IOV – Istituto Oncologico Veneto I.R.C.C.S. Integrated evaluation of PAM50 subtypes and immune modulation of pCR in HER2-positive breast cancer patients treated with chemotherapy and HER2-targeted agents in the CherLOB trial Maria Vittoria Dieci Università di Padova, Dipartimento di Scienze Chirurgiche, oncologiche e Gastroenterologiche IOV – Istituto Oncologico Veneto I.R.C.C.S.

BACKGROUND HER2+ breast cancer is clinically and biologically heterogeneous. Molecular intrinsic subtypes are not fully recapitulated by hormone receptor status. Prat A, JNCI 2014; Carey L JCO 2016

Intermediate/present 2016 Median % N None/absent Intermediate/present High All 4161 16 89 11 TN 1640 15 80 20 HER2+ 929 9 84 HR+ 2410 94 6

TILs in HER2+ BC patients treated with NACT Denkert C, SABCS 2016

Aim: To evaluate the relative contribution of tumor-related and immune-related diversity of HER2-positive disease on the response to neoadjuvant chemotherapy plus anti-HER2 agents. Exploratory translational evaluation of PAM50 molecular intrinsic subtypes, TILs and immune gene signatures in the context of the neoadjuvant prospective CherLOB trial.

METHODS – TILs and PAM50 Hematoxylin and eosin-stained (HES) slides from the diagnostic core biopsy were evaluated for Intratumoral (It) and Stromal (Str) TILs (%), according to predefined criteria [Salgado R, AoO 2014]. Lymphocite predominant breast cancer (LPBC): ItTILs and/or StrTILs >=60% RNA extracted from snap-frozen diagnostic core biopsy samples (Guarneri V, Oncologist 2012). The research-based, 50-gene prediction analysis of microarray (PAM50) subtype predictor was applied to classify into one of the following groups: Luminal A, Luminal B, HER2-enriched, Basal-like and Normal-like.

METHODS – Immune signatures unsupervised clustering analysis of the 4909 most variable genes across the 86 cherLOB patients with GE data. Criteria: at least 20 genes and a coefficient correlation among them >0.80 Immune2 Immune3 RibasImmune predictive of anti-PD1 therapy in patients with melanoma (Ribas A, ASCO 2015) T-helper cell Ten gene lists associated with different immune cell types identified from the literature (Bindea G, Immunity 2013; Galon J, Immunity 2013; Talmadge JE, Cancer Metastasis Rev 2007) aDC B-cell CD8-Tcell NK T-cell Th1-cell Th2-cell Treg-cell iDC StromalSAM 82-gene list associated with StrTILs (cherLOB patients) in a SAM quantitative analysis (FDR<below 10%), enriched for immune-related processes.

RESULTS: PATIENTS POPULATIONS

RESULTS: PATIENTS CHARACTERISTICS   CherLOB population N=121 TIL population N=105 GE population N=86 N (%) Mean (range) Age 121 49 (26-68) 105 50 (25-68) 86 49 (27-68) Clinical stage IIA 38 (31) 37 (35) 28 (32) IIB 61 (50) 51 (49) 41 (48) IIIA 22 (18) 17 (16) 17 (20) Histotype Ductal 115 (95) 101 (96) 83 (96) Lobular 6 (5) 4 (4) 3 (4) Grade 1-2 23 (23) 20 (23) 17 (24) 3 77 (77) 68 (77) 55 (76) ER ER- 50 (41) 40 (38) 36 (42) ER+ 71 (59) 65 (62) 50 (58) Ki67 113 29.6 (4-90) 101 28.3 (4-70) 82 29.4 (4-90) Arm A (CT+T) 36 (30) 32 (31) 22 (26) B (CT+L) 39 (32) 34 (32) 30 (35) C (CT+T+L) 46 (38) 39 (37) 34 (39) Evaluable for path response Yes No 118 84 2

RESULTS: TILs according to patients characteristics   StrTIL % LPBC phenotype, N(%) N (%) Median [range] P LPBC Non-LPBC All 105(100) 17 [0-100] - 17 (16) 88 (84) Age >50yrs 52 (50) 18 [0-100] 9 (17) 43 (83) <50yrs 53 (50) 15 [0-90] ns 8 (15) 45 (85) Stage IIA IIB IIIA 37 (35) 51 (49) 17 [0-90] 18 [2-100] 4 (11) 9 (18) 4 (23) 33 (89) 42 (82) 13 (77) Grade 1-2 3 20 (29) 68 (71) 10 [2-70] 20 [0-90] 2 (10) 14 (21) 18 (90) 54 (79) ER ER- 40 (38) 27.5 [0-100] 11 (27) 29 (73) ER+ 65 (62) 12 [0-90] 0.003 6 (9) 59 (91) 0.014 Ki67 <20% >20% 35 (35) 66 (65) 0.045 2 (6) 33 (94) 52 (79) 0.042 Arm A (CT+T) B (CT+L) C (CT+T+L) 32 (31) 34 (32) 39 (37) 17.5 [0-70] 20 [0-100] 15 [2-90] 5 (16) 6 (18) 6 (15) 27 (84) 28 (82) 33 (85) Similar results for ItTILs

RESULTS: TILs and association with pCR OR 1.03, 95% CI 1.02–1.05 (P < 0.001) for each 1% increase in Str-TILs OR 1.09, 95% CI 1.04–1.15 (P < 0.001) for each 1% increase in It-TILs Dieci MV, Ann Oncol 2016

RESULTS: PAM50 intrinsic subtypes ALL PATIENTS (N=86) ER+ (N=50) ER- (N=36) 8% 17.4% 16% 19.4% 12% 26.7% 2.8% 16.3% 8.3% 52.8% 26% 14% 38% 16.7% 25.6%

pCR according to PAM50 intrinsic subtypes RESULTS: pCR according to PAM50 intrinsic subtypes p=0.026 N=69 N=22 N=12 N=14 N=21

RESULTS: TILs vs PAM50 intrinsic subtypes HER2-enriched vs others: p=0.012 HER2-enriched vs others: p=0.015

RESULTS: Correlation between immune-related and tumor-related parameters The correlation coefficient among the various immune-related gene signatures, except those tracking Treg-FOXP3 and NK cells, was high (0.816). When TILs were included, the correlation among all immune variables was moderate (0.572). Weak correlation was found between PAM50 subtype with immune parameters in general.

Immune parameters and PAM50 subtypes: association with pCR Univariate analysis Corrected for PAM50 Dieci MV, Ann Oncol 2016

Conclusions The results are in line with previous reports showing that HER2-positive BCs encompass a spectrum of distinct molecular entities. The complexity of HER2+ BC relies on both tumor cell-related features and the stroma, both contributing to a significant extent to the modulation of treatment sensitivity. Immune gene signatures rather than TILs may be more descriptive of the complex immune microenvironment in HER2+ BC. However, TILs still represent the easiest and cheapest immune biomarker and the lack of independent association with pCR beyond PAM50 may be due to limited sample size.