Author: Anya Hotinski
Hotinski, Anya, 2021 Trisomy 12 in Chronic Lymphocytic Leukaemia, Flinders University, College of Medicine and Public Health
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Chronic Lymphocytic Leukaemia (CLL) is a common incurable haematological malignancy of B lymphocytes. Trisomy of chromosome 12 is a recurrent genomic abnormality in CLL with unique associations and clinical response but an unclear molecular pathogenesis. Comprehensive immunogenetic characterisation of a local cohort of trisomy 12 CLL was performed and confirmed the known association with cell surface expression of the integrin and poor prognostic maker, CD49d. Two trisomy 12 CLL cases with bimodal expression of CD49d were identified and extensively interrogated after flow-cytometry cell sorting of the CD49d+ and CD49d- CLL cell populations. One of these cases was comprised of two completely unique leukaemic clones with different immunoglobulin heavy variable gene (IGHV) usage and mutational status, different karyotypes, and different exome sequencing variants. The CD49d+ clone harboured trisomy 12, a hypermutated IGHV4-34and a lysine methyltransferase 2D (KMT2D) mutation on chromosome 12 that represents a putative novel driver of trisomy 12 CLL. The CD49d- clone had disomy 12, a sub-clonal deletion of chromosome 17p, an unmutated IGHV3-21 and a splicing factor 3b subunit 1 (SF3B1) mutation, a previously reported high-risk feature of CLL. Both clones harboured mutations in common that were not present in the non-malignant T cells which implies the existence of a pre-leukaemic progenitor cell prior to the IGHV gene rearrangement that occurs during lymphoid maturation and prior to the development of CLL. As well as the general implications for the clonal evolution of CLL, the case also demonstrated that the high-risk feature of CD49d expression does not necessarily associate with other high-risk features such as an unmutated IGHV and may not be a true driver of leukaemia (being normally expressed at high levels in mature B cells). Regulation of integrin subunit alpha 4, ITGA4 (the CD49d gene), was shown not to be dependent on the methylation status of the promoter, at least in this case, in contrast to previous reports.
A comparison of the transcriptome of the trisomy 12 and disomy 12 clones in this biclonal case of CLL was also performed to identify pathways that are differentially regulated in trisomy 12 CLL. RNAseq of the clones implicated the potential importance of toll-like receptor signalling (via toll-like receptor 4, TLR4) in trisomy 12 CLL. Furthermore, tumour necrosis factor alpha induced protein 3 (TNFAIP3), an inhibitor of the TLR4 pathway, was identified in the gene-set expression analysis and is itself a target of the epigenetic regulator, KMT2D, which was also found to be mutated in the trisomy 12 clone. The TLR4 pathway was stimulated via addition of lipopolysaccharide (LPS, an immunogenic component of bacterial cell walls) to a cohort of 7 primary trisomy 12 and 4 disomy 12 CLL samples. Cell viability and RNA and protein expression of a range of intermediaries in the TLR4 pathway were measured after a 48-hour incubation. There was significantly higher expression of TLR4 and CD14 (a component of the TLR4 complex) in the trisomy 12 group at baseline which approximated expression levels observed in normal B lymphocytes. No difference in cell viability or surface expression of TLR4, CD49d, CD14, however, was identified between the two groups following stimulation with LPS. In addition to this, there were no changes in mRNA expression of TNFAIP3, KMT2D, TLR4 or interleukin 8, IL8 (a downstream pro-inflammatory chemokine of TLR4 signalling), between the trisomy 12 and disomy 12 groups either at baseline or following stimulation. The stimulation assays were limited by low sample numbers of variable quality in terms of cell viability upon thawing, total viable cell numbers and RNA quality. A dependence of trisomy 12 CLL on TLR4 signalling could not be confirmed during this thesis.
In conclusion, this thesis presents evidence that trisomy 12 CLL cells more closely resemble normal B lymphocytes than their disomic CLL counterparts, demonstrates the importance of thorough investigation of unique individual cases to gain insights into clonal evolution and genomic complexity in CLL, and provides an avenue for future research in toll-like receptor signalling pathways to advance the understanding of early drivers of trisomy 12 CLL.
Keywords: chronic lymphocytic leukaemia, trisomy 12, CD49d, IGHV
Subject: Medical Science thesis
Thesis type: Doctor of Philosophy
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Bryone Kuss