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.

[Molecular biology examination in chronic lymphocytic leukemia]. / Molekuláris biológiai vizsgálatok krónikus lymphoid leukaemiában.

Chronic lymphocytic leukaemia (CLL) is the most common adult leukaemia characterised by the accumulation of monoclonal CD5 + B-lymphocytes. The pathogenesis and the biology of CLL is complex and many details are still unknown. Several molecular biological methods have been used in the investigation of CLL, among them the study of apoptosis appears to be one of the most important. Initial experiences obtained by the spontaneous and fludarabine induced apoptosis, multidrug resistance (MDR)-test and fluorescent in situ hybridization (FISH) are reported by the authors. Apoptosis of CLL cells could be induced by fludarabine, while more studies should be performed to determine the exact role of MDR-test and FISH.

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.

[Fluorescence in situ hybridization in the diagnosis and follow-up of chronic myelogenous leukemia]. / Fluoreszcencia in situ hibridizációval szerzett tapasztalataink a krónikus myeloid leukaemia diagnosztikájában és követésében.

Chronic myelogenous leukaemia is a clonal myeloproliferative stem cell disease. Its cytogenetical hallmark is the Philadelphia chromosome (Ph) or the BCR/ABL fusion gene. Their identification is important both in the diagnosis and the follow-up of the disease. In our department we have investigated the BCR/ABL gene arrangement in 21 patients with fluorescence in situ hybridization. The aim of the analysis in freshly suspected patients without any previous therapy was to confirm diagnosis and mapping the ratio of Philadelphia positive cells. In contrast to the 95-100% Ph-positivity of mononuclear cells by classical cytogenetical examinations we found BCR/ABL gene arrangement only in various but always lower proportions. Therefore the latter examination gives a better representation of residual normal hemopoesis. Out of 9 patients who had received interferon treatment for at least 6 months, 4 gave a major, 4 a minor cytogenetical answer and in 1 case there was no cytogenetical response. Seven patients reached a complete and 2 a partial hematological remission. Among 5 other patients receiving interferon treatment, in 2 cases with double Ph-positivity we found a rapid progression. The data of 3 patients had to be excluded from the evaluation due to the so far short following time.

Use of a platelet filter test in patients with thrombocytosis.

In the differential diagnosis of primary and secondary thrombocytosis platelet function tests may play an important role. We examined the applicability of a platelet filter test (shear-dependent platelet aggregation) as a tool, to differentiate primary thrombocytosis (cases with myeloproliferative disorders) from secondary (reactive) thrombocytosis. The test was carried out in 53 patients suffering from myeloproliferative disorders associated with primary thrombocytosis and in 21 patients with other diseases complicated by secondary thrombocytosis. Using citrate as anticoagulant, the sensitivity of the O'Brien's test proved to be 77.1%, and its specificity 94.4%. Using heparin as anticoagulant the sensitivity and specificity of the test were found to be also reliably high. Based on these studies we suggest the use of the O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated platelet count. In the case of normal results, the causes of reactive thrombocytosis should be clarified first, while with abnormal results, haematological examination of the patients should be performed.

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.

[Hairy cell leukemia: authors' own experience (1977-1998)]. / A "hajas" sejtes leukaemiáról: saját tapasztalatok (1977-1998).

Hairy cell leukaemia (HCL) is a rare, clinically and haematologically well characterised entity. The prognosis of patients with hairy cell leukaemia has significantly improved due to the new therapeutic approaches. Development of diagnostic and therapeutic methods, together with the analysis of their own hairy cell leukaemia patients, is reviewed by the authors. Between 1977 and 1998 twenty five patients (16 male, 9 female) were treated. The malignant cells were usually analysed by morphological and cytochemical methods and recently flow cytometric analysis could be performed in eight patients. Splenectomy with lethal outcome in six patients was performed in 21 cases. Approximately one third of patients received interferon, while 2-chlorodeoxyadenosine was given only to three patients. Favourable experiences obtained by splenectomy and efficacy of interferon treatment are emphasised, but according to the literature and their own results administration of purine analogues can be highly recommended in the future.

[The O'Brien filter test in the differential diagnosis of disorders with high-grade thrombocytosis]. / A nagy thrombocytaszámmal járó kórképek elkülönítése az O'Brien-féle filter-teszt segítségével.

In the differential diagnosis of primary and secondary thrombocytosis, platelet function test can be used. We have examined the possible role of O'Brien's filter test in the differentiation of primary and secondary thrombocytosis in 53 patients with myeloproliferative diseases with primary thrombocytosis and in 21 patients with other disorders complicated by secondary thrombocytosis. By using heparin as an anticoagulant, the sensitivity of O'Brien's filter test proved to be 75%, and it's specificity was 85.7%. In blood samples anticoagulated with citrate, the sensitivity was 100% and specificity 83.3%. Based on these studies we suggest the use of O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated (> 400 x 10(9)/L) platelet count. In case of normal results, the causes of reactive thrombocytosis should be cleared first, while with pathologic results, haematological examination of the patients should be performed.

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.

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.

[Pure red cell aplasia, associated with chronic lymphoid leukemia, successfully treated with closorine]. / Krónikus lymphoid leukaemiához társuló "pure red cell" aplasia sikeres cyclosporin kezelése.

Pure red cell aplasia (PRCA) is a rare disorder, which is characterized by severe anaemia associated with reticulocytopenia and absence of erythroid precursor cell from the bone marrow. Mostly immunological mechanisms have a role in its pathogenesis. Primarily the acquired, idiopathic type occurs in adults, however, it is rarely associated with other disorders (autoimmune-, and lymphoproliferative diseases, etc.). The authors present a patient with B-cell chronic lymphocytic leukemia associated with PRCA, which was successfully treated with cyclosporine. The pathogenesis and the therapy of the PRCA is also summarized.

[Fibrinolysis studied by an in vitro clot lysis test in polycythemia vera]. / A fibrinolysis vizsgálata in vitro alvadékoldási teszt segítségével polycythaemia verában.

Results obtained by a simple in vitro clot lysis test in polycythemia vera, polyglobulia, essential thrombocythemia and chronic immun thrombocytopenic purpura are reported. Increased lysis resistance of the platelet-rich clots was demonstrated in polycythemia and essential thrombocythemia, ostensibly caused by the high plasma level of plasminogen activator inhibitor-1. Following retraction of platelet-rich clots no further increase of lysis-resistance occurred in polycythemia and essential thrombocythemia. This phenomenon is most likely due to abnormal platelet function. The well known thromboembolic complications may at least partly result from the impaired fibrinolysis in diseases with high platelet counts.

[Hypocholesterolemia and other lipoprotein disorders in myelofibrosis]. / Hypocholesterinaemia és egyéb lipoprotein-rendellenességek myelofibrosisban.

In the recent years more and more data suggest a significant relationship between malignant diseases and cholesterol, respectively lipoprotein metabolism. It is a significant decrease of cholesterol in primary myelofibrosis (agnogenic myeloid metaplasia) and in secondary myelofibrosis. Similarly, the HDL and LDL cholesterol levels are also decreased in these diseases. On the contrary, the triglyceride level is significantly higher in these groups, 41 patients with agnogenic myeloid metaplasia and 16 patients with myelofibrosis following polycythaemia vera were examined. Decrease of serum cholesterol level was significant: 3.7 and 3.3 mmol/l and 5.2 mmol/l in 76 healthy controls, as the mean values.

[Eosinophilic leukemia: a rare form of Philadelphia chromosome negative chronic myeloid leukemia?]. / Eosinophil leukaemia: a Philadelphia chromosoma negatív krónikus myeloid leukaemia ritka formája?

Eosinophil leukaemia is a rare and poorly defined entity characterized by neoplastic proliferation of eosinophil cell line. This form of the hypereosinophilic state is considered to be a variant form of CML, although as a diseases entity is not generally accepted. A history of a patients is reported, whose clinical course is thought to fulfill the requirements of eosinophil leukaemia. On the basis of the initial results (pathological lymphogram, eosinophilia, Ph-negativity) lymphogranulomatosis was suspected and explorative laparotomy was performed. However, only marked eosinophilic infiltration of the spleen was detected. After splenectomy his disease was stable without treatment for six months when his leukocytosis and eosinophilia increased. Despite the administration of hydroxyurea the leukocyte count exceeded 100 x 10(9)/l (eosinophil cells 70%), and the bone marrow revealed massive (80%) eosinophilic infiltration. Neither Ph-chromosome, nor cabl and bcr gen rearrangement were demonstrated, but the expression and amplification of c-myc oncogene indicated disease progression. Interferon therapy produced long-term clinical and haematological improvement, but blastic transformation was developed in the second year of his disease. Autopsy showed multiple organ involvement characteristic of CML, but no marked eosinophilic infiltration was found. The feature of this case suggest that eosinophil leukaemia might represent an uncommon form of Ph-negative CML.

Co-expression of c-abl and c-myb oncogenes in Philadelphia chromosome-negative, bcr-negative chronic myeloid leukaemia.

Three cases of Philadelphia (Ph) chromosome-negative, bcr-negative chronic myeloid leukaemia (CML) have been investigated for oncogene expression by Northern blot and cytoplasmic RNA dot blot hybridization. Considerably high levels of expression of c-abl and c-myb were observed in all cases. In the Ph-negative cells the normal 6.0 and 7.0 kb c-abl and 3.8 kb c-myb transcripts were found. No amplification of c-abl or c-myb oncogenes was detected in the DNAs of Ph-negative CML cells. Data suggest that co-operation between the overexpressed c-abl and c-myb oncogenes is causally related to Ph-negative bcr-negative CML.

Prognostic significance of bone marrow reticulin fibres in idiopathic myelofibrosis: evaluation of clinicopathological parameters in a scoring system.

A clinicopathological scoring system was performed for obtaining a better estimate of prognosis in 50 patients with idiopathic myelofibrosis (IMF). Laboratory parameters including Hb-level, leukocyte and platelet counts, percentage of blast cells in peripheral blood, spleen and liver size, and a semiquantitative histological grading of reticulin fibre content of bone marrow biopsies taken at the time of initial diagnosis were analysed. Based upon these haematological and histological parameters three prognostic groups could be categorized with a significantly different survival (low-risk group with 21 patients = 75 months; medium-risk group with 18 patients = 51 months, and 11 patients in a high-risk group = 18 months). In an univariate (log rank test) and in a multivariate regression analysis the Hb-concentration, mild splenomegaly (less than 5 cm) and a higher grade of bone marrow reticulin content proved to be important prognostic parameters, whilst leukopenia, thrombocytopenia and the presence of peripheral blast cells were only of prognostic significance within the first 6 months from initial diagnosis. It was concluded that the increase of reticulin fibre deposition in bone marrow together with anaemia and mild splenomegaly could be responsible for a progressively worse life-expectancy of high-risk patients with idiopathic myelofibrosis.

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 maturation's 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).

HTLV-related markers in a Hungarian patient with adult T-cell leukemia.

Monoclonal integration of DNA sequences related to, but not identical to HTLV-I provirus was detected in the peripheral blood lymphocytes of a Hungarian male suffering from ATL. The patient and his parents showed serological cross-reactivity with both HTLV-I and HTLV-II group-specific antigens. Restriction enzyme analysis with EcoRI, PstI, BamHI, HindIII and SacI revealed structural similarity of the provirus integrated in the DNA of ATL cells to HTLV-I but not to HTLV-II. Data suggest that this provirus and HTLV-I are similar to each other along gag and pol regions, but they are different in the env region.