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Expression of novel immunotherapeutic targets in luminal breast cancer patients
Gargi Basu1, Ph.D., Anatole Ghazalpour1, Ph.D., Joanne Xiu, Ph.D., Randal Vader1, Sandeep Reddy1, M.D., Karen S. Anderson2, M.D., Ph.D., Ann McCullough3, M.D., Barbara Pockaj3, M.D.
1Caris Life Sciences, Phoenix, Arizona 2Biodesign Institute, Arizona State University, Tempe, Arizona 3Mayo Clinic, Phoenix, Arizona
Abstract
Background: The development of novel chemotherapeutic agents has
significantly improved the prognosis and survival of patients with breast cancer.
ER positive or luminal tumors represent around two thirds of all breast cancers
and these cancers are comprised of different histologies including differing gene
expression and mutational profiles. This study examined biomarkers involved in
immune evasion including PD-L1 and its association with other biological
pathways as potential treatment options for luminal breast cancer patients.
Methods: We analyzed 1311 breast samples using a multiplatform approach
including whole genome mRNA expression (HumanHT-12 v4 BeadChip Illumina
Inc., San Diego, CA), protein expression (immunohistochemistry), gene copy
number changes (in situ hybridization) and gene sequencing and an additional
304 breast samples were tested for PD-1 and PD-L1 by IHC. The mRNA
expression data was based on whole tumor and represents cell type
heterogeneity. Heat map analysis was done to look at differential gene
expression between the PD-L1 high vs. low luminal breast cancers.
Results: Based on expression of ER, PR and HER2 by IHC, we subdivided the data
into sub- cohorts. Elevated mRNA expression of immune markers including PDL1, CTLA4, B7H-3, and IDO1 was noted in the ER+HER2- luminal population
including ER+ PR- and ER+PR+ cohort. Positive correlation was found between
PD-L1 and other immune regulators including CTLA-4, B7-H3 and IDO1
(Spearman correlations of 0.49, 0.36 and 0.46). Protein expression of PD-L1 was
found to be specific to the HER2 negative cohort with no expression in the HER2
positive cohort regardless of ER status. Within the ER+HER2- cohort, PD-L1
expression was 5% {ER+PR+ was 5% (4/81) and the ER+PR- was 6% (2/34)}. In
contrast, PD-L1 expression was higher in the triple negative cohort, 17% (13/75).
The expression of PD-1 on the other hand was present throughout the different
cohorts. PD-1 expression ranged from 43% in the ER+HER2- cohort {41% (14/34)
in ER+PR-HER2-; 43% (35/81) in ER+PR+HER2-} to 33% in the ER+HER2+ cohort
{44% (4/9) in ER+PR-Her2+; and 17% (1/6) in ER+PR+HER2+}. In the ER-HER2+
cohort PD-L1 expression was 75%{6/8 in ER-PR-HER2+ and 0/0 in ER-PR+HER+}
and 63%(47/75) in the triple negative cohort. Pathway analysis of the PD-L1
negative vs. positive luminal population identified 127 genes involved in various
cancer pathways including EGFR and VEGF signaling network (P=7.47e-09).
Conclusions: The expression of immune regulatory targets in the breast cancer
population suggests that immune- targeted therapies with anti PD-1/PD-L1,
CTLA4, B7-H3 and IDO1 may be effective especially in the luminal cohort. Our
study further shows that ER+HER2- breast cancer patients may be better
candidates for immunotherapy with checkpoint inhibitors due to lack of PD-L1
expression in the HER2 positive cohort. Further validation of our findings is
ongoing.
Results
Full analysis includes additional tumors profiled since the abstract
submission
Figure 1: mRNA microarray revealed elevated immune markers including PDL1 (upper), CTLA-4 (middle) and IDO1 (lower). Positive correlations were
found between PD-L1 and CTLA-4 (Spearman correlation of 0.49), and IDO1
(Spearman correlation of 0.36 )
Figure 3: Heatmap of differentially expressed genes between the PD-L1 low and high
expressors as determined by Illumina gene expression microarray from the luminal
cohorts (defined as ER+ Her2-). The top and bottom 20th percentile population was
selected based on PD-L1 expression and a t-test run to determine differentially
expressed genes. The columns represent the high and the low PD-L1 expressor cases
utilized in this study. The rows are the significantly differentially expressed genes
between the two groups. Red color on the top bar labels the cases with high PD-L1
expression and the blue bar labels the low PD-L1 expressors. The blue color within the
heatmap indicates low expression and red indicates high expression.
Table 1: A total of 732 breast cancer tumors with PD-L1 and PD-1 staining
results were subdivided into 8 groups based on ER, PR and Her2 status. PD-1
and PD-L1 protein expression were evaluated by immunohistochemistry on
TIL (tumor infiltrating lymphocytes) and tumor cells, respectively.
Protein expression in breast cancer (total N=732)
ER+
ERPR+
PRPR+
PRHer2+ Her2- Her2+ Her2- Her2+ Her2Her2+ Her224
271
17
109
2
12
41
256
Total Patient N
N
12
110
6
55
1
4
23
151
PD-1 +
Percent 50.0% 40.6% 35.3% 50.5% 50% 33.3% 56.1% 59.0%
N
1
17
0
4
0
4
4
49
PD-L1+
Percent
4.2%
6.3% 0.00% 3.7% 0.00% 33.3%
9.8%
19.1%
Figure 2: PD-L1 expression in the luminal cohort (ER+Her2-) is higher than
the ER+Her2+ cohort. PD-L1 expression is significantly higher in triple
negative breast cancer than luminal breast cancer (Gatalica et al, 2014); PD-1
expression is found in a significant portion of breast cancer across different
subtypes (from a total N of 732).
Conclusions
•
•
•
•
Using immunohistochemistry, 6.0% (26/435) of HR+ (hormone receptor +) breast cancer
cases are positive for PD-L1 expression and 43% (188/435) are positive for PD-1
expression.
HR+Her2- breast cancer cases are more than twice as likely to overexpress PD-L1 as HR+
Her2+ breast cancer (25/392 or 6.4% vs. 1/43 or 2.3%; not significant possibly due to
low N, p=0.50); PD-1 expression is similar in these two cohorts.
Consistent with the finding by microarray analysis, HR+ breast cancer cases are
significantly less likely to have PD-L1 (26/435 or 6.0% vs. 49/256 or 19%, p<0.0001) or
PD-1 expression (188/435 or 43% vs. 151/25659%, p<0.0001) as tested by IHC when
compared to triple negative breast cancer.
The expression of immune regulatory targets tested by microarray in the breast cancer
population suggests that immune- targeted therapies with anti PD-1/PD-L1, CTLA4, B7H3 and IDO1 may be effective in luminal breast cancer.
Reference
•
Gatalica, Z, et al (2014) “Programmed Cell Death 1 (PD-1) and Its Ligand (PD-L1) in
Common Cancers and Their Correlation with Molecular Cancer Type.” Cancer Epidemiol
Biomarkers Prev. 1-6