László Rejto

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.

THE ASSOCIATION OF REDUCED ENDOTHELIUM DERIVED RELAXING FACTOR-NO PRODUCTION WITH ENDOTHELIAL DAMAGE AND INCREASED IN VIVO PLATELET ACTIVATION IN PATIENTS WITH DIABETES MELLITUS

The role of reduced endothelial production of EDRF-NO in the pathogenesis of diabetic angiopathy has received much attention, however, most of the rather conflicting data were gained from animal experiments. Limited human experience seems to be available in insulin dependent diabetes, calling attention to decreased EDRF-NO production. Hereby the clinical, as well as laboratory investigation (urinary and serum nitrate/nitrite, lipid peroxidation, glucometabolic parameters, endothelial and in vivo platelet activation markers, etc.) of 35 non-insulin dependent (NIDDM) and 15 insulin dependent diabetics (IDDM) patients are given. Urinary and serum nitrate/nitrite concentrations were proven to be reduced in both patients groups. This change was independent of diabetes duration, presence of macroangiopathy, coronary heart disease and the glucometabolic parameters, however, correlation was registered with lipid peroxidation (total antioxidant status). An inverse correlation of nitrate/nitrite excretion with endothelial markers (von Willebrand factor, soluble thrombomodulin) was documented in NIDDM, this correlation was much stronger in IDDM. Moreover, in IDDM patients reduced nitrate/nitrite excretion was strongly associated with elevated plasmatic beta-thromboglobulin levels. The data presented here support to the hypothesis, that EDRF-NO production is reduced in diabetes and this reduction seems to correlate with endothelial damage. In IDDM the decreased nitrate/nitrite excretion may also lead to increased in vivo platelet activation, which suggests that the reduced amount of EDRF-NO might play a role in the pathogenesis of angiopathy in IDDM.

Decreased Expression Levels of Tumor Suppressor MicroRNAs in Hairy Cell Leukemia

Hairy cell leukemia is a chronic, clonal disease of the hemopoetic stem cell. Although our knowledge on its pathogenesis has increased a lot during the previous years, further details are needed to provide a more personalized, and therefore more successful treatment for patients. New advancements in the growing field of epigenetics may contribute to the finding of novel therapeutic target molecules. Similarly to acute leukemia, hairy cell leukemia also possesses a unique microRNA expression pattern. Decreased levels of tumor suppressor microRNAs lead to the elevation of the expression levels of their oncogenic targets. During our investigations on the level of let-7b and miR-124 in the bone marrow sample of a patient suffering from hairy cell leukemia, decreased expression levels have been found in the case of both microRNAs. These alterations also influence the activity of the MAPK signaling pathway, resulting from the regulation of its members by microRNAs including those mentioned above. Our results support new details about the connection between altered microRNA expression levels and signal transduction pathways in hairy cell leukemia.

Effects of in vitro platelet activation on platelet derived nitric oxide production in healthy humans and in chronic myeloproliferative diseases with elevated platelet counts.

Intravascular EDRF-NO production is known to be impaired in some diseases, e.g., diabetes. This phenomenon may also contribute to the development of diabetic vascular disease. More recently the presence of NO synthase (ecNOS, iNOS) have been recognized in human platelets. Platelets produce NO only during activation, even though in minute amounts. This platelet derived NO seems to play an important physiological role, as it inhibits further platelet recruitment quite substantially. In the present report washed platelets isolated from healthy persons and patients with chronic myeloproliferative diseases (CMPD) were exposed to common and physiologically relevant activators (i.e., thrombin, collagen, epinephrine etc.). These tests were carried out in 20 healthy volunteers and 15 patients suffering from myeloproliferative disorders associated with thrombocytosis. As a consequence of pathological platelet function observed in CMPD, the in vitro platelet NO response is impaired in the patient group. One may assume, that reduced platelet NO response, at least in part, may contribute to platelet hyperfunction, angiopathy and thrombotic complications in some cases of CMPD.

Increased in vivo platelet activation and reduced intravascular endothelium-derived relaxing factor and nitrate/nitrite production in patients with insulin-dependent diabetes mellitus.

Limited information seems to be available about the role of reduced endothelial production of endotheliumderived relaxing factor (EDRF)-nitrate/nitrite (NO) in the pathogenesis of diabetic angiopathy in insulindependent diabetes. A report of urinary and serum nitrate/nitrite, glucometabolic parameters, endothelial and in vivo platelet activation markers of 22 insulin dependent diabetics (IDDM) patients are given. Urinary and serum nitrate/nitrite concentrations were reduced in IDDM. This was independent of disease duration, presence of angiopathy and the glucometabolic parameters. A significant and inverse correlation of nitrate/nitrite excretion with endothelial markers (von Willebrand factor, soluble thrombomodulin) was documented. Moreover, reduced nitrate/nitrite excretion was strongly associated with elevated plasmatic beta -thromboglobulin levels. EDRF-NO production is reduced in IDDM and this reduction correlates with endothelial damage. Decreased nitrate/nitrite excretion may also influence in vivo platelet function, which results in increased in vivo platelet activation and suggests that the reduced intravascular production of EDRF-NO might play a role in the pathogenesis of angiopathy in IDDM.

Chromosomal aberrations and CD38 expression in two siblings with B-cell chronic lymphocytic leukemia: A report of two siblings

In this study our purpose was to define chromosomal aberrations and CD38 expression in male siblings 69 and 66-years-old with B-cell chronic lymphocytic leukemia (B-CLL). Cells from peripheral blood were analysed by comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). The alteration detectable by CGH was the over-representation of the Y chromosome in both samples. Interphase FISH were performed using locus (13q14 and 17p53) and centromere (chromosome 12, 17 and Y) specific DNA probes. One brother (patient 1, 69 years of age) showed deletion of the 13q14 region, this alteration was associated with low CD38 expression, both predicting a favourable prognosis. However, the younger patients (patient 2, 66 years of age) cells expressed CD38 in high percent, which is considered as an indicator of poor prognosis, and deletion of the 13q14 was not seen. Other, relatively frequent chromosomal alterations including trisomy 12 and deletion of 17p53 were not present in any of the samples. The cytogenetic findings and the CD38 expression are in concordance with the clinico-pathological data of the siblings. Thus, we found the variability of these parameters described in B-CLL even in the familial form of the disease.

Increased platelet glycoprotein Ib receptor number, enhanced platelet adhesion and severe cerebral ischaemia in a patient with polycythaemia vera

The present study describes severe multiplex cerebral ischaemic laesions in a male patient being diagnosed with polycythaemia vera (PV). In contrast to previous publications, unique platelet receptor pattern with normal platelet count was identified. Glycoprotein Ib receptor number on the surface of resting platelets was increased two-fold and almost three-fold in case of activated platelets compared to the controls. More over, in an in vitro study when whole blood was circulated both at venous and arterial shear conditions and shear rate was adjusted according to the blood viscosity, platelet aggregate/thrombus formation was characteristic on surfaces covered with purified von Willebrand factor while in case of controls the surface was covered with single platelets or platelet monolayer. Similar results with pathological findings have not been published in PV until now. Our result undersigns the necessity of antiplatelet therapy of PV patients, even at normal platelet count.

Successful treatment of B-cell prolymphocytic leukemia (B-PLL) with FCR-Lite (fludarabine, cyclophosphamide, rituximab) protocol

B-cell prolymphocytic leukemia (B-PLL) is a rare disorder characterized by marked lymphocytosis in the peripheral blood, matured lymphocytic infiltration in the bone marrow and splenomegaly. It has a distinct immunophenotype (CD19, CD20, CD22, FMC7, intensive surface immunoglobulin positivity) which helps to differentiate from other lymphoproliferative malignancies. It has a poor prognosis and its treatment is unsettled. The authors present a case of a patient with typical B-PLL treated with FCR-Lite (fludarabine, cyclophosphamide, rituximab) protocol achieving complete hematological (and immunophenotypic) remission. The treatment was well tolerated. Neither major infective complication nor tumor lysis syndrome was observed. According to the authors experience the FCR-Lite protocol can not only be used in patients with CLL but it also can be effective in patients with B-PLL. No clinical experience has been reported yet in the literature with this protocol.

Treatment of mantle cell lymphoma with autologous stem-cell transplantation in a patient with severe congenital haemophilia-A and chronic (B and C virus) hepatitis

Mantle cell lymphoma (MCL) is a highly malignant disease with a median survival of 3–5 years. Most of the patients have advanced stage disease at the time of diagnosis. Combined chemo-immunotherapy, mainly rituximab and cyclophosphamide, vincristine, doxorubicin, dexamethasone altering with methotrexate and cytarabine [hyper-CVAD (cyclophosphamide, vincristine, doxorubicine, dexamethasone altering with methotrexate and cytarabine) programme], autologous stem-cell transplantation (ASCT) in first remission and allogeneic stem-cell transplantation (in patients with relapsed or refractory MCL) improved the outcome, resulting in longterm disease control [1]. We present a case of MCL associated with severe congenital haemophilia A as well as chronic B and C virus infection. It is the first report of ASCT in such a patient. The first (and so far only) ASCT in a patient with severe haemophilia A and AIDS-related lymphoma was described in 2007 [2]. Our patient was not infected with HIV. A 38-year-old man, with severe congenital haemophilia A [factor VIII (FVIII) <1%, inhibitor was not detectable] and chronic (B and C virus) hepatitis, presented with generalized lymphadenopathy. Viral infections were acquired via factor substitutions. Lymph node biopsy revealed MCL (blastoid variant) with bone marrow involvement (stage IV/A). Viral loads were not investigated at this time. Eight cycles of CEOP (cyclophophamide, etoposide, oncovin and prednisone) therapy were administered in a county hospital. Follow-up PET-CT revealed only slight response. Treatment was continued with two cycles of dexamethasone, high-dose Ara-C and cisplatin (DHAP) with poor response. Hyper-CVAD programme was started at our department with good partial response. As a result of chronic viral infection and absence of appropriate virological investigation, rituximab was not added. Stem-cell collection was performed after the third cycle of therapy; however, some weeks later, rapid enlargement of abdominal lymph nodes was detected. Despite the lymph node progression, he proceeded to ASCT. At this time, a detailed virological investigation was carried out: HBV DNA, determined by RT-PCR method, was negative. Other HBV assays were performed as follows: HBeAg: negative, anti-HBe: positive, antiHBc IgM: negative. HCV RNA was positive at high titre (5 650 000 IU mL), measured by HCV Amplicor (TaqMan Roche, Rotkreutz, Switzerland) assay. Transaminase levels were normal. The therapy was supplemented with rituximab. The conditioning regimen included total body irradiation (TBI) (4 · 3 Gy, on days )7 to )4), cyclophosphamide (2 · 60 mg kg, on days )3 to )2) and rituximab (375 mg m on day )1), where treatment was supplemented with bortezomib (1.3 mg m on day )3), followed by infusion of 8.25 · 10 CD34+ cells bwkg (day 0). Recovery of neutrophils (‡1.0 · 10 L) occurred on day 9. As abdominal lymphadenopathy was still observed subsequent to transplantation, steroid therapy (32 mg methylprednisolone daily), alfa-2b interferon (1.5 MU three times weekly) and rituximab treatment (375 mg m once a month) were initiated. Before stem-cell transplantation, he was on prophylactic therapy (3.000 IU octanate/octapharma/ twice weekly). He had no bleeding complication during the year before transplantation. Once platelet count dropped below 50 · 10 L because of chemotherapy, 1 · 40 IU bwkg FVIII concentrate was administered daily to achieve 17–35% plasma FVIII levels (12 h after the FVIII substitution). There was Correspondence: Laszlo Rejto, 2nd Dept. of Medicine, Health Sciences Center, University of Debrecen, Nagyerdei 98, Debrecen 4012, Hungary. Tel.: +36 52 314 410; fax: +36 52 255 152; e-mail: lrejto@dote.hu

Az urát-nephropathia patofiziológiája, klinikuma és kezelése

Tumor lysis syndrome is an oncologic emergency that is characterized by severe electrolyte abnormalities. The syndrome occurs in patients with lymphoproliferative malignancies, most often after chemotherapy, but also spontaneously. The pathophysiology involves tumor cell lysis resulting in the release of potassium, phosphate and uric acid. The deposition of uric acid and calcium phosphate crystals in the renal tubules may lead to acute renal failure. The treatment consists in hydration, correction of the acidosis and hyperkalemia, use of allopurinol and recombinant urate oxidase (rasburicase) for preventing urate nephropathy and haemodialysis. The authors report a case of a patient with acute myeloid leukemia, who developed severe tumor lysis syndrome after chemotherapy.