bluuebunny/crossref_metadata_2025_split huggingface dataset:
Patients with Lymphoma May Harbor Mutations of the Perforin Gene.
Authors: Clementi Rita , Locatelli Franco , Dupre Loic , Garaventa Alberto , Emmi Lorenzo , Bregni Marco , Cefalo Graziella , Moretta Antonia , Danesino Cesare , Comis Margherita , Pession Andrea , Ramenghi Ugo , Maccario Rita , Arico’ Maurizio , Roncarolo Maria Grazia 
Published: April 2020

Abstract
Perforin (PRF), a pore-forming molecule expressed in granules of cytotoxic effector lymphocytes, has been implicated in T and NK cell-mediated immune surveillance against viral infections and tumors. Indeed, increased lymphoma incidence has been associated with PRF deficiency in mice. In view of these experimental findings, we reasoned that also some patients with lymphomas might harbor mutations of the PRF gene and thus we investigated the presence of mutations of the gene in a group of lymphoma patients with pre-defined clinical characteristics. Bi-allelic mutations of the PRF gene were found in 4 out of the 30 patients examined. A female (19 y.) had been successfully cured of her T-cell lymphoblastic lymphoma, when her brother(22 y), 2 years later, developed HLH. The 2 siblings shared the same mutations. The first one, (272 C&gt;T), changes arginine at position 91 into valine; the second one (1122 G&gt;A), changes tryptophan at position 374 into a stop codon. The second patient (7 y), with EBV-positive Hodgkin lymphoma,three years later, developed a large B-cell non-Hodgkin lymphoma. Two mutations were identified: the first one, (1304 C&gt;T), changes threonine at position 435 into methionine. The second one, (1349C&gt;T), changes threonine at position 450 to methionine. The third patient (7 y), a female, presented, with multiple reddish non-ulcerated subcutaneous nodules and B-symptoms. Two surgical lesion biopsies were performed and a rare form of subcutaneous, panniculitis-like, T-cell lymphoma was diagnosed. Sequencing of the PRF gene disclosed the following 2 mutations: the first one, (272 C&gt;T), changes arginine at position 91 into valine; the second one, (1262 T&gt;G), changes phenylalanine at position 421 into cysteine. An female (18 y)presented with fever, malaise and massive hepato-splenomegaly. A peripheral T-cell lymphoma, associated with bone marrow infiltration, was diagnosed and 6 months after diagnosis, she was transplanted from an HLA-identical sibling. PRF gene sequencing showed two novel mutations: the first one (g914a) changes at position 305 glycine in aspartic acid, the second one (c1066a) changes arginine in tryptophan at position 356. Present data, point to a more complex disregulation of the immune system when PRF is absent, which may be associated with quite different clinical presentations indicating that the resulting effect of PRFmutations is likely to be more complex than previously anticipated and may vary among individuals, possibly depending on additional genetic and/or environmental factors.
Polymorphisms in the Hyaluronan Synthase 1 Gene May Be Predisposing Factors for Waldenstrom’s Macroglobulinemia.
Authors: Adamia Sophia , Treon Steven P. , Mant Michael J. , Larratt Loree M. , Reiman Tony , Belch Andrew R. , Pilarski Linda M. 
Published: April 2020

Abstract
The hyaluronan synthase 1 gene (HAS1), which maps to chromosome location 19q13.4 encodes a plasma membrane protein, which synthesizes hyaluronan (HA), an extracellular matrix molecule. Previously, in WM patients we detected up-regulation of HAS1 transcripts and identified aberrant splice variants of this gene, termed HAS1Va, Vb, and Vc. In patients with multiple myeloma, expression of HAS1Vb either alone or in combination with HAS1 and other variants strongly correlates with poor survival (P=0.001). Our gene expression analysis of the HAS1 family members demonstrated that 76–97% of individual CD20+ IgM+ WM cells obtained from BM aspirates and from blood (PBMC) express HAS1Va or HAS1Vb aberrant splice variants, often in the absence of full length HAS1 transcripts. Aberrant splicing of HAS1 is the result of activation of cryptic splice sites, which lead to exon skipping and/or intron retention. In turn, activation of cryptic donor and acceptor splice sites of the gene can be promoted by the mutations occurring upstream of these sites and/or at the branch point of slicing. We measured the frequency of a known polymorphism in the HAS1 gene of 16 BM and 30 PB samples obtained from WM patients, in comparison with PBMC samples from 33 healthy donors. Our results indicate that in healthy individuals, the frequency of the two alleles each reached 50% perhaps reflecting stabilizing selection. The majority of healthy donors (61%) are heterozygous for the HAS1 gene polymorphism. In contrast, 92% of analyzed WM patients are homozygous for this same polymorphism. An expression analysis of HAS1 and variants in BM cells and PBMC obtained from the same group of WM patients demonstrated that only those patients who were homozygous for the HAS1 polymorphism express HAS1 and/or its variants. The few WM patients who are heterozygous for this polymorphism have no detectable expression of aberrantly spliced HAS1 variants or full length of HAS1, a phenotype identical to that of healthy donors. Thus, our observations so far suggest that polymorphisms in HAS1 may contribute to aberrations such as exon skipping and/or activation of a new cryptic splice site leading to aberrant splicing of HAS. Based on the results obtained thus far, we speculate that individuals who are homozygous for HAS1 polymorphism(s) are at enhanced risk of developing WM due to a predisposition towards aberrant HAS1 splicing and expression of HAS1 variants, with predicted oncogenic consequences. However, the study of a much larger number of patients and healthy donors is needed to confirm these speculations and to evaluate the prognostic significance of these findings.
Development and Evaluation of a Novel Flow Cytometric Assay for Determination of Fcγ Receptor IIIa-158 V/F Dimorphism.
Authors: Boettcher Sebastian , Ritgen Matthias , Pott Christiane , Humpe Andreas , Kneba Michael 
Published: April 2020

Abstract
A recently described dimorphism at position 559 of FCGR3A gene results in two allotypes of Fcγ receptor IIIa (Fcγ RIIIa) with phenylalanine (F) or valine (V) at amino acid position 158. It has been shown that Fcγ RIIIa-VV homozygous patients with follicular lymphoma respond better to Rituximab as a single agent than F carriers. However, there is evidence that this disadvantage in F carriers can be overcome by higher Rituximab concentrations or by the use of defucosylated therapeutic antibodies. Therefore rapid, widely applicable, and cost-effective methods for the determination of this Fcγ RIIIa dimorphism are necessary. Currently available methods to determine this dimorphism are based on PCR techniques. To simplify the determination of Fcγ RIIIa-V/F dimorphism we developed a novel flow cytometric approach using known differences in epitope recognition of monoclonal antibodies (moabs) to Fcγ RIIIa. The moab MEM-154 recognizes Fcγ RIIIa-V only, whereas moab 3G8 recognizes Fcγ RIIIa irrespective of the dimorphism. We determined the expression level of epitopes recognized by 3G8 and MEM-154 in NK cells of 35 healthy donors (FF 14; V/F 17; VV 4) by three color flow cytometry and secondary immunofluorescence. Results were compared to genotypes determined by a TaqMan assay using allele specific fluorochrome labelled probes. Donors genotyped as Fcγ RIIIa FF, V/F, and VV demonstrated overlapping immunofluorescence levels detected by both 3G8 and MEM-154. However, the ratio of fluorescence measured using MEM-154 divided by the immunofluorescence measured using 3G8 was 100% accurate for predicting genotypes. Ratios below 0.05 were measured in Fcγ RIIIa FF individuals, ratios between 0.1 and 0.5 were measured in heterozygotes, whereas ratios higher than 0.6 were found in Fcγ RIIIa VV individuals only. Quantitative flow cytometry demonstrated a great variation in Fcγ RIIIa expression on NK cells between individuals with identical Fcγ R IIIa genotype explaining the failure to predict the genotype by a single Fcγ R IIIa moab only. This novel flow cytometric assay is cost-efficient, easy to implement, reliable and uses standard flow cytometric techniques. Compared to known methods to determine the dimorphism it is faster and applicable in laboratories without sophisticated PCR technology.
Phase I/II study of Bortezomib Alone and Bortezomib with Dose-Adjusted EPOCH Chemotherapy in Relapsed or Refractory Aggressive B-Cell Lymphoma.
Authors: Dunleavy Kieron , Janik John , Gea-Banacloche Juan , Shovlin Margaret , White Therese , Goldschmidt Neta , Grant Nicole , Koc Omer , Pittaluga Stefania , Jaffe Elaine , Staudt Louis , Wilson Wyndham H. 
Published: April 2020

Abstract
Activation of NF-kB is a possible mechanism of drug resistance in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Bortezomib is a proteasome inhibitor that inhibits NF-kB activation and has promising activity in some lymphoma subtypes. We hypothesized that bortezomib may be active in ABC DLBCL and enhance the efficacy of chemotherapy. The effect of bortezomib on NF-kB and other pathways in serial tumor samples will be examined by microarray and correlated with outcome. The study employs a sequential treatment design. Patients first receive bortezomib alone (Part A), unless they require chemotherapy due to disease, and then cross over to DA-EPOCH-bortezomib (Part B) if they do not achieve a CR. In Part A, the dose of bortezomib is 1.3 mg/m2 IV d 1, 4, 8, 11 q21days. In Part B, bortezomib is escalated over 4 dose levels, in an accelerated design, of 0.5, 1, 1.5 and 1.7 mg/m2 on d 1,4 with DA-EPOCH, q21days. Of 32 patients enrolled, there are 16 on Part A and 26 on Part B. Patient characteristics include median age (range) 54 (19–76); stage III/IV 29 (91%); median 4 (1–8) prior regimens and; 25 (83%) refractory to the last therapy. Part A response in 15 (1 TE) patients includes 1 PR, 3 SD and 11 (73%) PD. Toxicity over 35 cycles includes 2 (6%) grade 3 neutropenia; 2 and 3 grade 3/4 thrombocytopenia, respectively; and 3 grade 3 GI toxicities. Part B response in 25 (1 TE) patients includes 2 (8%) CR, 4 (16%) PR, and 20 (80%) NR. Twenty-three patients enrolled at bortezomib 1.7 mg/m2 (max dose). Toxicity over 58 cycles includes 31 (53%) grade 4 neutropenia; 13 (22%) grade 4 thrombocytopenia and; 12 (21%) fever/neutropenia. Transfusions were required with red cells on 22 (38%) and platelets on 18 (31%) cycles. Other toxicity included grade ≥ 3 sensory neurotoxicity in 2 (8%) patients and grade ≥ 2 GI toxicity on 32 (55%) cycles. Comparison of hematological toxicities of DA-EPOCH-bortezomib with fixed dose EPOCH in relapsed patients (JCO18:3633, 2000) was similar with fever/neutropenia 21% vs. 18%; grade 4 neutropenia 53% vs. 48% and; grade 3/4 thrombocytopenia 51% vs. 27%, respectively. GI toxicity appears higher with ≥ grade 2 on 55% vs. 14% of cycles. Neurotoxicity ≥ grade 2 was 8% vs. 22%, but patients on the present study received fewer cycles of treatment. Bortezomib alone is ineffective in relapsed/refractory DLBCL. Bortezomib can be safely combined with combination chemotherapy but does not appear to increase the efficacy of DA-EPOCH chemotherapy in relapsed/refractory DLBCL. However, compared to our previous study of EPOCH alone, most patients on the current study entered with chemotherapy-refractory disease. Results of microarray profiling are pending. Accrual continues.
Treatment with an Anti-C3b/iC3b Monoclonal Antibody as an Adjuvant to Rituximab Enhances the Killing of Non-Hodgkin’s Lymphoma and Chronic Lymphocytic Leukemia Cells.
Authors: Peng Wu , Zhang Xin , Li Juan , Inghirami Giorgio , Takeshita Kenichi , Pecora Andrew , Pincus Steve E. , Nardone Linda L. , Casey Leslie S. , Spitalny George L. 
Published: April 2020

Abstract
Complement-dependent cytotoxicity (CDC) is a key mechanism of Rituximab® (RTX) action in killing Non-Hodgkin’s Lymphoma (NHL) cells both in vitro and in vivo. Here we present studies of a mouse/human chimeric monoclonal antibody (Mab), ETI-210, specific for human complement C3 cleavage products C3b and iC3b, in enhancing RTX- mediated killing in vitro and ex vivo. Raji (NHL), primary NHL, and CLL cells were treated with RTX and ETI-210 in the presence of normal human serum (NHS) as a source of complement. Cell death was determined by propidium iodide staining or reduction of Alamar Blue®. After 1 hour of treatment, the killing of Raji cells by RTX plus ETI-210 plus NHS was observed to be 30–70% greater than that of RTX alone plus NHS by either detection method. The enhancing effect of ETI-210 was also seen in longer-term treatment (1 – 2 days). The killing by RTX plus ETI-210 was much lower in the absence of NHS than in the presence of NHS. ETI-210 was also tested ex vivo against primary cells from 12 NHL and 9 CLL patients. In 5 NHL samples, ETI-210 significantly enhanced RTX-mediated killing; in the remaining samples RTX alone already caused killing above 90%. The Mab also increased RTX-mediated killing in 4 out of 9 CLL samples. A pilot study examining the safety of administrating ETI-210 (5 and 30 mg/kg) with or without RTX was performed in cynomolgus monkeys. There were no cardiovascular changes. Clinical chemistry (potassium, glucose, sodium), liver function (alkaline phosphatase, ALT, albumin, total protein), kidney function (BUN, creatine, serum electrolytes) and hematological parameters (CBC profile, hemoglobin, MCV, MCH, MCHC) were not changed significantly. There was a transient reduction in CH50 values in the 30mg/kg ETI-210 group, which was independent of RTX administration. Serum concentrations of C3 and C4 were not affected. Because, ETI-210, a chimeric anti-C3b/iC3b Mab, was able to substantially enhance RTX-mediated killing of lymphoma cells via complement-mediated lysis and did not cause toxicity in monkeys, ETI-210 is being developed as a therapeutic agent for NHL and CLL treatment.
A Novel Method for Producing Custom-Made Idiotype Vaccines for Lymphoma Immunotherapy Using a Cell-Free Expression System.
Authors: Kanter Gregg , Yang Junhao , Voloshin Alexei , Swartz James R. , Levy Ronald 
Published: April 2020

Abstract
Idiotype vaccines have been used as a customized immunotherapy for lymphoma. This strategy takes advantage of a unique antigen (either immunoglobulin or T cell receptor) found on the lymphoma cells of each patient. To make this personalized vaccine feasible for widespread use a production method, which is rapid and relatively inexpensive will be required. In an E. coli-based cell-free protein expression system we were able to make ScFv proteins derived from the immunoglobulins of a mouse B cell lymphoma, 38C13, in high yields. We have used this cell-free system to make 38C13 ScFv proteins fused to either the cytokine mGM-CSF or a 9 amino acid peptide derived from hIL-1b. The 9 amino acid fragment of hIL-1b was chosen because it is known to retain immunostimulatory function while lacking pyrogenicity. We were able consistently to produce these proteins in high yield and to purify them using a histadine tag at the terminus of the molecule. The ScFv portion of the molecule is folded properly, as assayed by its binding to an anti-idiotype Ab. Similarly, the mGM-CSF fused to the ScFv can be recognized by an anti-mGM-CSF Ab. The mGM-CSF portion is biologically active as determined by its ability to support the growth a mGM-CSF dependant cell line. We immunized C3H mice with either the ScFc-hIL-1b or the ScFv-mGM-CSF fusion protein and we were able to induce an idiotype-specific Ab response, which recognizes the native idiotype protein in ELISA assays as well as the 38C13 tumor as detected by flow cytometry. Most importantly, in the highly aggressive 38C13 lymphoma model system, three bi-weekly vaccinations with either fusion protein protected mice from tumor challenge with the syngeneic 38C13 tumor. These data establish that a cell-free protein expression system can work for producing individualized idiotype vaccines for lymphoma. (This work was supported by NIH grant CA34233)
Targeted Overexpression of Mutant Activated N-ras Leads to Aberrant Plasma Cell Biology.
Authors: Linden Michael A. , Kirchhof Nicole , Van Ness Brian G. 
Published: April 2020

Abstract
The ras oncogene regulates a variety of cellular functions, and its dysregulation has been implicated in a variety of human cancers, including multiple myeloma. Indeed, activating ras mutations have been described in 35 – 50% of myeloma patients, 50% of human myeloma cell lines, and 12.5% of patients with monoclonal gammopathy of undetermined significance (MGUS). Given the higher incidence of activating ras mutations in myeloma compared to MGUS, the current models of myelomagenesis suggest that activating ras mutations are involved in the progression of MGUS to myeloma. While there has been a fairly extensive analysis of activating ras mutations in myeloma patients, there have been few studies to investigate the biology of an activated ras mutation in the context of B- and plasma cell development and tumorigenesis. We previously described a transgenic platform that uses the 3′ kappa immunoglobulin light chain enhancer (3′KE) to target transgene expression to B-cells in late developmental stages, including plasma cells (Blood103: 2779, 2004). To study the potential influence of elevated mutant ras expression on B- and plasma cell survival and proliferation, we used the 3′KE to generate a 3′KE/N-ras V12 transgenic mouse. We hypothesized that the presence of the mutant ras gene would affect normal B- and plasma cell homeostasis. Indeed, samples of mononuclear splenocytes from 4-week-old transgenic mice demonstrate a 70% increase in the number of B220+kappa+ B-cells and a 250% increase in the number of CD138+B220hi plasmablastic cells compared to littermate controls. While survival of the 3′KE/N-ras V12 mice appears similar to littermate controls and transgenic animals do not develop tumors at 35 weeks of age, aberrant lymphocyte biology was noted in multiple founder lines. All aged 3′KE/N-ras V12 transgenic founders demonstrated an immunoglobulinemia. Interestingly, the animal with the highest transgene copy number had the least pronounced immunoglobulinemia, while the animal with the lowest transgene copy number had the most pronounced immunoglobulinemia, suggesting an inversely dose-dependent relationship between over-expression of an activated Ras protein and immunoglobulinemia. We performed extensive necropsies and histopathological analyses on all founder mice and aged-matched littermate controls. While no tumors were found in any of the mice, three of the founder mice demonstrated abnormal accumulations of plasma cells in extramedullary sites, such as the kidney. These data indicate that an activated ras transgene can affect B- and plasma cell homeostasis, and this transgenic model could prove useful in studying the role of activating ras mutations in plasma cell tumorigenesis. We are currently using three targeted c-myc gene expression systems to elicit B- and/or plasma cell tumors by co-expressing c-myc and N-ras V12.
A Comprehensive Study of Chromosome 13 Abnormalities (Δ 13) in Multiple Myeloma Using High-Throughput, Array-Based Methods.
Authors: Baker Angela S. , Price-Troska Tammy , Bittner Michael L. , Van Wier Scott A. , Rempel Rachel , Ahmann Gregory J. , Pidgeon Tyler S. , Mousses Spyro , Futscher Bernie , Fonseca Rafael D. , Carpten John D. 
Published: April 2020

Abstract
Chromosome 13 abnormalities (Δ13), monosomy or deletion, are seen in greater than 50% in patients with Multiple Myeloma (MM) and are associated with poor survival and reduced response to therapy. Δ13 is suggestive of a tumor suppressor model. In this study, we use a comprehensive, high-throughput approach involving array-based technologies to identify genes on chromosome 13 that are important in the pathogenesis and/or progression of MM. Array-based Comparative Genomic Hybridization (aCGH) is being conducted to identify a minimum region of 13q loss in a series of MM cell lines and patient samples. DNA is isolated, labeled and hybridized with a differentially labeled normal DNA reference to determine gene/genomic copy number changes. Arrays are analyzed to search for the minimum region of loss based upon single copy loss for a series of nearby mapping transcripts in each cell line. Data will be cross-referenced to determine the true minimum region of 13q loss in MM. Also, to discover genes harboring potential inactivating mutations, we are using inhibition of nonsense-mediated RNA decay (NMD) coupled with cDNA microarray. This modification of the Gene Identification by NMD Inhibition (GINI) method, first introduced by Noensie and Dietz (2001), is used to identify genes on chromosome 13 that may be inactivated due to premature truncating codons (PTCs). This method is based upon a cell’s natural ability to survey for and degrade RNA species that contain PTCs by activation of the nonsense mediated RNA decay (NMD) pathway. Treating cells with the drug Emetine can inhibit NMD and increase the expression of genes likely harboring truncating mutations. Preliminary data have revealed several candidate genes, with increased ratios after Emetine treatment, which map to chromosome 13. Upon defining a minimum region of loss based upon aCGH data, we will prioritize genes from our NMD study for mutational analysis. It our hope to also perform an array-based CpG island screen to simultaneously analyze all CpG islands on chromosome 13 to detect genes possibly inactivated by hypermethylation. This comprehensive, organized approach incorporating high-throughput techniques for the detection of genomic loss, mutation, and methylation is the first of its kind to our knowledge and may maximize the potential for the identification of a 13q tumor suppressor in MM.