Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1-10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Residual vein thrombosis for assessing duration of anticoagulation after unprovoked deep vein thrombosis of the lower limbs: the extended DACUS study.

The safest duration of anticoagulation after idiopathic deep vein thrombosis (DVT) is unknown. We conducted a prospective study to assess the optimal duration of vitamin K antagonist (VKA) therapy considering the risk of recurrence of thrombosis according to residual vein thrombosis (RVT). Patients with a first unprovoked DVT were evaluated for the presence of RVT after 3 months of VKA administration; those without RVT suspended VKA, while those with RVT continued oral anticoagulation for up to 2 years. Recurrent thrombosis and/or bleeding events were recorded during treatment (RVT group) and 1 year after VKA withdrawal (both groups). Among 409 patients evaluated for unprovoked DVT, 33.2% (136 of 409 patients) did not have RVT and VKA was stopped. The remaining 273 (66.8%) patients with RVT received anticoagulants for an additional 21 months; during this period of treatment, recurrent venous thromboembolism and major bleeding occurred in 4.7% and 1.1% of patients, respectively. After VKA suspension, the rates of recurrent thrombotic events were 1.4% and 10.4% in the no-RVT and RVT groups, respectively (relative risk = 7.4; 95% confidence interval = 4.9-9.9). These results indicate that in patients without RVT, a short period of treatment with a VKA is sufficient; in those with persistent RVT, treatment extended to 2 years substantially reduces, but does not eliminate, the risk of recurrent thrombosis.

Drug-related cardiotoxicity for the treatment of haematological malignancies in elderly.

Several publications have focused on the cardiotoxicity of specific classes of hematological therapeutic agents such as antracyclines and cyclofosfamide. Cardiotoxicity of cancer chemotherapeutics is a problem for patients of all ages, but it increases with age. Toxicity can also develop months after the last chemotherapy dose, and late reactions can be seen years later when they present as new-onset cardiomyopathy. No data are available about the cardiotoxicity of non-chemotherapy agents currently used as preferred therapy for hematological malignancy in elderly. In this review we have provided a summary of the cardiovascular toxic effects produced by different drugs and therapeutic agents. Early identification of patients who are at risk for cardiotoxicity should be a primary goal for hematologists in the development of personalized antineoplastic therapeutic strategies or interventions. Thus, the discovery of new biomarkers to identify patients at a high risk for the development of these complications is a high priority. Although targeted therapies such as imatinib and anti-CD20 antibody such rituximab are considered less toxic and better tolerated by patients compared with classic chemotherapy drugs, certain cardiological complications can be very serious and as these agents have been in use for a limited period of time.

Relapsing or refractory idiopathic thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: the role of rituximab.

Idiopathic thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is a rare disease responsive to treatment with plasma exchange (PE) but with a high percentage of relapse or refractory patients. A severe deficiency of ADAMTS-13 (<5% of normal activity), congenital or caused by an autoantibody, may be specific for TTP and it has been proposed that severe ADAMTS-13 deficiency now defines TTP. B cells play a key role in both the development and the perpetuation of autoimmunity, suggesting that B-cell depletion could be a valuable treatment approach for patients with idiopathic TTP-HUS. This review of the literature focuses on the role of rituximab, a chimeric monoclonal antibody directed against CD20 antigen expressed by B lymphocytes, in patients with relapsing or refractory TTP-HUS with or without ADAMTS-13 deficiency, suggesting that rituximab may produce clinical remission in a significant proportion of patients. Rituximab therapy reduces plasma requirement and avoids complications related to salvage-immunosuppressive therapy. In conclusion, rituximab provides an effective, well-tolerated, and safe treatment option for patients with idiopathic TTP-HUS, thus giving an alternative approach to the current treatment based on PE.

Correlation between leukocytosis and thrombosis in Philadelphia-negative chronic myeloproliferative neoplasms.

The evidence that leukocytes may contribute to the pathogenesis of thrombosis in Chronic Myeloproliferative Neoplasms is increasing but not definitive. To further enforces whether an increased leukocyte count is associated with thrombosis and whether this effect can be modulated by cytoreductive therapy, we analyzed the clinical course of 187 patients with Polycythemia Vera (PV) and Essential Thrombocythemia (ET) followed at two Italian Institutions over a period of 7 years. The association was measured at diagnosis or before thrombotic events: a multivariable analysis was carried out using data at baseline and time-dependent covariates. We found that white blood cells (WBC) count above 9.5 x 10(9)/L at diagnosis (baseline analysis) was associated with thrombosis during the follow-up (Hazard Ratio [HR] of 1.8, p 0.03). At the time-dependent analysis, therapy with hydroxyurea (HU), lowering by 35% the baseline WBC level, reduced such strength of association giving a HR of 1.3 (p value non significant). We found a trend between WBC level and thrombosis in untreated low-risk patients (RR of 1.9, 95% CI 0.9 to 3.1); in high-risk patients treated with HU this correlation was clearly lost (RR 1.1, 95% CI 0.2 to 2.7). Finally, we could not identify the presence of JAK2 (V617F) as a risk factor for thrombosis. Properly designed prospective studies should corroborate such results.

[Serological screening for Leishmania infantum in asymptomatic blood donors and HIV+ patients living in an endemic area]. / Screening sierologico per Leishmania infantum su donatori di sangue e HIV+ asintomatici residenti in area endemica.

Visceral leishmaniasis (VL) is endemic in Sicily (48 new cases in 2004, of which nine were in Agrigento). In southern Europe between 25-70 per cent of adult VL cases are related to HIV infection. The HIV cases have a high risk (1.5-9%) of developing VL either as a new infection or as the revival of a latent infection. We therefore carried out serologic screening to detect antibodies against L. infantum by IFAT in 1449 blood donors in Agrigento and the surrounding area (May-December 2005) and in 120 HIV+ in western Sicily, all of whom were asymptomatic and had no history of VL. L. DNA was assessed by nested PCR in blood samples of some seropositive donors. Of the 1449 blood donors, 11 (0.75%) were positive by IFAT and three of them were also positive in PCR. L. infantum seropositivity is most probably the expression of recent infection because the clearance of serum antibodies is rather fast (6-12 months) after VL. This is why blood donation by Leishmania seropositive donors, whether positive or negative by PCR, could constitute an infection risk especially for immunosuppressed recipients, who should receive deleukocyted blood. Moreover it could be useful to monitor HIV/Leishmania coinfection cases to avoid the risk of slatentization of L. infection when CD4+ levels are very low.

Identification of a terphenyl derivative that blocks the cell cycle in the G0-G1 phase and induces differentiation in leukemia cells.

To further explore the SAR of resveratrol-related trans-stilbene derivatives, here we describe the synthesis of (a) a series of 3,5-dimethoxy analogues in which a variety of substituents were introduced at positions 2', 3', 4', and 5' of the stilbene scaffold and (b) a second group of derivatives (2-phenylnaphthalenes and terphenyls) that incorporate a phenyl ring as a bioisosteric replacement of the stilbene alkenyl bridge. We thoroughly characterized all of the new compounds with respect to their apoptosis-inducing activity and their effects on the cell cycle. One of the new derivatives, 13g, behaved differently from the others, as it was able to block the cell cycle in the G(0)-G(1) phase and also to induce differentiation in acute myelogenous leukemia HL60 cells. Compared to resveratrol, the synthetic terphenyl 13g showed a more potent apoptotic and differentiating activity. Moreover, it was active on both multidrug resistance and Bcr-Abl-expressing cells that were resistant to resveratrol.

Assessment of the frequency of additional cancers in patients with splenic marginal zone lymphoma.

OBJECTIVES: Solid second primary cancers (SPC) have become an issue of extensive research. The purpose of the present study was to estimate the standardised incidence ratio (SIR) and the absolute excess risk (AER) of SPC in patients with splenic marginal zone lymphoma (SMZL). METHODS: We investigated the incidence of additional cancers in 129 patients consecutively diagnosed with SMZL in three Italian haematological centres, asking the cooperating doctors for additional information on initial and subsequent therapies and on the onset and type of second cancers. RESULTS: Twelve SPC were recorded (9.3%); the 3- and 5-yr cumulative incidence rates were 5.5% and 18.3% respectively, with an SIR of 2.03 [95% confidence interval (CI): 1.05-3.56; P < 0.05; AER = 145.81]. Of 12 SPC observed, four were urinary tract neoplasms (SIR, 3.70; 95% CI: 1.01-9.48; P < 0.05; AER = 70.06), four were lung cancers (SIR, 9.16; 95% CI: 1.41-13.25; P < 0.05; AER = 85.50) and the other four were hepatic carcinoma, endometrial cancer, breast cancer and colorectal cancer. CONCLUSIONS: Our findings evidence a high frequency of additional cancers in patients with SMZL and suggest that the incidence rate of SPC is significantly different from that expected in the general population. The frequency of cases with urinary tract and lung malignancies in our series is higher than expected. Although confirmatory data are needed, it is our opinion that SMZL patients are at risk of second cancer and should be carefully investigated on diagnosis and monitored during the follow-up.

Deoxycoformycin (pentostatin) in the treatment of splenic marginal zone lymphoma (SMZL) with or without villous lymphocytes.

BACKGROUND: Splenic marginal zone lymphoma (SMZL) is an infrequent B-cell neoplasm that pursues an indolent course. Signs and symptoms, mostly related to hypersplenism, are successfully managed by splenectomy. However, the therapy of patients who are not fit for a surgical procedure or who relapse after splenectomy, is still an unsettled issue. PATIENTS AND METHODS: We report a phase-II study on 16 patients with SMZL, three therapy naïve and 13 pretreated, all showing systemic symptoms or progressive worsening of peripheral cytopenia, who were treated with pentostatin at a dose of 4 mg/m2 every other week for 6-10 wk. In relapsed patients, the median interval between diagnosis and treatment was 26 month (range: 8-49). RESULTS: Overall, 68% of the patients showed a clinical response. Two out three patients, who received pentostatin as first line therapy, attained a complete response (CR). One CR and seven minor or good haematological responses were recorded in relapsed patients. Treatment toxicity, mostly haematological, proved manageable. With a median follow-up of 35 month the median overall survival (OS) is 40 month and the median progression free survival (PFS) is 18 month. CONCLUSION: Our data show that pentostatin administered every other week has a good degree of activity in the treatment of SMZL and suggest that this schedule could be considered a possible therapeutic option for patients who are not fit for splenectomy or have relapsed.

Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells.

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Pterostilbene and 3'-hydroxypterostilbene are effective apoptosis-inducing agents in MDR and BCR-ABL-expressing leukemia cells.

Pterostilbene and 3,5-hydroxypterostilbene are the natural 3,5-dimethoxy analogs of trans-resveratrol and piceatannol, two compounds which can induce apoptosis in tumor cells. In previous studies we demonstrated the importance of a 3,5-dimethoxy motif in conferring pro-apoptotic activity to stilbene based compounds so we now wanted to evaluate the ability of pterostilbene and 3,5-hydroxypterostilbene in inducing apoptosis in sensitive and resistant leukemia cells. When tested in sensitive cell lines, HL60 and HUT78, 3'-hydroxypterostilbene was 50-97 times more potent than trans-resveratrol in inducing apoptosis, while pterostilbene appeared barely active. However, both compounds, but not trans-resveratrol and piceatannol, were able to induce apoptosis in the two Fas-ligand resistant lymphoma cell lines, HUT78B1 and HUT78B3, and the multi drug-resistant leukemia cell lines HL60-R and K562-ADR (a Bcr-Abl-expressing cell line resistant to imatinib mesylate). Of note, pterostilbene-induced apoptosis was not inhibited by the pancaspase-inhibitor Z-VAD-fmk, suggesting that this compound acts through a caspase-independent pathway. On the contrary, 3'-hydroxypterostilbene seemed to trigger apoptosis through the intrinsic apoptotic pathway: indeed, it caused a marked disruption of the mitochondrial membrane potential delta psi and its apoptotic effects were inhibited by Z-VAD-fmk and the caspase-9-inhibitor Z-LEHD-fmk. Moreover, pterostilbene and 3'-hydroxypterostilbene, when used at concentrations that elicit significant apoptotic effects in tumor cell lines, did not show any cytotoxicity in normal hemopoietic stem cells. In conclusion, our data show that pterostilbene and particularly 3'-hydroxypterostilbene are interesting antitumor natural compounds that may be useful in the treatment of resistant hematological malignancies, including imatinib, non-responsive neoplasms.

Heterocyclic and phenyl double-bond-locked combretastatin analogues possessing potent apoptosis-inducing activity in HL60 and in MDR cell lines.

Two new series of combretastatin (CA-4) analogues have been prepared. The alkenyl motif of CA-4 was replaced either by a five-membered heterocyclic (isoxazoline or isoxazole) or by a six-membered ring (pyridine or benzene). The new compounds have been evaluated for their effects on tubulin assembly and for cytotoxic and apoptotic activities. Five compounds (18b, 20a, 21a, 34b, and 35b) demonstrated an attractive profile of cytotoxicity (IC50 < 1 microM) and apoptosis-inducing activity but poor antitubulin activity. The isoxazoline derivatives 18b, 20a, and 21a, demonstrated potent apoptotic activity different from that of natural CA-4. Their ability to block most cells in the G2 phase suggests that these compounds could act on targets different from the mitotic spindle. This would indicate activation of both the intrinsic and the extrinsic apoptotic pathways. The data suggest unambiguously that structural alteration of the stilbene motif of CA-4 can be extremely effective in producing potent apoptosis-inducing agents.

Hepatitis B virus reactivation and alemtuzumab therapy.

Reactivation of hepatitis B virus infection in subjects receiving cytotoxic treatment for heamatological malignancies occurs in 21-53% of chronic HBsAg carriers and in an unknown number of HBsAg negative subjects harbouring occult HBV infection. Immunotherapy with alemtuzumab, a humanized monoclonal antibody against CD52 epitopes on lymphocytes cells produces deep immunosuppression. We describe two subjects with chronic lymphocytic leukaemia and occult HBV infection who developed a virological and biochemical flare of hepatitis B following immunotherapy with alemtuzumab. One of them developed full blown hepatitis with seroreversion from anti-HBs to HBsAg after four weeks of alemtuzumab therapy. Lamivudine (100 mg die) achieved a complete clinical recovery and HBV-DNA clearance from blood within 8 weeks. The second patient (HBsAg and HBV-DNA seronegative, anti-HBs and anti-HBc positive before treatment) was kept under prophylaxis with lamivudine up to three months after alemtuzumab. Two months after withdrawal of lamivudine, clinical and laboratory features of acute hepatitis B developed. Lamivudine therapy was restarted and a prompt recovery was obtained with HBsAg and HBV-DNA clearance.