Dataset Information

Clinical evaluation of a droplet digital PCR assay for detecting POLE mutations and molecular classification of endometrial cancer.

ABSTRACT:

Objective

We evaluated droplet digital polymerase chain reaction (ddPCR) method for detecting POLE mutations in endometrial cancer (EC) and guiding its molecular classification.

Methods

We reviewed 240 EC specimens from our hospital database. A ddPCR assay was used to identify POLE mutations at 5 known hotspots (P286R, S297F, V411L, A456P, and S459F). Expressions of p53 and mismatch repair proteins were identified using immunohistochemistry.

Results

The ddPCR assay identified POLE mutations in 10.8% of patients. The most common mutation was V411L (61.54%), followed by P286R (23.07%), S459F (7.69%), S297F (3.85%), and A456P (3.85%). Eight/one cases had positive ddPCR but negative Sanger sequencing/next-generation sequencing, respectively. Molecular classification revealed p53-mutated subtype as significantly more common for tumors with a high International Federation of Gynecology and Obstetrics (FIGO) grade, deep myometrial invasion, lymphovascular space invasion, advanced stage, and high/advanced risk groups; the POLE mutated group was more frequent in the low stage and low/intermediate risk group. Survival analyses revealed the poorest outcomes for p53-mutated EC, while mismatch repair-deficient and no specific molecular profile ECs had similar progression-free survival (PFS) outcomes, and POLE-mutated ECs had the best PFS outcome (p<0.001). When only intermediate, high-intermediate, and high-risk groups were analyzed for subgroups, molecular classification still showed differences both in PFS (p=0.003) and overall survival (p=0.017).

Conclusion

Hotspot POLE mutations can be detected using the ddPCR assay. We suggest simultaneously evaluating POLE mutation status using ddPCR and p53/mismatch repair protein expressions using immunohistochemistry, which can rapidly and accurately determine the molecular subtype of EC.

SUBMITTER: Kim G

PROVIDER: S-EPMC8899877 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

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Publications

Clinical evaluation of a droplet digital PCR assay for detecting <i>POLE</i> mutations and molecular classification of endometrial cancer.

Kim Gilhyang G Lee Song Kook SK Suh Dong Hoon DH Kim Kidong K No Jae Hong JH Kim Yong Beom YB Kim Hyojin H

Journal of gynecologic oncology 20211206 2

<h4>Objective</h4>We evaluated droplet digital polymerase chain reaction (ddPCR) method for detecting <i>POLE</i> mutations in endometrial cancer (EC) and guiding its molecular classification.<h4>Methods</h4>We reviewed 240 EC specimens from our hospital database. A ddPCR assay was used to identify <i>POLE</i> mutations at 5 known hotspots (P286R, S297F, V411L, A456P, and S459F). Expressions of p53 and mismatch repair proteins were identified using immunohistochemistry.<h4>Results</h4>The ddPCR ...[more]

PMID: 34910396

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Project description:IntroductionIt has been proposed that the copy number (CN) variation (CNV) in β-defensin genes (DEFB) on human chromosome 8p23 determines phenotypic differences in inflammatory diseases. However, no method for accurate and easy DEFB CN quantification is yet available.ObjectiveDroplet digital polymerase chain reaction (ddPCR) is a novel method for CNV quantification and has been used for genes such as CCL4L, CCL3L1, AMY1, and HER2. However, to date, no ddPCR assay has been available for DEFB CN determination. In the present study, we aimed to develop and evaluate such a ddPCR assay.MethodsThe assay was designed using DEFB4 and RPP30 as the target and the reference gene, respectively. To evaluate the assay, 283 DNA samples with known CNs previously determined using the multiple ligation-dependent probe amplification (MLPA) method, the current gold standard, were used as standards. To discover the optimal DNA template amount, we tested 80 to 2.5 ng DNA by a serial of one to two dilutions of eight samples. To evaluate the reproducibility of the assay, 31 samples were repeated to calculate the intra- and inter-assay variations. To further validate the reliability of the assay, the CNs of all 283 samples were determined using ddPCR. To compare results with those using quantitative PCR (qPCR), DEFB CNs for 48 samples were determined using qPCR with the same primers and probes.ResultsIn a one-dimensional plot, the positive and negative droplets were clearly separated in both DEFB4 and RPP30 detection channels. In a two-dimensional plot, four populations of droplets were observed. The 20 ng template DNA proved optimal, with either high (80 ng) or low (10, 5, 2.5 ng) template input leading to ambiguous or inaccurate results. For the 31 standard samples, DEFB CNs were accurately determined with small intra- and inter-assay variations (coefficient of variation < 0.04 for both). In the validation cohort, ddPCR provided the correct CN for all 283 samples with high confidence. qPCR measurements for the 48 samples produced noisy data with high uncertainty and low accuracy.ConclusionsddPCR is an accurate, reproducible, easy-to-use, cheap, high-throughput method for DEFB CN determination. ddPCR could be applied to DEFB CN quantification in large-scale case-control studies.