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Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28>D/E, P45>S, E55>Q, and S64>I changes (p < 0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

Deletion 11q22 [del(11q)] is among the most common chromosomal alteration in chronic lymphocytic leukemia (CLL). This anomaly is highly variable in size and may be distinguished in large and small deletions. The minimal deleted region on 11q usually includes ATM gene and can also encompass the BIRC3 locus, located 6 Mb centromeric to ATM, in approximately 80% of cases. In this study, we have evaluated BIRC3 and ATM losses and their association with adverse prognostic factors in CLL patients. Thirty-three out of 200 patients (16.5%) sequentially studied in our laboratory harbored del(11q). Our study shows a predominant presence of concomitant ATM and BIRC3 deletions but with differences in the percentage of abnormal cells: 54.5% of cases had similar ATM and BIRC3 percentages, 9.1% showed higher frequency of ATM deletion and 21.2% increased percentage of BIRC3 loss, indicating clonal evolution. Interestingly, 9.1% patients presented only BIRC3 deletion and 6.1% cases showed only ATM loss, suggesting the inclusion of BIRC3 analysis in the routine FISH laboratory screening. A strong association between ATM and BIRC3 deletions with TP53 loss (48.5%), complex karyotypes (36%), unmutated IGHV (72.7%) and intratumor heterogeneity (51.5% patients) was observed. A short time to first treatment (p

Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV4-34 is one of the most frequently used genes in CLL patients, which usually display an indolent outcome. In this study, we explored the mutational profile of CLL patients expressing IGHV4-34 within different stereotypes and their association with prognostic factors and clinical outcome. A multi-institutional cohort of unselected 1444 CLL patients was analyzed by RT-PCR and bidirectional sequencing. Cytogenetics and molecular cytogenetics analyses were also performed. We identified 144 (10%) IGHV4-34 expressing cases, 119 mutated (M), 44 of them with stereotyped B-cell receptors. Subset #4 was the most frequent (56.8% of cases) followed by subsets #16 (13.6%), #29 (6.8%), and #201 (2.3%), with different distribution among countries. Analysis of somatic hypermutation profile showed significant differences among stereotyped subsets for G28D/E, P45S, E55Q, and S64I changes (p0.01) and high frequency of disruption of the glycosylation motif in the VH CDR2 region. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 as a sole alteration was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in non-stereotyped vs. stereotyped M-IGHV4-34 patients (p = 0.034). Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of South American CLL patients.

Genomic instability is a hallmark of cancer, contributing to tumour development and transformation, being chromosome instability (CIN) the most common form in human cancer. Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. In this study, we have evaluated basal CIN in untreated patients with CLL by measuring chromosome aberrations (CAs) and micronucleus (MN) frequency and their association with different prognostic factors. Seventy-two patients and 21 normal controls were analysed. Cytogenetic and fluorescence in situ hybridisation (FISH) studies were performed. IGHV (immunoglobulin heavy chain variable region) mutational status was evaluated by reverse transcription polymerase chain reaction and sequencing. An increased number of CA in patients compared with controls ( P = 0.0001) was observed. Cases with abnormal karyotypes showed increased CA rate than those with normal karyotypes ( P = 0.0026), with a particularly highest frequency in cases with complex karyotypes. Among FISH risk groups, a significant low frequency of CA was found in patients with no FISH alterations compared to those with del13q14 and ≥2 FISH alterations ( P = 0.0074). When mean CA value (6.7%) was considered, significant differences in the distribution of low and high CA frequency between cases with normal and abnormal karyotypes ( P = 0.002) were observed. By MN analysis, higher frequency in patients compared to controls ( P = 0.0001) was also found, as well as between cases with ≥2 FISH abnormalities and those with no FISH alterations ( P = 0.026). Similarly, significant differences were observed when patients were divided according to mean MN frequency (2.2%; P ≤ 0.04). Interestingly, patients with high MN frequency had shorter time to first treatment than those with low frequency ( P = 0.024). Cases with mutated and unmutated IGHV status showed increased CA and MN frequencies compared to controls ( P ≤ 0.0007), but no differences between both groups were found. Our results support the strong interaction between CIN and genomic complexity as well as their influence on poor outcome in this pathology. Fil: Palmitelli, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Investigaciones Hematológicas "Mariano R. Castex"; Argentina Fil: Stella, Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Krywinski, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Bezares, Raimundo F.. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Dr. Teodoro Álvarez"; Argentina Fil: Gonzalez Cid, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Slavutsky, Irma Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina