scholarly journals HLA Alleles Repertoire in Russian CLL Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5462-5462
Author(s):  
Bella V. Biderman ◽  
Ekaterina G Khamaganova ◽  
Ekaterina B Likold ◽  
Alena R Abdrakhimova ◽  
Evgeny A Leonov ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Variable Region ◽  
The United States ◽  
Small Sample ◽  
Lymphocytic Leukemia ◽  
Hla Typing ◽  
Chi Square ◽  
Hla Alleles ◽  
Mutational Status ◽  
Healthy Donors

Introduction. Chronic lymphocytic leukemia (CLL) is one of the most common lymphoproliferative diseases in Europe and the USA. The mutational status of the immunoglobulin heavy chain variable region genes (IGHV) has long been known as an important factor for long-term prognosis in CLL. The variety of the rearranged IGHV structure variants in CLL cells is very limited and differs significantly from that in normal B cells. About 30% of all cases of CLL have immunoglobulin receptors with very similar nucleotide sequences. These quasi-identical receptors are called stereotyped. The most common 20 subtypes of stereotyped antigen receptors (SARs) are presented in 12% of all CLL cases. HLA-complex plays a central role in the formation of the immune response. Associations of specific HLA-alleles with different malignant, autoimmune and infectious diseases have been described. However, possible relationship of HLA phenotype to the development of CLL and the association of HLA alleles with disease are not completely understood. Aim. To study the repertoire of HLA alleles in Russian CLL patients with the most common SARs. Patients and methods. The study included 50 CLL patients with the most common IGHV genes and SARs, followed up at the National Research Center for Hematology from 2008 to 2019. Two groups of healthy donors were selected as control - one from Moscow (1507) and other from Eastern Siberia (296). The mutational status of IGHV and the SARs were determined according to European Research Initiative in CLL (ERIC) criteria. HLA-typing in 5 loci (HLA-A, -B, -C, -DRB1, -DQB1) was performed using Luminex 200 system (Luminex, TX, USA) with Lifecodes HLA SSO typing kits (Immucor, CT, USA). HLA frequencies were compared using chi-square test. ARLEQUIN software package, version 3.5 was used for statistical analysis. Results. All patients enrolled in the study had unmutated IGHV genes. In 43 of them, homology with the germline gene was 100%, and in 7 - it was in the range of 98.6-99.7%. IGHV genes of 46 patients (92%) belonged to the 1st family. In 14 (28%) patients CLL#1 SAR, associated with the most aggressive course of the disease, was expressed. Nine patients (18%) each belonged to the CLL#3 and CLL#6 subtypes. The remaining patients expressed CLL#5, 7H, 12 and 28A SARs. No significant differences in the frequency of HLA-A alleles were found between CLL patients and both donor groups. HLA-B*18, HLA-B*52 and HLA-C*12:02 were found significantly more often in CLL patients than in donors. In the DRB1 locus, differences were observed in the two allelic groups. In CLL patients HLA-DRB1*15 was twice more frequent than in healthy donors, HLA-DRB1*13, on the contrary, was twice less frequent. No significant differences were found for DQB1 locus. Most common HLA haplotype frequencies were also calculated. DRB1*15-DQB1*06 haplotype was significantly 2 times more frequent in CLL patients than in donors. Furthermore, A*02-B*07-C*07-DRB1*15-DQB1*06 and A*25-B*18-C*12-DRB1*15-DQB1* 06 were more frequent, but these data were not statistically significant probably due to the small patient sample (Table 1). No significant differences were found between the SAR subtypes of CLL patients in the studied cohort. Discussion. Despite a small sample, we were able to detect 4 predictive and 1 protective HLA allele for CLL patients. Our data disagree with the data of researchers from the United States and France and partially match those obtained by Iranian scientists. This might be explained by two reasons. First, HLA allele frequencies are differ between various populations. Second, we studied a small selected sample - all patients had unmutated IGHV genes, mainly from the same V gene family, and belonged to a cohort with an aggressive course of the disease. Further studies on extended samples of CLL patients are required to assess possible associations of HLA alleles with CLL subtypes and the course of the disease. Disclosures No relevant conflicts of interest to declare.

Transcriptional Mistargeting Of NFAT2 In CLL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4122-4122
Author(s):  
Melanie Märklin ◽  
Jonas S. Heitmann ◽  
David Worbs ◽  
Alexandra Poljak ◽  
Claude Evouna ◽  
...  
Keyword(s):  
B Cells ◽  
Signaling Pathway ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Resting Cells ◽  
Qrt Pcr ◽  
Mutational Status ◽  
Healthy Donors

Abstract Chronic Lymphocytic Leukemia (CLL) is a hematological malignancy of mature B cells and constitutes the most common leukemia in adults. It is characterized by a progressive accumulation of clonal B cells, which coexpress CD19, CD23 and CD5. The clinical course of CLL can be predicted by serveral prognostic markers like CD38, ZAP70 and cytogenetic abnormalities. While the treatment of CLL has significantly improved during recent years, it remains an essentially incurable disease and the molecular events that lead to its development are still largely elusive. NFAT is a family of highly phosphorylated transcription factors residing in the cytoplasm of resting cells. Upon dephosphorylation NFAT proteins translocate to the nucleus where they orchestrate developmental and activation programs in diverse cell types. NFAT is inactivated by a network of several kinases. Several recent studies have demonstrated that Ca2+/NFAT signaling is involved in the pathogenesis of a wide array of different tumor types including pancreatic adenocarcinoma, breast cancer and Non Hodgkin´s lymphoma. In this study we investigated the significance of the Ca2+/NFAT signaling pathway in B-CLL. For this purpose, we analyzed CLL cell lines (MEC-1, JVM-3) as well as primary blood samples from patients with CLL (n=30). The analyzed patient population exhibited a representative distribution of age, sex, Binet stage, WBC count, cytogenetics and IGVH mutational status. We detected a profound overexpression of NFAT2 mRNA as well as NFAT2 protein in all CLL samples. Using qRT-PCR we found that CD19+CD5+ CLL cells exhibited an at least three fold overexpression of NFAT2 as compared to CD19+ B cells isolated from healthy donors. In one case, NFAT2 expression in CLL cells was 200 times higher than in the corresponding controls. This profound overexpression of NFAT2 in CLL cells could be confirmed on the protein level using Western Blotting and Immunocytochemistry. We could further demonstrate that even under resting conditions significant amounts of NFAT2 protein had translocated to the nucleus in CLL cells, whereas virtually all NFAT2 was in the cytoplasm in healthy B cells. NFAT2 nuclear translocation could be inhibited using pretreatment with Cyclosporin A demonstrating that this process was still calcineurin-dependent in CLL cells. We could further show that nuclear NFAT2 in CLL cells was able to bind DNA using electrophoretic mobility shift assays (EMSA). To assess the transcriptional activity of NFAT2 in human CLL we determined the expression of the apoptosis regulators OX40L, osteopontin and PD-L2, which we previously identified as NFAT2 target genes in a gene expression analysis with CD19+CD5+ CLL cells from TCL1 transgenic mice with intact NFAT2 and NFAT2 deletion, respectively. Interestingly, qRT-PCR revealed a tremendous reduction of all three target genes in the analyzed CLL samples as compared to control B cells from healthy donors. This is particularly remarkable, since in the TCL1 mouse model we observed a similar reduction of the expression of these genes in CLL cells with NFAT2 ablation. In summary, these results provide strong evidence that the Ca2+/NFAT signaling axis is constitutively activated in CD19+CD5+ CLL cells. Our data suggest that the profound overexpression of NFAT2 in CLL cells leads to its targeting to aberrant genetic loci different from its phsiological target genes resulting in a consecutive knock out phenotype with respect to the expression of the apoptosis regulators OX40, osteopontin and PD-L2 in CLL. Further investigation is therefore warranted to decipher the therapeutic potential of modulating the Ca2+/Calcineurin/NFAT signaling pathway in this disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals HLA allele repertoire in Russian chronic lymphocytic leukemia patients with an unfavorable prognosis

2020 ◽  
Vol 65 (3) ◽  
pp. 312-320
Author(s):  
B. V. Biderman ◽  
E. B. Likold ◽  
A. R. Abdrakhimova ◽  
E. A. Leonov ◽  
E. G. Khamaganova ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Clone ◽  
Lymphocytic Leukemia ◽  
Hla Typing ◽  
Control Group ◽  
Antigen Receptors ◽  
Hla Alleles ◽  
Healthy Donors ◽  
Allele Group ◽  
Selection Of

Introduction. An unfavorable prognosis in chronic lymphocytic leukemia (CLL) is associated with unmutated status of rearranged IGHV genes. CLL is also characterized by a narrowing of the repertoire of IGHV genes and the formation of quasiidentical (stereotyped) receptors, which is probably associated with antigenic selection of the tumor B-cell clone in the pathogenesis of the disease. The HLA phenotype plays an important role in antigenic selection of B cells. On the other hand, the association of specifi c HLA alleles with various diseases has been described. Aim. To assess the frequencies of HLA alleles in CLL patients with unmutated IGHV genes and the most common stereotyped receptors (SARs). Materials and methods. The study included 100 CLL patients with unmutated IGHV genes - 50 with the most common stereotyped antigen receptors (SARs) and 50 with non-stereotyped antigenic receptors. Control group of healthy donors was also included. Results. Signifi cant differences in HLA-allele repertoire between this two groups of patients and groups of donors were found. B*18 allele group was found much more common in patients with SARs than in donors and in patients without SARs. HLA-B*39 was more frequent for patients with SARs compared to donors; in patients without SARs these alleles were not found. For all patients, the frequency of HLA-B*52 alleles was higher than for donors. HLA-C*12 allelic group was found more frequent in CLL patients than in donors. HLA-DRB1*15 in CLL patients with SARs was found twice as often as in healthy donors or patients without SARs, while HLA-DRB1*13, oppositely, was found twice as rare. HLA-DRB1*16 was signifi cantly more frequent in patients without SARs, compared with donors and the patients with SARs. No signifi cant differences were found in the HLA-A and HLA-DQB1 loci. Conclusion. The association of two HLA alleles with “unmutated” CLL and two others with CLL bearing prognostically unfavorable SARs was found. HLA typing of expanded samples of CLL patients with different prognosis and course of the disease will provide more information on the mechanisms of antigen selection in the pathogenesis of CLL and improve diagnostic and therapeutic approaches.

DETECTION of CHROMOSOMAL 14q32 ABNORMALITIES IN B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4385-4385
Author(s):  
Mariangela Mura ◽  
Alessandra Trojani ◽  
Silvia Soriani ◽  
Alessandra Tedeschi ◽  
Milena Lodola ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Chromosomal Abnormalities ◽  
Conflicts Of Interest ◽  
Small Sample Size ◽  
Variable Region ◽  
Small Sample ◽  
Lymphocytic Leukemia ◽  
Chromosome 14 ◽  
Chromosomal Breakage ◽  
Cpg Oligodeoxynucleotide

Abstract Abstract 4385 Introduction Conventional cytogenetic and FISH studies with the standard panel (13q14, 11q22.3, 17p13, 12, 6q23) are used to detect chromosomal abnormalities of clinical significance in patients with chronic lymphocytic leukemia (B-CLL). Recently, translocations and 5'IGH deletions involving chromosome14q32 are associated with B-CLL. Aim Our aim was to investigate the presence and the characteristics of chromosome 14q32 aberrations in a group of patients with B-CLL. Patients and Methods A total of 58 patients with B-CLL were investigated by conventional cytogenetic, in addition the cultures were stimulated with CpG oligodeoxynucleotide plus IL2. FISH studies with 14q32/IGH break-apart probe designed to detect chromosomal breakage of the IGH (14q32) locus, were done for 59 patients. Results Chromosomal abnormalities were detected in 60.3% of cases by cytogenetic. 14 patients showed complex cariotype while trisomy 12 was the most frequent anomaly found in 8 patients. Among the twelve other patients several chromosomal aberrations were noted: del(13)(q14), del(13)(q12q21), del(13)(q12q14), del(13)(q13q32), +21, t(11;13)(q23;q14), +12 t(1;4)(q31;p14), t(3;14)(p21;q22), del (11)(q12) +21, del(6)(q21), inv3(?p13q21), del(11)(q12). Abnormalities of chromosome 14 were found in 23.8% of patients. Deletion of 5'IGH, corresponding to the variable IGH segment, was the most frequent anomaly found in 8 patients. Interesting, a 3'IGH deletion was detected in two patients, while only one patient showed a complete deletion of chromosome 14 (47,XXY,add14q32). Three patients showed 14q32 translocations involving the IGH locus. Conclusions Based on our findings, deletions of the variable region of the IGH gene (IGHv) and 14q32/IGH translocations are involved in B-CLL. As these preliminary data are based on small sample size, our goal will be to study the cytogenetic profile of a large number of CLL patients. Future studies will permit the identification of 14q32 translocations and the type and the frequency of IGH rearrangements. Finally, chromosome 14 abnormalities will be correlated to other cytogenetic and FISH abnormalities, and associations with known prognostic markers, such as IGVH mutation status and ZAP-70 expression, will be investigated. Disclosures: No relevant conflicts of interest to declare.

Impaired Expression of p66Shc, a Novel Regulator of B-Cell Survival, in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 801-801
Author(s):  
Cosima T. Baldari ◽  
Nagaja Capitani ◽  
Orso Maria Lucherini ◽  
Elisa Sozzi ◽  
Micol Ferro ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Apoptosis ◽  
Conflicts Of Interest ◽  
Group Analysis ◽  
Lymphocytic Leukemia ◽  
Expression Of Genes ◽  
Mutational Status ◽  
Healthy Donors

Abstract Abstract 801 Intrinsic defects in the apoptotic circuitry underlie to a large extent the extended survival of malignant B cells in chronic lymphocytic leukemia (CLL) and are moreover believed to be responsible for their resistance to chemotherapy. We have recently demonstrated that p66Shc, a member of Shc family of protein adapters, acts as a promoter of apoptosis in T cells. Here we show that p66Shc uncouples the B-cell antigen receptor (BCR) from the Erk and Akt dependent survival pathways, thereby enhancing B-cell apoptosis. Expression of p66Shc was found to be profoundly and consistently impaired in CLL B cells compared to peripheral blood B cells form healthy donors. Moreover, significant differences in p66Shc expression were observed in patients with favorable or unfavorable prognosis, classified on the basis of the mutational status of the IGHV genes, with the lowest expression in the unfavorable prognosis group. Analysis of the expression of genes previously implicated in the apoptosis defects of CLL B cells revealed a selective alteration in the balance of pro- and anti-apoptotic members of the Bcl-2 family in these patients. Reconstitution experiments in CLL B cells, as well as data obtained on B cells from p66Shc-/- mice, showed that p66Shc expression correlates with a bias in the Bcl-2 family towards the pro-apoptotic members. Collectively, the data identify p66Shc as a novel regulator of B cell apoptosis which attenuates survival signals emanating from the BCR and modulates expression of the Bcl-2 family. They moreover provide evidence that the defect in p66Shc expression identified in CLL B cells may be causally related to the imbalance towards the anti-apoptotic Bcl-2 family members observed in these cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Akt Inhibitors Induce Apoptosis in Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1731-1731
Author(s):  
Mercè de Frias ◽  
Daniel Iglesias-Serret ◽  
Ana M Cosialls ◽  
Llorenç Coll-Mulet ◽  
Antonio F Santidrián ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Mrna Level ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Healthy Donors ◽  
Induced Apoptosis

Abstract Abstract 1731 Poster Board I-757 Phosphatidylinositol-3-kinase (PI3K)/Akt pathway has been described to be critical in the survival of chronic lymphocytic leukemia (CLL) cells. Here, we have analyzed the effect of two selective chemical inhibitors of Akt (Akti-1/2 and A-443654) in the survival of CLL cells. We studied by cytometric analysis the cytotoxic effects of Akt inhibitors on peripheral B and T lymphocytes from patients with CLL and from healthy donors. Both inhibitors induced apoptosis in CLL cells in a dose-dependent manner. Moreover, B cells from CLL samples were more sensitive to Akt inhibitors than T cells from CLL samples, and B or T cells from healthy donors. Survival factors for CLL cells, such as IL-4 and SDF-1a, were not able to block the apoptosis induced by both Akt inhibitors. We studied the changes induced by Akti-1/2 and A-443654 at mRNA level by performing reverse transcriptase multiplex ligation–dependent probe amplification (RT-MLPA). Akti-1/2 did not induce any change in the mRNA expression profile of genes involved in apoptosis, while A-443654 induced some changes, including an increase in NOXA and PUMA mRNA levels, suggesting the existence of additional targets for A-443654. We also studied the changes induced by both Akt inhibitors in some BCL-2 protein family members on CLL cells by Western blot. Both inhibitors induced an increase in PUMA and NOXA protein levels, and a decrease in MCL-1 protein level. Moreover, Akti-1/2 and A-443654 induced apoptosis irrespective of TP53 status. These results demonstrate that Akt inhibitors induce apoptosis of CLL cells and might be a new therapeutic option for the treatment of CLL. Disclosures No relevant conflicts of interest to declare.

Incidence of Genomic Aberrations and Associated Efficacy from a Phase III Study Comparing Alemtuzumab (CAMPATH®, MABCAMPATH®) vs Chlorambucil as First Line Therapy for B-Cell Chronic Lymphocytic Leukemia (BCLL).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2092-2092 ◽  
Author(s):  
Tadeusz Robak ◽  
Anna Dmoszynska ◽  
Raouf Fetni ◽  
Ying Wang ◽  
Malika Belkacz ◽  
...  
Keyword(s):  
Chromosomal Aberrations ◽  
Small Sample Size ◽  
Variable Region ◽  
Small Sample ◽  
Lymphocytic Leukemia ◽  
Phase Iii ◽  
Chain Gene ◽  
Fish Analysis ◽  
Myc Oncogene ◽  
Trisomy 12

Abstract CAM307 is a randomized Phase III trial comparing the efficacy and safety of alemtuzumab (CAM) with chlorambucil (CHLO). The trial enrolled 297 previously untreated patients (pts) requiring therapy according to NCI-WG criteria. Pts were randomized 1:1 to CAM (n=149) vs CHLO (n=148) using standard dosing regimens. Fluorescence in situ hybridization (FISH) on interphase nuclei of lymphocytes isolated from blood was analyzed for cytogenetic abnormalities prior to the start of therapy. FISH analysis was performed using 13 DNA probes to detect chromosomal aberrations in 17p13.1 (P53), 13q14 (RB1, D13S319 and D13S25), 11q22.3-11q23 (ATM and MLL), 6q27 (subtelomere), 6q21 (chromosome 6q21/alphasatellite 6 cocktail probe), trisomy 8q24 (c-myc), trisomy 12 (CEP12) and translocations involving the locus of immunoglobulin heavy chain gene (IGH, 14q32.33). Samples were analyzed in 282 pts (95%); chromosomal aberrations were detected in 231 pts (82%) while 51 pts (18%) exhibited a normal interphase FISH pattern. The most frequent abnormalities were deletions (del) at loci 13q (49%), sole del 13q (24%), 11q (19%), 17p (7 %), 6q (4 %), and trisomies 12 (14%) and 8q (5%). Translocations IGH, 14q32.33 were detected in 10 pts (4%). An exploratory analysis was performed to correlate time to event variables (assessed by an independent response review panel) with cytogenetics. Overall 165 pts (59%) revealed combination abnormalities. The most frequently observed chromosomal associations were: del 13q + del 14q (N=20, 12%), del 11q + del 13q (N=17, 10%), del 11q + del 13q + del 14q (N=11, 7%), del 11q + del 14q (N=7, 4%), trisomy 12 + del 13q (N=5, 3%), del 13q + del 17p (N=4, 2%), del 11q + trisomy 12 (N=3, 2%) and del 17p + del 6q (N=3, 2%). Coexistence of del 17p and del 11q was not observed. Although del 13q was observed with all chromosome abnormalities, nearly half of the cases del 13q14.3 (D13S25 and D13S319) coincided with an ATM deletion (11q22.3). FISH analysis has allowed the detection of uncommon abnormalities: tetraploidy (n=1), hyperdiploidy (n=1), trisomy 18 (n=1) and c-myc oncogene amplification (>15 copies per nuclei) (n=2). The latter is a well known abnormality in solid tumors but rarely seen in leukemia. In addition, del of the IGH variable region was detected in 70 pts. The biological and clinical significance of this abnormality is to be investigated. Conclusions: Overall, 82% of treatment naïve BCLL pts revealed cytogenetic aberrations and 59% were combination abnormalities. CAM307 demonstrates a significant improvement in PFS in pts treated with CAM vs CHLO who present with del 13q as the sole abnormality; no difference in pts with del 11q. However, a trend towards improved PFS was observed in pts with trisomy 12 and del 17p, which did not reach significance due to small sample size. Further investigation of CAM therapy in high risk cytogenetic subgroups is warranted.

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.

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.

Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2880-2880
Author(s):  
Martin Trepel ◽  
Fabian Muller ◽  
Mareike Frick ◽  
Janina Rahlff ◽  
Claudia Wehr ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Phage Display ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Specific Binding ◽  
Variable Region ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Fab Fragment

Abstract Abstract 2880 Background: The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL. Methods: The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins. Results: Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence. Conclusions: These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients. Disclosures: No relevant conflicts of interest to declare.

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