Status of the Immunoglobulin Heavy Chain and Light Chain Genes in Chronic Lymphocytic Leukemia and Related Disorders

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4161-4161
Author(s):  
Hirotaka Nakahashi ◽  
Masamitsu Karasawa ◽  
Akio Saito ◽  
Kohtaro Toyama ◽  
Takeki Mitsui ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Light Chain ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Antigen Receptors ◽  
Asian Countries ◽  
Western Countries ◽  
Ighv Gene

Abstract The incidence of chronic lymphocytic leukemia (CLL) is low in Asian countries including Japan, while it is the most common type of leukemia in western countries. It has been evident that the immunoglobulin heavy chain variable region (IGHV) gene mutation status can predict the prognosis in CLL; unmutated IGHV genes correlate with a worse prognosis than mutated genes. Over-representation of selected IGHV genes is noted in western CLL, in particular IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-21. Although their relative frequencies vary between cohorts, the most frequent gene in western countries is IGHV1-69, which is found in about 10–20% of all CLL patients. Several studies have shown very unusual Ig characteristics in CLL: highly restricted IGHV gene usage and very similar antigen-binding sequences (stereotyped antigen receptors), suggesting a role for antigen selection during the development and maintenance of the disease. For the purpose of clarifying the characteristics of CLL in the Japanese population, we analyzed both IGHV and Ig light chain (κ-chain, IGK and λ-chain, IGL) genes in 81 CLL cases and compared the findings with cases of 52 leukemic chronic lymphoproliferative disorders (CLPD) including 6 hairy cell leukemia (HCL), 1 prolymphocytic leukemia (PLL), 31 indolent lymphoma in leukemic phase (15 mantle cell lymphoma (MCL), 7 follicular lymphoma (FL), 5 splenic marzinal zone lymphoma (SMZL), and 4 lymphoplasmacytic lymphoma (LPL)) and 14 cases that could not be classified further. Of the 81 Japanese CLL patients, 17 (21.3%) had unmutated IGHV, and 63 (78.7%) had mutated IGHV. The number of CLL with mutated IGHV was at a higher frequency compared to previous reports from western countries. It may be partly explained by the fact that the commonly unmutated IGHV1-69 type was rare (1.2%), but the commonly mutated IGHV4-34 type was frequent (27.2%) in the Japanese CLL patients. We previously reported that IGHV1-69 CLL is rare in Japan (1/44), which is confirmed by the present study of newly diagnosed cases (0/37). Moreover, only 1 of 65 CLL patients was reported to use IGHV1-69 in China. These findings suggest that IGHV1-69 is extremely rare in Asia. Similar to reports from Scandinavian countries, IGHV3-21 cases showed biased λ-chain expression (5/6), but were not associated with overuse of IGLV3-21 (V2-14) in our cohort. Recently, studies of B-cell antigen receptors (BCRs) in patients with CLL identified that subsets of cases expressed almost identical BCRs. We also found a pair of CLL patients who had the same IGHV4-39, IGHD6-13, IGHJ5 (heavy chain) and IGKV1-39 (O12), IGKJ1 or 2 segment with remarkably similar H and L CDR3 sequences. The use of IGHV, IGKV and IGLV was significant different when compared between CLL and leukemic CLPD. IGHV4-34, which was the most preferentially used in CLL patients (21/81, 26.0%), was used rarely in CLPD patients (4/52, 7.7%, p = 0.007). Of the 4 CLPD patients with IGHV4-34, 3 were MCL (CD5+) and 1 was unclassified CLPD (CD5 −). As leukemic cells of all CLL cases were CD5+, only 1 of the 25 patients using IGHV4-34 had CD5 negative cells. In normal B-cell development, naive IGHV4-34 B-cells are positively selected and mostly restricted to the follicular mantle zone but these cells are largely excluded from the germinal centers. This mechanism may be relevant to IGHV4-34 usage being underrepresented in CLPD other than MCL, which mainly consisted of GC- or post-GC-derived lymphomas/leukemias. In CLPD patients, only 1 patient with SMZL used the IGHV1-69 gene. Interestingly, IGHV1-69 was associated with IGHD5-24, IGHJ3, IGKV3-20 and IGLKJ1, which have been previously identified to comprise one of the stereotypical BCR gene subsets in patients with CLL. In the CLL patients, IGKV3-11 (L6) and IGLV3-21 (V2-14) were the most frequent IGKV (7/43) and IGLV (11/35), respectively. However, in the CLPD patients, IGKV3-11 and IGLV3-21 were used by none (0/26, p = 0.03) and only 1 MCL patient (1/22, p = 0.002), respectively. To date little data has been obtained on CLL in Japan and other Asian countries, where the susceptibility to CLL is very low. Thus, it is important to investigate genetic and environmental differences between Asian and western countries to identify risk factors that give rise to this disease. In addition, a comparison of the disease features of CLL with other lymphoproliferative disorders will further elucidate the clinical and pathogenetic characteristics of CLL.

The Repertoire of Heavy Chain Immunoglobulin Genes in B-Cell Chronic Lymphocytic Leukemia in Russia and Belarus

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4579-4579
Author(s):  
Bella Biderman ◽  
Eugene Nikitin ◽  
Tatiana Sergienko ◽  
Alexandra Bakun ◽  
Irina Taras ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Lymphocytic Leukemia ◽  
Antigen Receptors ◽  
Mutation Status ◽  
The Uk ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 4579 Introduction. Mutation status of the heavy chain variable region genes is known as an important factor in long-term prognosis in B-cell chronic lymphocytic leukemia (B-CLL). A more detailed study of the gene sequences of immunoglobulin heavy chain (IgVH) led to the discovery of stereotyped antigen receptors (SAR) - receptors that have the same set of VH-, D- and JH- genes used. SARs have been found almost in a quarter of all B-CLL cases. Since this study there have been no data available concerning VH-gene usage and SARs in Russian and Belarusian B-CLL patients. Patients and methods. DNA or cDNA was amplified in 6 separated reactions using primers specific for VH-families, and a consensus JH primer [Campbell et al. 1992] or primer sets recommended by the BIOMED-2 [van Dongen et al. 2003]. PCR products were sequenced using family-specific primers and Big Dye Terminator v3.1 kit (Applied Biosystems). Sequences were analyzed with IgBlast (http://www.ncbi.nlm.nih.gov/igblast). 98% homology cutoff was used to discriminate between mutated and unmutated cases. Results. Total of 547 patients with B-CLL where analyzed; 192 patients with mutated IgVH-genes (35%) and 355 patients with unmutated ones (65%). We have identified 65 stereotyped receptors (SARs) in 198 of 491 Russian patients (40%). Twenty one SARs (confirmed) appeared in more than 3 patients (110 out of 198, 55%), 44 SARs (potential) found in 2 patients (88 out of 198 cases, 45%). The vast majority of confirmed SARs were found in the subgroup of patients without mutations in IgVH genes (95%). Among the potential SARs 15 pairs were from patients with and without mutations (34%), seven pairs from patients with mutations (16%) and 22 pairs from patients without IgVH mutations (50%). The most common SARs were: VH1–69/D3-3/JH6 (24 patients, 5%); VH1–69/D3–16/JH3 (8 patients); VH1–69/D2-2/JH6 (8 patients); VH1–69/D3–10/JH6 (6 patients). Among 56 Belarusian patients we have identified only 4 SARs, one confirmed SAR (4 cases) and 3 potential ones. Confirmed (VH1–69/D3-3/JH6) and one potential (VH1–69/D3–16/JH3) were also found in Russia while other two potential (VH1–69/D2–15/JH6 and VH2–5/D2-2/JH6) were not observed in Russia. Discussion. In Russia and Belarus, VH1–69 gene is found in 20% of all cases of B-CLL, and almost always (95%) in unmutated cases. This finding well correlates to the data obtained by [Kryachok et al. 2012] concerning high VH1–69 usage in Ukrainian patients. In other European countries, this gene is less common: about 14% of cases in Sweden, France and Spain, 11% in the UK and about 6–7% in Greece and Italy [Ghia et al. 2005; Tobin et al. 2004; Duke et al. 2003]. In all these countries, this gene is also prevalent in patients with unmutated VH-genes. Nordic countries are characterized by very frequent use of gene VH3–21 - 9%, while in Russia and the countries of central and southern Europe, it occurs at least 3 times less (about 3%), and was not found in Belarusian samples. This gene is also associated with poor prognosis of B-CLL, regardless of mutation status of IgVH genes. Interestingly, in Asia (China, Japan, Iraq) VH1–69 and VH3–21 genes are almost not observed [Nakamura et al. 1999; Farsangi et al. 2007; Lijuan Chen et al. 2008]. Narrowing of the repertoire of IgVH genes - specific feature of B-CLL indicatites that influence of antigen (at least in some cases) occurs during the development of the disease. Also, factors of genetic background as well as geographical environment could be important. It is possible that future treatment decisions will be based not only on the IgVH mutation status, but also on the characteristics of certain antigen receptor. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Therapeutic Targets in Chronic Lymphocytic Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3259
Author(s):  
Luca Laurenti ◽  
Dimitar G. Efremov
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Therapeutic Targets ◽  
Common Type ◽  
Lymphocytic Leukemia ◽  
Western Countries ◽  
B Cell Malignancy ◽  
Adult Leukemia ◽  
Cell Malignancy

Chronic lymphocytic leukemia (CLL) is a common B cell malignancy and is the most common type of adult leukemia in western countries [...]

Stereotyped patterns of somatic hypermutation in subsets of patients with chronic lymphocytic leukemia: implications for the role of antigen selection in leukemogenesis

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1524-1533 ◽  
Author(s):  
Fiona Murray ◽  
Nikos Darzentas ◽  
Anastasia Hadzidimitriou ◽  
Gerard Tobin ◽  
Myriam Boudjogra ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Germ Line ◽  
Somatic Hypermutation ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
Distinctive Features ◽  
Ighv Gene ◽  
Gene Usage

Abstract Somatic hypermutation (SHM) features in a series of 1967 immunoglobulin heavy chain gene (IGH) rearrangements obtained from patients with chronic lymphocytic leukemia (CLL) were examined and compared with IGH sequences from non-CLL B cells available in public databases. SHM analysis was performed for all 1290 CLL sequences in this cohort with less than 100% identity to germ line. At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged from the analysis of certain subgroups of CLL sequences defined by: (1) IGHV gene usage, (2) presence of stereotyped heavy chain complementarity-determining region 3 (HCDR3) sequences, and (3) mutational load. Recurrent, “stereotyped” amino acid changes occurred across the entire IGHV region in CLL subsets carrying stereotyped HCDR3 sequences, especially those expressing the IGHV3-21 and IGHV4-34 genes. These mutations are underrepresented among non-CLL sequences and thus can be considered as CLL-biased. Furthermore, it was shown that even a low level of mutations may be functionally relevant, given that stereotyped amino acid changes can be found in subsets of minimally mutated cases. The precise targeting and distinctive features of somatic hypermutation (SHM) in selected subgroups of CLL patients provide further evidence for selection by specific antigenic element(s).

scholarly journals Production of tumor necrosis factor-alpha by B-cell chronic lymphocytic leukemia cells: a possible regulatory role of TNF in the progression of the disease

Blood ◽  
1990 ◽  
Vol 76 (2) ◽  
pp. 393-400 ◽  
Author(s):  
R Foa ◽  
M Massaia ◽  
S Cardona ◽  
AG Tos ◽  
A Bianchi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tumor Necrosis Factor ◽  
B Cell ◽  
Tumor Necrosis ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Necrosis Factor Alpha ◽  
Factor Alpha ◽  
Necrosis Factor ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Tumor necrosis factor-alpha (TNF) is a cytokine that displays a pleomorphic array of effects on different cell populations. Evidence is presented that TNF may be constitutively produced by B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL) cells and that it may play a relevant role in these diseases. These conclusions are based on the presence of circulating levels of TNF in the serum of 20 of the 24 patients tested (83.3%), while undetectable values were found in normal sera. The suggestion that the increased serum levels were due to the leukemic cell population is strengthened by the evidence that purified B-CLL and HCL cells may constitutively release variable degrees of TNF. These levels markedly increase after incubation with interferon gamma or phytohemagglutinin (PHA) plus phorbol myristate acetate (PMA). The cellular release of TNF by primary B-CLL cells was significantly (P less than .001) higher in B-CLL stage O-I patients compared with stage II-III patients. The demonstration that, in B-cell chronic lymphoproliferative disorders, the pathologic cells may release TNF was further confirmed by the presence of the mRNA for this cytokine in primary and/or in pre-activated cells. Recombinant TNF was capable of inducing a proliferative signal only in a minority of cases (4/24); in most cases it was ineffective, and, in a few, it reduced the degree of proliferation. Furthermore, in costimulatory experiments with interleukin-2 and PHA plus PMA, TNF was ineffective. On the other hand, when primary B-CLL cells were incubated in the presence of an anti-TNF antibody, in 8 of 12 independent experiments a 2- to 15-fold increase in thymidine uptake was documented. Taken together, these results suggest that TNF may play a regulatory role in the progression of the neoplastic clone in B-cell chronic lymphoproliferative disorders and may be implicated in some of the side effects associated with these diseases.

CD5+ B-cell lymphoproliferative disorders: Beyond chronic lymphocytic leukemia and mantle cell lymphoma

2010 ◽  
Vol 34 (9) ◽  
pp. 1235-1238 ◽  
Author(s):  
Dragan Jevremovic ◽  
Roxana S. Dronca ◽  
William G. Morice ◽  
Ellen D. McPhail ◽  
Paul J. Kurtin ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mantle Cell Lymphoma ◽  
Cell Lymphoma ◽  
Lymphoproliferative Disorders ◽  
Lymphocytic Leukemia ◽  
Mantle Cell

Serum BAFF (B-Cell Activating Factor of the TNF Family) Compared with Immunoglobulin Heavy-Chain Mutation Status, ZAP-70 and CD38 as a Predictor of Time to First Treatment in Early B-Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3093-3093
Author(s):  
Stefano Molica ◽  
Gaetano Vitelli ◽  
Giovanna Cutrona ◽  
Giovanna Digiesi ◽  
Rosanna Mirabelli ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Immunoglobulin Heavy Chain ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
B Cell Activating Factor ◽  
Mutational Status ◽  
Time To First Treatment ◽  
Cell Chronic Lymphocytic Leukemia

Abstract We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently patients with Rai stage 0 (P=0.008) and mutated IgVH (P=0.03). In contrast, peripheral blood lymphocytosis (P=0.06), serum β2-m (P=0.159), LDH (P=0.333) and percentage of ZAP-70-positive (P=0.242) or CD38-positive B-CLL cells (P=0.142) did not reflect circulating levels of BAFF. The relationship among various bio-pathological parameters, analyzed by the multiple correspondence analysis (MCA), showed two different clinico-biological profiles. The first, characterized by higher BAFF serum levels (i.e., > 336 ng/mL), presence of mutation in the IgVH, low percentage of CD38-positive B-CLL cells (< 30%) and low LDH was associated with a stable pattern of disease generally not requiring therapy. The second, defined by lower BAFF levels, absence of mutation in the IgVH, high percentage of CD38- positive B-CLL cells and high LDH was associated with a more progressive pattern of disease and a shorter TFT. After a median follow-up time of 35 months (range, 2–120 months) 26 (37.6%) out of 69 patients experienced a need for chemotherapy. Kaplan-Meier estimates of patientsTFT, plotted after searching the best cut-off for BAFF (i.e., 336 ng/mL), demonstrated that low BAFF concentration was associated with a shorter TFT (median TFT 36 months) while median was not reached by patients with BAFF levels higher than 336 ng/mL (P<0.0001). Along with lower serum levels of BAFF (Hazard Ratio [HR], 0.19; P<0.0001), the univariate Cox proportional hazard model identified absence of mutation in IgVH (HR, 0.17; P<0.0001), CD38-positivity (HR, 3.32; P=0.01) and lower platelet count (HR, 0.19; P=0.03) as predictor of shorter TFT. Finally, in multivariate analysis only mutational status of IgVH (HR, 0.25; P=0.007) and serum concentration of BAFF (HR, 034; P=0.04) affected significantly TFT. Our results indicate that in early B-cell CLL clinico-biological profile including among other parameters BAFF may provide a useful insight into the complex interrelationship of prognostic variables and semplify their interpretation. The possible presence of BAFF isoform in B-CLL could peraphs account for the unexpected correlation between low soluble BAFF levels and poor clinical outcome in patients with early disease.

Usage of IGHV4-39 with Stereotypic B Cell Receptor Is An Independent Risk Factor of Chronic Lymphocytic Leukemia Transformation to Richter Syndrome

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 778-778
Author(s):  
Davide Rossi ◽  
Valeria Spina ◽  
Michaela Cerri ◽  
Clara Deambrogi ◽  
Lorenzo De Paoli ◽  
...  
Keyword(s):  
Multivariate Analysis ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Control Analysis ◽  
Ighv Gene ◽  
Gene Usage

Abstract Richter’s syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL) to aggressive lymphoma, mainly occurring as diffuse large B-cell lymphoma (DLBCL). The biology of CLL transformation to RS is poorly understood and knowledge on risk factors of RS development is scant. We tested whether IGHV gene usage and stereotypic B cell receptor (BCR) at CLL diagnosis have an impact on RS transformation. The first step of the study consisted of a case-control analysis comparing IGHV gene usage and prevalence of stereotypic HCDR3 in RS (n=69; all DLBCL) versus a control group (n=715) of CLL that had not transformed to RS. The second step consisted of an actuarial assessment of the impact of IGHV gene usage and stereotypic HCDR3 at CLL diagnosis, on the risk of subsequent transformation to RS in a cohort of 754 CLL, of which 39 had transformed to RS. Comparison of IGHV usage in unmutated RS versus unmutated control CLL documented that IGHV4-39 was the sole gene preferentially utilized (6/48, 12.5% vs 5/277, 1.8%, respectively, p=.002) by RS. Prevalence of stereotypic HCDR3 was significantly higher in RS compared to non-transformed CLL when considering all cases (RS: 50.7% vs non-transformed CLL: 22.2%; p<.000001), unmutated cases only (RS: 58.3% vs non-transformed CLL: 35.7%; p=.003), and mutated cases only (RS: 33.3% vs non-transformed CLL: 13.7%; p=.022). Compared to non-transformed CLL, RS preferentially utilized BCR belonging to a subset characterized by rearrangement of unmutated IGHV4-39/IGHD6-13/IGHJ5 genes (2/159, 1.2% vs 5/35, 14.3%, respectively; p=.002). All cases with stereotypic IGHV4-39 carried +12 as the sole FISH abnormality. After a median follow-up of 41.1 months, 39/754 CLL had transformed to RS. Univariate analysis documented: shorter time to transformation in CLL utilizing IGHV4-39 (5-year risk: 35.4%) compared to CLL utilizing other IGHV genes (5-year risk: 5.6%) (p<.000001); higher risk of RS in CLL utilizing stereotypic HCDR3 (5-year risk: 14.2%) compared to CLL without stereotypic HCDR3 (5-year risk: 3.9%) (p<.00001). CLL with stereotypic HCDR3 and IGHV homology 98% showed a significantly higher risk of transformation (5-year risk: 18.4%) compared to CLL with IGHV homology 98% but without stereotypic HCDR3 (5-year risk: 6.8%) (p=.006). Also, stereotypic HCDR3 identified a CLL subgroup that, despite presenting with IGHV homology <98%, showed an increased risk of RS (p=.040). This observation indicates that stereotypic HCDR3 is not a surrogate of IGHV homology for RS prediction. We then tested the independent predictive value for RS transformation of IGHV4-39 usage and of stereotypic HCDR3. Multivariate analysis selected IGHV4-39 usage (HR: 4.25; p=.002) and stereotypic HCDR3 at CLL diagnosis (HR: 3.08; p=.002) as independent predictors of RS transformation. The observation that all RS utilizing IGHV4-39 carried stereotypic HCDR3 prompted investigation of the interaction between IGHV4-39 usage and stereotypic HCDR3 in the model. Multivariate analysis selected the interaction between IGHV4-39 usage and stereotypic HCDR3 at CLL diagnosis as the strongest independent predictor of RS transformation (HR: 5.13; p=.001). The relevance of the interaction between IGHV4-39 and stereotypic HCDR3 was confirmed by bivariate log rank analysis. Accordingly, CLL utilizing both IGHV4-39 and stereotypic HCDR3 were identified as the disease category with highest risk of transformation (5-year risk: 68.7%). Transformation to RS and progression to symptomatic disease according to NCI Working Group guidelines are distinct events in CLL. Accordingly, neither IGHV4-39 usage nor stereotypic HCDR3 affected the risk of CLL progression occurring without transformation to RS. IGHV4-39 usage and stereotypic HCDR3 may be appropriate biological markers for RS prediction since: these markers predict RS in a fashion that is independent of other clinical and biological features; given the widespread use of IGHV sequencing for CLL prognostication, information on IGHV4-39 and stereotypic HCDR3 may be obtained at CLL diagnosis without additional testing; and importantly all CLL with concomitant IGHV4-39 usage and stereotypic HCDR3 ultimately transform to RS. A close monitoring and a careful biopsy policy may be of help for early recognition of RS transformation in patients carrying IGHV4-39 usage and stereotypic HCDR3.

Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2337-2337
Author(s):  
Lesley-Ann Sutton ◽  
Efterpi Kostareli ◽  
Anastasia Hadzidimitriou ◽  
Nikos Darzentas ◽  
Athanasios Tsaftaris ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
B Cell Receptors ◽  
Valuable Insight ◽  
Cell Receptors ◽  
Mutational Status ◽  
The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

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