Ig Lambda Light Chain Research Articles

Concomitant Deletion of Short Arm (del 1p) and Amplification or Gain (1q21) of Chromosome 1 By Fluorescence in Situ Hybridization (FISH) Is Associated with Poor Clinical Outcome

Introduction- Chromosome 1 abnormalities in multiple myeloma (MM) are increasingly recognized as high risk defined features. While there is robust data on 1q21 gain and amplification (amp), the clinical characteristics and outcome of patients with del 1p is less defined. Novel agents are incorporated into a backbone of multi-agent chemotherapy and tandem autologous stem cell transplantation (ASCT) in successive Total Therapy (TT) protocols for MM patients. We hereby report the prognostic value of del 1p by FISH at enrollment in subjects treated on TT protocols.Methods: (FISH was performed on bone marrow obtained at the time of first visit to our institution or initial diagnosis. FISH probes were generated from specific BAC DNA clones for AHCYL1 gene locus (1p13.3) and CKS1B locus (1q21). MM cells were identified post-hybridization using isotype specific antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) to stain Ig-Kappa or Ig-Lambda light chain in cytoplasm (cIg) of myeloma tumor cells. The FISH signals in 100 myeloma cells were recorded. For this analysis, 3 copies of 1q21 are considered as 1q21 gain and ≥ 4 copies as 1q21 amp. A 20% cutoff point was used for detection of significant abnormalities, i.e. del 1p and 1q21 gain and amp. A multivariable logistic regression model was used to examine the combined effects of clinical variables on progression free (PFS) and overall survival (OS).Results- A total of 1133 patients were included in this analysis. The median age was 60 (range 30.2-75), 434 (38.3%) patients were female and 106 (9.4%) were African Americans. ISS stage III disease accounted for 287 (25.3%). GEP70 high-risk was noted in 160/1133 (14.1%) of all patients. Of all patients, 1084 (95.7%) had at least one ASCT and 812 (71.7%) had tandem upfront ASCT. Metaphase cytogenetic abnormalities were noted in 548 (48.4%). While del 1p was detected in 220 (19.4%) patients, 1q21 gain or amplification were observed in 300 (26.5%) and 150 (13.2%) patients, respectively. Isolated 1q21 gain and amplification without del 1p were seen in 235 (20.7%) and 121 (10.7%) patients. Overall, there was enrichment of high-risk features such as ISS stage III disease (5.7% vs 10.9% p=0.049), GEP70 high-risk (8.4% vs 36.8%), GEP 70 subtypes such as MF (4.6% vs 8.2%), MS (10.5% vs 13.6%) and PR (11.3% vs 22.7%) and abnormal cytogenetic abnormalities (45.7% vs 59.5% p=<0.01) in the group with del 1p . The median progression free survival (57 vs 39m; p= <0.0004) and overall survival (131 vs 89 m; p=<0.0013) were inferior in the subgroup with del 1p. On multivariate analysis, presence of ISS stage III disease, GEP high-risk, 1q21 gain and amplification were significant while presence of del 1p did not emerge as independent predictors of PFS or OS. Next, we looked at concomitant 1q21 gain and amp in the context of del 1p. del 1p with 1q21 gain or amp was observed in 65 (5.7%) and 29 (2.5%) patients, respectively. The PFS of the group with combined del 1p/1q21 gain and del 1p/1q21 amp were 25m and 35m respectively compared to 44 m with 1q21 gain and 37m with 1q21 amp alone. The OS of the group with both 1 del 1p/1q21 gain and del 1p/1q21 amp were dismal at 59m and 84 m respectively compared to 108 m with 1q21 gain and 76 m with 1q21 amp alone (Fig 1).Conclusion: Deletion of short arm of chromosome 1p was observed in 19% of MM patients. Concomitant del 1p with 1q21 gain and /or amp was present in 8% of patients. The PFS and OS of patients with combined del1p/1q21gain abnormalities was significantly worse compared to del 1p alone and 1q21 gain alone and thus identifies a subset of patients with poor clinical outcome. [Display omitted] DisclosuresMohan: Medical College of Wisconsin: Current Employment.

Gastric Plasmacytoma with Immunoglobulin Lambda Light Chain Deposition in a Dog

A 12-year-old castrated male Jack Russell Terrier presented with intermittent vomiting. Abdominal ultrasonographic examination detected a thickened stomach wall with a mass measuring approximately 1.5cm in diameter. Computed tomography revealed a solitary mass measuring approximately 2.1cm in diameter between the submucosa and muscle layers in the greater curvature the pyloric region of the stomach, and a swelling in the hepatic lymph node. The gastric mass was composed of round neoplastic cells arranged in a diffuse pattern. The neoplastic cells had a round nucleus and a pale abundant cytoplasm. Multinucleated giant cells were often found. Hyalinized eosinophilic material, which did not stain with Congo red and had no affinity for thioflavin T, was also observed. Neoplastic cells were immunopositive for MUM1, CD79a and Ig lambda light chain but negative for CD3, CD20, BLA36, IgG and Ig kappa light chain. Stromal eosinophilic material was positive for Ig lambda light chain. The neoplasm was therefore diagnosed as a gastric plasmacytoma with non-amyloid Ig lambda light chain deposition.

Effect of iFISH defined 1q21 amplification/gain in multiple myeloma patients treated on total therapy protocols.

8023 Background: The amplification of the proximal 1q21 region has been reported by interphase FISH (iFISH) in about 40% of newly diagnosed patients, and in 70% of MM patients at relapse. We hereby report the prognostic value of 1q21 amplification/gain by iFISH at enrollment in subjects treated on total therapy (TT) 4, 5 and 6 protocols. Methods: TT4 protocol enrolled newly diagnosed patients with LR disease as defined GEP 70 model ≤ 0.66. TT5 was designed for newly diagnosed patients with HR MM.TT6 enrolled previously treated patients with both HR and LR disease. iFISH was performed on bone marrow samples obtained at the time of study enrollment. FISH probes were generated from specific BAC DNA clone for CKS1B gene locus (1q21) gene locus. MM cells were identified post hybridization using isotype specific antibody conjugated with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) to stain Ig-Kappa or Ig-Lambda light chain in cytoplasm (cIg) of MM cells. The iFISH signals in 100 of MM cells were recorded. A 20% cutoff point was used for detection of significant abnormalities i.e. gain of 1q21 (≥ 3 copies). Results: 607 patients were included in this analysis. With a median age of 61 years, 39% had high risk and 88% low risk disease. 1q21 abnormalities were present in 55% of patients at enrollment. 591 patients (97%) received at least one HDT ASCT. The analysis included 382 TT4 patients with a median PFS of 7.2 years and OS not yet reached. 75 patients enrolled on TT5 protocol experienced median PFS of 2.2 years and median OS of 5.6 years.150 TT6 enrolled patients experienced median PFS of 4.1 years and median OS of 7.5 years. Statistically significant difference in PFS (p &lt; 0.0001) and OS (p &lt; 0.0001) was observed between 1q21 defined groups (figure 1). No differences in survival were observed based on the 1q21 copy number &gt; 3. Conclusions: This retrospective analysis clearly showed that 1q21 amplification/gain detected by interphase iFISH in different stages of disease can identify patients with significant shorter progressive free and overall survival even if exposed to total therapy regimens. Clinical trial information: NCT00734877, NCT00869232.

Abstract 5074: Omentum immune microenvironment: Metastatic niche for ovarian cancer

Abstract The status of the tumor-immune microenvironment plays a critical role in determining the response to immunotherapy. The omentum, a multifunctional organ that serves as the central regulator of peritoneal homeostasis and plays a central role in immune surveillance, is the most common site of metastasis in early stage ovarian cancer. We analyzed the human omental immune microenvironment that support ovarian cancer metastases. In experimental models, ovarian cancer cells that colonize the omentum are invariably found in association with immune aggregates known as milky spots. We hypothesize that ovarian cancer cells exploit the unique environment of these omental-immune clusters to promote their own survival and growth. Thus, we formulated a systematic approach to evaluate normal and ovarian cancer-populated omental tissues to characterize the cellular components of human omental milky spots. We performed immunohistochemistry and flow cytometry to profile immune cell populations present in omental immune clusters. To facilitate the quantitation of immunohistochemical staining, we developed a computational image processing workflow that enables quantitation of multiple immune-biomarkers from serial sections of formalin-fixed paraffin-embedded tissue. In a complementary approach, we used comprehensive multi-color flow cytometry staining panels detecting 28 markers that collectively encompass all innate and adaptive immune cell populations. We analyzed eight human omental samples (four omenta involved with serous carcinomas and four omenta from benign disease) by immunohistochemistry and found that M2-subtype macrophage (CD163+CD68+) infiltration predominate in high-grade cancer-involved omentum. Conversely, T-regulatory cells (CD25+) predominate cell type in the uninvolved omentum. Hi-D flow cytometry of three cancer-involved omentum samples revealed tumor-associated macrophages (20-40%), PMN myeloid-derived suppressor cells (2-3%), “double positive” CD3+CD4+CD8+ T-cells (3-4%), and “double negative” CD3+CD4-CD8- T-cells (0.9-1%). In addition, we found omental T-cells (Th, Tc, Treg) express immune checkpoint receptors CTLA4 and PD-1, and that omental B-cells express IgD, IgA, and/or IgM, and may be enriched for Ig lambda light chain expression. As this study is ongoing, we expect to present data for omenta from a larger cohort of patients with either benign or neoplastic disease. However, our findings are already sufficient to show that activated T cells, B cells and macrophages infiltrate omental milky spots in patients with high grade serous ovarian cancers. Citation Format: Venkatesh Krishnan, Paul A. Raju, Koah Vierkoetter, Sonia Patel, Justin Youngyunpipatkul, Supreeti Tallapragada, Bruce Schaar, Ann K. Folkins, Leonore A. Herzenberg, Oliver Dorigo. Omentum immune microenvironment: Metastatic niche for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5074.

The Importance of an In-depth Study of Immunoglobulin Gene Rearrangements When Ascertaining the Clonal Relationship between Concomitant Chronic Lymphocytic Leukemia and Multiple Myeloma.

Chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) are hematological disorders that occur at different stages of B-cell development. It has been shown that CLL B-cells can differentiate into plasma cells in vitro and in vivo. CLL is the most frequent adult leukemia in the western world. It is a heterogeneous disease, characterized by clonal proliferation and the accumulation of mature CD5+ B lymphocytes (1). MM is a clonal plasma cell malignancy that accounts for more than 10% of all hematologic cancers (2). Although secondary cancers [particularly solid tumors (3–5)] can occur with CLL and MM, the concomitant occurrence of these two disorders in the same patient is rare [for a review of the few reported cases, see Ref. (6)]. The clonal relationship between these diseases has not always been clarified but is important in terms of understanding the pathogenesis and optimizing treatment. The clonal relationship between CLL and MM can be evaluated by (i) analyzing immunoglobulin (Ig) heavy chain and light chain (Ig kappa light chain and Ig lambda light chain) gene rearrangement, (ii) identifying and comparing somatic mutations, and (iii) studying chromosomic aberrations. Nevertheless, Ig rearrangements must always be interpreted in the light of specific phenomena such as allelic exclusion, B-cell receptor (BCR) revision (VH and DH gene replacement), BCR editing, and somatic mutations—events that were not considered in previous studies. These issues can be addressed by sequencing the rearranged Ig genes from sorted populations and interpreting the generated data. In the present study, we evaluated the putative clonal relationship between the two diseases by combining DNA copy number analysis with an assessment of Ig gene rearrangements [clonality assessment, V(D)J sequencing, and somatic hypermutation analysis] in highly enriched CD19+ CD5+ (CLL) and CD38+ CD138+ (MM) cell populations. Array comparative genomic hybridization data suggested a possible phylogenic progression from CLL to MM. Moreover, V(D)J sequencing indicated that both CLL and MM cells used the same VH and JH genes but different DH genes. However, in-depth analysis and interpretation of Ig gene rearrangements ultimately suggested that the two diseases had distinct clonal origins.

Characterization of immunoglobulin E plasma cells that are elevated in the upper airway mucosa of nonatopic patients with chronic rhinosinusitis without nasal polyps.

The immunologic mechanisms driving inflammation in the upper airways of patients with chronic rhinosinusitis (CRS) are poorly understood. Previous studies have shown that B cells and immunoglobulin E (IgE) levels are elevated in the nasal tissue of patients with atopic chronic rhinosinusitis without nasal polyps (CRSsNP). However, less is known regarding B cell subsets and IgE-producing plasmablasts in nonatopic CRSsNP patients. Human blood and ethmoid sinus mucosa samples were analyzed from control (n = 6) and nonatopic CRSsNP (n = 11) patients. Tissue samples were evaluated using high-dimensional flow cytometry. A population of IgE antibody secreting cells is significantly increased in situ within inflamed nasal tissue of nonatopic CRSsNP subjects when compared to control nasal tissue and the circulating peripheral blood (p < 0.05). This IgE plasma cell population displays ∼90% cell surface Ig lambda light chain, is mitotically active (Ki-67(+)), and displays intracellular IgE expression. The predominant B cell population expressing IgE are plasmablasts (CD38(high), CD138(-)) not typically found in the blood or peripheral tissue of these patients. The nasal mucosa from nonatopic CRSsNP patients demonstrate a significant regional spike in resident in situ IgE plasmablast cells not seen in control nasal tissue or peripheral blood from the same patient. The restricted expression of Ig lambda light chain in this mitotically active IgE plasmablast population supports the hypothesis of aberrant B cell proliferation in the context of CRS. These findings suggest the presence of a unique regional immune microenvironment for B cell priming and/or selection within chronically inflamed airway tissues.

The Use of Immunoglobulin Gene Rearrangement Polymerase Chain Reaction Assays for Detection of B-Cell Clonality for Plasma Cell Neoplasms Using Novel PCR Primers

We have designed a standardized protocol with multiplex-primer sets capable of detecting majority IG kappa (IGK) and IG lambda (IGL) light chain rearrangements in plasma cell neoplasms (PCN). Thirty primers were combined in three multiplexed PCR reactions to target IGK, KDE and IGL rearrangements. Variable region (V) primers were designed to prevent “primer dimers”, provide matching melting temperatures (Tm), minimize amplicon size, and optimize sequencing time. Amplicons were subjected to capillary gel electrophoresis for analysis. In a discovery series, we tested 37 plasma cells neoplasms PCN (28 PCNs at diagnosis and 9 PCNs post-treatment). The assay investigated an additional 52 prospective PCN cases in the validation series. Results were compared to bone marrow morphology, immunohistochemical (IHC), flow cytometry data, and standard IGH FRIII gene rearrangement assay. In the discovery series, the following sensitivities/specificities were obtained for mature B-cell neoplasms: IGH FRIII: 29.7%/100%, IGK: 80.4%/100%, KDE: 25.0%/100%, and IGL: 35.1%/96.8%. The combination of IGH FRIII, IGK, and KDE detected 83.8% (31/37) vs 67.3% (35/52) in the discovery vs validation series, respectively, for the PCN population. Interestingly, 21.2% (11/52) of the validation samples positive by IG clonality, were negative by IHC and flow cytometry. In IHC/flow cytometry positive cases with a PCN representing a tumor burden of >50%, 10% to 50%, 1% to 10%, 0% to 1% of cells, the combined sensitivity of the IG clonality assay was 100% (20/20), 72% (23/32), 53% (10/19) and 20% (1/5) respectively. This IGK/IGL clonality assay has good sensitivity at diagnosis.

Amplification of highly mutated human Ig lambda light chains from an HIV-1 infected patient

Isolation and characterization of anti HIV-1 broadly neutralizing antibodies (bNAbs) have elucidated new epitopes and sites of viral vulnerability. Anti-HIV-1 bNAbs typically show high levels of somatic mutations in their variable region genes. This feature potentially limits antibody identification, since the mutated antibody sequences are no longer complimentary to primers designed based on germline antibody sequences. Here we report a new set of primers for Igλ light chains that aligns to the 5′ end of the leader sequence and is highly efficient for the amplification of antibodies that contain mutations and deletions in the 5′ end of human Igλ.

Diversity of immunoglobulin lambda light chain gene usage over developmental stages in the horse

To further studies of neonatal immune responses to pathogens and vaccination, we investigated the dynamics of B lymphocyte development and immunoglobulin (Ig) gene diversity. Previously we demonstrated that equine fetal Ig VDJ sequences exhibit combinatorial and junctional diversity levels comparable to those of adult Ig VDJ sequences. Herein, RACE clones from fetal, neonatal, foal, and adult lymphoid tissue were assessed for Ig lambda light chain combinatorial, junctional, and sequence diversity. Remarkably, more lambda variable genes (IGLV) were used during fetal life than later stages and IGLV gene usage differed significantly with time, in contrast to the Ig heavy chain. Junctional diversity measured by CDR3L length was constant over time. Comparison of Ig lambda transcripts to germline revealed significant increases in nucleotide diversity over time, even during fetal life. These results suggest that the Ig lambda light chain provides an additional dimension of diversity to the equine Ig repertoire.

HIV-negative, HHV-8-unrelated primary effusion lymphoma-like lymphoma with genotypic infidelity and c-MYC expression

Dear Editor, HIVor HHV-8-related primary effusion lymphoma (PEL) is a rare and poor prognostic disease which presents as serous effusion without detectable tumor masses. PEL occasionally has both immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements, so-called genotypic infidelity [1, 2]. Recently, HIV-negative HHV-8-unrelated primary effusion lymphomalike lymphoma (PEL-LL) has been reported, and some cases are associated with c-MYC amplification. Although HHV-8 virus antigen can increase c-MYC stability in PEL [3], c-MYC expression in PEL-LL has not been confirmed so far. A 76-year-old male had suffered from esophageal cancer, which responded to chemoradiotherapy. Three years after treatment, however, he had experienced cough, general fatigue, and night sweats. Physical examination revealed no breath sounds on the right hemithorax. Chest X-ray showed severe pleural effusion in the right lung (Fig. 1a). However, no tumor mass or lymphadenopathy was detected. Gastrointestinal fiberscope showed no sign of esophageal cancer recurrence. Complete blood count and serum biochemistry studies were as follows: WBC 7,170/μl, hemoglobin 10.6 g/dL, platelet 402,000/μl, AST 20 IU/L, ALT 12 IU/L, LDH 377 IU/L, total bilirubin 0.4 mg/dL, and sIL-2R 525 U/ml. Serological tests for HIV, HTLV-1, and HCV were negative. Bone marrow was intact. In analysis of pleural effusion, there were many large atypical lymphocytes which were positive for CD19, CD20, CD30, CD79a, and Ig lambda light chain but negative for CD10 and CD56 (Fig. 1b). HHV-8 and EBVencoded RNA by immunochemistry were negative. There was a small fraction of normal T cells. Eighty percent of lymphocytes were Ki-67 positive. Southern blot analysis of tumor cells demonstrated Ig heavy chain (IgH) as well as TCR rearrangement (Fig. 1c). Karyotype analysis showed complex karyotype, including add(8)(q24) and c-MYC amplification, and these cells strongly expressed c-MYC protein by immunostaining (Fig.1d). He was diagnosed with PEL-LL and was treated with six courses of R-CHOP. He remains alive and well with slight pleural effusion without additional therapy for more than 18 months. There are only two cases of PEL-LL with both IgH and TCR rearrangements. The first case is a pleural cavity-based T cell-rich B cell lymphoma [4]. Actually, it was reported for this lymphoma to show the dual gene rearrangement [5]. It could be bi-clonal lymphoma, of which the T cell clone was derived from abundant background Tcells. The second case is post-liver transplant effusion lymphoma [6]. It demonstrated incomplete TCR-β rearrangements by PCR; wherein, these rearrangements can be found in some B cell lymphomas [7]. In our case, most cells in the effusion were B cells. In addition, we confirmed both rearrangements by southern blot analysis (Fig. 1c). Therefore, we believe that this is the first definitive case of genotypic infidelity in PEL-LL. In addition, we confirmed strong c-MYC expression on tumor cells along with T. Kashiwagi :K. Minagawa :H. Kawano : T. Hirata : S. Kashiwagi :Y. Nakagawa : S. Kusaka : T. Suzuki : T. Inagaki : M.Kishi :N.Miwa : S. Kimura :M. Takechi :M. Koide :M. Iwai : T. Matsui Department of Internal Medicine, Nishiwaki Municipal Hospital, Nishiwaki, Hyogo 677-0043, Japan

Rituximab Therapy Leads to Reduced Imprints of Receptor Revision in Immunoglobulin κ and λ Light Chains

Transient B cell depletion by rituximab (RTX) has become a specific treatment of rheumatoid arthritis (RA). Although phenotypic repopulation kinetics of B cell subsets are well documented, precise molecular analyses of the reconstituting immunoglobulin (Ig) genes encoding the B cell receptor in RA are sparse. A total of 708 individual CD19+CD27+ (memory) and CD19+CD27- (naive) B cells from 2 patients with RA were analyzed at baseline and 7 months after RTX at B cell repopulation. Ig light chain variable kappa (Vκ) and lambda (Vλ) light chain gene rearrangements were amplified, sequenced, and analyzed with a focus on receptor revision. The naive as well as the memory repertoire repopulated polyclonally with diverse use of variable light chain gene families and minigenes. During the reconstitution phase, B cells used significantly fewer Jκ distal Vκ genes (p = 0.0006), with a higher frequency of somatic hypermutation of rearrangements employing Jκ5 compared to baseline in memory B cells. The use of Vλ rearrangements in regenerating B cells was also biased toward use of Vλ genes of the proximal cassette. In general, reemerging CD27+ Ig light chain genes were substantially more highly mutated than before RTX therapy (p < 0.0001, baseline vs during reconstitution). Our data indicate that RTX therapy leads to generation of distinct Vκ/Jκ and Vλ/Jλ gene repertoires consistent with replenishment of antigen-experienced B cells by germinal centers. At baseline, the imprints of receptor revision appeared to be more striking, which indicates that receptor revision is active in patients with RA and can be reduced by RTX.

Allelic exclusion of immunoglobulin genes: models and mechanisms.

The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Ig kappa and Ig lambda light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.

Activation of Cyclosporin A Transport by a Novel λ Light Chain of Human Ig Surface Antigen-Related Gene inXenopus laevisOocytes

In the present study, we isolated and determined the pharmacological characteristics of a novel gene encoding the lambda light chain of human Ig surface antigen-related gene (IgLC-rG). The isolated cDNA consisted of 693 base pairs that encoded a 232-amino acid protein. Northern blot analysis revealed that the IgLC-rG mRNA is expressed in the adult spleen and small intestine but not in fetal tissues. When expressed in Xenopus laevis oocytes, IgLC-rG mediated the high-affinity transport of [(3)H]cyclosporin A (CsA) (K(m) = 189.7 +/- 123.5 nM) in a sodium-dependent manner; however, other organic solutes such as p-aminohippuric acid and TEA were not transported via IgLC-rG. The transport of [(3)H]CsA by IgLC-rG was sensitive to pH. The uptake of [(3)H]CsA was trans-stimulated by CsA and GSH. Immunohistochemical analysis revealed that the IgLC-rG protein is localized at the brush border membrane in the human small intestine. Although the isolated IgLC-rG gene is a member of the human Ig lambda light chain surface antigen superfamily, our findings suggest that IgLC-rG functions as a novel transport peptide responsible for CsA in the human body. Our results should provide insight into the novel function of membrane-bound proteins, such as Igs.

OH-2, a Hyperdiploid Myeloma Cell Line without An IGH Translocation, Has a Complex Translocation Juxtaposing MYC near MAFB and the IGK Locus

Despite heterogeneity of phenotypes, multiple myeloma (MM) can be classified into two major groups: hyperdiploid (HRD) tumors with 48–74 chromosomes, which typically have extra copies of at least four of the eight odd chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; and non-hyperdiploid (NHRD) tumors, which usually have immunoglobulin heavy chain (IGH) translocations and <46 or >74 chromosomes. Human myeloma cell lines (HMCL) with a typical HRD genotype are lacking. The OH-2 HMCL was derived from extramedullary myeloma, and retains the same HRD phenotype as the primary tumor, with extra copies of chromosomes 3, 7, 15, 19, and 21, as demonstrated by array comparative genomic hybridization. This provides a unique example of an HMCL and the corresponding primary tumor that share the same HRD phenotype. Also Spectral Karyotyping showed the same HRD phenotype. By fluorescence in situ hybridization it was shown that OH-2 does not have an IGH or Ig lambda light chain translocation. Instead, both the HMCL and the primary tumor have complex translocations involving chromosomes 2, 8, and 20. The translocation juxtaposes MAFB near MYC and the Ig kappa (IGK) enhancer. Both MYC and MAFB are highly expressed, as shown by microarray analysis, and are thought to be dysregulated by the IGK enhancer. Both OH-2 HMCL and the primary tumor cells express high levels of Cyclin D2, an expected consequence of the enhanced MAFB level. OH-2 has a phenotype similar to the 20% of HRD MM tumors that express increased levels of Cyclin D2 but not Cyclin D1 and could be a good in vitro model for studies of this subgroup of MM. OH-2 is dependent on medium containing human serum and growth-promoting cytokines like IL-6 or IL-21, reflecting the critical role of the microenvironment for growth of MM cells.

Positional cloning of the Igl genes controlling rheumatoid factor production and allergic bronchitis in rats

Rheumatoid factors (RF), autoantibodies that bind the Fc region of IgG, are one of the major diagnostic marker in rheumatoid arthritis (RA) but occur with lower frequency also in other infectious and inflammatory conditions. Through positional cloning of the previously described quantitative trait locus (QTL) Rf1 in congenic and advanced intercrossed rats, we identified the Ig lambda light chain locus as a locus that regulates the production of RF in rats. The congenic rats produce RF-Ig lambda and have significant higher levels of RF-IgG and RF-IgM in serum, while the DA rat has an impaired RF production and does not produces RF-Ig lambda. Thus, we could investigate the role of RF in pristane-induced arthritis (PIA) as well as ovalbumin-induced airway inflammation. We show that there was no difference in the development and severity of PIA between congenic and parental DA rats, suggesting that RF using lambda light chains have no impact on PIA. However, the RF producing congenic rats developed a more severe airway inflammation as indicated in the significantly increased number of eosinophils in bronchoalveolar lavage fluid as well as total IgE in serum. In addition, RF congenic rats had a significantly enhanced immune response toward OVA due to increased OVA-Igk but not OVA-Igl antibodies, suggesting a possible involvement of RF in the regulation of the humoral immune response.

Rearrangement of Mouse Immunoglobulin Kappa Deleting Element Recombining Sequence Promotes Immune Tolerance and Lambda B Cell Production

The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

Stereotypic IGHV3-21/IGLV3-21 Antibodies Expressed in High-Risk Chronic Lymphocytic Leukemia Bind Peptostreptococcus Magnus Protein L.

We examined a large cohort (N=2,457) of chronic lymphocytic leukemia (CLL) patients evaluated by the CLL Research Consortium (CRC) and found 63 (2.6%) used IGHV3-21. Comparing the Ig heavy chain third complementarity determining region (HCDR3) of the IGHV3-21 cases: 25/63 cases (39.7%) had a conserved amino acid motif (motif 1: DANGMDV) in the otherwise highly variable Ig HCDR3, as described by Tobin et al. Blood 2003. All but one of these Ig heavy chains (IgH) were paired with a lambda light chain encoded by IGLV3-21. In addition, we found that 3/63 cases (4.8%) had a previously unrecognized conserved HCDR3 amino acid motif (motif 2: DPSFYSSSWTLFDY). In contrast, these IgH invariably were paired with kappa immunoglobulin light chains (IgL) encoded by IGKV3-20. Similarly to that noted for CLL cases that use IgH encoded by unmutated IGHV1-69 (Widhopf et al. Blood Epub First Edition 2007), the pairing of IgH encoded by IGHV3-21 with IgL appears governed by the HCDR3. The non-stochastic pairing of IgH with IgL argues strongly that antigen plays a role in selecting the Ig expressed in CLL. To examine for the antigen(s) recognized by the most common Ig encoded by IGHV3-21, we isolated IgH and IgL genes expressed by IGHV3-21/IGLV3-21 CLL cases and generated recombinant antibodies, which we examined for binding to antigen(s) present on microarray of self or environmental antigens. We found that Ig encoded by IGHV3-21/IGLV3-21 had apparent specific binding for protein L, a multi-domain cell-wall protein isolated from Peptostreptococcus magnus, a Gram-positive commensal bacteria that comprise a large portion of the human bacterial gut flora. Prior studies identified that protein L is a superantigen capable of binding human Ig kappa light chains encoded by IGKV genes of the I, III, and IV subgroups, but not human Ig lambda light chains. The specific binding of IGHV3-21/IGLV3-21 to protein L suggested that protein L might play a role in the development of CLL cells that express such Ig. To test this hypothesis, we examined the capacity of various recombinant antibodies to bind protein L by ELISA. We found that lambda IgL encoded by IGLV3-21 could bind to protein L with similar activity, independent of whether this lambda IgL paired with the native IgH, IgH encoded by IGHV3-21 lacking the DANGMDV HCDR3 motif, or even irrelevant IgH encoded by IGHV4-39 that are not found paired with IGLV3-21 in the Ig expressed in CLL. Moreover, Ig formed by pairing IgH encoded by IGHV3-21 that has the DANGMDV HCDR3 motif with an IgL encoded by an IGLV that was irrelevant to IGLV3-21 did not bind protein L. These results reveal a previously unrecognized capacity of human IgL encoded by IGLV3-21 to bind the protein L superantigen of Peptostreptococcus magnus, a bacteria commonly found in the human gastrointestinal tract. However, because the binding of IGLV3-21 does not depend upon the non-stochaistic pairing of IgH and IgL observed in CLL, we reason that the capacity of IGLV3-21 to bind protein L cannot account for the selected Ig repertoire expressed in CLL, suggesting that it actually does not play a role in CLL leukemogenesis. This finding suggests that caution should be exercised when defining an antigen that is found capable of binding the restricted Ig expressed in CLL as the driving factor responsible for leukemogenesis.

Proteome profiles of mucosal immunoglobulin uptake in inflamed porcine gut

Acquisition of passive immunity by endocytosis of intact immunoglobulins (Ig) from colostrum is critical for prevention of intestinal and systemic diseases in neonatal mammals. We compared proteome patterns of healthy and inflamed gut tissues from pre-term piglets to investigate the effect of inflammation on acquisition of passive immunity. A clear difference in the two-dimensional gel electrophoresis protein patterns between healthy and inflamed intestinal tissues was observed, suggesting that inflamed tissues failed to absorb and transfer Ig from colostrum to epithelial cells. We have mapped and identified the Ig proteins that are taken up by healthy intestinal tissues, and found that isoforms of the IgA and IgG heavy chain and Ig kappa and lambda light chains were internalized. Our results indicate that colostrum protein uptake in the porcine gut is a selective process that is obstructed in inflamed pre-term gut.

The Tumor Microenvironment Measured by Flow Cytometry Predicts Overall Survival (OS) and Transformation Risk (TR) in Follicular Lymphoma.

Background: FL is an indolent but heterogeneous lymphoid neoplasm with a variable clinical course. Transformation into an aggressive lymphoma is a dominant clinical event that is frequently followed by shorter survival. There is no consistent biological prognostic marker for TR. Recent studies have highlighted the role of the microenvironment in helping to determine the prognosis of FL. However, its impact on TR is largely unknown. In this study we used diagnostic flow cytometry (FC) analysis to assess the role of non-malignant cells in determining OS and TR in FL.Methods: We identified 567 patients with FL diagnosed at the BCCA over a 5-year period between 1997 and 2001. Of these, 270 cases had diagnostic FC and histological review, of which 137 were nodal and had complete clinical data in our electronic database. FC results were re-analyzed. The antibodies studied included anti-CD3, CD4, CD5, CD8, CD10, CD14, CD19, CD20, CD23, CD45, FMC-7 and IG kappa and lambda light chains. To ensure that biopsies were representative, the sum of the % gated events for CD3 and CD20 had to equal 100% +/− 20%. Light chain restriction was present in all cases and established clonality. The estimate of neoplastic B cells was determined by examining CD19 and CD20 frequency and the ratio of clonal light chain vs non-clonal light chain. Non-neoplastic B cells were estimated using CD19/20 and the amount of non-clonal light chain × 0.5 (λ clonal) or × 2 (κ clonal). Clinical characteristics, different subsets of T cells and the cell content of reactive, non-neoplastic B cells were evaluated using SPSS® software.Results: The median age of the 137 patients was 57 years, 51.8% were male and 38.6% had a high IPI (4/5). There were 97 grade 1, 27 grade 2 and 13 grade 3a FL. The median ratio of CD4/CD8 was 4.4. Patients were given a variety of treatments, including observation if asymptomatic, precluding an analysis of progression-free survival. The median follow-up of the living patients was 5.8 years and the estimated 5-year OS and TR were 70% and 20%, respectively. The IPI was predictive of OS (p<0.0001). Two biological variables showed a significant impact on survival. Firstly, cases in which CD8+ cells represented more than 25% of the total (CD3+) T cells had shorter OS (p = 0.028) and increased TR (p = 0.013). The CD8 ratio (p = 0.026) affected OS independently of IPI (p = 0.046). Secondly, cases with a low content of reactive, non-neoplastic B cells, defined by the ratio between IG light chains >1/15 had shorter OS (p = 0.003) and increased TR (p=0.002). The impact of a reduction in normal reactive B cells (p = 0.014) on transformation risk was independent of the IPI (p = 0.05).Conclusion: Two features of the FL microenvironment studied by diagnostic FC demonstrated an impact on prognosis. The proportion of CD8+ T cells relative to the total T cells and the number of residual, non-neoplastic B cells were both predictors of OS. Importantly, both predict, independently of the IPI, the risk of transformation. These biomarkers are easily measured and may be used to better stratify patients, choose initial treatment options and predict transformation risk in patients with FL. [Display omitted]

SGN-70, a Humanized Anti-CD70 Antibody, Targets CD70-Expressing Hematologic Tumors.

Antigens expressed on malignant cells in the absence of significant expression on normal tissues are highly desirable targets for therapeutic antibodies. CD70 is a TNF superfamily member whose normal expression is restricted to activated lymphocytes but is aberrantly expressed in hematologic malignancies and solid tumors including non-Hodgkin's lymphoma (NHL), Hodgkin's disease, multiple myeloma (MM), Waldenstrom's macroglobulinemia, renal cell carcinoma, glioblastoma, and nasopharyngeal carcinoma. To target CD70-expressing hematologic malignancies, we have engineered a humanized IgG1 anti-CD70 antibody that mediates lysis of tumor targets via ADCC and CDC and facilitates antibody dependent cellular phagocytosis in vitro. In vivo, administration of SGN-70 prolonged survival of SCID mice bearing CD70+ disseminated human NHL or MM xenografts. Intravenous injection of the CD70+ MM cell line MM.1S resulted in disease as measured by onset of paralysis, presence of CD138+ MM cells in the bone marrow, and increasing levels of circulating human Ig lambda light chain. SGN-70 treatment of MM.1S-bearing mice significantly delayed onset of paralysis and reduced the monoclonal protein levels detected in serum approximately 4-fold compared to untreated or non-binding antibody control-treated mice. Whereas myeloma cells comprised 32±6.4 % of mononuclear cells in the long bone marrow of control mice, SGN-70 treatment reduced the myeloma cell fraction to 4.9±1.9% of mononuclear cells recovered. SGN-70 treatment also significantly extended the survival of mice bearing disseminated Raji tumors (NHL) compared to control mice. Survival benefit was absent when mice received an Fc-modified antibody deficient in effector functions, confirming that the activity of SGN-70 in these models was dependent upon Fc-FcγR interaction with host immune cells. Together, these data demonstrate that SGN-70 possesses effector cell-mediated antitumor activity and provides rationale for clinical study of SGN-70 in CD70+ hematologic malignancies.