Alessandro Bucalossi

Reduction of antithrombin III, protein C, and protein S levels and activated protein C resistance in polycythemia vera and essential thrombocythemia patients with thrombosis

Patients with polycythemia vera (PV) or essential thrombocythemia (ET) show a high frequency of thrombosis. The reduction of hematocrit after phlebotomy and normalization of platelet counts do not completely eliminate thrombotic risk. Some preliminary studies reported a reduction in the concentration of natural anticoagulants (NA) in this group of patients. For this reason we evaluated protein S (PS) total antigen, antithrombin III (AT III), and protein C (PC) activity in 81 patients with chronic myeloproliferative disorders (33 with PV and 48 with ET). Data were compared with those obtained in 70 healthy sex‐ and age‐matched subjects. Fifty‐seven percent of patients (46 out of 81) showed one or more thrombotic episodes at diagnosis or during follow‐up. Interestingly, we found a NA deficit in 43.5% of patients with thrombosis versus only 5.7% in the group of patients without thrombosis. These results may suggest new interpretations about the pathogenesis of thrombosis in PV or ET patients.

“Fludarabine Containing-Regimens May Adversely Affect Peripheral Blood Stem Cell Collection in Low-Grade Non Hodgkin Lymphoma Patients”

Fludarabine (FLUDA) based chemotherapy has shown promise in both initial and salvage treatment of low-grade non Hodgkins lymphomas (LG-NHL). Recently, more aggressive therapies followed by autologous hemopoietic progenitor cell rescue, have also been successfully employed in these patients. However, this procedure, due to several factors including previous therapeutic regimens, is often limited by an inadequate collection of peripheral blood stem cell (PBSC). At present, very little data is available on the effect of FLUDA containing regimens in PBSC collection. We report our preliminary experience showing a possible correlation between FLUDA based chemotherapy regimens employed before mobilization and inability to collect an adequate number of blood derived hematopoietic progenitors for autologous PBSC transplantation in LG-NHL patients.

Fludarabine, Arabinosyl Cytosine and Idarubicin (FLAI) for Remission Induction in Poor-Risk Acute Myeloid Leukemia

Progress in treatment of acute myeloid leukemia (AML) is slow and treatment intensification alone has limited effects, particularly in poor-risk cases. Poor-risk cases, that are identified mainly by prior history, leukemic cell mass and cytogenetic abnormalities, share multiple mechanisms of drug resistance that are responsible for treatment failure. Since Pgp-mediated resistance to anthracycline can be reduced with Idarubicin (IDA) and resistance to arabinosyl cytosine (AC) can be reduced with Fludarabine (FLUDA), we tested a combination of high dose AC (2000mg/sqm, 5 doses), FLUDA (30 mg/sqm, 5 doses) and IDA (12mg/sqm, 3 doses) for remission induction and consolidation in 45 consecutive cases of poor-risk AML. The complete remission (CR) rate was 71 % after the first course and 82% overall, with a projected 2-year survival and relapse-free survival of 44% and 50% respectively. Non-hematologic toxicity was very mild, that is very important in elderly patients, but hemopoietic toxicity was substantial, with a time to hematologic recovery of 3 to 4 weeks and two cases of death in CR. Peripheral blood stem cells (PBSC) could be mobilized and collected successfully only in 11 cases. This three-drug combination is effective and has a limited non-hematologic toxicity, but FLUDA may increase the difficulty of obtaining PBSC early after remission induction.

Recombinant α2a interferon and polycythemia vera: Clinical results and biological evaluation by means of Fourier‐transform infrared microspectroscopy

Polycythemia vera (PV) is a chronic myeloproliferative disease. The use of recombinant α2a Interferon (IFN) therapy in this disease is a novel approach. We applied Fourier‐transform infrared microspectroscopy (FT‐IR‐M) to investigate the behavior and therapeutic responsiveness of PV patients treated with IFN. A spectroscopic parameter (A1/A2) was used, corresponding to the ratio of the integrated areas of the bands at 1080 cm‐1 and at 1540 cm‐1 due to nucleic acids and proteic components, respectively, calculated on the spectra of single megakaryocytes (MKs). In previous studies, we have pointed out that MKs in PV have a surprisingly strong myeloproliferative impulse when compared to MKs from other chronic myeloproliferative diseases. Nine patients out of the 11 studied exhibited a satisfactory responsiveness to the IFN treatment. Ten patients were evaluated by the A1/A2 parameter. In 8 of these, a good agreement was seen between this parameter and the laboratory data commonly used for the assessment of this disease. The infrared parameter, which we propose, proves to be an original, reliable method for the evaluation of recombinant α2a IFN responsiveness in this disease.

Unusual discordant responses in two multiple myeloma patients during bortezomib treatment.

Background: Cases of discordant responses in multiple myeloma (MM) patients after thalidomide therapy have been sometimes reported, in which extramedullary masses progress or present de novo with a simultaneous serum monoclonal protein reduction. Patients and Methods: We hereby report, for the first time, on two cases of MM patients with extramedullary myeloma localizations that developed during Velcade (bortezomib, PS341) treatment with a concomitant serum monoclonal protein reduction. Results: We observed in both patients a very good response in the serum monoclonal protein level, while extramedullary lesions appeared in the central nervous system and subcutaneously. Conclusions: We discuss pharmacokinetics of bortezomib and physiopathology of this unusual event and review the literature.

Immunological Response to Influenza Virus Vaccine in B-Cell Chronic Lymphocytic Leukaemia Patients

Dr. Alessandro Bucalossi, Divisione di Ematologia, ViaTufi, 1, 1-53100 Siena (Italy) We read with interest the report by Gri-babis et al. [1] concerning the clinical utility of influenza virus vaccine in B-cell chronic lymphocytic leukaemia (B-CLL) patients. We also investigated the effects of influenza virus immunization in 30 B-CLL patients in different stages of disease. Nine patients were in stage A, 12 stage B and 9 stage C [2]. The patients (22 male and 8 female, with a median age of 69 years) and 10 healthy, sexand agematched subjects were vaccinated with the inactivated Inflexal Berna vaccine (Istituo Sieroterapico e Vaccinogeno Svizzero Berna, Berne, Switzerland) containing the following antigens: A/Beijing/32/ 92 (H3N2), A/Singapore/6/86 (H1N1) and B/Panama/45/90. Patients were vaccinated twice (on days 1 and 31); serum was collected and stored at -80°C before the first immunization and after 30 and 60 days. The patients were not treated with chemotherapy or steroids for at least 4 weeks. Antibody production was assessed by the haemagglutination-inhibition technique, and a titre > 1:10 was considered as a protective response. A significant antibody response was obtained in 16 out of 30 patients (53%) and in all the controls. The frequency of specific antibody, for the three antigens used, was similar for each group. Interestingly, 5 out of 16 responders showed a protective titre only after the second immunization and in 8 patients we observed an increase in antibody titre after the second inoculation. In agreement with the results published by Gribabis et al. [1], we found a significant difference in the response when we compared patients with normal or low IgG levels (1,016 ± 495 vs. 619 ± 258 mg/dl; p < 0.05). Moreover, we observed a correlation between the response and the stage of disease. For this analysis, we combined the patients in the low stages and in the intermediatehigh stages. We found a statistically significant difference with the staging system of Binet et al. [2] (9/9 patients in stage A vs. 7/21 in stages B and C; p < 0.05), but not with the system of Rai et al. [3] (10/13 patients in stage 0-1 vs. 6/17 in stage II-IV; p = 0.058). As Gribabis et al. [1], we observed no correlation with the pattern of bone marrow infiltration, lymphocyte doubling time, or absolute lymphocyte count. Furthermore, we did not find any

Massive intravascular hemolysis: a fatal complication of Clostridium perfringens septicemia in a patient with acute lymphoblastic leukemia

Vaiopoulos et al. [1] described a case of massive intravascular hemolysis secondary to Clostridium perfringens septicemia in a patient with acute myeloid leukemia. We report a similar case of a 50-year-old male with acute lymphoblastic leukemia relapsed after 4 years from allogenic bone marrow transplantation. The diagnosis was confirmed by peripheral blood and bone marrow studies; the bone marrow was infiltrated (90%) by blast cells that appeared to be lymphoblasts on cytology. Immunophenotyping showed positive expression of CD19, CD10, CD22, HLADR, CyIgM and TdT in lymphoblasts. A conventional cytogenetic analysis was performed on short-term unstimulated bone marrow cultures. Chromosomal abnormalities were described according to the International System for Human Cytogenetic Nomenclature (ISCN) [2]. The karyotype showed: 46,XY [11]; 47,XY, – 2, – 3,t(2; 6)(q12; q13), + 7, + 11, + 12, – 13, – 14, – 15, + 21, + 22, +mar [9]. The patient started a re-induction program and received 2 doses of vincristine, 1 of cyclophosphamide and a daily high dose of methylprednisolone. On day 9 after the start of therapy, while he was neutropenic, he developed diarrhea and mild fever. Two days later severe anemia was evident with a marked reduction in serum hemoglobin level down to 3.5 g/dl. On the blood sample, the serum appeared bright red and a peripheral blood smear revealed microspherocytosis, indicating intravascular hemolysis. Serum biochemistry showed increased creatinine and lactate dehydrogenase levels and the aptoglobin level was below measurable standards. Hemoglobinuria was also present. Suddenly the patient developed hyperkalemia with cardiac failure and shock, and unfortunately died within a few hours. Clostridium perfringens was isolated from all blood and stool cultures taken. We agree with Vaiopoulos et al. [1] about the possibility, in neutropenic patients, of Clostridium perfringens producing hemolysine sepsis with massive intravascular hemolysis. According to the literature, such a complication, with a few exceptions, seems restricted to acute myeloid leukemias [1]. However, we would like to add that this pattern can also be observed in acute lymphoblastic leukemia, even if the neutropenic condition can be considered the main predisposing factor. Furthermore, we suggest promptly starting empiric antibiotic therapy at the beginning of mild fever in patients treated with high-dose corticosteroids, as this therapy could hide systemic signs of infection.

Thrombotic microangiopathy and occult neoplasia.

Thrombotic thrombocytopenic purpura (TTP), which is typically characterized by fever and central nervous system manifestations and hemolytic uremic syndrome (HUS), in which renal failure is a prominent feature are the most common thrombotic microangiopathies (TMAs). TTP is usually associated with a severe deficiency of ADAMTS13 [a metalloproteinase involved in the degradation of von Willebrand factor (vWF) multimers], causing excessive accumulation of ultra-large vWF multimers and platelet aggregation with organ failure. By contrast, patients with HUS or other TMAs usually display a normal or at least detectable ADAMTS13 activity. A TMA may be occasionally developed in association with HELLP (haemolysis, elevated liver enzymes, and low platelet count) syndrome, infections, cancer and bone marrow transplantation. In cancer patients, TMA may be related to chemotherapeutic regimens or the malignant disease itself. Occasionally, TMA is the first manifestation of an occult cancer, and in large series approximately 3% of patients who were originally diagnosed with TTP, were in fact harboring an occult malignancy. The pathogenesis of cancer-associated TMAs is not completely elucidated, but probably the most important factor is endothelial damage. However, cancer-associated TMAs show some distinct features that should promptly lead to complementary investigations for an underlying malignancy. Weakness, cough and dyspnoea, fever, weight loss, bone and abdominal pain are the most common presenting symptoms. Generally, biochemistry reveals markedly increased LDH levels, increased alkaline phosphatase and the blood smear shows erythromyelemia. Bone marrow biopsy is a valuable tool in order to establishing malignant seeding. Treatment of the underlying neoplasia is the mainstay of therapy and there is no role for plasmapheresis or plasma infusions.

Increased serum lactate dehydrogenase isoenzymes in Ph-negative chronic myeloproliferative diseases: A metabolic adaptation?

Abstract We evaluated the significance of lactate dehydrogenase (LDH) isoenzymes in chronic myeloproliferative disorders (CMDs) by studying LDH isoenzymes in the serum of patients with secondary polycythemia (SP), polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) in different disease status. LDH activity and isoenzymes were evaluated retrospectively in serum samples from four groups of patients and compared with a control group. LDH activity and isoenzyme distributions of patients with SP and PV did not reveal significant variations with respect to controls. In the ET and IMF group LDH isoenzyme revealed significant variations: IMF showed significant increase of LDH2 and significant reduction of LDH5 isoenzyme, whereas ET showed significant decrease in LDH1 and increase of LDH3. These data suggest that LDH isoenzyme patterns may be a useful marker of CMDs, but this enzymatic pattern could be expression of a metabolic adaptation.

CD4+/CD45RA+ ‘Naive' T Cells and Immunological Response to Influenza Virus Vaccine in B-Cell Chronic Lymphocytic Leukaemia Patients

B-cell chronic lymphocytic leukaemia (B-CLL) is characterized by a high frequency of infections, including those of viral aetiology. Previous reports have demonstrated a specific immunologic response to influenza virus vaccine in B-CLL patients with normal IgG levels. In this study, we have evaluated different immunophenotypically defined B and T cell subsets in 18 B-CLL patients before immunization with killed-influenza-virus vaccine. A correlation between immunological response to vaccination and both absolute numbers of CD4+/CD45RA+ naive T cells and CD5– B cells was found. These data may suggest a supporting role of the CD4+/CD45RA+ T cell subset in the specific antibody response to vaccination with influenza virus vaccine in B-CLL patients.