Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer
Development of a multiparameter flow cytometric assay as a potential biomarker for homologous recombination deficiency in women with high-grade serous ovarian cancer
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DOI：
10.1186/s12967-015-0604-z
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发表时间：
2015-07-22
影响因子：
7.4
通讯作者：
Kohn, Elise C.
中科院分区：
文献类型：
--
作者：
Lee, Jung-Min;Gordon, Nicolas;Kohn, Elise C.
目的：PARP抑制剂（PARPi）是一类新型药物，对获得性或生殖系同源重组（HR）缺陷相关的高级别浆液性卵巢癌（HGSOC）患者具有活性。我们假设，测量γ H2AX作为DNA双链断裂（DSB）的指标，MRE 11或RAD 51作为DSB修复的指标，将反映HR状态并预测对PARPi治疗的反应。我们的目的是开发和使用高通量多参数流式细胞术来定量γ H2AX与MRE11或RAD51在PBMCsas a ready available surrogate. Methods：健康供体PBMCs用于测定开发和优化。我们通过用荧光团缀合的抗体染色来验证γ H2AX、MRE 11和RAD 51的诱导。多参数流式细胞术方法应用于PBMC样本从复发HGSOC患者谁是PARPi，奥拉帕尼和carboplatin. Results：刺激是必要的定量的DNA损伤反应奥拉帕尼/卡铂在健康供体PBMC。流式细胞术方案不能区分细胞质和细胞核RAD 51，错误地指示响应于损伤的激活。因此，选择MRE 11作为DSB修复的标志物。然后检查来自15名复发HGSOC患者的PBMC。对PARPi治疗无反应的患者治疗前γ H2AX水平显著较高（p = 0.01），γ H2AX/MRE 11比值较高（11.0 [3.5 - 13.2] v. 3.3 [2.8 - 9.9]，p <0.03）。我们成功地开发并应用了多参数流式细胞术测定来测量PBMC中的γ H2AX和MRE 11。将需要前瞻性研究来验证这种替代生物标志物测定作为基于PARPi的治疗的潜在预测生物标志物。
Objectives: 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 gamma H2AX 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 gamma H2AX 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 gamma H2AX, 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 gamma H2AX (p = 0.01), and a higher ratio of gamma H2AX/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 gamma H2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.