1. 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.

2. 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

3. 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

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

6. 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.

8. 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.

9. 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.

11. RESULTS: PATIENTS POPULATIONS

12. 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

13. 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

14. 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

15. 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%

16. 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

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

18. 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.

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

20. 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.