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

Wren, Dörte (2021) Investigation of mutations, copy number variations and translocations by capture-based Next-generation sequencing in patients with chronic B-cell lymphoproliferative disorders. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

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 <98% homology to the germline sequence. None of the IGHV rearrangements showed a CLL-stereotype. Preliminary analysis of copy-number variants showed a high frequency of trisomy 12 (17 cases), 17p deletion events in combination with TP53 variants as well as deletions of 7q, a recurrent feature of MZL. This prospective study clearly demonstrates the benefit of using NGS-panel approaches for the characterization of patient cohorts which based on routine 4 diagnostics evaluation cannot be assigned to a defined disease category. An additional 12 patients were given a formal diagnosis based on the molecular findings and further clinical review prompted by the results of this study will likely lead to an increase in this number. The detailed information obtained about the clonal structure, including the specific V(D)J-rearrangements and combinations of molecular aberrations in each case, will enable further research into the aetiology of these diseases. From a diagnostic laboratory perspective in particular, the NGS panel utilised here is capable of reducing the time and cost of the evaluations required by combining the analysis of clonality, single-nucleotide variants, copy-number variants and structural variants in a single assay. Given the breadth of results, this NGS panel is also a useful tool for the characterisation of cohorts in a research setting and the design can be extended in future as novel markers and areas of interest are described in the literature.