Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Corless, Broderick C.; Chang, Gregory; Cooper, Samantha; Syeda, Mahrukh M.; Shao, Yongzhao; Osman, Iman; Karlin-Neumann, George; Polsky, David

doi:10.1016/j.jmoldx.2018.09.003

Cited by 51 publications

(46 citation statements)

References 23 publications

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“…In addition, there is an important limitation in the analysis of TERT promoter mutations in NGS panels. Despite validated for FFPE NGS panels 38 , TERT promoter mutations have not performed well for cfDNA in NGS custom panels 39 , 40 due to its high GC content, and so, it is mostly described in droplet digital PCR approaches 41 , 42 . Furthermore, BRAF and NRAS mutations are mutually exclusive, as well as TERT C250T and C228T, and then, a maximum of two mutations will be tested in plasma for each melanoma patients, making feasible the dPCR approach proposed in this study.…”

Section: Discussionmentioning

confidence: 99%

“…Despite using 2 ml of plasma they tested samples in 4 replicates 43 . Corless and colleagues screened 12 replicates from plasma samples for TERT evaluation in ddPCR 42 . Some other studies isolated DNA from a larger volume of plasma, up to 5mL 41 , 44 .…”

Section: Discussionmentioning

confidence: 99%

“…TERT mutation occurs in up to 80% of cutaneous melanomas, which makes this gene extremely important to be part of the panel proposed 26 – 29 . However, it is established that the high GC content (approximately 80%) in the promoter region of the gene imposes difficulties in the design of an efficient and specific PCR assay to detect and quantify mutations in this region 42 . This was a technical challenge with TERT C228T mutation.…”

Section: Discussionmentioning

confidence: 99%

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Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

Marczynski

Laus

Reis

et al. 2020

Sci Rep

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BRAF, NRAS and TERT mutations occur in more than 2/3 of melanomas. Its detection in patient’s blood, as circulating tumor DNA (ctDNA), represents a possibility for identification and monitoring of metastatic disease. We proposed to standardize a liquid biopsy platform to identify hotspot mutations in BRAF, NRAS and TERT in plasma samples from advanced melanoma patients and investigate whether it was associated to clinical outcome. Firstly, we performed digital polymerase chain reaction using tumor cell lines for validation and determination of limit of detection (LOD) of each assay and screened plasma samples from healthy individuals to determine the limit of blank (LOB). Then, we selected 19 stage III and IV patients and determined the somatic mutations status in tumor tissue and track them in patients’ plasma. We established a specific and sensitive methodology with a LOD ranging from 0.13 to 0.37%, and LOB ranging from of 0 to 5.201 copies/reaction. Somatic mutations occurred in 17/19 (89%) patients, of whom seven (41%) had ctDNA detectable their paired plasma. ctDNA detection was associated with shorter progression free survival (p = 0.01). In conclusion, our data support the use of ctDNA as prognosis biomarker, suggesting that patients with detectable levels have an unfavorable outcome.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

Marczynski

Laus

Reis

et al. 2020

Sci Rep

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“…For most of the samples, the TERT p mutations were detected by the ddPCR technique according to protocol described by Corless et al [30], using the TERT C250T_113 Assay and C228T_113 Assay (unique assay ID dHsaEXD46675715 and dHsaEXD72405942, respectively; Bio-Rad). Both assays include FAM-labelled probes for the C250T and C228T mutations respectively, HEX-labelled wild-type (WT) probes, and primers for a 113-bp amplicon that encompasses the mutational sites.…”

Section: Methodsmentioning

confidence: 99%

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Salgado

Roelse

Nell

et al. 2019

Preprint

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AbstractThe telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. Contrary to common concepts, CpG methylation in the TERT promoter (TERTp), was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to assist binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and higher-order chromatin structure) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin and in melanoma cell lines (n=61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state throughout the promoter in TERTp-WT samples due to high methylation or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T/C250T mutations.Author summaryPvdV and RvD formulated research goals and aims and supervised the overall progress. Wet-lab experiments, preparation of the manuscript and statistical analysis were performed by CS and CR. CS designed the novel assays. RN was involved in the experimental setup. RvD, NG and PvdV were responsible for funding acquisition. CR, RN, NG, RvD and PvdV critically reviewed the manuscript.

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“…Mutation analysis using commercial TERT C250T and C228T mutation assays. For most of the samples, the TERTp mutations were detected by the ddPCR technique according to protocol described by Corless et al [43], using the TERT C250T_113 Assay and C228T_113 Assay (unique assay ID dHsaEXD46675715 and dHsaEXD72405942, respectively; Bio-Rad). Both assays include FAM-labelled probes for the C250T and C228T mutations respectively, HEX-labelled wild-type (WT) probes, and primers for a 113-bp amplicon that encompasses the mutational sites.…”

Section: Assessment Of Mutational Statusmentioning

confidence: 99%

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

et al. 2020

View full text Add to dashboard Cite

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

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Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples

Cited by 51 publications

References 23 publications

Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

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