Contributions
Abstract: EP625
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV (immunoglobulin heavy-chain variable region) mutational status displays strong prognostic value. About 40% of CLL patients carry quasi-identical B-cell receptor (BCR) immunoglobulins that can be assigned to distinct stereotyped subsets. IGHV4-34 is one of the most frequently used genes in CLL patients and mostly of them display an indolent outcome.
Aims
We here study the mutational profile of CLL patients expressing IGHV4-34 focusing on the different stereotypes that are present in this rearrangement and their correlation with prognostic factors and activation-induced cytidine deaminase (AID) expression, one of the hallmarks of disease progression in CLL.
Methods
A multi-institutional cohort of 1444 unselected CLL patients from Argentina (891), Brazil (358), Uruguay (116) and Venezuela (79) were analyzed. RT-PCR and bidirectional sequencing were done. Cytogenetics and molecular cytogenetics analysis were performed. IGHV sequences with <98% homology with respect to the germline counterpart were considered as mutated (M), whereas those with ≥98% homology were classified as unmutated (U). Silent (S) and replacement (R) mutations were analyzed per VH FR and VH CDR subregions. ARResT/AssignSubsets and ARResT/Teiresias were employed in order to identify major and minor/novel subsets, respectively. The study was approved by the local Ethics Committees of each Institution. All individuals provided their informed consent.
Results
We identified 144 (10%) IGHV4-34 expressing cases (78 males; mean age: 65.6 years; Rai stages: 0: 60.6%, I-II: 31.1%, III-IV: 8.2%); 119 M, 35 of them with stereotyped BCR. Subset #4 was the most frequent (71.4% of cases) followed by subsets #16 (17.1%), #29 (8.6%), and #201 (2.9%), with different distribution among countries. R mutations were more represented in VH FR2, VH CDR1 and VH CDR2 in subset #16 compared to subsets #4, #29, and non-stereotyped (NST) IGHV4-34 M-CLL with significant differences in VH FR2 between subset #16 and the NST IGHV4-34 M-CLL (p=0.0001). Asymmetric distribution in R/S mutations ratio among different VH subregions with higher ratio in VH CDR2 for subset #29 and, in VH CDR1 and VH FR2 for subset #16 was found. Analysis of somatic hypermutation profile showed significant differences in G28>D/E between subsets #4 and #16 (p<0.0001) and in P45>S for subsets #4, #16, and #29 vs NST M-IGHV4-34 (p=0.048). In addition, differences in E55>Q for subset #4 vs #16 (p=0.0376) or subset #16 vs NST (p=0.0001) or across subsets (p=0.0001) and, in S64>I for subset #29 vs NST (p=0.0001) were also observed. S64T was absent in subsets #4 and #16. High frequency of disruption of the glycosylation motif in VH CDR2 region was always present. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 single was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in NST vs stereotyped M-IGHV4-34 patients (p=0.034). AID overexpression was evaluated in 26 IGHV4-34 patients, 8 of them display expression, only 2 stereotyped. The remaining patients expressing AID were NST and 5/6 (83%) showed a U-IGHV status.
Conclusion
Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of our cohort and, reinforcing the role of the genetic background and environmental factors in CLL patients.
Keyword(s): Chronic lymphocytic leukemia, Immunoglobulin gene, Mutation status, Somatic hypermutation
Abstract: EP625
Type: E-Poster Presentation
Session title: Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background
Chronic lymphocytic leukemia (CLL) is the most common mature B-cell neoplasm in the West. IGHV (immunoglobulin heavy-chain variable region) mutational status displays strong prognostic value. About 40% of CLL patients carry quasi-identical B-cell receptor (BCR) immunoglobulins that can be assigned to distinct stereotyped subsets. IGHV4-34 is one of the most frequently used genes in CLL patients and mostly of them display an indolent outcome.
Aims
We here study the mutational profile of CLL patients expressing IGHV4-34 focusing on the different stereotypes that are present in this rearrangement and their correlation with prognostic factors and activation-induced cytidine deaminase (AID) expression, one of the hallmarks of disease progression in CLL.
Methods
A multi-institutional cohort of 1444 unselected CLL patients from Argentina (891), Brazil (358), Uruguay (116) and Venezuela (79) were analyzed. RT-PCR and bidirectional sequencing were done. Cytogenetics and molecular cytogenetics analysis were performed. IGHV sequences with <98% homology with respect to the germline counterpart were considered as mutated (M), whereas those with ≥98% homology were classified as unmutated (U). Silent (S) and replacement (R) mutations were analyzed per VH FR and VH CDR subregions. ARResT/AssignSubsets and ARResT/Teiresias were employed in order to identify major and minor/novel subsets, respectively. The study was approved by the local Ethics Committees of each Institution. All individuals provided their informed consent.
Results
We identified 144 (10%) IGHV4-34 expressing cases (78 males; mean age: 65.6 years; Rai stages: 0: 60.6%, I-II: 31.1%, III-IV: 8.2%); 119 M, 35 of them with stereotyped BCR. Subset #4 was the most frequent (71.4% of cases) followed by subsets #16 (17.1%), #29 (8.6%), and #201 (2.9%), with different distribution among countries. R mutations were more represented in VH FR2, VH CDR1 and VH CDR2 in subset #16 compared to subsets #4, #29, and non-stereotyped (NST) IGHV4-34 M-CLL with significant differences in VH FR2 between subset #16 and the NST IGHV4-34 M-CLL (p=0.0001). Asymmetric distribution in R/S mutations ratio among different VH subregions with higher ratio in VH CDR2 for subset #29 and, in VH CDR1 and VH FR2 for subset #16 was found. Analysis of somatic hypermutation profile showed significant differences in G28>D/E between subsets #4 and #16 (p<0.0001) and in P45>S for subsets #4, #16, and #29 vs NST M-IGHV4-34 (p=0.048). In addition, differences in E55>Q for subset #4 vs #16 (p=0.0376) or subset #16 vs NST (p=0.0001) or across subsets (p=0.0001) and, in S64>I for subset #29 vs NST (p=0.0001) were also observed. S64T was absent in subsets #4 and #16. High frequency of disruption of the glycosylation motif in VH CDR2 region was always present. All stereotyped IGHV4-34 cases showed normal karyotypes. Deletion 13q14 single was present in 42.8% of stereotyped cases with a different distribution among subsets. A shorter time to first treatment was found in NST vs stereotyped M-IGHV4-34 patients (p=0.034). AID overexpression was evaluated in 26 IGHV4-34 patients, 8 of them display expression, only 2 stereotyped. The remaining patients expressing AID were NST and 5/6 (83%) showed a U-IGHV status.
Conclusion
Our results add new information supporting the importance of recurrent amino acid changes at particular positions, contributing to refine the molecular characterization of our cohort and, reinforcing the role of the genetic background and environmental factors in CLL patients.
Keyword(s): Chronic lymphocytic leukemia, Immunoglobulin gene, Mutation status, Somatic hypermutation