Megakaryoblastic leukemia cell line MOLM-16 derived from minimally differentiated acute leukemia with myeloid/NK precursor phenotype

The megakaryoblastic leukemia cell line MOLM-16 was established at relapse from the peripheral blood of a 77-year-old Japanese woman with minimally differentiated acute myeloid leukemia (AML-M0). Immunophenotyping of the fresh leukemic cells revealed a myeloid/NK precursor phenotype being positive for CD7, CD13, CD33, CD34, and CD56. In addition, megakaryocyte-associated antigens CD41 and CD61 were found to be positive. The established cell line designated MOLM-16 was proliferatively responsive to the treatment with various cytokines including EPO, GM-CSF, IL-3, PIXY-321, and TPO. MOLM-16 revealed characteristics of the megakaryocytic lineage in terms of immunophenotyping being positive for CD9, CD31, CD36, CD41, CD61, CD62P, CD63, CD110, CD151, thrombospondin, von Willebrand factor (vWf), and fibrinogen. Electron microscopic analysis showed positivity for ultrastructural platelet peroxidase in the nuclear envelope. The karyotype analysis of MOLM-16 revealed various numerical and structural abnormalities including t(6;8)(q21;q24.3), t(9;18)(q13;q21) and marker chromosomes. The extensive immunological, cytogenetic and functional characterization of MOLM-16 suggests that this cell line may represent a scientifically significant in vitro model which could facilitate the evaluation of megakaryocytic differentiation.     

Proteasome inhibition in myelodysplastic syndromes and acute myelogenous leukemia cell lines

In this work, effects of bortezomib on apoptosis, clonal progenitor growth, cytokine production, and NF-κB expression in patients with MDS with cytopenias requiring transfusion support are examined. Bortezomib increased apoptosis in marrow mononuclear cells but had no effects on CFU-GM, BFU-E, or CFU-L content. No consistent effects on NF-κB activation in vivo were noted. To further define the role of bortezomib in AML and MDS, we examined it in combination with several targeted agents and chemotherapeutic agents in vitro. Combinations with arsenic trioxide, sorafenib, and cytarabine demonstrated synergistic in vitro effects in AML cell lines.     

Farnesyltransferase inhibitors in acute myeloid leukemia and myelodysplastic syndromes

Farnesyltransferase inhibitors were initially developed as Ras inhibitors as they inhibit the prenylation necessary for Ras activation. It is clear now that their mechanism of action is more complex and probably involves other proteins unrelated to Ras. At least 3 drugs within this family have been investigated in acute myeloid leukemia, myelodysplastic syndromes, and other leukemias. These are tipifarnib (R115777, Zarnestra), lonafarnib (SCH66336, Sarasar), and BMS-214662. The first 2 are administered orally, whereas BMS-214662 is given intravenously. These drugs are at different stages of development, and design of treatment schedules and methodology of the available studies are very different. Although most of the information is still preliminary, these agents have demonstrated clear evidence of clinical activity in these diseases and very favorable toxicity profiles. Several studies are still ongoing to better define the efficacy of these agents in the treatment of leukemias, as well as to determine the best schedules, the role of combination with other agents, and the role of these agents in different settings, such as the management of minimal residual disease. It is very possible that these agents will soon find their way to the ranks of established agents for the management of myeloid malignancies     

Advance care planning in older patients with acute myeloid leukemia and myelodysplastic syndromes

IntroductionOlder patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) have worse survival rates compared to younger patients, and experience more intense inpatient healthcare at the end of life (EOL) compared to patients with solid tumors. Advance care planning (ACP) has been shown to limit aggressive and burdensome care at EOL for patients with AML and MDS. The purpose of this study was to better understand ACP from the perspective of clinicians, older patients with AML and MDS, and their caregivers.Materials and MethodsWe conducted semi-structured interviews with 45 study participants. Interviews were audio-recorded and transcribed. Open coding and focused content analysis were used to organize data and develop and contextualize categories and subcategories.ResultsGuided by our specific aims, we developed four themes: (1) The language of ACP and medical order for life-sustaining treatment (MOLST) does not resonate with patients, (2) There is no uniform consensus on when ACP is currently happening, (3) Oncology clinician-perceived barriers to ACP (e.g., patient discomfort, patient lack of knowledge, and lack of time), and (4) Patients felt that they are balancing fear and hope when navigating their AML or MDS diagnosis.DiscussionThe results of this study can be used to develop interventions to promote serious illness conversations for patients with AML and MDS and their caregivers to ensure that patient care aligns with patient values.     

NPM1 mutations in myelodysplastic syndromes and acute myeloid leukemia with normal karyotype

Mutations at exon 12 of the nucleophosmin (NPM1) gene are the most frequent acquired molecular abnormalities in adult and pediatric acute myeloid leukaemia (AML) with normal karyotype. We screened 28 patients with new diagnosed primary AML with normal karyotype, 38 patients with myelodysplastic symdromes (MDS) and 19 healthy volunteer for mutations at exon 12 of NPM1 gene. NPM1 mutations were identified in four AML patients and two MDS patients, including one novel sequence variant. As far as we know, this is the first report of NPM1 mutation in patients with MDS in the English literature until now, and our primary data support that NPM1 mutations may be also involved in the pathogenesis of MDS.     

Properties of the K562 cell line,derived from a patient with chronic myeloid leukemia

The K562 cell line derived from a CML patient in blast crisis was examined for properties of B and T lymphocytes and cell lines. K562 lacks the B markers of immunoglobulin, Epstein-Barr virus (EBV) genome and associated nuclear antigen, and receptors for EBV. A low proportion of cells form rosettes with sheep erythrocytes, the frequency of which is considerably increased after neuraminidase treatment. Unlike B lines but like T lines, K562 cells are lysed rapidly by C'/Fc receptor-positive human blood leukocytes and do not stimulate MLC reactions. On the other hand, K562 lacks T antigen, high radiosensitivity and sensitivity to growth inhibition by thymidine. The cells do not contain N-APase, an enzyme found in all lines derived from lymphoid cells and in lymphoproliferative diseases. By scanning electron microscopy, K562 cells were seen to be rounded and relatively smooth, with small numbers of short microvilli resembling undifferentiated leukemic cells. A few cells had narrow ridge-like profiles and small ruffles similar to granulocytic leukemic cells. K562 is strongly positive for immunoglobulin Fc receptors and pinocytosis, but does not phagocytose or mediate antibody-dependent phagocytosis or cytolysis. Among histochemical stains, K562 is positive for esterase, lipid, and acid phosphatase. There seems to be no doubt that K562 is not a B cell line. While it has some T cell properties, these are not exclusive. Some of its characteristics indicate that it is probably not lymphoid. Due to its low level of differentiation, its nature cannot be stated with certainty. On the basis of the possible presence of the cellular marker of chronic myeloid leukemia, the Ph chromosome, it may be regarded as belonging to the granulocytic series of cells.     

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.     

Epigenetic modifications of splicing factor genes in myelodysplastic syndromes and acute myeloid leukemia

Somatic mutations in splicing factor genes have frequently been reported in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Although aberrant epigenetic changes are frequently implicated in blood cancers, their direct role in suppressing one or both alleles of critical splicing factors has not been previously examined. Here, we examined promoter DNA hypermethylation of nine splicing factors, SF3B1, SRSF2, U2AF1, ZRSR2, SF3A1, HNRNPR, MATR3, ZFR, and YBX3 in 10 leukemic cell lines and 94 MDS or AML patient samples from the Australasian Leukemia and Lymphoma Group Tissue Bank. The only evidence of epigenetic effects was hypermethylation of the YBX3 promoter in U937 cells in conjunction with an enrichment of histone marks associated with gene silencing. In silico analysis of DNA methylation data for 173 AML samples generated by the Cancer Genome Atlas Research Network revealed promoter hypermethylation of the gene encoding Y box binding protein 3, YBX3, in 11/173 (6.4%) AML cases, which was significantly associated with reduced mRNA expression (P < 0.0001). Hypermethylation of the ZRSR2 promoter was also detected in 7/173 (4%) cases but was not associated with decreased mRNA expression (P = 0.1204). Hypermethylation was absent at the promoter of seven other splicing factor genes in all cell lines and patient samples examined. We conclude that DNA hypermethylation does not frequently silence splicing factors in MDS and AML. However, in the case of YBX3, promoter hypermethylation‐induced downregulation may contribute to the pathogenesis or maintenance of AML.     

Low dose arabinosyl cytosine for treatment of myelodysplastic syndromes and subacute myeloid leukemia

Several agents, including arabinosyl cytosine (ARA-C) at a low concentration, can induce leukemic myeloblasts to mature to a variable extent. The therapeutic implications of this observation are worth investigating. A few case-reports have shown that low dose ARA-C can be useful for treatment of the myelodysplastic syndromes (MDS) and of acute myeloid leukemia (AML). However, no information is available yet on the proportion of patients who can be expected to respond. We treated by low dose ARA-C (20-30 mg/sqm/day i.v. or i.m. for 7-10 days) 20 consecutive patients. A complete remission of 5 months was obtained in one of nine cases of subacute myeloid leukemia (SAML). A partial remission (complete normalization of blood counts with a slight excess of marrow blast cells) was obtained twice in one of 11 cases of MDS. An increase of Hb level (more than 11.5 g/dl) was obtained and maintained for 12 months in a case of MDS. A short-lasting increase of granulocyte count was obtained in another two cases of MDS and SAML respectively. It is suggested that low dose ARA-C can advantageously modify the proliferation to maturation imbalance of leukemic cells by slowing down cell proliferation rate. However, the proportion of patients who respond is probably low. This treatment is at a very early experimental stage and should be probably limited to selected cases of MDS and subacute or acute myeloid leukemia.     

Epigenetic priming with decitabine followed by low dose idarubicin and cytarabine in acute myeloid leukemia evolving from myelodysplastic syndromes and higher-risk myelodysplastic syndromes: a prospective multicenter single-arm trial

Patients with acute myeloid leukemia (AML) evolving from myelodysplastic syndrome (MDS) or higher-risk MDS have limited treatment options and poor prognosis. Our previous single-center study of decitabine followed by low dose idarubicin and cytarabine (D-IA) in patients with myeloid neoplasms showed promising primary results. We therefore conducted a multicenter study of D-IA regimen in AML evolving from MDS and higher-risk MDS. Patients with AML evolving from MDS or refractory anemia with excess blasts type 2 (RAEB-2) (based on the 2008 WHO classification) were included. The D-IA regimen (decitabine, 20 mg/m² daily, days 1 to 3; idarubicin, 6 mg/m² daily, days 4 to 6; cytarabine 25 mg/m² every 12 hours, days 4 to 8; granulocyte colony stimulating factor [G-CSF], 5 μg/kg, from day 4 until neutrophil count increased to 1.0 × 10⁹/L) was administered as induction chemotherapy. Seventy-one patients were enrolled and treated, among whom 44 (62.0%) had AML evolving from MDS and 27 (38.0%) had RAEB-2. Twenty-eight (63.6%) AML patients achieved complete remission (CR) or complete remission with incomplete blood count recovery (CRi): 14 (31.8%) patients had CR and 14 (31.8%) had CRi. Six (22.2%) MDS patients had CR and 15 (55.6%) had marrow complete remission. The median overall survival (OS) was 22.4 months for the entire group, with a median OS of 24.2 months for AML and 20.0 months for MDS subgroup. No early death occurred. In conclusion, the D-IA regimen was effective and well tolerated, representing an alternative option for patients with AML evolving from MDS or MDS subtype RAEB-2.     

Effectiveness and safety of different azacitidine dosage regimens in patients with myelodysplastic syndromes or acute myeloid leukemia

We investigated the effectiveness and tolerability of azacitidine in patients with World Health Organization-defined myelodysplastic syndromes, or acute myeloid leukemia with 20–30% bone marrow blasts. Patients were treated with azacitidine, with one of three dosage regimens: for 5 days (AZA 5); 7 days including a 2-day break (AZA 5-2-2); or 7 days (AZA 7); all 28-day cycles. Overall response rates were 39.4%, 67.9%, and 51.3%, respectively, and median overall survival (OS) durations were 13.2, 19.1, and 14.9 months. Neutropenia was the most common grade 3–4 adverse event. These results suggest better effectiveness–tolerability profiles for 7-day schedules.     

Antigens present on human myeloid leukemia cell lines

Two human acute myelogenous leukemia cell lines have recently been established: KG-1 and HL-60. Both lines retain the morphology and cytochemistry of myeloid cells, are not infected with EB virus, and respond to colony-stimulating activity (CSA) with increased colony formation in soft-gel culture. The K 562 cell line is composed of undifferentiated blast cells which are rich in glycophorin and may be induced to produce human fetal and embryonic hemoglobin. We used a variety of antisera to characterize the antigen composition of the leukemic colony-forming cells (CFC) and the general population of leukemic cells. Cells were incubated with antisera and complement, washed, plated in agar, and the colonies counted after 12 days' incubation. Culture data were correlated with the results obtained using direct and indirect immunofluorescence and dye exclusion microcytotoxicity.The majority of KG-1 cells, including CFC, expressed the ‘Ia-like’ antigen. The HL-60 and K562 cells did not express the Ia antigen, although indirect evidence using cytogenetic and isoenzyme studies suggests that the HL-60 gene locus for the antigen was probably intact.We found that the leukemia CFC had the HLA-A and -B antigens as did the general leukemia cell population. The leukemia CFC did not express a T-lymphocyte antigen, certain erythrocyte antigens, nor antigens associated with acute lymphocytic leukemia. HL-60 cells underwent morphological and functional maturation in the presence of DMSO, but no change in surface antigenic composition was noted. All antigens expressed on the leukemia CFC were also present on the general cell population.     

KRN5500 induces apoptosis (PCD) of myeloid leukemia cell lines and patient blasts

The purpose of this study was to determine if KRN5500, a spicamycin derivative with a unique acyl tail, would induce programmed cell death (PCD) of myeloid leukemia cell lines and cryopreserved leukemic blasts from newly diagnosed children with acute leukemia (AL). Cells were incubated with varying concentrations (0-5 ng/ml) of KRN5500 and the percent PCD determined using a modified in situ end labeling (ISEL) technique with Klenow fragment. The percent PCD was calculated using the formula: Percent PCD (% PCD)=[number of apoptotic cells/(viable cells+apoptotic cells)]x100. DMSO (0.30% w/v) was added to the cells in culture as the positive control for PCD; the negative control was media or albumin. KRN5500 increased the amount of PCD significantly in all five of the tested cell lines; U937 41+/-1.8%, KG1a 40+/-0.3%, HEL 14+/-2.2%, HL-60 41+/-0. 9%, K562 36+/-2% (mean PCD+/-SD). Patient blasts exposed to KRN5500 had an increase in PCD when exposed to 2 ng/ml of agent from 2 to 8 h; acute myeloid leukemia patients 7.5+/-0.5% at 2 h to 43.5+/-1.6% at 8 h, and acute lymphocytic leukemia patients rose from 12.4+/-3.8% at 2 h to 29.9+/-11.6% after 8 h (mean+/-SE). Overall the PCD for the patient samples was 3.7 versus 28+/-4% at 2 and 8 h, respectively. PCD was proportional to the dose of KRN5500 and incubation time. Further pre-clinical and clinical studies are required.     

Chromosome analyses of lymphoid cell lines derived from patients with chronic lymphocytic leukemia

To study chromosome complements of chronic lymphocytic leukemia cells, six Epstein-Barr virus-transformed lymphoid lines were established from two patients with this disease who were heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate de-hydrogenase (G6PD). Immunoglobulin and G6PD were used as markers of the leukemic versus normal cell origin of the cell lines. The two lines, derived from putative normal cells, had no chromosomal changes. In contrast, chomosome abnormalities were found in each of the four cell lines of presumed leukemic cell origin. Although the chromosome aberrations are not as specific as the Philadelphia chromosome, there appears to be non-random involvement in chronic lymphocytic leukemia of some chomosomes, such as the No. 12.