Concurrent RB1 loss and BRCA-deficiency predicts enhanced immunological response and long-term survival in tubo-ovarian high-grade serous carcinoma

Abstract

Background:

Somatic loss of the tumour suppressor RB1 is a common event in tubo-ovarian high-grade serous carcinoma (HGSC), which frequently co-occurs with alterations in homologous recombination DNA repair genes including BRCA1 and BRCA2 (BRCA). We examined whether tumour expression of RB1 was associated with survival across ovarian cancer histotypes (HGSC, endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous carcinoma (LGSC)), and how co-occurrence of germline BRCA pathogenic variants and RB1 loss influences long-term survival in a large series of HGSC.

Patients and methods:

RB1 protein expression patterns were classified by immunohistochemistry in epithelial ovarian carcinomas of 7436 patients from 20 studies participating in the Ovarian Tumor Tissue Analysis consortium and assessed for associations with overall survival (OS), accounting for patient age at diagnosis and FIGO stage. We examined RB1 expression and germline BRCA status in a subset of 1134 HGSC, and related genotype to survival, tumour infiltrating CD8+ lymphocyte counts and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cell lines with and without BRCA1 mutations to model co-loss with treatment response. We also performed genomic analyses on 126 primary HGSC to explore the molecular characteristics of concurrent homologous recombination deficiency and RB1 loss.

Results:

RB1 protein loss was most frequent in HGSC (16.4%) and was highly correlated with RB1 mRNA expression. RB1 loss was associated with longer OS in HGSC (hazard ratio [HR] 0.74, 95% confidence interval [CI] 0.66–0.83, P = 6.8 ×10⁻⁷), but with poorer prognosis in ENOC (HR 2.17, 95% CI 1.17–4.03, P = 0.0140). Germline BRCA mutations and RB1 loss co-occurred in HGSC (P < 0.0001). Patients with both RB1 loss and germline BRCA mutations had a superior OS (HR 0.38, 95% CI 0.25–0.58, P = 5.2 ×10⁻⁶) compared to patients with either alteration alone, and their median OS was three times longer than non-carriers whose tumours retained RB1 expression (9.3 years vs. 3.1 years). Enhanced sensitivity to cisplatin (P < 0.01) and paclitaxel (P < 0.05) was seen in BRCA1 mutated cell lines with RB1 knockout. Among 126 patients with whole-genome and transcriptome sequence data, combined RB1 loss and genomic evidence of homologous recombination deficiency was correlated with transcriptional markers of enhanced interferon response, cell cycle deregulation, and reduced epithelial-mesenchymal transition in primary HGSC. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1.

Conclusions:

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

INTRODUCTION

Despite a high response rate to primary treatment, the progressive development of acquired drug resistance is common in tubo-ovarian high-grade serous carcinoma (HGSC), a histotype that is associated with approximately 70% of ovarian cancer deaths¹. The frequent acquisition of resistance-conferring alterations in HGSC^2–4 suggests that the development of drug resistance may be inevitable when curative surgery is not achieved in these patients. Countering that view, however, is the observation that a small subset of patients with HGSC advanced disease experience an exceptional response to treatment, survive well beyond a median of 3.4 years⁵, and in some cases, remain disease free^6,7. Interest in studying long-term cancer survivors is growing as they may assist the discovery of prognostic biomarkers, novel treatments, and approaches to limit the development of resistance⁸.

Several clinical and molecular factors that influence treatment response and overall survival (OS) in HGSC have been described. Complete surgical debulking is associated with a more favourable outcome compared to patients left with residual disease^9–11. Molecular subtypes defined by distinct gene expression patterns in primary HGSC are associated with different outcomes¹², including the poor survival C1/mesenchymal subtype that is more often seen in patients where complete surgical tumour resection cannot be achieved^13–15. By contrast, the C2/immunoreactive subtype is typified by extensive infiltration of intraepithelial T cells¹², a feature known to be strongly associated with improved survival^16,17. Tumours arising in individuals with germline or somatic alterations in BRCA1 or BRCA2 genes are typically more responsive to conventional chemotherapy and poly(ADP-ribose) polymerase (PARP) inhibitors, whereas those tumours with intact homologous recombination (HR) DNA repair are more often resistant to treatment^18–20. Patients with germline BRCA1 or BRCA2 pathogenic variants show more favourable survival at five years post-diagnosis compared to non-carriers, with BRCA2 mutation carriers retaining a long-term (>10 year) survival advantage^21–23. Although deleterious mutations in BRCA1, BRCA2 and other genes involved in HR DNA repair are associated with a favourable response to treatment, these are not sufficient alone to confer long-term survival and a large proportion of such patients experience a typical disease trajectory. A differential outcome in mutation carriers can in part be ascribed to alternative splicing²⁴ or retention of the wild-type BRCA allele in tumours²⁵, both of which appear to limit the effectiveness of chemotherapy.

We previously characterised a small series of HGSC exceptional survivors and found that co-occurring loss of function alterations in both BRCA and RB1 were associated with unusually favourable survival^7,26. Disruption of the RB pathway is found in many cancer types but with variable impacts on patient outcome. For example, co-loss of RB1 and BRCA is associated with shorter survival in breast and prostate cancer, possibly due to lineage switching and resistance to hormonal therapy^27–29. A transcriptomic signature of RB1 loss was recently described to be associated with poor outcomes across cancer types³⁰. We have previously found that chromosomal breakage is the most common mechanism of RB1 inactivation in HGSC³, accounting for approximately 80% of all RB1 alterations. In addition to its crucial role in cell cycle regulation, RB1 is involved in non-canonical functions in a context- and tissue-dependent manner^31–33, including HR mediated DNA repair. Loss of RB1 expression in HGSC has been associated with a survival benefit³⁴, including in the context of abnormal block-like p16 staining³⁵.

Factors underlying the association of RB1 loss with improved outcome in HGSC are unknown. Here, we contrast the pattern and clinical consequences of RB1 loss in HGSC with other epithelial ovarian cancer subtypes, investigate the relevance of co-occurring BRCA1 or BRCA2 mutations and RB1 loss in HGSC patients, and explore the functional effects of combined BRCA and RB1 impairment in HGSC cell lines.

PATIENTS AND METHODS

Patient cohorts

The study population consisted of 7436 patients diagnosed with invasive epithelial ovarian, peritoneal or fallopian tube cancer from 20 studies or biobanks participating in the Ovarian Tumor Tissue Analysis (OTTA) consortium³⁶ (Supplementary Fig. S1). Written informed consent or IRB approved waiver of consent was obtained at each site for patient recruitment, sample collection, and study protocols (Supplementary Table S1).

Whole-genome sequence and matched transcriptome sequence data of primary HGSC tumours were available from 126 patients from the Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG) study²⁶ (Supplementary Fig. S1). This cohort consisted of 34 short-term survivors (OS <2 years), 32 moderate-term survivors (OS ≥2 and <10 years) and 60 long-term survivors (OS ≥10 years) with advanced stage (IIIC/IV) disease, enrolled in the Australian Ovarian Cancer Study (AOCS), the Gynaecological Oncology Biobank at Westmead Hospital (Sydney) or the Mayo Clinic Study.

Molecular analyses

RB1 protein expression was determined by immunohistochemistry (IHC) staining and scoring of tissue microarrays (TMAs) from formalin-fixed paraffin-embedded (FFPE) tumour samples, using our previously described protocol⁷ (RB1 antibody clone 13A10, Leica Biosystems; Supplementary Material). Subsets of HGSC patients had additional molecular or immune data available (Supplementary Fig. S1), including tumour p53 protein expression status previously classified³⁷ as normal (wild-type) or abnormal (overexpression, complete absence, and cytoplasmic), germline BRCA1 and BRCA2 pathogenic variant status obtained from OTTA, RB1 mRNA tumour expression obtained using NanoString (ref³⁴ and unpublished data), transcriptional subtypes of tumours using NanoString³⁸ and CD8+ tumour infiltrating lymphocyte (TIL) density was previously classified³⁹ based on the number of CD8+ TILs per high-powered field: negative (no TILs), low (<3 TILs), moderate (3–19 TILs) or high (≥20 TILs).

The MOCOG whole-genome and transcriptome sequencing dataset of 126 short-, moderate- and long-term survivors was uniformly processed as previously described²⁶, and included detailed characterisation of each tumour sample for inactivating alterations in RB1 and HR pathway genes, including germline and/or somatic mutations in BRCA1, BRCA2, BRIP1, PALB2, RAD51C and RAD51D, or promoter methylation of BRCA1 and RAD51C. Homologous recombination deficiency (HRD) status was assessed using the CHORD (Classifier of Homologous Recombination Deficiency) method⁴⁰, which uses specific base substitution, indel and structural rearrangement signatures detected in tumour genomes to generate BRCA1-type and BRCA2-type HRD scores. Primary tumours were classified as either BRCA1-HRD & RB1 altered; BRCA1-HRD & RB1 wild-type; BRCA2-HRD & RB1 altered; BRCA2-HRD & RB1 wild-type; homologous recombination proficient (HRP) & RB1 altered, or HRP & RB1 wild-type. For details on differential gene expression analyses, see Supplementary Material.

Cell culture

The AOCS patient-derived cell lines (AOCS1, AOCS3, AOCS7.2 AOCS9, AOCS11.2, AOCS14, AOCS16, AOCS22, AOCS30) were established from ascites drained from patients with HGSC, as previously described⁴. All AOCS cell lines were authenticated against matched patient germline DNA using short tandem repeat markers (STR, GenePrint10 System, Promega). Commercial cell lines OAW28 and CAOV3, categorised as likely HGSC⁴¹, were purchased from the American Type Culture Collection (ATCC), and JHOS2 and OVCAR4 were obtained from the National Cancer Institute Repository. Commercial lines were authenticated by comparing STR profiles (GenePrint10 System, Promega) to those published by online repositories (Cancer Cell Line Encyclopaedia, The Cancer Genome Atlas) before use in experiments. Cell lines were confirmed to be free of Mycoplasma by PCR at each revival and after finishing experiments. For details on cell growth conditions, CRISPR-mediated gene knockout, and molecular and functional cell line characterisation, see Supplementary Material.

Statistical analyses

Cox proportional hazards models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) using the ‘coxph’ function of the R package survival (v3.2–7). Final models were fitted using Cox regression adjusted for age at diagnosis and FIGO stage. A spline function was used for age at diagnosis with degree of freedom (df) 5 to account for the non-linear effect of the continuous variable. Regression models were fitted separately by histotype. The HGSC regression models were also stratified by site of participant recruitment, and sites with fewer than 10 events within the study period were excluded. The ENOC regression model was not stratified by site due to the limited number of overall patients per site. The OTTA survival dataset was right censored at 10 years from diagnosis to reduce the number of non-ovarian cancer related deaths. In the final Cox regression model, there was evidence for deviation from the proportional hazard assumption, but the degree of deviation was not substantial when considered alongside the large sample size and Schoenfeld residuals. The Kaplan–Meier method was used to estimate and plot progression-free and overall survival probabilities, and the log-rank (Mantel–Cox) test used to compare the survival duration between subgroups. In the Kaplan-Meier curves, the number of patients at risk on the date of diagnosis (time = 0) may be fewer than subsequent time intervals, owing to left truncation of follow-up resulting from delayed study enrolment at some OTTA sites. Differences in proportions of categorical features were assessed by either the chi-square or Fisher’s exact test as indicated. Differences in continuous variables were assessed by either a Wilcoxon Rank Sum Test or a Kruskal-Wallis test. All in vitro assays were performed across at least three independent experiments, and data are expressed as mean ± standard error of the mean (SEM) as indicated, from a minimum of three independent measurements. All statistical tests were two-sided and considered significant when P < 0.05. Statistical analyses were performed using either Prism (v9.3.1) or R (v3.6.3).

RESULTS

Loss of RB1 expression is most frequent in HGSC

RB1 protein expression was assessed by IHC in tumour samples from 7436 ovarian cancer patients using TMAs from 20 centres participating in the OTTA consortium (Supplementary Tables S1 and S2). RB1 tumour expression was classified as either retained or lost in 6564 samples, with 872 samples excluded that had either subclonal loss (n = 66), cytoplasmic (n = 17), or uninterpretable results (n = 789) due to either sample drop out or the absence of an internal positive control (Fig. 1A, Supplementary Material).

Figure 1. Expression of RB1 and survival associations across ovarian cancer histotypes.

(A) Representative images of immunohistochemical 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 tumour samples by ovarian cancer histotypes. Chi-square P value reported for difference in proportions across all histotypes. HGSC, tubo-ovarian high-grade serous carcinoma; LGSC, low-grade serous carcinoma; MOC, mucinous ovarian cancer; ENOC, endometrioid ovarian cancer; CCOC, clear cell 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 overall survival in patients diagnosed with HGSC (D) and ENOC (E) stratified by tumour RB1 expression. (F) Loss of RB1 tumour expression is more common in germline BRCA1 and BRCA2 mutation carriers than retained RB1 expression. Chi-square P value is reported. (G) Kaplan-Meier estimates of overall survival in HGSC patients by combined germline BRCA and tumour RB1 expression status.

RB1 loss was most frequent in HGSC (16.4%), followed by endometrioid ovarian cancer (ENOC; 4.1%, Chi-square P < 0.0001, Fig. 1B). Loss of RB1 expression was less frequent in all other histotypes (1.8% to 2.8%). RB1 mRNA expression was also assessed by NanoString in a subset of HGSC tumours (n = 2552) and was significantly associated with RB1 protein expression (Fig. 1C, P < 0.0001).

RB1 loss is associated with longer survival in HGSC

Loss of RB1 protein expression was associated with longer OS in patients with HGSC (HR 0.74, 95% CI 0.66–0.83, P = 6.8×10⁻⁷; Table 1) following multivariate analysis adjusting for stage and age at diagnosis and stratified by study. Patients with HGSC were comparable in terms of stage regardless of RB1 loss or retained expression (P = 0.9246), however those with RB1 loss had a younger age at diagnosis (median 59 years versus 61 years, P = 0.0003; Supplementary Table S3). Median OS was 4.7 years for patients with RB1 loss compared to 3.6 years for those with retained RB1 expression (Fig. 1D).

Table 1.

Multivariate analysis of molecular alterations and overall survival in patients with HGSC and ENOC

Histotype	Feature	Category	No. patients (events, %)	HR (95% CI)	P	P for interaction
HGSCa,b	RB1	Retained	3453 (71.3)	1 [Reference]
		Loss	686 (61.1)	0.74 (0.66-0.83)	6.8 × 10−7
ENOCa	RB1	Retained	649 (22.7)	1 [Reference]
		Loss	28 (39.3)	2.17 (1.17-4.03)	0.014
HGSCa,b	RB1 and BRCA status	RB1 retained & non-carrier	714 (76.3)	1 [Reference]		0.24
		RB1 loss & non-carrier	135 (60.7)	0.74 (0.57-0.96)	0.023
		RB1 retained & BRCA carrier	159 (67.9)	0.69 (0.55-0.86)	0.001
		RB1 loss & BRCA carrier	70 (42.9)	0.38 (0.25-0.58)	5.2 × 10−6
ENOCa	RB1 and p53	RB1 retained & p53 normal	492 (17.5)	1 [Reference]		0.698
		RB1 retained & p53 abnormal	58 (36.2)	2.26 (1.38-3.71)	0.001
		RB1 loss & p53 normal	11 (27.3)	1.77 (0.56-5.65)	0.332
		RB1 loss & p53 abnormal	12 (58.3)	5.34 (2.43-11.8)	<0.001

^aAdjusted for stage and age at diagnosis.

^bStratified by study.

HR, hazard ratio, CI, confidence interval; HGSC, tubo-ovarian high-grade serous carcinoma; ENOC, endometrioid ovarian cancer.

In contrast to HGSC, loss of RB1 expression in tumours from patients with ENOC was associated with advanced stage (P = 0.0003) and poorer survival (HR 2.17, 95% CI 1.17–4.03, P = 0.0140; Table 1, Fig. 1E, Supplementary Table S4). RB1 loss and abnormal p53 protein expression, which is highly predictive of TP53 mutation⁴², were strongly correlated (chi-square P < 0.0001; Supplementary Fig. 2A). While TP53 mutation is known to be associated with inferior survival in patients with ENOC^37,43, we note that combined RB1 loss and abnormal p53 expression were associated with the shortest patient survival (median OS 3.0 years; Supplementary Fig. 2B), suggesting that loss of RB1 and TP53 mutation have a compounding negative impact on survival in patients with ENOC.

Combined RB1 loss and germline BRCA mutation is associated with exceptionally good survival

We previously observed that co-occurrence of somatic RB1 protein loss and BRCA1 or BRCA2 alteration (somatic or germline) was associated with longer progression-free survival (PFS) and OS in HGSC⁷. Here, germline BRCA1 and BRCA2 status was available for 1134 HGSC patients for which we had RB1 IHC data (Supplementary Fig. S1). Consistent with having a younger age of diagnosis, patients with RB1 loss were more likely to have concurrent germline BRCA1 or BRCA2 mutations than those with retained RB1 expression (Fig. 1F, Chi-square P < 0.0001). Patients with both RB1 loss and a germline BRCA mutation had a 62% reduced risk of death compared with non-carriers with retained RB1 (HR 0.38, 95% CI 0.25–0.58, P = 5.2×10⁻⁶; Table 1). The median OS of BRCA germline carriers with RB1 loss was three times longer than non-carriers with RB1 retained tumours (median OS 9.3 years vs. 3.1 years, respectively), while median OS was 5.2 years for BRCA carriers with retained RB1 expression and 4.5 years for non-carriers with RB1 loss (Fig. 1G; Supplementary Table S5).

Enhanced response to chemotherapy in cells with impaired BRCA and RB1 function

To investigate whether co-occurrence of RB1 and BRCA alterations enhances sensitivity to standard-of-care ovarian cancer drugs, nine patient-derived HGSC cell lines with confirmed pathogenic TP53 mutation and known RB1 and BRCA status were treated with cisplatin, paclitaxel and olaparib (Supplementary Fig. S3A–C). AOCS14, the only cell line with a germline BRCA1 mutation and concomitant loss of RB1 expression, showed the best response to cisplatin and olaparib, and was the second most sensitive cell line to paclitaxel. In contrast AOCS11.2, a line with BRCA1 promoter methylation and loss of RB1 expression, was relatively resistant to paclitaxel and olaparib. Among cell lines with intact RB1 protein expression and BRCA wildtype background, AOCS3 was resistant to cisplatin, paclitaxel and olaparib.

Except for the chemo-naïve cell lines AOCS30 and AOCS14, all other lines were derived from patients previously treated with chemotherapy. Since the evaluation of HGSC cell lines with existing RB1 mutations may have been confounded by their prior, differential exposure to chemotherapy we therefore characterised responses in isogenically matched lines deleted of RB1 and/or BRCA1. We first inactivated RB1 in two BRCA1-mutant (AOCS7.2, AOCS16) and one wild-type line (AOCS1) using CRISPR-Cas9 (Fig. 2A, Supplementary Fig. S4A). RB1 knockout clones of the BRCA1-mutant cell line AOCS7.2 had enhanced sensitivity to cisplatin and paclitaxel compared to RB1 wild-type clones, which was observed both in short-term drug assays (72 hours, Fig. 2B) and longer-term clonogenic survival assays (12 days, Fig. 2C). In this cell line, sensitivity to paclitaxel and olaparib was increased after RB1 knockout (paclitaxel IC50 92.0 nM versus 11.8 nM, P < 0.0001; olaparib IC50 6.1 versus 1.1 nM, P < 0.0001). Further, significantly fewer colonies grew in this BRCA1-mutant cell line after RB1 knockout upon treatment with cisplatin (P = 0.01), paclitaxel (P = 0.02) or a combination of both drugs (P = 0.067) in a clonogenic survival assay (n = 3). This effect was not apparent in the BRCA-wild-type line (AOCS1) or the other BRCA1-mutant line (AOCS16). Western blot and IHC analysis (Supplementary Fig. S4A) found that AOCS16 lacked expression of p16, which may functionally disrupt the RB1 pathway irrespective of an RB1 knockout⁴⁴.

Figure 2. Sensitivity to therapeutic agents in BRCA1-mutant cell lines with RB1 knockout.

(A) RB1 was knocked out using CRISPR/Cas9 in 3 patient-derived Australian Ovarian Cancer Study (AOCS) HGSC cell lines with either wild-type or mutant BRCA1 background. Representative Western Blots show protein levels of RB1 and phosphorylated RB1 (pRB1) compared to GAPDH loading control in single cell cloned, homozygous RB1 wildtype (WT) and knockout (KO) colonies in comparison to 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 hours), paclitaxel (72 hours) or olaparib (120 hours). Nonlinear regression drug curves are shown; P values of a curve fit, extra sum-of squares F test (ns, not significant; ** P < 0.01; **** P < 0.0001; 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 (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.

Given that RB1 plays a central role in the negative control of the cell cycle^44,45, we tested whether the enhanced chemosensitivity of RB1 knockout AOCS 7.2 cells was associated with increased cell division. Live cell imaging showed similar growth rates of RB1 wildtype and knockout clones of all three isogenically matched HGSC cell lines (Supplementary Fig. S4B). In both BRCA wild-type and BRCA1 mutant cell lines, RB1 knockout did not alter cell cycle distribution at baseline or after 24 hours of cisplatin treatment (Supplementary Fig. S4C). Paclitaxel treatment resulted in a larger proportion of cells with a tetraploid DNA content in RB1 knockout cells compared to RB1 wild-type cells, indicating arrest in the G2 or M phase of the cell cycle. This effect was observed in all cell lines independent of BRCA or p16 status, however the arrest was more profound in the AOCS7.2 cell line (AOCS1, G2/M difference 8.59% ± 4.73%, P = 0.144; AOCS16, G2/M difference 8.13% ± 4.45%, P = 0.142; AOCS7.2: G2/M difference 14.49% ± 3.99%, P = 0.022; Supplementary Fig. S4C).

We extended our analysis of isogenically matched pairs by inactivating BRCA1 and/or RB1 in the chemo-naïve cell line AOCS30. While we were readily able to establish RB1 knockout lines, all BRCA1 targeted clones were hemizygous for BRCA1 deletion and retained BRCA1 expression (Supplementary Table S6), suggesting that engineered homozygous loss of BRCA1 was cell lethal, even in a tumour type where BRCA1 loss is frequently observed⁴⁶.

Genomic and transcriptional landscape of HGSC with combined inactivation of BRCA and RB1

To further understand how RB1 loss may impact the biology of HGSC with co-loss of BRCA1 or BRCA2, we explored matched whole-genome and transcriptome data of primary HGSC tumours in the MOCOG cohort²⁶ of 126 short- (OS <2 years), moderate- (OS ≥2 to <10 years) and long-term (OS ≥10 years) survivor patients (Supplementary Fig. S1). Each tumour genome was classified according to their HRD and RB1 status, resulting in 6 groups: BRCA1-HRD & RB1 altered (n = 13); BRCA1-HRD & RB1 wild-type (n = 36); BRCA2-HRD & RB1 altered (n = 8); BRCA2-HRD & RB1 wild-type (n = 20); HRP & RB1 altered (n = 4), or HRP & RB1 wild-type (n = 45; Fig. 3A).

Figure 3. Genomic landscape of high-grade serous ovarian tumours with co-occurring BRCA and RB1 alterations.

(A) Pathogenic germline and somatic alterations in homologous recombination (HR) and DNA repair genes detected by whole-genome sequencing and DNA methylation analysis of 126 primary HGSC samples²⁶ are shown, as well as alterations in immune genes and CCNE1. Samples are grouped by HRD and RB1 status (wt, wild-type; mut, mutation). 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), tumour CHORD⁴⁰ scores, and the proportion of structural variant (SV) type (DUP, duplication; DEL, deletion; INV, inversion; ITX, intra-chromosomal translocation). (B) Kaplan-Meier estimates of progression-free and overall survival of patients with according to HR status (BRCA1-type HRD, BRCA2-type HRD or homologous recombination proficient tumours) and RB1 status (mut, mutation; wt, wild-type).

The cohort had been selected for a long-term survivor study²⁶ and hence was enriched for patients with very long survival. Among BRCA2-HRD patients, those with RB1 alterations had longer OS (median OS 17.0 years) compared with those without RB1 alterations (median OS 11.7 years, P = 0.0004; Fig. 3B). Similarly, BRCA1-HRD patients with RB1 alterations survived longer (median OS 10.4 years) than those with an intact RB1 gene (median OS 7.1 years). There were few HRP tumours with RB1 alterations, however these patients had a worse survival (median OS 1.4 years) compared to the HRP group with no RB1 alteration (median OS 2.4 years).

Examination of genomic features revealed relatively similar patterns within BRCA1-HRD and BRCA2-HRD groups, although there were a few discriminatory features identified between those with and without RB1 alterations (Supplementary Figs. S5 and S6). For example, the BRCA1-associated rearrangement signature Ovary_G⁴⁷ was more enriched in BRCA1-HRD tumours with RB1 alterations compared to those without (P = 0.039). Among BRCA2-HRD tumours, the mutational signatures DBS6 (unknown etiology) and SBS3 (associated with HRD)⁴⁸ were higher in RB1-altered tumours compared to non-altered tumours, although this was not significant (P = 0.082 and P = 0.1 respectively). Concordantly, the average BRCA1-type and BRCA2-type CHORD scores⁴⁰ were highest in BRCA1- and BRCA2-HRD tumours with RB1 alterations respectively, indicating a higher probability of HRD. As described previously⁴⁹, CCNE1 gene amplifications were absent in tumours with both HRD and RB1 alterations (P = 0.0006; Supplementary Fig. S7).

We hypothesised that tumours with combined HRD and RB1 loss may have unique transcriptional profiles. To explore this, we compared gene expression profiles between each HRD/RB1 group and the reference set of tumours that were HRP and RB1 wild-type (Supplementary Table S7, Supplementary Fig. S8). There was significant enrichment of MSigDB hallmark gene sets among genes differentially expressed in BRCA1-HRD tumours with RB1 alterations, the most prominent being interferon gamma response (up), interferon alpha response (up), oxidative phosphorylation (up), and E2F targets (up; adjusted P < 0.0001; Fig. 4A). The differentially expressed genes identified between BRCA2-HRD / RB1 altered tumours and the reference set were significantly enriched for the MSigDB hallmark gene sets: E2F targets (up), epithelial mesenchymal transition (down), G2M checkpoint (up), and TNF alpha signalling via NF-kB (up; adjusted P < 0.0001).

Figure 4. Characterisation of HGSC with co-loss of RB1 and BRCA.

(A) Gene set enrichment analysis indicating up- and downregulated pathways in tumours according to BRCA and RB1 status. HRP, homologous recombination proficient; HRD, homologous recombination deficient; RB1wt, RB1 wild-type; RB1m, RB1 altered. (B) Proportion of tumour infiltrating lymphocytes (TILs) in HGSC tumours grouped by RB1 expression and BRCA germline mutation status (Chi-square P value is indicated). (C) Proportion of tumours classified as each HGSC molecular subtype¹² grouped by RB1 expression and BRCA germline mutation status (Chi-square P value is indicated; C5.PRO, C5/proliferative subtype; C4.DIF, C4/differentiated subtype; C2.IMM, C2/immunoreactive subtype; C1.MES, C1/mesenchymal subtype).

Since enhanced tumour cell proliferation has been associated with long-term survival in HGSC^7,26, and loss of RB1 might accelerate proliferation³¹, we evaluated the expression of proliferation markers across the RB1 and BRCA subgroups. BRCA1-HRD tumours with RB1 alterations had significantly higher mRNA levels of the cell proliferation related genes PCNA (proliferating cell nuclear antigen) and MCM3 (minichromosome maintenance complex component 3) compared to BRCA1-HRD tumours without RB1 alterations (P < 0.0001, Supplementary Fig. S6). However, there were no significant differences in the proportion of Ki-67 positive cancer cell nuclei (P = 0.3297) across the subgroups (Supplementary Fig. S6), which was previously quantified by immunohistochemistry⁷ in a subset of primary tumours (n = 59).

Germline BRCA mutation carriers with somatic loss of RB1 tumour expression show elevated immune activity

Having observed that HGSC with combined RB1 loss and HRD have enrichment of transcriptional signatures associated with an enhanced immune response, we accessed existing immunohistochemical data³⁹ to determine the prevalence of CD8+ TILs in HGSC samples that also had RB1 protein expression and BRCA germline mutation status (n = 868). BRCA carriers with RB1 loss had a significantly higher proportion of tumours (79.6%) with moderate and high densities of CD8+ TILs, compared to BRCA carriers with retained RB1 (64.9%), non-carriers with RB1 loss (72.4%) and non-carriers with retained RB1 (63.6%, P = 0.0264; Fig. 4B). Tumours with complete absence of CD8+ TILs were the least frequent in BRCA carriers with RB1 loss (4.1%) compared to the other groups (13.8 % of BRCA carriers with retained RB1 tumour expression, 14.6% of non-carriers with RB1 tumour loss, 18.8% of non-carriers with retained RB1 tumour expression).

Gene expression-based molecular subtypes^12,38 also differed by RB1 and BRCA status (P = 0.0271, n = 601; Fig. 4C). As expected, there was enrichment for the C2/immunoreactive subtype, a subtype characterised by the presence of intratumoural CD8+ T cells and good survival, in germline BRCA carriers with RB1 loss (32.4%) compared to the other subgroups (between 19.8% and 23.4%). Additionally, tumours with RB1 loss were enriched for the C4/differentiated molecular subtype, a subtype characterised by cytokine expression and good survival, regardless of BRCA status (45.9% in BRCA carriers with RB1 loss, 50.0% in non-carriers with RB1 loss, 39.5% in BRCA carriers with retained RB1, 32.1% of non-carriers with retained RB1). BRCA carriers with RB1 loss also had the lowest proportion of the C5/proliferative molecular subtype (2.7% versus 17.2% to 20.3% in the other groups), a subtype associated with diminished immune cell infiltration and poor survival^12,19.

DISCUSSION

Identifying the determinants of long-term patient survival, particularly in cancers with a generally unfavourable prognosis such as HGSC, may reveal novel therapeutic targets and inform personalised treatment strategies⁸. Improved survival associated with RB1 loss has been described previously in HGSC^7,34,35,50 but the underlying factors contributing to this survival benefit have not been studied to date. We assessed tumour samples from a cohort of more than 7,000 women with ovarian cancer, including a subset with high resolution genomic data, to understand how RB1 loss may impact on therapeutic response and patient survival.

Alteration of the RB1 pathway is a frequent event in tumourigenesis, including loss of regulators such as p16, activation of D- and E-type cyclins and their associated cyclin dependent kinases, and loss of RB1 itself (reviewed in ⁵¹). Our study showed that RB1 loss is associated with longer survival in patients with advanced stage HGSC, but by contrast, loss of RB1 in ENOC was associated with a shorter survival, particularly in combination with p53 mutation. Similar to ENOC, in endocrine-driven breast and prostate cancer, RB1 loss is associated with poorer survival: early co-loss of BRCA2 and RB1 is associated with an aggressive, castration-resistant prostate cancer subtype (CRPC) characterised by epithelial-to-mesenchymal transition and shorter survival²⁹. RB1 loss facilitates lineage plasticity and, with p53-comutation, leads to an androgen-independent phenotype^52,53 and consequently resistance to anti-androgen therapy. In estrogen-receptor (ER) positive breast cancer, CDK4/6 inhibitor resistance is associated with RB1 loss and cyclin E2 activation^54,55.

Triple negative breast cancer (TNBC) provides an important contrast to the findings for RB1 loss in ER-positive breast cancer. In TNBC, RB1 loss is most common in the basal-like subtype, where BRCA1 mutation and promoter hypermethylation is associated with frequent RB1 gene disruption and RB1 loss²⁸. RB1 loss alone, as well as co-occurrence with BRCA1 promoter hypermethylation, is associated with a favourable chemotherapy response and outcome^27,56–58. Notably, TNBC and HGSC are more similar than the cancers that they are grouped with anatomically, sharing gene expression patterns, genetic drivers including BRCA1 and BRCA2, ubiquitous loss of TP53, extensive copy number variation, and susceptibility to platinum-based chemotherapy^59,60. Taken together, the relationship between RB1 loss and patient survival appears to be dependent on cancer type and molecular context⁶¹.

Some, but not all TNBC and early metastatic prostate cancers are associated with germline variants in BRCA1, BRCA2 and other genes involved in HR DNA repair. However, previous tumour studies of RB1 expression have not also defined the HRD status of individual samples. A strength of this study was the known BRCA germline status of 1134 of the HGSC patients for which we also had RB1 protein expression, and this revealed the strong association of co-mutation in either BRCA1 or BRCA2 and RB1 with survival. In addition to germline mutations in BRCA1 or BRCA2, germline or somatic mutations, and promoter methylation of other genes involved in HR DNA repair, such as RAD51C, can result in a similar molecular phenotype, characterised by distinct genomic scarring²⁶. Using whole-genome sequence data, we determined the likely tumour HRD status in a subset of 126 tumours using an algorithm that recognises genomic scarring associated with HRD (Fig. 3A), rather than simply designating BRCA mutation status, which does not account for all mechanisms of HR repair inactivation. Although the number of samples with RB1 loss and HR proficiency was small, the very poor outcome we observed with this group indicated that for RB1 to impart a survival benefit in HGSC, it must occur in an HRD background. Validation of this finding in a larger cohort may further inform how RB1 loss could favourably influence survival in certain histological and molecular contexts.

We have previously noted that enhanced proliferation in HGSC is associated with long-term survival^7,26 and it is reasonable to suggest that RB1 loss may be imparting an effect through deregulating the cell cycle. However, data on the effect of RB1 loss on proliferation in HGSC tumours and cancer cell lines is inconsistent. RB1 knockout in our HGSC cell lines did not cause cell cycle alterations in the absence of treatment, and despite differences in proliferative markers at the mRNA level, there was no significant difference in the proportion of Ki-67 positive nuclei between tumours with or without RB1 protein expression. In a recent OTTA study, Ki-67 expression was not associated with survival in HGSC; however, there was strong correlation between loss of RB1 and the proliferative marker MCM3⁶², which may provide a more accurate measure of tumour cell proliferation than Ki-67⁶³.

In addition to its role in driving progression through the G1 stage of the cell cycle, RB1 has non-canonical functions. RB1 has been shown to participate in HR DNA repair through interactions with BRG1 and ATM⁶⁴. A recent pan-cancer study⁶⁵ found that combined loss of TP53 and RB1 was associated with a particularly high genome-wide loss-of-heterozygosity score, one of the key elements of genomic scarring associated with HRD. In our whole-genome analysis, HGSC tumours with dual loss of HRD and RB1 did not exhibit overall higher mutation burden; however, we did observe elevated levels of mutational signatures associated with HRD, which may be evidence of compounding DNA repair defects. It remains possible that the combined inactivation of RB1 and HR genes contribute to enhanced chemotherapy response and/or an impaired ability for tumour cells to develop therapy resistance.

When we evaluated a set of patient derived HGSC lines, those with germline BRCA1 mutation and RB1 alteration were most sensitive to cisplatin and olaparib. Knockout of RB1 in the AOCS 7.2 cell line which had a pre-existing BRCA1 mutation, resulted in an increase in chemosensitivity, consistent with the notion that co-mutation enhances chemotherapy response⁷. Unfortunately, despite considerable efforts, we were unable to generate a larger series of isogenically matched cell lines with combinations of conditional knockouts of RB1 and BRCA1 as all surviving clones retained at least one BRCA1 allele. BRCA1 loss is embryonic lethal and engineered loss in cell lines has been reported as lethal elsewhere including in the human haploid cell line, HAP1⁴⁶.

Our data provides evidence of an enhanced immunogenicity in HGSC with RB1 loss, with higher CD8+ TIL counts and upregulated expression of IFN-γ signalling pathways. RB1 has been shown to inhibit innate IFN-β production in immunocompetent mice⁶⁶ and RB1 deficiency triggered an increased IFN-β and IFN-α secretion. Co-mutation of RB1 and TP53 was recently found to be associated with an enhanced response to the immune checkpoint inhibitor atezolizumab in metastatic urothelial bladder cancer⁶⁷. Similarly, a case report described a complete response to atezolizumab in heavily pre-treated, RB1-negative TNBC⁶⁸. This generates the hypothesis that RB1 loss could predict response to such therapies in HGSC, since this tumour type ubiquitously harbours TP53 mutations⁶⁹. However, a recent biomarker study in ovarian cancer patients treated with atezolizumab or placebo and standard chemotherapy found that deleterious mutations in RB1 were prognostic for a better PFS, regardless of the addition of atezolizumab⁷⁰. While it appears RB1 loss alone may not be predictive of response to the PD-L1 inhibitor atezolizumab, response rates to PD-1/PD-L1 pathway checkpoint inhibitors are generally quite low in HGSC, with the best objective response rates between 8% and 15%⁷¹. Our study has identified a subset of patients with combined RB1 and BRCA inactivation who demonstrate exceptional immune responses and may provide clues for the development of new immunotherapeutic strategies for HGSC that extend beyond targeting PD-L1/PD-1.

Our work highlights the importance of RB1 loss to treatment response and survival and focuses attention on other therapeutic opportunities in this subset of HGSC patients. Approximately 20 percent of HGSC patients have somatic loss of RB1 assessed using genomic data^3,26, a figure that is consistent with the immunohistochemical results obtained in the large patient cohort described here. Both approaches indicate that RB1 loss is generally clonal, enhancing its value as a therapeutic target if selective inhibitors can be identified. Casein kinase 2 (CK2) inhibitors have been reported to enhance the sensitivity of RB1-deficient TNBC and HGSC cells to carboplatin and niraparib⁷². In addition, Aurora kinase A and B inhibition is synthetically lethal in combination with RB1 loss in breast and lung cancer cells^73–75. Irrespective of HRD status, RB1 mutations correlate with sensitivity to WEE1 inhibition in TP53 mutant TNBC and HGSC patient-derived xenografts⁷⁶, indicating additional treatment options that exploit RB1 inactivation in these tumours. In this study, the BRCA1-mutant cell line AOCS7.2 with induced RB1 knockout was more sensitive to olaparib suggesting that RB1 loss may also predict responses to PARP inhibitors in HGSC. RB1 staining of tumour tissue by IHC is a relatively low-cost pathology-based assay that could be used in prospective studies to test whether RB1 expression is predictive of responses to PARP inhibitors, either alone or in combination with approved HRD tests.