Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma

Abstract

Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC).

Experimental design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss.

Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial-mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1.

Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.

Figure 1.

Expression of RB1 and survival associations across ovarian cancer histotypes. A, Representative images of IHC detection of RB1 expression in ovarian carcinoma tissues, showing examples of the three most common expression patterns: retained, lost, and subclonal loss. B, Proportion of patients with loss or retention of RB1 protein expression in tumor samples by ovarian cancer histotypes. χ²P value reported for difference in proportions across all histotypes. CCOC, clear cell ovarian cancer; LGSC, low-grade serous carcinoma; MOC, mucinous ovarian cancer. C, Boxplots show RB1 mRNA expression (NanoString) by RB1 protein expression status; lines indicate median and whiskers show range (Mann–Whitney test P value reported). Kaplan–Meier analysis of OS in patients diagnosed with HGSC (D) and ENOC (E) stratified by tumor RB1 expression. F, Frequency of germline BRCA wild-type (gBRCAwt) and germline BRCA pathogenic variants (gBRCAvar) in patients with HGSC stratified by RB1 protein expression. χ²P value is reported. G, Kaplan–Meier estimates of overall survival in patients with HGSC by combined germline BRCA and tumor RB1 expression status.

Figure 2.

Sensitivity to therapeutic agents in BRCA1-altered cell lines with RB1 knockout. A,RB1 was knocked out using CRISPR/Cas9 in three patient-derived Australian Ovarian Cancer Study (AOCS) HGSC cell lines with either wild-type or altered BRCA1 (BRCA1 var) background. Representative Western Blots show protein levels of RB1 and phosphorylated RB1 (pRB1) compared with GAPDH loading control in single-cell cloned, homozygous RB1 wild-type (WT) and knockout (KO) colonies in comparison with heterogeneous populations with a scramble single guide RNA (sgRNA). Independent blots were used for RB1 and pRB1. B, Cell viability was compared between RB1 WT and KO clones following treatment with cisplatin (72 h), paclitaxel (72 h), or olaparib (120 h). Nonlinear regression drug curves are shown; P values are shown in Supplementary Table S18 (n = 3). Error bars indicate ± SEM; for some values, error bars are shorter than the symbols and thus are not visible. C, Proportion of surviving colonies following 16 days of treatment with cisplatin, paclitaxel, or a combination of both (Cis/Pac; with half of the IC50 determined per drug and cell line respectively) relative to DMF vehicle control (n = 3 replicates). Data are presented as mean ± SEM. Mean values were compared by Student’s t test (ns, not significant; *, P < 0.05; **, P < 0.01). Representative scans of the fixed cell colonies stained with crystal violet are shown for each condition.

Figure 3.

Genomic landscape of high-grade serous ovarian tumors with co-occurring BRCA and RB1 alterations. A, Pathogenic germline and somatic alterations in HR and DNA repair genes detected by whole-genome sequencing and DNA methylation analysis of 126 primary HGSC samples (27) are shown, as well as alterations in immune genes and CCNE1. Samples are grouped by HR and RB1 status. Bars at the top indicate the number of alterations in each listed gene per patient. Patients are annotated with survival group (LTS, long-term survivor, OS > 10 years; MTS, mid-term survivor, OS 2–10 years; STS, short-term survivor, OS < 2 years), tumor CHORD (41) scores, and the proportion of structural variant (SV) type (DEL, deletion; DUP, duplication; INV, inversion; ITX, intra-chromosomal translocation). B, Kaplan–Meier estimates of progression-free survival (left) and overall survival (right) of patients according to HR status (BRCA1-type HRD; BRCA2-type HRD; or HRP tumors) and RB1 status (altered vs. wild-type).

Figure 4.

Characterization of HGSC with co-loss of RB1 and BRCA. A, GSEA indicating up- and downregulated pathways in tumors according to BRCA and RB1 status. RB1-alt, RB1 altered; RB1-wt, RB1 wild-type. B, Boxplots comparing GSVA pathway enrichment scores of the cGAS-STING and Toll-like receptor signaling pathways between molecular subgroups; points represent each sample, boxes show the interquartile range (25th–75th percentiles), central lines indicate the median, and whiskers show the smallest/largest values within 1.5 times the interquartile range. Colored boxes with black points indicate the HRD and/or RB1 altered groups, whereas the gray boxes with gray points indicate the HRP and RB1 wild-type group. P values were calculated using a two-sided Mann–Whitney-Wilcoxon test. Benjamini–Hochberg adjusted P values are shown above each pairwise comparison (*, P < 0.05; **, P < 0.01; ns, P ≥ 0.05). C, Bubble plot summary of HLA gene expression comparisons using DESeq2 between HGSC tumors grouped by HRD and/or RB1 status as shown. The size of the bubbles corresponds to the negative log₁₀ Benjamini–Hochberg adjusted P value (P_adj) and only values with P_adj ≤ 0.1 are shown. The color and intensity correspond to the log₂fold change. Genes are grouped by their classes. D, Proportion of TILs in HGSC tumors grouped by RB1 protein expression and BRCA germline status. χ²P value is indicated. E, Proportion of tumors classified as each HGSC molecular subtype (13) grouped by RB1 expression and BRCA germline status. χ²P value is indicated. C4.DIF, C4/differentiated subtype; C2.IMM, C2/immunoreactive subtype; C1.MES, C1/mesenchymal subtype; C5.PRO, C5/proliferative subtype.