Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors
Development of a Functional Assay for Homologous Recombination Status in Primary Cultures of Epithelial Ovarian Tumor and Correlation with Sensitivity to Poly(ADP-Ribose) Polymerase Inhibitors
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DOI：
10.1158/1078-0432.ccr-09-2758
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发表时间：
2010-04-15
影响因子：
11.5
通讯作者：
Curtin, Nicola J.
中科院分区：
文献类型：
--
作者：
Mukhopadhyay, Asima;Elattar, Ahmed;Curtin, Nicola J.
目的：聚腺苷二磷酸核糖聚合酶(PARP)抑制剂选择性地针对同源重组(HR)缺陷细胞，在BRCA1或BRCA2突变相关的遗传性乳腺癌和卵巢癌中表现出良好的临床活性。由于BRCA1/BRCA2或其他HR基因的遗传或表观遗传失活，很高比例(高达50%)的散发性卵巢上皮癌(EOC)可能存在HR缺陷。因此，如果能够确定HR状态，就有可能将PARP抑制剂的使用扩大到这些患者。我们开发了一种基于RAD51病灶形成的EOCs原代培养中HR状态的功能分析，该方法与强大的PARP抑制剂AG014699的敏感性很好地相关。实验设计：原代培养来自EOCs患者的腹水。用γ-H_2AX法检测HR状态，免疫荧光法检测RAD51病灶形成。结果：25种培养物的HR状态和对PARP抑制剂的细胞毒性进行了评价。在AG014699作用下，24个培养物中有9个(HR合格)RAD51病灶增加(36%)，而在24个培养物(%)中有16个(HR缺乏)没有增加(HR缺乏)。10 mU mol/L AG014699作用24小时后，16例HR缺乏样本中有15例(93%)观察到细胞毒作用，而9例HR正常样本中有9例未观察到细胞毒作用。结论：RAD51靶点形成法可以判断原发癌标本的HR状态，这与体外对PARP抑制的反应有关。现在需要在临床试验环境中对这种检测作为生物标记物的使用进行测试。临床癌症资源；16(8)；2344-51。(C)2010年AACR。
Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors selectively target homologous recombination (HR)-defective cells and show good clinical activity in hereditary breast and ovarian cancer associated with BRCA1 or BRCA2 mutations. A high proportion (up to 50%) of sporadic epithelial ovarian cancers (EOC) could be deficient in HR due to genetic or epigenetic inactivation of BRCA1/BRCA2 or other HR genes. Therefore, there is a potential for extending the use of PARP inhibitors to these patients if HR status can be identified. We developed a functional assay of HR status in primary cultures of EOCs based on Rad51 focus formation that correlates well with sensitivity to the potent PARP inhibitor AG014699.Experimental Design: Primary cultures were derived from ascitic fluid from patients with EOCs. HR status was investigated by gamma H2AX and Rad51 focus formation by immunofluorescence. Cytotoxicity to PARP inhibitors was tested by sulforhodamine B and survival assay.Results: Twenty-five cultures were evaluated for HR status and cytotoxicity to PARP inhibitor. Following exposure to AG014699, there was an increase in Rad51 foci (HR competent) in 9 of 24 (36%) but no increase (HR deficient) in 16 of 24 (64%) cultures. Cytotoxicity was observed in 15 of 16 (93%) HR-deficient samples but not in 9 of 9 HR-competent samples following 24-hour exposure to 10 mu mol/L AG014699.Conclusion: HR status can be determined in primary cancer samples by Rad51 focus formation, and this correlates with in vitro response to PARP inhibition. Use of this assay as a biomarker now needs testing in the setting of a clinical trial. Clin Cancer Res; 16(8); 2344-51. (C) 2010 AACR.