Abstract

We aimed to validate the prognostic association of p16 expression in ovarian high-grade serous carcinomas (HGSC) and to explore it in other ovarian carcinoma histotypes. p16 protein expression was assessed by clinical-grade immunohistochemistry in 6525 ovarian carcinomas including 4334 HGSC using tissue microarrays from 24 studies participating in the Ovarian Tumor Tissue Analysis consortium. p16 expression patterns were interpreted as abnormal (either overexpression referred to as block expression or absence) or normal (heterogeneous). CDKN2A (which encodes p16) mRNA expression was also analyzed in a subset (n = 2280) mostly representing HGSC (n = 2010). Association of p16 expression with overall survival (OS) was determined within histotypes as was CDKN2A expression for HGSC only. p16 block expression was most frequent in HGSC (56%) but neither protein nor mRNA expression was associated with OS. However, relative to heterogeneous expression, block expression was associated with shorter OS in endometriosis-associated carcinomas, clear cell [hazard ratio (HR): 2.02, 95% confidence (CI) 1.47–2.77, p < 0.001] and endometrioid (HR: 1.88, 95% CI 1.30–2.75, p = 0.004), while absence was associated with shorter OS in low-grade serous carcinomas (HR: 2.95, 95% CI 1.61–5.38, p = 0.001). Absence was most frequent in mucinous carcinoma (50%), and was not associated with OS in this histotype. The prognostic value of p16 expression is histotype-specific and pattern dependent. We provide definitive evidence against an association of p16 expression with survival in ovarian HGSC as previously suggested. Block expression of p16 in clear cell and endometrioid carcinoma should be further validated as a prognostic marker, and absence in low-grade serous carcinoma justifies CDK4 inhibition.

Introduction
CDKN2A (cyclin-dependent kinase inhibitor 2A) is located on chromosome 9p21.3 and encodes two proteins, p16 and p14ARF, that have different reading frames 1. p14ARF inhibits p53 function and p16 inhibits the CDK4/6 complex acting as a negative cell cycle regulator suppressing the transition from the Gap1 to DNA synthesis (G1/S) phase and arresting the cell cycle in the G1 phase 2. Normal cells express variable amounts of p16 protein that can be detected by immunohistochemistry (IHC) in both nuclear and cytoplasmic localizations (heterogeneous p16 expression pattern) 3. There are two abnormal p16 expression patterns: absent and overexpressed, the latter also referred to as block expression as recommended by the Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions (LAST) 4. In keeping with its role as a tumor suppressor, absence of p16 expression can occur due to various mechanisms including homozygous deletion, loss of function mutations, promoter hypermethylation and translational suppression 5. In ovarian carcinoma, homozygous deletion of CDKN2A has been detected in only 3% of high-grade serous carcinomas (HGSC) 6, 15% of low-grade serous carcinomas (LGSC) 7, and in 30% of mucinous carcinomas (MC) 8. In contrast, p16 block expression results from a variety of alterations in G1/S cell cycle transition as a compensatory effort to inhibit G1/S transition. p16 block expression is classically observed in human papillomavirus (HPV)-associated uterine cervical neoplasms, in which viral proteins (E7) inactivate pRB and promote G1/S transition 9, 10. IHC overexpression of p16 is routinely used in clinical diagnostics for identification of HPV-related neoplasms. Ovarian carcinomas are not associated with HPV infections, but alterations promoting G1/S transition are common, e.g. RB1, CCNE1, CCND1, or MYC 6.

Ovarian carcinoma is a biologically heterogeneous disease 11 composed of five main histotypes: HGSC, LGSC, clear cell carcinoma (CCC), endometrioid carcinoma (EC), and MC, which should be studied separately 12. Older studies combining all histotypes showed that either overexpression or complete absence of p16 were associated with unfavorable outcomes 13-15. Recently, histotype-specific studies also reported that normal heterogeneous p16 expression was significantly associated with longer progression-free and overall survival (OS) in two series of 334 and 115 women with HGSC 16, 17. Therefore, we hypothesized that heterogeneous p16 expression reflecting the normal G1/S transition status is associated with a favorable outcome in HGSC compared to absent or block expression reflecting abnormalities of the G1/S cell cycle checkpoint complex. The purpose of this study was to validate whether abnormal p16 expression is associated with an unfavorable OS in HGSC, and to explore prognostic associations in other histotypes using tissue microarrays (TMAs) from the Ovarian Tumor Tissue Analysis (OTTA) consortium 18, 19.
Methods

Immunohistochemistry

The study investigators obtained tissue from 7492 patients with a diagnosis of primary ovarian, fallopian tube, or peritoneal carcinoma from 24 study sites (Supporting Information, Table S1). Most of these patients also participated in previous OTTA studies 18-20, and all studies received ethics board approval for tumor profiling. TMAs were constructed containing 1–6 cores of 0.6–1.0 mm in diameter from formalin-fixed paraffin embedded tissue representing tumor from each patient. p16 IHC was performed centrally at two institutions: Genetic Pathology Evaluation Centre, University of British Columbia, and Calgary Laboratory Services, University of Calgary, Canada. TMAs were stained in five batches with three different protocols (Table S2) using the same antibody (clone E6H4, CINtec, mtm laboratories). Three staining patterns were recorded: absent, heterogeneous and block (Figure S1). Block expression was distinguished from heterogeneous staining by using the recommendation for p16 interpretation from LAST 4; that is, block expression is characterized by diffuse staining of tumor cells in nuclear and/or cytoplasmic compartment with at least moderate intensity with virtually no negative tumor cell clusters. Interobserver reproducibility between two observers (PR and MK) was assessed for a subset of 120 cases. Seventeen studies were scored by PR and the remainder by MK. Cases represented by more than one core and discordant cores were consolidated as heterogeneous if any of a given case score was heterogeneous.

CDKN2A mRNA analysis
A subset of 2280 cases had CDKN2A mRNA expression data from NanoString n-counter analysis. RNA was extracted from 10 μm sections from formalin fixed paraffin embedded (FFPE) tissue blocks, which were macrodissected to avoid adjacent benign tissue but included tumor stroma using the Qiagen miRNeasy (Qiagen Inc. Toronto, Ontario, Canada) FFPE protocol and quantitated on a Nanodrop spectrophotometer (Thermo-Fisher Scientific, Waltham, MA, USA). After mixing 500 ng of total RNA per sample with a custom codeset (NanoString Technologies Inc, Seattle, WA, USA) and hybridization buffer (NanoString), hybridization was performed using a Tetrad 2 thermal cycler (Bio Rad Laboratories Inc, Hercules, CA, USA) for 16 or 20 h and then analyzed on a nCounter Digital Analyzer (NanoString). Data was normalized to housekeeping genes (RPL19, ACTB, PGK1, SDHA, and POLR1B) and pre-processed to a reference of 3 pooled ovarian cancer specimens as described previously 21. We interrogated the cBioportal for associations of CDKN2A alterations with OS in HGSC from TCGA 22, 23.

Statistical tests
Morphology-based histotype was derived from pathology reports with or without review of reports or slides (Table S1). Because some HGSC were mistakenly classified as other histotypes in the past, we used the highly specific WT1(+)/p53(mutant) IHC combination to reclassify those to HGSC 24. We excluded 409 cases owing to diagnosis other than the five major histotypes, 393 cases being uninterpretable, 31 cases with a combination of absence and block staining, and 134 cases with missing clinical follow-up data. The final sample size was 6525 (Table 1). The median time from diagnosis to enrollment was 0 days (interquartile range 0–182 days). Patients (n = 331) with missing data for either age or time from diagnosis to enrolment were not part of the multivariate survival analysis.