Assessing the function of homologous recombination DNA repair in malignant pleural effusion (MPE) samples

Abstract

Background: Patients with malignant pleural effusions (MPEs) generally have advanced disease with poor survival and few therapeutic options. Cells within MPEs may be used to stratify patients for targeted therapy. Targeted therapy with poly(ADP ribose) polymerase inhibitors (PARPi) depends on identifying homologous recombination DNA repair (HRR)-defective cancer cells. We aimed to determine the feasibility of assaying HRR status in MPE cells.

Methods: A total of 15 MPE samples were collected from consenting patients with non-small-cell lung cancer (NSCLC), mesothelioma and ovarian and breast cancer. Primary cultures were confirmed as epithelial by pancytokeratin, and HRR status was determined by the detection of γH2AX and RAD51 foci following a 24-h exposure to rucaparib, by immunofluorescence microscopy. Massively parallel next-generation sequencing of DNA repair genes was performed on cultured MPE cells.

Results: From 15 MPE samples, 13 cultures were successfully established, with HRR function successfully determined in 12 cultures. Four samples - three NSCLC and one mesothelioma - were HRR defective and eight samples - one NSCLC, one mesothelioma, one sarcomatoid, one breast and four ovarian cancers - were HRR functional. No mutations in DNA repair genes were associated with HRR status, but there was probable loss of heterozygosity of FANCG, RPA1 and PARP1.

Conclusions: HRR function can be successfully detected in MPE cells demonstrating the potential to stratify patients for targeted therapy with PARPi.

Figure 1

Pleural effusion primary cultures (A) Primary cultures established from pleural effusion samples grew as a monolayer exhibiting a polygonal cell morphology and a cobblestone-like appearance at confluency. (B) Epithelial cell growth in primary cultures assessed by pancytokeratin staining; cell nuclei are visualised with DAPI. Cultures with >90% positive cells were analysed further. Example images are taken from PPE007, a pleural effusion from a breast cancer.

Figure 2

HRR status in primary cultures. (A) Example immunofluorescence microscopy in primary cultures following DSB induction with a 24-h exposure to rucaparib. In HRR-competent cultures (PPE012, NSCLC), increased levels of γH2AX (indicating DSB) and RAD51 (indicating HRR) foci are seen in the nucleus following rucaparib treatment; however, in cultures with dysfunctional HRR (PPE003, NSCLC), only increased levels of γH2AX foci are seen following rucaparib treatment (+) compared with untreated control (−). (B) HRR status of primary cultures. Average number of γH2AX and RAD51 foci per cell was determined by foci counting (ImageJ). Foci numbers were normalised to the control and expressed as a fold induction following rucaparib treatment. A 2-fold induction (dashed line) was set as the threshold for γH2AX and RAD51 induction. Data are representative of 2–3 independent experiments (PPE002, 003, 007, 008, 009, 012, 014), with error bars indicating the s.e.m., or a single experiment (PPE001, 006, 010, 013, 015) where sample sizes were small or cultures stopped proliferating at an early passage.

Figure 3

Summary of genetic data. The distribution of putative pathogenic variants (known mutations or variants with a population frequency of <1 : 1000) across the major DNA maintenance families is shown, together with probable loss of heterozygosity. As private aneuploidies may skew coverage for individual cancers, expected sequencing performance is represented by mean target coverage (exons +10 bp/−50 bp) at a depth >15 reads for diploid samples (n=13). Only two nonsense mutations were detected across the listed genes; both were heterozygous. Three genes demonstrated pLoH in HRR-defective tumours only – FANCG, RPA1 and, surprisingly, PARP1. There is no clear pattern of genetic changes associated with HRR status, although more subtle relationships may be detected using a larger sample number.