Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer

Abstract

ObjectivesPARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.MethodsHealthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.ResultsStimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5–13.2] v. 3.3 [2.8–9.9], p < 0.03) compared with responders.ConclusionsWe successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0604-z) contains supplementary material, which is available to authorized users.

Predictive biomarkers for triple negative breast cancer treated with platinum-based chemotherapy

“…74 Besides, a multiparameter flow cytometric assay has been investigated as a potential biomarker for HR deficiency in HG-OSC. 75 More prospective and large researches should be conducted to identify the value of these biomarkers and confirm their clinical utility. Combining these biomarkers as a composite marker that thoroughly evaluates the properties of TNBC can more accurately predict drug efficacy.…”

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“…Excitation and emission wavelengths for FITC-Annexin V were 485 nm and 535 nm respectively and for PI were 535 nm and 620 nm respectively. The FACS data were analyzed using Flowjo software, and different cell populations (necrotic, primary apoptotic and late apoptotic) were determined 14 .…”

The emerging role of homologous recombination repair and PARP inhibitors in genitourinary malignancies

“…For example, Larsen et al analyzed 55 familial BRCA1 or BRCA2 breast tumors and 128 sporadic breast tumors to identify a transcriptional signature of DNA repair deficiency . Others have been working to develop functional HR assays via immunostaining using surrogates such as RAD51 or MRE11 localization . One assay that combines tumor sequencing with DNA cytogenetics is the Myriad HRD test (Myriad Genetics, Salt Lake City, Utah).…”

“…105 Others have been working to develop functional HR assays via immunostaining using surrogates such as RAD51 or MRE11 localization. 106,107 One assay that combines tumor sequencing with DNA cytogenetics is the Myriad HRD test (Myriad Genetics, Salt Lake City, Utah). The Myriad HRD test is performed on formalin-fixed, paraffin-embedded tumor samples evaluating for 54,091 single-nucleotide polymorphisms as well as 43 genes involved in HR, including BRCA1 and BRCA2.…”