bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Iodine-131-Rituximab on B Cell Lymphoma In Vitro and In Vivo.
Authors: Luo Rongcheng , Zuo Qiang , Wei Li , Liang Wangjun , Zhen Dayong , Luo Yuling , Yan Xiao , Yang Yang
Published: November 2019

The purpose of this study was to investigate the cell specific cytotoxic effect of Iodine-131 Rituximab on CD20-positive B cell lymphoma in vitro and on Raji cell tumors grown in vivo. Rituximab was labeled with Iodine -131 by the iodogen method. Cultured Raji cells or the nude mice bearing Raji tumors were treated with various concentrations of Iodine-131-Rituximab or Iodine-131 alone or Rituximab alone. The results showed that

The lethal effect was found on Raji cells treated with Iodine-131-Rituximab in a dose-dependent manner; The proliferation rate of Raji cells was significantly lower in cells treated with Iodine-131-Rituximab, as compared to the cells treated with Iodine-131or Rituximab alone (P<0.05); Tumor inhibition was found to be greatest in the mice treated with Iodine-131-Rituximab through intratumor injection, as compared with Iodine-131-Rituximab i.p. injection or Rituximab alone (p<0.05).

We conclude that Iodine-131-Rituximab specifically inhibits the growth of Raji tumor cells in vitro and in vivo. Iodine-131-Rituximab is a promising agent for radioimmunotherapy that targets CD20-positive B cell lymphoma.
Combined Treatment of STI571 (Glivec) and Curcumin Synergistically Suppresses the Growth of K562 Cells Via Inhibition of Bcr-Abl Pathway.
Authors: Park Juwon , Bae Eunkyung , Yoon Sung-Soo , Kim Byoung K.
Published: November 2019

Curcumin, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory and anti-tumor properties. As such, curcumin caused dose-dependent apoptosis of K562 cells, which is preceded by the activation of caspase 3. In addition, phosphorylation of Erk was effectively inhibited by curcumin. These findings leaded us to investigate the effects of combined treatment of curcumin and STI571 on K562 cells. With combined treatments, expression of Bcl-2, -XL, and Bid were dramatically down-regulated. In addition, the activity of caspase-3 was 2 fold higher than compared with either treatment alone. Cyclin D1 and its related proteins were down-regulated. In addition, combined treatment of curcumin and STI571 dramatically inhibited phoryphorylation of abl and its downstream proteins such as Src kinases and MAPK in K562 cells stimulated by IL-3. Taken together, our data suggested that curcumin potentiated the effects of STI571 in CML.
Quantitative Monitoring of T315I BCR-ABL Mutation by the Invader Assay.
Authors: Kakihana Kazuhiko , Ohashi Kazuteru , Najima Yuho , Kikuchi Taku , Nagata Yasunobu , Ueki Toshimitsu , Kobayashi Takeshi , Yamashita Takuya , Akiyama Hideki , Yamaguchi Toshikazu , Sakamaki Hisashi
Published: November 2019

Although imatinib mesylate has a highly inhibitory effect against BCR-ABL kinase, primary and secondary refractoriness have been observed. Point mutation within the kinase domain of BCR-ABL is one of the most important drug-resistant mechanisms. Of 25 reported point mutations, T315I BCR-ABL mutation might be dismal in clinical settings because it mediates clinical resistance to imatinib, nilotinib, and dasatinib, and the only established therapeutic option is hematopoietic stem cell transplantation at this moment. However, there is no information about the kinetics of this mutated clone. Here we developed quantitative Invader assay to monitor T315I BCR-ABL transcript. By using fluorescent resonance energy transfer system, the amount of released 5′ flap is measured as signal intensity, which enables us to calculate the percentage of T315I products with standard curve. Using this assay, we serially monitored T315I BCR-ABL transcript in a CML patient whose BCR-ABL transcript was still detectable (more than 100 copies/microgram RNA) 3 months after starting imatinib therapy. Although we have continued to monitor T315I BCR-ABL transcripts in 13 patients (chronic phase) up to 10 months, there was no patients who appear to be apparently resistant to imatinib due to a given T315I mutant, so far. In contrast, in a case of Ph+ ALL was being treated with chemotherapy including imatinib, we serially monitored both wild type and T315I BCR-ABL transcripts and observed the increased level of T315I transcript during relapse (0%at the time of diagnosis and 54.8% at relapse). Thus, our new strategy could be a useful tool to study the kinetics of the mutant clone and the pharmacokinetics of the drug resistant to T315I mutation.
BCR-ABL Induces Autocrine IGF-1 Signaling.
Authors: Kumarylakshmikuttyamma Asha , Pastural Elodie , DeCoteau John F. , Geyer C. Ronald
Published: November 2019

BCR-ABL is responsible for the initial phase of chronic myelogenous leukemia (CML), which is effectively treated by the BCR-ABL tyrosine kinase inhibitor imatinib. Over time, patients become resistant to treatment and progress to blast crisis, an event that is driven by additional genetic and epigenetic aberrations. Recently, we showed that RIZ1 expression decreases in blast crisis and that re-expression of RIZ1 inhibits IGF-1 expression. IGF-1 signaling is required in many stages of hematopoiesis and inappropriate activation of autocrine IGF-1 signaling may facilitate transformation to blast crisis. In this study, we examined mechanisms used by CML blast crisis cells to activate IGF-1 expression. We monitored the effects of small molecule inhibitors and shRNAs of BCR-ABL, HCK, and STAT5b on IGF-1 expression, proliferation, and apoptosis in CML blast crisis cell lines. We used chromatin immunoprecipitation assays to confirm that STAT5b associates with the IGF-1 promoter in a BCR-ABL-dependent manner. We found that BCR-ABL activates autocrine IGF-1 signaling using HCK and STAT5b. Imatinib blocks HCK phosphorylation, STAT5b phosphorylation, and the association of STAT5b with the IGF-1 promoter. Inhibition of these signaling components using small molecule drugs or shRNA inhibits IGF-1 expression, decreases proliferation, and enhances apoptosis. The effects of imatinib are partially reversed by the addition of exogenous IGF-1. Furthermore, treatment of CML blast crisis cell lines with the IGF-1 receptor inhibitor AG1024 decreases proliferation and enhances apoptosis and this activity correlates with IGF-1 expression levels. Our study highlights autocrine IGF-1 signaling as an important event in the transformation to blast crisis and provides a novel mechanism to explain the activity of IGF-1R and HCK inhibitors in blocking blast crisis phenotypes. Together, our results provide further support for the therapeutic potential of IGF-1 signaling components in treating CML, especially in cases associated with elevated autocrine IGF-1 expression.
Nordic CML Study Group Quality and Standardization Rounds for Quantitative RT-PCR of BCR-ABL To Facilitate Reporting on the International Scale.
Authors: Boström Hans , Lundan Tuija , Andersen Morten Tolstrup , Barbany Gisela , Fioretos Thoas , Golovleva Irina , Jonasson Jon , Juvonen Vesa , Lindlöf Mikael , Olsen Mats Irgen , Pallisgaard Niels , Savolainen Eeva-Riitta , Schaffer Marie , Silye Aleksandra , Hjorth-Hansen Henrik , Magnusson Magnus K. , Nielsen Johann Lanng , Porkka Kimmo , Simonsson Bengt , Kairisto Veli
Published: November 2019

Measuring the level of BCR-ABL transcripts by real-time quantitative polymerase chain reaction (RQ-PCR) has been shown to be a highly sensitive and specific method for evaluating residual disease burden in chronic myeloid leukemia (CML). In the IRIS trial, a 3-log reduction of BCR-ABL transcripts from a baseline median pre-treatment level was termed a “major molecular response”, and achieving this degree of minimal residual disease (MRD) was related to a more favorable prognosis. Recently it was proposed that the residual disease scale used in the IRIS trial should be named “International Scale” (IS) and this scale is recommended to be used in future studies of CML treatment. Since the baseline in the IRIS trial can no longer be reproduced, a project for distributing reference material has been initiated by the original IRIS study reference laboratory in Adelaide and by a secondary European reference laboratory in Mannheim. Within the framework of the Nordic CML Study Group, RQ-PCR for BCR-ABL is routinely performed in 13 clinical laboratories located in Finland, Sweden, Norway and Denmark. Only two Nordic laboratories had an opportunity to receive reference material from Mannheim. Therefore it was necessary to complement this effort by performing a Nordic quality and standardization round based not only on the external reference material but also on local pre-treatment samples from CML patients. Thus each of the participating laboratories prepared samples representing minimal residual disease and also additional pre-treatment CML samples. These samples were divided into 13 identical aliquots and distributed to all other participants this summer. By this approach we can not only check reproducibility of measurements, but also evaluate different laboratory specific steps in the procedures, such as the importance of source cell material, RNA isolation method, control genes, model of thermocycler and methods/enzymes used in reverse-transcription and RQ-PCR. Based on the results, provisional conversion factors for adjustment of the BCR-ABL transcript levels to the International Scale will be produced for all participating laboratories. This work is an example on successful international RQ-PCR standardization, which is essential for clinical and research estimation of MRD in CML patients.
Mechanisms of 2-Methoxyestradiol-Induced Apoptosis on Myelodysplastic Syndrome MUTZ-1 Cell Lines.
Authors: Chen Bao-An , Xia Guo-Hua , Lu Hui-Xia , Shao Ze-Ye , Gao Chong , Ding Hua-Jia , Xiao Ping-Pei , Chen Ning-Na , Zhao Gang , Gao Feng , Chen Jian , Wang Jun
Published: November 2019

Objective: To study the mechanism of proliferation inhibition and apoptosis of MDS-RAEB MUTZ-1 cells by 2-methoxyestradiol(2-ME).

Methods: MUTZ-1 cells were treated with different concentrations of 2-ME;cellular proliferation was determined by MTT assay;apoptosis rate was determined with annexinV-FITC/PI double staining and cell cycle was analyzed by flow cytometry (FCM);the changes of morphologic features of MUTZ-1 cells were investigated by cytomorphology with Wright-Giemsa’s staining; lactate dehydrogenises (LD) was determined by Beckman Counter and agarose gel electrophoresis was used to verify whether 2-ME could induce apoptosis in MUTZ-1 cells. Moreover, the activity of telomerase in MUTZ-1 cells was examined by TRAP-ELISA.

Results: The results showed that 1∼4μmol/L 2-ME inhibited the proliferation of MUTZ-1 cells in a dose-and time-dependent manner; the typical apoptotic morphological features appeared in MUTZ-1 cells after being treated with 4μmol/L 2-ME for 12hours; the marked DNA ladder pattern of internucleosomal fragmentation was observed after being treated with 4μmol/L 2-ME for 24hours and the up-regulated production of LD was assayed after treatment of 2-ME for 36hours. The number of MUTZ-1 cells of G0/G1 phase and S phase decreased, while the number of G2/M phase increased after MUTZ-1 cells were incubated with 1,2 and 4μmol/L2-ME for 12 hours, respectively(P<0.05). The inhibition effect of telomerase activity was enhanced in a dose- and time- dependent manner. Moreover, telomerase activity was significant negatively correlated with increased apoptosis (r =−0.954, p=0.046) and the number of sustained G2/M phase(r=−0.979, p=0.021) at the corresponding time point, respectively.

Conclusions :It is concluded that the mechanism of proliferation inhibition and apoptosis of MUTZ-1 cells induced by 2-ME is probably related with inhibition of telomerase activity and G2/M cell cycle arrest; 2-ME may be a potentially useful, adjunctive anticancer drug in treating myelodysplastic syndrome.
Good Response to Decitabine in an Elderly Patient with MDS (Refractory Anemia with Excess Blasts RAEB-2) after Failure of Azacitidine.
Authors: Aziz Ashraf R.
Published: November 2019

Decitabine, A pyrimidine nucleoside analog of cytidine, 5-aza-2′-deoxycytidine strongly inhibits DNA methylation, is capable of inducing cell differentiation, In a phase II multicenter trial of 66 patients with MDS (median age 68 years, range: 38 to 84), DAC was given in a dose of 15 mg/m2 IV over 4 hours every eight hours for three consecutive days; cycles were repeated every six weeks. The overall response rate was 25, 48, and 64 percent for those in the intermediate-I, intermediate-II, and high IPSS risk groups, respectively. The median survival time from the start of treatment for the IPSS high risk patients was 1.2 years, considerably longer than the expected survival of 0.3 to 0.5 years for high risk patients treated with supportive care alone. Response to Decitabine after failure of Azaitadine was mentioned only in one abstract presented in the American society of hematology meeting 2006 in 22 patients. We present a case of an 83 y/o gentleman with RAEB-2 with IPSS of 1 Intermediate I with normal cytogenetics, trilineage pancytopenia, was heavily transfusion dependent with both red cells and platelets on a weekly bases. The patient was treated initially with Revlamide for 3 months, followed by Azacitadine for total of 4 cycles, along with Exjade as an iron chelator without any objective response or reduction in his transfusion requirement. The patient eventually was switched to Decitabine 20 mg SC daily for 5 days every 28 days. The patient started to have a response after the 4 th cycle with prolongation of his transfusion intervals. After the 5 th cycle the patient did not need transfusions with platelets within normal limits. Since there is paucity of data regarding the response to these new agents, our case may be added to the small number of patients that was presented to try to create a data pool that helps clinicians to manage this difficult disease.
Cyclosporine A without or with Thalidomide for the Treatment of Patients with Myelodysplastic Syndromes.
Authors: Xiao Zhijian , Xu Zefeng , Wang Jianxiang , Hao Yushu
Published: November 2019

The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. Recent clinical trials have also confirmed that thalidomide may improve anemia and, less frequently, other cytopenias, in a proportion of younger patients with low-riskMDS (11–56%, on intention-to-treat analysis). In the current study, we report the outcome and side-effects in patients with MDS treated with cyclosporine A without or with thalidomide. From October 2000 to October 2005, 32 patients with MDS (RCMD 22 cases, RCMD-RARS 3 cases, RAEB-I6 cases, 5q- symdrome,1 case)were treated with cyclosporine A(CSA)(3mg/Kg.d) as single agents (n=14, group A) or CSA combined with thalidomide (200mg–400mg/d)(n=18, group B). There were no significant difference in hematologic or clinical features, Karyotype anomalies, classification of FAB or WHO, International Prognostic Scoring System(IPSS) score between the two group patients. Four patients of group A(28.5%) and twelve of group B (66.7%, P=0.011) achieved hematologic improvement. Three patients achieved a HI-E (three major), three patients HI-P(two major, one minor)and three patients HI-N(two major, one minor)within the group A. Twelve patients achieved a HI-E (eleven major, one minor), seven patients HI-P(five major, two minor)and six patients HI-N(three major, three minor)within group B. Two patients(2/11 cases,18.2%) became transfusion independence within group A, nine patients(9/14 cases, 64.3%)became transfusion independence within group B. There was statistic difference between two groups(P=0.021). Nine patients emerged hepatic lesion(three in group A(21.4%), six in group B(33.3%), P=0.457), Three patients emerged nephrotoxicity(one in group A(7.1%), two in group B(11.1%), P=0.702). These patients all made a recovery by treatment. Our primary results showed that CSA combined thalidomide is highly effective and relatively well-tolerated treatment for patients with MDS.
A Comparison of Stem Cell Source in Recipients of T-Cell Depleted Myeloablative Transplants for Leukaemia: No Difference in Mortality Using BM or PBSC.
Authors: Shaw B.E. , Russell Nigel H. , Pagliuca A. , Apperley J. , Cook G. , Mackinnon S. , Madrigal J.A. , Marks D. , Morgan G.J. , Peggs K. , Potter M.N. , Towlson K. , Byrne J.L.
Published: November 2019

The use of GSCF-mobilised Peripheral Blood Stem Cells (PBSC) for unrelated donor (UD) transplantation has increased dramatically since 2000. The association of PBSC with more rapid engraftment and with an increase in chronic Graft versus Host Disease (GvHD), compared to bone marrow (BM) has been reported in a number of studies. More recently the use of PBSC has been associated with an increase in transplant related mortality (TRM) and decrease in survival (OS) in T-cell replete transplants. We sought to analyse the impact of PBSC compared to BM in a cohort of UD transplant recipients, where T-cell depleting agents (in-vivo campath in >90%) were included in the transplant conditioning. The study included 145 patients transplanted between January 2000 and March 2006: CML- 35 in 1CP; acute leukaemia (AML in 61, ALL in 49)-110 in CR1 or 2. All had myeloablative conditioning regimens and received grafts with 9–10/10 matched HLA alleles. 86 patients received BM and 59 PBSC. There were no associations between the stem cell source and any transplant variable (including disease and stage). There was a trend to an increased use of PBSC in patients with a single antigen mismatch (p=0.052). All evaluable patients achieved neutrophil engraftment, with a significantly faster time to engraft in recipients of PBSC compared to BM (16 vs 20 days; p=0.0003). The incidence of acute GvHD was 46% (grade I in 50%, II in 41%, III in 8%, IV in 2%). This was significantly higher in recipients of PBSC (60%) compared to BM (36%; p=0.006), however there was no increase in either II/IV (p=0.69) or III/IV (p=0.18) disease in PBSC recipients. In univariate analysis, the presence of a single HLA mismatch (p=0.026) was the only other variable to be associated with an increase in acute GvHD. In a logistic regression model including both these variables, the use of PBSC remained significantly associated with an increase in aGvHD (OR=2.3; 95% CI 1.1,4.7;p=0.020). The TRM was 14%, 27% and 39% at 100 days, 1 and 5 years respectively. At none of these time points was the stem cell source associated with a significant difference in TRM. The 5-year incidence of chronic GvHD was 58% (BM 55%, PBSC 60%; NS), extensive disease in one third, and of relapse was 61% (BM 60%, PBSC 62%; NS). The 5-years OS was 41% with a median follow-up of 3.4 years (0.5–7.1). This was 44% using PBSC and 40% using BM (NS). In conclusion, although we observed an increase in acute GVHD with PBSC this was only of grade 1 disease. We found no association between the use of PBSC and an increased risk of chronic GVHD or of a worse transplant outcome, when compared to BM, in recipients of T-cell depleted myeloablative transplants for leukaemia.
Treatment of Myelodysplastic Syndrome with Azacitidine and Erythropoietin.
Authors: Limaye Sewanti , Lipshitz Jay , Dulala Renuka , Patel Dilip
Published: November 2019

PURPOSE: Myelodysplastic syndrome (MDS) is a group of clonal bone marrow disorders leading to bone marrow failure and acute myeloid leukemia. With better understanding of the biology of MDS, newer treatment options like therapy with the hypomethylating agents, azacitidine and decitabine have proven beneficial. Major randomized controlled trials by CALGB and others have demonstrated the efficacy of azacitidine. Growth factor use was prohibited in these trials. However, in the community, most patients have continued to get growth factor support along with azacitidine therapy. Since there is lack of evidence backing such clinical practice, we attempted to review response to therapy in patients receiving combination therapy with azacitidine and erythropoietin.

METHODS: We identified MDS patients treated with concomitant azacitidine and erythropoietin at our institution over the last five years. Data collected from retrospective chart review of these patients was statistically analyzed.

RESULTS: Sixteen patients with concomitant azacitidine and erythropoietin therapy were identified. Complete remissions were seen in 31%(5/16) of the patients. Induction of partial remissions in our study was 25% (4/16). Thirty one percent (5/16) of patients had hematologic improvement not meeting criteria for partial remission. Thirteen percent of patients (2/16) did not benefit at all from azacitidine and erythropoietin support. The median number of cycles received for adequate response was six. A total response (complete remission, partial remission and hematological improvement) of 87%(14/16,) was seen with combination therapy, which is significantly higher than previously published total response of 44–60%.

CONCLUSION: Azacitidine with erythropoietin support provides important clinical benefits for patients with MDS. There was a trend towards a higher rate of complete and partial responses in these patients than reported earlier in patients who received azacitidine alone. Randomized controlled studies will be needed to validate this further.

Comparison of response to therapy in high risk MDS: Responses *CALGB-9221(azacitidine) **Combined analysis(azacitidine) Study patients treated with azacitidine and growth factors *JCO May 15 2002: 2429–2440.**JCO Aug 20 2006: 3895–3903. Complete Remission 7% 10–17% 31% Partial Remission 16% Rare 25% Hematological Improvement 37% 23–36% 31% Total response 60% 44% 87%
No Reversal of Demethylation after Azacitidine Treatment in Concordance with Poor Clinical Response.
Authors: Tran Huong Thi Thanh , Kim Hee Nam , Ahn JaeSook , Lee Il-Kwon , Yang Deok-Hwan , Kim Yeo-Kyeoung , Lee Je-Jung , Park Kyeong-Soo , Kim HyeoungJoon
Published: November 2019

The epigenetic gene silencing associated with promoter DNA methylation is as powerful as gene mutations in functionally inactivating tumor suppressor genes. Thus, a non-intensive treatment may be changed the natural history of MDS for the first time by the demethylating agent, 5-aza-deoxycytidine (Decitabine) with silenced gene expression by reversal of p15 hypermethylation and protein expression in the bone marrow in MDS. The MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) ME-001B probemix (MRC-Holland) containing 25 tumor suppressor genes has been used to detect the methylation level in the peripheral blood samples of 29 MDS before azacitidine (Vidaza) and only 6 MDS after 3–5 courses of therapy. Patients that hypermethylated at least 1 gene were 7 of 29, either the common hypermethylating genes as p15, ESR1 or the previously known FHIT in MDS also have occurred. Only two patients except one patient related to either methylation level-reducing gene or removal methylated gene (putative demethylation reversal) have in concordance with clinical response in hematological evidence. Interestingly, three other patients were high methylation level persistently or additional methylated gene after treatment (putative demethylation no reversal or more severe), two patients of these are correspond with no clinical response and one is propensity to progressing leukemia. With IGSF4 gene hypermethylation, to the best of our knowledge, there was no report in MDS. Our results suggest that methylation level possibly contributes to the dignosistic, prognosistic and a molecular monitoring marker after treatment of Azacitidine.
Pediatric Myelodysplastic Syndrome and Juvenile Myelomonocytic Leukemia in Korea: A Nation-Wide, Multicenter, Retrospective Study.
Authors: Kook Hoon , Han Dong Kyun , Kang Hyeong Jin , Cho Bin , Chung Nak Gyun , Im Ho Jun , Park Hyeon Jin , Park Jun Eun , Yoo Keon Hee ,   for MDS Committee of the Korean Society of Pediatric Hematology Oncology
Published: November 2019

Recent advances in the diagnosis, molecular pathogenesis, classification and therapy have been made in the field of myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML) in childhood. We report a retrospective analysis of children with MDS and JMML diagnosed between 2001 and 2006 in Korea. In total, 135 patients were enrolled from 19 major hospitals with pediatric hematology oncology clinics: MDS, 96 (primary MDS, 77; constitutional anomalies with MDS, 13; treatment-related MDS, 6) and JMML, 39. The incidence of MDS/JMML was around 22.5/year, which is about 6% of childhood leukemia. Various classification systems including FAB, WHO, IPSS, CCC system, and pediatric adjustment of the WHO classification were applied. The median ages at diagnosis were 68 and 10 months in MDS, and JMML, respectively. Males dominated in JMML. Cytogenetic abnormalities were observed in 43% of MDS (monosomy 7, 5; trisomy 8, 3) and in 10% of JMML. Treatment was chosen by each institute’s preference: 34 patients with MDS received AML-type intensive chemotherapy, with complete remission rate of 82.0%. The 5-year Kaplan-Meier overall survival rate was 54% each for MDS and JMML. Survival of MDS patients was influenced by the marrow blast % (P = 0.007) and disease category (P= 0.006). Stem cell transplantations (SCT) were undertaken in 56 patients (MDS, 29; JMML, 27). The sources of stem cells were: bone marrow, 36; umbilical cord, 18; peripheral blood, 2). Matched related transplants were 9 cases. Conditioning was various, but BuCy based regimen was used in 68.4%. Acute GvHD ≥ Grade II was found in 43.8% and chronic GvHD in 35.1%. The 5-year Kaplan-Meier overall survival rate was 55% for MDS, and 57% for JMML. Survival after unrelated transplant was comparable with that of matched related transplants. This analysis inspired the necessity of nation-wide prospective studies in Korea, including morphologic study by a central pathology review board, epidemiologic study, molecular pathophysiologic study, and therapeutic trials incorporating SCTs, or new drugs.
Decisional Flow with a Score System To Start Platelet-Lowering Treatment in Patients with Essential Thrombocythemia (ET): Long-Term Results.
Authors: Latagliata Roberto , Rago Angela , Spadea Antonio , Santoro Cristina , Carmosino Ida , Breccia Massimo , Napoleone Laura , Fama Angelo , Biondo Francesca , Volpicelli Paola , Petti Maria Concetta , Alimena Giuliana , Mazzucconi Maria Gabriella
Published: November 2019

Treatment of ET is still a matter of debate for the difficulty in deciding between platelet(plt)-lowering agents and a “wait and see” policy with anti-aggregating agents only: a main problem is to identify what patients are “at risk” to develop severe thrombotic events during the course of the disease. Thus, we prospectively tested in ET patients at diagnosis with no clear indication to plt-lowering treatment a score system based on the following 5 variables: age, plt level, previous thrombotic events, smoking, dismetabolic diseases: the score was reassessed in each patient every 3 months during follow-up. From 04/92 to 03/98, 168 consecutive adult patients (57 males and 111 females, median age 59.5 yrs, range 20.5 – 84.8, median plt value 1,010 x 109/l, range 587 – 2,714) with ET according to PVSG criteria were diagnosed at our Institution. Among them, a plt-lowering treatment was started in 32 patients considered “symptomatic” for the presence at diagnosis or within 6 months before of severe haemorrhages, vascular accident or severe disturbances of microcirculation; plt-lowering treatment was also started in all the 33 patients aged > 70 years at diagnosis. The remaining 103 patients were “asymptomatic” and aged < 70 years and were classified according to our score system: thirty-two patients with a score ≥ 4 started plt-lowering treatment with hydroxyurea early after diagnosis. The remaining 71 patients had a score < 4 at diagnosis and received anti-aggregating agents only: of them, 24 (33.8%) started plt-lowering treatment during follow-up (18 for a score increase to ≥ 4 and 6 for the occurrence of thrombotic complications or symptoms) after a median time from diagnosis of 28 months (range 3–130) while 47 (66.2%) did not start any plt-lowering treatment. Thrombotic complications occurred in 9/103 patients (8.7%) scored at diagnosis: in particular, they occurred in 4/32 patients (12.5%) with score ≥ 4 receiving plt-lowering treatment since diagnosis and in 5/71 patients (7%) with score < 4 under anti-aggregating agents only. Overall, 6 out of 103 scored patients died and 27 were lost to follow-up, with a 15-year cumulative survival of 88%. In conclusion, a decisional flow based on present score system appears effective in patients without a clear indication for plt-lowering treatment at diagnosis to discriminate subjects at different risk of thrombotic events and could be useful to decide when a plt-lowering therapy needs to be started.
JAK2 V617F Mutation Testing in Polycythemia Vera: Use and Impact in a Single Academic Practice.
Authors: Means Robert T.
Published: November 2019

The observation that a gain-of-function mutation in JAK2 (JAK2 V617F) is present in more than 90% of cases of polycythemia vera (PV) has substantially revised the diagnostic approach to this disease. However, most studies of the utility of JAK2 V617 derive from large PV populations: the majority of hematologists have relatively few PV patients in their individual practices. In order to assess the effect of JAK2 V617F analysis in practices with a small PV population, the utilization and impact of this test in 5 individual clinics in a single academic practice were analyzed. Sixteen patients (15 male) carrying a diagnosis of PV were identified. Five patients were followed in a clinic at a University medical center staffed by a single attending hematologist; one was followed by that attending physician in a clinic at the affiliated VA hospital; and 10 were followed by Hematology/Oncology Fellows in three continuity clinics at the affiliated VA hospital. In these latter clinics, the Fellow is primary provider, but reviews management with an attending physician. Median age of the overall population was 67.5 yrs; patients in the Fellow clinics were significantly older than those in the Attending clinics (80 yrs vs. 63 yrs; p < 0.01). Duration of disease was longer in the Fellow clinic patients than the Attending patients (8.5 yrs vs. 1.4 yrs; p=0.05). Nine patients were tested for JAK2 V617F mutation. The majority (6/9) were tested near diagnosis (median duration of erythrocytosis was 0.7 yrs for patients tested and 10 yrs for patients not tested; p = 0.02). Two of the three established patients tested did not meet PV diagnostic criteria fully. Five of the 7 not tested met WHO criteria for PV. Of the 9 patients tested, 7 had a mutation detected (5 newly diagnosed, 2 > 1 yr duration of disease). Results of mutation testing changed the diagnosis in two cases (one where it was detected and one where it was not detected). In addition, one newly diagnosed patient who met WHO criteria did not have JAK2 V617F detected and was felt to have true “JAK2 V617F-negative” PV. JAK2 exon 12 studies were not performed.

Conclusions: The use and impact of JAK2 V617F mutation testing has been reviewed in a PV population similar to what an individual or small group practice might follow. JAK2 V617F mutation testing was primarily used in the early evaluation of suspected PV, or in continuing patients whose diagnosis was not fully established. Mutation testing was infrequently used in continuing patients where the physician considered the diagnosis clearly established or where a change in diagnosis would not alter management. Negative results of mutation testing altered diagnosis if they were consistent with the patient’s clinical picture.
CD26 Expression in Mature B-Cell Neoplasia: Its Possible Role as a New Prognostic Marker in B-CLL.
Authors: Cro Lilla , Zucal Nadia , Fabris Sonia , Neri Antonino , Lionetti Marta , Morabito Fortunato , Cutrona Giovanna , Ferrarini Manlio , Molica Stefano , Deliliers Giorgio Lambertenghi , Baldini Luca
Published: November 2019

CD26 (dipeptidyl peptidase IV, DPP IV) is a multifunctional type II cell surface glycoprotein that is widely expressed on T and natural killer cells, as well as on epithelial, endothelial and acinar cells of different tissues; its expression on B cells is very low but it is greatly upregulated following activation. We evaluated, by means of flow cytometry, the expression of CD26 in various types of B-cell lymphoid tumors: Follicular Lymphoma (Fo-Ly, 12 cases), Mantle cell Lymphoma (MCL, 12 cases) Multiple Myeloma (MM, 20 cases), Hairy cell Leukemia (HCL, 12 cases), B-cell Chronic Lymphocytic Leukemia (B-CLL, 112 cases), CD5 negative B-cell Chronic Lymphoproliferative Diseases (CD5neg-B-CLPD, 20 cases) and Diffuse Large cell Lymphoma (DLCL, 12 cases). CD26 expression was absent or low of Fo-Ly and MCL, high on MM and HCL, variable on B-CLL, CD5neg-B-CLPD and DLCL. Fluorescence intensity of positive cells was dim in B-CLL and CD5neg-B-CLPD, heterogeneous in DLCL, and bright in HCL and MM. Interestingly, in CLL patients, CD26 expression was significantly correlated with CD49d and CD38 expressions (p<0.0001); moreover Spearman’s test revealed a significant correlation between CD26, CD38 or ZAP-70 expression and IgVH mutational status (p<0.0001). We also found a significant correlation between CD26 expression and the presence of FISH chromosomal abnormalities grouped on the basis of their prognostic relevance (p<0.0001). After a median follow-up of 36 months, 65/107 CLL pts were treated; taking 10% of positive cells as best cut-off, Kaplan-Meier curves showed that the time without therapy was significantly longer for CD26 negative cases compared with CD26 positive cases (P <0.0001). Moreover the concordant negativity of CD26 and CD38, or ZAP-70 and the combination of CD26 negativity with IgVH mutated status, identified subsets of CLL patients with a favourable outcome in terms of need of therapy.
Modulation of Gene Expression Profile and In Vivo Anti-Myeloma Activity Induced by Valproic Acid, a Histone Deacytylase Inhibitor.
Authors: Neri Paola , Calimeri Teresa , Di Martino Mariateresa , Rossi Marco , Eramo Orietta , Propato Mariagrazia , Perri Gino , Pietragalla Antonella , Ventura Monica , Bulotta Alessandra , Blotta Simona , Tagliaferri Pierosandro , Anderson Kenneth C. , Munshi Nikhil C. , Tassone Pierfrancesco
Published: November 2019

Valproic acid (VPA) is a well-tolerated anticonvulsant drug that has been recently recognized as powerful histone deacetylase (HDCA) inhibitor. VPA induces hyperacetylation of histone H3 and H4 and inhibits both class I and II HDCACs. Recently it has been shown that VPA exerts in vitro and in vivo anti-tumor activity against solid cancers and its in vitro anti-Multiple Myeloma (MM) activity has been previously reported. However, the molecular mechanisms are still unclear. Here we have investigated molecular changes induced by VPA as well as its in vivo activity in murine models of MM. We first studied the in vitro activity of VPA against IL-6 independent as well as IL-6 dependent MM cells. A time- and dose-dependent decrease in proliferation and survival of MM cell lines was observed (IC50 in the range of 1–3 mM). Gene expression profile following treatment with VPA at 2 and 5 mM showed down-regulation of genes involved in cell cycle regulation, DNA replication and transcription as well as up-regulation of genes implicated in apoptosis and chemokine pathways. The signaling pathway analysis performed by Ingenuity Systems Software identified the cell growth, cell cycle, cell death as well as DNA replication and repair as the most important networks modulated by VPA treatment. We next evaluated the in vivo activity of VPA using two xenograft models of human MM. A cohort of SCID mice bearing subcutaneous MM1s or OPM1 were treated i.p. daily with VPA (200 mg/kg, and 300 mg/kg, n=5 mice, respectively), or vehicle alone (n=5 mice) for 16 consecutive days. Tumors were measured every 2 days, and survival was calculated using the Kaplan Mayer method. Following VPA treatment, we found a significant (p=0.006) inhibition of tumor growth in mice bearing subcutaneous MM-1s cells treated with VPA at 200 mg/kg compared to control group, which translated into a significant (p= 0.002) survival advantage in the VPA treated animals. Similar results were obtained in animals bearing subcutaneous OPM1 cells. Flow cytometry analysis performed on retrieved tumor tissues from animals showed reduction of G2-M and S phase in tumor specimens following VPA treatment, versus untreated tumors, strongly suggesting in vivo effects of VPA on cell cycle regulation. Taken together, our data demonstrate the in vitro and in vivo anti-tumor activity of VPA, delineate potential molecular targets triggered by this agent and provide a preclinical rationale for its clinical evaluation, both as a single agent or in combination, to improve patient outcome in MM.
ITF2357, a Novel Histone Deacetylase Inhibitor, Demonstrates Significant Apoptotic Activity Against Human Multiple Myeloma Cells.
Authors: Kline Michael , Donovan Kathleen A. , Lust John A.
Published: November 2019

We have evaluated the efficacy of a novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor, ITF2357, to promote cell death in multiple myeloma (MM) cells. HDAC inhibitors, which promote histone hyperacetylation and increase gene expression, have been evaluated as candidate agents for combating malignancies because they impact the expression of genes related to proliferation, differentiation, and survival. Exposure of MM cell lines to 1 micromolar ITF2357 led to dramatically increased levels of histone acetylation at 4 hours and 8 hours by Western analysis. Sub-micromolar concentrations of ITF2357 promoted time- and concentration-dependent cell death in MM cell lines. Using 500 nM ITF2357, a concentration potentially achievable in vivo, viability of KAS-6/1 IL-6 dependent myeloma cells was reduced to 28% of control at 24 hrs and 2% of control at 48 hours (Figure 1). In contrast, viability of normal PBMCs was 100% at 24 hours and 80% at 48 hours (Figure 2). U266 and 8226 myeloma cells were found to be sensitive to ITF-2357 in a similar fashion with U266 being least sensitive. Cell death proceeded via apoptosis as measured using Annexin V/propidium iodide staining. ITF 2357 was superior to suberoylanilide hydroxamic acid (SAHA) at inhibition of stromal cell IL-6 production. IL-1beta (10 pg/ml) was used to stimulate bone marrow stromal cell IL-6 production (105 ng/ml) after 48 hours. Concentration of ITF2357:Stromal Cell IL-6 production after 48 hours were as follows - 10 nM: 78 ng/ml; 100 nM: 79 ng/ml; 1000 nM; 32 ng/ml. SAHA at similar concentrations showed no significant decrease in stromal cell IL-6 production compared with the no drug control. In summary, ITF2357 induces significant myeloma cell apoptosis and can inhibit stromal cell IL-6 production. It represents an attractive therapeutic candidate for MM clinical trials.

Figure Figure Figure Figure
Anti-Acute Myeloid Leukemia Activity of Chaetocin, a Novel Epigenetic Drug Inhibitor Inducing Oxidative Stress.
Authors: Chaib Hassiba , Prebet Thomas , Restouin Audrey , Castellano Remy , Opi Sandrine , Olive Daniel , Borg Jean Paul , Vey Norbert , Collette Yves
Published: November 2019

Recent studies have highlighted the importance of epigenetic modifications in the pathogenesis of Acute Myeloid Leukemia (AML). This results have been confirmed by the activity of new drug like DNA demethylating agents and histone deacetylase (HDAC) inhibitors in both in vivo and in vitro studies. Recently, Chaetocin, a natural fungal compound, has been identified as the first specific inhibitor of the histone methyltransferase SU(VAR)3–9 which plays a role in heterochromatin gene silencing. In this study, we decided to evaluate Chaetocin as a therapeutic agent in AML in vitro and to explore the related mechanisms. We show that Chaetocin induce dramatic cell death at nanomolar concentrations in U937 and HL60 (97.2% ± 0.4 and 91.6% ± 9 cell death at 100 nM chaetocin, respectively), and to a lesser extend in K562 (67.3% ± 1.6 cell death at 100 nM chaetocin), cell cultures. Cell death occurred at 24 h incubation time which correlated with induction of apoptosis as assessed by Annexin V/7-AAD staining and activation of downstream executioner caspase-3/7. Using transcription low-density array and quantitative RT- PCR, Chaetocin was showed to up-regulate gene transcription such as of the cell cycle inhibitor p21/WAF1 consistent with a role for the targeted SU(VAR)3–9 in heterochromatin gene silencing. In agreement with the recent report of Chaetocin being a promising new antimyeloma agent acting via imposition of oxidative stress, intracellular levels of oxidative species were increased in Chaetocin treated U937 cells in a time- and dose-dependent manner that correlated with induction of cell death. Furthermore, incubation of cells with N-acetyl cysteine, a cell-permeable precursor of intracellular glutathione reductant, prevented chaetocin-induced accumulation of oxidative species, transcription of selected genes (e.g. p21/WAF1), activation of caspase-3, and cell death. Finally, Chaetocin was found to increase the antileukemia activity of HDAC inhibitors and Aracytin, and thus appears as a promising agent for further study as a potential anti-AML therapeutic. Preliminary results obtained in vivo in xenograft models and ex vivo, using blasts of a panel of patients with AML, will be presented.
rNAPc2, a Novel Inhibitor of Tissue Factor/Factor VIIa Complex, Inhibits Tumor Growth and Metastasis in Mouse Models of Colorectal Cancer.
Authors: Zhao Jingsong , Aguilar Gerard , Imperiale Michael , Funk Walter , Abo Arie
Published: November 2019

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+ mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.
High Frequencies Of Switching To 2nd TKIs and Failure To Maintain Standard Imatinib Dose In Japanese CML Patients With BIM Genetic Variants
Authors: Katagiri Seiichiro , Tauchi Tetsuzo , Umezu Tomohiro , Ohtsuki Kazuhiro , Tadokoro Kenichi , Yamamoto Yoshinori , Ohyashiki Junko H , Ohyashiki Kazuma
Published: November 2019

Recently, it has been demonstrated that the proapoptotic protein BIM showed a deletion polymorphism at exon 3 in eastern Asian population, and some CML patients with the BIM deletion polymorphism are resistant to imatinib treatment (Ng et al. Nature Medicine, 2012). More recently, a BIM single nucleotide polymorphism (SNP) at exon 8 (c465C>T) has also been found in French CML patients and this SNP is associated with not only imatinib resistance but also the presence of BCR-ABL mutations (Mahon et al. ASH abstract, 2012).

We aimed to investigate a possible association between such genetic variations of BIM and clinical manifestation in Japanese CML patients who experienced undetectable minimal residual disease (UMRD: so-called CMR4.5). In this study, we newly analyzed BIM SNP (c465C>T) in 47 CML-UMRD patients with known BIM deletion polymorphism status (Katagiri et al. Br J Haematol, 2013). Twenty normal subjects were used as controls.

The frequency of either BIM SNP at exon 8 or BIM deletion polymorphism did not deviate from the normal subjects in the Japanese population (P = 0.7597 and P = 0.2880, respectively). None of the subjects showed both BIM SNP at exon 8 and BIM deletion polymorphism concomitantly. We then compared the clinical features among 3 CML-UMRD groups: patients with BIM SNP, patients with BIM deletion polymorphism, and patients who showed neither BIM SNP nor BIM deletion polymorphism (no genetic variations). The frequency of CML patients who maintained 400 mg imatinib dose until stopping was significantly higher in those without genetic variations than in those with BIM SNP or BIM deletion polymorphism (P = 0.0002). Moreover, the frequency of CML patients who switched to second tyrosine kinase inhibitors (2nd TKIs) was significantly higher in those with BIM SNP or BIM deletion polymorphism than in those without such polymorphisms (P = 0.0055).Number of CML patientsMaintained IM 400 mgChange of imatinib dose2nd TKIs switchingBIM SNP (c465C>T)11/474/115/112/11BIM deletion polymorphism6/471/63/62/6BIM SNP or deletion polymorphism17/475/178/174/17No BIM genetic variationss30/4725/305/300/30

This is apparently the first study to circumstantiate the BIM genetic variants in Japanese CML patients with UMPD. Although the number of patients is small, our results suggest that CML patients without BIM deletion polymorphism/SNP could be maintained under standard imatinib dose without switching to 2nd TKIs, and thereby, have a possibility to stop TKIs therapy.

Disclosures:

Ohyashiki: Norvartis KK: Research Funding, Speakers Bureau; Bristol Meyer Squibe KK: Research Funding, Speakers Bureau.