Combined CCNE1 high-level amplification and overexpression is associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma

Abstract

CCNE1 amplification is a recurrent alteration associated with unfavourable outcome in tubo-ovarian high-grade serous carcinoma (HGSC). We aimed to investigate whether immunohistochemistry (IHC) can be used to identify CCNE1 amplification status and to validate whether CCNE1 high-level amplification and overexpression are prognostic in HGSC. A testing set of 528 HGSC samples stained with two optimised IHC assays (clones EP126 and HE12) was subjected to digital image analysis and visual scoring. DNA and RNA chromogenic in situ hybridisation for CCNE1 were performed. IHC cut-off was determined by receiver operating characteristics (ROC). Survival analyses (endpoint ovarian cancer specific survival) were performed and validated in an independent validation set of 764 HGSC. Finally, combined amplification/expression status was evaluated in cases with complete data (n = 1114). CCNE1 high-level amplification was present in 11.2% of patients in the testing set and 10.2% in the combined cohort. The optimal cut-off for IHC to predict CCNE1 high-level amplification was 60% positive tumour cells with at least 5% strong staining cells (sensitivity 81.6%, specificity 77.4%). CCNE1 high-level amplification and overexpression were associated with survival in the testing and validation set. Combined CCNE1 high-level amplification and overexpression was present in 8.3% of patients, mutually exclusive to germline BRCA1/2 mutation and significantly associated with a higher risk of death in multivariate analysis adjusted for age, stage and cohort (hazard ratio = 1.78, 95 CI% 1.38-2.26, p < 0.0001). CCNE1 high-level amplification combined with overexpression identifies patients with a sufficiently poor prognosis that treatment alternatives are urgently needed. Given that this combination is mutually exclusive to BRCA1/2 germline mutations, a predictive marker for PARP inhibition, CCNE1 high-level amplification combined with overexpression may serve as a negative predictive test for sensitivity to PARP inhibitors.

Keywords: CCNE1; PARP inhibitor; amplification; cyclin E1; high grade serous carcinoma; ovarian cancer; prognosis.

Figure 1

CCNE1 DNA CISH and IHC. (A) Tubo‐ovarian high‐grade serous carcinoma without amplification (original total magnification ×400). (B) High‐level amplification of CCNE1 evident by clearly visible nuclear clusters of CISH signal (original total magnification ×400). (C) Tubo‐ovarian high‐grade serous carcinoma with low CCNE1 expression by IHC. (D) Tubo‐ovarian high‐grade serous carcinoma with CCNE1 overexpression (>60% of tumour cells staining with >5% strongly staining).

Figure 2

Multi‐step CCNE1 IHC assay standardisation. (A) IHC controls for 2 different IHC assays (clones EP126 and HE12). (B) Image analysis data (% positive cells, optical density) from two different IHC assays. (C) Western blot of inducible cell lines. (D) The distribution of percentage positive tumour cells and optical density of the maximum cores analysed by image analysis of IHC on the testing cohort using clone EP126 assay. (E) Determination of the optimal cut‐off for distribution of percentage positive tumour nuclei by IHC to predict CCNE1 high level amplification by CISH. Upper panel, all positive tumour cells; lower panel, only strongly staining tumour cells (3+).

Figure 3

Kaplan–Meier survival analysis of combined CCNE1 high‐level amplification and overexpression status in tubo‐ovarian high‐grade serous carcinomas. CCNE1^amp – CCNE1 high‐level amplification (>8 copies by CISH); CCNE1^nonamp – negative for CCNE1 high‐level amplification (≤8 copies by CISH); CCNE1^hi – CCNE1 protein overexpression by IHC with ≥60% positive tumour cells and ≥5% strongly staining cells; CCNE1^lo – negative for CCNE1 protein overexpression by IHC with <60% positive tumour cells or <5% strongly staining cells.