bluuebunny/crossref_metadata_2025_split huggingface dataset:
Short Hairpin RNA-Mediated Inhibition of Bcl-2 Gene Increases Susceptibility to Methotrexate and Suppresses Lymphoma Growth In Vitro and In Vivo.
Authors: Fang Baoying , He Dongmei , Zhang Yuan 
Published: November 2019

Abstract
A high level of expression of Bcl-2 is associated with resistance to chemotherapeutic agents and radiation in a number of tumor types, so that a drug to reduce the levels of this protein would be expected to promote apoptosis and would therefore be considered a promising chemotherapeutic agent. At present, gene repression can also be achieved in mammalian cells by using vectors to express small hairpin RNA (shRNA) with a U6 or H1 promoter under the direction of RNA polymerase III. In our lab, we have constructed a U6 promoter based vector expressing shRNA structure targeting against Bcl-2, which could effectively down-regulate Bcl-2 protein. We previously demonstrated that this Bcl-2 shRNA decreased cell proliferation and enhanced radiation-induced apoptosis in Raji cells. In this study, we investigated the synergistic effect of Bcl-2 shRNA combined with methotrexate (MTX) in Molt4, Raji cells and nude mice model bearing human lymphoma.Bcl-2 shRNA was transfected into Molt4 cells and Raji cells with Lipofectamine. At 24,48,72,96 hours after transient transfection, the expression levels of Bcl-2 mRNA and protein were detected by RT-PCR and Western blot. The inhibition of cell growth was assessed by MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytomertry. To examine the effect of Bcl-2 shRNA on proliferation and chemosensitivity against MTX in vivo, human Raji cell line was inoculated into the skin of BALB/c nude mice to establish lymphoma model. After Molt4 and Raji cells were transfected with Bcl-2 shRNA, protein and mRNA levels of Bcl-2 obviously were decreased. MTT assays indicated that the growth of cells transfected with Bcl-2 shRNA were significantly lower than those cells with negative control shRNA and untransfected cells, respectively (P<0.05). Bcl-2 shRNA combined with MTX significantly inhibited the growth of cells (P<0.05). There was no difference in cell survival between control shRNA / MTX combination and cells treated with MTX alone. Using Giemsa staining, cells treated with Bcl-2 shRNA combined with MTX displayed changes of apoptosis. Apoptotic rates of both Molt4 and Raji cells treated with Bcl-2 shRNA combined with MTX significantly increased (P<0.05), compared with either control shRNA / MTX combination or MTX-treatment cells alone. In the assay of s.c. tumors in nude mice, tumor growth was inhibited in Bcl-2 shRNA group compared with that in negative control shRNA, and immunohistochemistry showed that Bcl-2 protein level of tumor was also decreased. The inhibition rate of tumor growth was significantly higher in tumors treated with Bcl-2 shRNA combined with MTX than in either control shRNA / MTX combination or MTX-treatment group (P<0.05). These results suggest that Bcl-2 shRNA increases MTX-induced apoptosis in Molt4 and Raji cells. Moreover, the combination of Bcl-2 shRNA and MTX produces greater antitumor effect.
Restoration of Peroxiredoxins and Catalase Is Closely Correlated with the Level of Philadelphia Chromosome during Imatinib Therapy in CML.
Authors: Lee Kyoung-Eun , Woo Hyun-Ae , Yang Seoug-Ha , Huh Jung-Won , Choi Moon Young , Chang Hye Jung , Ahn Jee-Young , Oh Soo Ah , Mun Yeung-Chul , Yoo Eun-Sun , Rhee Sue Goo , Seong Chu-Myong 
Published: November 2019

Abstract
Background: Several investigators have recently shown that activated growth factor receptors increase the relative levels of intracellular ROS and that bcr/abl kinase induces the production of ROS in hematopoietic cells. In addition, bcr-abl kinase induces self-mutagenesis via ROS to encode IM resistance. Meanwhile, two members of the peroxiredoxin family, Prx 1 or Prx 2, efficiently lowered the intracellular level of H2O2 and blocked the induction of apoptosis by ceramide, suggesting that the Prx enzymes contribute to intracellular signaling by removing H2O2. In this study, we investigated the changse in the levels of H2O2-removing enzymes like Prxs, glutathione peroxidase 1 (Gpx1), and catalase during Imatinib (IM) therapy in CML.
Methods: Mononuclear cells(MNC) were isolated by standard Ficoll-Hypaque from the bone marrow aspiration at the time of diagnosis and during treatment with IM (Gleevec, Novartis, East Hanover, NJ). Standard cytogenetics analysis and RT-PCR for Philadelphia chromosomes were performed. For immunoblot analysis of antioxidant enzymes, cell lysates were fractionated by SDS-PAGE, and the separated proteins were transferred electrophoretically to a nitrocellulose membrane (Protran, Germany) and were probed with antibodies specific for Prx I, II, VI, GPx1, or catalase (AbFrontier, South Korea).
Results: Samples from the diagnosis CML patients showed significantly decreased levels of Prx I, Prx II, Prx IV, and Gpx I, but also revealed an increased level of catalase. Especially prominent was the diminished ratio of Prx II to catalase in CML patients when compared with those of normal individuals. As the level of Philadelphia chromosomes decreased to that of normal individuals as the result of IM treatment, the expression levels of Prx(s) (P=0.018) and catalase (P=0.009) were restored to the levels of normal individuals.
Conclusions: Decreased Prx 2 and elevated catalase levels at the time of diagnosis are closely correlated with the elevated bcr/abl kinase level in CML. The aberrant expression of those antioxidant enzymes returned to normal with IM treatment. Now, we will attempt to develop these method(s) to assess the changes of Prx(s) and catalase on the single cell level using immunohistochemistry with monoclonal Ab(s). Understanding the molecular mechanisms of changes on antioxidant might be a potent tool in developing more effective new drugs in CML.
Liposomal Cytarabine (DepoCyte) for Treatment of Myeloid CNS Relapse in CML Occurring during Therapy with Imatinib.
Authors: Aichberger Karl J. , Herndlhofer Susanne , Agis Hermine , Sperr Wolfgang R. , Rabitsch Werner , Esterbauer Harald , Knoebl Paul , Sillaber Christian , Jaeger Ulrich , Valent Peter 
Published: November 2019

Abstract
Central nervous system (CNS) relapse in chronic myeloid leukemia (CML) is rare and if recorded is usually found to occur in patients with lymphoblastic transformation or in those with a generalized myeloid relapse. The BCR/ABL tyrosine kinase (TK) inhibitor imatinib is highly effective in patients with CML, but hardly crosses the blood-brain-barrier. We report on two CML patients who developed a myeloid CNS relapse during treatment with imatinib. One patient was in major cytogenetic response at the time of CNS relapse. In both cases, the myeloid origin of neoplastic cells in the cerebrospinal fluid (CSF) was demonstrable by immunophenotyping, and their leukemic origin by detection of the BCR/ABL oncoprotein. No BCR/ABL kinase domain mutations could be detected. Both patients received intrathecal liposomal cytarabine (DepoCyte®) (50 mg each cycle; 6 cycles). In one patient, additional CNS radiation was performed, whereas in the other patient, consecutive treatment with dasatinib (70 mg per os twice daily) was started. In response to therapy, the clinical symptoms resolved and the leukemic cells in the CSF disappeared in both patients. After four months of observation, both patients are in complete cytogenetic and major molecular response, without evidence for a systemic or a CNS relapse. In conclusion, ‘anatomic’ resistance against imatinib in the CNS can lead to an (isolated) myeloid CNS relapse. Liposomal cytarabine with or without radiation is effective as local therapy in these patients. For treatment of patients with a systemic relapse involving the CNS and for prophylaxis, second-generation BCR/ABL TK inhibitors crossing the blood-brain-barrier such as dasatinib should be considered.
Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).
Authors: Mascarenhas Cintia C. , Cunha Anderson F. , Pagnano Katia B.B. , Silveira Rosana A. , Costa Fernando F. , Pasquini Ricardo , Clementino Nelma C.D. , De Souza Carmino A. 
Published: November 2019

Abstract
Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.
Anti-Erythroblast Autoimmunity in Pediatric Myelodysplatic Syndromes.
Authors: Caruso Roberta , Funaro Daria , Pansini Valeria , Zaninoni Anna , Imperiali Francesca G. , Barcellini Wilma , Zanella Alberto 
Published: November 2019

Abstract
Introduction: Myelodysplatic syndromes (MDS) are characterized by ineffective and dysplastic hematopoiesis and peripheral cytopenias. Moreover, autoimmune phenomena, mainly directed against RBC, are described in early MDS, i.e. refractory anemia (RA) and RA with ringed sideroblasts (RARS). We already reported an autoimmune reactivity against erythroblasts in 50% of RA and RARS adult patients, along with peripheral signs of hemolysis, increased BM erythroblasts; and efficacy of steroid therapy. It has become evident that there are significant differences between MDS in children and adults. Most importantly, the cure is the aim of therapy in all children with MDS, while therapeutic possibilities are often limited in adults. MDS and myeloproliferative disorders are rare in childhood and there is no widely accepted system for their diagnosis and classification. Aims of this study were:
To evaluate BM autoimmunity in 9 pediatric patients with MDS by the same method used in adults, named mitogen-stimulated-direct antiglobulin test (MS-DAT), to correlate BM MS-DAT positivity with clinical parameters and therapy.
Methods: MS-DAT was performed by stimulating BM with PMA and PHA and antibodies were detected in supernatants by competitive solid phase ELISA.
Results: We demonstrated the presence of anti-erythroblast autoimmunity in roughly half of pediatric MDS patients, as already reported in adult MDS. More precisely, 4 out of 9 patients showed positive MS-DAT in BM. At variance with adult MDS, positive patients do not show increased erythroblast counts and peripheral signs of hemolysis (i.e higher reticulocytes, indirect bilirubin, and LDH, and lower haptoglobin) compared with MS-DAT negative ones. One of the four patients with anti-erithroblasts autoimmunity has RA, the other three show peripheral and medullary signs of refractory cytopenia with multilineage dysplasia (RCMD). Two out of four were successfully treated with steroid therapy (with a follow up respectively of 6 and 1 months).
Discussion: Although the short follow up and the limited number of patients does not allow definitive conclusions, we retain useful to investigate anti-erythroid autoimmunity in all pediatric MDS in order to identify selected patients with low risk MDS who display autoimmune phenomena. In these cases immunosoppressive therapy may be an effective treatment option.
Prognostic Factors for Myelodysplastic Syndrome in the Veteran Population: Single Institution Experience.
Authors: Axelson Michael , Reddy Shirisha , Lumby Crystal , Sivess-Franks Sue , Dowell Jonathan , Parmar Simrit 
Published: November 2019

Abstract
Background: Myelodyplastic syndrome (MDS) is the disease of the elderly and increasingly common in the veteran population. Here we report a single institution experience with MDS at the Dallas VA Medical Center.
Patients and Method: From a period of 1998–2007, eighty three pts were identified out of which 54 pts had bone marrow (BM) biopsy proven diagnosis of MDS. Overall survival (OS) analysis with dependent variables (Age at diagnosis, IPSS Score, WHO morphologic diagnosis, number of blood and platelet transfusions required, Hb level, ANC, cytogenetics, blast percentage, BM cellularity at diagnosis) were conducted by selection method “foreward” and only these significant variables were used in the Cox regression for multivariate analysis. Methods of Kaplan and Meier were used to generate OS curves.
Results: The median age of diagnosis was 74 yrs with a median follow up time of 12.5 months. The WHO morphologic subtype was RA/RARS (n=13), Del5q (n=1), RCMD/RCMDRS (n=34), RAEB1 (n=3), RAEB2 (n=1), missing (n=2). The distribution of IPSS score was 0 (n=25); 0.5 (n=15); 1.0 (n=8), 1.5 (n=4), missing (n=2). Five pts had treatment related MDS and 3 pts transformed to AML. One patient had concurrent MGUS and one patient developed multiple myeloma. At diagnosis, 23 pts had a hemoglobin (Hb) value of less than 10g/dl. Only 4 pts had ANC less than 500; sixteen pts had ANC 500–1800 and 34 pts had normal counts. A majority of pts had normal cytogenetics (n=37), 5 pts had good risk, 5 pts had intermediate risk and 7 pts had poor risk cytogenetics. Six pts had hypocellular (<30%) BM at diagnosis whereas 16 pts had a hypercellular marrow (> 50%). Only 4 pts had more than 5% blast in the BM. Twenty nine pts eventually became blood transfusion dependent and 12 pts needed platelet transfusion at some point. Thirty six pts were treated with erythropoietin (with or without neupogen) and 13 pts received some type of disease modifying therapy (5-azacytidine/lenalidomide/ATG/clinical trial). The mean survival time was 106 months. Median survival was not reached at the time of analysis. In the univariate analysis, IPSS score (p=0.003), No. of blood transfusions (p=0.028), cytogenetics (p=0.0001) and blast percentage (p=0.0015), were statistically significant. BM cellularity (p=0.06) and Hb level (p=0.09) showed a trend towards significance. On multivariate analysis, Hb greater than 10 (HR 0.08; p=0.011), abnormal cytogenetics (HR 4.2; p=0.001), BM Blast > 5% (p=0.026) and BM cellularity < 30% (HR 4.6; p=0.033) emerged as the significant predictors of overall survival. IPSS score or Blood transfusion requirement did not pan out to be significant.
Conclusion: MDS in the veteran population may be different from general population and may have unique predictors of survival. A larger number of patients and longer duration of follow up is required to further evaluate these prognostic factors.
Prevalence of Polycythemia Vera and Essential Thrombocythemia.
Authors: Ma Xiaomei , Vanasse K. Gary J. , Selinger H. Andrew , Wang Yun , Cartmel Brenda 
Published: November 2019

Abstract
Polycythemia vera (PV) and essential thrombocythemia (ET) are two common types of myeloproliferative disorders (MPD). The prevalence of PV and ET in the United States (US) has not been well documented. Recent breakthroughs in the molecular etiology of these disorders and the accelerated development of targeted pharmacotherapeutics to treat the MPD have significantly increased the need to accurately define the affected population. In the present study, we obtained detailed demographic and health claims data from major commercial insurance payers in Connecticut and the Center for Medicare and Medicaid Services to estimate the prevalence of PV and ET. Health claims data from one payer and the actual diagnoses made by physicians who submitted claims with MPD-related ICD-9 codes were utilized to develop claim-based statistical algorithms to predict the probability that an individual with claims suggestive of MPD truly has PV or ET. Specifically, logistic regression was used to develop the algorithms, and area under the receiver operating characteristics curve (AUC) was used as the measure of goodness-of-fit for each model. Different models were fit, and the model with the highest AUC was selected. For PV, the best-fitting model included age as a continuous variable and the frequency of two PV-related ICD-9 codes (238.4 and 289.6 combined) as a continuous variable, and the AUC was 0.95. For ET, the best-fitting model included age as a continuous variable and the frequency of code 289.9 as a categorical variable (=0, =1, or ≥2), and the AUC was 0.72. For both models, the addition of gender or the frequency of the non-specific code 238.7 did not improve AUC. Subsequently, the algorithms were applied to health claims from multiple payers to estimate the number of PV and ET patients in Connecticut. The total number of Connecticut residents included in the study was close to 2,900,000, which represented about 83.2% of the estimated population of the entire state in July 2003. As of 2003, the age-standardized prevalence of PV and ET in Connecticut was 22 per 100,000 and 24 per 100,000, respectively. Applying the age-specific prevalence of PV and ET to the entire US population resulted in an estimated total of 65,243 patients with PV and 71,078 patients with ET in the US in 2003. This study is the first to assess the prevalence of PV and ET in a very large US population. Given the large number of individuals afflicted with these diseases and the fact that demographic changes alone will further increase the burden of these diseases in the foreseeable future, it is imperative to conduct more systematic research into the etiology and treatment of PV and ET.
Incidence of Polycythemia Vera in the Elderly.
Authors: Ma Xiaomei , Selinger H. Andrew , Wang Yun , Cartmel Brenda 
Published: November 2019

Abstract
Due to the lack of large population-based studies, the incidence of polycythemia vera (PV) has not been well documented in the United States. In 2001, PV became reportable to the Surveillance, Epidemiology, and End Results (SEER) program, which consists of high-quality population-based cancer registries supported and sponsored by the National Cancer Institute. In the present analysis, we accessed the SEER data to estimate the incidence of PV in the United States during 2001 – 2003. Since the addition of PV to SEER reporting is fairly recent, and the diagnosis of PV is different from that of many other types of cancer, especially solid tumors, under-reporting is possible. Therefore, we also used health claims data from Medicare services to estimate the incidence of PV among individuals 65 years and older. SEER data suggest that the age-adjusted incidence rate of PV was 0.87 per 100,000 per year (95% confidence interval 0.83 – 0.91) during 2001 – 2003, which is lower than the estimates from other studies (Ania BJ et al. Trends in the incidence of polycythemia vera among Olmsted County, Minnesota residents, 1935–1989. Am J Hematol1994;47(2):89–93. Johansson P. Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. Semin Thromb Hemost2006;32(3):171–3). The incidence of PV increased with age and was higher in men and white individuals than in women and other racial groups. The SEER-derived age-adjusted incidence rate of PV was 4.1 per 100,000 per year among individuals 65 years and older during 2003, which is considerably lower than the estimates made from nationwide Medicare claims data in the same age group during the same year, suggesting possible under-reporting to SEER. The addition of PV to SEER reporting represents a major step in generating population-based epidemiologic data for this understudied malignancy. However, it is critical to ensure the completeness in the ascertainment and reporting of PV so high-quality SEER data can be utilized to facilitate much needed research on the etiology and outcomes of PV, as well as other myeloproliferative disorders.
JAK2 Mutations Are Present in All Cases of Polycythemia Vera.
Authors: Wang Y. Lynn , Vandris Katherine , Jones Amy V. , Cross Nicholas C.P. , Christos Paul J. , Adriano Fernando , Silver Richard T. 
Published: November 2019

Abstract
The JAK2V617F point mutation has been described in 65 to 97% of patients with polycythemia vera (PV). Previously, 17 phenotypical cases of PV from a large group of patients from three institutions were initially reported as JAK2V617F negative. These cases were re-analyzed in detail to determine the cause, if any, of the JAK2V617F negativity. Reasons for this initial negativity included inaccurate clinical diagnosis, relatively insensitive laboratory techniques, insufficient material for re-testing, and possible treatment effect. It was predicted that when tested appropriately, a JAK2V617F mutation would be found in all new cases of PV. We developed a highly sensitive amplification refractory mutation system assay (ARMS) for the detection of JAK2V617F. The analytic sensitivity of the assay is 0.05-0.1% as defined by mixing JAK2V617F-containing HEL cells with normal peripheral blood mononuclear cells. At this level of sensitivity, the assay has a false positive rate of less than 1% (we found 1 positive case in 300 individuals without myeloid disorders). Pyrosequencing involves synthetic nucleotide extension by DNA polymerase. Unlike ARMS, the PCR pyrosequencing method reliably detects JAK2V617F only when the mutant allele is more than 5%. The sensitivity of the ARMS assay is therefore approximately 50 times greater than the pyrosequencing method. Of 105 PV cases diagnosed by Polycythemia Vera Study Group criteria, pyrosequencing detected the mutation in 96 cases. The ARMS technique detected JAK2V617F alleles in 8 of the remaining 9 cases. Of the 8, 2 had been treated by phlebotomy only, 4 had been on interferon for a median of 13 years, and 2 on imatinib for 3 years. In the last patient, who was negative by both assays, the JAK2 exon 12 deletion (E543-D544) was detected. This patient had been on imatinib for 3 years and remains in complete remission. We conclude that a sensitive assay is required to detect the JAK2 mutations in patients with polycythemia vera, especially in patients whose JAK2 mutation load might be affected by treatment.