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1.  Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas 
International Journal of Oncology  2014;46(3):953-962.
Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.
doi:10.3892/ijo.2014.2790
PMCID: PMC4324581  PMID: 25501347
BIOMED-2; clonality analysis; B-cell lymphomas; IGH rearrangement; IGK rearrangement
2.  PCR clonality detection in Hodgkin lymphoma 
Journal of Hematopathology  2009;2(1):34-41.
B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.
doi:10.1007/s12308-009-0024-1
PMCID: PMC2713492  PMID: 19669221
B cell; Hodgkin lymphoma; PCR clonality
3.  PCR-assays Detect B-lymphocyte Clonality in Formalin-fixed Paraffin Embedded Specimens of Classical Hodgkin Lymphoma without Microdissection 
Hodgkin lymphoma (HL) was shown to be a B cell malignancy using PCR-clonality studies of microdissected Reed-Sternberg cells. While methods for the detection of B cell clonality could aid in the diagnosis of HL, microdissection is not practical in most clinical settings. We assessed the standardized BIOMED-2 IGH and IGK PCR primers for the detection of clonality using 50 consecutively diagnosed formalin-fixed paraffin-embedded (FFPE) classic Hodgkin lymphoma specimens. Without microdissection, clonality was detected in 23/47 assessable cases. The IGK assay was significantly more sensitive than the IGH assay (18 vs. 10 positive results). These data, and two representative cases, demonstrate that PCR based B-cell clonality assays have utility when the histologic differential diagnosis of an FFPE specimen includes classic Hodgkin lymphoma.
doi:10.1309/AJCPK6SBE0XOODHB
PMCID: PMC3046780  PMID: 20551274
Hodgkin Lymphoma; immunoglobulin genes; PCR; clonality
4.  Polymerase Chain Reaction-Based Clonality Analysis of Cutaneous B-Cell Lymphoproliferative Processes 
Clinics  2010;65(1):53-60.
INTRODUCTION:
The differential diagnosis of B-cell lymphoproliferative processes remains a challenge for pathologists, dermatologists and oncologists, despite advances in histology, immunohistochemistry and molecular biology.
OBJECTIVE:
Evaluate aid and limitations of clonality analysis in the diagnosis of primary cutaneous B-cell lymphomas and B-cell pseudolymphomas.
METHODS:
This study included 29 cases of B-cell lymphoproliferative processes classified as primary cutaneous B-cell lymphomas (13), B-cell pseudolymphomas (6) and inconclusive cases (10) using histology and immunohistochemistry. The clonality analysis was performed by polymerase chain reaction analysis of immunoglobulin light chain and heavy chain rearrangements.
RESULTS:
DNA quality was shown to be generally poor; eight samples were inadequate for polymerase chain reaction analysis. The results showed monoclonality in eight of the primary cutaneous B-cell lymphomas and polyclonality in four of the B-cell pseudolymphomas. In addition, monoclonality was shown in two of the inconclusive cases by histology and immunohistochemistry, demonstrating the utility of polymerase chain reaction as an ancillary diagnostic tool for primary cutaneous B-cell lymphomas.
DISCUSSION:
The low quality DNA extracted from these cases demanded the use of an IgH protocol that yielded small fragments and IgK. Both methods used together improved detection.
CONCLUSION:
Use of the two protocols, immunoglobulin heavy chain FR3-trad and immunoglobulin light chain-Kappa Biomed protocols for clonality analysis improved diagnostic accuracy.
doi:10.1590/S1807-59322010000100009
PMCID: PMC2815283  PMID: 20126346
Cutaneous lymphoma; Polymerase chain reaction; Gene rearrangement; Clonality; B-cell; Lymphoproliferative processes
5.  Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3) in Thai malignant lymphoma by High Resolution Melting curve analysis 
Diagnostic Pathology  2010;5:31.
Abstract
Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan) has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR) followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM). The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma.
Introduction
Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population [1]. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year [2]. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR) and immunoglobulin heavy chain (IgH) by polymerase chain reaction (PCR) assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal lymphoproliferation [3,4]. Analyzing DNA extracted from formalin-fixed, paraffin-embedded tissues by multiplex PCR techniques is more rapid, accurate and highly sensitive. Measuring the size of the amplicon from PCR analysis could be used to diagnose malignant lymphoma with monoclonal pattern showing specific and distinct bands detected on acrylamide gel electrophoresis. However, this technique has some limitations and some patients might require a further confirmation test such as GeneScan or fragment analysis [5,6].
GeneScan technique or fragment analysis reflects size and peak of DNA by using capillary gel electrophoresis. This technique is highly sensitive and can detect 0.5-1% of clonal lymphoid cells. It measures the amplicons by using various fluorescently labeled primers at forward or reverse sides and a specific size standard. Using a Genetic Analyzer machine and GeneMapper software (Applied Bioscience, USA), the monoclonal pattern revealed one single, sharp and high peak at the specific size corresponding to acrylamide gel pattern, whereas the polyclonal pattern showed multiple and small peak condensed at the same size standard. This technique is the most sensitive and accurate technique; however, it usually requires high technical experience and is also of high cost [7]. Therefore, rapid and more cost effective technique are being sought.
LightCycler PCR performs the diagnostic detection of amplicon via melting curve analysis within 2 hours with the use of a specific dye [8,9]. This dye consists of two types: one known as SYBR-Green I which is non specific and the other named as High Resolution Melting analysis (HRM) which is highly sensitive, more accurate and stable. Several reports demonstrated that this new instrument combined with DNA intercalating dyes can be used to discriminate sequence changes in PCR amplicon without manual handling of PCR product [10,11]. Therefore, current investigations using melting curve analysis are being developed [12,13].
In this study, three different techniques were compared to evaluate the suitability of LightCycler PCR with HRM as the clonal diagnostic tool for IgH gene rearrangement in B-cell non-Hogdkin lymphoma, i.e. thermocycler PCR followed by heteroduplex analysis and PAGE, GeneScan analysis and LightCycler PCR with HRM.
doi:10.1186/1746-1596-5-31
PMCID: PMC2886000  PMID: 20482846
6.  Implementation of the standard strategy for identification of Ig/TCR targets for minimal residual disease diagnostics in B-cell precursor ALL pediatric patients: Polish experience 
Introduction:
Minimal residual disease (MRD), detected based on immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements as markers of residual leukemic cells, is currently the most reliable prognostic factor in acute lymphoblastic leukemia (ALL). A feasibility study is presented of the standard strategy for the identification of Ig/TCR targets for MRD diagnostics in Polish ALL patients by identifying Ig/TCR gene rearrangement pattern using standard primer sets and protocols.
Materials and Methods:
The PCR-heteroduplex approach based on BIOMED-1 and BIOMED-2 protocols (recommended as the European standard) was used to detect IGH, IGK-Kde, TCRD, TCRG, and TCRB rearrangements in 58 Polish B-cell precursor ALL patients. Sequencing and homology analysis between the obtained and germline Ig/TCR sequences enabled identification of the rearrangements. The U-Gauss test was used for statistical analysis of the Ig/TCR rearrangement pattern in Polish patients compared with relevant data on other nationalities.
Results:
The following pattern was identified: IGH: 83% (VH-JH: 74%, DH-JH: 9%), IGK-Kde: 41%, TCRD: 78% (incomplete TCRD: 55%, Vδ2-Dδ3: 45%, Dδ2-Dδ3: 21%, Vδ2-Jα: 35%), TCRG: 50%, and TCRB: 13%. Considerable convergence of the Ig/TCR pattern in Polish patients and those of other nationalities (mainly West Europeans) was demonstrated. Statistically relevant differences were only found between the incidence of DH-JH in Polish (9%) and Dutch patients (24%; p<0.05) and Polish and Italian patients (19%; p<0.05), VH-JH in Polish (74%) and Chilean patients (100%; p<0.05), and TCRG in Polish (50%) and Brazilian patients (69%; p<0.05).
Conclusions:
The convergence of Ig/TCR patterns in Polish and European patients indicates that the strategy for Ig/TCR target identification based on standard primers and protocols might be directly used for the construction of Polish standards and recommendations for MRD diagnostics.
doi:10.1007/s00005-008-0045-y
PMCID: PMC2805919  PMID: 19043668
minimal residual disease; Ig/TCR gene rearrangements; acute lymphoblastic leukemia; BIOMED-1; BIOMED-2
7.  Nodal involvement by marginal zone B-cell lymphoma harboring t(14;22)(q32;q11) involving immunoglobulin heavy chain and light chain lambda as the sole karyotypically recognizable abnormality in a patient with systemic lupus erythematosus 
Recurrent non-random balanced chromosomal translocation, usually involving the immunoglobulin heavy chain (IgH) gene or an immunoglobulin light chain gene and a proto-oncogene, which results in the overexpression of the latter under the control of an enhancer or promoter of the former, is a hallmark of many types of non-Hodgkin lymphoma (NHL) of B-cell origin. However, translocations between IgH and the immunoglobulin (Ig) light chain lambda gene (IgL), namely, a t(14;22)(q32;q11), have rarely been described in B-cell NHL. Herein we report the first case of marginal zone B-cell lymphoma harboring a t(14;22)(q32;q11) as its sole genetic abnormality in a patient with a 12-year history of systemic lupus erythematosus (SLE). Other interesting findings of this case include: 1) the neoplastic B-cells lack expression of both surface and cytoplasmic Ig light chain as revealed by flow cytometry and 2) monoclonal rearrangement of Ig light chain kappa (IgK) only due to k-deleting element (kde) recombination event. This case illustrates the necessity of utilizing a multi-modality approach in the diagnosis of B-cell NHL.
PMCID: PMC4152091  PMID: 25197401
t(14;22)(q32;q11); K-deleting element; marginal zone B-cell lymphoma; systemic lupus erythematosus
8.  Diagnostic Utility of a Clonality Test for Lymphoproliferative Diseases in Koreans Using the BIOMED-2 PCR Assay 
Korean Journal of Pathology  2013;47(5):458-465.
Background
A clonality test for immunoglobulin (IG) and T cell receptor (TCR) is a useful adjunctive method for the diagnosis of lymphoproliferative diseases (LPDs). Recently, the BIOMED-2 multiplex polymerase chain reaction (PCR) assay has been established as a standard method for assessing the clonality of LPDs. We tested clonality in LPDs in Koreans using the BIOMED-2 multiplex PCR and compared the results with those obtained in European, Taiwanese, and Thai participants. We also evaluated the usefulness of the test as an ancillary method for diagnosing LPDs.
Methods
Two hundred and nineteen specimens embedded in paraffin, including 78 B cell lymphomas, 80 T cell lymphomas and 61 cases of reactive lymphadenitis, were used for the clonality test.
Results
Mature B cell malignancies showed 95.7% clonality for IG, 2.9% co-existing clonality, and 4.3% polyclonality. Mature T cell malignancies exhibited 83.8% clonality for TCR, 8.1% co-existing clonality, and 16.2% polyclonality. Reactive lymphadenitis showed 93.4% polyclonality for IG and TCR. The majority of our results were similar to those obtained in Europeans. However, the clonality for IGK of B cell malignancies and TCRG of T cell malignancies was lower in Koreans than Europeans.
Conclusions
The BIOMED-2 multiplex PCR assay was a useful adjunctive method for diagnosing LPDs.
doi:10.4132/KoreanJPathol.2013.47.5.458
PMCID: PMC3830993  PMID: 24255634
Lymphoma, B-cell; Lymphoma, T cell; Gene rearrangement; BIOMED-2 multiplex PCR assay
9.  Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience 
Radiology and Oncology  2014;48(2):155-162.
Background
Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations.
Materials and methods.
With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays.
Results
The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions.
Conclusions
In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value.
doi:10.2478/raon-2013-0072
PMCID: PMC4078034  PMID: 24991205
BIOMED-2; clonality analysis; lymphomas; IGH rearrangement; TCR rearrangement
10.  Accuracy and coverage assessment of Oryctolagus cuniculus (Rabbit) Genes Encoding Immunoglobulins in the Whole Genome Sequence Assembly (OryCun2.0) and Localization of the IGH Locus to Chromosome 20 
Immunogenetics  2013;65(10):749-762.
We report analyses of genes encoding immunoglobulin heavy and light chains in the rabbit 6.51x whole genome assembly. This OryCun2.0 assembly confirms previous mapping of the duplicated IGK1 and IGK2 loci to chromosome 2 and the IGL lambda light chain locus to chromosome 21. The most frequently rearranged and expressed IGHV1 that is closest to IG DH and IGHJ genes encodes rabbit VHa allotypes. The partially inbred Thorbecke strain rabbit used for whole-genome sequencing was homozygous at the IGK but heterozygous with the IGHV1a1 allele in one of 79 IGHV-containing unplaced scaffolds and IGHV1a2, IGHM, IGHG and IGHE sequences in another. Some IGKV, IGLV and IGHA genes are also in other unplaced scaffolds. By fluorescence in situ hybridization, we assigned the previously unmapped IGH locus to the q-telomeric region of rabbit chromosome 20. An approximately 3 Mb segment of human chromosome 14 including IGH genes predicted to map to this telomeric region based on synteny analysis could not be located on assembled chromosome 20. Unplaced scaffold chrUn0053 contains some of the genes that comparative mapping predicts to be missing. We identified discrepancies between previous targeted studies and the OryCun2.0 assembly and some new BAC clones with IGH sequences that can guide other studies to further sequence and improve the OryCun2.0 assembly. Complete knowledge of gene sequences encoding variable regions of rabbit heavy, kappa and lambda chains will lead to better understanding of how and why rabbits produce antibodies of high specificity and affinity through gene conversion and somatic hypermutation.
doi:10.1007/s00251-013-0722-9
PMCID: PMC3780782  PMID: 23925440
Rabbit; Immunoglobulin Genes; Heavy Chains; Fluorescence in situ hybridization; Chromosome 20; Light Chains
11.  Follicular Lymphomas in children and young adults: A comparison of the pediatric variant with usual follicular lymphoma 
Follicular lymphoma (FL), a common lymphoma in adults, occurs rarely in pediatric and young adult patients. Most pediatric cases have been described as Grade 3, but the criteria to distinguish the pediatric variant of FL (PFL) from usual FL (UFL) in adults are not well defined. We undertook a study of FL in patients under age 30. We identified 63 cases, which were analyzed by morphology, immunohistochemistry, and PCR analysis of IGH@ and IGK@ clonality. These data were correlated with clinical findings including stage, treatment, and outcome. Among the 63 cases, 34 cases were classified as PFL; 22 presenting in lymph nodes, 8 in Waldeyer’s ring and 4 in testis. Clonal immunoglobulin gene rearrangement was detected in 97% of PFL cases, but FISH analysis showed an absence of the BCL2/IGH@ translocation in all cases tested. Twenty-nine cases were classified as UFL, 28 of which presented in lymph nodes. The nodal PFL cases were observed exclusively in males in both children and young adults with a median age of 15 years. They showed marked head/neck predilection, blastoid cytological features with a high proliferation rate, lack of BCL2 protein and t(14;18), low clinical stage at presentation, and good prognosis. PFL involving Waldeyer’s ring were distinguished by MUM1 expression, 50% (3/6) of which carried IRF4 breaks. BCL2 expression was common (63%) in the absence of BCL2/IGH@ translocation. UFL cases were more common in females, exclusively in young adult patients (median age, 24 years) with no patients under age 18. 25/29 were grade 1–2, and four cases were classified as grade 3A. They exhibited a higher clinical stage at presentation. 83% expressed BCL2. Our results indicate that histological and immunophenotypic criteria can reliably separate PFL and UFL, and that UFL is exceptionally rare in the pediatric age group. PFL associated with particular anatomic sites have distinctive features, and should be evaluated separately in future clinical and biological studies.
doi:10.1097/PAS.0b013e31826b9b57
PMCID: PMC3566339  PMID: 23108024
follicular lymphoma; follicular lymphoma; IRF4 (interferon regulatory factor 4); MUM1; BCL2; BCL6; Fluorescence in-situ hybridization (FISH); Waldeyer’s ring; testis; pediatric
12.  Illegitimate V(D)J recombination involving non-antigen receptor loci in lymphoid malignancy 
Genes, chromosomes & cancer  2012;51(6):525-535.
V(D)J recombination of antigen-receptor loci (IGH, IGK, IGL, TCRA, TCRB, TCRG, TCRD) is an essential mechanism that confers enormous diversity to the mammalian immune system. However, there are now at least six examples of intra-chromosomal interstitial deletions caused by aberrant V(D)J recombination between non-antigen receptor loci; five of these six are associated with lymphoid malignancy. The SIL-SCL fusion, and deletions of CDKN2A, IKZF1, Notch1, and Bcl11b are all associated with lymphoid malignancy. These interstitial deletions seem to be species specific, as the deletions seen in mice are not seen in humans; the converse is true as well. Nucleotide sequence analysis of these rearrangements reveals the hallmarks of V(D)J recombination, including site-specificity near cryptic heptamer signal sequences, exonucleolytic “nibbling” at the junction site, and non-templated “N” region nucleotide insertion at the junction site. Two of these interstitial deletions (murine Notch1 and Bcl11b deletions) have been detected, at low frequency, in tissues from healthy mice with no evidence of malignancy, similar to the finding of chromosomal translocations in the peripheral blood or tonsils of healthy individuals. The contention that these are mediated via V(D)J recombination is strengthened by in vivo assays using extra-chromosomal substrates, and chromatin immunoprecipitation-sequence (ChIP-Seq) analysis which shows Rag2 binding at the sites of rearrangement. Although the efficiency of these “illegitimate” recombination events is several orders of magnitude less than that at bona fide antigen receptor loci, the consequence of such deletions, namely activation of proto-oncogenes or deletion of tumor suppressor genes, is devastating, and a major cause for lymphoid malignancy.
doi:10.1002/gcc.21942
PMCID: PMC3323722  PMID: 22334400
lymphoid leukemia; V(D)J recombination; SCL; CDKN2A; IKZF1; NOTCH1; BCL11B
13.  On the genomics of immunoglobulins in the gray, short-tailed opossum Monodelphis domestica 
Immunogenetics  2009;61(8):581-596.
Annotated maps of the IGH, IGK, and IGL loci in the gray, short-tailed opossum Monodelphis domestica were generated from analyses of the available whole genome sequence for this species. Analyses of their content and organization confirmed a number of previous conclusions based on characterization of cDNAs encoding opossum immunoglobulin heavy and light chains and limited genomic analysis, including: i) the predominance of a single IGHV subgroup and clan; ii) the presence of a single IgG subclass; iii) the apparent absence of an IgD; and iv) the general organization and V gene complexity of the IGK and IGL light chain loci. In addition several unexpected discoveries were made including the presence of a partial germ-line joined IGHV segment, the first germline joined Ig V gene to be found in a mammal. In addition was the presence of a larger number of IGKV subgroups than had been previously identified. With this report, annotated maps of the Major Histocompatibility Complex, T cell receptor, and immunoglobulin loci have been completed for M. domestica, the only non-eutherian mammalian species for which this has been accomplished, strengthening the utility of this species as a model organism.
doi:10.1007/s00251-009-0385-8
PMCID: PMC2880577  PMID: 19609519
immunoglobulins; genomics; marsupials; Monodelphis domestica
14.  Improvements to B cell clonality analysis using PCR amplification of immunoglobulin light chain genes 
Molecular Pathology  2002;55(2):98-101.
Aims: Clonality analysis using polymerase chain reaction (PCR) amplification of the immunoglobulin heavy chain (IgH) gene is an important aid to the diagnosis of B cell lymphoproliferative diseases. However, the method has a relatively high false negative rate. In an attempt to improve detection rates simple PCR strategies for clonality analysis of B cell populations using amplification of Ig light chain genes have been developed.
Methods: Novel PCR protocols, designed to amplify Igκ and Igλ light chain genes, were evaluated using high molecular weight DNA samples from 28 selected cases of B cell lymphoma with known light chain expression and 12 reactive lymphoid specimens. Products were run on 10% polyacrylamide minigels using heteroduplex analysis. Conventional IgH PCR analysis was also performed. Twelve randomly selected formalin fixed, paraffin wax processed samples from cases submitted for molecular genetic analysis were also studied.
Results: Polyclonal products were seen in all reactive lymphoid samples. Using Igκ PCR, 24 of 28 lymphomas, including four of five IgH negative cases, displayed monoclonal patterns. Using Igλ PCR, eight of 12 Igλ expressing tumours, including two of five IgH negative cases, showed monoclonal patterns. Standard IgH PCR demonstrated monoclonality in 23 of 28 B cell lymphomas. The detection rate was improved to 27 of 28 lymphomas using heavy and light chain PCR. Efficient amplification was achieved using paraffin wax processed samples, seven of which showed monoclonality compared with eight using IgH PCR.
Conclusions: Ig light chain PCR, used in conjunction with heavy chain analysis, enables improved detection of B cell monoclonality using routine histological specimens and can provide additional clone specific markers for the study of the biology of B cell tumours.
PMCID: PMC1187157  PMID: 11950958
immunoglobulin light chain genes; polymerase chain reaction; clonality analysis
15.  Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production 
PLoS ONE  2014;9(3):e90383.
Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM−/−, bIGHML1−/−; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM−/−, bIGHML1−/− and bIGL−/−; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).
doi:10.1371/journal.pone.0090383
PMCID: PMC3946162  PMID: 24603704
16.  EBV-Positive Plasmacytoma of the Submandibular Gland—Report of a Rare Case with Molecular Genetic Characterization 
Head and Neck Pathology  2011;5(4):389-394.
Plasmacytomas are differentiated plasma cell tumors that present as a mass lesion in osseous or extraosseous sites. Although the most common site for extramedullary plasmacytomas (EMP) is in the upper respiratory tract, plasmacytomas initially presenting as salivary gland masses are very uncommon. We describe a case of an EBV-positive plasmacytoma presenting as a 7.7 cm submandibular mass in an elderly immunocompetent man which displayed an abundance of “naked nuclei” on fine needle aspiration cytology. The tumor showed lambda light chain restriction and positive expression for CD38, MUM1 and EBER. Subsequent investigation for myeloma revealed absence of M-protein and end-organ damage, except for a lytic lesion in the radial bone. An extensive fluorescent in situ hybridization analysis showed the tumor to be negative for the t(4;14) FGFR3/IGH translocation as well as translocations involving the IGH,IGL, IGK, CCND1, BCL2, BCL6 and C-MYC genes. KRAS genetic analysis did not reveal any mutations of codons 12, 13 and 61.
doi:10.1007/s12105-011-0257-z
PMCID: PMC3210227  PMID: 21442194
Plasmocytoma; Salivary gland; Epstein barr virus; Fluorescence in situ hybridization; CMYC; KRAS
17.  Association between the Igk and Igh loci mediated by the 3’ Igk enhancer induces decontraction of the Igh locus in pre-B cells 
Nature immunology  2008;9(4):396-404.
V(D)J recombination at Igh and Igk loci takes place sequentially during successive stages in B cell development. Using 3-dimensional DNA fluorescence in situ hybridization (FISH), we identified a lineage- and stage-specific interchromosomal association between these two loci which marks the transition between Igh and Igk recombination. Co-localization occured between pericentromerically located alleles in pre-B cells, and was mediated by the 3’ Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus decontraction of an Igh allele, and resulted in increased distal VH gene rearrangement. Our data indicate a role for the Igk locus and its 3’ enhancer in directing the Igh locus to a repressive nuclear subcompartment, and in rendering the Igh locus unable to contract.
doi:10.1038/ni1567
PMCID: PMC2583163  PMID: 18297074
18.  Translocations at 8q24 juxtapose MYC with genes that harbor superenhancers resulting in overexpression and poor prognosis in myeloma patients 
Blood Cancer Journal  2014;4(3):e191-.
Secondary MYC translocations in myeloma have been shown to be important in the pathogenesis and progression of disease. Here, we have used a DNA capture and massively parallel sequencing approach to identify the partner chromosomes in 104 presentation myeloma samples. 8q24 breakpoints were identified in 21 (20%) samples with partner loci including IGH, IGK and IGL, which juxtapose the immunoglobulin (Ig) enhancers next to MYC in 8/23 samples. The remaining samples had partner loci including XBP1, FAM46C, CCND1 and KRAS, which are important in B-cell maturation or myeloma pathogenesis. Analysis of the region surrounding the breakpoints indicated the presence of superenhancers on the partner chromosomes and gene expression analysis showed increased expression of MYC in these samples. Patients with MYC translocations had a decreased progression-free and overall survival. We postulate that translocation breakpoints near MYC result in colocalization of the gene with superenhancers from loci, which are important in the development of the cell type in which they occur. In the case of myeloma these are the Ig loci and those important for plasma cell development and myeloma pathogenesis, resulting in increased expression of MYC and an aggressive disease phenotype.
doi:10.1038/bcj.2014.13
PMCID: PMC3972699  PMID: 24632883
19.  Gene rearrangement study for minimal residual disease monitoring in children with acute lymphocytic leukemia 
Objective
To detect markers for minimal residual disease monitoring based on conventional polymerase chain reaction for immunoglobulin, T-cell receptor rearrangements and the Sil-Tal1 deletion in patients with acute lymphocytic leukemia.
Methods
Fifty-nine children with acute lymphocytic leukemia from three institutions in Minas Gerais, Brazil, were prospectively studied. Clonal rearrangements were detected by polymerase chain reaction followed by homo/heteroduplex clonality analysis in DNA samples from diagnostic bone marrow. Follow-up samples were collected on Days 14 and 28-35 of the induction phase. The Kaplan-Meier and multivariate Cox methods were used for survival analysis.
Results
Immunoglobulin/T-cell receptor rearrangements were not detected in 5/55 children screened (9.0%). For precursor-B acute lymphocytic leukemia, the most frequent rearrangement was IgH (72.7%), then TCRG (61.4%), and TCRD and IgK (47.7%); for T-acute lymphocytic leukemia, TCRG (80.0%), and TCRD and Sil-Tal deletion (20.0%) were the most common. Minimal residual disease was detected in 35% of the cases on Day 14 and in 22.5% on Day 28-35. Minimal residual disease on Day 28-35, T-acute lymphocytic leukemia, and leukocyte count above 50 x 109/L at diagnosis were bad prognostic factors for leukemia-free survival in univariate analysis. Relapse risk for minimal residual disease positive relative to minimal residual disease negative children was 8.5 times higher (95% confidence interval: 1.02-70.7).
Conclusion
Immunoglobulin/T-cell receptor rearrangement frequencies were similar to those reported before. Minimal residual disease is an independent prognostic factor for leukemia-free survival, even when based on a non-quantitative technique, but longer follow-ups are needed.
doi:10.5581/1516-8484.20130115
PMCID: PMC3832314  PMID: 24255617
Precursor cell lymphoblastic leukemia-lymphoma; Neoplasm, residual; Gene rearrangement; Polymerase chain reaction
20.  Role of polymerase chain reaction and immunocytochemistry in the cytological assessment of lymphoid proliferations 
Journal of Clinical Pathology  2006;59(11):1160-1165.
Background
Fine‐needle aspiration cytology (FNAC) is used as a screening test to evaluate lymphadenopathy. The combined use of genetic analysis and flow cytometry for immunophenotyping has increased the accuracy of diagnosis and correct categorisation of lymphomas on cytological preparations.
Aim
To show the utility of immunocytochemistry and polymerase chain reaction (PCR) in the evaluation of cytological preparations of lymph nodes.
Methods
Fine needle aspirates were obtained from 33 patients (initial presentation, n = 27; recurrence, n = 6). Routine examination was undertaken using immunocytochemistry and DNA PCR to detect clonality and specific translocations. The cytodiagnosis and subclassification of lymphoma was correlated with histological diagnosis in the available follow‐up biopsies.
Results
14 patients had a cytological diagnosis of non‐Hodgkin's lymphoma (NHL), 4 had suspected NHL, 2 had atypical lymphoid proliferation and 13 had reactive hyperplasia. A World Health Organization (WHO) subtype was suggested in 8 patients. Incorporating the results of immunoglobulin heavy chain (IgH) and T‐cell receptor (TCR) gene rearrangements enabled diagnosis of lymphoma in 17 patients, including 5 of the 6 patients suspected to have NHL or an atypical lymphoid proliferation. Identification of the translocations t (14;18) and t (2;5) helped WHO categorisation in 3 of the patients. The cytological findings were confirmed in 12 out of the 13 patients for whom histological follow‐up was available. Seven of the 18 lymphoma patients were managed without a subsequent biopsy. We made one false–positive diagnosis of B‐cell NHL on cytology.
Conclusion
The use of immunocytochemistry and PCR is valuable in the definitive diagnosis and subtyping of malignant lymphomas on cytological preparations. The use of these techniques may avoid lymph node biopsies in some cases and allow definitive treatment based on aspirate findings alone.
doi:10.1136/jcp.2005.032987
PMCID: PMC1860501  PMID: 16533955
21.  Clinicopathologic Features of CDK6 Translocation-Associated B-Cell Lymphoproliferative Disorders 
Cyclin-dependent protein kinase 6 (CDK6), in cooperation with cyclin Ds, drives cell cycle progression from G1 to S phase through phosphorylation and subsequent inactivation of Retinoblastoma 1 protein (Rb). Alteration of this pathway results in both non-hematologic and hematologic malignancies, which include a small subset of B-cell lymphoproliferative disorders (BLPDs). We identified 5 cases of BLPD that carried CDK6 chromosomal translocations and characterized their clinical, pathologic, immunophenotypic, and genetic features. Common clinical characteristics included marked neoplastic lymphocytosis, systemic lymphadenopathy, splenomegaly, and bone marrow involvement. Three patients were diagnosed with low-grade B-cell lymphoma and had an indolent clinical course, and 2 patients (one who transformed to large B-cell lymphoma and another who was initially diagnosed with a high-grade B-cell lymphoma) had an aggressive clinical course. Immunophenotypically, the neoplastic B-cells expressed CD5, CDK6, and cytoplasmic Rb in all cases, expressed phospho-RB, p27kip1, and cyclin D2 in most cases, and uniformly lacked expression of all other cyclins. In four cases, the CDK6 translocation partner was kappa immunoglobulin light-chain gene (IGK); and in the fifth case, the CDK6 translocation partner was unknown. These distinct clinicopathologic and cytogenetic features distinguish the CDK6 translocation-associated BLPDs (CDK6-BLPDs) from other mature B-cell lymphomas.
doi:10.1097/PAS.0b013e3181934244
PMCID: PMC2674112  PMID: 19145199
cyclin D; cyclin dependent protein kinase (CDK); chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL); mantle cell lymphoma (MCL); splenic marginal zone lymphoma (SMZL); and Retinoblastoma protein (Rb)
22.  Homologous recombination between transferred and chromosomal immunoglobulin kappa genes. 
Molecular and Cellular Biology  1988;8(10):4041-4047.
Homologous recombination between transferred and chromosomal DNAs provides a means of introducing well-defined, predetermined changes in the chromosomal genes. Here we report that this approach can be used to specifically modify the immunoglobulin genes in mouse hybridoma cells. The test system is based on the Sp6 hybridoma, which synthesizes immunoglobulin M (kappa) specific for the hapten 2,4,6-trinitrophenyl (TNP). As recipient cells, we used the Sp6-derived mutant hybridoma igk14, which has a deletion of the kappa TNP gene and consequently does not synthesize TNP-specific immunoglobulin M. igk14 retains the mu TNP gene and two additional rearranged kappa genes, denoted kappa M21B1 and kappa M21G. As a transfer vector, we used pSV2neo bearing the functionally rearranged TNP-specific V kappa segment. Following DNA transfer by electroporation, we isolated rare transformants which produced normal amounts of the functional kappa TNP chain. Analysis of the DNA of these transformants indicated that in all cases, a functional kappa TNP gene had been formed as the result of a homologous integrative recombination event with the igk14 kappa M21B1 gene. These results suggest that homologous recombination might be used for mapping and introducing immunoglobulin gene mutations and for more conveniently engineering specifically altered immunoglobulins.
Images
PMCID: PMC365472  PMID: 3141782
23.  Diagnosis of primary pulmonary T- cell/histiocyte-rich large B cell lymphoma with tissue eosinophilia via clinicopathological observation and molecular assay 
Diagnostic Pathology  2014;9(1):188.
Background
Primary pulmonary lymphoma (PPL) is rare and easily misdiagnosed because of the lack of typical clinical features. It most commonly involves elderly patients aged between 60 and 70 years, and pathological diagnosis depends mainly on chest surgery rather than bronchial mucosal biopsy. Via percutaneous needle aspiration biopsy of the lung of a 33-year-old woman, which had distinct tissue eosinophilia, we diagnosed a rare case of rapidly growing large B cell lymphoma.
Methods
Bronchial mucosal biopsy and computed tomography–guided percutaneous needle aspiration biopsy were performed to determine the nature of the lesion, and we identified its immunophenotype using immunohistochemistry. We used BIOMED-2 gene rearrangement PCR to determine lymphocyte clonality; laser microdissection was used to confirm the clonality of suspicious malignant lymphocytes.
Results
Morphologically, the lesion was composed of a large number of eosinophilic cells and a few lymphoid cells. Immunohistochemical staining revealed a few CD1α-positive cells, but they were S-100–negative. The small lymphoid cells predominantly expressed CD3; the large lymphoid cells expressed CD20 and some scattered large lymphoid cells expressed Pax5. However, molecular studies confirmed clonal immunoglobulin heavy chain (IGH)-D gene rearrangement in Pax5–positive large B lymphocytes.
Conclusions
This is the first recorded case of T- cell/histiocyte-rich large B cell lymphoma with tissue eosinophilia of the lung. It highlights the unusual morphological features of PPL that might be mistaken for eosinophilic granuloma or parasitic infection. In addition, IGH and T cell receptor gene rearrangement play important roles in differentiating rare B cell lymphoma from lung space–occupying lesions with abundant eosinophils or T cell infiltration.
Virtual Slides
The virtual slide(s) for this article can be found here: http://med.motic.com/MoticGallery/Slides/AC5C9A6F-46EC-4C71-A448-1312F6900C65?user=2C69F0D6-A478-4A2B-ABF0-BB36763E8025
doi:10.1186/s13000-014-0188-6
PMCID: PMC4207321  PMID: 25273521
Primary pulmonary lymphoma; B cell lymphoma; Tissue eosinophilia; Gene rearrangement
24.  Array-Based Comparative Genomic Hybridization Analysis Reveals Chromosomal Copy Number Aberrations Associated with Clinical Outcome in Canine Diffuse Large B-Cell Lymphoma 
PLoS ONE  2014;9(11):e111817.
Canine Diffuse Large B-cell Lymphoma (cDLBCL) is an aggressive cancer with variable clinical response. Despite recent attempts by gene expression profiling to identify the dog as a potential animal model for human DLBCL, this tumor remains biologically heterogeneous with no prognostic biomarkers to predict prognosis. The aim of this work was to identify copy number aberrations (CNAs) by high-resolution array comparative genomic hybridization (aCGH) in 12 dogs with newly diagnosed DLBCL. In a subset of these dogs, the genetic profiles at the end of therapy and at relapse were also assessed. In primary DLBCLs, 90 different genomic imbalances were counted, consisting of 46 gains and 44 losses. Two gains in chr13 were significantly correlated with clinical stage. In addition, specific regions of gains and losses were significantly associated to duration of remission. In primary DLBCLs, individual variability was found, however 14 recurrent CNAs (>30%) were identified. Losses involving IGK, IGL and IGH were always found, and gains along the length of chr13 and chr31 were often observed (>41%). In these segments, MYC, LDHB, HSF1, KIT and PDGFRα are annotated. At the end of therapy, dogs in remission showed four new CNAs, whereas three new CNAs were observed in dogs at relapse compared with the previous profiles. One ex novo CNA, involving TCR, was present in dogs in remission after therapy, possibly induced by the autologous vaccine. Overall, aCGH identified small CNAs associated with outcome, which, along with future expression studies, may reveal target genes relevant to cDLBCL.
doi:10.1371/journal.pone.0111817
PMCID: PMC4221131  PMID: 25372838
25.  Critical roles of the immunoglobulin intronic enhancers in maintaining the sequential rearrangement of IgH and Igk loci 
The Journal of Experimental Medicine  2006;203(7):1721-1732.
V(D)J recombination of immunoglobulin (Ig) heavy (IgH) and light chain genes occurs sequentially in the pro– and pre–B cells. To identify cis-elements that dictate this order of rearrangement, we replaced the endogenous matrix attachment region/Igk intronic enhancer (MiEκ) with its heavy chain counterpart (Eμ) in mice. This replacement, denoted EμR, substantially increases the accessibility of both Vκ and Jκ loci to V(D)J recombinase in pro–B cells and induces Igk rearrangement in these cells. However, EμR does not support Igk rearrangement in pre–B cells. Similar to that in MiEκ−/− pre–B cells, the accessibility of Vκ segments to V(D)J recombinase is considerably reduced in EμR pre–B cells when compared with wild-type pre–B cells. Therefore, Eμ and MiEκ play developmental stage-specific roles in maintaining the sequential rearrangement of IgH and Igk loci by promoting the accessibility of V, D, and J loci to the V(D)J recombinase.
doi:10.1084/jem.20052310
PMCID: PMC2118354  PMID: 16785310

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