Béla Telek

Calcein assay for multidrug resistance reliably predicts therapy response and survival rate in acute myeloid leukaemia

In this study, we evaluated the suitability of the calcein assay as a routine clinical laboratory method for the identification of multidrug‐resistant phenotype in acute leukaemia. This study presents the results of the calcein tests obtained in two large haematological centres in Hungary. Assays were performed with blast cells of 93 de novo acute leukaemia patients, including 65 patients with acute myeloid leukaemia (AML). Results were expressed as multidrug resistance activity factor (MAF) values. AML patients were divided into responders and non‐responders and MAF values were calculated for each group. In both centres, responder patients displayed significantly lower MAF values than non‐responders (P = 0·0045 and P = 0·0454). Cut‐off values were established between the MAFR + SEM and MAFNR − SEM values. On the basis of these cut‐off levels, multidrug resistance (MDR) negativity showed a 72% predictive value for the response to chemotherapy, whereas MDR positivity was found to have an average predictive value of 69% for therapy failure. MDR activity was a prognostic factor for survival rate and the test was suitable for detecting patients at relapse. The calcein assay can be used as a quantitative, standardized, inexpensive screening test in a routine clinical laboratory setting. The assay detects both P‐glycoprotein and multidrug resistance‐associated protein activities, and identifies AML patients with unfavourable therapy responses.

Alterations of P53 and RB genes and the evolution of the accelerated phase of chronic myeloid leukemia.

Using the single-strand conformation polymorphism and heteroduplex analyses, the P53 and RB genes were analyzed in cell samples from twenty-eight patients with chronic myeloid leukemia (CML) both at diagnosis and at the onset of accelerated phase (AP) of the disease. No alterations of the P53 or RB genes were found in any of the chronic phase (CP) samples. Structural abnormalities of the P53 gene were observed in ten of twenty-eight AP samples within exons 4, 5, 7 and 9. Of the ten cases of AP disease with altered P53 genes, five patients also suffered from the deletion of the other allele. Alterations of the RB gene could be detected in six AP samples, and aberrant band patterns were found in the analysis of exons 2, 3, 4, 6, 7, 13, 14, 17, 21 and 26. Among the six AP samples with structural abnormalities of the RB gene, two showed the loss of the other allele. It is of note that alterations of both P53 and RB genes were observed in two AP samples. Our data strongly suggest that abnormalities of the P53 and RB genes and acceleration of CML are linked events in some cases of AP.

Reduced In Vitro Clot Lysis and Release of More Active Platelet PAI-1 in Polycythemia Vera and Essential Thrombocythemia

Because platelets interact with fibrinolysis in a complex manner, it can be expected that with abnormal platelet numbers and quality this interference can be even more profound. The aim of this work was to study the lysis-resistance of platelet-rich clots in diseases with high platelet counts: polycythemia vera (PV), essential thrombocythemia (ET) and to make comparison with polyglobulia (PG). Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were analyzed by an in vitro clot lysis test. Plasminogen activator inhibitor-1 (PAI-1) activity was measured in plasma and in the supernatants of the washed and gel-filtered platelets after activation by thrombin. The lysis showed decreased speed of PPP-clots in PV and ET. This phenomenon was even more marked in PRP-clots from PV and ET, but further increased lysis resistance after retraction was not observed in PV and ET, most likely due to abnormal platelet functions. Our results suggest that the fibrinolytic activity is reduced in PV and ET, and may play a role both in the increased aptitude for venous thrombosis and in the arterial complications. These are partly caused by higher plasmatic PAI-1 activity as well as by more active platelet PAI-1. The PAI-1 activity was significantly higher in the supernatants of the washed and gel-filtered platelets of PV after activation by thrombin compared with controls. Other factors might have influenced the reduced fibrinolysis.

Frequent methylation of p16INK4A and p14ARF genes implicated in the evolution of chronic myeloid leukaemia from its chronic to accelerated phase.

The frequency and mechanism of p16(INK4A) and p14(ARF) gene alterations were studied in cell samples from 30 patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukaemia (CML), both at diagnosis and at the onset of the accelerated phase (AP) of the disease. No alterations in the p16(INK4A) or p14(ARF) genes were found in any of the chronic phase (CP) samples. DNA sequencing analyses detected p16(INK4A) or p14(ARF) mutations in 17 AP samples. All mutations were heterozygous without loss of the other allele. Aberrant methylation of the p16(INK4A) or p14(ARF) promoters was found in 14 of 30 AP samples. The most common situation was the simultaneous methylation of both promoters. Our data indicate that p16(INK4A) and p14(ARF) are primary targets for inactivation by promoter methylation in the acceleration of CML. Transcriptional silencing of the p16(INK4A) and p14(ARF) genes may be important in the conversion of CML from the CP to the AP.

Changes in Oncogene Expression Implicated in Evolution of Chronic Granulocytic Leukemia from its Chronic Phase to Acceleration

Expression of nine oncogenes was investigated in cell samples from fifteen patients with Philadelphia chromosome (Ph)-positive chronic granulocytic leukemia (CGL) both at diagnosis and at the onset of accelerated phase (AP) of the disease. The bcr-abl fusion gene, the H-ras gene and the c-myb gene were universally expressed. In comparison with the chronic phase (CP) of the disease, an increase in the levels of bcr-abl-, c-myb- and H-ras-related transcripts was found in three, two and three AP samples, respectively. Elevation of the bcr-abl-related message was associated with duplication of the Ph chromosome and amplification of the bcr-abl fusion gene in one AP sample. No CP samples were positive for c-myc or c-sis expression. On the contrary, c-myc and c-sis were expressed in three and four AP samples, respectively. The presence of c-myc-related transcript was associated with trisomy 8 with or without amplification of the c-myc oncogene in leukemia cells of two patients with CGL in AP. No changes of oncogene expression were found in four AP samples. However, we observed deletions of chromosome 13 and 17 or i(17q) in three of them, suggesting that tumor suppressor gene alterations may also be responsible for the development of AP of CGL. Our data indicate that heterogeneous alterations in oncogenes and tumor suppressor genes accompany the evolution of CGL-CP to the AP of the disease.

Successful control of massive coumarol-induced acute upper gastrointestinal bleeding and correction of prothrombin time by recombinant active factor VII (Eptacog-alpha, NovoSeven) in a patient with a prosthetic aortic valve and two malignancies (chronic lymphoid leukaemia and lung cancer).

Severe, life-threatening acute upper gastrointestinal bleeding may occasionally occur in patients receiving coumarol prophylaxis for prosthetic heart valves. These patients are exposed to two potential, serious risks: bleeding due to the severe blood loss induced by excessive anticoagulant effect or as a consequence of the cessation of anticoagulation subsequent thrombotic occlusion of the valve and loss of patency. Herein a short case report is presented. The elderly male patient had a prosthetic valve in the aortic position and also suffered from two malignant diseases: chronic lymphocytic leukaemia and a more recently developed lung cancer with metastatic spread into both lungs. The patient had a major gastrointestinal bleed, leading to a sudden fall of haematocrit (0.09), and to a collapse of peripheral circulation due to too excessive a coumarol effect (International Normalized Ratio > 8). An acute left ventricular failure developed during the early period of the emergency blood transfusion, so the correction of prothrombin time by fresh-frozen plasma (due to the large volume requirement) was not feasible. The patient received 50 microg/kg intravenous bolus of NovoSeven (recombinant active factor VII) in an almost desperate situation. The International Normalized Ratio changed to 2.1 in 30 min; bleeding had stopped immediately. There was neither evidence of disseminated intravascular coagulation (in spite of the age and underlying diseases) nor loss of valve patency or infective endocarditis during follow-up. This modest report may call attention to the potential use of recombinant active factor VII in the coumarol-induced severe bleeding episodes of prosthetic heart valve patients.

MEGAKARYOCYTE MARKERS IN MYELOPROLIFERATIVE DISORDERS

Identification of megakaryocyte precursors with immunohistochemical methods in bone marrow trephine biopsy specimens (embedded in a plastic resin, Immuno-Bed) was performed from patients with blastic phase of chronic granulocytic leukaemia (five cases), from chronic megakaryocytic-granulocytic myelosis (four cases) and from acute megakaryoblastic leukaemia (11 cases). In megakaryoblasts of bone marrow biopsies immunohistochemical reactions using the ABC method and monoclonal antibodies against von Willebrand antigen and GpIIb/IIIa (CD41) were visible in various percentages depending on the maturations degree of megakaryocyte precursors. The number of circulating blast cells determined by flow cytophotometry was nearly similar to those of observed in biopsies. The greatest bone marrow reticulin content could be detected in acute megakaryoblastic leukaemia cases. Despite the different clinicopathological entities, the presence of the same phenotype (megakaryoblasts) was associated with a short survival in these haematological malignancies (in CGL MKB phase 4.0, in CMGM MKB phase 4.2, and in AML M7 5.8 months, respectively).

Successful alemtuzumab treatment of a patient with atypical hairy cell leukaemia variant

Although hairy cell leukaemia and hairy cell leukaemia variant are characterized by much alike clinical features, these two diseases are disparate in nature and treatment. While hairy cell leukaemia responds quite well to 2-chlorodeoxyadenosine (cladribine) treatment, hairy cell leukaemia variant has much worse response rate and has no effective treatment option yet. With other treatment modalities, including monoclonal antibody treatment, we have less experience. Alemtuzumab (Campath-1H, MabCampath) treatment has been reported in a case with hairy cell leukaemia in relaps while there is no data with alemtuzumab therapy in the treatment of hairy cell leukaemia variant. The authors present their case of a 58 year-old male who has been diagnosed with hairy cell leukaemia variant upon clinical findings and lymphocyte phenotyping. Alemtuzumab treatment was started (3 x 30 mg/week s.c. for 12 weeks). After 8 weeks of treatment haematologic remission was achieved; flow cytometry has revealed only 1.5% malignant cells. Alemtuzumab treatment can be favourable in those cases of hairy cell leukaemia and hairy cell leukaemia variant which is dominated mainly by bone marrow infiltration and present no lymphadenomegaly or splenomegaly. In our case the p53 mutation had no influence on the outcome of alemtuzumab treatment.

Pros and Cons on How to Measure Multidrug Resistance in Leukemias

Drug resistance is one of the most significant challenges in the treatment of various types of malignancies, however most of the experimental and clinical data in multidrug resistance (MDR) has been obtained in leukemias. MDR is the term that describes innate or acquired resistance of tumor cells to a wide range of anticancer drugs. As its presence determines treatment outcome in several forms of leukemias, it is imperative that clinical laboratories provide the most useful data on its expression. Here, a brief review is provided on the pathomechanism and diagnostics of MDR. From the diagnostic point of view it is fortunate that MDR proteins display similar effluxing activity towards many dissimilar agents some of which can be used in fluorescent assays. These tests mimic the real clinical problem i.e. the extrusion activity of MDR proteins towards xenobiotics. Thus, we believe that functional assays when carried out in a standardized way and particularly combined with labeling for various surface markers can be recommended as a front-line test in MDR measurement.

Behçet's disease in a patient with myelodysplastic syndrome.

A 75-year-old man presented with painful oral and groin ulcers. The lack of any infections and the location of the ulcers suggested Behçets disease. Subsequently, pancytopenia developed and bone marrow examination revealed myelodysplastic syndrome. Cytogenetic examination revealed 7q- and 20q- but not 8+. Immunosuppressive therapy with cyclosporine and corticosteroid resulted in a dramatic improvement in both clinical signs and hematologic abnormalities.