ALBERT

Description: Background. There are known prognostic factors in Acute myeloid leukemia (AML) patients, being the cytogenetic analysis the strongest as single predictor of disease relapse or poor therapy response. Recently, alterations in FLT3 gene (Internal tandem duplications-ITD and D835/836 mutations) are frequently detected by PCR in 30–35% of AML patients (pts) and would be associated with aggressive disease. This study reports the molecular characterization of 82 AML pts from Argentina and Uruguay, mostly of Spanish-Italian origin, studied between 1996 to 2005. Design and Methods. This study was based on 82 pts: 71 adults, median age 36 yrs, (range 25–80) and 11 children (median age: 11 yrs, range. 3–17 yrs). Cytogenetic risk was established in 77 pts by kariotyping, PCR and FISH: 49% (n=38) with low risk, 38% (n=29) with standard risk, and 13% (n=10)with high risk. The FAB distribution (n=75) was: M0=2,7% (n=2), M1= 6,7% (n=5); M2=17,3% (n=13); M3=46,7% (n=35); M4=16% (n=12); M5=6,7% (n=5); M6=4% (n=3). Clinical endpoints and follow up were available for 45 pts and 56 pts, respectively. A total of 42 pts achieved complete remission (CR), 12 pts had relapse of disease, 10 pts underwent early death without completing induction (ED pts), and 7 pts died after treatment. Prognostic factors considered were: Age 〉 55 years, WBC average, WBC 〉 100 x 106/L, % Blasts in bone marrow, Secondary etiology (therapy related/MDS). JM and TKD domain coding sequences were amplified by PCR for characterization of ITD and D835/836 mutations, respectively. Results and Interpretation. FLT3 mutations could be demonstrated in 23% (19/82 pts): ITD =16% (13/82), D835/836 =7% (6/82). The median follow-up time was 36 months (range 1 – 96 m). A total of 48% (n=27) of pts. were still alive without relapse at the end of this study. Higher incidence of Flt3 mutations [ITD+ and D835/836+] were found in: 41,7% (n=5) of pts with no achievement of CR (n=12) Vs 7,1% (n=3) pts with CR (n=42) (p=0.01), and in 38% (n=5) of the death patients group (n=13) Vs. 7,4% (n=2) pts still alive without relapse (n=27) (p=0,027).The WBC average was significantly higher in the ITD+ group (69,38x106/L) Vs ITD(−) group (9,27x106/L) (p=0.001). ITD mutation was more frequent in pts with WBC 〉100x106/L (83,3%) Vs WBC 100.106/L. Both type of FLT3 mutations (ITD and D835/836) were associated with early death in the cohort. This colaborative study showed that FLT3 mutational status had to be considered as important tool in prognosis of AML pts, however further follow up with larger number of pts is required to fully address its association with poor clinical outcome.

Description: Thrombotic thrombocytopenic purpura (TTP) is a rare syndrome characterized by microangiopathic hemolytic anemia, thrombocytopenia, fever, renal failure and neurological manifestation. It is caused by a severe decreased of Von Willebrand factor cleaving protease activity (ADAMTS-13), leading to persistence of unusually ultra-large Von Willebrand multimers (ULVWF) in the circulation that bind to platelets, causing platelet aggregates, microangiopathic hemolysis and thrombocytopenia. A lack of ADAMTS-13 activity can be caused by autoimmune inhibitors or may be due to a constitutional deficiency of this protein. Recently, the ADAMTS-13 gene that encodes for the ADAMTS-13 protein was found. It was mapped to chromosome 9q34 and consists of 29 exons. Several mutations has been identified in the ADAMTS gene in patient with the congenital form of TTP. Although TTP usually occurs as an acquired form due to autoantibodies against ADAMTS-13. The determination of the activity of ADAMTS-13 and of antibodies against ADAMTS-13 are important part in the workup of patients with TTP. Plasma exchange (PE) with fresh frozen plasma replacement is the standard treatment in the acquired TTP. The efficacy of PE is likely due to the removal of both antibodies and ULVWF and the infution of ADAMTS-13. Additional treatment modalities include glucocorticoids, splenectomy, vincristine, cyclophosphamide, azathioprine, cyclosporin A, combination chemotherapy, intravenous immunoglobulins and, recently, rituximab, a monoclonal antibody against CD20 present on B-limphoid cells. We report a case of chronic relapsing acquired idiopathic TTP successfully treated with rituximab. The patient, an 50-year old woman, developed her first episode of TTP in May 2001. Remission was achieved after 12 sessions of PE, four dose of vincristine at dose of 0,02 mg/kg/die, corticosteroids at dose of 1 mg/kg/die and increased dose of prociclide from 10 to 60 mg /kg/die. From 2001 to 2004, she had six relapses responding to treatment with PE, vincristine, and corticosteroids. The relapse in 2004 was followed by a protracted course despite the addition of cyclosporine A and she become dependent on PE. On May 2004 she was treated with splenectomy. The postoperative course was uneventful. The inhibitors against ADAMTS-13 disappared, but after 8 months the patient relapsed and received six PE and corticosteroids, and then rituximab therapy (four doses of 375 mg/mq weekly). ADAMTS-13 activity and inhibitor levels were monitored. ADAMTS-13 activity was initially, pre-rituximab,

Description: HAART has improved the outcome of AIDS patients (pts). Thus high-dose chemotherapy followed by ASCT has been used for the treatment of HIV+Ly pts, similarly to HIV-neg-Ly pts. In a recent report we found delayed engraftment after ASCT in HIV+Ly compared with HIV-neg Ly pts (Exp.Hem33;487–494,2005). To test the role played by HAART on engrafment we conducted this study, comparing engraftment results on HIV+Ly pts that received HAART with those in wich HAART was withdrawn after ASCT. Patients and Methods: From June 2000 a total of 19 HIV+Ly pts (17 male) received an ASCT in a multicenter cooperative study; 5 were Hodgkin disease (HD) and 14 non-Hodgkin high-grade malignant lymphoma (NHL). Median age was 43 years (range, 31–61); adjusted IPI (aIPI) was: 0–1 in ten pts and 2–3 in nine more pts; advanced Ann Arbor stage (III–IV) was present in 14 pts.Ten pts were treated with HAART during ASCT (HAART+) and in nine pts HAART was withdrawn due to gastrointestinal toxicity (HAART neg). Both groups were similar for the most relevant clinical characteristics (p value Mann-Whitney test or chi-square test 〉 0.05): HD/NHL, Ann Arbor stage, aIPI, status at ASCT (CR-1/more than CR-1), age, number of lines of chemotherapy before ASCT, number of cycles of mobilization to obtain an adequate amount of CD34+ cells, number of CD34+ cells infused, day of start of GCSF and duration of GCSF-treatment. Results: Median time to reach PMN〉0.5 x 109/L was 12 days (9–17) for HAART neg group and 18 days (9–33) for HAART+ pts (p=0.069). Platelet engraftment (〉20x109/L) was achieved after a median of 20 days (11–28) and 26 days (11–455) respectively (p=0.25). Nevertheless, a multivariate regression analysis using as covariates HAART, GCSF (starting day) and amount of CD34+ cells infused, was performed. Accordingly no statisticaly significant results were found to relate with myeloid engraftment. Conclusions: Despite the HAART+ group shows longer interval to reach PMN and platelets engraftment following ASCT, we can not definitively conclude that HAART plays a negative influence on engraftment in this clinical setting. In order to clarify these results further recruitment of patients is needed.

Description: Patients with essential thrombocythemia (ET) and polycythemia vera (PV) have a high risk of thrombosis. Inherited thrombophilias are established risk factors for thrombosis. To determine if inherited thrombophilic abnormalities might contribute to the thrombotic risk in ET and PV, we investigated antitrombin III (ATIII), protein C (PC) and protein S (PS), polymorphisms of clotting factors V (FVQ506) and II (G20210A) and methylenetetrahydrofolate reductase (MTHFR) mutation in patients with ET and PV and we tried to correlate these results with prothrombotic markers such as bthromboglobulin (bTG), platelet factor 4 (PF4), prothrombin fragment 1+2 (F1+2) and d-dimer (DD). In addition, we analysed a possible association between inherited coagulopathy and thrombosis. ATIII, PC and PS antigen levels were assayed by ELISA as well as bTG, PF4 and F1+2. PCR-based assays were used for FVQ506, prothrombin and MTHFR. DD was measured by immunoturbimetric latex agglutination. The study involved 101 patients diagnosed with ET (71 patients, 30 men, 41 women, mean age 62.3 years) or PV (30 patients, 21 men, 9 women, mean age 65.4 years) according to conventional criteria. The duration of disease ranged between 1 to 12 years. Of 101 patients, 53 (52,47%) were non-carriers of mutations and among these 24 (45.28%) experienced thrombosis, 48 (47.52%) were carriers and 27 (56.25%) had thrombosis. With reference to carriers, 9/48 (14.58%) had combined mutations such as PC and PS (2/9), PS and G20210A (1/9), PC and MTHFR (4/9), G20210A and MTHFR (2/9). The prevalence of studied defects was 31.25% (15/48) and 18.75% (9/48) for deficiency of PC and PS, respectively, 8.33% (4/48) and 14.58% (7/48) for heterozygosity of FVQ502 and G20210A, respectively, and 45.83% (22/48) for MTHFR mutation. Deficiency of ATIII was not present. All patients had higher bTG, PF4 and F1+2 (365±654 IU/ml, 133±64 IU/ml and 2.6±2.5 nmol/L, respectively) than to controls (23.8±9.1 IU/ml, 5.5±2.8 IU/ml and 0.60.2 nmol/L, respectively) (p

Description: Background: Src Family Kinases (SFKs) play pivotal roles in normal B-cell development and in B-cell neoplasias. Our aim is to elucidate the roles of SFKs in signaling cascades resulting in cell proliferation or anti-apoptosis in multiple myeloma (MM). The pleiotropic cytokine interleukin-6 (IL6) is one of the major growth factors for MM cells. We have previously shown that IL6 induces the activation of the SFKs Hck, Lyn and Fyn and that Hck is associated with the IL6R beta chain (gp130) via an acid domain (AD) in gp130. Aim: We observed that an 18mer peptide (18AD), which is derived from the AD, inhibited the IL-6-dependent growth of myeloma cells. In order to develop a lead structure for small molecule inhibitors we wish to elucidate the cellular and molecular effects of peptide 18AD. Results: On the cellular level, 50–100μM of a membrane-permeable myristoylated peptide 18AD inhibited factor-dependent proliferation in 7TD-1 (mouse) and INA-6 (human) cells by ~75%. Taking the percentage of annexinV-stained cells after IL6-withdrawal as the reference value for the level of maximal apoptosis, treatment of 7TD1 and INA-6 cells with peptide 18AD resulted in 100 and 60 % of the respective percentages of apoptotic cells. A control peptide (18sc) with an identical amino acid composition but an arbitrarily scrambled sequence had no effect on proliferation and apoptosis. Similar effects of peptides 18AD and 18sc were observed in Baf-EH cells, which overexpress a chimeric erythropoietin receptor-gp130 and human Hck and grew in an erythropoietin dependent manner. Peptide 18AD had no significant growth inhibiting effects on BaF-HE cells cultured in the presence of IL3 or on factor-independent OKT1 hybridoma cells. In 7TD1 cells the expression of a kinase-inactive Hck mutant lead to a reduced proliferative response to IL6. On the molecular level, immunoprecipitation experiments in 7TD1 cells showed that peptide 18AD decreased the complex formation between Hck and gp130 in a concentration dependent way. Immunocomplex kinase assays in INA-6 cells showed that the IL6 induced activation of Hck, Lyn and Fyn was blocked by peptide 18AD, but not by peptide 18sc. Conclusion: We characterized peptide 18AD, which inhibits the association of Hck and gp130 as well as IL6-induced SFK activity. Thus, the observed anti-proliferative and pro-apoptotic effects of peptide 18AD may be due to inhibition of SFK-mediated pathways.

Description: Flow cytometric determination of peripheral blood CD34+ cells provides reliable measurements of circulating hemopoietic progenitors. Since the detection of the absolute number of circulating CD34+ cells has been found of clinical utility in the setting of chronic myeloproliferative disorders, we investigated whether peripheral CD34+ cells could play any role in the clinical work-up of B-cell chronic lymphocytic leukemia (B-CLL). In this view, we determined by flow cytometry the absolute number of circulating CD34+ cells in the peripheral blood of 28 patients (16 males and 12 females, median age 67 years) affected by typical B-CLL (Matutes score 5,4,3) and in different Rai stages of the disease (19 early stage: Rai 0, I, II; 9 advanced stage: Rai III, IV). Conventional and multiparameter flow cytometric analysis was performed utilizing a FACSCalibur cytometer (Becton Dickinson). Our data showed a significant increase in the number of circulating CD34+ cells in the peripheral blood of patients with B-CLL (median CD34+ cells:7.8mL) as compared to controls (median CD34+ cells 0.1mL) (p=0.008). No statistical difference between B-CLL patients in early versus advanced stage (p=0.5) and between untreated versus treated (p=0.7) was found, as well as there was no correlation with some of the clinical characteristics of B-CLL (WBC-count, LDH levels, Beta-2M). In 10 out of 28 B-CLL affected patients, circulating CD34+ cells were correlated with ZAP-70 and CD38 antigen but no correlation was found. In addition, we detected in the peripheral blood of 22 out of 28 patients small numbers of circulating CD34+ cells displaying the CD19+/CD5+ phenotype (median CD34+/CD19+/CD5+ cells:5.7mL) whereas these cells were absent in normal controls. This unexpected finding, whose significance remains to be clarified and still restricted to a small number of cases, could be directly correlated to the underlying lymphproliferative disease and might represent a pool of leukemic stem cells. However, further studies are warranted.

Description: Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a large clonal population of blood cells deriving from hematopoietic stem cells (HSCs) deficient in glycosylphosphatidylinositol (GPI)-anchored surface molecules. A current model postulates that PNH arises through negative selection against normal HSCs exerted by autoreactive T cells, whereas PNH HSCs escape damage. We have investigated the inhibitory receptor superfamily (IRS) system in 13 patients with PNH. We found a slight increase in the proportion of T cells expressing IRS. In contrast to what applies to healthy donors, the engagement of IRS molecules on T cells from patients with PNH elicited a powerful cytolytic activity in a redirected killing assay, indicating that these IRSs belong to the activating type. This was confirmed by clonal analysis: 50% of IRS+ T-cell clones in patients with PNH were of the activating type, while only 5% were of the activating type in healthy donors. Moreover, the ligation of IRS induces (1) production of tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) and (2) brisk cytolytic activity against cells bearing appropriate IRS counter-ligands. In addition, these IRS+ T cells show natural killer (NK)-like cytolytic activity to which GPI- cells were less sensitive than GPI+ cells. Thus, T cells with NK-like features, expressing the activating isoforms of IRS, may include effector cells involved in the pathogenesis of PNH.

Description: Apolipoprotein E (apoE) plays an important role in lipid metabolism. Its ɛ4 allele has been consistently associated with lipoprotein disorders but its connection to myocardial infarction (MI) is controversial. Because ɛ4 frequency decreases with age we thought that the contradictory results in different studies could be due to the wide age range of the subjects included. To test our hypothesis, ApoE genotyping was performed in 474 MI cases and an analysis was performed by percentiles of age. The frequencies of ɛ3ɛ4 genotype and ɛ4 allele in the MI group as a whole (subjects aged 31 to 92) were not significantly different from those in our area general population. However, significant differences were observed when comparing by group of age. The frequencies decreased as age increased. The ɛ3ɛ4 and ɛ4 frequencies were significantly higher in MI subjects aged 31 to 56 than in subjects over 74. The ɛ3ɛ4 genotype prevalence in an age and sex matched control group of subjects aged 31 to 56 was significantly lower than in the 31–56 year-old MI group. In conclusion, our data shows different ɛ3ɛ4 and ɛ4 frequencies depending on the age range of the subjects with MI, being significantly higher in the middle-aged group. This finding may help explain the discrepancies between studies analyzing association between apoE genotype and MI, and emphasizes the idea of considering apoE genotype for prevention at early age.