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.

DNA Damage Response Assessments in Human Tumor Samples Provide Functional Biomarkers of Radiosensitivity

“…Several groups have used ex-vivo assays in recent years to monitor DDR foci responses following treatment with IR or drugs 4,5,8,13,14,85 . This approach is based on the idea that tumor tissues can remain viable for several days if cultured properly following removal from the patient.…”

“…While some data suggest that IR-based ex-vivo assays will accurately predict PARP inhibitor sensitivity 5,112 , we are not aware of any studies that have exposed organotypic 3D cultures to PARP inhibitors. To this end, Curtin and colleagues established cell cultures from malignant pleural effusions obtained from 13 patients with various cancers 8 . Cultures were exposed to a PARP inhibitor, and RAD51 and γ-H2AX foci were scored, in parallel to next-generation sequencing of DNA repair genes.…”

“…There is increasing evidence from functional studies and recent genomic analyses that alterations of DNA repair exist in human tumors that may impact treatment sensitivities 2–8 . Therefore, identification of alterations in the DDR following DSB induction would be very useful for individualization of treatments.…”

“…Many investigators now believe that a functional assessment of the DDR in human tumor tissues can be used to predict treatment sensitivities 2,4,5,8 . Such functional assays most often rely on the detection of subnuclear foci of DDR proteins (reviewed in ref.…”

“…There exists a vast literature on foci responses in established cell lines and a considerably smaller but emerging body of data on conducting foci assays on tissues and cells from patients 4,5,8,11–14 . The focus of the current review is to provide an overview of the opportunities as well as technical challenges associated with the use of patient samples.…”