P606: MEASUREMENT OF INTRACLONAL DIVERSIFICATION REFINES THE PROGNOSTIC IMPACT OF IGHV MUTATIONS IN CLL

Background: Investigation of the mutational status of the heavy chain variable region of the immunoglobulin (IGHV) genes entered the clinical practice in chronic lymphocytic leukemia (CLL), due to its clinical relevance as prognostic/predictive marker. Despite the canonical categorization in mutated (M) and unmutated (UM) IGHV, some CLL may exhibit evidence of IGHV clonal evolution through the introduction of new mutations, in a process known as intraclonal diversification (ID). In NGS era, a rigorous approach for ID evaluation is still lacking. Aims: To develop a bioinformatic workflow for ID quantification in CLL and evaluate its clinical impact. Methods: The study included 983 CLL patients (759 with Time-to-first treatment, TTFT) analyzed at the time of diagnosis/first presentation. Lymphotrack assay was used to generate IGHV NGS libraries and sequencing data were analyzed with an original error-suppression pipeline to avoid ID overestimation. The inverse Simpson Index (iSI) was calculated to evaluate ID; a value set at 1.2 was chosen to discriminate between samples with ID (intraclonal) from samples without ID (clonal). IGHV analysis (n=52) utilizing Unique Molecular Identifier (UMI) as gold-standard revealed a very close correlation in iSI scores between samples processed with our pipeline vs. the UMI-based protocol (22 cases with ID and 30 cases without ID according to both the analyses, Fig.A). The pipeline was validated in lymphoproliferative disorders with different ID levels, including 14 hairy cell leukemia (HCL), 28 diffuse large B cell lymphoma (DLBCL), 40 follicular lymphoma (FL), and 43 mantle cell lymphoma (MCL). Results: By applying our UMI-independent pipeline, most of DLBCL (68%) and FL (72%) revealed ID (Fig.B), according to their germinal center origin. Moreover, about 50% of HCL had ID, in keeping with the heterogeneity of the disorder (Fig.B), and as expected from the naïve B cell origin, only a minority of MCL (22%) had ID (Fig.B). Among 983 CLL (iSI range 1.0-20.4), 144 CLL (14%) had ID while 839 were without ID (Fig.B). Based on IGHV mutations, 508 and 475 cases were either M- or UM-CLL, respectively. By combining ID and IGHV status, we observed 422 UM-CLL without ID, 417 M-CLL without ID, 53 UM-CLL with ID, and 92 M-CLL with ID, with a significant overrepresentation of cases with ID among M-CLL without correlation with specific IGHV families and genes. Moreover, CLL cells with ID overexpressed Activation-Induced cytidine Deaminase (AID; P=0.027), an enzyme responsible for the somatic hypermutation process, and revealed AID-specific mutational signatures (WRC/GYW). Strikingly, M-CLL patients with ID had significantly longer TTFT respect to M-CLL patients devoid of ID (P=0.015; Fig.C). A multivariate analysis, carried out on M-CLL, identified ID classification as independent variable along with Rai Stage, CD49d, and del 11p/del 17p (Fig.D). RNASeq performed on 14 M-CLL (8 with ID vs. 6 without ID) revealed that M-CLL with ID upregulated gene pathways related to BCR downstream signalling, T/NK cell activation and cellular apoptosis/programmed cell death, while downregulating genesets associated to increased protein synthesis and transcription, in keeping with their indolent clinical behavior (Fig.EF). Summary/Conclusion: Here we report a novel UMI-independent method to assess ID in CLL. By applying this approach, we provide evidence that: i) ID prevalently affects M-CLL; ii) patients affected by M-CLL with ID have better outcome than M-CLL cases without ID; iii) ID identifies a subset of M-CLL with specific molecular/biological features and clinical characteristics.Keywords: IGH, Prognostic factor, Chronic lymphocytic leukemia