bluuebunny/crossref_metadata_2025 huggingface dataset:
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

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

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

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

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

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

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

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.
Immunosuppression for Myelodysplastic Syndrome: Association between a Score Based on Presenting Features and Long-Term Survival.
Authors: Sloand Elaine M. , Saunthrararajah Yogan , Battiwalla Minoo , Nakamura Ryo , Boss Carol , Aliyu Zakari Y. , Young Neal S. , Barrett John
Published: April 2020

Immunosuppressive therapy (IST) can improve cytopenias in selected patients with myelodysplastic syndrome (MDS). Between 1998 and 2004, 133 patients with MDS (FAB classification RA, RA, or RARS ) were enrolled in NHLBI IST protocols: 74 received ATG (Pharmacia, 40mg/kg x 4 days); 43 received a combination of ATG and CsA Fourteen received cyclosporine (CsA) to maintain levels above 100 ng/ml for up to 6 months. Average follow-up was 41 months (range 1 week to 10 years). Previously, we reported that responses to ATG alone were most likely in HLA DR15 individuals of younger age and with a shorter duration of red cell transfusion-dependence. We applied a score based on these factors to assign patients into high probability (HP) and low probability (LP) of response to immunosuppression. Here, we use the score to analyze effectiveness of different IST and compare survival, response, and leukemic progression in HP and LP patients. Patients with HP were more likely to respond to any IST schedule than were patients with LP (85% vs 8%; p<0.0001) and they had longer survival when compared to LP patients (90% vs 38% alive at 3 years; P<0.0001). There were no differences in response or survival between HP patients treated with ATG or with ATG/CsA; CsA-alone treated patients were too few for survival evaluation. Other factors positively affecting survival were response to therapy (p=.0004) and female sex (p=0.03). Neither bone marrow cellularity nor cytogenetics independently affected response or survival; patients with monosomy 7 and 5q- were in the LP group while patients with trisomy 8 predominated in the HP group. In general, patients with trisomy 8 and 5q- survived comparably to patients with normal cytogenetics, but patients with monosomy 7 had statistically shorter survival (p=0.007) than patients with normal cytogenetics or trisomy 8. The scoring system further segregated 95 patients within IPSS int-1 into a HP group with 92% 3 year survival vs 39% for LP patients (p<.0001). Mortality in the HP group was a consequence of cytopenias, while most deaths in the LP were from leukemia: only 2/50 (4%) of patients in the HP group developed leukemia compared with 30% of LP patients followed for a mean of 57 months (hazard ratio = 11; 95% CI 2.566 to 12.18; p< P<0.0001). These findings suggest that immunological characteristics may be independent factors determining the outcome of IST in patients with MDS.

Figure Figure
Non-Myeloablative Allografting from HLA-Identical Sibling Donors for Treatment of CML.
Authors: Kerbauy Fabio R. , Lange Thoralf , Gooley Ted , Shizuru Judith , Radich Jerald , Maloney David G. , Agura Edward , Bruno Benedetto , Epner Elliot , Chauncey Thomas , Blume Karl , Storb Rainer , Niederwieser Dietger , Sandmaier Brenda M.
Published: April 2020

We retrospectively analyzed data from 24 patients with chronic myeloid leukemia (CML) treated with HLA-matched related donor hematopoietic cell transplantation (HCT) after non-myeloablative conditioning with 2 Gy total body irradiation (TBI), given either alone (n=8) or in combination with 3 days of fludarabine, 30mg/m2/day (n=16). Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. CML patients in first chronic phase (CP1) (n=14), second chronic phase (n=4) or accelerated phase (n=6) who were not candidates for conventional HCT, were enrolled in a median of 28.5 (range, 11–271) months after diagnosis. The median age at HCT was 58 (range, 27–71) years. Unmodified G-CSF mobilized peripheral blood stem cell grafts containing medians of 7.9 (range, 2–18.1) x106 CD34+ cells/kg and 3.4 (range 1.7–2.4) x 108 CD3+ cells/kg, respectively, were infused. The median number of days in which ANC was less than 0.5 x 109/L was 5 (range, 0–8) days and 12 patients maintained ANCs greater than 0.5 x 109/L after HCT. Only 2 patients developed platelet counts less than 20 x 109/L. All patients had initial donor engraftment (>5% donor T-cell chimerism) at day 28 after transplant, and 17 (71%) achieved sustained full donor chimerism (>95% donor T-cell chimerism). There were 4 rejections followed by autologous hematopoietic reconstitution among 8 patients not given fludarabine, and donor lymphocyte infusions (DLI) were ineffective to reverse rejections. Fifteen (60%) patients achieved CR and 13 (54%) had complete molecular remissions, (defined as RT-PCR negativity for bcr-abl). Thirteen of 24 patients (54%) were alive and in CR at a median of 36 (range, 4–49) months after HCT. There were 5 (21%) deaths from non-relapse causes, one of them (4%) during the first 100 days. The incidences of grade II, III and IV acute GVHD were 38%, 4%, and 8%, respectively. The 2-year incidence of chronic extensive GVHD was 32%. The 2-year estimated overall survivals for patients in CP1 (n=14) and beyond CP1 (n=10) were 70% and 56%, respectively. Ten patients in CP1 received conditioning with fludarabine in addition to 2 Gy TBI; all ten achieved CR and 9 complete molecular remissions. Seven of 10 patients are alive in molecular remissions with a median follow-up of 36 (range, 6–49) months. In summary, non-myeloablative HCT were well tolerated in a group of patients who were older and/or medically infirm and therefore ineligible for myeloablative HCT. This approach should be considered for patients who fail to respond to initial conventional therapy and are not eligible for myeloablative conditioning regimens. The effectiveness of non-myeloablative compared to conventional myeloablative HCT will need to be definitively assessed in Phase III clinical trials.
Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.
Authors: Weisel Katja C. , Gao Ying , Shieh Jae-Hung , Kanz Lothar , Moore Malcolm A.S.
Published: April 2020

The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.
Intrahepatic Portal Application of Autologous CD133+ Bone Marrow Stem Cells to the Liver - Early Experience with a Novel Concept To Support Hepatic Regeneration.
Authors: Esch Jan Schulte am , Knoefel Wolfram T. , Klein Michael , Ghodsizad Alireza , Fuerst Guenter , Poll Ludger W. , Saleh Andreas , Piechaczek Christoph , Feifel Nico , Stockschlaeder Marcus , Stoldt Volker , Stoecklein Nikolas , Tustas Roy Y. , Eisenberger Claus F. , Peiper Matthias , Haeussinger Dieter , Hosch Stefan B.
Published: April 2020

The liver has a large capacity for regeneration after surgical resection. However, below a critical level of future liver remnant volume, liver resection is accompanied by significant increase of postoperative liver failure and mortality. There is accumulating evidence for the contribution of hematopoetic stem cells to participate in liver regeneration. Here we report on three patients that were subjected to intra portal application of CD133+ bone marrow stem cells to support a standard concept of preoperative proliferation of the prospective liver mass. In all three cases, scheduled for right hepatic trisegmentectomy, isolated portal venous embolisation (PVE) of liver segments I and IV–VIII routinely utilized to expand left lateral hepatic segments II/III (prospective remaining liver mass) was questionably sufficient. Autologous bone marrow cells were enriched for stem cell marker CD133 utilizing an immunomagnetic device. For characterisation, each preparation of Cells was subjected to FACS-analyses for markers CD34, CD45 and CD133. Absolute numbers of 2.4 to 12.3 mio CD133+ cells were porto-venously applied to the left-lateral liver segments following PVE of contra lateral liver segments. The treatment with CD133+ was well tolerated without obvious side effects. CT-scan volumetry revealed 2.5 fold increased mean proliferation rates of left-lateral segments when compared to a group of three consecutive patients, treated with the same therapeutic concept - except the application of stem cells. Two to three weeks after intervention, future remnant liver volume was sufficient to perform expanded liver resection, which was applied safely to all patients. This first experience with a novel concept of portovenous application of CD133+ bone marrow cells subsequent to porto-venous embolisation encourages to carry out future controlled trials to evaluate the effectiveness of this approach to boost liver regeneration processes.
Myeloma Cells Suppress Osteoblast Differentiation by Secreting a Soluble Wnt Inhibitor, sFRP-2.
Authors: Oshima Takashi , Abe Masahiro , Asano Jin , Hara Tomoko , Kitazoe Kenichi , Sekimoto Etsuko , Tanaka Yoichi , Shibata Hironobu , Hashimoto Toshihiro , Ozaki Shuji , Kido Shinsuke , Inoue Daisuke , Matsumoto Toshio
Published: April 2020

Multiple myeloma (MM), a malignancy of plasma cells, develops in the bone marrow, and generates devastating bone destruction. Along with enhanced bone resorption, clinical evidence has also suggested suppression of bone formation as a contributing factor to the bone loss in MM. In contrast to recent understanding on mechanisms of osteolysis enahnced in MM, little is known about factors responsible for impaired bone formation. A canonical Wingless-type (Wnt) signaling pathway has recently been shown to play a critical role in osteoblast differentiation. Therefore, in the present study, we aimed to clarify mechanisms of suppression of osteoblast differentiation by MM cells with a particular focus on a canonical Wnt signaling pathway. Because several secreted Frizzled related protein (sFRP) and DKK family members are known as soluble Wnt antagonists, we first examined the expression of sFRP-1, 2 and 3 and DKK-1 in MM cell lines including U266, RPMI8226 and ARH77. All cell lines expressed sFRP-2 and sFRP-3 mRNA observed by RT-PCR. However, sFRP-1 was not expressed in any cell line, and Dkk-1 was expressed only in U266 cells at mRNA levels. We next conducted Western blot analyses for these factors and detected only sFRP-2 in immunoprecipitants of conditioned media as well as cell lysates of all these cell lines. However, no other factors were found at protein levels. Furthermore, sFRP-2 mRNA and protein expression was detected in most MM cells from patients with advanced or terminal stages of MM with bone destruction including plasma cell leukemia (3/4 and 8/10, respectively). In order to examine a biological role for sFRP-2, we added recombinant sFRP-2 to MC3T3-E1 cell culture together with BMP-2. Exogenous sFRP-2 partially suppressed alkaline phosphatase activity but almost completely mineralized nodule formation enhanced by BMP-2. Furthermore, sFRP-2 immunodepletion significantly restored mineralized nodule formation in MC3T3-E1 cells suppressed by RPMI8226 and ARH77 CM. These results suggest that sFRP-2 alone is able to suppress osteoblast differentiation induced by BMP-2 and that MM cell-derived sFRP-2 is among predominant factors responsible for defective bone formation in MM. Because MM cell-derived factors such as DKK-1, IGF-BP4 and IL-3 other than sFRP-2 have been implicated as an inhibitor of osteoblast differentiation, sFRP-2 may act alone or in combination with such other factors to potently suppress bone formation in MM. Taken together, MM cells may cause an imbalance of bone turnover with enhanced osteoclastic bone resorption and concomitantly suppressed bone formation, which leads to devastating destruction and a rapid loss of bone.
TNFRSF6 (Fas Antigen) Mutations in Patients with Sinus Histiocytosis with Massive Lymphadenopathy (Rosai-Dorfman Disease).
Authors: George Tracy I. , Ma Lisa , Nagy Peter L. , Natkunam Yasodha , Warnke Roger A. , Dorfman Ronald F. , Arber Daniel A.
Published: April 2020

Sinus histiocytosis with massive lymphadenopathy (SHML or Rosai-Dorfman disease) is a rare histiocytic proliferation of unknown etiology. There is a known association between SHML and immune disorders. Recently, histologic changes of SHML have been found in lymph nodes of patients with autoimmune lymphoproliferative syndrome (ALPS), a disorder most commonly associated with mutations in the TNFRSF6 gene that encodes the Fas antigen. We retrospectively analyzed tissue from 22 patients with SHML for TNFRSF6 gene mutations using isolated genomic DNA from paraffin-embedded tissue followed by screening of PCR products for the promoter regions and all exons (1–9) of the gene for possible mutation by high performance liquid chromatography. Mutations were confirmed by direct sequencing. We identified six patients (27%) with SHML and mutations of the TNFRSF6 gene as listed below, including three cases (14%) with potentially significant mutations (*) involving promotor and splice donor regions, and frameshift mutations. The samples with mutations had a 4:2 male:female ratio with a mean/median age of 39/43 years (range 7–68 years) compared to a 9:7 male:female ratio, mean/median age of 42/43 years (range 12–76 years) for the non-mutated samples. No differences in site of disease were identified in the mutated versus non-mutated groups. None of the samples had morphologic features to suggest ALPS. These results suggest that at least a subset of cases of SHML are associated with disruption of the Fas apoptotic pathway. Additional study of other components of the apoptotic pathway may be warranted in this disease.

Cases with TNFRSF6 mutations.

Case No. Nucleotide Change Localization Codon Amino acid change **Numbering for the promoter sequence, according to www.mutationdiscovery.com. All other numbering according to GeneBank accession number M67454. 1 c217+34A>G Intron 7 c217+40A>G Intron 7 2 T779C Exon 7 195 Val --> Ala 3 c217+1G>A Intron 7 Splice defect* T763C Exon 7 190 Val --> Ala c217+182A>G Intron 7 c217+183A>G Intron 7 C1425T Exon 9II 3′UTR * 4 597del T Exon 4 135 Frameshift* 5 T/C 4656** Promoter * c189+4A>T Intron 6 5′ consensus splice donor site* C729T Exon 6 179 Leu --> Phe 6 c218-84A>G Intron 7
A Phase I, Multi-Dose, Dose Escalation Study of SGN-40 (anti-huCD40 mAb) in Patients with Refractory or Recurrent Multiple Myeloma.
Authors: Hussein Mohamad A. , Niesvizky Ruben , Munshi Nikhil , Berenson James C. , Anderson Kenneth C. , Ryan Kate , Baumgartner Karen , Miller Dennis M. , McDonald Michael
Published: April 2020

CD40 is a type I transmembrane protein that upon binding to CD40 ligand regulates important biologic effects in the immune system. CD40 is also highly expressed on hematologic tumors, which has raised interest in the potential for its use as a tumor target for antibody-based cancer therapy. SGN-40 is a humanized monoclonal antibody that selectively binds to human CD40 and induces apoptosis and growth inhibition of a wide variety of B-cell derived cancer cell lines in vitro. Our preclinical work has confirmed the in vitro cytotoxicity of SGN-40 against human multiple myeloma (MM) cells via several mechanisms. These include induction of cytotoxic ligands of TNF superfamily; suppression of IL-6-induced proliferative and anti-apoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (Tai, et al, Cancer Research64, 2846–2852, April 15, 2004). Since ≥ 90% of MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treatment strategy. Based on these preclinical data, a phase I study is being conducted to define the toxicity profile, characterize the pharmacokinetics (PK), and evaluate antitumor effects of SGN-40 in patients with refractory or recurrent MM. Four weekly doses ranging from 0.5 to 16 mg/kg are planned to be administered to groups of at least three patients per cohort. Patients will be followed for up to 6 weeks post their last dose. Currently, a total of seven patients have been treated with SGN-40 at dose levels of 0.5 and 1.0 mg/kg. No grade 3 or 4 non-hematologic dose limiting toxicities have been observed. One patient experienced a transient Grade 3 decrease in hemoglobin. Decrease in CD19 positive B-cells were noted for patients treated at both dose levels. Changes in serum and urine M protein levels were measured to estimate potential anti-tumor effects of SGN-40. Of the seven patients evaluated, one patient at 0.5 mg/kg dose had stable disease, based on serum M protein, over the 10 week study period. Clinical evaluation with dose escalation of this agent continues and updated safety, PK and antitumor data will be presented.
BMS-354825 Potently Inhibits the Kinase Activity of KIT Activation Loop Mutations Associated with Systemic Mastocytosis and Induces Apoptosis of Mastocytosis Cell Lines.
Authors: Schittenhelm Marcus , Shiraga Sharon , Lee Francis Y. , Heinrich Michael C.
Published: April 2020

Activating mutations of the activation loop (AL) of KIT are reported in several human neoplasms including a subset of patients with AML or systemic mast cell disorders. The small molecule tyrosine kinase inhibitor, imatinib, is a potent inhibitor of wild type KIT and certain mutant KIT isoforms and has become the standard of care for treating patients with metastatic GI stromal tumors. However, KIT AL mutations such as D816V that are typically found in AML and systemic mast cell disease are insensitive to imatinib (IC50 > 5–10 micromolar). In addition, acquired AL mutations are associated with imatinib resistance in GI stromal tumors. Experimental models of these diseases suggests that targeted inhibition of AL-mutant KIT in such cells would result in decreased proliferation and induction of apoptosis. BMS-354825 is a synthetic small-molecule ATP-competitive inhibitor of SRC and ABL tyrosine kinases with potency in the low nanomoloar range. In addition, BMS-354825 also inhibits many of the acquired BCR-ABL mutations associated with imatinib-resistant CML. Notably, BMS-354825 binds to the ATP-binding site in ABL regardless of the conformation of the AL and is therefore able to potently inhibit the kinase activity of AL-mutants of BCR-ABL. BMS-354825 is currently being tested in phase I studies for safety and efficacy in the treatment of imatinib-resistant CML as well as for various solid tumors. We investigated the inhibitory potential of BMS-354825 against KIT AL-mutants expressed by different mastocytosis/mast cell leukemia cell lines. We first examined the activity of BMS-354825 against the kinase activity of wild-type KIT kinase expressed by M07e cells. BMS-354825 potently inhibited the kinase activity of SLF-dependent M07E cells with an IC50 of 1–10 nM for autophosphorylation and cellular proliferation. In contrast, the IC50 for inhibition of GM-CSF-dependent proliferation was ~5 micromolar. BMS-354825 also potently inhibited the proliferation of the human mast cell leukemia cell line HMC-1 that expresses a juxtamembrane KIT V560G mutant kinase (IC50 1–10 nM). Notably, BMS-354825 inhibited autophosphorylation of KIT D816V with an IC50 of ~100 nM. Similar potency for inhibition of proliferation and induction of apoptosis (IC50 150–400 nM) was seen using three different cell lines expressing human D816V or the homologous murine D814Y KIT kinase. We conclude that BMS-354825 potently inhibits the kinase activity of wild-type KIT and imatinib-resistant KIT AL-mutant kinases associated with AML and systemic mast cell disorders. If BMS-354825 proves to be safe and tolerable in ongoing phase I trials, it should be further tested in clinical studies targeting patients with human malignancies associated with imatinib-resistant KIT AL-mutations.
KIR-LIGAND Mismatched Natural Killer Cells Effectively Kill Primary Myeloma and Myeloma Cell Lines.
Authors: van Rhee Frits , Szmania Susann M. , Shi JuMei , Cottler-Fox Michele , Dyniewski Justin W. , Tricot Guido J.
Published: April 2020

Recent observations in haplo-identical, T-cell-depleted allogeneic transplantation have focused attention on the rapid and remarkable anti-tumor effect mediated by Killer Inhibitory Receptor (KIR) ligand mismatched natural killer (NK) cells. Most data are confined to the role of KIR-ligand mismatched NK cells for acute leukemia after allogeneic transplantation. In this study, we report both the ability of KIR mismatched NK cells to kill primary myeloma cells, and the effects of interleukin (IL)2 and IL15 on NK cells in vitro. NK cells were prepared from healthy donor PBMCs by depletion of T-lymphocytes using immunomagnetic beads conjugated to anti-CD3 followed by depletion of monocytes using a simple adherence step. After overnight incubation in cytokines (IL2/IL15 as indicated) standard chromium release assays determined the cytotoxicity of NK cells against target cells and 3H-thymidine incorporation assessed NK cell proliferation. KIR-ligand mismatched NK cells incubated overnight in 300 IU/ml IL2 killed primary MM cells from four individuals (76, 64,60,48% lysis) as well as MM lines U266, NCI H929, and the NK sensitive line K562 (95, 86, 76% lysis, respectively) at an E:T ratio of 20:1. NK cells were not cytotoxic towards autologous phytohemagglutinin (PHA) or allogeneic PHA-induced T-cell blasts. Increasing the IL2 concentration during overnight NK cell incubation from 300 to 1000 IU/ml did not significantly enhance the cytotoxicity against U266 myeloma cells or control K562 cells. IL15 was more potent than IL2 in inducing NK cell proliferation. The optimal IL15 concentration was 300 IU/ml (range tested: 0–3000 IU/ml). The use of both cytokines in concert was less effective than the use of IL15 alone, probably due to homology of the IL2 and IL15 b and g receptors. We conclude that T cell and monocyte depletion of PBMC results in a preparation significantly enriched for NK cells (±50%) that effectively kills KIR-ligand mismatched primary myeloma cells and myeloma cell lines. IL15 is the superior cytokine for enhancing NK cell proliferation. The exciting anti-leukemic effects mediated by KIR-ligand mismatched NK cells have thus far only been reported in the haplo-identical transplant setting. In view of our positive in vitro data in MM, we will evaluate in a phase II trial wheter the repeated transfusion of KIR-ligand-mismatched, T-cell depleted NK cells from haplo-identical donors after immunosuppressive treatment with fludarabine and dexamethasone (to avoid rejection of donor NK cells) and tumor reduction with melphalan followed by a delayed auto-transplant can improve outcome in patients who have high risk MM or who have relapsed after a previous auto-transplant. This will be the first clinical application of KIR-ligand-mismatched NK cells in autologous transplantation and the first such trial in myeloma.

Figure Figure
Caveolin-1 Is Required for VEGF- Triggered Multiple Myeloma Cell Migration and Is Targeted by Bortezomib (Velcade®).
Authors: Podar Klaus , Shringarpure Reshma , Tai Yu-Tzu , Simoncini Melissa , Sattler Martin , Ishitsuka Kenji , Richardson Paul G. , Hideshima Teru , Chauhan Dharminder , Kenneth Anderson C.
Published: April 2020

We recently demonstrated that caveolae, vesicular flask-shaped invaginations of the plasma membrane represent novel therapeutic targets in multiple myeloma (MM). In the present study, we demonstrate that vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130Cas phosphorylation and MM cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130Cas phosphorylation and VEGF-triggered MM cell migration. The proteasome inhibitor bortezomib, both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF- induced MM cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in HUVECs.

Taken together, these studies demonstrate the requirement of caveolae for VEGF-triggered MM cell migration and identify caveolin-1 in MM cells and HUVECs as a molecular target of bortezomib.
NY-ESO-1 Specific Antibodies Are Frequently Detected in Hight-Risk Myeloma.
Authors: van Rhee Frits , Szmania Susann M. , Pomtree Mindy , Zhan Frank , Moreno Amberly , Rosen Nancy , Fox Michele , Barlogie Bart , Shaughnessy John , Batchu Ramesh , Tricot Guido J.
Published: April 2020

We have previously reported that NY-ESO-1 is especially present in high-risk multiple myeloma (MM) as defined by abnormal metaphase cytogenetics. The presence of NY-ESO1 specific antibody (ab) response is an excellent surrogate marker for the presence of a CD4+ NY-ESO-1 specific T-cell response, which is essential to priming, expanding and sustaining CD8+ NY-ESO-1 specific T-cell responses. We therefore studied the sera of 56 MM patients enrolled in our Total Therapy II protocol for the presence of specific NY-ESO-1 ab by sandwich ELISA using highly purified recombinant NY-ESO-1 protein. NY-ESO-1 RNA expression of purified MM cells was determined by gene expression profiling. NY-ESO-1 specific ab were detected in 13/39 (33%) of patients with NY-ESO-1 positive MM. In contrast, patients with NY-ESO-1 negative MM (n=27) all tested negative for NY-ESO-1 ab by ELISA, as did normal donors (n=20). Antibodies to NY-ESO-1 were present in 9/19 (47.3%) of patients who were studied with frank, NY-ESO-1 positive MM (at diagnosis, progressive disease or at relapse). All patients with progressive/relapsed MM had previously received intensive chemotherapy and/or auto-transplantation. In NY-ESO-1 positive MM patients who had a very good partial or partial response to therapy NY-ESO-1 specific ab were detected in only 4 patients of 13 tested (30.7%). In contrast, no NY-ESO-1 specific ab were detected in 7 NY-ESO-1 positive MM patients who had achieved (near) complete remission. Nine of the 13 (70%) NY-ESO-1 ab positive patients had high-risk MM as defined by abnormal metaphase cytogenetics. We conclude that the presence of NY-ESO-1 positive MM, and thus antigenic stimulation, is the principal determinant of the ability to mount a humoral response to NY-ESO-1 protein despite the presence of an immune system compromised by the immunosuppressive effects of MM or (high dose) chemotherapy. Further, NY-ESO-1 specific ab are principally found in patients with high-risk MM. The presence of spontaneous humoral immune responses to NY-ESO-1 is important since it suggests that there is a reservoir of NY-ESO-1 specific CD4+ T-cells that could be expanded and utilized for the immunotherapy of high-risk MM by vaccinating with NY-ESO-1 peptides, proteins or DNA based vaccines.