Péter Batár

Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

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.

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.

Effective PAD (bortezomib, doxorubicine, dexamethasone) treatment of a patient with plasma cell leukaemia developed after autologous stem cell transplantation

A plazmasejtes leukemia a myeloma multiplex ritka es agressziv megjelenesi formaja. Kezelese megoldatlan, es kulonosen a szekunder plazmasejtes leukemiak eseten varhato gyors progresszio. A szekunder formaban ugyanis tobbnyire intenziv kemoterapiaban reszesult myelomas betegekben terminalisan kovetkezik be a leukemias transzformacio, mig a primer esetekben mar a diagnozis idejen kimutathatok a plazmasejtes leukemia tunetei. A szerzők nem szekretoros myeloma multiplex miatt periferias autolog őssejttranszplantacion atesett plazmasejtes leukemias beteguk kezeleset ismertetik. A bortezomib (proteaszomagatlo), a doxorubicin es a dexamethason kombinacioja (PAD-protokoll) komplett remissziot es kilenc honapos tulelest eredmenyezett. A tobbnyire betegismerteteseken alapulo irodalmi adatok szerint a PAD-protokoll, sajat tapasztalatunkhoz hasonloan, plazmasejtes leukemiaban is hatekony es jol toleralhato. Autolog es/vagy allogen transzplantacioval kombinalva a betegek tulelesenek tovabbi javulasa varhato. after autologous stem cell transplantation. The most agressive and rare manifestation of multiple myeloma is plasma cell leukaemia (PCL). While secondary form of PCL represents those heavily pretreated cases when leukaemic transformation developes terminally after intensive chemotherapy in patients with multiple myeloma, primary cases are characterized by leukaemic symptoms present at diagnosis. The secondary form has a rapid progression. The management of PCL is still unsolved. The authors present a case of a patient with non-secretory multiple myeloma who had developed plasma cell leukaemia after peripheral stem cell transplantation. PAD (bortezomib, doxorubicine, dexamethasone) treatment resulted in complete remission and 9-month survival of the patient. Previous case reports in the literature and our experience have revealed PAD protocol to be well tolerated and effective in PCL. Combination of PAD treatment with autologous and/or allogenic stem cell transplantation might further improve patients’ outcome.

Increased platelet activation and decreased fibrinolysis in the pathogenesis of aseptic necrosis of the femoral head.

Thrombotic events, increased tendency toward intravascular thrombosis and decreased fibrinolysis seem to be possible pathologic causes for aseptic necrosis of the femoral head (ANFH) in adults. This project was to study whether either increased platelet activation or decreased fibrinolytic activity and/or any other thrombogenic factor may be implicated in the evolvement of ANFH. The speed of the in vitro lysis was significantly lower in patients (both in primary and in secondary cases) compared with healthy controls. The platelet activation (measuring with beta TG) proved to be significantly higher in the primary group as well as in the secondary group compared with healthy controls. Lp(a) levels were elevated in primary and secondary cases. This alteration was more characteristic in the primary cases. Fibrinogen levels were also elevated in the primary group, but the difference was not significant. The data shown here may further support that hypofibrinolysis and increased thrombogenesis are major causes of ANFH. Early diagnosis of ANFH increases the chances of modifying the course of this disabling disease.

One hundred fifty autologous peripheral haemopoietic stem cell transplantations and their lessons

UNLABELLED Five years ago (in September, 2003), the activity of the 5th Haemopoietic Stem Cell Transplantation Centre of Hungary has begun. This centre has been registered as No 648. by the European Group for Blood and Marrow Transplantation-Centres. AIMS To supply the needs of stem cell transplantation regions in north-east Hungary and to develop an active co-operation with the Hungarian and international centres. METHODS Transplantations were made according to international criteria. RESULTS 150 autologous stem cell transplantations has been performed so far, including 74 patients with myeloma multiplex, 43 patients with non-Hodgkin lymphoma, 27 patients with Hodgkins disease, 4 patients with autoimmune disease, and one patient with leiomyosarcoma. The survival rates were similar to the previous Hungarian and international data. The centre played a role in other activities using stem cell therapy at the University of Debrecen (dendritic cell vaccine program, stem cell therapy in myocardial infarction, stem cell therapy in peripheral arterial- and autoimmune diseases). This centre performed the largest quantity of the conditioning protocol Zevalin, Bischloronitrosourea, Etoposide, cytosine-Arabinoside, Melphalan in non-Hodgkin lymphoma in Hungary; ten patients were treated with this protocol.

Comparison of coated-platelet levels in patients with essential thrombocythemia with and without hydroxyurea treatment.

Essential thrombocythemia (ET) is an acquired myeloproliferative disorder with sustained increase of platelet count. This disease may be associated with thrombotic or bleeding complications due to the altered number and function of platelets. Coated-platelets produced by a simultaneous activation of collagen and thrombin represent a subpopulation of activated platelets with high prothombinase activity and the retention of several α-granule-derived coagulation factors on their surface. There is a growing body of evidence for a relationship between variable levels of coated-platelets and different hemostatic alterations. However, no data are available on coated-platelet formation in the pathogenesis of ET in the presence or absence of treatment. The levels of coated-platelets in 43 ET patients (15 non-treated and 28 hydroxyurea-treated) without known thrombotic or hemorrhagic complications were analyzed using flow cytometry. These results were compared with data of 31 healthy individuals. In addition, platelet function was analyzed with PFA-100 analysis, and P-selectin (CD62) positivity was also measured by flow cytometry. Increased P-selectin expression was detected with prolonged PFA-100 closure times in the ET group; however, significantly lower levels of coated-platelets were found in non-treated ET patients compared to controls (23.1 ± 8.8% vs. 37.6 ± 12.7%, p = 0.0008). This tendency was more evident in patients with JAK2-V617F mutation. Patients on hydroxyurea treatment had elevated coated-platelet levels (34.1 ± 12.3%) close to the normal value. In conclusion, lower than normal levels of coated-platelets were generated in ET, which were significantly (p = 0.0008) increased by hydroxyurea treatment. We suppose that abnormal coated-platelet level may also contribute to platelet dysfunction in ET.

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

Dasatinib inhibits coated-platelet generation in patients with chronic myeloid leukemia

Abstract Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.