bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the Redox Function of Ape-1/ref-1 in Myeloid Leukemia Cells Results in Enhanced Sensitivity to Retinoic Acid Induced Differentiation and Apoptosis.
Authors: Robertson Kent A. , Colvin Edwin S. , Kelley Mark R.
Published: October 2019

Ape-1/ref-1 is a multifunctional base excision DNA repair protein that is involved in the repair of abasic sites in DNA. However, it also has a distinct role in the redox regulation of a variety of cellular proteins, such as Fos, Jun, p53, NFκB, PAX, HIF-1α, HLF, and others. Ape-1/ref-1 maintains these proteins in a reduced state thereby facilitating their DNA binding and transcriptional activation capability. HL-60 cells are known to respond to retinoic acid (RA) with terminal granulocytic differentiation and apoptosis, which is mediated through the RA receptors. Previous experiments suggested that elevated Ape-1/ref-1 expression is related to differentiation and apoptosis. To further define the role of the redox function of Ape-1/ref-1 in this relationship, redox function was blocked using two techniques. First, we used retroviral gene transduction to over-express a redox-inactive C-65 mutant of Ape1/ref-1 in HL-60 myeloid leukemia cells and examined the response to retinoic acid. In a second set of experiments we used an Ape-1/ref-1 specific small molecule inhibitor to pharmacologically block the redox function and again examined the response to retinoic acid. Differentiation was evaluated by morphologic change in differential cell counts and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis was examined with propidium iodide flow cytometry.

Results:

HL-60 cells expressing high levels of C-65 Ape-1/ref-1 responded to retinoic acid with a significantly higher level of differentiation and a moderate increase in apoptosis. Pharmacologic blockade of Ape-1/ref-1 redox function resulted in a profound increase in differentiation and a moderate increase in apoptosis compared to controls. dose dependent studies with retinoic acid demonstrated a similar degree of differentiation (CD11b expression) in cells treated with 10 μmolar retinoic acid and those treated with the redox inhibitor + 0.1 μmolar retinoic acid; alllowing HL-60 cells in the presence of the redox inhibitor to give a similar response to a 100 fold lower dose of retinoic acid.

The redox inhibitor alone did not induce differentiation and induced only a minimal amount of apoptosis but did increase the number of cells in S phase significantly. In conclusion, our data supports the contention that redox function of Ape-1/ref-1 may be important for controlling RA-induced myeloid differentiation and programmed cell death. The implication of these findings is that myeloid leukemia cells may be sensitized to retinoids by manipulation of the redox status of Ape-1/ref-1.
Importance of the Polymorphisms of the Thiopurine S-Methyltransferase Gene (TMPT) Determination in Mexican Mestizo Patients with Acute Lymphoblastic Leukemia (ALL).
Authors: Candelaria Myrna , Taja-Chayeb Lucia , Vidal Silvia , Gutierrez Olga , Dueñas-Gonzalez Alfonso
Published: October 2019

Background: Polymorphisms at the thiopurine S-methyltransferase coding gene (TPMT) determine enzyme activity and consequently, the development of toxicity secondary to thiopurines.

Methods: A total of 108 DNA samples from volunteer donors and 39 from patients with acute lymphoblastic leukemia (ALL) were analyzed. Genomic DNA from peripheral blood leukocytes was isolated by standard methods. Known (wild-type and polymorphic) sequenced polymerase chain reaction (PCR) fragments of the TPMT gene were used as controls. TPMT gene fragments were amplified for exons 5, 7 and 10. Thereafter, PCR products were analyzed by denaturating high performance liquid chromatography (DHPLC) for the most frequent mutant TPMT alleles, according to the method developed by Schaeffeler et al. on an analysis system from Transgenomics.

Results: No elution profiles on DHPLC analysis, different from those previously reported, were documented. Frequency of allele polymorphisms was 27.3% in healthy and 19.7% in ALL population. However, only 17.6% of all polymorphisms found are considered functional, the most frequent being *3A (n = 13; 4.4%), followed by *3B (n = 5; 1.7%), *3C (n = 5; 1.7%), and *2 (n = 3; 1.0%). From 39 ALL patients, 22 were treated with thiopurines, and particularly one patient, who was referred to our Hospital to investigate relapse, after developing pancytopenia while on treatment with thiopurine, had a TPMT*3C allele. He required broad spectrum antibiotics, platelets transfusions, as well as G-CSF. This example illustrates that TPMT polymorphisms must be determined before thiopurines are indicated.

Conclusions: DHPLC is a highly sensitive, rapid and efficient method to identify relevant TPMT gene mutations which allows the screening for genetic variability in the TPMT gene. This is the first analysis of the polymorphisms at this gene in Mexican population. The frequency of known polymorphisms was similar to those reported in other regions worldwide. Since a direct relation has been documented within functional polymorphisms and enzyme activity, and this methology is feasible to realize in any phase during the treatment of ALL patients, the routine typing of TPMT polymorphisms in the patients with ALL has been set in our Institution.

Table 1. Frequency of TPMT polymorphisms in mexican population Population TPMT*1 TPMT*1S TPMT*2 TPMT*3A TPMT*3B TPMT*3C * Acute Lymphoblastic Leukemia Healthy n(%) 50 (72.7) 42 (19.4) 2 (0.92) 7 (3.24) 5 (2.3) 3 (1.4) ALL* n(%) 24 (80.3) 5 (6.9) 2 (2.7) 6 (7.6) -- 2 (2.5)
Genetic Polymorphisms of DNA-Repair- and Detoxification Enzymes in Therapy-Related AML and MDS.
Authors: Fischer Stephanie , Seedhouse Claire , Schulz Thomas , Germing Ulrich , Truemper Lorenz H. , Haase Detlef
Published: October 2019

The most serious long-term complications of anti-tumour therapy are secondary malignancies. After treatment of haematological malignancies such as NHL, Hodgkin’ disease and multiple myeloma as well as solid tumours like breast cancer, lung cancer, soft tissue cancer and ovarian carcinoma therapy-related leukaemia occur in 0.5 to 10% of pts. Parameters which might allow an estimation of the individual risk to develop a therapy-induced neoplasia are urgently needed. Referring to recent findings defects in the DNA-repair-system and in detoxification genes are candidate predictive factors. We have examined 125 cases of t-AML/MDS in a retrospective study. The primary neoplasias were: breast cancer (33%), NHL (22%), M. Hodgkin (7%), multiple myeloma (8%) or other miscellaneous (30%). Cytogenetic data of therapy-induced malignancies were available in 116 of 125 pts. in 66% (n=78) of whom clonal abnormalities were detected. The most frequent single abnormalities were -7/7q- (15%), inv(16)(12%), +8 (8%), t(15;17) and del(20q) (5% each). Aiming to identify possible risk factors we used this patient cohort to study genetic polymorphisms influencing the activity of DNA-repair- and detoxification enzymes. For this purpose analyses of XRCC3, RAD51, GSTT1/M1/A1/P1 and NQO1 were performed. Our patient cohort (n=125) attracted attention that patients with a deficiency for GSTT1 or with combined defects of GSTT1 and M1 had a 1.7-fold (p=0.03625) and a 2.4-fold (p=0.00921) risk resp. of developing t-AML/MDS as compared to healthy controls (n=381). Noticeable is as well that the inactivating polymorphism of GSTA1*B was significantly more frequent in the control group (p=0.01847) in comparison to our patient cohort. Subsequently we generated different categories for statistical analysis according to the primary malignancy: As yet, focusing on GSTT1/M1 revealed an overrepresentation (p<0.0005) (OR=4.0) of double null genotypes for GSTT1/M1 in pts. with t-AML/MDS after adjuvant treatment for breast cancer (n=41). Preliminary results suggest that patients with tAML/MDS following NHL (n=22) who have a defect for XRCC3 have a 4.2-fold risk to develop a tAML/MDS (p=0.02960). The polymorphism of GSTA1*B was significant only in our control group in comparison to patients who showed NHL as a primary malignancy (n=22). We suppose that in this cohort the GSTA1*B polymorphism might protect against t-AML/MDS. This could be an explanation for the overrepresentation of GSTA1*B-defect in our control group as compared to our patient cohort. Other statistical analyses did not show any significance. Taken together polymorphism of GSTA1*B and XRCC3 might be associated with the risk to develop t-AML/MDS after NHL. Defects of GSTT1 and M1 can be taken as a first hint to a risk factor for t-AML/MDS following breast cancer.
Role of NOTCH1 in Aggressive NK-Cell Leukemia.
Authors: Makishima Hideki , Ota Masao , Suzuki Takefumi , Nakayama Kozo , Ishida Fumihiro
Published: October 2019

Back ground: Aggressive NK-cell leukemia (ANKL) is a malignant NK-cell proliferative disorder resistant to most chemotherapies and frequently shows rapidly progressive clinical course. ANKL is characterized with hepatosplenomegaly, hemophagocytic syndrome, and strong association with EBV, and is mostly observed among Asian populations. Oncogenic mechanism of ANKL is not clear. NOTCH1 is a gene encoding a transmembrane receptor that regulates T-cell development and is a major oncogene in T-cell lymphoblastic leukemia (T-ALL). Recently, the potential involvement of NOTCH signaling in the development of NK-cells has been reported. We discuss here that the NOTCH1 protein might play a significant role in the pathogenesis of ANKL.

Patients, materials, and methods: 6 ANKL cases, 7 Chronic NK-cell lymphocytosis (CNKL) cases, and 3 NK tumor cell lines (NKL, NK-92, and KHYG) derived from ANKL patients were enrolled. Chromosomal translocation (7;9) was not found in any subjects. The expression levels of NOTCH1 intracellular subunit (NOTCH-IC) were examined with flowcytometry and western blotting. NK-cell lines were cultured with g-secretase inhibitor (GSI) (L-685,458) for evaluation of the effect. Cell proliferation and apoptosis were measured with XTT assay and annexinV positivity, respectively. The mutation analysis of NOTCH1 genome was performed with nested PCR and direct sequencing. mRNA expression of NOTCH1 gene was measured with real time PCR using the corresponding TaqMan probes.

Results: NOTCH-IC was detected in CD2+CD3− fractions from healthy donors, CNKL patients, ANKL patients, NK-cell lines, and Cos7 cells transfected with NOTCH1 gene coding NOTCH-IC, but not in CD2-CD3− fractions. The expression levels were significantly high in ANKL patients, compared to healthy donors (P < 0.05). With western blotting, cleaved form (110kDa) of NOTCH1 protein was recognized in all NK-cell lines. NOTCH-IC expression was reduced with the treatment of GSI in NKL and NK-92, but not in KHYG. Cell proliferation was inhibited with GSI in NKL and NK-92, whereas the inhibitory effect was not seen in KHYG. There was no significant difference in the increase ratio of apoptotic cells after the treatment of GSI among 3 NK-cell lines. Mutations in HD, TAD, or PEST domain, where frequent mutations are detected in T-ALL, were not found in NK-cell lines, and no enhanced expression of NOTCH1 mRNA in ANKL cells, compared with healthy donor NK-cells.

Conclusion: These results indicate that NOTCH-IC was increased in ANKL cells and might be an important role in the proliferation. Genomic or transcriptional dysregulation would not be the cause for enhanced expression of NOTCH1 in ANKL contrast with T-ALL. Further investigation is required for the relationship with NOTCH1 ligands. In the future, NOTCH1 might be a novel therapeutic target in ANKL.
FLT3 Mutation in Patients with Acute Promyelocytic Leukemia Has No Effect on Therapy Response.
Authors: Xue Meng Xing , Qiu Hui Ying , Feng Yu Feng , Pan Jin Lan , Chen Su Ning , Wu De Pei
Published: October 2019

Fms-Like tyrosine kinase 3 (FLT3) mutations are one of the most frequent genetic changes in acute myeloid leukemia (AML) and are related to poor prognosis. However, in acute promyelocytic leukemia (APL) the prognostic significance of this mutation is not firmly established. We investigated FLT3 internal tandem duplications (FLT3/ITD) or point mutation of the activation loop domain (FLT3/ALM) in initial marrow samples in 160 APL patients, also studied the impact of FLT3 mutations on disease characteristics and clinical outcome. Some of FLT3/ITD+ samples were further examined to determine the ITD allelic ratio(ITD-AR) using Genescan analysis. FLT3/ITD and FLT3/ALM were detected in 31 (19.4%) and 17(10.6%) of the patients, 2(1.25%) showed both ITD and ALM mutations. 19 of 31 FLT3-ITD+ patients were examined to determine ITD-AR, which varied from 0.11 to 1.79, with a median of 0.92. ITD-AR in 8 patients were greater than 1.0. Both mutations were associated with higher white blood cell(WBC) count at presentation(P<0.05); Among patients with WBC counts greater than 10×109/L, FLT3/ITD and FLT3/ALM rates were 49.1%(26/53) and 22.6% (12/53) respectively, were both higher than those of patients with WBC counts lower than 10×109/L (P<0.01). For clinical outcome, the complete remission(CR) of FLT3-ITD+ patients was lower than that of FLT3-ITD− patients(64.5% vs 91.5%, P<0.01), however the CR rates were not significantly different between patients with and without FLT3-LAM(84.6% vs 100%, P>0.05). Six originally FLT3-ITD+ patients were not detectable mutation after remission; so were six patients with originally FLT3-LAM+; however FLT3-LAM in one originally FLT3-LAM+ patient reappeared after relapse. In conclusion, FLT3 mutations (FLT3/ITD or FLT3/ALM) were frequently identified in patients with newly diagnosed APL, and both mutations were associated with higher WBC count at presentation. FLT3/ITD is more frequent than ALM mutation, and predicts a poorer prognosis because of the lower CR rates, while, FLT3/ALM mutation did not show the same unfavorable prognostic effect. In our study, ITD-AR were not found significantly difference between CR and no remission patients (P>0.05)
Wilm’s Tumor 1 Gene Expression Is a Useful Marker for Minimal Residual Disease in Acute Myeloid Leukemia.
Authors: Hsu Jack W. , Liu Hong , Jamieson Katarzyna , Hou Wei , Chang Myron N. , Ahrens Kim P. , Braylan Raul C. , Wingard John R.
Published: October 2019

One of the difficulties in managing patients with acute myeloid leukemia (AML) is the detection of minimal residual disease. Only a fraction of AML patients have a distinct phenotype on flow cytometry or a genetic abnormality which can be followed as a sensitive marker for minimal residual disease (MRD). The Wilm’s tumor - 1 gene (WT1) is overexpressed in leukemic blasts, but not in normal blood or marrow cells, and thus can be used as a marker for MRD in leukemia patients who otherwise do not have a characteristic immunophenotypic or molecular marker that can be monitored. We prospectively measured serial peripheral blood WT1 levels by RQ-PCR in 54 patients with AML. Of the 54 patients, 23 patients were newly diagnosed. The remaining 31 patients were in complete remission prior to a planned allogeneic transplant. Subsequent WT1 levels were obtained during routine evaluation both during and up to three years after completion of either chemotherapy and/or transplant. Using regression analysis, we found that a normalized WT1 level greater than 10−4 (as compared to a level of 1 in K562 cells) indicates a higher likelihood of the patient having active leukemia with a sensitivity of 87.5% and a specificity of 89.1%. The positive and negative predictive values are 71.19% and 95.89% respectively. We also compared WT1 levels with peripheral blood and bone marrow blasts to determine whether a correlation existed. We found a strong correlation between WT1 expression and bone marrow blasts with a correlation coefficient of 0.77 (p< 0.0001). A weak correlation was found with peripheral blood blast count (correlation coefficient = 0.35, p<0.0001). Our data implies WT1 can be a useful marker for minimal residual disease in patients with acute myeloid leukemia, especially in patients who do not have an unusual phenotype or molecular marker to monitor MRD.
Prognostic Implications and Biological Roles of RhoH in Acute Myeloid Leukemia.
Authors: Iwasaki Toshihiro , Katsumi Akira , Kiyoi Hitoshi , Tanizaki Ryohei , Kobayashi Miki , Abe Akihiro , Matsushita Tadashi , Naoe Tomoki
Published: October 2019

The Rho family of small GTPases including Rho, Rac, and Cdc42 has been well characterized as molecular switches to transduce signals from plasma membrane to the downstream effectors. Rac1 and Rac2 are known to regulate engraftment and mobilization of hematopoietic stem cells. RhoH, a member of the Rho family, is specifically expressed in hematopoietic cells, and has been reported to inhibit the cell adhesion through regulating Rac and αLβ2 integrin. As RhoH is GTPase deficient and constitutively active, GTP-bound form, the activity of RhoH is directly related to the level of expression. Previous reports demonstrated the aberrant somatic hypermutation of RhoH gene as a novel mechanism of genetic lesion in diffuse large B-cell lymphoma, possibly through the deregulated expression although the role of the RhoH on leukemia is largely unknown. Here we have screened for the expression level of RhoH gene in the bone marrow samples from 90 previously untreated acute myeloid leukemia cases by using a real-time fluorescence detection method. The expression level of RhoH was neither related to the FAB classification, CR rate, nor WBC counts. In addition, the RhoH expression was not associated with the known gene mutations such as N-Ras, FLT3, and p53. However, the multivariate analysis demonstrated that low expression of RhoH was the independent unfavorable prognostic factor for overall and disease free survival (p=0.0028 and 0.003, respectively). RhoH did not affect the affinity modulation of α4β1 integrin, however, RhoH negatively regulate Rac activation in our system, suggesting that RhoH might work as a proapoptic molecule through Rac deactivation. Further investigations would be required to clarify the biological roles of RhoH on leukemic cells.
Retrospective Analysis of Post-Remission Modalities in AML Patients with Normal Karyotype.
Authors: Chae Yee Soo , Sohn Sang Kyun , Kim Jong Gwang , Cho Yoon Young , Moon Joon Ho , Ahn Byung Min , Kim Shi Nae
Published: October 2019

Background: Cytogenetics is considered as the most important factor to determine type of post-remission treatment in patients with AML However, there are controversy regarding to select consolidation modality in the intermediate risk group because of heterogeneity. We compared the outcome of patients with normal karyotype according to the different consolidation modalities.

Methods: AML patients with normal karyotype who achieved complete remission (CR) and did not have any contraindication for SCT, were included. The outcomes were retrospectively analyzed according to consolidation treatments.

Result: Among 51 patients who achieved CR, 35 patients underwent stem cell transplantation (SCT; 27 HLA-matched allogeneic and 8 autologous SCT), while 16 patients received high-dose cytarabine chemotherapy (HDAC) for consolidation therapy. No differences in the patient characteristics were observed. The estimated relapse rate at 3 years was significantly lower in the SCT group (38.6 vs. 75.6%, p=0.010). The DFS was significantly higher in the SCT group (HR=0.360; 95%CI=0.161–0.870; p=0.013), while OS was insignificantly higher in the SCT group (HR=0.489; 95%CI=0.199–1.205; p=0.120). There were no differences in DFS and OS between allogeneic and autologous SCT (p=0.714 and 0.515, respectively)

Conclusion: SCT showed a better survival outcome than chemotherapy alone as post-remission therapy in AML patients with normal chromosome. Considering late complications like GVHD, auto-SCT might be a feasible option for post-remission therapy in patients with normal karyotype.
Expression of p14ARF in De Novo AML with Normal Karyotype. Implication on Drug Resistance and Survival.
Authors: Paul Esbjorn O. , Deneberg Stefan , Lehmann Soren , Bengtzén Sofia , Nahi Hareth
Published: October 2019

The p53 protein exerts a significant role in growth control of cells and impaired function of p53 by mutation or otherwise is regarded to be important for development of many human cancers. The p53 protein is strongly regulated by the E3-ligase HDM-2 that specifically binds to p53 and causes proteosomal degradation. P14ARF encoded from the INK 4a/ARF gene locus of chromosome 9p, activates the p53 pathway by binding to and inhibiting HDM-2. The objective was to study if the level of mRNA for p14ARF in leukemic cells from patients with AML was related to in vitro drug resistance and clinical outcome. Leukemic cells (>90% pure) isolated from 46 adult patients with normal karyotype de novo AML were incubated with antileukemic drugs (daunorubicin, mitoxantrone, etoposide and Ara-C) cultured for 4 days. The effect was determined by bioluminiscence measuring of intracellular ATP compared to an untreated control. We also tested the activity against PRIMA (p53-dependent reactivation and induction of massive apoptosis), a novel, small molecule shown to activate down regulated p53. mRNA for p14ARF was determined by real time PCR.

Results: Patients whose leukemic cells expressed high level of p14ARF mRNA in the leukemic cells (≥0.2 compared to the housekeeping gene) had a significantly better survival compared to those with low level (<0.02). Median survival not obtained compared to 9 months. The mean activity of all tested conventional antileukemic drugs was higher on leukemic cell samples expressing p14ARF mRNA ≥0.2 compared to those with low levels. In contrast, PRIMA exerted significantly higher in vitro effect on leukemic cell samples with low levels of p14ARF mRNA. We conclude that low levels of p14ARF in leukemic cells from patients with normal karyotype AML is a marker for poor prognosis, which may depend on impaired p53 activity causing resistance against conventional antileukemic drugs. Treatment with p53 targeting drugs as PRIMA may be a future possibility to improve the outcome for these patients.
Predictive Value of Flow Cytometric MRD Analysis in Childhood Acute Lymphoblastic Leukaemia at the End of Remission Induction Therapy.
Authors: Motwani J. , Jesson J. , Sturch E. , Eyre L. , Short P. , Williams M.D. , Darbyshire P.J. , Hill F.G.H. , Lawson S.
Published: October 2019

Patients with acute lymphoblastic leukaemia (ALL) in morphological remission may still have up to 1010 residual malignant cells. Detection of minimal residual disease (MRD) at the end of induction therapy allows better estimation of the leukaemic burden and can help selection of appropriate therapeutic strategies. Flow cytometric (FC) detection of MRD is based on the identification of immunophenotypic combinations expressed on leukaemic cells but not on normal hematopoietic cells - leukaemia associated immunophenotypes (LAIPs). We prospectively analysed bone marrow samples from 77 patients who presented with ALL to our unit between 1999–2003 and attained morphological remission. These patients were treated on a standard protocol. Multiparameter FC identification of LAIPs was performed at various time points, as dictated by the treatment protocol. Our results show that flow cytometric MRD at the end of induction therapy is an independent and the most significant predictor of relapse, both on univariate and multivariate analysis. The relapse risk was 4% if day 28 MRD was <0.01% and 50% if day 28 MRD was >0.01% (p<0.05). We conclude that flow cytometric based MRD assays can be used to assess early response to treatment and predict relapse in a similar way to molecular MRD analysis at the end of induction therapy. Flow cytometric analysis of MRD offers the advantages of being cheaper, more widely available and has quicker turnaround times.
Influence of Maternal Folic Acid Supplementation on Hematopoietic Factors in Offspring.
Authors: Johnson Kimberly J. , Largaespada David A. , LeBien Tucker W. , Allaei Raha , Ross Julie A.
Published: October 2019

Observational studies of childhood acute lymphoblastic leukemia (ALL) have generally supported a protective role for maternal intake of foods or vitamins rich in folic acid during pregnancy. Using C57BL/6J mice, we evaluated the effect of dietary folic acid intake in the perigestational period on surface marker and gene expression in B-lineage cells as a potential model to further elucidate the role of maternal diet in the etiology of childhood ALL. Thirty-six female mice were randomized to one of three folic-acid varied diets (low=0.3, control=2.0, high=8.0 mg/kg) one month prior to mating and maintained on the diet until pup weaning at three weeks. Reproductive characteristics and offspring red blood cell (RBC) folate were recorded, and bone marrow lymphocytes were isolated for surface marker characterization by flow cytometry. For gene expression analyses, CD19 positive B-lineage bone marrow cells were purified from three pups per dietary group using anti-CD19 microbeads. Gene expression levels were interrogated using the Agilent mouse whole genome microarray. No statistically significant differences were found among dietary groups with respect to mating success, litter size, estimated 21 day offspring weights, or the percentage of male offspring. However, there were significant differences among the 3 diets in offspring survival to the end of the experiment (39%, 84%, and 30% for the 0.3, 2.0, and 8.0 mg/kg groups, respectively). Pup RBC folate values were positively correlated with dietary folic acid dose with mean values of 428, 615, and 724 ng/ml, respectively. Mice were euthanized at 3 weeks. There was no significant difference in the expression of CD19, IgM, or kappa light chains on bone marrow lymphocytes in the offspring among dietary groups. ANOVA analysis, using Genedata Expressionist Pro software, showed differential expression of 267 genes using a Benjamini and Hochburg false discovery rate cutoff of 10%. Three main patterns of gene expression between pups of different dietary groups were identified through unsupervised hierarchical clustering analysis; 152 and 86 genes were up- and down-regulated, respectively, in both the high and the low folic acid groups relative to the control group. Twenty-seven genes showed increasing expression with increasing dietary folic acid dose. In conclusion, dietary folic acid given to dams prior to conception through weaning had a significant effect on offspring survival and gene expression. Additional analyses are underway to determine the relevance of genes found to be differentially expressed with respect to risk of B-cell lineage leukemias followed by validation of relevant gene candidates through quantitative RT-PCR. These data will be used to help inform future etiologic studies of childhood ALL.
A Mouse Strain Which Responds Poorly to Angiogenic Stimuli Has a Higher Incidence of Irradiation-Induced Hematological Malignancies.
Authors: Osorio Catarina , Fragoso Rita , Igreja Catia , Dias Sergio
Published: October 2019

Genetic heterogeneity of murine strains to vasculogenic/ angiogenic stimuli has been suggested to correlate with the susceptibility to carcinogenesis and subsequent tumor growth. A more “angiogenic” strain would, likely, be more prone to carcinogenesis/ tumorigenesis stimuli. To study the importance of the endothelial compartment (vasculogenesis/ angiogenesis) during irradiation-induced hematopoietic malignancies (leukemia and lymphoma) we administered 3 cycles of irradiation (sub-lethally; 3.5 Gy), with one month of interval between them, to 2 angiogenically different mouse strains (12 high angiogenic Balb/c mice vs 12 low angiogenic C57BL-6 mice). Every month after the last irradiation, the levels of circulating endothelial progenitor cells (EPC: Flk-1+Sca+ cells) in peripheral blood were quantified by flow cytometry. At the end of the assay (∼6 months after last irradiation) the incidence of hematopoietic disease among the 2 mouse strains was scored and the levels of EPC (Flk-1+Sca+ cells), endothelial cells (Flk-1+ cells) and HSC/HPC (Sca+c-Kit+ cells) were measured in BM (bone marrow) samples of sick and healthy mice. Interestingly, the less angiogenic C57BL-6 mouse strain displayed a significantly higher leukemia and thymic lymphoma incidence (50% - 2x-fold higher than Balb-c). As determined by FACS analysis of BM cells, in both mouse strains, sick mice showed a reduction in the levels of HSC/HPC (hematopoietic progenitor cells) compared to their healthy counterparts. Nonetheless, this reduction was more pronounced in the less angiogenic C57BL-6 mouse strain (2.1 reduction in C57BL-6 vs. 1.4 reduction in Balb-c). Beside the difference in the hematopoietic compartment, C57BL-6 leukemic mice also evidenced a dramatic change in the BM endothelial compartment, where a significant decrease in EPC and endothelial cells was observed compared to leukemic Balb-c and healthy mice. In contrast, C57BL-6 leukemic mice showed a higher number of circulating EPC compared to healthy mice. Taken together, our data suggests that a higher incidence of hematopoietic malignancies observed in the less angiogenic mouse strain may be associated with a deficiency in the BM vascular compartment. Since it is known that whole body irradiation induces the turnover and the mobilization of HSC/HPC inside the BM, we hypothesize that the vascular compartment (EPC/ endothelial cells) regulates HSC/HPC turnover during irradiation-induced carcinogenesis. As such, BM vascular deficiency/ depletion after BM irradiation (particular evident in low angiogenic genetic backgrounds) may promote the turnover of HSC/HPC and increased susceptibility to the irradiation effects. These results may be particularly relevant in the context of irradiation/ chemotherapy-induced secondary tumors in myelodysplastic/ leukemia patients.
Detection and Production of Haptoglobin within Acute Myeloid Leukemic Blasts.
Authors: Lingamfelter Daniel , Yu Na , Quinn Tim , Williams Casey , Guan Xiuqin , McGuirk Joseph , Chen Yayan , Abhyyankar Stella , Arora Navanshu , Gaur Rakesh , Wiemen Jeffery , Yan Ying
Published: October 2019

While a directly proportional relationship between serum haptoglobin (Hp) levels and acute myeloid leukemia (AML) has been documented, the exact source of this elevated haptoglobin remains in dispute. Furthermore, no reported studies thus far have analyzed haptoglobin-targeted immunohistochemistry as a potential diagnostic tool for AML. In this study, we examined Hp expression in patients with AML. Previously collected blood samples were obtained, with informed consent, during routine diagnostic blood studies from twenty-one patients with AML (13 males, 8 females; age range 4–73 years). Cytospin samples were prepared, and immunohistochemistry was performed using a polyclonal anti-haptoglobin antibody. Total RNA was extracted from 19 AML patient samples as well as from a K562 leukemic cell line for the RT-PCR assay. Cytospin specimens derived from all twenty-one patients with confirmed AML showed consistent anti-haptoglobin staining of blast cells in a granular, cytoplasmic pattern. The K562 cell line, used as a positive control, stained in a similar fashion to the patient samples. Immunohistochemical co-labeling of blasts was achieved with the AML anti-podocalyxin marker in conjunction with the anti-haptoglobin antibody. All of the AML blasts as well as the K562 cells expressed Hp by the RT-PCR assay. The ratio index (RI) of Hp band density against β-actin serving as an internal control was used to compare the expression level among the AML cases. The RIs ranged from 0.89 to 0.02, indicating different Hp transcription levels among the AMLs. We conclude that haptoglobin is produced by AML blasts and stored within the cytoplasm. This may partly explain the increased serum level of haptoglobin observed in AML patients. Furthermore, immunohistochemistry consistently detects these intra-blastic haptoglobin stores, suggesting a potential for a novel diagnostic tool for AML.
Correlation of New Method for Measuring Plasma Myeloperoxidase with Clinical Behavior in Acute Myeloid Leukemia and Myelodysplastic Syndrome.
Authors: Abdool Adam , Wang Xiugiang , Jilani Iman , Zhou Weidong , Kantargian Hagop , Estey Elihu , Giles Francis , Albitar Maher
Published: October 2019

We previously reported that levels of myeloperoxidase (MPO) enzyme in plasma of patients with acute myeloid leukemia (AML) may correlate with aggressive disease. However, we found poor reproducibility and significant variability from lot to lot when commercially available kits are used. We developed a cytometric beads assay (CBA) for measuring plasma MPO that shows high reproducibility (CV<10%) with no significant variability with time. More importantly, MPO levels as measured using this CBA assay showed better correlation with clinical behavior in AML and myelodysplastic syndrome (MDS). Based on studying plasma levels of MPO in 98 patients with AML and 50 patients with advanced stage MDS, we found no significant difference in plasma MPO between AML and MDS patients. As expected MPO levels were significantly lower in patients with ALL (N=48) than in AML patient (P=0.04) and MDS (P=0.02). While MPO levels were significantly higher in patients with good cytogenetic abnormalities (P=0.004) in AML patients, MPO levels did not differ between intermediate and poor cytogenetic groups in patients with AML or MDS. Unexpectedly we found a difference between AML and MDS in their correlation with MPO levels and clinical outcome. Patients with advanced stage MDS and high levels of MPO (<300 ng/ml) had significantly longer survival than patients with lower levels (P=0.01). Multivariate analysis showed that this correlation with better survival in this group of MDS patients was independent of cytogenetic grouping. In contrast in AML, higher levels of MPO (>70 ng/ml) correlated with shorter survival (P=0.04). Similarly, patients with acute lymphoblastic leukemia (ALL) and high levels of MPO (>70 ng/ml) had shorter survival (P=0.05). Despite there was a correlation between the commercially available kit for measuring MPO and our CBA method (r=0.71, P<0.0001), the commercial kit did not provide robust data for clinical correlation. This data confirms that confirms that plasma levels of MPO play a role in the biology of the leukemia. Further studies are needed to establish if this role is a reflection of an inflammatory process or a reflection of disease load, although the finding of clinical relevance in ALL suggests that it is more likely reflecting an inflammatory process.

Figure Figure
Efficacy of Imatinib-Based Therapy in a Patient with Resistant NUP214-ABL1 T-Cell Acute Lymphoblastic Leukemia.
Authors: Delaunay Jacques , Abdelali Raouf Ben , Touzeau Cyrille , Chevallier Patrice , Thierry Guillaume , Le Gouill Steven , Ayari Sameh , Harousseau Jean-Luc , Macintyre Elisabeth , Loiseau Herve Avet , Dombret Herve , Talmant Pascaline
Published: October 2019

T-cell acute lymphoblastic leukemia (T-ALL) is a rare disease which represents 25% of adult ALL. In T-ALL, many cryptic abnormalities can only be detected by FISH or RQ-PCR. Herein, we describe the case of a 25 year-old man presenting with a NUP214-ABL1 T-ALL. This rearrangement is observed in about 5% of adult T-ALL, results in ABL1 amplification and has been associated with sensitivity to imatinib in vitro At diagnosis, the patient presented with multiple lymphadenopathies and a high WBC (120 G/L). Blast cells expressed T (CD3, CD5, CD7, and CD8) and myeloid (CD13 and CD33) markers. Karyotype was normal, while FISH analysis revealed HOX11L2 rearrangement and extrachromosomal ABL amplification. Presence of NUP214 exon32-ABL1 exon2 transcript and overexpression of HOX11L2 were both confirmed by RT-PCR. Treatment induction according to the GRAALL-2003 protocol, consisted of a corticosteroïd prephase (d1–7) followed by 5-drug induction (doxorubicin, cyclophosphamide, vincristine, corticosteroid and asparaginase) (d8–21). Because of ALL resistance and presence of severe infection, treatment was stopped at d21 and salvage therapy associating imatinib (400 mg, bid), vincristine (2 mg d1,d8,d15,d21) and dexamethasone (30 mg/m2 d1–2,d8–9,d15–16,d21–22) was initiated at d25. At d50, the patient had reached complete hematological response. He then received monthly consolidation courses combining imatinib (400 mg bid d1–28), vincristine (2 mg d1) and prednisolone (40mg/m2 d1–7). After the second course, he reached major molecular response at d120 (NUP214/ABL1 = 1x10-3 by RQ-PCR) but relapsed at d150. Karyotype was still normal and FISH analysis showed persistance of HOX11L2 rearrangement but loss of extrachromosomal ABL amplification and presence of a third ABL signal (cryptic rearrangement) in the majority of metaphases. Quantitative RT-PCR analysis detected a low level of NUP214/ABL1. We initiated a second salvage combining dasatinib (70 mg bid) and Hyper-CVAD, with achievement of a second hematological CR. After two consolidation courses, the patient is still in hematological CR (8 months) and complete molecular response. He will receive a double cord blood transplant In conclusion, we report here the first case of transitory imatinib efficacy in a patient with NUP214/ABL1 T-ALL, followed by achievement of a secondary remission with dasatinib, despite the partial loss of NUP214-ABL1, in favor of the use of ABL inhibitors in this specific T-ALL subset. We also raise the hypothesis that NUP214/ABL1 may be a secondary oncogenic event.
Adaptation of Augmented Berlin-Frankfurt-Munster (ABFM) Therapy in Adolescents and Young Adults with Acute Lymphoblastic Leukemia (ALL).
Authors: Rytting Michael , Thomas Deborah , Kantarjian Hagop , Cortes Jorge , Borthakur Gautam , Wierda William , Nunez Cesar , Kornblau Steven , Ravandi Farhad , Wells Robert , Brenda Muriera , Anna Franklin , O’Brien Susan
Published: October 2019

Comparisons of survival of adolescents up to age 21 years of age treated on either pediatric or adult ALL protocols so far show improved survival for patients (pts) treated with pediatric therapies. ABFM therapy has been shown to be effective therapy for teen-aged pts up to age 21, and is the standard therapy arm of the current Children’s Oncology Group high-risk ALL trial. We have initiated a trial of ABFM based therapy for pts up to the age of 30 with lymphoblastic leukemia. Pts receive four drug induction with prednisone 60 mg/m2 daily for 28 days, daunorubicin 25 mg/m2 weekly for four doses, vincristine 2 mg weekly for four doses, and a single dose of intravenous pegylated asparaginase (PEG-asp) in week one of therapy. Intrathecal (IT) cytarabine is given on day one, and IT methotrexate is given on days 8 and 29. IT therapy is intensified depending on the presence of spinal fluid blasts. Induction is extended by two weeks for patients who do not acheive a bone marrow morphologic remission (MR) by day 29. Pts that are in MR by day 15 are rapid early responders; they receive one phase of delayed intensification. Pts who are not in MR by day 15 but enter MR by day 29 or 42 are slow early responders; they receive two delayed intensifications. Upon completion of induction, pts continue with intensive phases of chemotherapy for approximately 6 months. They then start 24 months of maintenance therapy. 13 patients with newly diagnosed ALL have been enrolled with a planned enrollment of 80. The median age is 20 (range 14–28). 10(77%) have pre-B ALL and 3(23%) have T-ALL. 12(92%) are rapid early responders. All pts are in MR by day 29. Minimal residual disease (MRD) status is evaluated at day 29 and day 84 by four-color flow cytometry. 8(62%)pts are MRD negative by day 29. All pts so far are MRD negative by day 84. One pt has relapsed. There are no treatment related deaths. Treatment delays for bone marrow suppression are common. There has been 1 allergic reaction to PEG-asp and 2 cases of clinical pancreatitis. 2 pts have had stroke-like symptoms with MRI findings compatible with treatment toxicity; complete clinical resolution has occurred in both. 4 pts have had grade (Gr) 3–4 hyperglycemia. 2 pts have had Gr 3–4 hyperbilirubinemia. 2 patients have been non-compliant. One pt has had Gr 4 sepsis. Other infectious complications are not common. Early evaluation indicates that ABFM therapy is effective in inducing rapid MR in young adults with ALL. The regimen appears tolerable, but morbidity is frequent. Gr 3–4 toxicity occurs more often than recently reported for similar therapy in adults with ALL (Douer D, et al. Blood, 1 Apr2007, 2744050).
Potentiation of Reactive Oxygen Species Is a Marker for Synergistic Cytotoxicity of MS-275 and 5-Azacytidine in Leukemic Cells.
Authors: Gao Shan , Miller Claudia , Boklan Jessica , Chandra Joya
Published: October 2019

Epigenetic therapy, the reversal of aberrant epigenetic changes in tumor cells such as the silencing of tumor suppressor genes through means of DNA hypomethylation and histone deacetylation, has become a successful new mode for cancer treatment. Classes of drugs such as histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors are currently being used to target these epigenetic changes. Recently, numerous studies supporting the combination of HDACi and DNA methyltransferase inhibitors have emerged, encouraging early clinical trials of these agents together. MS-275 is a novel and highly active benzamide derivative HDACi that has exhibited in vivo and in vitro anti-proliferative activity toward pancreatic, gastric, lung, and ovarian cancer cells. In addition, MS-275 has been evaluated in Phase I/II clinical trials to treat acute leukemias and solid tumors. 5-azacytidine (Vidaza), a DNA methyltransferase inhibitor, has been shown to reactivate tumor suppressor and DNA repair genes through the hypomethylation of cytosines and is used for treatment of acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Here we show that MS-275 and 5-azacytidine display synergistic cytotoxicity in AML and ALL (acute lymphocyctic leukemia) cells. Loss of cell viability was increased in a synergistic manner in Jurkat and ML-1 cells treated with the combination of MS-275 and 5-azacytidine. Synergy was assessed using isobologram analysis employed by Calcusyn software. Quantitation of several distinct biochemical apoptotic events, such as DNA fragmentation, phosphotidylserine exposure and caspase-3 activity, also indicated synergistic interactions between MS-275 and 5-azacytidine. Hyperacetylation of histone H3 was also measurably stronger in cells treated with the combination versus either drug alone. Interestingly, intracellular production of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, also increased in a dose dependent and synergistic manner. Since this is a novel marker for this synergism in ALL cells, our data suggest that assessment of oxidative stress can serve as a marker of the concerted action of MS-275 and 5-azacytidine. Recent results from a clinical trial combining MS-275 and 5-azacytidine suggest that the hypomethylation of certain tumor suppressor genes, such as p15, CDH-1, DAPK-1 and SOCS-1, did not correlate with clinical response in myeloid malignancy patients. Thus, identifying new biological markers for clinical activity of these compounds will assist the development of these epigenetic strategies.
Microvesicle Information Transfer from Lung to Marrow Cells.
Authors: Aliotta Jason , Faradyan Sam , Dooner Mark , Dooner Gerri , Quesenberry Peter J.
Published: October 2019

A body of work in the literature has shown that after marrow transplantation into irradiated mice, there appear cells in the lung with an epithelial lung cell phenotype, but with markers of the donor marrow cells. After transplantation of green-fluorescent protein positive marrow cells into lethally irradiated mice, we have previously shown that there are from 3–5% non-hematopoietic cells bearing the donor marrow markers in the lung, most of these being cytokeratin positive (Aliotta et al., Exp Hematol., 34(2):230–41, 2006). We have utilized a cell impermeable double culture chamber to co-culture irradiated and non-irradiated lung across from marrow cells. With 2 or 7 days of co-culture, the marrow cells express the lung-specific mRNAs surfactant proteins B and C and Clara Cell specific protein at high levels. This phenomenon is most pronounced when the lung has been exposed to 500 cGy 5 days prior to initiating co-culture, although it is also seen with non-irradiated lung. Conditioned media from lung exerts the same effects and RNase treatment of the conditioned media markedly decreased this effect. Ultracentrifugation of the conditioned media pellets the converting activity, which appears to reside in microvesicles. These microvesicles were demonstrated to enter marrow stem cells and to enhance their capacity to convert to lung epithelial cells after transplantation. Most recently, we have also shown that the capacity to enter marrow cells varies both with the cell cycle status of lineage negative Sca-1+ murine marrow cells and with the source of the microvesicles, either from irradiated or normal lung. Altogether, these data indicate that microvesicle transfer from damaged tissue may be the basis of some forms of marrow plasticity, which have been previously reported.
Ubenimex Enhances the Gemtuzumab Ozogamicin-Induced Cytotoxicity Against Myeloid Leukemia Cells.
Authors: Takamatsu Yasushi , Jimi Shiro , Yoshimura Yuka , Suzumiya Junji , Tamura Kazuo
Published: October 2019

Gemtuzumab ozogamicin (GO) is a humanized anti-CD33 antibody conjugated to a derivative of a tumoricidal antibiotic calicheamicin. GO alone induces complete remission in 26% of AML patients in first relapse. One way to improve the response rate is to combine GO with other agents. A quantitative relationship has been shown between rates and degrees of CD33 expression and GO-induced cytotoxicity in vitro by using gene transfer to manipulate CD33 expression in myeloid cell lines. We first studied CD33 expression on myeloid leukemia cells by stimulating with several agents in vitro, and found that ubenimex, but not G-CSF, M-CSF, or ATRA, increased CD33 expression on both HL-60 and K562 cells. Ubenimex is an aminopeptidase inhibitor isolated from Streptomyces olivoreticuli and commercially available as an oral agent for clinical use for AML treatment. To investigate whether ubenimex enhances the cytotoxicity of GO, HL-60 and K562 cells were cultured with GO and/or ubenimex for 3 days. When GO was administered, cell viabilities of HL-60 and K562 were reduced to 31.5% and 90.3% as compared with control, respectively. Treatment with ubenimex alone did not influence viabilities of either HL-60 or K562. However, when cells were preincubated with ubenimex and then cultured with GO, cell viabilities decreased to 18.5% and 81.4% for HL-60 and K562, respectively, indicating that pretreatment with ubenimex enhanced GO-induced myeloid leukemia cell death in vitro. We next assessed the mechanism of cell death. The treatment with GO alone and ubenimex alone induced apoptosis in 39.2% and 2.9% of HL-60 cells, respectively. When HL-60 cells were preincubated with ubenimex and then cultured with GO, the number of apoptotic cells increased to 62.9%, demonstrating that ubenimex augments GO-induced apoptosis. Our data suggest that the priming of AML cells with ubenimex should improve the clinical efficacy of GO.