Investigation of mutations, copy number variations and translocations by capture-based Next-generation sequencing in patients with chronic B-cell lymphoproliferative disorders

Patients with leukaemic indolent B-NHL who could not be assigned to a specific disease category were identified and 108 cases underwent extensive molecular characterisation using a capture-NGS sequencing approach designed by the EuroClonality NGS consortium. Without a clear diagnosis such patients have no evidence-based, standardised treatment pathways and have no or limited access to clinical trials and novel therapies as eligibility and approval are based on distinct histological categories or WHO-defined disease entities. Well-characterised translocations were found in 10 cases supporting a diagnosis of Mantle cell lymphoma in six patients, follicular lymphoma in two patients and atypical CLL in a further 2 cases. In a further case a rare, TRA-CDK6 fusion was detected which was recently confirmed as a recurrent, diagnostic finding in SMZL. Combination of variants in KLF2, deletion of 7q together with IGHV1-2*04 gene usage confirmed the suspected diagnosis of SMZL in one case. The most frequently mutated genes were TP53 in 16/101 (15%) and the hotspot MYD88 p.(Leu265Pro) variant in 13/101 (13%) followed by variants in genes associated with NF-B signalling. Integration of the molecular findings together with other (histo)pathological and clinical findings may enable the distinction between LPL and MZL-related disease categories in these patients. NGS was capable of identifying clonal IG-rearrangements in all cases. Further stereotyping analysis using the VLeader protocol and Sanger sequencing showed most cases carried IGHV3 (52%) and IGHV4 (27%) genes with IGHV4-34 the most common (17%). Evidence of Somatic hypermutations were found in 90% of cases with 77% showing