in at least one of these pathways ATM NF1 CRAF BRAF KRAS RB1 CDNK2A CCNE1 CCND3 CCND2 CCND1 CDK6 AURKA CDK4 MET IGF1R EGFR PI3K/mTOR DNA Repair Ras/MAPK Cell Cycle GFRs FGFR4 FGFR1 KIT FGFR2 PI3K/mTOR inhibitors DNA-repair targeting agents RAF/MEK inhibitors Cell cycle/mitotic spindle inhibitors Targeted RTK inhibitors

33 Role of neoadjuvant therapy studies in discovery of mechanisms of drug resistance Provide high-quality tumor material in patients not achieving a path CR, where mechanisms of drug resistance can be investigated The molecular profiles of these tumors may provide leads for additional adjuvant therapy or (targeted) treatment at the time of recurrence Molecular profiling of the residual drug resistant tumor following neoadjuvant therapy should become common practice in the near future

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