bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epigenetic Modulation by Vidaza™ induces Apoptosis and Overcomes In Vitro Drug Resistance in Human Multiple Myeloma Cells.
Authors: Kiziltepe Tanyel , Catley Laurence , Hideshima Teru , Raje Noopur , Yasui Hiroshi , Vallet Sonia , Ikeda Hiroshi , Okawa Yutaka , Anderson Kenneth C. 
Published: April 2020

Abstract
Methylation of DNA is an epigenetic modification that plays an important role in the regulation of gene expression in mammalian cells. Although DNA methylation is required for normal cell development and function, aberrant methylation and the resulting aberrant expression of genes, such as tumor suppressor genes and oncogenes, contribute to the development of malignancies. Interestingly, aberrant DNA methylation has recently emerged as one of the most frequent molecular alterations in hematologic malignancies, providing a powerful rationale to use inhibitors of DNA methylation as a novel means of targeting hematologic malignancies. Vidaza™ [Pharmion Corporation] (5-azacytidine), an FDA approved drug for the treatment of myelodysplastic syndromes, is an inhibitor of DNA methylation. Multiple myeloma (MM) is currently an incurable hematological malignancy despite all the conventional and novel therapies, and we here examined the biological effects using Vidaza™ on human MM cells. We demonstrate here that Vidaza™ has significant cytotoxicity in both conventional therapy sensitive (MM1S, RPMI-8226) and resistant (MM1R, RPMI-Dox40, RPMI-LR5) MM cell lines, as well as freshly isolated patient MM tumor cells, with an IC50 of 1.25–4 mM at 72 hours in vitro. Importantly, no cytotoxic effects of Vidaza™ were detected in peripheral blood mononuclear cells (PBMNC) obtained from healthy volunteers at ≤20 mM, suggesting a therapeutic index. Moreover, Vidaza™ overcame the survival and growth advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells (BMSC). Vidaza™ induced apoptosis in MM cells, as determined by flow cytometric analysis using PI and Annexin V staining. Vidaza™-induced apoptosis was associated with PARP, caspase 8 and caspase 9 cleavage. Importantly, pan-caspase inhibitor zVAD-fmk, significantly, but only partially, inhibited apoptosis induced by Vidaza™, suggesting the involvement of both caspase dependent and independent pathways. Taken together, our studies therefore demonstrate that Vidaza™ induces apoptosis and overcomes in vitro drug resistance in MM cells. Ongoing studies are delineating the mechanism of action of Vidaza™ against MM cells to further provide the preclinical rationale for clinical evaluation of Vidaza™, alone or in combination with other agents, to improve patient outcome in MM.
Elimination of the Need for Urine Studies during Diagnostic Studies of Monoclonal Gammopathies by the Combined Use of Serum Immunofixation and Serum Free Light Chain Assays.
Authors: Katzmann Jerry A. , Dispenzieri Angela , Kyle Robert , Snyder Melissa R. , Plevak Mathew F. , Larson Dirk R. , Abraham Roshini S. , Lust John A. , Melton L. Joseph , Rajkumar S. Vincent 
Published: April 2020

Abstract
Due to the diagnostic sensitivity of serum free light chain quantitation for monoclonal light chain diseases, it has been suggested that urine assays no longer need be performed as part of the diagnostic algorithm for monoclonal proteins. We reviewed our experience to determine the relative diagnostic contribution of urine assays.
Methods: Patients with a monoclonal gammopathy and monoclonal urinary protein at initial diagnosis who also had a serum immunofixation and serum free light chain quantitation within 30 days of diagnosis were identified (n = 428). The laboratory results for serum protein electrophoresis, serum immunofixation, serum free light chain, urine protein electrophoresis, and urine immunofixation were reviewed.
Results: The patients in this cohort had diagnoses of multiple myeloma, primary amyloid, monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, solitary plasmacytomas, and other less frequently detected monoclonal gammopathies. By definition of the cohort, all 428 had a monoclonal urine protein. 86% had an abnormal serum free light chain K/L ratio, 81% had an abnormal serum protein electrophoresis, and 94% had an abnormal serum immunofixation. In only 2 patients, however, were all 3 serum assays normal. Both of these were patients with monoclonal gammopathy of undetermined significance (idiopathic Bence Jones proteinuria).
Conclusion: Discontinuation of urine studies and reliance on a diagnostic algorithm using solely serum studies (protein electrophoresis, immunofixation, and free light chain quantitation), missed 2 of the 428 monoclonal gammopathies (0.5 %) with urinary monoclonal proteins, and these 2 cases required no medical intervention.
BAX UTR G (-248) A Polymorphism Has No Impact on Survival in Patients with Multiple Myeloma.
Authors: Paneesha Shankaranarayana , Hawkins K. , Davies F. , Hooper L. , Starczynski J. , Pepper C. , Morgan Gareth , Fegan C. , Pratt Guy 
Published: April 2020

Abstract
Multiple myeloma is a malignant tumour of plasma cells that remains incurable for the vast majority of patients with a median survival of 2–3 years. The bax protein is a member of the Bcl-2 family and is an important pro-apoptotic protein. Low BAX expression has been shown to be associated with prolonged survival in patients with myeloma(1). A novel polymorphism, G(-248)A in the BAX promoter in patients with Chronic lymphocytic leukaemia (CLL) has shown to be associated with reduced Bax protein expression, progressive disease and treatment failure(2, 3). We report here the impact of this polymorphism on survival in 304 (M: 168; F: 136) patients with myeloma
Materials and Methods
This study included 158 (M: 89; F: 69) patients from MRC Myeloma VII trial and 146 (M: 79; F: 67) under our care from January 1992 and February 2006. DNA was extracted from myeloma patients and healthy volunteer’s peripheral blood using a standard salting out method. Analysis of the 5-UTR polymorphism was undertaken using method already published by our centre(3). Patients received either intensive or non intensive antimyeloma therapy as per standard practice. Statistical analysis was carried out using SAS software.
Results
The frequency of GG and GA/AA genotypes was 81.6% and 18.4% in myeloma patients as compared to 85.2% and 14.8% in normal controls. Median age at diagnosis was 58 yr (range 34–87 yrs). (GA/AA Group: 58 yrs (42–85); GG Group: 58 yr (34–87)). There were no significant differences in the sex distribution, β2 microglobulin, haemoglobin, bone lesions, calcium and age between the GA/AA & GG groups with p values 0.72, 0.46, 0.95, 0.61, 0.49 and 0.46 respectively. Abnormal creatinine levels were significantly few in GA/AA group (p value: 0.002). Median survival for the AA/GA group was 48 months (95% CI 36 – 61 months) compared with 54 months in the GG group (95% CI 46 – 63 months)[p=0.5 log-rank test] with a median follow up of 62 months (range 1 to 141).
Conclusions
Our results show that the GA/AA polymorphism has no impact on the overall survival in patients with myeloma. Further work is ongoing in order to assess the impact of this polymorphism on response in various treatment groups.
Interruption of Bisphosphonates (BP) Therapy in Multiple Myeloma (MM) Patients and Osteonecrosis of the Jaw (ONJ).
Authors: Badros Ashraf , Goloubeva Olga , Weikel Dianna , Rapoport Aaron P. , Akpek Gorgun , Yanovich Saul , Meiller Timothy , Sausville Edward 
Published: April 2020

Abstract
ONJ is a serious complication in MM patients; incidence is time-dependent with highest risk seen after long-term use of BP in older MM patients, often after dental surgical procedure (Badros, et al JCO 2006). Therapy has focused on debridement of necrotic bone, infection management, pain control and discontinuation of BP. The association between BP and ONJ remains provisional; theoretically and anecdotally, however there is evidence that BP withdrawal may allow for recovery of normal osteoclast function and reversal of antiangiogenic effects of the drug on the periosteum allowing for bone healing. We have observed an increased risk of skeletal complications in MM patients after long interruptions of BP therapy (6–12+months), even though patients had been on BP therapy for a median of 3 yrs (range: 2–10+yrs). Table below summarizes the complications that occurred off BP therapy in MM patients with ONJ (n=28) and without ONJ who stopped BP ahead of dental procedures (n=20). Skeletal complications included avascular necrosis of the hip (AVN), fractures, new lytic lesions on skeletal surveys and bone pain; all occurred in relapsed patients with active MM. These events highlighted the importance of BP cornerstone for treatment of skeletal complications of MM; of the 28 ONJ patients, 8 died from progressive MM and 3 patients had non-healing oral lesions and did not resume BP. Twelve of 17 ONJ patients were restarted on BP after complete healing of the oral lesions; all of whom had relapsed MM and bone disease. They received pamidronate every 3 months. So far 3 of 12 patients (25%) had recurrent ONJ and none had skeletal complications. Of the 20 patients who stopped BP before dental procedures, 3 (15%) went on to develop ONJ. Fifteen had restarted BP 2–3 months after the procedure site was well-healed; one patient developed ONJ at the healed extraction site 6 months after restarting BP therapy. This patient was in remission on maintenance thalidomide but with extensive myeloma bone disease. In conclusion, interruption of BP in MM patients with ONJ as well as before and after dental procedures had inconsistent results with regards to both healing and prevention of ONJ. On the other hand, the damage of holding BP therapy is evident with increased frequency of skeletal complications. Until further studies define the optimal frequency and duration of BP therapy, we propose continuous administration of BP therapy probably less frequently after 2 years of monthly infusions with careful assessment of the risk status of each individual patient.
Skeletal complications off bisphosphonate therapy FracturesΔ AVN New lytic lesions Bone pain Δ Fractures affecting long bones or ribs. λ Two had bone disease as first site of relapse. π One patient had a femoral Infarction and severe pain. ONJ; n=28 5 5 2 4 Dental Procedure; n=20 3 1 3λ 1π
Neutropenia-Associated Mutations in PRDM5, a Novel Epigenetic Regulator of Hematopoiesis.
Authors: Duan Zhijun , Huang Shi , Person Richard E. , Badolato Raffaele , Donadieu Jean , Horwitz Marshall S. 
Published: April 2020

Abstract
The growth factor independent 1 (Gfi1) oncoprotein is an intrinsic regulator of hematopoiesis, and its mutations are associated with congenital neutropenia. We performed a yeast two-hybrid screen with Gfi1, in order to begin dissecting the gene network through which it acts and also toward identifying other genes potentially responsible for cases of hereditary neutropenia lacking mutations in Gfi1 or ELA2 (encoding neutrophil elastase, the most common cause of hereditary neutropenia). From a limited number of proteins found to interact with Gfi1 in the yeast two-hybrid screen, we evaluated PRDM5, a previously uncharacterized zinc finger protein belonging to the PR domain (PRDI-BF1 and RIZ homology) -containing family of tumor suppressor genes. We screened for mutations in the gene encoding PRDM5 among 230 unrelated patients with otherwise genetically unexplained congenital neutropenia and discovered two with heterozygous amino acid sequence substitutions that were not detected in twice the number of controls. Consequently, we systematically characterized PRDM5, and found that it functions as a novel epigenetic regulator of hematopoiesis: First, PRDM5 is a sequence-specific DNA-binding transcription factor as revealed by random sequence selection. Second, by using chromatin immunoprecipitation (ChIP)-based cloning along with “ChIP-on-chip” assays, we identified approximately two hundred protein-coding genes (including hematopoietic transcription factors) and thirty-four microRNA genes as putative PRDM5 targets, a subset of which we confirmed by additionally performing gel retardation and transfection reporter assays. Significantly, one of the targets of PRDM5—just as is true of Gfi1—is ELA2. Third, depletion of PRDM5 by siRNA in both HeLa and HEK293 cells changed the cell cycle program, and, as revealed by microarray and real-time RT-PCR assays, PRDM5 knockdown in HEK293 cells altered the expression of ninety of the protein-coding targets and eight of the microRNA targets. Fourth, transcriptional profiling revealed that the expression of about five thousand genes, including many involved in the regulation of cell proliferation and differentiation, changed more than five-fold following PRDM5 knockdown in HEK293 cells. Fifth, we found that PRDM5 regulates transcription by associating with the histone deacetylase HDAC1 and the histone methyltransferase G9a and recruits them to its target genes. Treatment of cells with the histone deacetylase inhibitors trichostatin A (TSA) or 4-phenylbutyric acid (PBA) deregulates PRDM5 target genes. Finally, the two variants observed in patients with congenital neutropenia alter the DNA-binding activity of PRDM5 and interfere with its function as a transcriptional repressor in reporter assays, suggesting that, just as with Gfi1, rare mutations in a global regulator of hematopoiesis may contribute to the genetic mechanisms causing neutropenia.
Phenyhexyl Isothiocyanate, a Novel Inhibitor of Histone Deacetylation and Methylation, Induce Growth Arrest and Apoptosis of Myeloma Cells.
Authors: Lu Quanyi , Feng Jane , Seiter Karen , Ahmed Tauseef , Chiao Dicky J.W. , Liu Delong 
Published: April 2020

Abstract
Histone deacetylase (HDAC) inhibitors are emerging as a promising family of new agents in the therapy of hematologic malignancies. We have shown that phenyhexyl isothiocyanate(PHI) is a novel histone deacetylase inhibitor and can also modulate histone methylation in leukemia cells. In this study we investigated the effect of PHI on human myeloma cell line RPMI8226 in vitro.
We have observed that cell proliferation was inhibited by PHI in a dose and time dependent manner. Cell cycle analysis indicated an arrest in G0/G1 phase, and significant apoptosis was detected in PHI treated RPMI8226 cells. The accumulation of P21 and reduced Levels of PCNA were detected. We also examined the effect of PHI on vascular endothelial growth factor (VEGF) production by the myeloma cells. Treatment with 20μM PHI for 24hrs led to a decrease of VEGF concentration by 50% of that secreted by the control cells. VEGF production further decreased to 25% when the cells were exposed to PHI for 48hrs. These data suggest that myeloma cells are sensitive to the novel HDAC inhibitor, and PHI may become a novel agent in multiple myeloma therapy.
PHI Inhibit VEGF production by Myeloma Cells PHI Inhibit VEGF production by Myeloma Cells
Monokine Induced by Interferon-γ (MIG) Serum Levels Are a Marker of Disease Load and Correlate with Prognosis in Multiple Myeloma.
Authors: Schreder Martin , Koch Gudrun , Heintel Daniel , Strasser-Weippl Kathrin , Frischmuth Katrin , Jaeger Ulrich , Ludwig Heinz , Zojer Niklas 
Published: April 2020

Abstract
Background and Aims: Monokine-induced by interferon-γ (MIG) is a chemokine that is produced by monocytes and macrophages in response to interferon-γ and acts as a chemoattractant mainly to T-lymphocytes in inflammatory processes. MIG has also been suggested to act in an autocrine loop to stimulate tumour cells through its receptor CXCR3, which is known to be expressed in myeloma cells. However, it is presently unclear if MIG is of biologic significance in myeloma in vivo. We have recently shown that multiple myeloma oncogene 1 (MUM1) expression in myeloma cells correlates with prognosis in this disease (Heintel D et al., ASH 2005), and MUM1 was found to upregulate MIG gene expression in B cell malignancies (Uranishi M et al., Leukemia 2005). This led us to evaluate the potential prognostic significance of MIG serum levels in a series of well characterized myeloma patients.
Methods: 105 newly diagnosed multiple myeloma patients (median age 69.3 years, range 39.4–90.5) were enrolled. 18 patients presented with Durie/Salmon stage I disease, 9 had stage II and 78 had stage III. MIG serum levels were determined by a commercially available ELISA (R&D Systems). Serum samples from 17 MGUS patients and 37 age-matched healthy volunteers were used as controls.
Results: MIG serum levels were elevated in multiple myeloma patients (median 161.3 pg/ml, range 9.37–1966.0) compared to MGUS patients (median 92.7 pg/ml, range 6.29–1303.1) and healthy controls (median 106.2 pg/ml, range 51.0–390.6). For analysis of myeloma cases, a cut-off level for MIG of 200pg/ml (=95th percentile of MIG in controls) was chosen to identify low and high MIG expressers. Using the cut-off as defined above, 63 patients with low MIG serum levels and 42 high MIG expressers were identified in a population of 105 myeloma patients. MIG serum levels in myeloma patients showed strong correlations with several markers of tumour load including low albumin and high β2-microglobulin. Interestingly, no correlation was found with C-reactive protein levels, indicating that MIG is not associated with an inflammatory response in myeloma. Median survival was significantly shorter in patients with high MIG serum levels compared to patients with low MIG expression (median not reached vs. 17.0 months, p&lt;0.001; see figure).
Conclusions: MIG serum levels correlate with markers of disease burden in myeloma and high MIG levels are associated with a poor outcome in this disease.
Overall Survival: low vs. high MIG serum levels Overall Survival: low vs. high MIG serum levels
Significance of Serum Free Light Chain Estimation with Detectable Serum Monoclonal Protein on Immunofixation Electrophoresis.
Authors: Mehta Jayesh , Stein Regina , Vickrey Eric , Resseguie William , Singhal Seema 
Published: April 2020

Abstract
The serum free light chain (SFLC) assay is useful in detecting monoclonal protein when there no detectable M protein on immunofixation electrophoresis (IFE). There are limited data on its value when IFE is positive. In a small series of 5 patients achieving CR, normalization of SFKLR was found to precede IFE negativity by a few weeks (Moesbauer et al. ASH 2005). Results on 231 serum samples from myeloma patients (most on therapy) where serum IFE showed IgA κ (n=33), IgA λ (n=13), IgG κ (n=153), or IgG λ (n=32), and where simultaneous SFLC and immunoglobulin (Ig) estimation had been performed were analyzed. Samples with &gt;1 monoclonal band or multiple oligoclonal bands were excluded. The serum free κ:λ ratio (SFKLR; normal 0.26–1.65) was abnormal in 113 (49%) and normal in 118 (51%). IgG and IgA levels were compared in the context of normal versus abnormal SFKLR within each of the 4 isotypes (IgA κ, IgAλ, IgG κ, IgG λ). The table below shows that involved Ig levels were higher with abnormal than with normal SFKLR. However, uninvolved Ig levels were higher with normal than with abnormal SFKLR suggesting that normalization of SFKLR may mark a response to therapy - improved uninvolved Ig levels being evidence of response.
Monoclonal protein Immunoglobulin Abnormal SFKLR Normal SFKLR P IgA kappa IgA 1640 (190–4000) 515 (102–2230) 0.048 IgA kappa IgG 419 (118–1120) 404 (197–1740) 0.39 IgA lambda IgA 408 (159–696) 704 (180–779) 0.17 IgA lambda IgG 619 (495–1510) 1530 (533–1700) 0.025 IgG kappa IgA 42 (7–225) 94 (7–642) 0.0009 IgG kappa IgG 1490 (585–5560) 1260 (327–2690) 0.004 IgG lambda IgA 32 (7–121) 96 (19–562) 0.047 IgG lambda IgG 2060 (555–12300) 1050 (432–2830) 0.018
However, does normalization of SFKLR universally herald IFE negativity? This is an important unanswered question because SFKLR is normal in a high proportion of samples which still show monoclonal protein on IFE. The figures below show scatter plots of IgG and IgA for each of the 4 isotypes for normal vs abnormal SFKLR. Within each plot, there is no obvious pattern distinguishing normal (x) from abnormal (o) SFKLR. However, there are a number of normal SFKLR points with high involved and low uninvolved Ig levels where a normal SFKLR is difficult to explain.
Figure Figure Figure Figure
We conclude that the SFLC assay often reveals normal SFKLR even when there is a detectable monoclonal protein in the serum. Whether this always predicts eventual paraprotein clearance and achievement of IFE negativity in patients on therapy is unknown, and needs to be studied prospectively.
Characterization of Bortezomib Resistant Human Multiple Myeloma Cell Line (HMCL): A Clinically Relevant Model for Novel Drug Development in Multiple Myeloma (MM).
Authors: Chanan-Khan Asher Alban , Ersing Noreen , Pera Paula , Bernaki Ralph , Coignet Lionel , Goodrich David W. , Haung Raya , Porter Carl W. 
Published: April 2020

Abstract
Introduction: Bortezomib (B) is the first in class proteasome inhibitor that received FDA approval for the treatment of MM patients with relapsed or refractory disease. Despite impressive clinical activity all patients develop resistance to B. The underlying mechanism of resistance to B remains undetermined. Mechanisms to overcome B resistance or development of new therapeutic agent(s) with activity in context of B resistance are limited due to unavailability of established preclinical B resistant MM models. To overcome this challenge we developed a B resistant HMCL.
Methods: we chronically exposed the OPM-2 HMCL to sub-lethal doses of B. Surviving cells were harvested, re-cultured and dose of B incrementally increased over prolong period of time. The resulting resistant cells were further characterized using array Comparative Genomic Hybridization (aCGH), spectral karyotyping (SKY) and gene expression profiling.
Results: After several passages we were able to induce B resistance in this HMCL. Final clone (OPM-2BR) demonstrate resistance to 2 × IC50 dose of B. While SKY and aCGH analysis demonstrated significant differences when compared with parent OPM-2WT (wild type) HMCL, gene expression profiling of the resistant and parental lines demonstrated significant upregulation in the expression of a number of ATP-binding cassette transporters. For example, the breast cancer resistance protein (ABCG2) is upregulated 4-fold in the resistant cell line compared to the parental cell line. This data suggests that drug efflux mediated by drug transporters represents one potential mechanism of resistance to B.
Conclusion: Chronic in vitro B exposure results in induced resistance in HMCL-OPM-2BR. Resistant cell line demonstrates cytogenetic variability when compared to the parent cell line. Induction of resistance is stable and provides an important preclinical tool to investigate mechanism(s) of B resistance as well as new drug development for B resistant myeloma patients. Detailed analysis of this cell lines including therapeutic interventions investigating resistance reversal will be presented at the meeting.