Immunoglobulin Kappa Locus Research Articles

The t(2;12)(p12;p13), leading to the juxtaposition of the cyclin D2 CCND2 gene with the immunoglobulin kappa locus (IGK), defines a small subset of mantle cell lymphomas lacking cyclin D1 expression.

The t(2;12)(p12;p13), leading to the juxtaposition of the cyclin D2 CCND2 gene with the immunoglobulin kappa locus (IGK), defines a small subset of mantle cell lymphomas lacking cyclin D1 expression.

Applicated value of multi-parameter flow cytometry and Ig gene rearrangement in early diagnosis of multiple myeloma

Objective To explore the clinical value of multi-parameter flow cytometry and Ig gene rearrangement in early diagnosis of multiple myeloma. Methods 19 cases of multiple myeloma were detected and analyzed by BD flow cytometry and Ig gene rearrangement, the multi-parameter analysis of tumor cell markers were CD45, CD138, CD10, CD19, CD20, CD22, CD13, CD33, CD117, CD56, CD38, cell oar kappa light chain(cκ), cell plasma lambda light chain(cλ). Results Abnormal plasma cells ratio was 1.40% ~32.8% detected by flow cytometry, which gate using CD45 and CD138; the proportions of antigen positive expression were CD138: 100.0%, CD38: 100.0%, CD56: 63.1%, CD117: 42.1%, CD13: 0.0%, CD33: 26.3%, CD20: 5.2%, CD22: 0.0%, CD10: 0.0%, CD19: 0.0%, respectively. The naive and mature plasma cell ratio was 2.5%~75.5% detected by bone marrow morphology. The both methodologies above had a correlation(r=0.702, P<0.01). The restricted expression of immunoglobulin cytoplasm in MM patients, 9 cases of cκ(9/19), 10 cases of cλ(10/19). The subtypes were 4 cases IgG+ κ, 2 cases IgA+ κ, 4 cases IgG+ λ, 1 case IgA+ λ, 3 cases κ and 5 cases λ detected by immunofixation electrophoresis; Ig gene rearrangement shown 8 cases of IGH FR1-JH rearrangement, 11 cases of IGH FR2-JH rearrangement, 2 cases of IgH DH-JH rearrangement, 11 cases of IGK Vk-Jk rearrangement, 8 cases of IGK Vk-Kde and intron-Kde rearrangement; combined with IgH and IgK gene rearrangement, the positive rate of early MM was 89.5%(17/19). Conclusion Multi-parameter flow cytometry can be more accurate to set the required analysis of abnormal plasma cell population, abnormal plasma cells most of the expressions of CD138, CD38, CD56 antigen, while does not express CD19, CD10, CD13, CD22.The diagnostic value of Ig in the diagnosis of MM is the combination of cell morphology examination and immunohistochemistry electrophoresis and the application of BIOMED 2 gene rearrangement technique. Key words: Multiple myeloma; Flow cytometry; Gene rearrangement; Diagnosis

Relapsed anaplastic lymphoma kinase-positive large B-cell lymphoma expressed cluster of differentiation 4 and cytokeratin: An initially misdiagnosed case corrected by immunoglobulin κ locus gene rearrangement detection.

Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (LBCL) is a rare lymphoma subtype. The present study investigated a refractory nodal ALK-positive LBCL case in a 28-year-old Chinese male. It was initially misdiagnosed as ALK-positive anaplastic large cell lymphoma; however, the patient's lesions relapsed and spread widely following a short remission for chemotherapy and the patient succumbed to the disease 3 months' post-autologous stem cell transplantation; thus, a revision was performed. Histologically, the tumor cells exhibited a characteristic immunoblastic morphology with marked cellular pleomorphism. All lesions shared the same immunoprofiles, including granular cytoplasmic ALK staining patterns and a lack of cell lineage-associated markers, with the exception of cluster of differentiation (CD)45 and CD4. CD30 expression was revealed to be negative and CD138 staining was observed to be positive, additionally, cytokeratin was expressed aberrantly in a relapsed tumor biopsy. Fluorescence in situ hybridization studies demonstrated breakage and extra copies of the ALK gene in ≥30% of cells. Final clarification was provided by the detection of immunoglobulin κ locus (IGK) gene rearrangement in clonality studies [but notimmunoglobulin heavy locus (IGH) and immunoglobulin λ locus (IGL) genes]. This aggressive entity requires distinct modalities of standard treatment, and may be ignored owing to its rarity in routine pathology laboratories. BIOMED-2 polymerase chain reaction assays, including for IGH, IGK and IGL genes, are essential for the detection of gene rearrangement.

Application of Combined Detection of Fusion Gene and BIOMED-2 Standardized Ig Gene Rearrangement System in Childhood B-cell Acute Lymphoblastic Leukemia

To explore the application of combined detection of fusion gene and BIOMED-2 standardized immunoglobulin (Ig) gene rearrangement system in diagnosis and treatment of children with acute lymphoblastic leukemia (ALL). Multiplex-PCR amplifications and RQ-PCR of RNA/DNA were performed using ALL fusion gene detection kit and BIOMED-2 primer. The Ig gene rearrangements were analyzed by using PCR fragment analysis system. Out of 251 children with B-ALL, 77 cases were TEL-AML1(+) , 28 cases were E2A-PBX1(+) , 10 cases were MLL-AF4(+) , 11 cases were BCR-ABL(+) , the total positive rate was 50.2%, 82.5% showed IgH VH-JH rearrangement, 53.4% showed IgK rearrangement. The positive rate of combined detection of fusion gene and gene rearrangement was 99%. E2A-PBX1(+) and MLL-AF4(+) with IgK(+) gene rearrangement group was compared with negative control group, the difference was statistically significant (P < 0.001 or P = 0.005); 105 ALL fusion gene positive cases had been detected by fluorescence in situ hybridization (FISH) simultaneously, the accordance rate of fusion gene and FISH was more than 94%. The combined detection of ALL fusion gene and BIOMED-2 standardized clonality analysis system can improve the positive detected rate of B-ALL dramatically, and make the grouping of disease prognosis more accurately; this combined detection is a more faster and sensitive method than FISH.

Atypical Marginal Zone Hyperplasia Is a Mimic for Lymphoma in Pediatric Transplant Recipients: Report of Two Patients.

Atypical marginal zone hyperplasia (AMZH) of mucosa-associated lymphoid tissue (MALT) closely resembles lymphoma in that it shows expansion of the marginal zones with prominent intraepithelial B lymphocytes, is immunoglobulin light-chain restricted, and may show aberrant CD43 expression. However, unlike lymphoma, it does not show rearrangement of the immunoglobulin heavy chain gene (immunoglobulin H [IgH]) by polymerase chain reaction (PCR), and it behaves in a benign fashion. We identified AMZH in 2 pediatric solid organ transplant recipients who presented with adenotonsillar hypertrophy. To date, the patients have experienced a self-limited course in the absence of treatment or reduction of immunosuppression. Atypical marginal zone hyperplasia is a pitfall for posttransplant lymphoproliferative disorder and MALT lymphoma in the pediatric solid organ transplant population. In transplant patients with a lambda-restricted B-cell clone and marginal zone hyperplasia in native MALT sites, PCR for IgH and IgK gene rearrangement is essential to prevent misdiagnosis.

Evaluation of IGK and IGL molecular gene rearrangements according to the BIOMED-2 protocols for clinical diagnosis of Hodgkin lymphoma

Background: Although the analysis of molecular clonality rearrangements of the immunoglobulin light chains (IGK and IGL) is an alternative approach for diagnosis of B cell non-Hodgkin lymphomas (NHLs) using BIOMED-2 protocols, NHLs have not been extensively confirmed for Hodgkin lymphoma (HL) cases. We evaluated BIOMED-2 protocols in HL cases, which have been suggested previously as gold standard method for molecular clonality analysis on formalin fixed, paraffin-embedded (FFPE) tissue in NHL patients.Methods: We recruited 50 consecutive FFPE tissues of HL samples to evaluate IGK and IGL clonality gene rearrangements using BIOMED-2 and Heteroduplex methods.Results: Our findings revealed a total of 94% (47/50) positive clonality, which consisted of 70% (35/50) for IGK and 44% (22/50) for IGL. In three cases, clonality was not detected in any of the immunoglobulin gene segments.Conclusions: Analysis of clonality gene rearrangements in IGK and IGL genes using BIOMED-2 protocols could be implemented as a valuable method for improving clonality detection rate in HL cases and sensitivity (94%) and accuracy of HL diagnosis similar to that of the NHL samples will be increased.

Detection of B-Cell Clonality in Bone Marrow Is Independent Predictor of Outcome in De Novo Diffuse Large B-Cell Lymphoma Patients Treated with High-Dose Chemotherapy

Introduction: Detection of bone marrow involvement (BM) is a factor of poor prognosis for diffuse large B-cell lymphoma (DLBCL), since DLBCL BM is characterized by aggressive course and low response level to standard chemotherapy (CT), and 5-year overall survival (OS) rate for this group is less than 30% after ÑHOP-like CT. Intensification of therapy in this group of patients can improve the results, except patients with bone marrow involvement at diagnosis. Bone marrow involvement is a poor prognostic factor for patients treated with high-dose chemotherapy.Bone marrow involvement in DLBCL by histology is detected in 10-30% patients. The importance of B-cell clonality examination in bone marrow as prognostic and staging factor has not been described in the literature.Aim: To evaluate the significance of the immunoglobulin heavy chain gene rearrangement analysis performed by PCR for identification of the bone marrow involvement frequency and its value for staging and prognosis in patients with de novo DLBCL treated with high-dose chemotherapy (HDC).Patients and methods: We performed a pilot prospective trial, including 175 consecutive adult patients (median age 45 years, range 18–67) with newly diagnosed DLBCL who were enrolled in HDC protocol (mNHL-BFM-90 program or scheme R-EPOCH/R-HMA) since June 2007 till July 2014. Their clinical characteristics included such factors as IPI and phenotype DLBCL by immunohistochemical study. Out of the 175 patients, 85 had a GCB phenotype and 90 - non-GCB; 36 patients (20%) had low IPI risk, 40 patients (23%) had low-intermediate, 38 patients (22%) had high-intermediate, 61 patients (35%) had high risk.B-cell clonality was evaluated using PCR amplification by IGH (FR1, FR2, FR3) and IGK (Vκ-Jκ, Vκ/intron-Kde) gene rearrangements with multiplex BIOMED-2 primer sets and subsequent fragment analysis using ABI PRISM 3130 Genetic Analyzer (Applied Biosystems).In 105 of 175 patients the study by BIOMED-2 multiplex polymerase chain reaction protocol of B-cell clonality of bone marrow was fulfilled. Statistical analysis was done using JMP ver. 10.0 (SAS, Cary, NC).Results: Histological and immunohistochemical studies validated bone marrow involvement in 19 patients (10.8%), including: concordant in - 12 patients (63%), and discordant - in 7 patients (37%). In 14 of these patients the B-cell clonality detection was performed and confirmed immunoglobulin (IG) heavy chain gene rearrangement in all cases. In 26 out of 105 patients, bone marrow involvement (24.7%) was revealed: in 14 patients bone marrow involvement was identified by histological examination and in 12 patients only by clonality of bone marrow. 12 patients with only B-cell clonality in bone marrow were classified by IPI: 4 (33%) patients had high IPI, 6 (50%) patients - high-intermediate IPI and low-intermediate IPI - 2 (17%) patients. So, in 17% patients had to change the risk group according to bone marrow involvement. Thus, 31 patients with bone marrow involvement signs were identified.Univariate analysis of the entire cohort (n = 175) revealed that both IPI and phenotype were not of prognostic significance (p > 0.05). In multivariate analysis, detection of B-cell clonality in bone marrow was an independent predictor of OS and relapse-free survival (RFS) (p < 0.005). (Fig.1-2). OS, RFS in patients with histological detection bone marrow involvement and with only B-cell clonality in the bone marrow did not differ.Conclusion:Detection of B-cell clonality in the bone marrow seems to be an independent predictor of outcome in de novo DLBCL pts, treated with high-dose chemotherapy, while IPI and phenotype DLBCL cannot be considered as risk factors for this group of pts. It seems to be reasonable to include the detection of B-cell clonality in the bone marrow in primary diagnostics of the DLBCL for staging and predicting response in HDC. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reduction Of Immune Repertoire Diversity Through DNA Sequence Constraints At VDJ Junctions

B and T lymphocytes are effector cells of the adaptive immune system. These cells express surface receptors that bind a huge variety of antigens, and together they comprise a person's immune repertoire. A diverse repertoire is essential for mounting robust immune responses against a wide range of pathogens, and repertoire diversity affects the probability that DNA sequencing can uniquely tag a clonally expanded population of cells for the detection of minimum residual disease (MRD) during cancer treatment.Immune repertoire diversity arises partly through the combinatorial splicing of gene segments from the variable (V), diversity (D), and joining (J) regions of a B or T cell receptor locus. Much additional diversity is created through the stochastic insertion and deletion of nucleotides at the splice junctions, and by somatic hypermutation (SHM) in maturing lymphocytes. The generation of junctional diversity is an important part of this process, but it may be constrained by the underlying biological mechanisms.To explore the landscape of junctional diversity among immune receptor loci, we developed a likelihood model that can annotate VDJ junctions in the presence of SHM and compute the probability that a given receptor sequence was generated only once in a person's repertoire, which is essential for tracking MRD. Using high-throughput sequencing data from several individuals and a range of receptor loci, we identify mechanistic constraints that limit B and T cell receptor diversity. For example, we show that the usual variability in CDR3 length is reduced at the immunoglobulin kappa (IgK) locus, and we connect this finding to sequence motifs that constrain nucleotide deletion at the ends of IgK gene segments. Our findings will inform future genetic studies of the adaptive immune system, and they provide quantitative guidance for deciding which cancer clones can be tracked for reliable MRD detection. Disclosures:Howie:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Carlson:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties.

Next-Generation Sequencing and Real-Time Quantitative PCR For Quantification Of Low-Level Minimal Residual Disease In Acute Lymphoblastic Leukemia Of Adults

BackgroundDetection of minimal residual disease (MRD) in adult ALL is an independent risk parameter and early indicator of an impending relapse. MRD diagnostics have been incorporated in many ALL treatment protocols as a tool for pre-emptive treatment or risk group stratification. Currently, real-time quantitative (RQ)-PCR of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements is regarded as the most sensitive and standardized method to quantify MRD in BCR-ABL negative ALL. However, IG/TR oligoclonality and clonal evolution may lead to false negative results, while sensitivity varies depending on background amplification and is generally limited to 1E-05. Next-Generation Sequencing (NGS) of IG/TR gene rearrangements might overcome these limitations and potentially provide further increases in sensitivity, specificity, accuracy and reproducibility. We have performed a comparison of the two approaches on 130 diagnostic (DG), relapse (REL) and remission (FU) samples of 14 adult patients (pts) with B-cell precursor ALL. To test the NGS sensitivity, we partly selected follow-up samples for (low-level) RQ-PCR MRD positivity or a high pre-test probability of residual disease (either due to a later relapse or MRD detection in temporal relation to the analyzed sample). Patients and Methods130 samples (97 bone marrow, 31 peripheral blood, 2 stem cell apheresis; 15 DG, 107 FU, 8 REL samples) were analyzed from 14 pts with B-cell precursor ALL. IG/TR-based RQ-PCR was carried out within routine diagnostics in the context of prospective clinical trials [Brüggemann et al., Blood 2006], according to the EuroMRD criteria [van der Velden et al., Leukemia 2007] at the MRD reference laboratory in Kiel. NGS was performed at the Sequenta facilities in South San Francisco. Using universal primer sets, IGH, IGK, TRB, TRG, and TRD variable, (diversity), and joining gene segments were amplified from DG and REL DNA. PCR products were sequenced to obtain a high degree of coverage and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency in DG samples. The presence of the tumor-specific clonotype was then quantitated in FU samples. A quantitative and standardized measure of clone level among all leukocytes was determined using internal reference DNA. Comparability of MRD results by RQ-PCR and NGS was assessed using bivariate correlations between methods analysis (software R 3.0.1 with cor.test). ResultsSanger sequencing of multiplex PCR detected 60 clonal IGH, IGK, TRB, TRG and/or TRD gene rearrangements at diagnosis (1-6/patient), NGS identified 57 index sequences (1-6/patient). Using NGS, relapse samples were analyzed for clonal evolution: 16/19 index rearrangements of relapsing pts were stable at relapse while 3/19 rearrangements (2 IGH VH-JH and 1 IGK) were lost at relapse.Based on Sanger sequence information of clonal rearrangements 1-3 RQ-PCR assays/patient were established while NGS focused on 1-4 clonal markers/patient.MRD was quantified in 107 FU in remission using both tools. A mean of 223,086±48,030 cells were analyzed per target using RQ-PCR, whereas NGS used 575,776±304,653 cells. An excellent concordance between the two methods was observed (p<0.0001, r2 = 0.974) with 46/107 samples being MRD positive and 45/107 MRD negative with both tools. Sequencing detected MRD in 13 samples that were negative by RQ-PCR whereas RQ-PCR revealed MRD in 3 NGS negative samples. All discrepancies affected samples with low MRD levels (below quantitative range (RQ-PCR) and levels below 8E-06 (NGS)) being consistent with Poisson sampling. In addition, both methods showed quantitative and qualitative inter-target differences with 11/49 RQ-PCR and 32/59 NGS positive samples being negative for at least one target illustrating the importance of focusing on more than one index sequence for RQ-PCR and NGS. ConclusionsSignificant concordance was observed between NGS and RQ-PCR, with NGS having similar or slightly better sensitivity compared to standardized RQ-PCR. This can be partly attributed to the higher number of cells analyzed by NGS and points out that sensitivity advantages afforded by the NGS platform may be achieved by higher input cell amounts. Prospective analyses of unselected cases must be performed to verify the clinical impact of low level NGS-based MRD detection. Disclosures:Pepin:Sequenta Inc.: Employment, Stockholder Other. Carlton:Sequenta Inc.: Employment, Stockholder Other. Faham:Sequenta Inc.: Employment, Stockholder Other.

Activation-Induced Cytidine Deaminase Does Not Impact Murine Meiotic Recombination

Activation-induced cytidine deaminase (AID) was first described as the triggering enzyme of the B-cell−specific reactions that edit the immunoglobulin genes, namely somatic hypermutation, gene conversion, and class switch recombination. Over the years, AID was also detected in cells other than lymphocytes, and it has been assigned additional roles in the innate defense against transforming retroviruses, in retrotransposition restriction and in DNA demethylation. Notably, AID expression was found in germline tissues, and in heterologous systems it can induce the double-strand breaks required for the initiation of meiotic recombination and proper gamete formation. However, because AID-deficient mice are fully fertile, the molecule is not essential for meiosis. Thus, the remaining question that we addressed here is whether AID influences the frequency of meiotic recombination in mice. We measured the recombination events in the meiosis of male and female mice F1 hybrids of C57BL/6J and BALB/c, in Aicda+/+ and Aicda−/− background by using a panel of single-nucleotide polymorphisms that distinguishes C57BL/6J from BALB/c genome across the 19 autosomes. In agreement with the literature, we found that the frequency of recombination in the female germline was greater than in male germline, both in the Aicda+/+ and Aicda−/− backgrounds. No statistical difference was found in the average recombination events between Aicda+/+ and Aidca−/− animals, either in females or males. In addition, the recombination frequencies between single-nucleotide polymorphisms flanking the immunoglobulin heavy and immunoglobulin kappa loci was also not different. We conclude that AID has a minor impact, if any, on the overall frequency of meiotic recombination.

YY1 controls Igκ repertoire and B-cell development, and localizes with condensin on the Igκ locus

Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.

Molecular analysis of the t(2;8)/MYC–IGK translocation in high‐grade lymphoma/leukemia by long‐distance inverse PCR

Burkitt lymphoma and a subset of diffuse large B-cell lymphomas are characterized by chromosomal alterations affecting the MYC oncogene on 8q24. In most cases MYC is found juxtaposed to the immunoglobulin heavy chain (IGH) gene locus. Translocations to the immunoglobulin kappa (IGK) gene locus on 2p11 are observed in around 5-10% of cases. Little data exist on the molecular mechanisms leading to this aberration. The chromosomal breakpoints on chromosome 8 have been found dispersed over a large area 3' of MYC. In order to obtain a better understanding of this chromosomal translocation we developed a long-distance inverse (LDI) PCR method for the identification of chromosomal translocations affecting the IGK locus. We investigated a number of cytogenetically mostly uncharacterized high-grade lymphoma samples and identified a MYC-IGK juxtaposition in seven patients and three t(2;8)-positive cell lines. The chromosomal breakpoints were molecularly characterized and analyzed. The linear distance of the breakpoints on chromosome 8 to MYC ranged from some 100 bp to more than 0.5 MB. The reciprocal translocated allele could be characterized in the majority of cases. This study represents the largest series of t(2;8)-positive cases analyzed so far. The LDI PCR method developed here should also be useful for the analysis of chromosomal translocations affecting the IGK locus in general.

Evolution of the porcine (Sus scrofa domestica) immunoglobulin kappa locus through germline gene conversion

Immunoglobulin (IG) gene rearrangement and expression are central to disease resistance and health maintenance in animals. The IG kappa (IGK) locus in swine (Sus scrofa domestica) contributes to approximately half of all antibody molecules, in contrast to many other Cetartiodactyla, whose members provide the majority of human dietary protein and in which kappa locus utilization is limited. The porcine IGK variable locus is 27.9 kb upstream of five IG kappa J genes (IGKJ) which are separated from a single constant gene (IGKC) by 2.8 kb. Fourteen variable genes (IGKV) were identified, of which nine are functional and two are open reading frame (ORF). Of the three pseudogenes, IGKV3-1 contains a frameshift and multiple stop codons, IGKV7-2 contains multiple stop codons, and IGKV2-5 is missing exon 2. The nine functional IGKV genes are phylogenetically related to either the human IGKV1 or IGKV2 subgroups. IGKV2 subgroup genes were found to be dominantly expressed. Polymorphisms were identified on overlapping BACs derived from the same individual such that 11 genes contain amino acid differences. The most striking allelic differences are present in IGKV2 genes, which contain as many as 16 amino acid changes between alleles, the majority of which are in complementarity determining region (CDR) 1. In addition, many IGKV2 CDR1 are shared between genes but not between alleles, suggesting extensive diversification of this locus through gene conversion.

High Throughput Digital Quantification of Genomic Copy Number Alterations in Multiple Myeloma

Abstract 1830Multiple myeloma (MM) is a fatal disease characterized by clonal expansion of malignant plasma cells. The etiopathogenesis of MM is not fully understood. Several numerical and structural chromosomal aberrations have been identified as diagnostic markers and predictors of evolution in MM. Cytogenetic studies in MM patients are often not informative due to technical difficulties related to low proliferation of malignant plasma cells and outgrowth of non-malignant cells. Fluorescence in-situ hybridization (FISH) on CD138+ sorted plasma cells is probably the best method for maximizing diagnostic yield in MM, but is limited to the genomic regions queried. To overcome the limitations of the amount of clinical material available and to be able to interrogate large number of MM specific genomic aberrations, we developed and validated a MM genomic copy number signature. This signature comprised of 183 MM specific genes, was developed by pooling data from extensive meta-analyses on publically available raw data from ∼450 MM patients and copy number data generated by high-resolution SNP arrays (Affymetrix) from 39 MM patients in our cohort. To validate this signature of a large number of genes, we tested a recently developed innovative high throughput digital technology NanoString - nCounter assay. This technology captures and counts individual DNA molecules without enzymatic reactions or bias and is notable for its high levels of sensitivity, linearity, multiplex capability, and digital readout. It requires minimal input of DNA (∼300ng) making it a valuable tool for genomic copy number signature validation, diagnostic testing, and large translational studies, all of which often are limited by the very small amounts of clinical material available. Digital data was generated using nCounter analysis in 42 newly diagnosed, untreated MM patients. To identify the true acquired somatic copy number changes matched germline (skin) and tumor (sorted CD138+ cells) were analyzed from each of these MM patients. All of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The dynamic range in copy number calls with this assay is very large since there are no saturation issues and there is very low background. In this study, we were able to detect a maximum of 9 copies in some of the targets. We observed amplification of chromosomes 1q(51%), 3(65%), 5(65%), 7(70%), 9(56%), 11(72%), 15(56%), 19(53%), 21(42%), and deletion of chromosomes 1p(25%), 6q(28%), 8p(42%), 12p(40%), 13(47%), 14(26%) and 16q(49%). Interestingly, cytoband 2p11.2 and 14q32.33 consisting IGK and IGH genes were deleted in 75% and 93% of the patient population respectively. Overall, our results correlate well with the known pattern of genomic aberrations in MM. Additional analysis in an extended panel with clinically categorized samples is carried on to test the utility of this myeloma specific gene signature. To the best of our knowledge this is the first application of a high-throughput digital system to validate genomic copy number signature in cancer. Disclosures:No relevant conflicts of interest to declare.

Molecular Analysis of the t(2;8)/MYC-IGK Translocation in High-Grade Lymphoma/Leukemia by Long-Distance Inverse PCR

Abstract 4407 Background:Burkitt lymphoma and a subset of diffuse large B-cell lymphomas are characterized by chromosomal alterations affecting the MYC oncogene on 8q24. In most cases MYC is found juxtaposed to the immunoglobulin heavy chain (IGH) gene locus. Translocations to the immunoglobulin kappa (IGK) gene locus on 2p11 are observed in around 10% of cases. Little data exist on the molecular mechanisms leading to this aberration. The chromosomal breakpoints on chromosome 8 have been found dispersed over a large area 3' of MYC. Currently, molecular cytogenetics (FISH) and cytogenetics are the methods of choice to detect the t(2;8) translocation and until now there exists no PCR method to reliably detect it. Objectives:In order to obtain a better understanding of this chromosomal translocation we developed a long-distance inverse (LDI) PCR method for the identification of chromosomal translocations affecting the IGK locus. The LDI PCR method takes advantage of the fact that the chromosomal breaks occur on chromosome 2 in the vicinity of the IGK joining segments. Using this method we investigated a number of cytogenetically mostly uncharacterized high-grade lymphoma samples. Results:A MYC-IGK juxtaposition was identified in 7 patients and three t(2;8)-positive cell lines. The chromosomal breakpoints were molecularly characterized and analyzed. The linear distance of the breakpoints on chromosome 8 to MYC ranged from some 100 bp to more than 0.5 MB. The breakpoints appeared to be clustered in distinctive regions, however, the number is still to small to draw definitive conclusions. The reciprocal translocated allele could be characterized in the majority of cases and putative break mechanisms are proposed. No larger sequence homologies were detected at the break sites. A minority of breaks were located near putative recombination signal sequences (RSS). Conclusions:This study represents the largest series of t(2;8)-positive cases analyzed so far and the LDI PCR developed therein is the first universally applicable PCR-based method to detect this aberration. This LDI PCR should furthermore in general be useful for the molecular analysis of chromosomal translocations affecting the IGK locus. These aberrations have been detected in various lymphoma entities but are currently largely unexplored. Disclosures:No relevant conflicts of interest to declare.

Application of Microfluidic Technology to the BIOMED-2 Protocol for Detection of B-Cell Clonality

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.

BIOMED‐2 PCR assays for IGK gene rearrangements are essential for B‐cell clonality analysis in follicular lymphoma

B-cell clonality analysis is commonly performed by polymerase chain reaction (PCR) targeting the IGH genes although a high false-negative rate is recognized for germinal centre/post-germinal centre B-cell malignancies, especially follicular lymphoma. We assessed the diagnostic value of BIOMED-2 IGK assays and investigated the cause of IGH PCR failure in 77 patients with follicular lymphoma. Using the full set of BIOMED-2 reactions, clonal immunoglobulin gene rearrangements were detected in 74 (96%) cases. The clonality detection rate was 86% by two IGK reactions but only 68% by five IGH reactions (P < 0·001). Sequencing of the clonal PCR products showed significantly fewer somatic mutations in the rearranged IGKV (9/27 cases, 33%, mean mutation rate 0·5%) than IGHV (17/17 cases, 100%, rate 11·0%) (P < 0·01). All IGHV-IGHJ PCR failures occurred in cases with at least one mutation at the corresponding IGHV primer binding sites. t(14:18)(q32:q21)/IGH-BCL2 was detected in 50 of 71 (70%) cases and the presence of the translocation was not associated with the poor performance of IGH assays. Our results showed that BIOMED-2 IGK assays are significantly more sensitive than IGH assays in follicular lymphoma due to the fact that the rearranged IGKV is less frequently targeted by somatic hypermutation than IGHV, and therefore, are essential in routine clonality analysis of these lymphomas.

Genetic variation at immunoglobulin kappa locus is associated with hepatitis C–treatment–induced viral clearance in African Americans

Host genetic factors, especially genes of the immune system, are thought to contribute to the racial differences in response rates to therapy for hepatitis C virus (HCV) infection. The aim of the present investigation was to determine whether immunoglobulin gamma heavy chain marker (GM) and kappa light chain marker (KM) —were associated with sustained viral response (SVR) in patients treated with peginterferon-α-2a and ribavirin. DNA samples from 319 subjects with genotype-1 HCV infections were allotyped for alleles at four GM loci: GM3/GM17, GM23+/GM23−, GM5/GM21, GM6+/GM6− and the KM locus: KM1/KM3, using molecular methods. Noncarriage of KM1 allele, i.e., KM3 homozygosity, was associated with higher SVR in African Americans (odds ratio = 2.50, 95% confidence interval = 1.12–5.60). Consistent with this finding, the HCV RNA level in KM1 noncarriers was significantly (p = 0.013) lower than in carriers of this allele. Thus, the KM3 allele may be a marker for higher SVR in African Americans.

EBV-Positive Plasmacytoma of the Submandibular Gland—Report of a Rare Case with Molecular Genetic Characterization

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 7.7cm 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.

Molecular Characterization of a Chromosomal Translocation Linking CDK6 to the IGK Locus In CD5- Monoclonal B-Cell Lymphocytosis (MBL).

AbstractAbstract 1191Efforts to decipher the biological basis of lymphoid malignancy have recently been accentuated by the discovery of clonal B-cells in otherwise healthy individuals, an entity now referred to as monoclonal B-cell lymphocytosis (MBL). However, while considerable attention has been devoted towards characterizing this pervasive condition, the subgroup of MBL lacking CD5 expression has been largely overlooked. To address this imbalance, our group previously examined a series of cases presenting with persistent but non-progressing CD5- clonal lymphocytosis (Amato D et al, Am J Clin Pathol 2007). Herein, karyotypic analysis revealed a unique case harboring the translocation t(2;7)(p11;q22), a genetic abnormality which has been documented in association with splenic marginal zone lymphoma (SMZL). The present study set out to further characterize the t(2;7) breakpoint of this novel case study in order to test whether the translocation represents a common genetic link between CD5- MBL and SMZL. Using fluorescent in situ hybridization (FISH), the breakpoint on chromosome 7 was initially narrowed down to a 41 kilobase (kb) candidate region at 7q21.2, as inferred by a split signal from the cosmid probe cos130a6. Based on the proximity of the chromosome 2 breakpoint to the immunoglobulin kappa (IGK) locus on 2p11, long-range polymerase chain reaction (L-PCR) was subsequently performed according to the hypothesis that the t(2;7) translocation had arisen from aberrant VJ recombination. Primers targeting conserved sequences within the IGK variable genes were sequentially juxtaposed with primers on chromosome 7 located at 6 kb intervals across the 41 kb candidate region, and L-PCR conducted using both patient and reference DNA as a template. This process gave rise to a single instance of strong patient-specific amplification, thereby indicating successful targeting of the t(2;7) breakpoint. Sanger sequencing of the corresponding PCR product confirmed this result, revealing a breakpoint at 2p11.2 localized to the heptamer recombination signal sequence (RSS) of the IGK variable gene V3-15, alongside a breakpoint at 7q21.2 located 3.6 kb upstream of the transcription start site of the cell cycle regulator cyclin dependent kinase 6 (CDK6). Remarkably, the breakpoint on chromosome 7 showed perfect sequence homology to the immunoglobulin RSS heptamer, and was within 3 base pairs of a breakpoint previously characterized in association with SMZL (Corcoran M et al, Oncogene 1999). Hence, this result implicates the dysregulation of CDK6 during the clonal expansion observed in CD5- MBL, and highlights the potential role of aberrant VJ recombination during the formation of this genetic abnormality. By establishing a clear genetic link between CD5- MBL and SMZL, this study supports the notion of CD5- MBL as a precursor of overt malignancy within a ‘multi-hit’ model of oncogenesis. Finally, the sequence-level characterization of a novel translocation breakpoint by L-PCR strongly validates the potential utility of this approach during future studies of immunoglobulin-associated translocations. Disclosures:No relevant conflicts of interest to declare.