P631: A PCR-BASED ASSAY FOR IGLV3-21R110 SCREENING CONFIRMS ITS PROGNOSTIC VALUE IN AN INDEPENDENT COHORT OF 613 PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA.

Background: Recent studies have shown that expression of the immunoglobulin lambda light chain IGLV3-21 gene carrying a point mutation in the amino acid 110 (named IGLV3-21R110) defines a subset of chronic lymphocytic leukemia (CLL) with an intermediate epigenetic subtype and poor prognosis (Maity et al. PNAS, 2020; Nadeu et al. Blood, 2021). This mutation is found in up to 6.5% of patients with CLL at diagnosis and up to 18% of cases enrolled in clinical trials. In addition, IGLV3-21R110 seems to have prognostic value independently of the IGHV gene somatic hypermutation (SHM) status, stereotype subset #2, and disease stage, which makes it a promising prognostic biomarker. However, further studies on independent cohorts are needed to support its application in clinical practice. Aims: To develop a rapid detection method of the IGLV3-21R110 mutation and to assess its prognostic significance in a cohort of 613 previously unpublished CLL patients. Methods: A multiplex IGLV3-21R110 mutation-specific polymerase chain reaction (msPCR) assay was established in a cohort of 12 patients (including 6 IGLV3-21R110 mutated) and validated in 159 cases (including 7 mutated; Nadeu et al. Blood, 2021). This msPCR assay was applied on an independent cohort of 613 CLL patients (575 CLL, 38 monoclonal B-cell lymphocytosis). Clinical analyses were performed for time to first treatment (TTFT) and overall survival (OS) from time of diagnosis considering only cases diagnosed as CLL. Results: A msPCR approach was designed integrating two forward primers aligning to distinct regions of the IGLV3-21 gene and two R110-specific reverse primers matching the IGLJ1 and IGLJ2/3 genes, respectively. A third pair of primers targeting exon 9 of FBXW7 was used as an internal control. PCR conditions were set up on a cohort of 12 cases and subsequently validated on 159 previously published cases with 100% concordance. We then applied this msPCR assay to a cohort of 613 previously unpublished CLL patients and identified the R110 mutation in 22 (3.6%) cases, including 75% mutated IGHV (M-CLL) and 25% unmutated IGHV (U-CLL). Moreover, 84.6% of IGLV3-21R110 cases carried non-stereotyped immunoglobulin genes while the remaining stereotyped IGLV3-21R110 cases were subset #2. Ten of the IGLV3-21R110 cases were classified according to the epigenetic subtypes and all belonged to the intermediate CLL (i-CLL) subgroup. Clinically, M-CLL patients carrying the IGLV3-21R110 as well as i-CLL with IGLV3-21R110 had a shorter TTFT compared to M-CLL and i-CLL lacking IGLV3-21R110, respectively, and similar to U-CLL/naïve-like CLL (p<0.005). Multivariable analyses including IGLV3-21R110, disease stage, and IGHV gene SHM status or epigenetic subtypes confirmed the independent prognostic value of IGLV3-21R110 on TTFT (p<0.005). IGLV3-21R110 had also a prognostic impact on OS since M-CLL carrying the IGLV3-21R110 had shorter OS similar to that of U-CLL (Figure). Finally, we combined these novel data with those of our previously published 489 CLL cohort (Nadeu et al. Blood, 2021) (N total=1102). Clinical analyses of the whole cohort confirmed the independent prognostic value of IGLV3-21R110. Image:Summary/Conclusion: We have developed a reliable, easy-to-use msPCR assay suitable for IGLV3-21R110 screening in large cohorts. By applying this msPCR on a cohort of 613 CLL patients, our results corroborate the relevance of IGLV3-21R110 testing to improve the risk stratification of CLL patients in the clinical practice, especially within M-CLL and i-CLL subtypes.