Association and prognostic significance of BRCA1/2-mutation status with neoantigen load, number of tumor-infiltrating lymphocytes and expression of PD-1/PD-L1 in high grade serous ovarian cancer

Kyle C Strickland, Brooke E Howitt, Sachet A Shukla, Scott Rodig, Lauren L Ritterhouse, Joyce F Liu, Judy E Garber, Dipanjan Chowdhury, Catherine J Wu, Alan D D'Andrea, Ursula A Matulonis, Panagiotis A Konstantinopoulos, Kyle C Strickland, Brooke E Howitt, Sachet A Shukla, Scott Rodig, Lauren L Ritterhouse, Joyce F Liu, Judy E Garber, Dipanjan Chowdhury, Catherine J Wu, Alan D D'Andrea, Ursula A Matulonis, Panagiotis A Konstantinopoulos

Abstract

Immune checkpoint inhibitors (e.g., anti-PD-1 and anti-PD-L1 antibodies) have demonstrated remarkable efficacy against hypermutated cancers such as melanomas and lung carcinomas. One explanation for this effect is that hypermutated lesions harbor more tumor-specific neoantigens that stimulate recruitment of an increased number of tumor-infiltrating lymphocytes (TILs), which is counterbalanced by overexpression of immune checkpoints such as PD-1 or PD-L1. Given that BRCA1/2-mutated high grade serous ovarian cancers (HGSOCs) exhibit a higher mutational load and a unique mutational signature with an elevated number of larger indels up to 50 bp, we hypothesized that they may also harbor more tumor-specific neoantigens, and, therefore, exhibit increased TILs and PD-1/PD-L1 expression. Here, we report significantly higher predicted neoantigens in BRCA1/2-mutated tumors compared to tumors without alterations in homologous recombination (HR) genes (HR-proficient tumors). Tumors with higher neoantigen load were associated with improved overall survival and higher expression of immune genes associated with tumor cytotoxicity such as genes of the TCR, the IFN-gamma and the TNFR pathways. Furthermore, immunohistochemistry studies demonstrated that BRCA1/2-mutated tumors exhibited significantly increased CD3+ and CD8+ TILs, as well as elevated expression of PD-1 and PD-L1 in tumor-associated immune cells compared to HR-proficient tumors. Survival analysis showed that both BRCA1/2-mutation status and number of TILs were independently associated with outcome. Of note, two distinct groups of HGSOCs, one with very poor prognosis (HR proficient with low number of TILs) and one with very good prognosis (BRCA1/2-mutated tumors with high number of TILs) were defined. These findings support a link between BRCA1/2-mutation status, immunogenicity and survival, and suggesting that BRCA1/2-mutated HGSOCs may be more sensitive to PD-1/PD-L1 inhibitors compared to HR-proficient HGSOCs.

Keywords: BRCA1 and BRCA2 mutations; PD-1 and PD-L1; high grade serous ovarian cancer; homologous recombination DNA repair; immunogenicity.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest to report.

Figures

Figure 1. Neoantigen load in BRCA1/2-mutated, non-BRCA1/2-mutated/HR-deficient…
Figure 1. Neoantigen load in BRCA1/2-mutated, non-BRCA1/2-mutated/HR-deficient and HR proficient cohorts, and association with outcome in the TCGA dataset
(A) Predicted neoantigen load in BRCA1/2-mutated (n = 54) vs all remaining non-BRCA1/2-mutated tumors (n = 191). (B) Predicted neoantigen load in BRCA1/2-mutated (n = 54), HR deficient/non-BRCA1/2-mutated (n = 69) and HR proficient tumors (n = 122). (C) Predicted neoantigen load of HR-deficient (n = 123) vs HR-proficient (n = 122). (D) Predicted neoantigen load of BRCA1- versus BRCA2-mutated tumors. (E) Tumors in the lowest quartile of neoantigen load were associated with significantly lower overall survival compared to the remaining tumors. Of the 60 tumors in the lower quartile, 20 were HR deficient and 40 were HR proficient. (F) Tumors in the lowest quintile of neoantigen load were associated with significantly lower overall survival compared to the remaining tumors. Of the 47 tumors in the lower quintile, 19 were HR deficient and 28 were HR proficient.
Figure 2. Outline of our study cohorts
Figure 2. Outline of our study cohorts
(A) Prediction of neoantigen load in the TCGA dataset. (B) Determination of BRCA1/2-mutated and HR proficient subsets in our institutional cohort. The BRCA1/2-mutated group was comprised of 37 HGSOCs with BRCA1/2 germline mutations (29 with BRCA1 and 8 with BRCA2 mutations) identified by genetic testing (left). The HR-proficient (HR intact) group (i.e. group without HR alterations) comprised 16 ovarian cancers which were identified in a two-step process (right). First, NGS excluded tumors with mutations in HR genes and this analysis identified 17 such tumors. Tumor was excluded based on absent BRCA1 expression by immunohistochemistry.
Figure 3. Results of BRCA1 immunohistochemistry
Figure 3. Results of BRCA1 immunohistochemistry
(A) Positive BRCA1 IHC in a representative case. BRCA1 expression was positive by IHC in 16 of the 17 tumors without HR alterations identified by NGS. (B) BRCA1 IHC was negative in one tumor that did not harbor HR alterations by NGS. Focal BRCA1 positivity was present in lymphocytes. Interestingly, review of the NGS data for this case demonstrated that the tumor had a single copy deletion of the BRCA1 gene, suggesting that BRCA1 loss in this tumor was likely due to single copy deletion of BRCA1 and epigenetic silencing of the complementary allele. (C) Corresponding area of tumor on H & E stain demonstrates the presence of intratumoral lymphocytes. (D) The presence of intraepithelial lymphocytes was confirmed by a CD3 IHC.
Figure 4. CD3+, CD4+ CD8+ and CD20+…
Figure 4. CD3+, CD4+ CD8+ and CD20+ intraepithelial lymphocytes in BRCA1/2-mutated vs HR proficient tumors
(A) Photomicrographs of representative BRCA1/2-mutated and HR-intact tumors depicting H & E staining and immunohistochemistry for CD3, CD4, CD8 and CD20. (B) Quantification and comparison of CD3+ TILs from BRCA1/2-mutated and HR intact tumors. (C) Quantification and comparison of CD4+ TILs from BRCA1/2-mutated and HR intact tumors. (D) Quantification and comparison of CD8+ TILs from BRCA1/2-mutated and HR intact tumors. (E) Quantification and comparison of CD20+ TILs from BRCA1/2-mutated and HR intact tumors.
Figure 5. PD-1 and PD-L1 expression in…
Figure 5. PD-1 and PD-L1 expression in the intraepithelial and peritumoral immune cells of BRCA1/2-mutated versus HR-proficient tumors
(A) Photomicrographs of representative BRCA1/2-mutated and HR-intact tumors depicting H & E staining and immunohistochemistry for PD-1 and PD-L1. Photomicrographs depict cases from each study group that were scored as positive. (B) Bar graphs illustrating the number of tumors with increased PD-1 and PD-L1 positive intraepithelial and peritumoral immune cells, as well as the number of tumors positive for PD-L1 in tumor cells of BRCA1/2-mutated and HR intact cases.
Figure 6. Association of CD3+ TILs and…
Figure 6. Association of CD3+ TILs and BRCA1/2-mutation status with survival in our institutional cohort
(A) Overall survival of patients with BRCA1/2-mutated (red) versus HR intact (HRP, blue) tumors. (B) Overall survival of patients with tumors containing above median number of CD3+ TILs/HPF (red) versus the remaining tumors (blue). (C) Overall survival of patients with tumors containing ≥ 13 CD3+ TILs/HPF (red) versus those containing < 13 CD3+ TILs/HPF (blue). (D) Overall survival of patients with BRCA1/2-mutated tumors with a high number of TILs (≥ 13 TILs/HPF) (red), HR proficient tumors (HRP) with a low number of TILs (< 13 TILs/HPF) (blue), and BRCA1/2-mutated tumors with low TILs or HRP tumors with high TILs (green).

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