Droplet digital PCR assay and PNA-LNA clamp method for RHOA mutation detection in angioimmunoblastic T-cell lymphoma

Summary

Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of nodal peripheral T-cell lymphomas. 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 PNA-LNA clamp method for detecting the G17V RHOA mutations. The G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA-LNA clamp method both detected the G17V mutations in 4 samples in addition to those detected with NGS (31of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P<0.001). Three other RHOA mutations involving the p.Gly17 position (c.[49G>T;50G>T], p.Gly17Leu in PTCL198; c.[50G>T;51A>C], p.Gly17Val in PTCL216; and c.50G>A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and PNA-LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had the RHOA mutations at the p.Gly17Val position. In conclusion, our results suggested that combination of ddPCR/PNA-LNA clamp methods and NGS are best feasible to assist the diagnosis of AITL by detecting the RHOA mutations at the p.Gly17 position.

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