bluuebunny/crossref_metadata_2025 huggingface dataset:
ABCChips as New Diagnostic Tool to Monitoring Multidrug Resistance in Human Tumor Cells to Chemotherapeutics Agents by Expression Profiling of ATP-Binding Cassette Transporter Genes.
Authors: Gillet Jean-Pierre , Efferth Thomas , Steinbach Damiel , de Longueville Françoise , Bertholet Vincent , Remacle José , Sayer Herbert G.
Published: April 2020

A major problem in the treatment of tumors represents the development of resistance to chemotherapy. Many mechanisms are responsible for the failure of treatment, the main one being the activation of the ABC transporters. In the present investigation, we developed a low density DNA microarray which contains 38 ABC transporter genes. This tool has been validated with three different characterized multidrug-resistant sublines (CEM/ADR5000, HL60/AR, MCF7-CH1000) and their corresponding drug-sensitive parental cell lines (CCRF-CEM, HL60, MCF7). The multidrug-resistant cell lines used are known to overexpress either the MDR1, MRP1, or BCRP genes. Interestingly, we found not only the overexpression of these genes but also of other ABC transporter genes by using low density microarrays. These results were corroborated by quantitative real time RT-PCR. The microarray allowed the determination of the expression profile of ABC transporters in a single hybridization experiment and may, hence harbor the potential as diagnostic tool to detect drug resistance in the clinic.
Induction of Apoptosis and Cell Cycle Arrest in Acute Myeloid Leukemia Cells by Histone Deacetylase Inhibitor, Suberoyl Hydroxamic Acid (SAHA): Lack of Cross-Resistance to Cytosine Arabinoside and Etoposide.
Authors: Dreyer Sabine , Decker Thomas , Wagner Michaela , Peschel Christian , Licht Thomas
Published: April 2020

Inhibitors of histone deacetylases (HDAC) such as SAHA are being introduced into the clinical treatment of hematopoietic neoplasms. These compounds can induce apoptosis or cell cycle arrest by modification of the chromatin structure of malignant cells, thereby modulating the expression of target genes. We investigated the effects of HDAC inhibitors, SAHA and trichostatin A, in twelve acute myeloid leukemia (AML) cell lines. Cytotoxicity was determined with the use of a tetrazolium-based colorimetric assay. The 50% inhibition concentrations (IC50) of SAHA were in the micromolar, of trichostatin A in the nanomolar range. Three cell lines were 3–5 times more resistant to both HDAC-inhibitors than the other leukemias, namely the myelomonocytic leukemia ML-2, and the erythroleukemias HEL and K562. All investigated AML cells were, however, more sensitive to SAHA than three patient samples of normal CD34+ progenitor cells mobilized into peripheral blood. A close association between the IC50 of TSA and SAHA was noted within the panel of AML lines (r=0.78). In contrast, chemosensitivity to HDAC-inhibitors was not significantly correlated with IC50 for etoposide, cytosine arabinoside, or staurosporine, respectively. To distinguish between growth arrest and induction of apoptosis by SAHA, we analyzed the cell cycle status by staining with propidium iodide, and exposure of phosphatidylserine by an Annexin V assay. Cell cycle arrest in G1 phase was observed in four AML cell lines with an increase of G1 cells by 20–49% in comparison with untreated cells. One cell line, KG-1a, displayed G2 arrest. SAHA induced apoptosis in eight cell lines with one line displaying both apoptosis and G1 cell cycle arrest simultaneously. KASUMI-1 cells bearing the AML1/ETO gene fusion, a target for HDAC’s, underwent apoptosis upon exposure to SAHA. Constitutive expression of the cell cycle inhibitor p27Kip1, determined by Western blotting, was associated with increased numbers of G1 cells following treatment with SAHA (p=0.036, one-tailed Mann-Whitney test). Conversely, constitutive expression of cyclins A, B1, D3 and E, as well as p53 and p16INK4 was not predictive for induction of cell cycle arrest. Expression of P-glycoprotein (ABCB1) as assessed by flow cytometry was not correlated with the IC50 for SAHA. The anti-apoptotic proteins Bcl-2, Mcl-1 and XIAP were analyzed by Western blotting. No association with resistance to SAHA-induced apoptosis was noticable. In summary, AML cells from permanent lines were more sensitive to SAHA than normal hematopoietic progenitor cells. The potential role of p27Kip1 in modulation of the cytotoxicity of HDAC inhibitors requires further study. Lack of cross-resistance with drugs used in clinical treatment suggests that HDAC inhibitors may be useful for treatment of chemoresistant AML.
Differential Anti-Proliferation of Resveratrol on Human Hematopoietic Progenitor Cells and Leukemic Cells.
Authors: Tsang Kam Sze Kent , Li Karen , Tang Sheung Hang , Wong Angela Pui Yan , Leung Yonna , Ling Margaret Heung
Published: April 2020

Resveratrol is present naturally in a variety of fruits and medicinal plants. It imparts protective benefits to plants against pathogenic attack and environmental stress. In human, it was noted to be able to protect against cardiovascular disease and inhibit platelet aggregation. Its anti-oxidant activities were also suggestive in human cancer chemoprevention and chemotherapy. In this in-vitro study we investigated the anti-proliferative effect of resveratrol on human normal hematopoietic progenitor cells and leukemic cells. Resveratrol of 1 mM to 0.1 μM at 10-fold dilutions were applied to hematopoietic progenitor cultures. Duplicates of 5 x 104 mononuclear cells (n=7) and 5 x 103 CD34+ cells (n=3) derived from 10 umbilical cord blood (UCB) samples were cultured for two weeks in methylcellulose enriched with 100 ng/mL granulocyte-macrophage colony-stimulating factor, 20 ng/mL stem cell factor, 2 IU/mL erythropoietin and various concentration of resveratrol. There was no growth of colony-forming unit (CFU) in cultures supplemented with high-dose resveratrol (10 UCB: 100% in 1 mM, 90% in 100 μM and 10% in 10 μM), exhibiting an inhibitory effect on the proliferation of hematopoietic stem and progenitor cells in a dose-dependent manner. The numbers of CFU-GEMM, CFU-GM and BFU derived from cultures with 10 μM, 1 μM and 0.1 μM resveratrol demonstrated no significant difference as compared to that of control cultures without resveratrol. Ten μM resveratrol were added to 12 human leukemic cell lines (AML: KG-1a, Kasumi-1, HL60, NB4, Meg-01, CHRF 288-11; CML: K562; B-lineage ALL: 697, RS-411, REH; T-lineage ALL: Molt-3, CCRF-CEM) at 5 x 105/mL and cultured for four days in triplicate. Cell counts, trypan blue dye exclusion tests and flow cytometry were conducted daily on cultures to evaluate growth, viability and cell cycle phase distribution, respectively. There was a significant and progressive decrease in the numbers of viable cells in resveratrol-supplemented cultures (median [range]: 26.1% [14.3% – 50.3%] at 24 hours, 43.1% [18.2% – 54.6%] at 48 hours, 45.6% [9.3% – 67.6%] at 72 hours and 32.5% [15.7% – 55.3%] at 96 hours). Cell cycle analysis revealed a remarkably elevated proportion of apoptotic cells at pre-G1 phase in resveratrol-supplemented cultures on day one (with vs. without: 15.9% [2.0% – 41.2%] vs. 7.8% [2.4% – 17.6%]; p=0.011). Besides, a growth arrest of CHRF 288-11 (51.6% vs. 39.8%; p=0.032) and Molt-3 (70.2% vs. 54.2%; p=0.002) was noted in G1 phase, whereas an accumulation of KG-1a (25.8% vs. 19.6%; p=0.019) and CCRF-CEM (26.2% vs. 23.2%; p=0.026) in S phase was observed. Data of this study suggest that 10 μM resveratrol may exert an anti-proliferative effect against human leukemic cells but pose no apparent suppression to normal hematopoiesis.
Functional Analysis of a Cation Transporter OCT6: A Mediator of the Cellular Influx of Adriamycin.
Authors: Okitsu Yoko , Harigae Hideo , Takahashi Shinichiro , Abe Shori , Okabe Mitsunori , Unno Michiaki , Abe Takaaki , Kaku Mitsuo , Sasaki Takeshi
Published: April 2020

(Introduction) It is necessary for killing leukemic cells effectively to achieve a sufficient intracellular concentration of anti-cancer drugs. To date, overexpression of efflux pump such as MDR-1 and MRP-1, which leads to a decrease of cellular concentration of drugs, has been shown to attenuate the therapeutic effect of anti-cancer drugs and correlate with low remission rate in acute leukemia. If the expression of genes, which import anti-cancer drugs, can be induced specifically in leukemic cells, it would lead to an increase of intracellular concentration, resulting in an enhancement of the pharmacological effect. In this study, we performed the pharmacological characterization of a cation transporter gene, OCT6, and explore the possibility of its application for anti-cancer therapy.

(Methods) Tissue distribution of OCT6 was examined by Northern blot analysis. Expression in clinical samples that consist of 34 patients; 15 patients with acute lymphoblastic leukemia (ALL), 17 patients with acute myelogenous leukemia (AML), 2 patients with chronic myelogenous leukemia (CML) in blastic crisis, was examined by quantitative RT-PCR. The percentage of blasts in each sample was above 80%. For functional analysis, OCT6 cRNA was injected into Xenopus oocytes and pharmacological analysis was performed. In order to examine the transporting activity in hematopoietic cells, we established subclones of Jurkat, a T lymphocytic leukemia cell line, in which OCT6 gene is constitutively expressed, and the change of the sensitivity for adriamycin was examined.

(Result) By Northernblot analysis, 2.4kb OCT6 mRNA was detected in testis and bone marrow in adult tissues. OCT6 expressed in Xenopus oocytes transported not only tetraethlammonium but also adriamycin in a saturable and dose-dependent manner. The apparent Km value for [14C] adriamycin was 5.2±0.4μM. On the other hand, OCT6 did not transport either of methotrexate (MTX) and 5-fluorouracil (5-FU), which are not organic cations. After the exposure of adrimycin at the therapeutic concentration, the viability of OCT6 overexpressing Jurkat cells was significantly decreased compared to that of control cells. Consistent with this, the percentage of apoptosis cells became higher than that of control cells. When the expression level was examined in clinical samples, the average of the levels of patients was comparable to PBMCs. The level of patients, who achieved with complete remission (CR), was slightly higher than that of patients with non-response (NR), but the difference was not significant. Some patients exhibited the high expression of OCT6, but the distribution did not deviated to either CR or NR group.

(Conclusion) These results suggest that the induction of OCT6 in leukemic cells might be a novel strategy for leukemia treatment.
Cotreatment with Pioglitazone, a Synthetic Ligand for Peroxisome Proliferator-Activated Receptor γ (PPARγ), Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis of Human Leukemia Cells.
Authors: Park Joo-In , Han Hoon , Han Ji-Seon , Kwon Hyuk-Chan , Han Jin-Yeong , Jeong Jin-Sook , Bae Yoe-Sik , Kwak Jong-Young
Published: April 2020

Imatinib (STI571, Glivec) is the choice treatment for Bcr/Abl-positive malignancies. Emergence of resistance to Imatinib warrants the exploration of novel well-tolerated anticancer agents. Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor family, which mainly associates with the adipocyte differentiation, but also appears to facilitate cell differentiation or apoptosis in certain malignant cells. Previous studies imply that the PPARγ activation pathway may be a possible intervention mode for treatment of leukemia, which is resistant to imatinib (STI571). In this study, we investigated the effects of pioglitazone, a synthetic ligand for PPARg, on the cell growth and TRAIL-induced apoptosis in a novel imatinib (STI571) resistant acute myeloid cell line (SR-1), which we have established from an STI571 resistant blast crisis patient, as well as HL-60 cells. HL-60 and SR-1 cells are relatively resistant to TRAIL-induced apoptosis. Pioglitazone alone inhibited the cell growth of SR-1 and HL-60 cells, but did not induce the apoptosis of these cell lines. However, simultaneous exposure of cells to 100 ng/ml TRAIL with either 25 μM pioglitazone or 50 μM piogliazone resulted in a striking increase in apoptosis. To clarify the mechanism of pioglitazone to sensitize the leukemia cells to TRAIL-induced apoptosis, we investigated the change of the proteins related to cell cycle and apoptosis by western blot. As results, we observed the significant decrease of X-linked inhibitor of apoptosis (XIAP) and the increased expression of p21 by cotreatment of pioglitazone with TRAIL. Taken together, these findings indicate that pioglitazone may have promising activity in augmenting TRAIL-induced apoptosis of human acute leukemia cells including the imatinib (STI571) resistant acute myeloid cell line.
Cytotoxic Chemotherapy-Induced Changes in Plasma Mannan-Binding Lectin in Patients with Cancers.
Authors: Turp Claire , Joyce Hilary , Sivakumaran Muttuswamy
Published: April 2020

Mannan-binding lectin (MBL) plays an important role in the innate immune responses in primates. The immune function of MBL is mediated by its ability to function as an opsonin and to activate complement through lectin pathway. Recently, there has been an interest in investigating the role of plasma MBL in patients undergoing chemotherapy for leukaemia. Reports indicate that low plasma MBL levels predispose to more severe and frequent infections. There is, however, no clear published data regarding the potential effect of cytotoxic drugs on plasma MBL levels in the absence of any overt sepsis. We conducted a study to obtain this information by carrying out serial measurements of plasma MBL levels during chemotherapy. In addition, we made serial assays of salivary MBL levels to ascertain whether changes in salivary content of this lectin during chemotherapy predispose to mucositis. Ten patients with various non-haematogenous malignancies receiving 3 weekly 5-fluouracil treatment were studied. Blood and saliva samples were collected prior to starting chemotherapy (baseline) and then before subsequent cycles of treatment. ‘Normal range’ of plasma MBL was established by measuring 50 plasma samples obtained from healthy blood donors. Plasma MBL was measured by an ELISA technique using mouse monoclonal antibody IgG1 anti MBL. Salivary MBL level was measured by a modified technique using the same antibody. The results show that there is a marked variation in the plasma MBL levels in healthy individuals (11ng/ml- 9189ng/ml, with a mean of 3057ng/ml). Similarly, the plasma MBL levels of the patient cohort also showed marked variation (10ng/ml- 10896ng/ml with a mean of 3243ng/ml). However, within an individual patient the plasma MBL level rose markedly following first course of chemotherapy. The mean post-first dose rise was 59.2%. Nine out of ten patients exhibited this phenomenon. Interestingly, however, the changes in plasma MBL levels with subsequent treatments were more varied ie in some patients (3/10) the levels continued to rise while in others the levels decreased. Salivary MBL levels in all patients were below detection limit suggesting that polymeric MBL is not present in significant quantities in saliva. Based on the findings of this study we conclude that cytotoxic drugs such as 5-FU may cause significant changes in plasma MBL levels and, therefore, caution should be exercised in interpreting individual measurements following chemotherapy when making any correlation between post chemotherapy plasma MBL levels and infection risk.
Col-6, a Compound Found in Edible Plants, Induces Apoptosis of Human Leukemia Cells and Down-Regulate Bcl-2 Expression.
Authors: Ma Xudong , Beklemisheva A. , Liu Delong , Chiao J.W.
Published: April 2020

Col-6 is a compound found in certain edible plants. Col-6 has been synthesized, and structurely-characterized. This study investigated its effects on the growth of human promyelocytic leukemic cell line, HL-60.

Exposure of HL-60 cells to Col-6 at high concentrations (≥40 μM) resulted in cytolysis in 90% cells within 72 hours. At lower concentrations (<20 μM) the cell density and viability were reduced as compared to untreated cells. A 30% reduction in cell growth rate was seen at 10 μM within 48 hours. A reduction of the proliferating cells in S and G2M phases along with an increase of G1 were detected, indicating a G1 arrest. Multiple assays were used to characterize apoptosis, including DNA strand breaks by TUNEL assay, and the presence of sub-G1 peak by flow cytometry. Col-6 induced apoptosis of HL-60 cells regularly at a concentration as low as 0.1 μM. The degree of apoptosis was proportional to the concentrations and the length of exposure. Mononuclear cells isolated from normal peripheral blood (PBMC) and bone marrow (BMMC) were similarly exposed to Col-6. At a concentration as high as 40 μM, Col-6 did not cause apoptosis of PBMC nor BMMC, suggesting that Col-6 does not have the apoptotic effects on normal non-dividing cells. Apoptosis was further correlated with the increase of caspase 9, and the cleavage of PARP (substrate of caspases), as defined by Western blot analyses. The expression of bcl-2 was markedly decreased in the treated cells.

Conclusion: Col-6, a compound found in edible plants, induced apoptosis of HL-60 cells and down-regulation of bcl-2. Col-6 may be a potential novel anti-leukemia agent. Further in vivo study is indicated.
Decreased Expression of CCAAT/enhancer Binding Protein ζ (C/EBPζ) in Patients with Different Myeloid Diseases.
Authors: Qian Jun , Chen Zi-xing , Lin Jiang , Wang Wei , Cen Jian-nong
Published: April 2020

CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that have been implicated in diverse cellular functions such as cellular differentiation and proliferation, and inflammatory processes. C/EBPζ, also known as GADD153, CHOP10, DDIT3, has been found associated with the development of myxoid liposarcoma and with the progression of melanoma. Based on the results of gene expression profiling on MDS patients in our laboratory, the down-regulated C/EBPζ (DDIT3) gene was selected to be one of the “candidate genes” with most remarkable differential expression pattern in MDS patients in comparison with the normal control. To further investigate the correlation of C/EBPζ transcript levels with the development of leukaemia, samples from 187 patients with myelodysplastic syndrome (MDS), acute myeloid leukaemia (AML), and chronic myeloid leukaemia (CML) were detected for C/EBPζ mRNA using real-time quantitative PCR (RQ-PCR). RQ-PCR analysis demonstrated that the median levels of C/EBPζ were significantly decreased in MDS, AML, and CML patients compared to normal controls (1.40, 0.96, 2.60 versus 12.20, P<0.0001). Significant differences were also observed between patients with CML and with AML or MDS. These results suggest that the insufficient dosage of C/EBPζ might be involved in the development of acute leukemia.
Autologous Tumor Combined with a GM-CSF-Secreting Cell Line Vaccine (GVAX®) Following Autologous Stem Cell Transplant (ASCT) in Multiple Myeloma.
Authors: Borrello Ivan , Biedrzycki Barbara , Sheets Nicole , George Beena , Racke Frederick , Loper Kathy , Noonan Kimberly , Donnelly Amy , Hege Kristen , Levitski Hyam
Published: April 2020

Background: Preclinical models have demonstrated enhanced cancer vaccine efficacy when administered early following ASCT and accompanied by vaccine-primed lymphocyte infusion.

Methods: Patients with untreated myeloma were enrolled and underwent a bone marrow harvest to obtain tumor cells for vaccine processing. Patients eligible for ASCT following induction chemotherapy received a pre-transplant vaccination followed by leukapheresis to collect “primed” lymphocytes that were reinfused with the stem cell graft. Eight post-transplant vaccinations were administered at 3-week intervals starting 6 weeks posttransplant. The vaccine formulation consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting K562 cells (CG9962) at a ratio of 5:2, respectively. Three dose levels were assigned (1, 4, and 10 x107 tumor cells/vaccination) based on tumor cell yield from the bone marrow harvest.

Results: 22 patients underwent tumor harvest, 18 completed induction chemotherapy, and 16 proceeded to vaccination (1 at 1 x 107,4 at 4x107, and 13 at 10x107) and ASCT. At baseline, the median b2 microglobulin was 4 (2–14) and 35% had an IgA subtype. No grade 3/4 vaccine-related toxicities were observed. Median time to neutrophil engraftment (>500) and platelet engraftment (>50K) was 10 days. Local vaccine injection site reactions were observed in all patients. Delayed-type hypersensitivity (DTH) reactions to injections of irradiated autologous tumor cells were induced in only 1/15 patients after 4 post-transplant vaccines, but 4/10 were positive at one year post transplant. Induction of autologous tumor-reactive antibodies as well as antibodies reactive against CG9962 cells was observed. In vitro studies have demonstrated the induction and adoptive transfer of tumor-specific cellular immune responses that can be followed throughout the post-transplant period. Reproducible serum GM-CSF levels were achieved with repeated vaccinations suggesting no increased clearance of CG9962 cells due to induction of an allogeneic immune response. Of the 16 patients that have completed the ASCT, there were 6 complete and 5 partial remissions, for an overall response rate of 69%. Three patients with rising paraprotein levels early post-transplant have demonstrated paraprotein declines following initiation of post-transplant vaccines, suggesting a possible vaccine-mediated effect.

Conclusions: These data demonstrate the feasibility of this vaccine approach and show the therapeutic potential of K562 GVAX® vaccination in the ASCT setting in multiple myeloma.
Imatinib Mesylate for Refractory Acute Myeloblastic Leukemia Harboring inv(16) and C-KIT Exon 8 Mutation.
Authors: Matsuno Naofumi , Nanri Tomoko , Kawakita Toshiro , Mitsuya Hiroaki , Asou Norio
Published: April 2020

While approximately 50% of acute myeloblastic leukemia (AML) patients carrying favorable karyotypes show long-term survival, treatment outcome is not universally favorable in such patients. Recently, mutations in the C-KIT and FLT3 genes were frequently found in patients with inv(16) AML. Of 15 patients we examined, 2 had the FLT3-D835 mutations, 1 had a mutation in the second tyrosine kinase domain, 1 in the juxtamembrane domain, and 1 in the exon 8 of the C-KIT gene. These mutations are potential therapeutic targets for specific tyrosine kinase inhibitors. In this study, we present a case of inv(16) AML harboring C-KIT exon 8 mutation that was successfully treated with imatinib mesylate. After treatment with low-dose cytarabine, aclarubicin and granulocyte colony-stimulating factor (CAG) plus vincristine and prednisone (VP), a 58-year-old man with AML M4Eo showed second relapse with left inguinal lymph nodes involvement. The patient was treated with high-dose cytarabine and mitoxantrone only unsuccessfully. However, the following treatment with 400 mg of imatinib mesylate for 2 weeks in combination with CAG plus VP brought about complete hematological remission. This treatment was well tolerated, and no severe adverse events occurred. This observation suggests that imatinib mesylate can be an alternative treatment modality for AML with the mutation in exon 8 of the C-KIT gene, although further studies are required to confirm the efficacy of this approach for refractory AML.
Prognostic Value of Early Treatment Response in Children with Acute Lymphoblastic Leukemia: A Single Institution Experience in Shanghai, China.
Authors: Tie Li-Jun , Gu Long-Jun , Song De-Lian , Jiang Li-Ming , Xue Hui-Liang , Tang JingYan , Dong Lu , Pan Ci , Chen Jing , Wang Yao-Ping , Chen Jing
Published: April 2020

Early response to therapy is one of the most important prognostic factors in childhood ALL. In the early 1980s, assessment of early treatment response to therapy relied mainly on morphological examination of peripheral blood or bone marrow after single-agent or multi-agent remission induction therapy. More recently, measurement of minimal residual disease (MRD) by flow cytometric detection of aberrant immunophenotype or by polymerase chain reaction of clonal T-cell receptor or immunoglobulin gene rearrangement emerged as a more reliable prognostic indicator. We evaluated the prognostic significance of early treatment response in our recent clinical trial. Among the children with ALL who entered protocol of ALL-XH-99 from January 1998 to May 2003, 193 patients with newly diagnosed ALL comprise the basis for this report. We examined blast cell count in the bone marrow on day 19 of remission induction and at the end of 35 days of remission induction. Minimal residual disease was measured with the use of flow cytometry. Probability of event-free survival was estimated by Kaplan-Meier analysis and the distributions of pEFS were compared using the log-rank test. A Cox proportional hazards model was used to identify independent prognostic factors. Univariate analysis indicated the probability of 4-year event-free survival (pEFS) was significantly worse for patients with ≥ 5% lymphoblasts in the bone marrow on day 19 as compared to those with less than 5% lymphoblasts on that date (42.59%±14.28% vs 74.24%±6.67%, P=0.0006). The probability of 4-year event-free survival (pEFS) was significantly worse for patients with any amount of lymphoblasts in the bone marrow on the remission date as compared to that of other patients with no morphologically identifiable blasts (63.47%±9.23% vs 76.41%±6.09%, P=0.0130). Patients who achieved a minimal residual level < 0.01%, fared significantly better than those with a higher level. (23.81%±20.26% vs 94.44%±5.4%, P=0.001). Early treatment response as assessed by morphologic examination or minimal residual leukemia determination by flow cytometry has important prognostic significance, and can be performed in a resource-poor patient population.
Clinical Significance of FLT3 Internal Tandem Duplication in Patients with Acute Myeloid Leukemia Who Underwent Allogeneic Bone Marrow Transplantation.
Authors: Lee Je-Jung , Lee Yu-Ra , Kim Hee-Nam , Kim Nan-Young , Park Kyeong-Soo , Yang Deok-Hwan , Kim Yeo-Kyeoung , Kim Hyeoung-Joon
Published: April 2020

Since the introduction of high-dose cytarabine therapy, there are still controversies for the role of allogeneic or autologous stem cell transplantation (SCT) in acute myeloid leukemia (AML), but the SCT, especially allogeneic, has a benefit in AML patients with intermediate risk. However, it is needed to investigate for a new prognostic factor, in addition to the cytogenetics, to the further stratifying approach. Internal tandem duplications (ITD) within FLT3 are present in 20–30% of patients with AML and have a prognostic implication in the disease. However, there was no report in AML patients who underwent allogeneic bone marrow transplantation (BMT). In this study, we evaluated the prognostic relevance of FLT3/ITD in 42 patients with AML who underwent allogeneic BMT. FLT3/ITD mutations were studied on bone marrow samples at diagnosis using PCR. As a baseline study, we firstly analyzed the incidence of FLT3/ITD in BM samples at diagnosis of 214 patients with AML. Of the patients, FLT3/ITD were found in 68 patients (31.8%). In this study, 64% of the patients who underwent allogeneic BMT were positive for FLT3/ITD mutations. Methologically, FLT3/ITD detections by melting curve analysis showed higher sensitivity (66.7%) than those by gel electrophoresis (61.9%). There were no significant differences in the engraft periods and the incidence of acute and chronic GVHD according to the presence of FLT3/ITD mutations. When we analyze patient’s survival according to the presence or absence of FLT3/ITD, the probability of overall survival (OS) at 3 years in the AML patients with FLT3/ITD tended to be shorter than those lacking FL3/ITD (53.9 ± 9.9% vs. 60.6 ± 14.7%, P=0.46). In addition, the probability of disease free survival (DFS) at 3 years in the AML patients with and without FLT3/ITD was 57.3±10.3% and 85.7±13.2%, respectively (P=0.046). Among the cytogenetic risk group, low-risk and high-risk groups were no significant differences according to FLT3/ITD despite of the limited number of patients studied. However, patients with intermediate-risk were significantly shorter DFS in the presence of FLT3/ITD than those in the absence of FLT3/ITD (P=0.048). These findings suggest that the presence of FLT3/ITD mutations is a poor prognostic factor for disease free survival in AML patients, especially with cytogenetically intermediate-risk, who underwent allogeneic BMT and melting curve analysis to detect the presence of FLT3/ITD mutations is a useful tool with high sensitivity.
The C677T Polymorphism in the Methylenetetrahydrofolate Reductase (MTHFR)Gene Is an Important Determinant of Bone Mineral Density in Pediatric Acute Lymphoblastic Leukemia Patients.
Authors: van Beek Robert D. , de Muinck Keizer-Schrama Sabine M.P.F. , de Jonge Robert , Pieters Rob , van den Heuvel-Eibrink Marry M.
Published: April 2020

Introduction: Pediatric acute lymphoblastic leukemia (ALL) and its treatment have adverse effects on growth, bone mineral density (BMD) and body composition. Methylenetetrahydrofolate reductase (MTHFR) is involved in folate and homocysteine metabolism. Several studies showed a higher incidence of methotrexate (MTX) related toxicity among carriers of polymorphisms in the (MTHFR) gene.

Methods: We studied whether polymorphisms in the MTHFR gene influence the risk of therapy-induced side effects in pediatric ALL. C677T and A1298C polymorphisms in the MTHFR gene were studied in 83 pediatric ALL patients (47 male, 36 female) using real-time PCR and hybridization probes (LightCycler system). Mean age at diagnosis was 7.5 yr (1.5 – 16.8 yr). All patients were treated with a dexamethasone-based treatment protocol, without cranial irradiation. BMD of lumbar spine (LS) and total body (TB) and body composition were measured using DEXA-scan four times during therapy and once one year after therapy; results are compared with healthy age- and sex-matched controls and expressed as standard deviation scores (SDS). Bone mineral apparent density of the lumbar spine (BMAD) was calculated to correct for bone size.

Results: In all patients, lean body mass (LBM) was already reduced at baseline and remained low during therapy, whereas percentage body fat increased during therapy. Height SDS was reduced during therapy and remained low during the first year after therapy. Carriers of the 677 T allele showed a reduced BMDLS as compared to healthy controls both at baseline (SDS −0.81; p<0.01) as well as during therapy and one year after cessation of therapy, whereas BMDTB and BMAD were normal at baseline in 677 T carriers as compared to healthy controls. After the first 32 weeks of therapy both BMDTB and BMAD were significantly reduced as compared to controls (SDS −0.90; p<0.001 and SDS −0.60; p<0.01 respectively). In carriers of the 677 T allele BMDTB remained low until the end of therapy and also one year after cessation of therapy as compared to healthy controls. In contrast, patients carrying the 677 CC wild-type variant had a normal BMD at baseline, which remained normal throughout therapy. The difference (diff.) between the carriers and non-carriers of the 677 T-allele was significant for BMDTB at baseline (diff. −0.83 SDS, p=0.05), after 32 weeks of therapy (diff. −0.94 SDS, p<0.01) and after 1 year of therapy (diff. −0.94 SDS, p<0.01).We did not find any effect of the MTHFR A1298C polymorphism on height, BMD, body composition. The MTHFR C677T and A1298C polymorphisms did not affect fracture rate.

Conclusions: We identified the MTHFR C677T polymorphism as a determinant of bone mineral density in ALL patients especially in the first year of therapy, and as a risk factor for treatment-related loss of bone mass.
Fusion of SFQP to ABL in a Patient with a T(1;9)(p34;q34) and Acute Lymphocytic Leukemia.
Authors: Cross Nicholas C.P. , Chase Andrew J. , Drachenberg Milton , Roberts W. Mark , Finklestein Jerry Z. , Grand Francis H.
Published: April 2020

We have investigated a child who presented with pre-B ALL and an acquired t(1;9)(p34;q34). BCR-ABL was not detected by RT-PCR or FISH analysis, however FISH did indicate that the ABL gene at 9q34 was disrupted. To identify the putative partner locus in this case, a modified 5′RACE strategy was employed that selected against normal ABL transcripts. Several clones were recovered in which ABL was fused to SFPQ (also known as PSF), a gene mapping to 1p34 that encodes a polypyrimidine tract-binding protein-associated splicing previously identified as a fusion partner of the helix-loop-helix transcription factor TFE3 in papillary renal cell carcinomas. Both SFPQ-ABL and reciprocal ABL-SFPQ transcripts were detectable by RT-PCR, and disruption of these two genes was further confirmed by amplification and sequencing of the forward genomic breakpoint. SFPQ-ABL, the likely oncogenic product, is predicted to encode a protein that retains the coiled coil domain of SFPQ and the entire tyrosine kinase domain and C-terminal sequences of ABL. The breakpoint in ABL was downstream of that seen for other ABL fusion genes and the chimeric protein is predicted to lack the ABL-encoded SH3 domain and part of the SH2 domain. The patient was treated according to the Children’s Cancer Group Protocol 1961 and subsequently received augmented BFM therapy with doxorubicin and double delayed intensification. He achieved complete remission but suffered extramedullary testicular relapse at 4.5 years. Following orchiectomy and intensive chemotherapy he remains in complete remission more than 6 years after diagnosis. We conclude that SFPQ-ABL is a novel fusion gene associated with ALL. Although the patient here responded to conventional chemotherapy, SFPQ-ABL is likely to be sensitive to imatinib and use of this agent might be considered in further cases.
Hypergranular Non-M3 Acute Myelogenous Leukemia: A Potential Pitfall in Flow Cytometric Diagnosis.
Authors: Gorczyca Wojciech , Dong Henry , Weisberger James , Liu Zach
Published: April 2020

Background

Flow cytometry (FC) is an important tool in the diagnosis, subclassification and clinical management of acute leukemias. CD45 vs. orthogonal or side scatter (SSC) gating criteria allows easy identification of non-M3 blasts, which form a coherent population in the low SSC/moderate CD45 blast gate. The vast majority of acute myeloid leukemias (AML) of non-M3 type express pan-myeloid antigens CD13 and CD33, HLA-DR, blastic markers CD34 and CD117. AML M3 (acute promyelocytic leukemia, APL) often lacks CD34 and HLA-DR expression, and has characteristically high SSC/moderate CD45 expression, which overlaps with the granulocyte gate, but is CD117-positive. We report nine cases of non-M3 AML (hypergranular AML) with increased side scatter, moderate CD45 expression, and lack of expression of blastic markers, which mimic APL.

Design

Multiparameter 4-color FC analysis was performed on peripheral blood and/or bone marrow aspirate samples using the following antibody panel: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11b, CD11c, CD13, CD14, CD16, CD19, CD20, CD33, CD34, CD38, CD45, CD56, CD64, CD79a, CD117, TdT, and HLA-DR. Fresh bone marrow aspirate and/or peripheral blood films and cytospin preparations from FC samples were stained with Wright Giemsa and reviewed in each case.

Results

Instead of a coherent population of blasts in the low SSC/moderate CD45 blast gate, the blasts in all 9 cases displayed increased granularity, which placed them in the granulocyte gate on FC display. The immunophenotype was as follows: HLA-DR+ in 5/9, CD4+ in 6/9, CD10+ in 1/9, CD11b+ in 4/9, CD11c+ in 8/9, CD13+ in 8/9, CD14+ in 0/9, CD16+ in 0/9, CD19+ in 0/9, CD33+ in 9/9, CD34+ in 0/9, CD38+ in 9/9, CD56+ in 5/9, CD64+ in 9/9 and CD117+ in 0/9 cases. The cytologic preparations revealed type II and type III cytomorphology in all 9 cases; none contained Auer rods.

Conclusion

Hypergranular non-M3 AML is difficult to diagnose by FC by pattern recognition because the blasts overlap with maturing granulocytes by SSC/CD45 gating criteria, and are not located in the blast gate. In this respect, they mimic APL and regenerative marrows, including those following G-CSF therapy. Correlation with cytomorphology and the expression of CD10, CD11b, CD16 and CD56 helps to differentiate between hypergranular AML, benign processes, APL, and MDS. Specifically, benign maturing myeloid cells show graduating positivity for CD10, CD11b and CD16, and are negative for CD56. This is the inverse pattern of the majority of cases of hypergranular AML. These cases differ from APL by expression of HLA-DR (in a majority of cases), lack of CD117, and different cytomorphology. Myelodysplastic syndromes, which may display similar aberrant myeloid expression of CD10, CD11b, CD16 and CD56, are most often low in SSC, and blastic cells will be <20% cytologically on smear review.
Interactions between the Protein Kinase C (PKC) and the Calcineurin-Phosphatase (CP) Pathways in Differentiation of HL-60 Promyeloblasts into Dendritic Cells (DC).
Authors: Li Qian , Ozer Howard , Kharfan-Dabaja Mohamed A.
Published: April 2020

Continuing success in identifying tumor-specific antigens supports further implementing and refining immunologic strategies to target cancer more specifically. Intracellular signals responsible for differentiation and lineage commitment of pluripotent hematopoietic stem cells remain largely undefined. We have previously reported that blockade of PKC with 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide, also known as bisindolylmaleimide- I inhibits morphologic, phenotypic and functional differentiation of HL-60 myeloid blasts into DC by calcium mobilization with the calcium ionophore A23187. To further analyze the interdependence between the PKC and the CP pathways, we evaluated whether blockade of the CP pathway with cyclosporine A (CSA) affects the ability of the PKC agonist phorbol 12-myristate 13-acetate (PMA) to induce DC differentiation of HL-60 myeloblasts. We pre-treated the HL-60 myeloblasts with the CP inhibitor CSA at a dose of 50 ng/ml x 24 hrs, followed by the PMA at 10 ng/ml x 24 hrs or 48 hrs. On a pilot dose evalaution, we noted that a majority of HL-60 myeloblasts pre-treated with CSA x 24 hrs died upon exposure for ≥48 hrs PMA afterwards; therefore, PMA x 24 hrs was selected. Controls consisted of media, CSA (50 ng/ml x 24 hrs) and PMA (10 ng/ml x 24 hrs). We noted that pre-treatment of HL-60 myeloblasts with CSA→PMA, failed to inhibit development of DC-morphologic features; and on phase-contrast microscopy they were similar to those treated with PMA alone. Media and CSA (alone) treated myeloblasts did not develop DC morphology. Addidtionally, we evaluated the ability of HL-60 derived DC to activate allogeneic T-cells by CD69 activation. We noted that blasts treated with PMA upregulated expression of CD69 in allogeneic T-cells suggesting T-cell activation. Interestingly, pre-treatment of HL-60 blasts with CSA→PMA did not prevent T-cell activation (similar to PMA alone). Media and CSA (alone) treated promyeloblasts failed to induce T-cell activation. Our findings suggest that inhibition of the calcineurin-phosphatase pathway with CSA does not inhibit morphologic and functional differentiation of HL-60→ DC by direct PKC signaling with PMA.
Coagulopathy in HLA-DR Antigen-Negative Acute Myeloid Leukemia.
Authors: Uchiumi Hideki , Matsushima Takafumi , Yamane Arito , Handa Hiroshi , Irisawa Hiroyuki , Doki Noriko , Sakura Tohru , Tsumita Yuki , Jinbo Takahiro , Tsukamoto Norifumi , Karasawa Masamitsu , Murakami Hirokazu , Miyawaki Shuichi , Nojima Yoshihisa
Published: April 2020

Background: HLA-DR antigen is present on hematopoietic progenitors and granulocyte/monocyte, erythrocyte and megakaryocytic precursors but absent at the promyelocytic stage during myeloid cell maturation. In accordance with this, majority of promyelocytic leukemia (APL) cells were negative for HLA-DR. Meanwhile, some of non-APL acute myeloid leukemia (AML) cells is found to express HLA-DR. However, the clinical significance of HLA-DR antigen on AML cells is currently unclear.

Purpose: We sought to determine the prevalence and clinical characteristics of negativity in HLA-DR expression by retrospectively analyzing 181 consecutive patients with de novo adult AML.

Patients and Methods: AML patients examined in the current study (aged 15–86 years) had been diagnosed between August 1995 and July 2004, and categorized to M0 (8 patients), M1 (35), M2 (74), M3 (20), M4 (25), M5 (15), and M6 (4), based on the FAB classification. Median follow-up time was 19.3 months. Phenotypic analyses of leukemic cells were performed using CD45 gating methods. HLA-DR-negative AML was defined as HLA-DR expression less than 20% of cells in the CD45 leukemic cell gate.

Results: Among 181 patients, HLA-DR antigens were not detected on AML cells from 46 patients; 20 with APL and 26 with non-APL (non-APL/DR(−)), the latter of which included M0 (2 patients), M1 (15), M2 (7), M4 (2). Leukemic cells from other non-APL patients were HLA-DR-positive (non-APL/DR(+)). None of non-APL/DR(−) patients had t(15;17) nor PML/RARa rearrangement on cytogenetic analysis. Twenty out of 26 patients with non-APL/DR(−) had normal chromosome, and 6 had abnormal karyotypes. In the non-APL/DR(−) group, various degrees of nuclear folding, convolution, or lobulation were observed in 9 patients. Although treatment response and overall survival rate were similar in the three groups (APL, non-APL/DR(−), and non-APL/DR(+)), both FDP levels at diagnosis (57.3 vs 13.2, p<0.05) and maximal FDP levels (232.6 vs 43.8, p<0.01) were significantly higher in non-APL/DR(−) compared with non-APL/DR(+). The maximal FDP levels in the non-APL/DR(−) patients were comparable to those in the APL patients. FDP levels greater than 40 mg/ml were significantly more prevalent in the non-APL/DR(−) than in the the non-APL/DR(−) group. Logistic regression analysis demonstrated that low HLA-DR expression was an independent risk factor for FDP > 40 mg/ml.

Conclusion: Our study suggests that AML with negative HLA-DR antigen tend to be associated with abnormality in coagulation and fibrinolysis even if they are genetically non-APL. We propose that more attention should be paied for HLA-DR expression to avoid a devastating coagulopathy which carries a high risk of mortality unless specifically addressed.
Multi-Dimensional Flow Cytometric Analysis of Acute Myelomonocytic Leukemia: Evaluation of Disease Complexity and Effects on Host Hematopoiesis.
Authors: Katz Ben-Zion , Kam Zvi , Deutsch Varda , Bar-On Shoshana , Ben-Tal Ofira , Cohen Nir , Naparstek Elizabeth , Geiger Benjamin , Zamir Eli
Published: April 2020

The study of normal or malignant hematopoiesis requires the analysis of heterogeneous cell populations using multiple morphological and molecular criteria. Flow cytometry has the capacity to provide multi-parameter information of large hematopoietic cell populations, utilizing various combinations of over 200 molecular markers (CDs). However, current flow cytometry analyses are based on serial gating of two-parametric scatter plots - a process that is inherently incapable to discriminate all subgroups of cells in the data. Here we studied the cellular diversity of normal and leukemic bone marrow samples using multi-dimensional cluster analysis of six-parametric flow cytometry data (CD13, CD33, CD34, CD45, forward scatter and side scatter), focusing mainly on the myeloid lineage. Twenty-three subclasses of cells were identified within normal bone marrow samples, many of them inseparable even when examined at all possible two-parametric scatter plots. Seven subclasses of cells expressing the immature CD34 marker were identified. The multi-dimensional analysis could identify the hematopoietic progenitors, and readily distinguish this population from other types of immature cells. We further addressed the compositional variability of bone marrow samples by designing a classifier that assigns cell samples to subclasses, based on robust six-dimensional characteristics. We then applied this classifier to assess the population diversity in nineteen samples of acute myelomonocytic leukemia (AML M4). The myeloid classifier analyzed simultaneously normal cellular populations (e.g. lymphocytes, garnulocytes), and the diverse populations of AML cells. Most of the AML M4 samples contained an immature population assigned to a myeloid immature subclass distinct from the normal hematopoietic progenitors. Nearly 50% of the AML M4 samples contained a combination of immature myeloid cells and a significant population of immature monocytes. The presence of immature monocytic sub-population correlated with lower leukemia cell burden, and higher platelet counts. In summary, our data indicate that AML M4 may be separated into two distinct types, one restricted to an immature myeloid state, and the other with limited monocytic differentiation capacity. The sub-population composition of the leukemia may affect normal hematopoiesis. Multi-dimentional analysis using the myeloid classifier provides a novel method to accurately define both the increasing numbers of hematopoietic cellular markers and sample heterogeneity in AML. This method should further expand our ability to study normal hematopoiesis, and to identify and monitor hematopoietic diseases.
Ligation of CD44 by A3D8 Monoclonal Antibody Induces Terminal Differentiation of THP-1 Leukemia Cells by Promoting Cross-Talk between Extracellular Signal-Regulated Kinase and Phosphoinositide-3 Kinase/Akt Signaling Pathway.
Authors: Min Yoo-Hong , Kim Ji Yeon , Jeung Hoi Kyung , Lee Seung Tae , Cheong June-Won
Published: April 2020

Ligation of CD44 with anti-CD44 monoclonal antibody A3D8 induces terminal differentiation of human leukemia cells. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the importance of MEK/ERK, p38 MAPK, and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in A3D8-induced monocytic differentiation of THP-1 leukemia cells. THP-1 cells displayed cytologic changes typical of mature monocytes and an increased expression of monocyte-specific antigen CD14 (49% ± 4%) and of myeloid-specific antigen CD11b (68% ± 6%) after 3 days of A3D8 treatment. The level of CD15 was also increased. The increase in the expression of these differentiation antigens was dose- and time-dependent. We found that CD44 ligation with A3D8 led to rapid and sustained activation of the essential kinases in the extracellular signal-regulated kinase pathway such as phospho-Raf-1, phospho-MEK1/2, and phospho-ERK1/2. However, the total levels of these kinases were not affected during the course of differentiation. Ser473 Akt phosphorylation was also observed shortly after the cells were exposed to A3D8 and sustained thereafter. In contrast, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase were not increased by CD44 ligation. Pretreatment of cells with 20mM of MEK1 inhibitor PD98059 abrogated the A3D8-induced activation of MEK1/2 and ERK1/2 and potently inhibited the THP-1 differentiation. However, a p38 MAPK inhibitor SB203580 did not abolished the CD44 ligation-induced terminal differentiation of THP-1 cells. Pretreatment of cells with PI3-K inhibitor LY294002 resulted in a nearly complete inhibition of A3D8-induced terminal differentiation. Overexpression of dominant-negative Akt also reduced the A3D8-induced THP-1 differentiation, suggesting that A3D8-induced THP-1 differentiation is coupled to PI3-K/Akt activation. Surprisingly, the pharmacological inhibition of PI3-K blocked not only A3D8-induced Akt activation but also A3D8-induced activation of Raf, MEK and ERK pathway. By contrast, pretreatment of cells with PD98059 did not inhibit the A3D8-induced Akt activation, suggesting that PI3-K/Akt is the upstream of Raf/MEK/ERK pathway in A3D8-induced terminal differentiation of THP-1 leukemia cells. Taken together, our findings demonstrated that the cross-talk between the activation of A3D8-inducible PI3-K/Akt pathway and the Raf/MEK/ERK pathway in THP-1 cell exists, and plays a critical role during CD44 ligation-induced THP-1 differentiation.