Clinical significance of productive immunoglobulin heavy chain gene rearrangements in childhood acute lymphoblastic leukemia
Κaterina KatsibardiMaria BraoudakiChrissa PapathanasiouKalliopi KaramolegouFotini Tzortzatou‐Stathopoulou
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We analyzed the CDR3 region of 80 children with B-cell acute lymphoblastic leukemia (B-ALL) using the ImMunoGeneTics Information System and JOINSOLVER. In total, 108 IGH@ rearrangements were analyzed. Most of them (75.3%) were non-productive. IGHV@ segments proximal to IGHD–IGHJ@ were preferentially rearranged (45.3%). Increased utilization of IGHV3 segments IGHV3-13 (11.3%) and IGHV3-15 (9.3%), IGHD3 (30.5%), and IGHJ4 (34%) was noted. In pro-B ALL more frequent were IGHV3-11 (33.3%) and IGHV6-1 (33.3%), IGHD2-21 (50%), IGHJ4 (50%), and IGHJ6 (50%) segments. Shorter CDR3 length was observed in IGHV@6, IGHD7, and IGHJ1 segments, whereas increased CDR3 length was related to IGHV3, IGHD2, and IGHJ4 segments. Increased risk of relapse was found in patients with productive sequences. Specifically, the relapse-free survival rate at 5 years in patients with productive sequences at diagnosis was 75% (standard error [SE] ±9%), whereas in patients with non-productive sequences it was 97% (SE ±1.92%) (p-value = 0.0264). Monoclonality and oligoclonality were identified in 81.2% and 18.75% cases at diagnosis, respectively. Sequence analysis revealed IGHV@ to IGHDJ joining only in 6.6% cases with oligoclonality. The majority (75%) of relapsed patients had monoclonal IGH@ rearrangements. The preferential utilization of IGHV@ segments proximal to IGHDJ depended on their location on the IGHV@ locus. Molecular mechanisms occurring during IGH@ rearrangement might play an essential role in childhood ALL prognosis. In our study, the productivity of the rearranged sequences at diagnosis proved to be a significant prognostic factor.Keywords:
IGHV@
Immunoglobulin heavy chain
IGHD
Gene rearrangement
clone (Java method)
Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.
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We analyzed the CDR3 region of 80 children with B-cell acute lymphoblastic leukemia (B-ALL) using the ImMunoGeneTics Information System and JOINSOLVER. In total, 108 IGH@ rearrangements were analyzed. Most of them (75.3%) were non-productive. IGHV@ segments proximal to IGHD–IGHJ@ were preferentially rearranged (45.3%). Increased utilization of IGHV3 segments IGHV3-13 (11.3%) and IGHV3-15 (9.3%), IGHD3 (30.5%), and IGHJ4 (34%) was noted. In pro-B ALL more frequent were IGHV3-11 (33.3%) and IGHV6-1 (33.3%), IGHD2-21 (50%), IGHJ4 (50%), and IGHJ6 (50%) segments. Shorter CDR3 length was observed in IGHV@6, IGHD7, and IGHJ1 segments, whereas increased CDR3 length was related to IGHV3, IGHD2, and IGHJ4 segments. Increased risk of relapse was found in patients with productive sequences. Specifically, the relapse-free survival rate at 5 years in patients with productive sequences at diagnosis was 75% (standard error [SE] ±9%), whereas in patients with non-productive sequences it was 97% (SE ±1.92%) (p-value = 0.0264). Monoclonality and oligoclonality were identified in 81.2% and 18.75% cases at diagnosis, respectively. Sequence analysis revealed IGHV@ to IGHDJ joining only in 6.6% cases with oligoclonality. The majority (75%) of relapsed patients had monoclonal IGH@ rearrangements. The preferential utilization of IGHV@ segments proximal to IGHDJ depended on their location on the IGHV@ locus. Molecular mechanisms occurring during IGH@ rearrangement might play an essential role in childhood ALL prognosis. In our study, the productivity of the rearranged sequences at diagnosis proved to be a significant prognostic factor.
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Immunoglobulin heavy chain
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Gene rearrangement
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Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing 'truly unmutated' IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a 'truly unmutated' CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3'IGHV, IGHD, 5'IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying 'truly unmutated' IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.
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Background:In vivo Class Switch recombination (CSR) has previously been reported for a fraction of patients with chronic lymphocytic leukemia (CLL) expressing unmutated IGHV genes (U-CLL) and high mRNA levels of activation-induced cytidine deaminase compared to patients expressing mutated IGHV genes (M-CLL). This observation is certainly intriguing, however inherently limited due to the low-sensitive, Sanger-based approach used for the immunogenetic characterization. Aims: Here, we sought to overcome the aforementioned limitation and obtain more insight into in vivo CSR in CLL using a highly-sensitive, next generation sequencing (NGS) methodology. Methods: We studied 73 patients (48 U-CLL, 25 M-CLL) sampled at diagnosis. Case selection was based on the dominant expression of the IgM isotype, as determined by flow cytometry. IGHV-IGHD-IGHJ gene rearrangements were RT-PCR amplified for the μ, γ, and α transcripts utilizing IGHV Leader and IGHC-specific primers. All obtained PCR products (n=215) were subjected to paired-end NGS. High-quality, productive IGHV-IGHD-IGHJ gene rearrangements (n=23,217,561; median= 97,932/sample) were annotated with IMGT/HighV-QUEST. Clonotypes were defined as rearrangement sequences expressing the same IGHV gene and identical complementarity-determining region 3 (VH CDR3) amino-acid (AA) sequence. Clonotype variants were defined as different nucleotide sequences clustered in the same clonotype. Results: The IGH gene repertoire of μ transcripts was clearly monoclonal in all cases, showing a dominant CLL clonotype (DCC) with a median frequency of 93.5% for either U-CLL or M-CLL. Subsequently, the IGH gene repertoires of γ and α transcripts were scanned for the presence of the DCC. In U-CLL, 46/48 cases expressed switched transcripts with median frequencies of 14% (range 0.01-97.2%) and 21.3% (range 0.1-95.3%) for γ and α transcripts, respectively. In M-CLL, 24/25 patients expressed switched transcripts albeit at significantly lower levels than U-CLL (p=0.01) (median frequencies: 0.2% and 13.6%; ranges 0.03-94% and 0.04-91.2% for the γ and α transcripts, respectively). Interestingly, within U-CLL, cases belonging to stereotyped subset #1 (n=21) and its satellite subset #99 (n=2) exhibited significantly higher levels of switched transcripts (median frequencies: 37.9% and 37.6% for the γ and α transcripts, respectively) than all other cases (p=0.04 for γ transcripts, p=0.002 for α transcripts). Analysis of somatic hypermutation (SHM) in the DCC within the repertoires of μ, γ and α transcripts of U-CLL cases revealed the presence of shared SHMs (even among cases with 100% IGHV gene identity) but also distinct SHMs shared by the γ and α clonotypic transcripts but not the IgM, indicative of common selection pressure post-CSR. Interestingly, M-CLL patients displayed less intraclonal diversity in the γ and α clonotypic transcripts, despite the significant burden of SHM within the IgM DCC. Summary/Conclusion: In conclusion, our data reveal that the B cell receptor IGH gene repertoire is highly dynamic and interconnected in both U-CLL and M-CLL, albeit considerably more pronounced in the former. This is especially the case for patients assigned to the clinically aggressive stereotyped subsets #1 and #99, prompting speculations regarding the precise nature of the respective affinity maturation process. Shared SHMs between clonotype variants only among clonotypic γ and α transcripts rather than the μ transcripts indicate that CSR likely precedes SHM, particularly in U-CLL.
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Immunoglobulin heavy chain
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Background: The mutational status of the variable region of the Immunoglobulin heavy-chain (IGHV) gene is one of the most robust prognostic markers in Chronic Lymphocytic Leukemia (CLL), allowing the identification of 2 groups with different clinical behavior. Those with no or few somatic mutations (unmutated CLL, UM-CLL) with a more aggressive course of their disease, and those with a heavier load of mutations (mutated, M-CLL), with a more indolent disease. European Research Initiative on CLL (ERIC) has defined clear guidelines for Immunoglobulin gene sequence analysis. Materials and Methods: Patients with CLL in our institution were studied for productive IGHV rearrangements and its mutational status. There were no exclusion criteria for the analysis of this prognostic marker. IGHV-D-J gene rearrangements and mutational status were analyzed between 6/2016 and 2/2019. Nucleic acid used was genomic DNA (gDNA) and ERIC guidelines were followed. High molecular weight DNA was isolated from all patients and amplified using forward Leader primers combined with JH reverse consensus primer. In those cases, in which leader primers were unsuccessful at providing a product that could be sequenced, VH FR1 primers were used. Direct bidirectional sequencing was performed and Stereotype B cell receptors were analyzed. Results: From 150 samples received and analyzed, 138 productive IGHV-IGHD-IGHJ rearrangements were identified, 12 patients had insufficient leukemic cells for the analysis. Of 138 patients 54 (39%) were UM-CLL and 84 (61%) were M-CLL. Within this last group, 12 were considered “borderline” M-CLL. The most frequent IGHV family in this series was IGHV3, followed by IGHV4 and IGHV1. Double rearrangements were detected in 16 of the cases. Stereotype B cell receptors were found in 16 patients. Conclusion: These results show a slightly higher incidence on M-CLL than the published data. We believe this is due to a high proportion of asymptomatic patients with no need of treatment, in this cohort. The most frequent used IGHV family was IGHV-3 while IGHV4 and IGHV1 were in second and third place, respectively. The frequency of Stereotyped BcRs was lower from those observed in western countries cohorts. These results support previous published experience in other Latin American countries. Longer follow up will be necessary to determine the impact of this molecular factor in the clinical behavior of this group of patients. Keywords: gene rearrangement; immunoglobulins (Ig).
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Survival of patients with B cell chronic lymphocytic leukemia (B-CLL) can be predicted by analysis of mutations in the immunoglobulin heavy chain variable gene (IGHV). Patients without mutations (unmutated [UM]) are at greater risk for disease progression and death than patients with mutations (M). Despite this broad prognostic difference, there remains wide intragroup variation in the clinical outcome of UM patients, especially those with low/intermediate Rai risk disease. We evaluated UM B-CLL patients with low/intermediate Rai risk to determine the relationship between IGHV, IGH diversity (IGHD), and IGH joining (IGHJ) gene usage and time to treatment (TTT). Irrespective of IGHV usage, UM patients whose B-CLL cells expressed the IGHD3-3 gene had a significantly shorter TTT than other UM B-CLL patients, and specifically, use of the IGHD3-3 gene in reading frame 2 (RF2) predicted shorter TTT. As expected, Rai risk was the best single prognostic factor for TTT; however, IGHD usage was also a significant variable for TTT. Therefore, both IGHD gene and IGHD RF usage have prognostic relevance in UM B-CLL patients with low/intermediate Rai risk disease. In addition, these data support the concept that antigen-driven selection of specific Ig receptors plays a role in the clinical course of B-CLL.
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ABSTRACT The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus . We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflect differences in subspecies origin. To investigate this hypothesis, we conducted high-throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ), and diversity (IGHD) genes in four inbred wild-derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ, and PWD/PhJ), and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild-derived strains, including 247 not curated in the International Immunogenetics Information System. In contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. In contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand inter-strain variation in models of antibody-mediated disease.
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