title: Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer.
abstract: OBJECTIVE
  PARP 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.
  
  
  METHODS
  Healthy 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.
  
  
  RESULTS
  Stimulation 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. leej6@mail.nih.gov.
authors.forename: Jung-Min
authors.initials: JM
authors.lastname: Lee
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. nick.gordon24@gmail.com.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gordon
authors.affiliation: Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892, USA. jt66h@nih.gov.
authors.forename: Jane B
authors.initials: JB
authors.lastname: Trepel
authors.affiliation: Developmental Therapeutics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, 20892, USA. leemj@mail.nih.gov.
authors.forename: Min-Jung
authors.initials: MJ
authors.lastname: Lee
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. yum@intra.nci.nih.gov.
authors.forename: Minshu
authors.initials: M
authors.lastname: Yu
authors.affiliation: Women's Malignancies Branch, Center for Cancer Research, National Cancer Institute, 10 Center Dr. MSC1906, Building 10, Room 12N/226, Bethesda, MD, 20892-1906, USA. kohne@mail.nih.gov.
authors.forename: Elise C
authors.initials: EC
authors.lastname: Kohn
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000378:metabolism*; D016190:Carboplatin / Q000627:therapeutic use; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D000076228:MRE11 Homologue Protein; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000378:metabolism*; D010793:Phthalazines / Q000627:therapeutic use; D010879:Piperazines / Q000627:therapeutic use; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000627:therapeutic use; D051135:Rad51 Recombinase / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C121766:MRE11 protein, human; D010793:Phthalazines; D010879:Piperazines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D016190:Carboplatin; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000076228:MRE11 Homologue Protein; C531550:olaparib