VDJ Gene Rearrangement Research Articles

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection.

Advancements in high-throughput sequencing (HTS) of antibody repertoires (Ig-Seq) have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale. However, currently, only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions, possibly limited by inadequate sequencing depth and throughput. To better understand how HIV-1 infection would impact humoral immune system, in this study, we systematically analyzed the differences between the IgM (HIV-IgM) and IgG (HIV-IgG) heavy chain repertoires of HIV-1 infected patients, as well as between antibody repertoires of HIV-1 patients and healthy donors (HH). Notably, the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries, and the diversity of unique clones in HIV-IgG remarkably reduced. In aspect of somatic mutation rates of CDR1 and CDR2, the HIV-IgG repertoire was higher than HIV-IgM. Besides, the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire, presumably caused by the great number of novel VDJ rearrangement patterns, especially a massive use of IGHJ6. Moreover, some of the B cell clonotypes had numerous clones, and somatic variants were detected within the clonotype lineage in HIV-IgG, indicating HIV-1 neutralizing activities. The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Clonal relationships in recurrent B-cell lymphomas.

Immunoglobulin (Ig) gene rearrangements remain largely unmodified during the clonal expansion of neoplastic cells. We investigated the clonal relationships between lymphoma components at diagnosis and at relapse by analyzing Ig gene rearrangements. A BIOMED-2 multiplex polymerase chain reaction (PCR) assay was performed in 27 patients using formalin-fixed paraffin embedded tissues, with subsequent cloning and sequencing of the amplified Ig genes in 17 patients. All 27 cases of primary and corresponding relapsed tumors showed monoclonal rearrangements of the Ig genes by BIOMED-2 PCR. Whereas IgVH or IgVK fragment lengths were identical in 8/27 pairs (30%), fragment lengths differed in 19/27 pairs (70%). In 17 cases analyzed by sequencing, an identical VDJ gene rearrangement was confirmed in 4/4 pairs (100%) with the same fragment lengths and in 10/13 pairs (77%) with different fragment lengths. Four of 17 primary lymphomas had multiple VDJ rearrangements, and three of them showed an unrelated relapse. Unrelated relapse was observed in 1/8 mantle cell lymphomas, 1/5 diffuse large B-cell lymphomas, and a large B cell lymphoma developed in a patient with a small lymphocytic lymphoma. Unrelated relapses developed after a longer disease-free interval and tended to show poorer outcome compared with related relapse. In summary, relapse of a lymphoma from an unrelated clone is uncommon, but can occur in B-cell lymphomas. Clonal relationships should be determined by sequencing of the Ig genes, and not just by comparing the PCR product size.

Abstract 1521: High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance

Abstract The bulk of current knowledge on Multiple myeloma (MM) disease is mainly based on the molecular characterization of 138+ plasma cells, but we become aware that MM heterogeneity has more deepen roots. Recent studies support that Myeloma Propagating Cells (MPCs) might be responsible of relapses and they residing in the 138- compartment. In order to molecularly characterize mature and precursor cell fractions, we collected the 138+ and 138-19+27+ cell fractions from bone marrow (BM) and peripheral blood (PBL) of 50 newly diagnosed MM patients (pts). Clonogenic assays were performed by plating cell fractions of MM cell lines. The molecular characterization included: IgH rearrangement sequencing, analysis of genomic aberrations and gene expression profiling by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray. Clonogenic assays showed that 138- cells were able to form colonies more efficiently than 138+ cells. By VDJ gene rearrangement sequencing, a clonal relationship between the 138+ clone and precursor ones was confirmed. SNPs arrays showed that both BM and PBL 138+ cell fractions carried the same genomic macro-alterations. On the contrary, in the 138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations unique of the memory B cells clone were highlighted. An enrichment analysis revealed that genes are involved in DNA repair mechanisms, transcriptional regulation, negative regulation of apoptosis and angiogenesis. Interestingly, KRAS, WWOX and XIAP genes are located in the amplified regions of immature cells. Moreover, we observed several LOH regions that covered important tumor suppressor genes, including TP53, CDKN2C and RASSF1A. The involvement of immature cells in MM pathogenesis was confirmed by the analysis of the 19+ cells’ transcriptome profiles. 20 MM 19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature 138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM 19+ clones, respectively (2; FDR = 0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed and Hedgehog pathway overall down-regulated. Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p = 7,25E-14; FDR = 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC = -18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells. Presented data support the emerging role of the immature cell compartment in the MM disease course, where the putative 19+ MPCs might be involved in the neoplastic clone supply. Citation Format: Marina Martello, Angela Flores Dico, Daniel Remondini, Barbara Santacroce, Enrica Borsi, Mauro Procacci, Lucia Pantani, Elena Zamagni, Paola Tacchetti, Anna Maria Brioli, Serena Rocchi, Giulia Marzocchi, Giovanni Martinelli, Michele Cavo, Carolina Terragna. High-throughput molecular profiling of Multiple Myeloma clonotypic CD19+ B cells highlights pathways potentially involved in the disease endurance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1521. doi:10.1158/1538-7445.AM2015-1521

High-Throughput Molecular Profiling of Multiple Myeloma (MM) Clonotypic CD19+ B Cells Highlights Pathways Potentially Involved in the Disease Endurance

Background Although remarkable advances have been achieved in MM therapy, mainly thanks to the introduction of novel-agent-based regimens, the disease remains incurable. Neoplastic CD138+ plasma cells are the hallmark of MM: both their expansion in the bone marrow (BM) and the production of monoclonal immunoglobulin (Ig) are responsible for the clinical manifestation of the disease. However, the existence of a Myeloma Propagating Cells (MPCs) has been proposed as a major cause of MM drug-resistance, leading to relapse. Several studies support the hypothesis that MPCs are phenotypically close to memory B cells residing in the CD138- compartment; however, very little is known concerning their molecular characteristics.Here we present an extensive molecular characterization of clonotypic CD19+ B cells clones obtained from newly diagnosed MM patients (pts), in order to recognize biological pathways possibly explaining the malignant clone’s persistence.Methods CD138+ and CD138- cell fractions were collected from BM and peripheral blood (PBL) of 50 newly diagnosed MM pts. CD19+ B cell and CD27+ memory B cell populations were isolated from CD138- cell fraction. Clonogenic assays were performed by plating cell fractions obtained from RPMI-8226 and NCI-H929 cell lines. The molecular characterization included: IgH gene rearrangement Sanger sequencing; analysis of the whole spectrum of genomic aberrations and gene expression profiling, by Affymetrix 6.0 SNPs array and HG-U133 Plus 2.0 microarray, respectively.Results Clonogenic assays showed that CD138- cells, plated on conditioned media, were able to form colonies after two weeks of culture more efficiently than CD138+ cells.By VDJ gene rearrangement sequencing, a clonal relationship between the CD138+ clone and the memory B ones was confirmed.SNPs arrays showed that both BM and PBL CD138+ cell fractions carried exactly the same genomic macro-alterations. On the contrary, in the CD138-19+27+ cell fractions from BM and PBL any macro-alteration was detected, whereas several micro-alterations (median number per sample: 32 amplifications and 16 losses, range: 8-122 Kb, average markers per region: 50) unique of the memory B cells clone were highlighted. An enrichment analysis revealed the involvement of genes affected by losses (17 genes) in both DNA repair mechanisms and transcriptional regulation and the involvement of genes affected by gains (46 genes) in both the negative regulation of apoptosis and the angiogenesis. Interestingly, KRAS, WWOX and XIAP genes, renown to be involved in MM pathogenesis, are located in the amplified regions in the immature cells. Moreover, several LOH regions were described, which covered at least 106 tumor suppressor genes involved in MM and leukemia (including TP53, CDKN2C and RASSF1A).Transcriptome profiles analysis of the CD19+ cell fractions highlighted pathways suggesting a possible involvement of immature cells in MM pathogenesis. The gene expression profiles of 20 MM CD19+ cells samples (12 from PBL, 8 from BM) were compared both to their normal counterpart and to the mature CD138+ cell fractions. In particular, unsupervised analysis by hierarchical clustering discriminated the differential expression of 11480 and 11360 probes in the PBL and BM CD19+ clones, respectively (<-2FC>2; FDR=0,05; p <0,05). An overall de-regulation of pathways involved in self-renewal mechanisms was highlighted, with Notch and Wnt signaling over-expressed in every analyzed cell compartment; on the contrary, Hedgehog pathway was overall down regulated.Interestingly, the protein homeostasis deregulation possibly caused by ER stress, resulted particularly evident in the BM 19+ cells (p=7,25E-14; FDR= 2,98E-11); moreover, the down-regulation of genes related to the unfolded protein response (e.g. IRE1α and XBP1 FC=-18,0; -19,96. p<0,05) suggests the expression of a proteasome inhibitor-resistant phenotype of these cells.Conclusions Presented data support the emerging role of the immature cell compartment in the MM disease course, where the MM CD138+ clone might resume the end of the complex process of tumorigenesis, whereas the putative CD19+ MPCs, by displaying peculiar genomic micro-alterations and a unique transcriptional profile, might be involved in the neoplastic clone supply.Supported by ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; Ariad: Consultancy. Cavo:BMS: Consultancy, Honoraria; Millenium: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

In non-transplant patients with multiple myeloma, the pre-treatment level of clonotypic cells predicts event-free survival

BackgroundIn multiple myeloma (MM), the immunoglobulin heavy chain VDJ gene rearrangement is a unique clonotypic signature that identifies all members of the myeloma clone independent of morphology or phenotype. Each clonotypic MM cell has only one genomic copy of the rearranged IgH VDJ.MethodsPre-treatment bone marrow aspirates from myeloma patients at diagnosis or in relapse were evaluated for the number of clonotypic cells using real time quantitative PCR (RPCR). RPCR measured the level of clonal cells, termed VDJ%, in 139 diagnosis and relapse BM aspirates from MM patients.ResultsPatients with a VDJ% below the median had a significantly longer event free survival (EFS) then those with a VDJ% higher than the median (p=0.0077, HR=0.57). Further, although the VDJ% from non-transplant patients predicted EFS (p=0.0093), VDJ% failed to predict outcome after autologous stem cell transplant (p=0.53).ConclusionsOur results suggest that for non-transplant patients, the tumor burden before treatment, perhaps reflecting cancer stem cell progeny/output, is an indirect measure that may indicate the number of MM cancer stem cells and hence event free survival.

Chemokine-mediated B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (160.15)

Abstract Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells generate a diversified primary antibody repertoire by mutating their VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensals. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained the chemokine receptors CXCR4, CCR6, CXCR5 and CCR7 on appendix B cells from two- through seven-day-old rabbits with fluorescence-labeled antibodies or Ig-fusion proteins of their ligands and analyzed them by flow cytometry. The changes in relative chemokine receptor expression levels we observed during the first week of life suggest that B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes to generate the primary antibody repertoire.

The Inference of Phased Haplotypes for the Immunoglobulin H Chain V Region Gene Loci by Analysis of VDJ Gene Rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.

Mechanism for Pre-B Cell Loss in VH-Mutant Rabbits

Pre-BCR signaling is a critical checkpoint in B cell development in which B-lineage cells expressing functional IgH μ-chain are selectively expanded. B cell development is delayed in mutant ali/ali rabbits because the a-allotype encoding V(H)1 gene, which is normally used in VDJ gene rearrangements in wt rabbits, is deleted, and instead, most B-lineage cells use the a-allotype encoding V(H)4 gene [V(H)4(a)], which results in a severe developmental block at the pre-B cell stage. We found that V(H)4(a)-utilizing pre-B cells exhibit reduced pre-BCR signaling and do not undergo normal expansion in vitro. Transduction of murine 38B9 pre-B cells with chimeric rabbit-VDJ mouse-Cμ encoding retroviruses showed V(H)4(a)-encoded μ-chains do not readily form signal-competent pre-BCR, thereby explaining the reduction in pre-BCR signaling and pre-B cell expansion. Development of V(H)4(a)-utilizing B cells can be rescued in vivo by the expression of an Igκ transgene, indicating that V(H)4(a)-μ chains are not defective for conventional BCR formation and signaling. The ali/ali rabbit model system is unique because V(H)4(a)-μ chains have the capacity to pair with a variety of conventional IgL chains and yet lack the capacity to form a signal-competent pre-BCR. This system could allow for identification of critical structural parameters that govern pre-BCR formation/signaling.

Chemokine-directed B cell trafficking in rabbit GALT during diversification of the primary antibody repertoire (149.4)

Abstract Rabbits preferentially rearrange the 3’-most VH gene segment during VDJ gene rearrangement. Early in life, rabbit B cells diversify these VDJ genes in gut-associated lymphoid tissue (GALT) in response to signals acquired from intestinal commensal bacteria, and thus generate a diversified primary antibody repertoire. To gain insight into the cell-cell and cell-microbe interactions required for VDJ gene diversification, we examined B cell surface expression levels of four chemokine receptors that mediate trafficking to important sites within GALT during repertoire diversification. We stained B cells from two- through seven-day-old appendix with fluorescence-labeled Ig-fusion proteins of the chemokines CXCL12, CCL20, CXCL13 and CCL21 and analyzed them by flow cytometry. The changes we observed in relative chemokine receptor expression levels during the first week of life suggest a model in which B cells, upon entering appendix follicles, first traffic to the follicle-associated epithelium, where they likely encounter commensal bacteria sampled from the intestinal microbiota. B cells subsequently migrate to, and likely receive additional signals from, the follicular dendritic cell network. Upon becoming activated, B cells then traffic to the basal region of the follicle, where they proliferate and diversify their VDJ genes, thereby generating the primary antibody repertoire.

Abstract 12: Metastatic neuroblastoma cancer stem cells display a mixed phenotype of tumor and niche origin required for survival

Abstract Neuroblastoma (NB) is a pediatric tumor of neural crest origin, and is the most common cancer of infancy. 50% of patients have metastases at diagnosis, of which 85% will die after multiple relapses from metastatic disease. We identified tumor-initiating cells (TICs) from bone marrow (BM) metastases of high-risk patients that are propagated as spheres and that have many properties of cancer stem cells, and form NB in mice with as few as 1 cell. To understand patient relapse and disease progression, we compared NB-TICs from BM with those from tumor and brain metastases and SKPs, a normal pediatric stem cell counterpart, by cDNA microarray, flow cytometry, and whole genome shotgun sequencing transcriptome analysis. BM-derived NB-TICs expressed primitive neural crest and neuronal markers as well as hematopoietic markers from primitive, myeloid, and B-cell lineages and contained VDJ gene rearrangements, which are normally associated with B-cell leukemias. Hematopoietic genes were not expressed or expressed at very low levels in tumor-derived sphere lines and a line from an NB brain metastasis, however brain metastasis-derived TICs expressed CD133, while BM-derived NB-TICs did not. Furthermore, we found that shRNA to CD74, which is upregulated in B-cell lymphoma and multiple myeloma and is a therapeutic target for those cancers, induced the rapid death of NB-TICs but not normal SKPs. Interestingly cells co-staining for the hematopoietic markers CD45 or CD74 and the neural neural progenitor marker nestin were found in BM smears of patients with relapsed NB in the bone marrow. We suggest that metastatic TICs from some tumors adopt resident niche-specific gene expression signatures, which may aid diagnosis and the development of novel treatments. We hypothesize that drugs used for leukemia may be efficacious therapeutics for metastatic NB, and are currently testing this hypothesis in mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 12.

Role of CXCL12 and its receptor, CXCR4, in diversification of the rabbit primary antibody repertoire (133.3)

Abstract The antibody repertoire of early postnatal rabbits is of limited diversity due to preferential V gene usage during VDJ gene rearrangement. Early in life, rabbit B cells expand this repertoire by diversifying their VDJ genes in gut-associated lymphoid tissue. VDJ gene diversification begins around 1 week of age, in B cells located in the basolateral region of nascent follicles. Because these B cells share similarities with B cells in germinal center dark zones, we hypothesized that activated B cells localize to the basolateral region in response to the chemokine CXCL12. To test this hypothesis, we examined B cell expression of CXCR4, the CXCL12 receptor, by in situ hybridization. We found that B cells in the basolateral region, but not those located elsewhere in the follicle, expressed CXCR4. Because antibody repertoire diversification requires signals derived from intestinal commensals, we further examined CXCR4 expression in appendices that were kept sterile by surgical ligation at birth. We found that B cells in these appendices did not express CXCR4 and did not diversify their VDJ genes. By contrast, we detected CXCL12 expression in both conventional and sterile appendices, demonstrating that it is not dependent on commensal-derived signals. Our results suggest that B cells activated by microbial-derived signals upregulate CXCR4 and migrate to the basolateral region of the follicle, likely in response to CXCL12, where they proliferate and diversify their VDJ genes.

Molecular Basis for Loss of VH4-encoded pre-B cells in Mutant Rabbits (36.7)

Abstract Developing B cells in mutant ali/ali rabbits predominantly utilize VH4 during VDJ gene rearrangement. However, most of the VH4-utilizing B cells encounter a severe developmental block at the pre-B cell stage. In contrast, wildtype rabbit B cells predominantly rearrange VH1, a gene &amp;gt;90% identical to VH4, and develop normally. We seek to determine how these nearly identical VH genes have profoundly different effects on B cell development. We hypothesize that VH4-utilizing cells are blocked at the pre-B stage due to weak interaction of the μ-chain with surrogate light chain (SL). We expressed VH4-utilizing μ-chains in a pro-B cell line that expresses SL and found that VH4-encoded μ-chains exhibit a 2 fold reduction in preBCR surface localization compared to control VH1-encoded μ-chains. Remarkably, a single amino acid substitution in VH4-framework region 2 (FR2) from tyrosine to an evolutionarily conserved tryptophan, found in VH1 and most VH genes of other species, restored surface preBCR expression. We are testing if this tryptophan substitution in FR2 of VH4 is sufficient to rescue pre-B cell development. These studies should identify evolutionarily conserved VH structural regions required for efficient preBCR formation and B cell development.

Diagnosis of intraocular lymphoma by polymerase chain reaction analysis and cytokine profiling of the vitreous fluid

To determine whether a diagnosis of intraocular lymphoma (IOL) can be made using a combination of polymerase chain reaction (PCR) analysis to detect gene rearrangement of immunoglobulin and cytokine concentrations in the vitreous fluid. Vitreous samples from 22 patients with clinically suspected IOL and ten control patients with acute retinal necrosis or cytomegalovirus retinitis were examined by PCR analysis and cytokine measurements. Genomic DNA was extracted from the cells in the vitreous, and the immunoglobulin heavy chain (IgH) gene was amplified by two PCR procedures: (1) microdissection and PCR to detect IgH gene rearrangement and (2) qualitative PCR to detect IgH VDJ gene rearrangement. The supernatants of the vitreous samples were used for enzyme-linked immunosorbent assay to determine interleukin (IL)-10 and IL-6 levels. PCR examinations detected IgH rearrangement in the vitreous in 21 of the 22 IOL patients (95.5%) and in none of the ten control patients. Elevated IL-10 concentrations (>100 pg/ml) and the IL-10/IL-6 ratio (>1.0) were positive in 18 of the 22 IOL patients (81.8%), but negative in all of the control patients. Sensitivity, specificity, positive predictive value, and negative predictive value of PCR for the diagnosis of IOL were calculated to be 0.955, 1.000, 1.000, and 0.909, respectively, and those of the cytokine concentration assay to be 0.818, 1.000, 1.000, and 0.714, respectively. When both the intravitreal cytokine assay and PCR analysis of the vitreous samples are used, as well as diagnostic criteria of IOL defined as a positive outcome from one of the two assays together with clinical signs, the sensitivity and specificity of the criteria were 1.000. A combination of PCR assay to detect gene rearrangement of IgH and cytokine profiling (IL-10 and IL-6) is extremely useful for the diagnosis of intraocular lymphoma.

VH Gene Usage and Restricted B cell Development (138.4)

Abstract Signaling through the preBCR is an important checkpoint in B cell development in which the capacity for μ-chain to pair with surrogate light chain (SLC) is tested. Mutant ali/ali rabbits that lack the predominantly rearranged gene, VH1, use VH4 in most VDJ gene rearrangements. Surprisingly, the periphery of these rabbits is initially dominated by VHn-utilizing B cells, not VH4-utilizing B cells. We hypothesize that development of VH4-utilizing B cells is blocked in the bone marrow at the preB cell stage due to weak interaction of the μ-chain with SLC. The IgH CDR3 is important for formation of, and for signaling through the preBCR. Previous crystal structure analysis demonstrated that IgH CDR3 interacts with both components of the SLC, and raises the possibility that VH4-utilizing μ-chains require CDR3 of a particular size and/or amino acid sequence for SLC pairing. We compared IgH CDR3 sequences of VH4- and VHn-utilizing B cells and although we found no obvious differences in amino acid sequences, we did find that the CDR3 of VH4-utilizing B cells was significantly shorter than those found associated with VHn VDJ genes. We hypothesize that long IgH CDR3s encoded by VH4-utilizing VDJ genes prevent pairing with SLC. We are using in vitro experiments to test if pairing of VH4-derived μ-chains with SLC is restricted by CDR3 length. These studies will further identify the structural parameters required for preBCR formation.

Molecular Quantification of Pre-Treatment Bone Marrow Clonal Cells in Multiple Myeloma Is a Novel Predictive Indicator of Remission Length.

Multiple myeloma (MM) is a clonal B-cell malignancy with an accumulation of plasma cells in the bone marrow (BM). Newer treatments continue to increase remission occurrence and decrease side effects, however, relapse invariably occurs. Current diagnostic and monitoring criteria such as bone marrow plasmacytosis (BMPC) and serum M-protein levels (Mpr) fail to predict the length of remission for a given patient. The clonal IgH VDJ gene rearrangement provides a molecular method of measuring the MM disease burden. This unique biomarker can identify and quantify the number of malignant cells through analysis of genomic DNA with quantitative PCR. Here we use SYBR Green real-time quantitative PCR (rqPCR) to measure the level of clonal cells, termed VDJ%, in 91 previously untreated and 51 relapsed pre-treatment BM aspirates from MM patients. Kaplan-Meier analysis showed that VDJ%s lower than the median were associated with longer event free survivals (EFSs) (p=0.0077, HR=0.57). These patients were treated with a variety of different regimens (including VAD, ASCT, Melphalan, Prednisone, Thalidomide, Dexamethasone, Velcade or Revlimid). A modest correlation between the VDJ% and the BMPC was observed (p=0.0003, r2=0.099) but, the BMPC was not able to stratify these patients into groups with significantly different EFSs (p=0.1153, HR=0.6521) as was the VDJ%. This result confirms the BMPC's poor association with outcome. The Mpr was not significantly correlated with VDJ% (p=2312, r2=0.012). The IMWG response subcategories correlated well with EFS where the poorer the response the shorter the EFS. However, there was no significant correlation between the pre-treatment VDJ% and the response subcategories. In addition to the pre-treatment BM samples, the tumor burden in 30 remission BM samples was quantified. To date, neither the remission VDJ% nor the reduction in VDJ% relative to the pre-treatment VDJ% was associated with EFS in a Kaplan-Meier analysis. The remission VDJ% had a significant yet modest correlation with the remission BMPC (p=0.014, r2=0.27) and nadir Mpr (p=0.0001, r2=0.61). Additionally, the IMWG response subcategory correlated with the proportionate reduction of the remission BM (p=0.036). Taken together these results indicate that VDJ% in pre-treatment BMs, but not the matched BMPC or Mpr, is a significant measure of outcome. The percent of clonal cells as measured in BM aspirates with rqPCR could be developed as a novel tool for predicting outcome by placing patients into a high risk or low risk category for EFS. Patients with a higher than median VDJ% have a shorter EFS than patients with a VDJ% below the median. RqPCR enumerates all cells containing the clonal sequence and thus may include relevant cells types not counted in the BMPC or reflected in the protein electrophoresis and not proportionally represented in remission BMs. The predictive value of pre-treatment VDJ% occurred despite varying types of therapy and tumor response, suggesting that these factors might provide independent prognostic information.

Microfluidic Chips for Detecting the t(4;14) Translocation and Monitoring Disease during Treatment Using Reverse Transcriptase-Polymerase Chain Reaction Analysis of IgH-MMSET Hybrid Transcripts

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

Molecular Characterization of Clonotypic VDJ Sequences in Waldenstrom's Macroglobulinemia Suggests Independent Transformation Events in the Development of Biclonal B Cells.

Malignant B lineage cells in Waldenstrom's macroglobulinemia (WM) express a unique clonotypic VDJ associated with IgM. Studies of WM patients revealed a frequent incidence of biclonal B cells (16%) as defined by the presence of two distinct clonotypic VDJ sequences. This is the first report to estimate the frequency of biclonality in WM. Four WM cases are reported: two with distinct IgM clones (WM1-19 and WM1-09), one with distinct IgM/IgA clones (WM1-18), and one with related but diversified IgM/IgG clones (WM10). In 2 cases (WM1-19 and WM10), the two clonotypic signatures were found to be abundant in bone marrow (BM) but less frequent in blood, reminiscent of monoclonal WM cases. In WM1-19, single cell analysis showed that only one partner VDJ was expressed per cell, excluding the possibility of aberrant biallelic rearrangements. The distribution ratio between the two tumor clones was 2:1, suggestive of their mutual role in the clinical manifestation. In the other 2 cases (WM1-09 and WM1-18), partner clones were shown by CDR3 fragment analysis to be anatomically distinct, with one BM clonotypic signature and one in blood. In these 2 patients, the BM clone was hypermutated (6.2% and 3.8%) while the blood clone was germline or minimally mutated (0% and 1.0%). Partner clones lacked intraclonal diversity and Ig class switching, characteristic of malignant WM clones, suggesting relatively frequent transformation events throughout B lineage differentiation. The biological events leading to the appearance of clones in two different anatomic sites and the clinical implications remain to be understood. CDR3 fragment analysis in the longitudinal studies of WM1-09 and WM1-18 suggested that the repertoire of blood B cells may recover some level of diversity after successive cycles of treatment (WM1-18), while the persistence of the monoclonal peak (WM1-09) likely reflects a tumor that does not respond to treatment. In WM10, biclonal IgM/IgG B cell clones shared common VDJ gene rearrangement. The IgG clone displayed a higher mutation rate than did the IgM clone (9.7% vs. 6.1%). Increased somatic hypermutation in IgG consists mostly of replacement mutations that are clustered within the CDR regions, strongly supporting a contention that the IgG clone has undergone affinity maturation. There are 11 point mutations in the IgM that are not present in the IgG, suggesting that the two clones are distinctly different and belong to different branches of the genealogical tree. These distinct mutational signatures suggest that biclonal IgM/IgG clones are unlikely derived from clonal evolution. Overall, our results suggest that for the four biclonal WM evaluated here, the partner B cell clones appear to have undergone separate transformation events in the development of biclonality. The extent to which each partner clone contributes to disease progression and death, whether separately or in synergy, is as yet unknown.

Quantitative Detection Using SYBR Green of Residual Disease in Myeloma Patients Treated with Revlimid or Velcade.

The treatment of multiple myeloma (MM) with new drugs like Velcade and Revlimid shows promise in the clinic. These two drugs give excellent partial to complete remissions, but the disease invariably returns indicating escape of the malignant clone. The current clinical practices for quantifying minimal residual disease (MRD), such as plasma cell counts, lack needed sensitivity. PCR based assays amplify the clonotypic IgH VDJ gene rearrangement in myeloma and identify all compartments of the malignant clone, providing a unique molecular signature to monitor disease. Every myeloma cell carries one copy of the specific IgH VDJ rearrangement and provides a quantitative measure of the number of malignant cells remaining in each patient. No previous work has quantified MRD after treatment with either Revlimid or Velcade. TaqMan real-time quantitative PCR (rqPCR) assays are able to quantify the level of disease burden but probes are expensive and difficult to design, making this assay unsuitable for routine clinical use. SYBR green, however, is an inexpensive alternative to probes and utilizes the same unique molecular VDJ signature with consistency and reliability (r2=0.9997). PCR product specificity is confirmed using melting curve analysis. This is the first report of the successful use of SYBR green in quantifying the level of malignant disease in MM. The specific clonal rearrangement is identified for each patient. Patient specific primers are designed and verified as being present in the majority of plasma cells using single cell PCR. A cloned IgH VDJ product for each patient provides a standard curve for each patient-specific rqPCR. A cloned B2Microglobulin PCR product provides an independent standard curve to quantify cell number. After correcting for copy number differences, the percentage of malignant cells is calculated by dividing the number of VDJ molecules by the number of B2M molecules. For all tests, the level of residual disease in bone marrow was measured using 150ng of DNA. In patients achieving complete clinical remission during treatment with Revlimid, the SYBR green method detected 0.104% clonal cells with a range of 0.014%–0.26%. In patients with a partial remission, 3.034% clonal cells were detected, with a range of 0.62%–7.79%. A subsequent bone marrow from one Revlimid treated patient revealed no clonal cells this using SYBR Green method. In patients treated with Velcade at the time of relapse, the remaining clonal cells averaged 1.042% with a range of 0.00075%–3.11%. When Velcade was used as a frontline therapy followed by a stem cell transplant, 0.10% clonal cells remained with a range of 0%–0.287%. In one dexamethasone treated patient who achieved a complete remission, 0.018% clonal cells were found. The SYBR green rqPCR assay for patient specific clonal VDJ was shown to reliably quantify residual disease. To date, our results suggest that frontline treatment with Velcade may lead to lower levels of residual disease than treatment at the time of relapse. In Revlimid treated patients, the extent of residual disease correlates with clinical classifications of complete or partial remission. The significance of these low numbers of malignant cells remains to be established, but the persistent occurrence of relapse suggests they are clinically relevant.

An evolutionarily mobile antigen receptor variable region gene: Doubly rearranging NAR-TcR genes in sharks

Distinctive Ig and T cell receptor (TcR) chains define the two major lineages of vertebrate lymphocyte yet similarly recognize antigen with a single, membrane-distal variable (V) domain. Here we describe the first antigen receptor chain that employs two V domains, which are generated by separate VDJ gene rearrangement events. These molecules have specialized "supportive" TcRdeltaV domains membrane-proximal to domains with most similarity to IgNAR V. The ancestral NAR V gene encoding this domain is hypothesized to have recombined with the TRD locus in a cartilaginous fish ancestor >200 million years ago and encodes the first V domain shown to be used in both Igs and TcRs. Furthermore, these data support the view that gamma/delta TcRs have for long used structural conformations recognizing free antigen.