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.
Subject(s)
BRCA1 Protein , BRCA2 Protein , Cystadenocarcinoma, Serous , Ovarian Neoplasms , Retinoblastoma Binding Proteins , Humans , Female , Ovarian Neoplasms/genetics , Ovarian Neoplasms/mortality , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , BRCA2 Protein/genetics , BRCA2 Protein/deficiency , BRCA1 Protein/genetics , BRCA1 Protein/deficiency , Cystadenocarcinoma, Serous/genetics , Cystadenocarcinoma, Serous/pathology , Cystadenocarcinoma, Serous/mortality , Cystadenocarcinoma, Serous/immunology , Retinoblastoma Binding Proteins/genetics , Prognosis , Ubiquitin-Protein Ligases/genetics , Neoplasm Grading , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Middle Aged , Germ-Line Mutation , Gene Expression Regulation, Neoplastic , Aged , Biomarkers, Tumor/genetics , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolismABSTRACT
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.
ABSTRACT
BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.
Subject(s)
Carcinoma , Cystadenocarcinoma, Serous , Ovarian Neoplasms , Female , Humans , Ovarian Neoplasms/pathology , Transcription Factors/genetics , RNA, Messenger , Cystadenocarcinoma, Serous/genetics , Oncogene Proteins/genetics , Oncogene Proteins/therapeutic use , Cyclin E/geneticsABSTRACT
PURPOSE: Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. EXPERIMENTAL DESIGN: Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). RESULTS: Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04-7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04-1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01-1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). CONCLUSIONS: An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.
Subject(s)
Adenocarcinoma, Mucinous , Gastrointestinal Neoplasms , Ovarian Neoplasms , Female , Humans , Neoplasm Staging , Ovarian Neoplasms/metabolism , Carcinoma, Ovarian Epithelial/pathology , Adenocarcinoma, Mucinous/genetics , Adenocarcinoma, Mucinous/diagnosis , Prognosis , Gastrointestinal Neoplasms/metabolismABSTRACT
Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36-0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.
Subject(s)
Cystadenocarcinoma, Serous , Minichromosome Maintenance Complex Component 3 , Ovarian Neoplasms , Biomarkers, Tumor/analysis , Cell Proliferation , Cystadenocarcinoma, Serous/pathology , Female , Humans , Ki-67 Antigen , Minichromosome Maintenance Complex Component 3/genetics , Ovarian Neoplasms/pathology , RNA, Messenger , Survival RateABSTRACT
BACKGROUND/AIMS: Receptor-mediated endocytosis of albumin by the renal proximal tubule requires a number of proteins including megalin/cubilin, sodium/hydrogen exchanger isoform 3 (NHE3) and ClC-5, as well as the PSD-95/Dlg/Zo-1 (PDZ) scaffold sodium/hydrogen exchanger regulatory factor 2 (NHERF2). Despite members from the AGC kinase family, v-Akt Murine Thymoma Viral Oncogene (Akt or Protein Kinase B) and Serum/Glucocorticoid regulated Kinase 1 (Sgk-1) regulating a number of essential proteins in the albumin handling pathway, their role in uptake is largely unknown. METHODS: Opossum kidney (OK) cells were exposed to Texas-Red albumin, in the presence of silencing constructs against Sgk-1, Akt and NHERF2, in addition to the NHE3 inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and NHE3 activator dexamethasone. Target protein was also measured by Western blot analysis in OK cells following exposure to dexamethasone and albumin. RESULTS: Silencing Sgk-1 or overexpression of a dominant negative mutant (DN-Sgk-1) led to a significant reduction of albumin endocytosis compared to control. Conversely, over-expression of wildtype (WT) or constitutively active (CA) Sgk-1 significantly increased uptake. Previous reports have shown Sgk-1 can activate NHE3 through an interaction mediated by NHERF2. We found that silencing both Sgk-1 and NHERF2 demonstrated no additive effect on uptake, suggesting signaling via similar endpoints. Treatment with dexamethasone increased Sgk-1 protein levels and increased albumin endocytosis in OK cells. Interestingly, silencing Akt also lead to a reduction in albumin endocytosis, however in cells silenced for both Sgk-1 and Akt, the additive change in albumin uptake demonstrated that these proteins may act via separate pathways. CONCLUSIONS: We have characterized a Sgk-dependent pathway that regulates albumin uptake in the proximal tubule which also includes NHE3 and NHERF2. These data provide further insights into this essential tubular process.