IgH Rearrangements and Mutational Status in Class-Switched IgG B-CLL.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2793-2793 ◽  
Author(s):  
James A.L. Fenton ◽  
David Gonzalez ◽  
Andy C. Rawstron ◽  
Gareth J. Morgan ◽  
Andrew S. Jack ◽  
...  
Keyword(s):  
B Cells ◽  
Mutational Analysis ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Specific Antigen ◽  
Distinct Pattern ◽  
Published Data ◽  
Molecular Features ◽  
Mutational Status ◽  
Igh Genes

Abstract Analysis of immunoglobulin heavy chain (IgH) rearrangements in B-CLL differentiates subgroups of patients with significantly different clinical outcomes. Cases can be categorised according to mutational status of the variable (V) segment with unmutated VH regions linked to a worse prognosis. A restricted pattern of use of specific VH, DH and JH gene segments has also been reported in B-CLL. It has been hypothesised that B-CLL originates as a clonal expansion of B-cells that have been selected and activated by contact with self or foreign antigens, leading to those small clones to proliferate, mutate their IGH genes, acquire genetic lesions and eventually become malignant. B-CLL cells normally express low levels of Ig on the surface, normally IgM, although a proportion of patients express IgG or IgA, following the class-switch recombination (CSR) process. We have analysed the pattern of SHM and gene segment usage in this particular subgroup for 44 patients with IgG B-CLL. Successful PCR amplification of recombined Smu-Sgamma switch region DNA was achieved in 40 patients, confirming the presence of IgG class-switching. Mutational analysis of IgH V genes revealed 80% of patients contained more than 2% somatic hypermutation (SHM), with 63% of samples having a greater than 5% SHM rate. For VH gene segment usage, a significant predominance of the VH4 family was seen in 22 cases (50%), followed by VH3 in 17 cases (39%), while VH1 family was found in only 3 of 44 samples, this differs from classical IgM B-CLL where VH3 family usage predominates. Overall, VH4-34 was the most frequently used gene segment (34%), followed by VH3-07 (14%) and VH4-39 (9%). DH6-13 was the most frequently used DH segment (21%), followed by DH6-19 (13%). JH gene segment usage did not differ from normal B-cells, with JH4 being the most frequently used, followed by JH6 and JH5. There was a significant association between VH4-39, DH6-13 and JH5 in three samples all containing unmutated sequence. Together this data reveals a distinct pattern of IGH VDJ rearrangements in IgG B-CLL compared to classical IgM B-CLL. Firstly, the rate of SHM in IgG B-CLL (80%) is significantly higher than the 50% observed in IgM B-CLL. Secondly, VH segment usage pattern differs between the two subgroups with a significant under-representation of VH1 as well as an over-representation of VH4 family members in the IgG subgroup. Finally, there is a striking association between VH4-39 and DH6-13/JH5 in the very few unmutated rearrangements. This could be indicative of a different clonal history of these particular B cells in B-CLL. Together with recent published data, this latter finding suggests that this is a further sub-category exclusive to IgG B-CLL, where selection of a specific antigen receptor may have lead to B-CLL development in such cases. We conclude that class-switched IgG B-CLL contains different molecular features in the IgH genes compared with classical IgM B-CLL, and other B-cell malignancies. The clinical implications of these differences, especially the relationship between the mutational status of the VH genes and outcome in IgG B-CLL, will be further investigated.

Analysis of IgVH, BCL-6, PIM, RHO/TTF and PAX5 Mutational Status in Splenic and Nodal Marginal Zone B Cell Lymphoma Suggests a Particular B Cell Origin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 162-162 ◽  
Author(s):  
Alexandra Traverse-Glehen ◽  
Aurelie Verney ◽  
Lucille Baseggio ◽  
Pascale Felman ◽  
Evelyne Callet-Bauchu ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Somatic Mutations ◽  
Marginal Zone ◽  
Somatic Hypermutation ◽  
B Cell Lymphoma ◽  
Marginal Zone B Cells ◽  
Mutational Status ◽  
Frequent Absence

Abstract Background and Objectives Splenic and nodal marginal zone B cell lymphoma (SMZL and NMZL) have been recently identified as distinct clinicopathological entities in the WHO classification. These lymphomas entities may have a common origin in the marginal B-cell compartment of the lymphoid organs. However the precise cell of origin of marginal zone B cells, its status in the B cell differentiation pathway and the mechanisms involved in lymphomagenesis remain unclear. The most widely held view is that marginal zone B cells are mostly memory B cells. But the origin of these cells, especially the transit through germinal center pathway, remains contradictory. Somatically mutated variable-region of immunoglobulin genes and bcl-6 gene represent at this time faithful markers for exposure to the germinal center. In addition, aberrant somatic hypermutations have been suggested to contribute to the development of B-cell lymphomas, occurring in the 5′ sequence of several proto-oncogenes. Interestingly those mutation do not occur in normal germinal center B cells. Design and Methods: IgVH, BCL-6, PIM1, Rho/TTF and PAX 5 genes, highly mutated in DLBCL and other indolent lymphoma such as B-CLL, were analysed for the presence of somatic mutations from 50 marginal zone lymphoma tissue and blood samples (21 NMZL and 29 SMZL including 10 cases with numerous villous lymphoma cells in peripheral blood). According to the morphological and immunophenotypical analysis, the fraction of malignant cells in the specimen was 70% or more in all cases. Mutational analysis was restricted to the regions previously shown to contain more than 95% of mutations in DLBCL. PCR products were directly sequenced on both sides and perfomed in duplicate in two independent reactions. Results: Out of 18 NMZL cases analysed for IgVH mutational status (3 cases not analysed for IgVH) 15 cases were mutated and 21 out of 28 in SMZL cases. Mutation of BCL-6 was detected in only 1 NMZL patients (1/21) and 1 SMZL patients (1/29). For RhoH/TTF, PIM1, PAX5 the mutation average was also low with only 1 case mutated per group and per gene, with a different case mutated in each for each gene. Conclusion In summary, we demonstrate the low frequency of aberrant somatic mutations in SMZL and NMZL, suggesting that this process is probably not a major contributor to lymphomageneis. However the frequent absence of mutation in BCL6 suggest a particular differentiation pathway, as suggested before in normal marginal zone B cells, possibly without transit through the germinal center. Interestingly the relatively high frequency of VH mutated cases compared with the frequent absence of mutation of BCL6, considered as a specific germinal center tag, could suggest somatic hypermutation outside the germinal center. In addition the absence of hypermutation could be linked with the absence of recurrent translocation in SMZL and NMZL, the translocation process haveing been associated with somatic hypermutation dysfunction.

Comparison of IgH Gene Rearrangement Configuration between Hairy Cell Leukemia (HCL) and Hairy Cell Leukemia Variant (HCL-v).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4997-4997
Author(s):  
David Gonzalez ◽  
Ricardo Morilla ◽  
Alison Morilla ◽  
Monica Else ◽  
Nnenna Osuji ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Hairy Cell Leukemia ◽  
Marginal Zone ◽  
Gene Segment ◽  
Variant Form ◽  
Splenic Marginal Zone Lymphoma ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Mutational Status

Abstract Hairy cell leukemia (HCL) is a rare B-cell disorder, with a variant form, which differs from the former in clinical behaviour, morphology and immunophenotype. The resemblance of these malignancies to splenic marginal zone lymphoma (SMZL), has led to the suggestion that they may share a common clonal progenitor that homes within the marginal zone of the splenic germinal centres. The immunophenotype and recent global gene expression results offer some support for this view, with a similar profile seen in HCL, and a subset of memory B-cells that are localized to the marginal zone. However, studies of the mutational status of the Ig genes are controversial. The majority of HCL cases are mutated, whereas up to 30% of SMZL cases are unmutated. Further investigation to understand these differences and to identify the cell of origin in these disorders is needed. In addition, there are no data on gene expression or VH mutation in HCL-variant (HCL-v), an entity with a poorer outcome and for which there is as yet no effective therapy. We have analysed a series of 13 HCL and 23 HCL-v, for the configuration of the Ig heavy chain rearrangements. VDJH rearrangements were PCR-amplified and sequenced using an automated 3130xL sequencer (Applied Biosystems, Warrington, UK). The sequences obtained were compared to the closest germline segments using V-base (CRI, Cambridge, UK) in order to study the level of somatic hypermutation as well as gene segment usage in IgH rearrangements. The majority of the HCL (92%) showed more than 2% deviation from the closest germline VH gene segment, compared to only 17/23 (74%) in the HCL-v cases. In the mutated cases, the percentage of mutation was similar for HCL and HCL-v (mean 5.8% and 5.9%, respectively), and none of the cases appeared to show ongoing hypermutation. Within the HCL-v group, 4 out of the 6 unmutated cases used VH4-34 segment, and the remaining 2 used VH1-03. Both genes are known to be associated with autoimmune diseases, and they are excluded from the normal and malignant memory plasma cell repertoires. Also, JH5 segment was found in 47% of the HCL-v, while it was absent in the HCL repertoire. This finding is particularly interesting, as JH5 segment is normally under-represented in most normal and malignant B-cells, except for a characteristic group of IgG-expressing chronic lymphocytic leukemias. Our data suggest that HCL-v is, unlike HCL, heterogeneous regarding IgVH mutations, but has restricted IgH repertoire that, at least in some cases, might be derived from activated, self-reactive B-lymphocytes that have not experienced the germinal-centre reaction.

scholarly journals A broad atlas of somatic hypermutation allows prediction of activation-induced deaminase targets

2018 ◽  
Vol 215 (3) ◽  
pp. 761-771 ◽  
Author(s):  
Ángel F. Álvarez-Prado ◽  
Pablo Pérez-Durán ◽  
Arantxa Pérez-García ◽  
Alberto Benguria ◽  
Carlos Torroja ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Mutagenic Activity ◽  
Predictive Tool ◽  
Chromosome Translocations ◽  
Molecular Features ◽  
Antibody Diversification ◽  
Base Excision ◽  
Activation Induced Deaminase

Activation-induced deaminase (AID) initiates antibody diversification in germinal center (GC) B cells through the deamination of cytosines on immunoglobulin genes. AID can also target other regions in the genome, triggering mutations or chromosome translocations, with major implications for oncogenic transformation. However, understanding the specificity of AID has proved extremely challenging. We have sequenced at very high depth >1,500 genomic regions from GC B cells and identified 275 genes targeted by AID, including 30 of the previously known 35 AID targets. We have also identified the most highly mutated hotspot for AID activity described to date. Furthermore, integrative analysis of the molecular features of mutated genes coupled to machine learning has produced a powerful predictive tool for AID targets. We also have found that base excision repair and mismatch repair back up each other to faithfully repair AID-induced lesions. Finally, our data establish a novel link between AID mutagenic activity and lymphomagenesis.

scholarly journals Molecular features responsible for the absence of immunoglobulin heavy chain protein synthesis in an IgH− subgroup of multiple myeloma

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1087-1093 ◽  
Author(s):  
Tomasz Szczepański ◽  
Mars B. van 't Veer ◽  
Ingrid L. M. Wolvers-Tettero ◽  
Anton W. Langerak ◽  
Jacques J. M. van Dongen
Keyword(s):  
Multiple Myeloma ◽  
Heavy Chain ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Immunoglobulin Heavy Chain ◽  
Polymerase Chain Reaction Analysis ◽  
Class Switch ◽  
Molecular Features ◽  
Igh Genes ◽  
Polymerase Chain

Abstract This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic Jh gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the Jh and Cμ regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal Jhgene rearrangements. Only 4 of the joinings were complete Vh-(D)-Jhrearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete Dh-Jh joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame Vκ-Jκgene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of Ch probes. In 5 patients the rearrangements in the Jh and Ch regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32.3). These data indicate that in many IgH− MM patients illegitimateIGH class switch rearrangement or illegitimate deletion of the functional Vh-(Dh)-Jhallele are responsible for IgH negativity. For example, the exclusive presence ofDh-Jhrearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH− MM patients.

scholarly journals Self-reactive VH4-34–expressing IgG B cells recognize commensal bacteria

2017 ◽  
Vol 214 (7) ◽  
pp. 1991-2003 ◽  
Author(s):  
Jean-Nicolas Schickel ◽  
Salomé Glauzy ◽  
Yen-Shing Ng ◽  
Nicolas Chamberlain ◽  
Christopher Massad ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Healthy Donor ◽  
Somatic Hypermutation ◽  
Gene Segment ◽  
Commensal Bacteria ◽  
Healthy Individuals ◽  
Cell Repertoire ◽  
B Cell Repertoire ◽  
Variable Heavy

The germline immunoglobulin (Ig) variable heavy chain 4–34 (VH4-34) gene segment encodes in humans intrinsically self-reactive antibodies that recognize I/i carbohydrates expressed by erythrocytes with a specific motif in their framework region 1 (FWR1). VH4-34–expressing clones are common in the naive B cell repertoire but are rarely found in IgG memory B cells from healthy individuals. In contrast, CD27+IgG+ B cells from patients genetically deficient for IRAK4 or MYD88, which mediate the function of Toll-like receptors (TLRs) except TLR3, contained VH4-34–expressing clones and showed decreased somatic hypermutation frequencies. In addition, VH4-34–encoded IgGs from IRAK4- and MYD88-deficient patients often displayed an unmutated FWR1 motif, revealing that these antibodies still recognize I/i antigens, whereas their healthy donor counterparts harbored FWR1 mutations abolishing self-reactivity. However, this paradoxical self-reactivity correlated with these VH4-34–encoded IgG clones binding commensal bacteria antigens. Hence, B cells expressing germline-encoded self-reactive VH4-34 antibodies may represent an innate-like B cell population specialized in the containment of commensal bacteria when gut barriers are breached.

scholarly journals Different superantigens interact with distinct sites in the Vbeta domain of a single T cell receptor.

1996 ◽  
Vol 183 (4) ◽  
pp. 1437-1446 ◽  
Author(s):  
S C Hong ◽  
G Waterbury ◽  
C A Janeway
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mhc Class Ii ◽  
Mutational Analysis ◽  
Gene Segment ◽  
Class Ii ◽  
Cd4 T Cell ◽  
Published Data ◽  
Antigenic Peptides ◽  
Mhc Class Ii Molecules

CD4 T cell receptors (TCRs) recognize antigenic peptides presented by self major histocompatibility complex (MHC) class II molecules as well as non-self MHC class II molecules. The TCRs can also recognize endogenous retroviral gene products and bacterial toxins known collectively as superantigens (SAGs) that act mainly on the Vbeta gene segment-encoded portion of the Vbeta domain; most SAGs also require MHC II class for presentation. We have studied the interaction of the TCR from a well-characterized CD4 T cell line with SAGs by mutational analysis of its Vbeta domain. This appears to separate viral (v)SAG from bacterial (b)SAG recognition. T cells having a TCR with glycine to valine mutation in amino acid residue 51 (G51V) in complementarity determining region 2 of the TCR Vbeta domain fail to respond the bSAGs staphylococcal enterotoxin B (SEB), SEC1, SEC2, and SEC3, whereas they retain the ability to respond to non-self MHC class II molecules and to foreign peptides presented by self MHC class II molecules. It is interesting to note that T cells expressing mutations of both G51V and G53D of V beta regain the response to SEB and partially that to SEC1, but do not respond to SEC2, and SEC3, suggesting that different bacterial SAGs are viewed differently by the same TCR. These results are surprising, because it has been generally believed that SAG recognition by T cells is mediated exclusively by hypervariable region 4 on the exposed, lateral face of the TCR Vbeta domain. Response to the vSAG Mtv-7 was generated by mutation in Vbeta residue 24 (N24H), confirming previously published data. These data show that the vSAG Mtv-7 and bSAGs are recognized by different regions of the TCR Vbeta domain. In addition, various bSAGs are recognized differently by the same TCR. Thus, these mutational data, combined with the crystal structure of the TCR beta chain, provide evidence for distinct recognition sites for vSAG and bSAG.

scholarly journals Molecular features responsible for the absence of immunoglobulin heavy chain protein synthesis in an IgH− subgroup of multiple myeloma

Blood ◽  
2000 ◽  
Vol 96 (3) ◽  
pp. 1087-1093 ◽  
Author(s):  
Tomasz Szczepański ◽  
Mars B. van 't Veer ◽  
Ingrid L. M. Wolvers-Tettero ◽  
Anton W. Langerak ◽  
Jacques J. M. van Dongen
Keyword(s):  
Multiple Myeloma ◽  
Heavy Chain ◽  
Plasma Cells ◽  
Somatic Hypermutation ◽  
Immunoglobulin Heavy Chain ◽  
Polymerase Chain Reaction Analysis ◽  
Class Switch ◽  
Molecular Features ◽  
Igh Genes ◽  
Polymerase Chain

This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic Jh gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the Jh and Cμ regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal Jhgene rearrangements. Only 4 of the joinings were complete Vh-(D)-Jhrearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete Dh-Jh joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame Vκ-Jκgene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of Ch probes. In 5 patients the rearrangements in the Jh and Ch regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32.3). These data indicate that in many IgH− MM patients illegitimateIGH class switch rearrangement or illegitimate deletion of the functional Vh-(Dh)-Jhallele are responsible for IgH negativity. For example, the exclusive presence ofDh-Jhrearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH− MM patients.

Stereotyped Patterns of Somatic Hypermutation (SHM) in Subsets of Patients with Chronic Lymphocytic Leukemia (CLL): Implications for the Role of Antigen Selection in Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 744-744
Author(s):  
Fiona Murray ◽  
Nikos Darzentas ◽  
Anastasia Hadzidimitriou ◽  
Gerard Tobin ◽  
Myriam Boudjogra ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Protein A ◽  
Specific Antigen ◽  
Lymphocytic Leukemia ◽  
Binding Motif ◽  
Staphylococcal Protein A ◽  
Binding Motifs ◽  
Mutational Status ◽  
Identity Groups

Abstract We examined SHM features in 1967 IGH rearrangements from 1939 patients with CLL. The sequences were divided into four “identity groups”; “truly unmutated” (100% identity to germline; 677 sequences), “minimally mutated” (99–99.9% identity; 133 sequences), “borderline mutated” (98–98.9% identity; 93 sequences) and “mutated” (<98% identity; 1064 sequences). At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged on analysis of subgroups of sequences. In particular, the IGHV repertoire of the four “identity groups” differed considerably, with the IGHV1-69 and IGHV1-2 genes predominating among “truly unmutated” and “minimally mutated” sequences, respectively. In contrast, other genes were mostly used in “mutated” rearrangements (eg, IGHV4-34/3-23/3-7). Of note, IGHV3-21 and IGHV3-48 had the highest proportion of “borderline mutated” rearrangements. In selected groups of sequences, a remarkable preservation of the germline configuration was observed in superantigenic binding motifs, prompting speculation that subsets of CLL cells could also receive stimulation signals by superantigenic-like interactions. In detail, the vast majority of IGHV4-34 sequences retained germline conformation at the four FR1 positions of the IGHV4-34-specific I/i binding motif. Similarly, IGHV3-21 sequences displayed remarkably few alterations of the IGHV3-specific staphylococcal protein A binding motif. Following established criteria, we identified 530/1967 sequences (27%) as belonging to 113 different subsets with stereotyped HCDR3. The distribution of sequences among subsets differed significantly according to mutational status: 43% of “truly unmutated” sequences belonged to a subset, compared to only 16% of the “mutated” group (p<0.001). Of note, among certain IGHV genes (ie, IGHV1-2/3-21/4-34/4-4), shared “stereotyped” amino acid (AA) changes (i.e. the same AA replacement at the same position) occurred across the entire IGHV sequence significantly more frequently in cases with stereotyped vs. heterogeneous HCDR3s and therefore, could be considered as “subset-biased”. Stereotyped AA changes were also observed in subsets of minimally mutated cases, indicating that even a low level of mutations may be functionally relevant. Comparison to public-database non-CLL sequences revealed that certain stereotyped AA changes were over-represented in CLL and thus could also be considered as “CLL-biased”. The very precise targeting and distinctive features of SHM in subgroups of CLL patients provide further evidence for the important role of selection by specific antigen(s) in CLL leukemogenesis.

scholarly journals Identification of the Antigenic Targets of the Major CLL-Derived Stereotyped BCRs and First Demonstration of Sub-Subsets

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 298-298
Author(s):  
Klaus-Dieter Preuss ◽  
Natalie Fadle ◽  
Evi Regitz ◽  
Maria Kemele ◽  
Manuela Wölfle ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Specific Antigen ◽  
Fetal Brain ◽  
Cell Receptor ◽  
Published Data ◽  
Activated T Cells ◽  
Chronic Antigenic Stimulation ◽  
Papain Digestion ◽  
Antigenic Targets

Abstract Background: The B-cell receptor (BCR) is the hallmark of mature B cells and a clonal BCR is expressed on the neoplastic cells of B-cell malignancies. The BCR mediated signalling provides proliferative and anti-apoptotic signals for the neoplastic B cells and represents an attractive therapeutic target. The BCRs of about 1/3 of CLL cases display highly homologous complementary determining regions 3 (CDR3), on the basis of which stereotyped subsets have been defined. The homologies of the BCR of a given subset suggest selection by shared antigen(s), which might play an important role in the pathogenesis of the respective B-cell neoplasm by chronic antigenic stimulation. Methods: Tumor cells from CLL patients belonging to subsets 2, 3, 4, 5, 6, 8 were studied. Natural Fabs (nFab) were obtained by papain digestion of membrane-bound Igs. Subset-derived nFabs were used to screen for reactivity with proteins represented in two high-density protein macroarrays, one derived from human fetal brain cDNA and a second derived from a mixture of expression libraries derived from activated T-cells, lung and colon. Identified antigens were analysed by standard techniques. All methods used (nFab generation by papain digestion, BCR cloning, membrane screening, epitope mapping etc.) are described in detail in Zwick C et al., Blood 2013;121(23):4708-4717. Results: Our ongoing systematic search for CLL subset-specific antigens together with our previously published data (Zwick et al) led to the identification and epitope characterization of the antigenic targets for the 6 most common CLL subsets: MARK3 as antigen for subset 2, NCOR2 as antigen for subset 3, CaCyBP as antigen for subset 4, FAM32A protein as antigen for subset 5 and GLDC as antigen for subset 8. In addition we could confirm MYHIIA as antigen for subset 6 (in accordance with Chu CC et al, Blood 2010;115(19):3907-3915). The analysis of the BCR binding revealed (except for oxLDL) in minimum 2 different epitopes for each subset-specific antigen, thus defining sub-subsets with BCRs binding to distinct epitopes. Sequence analysis of the respective BCR suggests that this epitope-spreading is due to antigen-driven mutation/maturation of BCRs with a highly homologous CDR3. Conclusions: The antigenic targets (and stimulus) for the clonal BCRs of the major sterotyped subsets have been identified providing for the first time experimental evidence for the hypothesis that BCRs of a given subset indeed target the identical antigen. Moreover, based on the BCR-binding epitopes, sub-subsets of stereotypes have been defined by BCRs obviously originating from the same germline, but having undergone different antigen-driven mutations. Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. Disclosures Stilgenbauer: Pharmacyclics, Janssen: Honoraria, Research Funding.

Expression of Specialized Error-Prone DNA Polymerases in Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2941-2941
Author(s):  
Marta Crespo ◽  
Eva Gine ◽  
Ana Muntanola ◽  
Neus Villamor ◽  
Emili Montserrat ◽  
...  
Keyword(s):  
B Cells ◽  
Peripheral Blood ◽  
Somatic Hypermutation ◽  
Dna Polymerases ◽  
Burkitt’S Lymphoma ◽  
Mean Value ◽  
Dna Lesions ◽  
Burkitt's Lymphoma ◽  
Prolymphocytic Leukemia ◽  
Mutational Status

Abstract Specialized DNA polymerases are low fidelity enzymes required to bypass DNA lesions that are also involved in somatic hypermutation. Deregulation of these enzymes can lead both to an increased mutation rate and to an enhanced translesion synthesis capability. DNA polymerase Kappa (DNA pol Kappa) has been found to promote tumorigenesis and to be overexpressed in some cancers. Expression levels of specialized DNA polymerases Iota, Eta, Zeta and Mu were analyzed in primary B-cells from 12 CLL patients (6 with IgVH mutated genes [M] and 6 with unmutated [UM] genes). Expression of DNA pol Kappa was also assessed in 27 CLL samples (13 M and 14 UM), in B-cell lines derived from pre- and post-germinal center malignancies (NALM-6 [preB-acute lymphoblastic leukemia], JVM-2 [prolymphocytic leukemia], Granta, NCEB and REC [mantle-cell lymphoma] and Daudi [Burkitt’s lymphoma] and in normal B-cells from peripheral blood and tonsil. Expression of DNA polymerases was measured by Quantitative RT-PCR using Ramos (a constitutively hypermutating Burkitt’s lymphoma cell line) as a reference, considering an arbitrary unit of 1 for each gene. Although somatic hypermutation is absent in UM CLL cells, there were no differences in the expression of the DNA polymerases analyzed according to the mutational status. Expression of DNA pols Iota and Zeta was similar in CLL cells and Ramos (mean value: 0.95 and 1.3, respectively), whereas expression of DNA pol Mu was slightly higher (mean 3.24) and DNA pol Eta was underexpressed (mean 0.19). In contrast, among all the hematological malignancies analyzed, significant overexpression of DNA pol Kappa was only found in CLL samples (mean value; 8.43;range: 1.42–19.47). Induction of AID expression by stimulating CLL cells with CD40 ligand and IL-4 did not modulate DNA pol Kappa expression. Finally, expression of DNA pol Kappa in normal B cells from peripheral blood was similar to that in Ramos (0.87) and slightly higher in B cells from tonsil (2.02). In conclusion, no differences in DNA polymerases were observed between M and UM CLL. As previously described in some solid tumors, CLL cells showed an increased expression of DNA pol Kappa, regardless the mutational status of the IgVH genes, suggesting that the expression of this polymerase is not related to the process of somatic hypermutation that occurs in some CLL cells. Taken together, these results support the concept that DNA pol Kappa may behave as an oncogene in CLL and that its regulation may be crucial for maintaining genomic stability.

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