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Title: Cytogenetic and genomic assessment of selected lymphoproliferative disorders
Other Titles: Cytogenetische en genomische evaluatie van geselecteerde lymfoproliferatieve aandoeningen
Authors: Put, Natalie
Issue Date: 3-Sep-2012
Abstract: Chronic mature B-cell lymphoproliferative disorders, i.e. B-cell chronic lymphocytic leukemia (CLL) and plasma cell dyscrasias such as multiple myeloma (MM), have a variable disease course. Clinical staging systems and several biological parameters have been used to estimate tumor burden and predict prognosis. In addition, cytogenetic aberrations have prognostic significance and are therefore investigated in the routine evaluation of these diseases. Identifying the recurrent lesions underlying malignant transformation, is expected to lead to recognition of novel clinico-biological entities within the B-cell lymphoproliferative disorders, to improved insights in their pathogenesis and, hence, to identification of novel therapeutic targets. First, we evaluated available techniques, which can be applied to detect cytogenetic abnormalities in the routine investigation of CLL and MM: conventional cytogenetic analysis (CCA), fluorescence in situ hybridization (FISH) and high resolution single nucleotide polymorphism (SNP)-arrays [Affymetrix Cytogenetics Whole Genome 2.7M Arrays (2.7M arrays)]. Peripheral blood or bone marrow samples are cultured in order to obtain metaphases for CCA. However, CLL lymphocytes and plasma cells have a poor mitotic index in vitro and bone marrow (BM) infiltration may be limited. For CLL, several mitogens and cytokines can be added to the culture medium to overcome this problem. We performed a direct comparison of karyotyping after stimulation with the new CpG/IL-2 combination on one hand and the conventional mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) on the other hand. We observed higher abnormality detection rates, higher percentages abnormal nuclei and in particular higher detection rates of del(13q) and translocations, using CpG/IL-2 vs. TPA. Therefore, CpG/IL-2 should be preferred for routine conventional cytogenetic analysis of CLL. In MM, addition of cytokines, i.e. interleukin-6, and prolonged culture times do not significantly improve the number of informative karyotypes. As a consequence, analysis of nondividing cells, i.e. interphase FISH is widely used and has become the standard technique for the cytogenetic evaluation of MM. We have compared interphase FISH on cultured whole bone marrow with FISH on uncultured, selected plasma cells and observed a higher abnormality detection ratio, independent of the extent of bone marrow infiltration by plasma cells, a higher number abnormalities per case and higher percentages aberrant nuclei with the latter technique. This study validated the recommendation, initially based on a consensus agreement, that FISH should be performed after plasma cell selection in the routine investigation of MM. Until now the use of SNP-arrays was restricted to the research setting. Therefore we aimed to evaluate high resolution SNP-arrays for routine application in CLL, i.e. the Affymetrix 2.7M arrays which have a whole-genome coverage and contain more than 2 million copy number markers. Unfortunately, we found the 2.7M arrays not suitable for routine investigation of CLL. However, whole genome approaches are valuable for the analysis and prognostic stratification of CLL. Therefore, either the combination of CCA and FISH or another genomic array-platform (e.g. the 250K array) should be considered for the routine investigation of CLL. Second, we characterized cytogenetic entities in CLL, in particular translocations involving immunoglobulin genes (IG) and the proto-oncogenes BCL2 and MYC, and correlated the genetic findings with clinical characteristics. Forty and 30 patients with respectively a BCL2- and MYC-translocations were studied. While BCL2-translocations were associated with early stage disease, typical morphology and immunophenotype, patients with a MYC-translocation presented with more advanced disease stages and the presence of prolymphocytes and even prolymphocytic transformation was observed. Moreover MYC-translocations were associated with poor prognostic markers i.e. del(17p), del(11q) and complex karyotype. This contrasts with BCL2-translocations, in which an association with trisomy 12, absence of del(11q) and mutated IGHV was observed. Finally, patients with MYC-translocations had shorter overall survival and treatment-free survival, compared with patients with BCL2-translocations, indicating that the identification of the specific IG-translocation and the cytogenetic partner allows improved patient stratification. Finally, we focused on clonal evolution in CLL, in order to identify new (cyto)genetic aberrations or patterns relevant for the transition from indolent to clinically progressive and therapy-requiring CLL. Therefore, we analyzed 106 paired consecutive samples of 53 untreated patients by karyotyping, FISH and 2.7M arrays. Although, the genome of CLL appeared to be quite stable over time, clonal evolution did occur in the early follow-up of untreated patients, with a prevalence of 32%. The median number copy number aberrations detected by 2.7M array, did not change over time. The most frequent aberration at initial presentation and acquired aberration at evolution was the del(13q). Copy number neutral loss of heterozygosity (cnLOH) of the entire 13q arm was recurrent and stable over time. Presence of either del(17p), del(11q) or trisomy 12, were significantly more observed in patients with clinical evolution, as were unbalanced translocations and the presence of at least one large CNA (>5Mb). Finally, clonal evolution observed with CCA, FISH and SNP-arrays, was not associated with a higher risk for clinical evolution, indicating that aberrations driving clinical evolution were either present at the first time of analysis or were not detected with the applied techniques. Very recently, the application of other newly developed techniques, such as next-generation sequencing, led to the identification of novel recurrent genetic abnormalities and pathways involved in clinical evolution of the patient and are therefore promising in the detection of potential therapeutic targets.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Department of Human Genetics - miscellaneous
Laboratory for Genetics of Malignant Disorders

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