bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved Prognosis of Children and Adolescents with Acute Lymphoblastic Leukemia (ALL) and Very-High Minimal Residual Disease (MRD) and the End of Induction therapy (EOI)? Preliminary Results of the FRALLE 2000 Protocol
Authors: Cayuela Jean-Michel , Beldjord Kheira , Fasola Sylvie , Auclerc Marie-Françoise , Brethon Benoît , Auvrignon Anne , Mechinaud Francoise , Gandemer Virginie , Margueritte Geneviève , Blouin Pascale , Grardel Nathalie , Cavé Hélène , Macintyre Elizabeth , Leverger Leverger , Baruchel Andre
Published: April 2020

MRD at EOI is a powerful prognostic indicator in childhood ALL. Cave et al (N Engl J Med 1998) and van Dongen et al (Lancet 1998) have shown that a very high EOI-MRD (≥ 10−2) is associated to a dismal prognosis (5y EFS: 15–20%). From December 2000 to July 2007 1496 children and adolescents (1–20 years, no Ph+) have been included in the ongoing FRALLE 2000 protocol. EOI-MRD at D35–42 was measured by competitive PCR of Ig/TCR markers and Gene Scan analysis and was decisional for further treatment intensification if ≥ 10−2. 1017 pts are fully evaluable (monitored data, no induction failure, evaluable EOI-MRD, outcome). MFU is 36 months. Three risk-groups have been defined. A (n=620): NCI standard risk BCP-ALL, B (n=265): NCI high risk-ALL, T (n=132): T-cell ALL. Fifty-eight pts (5.7%) have a very high EOI-MRD, 2.6% in group A, 9% in group B, 14% in group T. These pts were more likely than pts with lower MRD (< 10−2) to have a D8 poor prednisone response (PPR) (31% vs 10%, p <.001), a slow marrow response (D21M2 or M3, 24% vs 5%, p <.001) or both (22% vs 1.5%, p <.001). A very high MRD only (no high-risk cytogenetic features, no D8 PPR, no D21 M2/M3 marrow) was found in 33 out the 58 pts (57%), i.e. 3% of all pts. A group pts (n=16) with very high EOI-MRD received a 3 block-consolidation plus a double delayed intensification (DDI) while B or T group pts received a 3 block-consolidation plus DDI (24 pts) or HSCT (18 pts). 5y EFS and OS where 50±10% and 74±7% respectively for the 58 pts. A similar 5 y EFS was found for pts with BCP-ALL or T-cell ALL with very high MRD (50±12% and 48±12%, respectively). There is a trend for a better EFS for pts with very high EOI-MRD only compared to those with other associated high-risk features (60±11% vs 39±11%, p=.10).

Conclusion: these results compare favourably with published results suggesting that intensification of therapy may improve the outcome of pts with bad early response to initial chemotherapy.
Distinct Gene Expression Profiling in AML in Elderly Versus Younger Patients
Authors: de Jonge Henk-Marijn , de Bont Evelina S , Valk Peter , Schuringa Jan Jacob , Kies Marcel P , Woolthuis Carolien M. , Delwel Ruud , Vellenga Edo , Lowenberg B. , Huls Geert A
Published: April 2020

Acute myeloid leukaemia (AML) has a different clinical and biological behaviour in elderly patients. We used Affymetrix HGU133Plus2.0 gene expression data of 525 patients with AML -who were treated on various HOVON/SAKK studies-to ascertain the difference of gene expression profiles in AML samples of older patients compared to AML samples of younger patients. Differentially expressed genes were identified for 175 youngest with a median age of 31-years vs. 175 oldest AML patients with a median age of 59-years using a multivariate permutation test in Biometric Research Branch ArrayTools (BRB ArrayTools). This analysis revealed that 478 probes were up-regulated and 492 probes were down-regulated with increasing age at the significance level of P <1.0×10−5. Biological processes (represented by GO-categories) enriched among the 478 (up) and 492 (down) differentially expressed probes were identified. Processes as regulation of cell proliferation, negative regulation of apoptosis as well as response to stress were up regulated with increasing age, whereas processes as chromatin modification, programmed cell death as well as transcription repressor activity were found to be down regulated with increasing age. It was striking that the tumour suppressor gene p16INK4a was identified as one of the strongest down-regulated genes with age. In contrast, various studies have demonstrated an increasing expression of p16INK4a during physiological ageing. Downregulation of p16INK4a with increasing age was confirmed in an independent cohort of CD34+ cells of 54 AML patients, whereas in CD34+ cells of 23 healthy volunteers p16INK4a was found to be upregulated with increasing age. In conclusion, gene expression profiles of AML samples of young and old patients differ significantly. That these differences in gene expression profiles not simply reflect an ageing phenomenon is illustrated by the down regulation of p16INK4a in samples of elderly AML patients. These data suggest that counteracting the senescence-promoting increase in p16INK4a, which is associated with physiological ageing, plays an important role in the development of AML in elderly patients.
Characterization of Gene p306 Expressed in Monocyte Derived Dendritic Cells
Authors: Bringmann Ani ta , Hilgendorf Ingo , Appel Silke , Held Stefanie , von Schwarzenberg Karin , Grünebach Frank , Brossart Peter
Published: April 2020

Dendritic cells (DCs) play a significant role in the initiation and maintenance of primary immune responses. The identification of new genes that are involved in DC biology is crucial for a better understanding of the unique DC functions. We approached this issue by generating a substractive cDNA library based on suppression hybridization between monocyte derived DCs (mDCs) cDNA and the reference monocyte cDNA. Among various differentially in mDC expressed genes, we identified the gene p306 with unknown functions. In the following, we characterized the gene p306 and the translated protein splice variants. We confirmed the p306 expression in mDCs on mRNA level by RACE and RT-PCR with subsequent sequencing. Besides the 2 known splice variants, we found a third novel form. Two splice variants were expressed in activated myeloid and plasmacytoid DCs sorted from blood. To characterize the p306 protein forms, we analyzed the hypothetical proteins by various prediction programs. We found that the N-terminus of the protein isoforms is homologue to a DNA-binding protein. Structural features are an oligonucleotide-binding fold with an integrated zinc ribbon. To detect p306 protein expression, we generated a polyclonal antibody and used it in Western blotting experiments. We were able to detect 3 protein bands with sizes of 36 kDa, 84 kDa and about 200 kDa, which differed from the expected sizes of 20 kDa and 111 kDa. We found that the unexpected sizes are in part due to N-glycosylation using the inhibitor tunicamycin. When analyzing cell lysates and nuclear extracts, we found that the 36 kDa protein form is expressed exclusively in the nucleus, while other forms could be detected in nucleus and cytosol. Interestingly, activation of mDCs with Toll like receptor ligands Pam3Cys, Poly I:C, LPS and R848 selectively upregulated the expression of the 84 kDa form in the nucleus. DC treatment with IL-10 or Imatinib, compounds that inhibit DC differentiation and function, abolished the expression of all 3 protein forms. In order to analyze the possible effect of proteases on the post-transcriptional regulation of the protein expression and generation of the identified splice variants, mDCs were treated with several protease inhibitors. DC treatment with cathepsin/subtilisin inhibitor led to the downregulation of all 3 protein variants, indicating that p306 might be involved in the regulation of apoptosis induction. The pan-caspase inhibitor zVAD caused the upregulation of all protein forms. In conclusion, we identified a novel gene p306 that is differentially expressed in mDC, which encodes for at least 3 different splice variants and protein forms with diverse cellular localization. Although the functions of the p306 proteins still have to be determined, the gene p306 is probably involved in binding nucleic acids and in regulating apoptosis induction.
Chemotherapy Alone for Localized Non-Bulky Hodgkin Lymphoma
Authors: Canellos George P.
Published: April 2020

Combined modality therapy with radiation and chemotherapy has been the standard treatment for limited-stage HL. Long-term toxicities, including cardiac and disease and secondary cancers, s have been reported in multiple series with long follow-up. Lower doses and smaller fields may be less toxic, but mediastinal/cardiac radiation is unavoidable in most cases. The Dana-Farber Cancer Institute and Massachusetts General Hospital lymphoma teams have now treated 74 patients with localized, non-bulky classical HL with chemotherapy as the only therapy. The median age of the series was 29 years (17–42). The M/F ratio was 33/41. The clinical stages were IA (10), IIA (56), IIB (9). The treatment consisted of ABVD × 6 cycles (70 pts), ABVD × 4 cycles (3 pts), and variants of ABVD (2 pts). The median F/U is 48+ months (4+–174+). One patient had primary refractory disease. Seven patients (median age 30, range 20–38) have relapsed (6, 10, 10, 11, 14, 20 and 63 months) whose original stage was IA (1), IIA (3), IIB (3). The progression-free survival at 4 years is 91%. All patients are alive. Six of seven patients received salvage RT and autologous stem cell transplantation, and only one has relapsed to date. In retrospect, all seven patients who relapsed from CR or CRu were FDG-PET negative at mid-cycle. Thus far, there has been no long-term, chemotherapy-related toxicity in the patients who remain in remission. 94% (62/66) asymptomatic patients remain in remission compared to 6/9 (67%) IIB patients.

Conclusion: In young adults with non-bulky, localized HL chemotherapy alone with ABVD can achieve excellent progression-free and overall survival, especially those asymptomatic at diagnosis. These data and previously published results indicate an equivalent long-term outcome when compared to combined modality trials but without the long-term risks of radiation therapy. Avoidance of radiation therapy may be even more important in the pediatric age group.
Direct Inhibitory Effects of Lenalidomide on the Proliferation and VEGF Production of Non-Hodgkin Lymphoma Cells Are Associated with Increased SPARC Expression
Authors: Zhang Ling-Hua , Schafer Peter H , Muller George , Stirling David , Bartlett Blake
Published: April 2020

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug approved for the treatment of Low- or Intermediate-risk myelodysplastic syndrome associated with a del(5q) cytogenetic abnormality, and in combination with dexamethasone for previously treated multiple myeloma. Early clinical results suggest potential clinical efficacy in B-cell non-Hodgkin lymphoma (NHL). In this study, we investigated lenalidomide-mediated inhibition of cell proliferation and angiogenic factors in the following NHL subtypes: mantle cell lymphoma (MCL), diffuse large-B-cell lymphoma, and follicular lymphoma (FL). The effect of lenalidomide on these tumor cells was determined after 1–3 days of treatment by 3H-thymidine incorporation, Luminex-based microbead array, and real-time qRT-PCR. We also assessed the expression of tumor suppressor genes such as p21cip1 and secreted protein acidic and rich in cysteine (SPARC). We found that lenalidomide induced direct anti-proliferative effects on each NHL subtype, with MCL cells being the most sensitive. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) were expressed at a high level in all lymphoma cell lines. Lenalidomide inhibited VEGF production at much lower concentrations than required for anti-proliferative effects, particularly in MCL and FL cell lines. Addition of recombinant human VEGF or neutralizing anti-VEGF antibody had no effect on MCL cell proliferation, suggesting that these effects are independent. Mechanistic studies indicated that lenalidomide strongly increased the gene expression of the tumor suppressor genes p21cip1 and SPARC to varying degrees. Elevation of SPARC mRNA significantly correlated with both the anti-proliferative and the VEGF-suppressive effects of lenalidomide on MCL cells (p < 0.05). Transfection of tumor cells with SPARC siRNA led to significant resistance to lenalidomide suggesting that this effect is mediated at least in part through the up-regulation of SPARC. In conclusion, lenalidomide demonstrates anti-proliferative activity against multiple NHL cell subtypes with greatest potency against MCL. The potent anti-VEGF activity of lenalidomide supports the anti-angiogenic potential of the drug. Thus, lenalidomide-induced up-regulation of SPARC mRNA correlates with MCL sensitivity and may have biomarker potential.
The “Side-Population” of Human Lymphoma Cells Have Increased Chemo-Resistance, Stem-Cell Like Properties and Are Potential Targets for Immunotherapy
Authors: Shafer Jessica Ann , Leen Ann M. , Cruz Conrad Russell , Craddock John A , Heslop Helen E. , Rooney Cliona M. , Brenner Malcolm K , Bollard Catherine M. , Foster Aaron
Published: April 2020

We have previously demonstrated that hematological malignancies contain a distinct “side-population” (SP), which is characterized by the active transport of Hoechst fluorescent dye. The mechanisms responsible for Hoechst efflux also contribute to resistance of the SP to chemotherapy. This population has also been shown to be enriched for tumor initiating cells in several human cancer models. We hypothesize that targeting the cancer “SP” cells using immunotherapy may prevent relapse by eliminating the cell population that:

is least sensitive to chemotherapy and has tumor repopulating potential.

In this study we characterized SP cells in ten human lymphoma cell lines. Five of the 11 lymphoma cell lines tested (HDLM2, L428, Ramos, Toledo, Karpas) had a distinct SP ranging from 0.8–2% of total cells. We subsequently found SP cells with tumor associated markers in biopsy samples from patients with Hodgkin’s Lymphoma, T-cell Lymphoma, and Follicular lymphoma. Culture of the sorted cell lines show that SP but not the non-SP cells produced progeny that were both SP and non-SP cells. Gene expression analysis of the SP showed increased expression of ABCG2 and other ABC transporters that mediate both the transport of Hoechst dye and selected chemotherapeutic agents out of the cell. We evaluated whether lymphoma cell lines with an SP are resistant to the chemotherapeutic drug gemcitabine. The viability of lymphoma lines containing SP cells was significantly higher (mean 59.8%; range 37.4–87.8%%) than the viability of lines without an SP (mean 9.3% range 3.0%–12.2%) when cultured with 10nM gemcitabine for 3 days. The SP component of the lymphoma cells became enriched 10 fold when cultured with gemcitabine. Furthermore, when equal numbers of SP and non-SP cells were grown in separate fractions with gemcitabine, there was reduced viability of the non-SP (2,247 +/−294 mean counts per minute (cpm)) by thymidine assay. In contrast, the viability of the SP was preserved (13,200 +/− 7500 mean cpm). There was no difference in the viability between the untreated SP and Non SP, so that the gemcitabine rather than the Hoechst dye accounts for the differences in viability. Although lymphoma SP cells are resistant to chemotherapeutic agents, they also express higher levels of tumor associated antigens that are known targets for cytotoxic T cells. Hence RT-PCR and immunohistochemistry both show that SP cells are HLA positive and have increased expression of MAGE-4, SSX2, Survivin and NY-ESO transcripts and protein compared to non-SP cells from the same tumor. In IFNγ ELISPOT assays, we have shown that T cells secrete IFNγ in response to SP targets. Hence, lymphoma cell lines and primary lymphoma tissue contain chemotherapy-resistant SP cells which express tumor associated antigens and may be targeted by specific T cells.
Phosphoproteomic Analysis of Primary Acute Myeloid Leukemia Cells Reveals Redundant Roles for Src Family Kinases in AML Survival
Authors: Dierov Jamil K. , Kim Tae Kon , Yang Xiaowei , Perl Alexander , Burke Beth A , Wang Zhu , Gu Ting-Lei , Gewirtz Alan M. , Corey Seth J. , Polakiewicz Roberto , Carroll Martin
Published: April 2020

Recent results have demonstrated that multiple signal transduction pathways are activated in acute myeloid leukemia (AML) cells, however, the tyrosine kinase(s) that phosphorylates these signaling proteins is not identified. We have analyzed AML cells using a phosphoproteomics screen and demonstrate that the Src family kinases, Lyn, Lck and Fgr, are phosphorylated on their activation sites in AML samples. Expression and activation of Lyn has been previously confirmed. Evaluation of Lck demonstrated that Lck is expressed to a variable degree but consistently in AML samples (n=20). Lck kinase assays show activation of Lck in 17/20 samples tested at levels above the level of activation detectable in normal CD34+ progenitor cells. Lyn and Lck both contribute to AML cell growth as siRNA depletion of either kinase leads to decreased leukemia colony forming activity. Interestingly, both Lyn and Lck contribute to phosphorylation of STAT5 as STAT5 phosphorylation is decreased but not abrogated by siRNA modification of either kinase alone. Consistent with the necessity for this signaling pathway for optimal AML cell growth, siRNA knockdown of STAT5 leads to decreased expression of both STAT5A and STAT5B, decreased expression of the STAT5 target protein, Bclxl and decreased AML colony forming ability. Based on this data, we have studied the FDA approved compound, Dasatinib, and demonstrate that Dasatinib decreases AML colony formation in 4 out of 5 samples tested. Overall, these results demonstrate that SFK’s act redundantly to regulate STAT5 phosphorylation and AML cell growth in primary cells and that phosphoprotein analysis is a robust approach to identify new targets for therapy of malignancy. Src family kinase inhibitors may be valuable in the therapy of AML.
Characterization of the Effects of Pim Kinase Inhibition on Multiple Oncogene-Driven Cell Lines
Authors: Gross Stefan David , Robinson John E , Allen Shelley , Cox April , DeWolf Walt E , Galbraith Sarah , Lee Patrice , Lemieux Christine , Lyssikatos Joseph P , Marmsater Fredrik , Martinson Matthew , Munson Mark C , Wallace Ross D , Woessner Rich , Zuzack John , Winkler James , Koch Kevin
Published: April 2020

We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound’s anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.
Modulation of Cancer Cell-Associated Procoagulant Activity (PCA) by a Novel, Small Molecule, Orally Active Inhibitor of Oxidative Stress Pathways
Authors: Mousa Shaymaa S. , Patil Ghanshyam , Lansing Lawrence , Dier Emmy , Kannanayakal Theresa , Rickles Frederick , Mousa Shaker S
Published: April 2020

Background: The association between malignancy and venous thromboembolism (VTE) is well-recognized. VTE is associated with a systemic hypercoagulable state that is secondary to cancer, cancer-associated therapy and the host inflammatory response. We have tested the ability of OT-304, which is a dual inhibitor of reactive oxygen species (ROS)-mediated activation of both the EGR-1 and NF-κB pathways, to inhibit the PCA of tumor cells.

Methods: Molecular measurements of NF-kB (p50/p65), IkBa, TNF-a, EGR1 and tissue factor (TF) were performed on the human monocytic cell line THP-1, and human umbilical vein endothelial cells (HUVECs) using Affymetrix micro-array, RTPCR, Western blot, ELISA and gel shift assays. The cell-based, anticoagulant efficacy of OT-304 at 1–30 mM was determined in in vitro models of both pro-inflammatory and cancer cell-associated hypercoagulable states, using thrombelastography (TEG) in human blood and in a rabbit model of cell-based coagulation activation.

Results: OT-304 effectively inhibited LPS-mediated TNF-α production in human blood and in THP-1 cells, with an IC50 of 6.7 mM. Similarly, in mice, in a dose-dependent manner, OT-304 at 0.1–50mg/kg IP or 1.0–150 mg/kg oral, inhibited LPS-induced NF-κB activation-associated up-regulation of various chemokines and cytokines. At the molecular level, OT-304 demonstrated effective inhibition of LPS-induced NF-κB activation, nuclear translocation of p50/p65, degradation of IKBa, without any effect on nuclear p50/p65 DNA binding. OT-304 appeared to induce modest down-regulation of the key procoagulant mediator TF, presumably via the inhibition of EGR1. In human whole blood subjected to TEG, OT-304 inhibited the procoagulant effect induced by LPS, lipid hydroperoxide, and ceramide, without directly affecting factor Xa, factor IIa or other coagulation factors. Additionally, OT-304 at 0.1–10 mM inhibited the PCA of glioma, lung, and pancreatic cancer cells. In comparison to the low-molecular-weight heparin, enoxaparin, OT-304 (1–30 ug/ml) was without any significant effect on soluble blood coagulation, as measured by the activated partial thromboplastin time. Repeated administration of OT-304 to mice (10 mg/kg, IP daily for up to 14 days), was without any hemorrhagic toxicity as compared to enoxaparin administered at the same dose. In a mouse xenograft model of human glioma, OT-304 (30 mg/kg, IP, daily for 15 days) resulted in significant inhibition of tumor growth, tumor angiogenesis, and glioma-associated elevation of plasma D-dimer. In rabbits, OT-304 (10 mg/kg IP) resulted in significant inhibition of LPS-induced PCA.

Conclusions: OT-304, by virtue of its ability to target and modulate more than one pathway, may represent a promising anti-cancer therapy, capable of inhibiting tumor growth, inflammation and cancer-associated thrombosis without compromising hemostasis.
Accelerated Cellular Senescence in Myelodysplastic Syndrome
Authors: Wang Yuanyuan , Chen Zixing , Cen Jiannong , Shen Hongjie , Qi Xiaofei , He Jun , Liu Dandan , Yao Li
Published: April 2020

Myelodysplastic syndrome (MDS) is a group of hematopoietic stem/progenitor cell clonal disorders characterized by dysplastic morphology of at least one cell lineage and high risk of evolution to acute myeloid leukemia (AML). Thus, it could be viewed as a preneoplastic stage prior to development of overt acute myeloid leukemia (AML). A number of studies indicated that oncogene-induced senescence as a crucial cellular response at premalignant stage can protect cells from tumor formation. Senescent cells emerged in premalignant cell population but not in malignant ones. p16INK4a have been identified as a cellular senescence-associated molecular marker. In present study, we have determined and compared the expression level of p16INK4a by quantitative RT-PCR and western blot in BM mononuclear cells (BMMNCs)/CD34+ cells among 53 patients of MDS and 12 of AML as well as 11 healthy controls. A significantly upregulated expression level of p16INK4a in MDS, while an significantly lower p16INK4a expression level in AML had been detected in comparison with that in healthy controls(<0.05). The IPSS score of these MDS patients negatively correlated with the p16INK4a expression level. To further confirm the senescent status, the cells of MDS, AML patients and healthy controls were examined by histochemical assays for the activity of senescence-associated beta galactosidase (SA-β-Gal). A weak blue stain reflecting the SA-β-Gal activity could be found in less than 10% cells from healthy controls after four days’ culture. On the contrary, a much stronger blue stain for SA-β-Gal had been detected in about 30–40% cells from MDS, and a little-to-no blue stain in AML cells. The semi-quantitative RT-PCR had demonstrated a similar pattern of SA-β-Gal expression among these three groups. Our preliminary results raise the question whether an accelerated cellular senescence in addition to apoptosis may contribute to the hematopoietic ineffectiveness and dysplastic morphology of cells in MDS, and if it could be of any implications for the progression of the disease.
Sustained Response Duration Seen after Treatment with Single Agent Blinatumomab (MT103/MEDI-538) in the Ongoing Phase I Study MT103- 104 in Patients with Relapsed NHL
Authors: Bargou Ralf C , Kufer Peter , Goebeler Mariele , Knop Stefan , Einsele Hermann , Noppeney Richard , Viardot Andreas , Baeuerle Patrick A , Reinhardt Carsten , Schmidt Margit , Klappers Petra , Nagorsen Dirk , Zugmaier Gerhard
Published: April 2020

Introduction: Blinatumomab (MT103/MEDI-538), a BiTE antibody targeting the CD19 antigen, is a member of a novel class of molecules that redirect T cells for lysis of target cells. A Phase 1 dose escalation study is being conducted in patients with advanced NHL.

Methods: Relapsed NHL patients requiring treatment were included. Most patients were pre-treated, with a median of 3 previous chemo/immunotherapy regimens. To date, 7 dose levels ranging from 0.0005 to 0.09 mg/m2/24 h have been tested. Blinatumomab was continuously infused as single agent over a period of 4–8 weeks. Objective responses were assessed by Cheson criteria and centrally reviewed.

Results: To date 39 patients have been treated. Most common adverse events (AEs) included lymphopenia, pyrexia, and leukopenia. The majority of AEs (>95%) improved or resolved during treatment. Permanent treatment discontinuation due to AEs occurred in a total of 8 patients, of which 6 had fully reversible CNS events. One patient with a history of nearly fatal sepsis, pre-existing hypogammaglobulinemia and bone marrow affection by chemotherapy, experienced a fatal sepsis 5 weeks after treatment start. Dose-dependent activity was observed in mantle cell lymphoma, follicular lymphoma and CLL with responses observed in 11 out of 27 patients treated at doses of 0.015 mg/m2/24 h and higher. Five of those patients had complete and six had partial responses. At the dose level of 0.060 mg/m2/24 h, 7 out of 7 patients have shown objective responses. Beside one relapse after 14 months, no treatment failure has so far been observed for responders at dose levels of 0.030 mg/m2/24 h and 0.060 mg/m2/24 h. Five patients at these dose levels have ongoing responses for more than 6 months. Interestingly, partial remissions converted into complete remissions in two patients four weeks after end of infusion suggesting either reduction in lesion size due to efflux of a previously expanded T cell pool or prolonged T cell activity.

Conclusions: Blinatumomab as single agent induced apparently durable responses in pre-treated B-NHL patients with the highest response rate at a dose level of 0.06 mg/m2/24 h. Recruitment is ongoing.
Growth Differentiation Factor 15 Plasma Level in patients with Multiple Myeloma: A Study of Intergroupe Francophone Du Myelome
Authors: Corre Jill , Roussel Murielle , Huynh Anne , Gadelorge Melanie , Labat Elodie , Laharrague Patrick , Moreau Philippe , Facon Thierry , Avet-Loiseau Hervé , Fournié Jean-Jacques , Attal Michel , Bourin Philippe
Published: April 2020

We previously reported that bone marrow mesenchymal stem cells (BMMSCs) from multiple myeloma (MM) patients were abnormal. In particular, we showed that Growth Differentiation 15 (GDF15) expression was higher in MM BMMSCs than in normal BMMSCs and that GDF15 was a growth factor for the MOLP-6 stromal cell-dependant myeloma cell line. GDF15 is a divergent member of the human TGFβ superfamily. In normal human subjects, it is highly expressed in the placenta and its serum concentration increases during pregnancy. GDF15 concentration is increased in the serum of patients with prostate, colorectal or pancreatic cancers. However, GDF15 has never been studied in haematological malignancies. The first objective of this study was to evaluate the relashionship between GDF15 plasma concentration (pGDF15) of 131 MM patients and main prognostic factors of the disease. We found a significant relationship with ISS (p<0.003) and DS stage (p<0.02), b2 microglobulin, haemoglobin, creatinin, calcemia (p<0.001), deletion of chromosome 13 (p<0.004), serum monoclonal protein, bone status (p<0.02), and albumin (p<0.05). The second objective of this study was to appreciate the potential relationship between pGDF15 and survival. We demonstrate such a relationship. Indeed, in the group of 81 patients with high pGDF15, the probabilities of event-free and overall survival 30 months after diagnosis is 50 percent and 75 percent, respectively. In the group of 50 patients with low pGDF15, the probabilities of event-free and overall survival 30 months after diagnosis is 80 percent and 97 percent, respectively (p<0.0045 and p<0.013, respectively). On the opposite, pGDF15 is not related to response to treatment. We analysed impact of known prognostic factors and pGDF15 on event-free survival for all the 131 patients. In univariate analysis, event-free survival was significantly related to age (p=0.003), ISS (p=0.09), b2-microglobulin (p=0.02) and pGDF-15 (p=0.003). In multivariate analysis, event-free survival was significantly related to age (p=0.001) and pGDF15 (p=0.04). In conclusion, our study demonstrates a significant relationship between GDF15 and MM survival. This result strongly suggests the implication of microenvironment in the disease evolution. The exact role of GDF15 in MM physiopathology (directly responsible or simple reflect of an abnormal microenvironment) is still unclear and should be investigated.
Bone Morphogenic Protein 6: A Prognostic Factor Expressed by Normal Plasma Cells and Multiple Myeloma Cells Inhibiting Their Proliferation and Angiogenesis Induction
Authors: Seckinger Anja , Meißner Tobias , Moreaux Jérôme , Goldschmidt Hartmut , Benner Axel , Hundemer Michael , Rème Thierry , Shaughnessy John D. , Barlogie Bart , Bertsch Uta , Hillengass Jens , Ho Anthony D. , Pantesco Véronique , Jauch Anna , De Vos John , Rossi Jean François , Möhler Thomas , Klein Bernard , Hose Dirk
Published: April 2020

BACKGROUND: Pathogenesis of multiple myeloma is partly attributed to an aberrant expression of proliferation-, pro-angiogenic and bone-metabolism modifying factors by malignant plasma-cells.

AIM. Given the long and variable time-span from first diagnosis of early-stage plasma-cell dyscrasias to overt myeloma and the low proliferation rate of malignant plasma-cells, we hypothesize these to concomitantly express a novel class of anti-proliferative factors of potential prognostic relevance. Here, bone morphogenic proteins (BMPs) represent possible candidates, as they inhibit proliferation, stimulate bone formation, and have an impact on the survival of cancer patients.

PATIENTS AND METHODS. We assessed expression of BMPs and its receptors by Affymetrix DNA-microarrays (n=434) including CD138-purified primary myeloma-cell-samples, normal bone-marrow plasma-cell-samples, polyclonal plasmoblasts-samples, human myeloma-cell-lines (HMCL), and whole bone-marrow. Presence and differential gene expression was determined by PANP-algorithm and empirical Bayes statistics. Event-free (EFS) and overall survival (OAS) were investigated for the 168 patients undergoing high-dose chemotherapy (HM-group) using Cox’s proportional hazard model. Findings were validated using the same strategy on an independent group of 345 patients from the Arkansas-group. For validation, quantitative real-time PCR and flow cytometry were performed. In vitro induction of angiogenesis was assessed using the AngioKit-assay. Effect of BMP6 on proliferation of HMCL was assessed by 3H-thymidine uptake.

RESULTS. BMP6 is the only BMP expressed by normal- (13/14 samples) and malignant plasma-cells (228/233 samples). It is significantly lower expressed in proliferating non-malignant plasmablastic cells and human myeloma cell-lines. In vitro, BMP6 significantly inhibits proliferation of myeloma-cell-lines with an IC50 ranged from 0.08–2.15μg/ml, survival of primary myeloma-cells, and in vitro tubule formation down to the level of the negative control. High BMP6-expression in malignant plasma cells delineates significantly superior overall-survival for patients undergoing high-dose chemotherapy in both independent series of patients (n=168, P=.02 and n=345, P=.03, respectively, see below).

CONCLUSION. With BMP6 we report for the first time the autocrine expression of a prognostically relevant anti-angiogenic and anti-proliferative factor and its receptors by normal and malignant plasma-cells.

Figure Figure
Hepatitis C Virus (HCV) Infection, Monoclonal Immunoglobulin Specific for HCV Core Protein, and Plasma-Cell Malignancy
Authors: Bigot-Corbel Edith , Gassin Michelle , Corre Isabelle , Le Carrer Didier , Delaroche Odile , Hermouet Sylvie
Published: April 2020

Hepatitis C virus (HCV) infection can lead to B-cell malignancy via direct infection and transformation of B-lymphocytes, or via indirect transformation by chronic antigen-driven stimulation. Both mechanisms may occur simultaneously, as we previously reported in a case of HCV infection followed by plasma-cell leukemia (PCL), where blasts were infected with HCV and the monoclonal immunoglobulin (Ig) they produced was directed against the core protein of the virus (New Engl J Med, 2003; 348:178). Approximately 10% of HCV-positive patients develop a monoclonal Ig, the specificity of which is usually unknown. To evaluate the link between chronic HCV-antigen driven stimulation and plasma–cell transformation, we studied the specificity of monoclonal Ig developed in the context of HCV infection. Over a period of 13 months, sera from patients consulting or hospitalised at the Centre Hospitalier Universitaire of Nantes found positive for monoclonal Ig, were systematically tested for the presence of HCV RNA and anti-HCV Ig. Among the 700 patients thus studied, 10 (1.4%) were found positive for HCV. Purification of the monoclonal Ig was achieved for 7/10 patients. Using immunoblotting, the purified monoclonal Ig (2 IgG, 1 IgA, 1 IgM) of 4 patients, all with HCV genotype 2, recognized the C22–3 fragment of HCV-core protein; 2 (IgG) recognized NS-4 and 1 did not recognize HCV. Among the 4 patients with anti-HCV-core monoclonal Ig, two presented with mixed (type II) cryoglobulinemia and one was diagnosed with multiple myeloma. Hence, 2/5 patients with anti-HCV core monoclonal Ig developed plasma-cell malignancy. Anti-HCV treatment resulted in the disappearance of the monoclonal Ig for 3/3 treated patients. In summary, in the context of HCV infection monoclonal Ig were typically directed against the virus, and could distinguish patients with increased risk of plasma-cell malignancy. Efforts should be made to identify such patients, as anti-viral therapy should help eradicate the HCV-driven plasma-cell clone.
CD28 Supports Normal Plasma Cell Survival and Interactions with Microenvironment
Authors: Rozanski Cheryl H , Nair Jayakumar , Carlson Louise , Lee Kelvin P.
Published: April 2020

The long term generation of protective antibodies (Abs) requires the continuous survival of long-lived plasma cells that are maintained within specialized bone marrow niches by complex interactions that remain largely uncharacterized. Previous studies have shown that the T cell costimulatory receptor CD28 is expressed on normal and transformed (murine plasmacytoma, human multiple myeloma) plasma cells – however, its role in the B cell lineage remained unclear. We have recently shown that CD28 expressed on transformed human plasma cells (multiple myeloma cells) directly delivers pro-survival signals to the myeloma cells and protects them against intrinsically and extrinsically induced death (Bahlis et al, 2007). Furthermore, myeloma cells directly interact with dendritic cells (DC, both in vitro and in patient bone marrow biopsies), and the DC provide the ligands (i.e. CD80 and CD86) for myeloma-CD28. Others studies utilizing competitive bone marrow reconstitution have indirectly suggest a role for CD28 in the function and/or survival of normal murine plasma cells (Delogu et al, 2006). These observations led us to directly investigate the role of CD28 in normal plasma cell survival as well as cell-cell interactions with CD80/CD86+ bone marrow derived dendritic cells (BMDC). In vitro serum starvation experiments, direct activation of CD28 by an agonistic anti-CD28 mAb increased survival of serum-starved PC by 63% (p<0.001). Addition of BMDC improved the survival of PC by 20% over that seen with media alone, and resulted in a significant increase in IgG production (p<0.01). We and others have shown that CD28 binding to CD80/CD86 on DC also “backsignals” to the DC to produce the PC survival factor IL-6. We found that co-culture with the murine plasmacytoma cell line S194 induced 155 pg/ml of IL-6 from BMDC (p<0.01 vs. BMDC alone and S194 alone), and primary plasma cells isolated from bone marrow induced 290 pg/ml of IL-6 from BMDC (p<0.001 vs. BMDC alone). Induction of BMDC production of IL-6 by both primary and transformed PC was significantly inhibited (p<0.05) by antibody blockade of CD80 and CD86. Our data demonstrates that signaling through CD28 directly supports the survival of normal bone marrow plasma cells, and that “backsignaling” through PC-CD28 engagement of DC-CD80/CD86 induces DC to secrete the pro-survival cytokine IL-6. These findings suggest that CD28 is a key molecular bridge that connect normal plasma cells to the supportive microenvironment.
Prognostic Significance of Both the Cytoplasmic and Nuclear Overexpression of Yin-Yang 1 (YY1) among Patients with Multiple Myeloma (MM)
Authors: Huerta-Yepez Sara , Rivera-Pazos Clara , Libra Massimo , Baritaki Stavroula , Chen Haiming , Berenson James R. , Bonavida Benjamin
Published: April 2020

Objective and Rationale Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein, which can activate, repress or initiate transcription depending on the context in which it binds. In addition, YY1 can modulate protein levels or activity through protein-protein interaction. YY1 has been identified as a potential repressor factor for several genes. We have reported that YY1 can act as a transcription repressor for both Fas and TRAIL DR5. In addition to YY1-mediated regulation of tumor cell resistance to cytotoxic immunotherapy, it also has been shown to regulate resistance to chemotherapy [Baritaki et al., J Immunol 80:6199,2008]. Overexpression of YY1 has been shown to be of prognostic significance in prostate cancer [Seligson et al., Int J Oncol 27:131,2005].

Hypothesis In this study, we have examined the expression of YY1 in MM (cell lines and patients’ bone marrow [BM]) by hypothesizing that the resistance of MM cells to various cytotoxic agents may be, in part, regulated by overexpression of YY1 and that YY1 may also be of prognostic significance.

Designs and Methods MM cell lines and fresh BM samples from MM patients (n=21) were examined for YY1 expression, cytoplasmic and nuclear, by immunohistochemistry and by Western. The specificity of the anti-YY1 antibody was demonstrated by the competitive inhibition with a peptide used for immunization. Control isotype IgG did not show any staining on the cells.

Results First, we found that various MM cell lines (RMPI 8226, IM-9, U266) overexpress YY1 both in the cytoplasm and the nucleus. Next, we examined the expression of YY1 in BM derived from MM patients by immunohistochemistry. BM from MM patients showed overexpression of YY1 as compared to normal bone marrow samples. Furthermore, analysis of both the intensity and frequency of cells expressing YY1 in both the cytoplasm and nucleus was shown to be significantly higher among patients with progressive disease as compared to patients with stable or responsive disease.

Conclusions and Implications These findings show that the expression of YY1 among patients with MM may correlate with progression and also suggest the prognostic significance of YY1 in MM patients. Inhibition of YY1 by various agents (example: low-dose chemotherapy, proteasome inhibitors, NO donors, NF-kB inhibitors, etc.) all result in the reversal of resistance to various cytotoxic agents (eg CDDP, TRAIL) therefore, the findings also imply that agents that can inhibit YY1 expression in MM patients may be of therapeutic potential when used in combination with conventional therapeutics.
Immunoglobulin Variable Light Chain Restriction, Cytokine Expression and Plasma Cell-Stromal Cell Interactions in POEMS Syndrome Patients
Authors: Aravamudan Bharathi , Tong Caili , Lacy Martha Q. , Hayman Suzanne R. , Buadi Francis , Gertz Morie A. , Kumar Shaji , Dispenzieri Angela
Published: April 2020

POEMS syndrome is defined by the presence of peripheral neuropathy (P), organomegaly (O), endocrinopathy (E), monoclonal protein expression (M), and skin changes (S). In addition, patients also have sclerotic bone lesions, papilledema, extravascular volume overload, thrombocytosis, and restrictive lung disease/pulmonary hypertension. A striking but inexplicable feature of the clonal plasma cells in patients with POEMS is that they are virtually always lambda restricted. More surprising is that of the 81 functional immunoglobulin light-chain variable-region genes (IgVL) available in the plasma cell/B cell repertoire, among the 13 cases in which variable gene light chain usage has been reported, all have been either IgVL1-40 or IgVL1-44. The molecular mechanisms triggering the initiation and the progression of POEMS syndrome or the clonal expansion of plasma cells marking this condition remain unclear. We have analyzed the immunoglobulin heavy and light chains sequences in the plasma cells of POEMS patients at the Mayo Clinic Rochester. We found that in 8 out of the 9 patients tested, we could document heavy chain clonality in any given patient; however, no restriction of gene usage among patients was observed in the variable regions of these chains. In the case of light chains, in 14 out of 17 patients we could document not only clonality but also restriction of light chain variable gene usage to IgVL1-40 or IgVL1-44. Our goal is to understand the relevance of this restricted expression of light chain variable region, and its effect on the communication between plasma cells and the neighboring stromal cells that might provide more insight into the disease pathology. To this end, we are generating viral vector constructs that will harbor the germline or mutant versions of IgVλ1-40 and IgVλ1-44 genes and introduced into pre-established malignant plasma cell lines (i.e. myeloma cell lines). We will assay the effects of this over expression in terms of alterations in the expression of cytokines such as VEGF and TNF, and on plasma cell-stromal cell interaction (in a co-culture system). Results from these assays will be discussed.
RGB 286638, a Novel Multi-Targeted Small Molecule Inhibitor, Induces Multiple Myeloma (MM) Cell Death through Abrogation of CDKDependent and Independent Survival Mechanisms
Authors: Cirstea Diana , Hideshima Teru , Pozzi Samantha , Vallet Sonia , Ikeda Hiroshi , Santo Loredana , Loferer Hannes , Vaghela Nileshwari , Okawa Yutaka , Perrone Giulia , Gorgun Gullu , Calabrese Elisabetta , Munshi Nikhil C. , Anderson Kenneth C. , Raje Noopur
Published: April 2020

Uncontrolled proliferation and development of drug resistance in multiple myeloma (MM) cells are consequences of the numerous genetic aberrations which are further stimulated in the context of bone marrow microenvironment. Thus, inhibition of complementary pro-survival signaling and transcriptional networks rather than individual pathway is required for induction of optimal cytotoxicity in MM cells. Recent studies have shown that cyclin-dependent kinases inhibitors (CDKIs), designed to block cell cycle progression through inhibition of CDK/cyclin complexes, block transcription through suppression of RNA polymerase II phosphorylation at its C-terminal domain (CTD), resulting in downregulation of cell proliferation (cyclins: D, A, B1, pRb) and anti-apoptotic proteins (i.e. Mcl-1, survivin, XIAP). Here we examined the anti-MM activity of RGB 286638, a novel multi-targeted small molecule inhibitor, originally designed to induce broad cell cycle suppression via multiple CDK inhibition. Treatment with RGB 286638 triggered a dose-dependent cytotoxicity in conventional drug-sensitive (MM.1S, RPMI 8226, U266, OPM2), and resistant (MM.1R, Dox-40, LR5) MM cell lines, as well as primary tumor cells from MM patients. Induction of apoptosis was evidenced by Annexin V/PI staining, and confirmed by PARP and caspase cleavage. Additionally, RGB 286638 overcame the proliferative advantage conferred by MM patient-derived bone marrow stromal cells (BMSCs) and cytokines (IL-6, IGF-1) on MM cells. To determine molecular mechanisms responsible for RGB 286638-induced cytotoxicity, we assessed the cell cycle profile, which revealed G2/M arrest followed by increased sub-G1 phase. Importantly, RGB 286638 inhibited phosphorylation of RNA polymerase II in a dose- and a time-dependent fashion, followed by suppression of CDK1/cyclin B, CDK4, 6/Cyclin D1, D3, and CDK2/Cyclin E complexes associated with rapid down-regulation of Rb phosphorylation. Interestingly, RGB 286638 effectively reduced IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (Tyr705) and Janus kinase 2 (JAK2) phosphorylation (Tyr 1007/1008), suggesting that RGB 286638 is a possible JAK2 inhibitor. Based on sufficient in vitro cytotoxicity, we examined anti-tumor activity of RGB 286638 in vivo using a human MM cell xenograft model in SCID mice and demonstrated that RGB 286638 inhibited tumor growth and prolonged survival. In conclusion, our data demonstrate preclinical activity and provide the rational to test RGB 286638 in the treatment of MM.