Identification of chromosomal breakpoints of cancer-specific translocations by rolling circle amplification and long-distance inverse PCR

We describe the use of rolling circle amplification and long-distance inverse polymerase chain reaction (LD-PCR) to identify chromosomal breakpoints and fusion genes in cancer cells carrying acquired translocations. This approach produced enough template for 100 inverse PCR reaction from as little as 20 ng of patient DNA, consequently enabling the use of up to 500 times less patient DNA compared to standard inverse PCR. The method is based on identifying restriction sites in a putative breakpoint area in a cancer-specific translocation, followed by circularization and amplification of the restriction DNA products by using T4 DNA ligase and Phi29 enzyme, respectively. The amplified DNA thus obtained is used as a template in long-distance inverse PCR to amplify and detect the precise breakpoint of the chromosomal rearrangements in question by sequencing of the obtained PCR products. We demonstrate the feasibility of this approach by identifying fusion genes TAF15-ZNF384 (brought about by a (12;17)(p13;q21) translocation) and BCR-ABL1 (produced by a (9:22)(q34;q11.2) translocation) in five leukemia samples. The application of rolling circle amplification before inverse PCR may be particularly useful in the search for chromosomal breakpoints and fusion genes brought about by new translocations when only minute amounts of DNA are available from the sampled malignant lesion.     

Isolation and characterisation of recombination events involving immunoglobulin heavy chain switch regions in multiple myeloma using long distance vectorette PCR (LDV-PCR).

Immunoglobulin class switching occurs as a result of recombination between pairs of switch region sequences located 5' to each constant heavy chain gene except Cdelta. In the B cell neoplasm multiple myeloma, tumour cells have generally undergone class switching and often contain oncogenic sequences translocated into switch regions, presumably as a result of aberrant switch recombination. We have developed a method (LDV-PCR) which combines long distance PCR with one-sided vectorette PCR that is capable of detecting and isolating both normal and aberrant switch recombination breakpoints from multiple myeloma cell lines and primary multiple myeloma tumour material. Using LDV-PCR we have directly cloned the translocation breakpoints present in two multiple myeloma cell lines and isolated a normal productive switch recombination event from a primary tumour. Furthermore, we have isolated a novel translocation t(14;22)(q32;q12) from a primary tumour sample and have demonstrated that internal deletions within switch regions can occur in multiple myeloma cells. Compared to a Southern blotting approach, LDV-PCR is simpler and more rapid to perform, allows the simultaneous detection and isolation of recombination events, and can also be applied to amounts of DNA which are too low to permit the conventional cloning of recombination breakpoints.     

DNA sequence of immunoglobulin heavy chain variable region gene in thyroid lymphoma.

Patho-epidemiological studies have shown that thyroid lymphoma (TL) develops in thyroid affected by chronic lymphocytic thyroiditis (CLTH). CLTH is categorized as an organ-specific autoimmune disease, in which activated B-lymphocytes secrete a number of autoantibodies. Because antigenic stimulation might be involved in the pathogenesis of TL, the variable region in heavy chain (V(H)) genes was characterized in 13 cases with TL and 3 with CLTH. Clonal rearrangement of the V(H) gene was found in 11 cases of TL, and cloning study with sequencing of complimentary determining region (CDR) 3 revealed the presence of a major clone in 4. Three of the 4 cases used V(H) 3 gene, with the homologous germline gene of V3-30 in two cases and VH26 in one case. A biased usage of V(H) 3 and V(H) 4 genes with the homologous germline gene of VH26 in V(H) 3 gene was reported previously in cases with CLTH. A high level of somatic mutation (1-21%, average 12%) with non-random distribution of replacement and silent mutations was accumulated in all cases. The frequency of the occurrence of minor clones ranged from 29-44% per case, indicating the presence of on-going mutation. DNA sequencing of immunoglobulin V(H) gene suggests that TL develops among activated lymphoid cells in CLTH at the germinal center stage under antigen selection.     

Analysis of clonal immunoglobulin heavy chain rearrangements in ocular lymphoma

BACKGROUND: The morphologic diagnosis of primary and metastatic intraocular lymphoma (IOL) was made difficult by the paucicellular specimens with fragile populations of lymphocytes retrieved through pars plana vitrectomy (PPV). The analysis of immunoglobulin heavy chain (IgH) gene rearrangements (AIGHR) was used as an adjunct to cytopathology and flow cytometry in systemic lymphoma. In IOL, the sensitivity and specificity of AIGHR are unknown. METHODS: The authors reviewed the clinical records of patients who underwent PPV for suspicion of IOL at the Immunology and Uveitis Service of the Massachusetts Eye and Ear Infirmary (Boston, MA) between 2000 and 2002. AIGHR was performed as a routine diagnostic test on cell lysates isolated from < 0.5 mL of vitreous fluid. The authors used seminested polymerase chain reaction (PCR) with consensus primers for the VDJ-region of the IgH gene. PCR products were analyzed by polyacrylamide gel electrophoresis. RESULTS: Thirty patients (37 specimens) with chronic vitritis and 17 patients (23 specimens) with IOL were included. The specificity of vitreous fluid cytopathology, flow cytometry, and AIGHR was 1.0, and the sensitivity values were 0.24, 0.36, and 0.64, respectively. AIGHR was negative in two patients for whom cytopathology or flow cytometry revealed the diagnosis of lymphoma. Clonal IGHR was found in four specimens classified as negative for lymphoma based on cytopathology and flow cytometry. CONCLUSIONS: AIGHR supplemented cytopathology and flow cytometry to increase the diagnostic yield in IOL.     

Plasma vitamin D-binding protein (GC) factors,immunoglobulin G heavy chain (GM) allotypes and immunoglobulin kappa light chain (KM1) allotype in patients with sarcoidosis and in healthy control subjects

BACKGROUND AND AIM: Sarcoidosis is an immune disease with abnormalities in the production of vitamin D and immunoglobulins. The aim was to examine whether the distribution of plasma vitamin D-binding protein = group-specific component (GC) allotypes, immunoglobulin G heavy chain (GM) allotypes and immunoglobulin kappa light chain (KM) allotype differed significantly from the distribution in healthy subjects. METHODS: GC 1S, 1F, 2 allotypes, GM 1, 2, 5 allotypes, and KM1 allotype were assessed in 44 patients with sarcoidosis and in healthy control subjects. RESULTS: There were no significant differences between the frequencies of the GC, GM and KM allotypes in sarcoidosis patients and in control subjects. Furthermore, there was no relationship between the presentation or course of the sarcoid disease and GC, GM or KM allotypes. CONCLUSIONS: GC, GM and KMI allotypes do not appear to play any major role in the pathogenesis of sarcoidosis.     

Detection of immunoglobulin gene rearrangement in B-cell lymphomas by polymerase chain reaction gene amplification.

This is a report on our attempt to use polymerase chain reaction (PCR) to detect rearrangement of the immunoglobulin gene in the tissue specimens obtained from 30 patients with non-Hodgkin's lymphomas. There were 20 B-cell lymphomas and 10 T-cell. All 20 B-cell lymphomas but none of the 10 T-cell lymphomas had JH rearrangement by Southern analysis. Two pairs of primers (V670/OL-4 and VH26/OL-4) were designed to amplify the CDR3 region of the immunoglobulin gene heavy chain. The PCR analysis was positive using either one or both pairs of primers in 11 of the the 20 cases (55 per cent) of B-cell lymphomas which all had positive rearrangement by Southern analysis. The two pairs of primers seemed to produce complementary results as the specimens may be positive to one pair but negative to the other. The false negative rate of 45 per cent is however much higher than the respective figures of 18 per cent and 0 per cent observed in our patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia in a previous study. Peripheral blood and bone marrow biopsy specimens obtained at the time of initial diagnosis were available from 10 patients with B-cell lymphomas whose lymph node biopsy specimens at the time of diagnosis were positive by both Southern analysis and PCR. All these peripheral blood and marrow specimens had no microscopic evidence of involvement by lymphoma cells and JH rearrangement was not detected by Southern analysis. However, rearranged bands identical to that of the lymph node biopsy specimen were detected by PCR in the peripheral and marrow blood of one of them. This PCR technique has been shown to have a sensitivity of 0.1 per cent in our previous report and may be more useful than morphology alone or Southern analysis in detecting minimal lymphomatous involvement in the peripheral blood and bone marrow at the time of initial diagnosis. Further clinical correlation is required to confirm the finding.     

Rapid and sensitive nonradioactive detection of mutant K-ras genes via 'enriched' PCR amplification.

We have developed a rapid and highly sensitive nonradioactive method for the detection of a mutant codon 12 human c-K-ras allele in the presence of as many as 10(4) copies of the wild type codon 12 allele. This sensitivity is achieved by selective polymerase chain reaction (PCR) amplification of mutant K-ras gene sequences employing a two stage procedure. The first stage entails the amplification of both K-ras mutant and wild type codon 12 sequences, followed by a selective restriction enzyme digestion of only wild type sequences. The second stage involves a subsequent amplification of undigested amplified fragments, enriched in mutant codon 12 sequences. These products are subject to restriction length polymorphism analysis for the detection of point mutations at codon 12. This technique is rapid, nonradioactive, and eliminates the need for either oligonucleotide hybridization or DNA sequencing. Variations of this selective amplification procedure may prove promising for the detection of specific point mutations in heterogenous cell populations.     

Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.     

T-lymphoblastic lymphoma terminating as malignant histiocytosis with rearrangement of immunoglobulin heavy chain gene.

A 19-year-old man presented with cutaneous, mediastinal and intrapleural localization of a T-lymphoblastic non-Hodgkin's lymphoma (NHL) of immature phenotype. Two weeks after mediastinal irradiation the T-lymphoblasts had disappeared from the pleural effusion, but a clonal monocytic cell population was detected, as documented by immunological marker analysis and the presence of t(10;11), a cytogenetic aberration often associated with monocytic malignancies. Intensive chemotherapy induced a complete remission of the T-lymphoblastic NHL. However, the patient died from massive infiltration of lympho-hemopoietic tissue by cells with the morphology and immunological phenotype of macrophages. Southern blot analysis revealed the presence of a clonally rearranged immunoglobulin heavy chain (lgH) gene in tumorous tissue obtained at autopsy. The same clonally rearranged lgH was detectable in the post-irradiation pleural fluid 2 weeks after initial diagnosis. The observed germline configuration of T-cell receptor beta-genes and both lg light chain genes in this monoclonal proliferation provides additional evidence for the true histiocytic nature of the fatal disease. Therefore we conclude that a true histiocytic NHL with one rearranged lgH gene was most probably already present at initial diagnosis when the patient presented with the T-lymphoblastic NHL and that this true histiocytic NHL further developed despite the cytostatic treatment.     

Treatment of immunoglobulin light chain (primary or AL) amyloidosis

Not all forms of amyloidosis are systemic. Some patients may present with a localized form and should not be treated with chemotherapy. Some patients with systemic amyloidosis may have secondary, familial, or dialysis-related types.These types are not responsive to chemotherapy. Immunoglobulin light chain (primary or AL) amyloidosis is a plasma cell dyscrasia. Suppression of light chain production translates to organ response, improved organ function, and improved quality of life. This review of the various available options for the treatment of systemic amyloidosis is designed to help the clinician determine which patients are candidates for stem cell transplantation and which should be treated with conventional chemotherapy. The role of the recently introduced novel agents in management of amyloidosis is also reviewed.     

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines.

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.     

Mechanisms of disease: monoclonal immunoglobulin deposition. Amyloidosis, light chain deposition disease, and light and heavy chain deposition disease.

All forms of MIDD are related to the presence of an expanded clone of B-cell origin that is producing an Ig product, usually, but not exclusively an L-chain, which is predisposed to deposit in tissues, with or without some degree of processing. The nature of the processing is currently unclear, although limited proteolysis is likely to play a major role in most, but not all, patients. Diagnosis is made by the identification, using immunohistochemical techniques, of the monoclonal Ig nature of the deposited material, which may be fibrillar and Congo red-positive (AL and AH), or more amorphous and Congo red-negative (LCDD and LCHDD). Present modalities of therapy are similar or identical to those employed in multiple myeloma, attempting to eliminate the monoclonal cell population responsible for the production of the precursor of the deposited protein. A variety of ancillary therapeutic measures may be employed to treat problems associated with the failure of specific organs produced by the deposition. The details of how the uniformly soluble precursor molecule is converted to an essentially insoluble aggregate that compromises the function of the tissue in which it is formed are not yet known. It is still not possible to construct a potential "unified field theory" governing the deposition of intact Igs or their fragments. It is likely, as appears to be the case in other forms of amyloid unrelated to Ig, that many proteins contain, within their sequence, peptides that are capable of forming insoluble beta sheet-like structures. When these peptides are isolated from their surrounding molecular environment--either by proteolysis in the test tube, by a mutational change that predisposes them to limited proteolysis; or by a point mutation, deletion, or some other structural modification (as glycosylation), which alters their molecular context without proteolysis--and are present in sufficient concentration, they become less soluble under physiologic conditions. It is likely that the site of deposition depends upon the site of synthesis, but to a lesser extent than the protease profile and the physicochemical make-up of the affected tissues. Better understanding of the latter factors is necessary for the development of better modes of treatment.     

Analysis of immunoglobulin heavy and light chain variable genes in post-transplant lymphoproliferative disorders

Post-transplant lymphoproliferative disorders (PTLD) derive from antigen-experienced B-cells and represent a major complication of solid organ transplantation. We characterized usage, mutation frequency and mutation pattern of immunoglobulin variable (IGV) gene rearrangements in 50 PTLD (polymorphic PTLD, n=10; diffuse large B-cell lymphoma, n=35; and Burkitt/Burkitt-like lymphoma, n=5). Among PTLD yielding clonal IGV amplimers, a functional IGV heavy chain (IGHV) rearrangement was found in 40/50 (80.0%) cases, whereas a potentially functional IGV light chain rearrangement was identified in 36/46 (78.3%) PTLD. By combining IGHV and IGV light chain rearrangements, 10/50 (20.0%) PTLD carried crippling mutations, precluding expression of a functional B-cell receptor (BCR). Immunohistochemistry showed detectable expression of IG light chains in only 18/43 (41.9%) PTLD. Failure to detect a functional IGV rearrangement associated with lack of IGV expression. Our data suggest that a large fraction of PTLD arise from germinal centre (GC)-experienced B-cells that display impaired BCR. Since a functional BCR is required for normal B-cell survival during GC transit, PTLD development may implicate rescue from apoptosis and expansion of B-cells that have failed the GC reaction. The high frequency of IGV loci inactivation appears to be a peculiar feature of PTLD among immunodeficiency-associated lymphoproliferations.     

Identification of cryptic aberrations and characterization of translocation breakpoints using array CGH in high hyperdiploid childhood acute lymphoblastic leukemia.

High hyperdiploidy, characterized by non-random trisomies, is the largest cytogenetic subgroup in childhood acute lymphoblastic leukemia (ALL). It is not known whether the gained chromosomes are sufficient for leukemogenesis or if additional genetic aberrations are necessary. However, the suboptimal chromosome morphology of hyperdiploid ALLs makes detection of structural abnormalities difficult if using cytogenetic techniques; alternative methods are, therefore, needed. We performed array comparative genome hybridization (CGH) analyses, with a resolution of 100 kb, of eight cases of high hyperdiploid childhood ALL to characterize structural abnormalities found with G-banding/multicolor fluorescence in situ hybridization (FISH) and to detect novel changes. The non-centromeric breakpoints of four rearrangements, including three translocations and one 1q duplication, were narrowed down to <0.2 Mb. Furthermore, four submicroscopic imbalances involving 0.6-2.7 Mb were detected, comprising two segmental duplications involving 1q22 and 12q24.31 in one case and two hemizygous deletions in 12p13.2-31 - including ETV6 - and in 13q32.3-33.1 in another case. Notably, FISH analysis of the latter revealed an associated reciprocal t(3;13)(q?;32.2-33.1). In conclusion, the array CGH analyses revealed putative leukemia-associated submicroscopic imbalances and rearrangements in 2/8 (25%) hyperdiploid ALLs. The detection and characterization of these additional genetic aberrations will most likely increase our understanding of the pathogenesis of high hyperdiploid childhood ALL.     

Detection of the T(14;18) translocation in follicle centre cell lymphomas in South African ethnic groups using PCR.

PCR was used to detect the t(14;18) translocation in 64 South African cases with follicle centre cell lymphoma. DNA was purified from paraffin-embedded tissue collected from different ethnic groups namely white, black and "mixed" race patients, and primers used to detect both mbr and mcr. The overall incidence of the translocation was 45%, which is similar to that of Caucasian and Chinese patients. The ratio of rearrangements occurring at the mbr and mcr was 7:1 which may be an overestimation. The ratio was three times higher for the "mixed" race group compared to whites, and this suggests that there may be ethnic variation in breakpoints in South African patients.     

检测IgH和TCR γ基因重排对NHL分期参考价值的探讨

目的：探讨以IgH和TCR γ基因重排为标志对NHL患者临床分期的参考价值。方法：多聚酶链反应(PCR)技术结合限制性酶谱分析患者骨髓和淋巴结细胞DNA IgH和TCR γ基因重排的克隆性。结果：形态学无骨髓浸润的23例NHL患者发现6例(26．1％)具单克隆基因重排。结论：IgH和TCR γ基因重排作为分子标志对形态学检查不能确认骨髓浸润的NHL患者的分期诊断有一定参考价值。