Methods
Patients and clinical data
The flow chart of the study population was shown in Figure 1.Patients included in this study were diagnosed in Qilu Hospital of Shandong University, Cancer Hospital of Beijing Academy of Medical Sciences, Cancer Hospital affiliated to Sun Yat-sen University, Shanghai General Hospital, Fudan University Shanghai Cancer Center, Shandong Cancer Hospital, and the Fourth Hospital of Hebei Medical University from 2010/02/01 to 2018/09/24, and all carried the germline BRCA1/2 pathogenic mutation. The patient did not receive any maintenance regimen, such as PARPi, bevacizumab, etc., after first- and second-line platinum therapy. The secondary PFI was more than 6 months in all patients. Patients enrolled in the study group were treated with PARPi monotherapy after secondary platinum-sensitive relapse and continued treatment until disease progression, demonstrating resistance to PARPi. Patients in control group were treated with platinum-based chemotherapy after secondary platinum-sensitive relapse, without restriction on the specific type and dose of platinum. After progression of PARPi, patients treated with subsequent platinum-based chemotherapy were enrolled in study group 2; Among patients in control group, patients with PFI≥6 months after third-line chemotherapy, and receiving subsequent platinum-based chemotherapy were enrolled in control group 2, and patients with PFI<6 months were enrolled in control group 3.
The determination of response of PARPi and platinum-based chemotherapy were in accordance with Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria; If clinical data were insufficient for RECIST criteria assessment, the GCIG’s CA125 criteria were used as an alternative12.
PFS was defined as third-line therapy (including PARPi monotherapy and platinum-based chemotherapy) to disease progression or death, TFST was defined as the time from fourth-line chemotherapy to next subsequent therapy or death. PRS refers to the survival time after secondary platinum-sensitive relapse. Additional clinical data were collected including age at diagnosis, primary tumor location, CA-125 level at secondary relapse, neoadjuvant chemotherapy, pathological type, BRCA1/2 germline mutational status, platinum-based chemotherapy regimens, PARPi duration, efficacy and toxicity, primary and secondary PFI, and survival.
Germline BRCA1/2 detection
The protocol of germline BRCA1/2 detection based on NGS technology included the following six steps, namely, sample acquisition and processing, nucleic acid extraction, library construction, sequencing, data analysis and mutation interpretation, and each step included corresponding quality control steps. For amplicon based and hybridization capture methods, the detection regions included the entire exon coding region of BRCA1/2 gene and the exon-intron interface region (±20 base pairs). The average depth of each run was over 200×. Sanger DNA sequencing was performed for all reported variations using specific gene primers. All point mutations and small indels were confirmed by sanger DNA sequencing using specific gene primers, and the large fragment rearrangements were detected by multiplex ligation-dependent probe amplification (MLPA) methods. Variants were named according to HGVS nomenclature, and guidelines for the interpretation of sequence variants into 5-class system adapted from the International Agency for Research on Cancer13, 14.
Statistical analyses
Student’s t-test was used to compare differences in continuous variables with normal distribution. Differences in clinical characteristics and survival between defined groups of patients were assessed using chi-square test and Kaplan–Meier methods, where appropriate, and hazard ratios (HR) with 95% confidence intervals (CI) were calculated. In univariate analysis, p value 0.10 was defined as the upper limit for inclusion in multivariate analysis, in the latter, p<0.05 was considered significant. The SPSS program (version 16.0) was used for all statistical analysis. The significance levels were * p < 0.05 and ** p < 0.01, respectively.