bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Parvovirus B19 and Herpesvirus-6 Serology in Brazilian Children with Common B Cell Precursor Acute Lymphoblastic Leukemia
Authors: Silva Fernanda A , Vasconcelos Gisele M , Cordeiro Synara NS , Sant’Ana Mariana , de Carvalho Silvia Maia Farias , Pombo-de-Oliveira Maria S.
Published: April 2020

Background. Indirect epidemiologic evidences have suggested that viral infections may represent an important risk factor in the etiological mechanisms of B cell precursor lymphoblastic leukemia (BpALL), specially common-ALL. Parvovirus B19 (PvB19) is associated with aplastic crisis, thrombocytopenia, and congenital anemia. Five studies have reported the concurrent PvB19 infection in pediatric ALL. In a view of the increasing contest about the direct or indirect role of viral infection in ALL, we evaluated the possible associations of PvB19 and herpes virus 6 (HHV-6) in samples from children suffering from hematological disorders in order to estimate the magnitude of risk of association with common-BpALL.

Material and Methods. A total of 659 blood samples were randomly selected to perform the serological tests. They were part of diagnostic samples referred to CPq-INCA-Rio de Janeiro, Brazil, from 2002–2006, for diagnostic purpose. Blood samples were exclusively selected from patients in whom samples were taken at the time of the onset of sickness. The diagnosis of BpALL was based on immunophenotyping methods according to EGIL criteria. The presence of serum antibodies for PvB19 and HHV-6 were determinate by enzyme-linked immunosorbent assay. Statistical analyses were conducted using SPSS for Windows 11.0. All p values were two sided; p<0.05 was considered statistically significant.

Results. Common-ALL (n=176) and non-leukemic samples (n=107) considered as control group were stratified according to age-groups at diagnosis. Age distribution was:

less than 2 years (15.5%); 2–6 years-old (46.0%), and 7–12 years-old (17.7%).

The estimation of associated risk PvB19 IgG+ antibodies with common-BpALL was OR 0.53 95% CI (0.52–0.54); whereas HHV-6 IgG was OR 2.36, 95% CI (1.08–5.17).

Conclusions. We find out a higher prevalence of HHV-6 antibodies in patients with common-BpALL, suggesting an association between HHV-6 infection e common-BpALL in Brazilian children. There is a long history in developed countries regarding the role of infections in leukemogenesis process. Further tests are necessary to confirm these results.
Environmental and Socioeconomic Factors in Children with Acute Lymphoblastic Leukemia
Authors: Timur Cetin , Yilmaz Oznur , Yoruk Asim , Erguven Muferet , Imdadoglu Timucin , Kavakli Kaan
Published: April 2020

In our study, we aimed to evaluate environmental and socio-economic conditions in children with Acute Lymphoblastic Leukemia (ALL) and to point at possible etiologic factors that can affect leukemia risk. The parents of 105 children diagnosed and treated as ALL between the years 1997 –2007 in our clinic of Pediatric Hematology-Oncology were questioned in terms of environmental and socio-economical factors and results were compared with control group that consisted of 102 healthy children with similar age and gender. Educational level and monthly income were similar between the groups. Occupational exposure of fathers to dust and chemicals were significantly higher in leukemia group (OR:2.00; %95 CI=1.41–3.50, p:0.015). Living near transformer stations (OR: 4.08; %95 CI= 1.3–12.76, p: 0.034) and high-voltage power lines (OR: 2.43; %95 CI= 1.05–5.63, p:0.01) is found to be associated with increased risk of leukemia in children. There was no significant difference in terms of living near base stations (p>0.05). Exposure to industrial air pollution was significantly higher in leukemia group and was related to an elevated risk of ALL (OR: 26.77; %95 CI= 3.53–202.80, p:0.001). There was no significant difference in terms of exposure to insecticides and pesticides between the groups (p:>0.05). In conclusion, leukemia is a disease with multi-factorial etiology that occurs as a result of interactions of genes and environment. The list of possible chemical, physical and biologic agents suspected to play a role in its etiology increase with developing technology and environmental pollution. However there are no sufficient data and more extended studies have to be carried out.
Histone Deacetylase Inhibition Improves Differentiation of Dendritic Cells from Leukemic Blasts of Patients with TEL/AML1-Positive Acute Lymphoblastic Leukemia
Authors: Schmidt Kerstin , Seeger Karl , Scheibenbogen Carmen , Bender Roderich , Süßmilch Sina , Salama Abdulgabar , Moldenhauer Anja
Published: April 2020

Histone deacetylase inhibitors (HdI) could potentially improve the differentiation of leukemic dendritic cells (DC). Therefore, 100 bone marrow samples from patients with acute lymphoblastic leukemia (ALL) were cultured in the presence of TNFα, GM-CSF, c-kit ligand and FLT3 ligand with or without interleukin-3 and –4, and after administration of the HdI valproic acid (VAL), suberoylanilide hydroxamic acid (SAHA), isobutyramid or trichostatin A. Twenty-six of these ALL samples were positive for the t(12;21) translocation encoding the fusion gene TEL/AML1. SAHA increased CD83 expression of TEL/AML1- positive blasts in conditions without interleukins (36±6.5% versus 26.3±7.1%, p< 0.05), while SAHA and VAL increased the number of CD86(+)80(−) cells in the presence of interleukins (12.5±2.8 ×104 versus 6.3±1.1 ×104 cells). VAL and isobutyramid supported the allostimulatory capacities of TEL/AML1-positive leukemic DC (48.5±6.4 ×103cpm and 50.3±7.1 ×103cpm) and reduced those of TEL/AML1-negative DC (41.8±6.8 ×103 cpm versus 52.8±11.2 ×103cpm, p= 0.05). Cytotoxic T-cells sensitized with leukemic DC produced more INFγ and TNFα upon presentation of the TEL/AML1 peptide. They also induced the cytotoxic lysis of non-differentiated blasts, which was enhanced when TEL/AML1-positive DC had developed after addition of VAL or SAHA. In conclusion, the use of HdI in the differentiation of leukemic DC from patients with TEL/AML1-positive ALL is recommended.
Adults with Acute Lymphoblastic Leukemia and T(1;19) Abnormality Have a Favorable Outcome with Hyper-CVAD Chemotherapy
Authors: Garg Ravin Jain , Kantarjian Hagop M , Thomas D. A , Faderl Stefan , Ravandi Farhad , Lovshe Denise , Pierce Sherry , O’Brien Susan
Published: April 2020

Among the well-described cytogenetic abnormalities in adults with acute lymphoblastic leukemia (ALL), a translocation involving chromosomes 1 and 19 [t(1;19) (q23;p13)] occurs in a small subset but has varyingly been associated with a good or bad prognosis in different studies.

Adults with ALL and t(1;19) treated at M.D. Anderson Cancer Center were reviewed. Their clinical features and outcome were compared to those with other cytogenetic abnormalities. Endpoints included complete remission rate (CR), complete response duration (CRD) and overall survival (OS).

Of 411 adults with pre-BALL, 12 patients had t(1;19). Ten of the 12 patients with t(1;19) received Hyper-CVAD (Cyclophosphamide, Vincristine, Adriamycin, Dexamethasone alternating with Methotrexate and high-dose Cytarabine); the other 2 were treated with VAD (Vincristine, Adriamycin, Dexamethasone). All 12 patients achieved CR; the 3-year survival rate was 73%. Patients with t(1;19) had significantly better CRD and OS when compared to all other patients combined as well as individually to patients with Ph+, t(4;11), and lymphoma-like abnormalities [6q(−), 14q+, t(11;14), t(14;18)].

Adults with ALL and t(1;19) have an excellent prognosis when treated with the Hyper-CVAD regimen.

Outcome of patients by cytogenetic group: t(1;19) vs. individual cytogenetic groups

OVERALL SURVIVAL N Fail 3-Year % Median (weeks) P-value T(1,19) 12 3 73 Not recorded Diploid 138 72 52 179 0.09 Lymphoma-like 20 17 35 54 0.008 Ph+ 117 88 23 68 0.0002 Miscellaneous 112 56 56 236 0.17 T(4,11) 12 10 0 58 0.002 COMPLETE RESPONSE DURATION (CRD) N Fail 3-Year % Median (weeks) P-value T(1,19) 12 2 80 Not recorded Diploid 133 59 54 177 0.06 Lymphoma-like 16 13 34 89 0.009 Ph+ 102 52 42 63 0.006 Miscellaneous 100 41 59 401 0.16 T(4,11) 11 7 NR 45 0.018
NOD/SCID/γcnull mice provide a Unique Model to Investigate Childhood Haematopoietic Malignancies
Authors: Kato Itaru , Niwa Akira , Umeda Katsutsugu , Ito Mamoru , Heike Toshio , Nakahata Tatsutoshi
Published: April 2020

Remarkable improvements in the outcome for childhood haematopoietic malignancies have been made during the past few decades. However, haematopoietic malignancies are the most common pediatric malignancy, and outcome after relapse is generally poor. Recent reports suggest that at least some cases of human leukemia and cancer are arranged in a hierarchical fashion and maintained by rare “tumor-propagating cells”. However, whether the malignant clone arises from such a leukemic stem cell fraction has not been clarified in most haematopoietic malignancies other than acute myelogenous leukemia (AML). Establishment of an assay capable of generating of childhood haematopoietic malignancies from patient oriented blast cells will provide new insight into the haematopoietic malignancies biology and the mechanism of the development of haematopoietic malignancies in vivo. The recently described highly immunodeficient NOD/SCID/gamma null (NOG) mice provide a number of advantages. This strain, with life expectancy more than 90 weeks, is more robust than strains such as NOD/SCID and support generation of haematopoietic malignancies from transplanted human blast cells. In the present study, childhood haematopoietic malignancies cells, such as acute lymphoblastic leukemia(ALL; n=7), AML (n=4), juvenile myelomonocytic leukemia(JMML; n=1), Lymphoma(n=1), and Transient Myeloproliferative Disorder(n=1), are transplanted into 2.4Gy irradiated or non-irradiated NOG mice. The transplanted mice have splenomegaly, hepatomegaly, nephromegaly and testicular enlargement and developed haematopoietic malignancies which is the same flow cytometric immunophenotype and pathological pattern of the patient from 4 weeks after intravenous injection of 5×102-1×108 blast cells. The transplanted mice can live as long as 24 weeks after transplantation with more than 95% blasts in the bone marrow. The blast cells harvested from the bone marrow and the spleen generated the haematopoietic malignancies in tertiary transplantation especially in ALL. We report here examination results of reconstituted childhood haematopoietic malignancies in NOD/SCID/gamma null mice after the transplantation of patient oriented blast cells.
Outcome of Adults with Acute Lymphocytic Leukemia (ALL) with Second Salvage Therapy
Authors: O’Brien Susan , Thomas Deborah , Ravandi-Kashani Farhad , Faderl Stefan , Cortes Jorge , Borthakur Gautam , Pierce Sherry , Manero Guillermo Garcia- , Kantarjian Hagop
Published: April 2020

Background and Aims. The outcome of adults with ALL undergoing second salvage therapy is poorly characterized. The aim of this study was to determine the outcome and prognostic factors associated with second salvage therapy in patients with ALL.

Study Group and Methods. A total of 288 patients treated for ALL in second salvage at our institution were analyzed.

Results. 53 patients (18%) achieved complete remission (CR). The median remission duration was 7 months. The median survival was 3 months. By multivariate analysis, prognostic factors independently associated with achievement of CR were: duration of first CR, and platelet count. Patients with a first CR < 36 months and a platelet count < 50 × 109/L had an expected CR rate of 7%. By multivariate analysis, prognostic factors independently associated with survival were: duration of first CR, percent of marrow blasts, platelet count, and albumin levels. Expected 12-month survival rates for patients with 0–1, 2, 3, or 4 adverse factors were 33%, 14%, 8%, and 0%, respectively. A repeat multivariate analysis using landmark assessment at 6 weeks selected achievement of CR as adding significantly to the survival benefit (p = 0.0001, hazard ratio 0.51). Only 22 patients (8%) were able to undergo allogeneic stem cell transplant as second salvage therapy; their one-year survival rate was 18%.

Conclusions. The outcome of adults with ALL undergoing second salvage therapy is poor. Novel effective therapies against ALL are needed in this subset of patients.
Resveratrol Inhibits Proliferation and Induces Apoptosis of Mouse Pten Null T-Cell Acute Lymphocytic Leukemia Cells
Authors: Liu Qing , Gao Xiuhua , Yin Cheng C. , Ling Chen , You M. James
Published: April 2020

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a plant antibiotic produced in a wide variety of plants (including grapes, peanuts, and mulberries) in response to stress, injury, UV irradiation, and fungal infection, has been shown to inhibit cell growth of several types of cancer. Here, we have investigated the effects of Resveratrol on the proliferation and apoptosis in T-cell lymphocytic leukemia (T-ALL) cells. These T-ALL cells were derived from T-ALL of mice with the T-cell specific deletion of the Pten tumor suppressor gene. Flow cytometric analysis demonstrated that the T-ALL cells are positive for CD4 and negative for CD8. Morphologically, the T-ALL cells strikingly mimic human counterparts. Western blot analysis confirmed the absence of Pten expression and high expression level of phosphorylated Akt of the T-ALL cells. A single dose treatment of the T-ALL cells with Resveratrol (ranging from 3 to 50 mM) resulted in significant decrease in cell growth. In addition, Resveratrol treatment with the above concentrations showed a remarkable increase in apoptosis. The vehicle treated T-ALL cells grew exponentially and had minimal apoptotic cell death. Further investigation of the mechanisms of the Resveratrol’s efforts on inhibiting T-ALL cell growth and inducing apoptosis is on-going. In addition, combination of Resveratrol with conventional chemotherapy reagents for treating T-ALL is also being evaluated.
Prognosticating of Relapse Risk Based on Multiparameter Flow Cytomeric Assessment of Minimal Residual Disease in Patients with Acute Myeloid Leukemia
Authors: Wan Suigui , Qiu Lugui , Sun Xuejing , Zhao Hong , Su Li , Xu Juan
Published: April 2020

Abstract Objective: To investigate the prognosticating of relapse risk based on assessment of minimal residual disease(MRD) in patients with acute myeloid leukemia(AML).

Methods: Multiparameter flow cytometry (MPFC) analysis were used to detect the leukemia-associated aberrant immunophenotype (LAIP)of the pretreated patients with AML and assess the level of MRD after remission induction(Post-Ind MRD) and consolidation therapy(Post-Cons MRD).

Results: The results showed that definite LAIP could be detected in about 94.3%of patients (115/122) with AML (except APL). Only one LAIP was identified in 15 cases (13.0%), but in other 100 cases (87.0%), two or more LAIP were identified. The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0% and 34.8% respectively. MRD frequency was monitored in 41 CR patients with AML after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD+ and 17 patients were Post-Ind MRD−. The percentages of relapse in Post- Ind MRD+ and Post-Ind MRD− were 75.0%(18/24) and 29.4%(5/17) respectively after consolidation chemotherapy. The relapsed free survival(RFS) of the patients with Post-Ind MRD+ and Post-Ind MRD− was 49.06±6.53 months and 11.92±1.64 months(P<0.0001) respectively. 18 patients were Post-Cons MRD+ and 23 patients were Post-Cons MRD−. The percentages of relapse in Post-Cons MRD+ and Post-Cons MRD− patients were 100%(18/18) and 21.7%(5/23) respectively after consolidation chemotherapy. The RFS of the patients with Post-Cons MRD+ and Post-Cons MRD− was 41.74±5.52 months and 10.06±1.72 months (P<0.0001) respectively.

Conclusions: The level of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. MRD measured by MPFC provided prognostic information in AML patients.
Polymorphisms in Myeloid Cell Leukemia-1 and the Risk for Acute Myeloid Leukemia
Authors: Kim Hee Nam , Yu Li , Kim Nan young , Lee Il-Kwon , Kim Yeo-Kyeoung , Yang Deok-Hwan , Lee Je-Jung , Kim Hee-je , Min Woo-Sung , Park Moo-Rim , Jo Deog-Yeon , Won Jong-Ho , Kwak Jae-Yong , Kim Hyeoung- Joon
Published: April 2020

The essential pathogenic mechanism in acute myeloid leukemia (AML) is a heterogeneous group of malignant diseases arising as the result of progressive genetic damage occurring in hemopoietic progenitor cells. Mcl-1 (myeloid cell leukemia-1), an antiapoptotic protein of the Bcl-2 family, is located on chromosome 1 and has three exons and two introns. Overexpression of Mcl-1 delays apoptosis induced by cytotoxic agents, c-myc overexpression, and growth factor withdrawal in hematopoietuc cells. To evaluate the association between genetic variants of Mcl-1 gene and risk of AML, we genotyped two polymorphisms in promoter of Mcl-1[rs3738484; −324 C>A and rs3831987; −284 insertion(6bp or18bp)/deletion]. A case-control study of 728 controls and 660 cases was conducted in Chonnam National University Hwasun Hospital, Korea. The Mcl −324 CA and combined CA/AA genotype was significantly associated with a decreased risk for AML [odds ratio (OR) CA = 0.75; 95% confidence interval (CI) = 0.60–0.95; ORCA/AA = 0.76; 95% CI = 0.61–0.96]. There was no association with the Mcl -284 insertion/deletion and AML. The haplotype A-ins18bp is significantly associated with the risk of AML. Using subjects with the haplotype A- del, the OR of haplotype A-ins18bp is 5.87 (95% CI 1.7–20.06 p= 0.006). Our results suggest that antiapoptotic protein, Mcl-1 polymorphism may influence susceptibility to AML.
Expression and Prognostic Significance of MDR and IAP-Family Genes in Patients with Acute Myeloid Leukemia
Authors: Cioch Maria B. , Kocki Janusz , Kotwica Katarzyna , Gromek Tomasz , Dmoszynska Anna
Published: April 2020

Introduction. Refractory Acute Myeloid Leukemia (AML) is associated with very bad prognosis. One of the major reason of induction chemotherapy failure is the expression of genes and proteins responsible for multidrug resistance (Multidrug Resistance 1 - MDR1, Multidrug Resistance-Related Protein 1 - MRP1, Breast Cancer Resistance Protein-BCRP, Lung Cancer Related Protein - LRP) and apoptosis (IAP-family genes: XIAP, cIAP1, cIAP2, survivin). The aim of the study was the evaluation of the influence of MDR and IAP-family genes expression on results of chemotherapy in patients (pts) with AML.

Material and methods. The study was performed in 45 pts (20 females and 25 males; ranged from 18 to 82 years - mean 51.2) with newly diagnosed AML. There were following types acc. FAB classification: M0–1, M1–5, M2–13, M3–4, M4–16, M5–6 and cytogenetic risk groups: favorable-8, intermediate-29 and unfavorable-8 pts. The genes expression in leukemic cells was detected by RT-PCR method. All pts were treated with induction and consolidation therapy acc. Polish Adult Leukemia Group (PALG).

Results. The expression of MDR1 gene was detected in 47%, MRP1 in 21%, BCRP in 10%, LRP in 26% and IAP-family genes in 78% of pts. Expression of MDR1 was significantly higher in M5 (p=0.02), but expression of cIAP1 and cIAP2 in M4 type (p=0.01). There was significant difference in MDR1 expression between unfavorable and good risk cytogenetic group (p=0.03), but there was none difference between cytogenetic groups in expression of IAP-family genes. There was significant difference in MDR1 expression between pts who achieved complete remission (CR) and pts without CR (p=0.01). When the expression of MDR1 gene was associated with the expression of BCRP and LRP, CR was never obtained. In all pts with complex genetic changes (MDR1+MRP1, MDR1+BCRP+LRP, MDR1+MRP1+BCRP+LRP) CR after one course of induction chemoterapy was not obtained. There was significant difference between CR rate after one course of chemotherapy in pts without MDR genes expression or expression only one gene and in pts with expression of two or more genes (p=0.02). There was no correlation between IAP-family genes expression and CR rate. Despite of differences in post-consolidation therapy (bone marrow transplantation in some pts) expression of MDR1 and survivin genes correlated with overall surival rate (p=0.05 and 0.03, respectively).

Conclusions. Results of our studies on expression of MDR and IAP-family genes in pts with AML showed that expression of MDR genes better predicted response on induction chemotherapy than IAP genes. After confirmation on larger group of pts these findings may have important significance in defining of prognosis in AML and individualization of induction and postremission therapy, eventually with new generation of MDR modulators.
Apoptosis Was Induced by Casticin in Acute Myelocytic Leukemia Cells
Authors: Jin Jie , Shen Jia-Kun , Du Hua-ping , Yang Min , Wang Yun-Gui
Published: April 2020

Casticin, a component from Vitex rotundifolia wich was widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities in lung cancer and breast cancer. There are yet no reports on roles against acute myelocytic leukemia (AML). This study aims to elucidate the anti-leukemic activity of casticin on AML cells. We investigated the efficient efficacy and the mechanisms by which casticin triggers cell death in AML cells by analyzing cell cycle perturbations, apoptosis-related marker expression. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry and AV-PI assay. Western blot was performed to measure the apoptosis-related marker. Concentration-dependant cell deaths were observed in AML cell lines including K562, U937 and THP-1, with IC50 values of 24h (hours) being 47.4μM, 67.8μM and 61.7μM, respectively. Time-dependant cell deaths were also observed. At the concentration of 20μM casticin, 45.7%, 76.1% and 80.9% of K562 cells were inhibited at 24h, 48h and 72h, respectively; 24.7%, 30% and 61% of U937 cells were inhibited at 24h, 48h and 72h, respectively; while for THP-1, 29%, 41.8% and 53.9% were inhibited at 24h, 48h and 72h, respectively. Apoptosis was found using AV-PI staining by flow cytometry analysis. We observed an obvious G2/M phase increase prolongation in casticin treated K562 cells. BThe distribitions of G2/M phase were 2.9%, 33.6%, 75.3%, 54.9%, 29.7% and 27.0% in K562 cells after treated by 20μM casticin for 0h, 6h, 12h, 24h, 36h and 48h, respectively. Furthermore, apoptosis-related proteins, PARP and caspase 3, were cleaved in casticin treated K562 cells. Taken together, these results demonstrated that casticin can induce leukemic cell death through apoptosis, suggesting that casticin could be a promising therapeutic agent against acute myeloid leukemia.
Inactivation of JAK/STAT Cell Signaling by SK-7041, a Novel HDAC Inhibitor, Effectively Inhibits Growth of Acute Myeloid Leukemia Cells
Authors: Kim Byung-Su , Lee Chansu , Park Juwon , Ahn Kwang-Sung , Kim Byoung Kook , Park Seonyang , Lee Young-Yiul , Yoon Sung-Soo
Published: April 2020

Activation of the JAK/STAT pathway appears common in AML, occurring in up to 70% of AML patients. Therefore, JAK/STAT signal inhibitors are promising as candidate anti-cancer agents in AML. Recently, we reported that SK-7041, an HDAC inhibitor, inhibited the growth of AML cells via activation of caspase-3 and down-regulation of cyclin D1. These findings lead us to further examine whether SK-7041 inhibits the growth of KG1 AML cells via inactivation of JAK/STAT signals. Multi-immunoblotting technique (Kinetworks™ analysis) showed that expression of p-STAT-3, p-STAT-5, and p-Erk was down-regulated in KG1 cells treated with SK-7041. These results were confirmed by individual western blot analysis. In addition, IL-6-induced activation of STAT-3 and Erk was inhibited by treatment of SK-7041. Combined treatment of SK-7041 and JAK inhibitor (AG490) showed additive anti-leukemic effect as evidenced by caspase-3 activation, down-regulation of cyclin D1 (cMYC), and inhibition of phosphorylation of STATs (−1, −3). These results suggest that HDAC inhibitor, SK-7041, inhibited AML cell growth via inactivation of JAK/STATs pathway.
Pemetrexed Treatment of Breast Relapse in AML
Authors: Wang Chunyan , Tan Huo , Peng SiDa , Huang ZhenQian
Published: April 2020

Extramedullary relapse is a pattern of acute myeloid leukemia (AML) relapse post-allogeneic hematopoietic stem cell transplantation (alloHSCT). We present the case of a 43-year-old female patient with acute mix cell leukemia who had isolated extramedullary relapse develop in both breasts 2 years after alloHSCT, the first reported case in adult patients in the literature to the best of our knowledge. She received further chemotherapy with pemetrexed (Alimta) intravenous injection (500mg/D1, every 3 weeks is one course of treatment). After 2 courses of treatments, her swelled breasts were shrunk remarkably and no obvious side effects were found. We therefore conclude that pemetrexed treatment may be an effective regimen for breast extramedullary leukemia. Review of the literature about extramedullary relapse and breast involvement as well as the treatment of these cases were further discussed.
Clofarabine/Cyclophosphamide/Etoposide Combination Chemotherapy Well Tolerated in An Infant with Refractory AML
Authors: Rahman Shahera , Connor Philip P , Moppett John , Jenney Meriel , Cummins Michelle , Cornish Jackie
Published: April 2020

Refractory AML in children under one year of age is rare. Generally the high induction remission rate is offset by higher treatment-related mortality. We report a male infant who presented with generalised lymphadenopathy and hepatosplenomegaly aged 3 months. Morphology was consistent with M5 AML and cytogenetics showed a t(9;11)(p22;q23) MLL rearrangement. He was started on standard AML induction treatment for children under 1 year of age according to the AML15 protocol (daunorubicin 37.5mg/m2 days 1, 3, 5 with etoposide 75mg/m2 days 1 through 5 and cytarabine 75mg/m2 twice daily for 10 days). He never demonstrated count recovery and by day 25 post treatment, lymphadenopathy had returned. A bone marrow aspirate showed M5 morphology with 80% of cells showing MLL rearrangement by FISH. He was therefore taken off protocol and administered fludarabine 30mg/m2 and cytosine 2g/m2 once daily for 5 days and gemtuzamab ozogamicin 3g/m2 on day 1 (FLA-GO). At day 21 post chemo, his platelet count rose into the normal range but he remained neutropenic. A bone marrow showed some normal haematopoiesis but 33% of cells were blasts with MLL rearrangement by FISH. It was decided to repeat the FLA-GO but by day 25 post chemotherapy lymphadenopathy and organomegaly had returned and marrow examination showed 50% blasts with MLL rearrangement. At this point it was decided to administer clofarabine 25mg/m2 on days 1 to 4 with etoposide 75mg/m2 and cyclophosphamide 340mg/m2 on days 1 to 5. He tolerated the regimen well, the only side effect being myelosupression. By day 24 he had recovered peripheral counts, had no lymphadenopathy or organomegaly and a marrow showing 9% blasts with MLL rearrangement. He was able to proceed with allogeneic stem cell transplantation. This shows that clofarabine-containing combination regimens can be well tolerated in the very young.
Nature Products Enhance NKG2D Ligands Expression of CD123+CD34+CD38− Leukemia Stem Cells for Stimulating Cytotoxicity of NKG2D+ Cells to Themselves
Authors: Kunyuan Guo
Published: April 2020

CD123+CD34+CD38− leukemia cells regarded as leukemia stem cells, not only refractory to chemotherapeutics but also resistant to immune response such as cytotoxicity of prime natural killer cells, usually lead leukemia to relapse. It is essential to eliminate leukemia stem cells at utmost for cure. Here we would observe whether nature products could enhance CD123+CD34+CD38− leukemia cells to express NKG2D ligands for arousing cytotoxicity of NKG2D+ cells to thoroughly eliminate leukemia stem cells. First, CD123+CD34+CD38− leukemia cells, isolated from human acute leukemia cell line KG1a, whose MLL gene was broken apart and rearranged, of which over 60% were in G0 phase of cell cycle, which could form about 80 colonies per 1000 cells in methylcellulose after 21 days. What’s more, they were worse than CD123+CD34+CD38+ leukemia cells on resistance to cytotoxicity of natural killer cells(Killed ratio <19%). CD123+CD34+CD38− leukemia cells from KG1a had characteristic of defined leukemia stem cells. Then, NKG2D+ cells from human peripheral blood monocytes were stimulated by cytokines and CpG, whose cytotoxicity against K562 cells reached (96.5±1.23)% analyzed by LDH releasing assay, suggesting NKG2D+ cells had better cytotoxicity than prime natural killer cells (69.31±8.45)%. However, cytotoxicity of NKG2D+ cells against CD123+CD34+CD38− leukemia stem cells was only(49.17±3.90)%, still higher than of prime natural killer cells (19±2.63)%. Therefore, whether to arouse cytotoxicity of NKG2D+ cells to deplete leukemia stem cells become essential. Finally, three kinds of nature products would be selected, including resveratrol (RV, a polyphenol found in berries), matrine and arsenic trioxide (ATO), which could decrease proliferation and survival of CD123+CD34+CD38− leukemia cells and whose 50% effective concentration (EC50) were respectively 56μM, 500μg/ml and 4.0μM. Importantly, EC50 of resveratrol could up regulate NKG2D ligands ULBP1 to (53.28±8.60)%, ULBP2 to (36.54±11.45)% and ULBP3 to (65.08±14.44)% on the surface of CD123+CD34+CD38− leukemia cells, and stimulate cytolytic ability of NKG2D+ cells, so that cytotoxicity of NKG2D+ cells against them reached from (49.17±3.90)% to (92.33±2.63)%. Similarly to matrine and arsenic trioxide, they could each up regulate ULBP2 from to 9.71±2.37% and ULBP1 to 24.38±3.65%, resulting in cytotoxicity of NKG2D+ cells against them reaching to 73.20±3.41% and 55.70±0.53%. In conclusions, cytotoxicity of NKG2D+ cells was better than of prime natural killer cells against not only K562, but also CD123+CD34+CD38− leukemia cells, which had characteristic of leukemia stem cells, even to resist killing of prime natural killer cells. Nature products, resveratrol, matrine and arsenic trioxide, could enhance NKG2D ligands expression of CD123+CD34+CD38− leukemia stem cells for stimulating cytotoxicity of NKG2D+ cells to leukemia stem cells. This finding could provide a new strategy for eliminate leukemia stem cells.
Rapid Genotyping of SNPs Influencing Warfarin Drug Response by SELDI-TOF Mass Spectrometry
Authors: Yang Shangbin , Xu Lihui , Wu Haifeng
Published: April 2020

The anticoagulant drug Warfarin exhibits significant inter-individual variability in dosing requirements. Different responses to Warfarin therapy are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either Warfarin drug action (VKORC1) or drug metabolism (CYP2C9). Rapid genotyping of these SNPs is essential for clinicians to choose appropriate initial doses in order to quickly achieve anticoagulation effects and to prevent the complications associated with Warfarin overdoses. In this study, we explore the utility of surface-enhanced laser desorption and ionization time-of- flight (SELDI-TOF) mass spectrometry in the rapid genotyping of SNPs that control Warfarin drug sensitivity. The DNA containing the targeted SNPs is first amplified by polymerase chain reactions and then underwent the single base extension to generate specific SNP product. Afterwards, genetic variants displaying different masses are bound to Q10 anionic proteinChips and genotyped using a SELDI-TOF mass spectrometer in a multiplexed fashion. SELDI-TOF mass spectrometer offers a unique property of on-chip sample enrichment and clean-ups. Therefore, this genotype method eliminated many tedious experimental steps, such as sample desalting and concentrating, that are required prior to detection by standard mass spectrometers. The turnaround time for genotyping three known Warfarin sensitivity SNPs, CYP2C9*2, CYP2C9*3, VKORC1, is less than five hours. The analytical accuracy of genotyping detected by SELDI mass spectrometer has been confirmed by DNA sequencing. In summary, we have devised a novel multiplex genotyping method using a SELDI-TOF mass spectrometer. This test is fast, accurate, and therefore provides a superb clinical laboratory platform to promote the personalized health care in Warfarin therapy.
Practical Application of the 10 Mg Warfarin Initiation Nomogram
Authors: Wells Philip , Le Gal Gregoire , Tierney Sarah , Carrier Marc
Published: April 2020

Introduction: Initiation of warfarin therapy is a clinical challenge. A 10 mg warfarin initiation nomogram was recently validated in a randomized controlled trial. We sought to determine the efficacy and safety of this 10 mg warfarin initiation nomogram in “real-life” daily practice.

Methods: A retrospective cohort including all outpatients beginning concurrent treatment with warfarin and low-molecular-weight-heparin (LMWH) over a 24 month period in our Thrombosis Unit was reviewed.

Results: 841 patients were included, of them 640 (76.1%) were started on the nomogram. The nomogram was entirely followed in 324 patients (38.5%). The efficacy and safety profile was similar to that observed in the original clinical trial: 86% of patients managed according to the nomogram reached the INR target of 2.0 to 3.0 within 5 days. Mean duration of LMWH treatment was 6.0 ± 1.9 days, and 3.7% of patients had an INR ≥ 5.0 in the first four weeks of treatment.

Conclusion: The 10-mg nomogram effectively results into an early therapeutic INR with a good safety profile in “real life” daily practice.
Oncological and Hematological Neoplasms: Concomitant Oral Anticoagulation Therapy
Authors: Rupoli Serena , Lio Lidia Da , Scortechini Anna Rita , Ravaglia Francesca , Pulini Stefano , Mulattieri Simonetta , Picardi Paola , Canafoglia Lucia , Stronati Andrea , Tomassetti Simona , Tocchini Massimo , Leoni Pietro
Published: April 2020

The oral anticoagulant therapy (OAT) in oncologic patient is often problematic because both the haemorragic risk and the thrombotic risk, i.e. the recurrence of venous thromboembolism, are increased. Many studies have been conducted in patient with solid neoplasms; it remains still unclear whether these results could be entirely extrapolated to patients with haematological malignancies. Our aim was to analyse the clinical outcomes and the control of anticoagulation, measured as % time in target INR range, in the patients with cancer compared to patients without cancer; moreover we focused on patients with haematological neoplasms. A total of 500 patients were recruited in 6 years, regardless of the indication for OAT; 146 (29%) of them were affected by cancer (47 haematological and 99 solid neoplasms); those being treated with warfarin for less than 1 month were excluded from this analysis. It resulted that patients affected with cancer had a worse anticoagulation control, compared to those without cancer (55% vs 60%; p<0.001). The rate of thrombosis was similar (6/146=4.1% vs 14/354=3.9%; OR 1.1); moreover the former had a tendency toward a higher rate of major bleeding complications (3/146=2.1% and 5/354=1.4% respectively; OR 1.5). As regarding hematologic patients we noted that the quality of anticoagulation control did not differ compared with solid malignancies (54% vs 55%, p = ns); no significant differences were found in the major bleeding (1/47=2.1% and 2/99=2.0% respectively; OR 1.05) and thrombotic complications (2/47=4.3% and 4/99=4.1% respectively; OR 1.05). The cancer patients discontinued warfarin primarily for surgical, endoscopic diagnostic or therapeutic procedures (32% vs 19%; p<0.001); in particular the surgical procedures more frequently were the reason for discontinuation (27% vs 4% of the haematological patients, p<0.001). The 47 haematological patients showed a younger median age compared to those with solid neoplasms (67 vs 72 years, p<0.001), and discontinued OAT often because of invasive procedures (45% vs 26% of cancer patients, p<0.02). In this experience the haematological patients seem not to have an increased risk of thrombosis recurrence and bleeding during OAT. Such a result probably reflects a good management of OAT even in presence of risk factors like central vein catheter or thrombocytopenia. Moreover, an appropriate initial decision of employing low-molecular-weight heparins (LMWH) rather than OAT in particular subsets of haematological patients (for example those with life-expectancy less than 2–3 months or undergoing high-dose chemotherapy for stem cell transplantation) can reduce the hemorragic risk.
The Effect of Sample Storage on Impedance and Optical Platelet Counts
Authors: Munro Roger C , Tranter Jeremy , Hart Laura , Jones Ruth , Benton Ann , Worthing Anita , Fitzpatrick Caroline , Al-Ismail Saad , Wakeman Lisa J
Published: April 2020

Introduction: Processing blood samples for full blood count may be delayed for a multitude of reasons. There is little published information on the effect of sample storage on platelet count. This study compares the effect of storing normal samples at room temperature (RT) and at 4°C prior to processing using different analysers with different technologies. Sysmex SF 3000 and XE2100 analysers are capable of providing impedance (IMP) platelet counts and the latter can also perform fluorescence based optical (OPT) platelet counts. The Coulter LH750 and the ABX Pentra DX120 use IMP technology with enhanced data extraction techniques and algorithms to eliminate interference.

Method: Intravenous blood samples for routine blood counts were taken into 4ml K2 EDTA Greiner Vacuette containers from 20 out-patients and the platelet counts measured at 0, 6 and 24 hrs on Coulter LH750 (IMP), ABX Pentra DX120 (IMP), Sysmex SF3000 (IMP) and Sysmex XE2100 (both IMP and OPT) blood count analysers. Samples were stored at RT. Samples were also processed on the XE2100 following storage at 4°C.

Results: Mean changes in platelet counts (n = 20) expressed as percentages of the original value at 0 hrs and paired t-test values of significance are shown in the Table. The most significant changes occurred with the XE2100 where the IMP value increased by 7% over 24 hrs but the OPT value fell by 5%. No significant changes occurred with the Coulter or the ABX analysers at 0 and 24 hrs or with the SF3000 at 6 hrs.

6 hr v 0 hr 24 hr v 0 hr % change p % change p Coulter RT + 0.19 0.873 −0.12 0.908 ABX Pentra DX120 RT + 0.02 0.985 + 0.57 0.380 SF 3000 RT + 1.25 0.281 + 2.52 <0.05 XE 2100 Imp RT + 5.26 <.05 + 7.06 <0.05 XE 2100 Imp 4°C + 3.31 <0.05 + 5.06 <0.05 XE 2100 Opt RT −2.20 <0.0 −5.32 <0.05 XE 2100 Opt 4°C + 2.30 <0.05 −0.40 0.750

EDTA exposure and the formation of membrane lesions during sample storage leads to changes in platelet structure, volume, function and shape due to decreased optical density and the beginning of degranulation. All these have a potential impact on the accurate assessment of platelet numbers. If these numbers are to be used accurately as thresholds for platelet transfusion or for monitoring progressive loss or increases of platelets associated with underlying pathology, it is vital that the pre-analytical limitations of current analysers are fully understood. The way in which discriminators are used on various analysers to make distinction between the characteristics of platelets and other blood cells in individual, stored samples will vary in their effectiveness.

Conclusions: Our data indicate that various combinations of pre-analytical sample storage and analyser technology should be considered as potential influences on the outcome of platelet counting in the clinical laboratory.
Studies on Platelet Storage Biomarkers: Effect of Different Protein Extraction Buffers on Platelet Gelsolin and B-Actin Profiling
Authors: Kulkarni Sandhya , Kannan Meganathan , Atreya Chintamani D
Published: April 2020

Platelet concentrates (PCs) are one of the blood cell components that play a major role in transfusion medicine. The shelf-life of PC is 5–7 days at 22°C. During storage PCs undergo certain morphological changes as well as physiological changes such as loosing of its surface glycoproteins, and reduced aggregation response to its agonists, which are collectively referred to as “storage lesions”. To date there is no in vitro “gold standard” that unequivocally serves as a surrogate marker to predict PC quality in vivo following transfusion. A comprehensive survey of reports in this field clearly indicate that there is some confusion with regards to the determination of which platelet protein or a panel of proteins could serve as potential markers associated with PC storage. Following a careful review of all available information, we recognized that perhaps one core issue to the observed discrepancies could be the buffer system that is being utilized by individual laboratories in extracting proteins in profiling protein alterations in PCs during storage. To address this, in the present study we analyzed two platelet proteins, b-actin and gelsolin-related 40 kDa protein. Profiles of these two proteins were previously reported by others to be altered during PC storage and hence implicated to be useful as biomarkers of platelet storage. By employing 3 different protein extraction buffer systems to isolate proteins from platelets at three storage intervals (Table 1), our results indicate that

b-actin and gelsolin 40 kDa protein levels do not appear to change with time in platelet storage and therefore may not be useful as platelet storage markers and Buffer systems do make a difference in extracting different platelet proteins for analysis, for example 40 kDa gelsolin was not extractable in buffer 3 (Table 1), suggesting that one should be careful in selecting a buffer system for platelet proteomics to have meaningful results.

Same level of 40 kDa Gelsolin Same level of b-actin Buffer system Day 0 Day 5 Day 16 Day 0 Day 5 Day 16 Buffer 1 Not identified Yes Yes Yes Yes Yes Buffer 2 Not identified Yes Yes Yes Yes Yes Buffer 3 Not identified Not identified Not identified Yes Yes Yes

The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.
Effects of TAK-442, a Direct FXa Inhibitor, and Fondaparinux on Platelet-Associated Prothrombinase in the Balloon Injured Artery of Rats
Authors: Kawamura Masaki , Konishi Noriko , Katsuhiko Hiroe , Imaeda Yasuhiro , Fujimoto Takuya , Kubo Keiji
Published: April 2020

PCI patients routinely experience arterial injury during balloon angioplasty and stent deployment. At the injured arterial site, endothelial damage triggers platelet adhesion to the vascular wall and initiates platelet-associated prothrombinase complex (factor Xa [FXa]-factor Va-phospholipids-calcium) formation, resulting in the progression of thrombus formation. Recently, it was demonstrated that treatment with fondaparinux reduced the risk of death or recurrent myocardial infarction without an increase in the incidence of major bleeding compared to standard therapy in patients with STEMI. However, fondaparinux showed no superiority to placebo or heparin in patients undergoing PCI. On the other hand, small molecule direct FXa inhibitors, which are still in early stages of clinical development for the prevention of ACS, have been reported to strongly block reconstituted prothrombinase. Here, we compared the effects of TAK-442, a novel direct FXa inhibitor on platelet-associated prothrombinase activity, with fondaparinux. TAK- 442 and fondaparinux inhibited endogenous FXa activity in platelet-poor plasma from humans (IC50: 53 nM, TAK-442; 11 nM, fondaparinux) and rats (IC50: 32 nM, TAK-442; 19 nM, fondaparinux). When TAK-442 or fondaparinux were preincubated with free FXa, they each inhibited in vitro reconstituted prothrombinase activity, with IC50 values of 28 nM and 3.9 nM, respectively. In the absence of free FXa preincubation, TAK-442 retained this inhibitory effect (IC50, 51 nM); the inhibitory effect of fondaparinux, however, decreased dramatically (IC50, 1700 nM). In a rat model of balloon injury, thrombin activity on the surface of injured vessels increased to 3.3-, 20- and 5.7-fold that of intact aorta at 5 min, 1 hr, and 24 hrs after the injury, respectively. At 1 hr after the injury, 300 nM TAK-442 significantly (p<0.025) inhibited platelet-associated thrombin generation on the surface of injured aorta by 99% (IC50, 19 nM), whereas the same concentration of fondaparinux showed no significant (p=0.076) inhibition (IC50, >300 nM). These results highlight a limitation of fondaparinux in inhibiting platelet-associated prothombinase activity that leads to thrombus formation compared with TAK-442, and suggest that TAK-442 may be more effective in preventing arterial thrombosis in patients undergoing percutaneous coronary interventions.
Fibrinokinetic Deficit in Chronic Kidney Disease and End Stage Renal Disease Patients Contributes to the Hemostatic Abnormalities
Authors: Adiguzel Cafer , Bansal Vinod , Cunanan Josephine , Litinas Evangelos , Hoppensteadt Debra , Fareed Jawed
Published: April 2020

An increase in bleeding is observed in patients with chronic kidney disease (CKD) and end stage renal disease (ESRD) despite the normal coagulation profile and fibrinogen level. The hemostatic deficit in these patients may due to the defects in fibrin formation. The fibrinokinetic profile of CKD (n=50) and ESRD patients on hemodialysis prior to and after dialysis was measured. Citrated plasma from each patient was supplemented with 25 μl of 5 U thrombin and 25 μl of 0.05 M CaCl. The rate of fibrin formation was measured by monitoring the optical density (OD) at 405 nM. After reaching steady state, 50 μl of urokinase (30,000 U/ml) was added to measure the fibrinolytic profile. Forty normal male and female individuals were also analyzed. In addition PT, aPTT and fibrinogen levels were also measured. The normal male and female fibrinokinetic profile showed a strong clot formation with the clot reaching an average OD of 1.2±0.3 (range 0.7–1.4). In the CKD patients a much weaker clot was formed with an average OD of 0.21±0.13 (range 0.05–0.41). In the ESRD patients on maintenance hemodialysis, the pre-dialysis sample showed a weaker fibrinokinetic profile reaching near normal levels with a clot density of 1.3±0.4 (range 0.8–1.6). In the urokinase induced fibrinolysis assay, the CKD patients plasma exhibited a much stronger fibrinolytic index in comparison to the normal population (80% clot lysis vs. 20%). In the ESRD patients the clot lysis profile was relatively weaker in comparison to the CKD patients, suggesting that hemodialysis may contribute to impaired clot formation. These results are contrary to the reported observations that dense clot resistant to fibrinolysis are formed in CKD and ESRD patients. Furthermore the clot sobserved in these patients were highly susceptible to lysis. The maintenance hemodialysis results in improving the fibrinokinetic and fibrinolytic profile in the ESRD patients. Therefore maintenance hemodialysis contributes to improved hemostasis in ESRD patients.