bluuebunny/crossref_metadata_2025 huggingface dataset:
High Expression Levels of Iaps Predict Poor Overall Survival in Childhood De Novo Acute Myeloid Leukemia.
Authors: Tamm Ingo , Richter Stephan , Oltersdorf Doreen , Creutzig Ursula , Harbott Jochen , Scholz Frank , Karawajew Leonid , Ludwig Wolf-Dieter , Christian Wuchter
Published: April 2020

Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare. Using flow cytometry and western blot analysis, we therefore investigated 45 leukemic cell samples of children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins (CD95, Bcl-2, Bax, Bcl-xL, Procaspase-3, XIAP, cIAP-1, Survivin). XIAP (p<0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by FAB morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (p<0.01) and Bcl-xL (p<0.01) expression was lower in patients with favorable than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP {30 (n=10) vs. 41 months (n=34); p<0.05} and Survivin {27 (n=10) vs. 41 months (n=34); p<0.05}. We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics and poor overall survival should be confirmed within prospective pediatric AML trials.
Real-Time PCR Gene Expression Profiling of Microarray Gene Signatures in Acute Leukemia.
Authors: Sakhinia Ebrahim , Farahangpour Mahboubeh , Yin John A. Liu , Brady Gerard , Hoyland Judith A. , Byers Richard J.
Published: April 2020

Cancer subtype discovery and classification using microarray gene signatures has the potential to transform pathological diagnosis but measurement of indicator genes in routine practice remains difficult. We tested use of real-time PCR measurement of indicator genes for AML and ALL (Golub et al, Science, 1999) as a method for validation and application of microarray gene signatures. Mononuclear cells (MC) were isolated from whole bone marrow (BM) aspirates by density gradient centrifugation and sorted into unselected (total), CD34+ve and CD34-ve fractions. The mRNA in each fraction was globally amplified using a PolyA PCR method. We measured the expression profile of the 17 top ranked genes (cystatin C, leptin receptor, fumarylacetoacetate, CD33, HoxA9, adipsin, proteoglycan 1, LTC4 synthase, LYN, C-myb, MB-1, cyclin D3, SNF2, RbAp48, proteasome iota, HkrT-1 and E2A) from Golub et al (1999) by real-time PCR. All values were calibrated against control standards and normalized to the mean of three housekeeping genes (IF2-beta, GAPDH and human ribosomal protein S9). Data for all 17 genes were obtained for 4 (ALL), 26 (AML), 12 (AML remission) and 9 (morphologically normal) BM samples, each fractionated into three fractions (total MC, CD34+ve MC & CD34−ve MC). There was no significant difference in the mean of three housekeeping gene expression levels between the diagnostic groups. Comparison of the expression level of the other genes confirmed ability to separate AML and ALL, whilst the direction of expression change (increased or decreased) for each gene between AML and ALL was the same as found by Golub et al. In particular, c-myb showed largest significant increase in ALL vs AML in the total BM fraction, whilst cystain c was increased in AML in the CD34−ve fraction. hSNF2b was significantly increased in the ALL total B.M fraction and Hox-A9 was significantly increased in the AML CD34+ve B.M fraction. Furthermore expression level of LYN and CD33 was significantly increased in AML compared to remission AML, indicating ability of the method to determine activity status of disease. In addition, several of the genes provided better separation between AML and ALL when measured in the CD34+ve and −ve fractions indicating more prominent expression in cells of different maturity and that prior fractionation is diagnostically more informative. The results demonstrate ability of the method to validate gene expression signatures by an independent method, which is simple, sensitive and robust, allowing translation to routine clinical use. Whilst the present study used AML and ALL, in principle the method could be extended to any other tumor type for which gene signatures exist.
Evaluation of MRD after Administration of Imatinib by Measurement of Real-Time Quantitative PCR-Based Major bcr/abl mRNA.
Authors: Takahata Mutsumi , Hashino Satoshi , Fujisawa Fumie , Kondo Takeshi , Ota Syuichi , Kobayashi Sumiko , Tanaka Junji , Imamura Masahiro , Asaka Masahiro
Published: April 2020

Evaluation of minimal residual disease (MRD) provides prognostic information on various hematological malignancies. We describe here the efficacy of real-time quantitative polymerase chain reaction (RQ-PCR)-based analysis of major bcr/abl mRNA in cases of chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ALL), especially in cases administered imatinib mesilate (imatinib) followed by stem cell transplantation (SCT). Twenty-one patients with CML or Ph+ALL were enrolled as subjects to determine the usefulness of RQ-PCR-based measurement of bcr-abl/abl ratios. Seven of the 21 patients were administered imatinib before allogeneic SCT. Hematological relapse or failure of treatment by SCT was observed in 2 out of those patients who showed bcr-abl/abl ratios of more than 0.002%, and 5 of the 7 patients showed both RQ-PCR and RT-PCR negativity immediately after SCT. All of the 5 patients who were not administered imatinib before allogeneic SCT showed RQ-PCR negativity immediately after SCT, but the results of RT-PCR were positive in 3 patients at the same time points, and the results became negative by donor lymphocyte infusion (DLI) or appearance of graft-versus-host disease (GVHD). Administration of imatinib before SCT was thought to result in an earlier remission.

On the other hand, 8 patients who were administered imatinib without SCT showed a remarkable decrease in bcr-abl/abl ratios. However, the ratio gradually rose in one patient with Ph+ALL, enabling prediction of hematological relapse preceding detection by fluorescence in situ hybridization (FISH) analysis. Standardization of RQ-PCR analysis of bcr-abl mRNA will help prediction of early hematological relapse in patients with MRD.

In conclusion, RQ-PCR positivity immediately after SCT in patients pre-treated with imatinib suggests risk of recurrence.
Significant Correlation between Immunophenotypings and Cytogenetic Features by Analysis of High Resolution Flow Cytometry with WHO Classification in Adult Acute Myeloid Leukemia in China.
Authors: Wang Xingbing , Zheng Jine , Yao Junxia , Liu Jun , Yang Jin , He Yanli , Yu Jingming , Wei Jun , Liu Zhongping , Zhao Hongzhou , Li Hongrui , Du Wen , Zhang Jiahua , Chen Xiangjun , Liu Wei , Huang Shiang
Published: April 2020

New WHO classification has been rapidly used in leukemic diagnosis. Based on the coexpression and correlation of lineage-associated antigens with multidimensional data analysis, high-resolution flow cytometry has been developed to precisely identify lineage characteristics of leukemia. In this study, 116 cases of acute myeloid leukemia at primary diagnosis with WHO classification were analyzed by high-resolution flow cytometry with a panel of 25 different monoclonal antibodies. The correlation of immunophenotypings with cytogenetic abnormal changes (88 available cases), clinical features and FAB classification were further investigated. The results demonstrated that CD33, CD38 and CD13 were the most commonly expressed antigens (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%)and CD2(15%)As compared with all other AMLs, some antigens including lymphoid lineage are significantly correlated with FAB subtypes, such as increasing expression of CD2 in M3; HLA-DR, CD34 and CD56 absent expressions in M3; increased frequency of CD19 in M2; increasing expressions of CD14 and CD56 in M5 and lack of MPO expression in M0. Cytogenetic abnormal features were detected in 48 cases (54.5%). CD22, CD56 and TdT expressions are remarkably associated with chromosomal abnormalities. All six cases with CD22 expression in AML demonstrated various cytogenetic abnormal changes. Significant associations between immunophenotypings and genetic features are observed: all cases with t (8; 21) highly expressed CD15, CD19, CD34 and CD56. No lymphoid lineage antigens were detected in AML-M3 with t (15; 17). In addition, the expression of both CD7 and CD14 was significantly correlated with higher WBC count; CD4 or TdT expression with increased age; CD4, CD14 or CD56 expression with higher platelet count. No significant correlation was found between hemoglobin level and markers. The relationships of immunophenotypings and genetic features with treatment and prognosis are currently being studied. Conclusion: 1. Significant correlation between immunophenotypings and cytogenetic features in AML strongly suggested that the abnormal antigen expression is tightly linked with aberrant genetic changes; 2. Analysis of high resolution flow cytometry with WHO classification is a powerful tool to be used in primary leukemia diagnosis as well as in study of understanding leukemia malignancy.
Overexpression of EVI1 in Translocation t(3;8)(q26;q24) in a Patient with Trilineage AML.
Authors: Wernli Martin , Fernandez Paula C. , Roethlisberger Benno , Heijnen Ingmar , Burger Johanna , Muehlematter Dominique , Jotterand Martine , Huber Andreas R. , Bargetzi Mario J.
Published: April 2020

EVI1 is a transcription factor that is targeted for de-regulation in 10% of patients with acute myeloid leukemia (AML). Overexpression of EVI1 is associated with a particularly poor prognosis. Various karyotypic abnormalities involving chromosomal region 3q26 have been shown to inappropriately activate expression of EVI1, the most frequent alterations being inv(3)(q21q26)and t(3;3)(q21;q26). Rearrangements of 3q are frequently associated with trilineage myelodysplasia, especially dysmegakaryocytosis. So far, translocation t(3;8)(q26;q24) has been reported in one patient with MDS and two cases of AML without further molecular analysis. No other recurrent chromosome aberration with a breakpoint in 8q24 has been shown in AML. We report a case of a 58 year old male patient with a multilineage AML featuring a t(3;8)(q26;q24) as the sole chromosomal aberration in 80% of bone marrow cells analysed by conventional cytogenetics. The normocellular bone marrow aspirate showed 25–50% atypical blasts, dyserythropoiesis, dysplastic myelopoiesis and hyperactivated megakaryopoiesis with many micromegakaryocytes. Immunophenotyping of the blast population by flow cytometry confirmed the myeloid nature of the cells with CD13+, CD33+, CD64+, CD117+, HLA−DR+ phenotype and variable expression of CD14 and CD34. Persisting pancytopenia occurred after two cycles of chemotherapy and a relapse was evident 4 months later. By real time quantitative RT-PCR (RQ-PCR) we showed that EVI1 is highly overexpressed (>100:1) in this patients bone marrow cells as compared with an AML patient without 3q26 involvement. Fluorescence in situ hybridisation (FISH) is currently being performed to characterize the 8q24 and 3q26 breakpoints in more detail.

The chromosomal translocation t(3;8)(q26;q24) leads to overexpression of EVI1 and, as in other aberrations involving 3q, shows a poor prognosis in AML.
Sensitivity of B-CLL to Cell Death Induction by Anticancer Drugs Is Independent of the Immunoglobulin Heavy Chain Variable Genes (IgHv) Mutation Status.
Authors: Sturm Isrid , Bosanquet Andrew G. , Hummel Michael , Dorken Bernd , Daniel Peter T.
Published: April 2020

B-type chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease. Unmutated immunoglobulin (Ig) VH genes, i.e. a post-germinal center genotype, are associated with a more aggressive natural course of disease. To explore the impact of Ig variable region hypermutation on response to cytotoxic therapies we analyzed the sensitivities to a panel of cytotoxic modalities by use of the DiSC assay that measures cell death. These data were compared to the Ig VH mutation status in 113 B-CLL patients. 29 patients exhibited IgHV hypermutation (age 66.3±1.8 years, 18 male, 11 female) whereas 84 patients (age 62.2±1.1, 66 male, 18 female) were of pre-germinal center B-cell origin. Hypermutated samples were from Binet stage A in 11 cases (unmutated 12), Binet stage B in 5 cases (14), Binet stage C in 10 cases (44), and in stage A/B in 2 cases (10 cases), Chi-square test p=0.02. 11/29 patients with Ig VH hypermutation and 54 of the 84 of the patients with unmutated B-CLL had received prior chemotherapy. Ig VH hypermutation had no impact in the percentage of surviving cells after 92 hours for ionizing irradiation (IR, 2 Gy by a 137Cs source) (hypermutated, 35.3%±8.2, unmutated 34.2%±4.5). Furthermore, there was no significant difference for the log LC90 values for chlorambucil (Mann-Whitney U test, p=0.22), doxorubicin (p=0.86), fludarabine (p=0.36), and mafosfamide (p=0.49). Interestingly, the log LC90 value for methylprednisolone showed a significant difference and was lower in the unmutated (n=83, mean±SEM 0.8±0.13) as compared to the hypermutated samples(n=29, 1.36±0.2; p=0.038). Conclusions: The mutation status of the IgHV gene segment does not appear to be responsible for differences in the response to various drug treatments in B-CLL. Vice versa, differences in gene expression that were described in hypermutated versus unmutated B-CLL do not seem to impact on drug sensitivity profiles. Thus, both DiSC assay result and VH Gene hypermutation are valuable independent prognostic markers.
Application of Multiplex RT-PCR for Identification of Leukemia Associated Gene Abnormalities.
Authors: Lee Hyun Woo , Jung Youn Mu , Jang Jun Ho , Park Jun Seong , Cho Sung Ran , Choi Jin Hyuk , Lim Ho Yeong , Lee Jong Uk , Kim Hugh C.
Published: April 2020

The best prognostic predictor for acute leukemia is known to be the finding of genetic abnormalities of leukemic cells. Methods for detecting the genetic abnormalities include chromosomal studies for karyotyping, FISH(Fluorescence in situ hybridization) and RT-PCR. However, each methods have limitations i.e. low sensitivity in karyotyping, uncertainty of molecular probe to be used in FISH or RT PCR methods. Multiplex RT-PCR (MRT-PCR) allows simultaneous detection of 28 fusion genes, more than 80 breakpoints and splice variants associated with leukemia. Therefore, this method can be used for detection of molecular abnormality in fresh unknown leukemic cases, as well as for molecular remission in follow-up cases. The aim was to demonstrate whether MRT-PCR system might be successfully used to screen a large number of patients with acute leukemia and compare the result with that of chromosome studies. Frozen bone marrow cells from 78 patients, who were diagnosed with acute leukemia at Ajou university hospital between September 1994 and February 2004, were used for MRT-PCR. In all samples with a known conventional cytogenetic results, we performed MRT-PCR and to compared with conventional cytogenetic study regarding the concordance rate and analyzed discordant cases regarding their types. 78 samples(40 male and 38 female patients) were analyzed, and there were 59 AML patients and 19 ALL patients. We successfully obtained the mRNA from all frozen samples. In 21 cases with gene abnormalities by chromosome studies most of them (15/21) showed the same abnormalities with MRT-PCR. In 57 patients with normal karyotype by cytogenetic technique, we identified 18 translocations of clinical significance by MRT-PCR method. In 18 discordant cases, there were 4 cases with t(15;17), 4 cases with t(8;21), 3 cases with t(9;22), 2 cases with t(11;19) and 4 others [t(9;11),t(9;9),t(3;11),inv(16)]. Conventional cytogenetics detected 10 cases of good prognostic gene abnormalities [t(15;17),t(8;21),inv(16)], but MRT-PCR method detected 10 additional cases of good prognostic gene abnormalities which might change the treatment paln. The twenty good prognostic cases with MRT-PCR showed better overall survival than others. (median f/u = 10.6 month, p=0.0443) There were 69% concordance rate between cytogenetic technique and MRT-PCR. Furthermore clinically significant translocations were detected by MRT-PCR in 18 of 57 normal karyotype patients, indicating improved sensitivity and prognostic value with MRT-PCR. Further investigations are needed to ascertain the usefulness of MRT-PCR for the screening tool of leukemic gene abnormalities.
Loss of PTEN Expression Induces NF-kB Via PI3K/Akt Pathway Involving Resistance to Chemotherapy in Acute Lymphoblastic Leukemia Cell Lines.
Authors: Gu Lubing , Zhu Ningxi , Findley Harry W. , Zhou Muxiang
Published: April 2020

PTEN is a tumor suppressor gene responsible for downregulating the phosphoinositide 3-kinase (PI3k)/Akt pathway. Loss of PTEN expression frequently occurs in human cancer leading to high Akt activation, which consequently confers neoplastic cell survival and resistance to chemotherapy-induced apoptosis. Here we report a mechanism by which loss of PTEN expression activates the transcription factor NF-kB through the PI3k/Akt pathway that induces activation of the IkBa kinase (IKK). Activation of NF-kB by loss of PTEN expression results in resistance to doxorubicin in acute lymphoblastic leukemia (ALL) cells. Initially, we examined 27 leukemia cell lines derived from children with ALL for the expression of PTEN and constitutive activation of NF-kB to evaluate whether there is a correlation between these two events. We found that 12 of the 27 lines lacked PTEN expression (PTEN-). Of 12 PTEN- ALL lines, 10 lines expressed constitutive NF-kB activation. In contrast, 11 of the 15 PTEN positive (PTEN+) lines were defect of NF-kB activation. Treatment of PTEN- line with PI3k kinase inhibitor Ly294002 caused downregulation of Akt activity accompanied by reduced activation of IKK and inhibition of constitutive NF-kB activation, resulting in increased sensitivity to doxorubicin-induced apoptosis. Similar to treatment with Ly294002, transfection of the PTEN expression plasmid into the PTEN- lines attenuated constitutive activation of both Akt and NF-kB, thereby sensitizing these cells to doxorubicin. These results suggest that both constitutive and inducible activation of NF-kB play an important role in chemotherapy resistance, and that loss of PTEN expression is at least one reason for the constitutive activation of NF-kB in ALL cells.
Single Nucleotide Polymorphisms of DNA Synthetic and Repair Enzymes and Risk of Childhood Acute Lymphoblastic Leukemia.
Authors: Pakakasama Samart , Sirirat Thanasan , Kajanachumpol Saowanee , Chaunsumrit Ampaiwan , Hongeng Suradej
Published: April 2020

Although the outcome of children with acute lymphoblastic leukemia (ALL) has been improving, the origin of this disease is still unknown. It is likely that individual genetic susceptibility and environmental exposure play a role in carcinogenesis. To maintain normal hematopoiesis, DNA synthesis and repair systems need to be intact. Folate metabolism, producing purines and pyrimidines, is the source of nucleotides while base excision and nucleotide excision repair enzymes, i.e. XRCC1 and XPD, have important roles in protection of DNA damage. Single nucleotide polymorphisms (SNPs) of enzymes involving these processes alter their enzymatic structures and functions. We hypothesized that SNPs of folate metabolic enzymes including methylenetetrahydrofolate reductase (MTHFR 677 C>T; 1298 A>C), methionine synthase (MS 2756 A>G), and tandem repeats of thymidylate synthase (TS 2R or 3R) and of DNA repair enzymes (XRCC1 26304 C>T; 27466 G>A; 28152 G>A and XPD 23591 G>A; 35931 A>C) could effect the risk of ALL in children. We performed genotyping of these SNPs in 108 ALL and 317 normal controls by using PCR RFLP method. Genotype frequencies of folate metabolic enzymes SNPs were not different between cases and controls. However, TS 2R allele showed a trend to decrease risk of ALL (OR=0.66; 95% CI 0.41–1.07). We observed a 4.5 fold reduction in the risk of ALL in individuals with the XRCC1 26304 TT genotype (OR=0.22; 95% CI 0.5–0.96). XRCC1 26304 T allele showed a protective effect to this disease (OR=0.67; 95% CI 0.47–0.97). In contrast, individuals with XRCC1 28152 A allele had an increased risk of ALL (OR=1.67; 95% CI 1.2–2.33) compared to those with G allele. XRCC1 28152 GA and GA+AA genotypes were significantly higher among patients with ALL compared to controls (OR=2.17; 95% CI 1.37–3.45 and OR=2.18; 95%CI 1.38–3.42). No association between genotype or allele frequencies of both XPD SNPs and risk of ALL was shown in this study. In conclusion, our data demonstrated that SNPs of DNA repair enzymes (XRCC1 26304 C>T and 28152 G>A) but not folate-dependent DNA synthetic enzymes may influence risk of childhood ALL.
Akt/PKB Activation-Induced Cytoplasmic Mislocalization of p27Kip1 Protein Is Mediated by Jab1 Shuttling in Human Myeloid Leukemia Cells.
Authors: Min Yoo-Hong , Eom Ju In , Choi Ae Jin , Lee Jee Young , Kim Jin Seok , Lee Seung Tae , Cheong June-Won
Published: April 2020

We previously demonstrated the cytoplasmic mislocalization of p27Kip1 protein was highly correlated with constitutive Akt/PKB phosphorylation and poor prognosis in primary acute myeloid leukemia. However, mechanism involved in the Akt/PKB-mediated nuclear export of p27Kip1 in leukemia cells is not fully understood. Jun activation domain-binding protein 1 (Jab1)/CSN5 is the fifth component of the COP9 signalosome complex, which is involved in modulating signal transduction, ubiquitin-proteasome pathway, and nuclear transport. We examined the effects of Akt/PKB activation on the phosphorylation and localization of p27Kip1 protein in relation to Jab1 shuttling in K562 leukemia cells. Akt/PKB activation did not affect the phosphorylation at threonine 187 residue (T187) of p27Kip1 protein in K562 cells. However, the phosphorylation of p27Kip1 at the serine 10 (S10) and threonine T157 (T157) residue was markedly increased in the constitutively active Akt/PKB cDNA-transfected K562 (Myr-Akt/PKB-K562) cells. In the Myr-Akt/PKB-K562 cells, the S10- and T157-phosphorylated form of p27Kip1 localized almost exclusively to the cytoplasm. Leptomycin-B (LMB) pretreatment of Myr-Akt/PKB-K562 cells led to restoration of S10- and T157-phosphorylated p27Kip1 to the nucleus, indicating that Akt/PKB activation-induced cytoplasmic translocation of S10- and T157-phosphorylated p27Kip1 was mediated by CRM1-dependent pathway. We found that Akt/PKB activation did not alter the total cellular levels of Jab1 protein. However, Jab1 protein was exclusively localized to the cytoplasm in the Myr-Akt/PKB-K562 cells, whereas it was preferentially localized to the nucleus in the dominant negative Akt/PKB cDNA-transfected K562 cells. In Myr-Akt/PKB-K562 cells pretreated with LMB, the localization of the Jab1 was essentially nuclear. Jab1 silencing led to an accumulation of S10- and T157-phosphorylated p27Kip1 to the nucleus in the Myr-Akt/PKB-K562 cells, which was accompanied by a prominent decrease in the cytoplasmic level of p27Kip1. Taken together, we demonstrate that Akt/PKB activation-induced cytoplasmic mislocalization of S10- and T157-phosphorylated p27Kip1 protein is mediated by Jab1 shuttling in human myeloid leukemia cells. Apart from our understanding of the mechanism implicated in the regulation of phosphorylation and localization of p27Kip1 protein in leukemia cells, all these data might suggest possibilities for designing leukemia therapy since it should be possible to restore the function of a misplaced p27Kip1 protein.
Arsenic Trioxide (ATO) Downregulates the Expression of the AML1/MDS1/EVI-1 (AME) Oncoprotein and Induces Differentiation and Apoptosis in AME-Leukemic Cells.
Authors: Shackelford David , Waxman Samuel , Ren Ruibao
Published: April 2020

Arsenic is an ancient drug, used for many centuries to treat a broad range of ailments including cancer but was largely abandoned in the early 20th century. In the 1990s arsenic trioxide (ATO) was rediscovered as an effective treatment for patients suffering from acute promyelocytic leukemia (APL). ATO has been shown to specifically target the fusion protein resulting from the t(15;17) translocation, PML/RARα, and subsequently stimulate differentiation and apoptosis of APL cells both in vitro and in vivo. The success in using ATO as a targeted therapy for APL underscores the importance of matching the right drug to patients with appropriate molecular targets. We previously reported that expression of the AML1/MDS1/EVI1 (AME) transcription factor fusion protein [a product of the human t(3;21)(q26;q22) translocation found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in some patients with de novo and therapy-related myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML)] in mouse bone marrow cells by retroviral transduction impaired hematopoiesis and eventually induced an AML in mice. Our results established a causal role of AME in t(3;21)-positive myeloid malignancies. We went on to examine whether ATO could target AME. We found that ATO treatment downregulated forced-expressed AME protein in Bosc23 (a cell line derived from human 293 kidney cell line) and BM6-4 cells, a primary myeloid blast cell line derived from bone marrow of a mouse with AME-induced AML, in a dose dependent manner (within therapeutic relevant dosage range). We also found that ATO inhibited proliferation of BM6-4 cells, induced these cells to differentiate into macrophages and induced apoptosis in a dose dependent manner. Our results suggest that ATO could be used as a targeted therapy for t(3;21)-positive MDS and AML. The in vivo effect of ATO in the AME mouse model and mechanism of ATO-induced AME downregulation is under investigation. Such research may extend ATO therapy to other hematological malignancies and help to identify new therapeutic targets for human cancer.
Fibroblast Growth Factor Receptor-1 Expression and Signaling in Acute Myeloid Leukemia.
Authors: Karajannis Matthias A. , Vincent Loïc , Zhang Fan , Shmelkov Sergey V. , Cheung Joseph , Bohlen Peter , Zhu Zhenping , Kussie Paul , Sun Haijun , Rafii Shahin
Published: April 2020

Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors affecting cell proliferation, motility and survival. Fibroblast growth factor 2 (FGF2, basic FGF) represents the prototype FGFR ligand. Expression of FGFRs has been demonstrated in a subset of acute myeloid leukemias (AMLs) and FGF2 is overexpressed in the bone marrow of AML patients. The role of FGF/FGFR signaling in AML however is controversial, and downstream signaling cascades activated by FGFR1 are not known. Therefore, we hypothesized that subsets of primary acute leukemic cells may express functional FGFR1 mediating survival and proliferation of leukemic cells. We examined the role of the FGF/FGFR axis in primary AML blasts isolated from a 74 year old male (designated R81). Using RT-PCR we showed that FRFR1 was expressed in leukemic cells. To assess whether FGF2 stimulates FGFR1 signaling, R81 blasts were incubated in serum free medium with and without 20 ng/ml of FGF2 and/or a with 10 μg/ml of neutralizing monoclonal antibody to FGFR1. Viable cells were counted at 48 and 96 hours with trypan blue exclusion (see figure). The results show that FGF2 stimulates growth and/or survival of R81 AML blasts and this effect is abrogated by FGFR1 antibodies. Specifically, FGF2 induced a two-fold increase in the proliferation of leukemic blasts after 48 hours. To identify downstream signaling pathways involved in FGFR1 signaling, proliferation and survival of FGF2-treated R81 cells were examined in the presence or absence of selective PI3 kinase and MAP kinase inhibitors, LY-294002 and PD098059, respectively. The results indicated that FGF2 signaling exerts its proliferative and pro-survival effects primarily through PI3 kinase activation. In summary, we show that FGF/FGFR1 signaling plays a role in a subset of AMLs and may be blocked by a FGFR1 specific antibody. Currently, we are further elucidating the mechanisms involved in FGFR signaling and its effects on cell proliferation, migration, and apoptosis. In addition, animal experiments are underway to assess a possible therapeutic role of anti-FGFR antibodies for the treatment of FGFR-positive AML in murine xenotransplant models.

Figure Figure
TPCK Induces Apoptosis in Human Acute Myeloid Leukemia U-937 Cells.
Authors: Mazar Julia , Lichtenstein Alexandra , Katz Leora , Shpilberg Ofer , Levi Itai , Nathan Ilana
Published: April 2020

Many types of antitumor therapy in general and AML in particular exert their effect by activating apoptosis. Apoptosis of AML cells can be induced by cytostatic drugs, corticosteroids, and radiation. Recently, the role of different proteases as possible targets for chemotherapy was described. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like protease (CLP) inhibitor was shown to exert a dual effect on leukemic cells: proapoptotic and antiapoptotic. In the present study the mechanism of its proapoptotic effect was addressed. It was found that the CLP inhibitors, TPCK and 3,4 dichloroisicoumarine induced apoptosis in a time- and concentration-dependent manner. Apoptosis was accompanied by a decrease in mitochondrial membrane potential, cytochrome c release, caspase-3 (but not caspase-8) activation, PS flip-flop (measured by Annexin-V staining followed by flow cytometry analysis) and chromatin condensation, but not fragmentation (detected by acridine orange/ethidium bromide staining).

All apoptotic processes induced by TPCK were completely inhibited by cycloheximide. The ability of cycloheximide to inhibit TPCK-induced cell death suggests that protein synthesis plays a role in TPCK-induced apoptosis. Additionaly, the proapoptotic effect of TPCK was abolished by elimination of glucose from the medium. The data supports the role of mitochondria in this process.

In the present study the apoptotic pathway driven by inhibition of CLP was demonstrated. Moreover, these findings suggest possible ways of preventing the proapoptotic activity of TPCK and thereby enhancimg its antiapoptotic action.
Analysis of Cytokine Profiling in Hepatitis-Associated Aplastic Anemia.
Authors: Lu Jun , Wong Susan , Basu Atanu , Young Neal S. , Brown Kevin E.
Published: April 2020

In hepatitis-associated aplastic anemia (HAA), bone marrow failure follows an acute attack of seronegative hepatitis. Aplastic anemia (AA) is also associated with orthotopic liver transplantation for non-A, non-B, non-C hepatitis in young patients. Although the etiology of HAA is unknown, a clinical response to immunosuppressive therapy is observed in most clinical cases. The liver is the potential initial target organ of the immune response. We have previously determined the T-cell repertoire in the liver and peripheral blood lymphocytes in patients with HAA (Lu et al. Blood. 2004 Jun 15;103(12):4588–93). An antigen-driven T-cell expansion was implicated in the acute stage before bone marrow failure. In the present study, we measured serum cytokine levels in 22 HAA serum samples. Only interleukin (IL)-10 levels showed a significant increase compared to healthy controls (p<0.02); IFN-γ , TNF-α and IL-1, 2, 4, 5, 6, 8, 12 levels showed no significant difference. To further characterize the immune response in the liver, we performed RNAse protection assays and realtime PCR for RNA transcripts. In 4 HAA liver samples, 2 demonstrated increased IFN-γ by RNAse protection; TNF-α , IL-2, 3, 4, 5, 10, 15 transcripts were not different compared to transcript levels in 4 hepatitis B and/or C livers and 2 fulminant hepatitis (FH) samples. To confirm our preliminary data, we characterized the cytokine profile including CD4, CD8, CD69, TNF-α , IFN-γ and interleukins and compared them among the hepatitis B/C, FH and HAA liver samples by quantitative PCR for RNA transcripts. In 10 HAA liver samples, CD8 transcripts were increased and the CD4/CD8 ratio was decreased, but there was no significant difference between HAA and hepatitis B/C (p>0.05), confirming the important role of cellular immunity in HAA. IFN-γ transcripts were significantly overexpressed in both HAA and FH samples compared to hepatitis B/C samples (p<0.05), but without a difference between HAA and FH. CD69 transcripts were reduced in HAA but not significantly lower than in hepatitis B/C (p<0.05). There were no difference of TNF-α transcripts among HAA, hepatitis and FH samples (p>0.05). In summary, our data confirm that enhanced cellular immunity (increased IFN-γ level) is implicated in the pathogenesis of HAA, similar to that observed in the bone marrow of idiopathic AA, and in contrast to other liver diseases such as chronic hepatitis B/C. Quantitation of IFN-γ levels in acute hepatitis samples might be a predictive marker for the development of bone marrow failure after liver transplantation following seronegative hepatitis and FH.
A Small Number of PNH-Type Cells Predict Good Prognosis in Patients with Aplastic Anemia.
Authors: Sugimori Chiharu , Mochizuki Kanako , Yamazaki Hirohito , Feng Xingmin , Nakao Shinji
Published: April 2020

To determine the clinical significance of a minor population of glycosylphosphatidylinositol-anchored membrane protein-negative (GPI-AP−) cells in acquired aplastic anemia (AA), we quantified CD55− CD59− granulocytes and red blood cells (RBCs) in peripheral blood from 122 patients with recently diagnosed AA and correlated numbers of GPI-AP− cells and responses to immunosuppressive therapy (IST). The highly sensitive flow cytometry detected 0.005% to 23.1% of GPI-AP− cells in 68.0% of AA patients. Sixty-eight of 83 (91%) patients with an increased ratio of GPI-AP− cells (PNH+) responded to antithymocyte globulin (ATG) + cyclosporin (CsA) therapy, whereas 18 of 39 (48%) without such an increase (PNH−) responded. Failure-free survival rates were significantly higher (64%) among PNH+ than PNH− patients (12%) at 5 years (Figure) although overall survival rates were comparable between the groups. Numbers of GPI-AP− and GPI-AP+ cells increased in parallel among most PNH+ patients who responded to IST, suggesting that these cells are equally sensitive to immune attack. These results indicate that a minor population of GPI-AP− cells represents a reliable marker of a positive IST response and a favorable prognosis among patients with AA. Furthermore, immune attack against hematopoietic stem cells that allows PNH clonal expansion might occur only at the onset of AA.

Figure Figure
Daclizumab-Associated Erythroderma Is Associated with Rebound Increases in CD25-Positive CD4 Cells Following Treatment of Aplastic Anemia and Pure Red Cell Aplasia.
Authors: Lemery Steven J. , More Kenneth F. , Young Neal S. , Sloand Elaine M.
Published: April 2020

We previously have reported success in treating patients with moderate aplastic anemia with daclizumab, a humanized monoclonal antibody that recognizes the interleukin-2 receptor (Blood.2003; 102(10):3584–6). Since our first report we have treated eighty-eight patients with moderate aplastic anemia, pure red cell aplasia and relapsed severe aplastic anemia with 1 mg/kg of monoclonal antibody every 2 weeks for 3 months. Although this drug has been used extensively in the management of solid organ rejection, erythroderma has not been reported. In our study, five patients developed a severe generalized erythema in a period of 20 to 70 days following the last dose of treatment. The cutaneous eruptions were characterized by severe pruritis and intense erythematous inflammation with spongiotic papules and plaques. Pathologic evaluation was possible for four of five patients and described focal spongiosis, dermal lymphocytic infiltration including some eosinophils, and no evidence of infection. All patients were negative for anti-nuclear antibody and had no arthritis, fever, or other systemic signs. HLA types were available on 83 of the 88 patients. HLA DR13 was more common in the patients that developed a rash; 80% versus 18% in those who did not develop rash (p = .0071, Fisher’s Exact Test). The rash was associated with a two to ten fold increase in the proportion of CD25-positive CD4 cells compared to pre-drug levels (p<0.05). All patients required treatment with oral corticosteroids for periods of 2–6 weeks. Three patients who had partial or minimal responses to daclizumab elected to undergo a second course. The three patients who had a mild rash after the first course of treatment developed a more severe eruption with subsequent administration of the drug; rashes were delayed after each therapeutic administration of daclizumab. Two additional patients developed other autoimmune symptoms including inflammatory arthritis about three months following daclizumab administration. The absence of autoimmune complications following use of daclizumab in solid organ or allogeneic stem cell transplant recipients may be related to additional immunosuppression frequently administered to these patients. Our working hypothesis is that daclizumab associated rash is caused by an immune phenomenon. This is supported by the findings that CD4+CD25+ (effector) T cells are expanded at the time of the rash, the presence of a lymphocytic dermal infiltrate, and the relative preponderance of HLA-DR13 in the affected group. It is possible that treatment with daclizumab (an anti-CD25 antibody) results in a relative depletion of CD4+CD25+ regulatory T cells thereby permitting expansion of CD4+CD25+ effector T cells.
Prospective, Randomized, Multicenter Study Comparing Antithymocyte Globulin (ATG) Alone with ATG Plus Cyclosporine (CsA) for Treatment of Children with Nonsevere Aplastic Anemia (nSAA).
Authors: Yagasaki Hiroshi , Yabe Hiromasa , Ayukawa Hiroshi , Noguchi Yasushi , Ijichi Osamu , Kobayashi Ryoji , Tsuchida Masahiro , Mugishima Hideo , Ohara Akira , Morimoto Akira , Oga Shouichi , Bessho Fumio , Nakahata Tatsutoshi , Tsukimoto Ichiro , Kojima Seiji
Published: April 2020

In contrast to treatment for childhood severe aplastic anemia (SAA), there is no consensus on the treatment of childhood nSAA. Two thirds of patients progress to SAA, suggesting the need to consider early immunosuppressive therapy (IST). ATG+CsA was superior to ATG alone in a German randomized trial that enrolled both patients with SAA and nSAA. However, subgroup analysis revealed no advantage of adding CsA for patients with nSAA. These findings formed the basis for this prospective, randomized study comparing ATG alone with ATG+CsA for treatment of childhood nSAA. From 1997 to 2004, 62 children with newly diagnosed nSAA were enrolled. Interim analysis was done in April 2005. Patients were randomized to receive either horse ATG (Lymphoglobuline, 1.5vial/10kg/day for 5 days; n=31) or ATG+CsA (6mg/kg/day; n=31). CsA was added for non-responders at day+90 in ATG group. The endpoints were hematologic response at 6 months and failure-free survival (FFS). Complete response (CR) was defined as a neutrophil count > 1.5x 109/L, a platelet count > 100x109/L, and a hemoglobin level > 11.0g/dl. Partial response (PR) was defined as a neutrophil count > 1.0x 109/L, a platelet count > 30x109/L, and a hemoglobin level > 8.0g/dl. FFS was defined as survival with a response. Clinical profiles of two treatment groups were comparable. Median age was 11 years and 10 years in ATG+CsA group and ATG group, respectively. Median time from diagnosis to treatment was 20 days and 23 days in ATG+CsA group, and ATG group, respectively. After 6 months, CR was observed in ATG+CsA group in 5 patients (16%) and PR in 12 patients (39%). In ATG groups, 2 patients (6%) achieved CR and 13 patients (42%) achieved PR. However, 19 patients in the ATG group received additional CsA. There were 22 failures in ATG group and 14 failures in ATG+CsA group. A total of 10 patients who failed initial therapy (7 in ATG group and 3 in ATG+CsA group) received a second IST. Bone marrow transplant (BMT) was attempted in 4 non-responders in ATG group and 6 non-responders in ATG+CsA group. Requiring CsA was a cause of failure in 8 patients of ATG group. The probability of FFS was 54.8% in ATG+CsA group compared with 26.7% in the ATG group (p=0.001). There was 1 death in each group: 1 from BMT-related toxicity in ATG+CsA group and 1 from bacterimia in ATG group. The 5-year survival rate was 94.8% in ATG+CsA group and 96.7% in ATG group. Conclusion; the hematologic response rate at 6 months and overall survival rate are comparable between the two groups, however the combination of ATG and CsA is superior to ATG alone in term of FFS for children with nSAA.
RBC Hemolysis and Platelet Activation in Paroxysmal Nocturnal Hemoglobinuria Are Mediated by Oxidative Stress That Can Be Ameliorated by Antioxidants.
Authors: Fibach Eitan , Zelig Orly , Amer Johnny
Published: April 2020

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired stem cell disorder characterized by the unique triad of intravascular hemolysis, bone marrow failure and thrombosis. Complement-mediated hemolysis in PNH is explained by a deficiency in glycosylphosphotidylinositol (GPI)-anchored proteins, CD55 and CD59 on the RBC surface.

Using flow cytometry for simultaneous measurement of oxidative-state markers in RBC and platelets, we studied blood samples obtained from normal donors and PNH patients. Reactive oxygen species (ROS), reduced glutathione (GSH), membrane lipid peroxidation and phosphatidylserine (PS) exposure were measured by staining with dichlorofuorescein, mercury orange, fluor-DHPE and Annexin V, respectively, followed by gating based on size and granularity. The results of blood samples obtained from normal donors (N=25) and PNH patients (N=11) indicated that the RBC from the patients had higher levels of ROS (1.4-fold) and lipid peroxides (3.5-fold) and lower GSH levels (two-fold) than those of normal RBC. Similar results were obtained with platelets from the same samples, indicating that both cell types are in a state of oxidative stress. Within each cell type, cells with the PNH immunophenotype (CD55− CD59−) had higher ROS levels and PS exposure than cells with the normal (CD55+CD59+) phenotype. These results support the direct relationship between the PNH phenotype and intracellular oxidative stress. Oxidants, such as hydrogen peroxide, phenylhydrazine, hemin, ferric ammonium citrate, increased the oxidative status of the cells and caused concentration-dependent RBC lysis and platelet activation. Complement-containing plasma had a similar effect. The RBC lysis and platelet activation were preceded by a burst of ROS and were more prominent in cells with the PNH phenotype. Incubation of PNH-cells with antioxidants such as N-acetyl cysteine, vitamin C and tocotrinol, prevented the complement-mediated lysis.

We previously reported that cellular oxidative stress, prevalent in conditions such as thalassemia, may be associated with RBC hemolysis, platelet activation and a decreased PMN antibacterial potential. Oxidative stress in RBC, platelets and neutrophils in PNH could in part account for clinical manifestations such as hemolysis, a hypercoagulable state and recurrent bacterial infections. Our present results, indicating that incubation with antioxidants ameliorates such stress and its adverse consequences, suggest the possibility of using antioxidants for treatment of PNH and similar conditions.
Unexpected Fanconi Anemia Diagnosis in Patients with Bone Marrow Failure.
Authors: Pinto Fernando O. , Leblanc Thierry , Le Roux Gwenaelle , Dastot Helene , Santos Moema , Larghero Jerome , Rocha Vanderson , Esperou Helene , Socie Gerard , Baruchel Andre , Gluckman Eliane , Soulier Jean
Published: April 2020

Early diagnosis of Fanconi Anemia (FA) in patients with bone marrow failure is critical for optimal clinical management. However, the remarkably high clinical variability and the potential emergence of revertant hematopoietic cells (somatic mosaicism) can obscure and delay the diagnosis of FA. Here we addressed FA diagnosis in a prospective series of adult and pediatric patients who presented with bone marrow failure without clear overall clinical picture of FA.

Sixty-six patients were classified into three groups: (1) bone marrow failure likely to be congenital, based on dysmorphic features or a family history [n=18], (2) aplastic anemia likely to be idiopathic [n=32], (3) patients with intermediate clinical features not classified into the former groups [n=16]. Of note, FA patients with typical clinical features were not included in the present study. FA diagnosis was evaluated using chromosome breakage test and FANCD2 immunoblot in PHA-stimulated-PBL. In addition, skin primary fibroblasts were analysed in order to overcome potential hematopoietic FA reversion. For that purpose, and considering that chromosome breakage tests are barely efficient in fibroblasts, we used FANCD2 immunoblot and also developped a new flow cytometry test based on MMC-sensitivity in fibroblasts (to detect downstream FA/BRCA groups). Using these approaches, we detected FA in 4 previously undiagnosed patients: a 35-years old patient from the congenital-like group; a 10-years old patient presenting as an idiopathic aplastic anemia without any FA signs; and two patients from the intermediate group: a 10-years old patient with an isolated thrombocytopenia, and a 50-years old patient presenting with pancytopenia/MDS and complete hematopoietic reversion. Importantly, FA diagnosis was definitely excluded in all other patients. In conclusion, we could identify a few unexpected FA cases in a series of patients with bone marrow failure. Therefore, the comprehensive use of a large set of tests is useful for accurate FA diagnosis. Classical chromosomal breakage tests in PBL appeared to be sufficient to exclude FA in idiopathic aplastic anemia, whereas fibroblast analysis can be necessary to definitely diagnose or exclude FA in other patients.