Droplet digital polymerase chain reaction assay and peptide nucleic acid‐locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T‐cell lymphoma

摘要

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 (ddPCR) 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 style="fixed-case">NGS. dd style="fixed-case">PCR and style="fixed-case">PNA‐ style="fixed-case">LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with style="fixed-case">NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with dd style="fixed-case">PCR and those with style="fixed-case">NGS showed high concordance (P < .001). Three other style="fixed-case">RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in style="fixed-case">PTCL198; c.[50G > T;51A > C], p.Gly17Val in style="fixed-case">PTCL216; and c.50G > A, p.Gly17Glu in style="fixed-case">PTCL223) were detected using style="fixed-case">NGS. These sequence changes could not appropriately be detected using the dd style="fixed-case">PCR assay and the style="fixed-case">PNA‐ style="fixed-case">LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 style="fixed-case">PTCL samples (50.7%) had style="fixed-case">RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of dd style="fixed-case">PCR/ style="fixed-case">PNA‐ style="fixed-case">LNA clamp methods and style="fixed-case">NGS are best method to assist the diagnosis of style="fixed-case">AITL by detecting style="fixed-case">RHOA mutations at the p.Gly17 position.