Characterizing Features of Human Circulating B Cells Carrying CLL-Like Stereotyped Immunoglobulin Rearrangements
Davide BagnaraMonica ColomboDaniele ReverberiSerena MatisRosanna MassaraNiccolò CardenteGianluca UbezioVanessa AgostiniLuca AgnelliAntonino NeriMartina CardilloStefano VerganiFabio GhiottoAndrea Nicola MazzarelloFortunato MorabitoGiovanna CutronaManlio FerrariniFranco Fais
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Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of monoclonal CD5+ B cells with low surface immunoglobulins (IG). About 40% of CLL clones utilize quasi-identical B cell receptors, defined as stereotyped BCR. CLL-like stereotyped-IG rearrangements are present in normal B cells as a part of the public IG repertoire. In this study, we collected details on the representation and features of CLL-like stereotyped-IG in the IGH repertoire of B-cell subpopulations purified from the peripheral blood of nine healthy donors. The B-cell subpopulations were also fractioned according to the expression of surface CD5 molecules and IG light chain, IGκ and IGλ. IG rearrangements, obtained by high throughput sequencing, were scanned for the presence of CLL-like stereotyped-IG. CLL-like stereotyped-IG did not accumulate preferentially in the CD5+ B cells, nor in specific B-cell subpopulations or the CD5+ cell fraction thereof, and their distribution was not restricted to a single IG light chain type. CLL-like stereotyped-IG shared with the corresponding CLL stereotype rearrangements the IGHV mutational status. Instead, for other features such as IGHV genes and frequency, CLL stereotyped-IGs presented a CLL-like subset specific behavior which could, or could not, be consistent with CLL stereotyped-IGs. Therefore, as opposed to the immuno-phenotype, the features of the CLL stereotyped-IG repertoire suggest a CLL stereotyped subset-specific ontogeny. Overall, these findings suggest that the immune-genotype can provide essential details in tracking and defining the CLL cell of origin.Keywords:
CD5
IGHV@
Surface Immunoglobulin
Immunophenotyping
Immunoglobulin heavy chain
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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IGHD
Gene rearrangement
clone (Java method)
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Summary. Background: A number of epidemiological studies have shown an elevated radiation-associated risk for chronic lymphocytic leukemia (CLL). The aim of the paper was to analyze immunoglobulin heavy variable chain (IGHV) rearrangement and IGHV usage in CLL cases associated with ionizing radiation (IR) exposure. Materials and Methods: Samples of 76 clean-up workers of Chornobyl Nuclear Power Plant accident of 1986 (the main group) and 194 non-exposed patients (the control group) were analyzed. Two groups of CLL patients were comparable by gender (all patients were male), age, and place of residence (rural or urban). Results: Some features of IR-associated CLL cases as compared to CLL cases in patients without history of IR exposure were revealed. Among unmutated IGHV sequences, IGHV1 genes were less commonly used (29.4% vs 48.6%; p = 0.018), while the frequency of IGHD6 genes was higher (23.5% vs 10%; p = 0.029). The unmutated IGHV sequences did not use IGHD3-16 gene (0% vs 7.9%, p = 0.038). Mutated IGHV sequences were less frequently expressed IGHV3 genes (44% vs 68.5%; p = 0.037) due low representation of IGHV3-21 (4% vs 11.1%) and IGHV3-23 (0% vs 11.1%) genes; did not use IGHD3-22 gene (0% vs 18.5%, p = 0.025); and have signs of positive selection in the HCDR regions (Σ = 0.5029 ± 0.155 vs –0.0539 ± 0.14; p = 0.013). Conclusions: The revealed differences in IGHV gene usage and B-cell receptor structure in the main and the control groups of CLL patients indirectly indicate a change in the spectrum of antigens associated with CLL under IR exposure. The possible antigenic drivers associated with CLL associated with IR exposure are discussed.
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Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research Background: Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B-cells that weakly express a B-cell receptor (BCR). The mutational status of the variable region (IGHV) on the immunoglobulin (Ig) heavy-chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLLs (mCLLs) are genetically imprinted by activation induced-cytidine deaminase (AID). In normal condition, AID is required for IGH rearrangements in activated B-cells: the class switch recombination (CSR) and the recombination between switch Mu (Sµ) and the 3’ regulatory region (3’RR) of the IGH locus (Sµ-3’RRrec) (Peron Science 2012). The fact that most CLL cells express an IgM raises the question of CSR blockade in this B-cell cancer. Low levels of CSR can be observed in a small fraction of CLL tumor cells and seems to correlate with AID expression in these intra-clonal switched CLL cells. AID has been repeatedly detected in CLL B-cells independently of the IGHV mutational status. AID likely contributes to CLL evolution and seems to generate intraclonal diversity targeting the IGH locus and non-Ig off-targets. Aims: In this study, we raised the question of IGH switch blockade in CLL. For that purpose, we analyzed both CSR and Sµ-3’RRrec junctions as reflections of putative switch activity. Methods: We analyzed counts of CSR and Sµ-3’RRrec in 47 DNA sample CLLs diagnosis high blood tumor infiltration (>90%). Results were compared to results from mononuclear cells from peripheral blood from 9 healthy volunteers (HVs) and from 24 pediatric tonsils. The project was conducted according to the guidelines of the Declaration of Helsinki. Results: Our results separated CLLs into two groups on the basis of Sµ-3’RRrec counts per sample, Sµ-3’RRrecHigh cases (mostly unmutated IGHV CLLs) and Sµ-3’RRrecLow cases (mostly mCLLs), but not based on CSR junction counts. Sµ-3’RRrec appeared to be ongoing in Sµ-3’RRrecHigh CLL cells and comparison of Sµ-3’RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3’RRrecHigh CLLs harbor a non- germinal center (GC) experienced B-cell imprint while Sµ-3’RRrecLow CLLs are from AID-experienced B-cells from a secondary lymphoid organ. Moreover, we observed Sµ-3’RRrecHigh patient homogeneously exhibited telomeres that were shorter than HVs, reflecting increased proliferation cycles, and increased MYC expression. Questioning the impact of c-MYC on IGH recombination, we used the murine B-cell lymphoma CH12F3 cell line and its AID knock-out counterpart stably transfected or not with a MYC overexpression vector and stimulated in vitro to undergo CSR and Sµ-3’RRrec. Results show that c-MYC potentiated both CSR and Sµ-3’RRrec when AID was expressed. Even at low frequency, Sµ-3’RRrec was also possible in the absence of AID, in the presence of c-MYC overexpression. Summary/Conclusion In addition to the proposals already made concerning the CLL cell of origin, our study highlights that the IGH recombinatory activity allows the separation of CLL cases from different origins: the CLL Sµ-3’RRrecLow group that would originate from GC-experienced B-cells and the Sµ-3’RRrecHigh CLLs with a probable non-GC experienced B-cell with different prognoses. Finally, on-going Sµ-3’RRrec in Sµ-3’RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3’RRrec, even in the absence of AID. Keywords: c-myc, IgH rearrangment, B cell, Chronic lymphocytic leukemia
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Immunoglobulin heavy chain
Gene rearrangement
Activation-induced (cytidine) deaminase
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Objective: To investigate the mutational features of the immunoglobulin heavy chain variable region (IgHV) gene in patients with chronic lymphocytic leukemia (CLL) using immunophenotypic and molecular genetic methods. Methods: The laboratory results of 266 CLL patients who underwent IgHV gene examination at Sino-US diagnostics laboratory from February 2020 to February 2021 were analyzed for the IgVH mutational status and presence of specific IgVH fragments. In addition, their immunophenotypic, molecular, chromosomal karyotypic, and FISH profiles were investigated and correlated with the IgVH mutational status. Results: Among 266 patients, 172 were male and 94 were female, with a media age of 67 years (20-82 years).There were more patients with mutated IgHV (m-IgHV) than unmutated IgHV (un-IgHV) (69.2%∶30.8%). There was association of VH family and the presence of gene fragments: the overall incidence of VH families including VH3 family (142/266, 53.4%), VH4 family (75/266, 28.2%), and VH1 family (34/266, 12.8%) was about 95%, among which the proportion of VH4-34 (26/266, 9.8%), VH3-23 (25/266, 9.4%), VH3-7 (24/266, 9.0%), and VH4-39 (16/266, 6.0%) was about 35%. VH3-20 and VH3-49 only occurred in un-IgHV (P<0.05). In addition, the expression rates of CD38 (26.3% vs. 3.0%), CD79b (71.1%∶45.5%) and 11q deletion (25.5%∶5.3%) were higher in un-IgHV, and single trisomy 12 (37.9%∶5.6%) were more commonly found in m-IgHV (P<0.05). MYD88 was one of the major mutation genes in m-IgHV, while ATM had the highest mutation rate in un-IgHV. Conclusion: CLL patients have differential expression in terms of IgHV gene mutations, correlating to their immunophenotype and genetics characteristics.目的： 探讨慢性淋巴细胞白血病（CLL）患者的免疫球蛋白重链可变区（IgHV）突变和未突变患者的实验室检查特点。 方法： 对2020年2月至2021年2月天津见康华美医学诊断中心266例行IgHV基因检测的CLL初诊患者的实验室检查结果进行总结，分析IgHV基因突变状态、基因片段的使用特征；对突变与未突变患者的免疫表型、基因突变筛查、染色体核型及荧光原位杂交（FISH）结果进行差异性比较。 结果： 患者男性172例，女性94例，中位年龄67岁（20~82岁）。（1）IgHV突变患者比例高于IgHV未突变患者（69.2%∶30.8%）。（2）VH家族以及基因片段的使用均存在明显的偏颇性：VH3家族（142/266，53.4%）、VH4家族（75/266，28.2%）和VH1家族（34/266，12.8%）的发生率总和约为95.0%；具体到基因片段，使用VH4-34（26/266，9.8%）、VH3-23（25/266，9.4%）、VH3-7（24/266，9.0%）、VH4-39（16/266，6.0%）的比例总和约为35.0%，其中VH3-20片段和VH3-49片段仅发生于IgHV未突变患者（P<0.05）。（3）IgHV未突变患者中CD38（26.3%∶3.0%）和CD79b（71.1%∶45.5%），以及11q缺失（25.5%∶5.3%）检出率较高，IgHV突变患者中单独的第12号染色体三体（37.9%∶5.6%）常见（P<0.05）。MYD88为IgHV突变患者的主要突变基因之一，而ATM在IgHV未突变患者突变率最高。 结论： CLL患者IgHV基因具有独特的表达特点，突变和未突变CLL患者的免疫表型、分子生物学和遗传学检查结果具有一定规律。.
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Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sμ) and the 3’ regulatory region (3’RR) (Sμ-3’RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sμ-3’RRrec counts per sample: Sμ-3’RRrecHigh cases (mostly unmutated CLL) and Sμ-3’RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sμ-3’RRrec appeared to be ongoing in Sμ-3’RRrecHigh CLL cells and comparison of Sμ-3’RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sμ-3’RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sμ-3’RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sμ-3’RRrec in Sμ-3’RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sμ-3’RRrec, even in the absence of AID for the latter.
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Immunoglobulin heavy chain
Gene rearrangement
Activation-induced (cytidine) deaminase
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Актуальность. К настоящему времени известно, что в патогенезе хронического лимфоцитарного лейкоза (ХЛЛ) важную роль играют генетические нарушения. Их выявление может быть использовано для оценки прогноза заболевания, что является актуальной задачей, поскольку по клиническому течению ХЛЛ крайне разнороден. Цель исследования. Изучить молекулярно-цитогенетические нарушения в локусе IGH у больных ХЛЛ и их сочетание с нарушениями, выявленными при использовании стандартной для ХЛЛ цитогенетической панели. Материалы и методы. Исследование выполнено методом интерфазной in situ гибридизации (I-FISH) на архивных образцах нестимулированных лимфоцитов 89 больных с впервые выявленным ХЛЛ и 15 клинически здоровых доноров в качестве контрольной группы. Результаты. Нарушения в локусе тяжелых цепей иммуноглобулинов обнаружены у 64% больных. IGH транслокации выявлены у 19% больных, частичная/полная потеря одной копии IGHV региона - у 58% больных ХЛЛ. Мы разделили больных на 3 группы и оценили встречаемость в них нарушений в локусе 14q32. В группу неблагоприятного прогноза вошли 35 человек (16 больных с del17p13 и 19 больных с del11q22), в группу промежуточного прогноза вошли 18 человек (10 больных с трисомией хромосомы 12 и 8 больных без выявленных цитогенетических нарушений, и в группу благоприятного прогноза - 36 больных с del13q14 как единственной. Статистически значимых различий между группами прогноза по встречаемости больных с IGH транслокациями не выявлено. Установлено, что у больных с del17p13 и с del11q22 IGHV делеции встречались достоверно чаще, чем у больных с del13q14 как единственной (74% vs 44%, p = 0,011). В группах неблагоприятного и промежуточного прогноза по сравнению с группой благоприятного прогноза почти в 4 раза (40% и 39% vs 11% соответственно) больше больных с высокой (50% и выше) долей клеток с IGHV делециями (p < 0,05). Заключение. Нарушения в IGH локусе занимают второе место по встречаемости среди нарушений, выявляемых методом FISH при ХЛЛ. Эти нарушения могут быть значимыми, поскольку они достоверно чаще встречаются у больных в группе неблагоприятного прогноза. Дальнейшее наблюдение за больными c нарушениями в IGH локусе позволит оценить их клинико-биологические особенности и прогностическую значимость при ХЛЛ. Возможно, гетерогенность течения заболевания внутри группы больных с благоприятным прогнозом, выделенной на основании иерархической модели, обусловлена наличием дополнительных цитогенетических нарушений, в том числе и нарушений в IGH локусе. Background. Genetic disorders play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Identification of these disorders can be used for prognosis of the disease. This is an urgent task since the clinical course of CLL is extremely heterogeneous. The aim of this study was to explore cytogenetic aberrations of the immunoglobulin heavy chain (IGH) locus and their association with a common FISH panel in patients with chronic lymphocytic leukemia (CLL). Materials and methods. The study was performed by a retrospective analysis of samples of non-stimulated lymphocytes from 89 patients with newly diagnosed CLL and 15 clinically healthy donors as a control group using interphase fluorescence in situ hybridization (I-FISH). Results. Rearrangements of the IGH locus were found in 64% of CLL patients. IGH translocations were observed in 19% of patients whereas 58% of patients had total or partial IGHV deletions. The patients were divided into three groups and evaluated for occurrence of aberrations in the locus 14q32. The group of unfavorable prognosis included 35 patients (16 patients with del17p13 and 19 patients with del11q22); the group of intermediate prognosis included 18 patients (10 patients with chromosome 12 trisomy and 8 patients without identified aberrations); and the group of favorable prognosis consisted of 36 patients with del13q14 as the sole abnormality. There were no statistically significant differences between groups with different prognosis in the occurrence of patients with IGH translocations. IGHV deletions were significantly more frequent in patients with del17p13 and del11q22 than in patients with del13q14 as the sole abnormality (74% vs. 44%, p=0.011). Patients with a high (50% and more) proportion of cells with IGHV deletions occurred approximately 4 times more frequently in groups of unfavorable and intermediate prognosis compared to the group of favorable prognosis (40% and 39% vs. 11%, respectively, p<0.05). Conclusion. Rearrangements of the IGH locus detected using the FISH method is the second among all aberrations in CLL. These abnormalities may be important as they are significantly more frequently observed in patients with an unfavorable prognosis. Further observation of CLL patients with IGH aberrations will allow to identify clinical and biological features and prognostic importance of these aberrations. Probably, the intergroup heterogeneity of the clinical course in patients with a favorable prognosis determined on a hierarchical model is due to additional cytogenetic aberrations including IGH locus abnormalities.
IGHV@
Immunoglobulin heavy chain
Trisomy
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IGHV@
CD5
clone (Java method)
Immunoglobulin heavy chain
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CD5
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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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Immunoglobulin heavy chain
IGHD
genomic DNA
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IGHV@
Immunoglobulin heavy chain
Primer (cosmetics)
Gene rearrangement
Multiplex
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