Myelogenous Leukemia Patients Research Articles

Population pharmacokinetic (PK) analysis of ABT-869 in solid tumors and acute myelogenous leukemia (AML) patients

3567 Background: ABT-869 is an orally bioavailable, potent and specific inhibitor of all vascular endothelial growth factor and platelet derived growth factor family receptor tyrosine kinases. The objectives of this analysis were to understand the population pharmacokinetics of ABT-869 and explore the effect of several demographic/disease state covariates influencing ABT-869 disposition. Methods: A population PK analysis of 181 patients (pts) enrolled in two phase 1 (multiple types of solid tumors and AML) and three phase 2 monotherapy studies (non-small cell lung cancer, hepatocellular carcinoma [HCC] and renal cell carcinoma) was conducted. Approximately 90% of pts received ABT-869 based on body-weight dosing while the remaining pts had flat dosing. Available plasma concentrations obtained after intensive and sparse pre-dose PK sampling were analyzed by population PK using the non linear mixed effects modeling (NONMEM) approach. Potential covariates including body weight, body surface area (BSA), age, sex, creatinine clearance (CrCL) and disease state (HCC vs. non-HCC pts) were tested. Results: The mean body weight of enrolled pts was 71 kg and 57% were Asian, 36% Caucasian and 7% other races. The ABT-869 plasma concentration time profile was well described by a one-compartment model with first order absorption and elimination process. Oral clearance (CL/F) was not affected by body weight (range 35–177 kg); however, apparent volume of distribution (V/F) increased by 6L per 0.1 mg/m2 increase in BSA. CrCL (39.9–290.3 ml/min) was not a significant covariate on V/F and CL/F suggesting renally impaired pts do not require a different dose/dosing regimen. HCC pts had ∼40% lower CL/F values than pts with other malignancies suggesting a lower dose would be appropriate for HCC (Child Pugh A and B) pts. Conclusions: Population PK analysis showed that ABT-869 PK can be well described by a one-compartment model with first order absorption and elimination. Race and impaired renal function does not appear to alter PK. HCC pts had lower CL/F value therefore a lower dose may be recommended in these patients. Implications of increased V/F with increasing body size and appropriate dosing strategy are undergoing further analysis. [Table: see text]

Out of frame peptides from BCR/ABL alternative splicing are immunogenic in HLA A2.1 transgenic mice

New, potentially tumor-specific antigens have been described in Bcr/Abl positive leukemias. Besides the main BCR/ABL hybrid fusion transcripts, a small number of transcripts derived from alternative splicing between BCR exons 1, 13, and 14 with ABL exons 4 and 5 have been identified. These variants are expressed in chronic myelogenous leukemia and acute lymphocytic leukemia patients. The transcriptional products were characterized at their C-terminus by a large amino acid portion derived from out of frame (OOF) reading of the ABL gene. This OOF peptide is expressed only in leukemic cells and has no homology with known human proteins. In order to study an in vivo model, three 39-amino acid peptides, each corresponding to a third of the whole human OOF peptide sequence, were tested for their capacity to elicit specific immune responses in HLA A2.1 transgenic mice. Peptides A and B, but not C, induced the production of specific antisera, while A and C induced the generation of specific cytotoxic T lymphocytes.

Down-Regulation of Notch1 Expression is Involved in HL-60 Cell Growth Inhibition Induced by 4-Hydroxynonenal, a Product of Lipid Peroxidation

The role of the Notch1 pathway has been well assessed in leukemia. Notch1 mutations are the most common ones in T acute lymphoblastic leukaemia patients which carry either oncogenic Notch1 forms or ineffective ubiquitin ligase implicated in Notch1 turnover. Abnormalities in the Notch1-Jagged1 system have been reported also in acute myelogenous leukaemia (AML) patients where Jagged1 is frequently over-expressed. Moreover, activating Notch1 mutations, as well, can occur in human AML and in leukemia cases with lineage infidelity. As a result, Notch1 signalling inhibition is an attractive goal in leukaemia therapy. Blockage/delay in cell differentiation and/or increase of proliferation are the main results of Notch1 signalling activation in several leukemic cell lines. Moreover, specific genes involved in cell growth control have been identified as Notch1 transcriptional targets, i.e. Cyclin D1 and c-Myc. 4-Hydroxynonenal (HNE), an aldehyde produced during lipid peroxidation, is involved in several pathological and physiological conditions, including inflammation; atherosclerosis; and neurodegenerative and chronic liver diseases. Moreover HNE has an antiproliferative/ differentiative effect in several cell lines, by affecting the expression of key genes, such as oncogenes (e.g. c-Myc, c-Myb), cyclins and telomerase. This prompted us to study the effect of HNE on Notch1 expression and its related signalling in HL-60 cells, a leukemic cell line widely used for differentiation studies. RT-PCR as well as Western blot assay showed Notch1down-regulation in HNE-treated HL-60 cells. The expression of Hes1, a Notch1 target gene, was concomitantly down-regulated by HNE treatment, reflecting Notch1 signalling inhibition. DAPT, an inhibitor of Notch activity, when added contemporary to HNE, further increased cell growth inhibition, without affecting apoptosis. Moreover, DAPT treatment reversed the HNE-induced differentiation. Overall these results suggest that Notch1 is a target for HNE and its down regulation is a key event in HNE-mediated inhibition of cell proliferation in the HL-60 cell line. By contrast our data do not support a role for Notch1 in HNE- induced differentiation or apoptosis.

Secondary Acute Myelogenous Leukemia in Patients with Retinoblastoma: Is Chemotherapy a Factor?

Secondary Acute Myelogenous Leukemia in Patients with Retinoblastoma: Is Chemotherapy a Factor?

Cost-Effectiveness Analysis in Canada of Dasatinib Versus Imatinib for the Treatment of Chronic Myelogenous Leukemia in Patients with Imatinib Resistance.

Objective: To estimate the cost-utility of dasatinib versus high-dose imatinib (HDI, imatinib 800mg/day) for chronic myelogenous leukemia (CML) in patients with resistance to imatinib at standard doses.Methods: We adapted to the Canadian setting a Markov model with a monthly cycle length that followed hypothetical patients until death. Cost effectiveness was evaluated during three phases of CML: chronic (86% of Ontario patients in 2006), accelerated (9%) and blast crisis (5%). Efficacy of dasatinib and HDI (cytogenetic and hematologic response) and adverse events (AEs) were taken from randomized trials. Canadian costs of medications, laboratory tests, professional fees, hospitalizations, and AEs were obtained from published sources. Societal utility scores were estimated from a Canadian time trade-off study. Analyses were performed from the perspective of the Ontario Ministry of Health. Costs (2006 CDN$) and outcomes (QALYs) were discounted at 5% annually after the first year. Univariate and probabilistic sensitivity analyses were carried out to quantify uncertainty for the incremental cost-effectiveness ratios (ICERs).Results: Dasatinib dominated HDI in chronic phase patients with additional QALYs (3.92 vs. 3.47) and a lower lifetime cost ($379,678 vs. $444,934). The ICERs of dasatinib vs. HDI in accelerated and blast phase were $88,098/QALY and $173,922/QALY respectively. The higher cost/QALY of dasatinib in the advanced phases is explained primarily by the increased predicted survival of dasatinib patients and therefore the longer duration of drug therapy for these patients. Parameters with the greatest influence on results were the time horizon, drug costs and utility values. The interpretation of results remained robust in univariate and probabilistic sensitivity analyses.Conclusion: From an economic perspective dasatinib is an attractive treatment choice for the majority of imatinib resistant CML patients and provides better value for money than HDI.

Sustained remissions and low rate of BCR‐ABL resistance mutations with imatinib treatment chronic myelogenous leukemia in patients treated in late chronic phase: A 5‐year follow up

The introduction of Imatinib (IM) has significantly altered the treatment for CML, although only limited follow-up results are available. As failure of Interferon-alpha had been associated with poor prognosis and results of IM-treatment in this patient group may allow earlier estimation of long-term benefits for early chronic phase patients. Therefore we prospectively analyzed the quality and duration of remissions and the rate of BCR-ABL resistance mutations occurring in patients treated with IM, if they were intolerant or refractory to interferon. Fifty-nine patients were included and median follow up is 4.75 years. Haematologic remission rate was 92% and 62% of patients achieved at least major cytogenetic remission. There were no major differences between patients failing or being refractory to IFN. Major molecular response was observed in 67% of patients evaluable. For the entire group, median PFS was 4.3 years and OS was 82% at 4.75 years. Haematologic and complete cytogenetic remissions, but not molecular responses were correlated with improved PFS and OS. In patients with progression or inadequate response, BCR-ABL kinase domain mutations were detected in 3/28 patients. IM resulted in prolonged remission rates in a substantial proportion after IFN-failure/intolerance, with no differences in these patient groups. Quality of hematologic and cytogenetic, but not molecular response was associated with duration of remissions and survival. A low rate of resistance-mutations was detected, suggesting that alternative mechanism may play an important role in disease progression.

Secondary Acute Myelogenous Leukemia in Patients with Retinoblastoma: Is Chemotherapy a Factor?

To describe a series of patients with secondary acute myelogenous leukemia (sAML) and retinoblastoma (RB). Retrospective observational cases series. Ocular and pediatric oncologists at referral centers in Europe and the Americas and the RB databases at the National Institutes of Health and the Ophthalmic Oncology Service at Memorial Sloan-Kettering Cancer Center. Physician survey, retrospective database review, and literature search. History of RB and development of sAML, management of RB (surgery, radiotherapy, chemotherapy), age at diagnosis of RB and leukemia, French-American-British (FAB) subtype, and current status of patient (alive or dead). Fifteen patients with sAML were identified; 13 occurred in childhood. Mean latent period from RB to AML diagnosis was 9.8 years (median, 42 months). Nine cases were of the M2 or M5 FAB subtypes. Twelve patients (79 %) had received chemotherapy with a topoisomerase II inhibitor, 8 (43%) had received chemotherapy with an epipodophyllotoxin. Ten children died of their leukemia. Acute myelogenous leukemia is a rare secondary malignancy among retinoblastoma patients, many of whom were treated with primary or adjuvant chemotherapy. Additional studies are needed to assess potential risk factors contributing to sAML development in this cohort.

Modulation of P-glycoprotein and proinflammatory cytokines with HMG-CoA reductase inhibitor simvastatin (SV) in chronic myelogenous leukemia (CML) patients with imatinib mesylate (IM) resistance

17528 Background: Primarily monocyte-derived cytokines and soluble interleukin-2 receptor (sIL-2R) form a group of proinflammatory cytokines and mononuclear cell activaton respectively which are significantly elevated in patients with CML-chronic phase (CML-CP) or CML-blastic crisis (CML-BC).C-reactive protein (CRP) correlates with IL-6. IM is a tyrosine kinase inhibitor that specifically acts intracellularly by blocking the ATP-binding site of the kinase domain of Bcr/abl in CML. Acquired drug resistance in CML-CP patients treated with IM may occur by upregulation of P-glycoprotein (P-gp) drug efflux with P-gp+ leukemic cells and P-gp dependent decline of intracellular IM levels with retained phosphorylation Bcr/abl pattern and loss of IM effect on apoptosis and cellular proliferation. Modulation of P-gp with HMG-CoA reductase inhibitor (SV), a potent inhibitor of P-gp transport, readily overcame IM resistance. Methods: We present two cases of CML: CML-CP and CML-BC with evidence of IM resistance. After one year of IM therapy an 86 yo female with CML-CP stage II, developed reactivation of Bcr/abl signaling due to excretion of IM from the cell transmembrane transporters consistent with increased proinflammatory cytokines, sIL-2R, CRP, acute coronary syndrome but normal neopterin.Results: Modulation of proinflammatory cytokines with SV, IM dose adjustments, serial measurements of Bcr-abl/abl ratio didnot exceed 0.02% on three occasions or 0.05% on two occasions after changing from 0.001% to 0.003% and now after two years of IM plus SV, real-time Bcr-abl/abl ratio is 0.000 (a 5 log reduction).sIL-2R levels normalized from 31,709.55 pg/ml to 5,345.4 pg/ml (Normal 1770–9753 pg/ml). CRP decreased to 6.23 mg/dl from 16 mg/dl. Conclusions: IM an intracellular drug demonstrating high activity against Bcr/abl gene positive CML-CP or CML-BC patients may develop resistance and loss of cellular proliferation via upregulated P-gp-mediated drug efflux mechanism. Modulation of P-gp by HMG-CoA reductase (statins) inhibitor simvastatin, a potent inhibitor of P-gp, may allow intracellular levels of IM to restore its cytotoxicity and overcome resistance mechanism. No significant financial relationships to disclose.

First and second line imatinib treatment in chronic myelogenous leukemia patients expressing rare e1a2 or e19a2 BCR–ABL transcripts

During the formation of the Philadelphia (Ph) chromosome, in the majority of chronic myelogenous leukemia (CML) patients, the chromosome 22 breakpoint is located in the major breakpoint cluster region of the BCR gene (M-bcr). Minor and micro breakpoint cluster regions (m-bcr with e1a2 transcript and micro-bcr with e19a2 transcript) are rarely affected and have been suggested to be associated with peculiar CML phenotypes. Despite the different clinical characteristics, it is currently not established, whether different therapeutic options are preferably recommended for the treatment of e1a2 or e19a2 CML. Here we report two patients with e1a2 and one patient with e19a2 translocations, treated with different approaches including imatinib. First and second line imatinib treatments induced haematologic response in all of the three patients, and major cytogenetic response in one patient with e1a2, as well as in the patient with e19a2 CML. However, relapse occurred in the patient with e19a2 CML, possibly caused by the presence of additional chromosomal abnormalities such as an extra Ph chromosome, and loss of chromosome Y. Stem cell transplantation (SCT) therapy caused complete haematologic response with molecular remission; however, the patient died of infectious complication. We conclude that in patients with rare BCR-ABL variants, the effectiveness of imatininb treatment may be influenced by the CML stage besides the actual molecular type of the rare transcript. However, this conclusion cannot be generalized to larger patient groups with rare BCR-ABL variants for which further studies may be needed.

Monoclonal antibody-based therapeutics for leukemia

Monoclonal antibodies (mAbs) represent a unique class of therapeutics because of their exquisite antigen specificity and effector mechanisms. Clinically, antibody-based therapy has increasingly become an established modality for the treatment of cancer. Among the nine mAbs and immunoconjugates approved by the FDA for therapeutic indications in oncological diseases, five are for use in hematological malignancies. Alemtuzumab is approved for B cell chronic lymphocytic leukemia; rituximab, an anti-CD20 antibody approved for the treatment of B cell non-Hodgkin’s lymphoma, is being tested in patients with chronic lymphocytic leukemia; and gemtuzumab ozogamicin is marketed for relapsed acute myelogenous leukemia in patients of > 60 years of age. A number of other mAbs are in different stages of clinical development for antileukemia therapy. This article discusses the general aspects and mechanisms of action of therapeutic mAbs, and highlights the development of antibody-based therapies in leukemia.

The investigation of JAK2 V617F point mutation in myeloproliferative disorders by allele-specific polymerase chain reaction in combination with sequence analysis

To study the JAK2 V617F point mutation in myeloproliferative disorders (MPD) and explore the clinical significance. We used Allele-specific polymerase chain reaction (AS-PCR) in combination with sequence analysis to detect the mutation in genomic DNA of peripheral blood mononuclear cells from 20 chronic myelogenous leukemia (CML) patients, 23 polycythaemia vera (PV), 40 essential thrombocythaemia (ET), 8 idiopathic myelofibrosis (IMF), 3 hypereosinophilic syndrome (HES). JAK2 V617F was found in 38 (51.4%) of 74 BCR/ABL-negative MPD including 16 PV, 18 ET, 3 IMF and 1 HES patients. All positive samples and 10 negative samples identified by AS-PCR were confirmed by sequence analysis. Mutation-positive patients with ET had significantly increased hemoglobin, hematocrit, and neutrophil proportion than those without the mutation. JAK2 V617F mutation is the key molecular genetics feature of BCR/ABL-negative MPD. Detection of JAK2 V617F mutation will bring about a major impact to the diagnosis, classification and treatment of MPD.

The Association between Cytogenetic Abnormalities and Clinical Outcomes Based on Prognostic Factors of the Children Cancer Group (CCG) in Pediatric Patients with Acute Leukemia: Two Institutional Retrospective Studies

Results: The incidence of cytogenetic abnormalities was 51 (51.5%) in 99 patients, and 27 (39.7%) in acute lymphoblastic leukemia (ALL) patients and 24 (77.4%) in acute myelogenous leukemia (AML) patients. The most frequent cytogenetic abnormality was hyperdiploidy and t(8:21) in the ALL and AML patients, respectively. The overall survival rate (OS)/disease free survival rate (DFS) of the ALL patients was 74.0%/73.9%. The OS/DFS of the standard risk group (88.8%/85.2%) was significantly higher than that of the high-risk group (49.4%/39.3%) in the ALL patients (P=0.0005/P<0.0001). There was no significant difference in the survival rates according to the type of cytogenetic abnormalities among the ALL patients for the standard/high risk groups, based on the CCG prognostic factors. The OS/DFS of the AML patients were 43.4% and 41.7%, respectively, without significant differences of the survival rates according to the type of chromosomal abnormalities. Conclusion: There were significant differences of OS/DFS based on the risk groups in ALL patients when evaluated with the CCG prognostic factors (standard/high) and chromosomal abnormalities (good/ poor), respectively. However, there was no significant correlation between type of cytogenetic abnor- malities and clinical outcomes based on the CCG prognostic factors in children with ALL as well as with AML.

Reticulated platelets are increased in chronic myeloproliferative disorders, pure erythrocytosis, reactive thrombocytosis and prior to hematopoietic reconstitution after intensive chemotherapy

Reticulated platelets with a high RNA content represent the most recently released platelets and are regarded to reflect thrombopoiesis. In the present study we used flow cytometric analysis to determine the percentage of reticulated platelets in peripheral blood for patients with chronic myeloproliferative disorders (polycythemia vera, essential thrombocytosis) and acute myelogenous leukemia (AML) patients with severe chemotherapy-induced thrombocytopenia. Patients with essential thrombocytosis and polycythemia vera showed increased levels of reticulated platelets compared with healthy controls, and these levels persisted after normalization of the platelet count by hydroxyurea or interferon-α treatment. Patients with reactive thrombocytosis or thrombocytopenia with increased platelet turnover often had higher levels of circulating reticulated platelets than patients with myeloproliferative disorders. Furthermore, AML patients with severe chemotherapy-induced cytopenia showed low levels that started to increase 1–9 days prior to hematopoietic reconstitution. To summarize and conclude: (i) circulating reticulated platelets are increased in patients with chronic myeloproliferative disorders, reactive thrombocytosis and thrombocytopenia due to increased platelet turnover; (ii) patients with pure erythrocytosis often have additional abnormalities in the thrombopoiesis; and (iii) the levels of reticulated platelets seem to predict hematopoietic reconstitution for patients receiving intensive AML therapy.

Possible association between Budd‐Chiari syndrome and gemtuzumab ozogamicin treatment in a patient with refractory acute myelogenous leukemia

Gemtuzumab ozogamicin (GO; CMA-676; Mylotarg) is a chemotherapeutic agent approved for the treatment of CD33-positive acute myelogenous leukemia in patients of age 60 years or older after first relapse. Hepatic veno-occlusive disease has been reported to develop as a late complication of gemtuzumab ozogamicin treatment. A patient who developed Budd-Chiari Syndrome with hepatic vein thrombosis following treatment with GO is presented. This complication has not been previously reported, and it deserves to be considered as a possible adverse effect of gemtuzumab ozogamicin.

Coexistence of different clonal populations harboring the b3a2 (p210) and e1a2 (p190) BCR-ABL1 fusion transcripts in chronic myelogenous leukemia resistant to imatinib

In this study, we report the case of a Philadelphia (Ph) positive chronic myelogenous leukemia (CML) patient with the presence of p190 and p210 BCR-ABL1 mRNA fusion transcripts derived from e1a2 and b3a2 BCR-ABL1 genomic rearrangements, respectively. The presence of e1a2 BCR-ABL1 genomic rearrangement was seen in 2 different clones, one with the rearrangement and another one with the rearrangement and deletion of the BCR gene of the non-rearranged chromosome 22. After treatment with imatinib, the p210 transcript could not be detected, whereas p190 was still present 6 months after initiation of imatinib therapy and progression to blast phase. The absence of p210 transcript post treatment indicates that the clone with b3a2 responded to imatinib and that the observed resistance was associated to cells harboring the e1a2 genomic rearrangement. Despite resistance of this patient to imatinib, no evidence of mutations in the kinase domain of ABL1 was found. Loss of normal BCR in one cell clone may contribute to the resistance to imatinib due to the lack of BCR mediated inhibition of BCR-ABL1.

CD8+ T-cell-depleted, matched unrelated donor, allogeneic bone marrow transplantation for advanced AML using busulfan-based preparative regimens

The role of T-cell depletion (TCD) to prevent graft-versus-host disease (GVHD) after matched unrelated donor allogeneic bone marrow transplant (MUD BMT) remains undefined. Most studies employ total body irradiation and pan TCD. Between March 1993 and June 2002, we treated 33 relapsed acute myelogenous leukemia (AML) patients with busulfan-based preparative regimens and selective TCD. The preparative regimen consisted of busulfan 14 mg/kg, cyclophosphamide 120 mg/kg and VP-16 50 mg/kg in all but one patient who only received busulfan and cyclophosphamide. Donor marrow was depleted of CD8+ T cells by immunomagnetic bead separation. The patients were also treated with cyclosporine and methylprednisolone or FK-506 and mini-dose methotrexate. Four (15%) of 33 patients developed graft failure or rejection. However, three of these patients were serologically mismatched at HLA-Cw. Although 67% of evaluable patients developed acute GVHD, severe grade III-IV acute GVHD only developed in 19%. The severity of acute GVHD correlated with the degree of CD8+ TCD. Median relapse-free survival was 5 months among 20 patients treated with active AML, and 28 months among 13 patients treated in complete remission. Our results confirm that MUD BMT with CD8+ TCD for AML is a potentially curative treatment option.

Therapy-related myelodysplastic syndrome–acute myelogenous leukemia in patients treated for acute promyelocytic leukemia: an emerging problem

AbstractThe use of all-trans retinoic acid (ATRA) in combination with chemotherapy has markedly improved the prognosis for patients with acute promyelocytic leukemia (APL); the higher complete remission (CR) and survival rates now reported in this disease almost approach those obtained for other highly curable hematologic malignancies. Of 77 patients with APL who were consecutively treated at a single institution and who achieved CR after induction and consolidation therapy, 5 (6.5%) acquired therapy-related myelodysplasia (tMDS), acute myelogenous leukemia (AML), or both (tMDS–AML). Of these, 3 of 46 (6.5%) patients received front-line chemotherapy with or without ATRA and acquired tMDS–AML while in first remission of APL. Two underwent repeated chemotherapy cycles with ATRA because of APL relapse and acquired tMDS–AML while in the second or third remission of APL. In 2 patients, clinical and biologic characteristics of tMDS–AML were as expected for postalkylating forms (long latency, MDS phase preceding AML, karyotypic aberrations involving chromosomes 5 or 7), even though one of them had not previously received alkylating drugs. Three of the 5 patients died shortly after tMDS–AML diagnosis, one is alive with tMDS, and one is alive and in CR after allogeneic bone marrow transplantation. The occurrence of tMDS–AML after successful therapy for APL is an emerging problem. The availability of prognostic score systems at initial diagnosis and monitoring of residual disease by polymerase chain reaction might allow better tailoring of treatment intensity in APL to spare unnecessary toxicity and to minimize the risk for tMDS–AML in patients who are presumably cured.

Secondary acute myelogenous leukemia in patients previously treated for childhood renal tumors: a report from the National Wilms Tumor Study Group.

This review characterized cases of secondary acute myelogenous leukemia (AML) occurring after treatment of renal neoplasms on protocols of the National Wilms Tumor Study Group (NWTSG) between October 1969 and December 1991. The NWTSG database was reviewed for cases of secondary AML and for WT1 status of the affected patients. Referring institutions were contacted by a confidential letter requesting pathology reports, results of immunophenotyping, cytogenetic, and molecular analyses, and details concerning treatment of AML. Of the 5,278 patients treated during the study period, 43 had second malignant neoplasms, and 7 of these 43 had AML. At the time of diagnosis of Wilms tumor, the median age of the seven patients (4 boys) was 3.2 years. Five of the seven renal neoplasms had favorable histologic characteristics. The most common French-American-British morphology was M5. One patient had bilateral tumors, and two were treated for recurrent Wilms tumor. All patients received chemotherapy regimens that included doxorubicin (6) or etoposide (1), and six were treated with infradiaphragmatic irradiation. The median latency period from initial diagnosis of the renal neoplasm to development of secondary AML was 3 years (range, 1.2-4 yrs). One patient had the translocation t(9:11)(p22;q23); WT1 status was not noted for any of the seven patients. The development of secondary AML in this subset of patients after treatment of renal neoplasms may reflect the interaction of the effects of treatment and possible genetic predisposition toward cancer.

Early detection of relapse by hypermetaphase fluorescence in situ hybridization after allogeneic bone marrow transplantation for chronic myeloid leukemia.

Standard G-band cytogenetic analysis (CG) provides information on approximately 25 metaphases for monitoring the presence of Philadelphia chromosome positive (Ph+) cells in chronic myelogenous leukemia (CML) patients, making the detection of a low frequency of Ph+ cells problematic. The purpose of this study was to improve the detection of a low frequency of Ph+ cells. We combined fluorescence in situ hybridization (FISH) with long-term colcemid exposure, capturing several hundred metaphases in bone marrow cultures (hypermetaphase FISH [HMF]). Using probes that identify Ph+ cells, HMF was compared with CG analysis in the follow-up evaluations of 51 patients with CML at various time points after allogeneic bone marrow transplant (BMT). Thirty-five patients never showed the presence of Ph+ cells by either method. In four patients, high frequencies of Ph+ cells were detected by both methods. In the remaining 12 patients, Ph+ cells were detected by HMF at time points after BMT when they were not detected by CG. In seven of the 12 patients, low but statistically significant frequencies of Ph+ cells (0.37% to 5.20%) were detected 3 months or later after BMT, and when no intervention was initiated, all seven patients later relapsed. Based on those data, an eighth patient with mixed chimerism and a similar HMF-detected Ph+ frequency (1.8% at 27 months after BMT) was reinfused with donor lymphocytes and achieved remission with 0% Ph+ cells studied by HMF (up to 50 months after BMT). Ph+ cells detected by HMF but not by CG less than 3 months after BMT disappeared on later examination in two of four patients. After detection of Ph+ cells by HMF only, the median time to cytogenetic progression (detection of Ph+ cells by CG) was 101 days. The results demonstrate the ability of HMF to detect low but clinically relevant levels of leukemic cells not detected by CG in transplant patients. The data indicate that HMF can detect low levels of Ph+ cells before standard cytogenetics at a time that may be useful in monitoring disease status and planning clinical interventions.

Changes in the expression of voltage-gated K + currents during development of human megakaryocytic cells

We distinguished four distinct groups of megakaryocytic cells on the basis of their voltage-gated membrane currents. One group of 32 cells (15%), exhibited an inward rectifying current and had a diameter of 12±3.5 μm (mean±S.D.). A large group of 85 cells (39%) exhibited only a `leakage-like' current and had a diameter of 15.8±3.7 μm. The other two groups of cells exhibited voltage-gated outward currents. One group consisted of 43 `I-type' cells (19%), with a diameter of 22.3±3.4 μm, for which the maximal outward current occurred for a voltage step from −60 to either 0 or +20 mV. For the last group of 60 `M-type' cells (27%), which had a diameter of 26.7±2.9 μm, the maximal outward current occurred for a voltage step from −60 to +80 mV, the largest voltage step used. The currents recorded in this study, from megakaryocytes having `leakage-like' currents and `I-type' currents, were indistinguishable from the voltage-gated currents of the megakaryocytes from myelogenous leukemia patients, in which voltage-gated currents were suppressed (Kapural, L., O'Rourke, F., Feinstein, M.B. and Fein, A. (1995) Blood 86, 1043), suggesting that the megakaryocytes from the myelogenous leukemia patients are a dedifferentiated or less mature form of megakaryocyte.