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

BACKGROUND: 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...

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Autores principales: McEvoy, Ashleigh C., Calapre, Leslie, Pereira, Michelle R., Giardina, Tindaro, Robinson, Cleo, Khattak, Muhammad A., Meniawy, Tarek M., Pritchard, Antonia L., Hayward, Nicholas K., Amanuel, Benhur, Millward, Michael, Ziman, Melanie, Gray, Elin S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668006/
https://www.ncbi.nlm.nih.gov/pubmed/29108273
http://dx.doi.org/10.18632/oncotarget.20354
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author McEvoy, Ashleigh C.
Calapre, Leslie
Pereira, Michelle R.
Giardina, Tindaro
Robinson, Cleo
Khattak, Muhammad A.
Meniawy, Tarek M.
Pritchard, Antonia L.
Hayward, Nicholas K.
Amanuel, Benhur
Millward, Michael
Ziman, Melanie
Gray, Elin S.
author_facet McEvoy, Ashleigh C.
Calapre, Leslie
Pereira, Michelle R.
Giardina, Tindaro
Robinson, Cleo
Khattak, Muhammad A.
Meniawy, Tarek M.
Pritchard, Antonia L.
Hayward, Nicholas K.
Amanuel, Benhur
Millward, Michael
Ziman, Melanie
Gray, Elin S.
author_sort McEvoy, Ashleigh C.
collection PubMed
description BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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spelling pubmed-56680062017-11-04 Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma McEvoy, Ashleigh C. Calapre, Leslie Pereira, Michelle R. Giardina, Tindaro Robinson, Cleo Khattak, Muhammad A. Meniawy, Tarek M. Pritchard, Antonia L. Hayward, Nicholas K. Amanuel, Benhur Millward, Michael Ziman, Melanie Gray, Elin S. Oncotarget Research Paper BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. Impact Journals LLC 2017-08-18 /pmc/articles/PMC5668006/ /pubmed/29108273 http://dx.doi.org/10.18632/oncotarget.20354 Text en Copyright: © 2017 McEvoy et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Paper
McEvoy, Ashleigh C.
Calapre, Leslie
Pereira, Michelle R.
Giardina, Tindaro
Robinson, Cleo
Khattak, Muhammad A.
Meniawy, Tarek M.
Pritchard, Antonia L.
Hayward, Nicholas K.
Amanuel, Benhur
Millward, Michael
Ziman, Melanie
Gray, Elin S.
Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title_full Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title_fullStr Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title_full_unstemmed Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title_short Sensitive droplet digital PCR method for detection of TERT promoter mutations in cell free DNA from patients with metastatic melanoma
title_sort sensitive droplet digital pcr method for detection of tert promoter mutations in cell free dna from patients with metastatic melanoma
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5668006/
https://www.ncbi.nlm.nih.gov/pubmed/29108273
http://dx.doi.org/10.18632/oncotarget.20354
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