RHOA mutations detected in peripheral blood. (A) The results of targeted deep sequencing from cfDNA of RHOA exon 2 by Illumina CAPP-seq. Mutations were detected at presentation (n = 5) at c.50G>T (p.Gly17Val), c.73A>G (p.Phe25Leu), and c.48A>T (pCys16*). Diagnosis (either AITL or PTCL-NOS) is indicated. In all cases, the malignant T-cells expressed PD-1. (B) ddPCR assays from cfDNA for the 3 RHOA mutations. Diagnosis and PD-1 status are shown. Red shading indicates PD-1 expression; for cases with gray shading, PD-1 is negative, and for those with a diagonal line, PD-1 is not determined. (C) Mutational frequency by ddPCR results was correlated with VAF from Ion Torrent sequencing for the 3 RHOA mutations in samples at presentation and during the course of treatment (28 samples from 21 patients). Linear regression (red line) and 95% confidence interval are shown. For c.50G>T (p.Gly17Val), R² = 0.65 and P = .0001; for c.73A>G (p.Phe25Leu), R² = 0.974 and P = .0001; and for c.48A>T (pCys16*), R² = 0.862 and P = .0001. (D) Sanger sequencing fluorograms from cloned PCR product showing the 3 RHOA mutations c.50G>T, c.73A>G, and c.48A>T (pCys16*). Mut, mutated; WT, wild-type.