Abstract
High-grade serous ovarian cancer (HGSOC) is the most common subtype of epithelial ovarian cancer and early detection is challenging. TP53 mutations are a hallmark of HGSOC and detection of these mutations in liquid-based Pap samples could provide a method for early diagnosis. Here we evaluate the use of IBSAFE, an ultra-sensitive droplet digital PCR (ddPCR) method, for detecting TP53 mutations in liquid-based Pap samples collected from fifteen women at the time of diagnosis (diagnostic samples) and/or up to seven years prior to diagnosis (archival samples). We analysed tumours for somatic TP53 mutations with next generation sequencing and were able to detect the corresponding mutations in diagnostic samples from six of eight women, while one patient harboured a germline mutation. We further detected a mutation in an archival sample obtained 20 months prior to the ovarian cancer diagnosis. The custom designed IBSAFE assays detected minor allele frequencies (MAFs) with very high assay sensitivity (MAF = 0.0068%) and were successful despite low DNA abundance (0.17–206.14 ng, median: 17.27 ng). These results provide support for further evaluation of archival liquid-based Pap samples for diagnostic purposes and demonstrate that ultra-sensitive ddPCR should be evaluated for ovarian cancer screening in high-risk groups or in the recurrent setting.
Highlights
High-grade serous ovarian cancer (HGSOC) is believed to arise in the fallopian tube epithelium[9], and mutations in the tumour suppressor gene TP53 are believed to be a very early event in the carcinogenesis of HGSOC10
The time from the collection of archival samples to the time of HGSOC diagnosis ranged from 20 to 95 months; two patients had more than one archival sample and the remaining patients had a single archival sample
In this study we evaluated the ability of IBSAFE, an ultra-sensitive Droplet Digital PCR (ddPCR) method, to detect known TP53 mutations in Pap samples from patients with HGSOC collected at the time of diagnosis and approximately two to seven years prior to diagnosis
Summary
HGSOC is believed to arise in the fallopian tube epithelium[9], and mutations in the tumour suppressor gene TP53 are believed to be a very early event in the carcinogenesis of HGSOC10. A promising development occurred in 2013, when Kinde et al showed that somatic mutations in DNA shed from endometrial and ovarian cancers could be detected in standard liquid-based Pap test specimens by massively parallel sequencing[15]. Subsequent studies from this research group in collaboration with others have attempted to increase the sensitivity for detection of ovarian cancer by introducing new procedures including lavage of the uterine cavity[16], by combining Pap test and plasma sampling and by complementing the mutation assay with an assay for aneuploidy[17]. Clinical sensitivity remains a challenge with this approach, and will require extensive modelling before application in clinical diagnostics[18,19,20] These approaches have only been applied in symptomatic patients, at the time of diagnosis, and have so far not been evaluated in pre-symptomatic women prior to the time of diagnosis. The analysis of liquid Pap samples from pre-symptomatic women subsequently diagnosed with HGSOC has, to our knowledge, not been previously reported
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