scholarly journals Clonotypic Analysis of Immunoglobulin Heavy Chain Sequences in Patients with Waldenström’s Macroglobulinemia: Correlation withMYD88L265P Somatic Mutation Status, Clinical Features, and Outcome

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Loizos Petrikkos ◽  
Marie-Christine Kyrtsonis ◽  
Maria Roumelioti ◽  
George Georgiou ◽  
Anna Efthymiou ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Somatic Mutation ◽  
Gene Rearrangement ◽  
Somatic Hypermutation ◽  
Malignant Cell ◽  
Clinical Findings ◽  
Mutation Status ◽  
Mutational Status ◽  
Igh Genes

We performedIGHclonotypic sequence analysis in WM in order to determine whether a preferentialIGHgene rearrangement was observed and to assessIGHVmutational status in blood and/or bone marrow samples from 36 WM patients. In addition we investigated the presence ofMYD88L265P somatic mutation. AfterIGHVDJ locus amplification, monoclonal VDJ rearranged fragments were sequenced and analyzed.MYD88L265P mutation was detected by AS-PCR. The most frequent family usage wasIGHV3(74%);IGHV3-23andIGHV3-74segments were used in 26% and 17%, respectively. Somatic hypermutation was seen in 91% of cases.MYD88L265P mutation was found in 65,5% of patients and absent in the 3 unmutated. These findings did not correlate with clinical findings and outcome. Conclusion.IGH genes’repertoire differed in WM from those observed in other B-cell disorders with a recurrentIGHV3-23andIGHV3-74usage; monoclonalIGHVwas mutated in most cases, and a high but not omnipresent prevalence ofMYD88L265P mutation was observed. In addition, the identification of 3 patients with unmutatedIGHVgene segments, negative for theMYD88L265P mutation, could support the hypothesis that an extra-germinal B-cell may represent the originating malignant cell in this minority of WM patients.

Lymphoma of the Breast

1999 ◽  
Vol 123 (12) ◽  
pp. 1208-1218 ◽  
Author(s):  
Margarita Topalovski ◽  
Domnita Crisan ◽  
Joan C. Mattson
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Gene Rearrangement ◽  
Cell Lymphoma ◽  
Bone Marrow Involvement ◽  
B Cell Lymphomas ◽  
Breast Lymphoma ◽  
Gene Rearrangement Analysis ◽  
Lymphoid Aggregates ◽  
Large B Cell

Abstract Background.—Primary lymphomas of the breast are rare, accounting for 1.7% to 2.2% of extranodal lymphomas and 0.38% to 0.7% of all non-Hodgkin lymphomas. Although secondary breast lymphomas are also rare, they represent the largest group of metastatic tumors of the breast. Objectives.—To investigate the clinicopathologic and immunophenotypic characteristics of breast lymphomas, the relative frequency of primary and secondary mammary lymphomas, and in selected cases, the role of gene rearrangement analysis in diagnosis and staging of these lymphomas. Results.—We conducted a retrospective review of 22 cases of breast lymphoma diagnosed at William Beaumont Hospital, Royal Oak, Mich, during a 30-year period (1963–1994). Eleven of the 22 cases fulfilled the criteria for primary breast lymphoma; these cases represented 0.6% of all non-Hodgkin lymphomas seen in our hospital. Of the 11 cases, 5 were diffuse large B-cell lymphomas, 2 were follicle center lymphomas, 2 were marginal zone B-cell lymphomas (mucosa-associated lymphoid tissue type), 1 was a lymphoplasmacytoid lymphoma, and 1 was a peripheral B-cell neoplasm, unclassified. Using a panel of immunohistochemical stains (CD45RO, CD45RA, CD43, CD3, CD20, CD30, CD68, and HLA-DR), 8 cases demonstrated unequivocal B-cell phenotype and 3 cases had equivocal or weak staining patterns for B-cell markers. We identified no cases of T-cell lymphoma. Of 7 cases that had bone marrow biopsies for staging, 3 were positive morphologically for bone marrow involvement. Molecular analysis of B- and T-cell gene rearrangement was used to exclude bone marrow involvement in one case with bone marrow lymphoid aggregates and to confirm negativity in a case that was morphologically negative. Of the 11 secondary breast lymphomas, 5 were diffuse large B-cell lymphomas; 1 was diffuse large B-cell, primary mediastinal subtype; and 5 were follicle center lymphomas. Conclusions.—Breast lymphomas represented 1.2% of all non-Hodgkin lymphomas in this study; the frequency of primary and secondary cases was equal. In both groups, right breast lesions were predominant, and the most frequent morphologic type was diffuse large B-cell lymphoma. Gene rearrangement analysis is helpful in selected cases to rule out bone marrow involvement, especially in older patients, in whom lymphoid aggregates are common.

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.

miRNA Expression Profile of B-SLL Consistent with Normal Memory B Cells:BIC/miR–155 Specific Location in Proliferation Center.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2081-2081
Author(s):  
Miao Wang ◽  
Jan-Lukas Robertus ◽  
Lu-ping Tan ◽  
Geert Harms ◽  
Tjasso Blokzijl ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Profile ◽  
Mirna Expression ◽  
Direct Sequencing ◽  
Memory B Cells ◽  
Lymphocytic Leukemia ◽  
Chain Gene ◽  
Mutation Status ◽  
Igh Genes

Abstract Introduction: B cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by the accumulation of nonproliferating mature-appearing lymphocytes in the blood, marrow, lymph nodes, and spleen. B-CLL behaves like leukemia and is found mostly in the blood. The expression of ZAP-70 and mutation status of the immunoglobulin heavy-chain gene (IgH) can serve as prognostic markers in B-CLL. ZAP-70 positive cases usually present with unmutated IgH genes and have a bad prognosis, whereas ZAP-70 negative cases mostly present with mutated IgH genes and have good prognosis. Gene expression studies of B-CLL indicated that the profile of IgH mutated and unmutated cases are similar to normal memory B cells. Several studies showed that miRNAs play important roles in pathogenesis of B-CLL and some miRNAs correlate with the prognosis in B-CLL. B-SLL is considered to be the same disease entity as CLL, but this variant is found mostly in bone marrow and the lymphatic system. The most aggressive type of B-SLL is characterized by neoplastic cells that are more responsive to B-cell receptor signaling and are characterized by proliferation centers (PCs), a potentially important site of neoplastic cell stimulation. Until now, only a few reports have been published about the ZAP-70 expression and IgH mutation status and no data are available about the microRNA expression profile. Methods: 33 B-SLL cases were retrieved from the pathology files. ZAP-70 expression was analyzed by using immunohistochemistry. IgH mutation status was determined using PCR followed by direct sequencing. Cases with homology of ≥98% with germline sequences were considered as unmutated and cases with homologies <98% as mutated. Levels of 15 miRNAs were determined by qRT-PCR using U6 as a housekeeping gene in 28 B-SLL cases with good quality RNA. As a control we also analyzed miRNA levels in normal naïve, GC and memory B cell subsets. RNA in-situ hybridization (ISH) was used to localize the most abundantly expressed miRNAs in B-SLL tissues. Results: 16 B-CLL cases were ZAP70+ with the vast majority of tumor cells staining positive and 17 were negative. Of the ZAP70+ cases 10 carried unmutated and 3 mutated IgH genes and 3 cases failed due to bad quality DNA. 14/17 ZAP-70- cases carried mutated IgH genes and 3 cases failed. miR-150, miR-21, miR-16, miR-92 and miR-155 were expressed at high levels in all B-SLL cases independent of the ZAP70 and IgH mutation status. The miRNA expression pattern in B-SLL was very similar to normal memory B cells. RNA-ISH for BIC, the primary transcript of miR-155, demonstrated the most abundant expression in the proliferation centers of B-SLL cases. Conclusion: In B-SLL there is significant correlation between ZAP-70 expression and IgH mutation status similar to B-CLL cases. miRNA expression levels in B-SLL did not correlate with ZAP-70 or IgH status. The overall expression profile is very similar to normal memory B cells. BIC/miR-155 expression is observed specifically in the proliferation center of B-SLL tissues.

Impaired Precursor B-Cell Maturation/Production In The Bone Marrow: Association With Molecular and Immunophenotypic Markers Of Myeloid Dysplasia In Suspected Low-Grade MDS

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4943-4943
Author(s):  
Charles Repetti ◽  
Hsueh-Hua Chen ◽  
Yongbao Wang ◽  
Vanessa A Jones ◽  
Albert K Ho ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Somatic Mutation ◽  
Conflicts Of Interest ◽  
Statistical Significance ◽  
Sequence Variant ◽  
Low Grade ◽  
Precursor B Cells

Abstract Rationale Myelodysplastic syndromes (MDS) are clonal stem cell disorders that disrupt orderly maturation of multiple hematopoietic lineages. Several studies have suggested that maturation of precursor B cells (hematogones) is also abnormal in MDS. As a result, the presence of normal numbers or increased precursor B cells in bone marrow (BM) is frequently used as a diagnostic feature arguing against a diagnosis of MDS. We compared the presence of myeloid-associated gene mutations and myeloid maturation abnormalities with qualitative and quantitative precursor B cell findings in BM samples submitted for workup of cytopenias or MDS. Methods Seventeen BM aspirate samples with <5% blasts submitted for cytopenia or MDS evaluation were compared with 10 samples having 5% or more blasts and changes diagnostic of MDS or AML. Mutation analysis was performed on genomic DNA using a targeted exome sequencing assay. This assay employs a TruSeq custom amplicon design on the MiSeq platform (Illumina, San Diego, CA). The assay covers the commonly mutated areas of 19 myeloid-associated genes. Somatic mutation status was assigned based on mutation levels, previous association with myeloid neoplasia, and no prior identification in public or internal databases as a normal sequence variant. Flow cytometry using 6-color (CD19/CD34) and 8-color (CD19/10) formats was used to assess lymphoblasts; CD34/13 was used to assess myeloblasts; and CD11b, CD13, CD16, and CD38 were used to assess abnormalities in myelopoiesis. Results  Among the 17 BM samples submitted for cytopenia or MDS evaluation that had <5% blasts, 7 (41%) had immunophenotypic myeloid maturation abnormalities. Ten (59%) of the 17 cases had at least one myeloid-associated somatic mutation, with TET2 and ASXL1being the most commonly mutated genes. The ratio of myeloblasts to B-lymphoblasts, calculated using either CD10 or CD19, was >10:1 in 10/17 (59%) cases. Nine of the 17 (53%) cases had virtually no precursor B cells detected. Discrete abnormalities in more mature myeloid forms were seen in 7/10 (70%) cases with low numbers of B-lymphoblasts but in none of the 7 cases with significant numbers of B-lymphoblasts. MDS-associated mutations were more common in cases with rare B-lymphoblasts (7/9) than in those with higher percentages of precursor B cells (3/8), but the difference did not reach statistical significance (P = 0.15).  Genes mutated in the group with B-lymphoblasts present included ASXL1 (3 cases), DNMT3A (2), TET2 (1) and TP53 (2). Two of these mutated cases presented with isolated thrombocytopenia. By comparison, myeloblast/lymphoblast ratios were >50:1 in all 10 unequivocal MDS/AML samples (>5% blasts); 8 (80%) of these cases had MDS-associated mutations, and 4 (50%) had mutations in multiple genes. Conclusions Decreases in BM precursor B cells in cases of possible low-grade MDS were usually, but not always, associated with the presence of MDS-associated mutations. However, cases with normal or increased precursor B cell numbers also showed MDS-associated mutations although immunophenotypic evidence of myeloid maturation abnormalities was not seen in this group. The identification of a subgroup of cytopenic patients with likely pathogenic mutations in bone marrow precursors but minimal phenotypic evidence of myeloid dysplasia may indicate clonal abnormalities primarily located outside the granulocyte or common stem precursor populations, e.g. restricted to the megakaryocytic lineage. Therefore, the presence of intact precursor lymphoblast and myeloid maturation by higher-dimensional flow cytometry as a primary criterion to argue against a diagnosis of low-grade MDS needs further evaluation, especially when granulocytopenia is absent. Disclosures: No relevant conflicts of interest to declare.

Mutational Status of Splenic Diffuse Red Pulp Small B-Cell Lymphoma Revealed By Whole Exome Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1448-1448
Author(s):  
Nerea Martinez ◽  
Carmen Almaraz ◽  
Manuela Mollejo ◽  
Yolanda Campos-Martin ◽  
Sophia Derdak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tissue Samples ◽  
Mutational Status ◽  
Whole Exome ◽  
Red Pulp

Abstract Background: Splenic diffuse red pulp lymphoma (SDRPL) is a rare small B cell neoplasm provisionally included in a category of unclassifiable splenic B-cell lymphoma/leukemias in the 2008 WHO classification. SDRPL is characterized by a diffuse pattern of involvement of the splenic red pulp by small monomorphous B lymphocytes. Patients are normally diagnosed at stage IV when spleen, bone marrow and peripheral blood are involved. This indolent but incurable disease is more common in aged males and it shows with splenomegaly and moderate lymphocytosis. The differential diagnosis with other splenic lymphomas such as marginal zone lymphoma, hairy cell lymphoma and its variant is not always easy, due to the similar clinical presentation and the absence of specific molecular markers. Here we studied the mutational status of 15 SDRPL patients using Whole Exome Next Generation Sequencing. Methods: Genomic DNA was extracted from FFPE/FF splenic tumor or bone marrow samples. When available, DNA from oral mucosa was obtained as the corresponding non-tumor control. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). Validation of variants was performed by PCR based targeted resequencing using a MiSeq instrument (Illumina Inc., San Diego, CA). We performed paired-end-76pb whole exome sequencing on DNA from 15 SDRPL patients. The corresponding normal counterpart from 3 of the patients was sequenced. From one patient FFPE and bone marrow DNA was available for comparison. In total 9 FFPE tissue samples, 3 FF tissue samples, and 4 bone marrow samples were sequenced. Almost 95% of the selected variants were validated by PCR based resequencing in 9 of the patients, while from 6 of the patients no tissue was available for validation. Results: 290 substitutions and 26 indels were obtained after filtering. Whole exome sequencing permitted us to identify variations in several genes of relevant pathways in lymphomas, such as NFkB pathway (IkBKB, TRAF, TANK, SYK), Apoptosis (BAD, DCPS, BCLAF1), MAPK (CXCR4, TCF3, NF1, MAP3K5), Cell cycle (CCND3, POLD3, BUB1), Chromatin (CREBBP, ARID1A, ARID1B, ARID3A, MLL3), MYC regulators (AKAP10, CTCF, EP400) or WNT signaling (SALL1, WNT5B, GPC6). Moreover, CCND3 and MLL3 were recurrently mutated in 2 different patients. Genes specifically found mutated in other splenic malignancies, such as NOTCH2, BRAF, MAP2K1, and KLF2 were not found mutated in this series of SDRPL patients. Conclusions: SDRPL samples contain somatic mutations involving genes regulating relevant pathways for cell survival, such as NFkB, apoptosis, cell cycle, chromatin, or WNT. The mutational signature of the series studied here may indicate that SDRPL is a distinct entity with specific molecular features different to other lymphoid splenic malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals Small clonal B-cell population in the bone marrow as a possible tool in the diagnosis of occult primary parotid lymphoma

2016 ◽  
pp. 59-62 ◽  
Author(s):  
Martha Romero ◽  
Guido R. González-Fontal ◽  
Mónica Duarte ◽  
Carlos Saavedra ◽  
Andrés F. Henao-Martínez
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Pulmonary Thromboembolism ◽  
Clinical Findings ◽  
Small Extent ◽  
Past Medical History ◽  
Warthin Tumor ◽  
Oncocytic Papilloma ◽  
New Onset

Case Description: An 82-years old Hispanic woman with a past medical history significant for pulmonary thromboembolism on oral anticoagulation, rheumatoid arthritis, and hypertension developed a new onset thrombocytopenia. Clinical Findings: Small clonal B-cells populations (SCBP) also known as monoclonal B-cell lymphocytosis was found as part of the workup for an idiopathic thrombocytopenia and lead ultimately to the diagnosis of parotid primary follicular lymphoma coexisting with Warthin tumor involving the bone marrow in a small extent and oncocytic papilloma located in the maxillary sinus. Treatment and Outcome: Patient was treated with Rituximab monotherapy with improvement on her platelet count. Clinical relevance: Although it is unclear the role of this clonal cells, they may work as a possible diagnostic tool for occult lymphomas. Further prospective studies are needed to confirm this possible association.

Significant Differences in the IGVH and BCL6 Mutation Status in Aggressive B-Cell Lymphomas with and without MYC Breakpoints.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 161-161
Author(s):  
Christiane Pott ◽  
Heiko Trautmann ◽  
Lana Harder ◽  
Michael Kneba ◽  
Reiner Siebert ◽  
...  
Keyword(s):  
B Cell ◽  
Large Scale ◽  
Mutation Frequency ◽  
Molecular Mechanisms ◽  
Somatic Hypermutation ◽  
Direct Sequencing ◽  
Chromosomal Translocations ◽  
Coding Region ◽  
B Cell Lymphomas ◽  
Mutation Status

Abstract Somatically mutated IGVH regions are a hallmark of germinal center (GC) B-cells. Moreover, aberrant somatic hypermutation (SHM) of oncogenes, like changes in the 5′ non-coding region of the BCL6 gene occurring in 75% of DLBCL, are a mechanism of oncogene activation independently of chromosomal translocations. Large scale mutational screening using DHPLC and direct sequencing was applied to determine the somatic hypermutation status of clonal IGVH as well as several oncogenes like BCL6 and MYC in a series of more than 250 aggressive B-cell lymphomas included in the Deutsche Krebshilfe funded network “Molecular Mechanisms in Malignant Lymphoma”. Mutation patterns were correlated with the results of molecular cytogenetic analyses. MYC breakpoints detected by FISH clearly differentiated two groups of aggressive B-NHL with significantly different VH mutation status. MYC-positive lymphomas by FISH carried VH genes with a mutation frequency significantly lower compared to aggressive lymphomas lacking these features (median 4.8% vs.11.,0%, p&lt;0.0001). Similarly, the mutation frequency of BCL6 was significantly lower in MYC-positive than in MYC-negative lymphomas (median 0.13% vs 0.25% p=0,020). We observed a bias in VH gene usage in both groups with an overrepresentation of VH4 (40% in both groups) and VH3 gene (24% in MYC-positive, 40% in MYC-negative). The group of MYC-positive lymphomas was heterogeneous with regard to the pattern of chromosomal aberrations. (IGH, MYC, BCL2, BCL6, MALT1 and REL loci). Lymphomas with IG-MYC fusion lacking breakpoints in BCL2, BCL6, MALT1 or REL loci as well as non-MYC associated IGH translocations displayed a median IGVH mutation frequency of 4,8% vs. 12,2% in those cases with additional chromosomal translocations (p=0.0060). Molecular classification of aggressive B-cell lymphomas according to MYC breakpoints distinguishes subgroups with significant differences in the VH and BCL6 mutation frequencies.

Activation-Induced Cytidine Deaminase Accelerates Clonal Evolution of BCR-ABL1-Driven B Cell Lineage Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 181-181
Author(s):  
Tanja Gruber ◽  
Mi Sook Chang ◽  
Richard Sposto ◽  
Markus Müschen
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
B Cell ◽  
Genetic Instability ◽  
Tumor Suppressor Genes ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Activation Induced Cytidine Deaminase

Abstract Abstract 181 Activation-Induced Cytidine Deaminase (AID) is required for somatic hypermutation and immunoglobulin (Ig) class switch recombination in germinal center (GC) B cells. Occasionally, AID can target non-Ig genes and thereby promote GC B cell lymphomagenesis. We recently demonstrated that the oncogenic BCR-ABL1 kinase induces aberrant expression of AID in pre-B acute lymphoblastic leukemia (ALL). Compared to other ALL subtypes, BCR-ABL1 ALL is considered high risk and is characterized by a high degree of genetic instability. Because aberrant mutational activity of AID is associated with malignant transformation in B cell lymphoma, we sought to determine whether aberrant AID expression contributes to clonal evolution and genetic instability in Ph+ ALL. To investigate the function of AID expression in Ph+ ALL, we established a genetic loss-of-function model for Ph+ ALL: Bone marrow cells from AID−/− mice and AID+/+ controls were transformed by retroviral transduction with BCR-ABL1 under B lymphoid culture conditions and subsequently injected into lethally irradiated congenic recipients. Mice transplanted with AID−/−BCR-ABL1 ALL had prolonged median survival as compared to mice transplanted with leukemia cells generated from AID+/+ bone marrow (AID−/− 34 days (n=18) vs AID+/+ 13 days (n=21); p<0.0001). In secondary and tertiary transplant experiments, however, the difference between AID−/− and AID+/+BCR-ABL1 ALL narrowed as determined by a decreasing hazard ratio (from 25.5 in the primary transplant to 5.1 in the secondary and 2.9 in the tertiary transplantation). These findings suggest that aberrant AID expression accelerates clonal evolution of Ph+ ALL, but AID-independent factors exist that are sufficient for transformation. In support of enzymatic activity of AID in BCR-ABL1-transformed ALL cells, we observed that aberrant somatic hypermutation of non-immunoglobulin genes in these leukemias was largely dependent on AID: mutations in the known hypermutation target genes Pax5 and Rhoh were increased in AID+/+ but not AID−/−BCR-ABL1 ALL cells. Mutations in the first intron of Rhoh as observed here are relevant because they interfere with Rhoh transcription. Indeed, we found that Rhoh mRNA levels are significantly higher in AID−/− compared to AID+/+BCR-ABL1 ALL cells. Rhoh is a hematopoietic specific GTPase that negatively regulates Rac-mediated signaling downstream of the oncogenic BCR-ABL1 kinase. AID-dependent mutation and transcriptional inactivation of Rhoh in BCR-ABL1 ALL therefore likely augments oncogenic BCR-ABL1 signaling. Consistent with a causative role of AID in genetic instability, AID−/− leukemia had a lower frequency of amplifications (17+2 vs 45+7; p=0.002) and deletions (11+2 vs 40+7; p=0.003) as compared to AID+/+ leukemias. AID−/− and AID+/+ ALL cells showed a markedly distinct gene expression pattern with 2,365 differentially expressed genes (p=0.003; FDR 0.05). A detailed analysis of these differences in gene expression revealed that AID−/−BCR-ABL1 ALL cells failed to downregulate a number of tumor suppressor genes including p53, Rhoh, Cdkn1a (p21), and Blnk (SLP65). AID-dependent downregulation of p53 in BCR-ABL1 ALL cells is of particular importance, because previous work demonstrated that transcriptional repression of p53 in normal GC B cells is required to make these cells permissive to high levels of AID expression. AID-induced DNA damage would otherwise activate p53 and rapidly induce apoptosis. Compared to AID-deficient BCR-ABL1 ALL, AID+/+BCR-ABL1 ALL cells are more resistant to Imatinib-treatment. However, acquisition of BCR-ABL1 kinase domain mutations does not appear to be the main cause of drug-resistance in this experiment, since only one relevant mutation was amplified from AID+/+ ALL cells (no mutations in AID−/− ALL cells). We conclude that AID accelerates clonal evolution in BCR-ABL1 ALL by enhancing genetic instability, aberrant somatic hypermutation, and by negative regulation of tumor suppressor genes. Disclosures: No relevant conflicts of interest to declare.

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