Biomedical subjects

Mark Minden

Publications and source records attributed to Mark Minden.

12 recordsLinked to original sources

PubMed2006

A single nucleotide polymorphism chip-based method for combined genetic and epigenetic profiling: validation in decitabine therapy and tumor/normal comparisons.

Progress on several unresolved issues in cancer epigenetics will benefit from rapid and standardized methods for profiling DNA methylation genome-wide. In the area of epigenetic therapy, the demethylating drug decitabine (5-aza-2'-deoxycytidine) is increasingly used to treat acute myelogenous leukemia and myelodysplastic syndrome, but the mechanisms of its anticancer activity have remained unclear. Given the clinical efficacy of decitabine and the uncertainties about its mode of action, it will be useful to optimize methods for following DNA methylation as a biochemical response in individual patients. Here, we describe a single nucleotide polymorphism (SNP) chip-based method (MSNP) for profiling DNA methylation. Using this procedure, the extent of demethylation in bone marrow aspirates from patients with leukemia receiving decitabine can be assessed genome-wide using commercially available (Affymetrix) SNP chips. We validated the accuracy of MSNP by comparing the results with combined bisulfite restriction analysis and by sequencing cloned PCR products from bisulfite-converted DNA. We further validated MSNP in a Wilms' tumor/normal kidney comparison, comparing the results with methylation-sensitive Southern blotting. MSNP simultaneously detects aberrations in DNA copy number and loss of heterozygosity, making it a generally useful approach for combined genetic and epigenetic profiling in tissue samples from cancer patients.

Antimetabolites, Antineoplastic↗

PubMed2006

Gene expression profiles in acute myeloid leukemia with common translocations using SAGE.

Identification of the specific cytogenetic abnormality is one of the critical steps for classification of acute myeloblastic leukemia (AML) which influences the selection of appropriate therapy and provides information about disease prognosis. However at present, the genetic complexity of AML is only partially understood. To obtain a comprehensive, unbiased, quantitative measure, we performed serial analysis of gene expression (SAGE) on CD15(+) myeloid progenitor cells from 22 AML patients who had four of the most common translocations, namely t(8;21), t(15;17), t(9;11), and inv(16). The quantitative data provide clear evidence that the major change in all these translocation-carrying leukemias is a decrease in expression of the majority of transcripts compared with normal CD15(+) cells. From a total of 1,247,535 SAGE tags, we identified 2,604 transcripts whose expression was significantly altered in these leukemias compared with normal myeloid progenitor cells. The gene ontology of the 1,110 transcripts that matched known genes revealed that each translocation had a uniquely altered profile in various functional categories including regulation of transcription, cell cycle, protein synthesis, and apoptosis. Our global analysis of gene expression of common translocations in AML can focus attention on the function of the genes with altered expression for future biological studies as well as highlight genes/pathways for more specifically targeted therapy.

Apoptosis↗

PubMed2006

Nuclear localizing sequences promote nuclear translocation and enhance the radiotoxicity of the anti-CD33 monoclonal antibody HuM195 labeled with 111In in human myeloid leukemia cells.

UNLABELLED: Our objective was to evaluate the toxicity of the anti-CD33 monoclonal antibody HuM195 modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localizing sequence (NLS; underlined) of simian virus 40 large T antigen and labeled with (111)In against acute myeloid leukemia (AML) cells. METHODS: HuM195 was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (sulfo-SMCC) to introduce maleimide groups for reaction with NLS-peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with (111)In. The immunoreactivity of NLS-HuM195 was evaluated by its ability to displace the binding of (111)In-HuM195 to HL-60 leukemia cells. Nuclear localization was measured in HL-60 cells by subcellular fractionation. The antiproliferative effects of (111)In-NLS-HuM195 and (111)In-HuM195 on HL-60, U937, or K562 cells with high, intermediate, or minimal CD33 expression, respectively, were studied. The survival of HL-60 cells or patient AML specimens treated with (111)In-NLS-HuM195 or (111)In-HuM195 was studied. Normal tissue toxicity was evaluated in BALB/c mice injected intravenously with of 3.7 MBq (22 microg) of (111)In-NLS-HuM195 or (111)In-HuM195. RESULTS: NLS-HuM195 exhibited relatively preserved CD33 binding affinity (dissociation constant [K(d)] = 4.3 +/- 1.7 x 10(-9) mol/L to 6.9 +/- 1.3 x 10(-9) mol/L). Nuclear uptake increased from 10.5% +/- 0.5% for (111)In-HuM195 to 28.5% +/- 4.1% or 65.9% +/- 1.5% for (111)In-HuM195 substituted with 4 or 8 NLS-peptides, respectively. The inhibitory concentrations of 50% (IC(50)) and 90% (IC(90)) for HL-60 cells treated with (111)In-NLS-HuM195 were 37 kBq per 10(3) cells and 77-81 kBq per 10(3) cells, respectively. The IC(50) and IC(90) values for (111)In-HuM195 were 92 kBq per 10(3) cells and 203 kBq per 10(3) cells. Growth inhibition was correlated with the level of CD33 expression. The survival of HL-60 cells was reduced from 232 +/- 22 colonies (control) to 7 +/- 1 colonies with 1.48 mBq per cell of (111)In-NLS-HuM195; no colonies were found at 3.33 mBq per cell. The surviving fraction decreased >2-fold in 7 of 9 AML specimens treated with an excess of (111)In-NLS-HuM195 and >10-fold in 2 of these specimens. There were no decreases in body weight or hematologic parameters or increases in alanine aminotransferase or creatinine in mice administered 3.7 MBq (22 microg) of (111)In-NLS-HuM195 or (111)In-HuM195. There was no morphologic damage to the liver or kidneys. CONCLUSION: We conclude that NLS-peptides routed (111)In-HuM195 to the nucleus of AML cells, where the emitted Auger electrons were lethal. (111)In-NLS-HuM195 is a promising targeted radiotherapeutic agent for AML.

Active Transport, Cell Nucleus↗

PubMed2005

Disease biology rather than age is the most important determinant of survival of patients > or = 60 years with acute myeloid leukemia treated with uniform intensive therapy.

BACKGROUND: The objectives of the current study were to evaluate the outcome of patients > or = 60 years with acute myeloid leukemia (AML) treated uniformly with high-dose daunorubicin containing induction and modified high-dose cytosine arabinoside containing postremission therapy, and to identify factors predictive of complete disease remission (CR) and survival. METHODS: Between 1998 and 2002, the authors treated 117 newly diagnosed patients (acute promyelocytic leukemia excluded) with AML > or = 60 years (median, 67 years; range, 60-82 years). Karyotype (Medical Research Council classification) at diagnosis was categorized as good risk (n = 3), intermediate risk (n = 69), adverse risk (n = 26), and suboptimal/not done (n = 19). A normal karyotype was seen in 41 patients and 40 (34%) had secondary AML. RESULTS: The outcome of induction included the following: CR, 62 (53%); early death, 5 (4%); death during hypoplasia, 14 (12%); and resistant disease, 36 (31%). The 3-year event-free (EFS) and overall survival (OS) rates were 9% (95% confidence interval [95% CI], 3-16%) and 17% (95% CI, 9-29%), respectively. In a univariate analysis, cytogenetics, lactate dehydrogenase level, leukocyte count, and performance status were the significant factors for EFS and OS. Age was not a significant prognostic factor for either CR or survival. In a multivariate model, adverse-risk cytogenetics, previous history of myelodysplastic syndrome or antecedent hematologic disorder, and high leukocyte count (> 30 x 10(9)/L) were independent adverse prognostic factors for survival. The impact of adverse karyotype on EFS and OS was time dependent and was observed after 50 and 150 days, respectively. CONCLUSIONS: The authors concluded that candidacy for intensive therapy in older patients should be based on biologic features of disease and fitness, rather than on age.

Acute Disease↗

PubMed2005

The oncogene PDGF-B provides a key switch from cell death to survival induced by TNF.

Tumor necrosis factor (TNF) induces both cell death and survival signals. NF-kappaB, a transcription factor activated by TNF, is critical for controlling survival signals through trans-activation of downstream target genes. However, few NF-kappaB target survival genes have been identified with direct roles in oncogenesis. We report that platelet-derived growth factor B (PDGF-B), an oncogene and growth factor, is highly induced by TNF in fibroblasts in an NF-kappaB-dependent manner. PDGF-B can rescue NF-kappaB-deficient fibroblasts from TNF-mediated killing, and inhibition of PDGF-B signaling sensitizes wild-type cells to TNF-induced death. Interestingly, PDGF-B-transformed NIH-3T3 cells are even more highly sensitized to TNF-induced cell death with PDGF-B inhibition. Our results suggest that while normal cells contain multiple TNF-induced survival signals, tumor cells may favor a specific survival gene that is abnormally upregulated in order to evade death signals.

3T3 Cells↗

PubMed2005

Fungal liver infection in immunocompromised patients: depiction with multiphasic contrast-enhanced helical CT.

PURPOSE: To retrospectively assess multiphasic (nonenhanced, arterial phase, and portal venous phase) computed tomography (CT) of the liver for depiction of hepatic fungal infection in immunocompromised patients. MATERIALS AND METHODS: The institutional ethics review board approved the study and waived the requirement for informed consent. Sixty multiphasic hepatic CT examinations were performed in 39 immunocompromised patients who fulfilled the criteria for having probable or proved fungal liver infection. The detection and conspicuity of focal liver lesions were assessed on scans obtained during each CT phase. The lesion enhancement pattern was determined, and, accordingly, lesions were stratified into two groups: lesions suggestive of infection (with ring enhancement patterns or high attenuation) and nonspecific hypoattenuating lesions. Statistical analyses were performed by using logistic regression with generalized estimating equations. RESULTS: A total of 536 liver lesions detected at 36 CT examinations with results positive for fungal infection were assessed. All 36 (100%) examinations yielded positive results during the arterial phase, whereas 25 (69%) of them yielded positive results during the portal venous phase (P < .001). At lesion-by-lesion analysis, the arterial phase scans depicted significantly more lesions (483 of 536 [90%]) than the portal venous phase (329 of 536 [61%]) and nonenhanced (265 of 465 [57%]) scans (P < .001 for both comparisons). In addition, on arterial phase scans, 386 of 483 lesions, as compared with 134 of 329 lesions on portal venous phase scans (P < .001), were judged to have an enhancement pattern suggestive of infection. The CT phases did not differ significantly in terms of the conspicuity of detected lesions. CONCLUSION: In patients suspected of having hepatic fungal infection, arterial phase CT depicts significantly more hepatic lesions than does CT performed during the other phases, and it reveals more lesions with enhancement patterns suggestive of infection. Arterial phase CT should be performed in addition to portal venous phase CT in patients suspected of having hepatic fungal infection.

Adolescent↗

PubMed2005

Nonmyeloablative stem cell transplantation for myelodysplastic syndrome or acute myeloid leukemia in patients 60 years or older.

We analyzed the outcomes of 24 consecutive patients aged >or=60 years with poor-prognosis myelodysplastic syndrome or acute myeloid leukemia undergoing transplantation with nonmyeloablative conditioning using fludarabine (125 mg/m2) and low-dose total body irradiation (2 Gy) followed by allogeneic peripheral blood stem cell grafts from HLA-identical sibling donors. Graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and mycophenolate mofetil. The median age of the patients was 64 years (range, 60-71 years). In addition to age, 88% of patients had 1 or more adverse biological features of the disease. With a median follow-up of 21 months, 12 patients are alive, 11 of whom are disease free. The probabilities of 2-year overall and progression-free survival were 52% and 44%, respectively. The cumulative probabilities of relapse and of acute and chronic GVHD were 27%, 45%, and 74%, respectively. Nonrelapse mortality at 100 days and 2 years was 8% and 25%, respectively. Of the 15 patients with extensive chronic GVHD, 1 patient relapsed. These data suggest that nonmyeloablative stem cell transplantation is a feasible treatment option in patients aged >or=60 years with poor-prognosis myelodysplastic syndrome or acute myeloid leukemia. The reasonable disease control with nonmyeloablative transplantation in this high-risk group of patients merits further investigation.

Acute Disease↗

PubMed2005

Decitabine in acute myeloid leukemia.

Standard induction chemotherapy, depending on patient tolerability, is at present the treatment of choice in patients with acute myeloid leukaemia (AML) under the age of 75 years. Since AML is a disease primarily of the elderly, a large proportion of patients do not receive this therapy; therefore, novel treatment modalities are warranted. In addition, younger patients with poor-risk cytogenetics often have unsatisfactory results with conventional chemotherapy, so novel treatment options would represent a significant advancement in the management of this disease. The encouraging results of low-dose schedules of demethylating agents in myelodysplastic syndromes (MDS) have stimulated interest in the use of these agents in patients with AML. The DNA methylator phenotype of MDS and AML offers a biological rationale for treating these patients with doses of demethylating agents which act by reverting the hypermethylated state to an unmethylated state rather than by "classic" cytotoxicity mechanisms typically associated with higher doses. However, many issues surrounding this therapy need to be unravelled, including the role of different target genes that may become demethylated, and the potential use of combination therapy of hypomethylating agents with other drugs. This review will provide a summary of epigenetic processes in AML and the role of the demethylating agent 5-aza-2'-deoxycytidine (decitabine; Dacogen, MGI Pharma, Inc, Bloomington, MN).

Acute Disease↗

PubMed2004

Cryptic insertion of MLL gene into 9p22 leads to MLL-MLLT3 (AF9) fusion in a case of acute myelogenous leukemia.

The formation of a leukemogenic fusion product in hematopoietic malignancies is commonly achieved by chromosomal translocation. Alternate and cytogenetically undetectable mechanisms of fusion transcript generation have been documented for BCR-AB1, AML1-ETO, PML-RARA, NPM/ALK, and MLL-MLLT2 (AF4). Here, we report the investigation of a cryptic rearrangement leading to MLL-MLLT3 transcript formation. Cytogenetic analysis of peripheral blood from a 50-year-old acute myeloid leukemia patient yielded a karyotype of 47,XY,+8,del(11)(q21q23) in all metaphase cells examined. Metaphase fluorescence in situ hybridization analysis using the MLL probe at 11q23 revealed that the 5' portion of the MLL gene was inserted into chromosome 9 at band p22, whereas the 3' region of the MLL gene remained on chromosome 11. Whole-chromosome paint analysis confirmed the cryptic transfer of chromosome 11 material to 9p22. With this information, the karyotype was reassigned as 47,XY,+8,der(9)ins(9;11)(p22;q23q23),del(11)(q21q23). RT-PCR was used to show that the cryptic rearrangement in this patient led to the fusion of the MLL and MLLT3 transcripts on the der(9). The presence of the MLL-MLLT3 transcript is consistent with the clinical findings in this patient.

Acute Disease↗

PubMed2004

Low-dose prophylactic platelet transfusions in recipients of an autologous peripheral blood progenitor cell transplant and patients with acute leukemia: a randomized controlled trial with a sequential Bayesian design.

BACKGROUND: Prophylactic platelet (PLT) transfusions are standard treatment for patients receiving high-dose chemotherapy, but the optimal dose is not known. A randomized controlled trial was undertaken to examine the effectiveness of low-dose PLT transfusions and to determine the need for further studies. STUDY DESIGN AND METHODS: Patients (n = 111) with acute leukemia or undergoing autologous peripheral blood progenitor cell (PBPC) transplantation were randomly assigned to receive low-dose (3 PLT units) or standard-dose (5 PLT units) prophylactic PLT transfusions and were monitored daily for bleeding. Using a sequential Bayesian design, the difference in major bleeding events was determined. RESULTS: The percentage of patients with major bleeding events was 10.7 percent (95% credible region, 5.1%-21.2%) in the low-dose PLT group and 7.3 percent (95% credible region, 2.9%-17.2%) in the standard-dose PLT group. The two additional events in the low-dose group occurred when the PLT count exceeded 100 x 10(9) per L. There is an 89 percent probability that the absolute increase in major bleeds is less than 10 percent with low-dose PLT transfusions. The number of minor bleeding events was higher in the standard-dose group. Patients receiving low-dose PLT transfusions received 25 percent fewer PLT units. There was an 89 percent probability that low-dose transfusions reduced PLT utilization in patients with acute leukemia and a 60 percent probability in patients undergoing PBPC transplantation. CONCLUSION: Low-dose PLT transfusions appear to be safe and effective and reduce PLT utilization. They should be further evaluated in clinical trials designed to evaluate equivalency.

Acute Disease↗

PubMed2002

Molecular cytogenetic characterization of non-Hodgkin lymphoma cell lines.

Spectral karyotyping (SKY) and comparative genomic hybridization (CGH) have greatly enhanced the resolution of cytogenetic analysis, enabling the identification of novel regions of rearrangement and amplification in tumor cells. Here we report the analysis of 10 malignant non-Hodgkin lymphoma (NHL) cell lines derived at the Ontario Cancer Institute (OCI), Toronto, designated as OCI-Ly1, OCI-Ly2, OCI-Ly3, OCI-LY4, OCI-Ly7, OCI-Ly8, OCI-Ly12, OCI-Ly13.2, OCI-Ly17, and OCI-Ly18, by G-banding, SKY, and CGH, and we present their comprehensive cytogenetic profiles. In contrast to the 52 breakpoints identified by G-banding, SKY identified 87 breakpoints, which clustered at 1q21, 7p15, 8p11, 13q21, 13q32, 14q32, 17q11, and 18q21. G-banding identified 10 translocations, including the previously described recurring translocations, t(8;14)(q24;q32) and t(14;18)(q32;q21). In contrast, SKY identified 60 translocations, including five that were recurring, t(8;14)(q24;q32), t(14;18)(q32;q21), t(4;7)(p12;q22), t(11;18)(q22;q21), and t(3;18)(q21;p11). SKY also identified the source of all the marker chromosomes. In addition, 10 chromosomes that were classified as normal by G-banding were found by SKY to be rearranged. CGH identified seven sites of high-level DNA amplification, 1q31-32, 2p12-16, 8q24, 11q23-25, 13q21-22, 13q32-34, and 18q21-23; of these, 1q31-32, 11q23-25, 13q21-22, and 13q32-34 have previously not been described as amplified in NHL. This comprehensive cytogenetic characterization of 10 NHL cell lines identified novel sites of rearrangement and amplification; it also enhances their value in experimental studies aimed at gene discovery and gene function.

Chromosome Aberrations↗

PubMed2002

Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging.

OBJECTIVE: The aim of this study was to assess the potential of store-operated Ca(2+) channel (SOC) antagonists as purging agents for leukemia cells. MATERIALS AND METHODS: Clonogenic, limiting dilution, and nuclear condensation assays were used to evaluate SOC antagonist efficacy. SOC activity and endoplasmic reticulum Ca(2+) content were measured by flow cytometry. Murine bone marrow transplantation was used to determine purging efficacy and effects on hemopoietic reconstitution. RESULTS: Econazole (Ec) and ketotifen (Ke) were variably effective against human and murine leukemia cell lines after 24 hours of incubation. However, a 2-hour serum and bovine serum albumin-free treatment protocol with Ec was found to maximize differential sensitivity between leukemic cells and normal hemopoietic progenitors. Primary acute myelogenous leukemia blast cell viability was reduced 4.2 to 5.1 logs by 2-hour Ec treatment as measured by limiting dilution. An inverse relationship between endoplasmic reticulum Ca(2+) content and Ke sensitivity in leukemia and untransformed cells was observed. Nuclear condensation, an index of apoptosis, which occurred after 24-hour treatments with either Ec or Ke, was not observed after 2-hour serum- and bovine serum albumin-free Ec exposures; however, condensed nuclei were observed after an additional 10-hour incubation in growth medium without drug. Using bone marrow deliberately contaminated with 1% P815 cells, we showed that highly effective in vitro purging can be accomplished using Ec with no adverse effects on bone marrow reconstitution in mice. CONCLUSIONS: These studies suggest that SOC antagonists have potential as purging agents for residual leukemia cells present in bone marrow in the context of high-dose chemotherapy and autologous transplantation for leukemia.

Animals↗