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Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma.

ABSTRACT: Currently mainly BRAF mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. TERT promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of BRAF wild-type cases. Therefore, a sensitive method for detection of TERT promoter mutations would increase the number of patients that could be monitored through ctDNA analysis.A droplet digital PCR (ddPCR) assay was designed for the concurrent detection of chr5:1,295,228 C>T and chr5:1,295,250 C>T TERT promoter mutations. The assay was validated using 39 melanoma cell lines and 22 matched plasma and tumor samples. In addition, plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations.The established ddPCR assay detected TERT promoter mutations with a lower limit of detection (LOD) of 0.17%. Total concordance was demonstrated between ddPCR and Sanger sequencing in all cell lines except one, which carried a second mutation within the probe binding-site. Concordance between matched plasma and tumor tissue was 68% (15/22), with a sensitivity of 53% (95% CI, 27%-79%) and a specificity of 100% (95% CI, 59%-100%). A significantly longer PFS (p=0.028) was evident in ctDNA negative patients. Importantly, our TERT promoter mutations ddPCR assay allowed detection of ctDNA in 11 BRAF wild-type cases.The TERT promoter mutation ddPCR assay offers a sensitive test for molecular analysis of melanoma tumors and ctDNA, with the potential to be applied to other cancers.

SUBMITTER: McEvoy AC

PROVIDER: S-EPMC5668006 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Publications

Sensitive droplet digital PCR method for detection of <i>TERT</i> promoter mutations in cell free DNA from patients with metastatic melanoma.

McEvoy Ashleigh C AC Calapre Leslie L Pereira Michelle R MR Giardina Tindaro T Robinson Cleo C Khattak Muhammad A MA Meniawy Tarek M TM Pritchard Antonia L AL Hayward Nicholas K NK Amanuel Benhur B Millward Michael M Ziman Melanie M Gray Elin S ES

Oncotarget 20170818 45

<h4>Background</h4>Currently mainly <i>BRAF</i> mutant circulating tumor DNA (ctDNA) is utilized to monitor patients with melanoma. <i>TERT</i> promoter mutations are common in various cancers and found in up to 70% of melanomas, including half of <i>BRAF</i> wild-type cases. Therefore, a sensitive method for detection of <i>TERT</i> promoter mutations would increase the number of patients that could be monitored through ctDNA analysis.<h4>Methods</h4>A droplet digital PCR (ddPCR) assay was desi ...[more]

PMID: 29108273

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Project description:Somatic mutations in the telomerase reverse transcriptase (TERT) promoter regions are frequent events in urothelial cancer (UC) and their detection in urine (supernatant cell-free DNA or DNA from exfoliated cells) could serve as putative non-invasive biomarkers for UC detection and monitoring. However, detecting these tumor-borne mutations in urine requires highly sensitive methods, capable of measuring low-level mutations. In this study, we developed sensitive droplet digital PCR (ddPCR) assays for detecting TERT promoter mutations (C228T, C228A, CC242-243TT, and C250T). We tested the C228T and C250T ddPCR assays on all samples with sufficient quantity of urinary DNA (urine supernatant cell-free DNA (US cfDNA) or urine pellet cellular DNA (UP cellDNA)) from the DIAGURO (n = 89/93 cases and n = 92/94 controls) and from the IPO-PORTO (n = 49/50 cases and n = 50/50 controls) series that were previously screened with the UroMuTERT assay and compared the performance of the two approaches. In the DIAGURO series, the sensitivity and specificity of the ddPCR assays for detecting UC using either US cfDNA or UP cellDNA were 86.8% and 92.4%. The sensitivity was slightly higher than that of the UroMuTERT assay in the IPO-PORTO series (67.4% vs. 65.3%, respectively), but not in the DIAGURO series (86.8% vs. 90.7%). The specificity was 100% in the IPO-PORTO controls for both the UroMuTERT and ddPCR assays, whereas in the DIAGURO series, the specificity dropped for ddPCR (92.4% versus 95.6%). Overall, an almost perfect agreement between the two methods was observed for both US cfDNA (n = 164; kappa coefficient of 0.91) and UP cellDNA (n = 280; kappa coefficient of 0.94). In a large independent series of serial urine samples from DIAGURO follow-up BC cases (n = 394), the agreement between ddPCR and UroMuTERT was (i) strong (kappa coefficient of 0.87), regardless of urine DNA types (kappa coefficient 0.89 for US cfDNA and 0.85 for UP cellDNA), (ii) the highest for samples with mutant allelic fractions (MAFs) > 2% (kappa coefficient of 0.99) and (iii) only minimal for the samples with the lowest MAFs (< 0.5%; kappa coefficient 0.32). Altogether, our results indicate that the two methods (ddPCR and UroMuTERT) for detecting urinary TERT promoter mutations are comparable and that the discrepancies relate to the detection of low-allelic fraction mutations. The simplicity of the ddPCR assays makes them suitable for implementation in clinical settings.

Dataset Information

Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma.

Publications

Sensitive droplet digital PCR method for detection of <i>TERT</i> promoter mutations in cell free DNA from patients with metastatic melanoma.

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