Analysis of Stereotyped IGHV Distribution In a Series of 1133 Chronic Lymphocytic Leukemia Patients: The Experience of a Multicenter Italian Study Group

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2423-2423
Author(s):  
Francesco Maura ◽  
Giovanna Cutrona ◽  
Massimo Gentile ◽  
Serena Matis ◽  
Monica Colombo ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Pairwise Alignment ◽  
Immunoglobulin Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Study Group ◽  
Data Set ◽  
Italian Study ◽  
Mutational Status

Abstract Abstract 2423 Chronic lymphocytic leukemia (CLL) is characterized by an extremely variable clinical course. Mutational status of the immunoglobulin heavy-chain variable (IGHV) region defines two disease subsets with different prognosis. A fraction of CLL cases carries highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes and heavy-chain complementarity determining region-3 (HCDR3). We performed sequence analysis to characterize IGHV regions in a panel of 1133 CLL patients investigated by a multicenter Italian study group. A total of 1148 rearrangements were identified; the analysis of stereotyped subsets was performed based on previously reported criteria (Messmer et al, J Exp Med 2004; Stamatopuolos et al, Blood 2007). Specifically, we compared all our sequences with those found in three different publicly available data sets (Stamatopoulos et al, Blood 2007; Murray et al, Blood 2008 and Rossi et al, 2009 Clin Cancer Res). In addition, a pairwise alignment within all sequences was performed in order to discover novel potential subsets (HCDR3 identity > 60%). Based on the 2% cut-off used to discriminate between Mutated (M) and Unmutated (UM) cases, 777 sequences (67.59%) were classified as M, while 371 sequences (32.3%) as UM. The most represented IGHV genes within mutated cases were IGHV4-34 (104/118) and IGHV3-23 (85/96), whereas IGHV1-69 (97/112) was the most frequently used in the UM group. Interestingly, the IGHV3-21 gene, reported to be frequently expressed in CLL patients from Northern Europe, was present in only a small fraction of cases (24; 2.07%), confirming a previous finding reported by Ghia et al (Blood 2005) in a smaller panel. In our series, stereotyped HCDR3 sequences were found in 407/1148 (35.45%) patients, 177 of whom were M and 230 were UM cases. Overall, we observed that stereotyped sequences were significantly associated with UM IGHV status (Fisher's exact test, P<0.0001). Among the 407 stereotyped HCDR3 sequences, 345 belong to the clusters reported by Murray et al and 14 to those described by Rossi et al., 2009 Clin Cancer Res. The most frequent stereotyped subsets identified in our panel were #1 (35 cases), #7 (28 cases), #4 (24 cases), #3 and #9 (16 cases), #28 (13 cases), and #2 (12 cases), together with subsets #5, #8, #10, #12, #13, #16 and #22 (all ranging from 6 to 9 cases). Finally, we were able to identify by auto-matching analysis 48 sequences potentially specific for 23 novel putative stereotype subsets. In our series we identified 407/1148 (35.45%) stereotyped HCDR3 sequences. The percentage was higher than that reported by Stamatopoulos et al and Murray et al. This discrepancy may partially be due to the different approach used in our analysis, namely the matching to a general data set including all published stereotyped subsets instead of the auto-matching performed by those Authors. We demonstrated a significant association between IGHV status and stereotyped sequences and confirmed the finding that #1 is the most frequent subset identified so far. Finally, we were able to identify a series of 23 novel putative subsets that will require further confirmation. Disclosures: No relevant conflicts of interest to declare.

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.

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.

scholarly journals Preferential Usage of Specific Immunoglobulin Heavy Chain Variable Region Genes with Unmutated Profile and Advanced Stage at Presentation Are Common Features in Patients with Chronic Lymphocytic Leukemia from Senegal

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 19-19
Author(s):  
Abibatou Sall ◽  
Teresa Amato ◽  
Alessandro Gozzetti ◽  
Awa Oumar Touré ◽  
Saliou Diop ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Mononuclear Cells ◽  
Variable Region ◽  
Immunoglobulin Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear ◽  
Mutational Status ◽  
Igh Genes ◽  
Variable Region Genes

Introduction : Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with Causasien patients. In this study, we aimed to identify genetic factors that may account for this difference Methods: We collected peripheral blood mononuclear cells (PBMCs) from a total of 75 patients with CLL, 25 from Senegal (Africa), and 50 from Siena. Since it is well known that there are differences in germline IGH repertoires between different populations, we also collected PBMCs from five healthy Senegalese individuals as control. We analyzed immunoglobulin heavy chain (IGH) genes mutational status by performing next-generation sequencing in these 2 groups of patients. Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series. Conclusion: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background. Disclosures Gozzetti: Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding.

The Expression of BAG3 Gene in Chronic Lymphocytic Leukemia and Its Clinical Significance

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4605-4605
Author(s):  
Huayuan Zhu ◽  
Peng Liu ◽  
Jianyong Li
Keyword(s):  
Prognostic Factors ◽  
Chronic Lymphocytic Leukemia ◽  
Lymph Nodes ◽  
Heavy Chain ◽  
Internal Control ◽  
Conflicts Of Interest ◽  
Immunoglobulin Heavy Chain ◽  
Lymphocytic Leukemia ◽  
Expression Level ◽  
Leukemic Cells

Abstract Abstract 4605 Objective: BAG3 (BCL2-associated athanogene 3) is a member of BAG family. Previously, many reports indicated that BAG3 was over-expressed in different human cancer tissues, such as thyroid carcinoma, pancreatic cancer, prostate cancer, and leukemic cells. However, there is no or low expression of BAG3 in normal tissues. These imply that BAG3 maybe have some important roles in the human cancers. This study was aimed to investigate the expression level of BAG3 mRNA in chronic lymphocytic leukemia (CLL) and its significance in evaluation of CLL prognosis. Methods: 100 CLL human patients and 20 healthy controls were enrolled in this study. The BAG3 mRNA expressions were measured by real-time PCR with fluorescent dye SYBR Green I, the β-actin was used as internal control. The relative quantitative value of BAG3 expression was calculated by means of 2 (–ΔCt). We analyzed the results with established CLL prognostic factors, overall survival (OS) and treatment-free survival (TFS). Results: The expression of BAG3 mRNA in 100 CLL patients was significantly higher than 20 normal control (p = 0.038). Furthermore, The BAG3 expression was obviously increased in CD38 positive patients CD38 (n = 31) than CD38 negetive patients it (n = 61, p = 0.0238). We also found that the expression of BAG3 in the patients with unmutated immunoglobulin heavy-chain (n = 29) was markedly higher than those with mutated immunoglobulin heavy-chain (IgHV) (n = 62, p = 0.0326). It is also found that the patients with enlarged lymph nodes (n = 24) have higher BAG3 expression, compared with those without enlarged lymph nodes (n = 76, p = 0.0004). The increased BAG3 level can also be found in the patients with splenomegaly (n = 32),compared with those without splenomegaly (n=68, p = 0.0393). No association was found between BAG3 expression and patient clinical baseline information (gender and age), as well as other established prognostic factors (lymphocyte count, cytogenetics analysis and p53 mutation status). 25 of these patients were treated with fludarabine-based therapy, interestingly, BAG3 expression level was significantly increased in patients who were relapse and/or refractory to fludarabine-based treatment (n = 10, p = 0.019). However, there are no differences in OS and TFS between patients with low BAG3 expressions and high expression, which indicated BAG3 didn’t affect the patients’ OS and TFS. Conclusion: These results showed that the BAG3 expression in CLL patients is markedly higher than normal controls. CD38, mutation of IgHV, lymph nodes and splenomegaly are related to the expression of BAG3 in CLL. Furthermore, the patients, who were relapse and/or refractory to fludarabine, have a higher expression of BAG3. These imply that BAG3 may be involved in the pathogenesis of CLL and the roles of BAG3 in CLL need further investigation. Disclosures: No relevant conflicts of interest to declare.

Distinct IGHV Repertoire Features In Chinese Chronic Lymphocytic Leukemia Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5279-5279
Author(s):  
Yi Xia ◽  
Wei Xu ◽  
Lei Fan ◽  
Chun Qiao ◽  
Li Wang ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Heavy Chain ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Western World ◽  
Antigenic Difference ◽  
Mutational Status ◽  
Ighv Gene ◽  
Variable Heavy ◽  
Gene Usage

Abstract Mounting evidence indicates that immunoglobulin variable heavy-chain (IGHV) repertoire and mutational status in chronic lymphocytic leukemia (CLL) patients are prognostically relevant. However, rare data is available in Chinese CLL population. Our group investigated 270 Chinese CLL patients for their IGHV sequences and discovered significant differences between Chinese and European CLL patients. First of all, 169 (62.6%) patients in our group got mutated IGHV and 101 (37.4%) were unmutated, rendering a considerable higher percentage of mutated subgroup compared with European patients (55%) (Figure 1). While IGVH3 is still the most frequently used IGVH gene in Chinese CLL patients (135/270, 50%), discrepancy occurs in the usage of IGVH1 gene, which only presents in 13.7% (37/274) patients in our cohort whereas 23.79% for European (Figure 2). Regarding IGHV subgroups, IGHV3-23 and IGHV4-34 are more often used in Chinese CLL patients (10.7% and 10.4%, respectively). Remarkably, IGHV1-69, the most prevalent IGHV subgroup in European CLL patients (12.81%), only accounts for 5.2% (14/270) Chinese cases.Figure 1Higher percentage of mutated IGHV in Chinese CLL patientsFigure 1. Higher percentage of mutated IGHV in Chinese CLL patientsFigure 2Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 2. Different IGHV gene usage between Chinese and European CLL patients, with IGVH1 gene accounts for 23.79% of European CLL patients and for only 13.70% of Chinese CLL patients.Figure 3IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively).Figure 3. IGVH1-69 is the most prevalent IGHV gene among European CLL patients(12.81%), however, only 5.20% Chinese CLL patients use VH1-69. IGVH4-39 and IGVH4-59 are more often used in Chinese CLL patients (7.80% vs 3.73% and 5.60% vs 2.75%, respectively). We further studied the distribution of stereotyped BCR in our cohort. Thirty-eight patients (14.07%) with stereotyped BCR that belonged to 21 subsets were identified, with 1 to 7 sequences contained each. Among them, subset 1 and subset 8 are the most common types with 6 and 7 cases respectively. Three new subsets were discovered (Table 1). Notably, only 1 case belonged to subset 2, the subset with largest group size in western world. Hence, we conclude that Chinese CLL patients show unique IGHV repertoire features compared to patients from western countries. While the mechanism within remains unknown, the discrepancy might due to antigenic difference in geographically remote areas.Table 1Three new subsets of BCR stereotypy in Chinese CLL patientsNO.IGHVIGHDIGHJM/UMIdentityHCDR3 AA sequenceLengthNovel 1NJ-15IGHV4-59*083-22*016*03UM100,00%ARGNYYDSSGYYYVGYYYYYMDV23NJ-31IGHV4-59*013-22*016*03UM99,65%ARGDYYDSSGYYYVGYYYYYMDV23Novel 2NJ-186IGHV3-23*013-22*014*02M96.60%AKGYRDNYDGDQSSVFDS18NJ-23IGHV3-23*012-21*014*02M96,53%AKGYRDNYDGDQSSVFDS18Novel 3NJ-36IGHV4-34*016-6*015*02M93,33%AKLMAGRPNWFDP13NJ-123IGHV4-34*016-6*015*02M91,67%AKLMAGRPNWFDP13 Disclosures: No relevant conflicts of interest to declare.

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