TUMOR DYNAMICS OF CHRONIC LYMPHOCYTIC LEUKEMIA CASES WITH MULTIPLE PRODUCTIVE IGHV-IGHD-IGHJ REARRANGEMENTS

Sborník

Background. Due to the currently routine testing of IGHV mutational status in chronic lymphocytic leukemia (CLL) patients, evidence of cases with multiple productive immunoglobulin heavy genes rearrangements (MP-IGH) has emerged. Several mechanisms, such as lack of allelic exclusion at the IG loci, or the presence of two clonal populations have been linked with the MP-IGH phenomenon. However, the true biological and clinical implications are currently unknown. Moreover, over-time clonal drift, a dynamic process of alterations in malignant clones recently observed in other lymphoid malignancies, has not been studied in these cases. Aims. Our study aimed to obtain molecular insight into the biological causes of MP-IGH in CLL and to perform a systematic study of the clonal drift in such cases. Methods. Separated B-lymphocytes from peripheral blood of 31 patients with MP-IGH were obtained. IGHV-IGHD-IGHJ, IGKV-IGKJ and IGLV-IGLJ rearrangements were analyzed from cDNA following described protocols. In available samples, PCR was repeated using gDNA; partial IGHD-IGHJ gene rearrangements and KDE rearrangements inactivating IGK loci were assessed. Detailed immunophenotypization was also performed. For the molecular monitoring of clonal dynamics, allele-specific oligonucleotide PCR assays (ASO-qPCR) were used. Results. Thirty one CLL cases with MP-IGHs were analyzed (two or three IGH rearrangements were detected in 26 and 5 patients, respectively). Firstly, we sought to unravel the possible causes underlying the presence of MP-IGH by performing a detailed immunophenotypic and molecular profiling. By coalescing all such results, we categorized the MP-IGH cases as follows: (i) definite co-existence of two clonal B-cell populations: 9/31 cases, with differing immunophenotypic light chain restriction; (ii) highly likely co-existence of more clonal populations with homogenous phenotype: 16/31 cases, where the number of detected IG rearrangements exceeded genomic capacity of single cell IG loci; (iii) indeterminate as to one or more CLL-like populations: 6/31 cases, in which we failed to obtain conclusive evidence of more than one clone. Furthermore, repeated MP-IGH analysis was performed in 23/31 patients (median interval from first to last analysis 24 months, range 8-74 months) to evaluate over-time changes in proportion of clones. Based on fragment analysis, detected IGH were considered as (i) persistent (stable in subsequent samples), (ii) appearing (originally undetectable), or (iii) diminishing (decreasing proportion in subsequent samples). In 11/23 patients, ASO-qPCR was used to confirm and quantify such changes. Interestingly, we observed clonal drift in MP-IGH cases: appearing of a new clone in three patients; diminishing of a clone in 18 patients. Moreover, analysis of molecular features of detected IGH revealed that selection of IGH tends to favour (i) higher IGHV identity to germ-line and/or (i) longer HCDR3. This tendency was more striking in cases where stereotyped BCR was selected. Summary: Our study allowed us to obtain novel insight into the biological causes of MP-IGH in CLL by categorizing patients according to the evidence of more than one B-lymphocyte clones. Importantly, we observed clonal drift favouring specific BCR molecular features, which might eventually provide further insight into disease evolution with implications for patient monitoring and therapy. Supported by CZ.1.05/1.1.00/02.0068, CZ.1.07/ 2.3.00/20.0045, CZ.1.07/2.4.00/17.0042, MSM0021622430, MUNI/A/0784/ 2011, GACR204/09/H058

Haematologica, 2012; 97(s1):  48