Open AccessDroplet digital polymerase chain reaction assay and peptide nucleic acid‐locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T‐cell lymphoma
Author(s) -
Tanzima Nuhat Sharna,
SakataYanagimoto Mamiko,
Komori Daisuke,
Hattori Keiichiro,
Suehara Yasuhito,
Fukumoto Kota,
Fujisawa Manabu,
Kusakabe Manabu,
Matsue Kosei,
Wakamatsu Hirotake,
Shimadzu Mitsunobu,
Chiba Shigeru
Publication year - 2018
Publication title -
cancer science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.035
H-Index - 141
eISSN - 1349-7006
pISSN - 1347-9032
DOI - 10.1111/cas.13557
Subject(s) - rhoa , locked nucleic acid , microbiology and biotechnology , peptide nucleic acid , angioimmunoblastic t cell lymphoma , nucleic acid , digital polymerase chain reaction , mutation , polymerase chain reaction , biology , chemistry , dna , gene , genetics , t cell , oligonucleotide , immune system , signal transduction
Angioimmunoblastic T‐cell lymphoma ( AITL ) is a subtype of nodal peripheral T‐cell lymphoma ( PTCL ). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL . Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL . However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing ( NGS ), droplet digital PCR (dd PCR ) and peptide nucleic acid‐locked nucleic acid ( PNA ‐ LNA ) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS . dd PCR and PNA ‐ LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with dd PCR and those with NGS showed high concordance ( P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL 198; c.[50G > T;51A > C], p.Gly17Val in PTCL 216; and c.50G > A, p.Gly17Glu in PTCL 223) were detected using NGS . These sequence changes could not appropriately be detected using the dd PCR assay and the PNA ‐ LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of dd PCR / PNA ‐ LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.