IGLV3-21^R110 detection through a mutation-specific polymerase chain reaction assay and its clinical value in 2 independent CLL cohorts. (A) Primer design combining 2 forward primers aligning to distinct regions of the IGLV3-21 gene (green arrows) and 2 R110-specific reverse primers matching the IGLJ1, and IGLJ2/3 genes, respectively (red and orange arrows). A third pair of primers targeting exon 9 of FBXW7 (black arrows) was used as the internal control (top). QIAxcel Advanced System (QIAGEN) capillary electrophoresis image of msPCR analysis. The black arrow indicates the amplification of the internal control (FBXW7 exon 9). The middle and bottom arrows (in green) indicate the 2 bands amplifying in IGLV3-21^R110 mutated tumors. (B) Schema of the cohorts studied. (C) Frequency of IGLV3-21^R110 mutations in CLL subtypes in cohort 1 (top) and cohort 2 (bottom). (D) Distribution of the IGHV mutational load (% IGHV identity) and epigenetic subtypes within M-CLL, IGLV3-21^R110 CLL, and U-CLL. (E) Comparison of TTFT among patients with CLL stratified according to epigenetic subtypes and IGLV3-21^R110 in cohort 1 (left) and cohort 2 (right). C+, positive control; C–, negative control; DFCI, Dana-Farber Cancer Institute (∗120 CLL from DFCI has been previously published in reference¹⁴; Nadeu et al⁶); n-CLL, naïve-like CLL; R110, sample positive for IGLV3-21^R110; UHH, University Hospital Heidelberg (cohort described in reference¹⁵; no IGLV3-21^R110 analysis performed in this previous publication); WT, wild type (ie, sample negative for IGLV3-21^R110).