Immunoglobulin Gene Translocation Research Articles

Myeloma immunoglobulin rearrangement and translocation detection through targeted capture sequencing.

Multiple myeloma is a plasma cell neoplasm characterized by clonal immunoglobulin V(D)J signatures and oncogenic immunoglobulin gene translocations. Additional subclonal genomic changes are acquired with myeloma progression and therapeutic selection. PCR-based methods to detect V(D)J rearrangements can have biases introduced by highly multiplexed reactions and primers undermined by somatic hypermutation, and are not readily extended to include mutation detection. Here, we report a hybrid-capture approach (CapIG-seq) targeting the 3' and 5' ends of the V and J segments of all immunoglobulin loci that enable the efficient detection of V(D)J rearrangements. We also included baits for oncogenic translocations and mutation detection. We demonstrate complete concordance with matched whole-genome sequencing and/or PCR clonotyping of 24 cell lines and report the clonal sequences for 41 uncharacterized cell lines. We also demonstrate the application to patient specimens, including 29 bone marrow and 39 cell-free DNA samples. CapIG-seq shows concordance between bone marrow and cfDNA blood samples (both contemporaneous and follow-up) with regard to the somatic variant, V(D)J, and translocation detection. CapIG-seq is a novel, efficient approach to examining genomic alterations in myeloma.

Colorectal diffuse large B-cell lymphoma: molecular subclassification and prognostic significance of immunoglobulin gene translocation

Primary colorectal diffuse large B-cell lymphoma (DLBCL) is rare, and its clinicopathological and genetic features are poorly understood. The aim of our study was to elucidate the frequency and prognostic significance of molecular subgroups in colorectal DLBCL. We examined 25 cases of colorectal lymphoma with DLBCL-like morphology and classified them into germinal center B-cell like (GCB)/non-GCB subgroups by immunohistochemistry (IHC) for CD10, bcl-6 and MUM1, or into double-expressor (DE)/non-DE subgroups by IHC for bcl-2 and c-myc. Translocations involving BCL2, BCL6, MYC, IGH, IGK, IGL, and MALT1 were also investigated using break-apart fluorescence in situ hybridization (FISH). The 25 cases were classified into two entities-DLBCL, not otherwise specified (NOS) (n = 23; 92%) and high grade B-cell lymphoma, double hit (n = 2; 8%)-according to the recent WHO classification. None of them showed histological evidence of Epstein-Barr virus infection or high-grade transformation from low grade B-cell lymphoma. Ten cases were GCB-type and four cases were DE-type, but these subtypes did not contribute to clinicopathological differences. Translocations involving BCL2, BCL6, MYC, IGH, IGK, IGL, and MALT1 were detected in 3 (12%), 3 (12%), 10 (40%), 14 (56%), 3 (12%), 3 (12%), and 0 (0%) of 25 cases, respectively. Of note, the presence of IGH translocation was significantly associated with better overall survival (P = .0053) and progression free survival (P = .0259). Similarly, the translocation involving at least one of the IGs (IGH, IGK, and/or IGL) was associated with more favorable prognosis in DLBCLs or even in DLBCL, NOS. This is the first report to reveal that a small subset of colorectal DLBCL corresponds to double-hit lymphoma. In addition, translocations involving at least one of the IGs may be a favorable prognostic factor in colorectal DLBCL. Testing the translocation involving rearrangement of IGs as well as MYC and BCL2/BCL6 may thus be useful for diagnosis and prognosis.

Immunoglobulin gene translocations in chronic lymphocytic leukemia: A report of 35 patients and review of the literature.

Chronic lymphocytic leukemia (CLL) represents the most common hematological malignancy in Western countries, with a highly heterogeneous clinical course and prognosis. Translocations involving the immunoglobulin (IG) genes are regularly identified. From 2000 to 2014, we identified an IG gene translocation in 18 of the 396 patients investigated at diagnosis (4.6%) and in 17 of the 275 analyzed during follow-up (6.2%). A total of 4 patients in whom the IG translocation was identified at follow-up did not carry the translocation at diagnosis. The IG heavy locus (IGH) was involved in 27 translocations (77.1%), the IG κ locus (IGK) in 1 (2.9%) and the IG λ locus (IGL) in 7 (20.0%). The chromosome band partners of the IG translocations were 18q21 in 16 cases (45.7%), 11q13 and 19q13 in 4 cases each (11.4% each), 8q24 in 3 cases (8.6%), 7q21 in 2 cases (5.7%), whereas 6 other bands were involved once (2.9% each). At present, 35 partner chromosomal bands have been described, but the partner gene has solely been identified in 10 translocations. CLL associated with IG gene translocations is characterized by atypical cell morphology, including plasmacytoid characteristics, and the propensity of being enriched in prolymphocytes. The IG heavy chain variable region (IGHV) mutational status varies between translocations, those with unmutated IGHV presumably involving cells at an earlier stage of B-cell lineage. All the partner genes thus far identified are involved in the control of cell proliferation and/or apoptosis. The translocated partner gene becomes transcriptionally deregulated as a consequence of its transposition into the IG locus. With the exception of t(14;18)(q32;q21) and its variants, prognosis appears to be poor for the other translocations. Therefore, searching for translocations involving not only IGH, but also IGL and IGK, by banding and molecular cytogenetics is required. Furthermore, it is important to identify the partner gene to ensure the patients receive the optimal treatment.

Molecular pathogenesis of multiple myeloma: basic and clinical updates

Multiple myeloma is divided into two distinct genetic subtypes based on chromosome content. Hyperdiploid myeloma is characterized by multiple trisomies of chromosomes 3, 5, 7, 9 11, 15, 19 and 21, and lacks recurrent immunoglobulin gene translocations. Non-hyperdiploid myeloma in contrast is characterized by chromosome translocations t(4;14), t(14;16), t(14;20), t(6;14) and t(11;14). A unifying event in the pathogenesis of multiple myeloma is the dysregulated expression of a cyclin D gene, either directly by juxtaposition to an immunoglobulin enhancer, as a result of ectopic expression of a MAF family transcription factor, or indirectly by as yet unidentified mechanisms. Secondary genetic events include rearrangements of MYC, activating mutations of NRAS, KRAS or BRAF, a promiscuous array of mutations that activate NFkB and deletions of 17p. Among the poor-risk genetic features are t(4;14), t(14;16), t(14;20), del 17p and gains of 1q. Available evidence supports the use of a risk-stratified approach to the treatment of patients with multiple myeloma, with the early and prolonged use of bortezomib particularly in patients with t(4;14) and del 17p.

Many Multiple Myelomas: Making More of the Molecular Mayhem

Multiple myeloma (MM) is malignancy of isotype-switched, BM-localized plasma cells that frequently results in bone destruction, BM failure, and death. Important molecular subgroups are identified by three classes of recurrent immunoglobulin gene translocations and hyperdiploidy, both of which affect disease course. From a clinical standpoint, it is critical to identify MM patients carrying the t(4;14) translocation, which is present in 15% of myelomas and is associated with dysregulation of WHSC1/MMSET and often FGFR3. These patients should all receive bortezomib as part of their initial induction treatment because this has been shown to significantly prolong survival. In contrast, patients with translocations affecting the MAF family of transcription factors, del17p, or gene-expression profiling (GEP)-defined high-risk disease appear to have a worse prognosis that is not dramatically improved by any intervention. These patients should be enrolled in innovative clinical trials. The remaining patients with cyclin D translocations or hyperdiploidy do well with most therapies, and the goal should be to control disease while minimizing toxicity.

Abstract PL3-1: Biology of myeloma: Basis for novel drugs

Abstract The outcome of Multiple Myeloma (MM) patients has significantly improved over the last decade, and this is mainly due to the efficacy of novel drugs, such as Thalidomide, Lenalidomide, and Bortezomib. Nevertheless, the majority of patients relapses and become eventually refractory to all available treatments. Therefore, drugs with novel mechanism of action are urgently needed in order to improve the outcome of relapsed MM patients. The increase knowledge in MM biology is already contributing to a more specific drug design, and we have recently learned that in the pathogenesis of MM, as important as the malignant cells themselves, is their interaction with the microenvironment. MM requires a multistep transformation process that implies the sequential generation of primary Ig translocations, chromosomal instability (including mutations –i.e: RAS-, and deletions – i.e: RB-), and secondary translocations. Most primary immunoglobulin gene translocations occur early in the pathogenesis of MM. These translocations, which are mediated by errors in immunoglobulin heavy-chain switch recombination, result in the juxtaposing of an immunoglobulin enhancer and oncogene. On the basis of IgH translocations, MM patients can be divided into 5 subgroups: 1) D-type cyclins: Cyclin D1 on 11q23, Cyclin D3 on 6p21 and Cyclin D2 on 12p13 (25% of cases); 2) MMSET/FGFR3 proteins (4p16.3) (15%); 3) B-zip transcription factors: c-maf on 16q23 and mafB on 20q11 (15%); 4) other IgH translocations (20%); and 5) No IgH translocations (25%). Secondary oncogenic events may involve genes different from the Ig locus, as well as the 14q32 region, as occur in the c-myc translocations: t(8;14), t(2;8), t(8;22), t(14;20). Some of these molecular events represent potential therapeutic targets. Thus the t(4;14) translocation generates a constitutive activation of the oncogenic receptor tyrosine kinase FGFR3 with subsequent phosphorylation of the antiapoptotic STAT3 signalling pathway. Therefore, the use of inhibitors of the FGFR3 tyrosine kinase as well as inhibitors of cyclin dependent kinases could be attractive therapeutic targets. Similarly C-maf, that is over expressed in MM patients with t(14;16) as well as in some MM cases lacking this translocation, also represents a potential target. The second area of MM pathogenesis that has important implications for treatment intervention is the interaction between the malignant cell and the bone marrow microenvironment. MM cells adhere to the extra cellular matrix proteins and bone marrow stromal cells through a series of adhesion molecules, such as the β1-integrin family (VLA-4, VLA-5 and VLA-6) as well as ICAM-1 and VCAM-1. Adhesion of myeloma cells to BM microenvironment induces a cell-adhesion-mediated drug resistance. Interruption by downregulating the interactions between the tumor cell and its microenvironment can potentially halt MM cell growth and proliferation, and be of benefit to patients with MM. The binding of MM cell to BM microenvironment also induces the transcription and secretion of cytokines (TNFa, IL-6, IGF-1, SDF1a, VEGF), by both the plasma cells and BMSC, triggering signalling pathways (such as the RAF/MEK/MAPK, PI3K/AKT, and JAK/STAT pathways), that promote cell proliferation and prevent apoptosis. In this area several targeted oriented drugs are already at early phases of clinical investigation, and these includes: 1. Agents against receptors present in plasma cells (PC) which can be targeted by specific drugs or monoclonal antibodies: cell death receptors (such as DR4-6, FAS and BCL2); TK receptors, VEGFR; TACI, IL6-R, IGF-1R or the CD56 or CD40 antigens; 2. Inhibitors directed to signalling pathways including Farnesil Transferase (Tipifarnib), RAF (RAF 265), STAT3 (Atiprimod), mTor (RAD001) or AKT (Perifosine); and 3. Drugs that interfere with the interactions between PC and microenvironment such as the new Proteasome inhibitors, such as NPI-0052 and PR-171 (Carfilzomib), novel IMIDs (Pomalidomide), HDAC and Hsp90 inhibitors. Unfortunately, the expectations raised by some of these agents have not been so far confirmed in the clinic. It is probable that these targeted directed drugs will be more effective in science based combinations with other agents which have already shown clear efficacy in MM. Citation Information: Clin Cancer Res 2010;16(7 Suppl):PL3-1

Immunoglobulin Gene Translocations Are Frequent in Unclassifiable CD5+ Small B-Cell Leukemias and in Particular, a Novel Subtype Characterised by t(14;19)(q32;q13) and Deregulation of BCL3 Is Described.

We describe the features of 24 cases of atypical CD5+ small B-cell leukaemia unclassifiable by current methods. The male/female ratio was 3 (18M/6F), with a median age at diagnosis of 58 years (range 39–84 years). All cases were CD5 positive, but with an immunophenotype and morphology atypical for CLL. All cases were negative for t(11;14) and other B cell malignancies were excluded. FISH analysis was carried out using probes for IGH break apart, t(14;19) involving BCL3, trisomy 12, 13q14, 11q22–23 (ATM) and 17p13 (p53 locus). IgVH somatic mutations were analysed using direct sequencing (2% cut off). FISH using IgH break apart probe showed 8 (32%) cases with a split at 14q32. These 8 cases were further investigated using a FISH probe for t(14;19). Four cases (16%) exhibited t(14;19) ie. showed the IgH/BCL3 rearrangement in >70% of cells. Further characterisation of the 24 cases, at additional loci showed that 4 (16%) patients had 17p deletion, 3 (12%) 11q deletion, 3 (12%) 13q deletion, and 14 cases (56%) trisomy 12. The 8 cases showing a split IgH signal did not show deletions of 17p, 11q, or 13q but trisomy 12 was present in 7/8 (88%) patients. Sixteen out of 24 (67%) cases had unmutated IgVH genes with VH3 and VH4 families being used in 91% (22/24). Interestingly, unlike in chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL), the VH1 family was not used in this group of patients. All 4 cases with t(14;19) showed unmutated IgVH genes. Of the sixteen patients without involvement of 14q32, only 2 patients have died with a median follow up of 5.5 years. Of the 4 cases with the t(14;19) (IgH/BCL3 rearrangement), 3 required treatment within 2.5 years of diagnosis and two out of four patients died 5 years after diagnosis, following Richter's transformation suggesting that this rearrangement is a poor prognostic factor and defines a group with a distinct clinical outcome. Our findings show a high frequency of 14q abnormalities in atypical CD5+ small B-cell leukaemia. BCL3 rearrangement is seen in a significant subset of these cases. The BCL3 gene encodes a nuclear protein that belongs to the IkB family of inhibitors of NFkB. It is involved in the regulation of a variety of transcription factors and it is able to stimulate the expression of cyclin D1. The dysregulation of BCL3 via its juxtaposition to the regulatory element of the IgH locus in our cases suggests a pathogenic role for BCL3 in these B cell disorders and may define a group of patients with a more aggressive clinical course. Further, we have examined these cases for signatures, defined in myeloma, characteristic of NFkB deregulation. We suggest that in cases of CD5 positive B-cell disorders with an immunophenotype and morphology atypical for CLL, and negative for t(11;14), rearrangements involving the IgH locus are frequent and have allowed us to identify cases with poor prognosis and with a pathogenesis that involves deregulation of NFkB.

Waldenström Macroglobulinaemia: The Impact of Cytogenetic Analysis.

Background Waldenström macroglobulinaemia (WM) is an uncommon indolent B-cell lymphoproliferative characterized by bone marrow infiltration by lymphoplasmacytic cells and an IgM paraproteinemia. This study analyzed the impact of cytogenetics in WM and the pattern of evolution in 73 cases from the Mitelman database and 14 cases evaluated at the Mayo Clinic Arizona (MCA).Patients and Methods The Mitelman database of Chromosome aberrations in cancer (Mitelman et al., 2007) was searched, and 73 WM karyotypes were selected. We also evaluated 14 cases seen at MCA with karyotype analysis at diagnosis. An Excel spreadsheet in which the rows of the first column were labeled to represent each band of a 360-band human chromosome ideogram was created as previously described (Hoglund et al., 2001; Horsman et al., 2003) All karyotypes were scored individually in successive columns for net gain and loss of each chromosome band (loss=0 and gain=1). Recurrent abnormalities were defined as those that were involved in >5% of cases. Histograms of the number of abnormalities per tumor (NAPT) were then plotted, and the modal value extracted as variable called time to occurrence. Statistics correlation between the presence and absence of different abnormalities was analyzed using Spearman correlation matrices. P value of <0.05 was considered as statistical significanResults In total 73 cases of newly diagnosed WM from the Mitelman database were included in the analysis. The median chromosome number was 46 (Range 40 to 49).The most common abnormalities were loss of 6q21 (10%) (Fig 1), +3 (7.1%), loss of X (6%) and −10, −20, −21, +12, +18 and +5 (5.71% respectively). The most common translocation was t(8;14) (4%).The most recurrent breakpoints in our series include: 14q32 (6%), 6q21 (5%), 8q24 (5%), 18q21 (4%) and 19q13 (4%). In the MCA cohort, 14 karyotypes were reviewed and only 3 cases exhibited an abnormal result. Deletion of 6q21, 20q11 and a Robertsonian translocation t(13;14) were reported (3/11, 21%). In the 6q21 case, FISH analysis confirmed the deletion of BLIMP1 on 6q. One case exhibited a 20q deletion in a WM with kidney amyloidosis patient.Discussion In this comprehensive analysis we show a very small complexity of recurrent abnormalities expressed by conventional karyotype analysis in WM. The incidence of 6q21 deletion was 10% by conventional cytogenetics and it is been reported as high as 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Interestingly we report the case of a patient with 20q deletion at diagnosis in the MCA cohort. To date, only a limited number of cases with del(20q) as a sole cytogenetic anomaly have been reported in plasma cell dyscrasias, either at diagnosis or later in the disease course. The recurrent immunoglobulin gene translocation is known to be involved in the initial oncogenetic events in plasma cell dyscrasias and, so it is important to identify whether there are any masked translocations involving immunoglobulin genes and 20q in these cases. [Display omitted]

Treatment of Newly Diagnosed Multiple Myeloma Based on Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART): Consensus Statement

Treatment of Newly Diagnosed Multiple Myeloma Based on Mayo Stratification of Myeloma and Risk-Adapted Therapy (mSMART): Consensus Statement

Early Genetic Events Provide the Basis for a Clinical Classification of Multiple Myeloma

Multiple myeloma is a tumor of somatically mutated, isotype-switched plasma cells that accumulate in the bone marrow leading to bone destruction and bone marrow failure. The germinal center processes of somatic hypermutation and switch recombination are implicated in the development of recurrent immunoglobulin gene translocations in 40% of patients. These affect five loci: 11q13, 6p21, 4p16, 16q23 and 20q11, leading to dysregulation of CCND1, CCND2, FGFR3/MMSET, c-MAF and MAFB respectively. The remaining 60% of patients can be divided into four groups based on their expression of CCND1 and CCND2. The largest group (40%) ectopically express CCND1 bi-allelically and have hyperdiploidy with multiple trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21. The translocation and cyclin D (TC) groups identify patients with different genetics, biology, clinical features, prognosis and response to therapy.

B-cell activation and lymphoma in patients with HIV.

The risk of developing non-Hodgkin lymphoma (AIDS lymphoma) is greatly increased in HIV infection. Disruption of immune function by HIV infection may contribute to lymphomagenesis by inducing (1) loss of immunoregulation of Epstein-Barr virus-infected B cells [immunoblastic and central nervous system (CNS) lymphoma] caused by loss of T-cell function, and (2) chronic B-cell hyperactivation enhancing the generation of genetic lesions (c- :immunoglobulin gene translocation, -6 overexpression) associated with some forms of AIDS lymphoma (Burkitt lymphoma-like small noncleaved cell lymphoma and large noncleaved cell lymphoma). Also, the overproduction of B-cell-stimulatory cytokines (interleukin 10 and 6) has the potential to contribute to tumor development by supporting the growth and viability of nascent lymphoma cell clones. Therefore, HIV infection-associated B-cell hyperactivation, including direct activation of B cells by various mechanisms, and chronic overproduction of B-cell-stimulatory cytokines have the potential to contribute to the development and growth of AIDS lymphoma. Several recent reports are discussed in this review, including recent work relevant to understanding the potential of a virus-encoded cytokine-like molecule, HHV8 vIL6, to induce B-cell hyperactivation in HIV-infected people, work pointing to the potential role of a chemokine (stromal cell-derived factor 1) in lymphomagenesis, and studies on phenotypic changes in circulating B cells in HIV infection.