bluuebunny/crossref_metadata_2025 huggingface dataset:
The Molecular Basis for BCL-2 Oncogene Addiction in CLL.
Authors: Letai Anthony , Certo Michael , Brown Jennifer R. , Moore Victoria
Published: April 2020

CLL cells consistently express BCL-2 at a high level. Using a compound demonstrated to antagonize BCL-2 function, ABT-737, we show that CLL cells in short-term primary culture are uniformly sensitive to single-agent BCL-2 antagonism, with EC50’s in the 10 nM range. To understand the mechanism of CLL sensitivity to this compound, we studied mitochondria from these primary CLL cells. We employed a panel of peptides derived from the BH3 domain of pro-death BH3-only proteins, certain of which selectively inhibit BCL-2 function in vitro. We demonstrate that those with activity against BCL-2 in vitro induce cytochrome c release, a hallmark of the mitochondrial dysfunction during apoptosis. A non-malignant cellular model of BCL-2 dependence revealed that binding of the pro-apoptotic protein BIM to BCL-2 correlated with sensitivity to BCL-2 inhibitors. We likewise discovered that BCL-2 binds and sequesters BIM in CLL cells. In contrast to the BCL-2 dependent CLL cells, we examined myeloma cell lines with defined MCL-1 dependence and relative BCL-2 independence. These cell lines were 100 fold less sensitive to ABT-737. Furthermore, in mitochondrial assays only BH3 peptides which interact with MCL-1 induce cytochrome c release. These experiments reveal that cellular requirement for an anti-apoptotic protein function can be deduced from a functional mitochondrial requirement determined using our panel of BH3 peptides. In CLL, and perhaps other malignancies, it appears that BCL-2 maintains survival by sequestering pro-apoptotic proteins like BIM. Inhibition of this sequestration provokes cell death ex vivo, even with a single agent BCL-2 inhibitor. Reliance on BCL-2 for survival may be an Achilles heel for CLL and other cancers.
Hypercalcemia in B-Cell Chronic Lymphocytic Leukemia: Report of a Case and Review of the Literature.
Authors: Radhakrishnan Neetu , Hoffman Mark A.
Published: April 2020

A 53 year old man with Rai stage IV CLL was being treated with R-HyperCVAD when he presented between cycles with fatigue, lethargy and pancytopenia. Clinical examination revealed diffuse adenopathy and splenomegaly. Serum calcium was 13.6 mg/dl, phosphorus level was 2.7 mg/dl, and alkaline phosphatase was 54 U/L. PTH was 4 Units/L (10–65), PTHrP < 0.2 pmole/L (0–1.9), 1–25 (OH) Vitamin D < 10pgm/ml (22–67). Quantitative immunglobulins: IgA 10 mg/dl, IgG 252 mg/dl, IgM 50 mg/dl. Immunofixation revealed a faint IgG lambda paraprotein. There were no lytic lesions on skeletal survey. Bone marrow biopsy revealed focal large cell transformation (Richter’s syndrome). Cytogenetics revealed 3 metaphases with complex cytogenetic abnormalities, indicating clonal evolution. The hypercalcemia resolved with appropriate therapy, but despite subsequent treatment with CAMPATH, he died 2 weeks after diagnosis.

A review of reported patients with CLL and hypercalcemia in the literature was performed from 1980 onwards using MEDLINE and PubMed; only those cases in which clinical aspects, biochemistry, PTH levels, imaging studies and concurrent pathology (if obtained) were documented, are summarized in this analysis (n=13).

Rai stage: I n=1, II n=4, III n=3, IV n=5. Immunoreactive PTH levels were low or normal in 100% of patients. In 5 cases in which it was measured, 1–25 (OH) Vitamin D levels were not elevated. PTHrP was normal in 2 cases and elevated in 1. In nine patients, multiple lytic bone lesions were present on skeletal radiology. Two patients had osteopenia without lytic lesions. Two had no lytic lesions.

Six of ten patients had evidence of transfomation on lymph node and/or bone marrow biopsy performed at the time of evaluation for hypercalcemia.

Prognosis was poor (range 0.5–12 months) with only one patient surviving post allo-transplant.

In conclusion, hypercalcemia in CLL is rare. Osteolytic lesions are present in the majority of cases. PTH levels are low, and thus this hormone is not mediating the hypercalcemia. The evidence is also against a role of elevated Vitamin D. Histological transformation is seen in half of the cases. Survival is poor after diagnosis of hypercalcemia. The mechanisms(s) of the osteolysis and hypercalcemia remain to be defined.
Influence of Genetic Single Nucleotide Polymorphisms in CYP3A4, CYP3A5, GSTP1, GSTM1, GSTT1 and MDR1 Genes on Survival and Treatment Related Toxicity in Patients with Multiple Myeloma Treated in the HOVON 24 Trial.
Authors: Sonneveld Pieter , Schilthuizen Celine , Lokhorst Henk , Vellenga Edo , Raymakers Reinier , de Knegt Yvonne , Kamst Eric , van der Holt Bronno
Published: April 2020

Cytochromes P450 (CYP 450) and gluthatione-S-transferases (GSTs) are drug metabolizing enzymes involved in the detoxification of numerous chemotherapeutic agents.The Multidrug Resistance gene 1 (MDR1, ABCB1) is a transmembrane drug transporter present in tumor cells and mucosal epithelium. Genetic single nucleotide polymorphisms (SNP’s) in these genes are frequent and may alter the metabolism of certain anti-cancer drugs. The CYP3A4*1B (A-290G) polymorphism has not yet been proven to alter enzymatic activity. Polymorphisms in the CYP3A5*3 gene (A6986G) and the GSTP1 gene (Ile105Val) lead to decreased enzymatic activity. Homozygous deletions in the GSTM1 and GSTT1 genes lead to complete absence of the enzyme. Genetic polymorphism of MDR1 (C3435T) are frequent and associated with enhanced efflux function. We hypothesized that SNP haplotypes of these drug metabolizing enzymes may determine the interindividual differences of patients to treatment response and toxicity. We have investigated the presence of SNP’s of 211/345 previously untreated patients with multiple myeloma from whom diagnostic material was available and who were treated according to the HOVON-24 protocol, a clinical trial comparing myeloablative therapy with stem cell rescue added to intensified chemotherapy to intensified chemotherapy alone. Peripheral blood or bone marrow samples were analyzed by PCR or multiplex PCR. Patients with different haplotypes of CYP3A4, GSTP1, GSTM1, GSTT1 genes did not have significant differences of overall survival (OS), time to progression (TTP), progression free survival (PFS), event free survival (EFS), partial remission (PR), complete remission (CR) as determined by multivariate analysis with all clinical variables. Patients with mutant GSTP1(Ile/Val) had significantly more toxicity following high-dose melphalan (p=0.003). Patients with MDR1 C3435T mutation or absence of GSTP1 Ile105Val mutations had a lower probability to achieve at least a partial response(p=0.01). Patients genotyped homozygously for the mutant allele CYP3A5 gene had increased toxicity (p=0.040), improved OS (p=0.01) and PFS (p=0.04) and a decrease in TTP (p=0.03).

This study is part of an ongoing analysis and the results will be validated in the next HOVON trial. We conclude that in patients with multiple myeloma who are treated with intensive chemotherapy and stem cell transplantation, analysis of genetic polymorphisms of metabolic enzymes may improve our understanding of treatment associated toxicity and treatment outcome.
PPARβ-Mediated Suppression of the Growth and Survival in Human Myeloma Cells Conteracting NF-kB Activity.
Authors: Otsuyama Ken-ichiro , Ma Zi , Liu Shangqin , Abroun Saeid , Asaoku Hideki , Kawano Michio M.
Published: April 2020

PPAR(peroxisomal proliferators-activated receptor)β is considered to be involved in the lipid metabolism and regulating the inflammatory response and in human myeloma cells PPARβis predominantly expressed among these PPARs. However, it remains to be clarified what is the functional role of PPARβ in myeloma cells. In order to identify what are the ligands for PPARβ, we constructed the PPREβ-luciferase reporter gene and performed the reporter assay in Hela cells. Since we have already identified several reagents (DHEA (dehydroepiandrosterone), DHEA-S, baicalein, baicalin, dexamethasone) that suppressed the growth or survival of human myeloma cells (Cancer Res65:2269,2005; Blood105:3312,2005), we analyzed whether these reagents augmented the expression of reporter gene. DHEA-S, baicalein and dexamethasone showed the possibility of their PPARβ binding. Furthermore, these reagents could induce or upregulate the expression of PPREβ-target genes such as ILK and COX2 in myeloma cell lines. We’ve already confirmed that DHEA-S inhibited the proliferation and survival of primary myeloma cells as well as myeloma cell lines, and downregulated the activity of NF-kB, also baicalein showed the growth suppression through the downregulation of NF-kB. Therefore, it is possible that PPARβ-binding reagents such as DHEA-S and baicalein could counteract NF-kB activity to suppress the growth or survival in myeloma cells, while the exact mechanism is under investigation.
Characterization of Oncogene Dysregulation in Multiple Myeloma by Combined FISH and RT-PCR Analyses.
Authors: Qian Shenxian
Published: April 2020

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus and various partner loci frequently are associated with multiple myeloma (MM). We investigated the expression profiles of the FGFR3/MMSET, CCND1, CCND3, MAF genes, which are involved in t(4;14)(p16.3;q32), t(11;14)(q13;q32), t(6;14)(p21;q32), and t(14;16)(q32;q23), respectively. The analysis was performed by RT-PCR from purified plasma cell populations from 57 MMs and we compared the results with the presence of translocations as assessed by dual-color FISH. A t(4;14) was found in 11MMs, t(11;14) in 9 MMs, t(6;14) in 5 MM, and t(14;16) in 4 MMs. In all cases, the translocations were associated with the spiked expression of target genes. Furthermore, gene expression profiling enabled the identification of putative translocations causing dysregulation of CCND1 (1 MM) and MAF (1 MM) without any apparent involvement of immunoglobulin loci. Notably, all of the translocations were mutually exclusive. IGH-MMSET hybrid transcripts were found in 10 of the 57 (17.7%) MM samples. There was complete concordance between the findings of RT-PCR and FISH analyses of the MM samples, with 19.2% (11/57) t(4; 14) detected by FISH. Samples were separated further into three major groups based on the size of the RT-PCR product. The 1064bp, 438bp, and 275bp of IGH-MMSET were found in 7, 2, and in 1 sample, respectively. We then screened all 57 MM samples for the expression of FGFR3 using RT-PCR, with primers amplifying the 283bp fragments. Specific transcripts were detected in 11 (19.2%) samples that validate the t(4; 14) from cytogenetic studies. In the remaining 46 MM patients without t(4; 14), and 10 normal bone marrow controls, the FGFR3 amplified transcript was barely detectable. Only one patient sample without t(4; 14) revealed detectable levels of FGFR3 expression. Thus, RT-PCR assay for FGFR3 expression can detect all cases with evident or cryptic t(4; 14) translocation (P< 0.01). Using the primers corresponding to 7–10 exon in 11 cases of MM patients with overexpression of FGFR3, we directly sequenced the FGFR3 cDNA fragments amplified by PCR. Polymorphism (GGC>GGT) was detected in nine of the 11 patients. This polymorphism was tightly associated with higher expression of FGFR3. No FGFR3 mutations were found in the remaining 2 MM patients with overexpression of FGFR3. Our data indicate that RT-PCR is a sensitive and reliable method for the detection of FGFR3 and IGH-MMSET. Translocation t(4; 14) in MM detected by FISH can be validated by RT-PCR method. We examined our result by the Chi-Square test and revealed 90% sensitivity and 100% specificity. The Youden Index remains 0.9. This rapid and reliable detection of FGFR3 and IGH-MMSET overexpression may have practical clinical utility in the analysis and monitoring of the disease in MM patients with t(4; 14). Our data suggest the importance of using combined molecular cytogenetic and gene expression approaches to detect genetic aberrations in MM.
Circulating Endothelial Cells (CEC) and Angiogenesis-Related Factors in Multiple Myeloma (MM).
Authors: Jobe Dean A. , Asp Krista E. , Callister Steven M. , Cole Craig E. , Meyer Lori A. , Mathiason Michelle A. , Bottner Wayne A. , Farnen John P. , Go Ronald S.
Published: April 2020

Background: The profile of CEC can potentially be a biomarker of angiogenesis. As angiogenesis is increased in patients with MM, we studied the CEC and their relationships to angiogenesis-related factors in the blood and disease activity.

Methods: We prospectively quantitated (CD45-/CD146+/CD31+) and characterized (apoptotic [annexin stain], activated [CD106]) the CEC in the whole blood of 20 patients with MM (7 untreated [4 newly diagnosed, 3 smoldering], 9 receiving therapy, and 4 treated previously) using four-color flow cytometry and compared the findings to those from 20 normal volunteers. In addition, we determined the associations between CEC and disease activity and the plasma levels of several angiogenesis-related factors (vascular endothelial growth factor [VEGF], fibroblast growth factor-2 [FGF-2], hepatocyte growth factor [HGF], and thrombospondin-1 [TSP-1]) pertinent to MM.

Results: The median level of CEC was higher (P<0.001) in MM patients (124.8 cells/mL [range, 15–572] versus 21.9 cells/mL [range, 4–66]). However, only 6 (30%) MM patients had levels that were greater than 3 standard deviations from the mean of normal. In general, more apoptotic CEC were present in MM patients (41.5% [range, 0–100] vs 25.9% [range, 0–57.1]; P=0.028), while activated CEC were similar in both groups. There were no significant associations between CEC levels and disease stage, disease status (progressive versus stable/responding), or serum monoclonal protein levels. MM patients had higher levels of HGF (1159 pg/mL [range, 628–5401] vs 524.5 pg/mL [range, 260–938]; P<0.001) but lower levels of TSP-1 (43.9 mcg/mL [range, 3.9–134.1] vs 66.5 mcg/mL [range, 6.4–214.4]; P=0.021), and FGF-2 (20 pg/mL [range, 0–52] vs 34.5 pg/mL [range, 8–82]; P=0.018) in the plasma. In the subgroup of MM patients with elevated CEC, VEGF level was significantly higher than control (211 pg/mL [range, 13–1156] vs 61 pg/mL [0–401]; P=0.012).

Conclusions: Increased levels of CEC can be detected in some patients with MM. In these patients, measurement of CEC may be useful as a biomarker of angiogenesis. As we did not find a significant association between CEC and disease activity, additional studies are necessary to determine the clinical significance of increased CEC in MM.
CD 138 Immunostaining of Bone Marrow Trephine Specimens Is the Most Sensitive Method for Quantifying Marrow Involvement in Patients with Plasma Cell Dyscrasias.
Authors: Ng Ashley P. , Wei Andrew , Bhurani Dinesh , Chappell Peter , Feleppa Frank , Juneja Surender
Published: April 2020

AIM In this study plasma cell quantitation comparing four modalities comprising morphology of the aspirate, flow cytometry, HEtrephine section examination and bone marrow immunohistology was undertaken to determine the most sensitive technique.

METHODS 70 patients with a plasma-cell dyscrasia were retrospectively analysed. Plasma-cell quantitation was performed on Romanowsky-stained aspirate slides by a 200-cell differential. Flow cytometric quantitation of plasma-cell burden was performed by identifying CD38/138 co-expressing cells. Trephine specimens were analysed after HEand CD138 staining. Statistical analysis was performed using a two-tailed paired t-test.

RESULTS See Table 1.

DISCUSSION Significant discrepancy was noted between the four methods for determining plasma-cell infiltration. CD138 immunohistochemical staining of paraffin embedded trephine specimens was the most sensitive modality. Flow-cytometric analysis underestimated the degree of marrow infiltration. Morphology of the aspirate sample may also underestimate plasma-cell burden as sampling may be affected by patchy marrow infiltration as well hypoplastic, fibrotic marrows or clotted specimens. Quantification of plasma cells based on HEstained trephine specimens is less sensitive, and is dependent on the technical quality of the specimen and observer experience. Immunostaining has the advantages of improved plasma-cell identification compared to HEsections and avoids the problems inherent in the analysis of aspirate samples. In conclusion, CD138 immunostaining is the most sensitive method for quantifying plasma-cell burden in the bone marrow.

Two tailed Paired t-test analysis of Quantitation of Plasma Cell burden by various modalities Flow Cytometry Aspirate Hematoxylin & Eosin Immunostaining Mean % of plasma cells 5.6 18.9 23.6 29.7 Paired t-test (two tailed) Mean Difference compared to CD138 immunostaining −24.12 −11 −6.14 - 95% C.I. −30.5 to −17.8 −15.8 to −6.1 −8.34 to −3.9 - p-value p<0.0001 p<0.0001 p<0.0001 -
CD66 Expression on Malignant and Normal Plasma Cells: A Potential Target for Therapy.
Authors: Richardson Deborah , Hodges Elizabeth , Mani Adnan , Orchard Kim
Published: April 2020

CD66, a member of the carcinoembryonic antigen family, is known to be expressed on cells of myeloid and monocytic origin and has also been demonstrated on blasts from patients with B-lineage acute lymphoblastic leukaemia. An analysis of CD66 expression has been undertaken in bone marrow samples from patients with plasma cell disorders. Diagnostic bone marrow aspirate samples from 53 patients with multiple myeloma or monoclonal gammopathy were examined by multiparametric flow cytometry in order to demonstrate and quantitate plasma cells. Samples were analysed initially using a primary screening panel of antibodies including a combination of antiCD19 fluoroscein isothiocyanate (FITC)(Pharminogen), antiCD5 phycoerythrin (PE)(in-house), antiCD45 peridin chlorophyll protein (PerCP)(Becton Dickinson) and antiCD38 allophycocyanin (APC)(Phar). Samples shown to contain a CD45 negative, CD19 negative, CD38 positive population, consistent with the presence of plasma cells were then examined with a myeloma panel, including antiCD66. Samples from 41 patients were analysed using antiCD66 naked antibody (TheraPharm, GmBH) double-layered with sheep anti-mouse IgG F(ab’)2 fragment conjugated to FITC, antiCD138 PE, antiCD38 APC and antiCD45 PerCP. The correlation between CD38/CD138 and CD38/CD66 dual expression was 0.997. A further 13 samples were analysed using a commercial antiCD66 antibody conjugated to FITC (Dako). Again the correlation between CD38/CD138 and CD38/CD66 dual expression was 0.990. All patients apart from one, with morphologically detectable plasma cells, co-expressed CD66 with CD38. One patient with highly plasmablastic morphology did not express either CD38/138 or CD38/66 on plasma cells.The bone marrow of a normal individual was also examined and found to contain plasma cells which co-expressed CD19, CD138, CD38 and CD66. Plasma cells express CD66 in almost all patients with plasma cell disorders and expression has also been demonstrated on plasma cells in a normal individual. CD66 is therefore an attractive target for immunotherapy. A clinical trial using anti-CD66 targeted radiotherapy as part of the conditioning regimen for patients undergoing autologous or allogeneic stem cell transplantation, as therapy for multiple myeloma, is currently being conducted at our institution.
The Immunomodulatory Action of Thalidomide in Patients with Multiple Myeloma Involves a Clonal Expansion of Late-Differentiated Cytotoxic Effector Cells.
Authors: Brown Ross D. , Murray Allan , Sze Daniel M. , Spencer Andrew , Ho Phoebe Joy , Gibson John , Joshua Douglas E.
Published: April 2020

Although thalidomide has been reported to increase T cell activity and NK cell cytotoxicity in the blood of patients with multiple myeloma (MM) it is not known whether thalidomide stimulates tumour-specific T cells or causes a general lymphocytosis. We investigated the immunodulatory effects of thalidomide by studying peripheral blood samples from 64 patients in the Australian ALLG MM6 trial, a multicentre randomised phase III study of low-dose thalidomide, prednisolone and Zometa versus prednisolone and Zometa used as post-autologous stem cell transplant (ASCT) maintenance therapy in patients with MM. We have previously demonstrated that TCR Vβ clonal expansions are present in approximately 50% of patients with MM and that their presence correlates with a good prognosis. Sequencing has confirmed that these cells are a clonal expansion of CD3+CD8+CD57+CD28−CD27− late-differentiated cytotoxic effector cells. Flow cytometric analysis of TCR Vβ expansions (24 families) was performed on blood samples from 64 patients enrolled in the MM6 trial both prior to and 12 months post transplant. Prior to transplant, 59% of the 64 patients had TCR Vβ expansions. After thalidomide, T cell expansions were found in a further 8/34 patients compared with 3/30 in the control, no thalidomide group. In the thalidomide group, 70% of the patients had an expansion of more than one T cell clone compared with 32% in the control group (chi2=28.8; p<0.01). In the thalidomide treated patients there was a total of 32 new clones compared with 22 in the patients who did not receive thalidomide. There was a trend for an increase in NK and NKT cells following thalidomide. Further patients will be studied and the prognostic significance will be evaluated but these preliminary studies provide further evidence that thalidomide has an immunomodulatory action in patients with MM and that this is due, at least in part, to a clonal expansion of late-differentiated cytotoxic effector cells.
BP1 Expression in a Myeloma Cell Line: Modulation by Arsenic Trioxide and Ascorbic Acid.
Authors: Do Khanh , Berg Patricia , Mincheff Milcho , Ascensao Joao L.
Published: April 2020

BP1 belongs to a family of homeobox genes which are not expressed in normal bone marrow cells but are seen in multiple leukemic cell lineages including erythroid, myeloid, and T- and B-cell lineages, and whose modulation is important in differentiation and oncogenic transformation (Shen et al. EMBO J 1992). Multiple Myeloma (MM) is a clonal B-cell neoplasm marked by proliferation of plasma cells within bone marrow. BP1 expression has not previously been demonstrated in MM cell lineages. Recent unpublished data demonstrated elevated BP1 expression in the bone marrow of Acute Promyelocytic Leukemia (APL) patients and its down regulation with the differentiating agent ATRA. With recent data demonstrating arsenic trioxide (As2O3) induced remission in chemorefractory APL and MM (Grad et al. Blood 2001), we hypothesized BP1 may share a role in this response conversion.

In the current study, we treated ARH77 cells - a multiple myeloma cell line, with 0.25 – 3 uM As2O3 and adjuvant ascorbic acid (AA) for 24–96hrs; AA had been previously documented to potentiate As2O3-mediated cell death in multiple myeloma cells (Grad et al. Blood 2001). Viability, cell numbers and mRNA expression were determined at each time point. The % of viable cells at 72 and 96 hrs decreased to less than 10% of the control in cultures flasks containing 2 and 3uM; 0.25 uM had minimal effect in this assay. We then used 0.5 and 1uM, which caused growth arrest compared to controls at each interval. 1 uM caused a decrease of 12% viability at 72 hrs. A significant reduction in the expression of BP1 was seen in these cultures when balanced with beta-actin levels. Myeloma cells are marked by a myriad of antiapoptotic signaling mechanisms, which account for their acquired resistance to current chemotherapy. A better understanding of signaling pathways and genetic events may provide potential therapeutic targets and aid in correlation between genetic abnormalities and clinical outcomes.
Type I MAGE Proteins: Novel Markers of Proliferation in Multiple Myeloma Progenitor Cells.
Authors: Cho Hearn J. , Caballero Otavia , Jungbluth Achim A. , DiLiberto Maurizio , Niesvizky Ruben , Old Lloyd J. , Chen-Kiang Selina
Published: April 2020

CT7 (MAGE-C1) and MAGE-A3, two members of the type I MAGE family of Cancer-Testis antigens, are commonly expressed at both the mRNA and protein levels in primary tumor specimens from multiple myeloma patients. In previous analyses, tumors with higher percentages of type I MAGE-expressing cells had a positive correlation with abnormally elevated plasma cell proliferation. These data support the hypothesis that type I MAGE proteins are molecular markers of proliferating myeloma progenitor cells (the so-called “myeloma stem cell”) and may play a role in the pathobiology of this disease. To test this hypothesis, we examined the expression of type I MAGE in proliferating myeloma cells by flow cytometry. Human multiple myeloma cell lines U266, RPMI-8226, and KMS-11 were co-cultured for 12 or 24 hours with the nucleoside analog bromodeoxyuridine (BrdU), then fixed, permeabilized, and stained with CT7-33, a monoclonal antibody (mAb) to CT7, or M3H67 (to MAGE-A3), followed by a phycoerythrin (PE)-conjugated secondary mAb. The cells were then treated with DNAse and stained with a fluoroisothiocyanate (FITC)-conjugated mAb against BrdU. Proliferating cells that incorporated BrdU into their DNA exhibited high FITC fluorescence. For mAb M3H67, dual color analysis of this population showed that greater than 99% demonstrated a significant shift in PE fluorescence in all three of these cell lines as measured by Mean Fluorescence Index (MFI= geometric mean fluorescence [specific primary antibody]/mean fluorescence [no primary antibody], table 1). For CT7-33 mAb, greater than 85% demonstrated a shift in two of three lines (U266 and KMS-11), but not in RPMI-8226. For all three of these cell lines, dual color analysis of the BrdU-low population demonstrated less than 65% staining with either type I MAGE mAb. Interestingly, RT-PCR with CT7-specific primers of total RNA from RPMI-8226 revealed a product of lower molecular weight than expected, suggesting that a gene deletion occurred in this cell line possibly resulting in a stop codon, decreased translation, or decreased protein stability. This PCR product is being sequenced to determine the nature of the deletion. These results demonstrate that type I MAGE proteins are expressed in proliferating myeloma cells and are molecular markers of this population. These data suggest that novel therapeutics such as vaccines that target type I MAGE may preferentially eliminate the cycling myeloma cells, resulting in long-term cures.

Table 1. Type I MAGE expression in proliferating (BrdU+) myeloma cells Cell line MFI CT7-33 MFI M3H67 U266 65.8 62.5 KMS-11 9.9 48.4 RPMI-8226 3.1 12.3
Lovastatin and Thalidomide Have an Synergic Effect on the Rate of Multiple Myeloma Cell Apoptosis in Short Term Cell Cultures.
Authors: Podhorecka Monika , Klimek Piotr , Grzasko Norbert , Dmoszynska Anna
Published: April 2020

Multiple myeloma is characterized by an accumulation of plasma cells in the bone marrow. Despite many therapeutic regimens introduced recently, the prognosis for patients suffering from treatment-resistant or relapsing multiple myeloma is still very poor. Thus, there is an urgent medical need for novel innovative drugs. Thalidomide is successfully used in myeloma patients being reported to induce apoptosis or G1 growth arrest of plasma cells, regulate microvessel density and cytokine secretion. Statins, largely used for the treatment of hypercholesterolemia, seem to be promising drug in multiple myeloma also. High dose of lovastatin has been shown to have antiproliferative effect by inhibition of malignant cell proliferation and inducing programmed cell death. The aim of this study was the assessment of multiple myeloma cells apoptosis induced by mixture of lovastatin and thalidomide in short-term cell cultures. We analyzed plasmocytes of bone marrow samples obtained from 10 patients with treatment-resistant or relapsing multiple myeloma. To assess apoptosis we used Annexin V and propidium iodide binding. We also examined the regulation of BCL-2 and BAX protein expression in the population of CD138+ plasmocytes. The cells were analyzed with use of flow cytometry technique. The experiments were done before and after 72 hours of cell culture. We observed an increase of apoptotic cell number in all cultures supplemented with analyzed drugs in comparison to 0 h culture and to 72 h control. The percentage of Annexin V positive cells in culture with lovastatin and thalidomide mixture was significantly higher in comparison to culture with lovastatin or thalidomide alone (the mean percentages were 33.40 versus 27.04 and 26.49, respectively, p<0.05). The BCL-2/BAX ratio was lower in cell cultures supplemented with mixture of lovastatin and thalidomide (mean ratio 0.95) in comparison to cultures supplemented with lovastatin or thalidomide alone (mean ratio 1.25 and 1.17, p=0.06 and 0.05, respectively) indicating the tendency to apoptosis induction in analyzed cells. Basing on these results we can conclude that lovastatin and thalidomide may have an synergic effect on the rate of multiple myeloma cell apoptosis and may act together on BCL-2 and BAX regulation. Thus, further research should establish both the precise mechanism of this synergic action of statins and thalidomide and the new therapeutic option for myeloma patients.
Combination Therapy of Bortezomib with Low-Dose Bendamustine in Elderly Patients with Advanced Multiple Myeloma.
Authors: Hrusovsky Irena , Heidtmann Hans-Heinrich
Published: April 2020

Bortezomib has been approved for recurrent or therapy-resistent multiple myeloma in 2003. There is growing evidence that combination therapy of bortezomib with other agents considerably improves results (Richardson P, ASH 2004). Bendamustine is an alkylating agent widely used in Germany for treatment of multiple myeloma and indolent lymphoma. There is no cross-resistance to other alkylating agents. Weekly low dose application is well tolerated and highly effective in elderly patients with pretreated indolent lymphoma (Bremer K, ASCO 2003, Abstract 2410).

Treatment modalities: We treated 17 pts (8 female, 9 male, median age 70 yrs (range 51–82) with relapsed multiple myeloma. Previous therapies ranged from 2 to 7 (median 4), and included 15 x thalidomide, 2 x tandem high dose melphalan, 2 x bortezomib/dexamethasone.

Therapy regimen: bortezomib 1 – 1.3 mg/m2 d 1,4,8,11; bendamustine 60 mg/m2 d 1+8, dexamethasone 3x8 mg orally d 1–3 and d 8–10: ondansetrone 8 mg intravenously d 1,4,8,11. Cycles were repeated q3w until best response. The median number of cycles applied was 4 (range 3 – 6).

Results: Outcome was evaluated according to SWOG criteria.

ORR: 88%; CR (neg. immunofixation): 12% (n=2); PR 58% (n=10); MR 18% (n= 3); NC 12% (n=2). Median remission duration was 6+ months (range 3–12+ months). Until today, we observed 4 recurrences after 3 – 8 months.

Toxicity comprised mild fatigue and thrombopenia. Lowest platelet count was 55/pL without bleeding, spontaneously reversible within 1 week. Three cases of neuropathia occurred, with controllable symptoms, in patients pretreated with thalidomide or bortezomib mono.

Conclusion: Combination therapy of bortezomib with low dose bendamustine is well tolerated, induces a high rate of responses in spite of unfavourable patient characteristics, leads to early responses and can be limited to 3 months (i.e., 4 cycles), and can be done in an outpatient setting. Thus, bendamustine is a very promising agent to enhance bortezomib action, which warrants further study.
Trisenox (Arsenic Trioxide) in the Treatment for Multiple Myeloma after Bone Marrow Transplantation.
Authors: Gesundheit Benjamin , Shapira Michael Y. , Resnick Igor , Bitan Menachem , Ben-Yehuda Dina , Slavin Shimon , Or Reuven
Published: April 2020

Multiple myeloma (MM) remains an incurable disease, with median survival of 4–6 years despite various new therapeutic approaches including autologous and allogeneic bone marrow transplantation (BMT). New agents are studied and Arsenic Trioxide (ATO) seems to be a promising additional agent against progressive and refractory MM. Various mechanisms are described for the therapeutic effect of ATO: growth inhibition of myeloma cells; induction of apoptosis of drug resistant MM cell lines; inhibition of angiogenesis by blocking VEGF; interaction with BM microenvironment; immunologic mechanisms by a marked increase in lymphokine-activated Killer (LAK)-mediated killing and up-modulation of CD38 and CD54; inhibition of myeloma cells to stick to BM stromal cells by inhibiting IL-6. ATO is a form of arsenic, a naturally occurring element (As2O3), which was used for various therapeutic purposes for more than 2000 years. ATO was first approved by the FDA in 2000 for acute promyelocytic leukemia (APL), and is investigated for myelodysplastic syndrome (MDS) and other indications for ATO are currently under investigation.

We report our preliminary data from our clinical experience with ATO for 4 patients with MM after BMT, who had evidence of active and progressive disease. Other therapeutic approaches failed or were not relevant. ATO was given at low doses of 0.15 mg/kg/dose twice weekly and after respond at 0.3 mg/kg/dose once weekly. ATO was given intravenously in slow infusion over 2 hours together with intravenous lipids 500 ml (20%) to reduce ATO toxicity. ECG, markers for myeloma and possible adverse effects were monitored carefully. Oral ascorbic acid 500 mg twice daily was added to improve the effect of ATO. Our patients showed evidence of response to ATO both at the level of residual tumor mass and M-protein secretion. Furthermore, in one patient with severe GvHD we observed significant improvement of the GvHD manifestations concomittantly with Graft versus Myeloma effect.

Based on our data and the review of the literature regarding the use of ATO for MM, ATO might play in the future an established role in the treatment for MM. ATO might induce and augment immunomodulatory effects of Graft versus Myeloma. We discuss future research for the use of ATO in the treatment of MM.
A Mutation in Sec15l1 Disrupts the Transferrin Cycle and Causes Anemia in Hemoglobin Deficit (hbd) Mice.
Authors: Lim Jackie E. , Morgan Kelly , Wang Fudi , Fleming Mark D. , Andrews Nancy C.
Published: April 2020

Mouse mutants have been used to identify and characterize genes involved in iron homeostasis. The mouse mutant hemoglobin deficit (hbd), has inherited hypochromic, microcytic anemia and elevated zinc protoporphyrin in the setting of replete iron stores, consistent with a selective defect in erythroid iron assimilation. Circulating soluble transferrin receptor, a marker of iron-restricted erythropoiesis, is elevated out of proportion to the anemia. A mapping population of >3300 backcross animals allowed assignment of the hbd locus to a 0.5 Mb region on chromosome 19. Seven candidate genes were identified in mouse genome databases. Only Sec15l1, encoding one of two mouse homologs of yeast Sec15p, carried a mutation unique to hbd. The mutation deleted 4.5 kb of Sec15l1 genomic DNA including a 69 bp exon. Two distinct antibodies elicited against murine Sec15l1 demonstrated that there was little or no Sec15l1 protein detectable in tissues from hbd animals, suggesting that the mutant form was unstable in vivo. To confirm that mutation of Sec15l1 was responsible for the hbd phenotype, hbd bone marrow cells were transduced with a retrovirus expressing wild type Sec15l1 and transplanted into hbd mice. Expression of the wild type protein partially corrected the anemia. For further confirmation, mice carrying a different, targeted mutation in Sec15l1 were bred with hbd mice. Compound mutant mice carrying both the targeted and hbd alleles recapitulated the hbd phenotype. Sec15l1 functions as a component of the mammalian multi-protein exocyst complex. Previous studies in model organisms suggested that the exocyst is critical for a variety of cellular functions; loss of other exocyst proteins caused early lethality. We speculate that Sec15l2, a mammalian homolog of Sec15l1, can substitute in non-erythroid tissues. Expression analysis showed that little or no Sec15l2 was present in hbd spleen, which is massively infiltrated with erythroid precursors. The exocyst is an effector for Rab11, a GTPase involved in recycling of transferrin cycle endosomes. In S. cerevisiae, Sec15p interacts both with Sec10p, another exocyst component, and with a yeast homolog of Rab11. Preliminary studies suggest that the hbd form of mammalian Sec15l1, expressed in yeast, retains its ability to interact with both of its mammalian partner proteins - Rab11 and Sec10. We conclude that depletion of Sec15 in hbd mice alters transferrin receptor recycling and, possibly, enhances transferrin receptor shedding. As a consequence, erythroid iron assimilation is impaired. Sec15L1 should be considered a candidate gene in patients with inherited iron-resistant iron deficiency anemia.
Treatment with Arsenic Trioxide, Ascorbic Acid and Dexamethasone in Advanced Myeloma Patients: Preliminary Findings of a Multicenter, Phase II Study.
Authors: Wu K. , van Droogenbroeck J. , Beksac M. , de Knegt Y. , Sonneveld P.
Published: April 2020

Recent studies have shown that arsenic trioxide (Trisenox, ATO) can induce apoptosis in myeloma cells and overcomes anti-apoptotic effects of interleukin-6. Pre-clinical studies reported profound anti-myeloma effects when ATO, ascorbic acid (AA) and dexamethasone are combined. Single nucleotide polymorphism (SNP) in metabolic enzymes may influence susceptibility of a treatment and the prognosis of multiple myeloma (MM).

We performed a multicenter phase II trial of ATO in patients with advanced MM. Patients with progressive disease following more than one line chemotherapy were included. The treatment regimen consists of four 28-days cycles. In 1st cycle, during 1st week: a loading-dose with ATO 0,25mg/kg IV, AA 1000mg IV and Dex 40mg orally on day 1–5. During 2nd and 3rd week: a maintenance-dose of twice weekly ATO 0,25mg/kg IV, AA 1000mg IV and Dex 20mg orally. During the 4th week no treatment is given. In the 2nd, 3rd and 4th cycle: a maintenance-dose of twice weekly ATO 0,25mg/kg IV, AA 1000mg IV and Dex 20mg orally in 1st, 2nd and 3rd week was administered followed by the 4th week as a rest period. Patients with a clinical response after the 4th cycle received two additional treatment cycles.

Preliminary results: The ongoing study currently has enrolled 20 pts, median age 63 yrs (range 42–76), who had received a median of 4 lines of prior treatments (range 1–6). There were 2 early deaths from pneumonia and gastro-intestinal bleeding. Fifteen patients were evaluable for response; PR in 2 pts, MR in 3 pts, SD in 3 pts and 7 pts had PD. Six patients died from progressive disease. Grade 3 or 4 adverse events included fluid retention (3), thrombocytopenia (2), neutropenia (1), increased ALT (2), peripheral neuropathy (1), bacterial pneumonia (2). In addition to standard clinical observations we did an analysis for Genetic Single Nucleotide polymorphisms of relevant metabolic enzymes in order to assess the relevance for toxicity and effect of ATO. A polymerase chain reaction followed by restriction enzyme digestion for GSTP1, CYP3A4, CYP3A5, MDR-1, GSTT1 and GSTM1 was performed. Responding patients vs non-responding patients showed the following wild-type(ww) / mutant (mm) / heterozygous (wm/mm) haplotype: GSTP1 1 / 2 / 1 vs 2 / 3 / 3; CYP3A4 1 / 0 / 3 vs 1 / 0 / 7; CYP3A5 0 / 4 / 0 vs 0 / 8 / 0; MDR1 0 / 3 / 1 vs 0 / 1 / 7 and deletion / normal genotype: GSTT1 1 / 3 vs 2/ 6; GSTM1 1 / 3 vs 4 / 4.

Conclusion: These preliminary results suggest that the ATO-AA-Dex regimen shows efficacy, with limited toxicity in advanced MM patients. An extended genotype analysis will be performed to characterize the role of metabolic enzymes in the effect of this regimen.
Identification and Expansion of Myeloma-Specific Cytotoxic T Cells In Vitro.
Authors: Michalek Jaroslav , Ocadlikova Darina , Zahradova Lenka , Kovarova Lucie , Penka Miroslav , Hajek Roman
Published: April 2020

Multiple myeloma (MM) has been considered as low immunogenic incurable disease. The attempts has been made to invert the immune status to recognize myeloma cells by T cells or other cells of the immune system. Here we studied biology of myeloma-specific T cells in vitro.

Irradiated myeloma cell line ARH 77 has been used as tumor antigen to stimulate peripheral blood mononuclear cells (PBMC) of 8 healthy volunteers. Dendritic Cells loaded by irradiated autologous MM cells has been used to stimulate PBMC of 10 MM patients. Activated responder T cells have been immunomagnetically separated based on surface expression of interferon gamma (Miltenyi Biotech) and expanded in 5 cases by phytohemaglutinin and repeated high-doses of interleukin 2. Cytotoxicity against the original myeloma cells has been tested after the expansion using propidium iodide or 7-amino actinomycin D. Responder T cells were labeled by CFSE to distinguish them from myeloma cells. Third-party PBMC and interferon gamma negative fraction of T cells served as controls.

The percentage of interferon gamma positive cells in healthy donors has been enriched from 2.8±0.9 and 2.6±0.8 to 48.6±23.4 and 73.2±25.9 of CD3+CD4+ and CD3+CD8+ T cells, respectively, by immunomagnetic separation. Interferon gamma positive T cells have been further expanded in vitro from 0.54x106 ± 0.05x106 to 214.00x106 ± 103.46x106 within 4 weeks. A specific cytotoxicity has been tested after expansion. The killing of myeloma cells by expanded IFN g+ T cells reached 68.1±14.2%, while interferon gamma negative fraction killed only 0.8±0.3% of myeloma cells. As another control, killing of third-party PBMC by expanded interferon gamma positive T cells was 6.9±2.5%. Similar observations were made in MM patients and will be presented at the meeting.

These data demonstrate a specific cytotoxic effect of expanded interferon gamma positive T cells against myeloma cell line ARH 77 and open the possibility for clinical use of tumor-specific T cells in MM.
Identification and Characterization of an Erythrocyte Endosomal Ferrireductase Critical for Transferrin Dependent Iron Uptake.
Authors: Ohgami Robert S. , Campagna Dean R. , Antiochos Brendan , Greer Eric , Fleming Mark D.
Published: April 2020

The transferrin cycle is a critical pathway for iron uptake by developing red blood cells. Defects in the cycle typically result in a hypochromic microcytic anemia due to ineffective hemoglobin synthesis. Recently we described a new microcytic mouse mutant, nm1054, with an autosomal recessive defect in transferrin mediated iron uptake. We now have identified the gene responsible for the defect through positional cloning and complementation experiments. The gene, mammalian ferrireductase 1 (Mfre1) encodes a predicted six pass transmembrane protein with homology to oxidoreductases of archaea and bacteria as well as homology to the yeast family of FRE proteins. Mfre1 is expressed highly in hematopoietic tissues and co-localizes with the transferrin cycle endosome of cells. Over-expression of Mfre1 in cultured cells stimulates reduction of iron while reticulocytes deficient in Mfre1 are defective in conversion of ferric iron to a ferrous form. Here we comprehensively describe the role of Mfre1 in iron metabolism and speculate on the role of several other novel homologues in iron homeostasis.
Osteomyelitis of the Jaw in Multiple Myeloma Patients as a Complication of the Treatment with Biphosphonates.
Authors: Nemets Anatoly , Bumental Rima , Ruchlemer Rosa , Marilus Rafael , Bennet Michael , Sofer Enat , Gordin Josef , Lugassy Gilles
Published: April 2020

Biphosphonates (zoledronate, pamidronate and clodronate) are frequently used for bone disease prevention and treatment in patients with multiple myeloma. Monthly biphosphonate therapy improves quality of life and in some reports survival. However, biphosponates are associated with adverse effects such as renal impairment and osteonecrosis of the jaws. We have recently described 3 myeloma patients who developed oseomyelitis of the jaw during chronic biphosphonate therapy. In a national survey we found 11 patients from 6 centers in Israel with osteomyelitis of the jaws probably due to prolonged therapy with intravenous biphosphonates. Nine were female. Median age at the time of diagnosis of myeloma was 67 years (range 59–86). nine patients suffered from IgG myeloma, and 2 had light chain disease. In five patients MGUS was known during 4 to 9 years before development of overt myeloma. All patients had received chemotherapy, and 2 underwent ABMT. Median time from diagnosis of myeloma to diagnosis of osteomyelitis of the jaw was 30 months (range 15–48). In six cases osteomyelitis was provoked by dental procedure. In all cases the diagnosis of osteomyelitis was confirmed by bone biopsy and in 7 colonies of Actinomyces Israeli were demonstrated. In one patient Pseudomonas Aeruginosa was also found. All patients had been treated with biphosphonates before diagnosis of osteomyelitis (13 to 48 months): eight with pamidronate, and 3 with zoledronate. The mean total dose of pamidronate was 3190 mg (range 1690–4680), and zoledronate- 72mg (range 16–96). Therapy for osteomyelitis included prolonged antibacterial treatment, surgical debridment, and hyperbaric oxygenation. Acute, and chronic, osteomyelitis of the jaws secondary to biphosphonate therapy, is frequently encountered among myeloma patients and should be reported separately from another complication of biphosphonates, osteonecrosis of the jaws.
CD56 Expression Is the Potent Prognostic Marker of the Thalidomide Efficacy in the Patients with Multiple Myeloma.
Authors: Mori Akio , Tsutsumi Yutaka , Hashino Satoshi , Kanamori Hiroe , Ibata Makoto , Mashiko Shinobu , Masauzi Nobuo , Asaka Masahiro , Imamura Masahiro
Published: April 2020

Thalidomide (Thal) alone or in combination with steroids achieves responses even in the setting of refractory multiple myeloma (MM), however, responses are still limited. The precise mechanism of Thal action is unknown, further, no distinct marker, which could prognosticate the efficacy of Thal, is known. Therefore, we evaluated the correlation between the efficacy of Thal and the potent prognostic factors in patients with refractory MM. Ten patients with refractory MM received Thal at doses of 50 or 100 mg per day and steroids, either dexamethasone (Dex) or prednisolone (PSL). Dex was administrated 20 mg per day, 4 days every 28 days, and PSL was administrated 10 mg per day. The median age was 71.5 years (range, 62–79 years) and 20 % were man, and all patients were diagnosed as clinical stage IIIA based on the Durie and Salmon classification. The therapeutic response was assessed according to the modified criteria of Southwest Oncology Group (SWOG). Among 10 patients, 7 patients were the responders; 2 had complete remission, 3 had partial remission, and 2 had minimal remission. There were no differences in the pretreatment characteristics of responders and nonresponders (age, sex, type and concentration of serum and/or urine monoclonal component, international prognostic index, presence of bone lesion, and chromosomal abnormalities). However, flow cytometric evaluation of the myeloma cells revealed that CD56, which is one of the adhesion molecules N-CAM, expressed more than 45 % in all responders, while those expressed less than 5 % in all nonresponders (84 ± 19 (±SD) % v/s 4 ± 2 %, P=0.017). Furthermore, CD56 expression of the myeloma cells was reduced from 84% to 70 ± 32 % after Thal therapy in all evaluated responders (P =0.048). These results suggest that CD56 expression of the myeloma cells could be the potent prognostic marker of the Thal efficacy. Moreover, it was reported that Thal reduced the expression of cell adhesion molecules, such as LFA-1 and ICAM-1, and abrogated the binding of MM cells to bone marrow stromal cells, that triggered the secretion of interleukin-6 and vascular endothelial growth factor. Taken together, it was suggested that Thal reduced the expression of CD56 and altered the MM cell adhesion to bone marrow stromal cells, and that could be one of the pathogenesis of anti-MM activity of Thal.