Optimised ARID1A immunohistochemistry is an accurate predictor of ARID1A mutational status in gynaecological cancers.

Saira Khalique,James Campbell,Rachael Natrajan,Susana Banerjee,Divya Kriplani,Nicholas Matthews,Kalnisha Naidoo,Thomas Jones,Kerry Fenwick,Ayoma D Attygalle,Anne Lowe,Frances Daley,Michael Hubank,Christopher J Lord,Alistair G Rust

doi:10.1002/cjp2.103

Abstract

ARID1A is a tumour suppressor gene that is frequently mutated in clear cell and endometrioid carcinomas of the ovary and endometrium and is an important clinical biomarker for novel treatment approaches for patients with ARID1A defects. However, the accuracy of ARID1A immunohistochemistry (IHC) as a surrogate for mutation status has not fully been established for patient stratification in clinical trials. Here we tested whether ARID1A IHC could reliably predict ARID1A mutations identified by next‐generation sequencing. Three commercially available antibodies – EPR13501 (Abcam), D2A8U (Cell Signaling), and HPA005456 (Sigma) – were optimised for IHC using cell line models and human tissue, and screened across a cohort of 45 gynaecological tumours. IHC was scored independently by three pathologists using an immunoreactive score. ARID1A mutation status was assessed using two independent sequencing platforms and the concordance between ARID1A mutation and protein expression was evaluated using Receiver Operating Characteristic statistics. Overall, 21 ARID1A mutations were identified in 14/43 assessable tumours (33%), the majority of which were predicted to be deleterious. Mutations were identified in 6/17 (35%) ovarian clear cell carcinomas, 5/8 (63%) ovarian endometrioid carcinomas, 2/5 (40%) endometrial carcinomas, and 1/7 (14%) carcinosarcomas. ROC analysis identified greater than 95% concordance between mutation status and IHC using a modified immunoreactive score for all three antibodies allowing a definitive cut‐point for ARID1A mutant status to be calculated. Comprehensive assessment of concordance of ARID1A IHC and mutation status identified EPR13501 as an optimal antibody, with 100% concordance between ARID1A mutation status and protein expression, across different gynaecological histological subtypes. It delivered the best inter‐rater agreement between all pathologists, as well as a clear cost‐benefit advantage. This could allow patients to be accurately stratified based on their ARID1A IHC status into early phase clinical trials.

Highlights

Recent studies have highlighted that somatic loss of function mutations in the tumour suppressor gene ARID1A underpin 57% of ovarian clear cell carcinomas (OCCC) [1,2], 40% of uterine endometrioid carcinomas [3] and between 20 and 36% of uterine carcinosarcomas [4,5,6]; they are rare in high-grade serous ovarian carcinoma (HGSOC)[1,2]
ARID1A forms a key DNA binding subunit in the ATP-dependent SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodelling complex that modulates the winding of DNA around histone cores allowing access to the DNA to enable transcription, DNA repair, and replication [11,12]; loss of function of this protein leads to aberrant cell cycle and loss of proliferation control [13]
In order to assess if IHC could be used as a biomarker of mutation status, we evaluated the concordance of ARID1A IHC with the validated mutations

Summary

Introduction

Recent studies have highlighted that somatic loss of function mutations in the tumour suppressor gene ARID1A underpin 57% of ovarian clear cell carcinomas (OCCC) [1,2], 40% of uterine endometrioid carcinomas [3] and between 20 and 36% of uterine carcinosarcomas [4,5,6]; they are rare in high-grade serous ovarian carcinoma (HGSOC)[1,2]. Loss or significant reduction of ARID1A protein expression is associated with heterozygous ARID1A mutations [1,8,9], suggesting a dominant-negative tumour suppressor role (see [10] for a comprehensive review). Two genetically engineered mouse models (GEMM) have been created with ARID1A alterations that led to tumour formation with loss of ARID1A protein expression observed by IHC. ARID1A mutations are detected at high frequencies in other solid tumours including 23% of pancreatic ductal adenocarcinoma, 23% of advanced urothelial carcinomas, 18% hepatocellular carcinomas, and 6% of metastatic castrateresistant prostate cancers [16,17,18,19,20]

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