bluuebunny/crossref_metadata_2025 huggingface dataset:
A Rapid Method for Comparing the Oncogenic Potential of Retroviral Vectors.
Authors: Hendrie Paul C. , Russell David W.
Published: October 2019

Retroviral gene transfer has successfully cured patients with X-linked severe combined immunodeficiency (X-SCID). Unfortunately, three of the treated patients developed leukemia at least in part due to insertional activation of a neighboring gene. Further advances of this exciting therapeutic modality will require safer viral vectors. Genotoxicity experiments in animals, although an important preclinical study, are not practical for screening large numbers of vectors. We have developed a rapid plasmid-based assay to compare downstream gene activation by retroviral vectors. We constructed a plasmid, pACT, that contains a minimal CMV promoter, an internal ribosome entry site (IRES) and a luciferase reported gene followed by a polyadenylation site. Four different proviral constructs containing identical internal PGK-EGFP expression cassettes were inserted upstream of the minimal promoter and nucleofected (Amaxa Biosystems) into K562 cells. The relative luciferase activation of each provirus was determined and compared to cells nucleofected with the basic pACT plasmid. The relative luciferase activation by a plasmid containing a Moloney murine leukemia virus (MLV) vector provirus with full length long terminal repeats (LTRs) was 144, 1230, and 4525 at 1, 2 and 4 days post nucleofection, respectively. Changing the orientation of the proviral DNA did not significantly change the results, consistent with an enhancer effect from the proviral LTRs. In comparison, the relative luciferase activation by a plasmid containing a self inactivating (SIN) MLV vector provirus with LTR deletions was only 59, 90 and 170, respectively, and was reduced by greater than 90% with the reverse orientation consistent with residual activation by read-through transcription. For plasmids containing the HIV SIN vector provirus, the relative luciferase activation was 48, 45 and 52, respectively at 1, 2 and 4 days post nucleofection, and decreased by >90% with the inverse orientation. The relative luciferase activation for plasmids containing the Foamy Virus (FV) vector provirus, also a “SIN” vector, was the least, only 3, 2 and 6, respectively, at 1, 2 and 4 days post nucleofection. Therefore, all three SIN vectors (MLV, HIV and FV) produced significantly less downstream gene activation than a conventional MLV vector, with the FV vectors being the lowest. Additional studies comparing various internal promoters and vectors will also be presented. These results demonstrate the usefulness of the pACT assay system to identify safer retroviral vectors.
Defects in Management of Labile Iron and Oxidative Stress Underpin Red Cell Enucleation Defects in Rb Null Mice.
Authors: Macleod Kay F. , Spike Benjamin T.
Published: October 2019

The Rb tumor suppressor is critically required for end-stage red cell maturation under conditions of oxidative stress, including in the developing fetal liver, in the bone marrow of aging mice, in the spleen and bone marrow of young mice treated with phenylhydrazine to induce hemolytic anemia, and in lethally irradiated mice reconstituted with donor tissue [1]. Loss of Rb resulted in a failure of end-stage red cells to enucleate, accumulation of red cells with a 4N DNA content and aberrant chromatin structure [1]. The molecular basis of these defects is not defined nor do we understand the reasons why pRb should be required under stress conditions, but not during normal “steady-state” erythropoiesis. The work presented will address both of these questions.

In determining why pRb is critically required for stress erythropoiesis but not for steady-state erythropoiesis, we have demonstrated increased levels of reactive oxygen species (ROS) and labile iron in Rb null erythroblasts relative to wild-type control erythroblasts derived from E12.5 fetal liver. Furthermore, we show that quenching of ROS in Rb null erythroblasts by treatment of mice with the anti-oxidant N-acetyl cysteine (NAC) rescued aspects of the erythroid defect, including red cell enucleation and also extended the lifespan of Rb null mice. Similarly, chelation of labile iron with desferroxiamine restored enucleation capacity to Rb null erythroblasts. Furthermore, we show that the transferrin receptor (CD71) is transcriptionally repressed by pRb/E2F and examine whether deregulated expression of CD71 contributes to increased labile iron and oxidative stress in Rb null erythroblasts. These results suggest that loss of pRb limits the ability of erythroblasts to manage labile iron and oxidative stress, in part through deregulated expression of CD71, and that this contributes to the enucleation defect observed in Rb null mice. Given that pRb is itself regulated by ROS, we present a model in which the timely induction and repression of the CD71 receptor in differentiating erythroblasts is required to manage labile iron, oxidative stress and to coordinate cell cycle exit with end-stage maturation.
Inhibitory Effect of Monoclonal Antibodies Against CXCR3-B NH2-Terminus on the Proliferation of U937 Cells.
Authors: Gao Yuan , Chen Zhi-Cheng , Liu Li , Yang Jin-Ju , Liu Xiao-Lan , Chen Yong , Gao Jian-En , Liu Chaoyan , Visentin Gian Paolo , Sun Qi-Hong
Published: October 2019

The role of CXCR3 in mediating the angiostatic activity of IFN-inducible ELR−CXC chemokines has been further defined with the finding that CXCR3 exists as two alternatively spliced variants, termed CXCR3A and CXCR3B respectively. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, but only binds to its specific receptor CXCR3B for the angiostatic activity. CXCR3B, a novel chemokine receptor, is 51 amino acid residues longer at the NH2-terminus than CXCR3A. Unlike activation of CXCR3A, which is pertussis toxin-sensitive and activates cellular proliferation, activation of CXCR3B is pertussis toxin-resistant and leads to inhibit cellular proliferation. Thus, the 51 amino acid residues at CXCR3B NH2-terminus are very important for the CXCL4/PF4 binding and for the CXCR3B inhibitory effect. In the present studies, a GST-tagged fusion protein of CXCR3B (a.a 1-51) was used as the immunogen for generation of monoclonal antibodies (mAbs) against CXCR3B (a.a. 1–51), and three peptides derived from CXCR3B NH2-terminus (a.a. 1–19, a.a. 17–35, and a.a. 33–51) for epitope mapping of the established mAbs. Nine hybrodoma cell lines secreting mAbs against CXCR3B were established, and we found that two mAbs reacted specifically with the CXCR3B a.a. 1–19, and one antibody with the CXCR3B a.a. 33–51. Three of these antibodies could also bind to CXCR3B on the surface of human intestinal epithelial Caco2 cells, and CXCR3B recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues. The data from Cell proliferation assay showed that three mAbs could inhibit the growth of U937 cells, which were found to express CXCR3B as well. We conclude that the novel panel of CXCR3B mAbs could be useful tools for the CXCR3B functional studies. Meanwhile, CXCR3B may be not only the inhibitory target on endothelial cells, but also the new directly reactive site of CXCR3B-positive tumor cells such as promonocytic U937 cells.
Long-Term Effect of Bleomycin on Respiratory Function Tests in Pediatric Cancer Patients.
Authors: Aydogan Gonul , Bilgic E. , Akici F. , Salcioglu Z. , Turhan P. , Tugcu D.
Published: October 2019

Chemotherapy and radiotherapy which are used in the management of cancer have acute and chronic adverse effects on various organs. Patients develop pulmonary damage due to mantle zone irradiation and bleomycin in the short or long-term. In this study, after the completion of treatment long-term pulmonary complications of the teatment were investigated in 30 pediatric patients (19 male, 11 female) with cancer aged between 72–229 months. Twelve of the patients used also chemotherapy in addition to 1020–3570 rad. of mantle zone irradiation (median 2177 rad.), whereas the remaining 18 had chemotherapy alone. All of the patients had completed their treatment approximately 3,8 years ago, and the total dose of bleomycin they used was 12–180 mg. (median 69,83). All of the patients underwent physical examination, and complete blood count, chest radiograph and spirometric tests to measure lung volumes were also performed. Additionally, in 20 patients single breath technique to measure the diffusing capacity for carbon monoxide was carried out. Physical examination of cardiopulmonary system revealed normal findings and none of our patients had a cardiopulmonary symptom. Chest radiograph was normal in 25 (83,3%) but it was abnormal in 5 patients.

Lung volume measurements were normal in 21 (71%), but were abnormal in 9 (29%) patients. Of these 9 patients 23% had restrictive, 3% had combined and 3% had obstructive disorders. In our study, no correlation could be shown between the impaired respiratory function tests and irradiation and bleomycin doses used. In 3 (10%) out of 20 patients in whom the diffusing capacity test for carbon monoxide was performed, decreases in diffusing capacity for carbon monoxide were found. One of these patients had both high dose bleomycin (156 mg.) and mantle zone irradiation (2160 rad.), whereas the remaining 2 had low dose bleomycin (30–63 mg.) alone and received no radiotherapy.

The diffusing capacity test for carbon monoxide which returns to normal values in short -term is less valuable than the measurement of lung volumes for the long-term follow-up evaluation of respiratory functions in these patients. Generally, in our late phase study (3,8 years after the completion of therapy), there was a low rate of impairment in diffusing capacity test for carbon monoxide, but the rate of impairment in lung volumes was much higher. This finding was compatible with most of the related literature.

In conclusion, in pediatirc cancer patients respiratory function tests should be performed during the treatment and also after the completion of treatment in order to follow the course of pulmonary function impairment.
Patterns of Relapse and Toxicity in Patients of CML (Chronic Phase) on Imatinib Therapy.
Authors: Aggarwal Shyam , Gogia Atul , Lal Meena , Dutt Sarjana , Bhargava Manorama
Published: October 2019

44 patients of CML (in chronic phase) were evaluated for their hematological, cytogenetic and molecular response (by doing quantitative PCR- for bcr-abl/abl), over a period of last 4 years. The toxicity profile of these patients was also evaluated. The mean age of study group was 54.5 years, with a male: female ratio of 1.8:1 (28 males and 16 females). 13/44 (29.3%) patients were treated with interferon prior to imatinib therapy, while the rest (31) were started on imatinib (400mg/day) upfront. 43/44 (97.7%) patients achieved a complete hematological response. 1 patient who did not respond to imatinib therapy had long duration of disease (111 months). 24/44 (54.6%) had complete cytogenetic response, 4 (9.1%) had major and 5 (11.3%) had minor cytogenetic response. 7/44 (15.9%) had hematological relapse while on Imatinib therapy. 3/44 (6.8%) had cytogenetics / molecular relapse only. Out of the 13 patients who had received prior interferon, 4 developed hematological relapse and of these 4 patients, 1 developed blast crisis and other 3 are in accelerated phase. 3/31 (9.7%) patients who received imatinib therapy upfront developed hematological relapse, while 6/31 (19.2%) developed cytogenetic/molecular relapse including the 3 patients who developed hematological relapse. A median of 17 months of imatinib therapy was given in patients who did not receive prior interferon. 36/44 (81%) developed hypopigmentation 19/44 (43%) had fluid retention, 9/44 (20%) had muscle cramps and 3/44 (6.8%) developed myelosupression. The relapse rate in patients of CML in chronic phase is more in those who have received interferon prior to imatinib therapy (37.7%). At a median follow up of 17 months the relapse rate in patients who received imatinib de novo was 19.2%.
The JAK2 V617F Mutation Is Uncommon in Patients with Juvenile Myelomonocytic Leukemia.
Authors: Yoshida Nao , Yagasaki Hiroshi , Xu Yinyan , Yamamoto Tomoko , Kudo Kazuko , Kato Koji , Horibe Keizo , Manabe Atsushi , Kojima Seiji
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is a rare, clonal myeloproliferative disorder afflicting young children and is characterized by specific hypersensitivity of JMML cells to GM-CSF in vitro. The pathogenesis of JMML seems to arise from dysregulation of GM-CSF signal transduction. Approximately, 75% of patients with JMML exhibit dysregulation due to mutations in RAS, NF1 or PTPN11, all of which are positioned in the GM-CSF pathway. Potential causative mutations in remaining patients are anticipated to be in signaling components downstream of the GM-CSF receptor. Recently, a somatic point mutation (JAK2 V617F) in the JAK2 gene, which plays a major role in the development of hematopoietic cells by transducing signals from growth factor receptors including GM-CSF receptor, was found commonly in polycythemia vera, essential thrombocythemia and idiopathic myelofibrosis. All these myeloproliferative disorders are also characterized by hypersensitivity of hematopoietic progenitor cells to several growth factors and cytokines. Because the frequency of JAK2 V617F mutation in JMML remains unknown, we performed mutational analysis of JAK2 in 39 Japanese children (aged 1 to 69 months) with JMML to assess the pathogenetic relevance of this mutation in JMML, in addition to mutational analysis of PTPN11 and N-RAS. Genomic DNA were prepared from bone marrow or peripheral blood samples taken at initial diagnosis. Mutations of JAK2 (exon 12), PTPN11 (exon 3 and 13) and N-RAS (exon 1 and 2) were screened by direct sequencing. PTPN11 or N-RAS mutations were found in 19 (49%) or 3 (8%) patients respectively, and this is compatible with previous reports. In this study, no JAK2 V617F mutations were detected in any of the 39 JMML patients. In conclusion, we confirmed the presence of PTPN11 and N-RAS mutations in JMML. However, the JAK2 V617F mutation appears to be uncommon in patients with JMML, while it is extraordinarily common in classic MPD. Future investigation should focus on other signaling components in the same signaling pathway as JAK2, or on other parallel signal transduction cascades of the GM-CSF receptor.
Bone Marrow Fibrosis in Chronic Idiopathic Myelofibrosis Is Associated with Megakaryocyte Expression of Osteoprotegerin.
Authors: Centkowski Piotr , Sawczuk-Chabin Joanna , Prochorec-Sobieszek Monika , Dobrzanska Joanna , Seferynska Ilona , Solarska Iwona , Majewski Miroslaw , Warzocha Krzysztof
Published: October 2019

A feature that distinguishes chronic idiopathic myelofibrosis (CIM) from other chronic myeloproliferative disorders is the progression to bone marrow fibrosis and osteosclerosis. Because osteoclast formation can be inhibited by osteoprotegerin (OPG), we investigated its megakaryocytes’ expression and serum concentration in patients (pts) with CIM (n=20, median age 64 years, range 46–81) and in 20 controls showing no evidence of neoplastic disease (median age 62 years, range 46–82). The study group comprised 10 pts with prefibrotic cellular CIM and 10 with severe fibrosis. Assessment of OPG concentration in serum was performed using Osteoprotegerin ELISA Kit (Biomedica, Austria). EnVision (DAKO) kit with monoclonal anti-human osteoprotegerin/TNFRSF11B antibody - MAB8051 (R&D Systems, USA) was used to evaluate OPG expression in megakaryocytes. Gomori silver staining technique was applied for semi-quantitative assessment of bone marrow fibrosis according to increased amount of reticulin fibers. OPG serum levels in pts with CIM were significantly higher than in controls (105.78 vs 85.14 pg/ml, p=0.02), and showed positive correlation with age in both groups (r=0.61, p=0.0037 and r=0.55, p=0.012, respectively). Serum levels of OPG in pts with CIM patients were not associated with erythrocyte, leukocyte, platelet numbers and with the Visani, Lille or Cervantes prognostic scores. There was no correlation between serum levels of OPG and its expression in megakaryocytes in pts and controls. In megakaryocytes derived from patients with CIM as well as from controls, OPG expression was detected, but in those derived from CIM hematopoiesis (at any stage of the disease) were significantly higher. OPG expression in megakaryocytes derived from prefibrotic CIM was significantly lower than from advanced stages of the disease (p=0.007).

We conclude that OPG appears to be involved in bone marrow fibrosis in CIM through overexpression by megakariocyte.
Prognostic Significance of Biochemical Markers of Bone Metabolism in Patients with Bone Lesions from Multiple Myeloma (MM).
Authors: Cook Richard J. , Coleman Robert E. , Major Pierre , Lipton Allan , Brown Janet E. , Lee Ker-Ai , Smith Matthew , Saad Fred , Chen YinMiao , Hei Yong Jiang
Published: October 2019

BACKGROUND: Biochemical markers of bone metabolism are useful indicators of bone turnover in pts with malignant bone disease. Elevated N-telopeptide (NTX) levels were correlated with increased risks of skeletal-related events (SREs), disease progression, and death in pts with primary bone lesions from MM (Coleman RE, et al. J Clin Oncol. 2005). The prognostic value of other markers (eg, pyridinoline [PYD] and deoxypyridinoline [DXP]) in pts with MM is unknown.

METHODS: This study investigated the association between NTX, PYD, DXP, and bone alkaline phosphatase (BAP) levels and risk of SRE or pathologic fracture in pts with MM who were treated with 4 mg zoledronic acid in a phase III clinical trial (Rosen LS, et al. Cancer J. 2001). Cut-off values for low, moderate, and high marker levels were based on prior reports and the investigators’ experience with bone marker data. Both univariate and multivariate analyses were performed and risk ratios were derived for pts with elevated levels of each bone marker relative to pts with low marker levels.

RESULTS: By univariate analysis, moderate (50 to 100 nmol/mmol creatinine) and high (> 100 nmol/mmol creatinine) NTX levels corresponded with significantly increased risks of any SRE, onset of first SRE, or pathologic fracture. Elevated levels of PYD corresponded with a significantly increased risk of SREs, the onset of SREs, and pathologic fracture. Although there were some significant correlations, the association between DXP and BAP levels and risk of skeletal morbidity was inconsistent (Table). In multivariate models, only NTX and DXP were significant, and NTX had a stronger effect.

Risk Ratios By Univariate Analysis for SRE Compared With the Respective SRE Pathologic fracture Any Onset (1st) Any Onset (1st) NTX ≤ (vs 50 nmol/mmol creatinine) > 50 – 100 2.26 (.003) 2.76 (.012) 2.98 (< .001) 3.78 (.001) > 100 4.01 (.001) 6.80 (< .001) 5.35 (< .001) 8.87 (< .001) DXP ≤ (vs 15 nmol/mmol creatinine) > 15 – 30 1.95 (.016) 2.14 (.015) 2.30 (< .001) 2.29 (.009) > 30 1.89 (.041) 3.58 (.006) 1.74 (.134) 2.88 (.046) PYD ≤ (vs 62 nmol/mmol creatinine) > 62 1.89 (.009) 2.06 (.006) 1.74 (.028) 1.97 (.017) BAP ≤ (vs 146 U/L) > 146 1.48 (.103) 1.89 (.042) 1.69 (.042) 1.86 (.063)

CONCLUSIONS: Elevated baseline PYD, DXP, and BAP levels are significant predictors of an increased risk of some SREs and may therefore have clinical value. Elevated NTX levels are a consistent significant predictor of an increased risk of SREs and pathologic fractures. NTX levels may therefore aid in identifying patients at risk for skeletal complications.
Rituximab Added to Cyclophosphamide, Vincristine and Prednisolone in Heavily Pretreated Chronic Lymphocytic Leukemia.
Authors: Coelho Henrique , Gonçalves Cristina , Casais Claudia , Guerra Marisol , Mota Alexandra , Ribeiro Antonio P. , Coutinho Jorge
Published: October 2019

Purpose: Although patients with chronic lymphocytic leukemia (CLL) typically respond to available front-line chemotherapy, the response is often temporary, and patients frequently undergo relapse. The authors report the beneficial clinical activity and tolerability of an immunochemotherapeutic regimen consisting of rituximab added to cyclophosphamide, vincristine and prednisolone (R-CVP), in patients with heavily pretreated CLL.

Methods: The therapeutic regimen consisted of: cyclophosphamide 750 mg/m2 iv on day one, vincristine 1.4 mg/m2 iv on day one and prednisolone 100 mg po for five consecutive days every 4 weeks, combined with rituximab at a dose of 375 mg/m2 on day 1 of each cycle. Patients were evaluated after three cycles, with responding patients receiving up to eight cycles. No infectious prophylaxis was administered.

Results: Five patients have been treated with R-CVP: median age 65 years (range 52–68), advanced Binet stage (four stage C and one stage B) and heavily pretreated (range 1–3 prior therapies). All patients received at least three cycles of R-CVP. Clinical and hematological response was achieved in two patients. Three patients had stable disease. One patient was re-induced once after disease progression. Data from a median follow-up of 112 months (range 52–128 months) showed that three patients remained free of disease progression 8 months after the first cycle of R-CVP (range 2–14 months). The principle treatment complication was pulmonary aspergillosis.

Conclusion: These promising results suggest salvage therapy with R-CVP is beneficial in patients with heavily pretreated CLL.
Pentostatine Is a Safe and Active Agent in Chronic Lymphocytic Leukemia CLL with Minimal Toxicity.
Authors: Jajeh Ahmad , Tamkus Deimante , Berko Ebinezer , Ogandipe Alusola , Osafo David , Yim Barbara , Zalzaleh Ghassan
Published: October 2019

Pentostatin is a nucleoside analogue, it is a potent irreversible inhibitor of adenosine diaminase ADA. The triphosphate form of pentostatin is incorporated into DNA strand breaks. This effect is potentiated by the presence of an alkylating agent such as Cytoxan. Combination approaches will maximize the inhibition of DNA repair with less myelosuppression. A total of twenty five patients diagnosed with chronic lymphocytic leukmia treated with pentostatin combination in the last four years, nineteen patients had refractory CLL. Six patients has newely diagnosis with adverse features such as high risk cytogenetic/FISH abnormality, elevated serum Beta 2 microglobuline, elevated LDH, presence of the B symptoms, doubling time less than a year and progressive enlargment of spleen and lymph nodes. All patients had stage III-IV Rai classification. Fifteen males and ten females, eighty five percent were african american. Mean age is 52 years (range 46–70). Median follow up was eighteen months (range4–42 months). Thirteen patients recieved the combination of Pentostatin, Cytoxan and Rituxan. Rituxan or Rituximab is a chiemeric human monoclonal antibodies against CD expressing B cell lymphocytes. Pentostatin 4 mg/M2, Cytoxan 300 mg/M2 and Rituxan 375 mg/M2 given one day one every 28 days. Three patients recieved pentostatin and cytoxan. Nine patients recieved pentostatin and rituxan. Eighty percent of the patients recieved a minimum of six cycles. Complete response CR and near complete response nCR were confirmed by peripheral blood flowcytometric study. Ten patients achieved CR, nCR and fifteen patients achieved PR. One patient had short PR response less than six months post completing therapy. Five patients had autologous bone marrow transplant post CR, nCR response. Median time to progression is not reached. Toxicity observed with pentostain combination was minimal as compaired to retrospective analysis of Fludarabin whether as a single agent or in combination with cytoxan or rituxan. Bone marrow necrosis and tumor lysis syndrome were not seen. Neutropenic fever that required hospitaliztion is less than 10% as compaired to Fludarabin 40%. Growth factor G-CSF required less with pentostin combination. However, warm autoimmune hemolytic anemia was seen more during pentostatin therapy rather than prior to initiating therapy.

Conclusion: Pentostatin has less myelosuppressive effect and equal response as compaired retrospectively to Fludarabine. Head to head randomized prospective study is needed.
Serum Levels of OPG and MIP-1α in Untreated Multiple Myeloma Patients. Correlations with Staging, Survival and Bone Disease.
Authors: Papadogiannis Argyro , Kyrtsonis Marie-Cristine , Vassilakopoulos Theodoros P. , Tzenou Tatiana , Antoniadis Antonios G. , Kalpadakis Christina , Sachanas Sotirios , Dimopoulou Maria N. , Siakantaris Marina P. , Kokoris Styliani I. , Dimitriadou Evangelia M. , Angelopoulou Maria K. , Plata Eleni , Tsaftaridis Panagiotis , Pangalis Gerassimos
Published: October 2019

Cytokines, such as MIP-1a (macrophage inhibiting factor) and OPG (osteoprotegerin) are assumed to play a role in MM disease biology and bone disease, by mechanisms that are still under investigation. MIP-1a is constitutively secreted by myeloma cells, plays a possible role in the development of MM bone lesions, enhance MM cell adhesion to stromal cells and its serum levels have been recently related to survival in MM. OPG is a soluble decoy receptor which acts as a soluble receptor antagonist that prevents osteoclasts activation and the development of bone disease. Reported findings on serum OPG levels in MM patients are controversial as well as its possible role in the biology of the disease. In order to investigate the possible relationship of MIP-1a and OPG levels in MM patients with prognosis and bone disease, we determined by ELISA serum MIP-1a and OPG levels in 20 MGUS, 82 MM patients and 27 healthy individuals (HI). Both cytokines were determined by ELISA (R&D, Quantikine, USA) in frozen sera collected at dignosis, before treatment. The median age of MM patients was 69 years (44–84) and 50% were males. MM patients’ stage was as follows: 23 stage I, 28 stage II, 31 stage III according to Durie-Salmon staging system and 27 stage I, 17 stage II, 35 stage III according to the International Scoring System (ISS). In HI median MIP-1a was 28 pg/ml (15–54) and median OPG 1600 pg/ml (450–4600). In subjects with MGUS, median MIP-1a was 34 pg/ml (17–58) and median OPG 2300 pg/ml (820–6200). In MM patients, median pretreatment serum MIP-1a was 32 pg/ml (16–100) and OPG 3000 pg/ml (820–25000). No statistical significant difference was observed between HI, MGUS and MM patients with regard to MIP-1a levels but for OPG levels differences between HI and MM patients and between MGUS and MM patients were both significant (0.01 and 0.05 respectively). No relationship was found between MIP-1a or OPG levels and bone disease. However, there was a trend for longer survival in patients with MIP-1a or OPG levels lower than median (5-year overall survival 60 ± 12 vs 38 ± 14, p=0.08 and 66 ± 13 vs 29 ± 13, p=0.07 respectively). In addition MIP-1a levels were correlated with ISS stage: MIP-1a levels were 28.3±11.3 in ISS stage 1, 29.8±11.1 in ISS stage 2, 39±19.2 in ISS stage 3 (p=0.02). In conclusion, in our experience serum OPG levels are higher in MM patients than in MGUS or HI, MIP-1a levels are correlated with the ISS stage and both high serum MIP-1a and OPG levels at diagnosis are related with a shorter survival.
Interleukin-6 Promoter and Receptor Polymorphisms, Body Mass Index and Risk of Multiple Myeloma.
Authors: Cozen Wendy , Gebregziabher Mulugeta , Vandenberg David , Conti David , Coetzee Gerhard , Bernstein Leslie , Wang Sophia S. , Rothman Nathaniel , Hartge Patricia , Wang Wei , Coetzee Simon , Ingles Sue Ann
Published: October 2019

Interleukin-6 (IL-6) is a cytokine necessary for successful differentiation of plasma cells from B lymphocytes and high levels have been linked to prognosis of multiple myeloma. Several studies have examined a common functional single nucleotide polymorphism in the promoter region of the IL-6 gene (−174G→C) with null results. Terry et. al. reported that at least three promoter polymorphisms contribute to regulation of IL-6 levels (Terry, 2000). We evaluated the four promoter polymorphisms described by Terry et. al. plus an IL-6 receptor polymorphism in a case-control study to determine if any were associated with multiple myeloma risk. Blood samples and questionnaires were obtained from incident cases (n=150) ascertained from the population-based cancer registry in Los Angeles County. Similar samples were collected from two control groups consisting of siblings or cousins of cases (n=114) and population-controls obtained by random digit dialing (n=131). DNA was extracted and amplified by PCR and the −174 G→C, −572 G→C, −597 G→A promoter polymorphisms determined using Taqman. The −373 AnTn VNTR was determined by direct sequencing and IL-6 receptor A→D polymorphism was determined using RFLP. When cases were compared to relative and population controls, risk of multiple myeloma increased by 71% and 67% in persons carrying the −572 GC genotype compared to those with the GG genotype, respectively (odds ratio [OR] for cases v. relatives = 1.71, 95% Confidence Interval [CI ]= 1.10–2.78; OR for cases v. population-based controls =1.67, 95% CI=1.10–2.70). The effect was stronger among African-Americans, but not statistically significant (OR for cases v. relatives = 3.07; OR for cases v. population controls = 2.52). The other IL-6 promoter polymorphisms and receptor polymorphism had no effect on multiple myeloma risk. Increased body mass index was a statistically significant predictor of multiple myeloma risk when cases were compared to population controls but not when compared to relatives, and the effect was stronger in whites than African-Americans. We conclude that the −572 IL-6 promoter polymorphism is associated with multiple myeloma risk in both African-Americans and whites. Increased body mass index may be a risk factor for multiple myeloma in some groups. Finally, since the estimates of risk were very similar for the two comparison groups, relative controls offer a good alternative for genetic studies when population controls are difficult to recruit or are not available.

IL-6 Promoter and Receptor Polymorphisms and Multiple Myeloma Risk −174 G>C −572 G>C −597 G>A −373 AnTn Receptor A>D OR OR OR OR OR (95% CI) Odds Ratio [OR] adjusted for race, age, gender and body mass index Population Controls 0.98 1.67* (p<0.05) 0.90 1.10 0.92 Relative Controls 0.97 1.71* (p<0.05) 0.95 1.10 1.03
Thalidomide Response in African American Patients with Multiple Myeloma.
Authors: Jajeh Ahmad , Catchatourian Rose , Tamkus Deimante , Osafo David , Zalzaleh Ghassan , Yim Barbara
Published: October 2019

African american are not well presented in clinical trials. This is a retrospective analysis studying Thalidomide response in predominantly african american patients with multiple myeloma from January 2000 till August 2005. A total of fifty patients diagnosed with multiple myeloma started on Thalidomide which was given through the S.T.E.P. Program. Twenty one males and twenty nine females. Thirty six african americans (72%), eleven hispanics (22%), three whites and one other. Mean age at starting treatment is 63 years (range 41–76). All were stage II-III. Mean serum Albumin is 3.4mg/dL (range 2.2–4.7) and Beta 2 microglobulin is 3.8 (range 0.60–13.6), the normal range is 0–0.63 mg/dL. Twenty four patients with IgG kappa, twelve patients with IgG lambda, three with lambda light chain, three with kappa light chain, seven patients with IgA and one patient with non secretory MM. Mean Thalidomide dose is 100 mg Oraly, daily (range 50–200 mg). Medium duration of therapy is one year (range 3 months- three and a half years). Two patients stopped in the first few weeks because of intolerance due to side effects. Eighty percent of patients given treatment after failure to prior therapies. Fifty percent of patients recieved Thalidomide with Dexamethason. Response were evaluated as of Blade criterae. Seven patients (14%) achieved complete response CR or very good partial response. Partial response PR was achieved in nine patients (18%), minimal response was achieved in three patients (6%) and stable disease in six (12%). Total response rate of fifty percent(50%). Fifteen patients (30%) achieved a rise in back ground depressed serum level of IgA (range 1–3 folds of normal value). Two patients(4%) achieved arise in the back ground depressed level of IgG in IgA MM. Median time to progression in patients with CR and PR was not reached. However most of the progression occured at one and a half years from response.

Conclusion: Thalidomide alone or in combination with Dexamethasone is a well tolerated regimen. Low dose as 50 to 100mg seem to be effective and tolerated in most of the african american patients. The rise in IgA level is an interesting phenomena. This is particularly seen in CR and very good partial response. Whether this is an immune response or an epiphenomena need to be investigated.
Class Prediction Models of Thrombocytosis Using Gene Profiling.
Authors: Gnatenko Dmitri V. , Zhu Wei , Dhundale Anilkumar , Bahou Wadie F.
Published: October 2019

Human blood platelets contain unique and reproducible molecular signatures consisting of relatively small number of individual mRNA transcripts. Towards the goal of developing a molecular classification of platelet disorders, we have now developed and applied a unique microarray platform for the study of thrombocytosis. A 432-member gene list encompassing a subset of platelet- and leukocyte- restricted transcripts (with Arabidopsis probe elements as normalization controls) was used for the manufacturing of a customized platelet oligonucleotide (70-mer) gene chip (POC). In silico gene rank-intensity plots from sample RNAs [normal platelets (N=5); essential thrombocythemic platelets (ET; N=6); leukocytes (N=3)] hybridized to the Affymetrix HU133a gene chip validated the discriminatory capacity of the POC, which was then applied to a broader patient cohort. Gel-filtered platelets from normal controls (N=28), patients with ET (N=18), or patients with reactive thrombocytosis (RT; N=17) were used for RNA isolation, amplification, and Cy3:Cy5 co-hybridization using human universal RNA as reference. Genotyping for the presence of the JAK2 V617F mutation was established by pyrosequencing analysis using both genomic (leukocyte) DNA and platelet-derived mRNA. Initial two-way non-parametric ANOVA identified a 131-member gene list of differentially-expressed transcripts among the three subgroups (p<0.05), with no gender differences in healthy controls. Stepwise discriminant analysis delineated a 14-gene subset that appropriately classified 94% of original cases. Leave-one-out cross-validation using 3 of the 14 markers correctly classified 94% ET and 100% RT patients, independent of the presence of the JAK2 V617F mutation. Leave-one-out cross-validation using 6 of the 14 markers also correctly subclassified 88% ET patients with JAK2 V617F mutation (N = 8) and 90% of ET patients without such mutation (N = 10). These data (1) provide the first evidence for the existence of discriminatory molecular signatures among etiologically-diverse causes of thrombocytosis, (2) demonstrate the presence of distinct ET transcriptomic profiles substratified by JAK2 V617F genotype, and (3) provide the foundation for development of a clinically-useful classification scheme of genetic platelet disorders.
A Critical Role for CCL Chemokines in the Immuno-Protection Induced by Type I Interferons and IRF8/ICSBP Against Bcr/Abl-Induced Leukemia.
Authors: Nardi Valentina , Naveiras Olaia , Azam Mohammad , Daley George Q.
Published: October 2019

Until recently, the mainstay of Chronic Myelogenous Leukemia (CML) therapy was Interferon (IFN) alpha, which in a minority of patients induces long lasting cytogenetic remission. While the exact mechanism of action of IFN alpha in CML is still obscure, it is clear that the clinical response to IFN alpha correlates with immune system reactivity against leukemic clones. As minimal molecular disease often persists despite the use of imatinib and new Bcr-Abl inhibitors, immunotherapy remains an appealing adjunct to molecularly targeted inhibitors in CML therapy. We have shown that IRF8/ICSBP (Interferon Consensus Sequence Binding Protein) expression in Bcr-Abl transformed cells prevents their capacity to form a lethal leukemia when injected into mice, and that this protection is mediated by a long-lasting and potent CD8+ response against unknown epitopes on the leukemic cells. We hypothesized that the protection mediated by IRF8/ICSBP might be related to the anti-leukemic effects of IFN alpha. We now find that Type I IFNs like IFN alpha regulate IRF8/ICSBP expression in mouse and human cells and in Bcr-Abl transformed cells. Furthermore, type I IFNs can substitute for ICSBP in inducing the anti-leukemic immunity against Bcr-Abl transformed cells. Transcriptional profiling of cells expressing ICSBP, Bcr-Abl, or both ICSBP and Bcr-Abl identified two chemokines, CCL6 and CCL9, which were associated with the immune protection induced by IRF8/ICSBP expression. Type I IFNs and IRF8/ICSBP induce the expression of these chemokines in cells transformed with Bcr-Abl. RNAi-mediated inhibition experiments in our mouse model of CML show that these chemokines are required for the IRF8/ICSBP-mediated CD8+ anti-leukemic response to the Bcr-Abl transformed cells, suggesting that these chemokines could be exploited for immunotherapy in combination with existing Bcr-Abl peptide vaccines.
Degrasyn-Induced Trafficking of BCR-ABL as a Novel Mechanism of Kinase Inactivation.
Authors: Donato Nicholas , Kapuria Vaibhav , Sun Hanshi , Maxwell David , Bartholomeusz Geoffrey , Bornmann William , Peng Zhenghong , Pal Ashutosh , Talpaz Moshe
Published: October 2019

Inhibitors that inactivate specific tyrosine kinases have proven to be a very effective form of therapy of many leukemias and hematopoetic disorders. Most inhibitors function by competing for the ATP-binding pocket or by preventing association with protein substrates. However, clinical and molecular studies have shown that small changes in the structure of the target kinase (point mutations, post-translational modification) affect inhibitor binding affinities, resulting in resistance to this class of inhibitor. Therefore, development of agents that reduce the activity of leukemogenic kinases through alternate mechanisms are of great interest. We previously described a novel class of compounds, termed degrasyns, which reduced BCR-ABL (and Jak2) kinase activity through a unique mechanism. Degrasyn treatment of CML or BaF3 cells expressing wild-type or mutant (T315I) BCR-ABL resulted in a reduction of cytoplasmic BCR-ABL protein levels and loss of downstream signaling without a direct effect on BCR-ABL enzymatic activity. Biochemical studies demonstrated that degrasyn induced a rapid translocation of BCR-ABL from the cytosol to the cytoskeletal fraction (complete within 60 min) and this event correlated with loss of BCR-ABL signaling and initiation of apoptosis. A GFP-fusion protein composed of wild-type or T315I mutant BCR-ABL demonstrated that degrasyn induced high density translocation to the cytoskeletal fraction, as determined by direct fluorescence imaging. Translocation was specific for BCR-ABL and not other kinases (except Jak2) or signaling proteins. Translocation correlated with degrasyn-mediated tyrosine phosphorylation of a subset of specific proteins, including the tyrosine kinase Lyn, in the insoluble cellular fraction. CML cells expressing high levels of Lyn were more sensitive to degrasyn-mediated apoptosis suggesting that Lyn plays a role in degrasyn activity. Together these results suggest that degrasyn inactivates BCR-ABL by inducing its translocation to a cellular compartment that prevents its participation in oncogenic signaling. Degrasyn-induced BCR-ABL translocation was not effected by mutations that block dasatinib and imatinib activity and shows greater activity against some forms of imatinib resistance (Lyn overexpression). Animal studies demonstrated that degrasyn has anti-leukemic activity and functions through a unique mechanism of action. Development of inhibitors with this mode of action may be of significance for CML patients that fail therapy with ATP-binding pocket-directed tyrosine kinase inhibitors.
Upregulation of CXCR4 Is Essential for the Migration and Survival of Quiescent CML Progenitor Cells in the Bone Marrow Microenvironment after Imatinib Treatment.
Authors: Tabe Yoko , Jin Linhua , Konoplev Sergej , Hongbo Lu , Kimura Shinya , Rios Mary-Beth , Kantarjian Hagop M. , Andreeff Michael , Konopleva Marina
Published: October 2019

Chronic myeloid leukemia (CML) is driven by constitutively activated Bcr-Abl tyrosine kinase, which causes the defective adhesion of CML cells to bone marrow (BM) stroma. The overexpression of p210Bcr-Abl was reported to down-regulate CXCR4 expression, resulting in cell migration defects in CML. We proposed that the tyrosine kinase inhibitors imatinib or INNO-406 may restore CXCR4 expression and cause the migration of CML cells to BM microenvironment niches which results in acquisition of stroma-mediated chemoresistance of CML progenitor cells. In KBM5 and K562 cells, imatinib or INNO-406 increased CXCR4 expression and migration. Imatinib induced a G0/1 cell cycle block in CML cells, which was further enhanced in a bone marrow-derived stromal cell (MSC) co-culture system. Quiescent KBM5 cells migrating to stromal cells were resistant to Imatinib-induced cell death. These anti-apoptotic effects were abrogated by inhibition of CXCR4 (AMD3465) or Integrin-linked kinase (QLT0267). BM CD34+ cells from newly diagnosed (n=15) or blast crisis CML patients (n=5) expressed markedly lower CXCR4 levels compared with normal CD34+ BM progenitor cells (n=12) (p<0.05), and was associated with deficient migration of CD34+ CML cells. A longitudinal analysis of peripheral blood samples from CML patients in blast crisis treated with imatinib (n=5) revealed induction of CXCR4 in peripheral blood CD34+ progenitor cells within the first few days after initiation of treatment, which coincided with decreased WBC and absolute blast counts. After prolonged (3–10 day) exposure to imatinib, however, CXCR4 levels of CD34+ PB CML cells decreased, frequently below the starting levels. CXCR4 immunostaining on blasts/immature cells in BM biopsy sections from patients demonstrated that 5 of 7 patient samples were CXCR4-negative prior to imatinib therapy; 4 of these 5 became CXCR4 positive after imatinib treatment. While patients in blast-phase had short-lived hematologic responses to imatinib, Ph+ clone remained predominant in the bone marrow, and all patients ultimately relapsed with recurrence of blasts in the BM. Altogether, these findings suggest that the up-regulation of CXCR4 by imatinib promotes migration of CML cells to BM stroma, causing G0/1 cell-cycle arrest and enhancing the survival of quiescent CML progenitor cells within bone marrow microenvironment. This provides the rationale for interfering with the protective effects of BM stroma cells by inhibiting CXCR4 and/or integrin signaling, which could be of benefit in eradicating residual quiescent CML cells.
Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.
Authors: Albers Corinna , Illert Anna L. , Miething Cornelius , Peschel Christian , Duyster Justus
Published: October 2019

Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.