ALBERT

Beschreibung: Background CMV infection represents one of the main cause of morbility and mortality after stem cell transplantation. Type III interferons (IFNs), including IFNl1 (IL29), IFNl2 (IL28A) and IFNll3 (IL28B), are thought to display potent antiviral and immunemodulatory properties in vivo, which may overlap partially with those exerted by type I IFNs. Type I and Type III IFNsl both generate an antiviral state by triggering the JAK-STAT pathway, ultimately upregulating the expression of interferon-stimulated genes. Rs12979860 single nucleotide polymorphism (SNP) in IL28B gene region is well known to influence the spontaneous and treatment-induced clearance in HCV infection. Data on the relevance of such a SNP in other viral infections is still debated even, Bravo et al. recently documented a protective effect of the T allele against CMV infection in the Allogenic stem cell transplantation (Allo-SCT) (Journal of Medical Virology 2014.86:838). Aim of the study: the current study was aimed at investigating whether the IL28B polymorphism rs12979860 may effect on the incidence rate of clinically-relevant active CMV infection in the Autologous stem cell transplantation setting. Patients and methods: From October 2014 45 patients were included in the study because underwent a autologous stem cell transplantation for hematological diseases. The median age of the patients was 56 years (16-66 years). Patients were distributed according to Hematologic disease as following: 73% of the patients had Multiple Myeloma, 20% non Hodgkin Lymphoma, 5% Hodgkin Lymphoma e 2% Acute Myeloid Leukemia. The rs12979860 IL28B SNP (C/T) genotype was determined by Melthing analysis on DNA derived from peripheral blood samples. CMV DNAemia was determined by quantitative Real-Time PCR with a limit detection of 50 copies/mL (Artus, Qiagen). Patients were monitored for CMV DNAemia weekly for the three months after stem cell transplantation. RESULTS: CC genotype was detected in 51% of patients, CT genotype in 35.5% and TT genotype only in 13.5% of patients according to the lowest frequency of TT genotype harboring in general population. A clinically-active CMV infection was documented respectively in 66,6%, 17,4% and 12,5% of patients carrying TT, CT and CC genotype. A trend towards a higher incidence of clinically-active CMV infection was noted in the TT population with respect to CT and CC population (TT vs CC: P= 0.03 and TT vs CT: P=0.02). The duration and peak of CMV-DNAemia levels tended to be higher in patients carrying the TT genotype then in ones with CC or CT genotype, although statistical significance was not reached. A positive correlation was observed between day 7 post ASCT CMV-DNAemia and the monocytes and neutrophils count. By the contrast, a negative correlation was found between day 21 post ASCT CMV-DNAemia levels and the monocytes count on 35th and 45th days after ASCT. Conclusions In conclusion, our data suggest that patients with TT genotype have higher incidence of clinically-active CMV infection in Auto-SCT setting. Even though these results should be confirmed by a larger sample size, the lowest prevalence of TT genotype in general population and higher (66.6%) clinically-active CMV infection in TT genotype patients strongly support our data. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 3192 Autoimmune hemolytic anemia (AIHA) is a rare disease, with an incidence of 1:100,000 for warm AIHA and of 1:1,000,000 for cold AIHA. AIHA can occur without any evidence of an underlying disorder (idiopathic or primary AIHA) or can be diagnosed in association with another disease (secondary AIHA), such as a malignancy, a lymphoproliferative disorder (LD), an autoimmune disorder, an infection. A successful treatment of secondary AIHA is based upon the treatment of the associated disease. In particular, the detection of a possible underlying LD is a relevant issue since a successful treatment approach for both conditions includes monoclonal antibodies that target B lymphocytes producing the anti-erythrocyte autoantibody (AeAb). With the aim of unravelling the presence of an associated disease, between January 2000 and January 2012, patients with an AIHA diagnosis defined as “primary” on clinical grounds and observed at our institution were included in this study. Patients were required to show anemia, serological evidence of an AeAb by a positive direct antiglobulin test, clinical and laboratory signs of hemolysis, no previous history and/or clinical signs of an associated malignancy, LD, autoimmune disorder, infection or a prior use of drugs commonly implicated in the pathogenesis of AIHA. Before starting treatment, a diagnostic work-out including the following examinations was carried out: autoantibody profile (anti-nucleus; anti-cardiolipin; anti-gastric parietal cells; anti-beta 2-glycoprotein-I; anti-peroxidase and anti-thyroglobulin); HBV, HCV and HIV serology; total body CT scan or chest X-ray associated with an abdomen ultrasound, bone marrow (BM) aspirate and biopsy, peripheral blood (PB) and BM immunophenotype by 4 color flow-cytometry analysis (CD19/CD5/CD3/CD20; CD23/CD10/CD19/CD20; Igκ/Igλ; CD19/CD5; CD3/CD4/CD45/CD8). Sixty-four patients were included in the study, 23 males and 41 females with a median age of 65.5 years (range: 20–83). The median Hb value was 7.9 g/dl (range: 4–11 g/dl), the median value of indirect bilirubin 2.2 mg/dl. The AeAb isotype form was an IgG in 38 cases (59%), IgM in 25 (39%; cold IgM associated with IgG, 4 cases; warm IgM, 1 case) and IgA in 1. After the initial screening, a primary AIHA diagnosis was confirmed in 38 cases (59%), while in 21 patients (33%) an underlying LD was detected, in 2 (3%) a cancer (breast, larynx), in 2 (3%) an autoimmune disorder (Hashimoto's thyroiditis, athrophic gastritis), in 1 an active HCV hepatitis. In the majority of cases (19/21, 90.5%) the diagnosis of LD was made on the basis of the BM biopsy. In all cases with BM involvement, PB flow-cytometry revealed the presence of monoclonal B lymphocytes (median number of clonal lymphocytes, 0.405×109/L) displaying most frequently a lymphoma-like phenotype (CD5−/CD20+/CD23±). As expected, the group of patients with the evidence of a LD included a significantly higher rate of cases with an IgM AeAb as compared to the group with “true” primary AIHA (67% vs 12.5%; p= .005). Taken together, the results of this study demonstrate that in 41% of patients with an AIHA defined as primary on clinical grounds, an extended diagnostic work-out allowed to identify an associated disease, most frequently a LD involving the BM associated with a clonal B population in PB. These data suggest that in patients with a diagnosis of AIHA, an extended screening including a BM biopsy and a PB immunothenotype should be considered in order to identify (or exclude) the presence of a LD and to address the appropriate treatment. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1377 Targeting the CLL microenvironment with immunomodulatory agents like lenalidomide results in antileukemic activity. Addition of lenalidomide to an effective regimen such as fludarabine and cyclophosphamide (FC) could further increase therapeutic activity. On this basis, we designed a study aimed at evaluating the activity and safety profile of FC combined with lenalidomide (FCL) in previously treated CLL patients. Treatment consists of 6 monthly courses of FC (F, fudarabine: 30 mg/m2 iv and C, cyclophosphamide: 250 mg/m2, days 1–3) combined with 14 consecutive days of lenalidomide administration (days1-14). The first phase of this study was focused on defining the maximum tolerated dose (MTD) of lenalidomide given in combination with FC. In all patients, lenalidomide was given at the starting dose of 2.5 mg daily during the first course. For the subsequent courses the dose of lenalidomide was progressively escalated to reach a maximum daily dose of 5 mg, 10 mg and 15 mg, respectively, in each 3 cohorts of 3 patients, unless a dose limiting toxicity (DLT) was experienced. DLT was defined as persistent and severe hematologic toxicity, grade ≥2 tumor lysis syndrome (TLS), grade ≥3 tumor flare reaction (TFR) or other grade ≥3 toxicities. Supportive treatment included primary prophylaxis of granulocytopenia (G-CSF, days 6–11), PC and HVZ prophylaxis, thromboembolic prophylaxis with low molecular weight heparin (enoxaparin, 4000 units/sc/day) and TLS prophylaxis (oral hydration, allopurinol 300 mg/day, days -3 - 90). Strict contraceptive measures were required for all males and females with childbearing potential. At present, 9 relapsed CLL patients with advanced disease have been included in the first phase of the study focused on identifying the MTD for lenalidomide given in combination with FC. The median age was 63 years (range: 50–68); the median number of prior treatments was 2 (range: 1–2). All patients but 1 had been previously treated with the FC/FCR regimens; the majority showed a considerably high white blood count (range: 33–216 × 109/L) and 2 had a bulky nodal disease. Adverse cytogenetic abnormalities were detected by FISH in 5 cases (17p-, 2 cases; 6q-, 3 cases). While no DLT was observed in 3 patients who reached the 5 mg dose of lenalidomide given in combination with FC, a DLT was recorded in 2 of 3 patients who received 10 mg of lenalidomide (pneumonia; grade 3 hepatic toxicity). The 5 mg dose of lenalidomide was tested in 3 further patients with no DLT. Thus, 5 mg of lenalidomide was defined as the MTD of lenalidomide given in combination with FC. A marked reduction of white blood count was observed within the first 2 weeks of treatment in all cases but 1. A response was observed in 6 patients (67%; CR, 3; PR, 3), while a treatment-failure was recorded in 3 cases (SD, 2; Richter's syndrome,1). Despite G-CSF prophylaxis, the majority of patients experienced transient grade 3–4 neutropenia. Grade 1 fatigue and constipation were frequently observed during the first course of treatment. A transient grade 1 TFR was documented, while no episodes of TLS and thrombosis were recorded. Our findings indicate that 5 mg daily is the MTD of lenalidomide given in combination with FC. Initial responses suggest that FCL is a promising investigational regimen for patients with CLL who have failed prior treatment with fludarabine-based combinations. In the ongoing phase 2 of this study 32 patients are planned to receive 5 mg of lenalidomide in combination with FC. Disclosures: Foà: Celgene: Membership on an entity's Board of Directors or advisory committees.

Beschreibung: Abstract 4373 In chronic lymphocytic leukemia (CLL), the distribution and prognostic impact of genetic and molecular markers have been mostly investigated in retrospective studies including patients in different phases of the disease. From November 2002 to May 2009, we prospectively assessed the clinical and biologic features of 219 young CLL patients ('65 years), distributed in three different cohorts: 124 cases at diagnosis, of which 99 in Binet stage A (group 1), 68 at first progression (group 2), 27 at progression after one or more therapies (group 3). Median age was 52 years for group 1, 57 years for group 2 and 61 years for group 3. Advanced disease defined as Rai stage III/IV was present in 6%, 19% and 48% patients and as Binet stages B/C in 19%, 68% and 78% patients in the 3 cohorts, respectively. The proportion of unmutated IgVH cases progressively and significantly increased, being 36% in group 1, 48.5% in group 2 and 75% in group 3 (p 0.001). The same trend was found for CD38 (p 0.01). The incidence of ZAP-70 expression and p53 mutations showed a trend towards a progressive increase among the three groups (41%, 55% and 56.5% for ZAP-70 and 4%, 7% and 12.5% for p53 mutations), without however reaching significance. The incidence of del(17p) ≥20% raised progressively from group 1, to groups 2 and 3 (2%, 4.5% and 18.5%, respectively; p 0.001). The incidence of del(11q) increased from group 1 to 2 (10% and 16%; p 0.18), though it was not higher in group 3 (7.5%). Focusing the comparison to groups 1 and 2 only, i.e. patients with CLL at diagnosis in all stages and patients at first progression of the disease requiring therapy, the distribution of prognostic markers did not differ significantly, except for a lower proportion of cases expressing CD38, although the proportion of del(17p) 〉20% and 〉10% and of +12 doubled. When the analysis was restricted to stage A CLL at diagnosis, there was a highly significant difference in the lower proportion of unmutated IgVH (0.003), CD38 expression (p 0.009), ZAP-70 expression (p 0.004), del(17p) (p 0.034) and in the higher proportion of del(13q) (p 0.028) in comparison with CLL at first progression. No patient with stage A CLL at diagnosis showed del(17p) ≥20%, whilst 2% showed del(17p) 〉10% and 3% harbored p53 mutations. Of the 40 patients evaluated by FISH at multiple time points, 35% showed clonal evolution. Only previous treatment was significantly associated with the development of clonal evolution, whilst no correlation with the IgVH mutational status, ZAP-70 or CD38 expression was found. Patients of group 1 required treatment after a median time of 49.6 months from diagnosis. Treatment-free interval (TFI) was significantly shorter in cases with IgVH unmutated vs. mutated (at 48 months, 16.5% and 68.3%, respectively p

Beschreibung: Background Although in some areas of the world a chance of finding a related HLA- matched non sibling donor could be as high as 13% to 18%, and approximately 13% of patients could have a one-antigen mismatched related donor, the role of such alternative donor HSCT in thalassemia is not well established. Our previous study (BMT 2000,25:815) examining alternative related BMT for thalassemia was not successful due to a high graft failure, GVHD, and low disease-free survival rates. In 2005 we adopted a new transplant approach for alternative related donor transplantation in thalassemia and here we report transplant outcomes. Patients and Methods Here we used a new treatment protocol (Pc 26.1) in 16 thalassemia patients (median age 9.6 years; range 1.4-24 years) to perform BMT using phenotypically HLA-identical or one-antigen mismatched related donors (related donors-RDs). We compared these results with HLA matched sibling donor (matched sibling donors-MSDs) BMT in 66 patients (median age 10 years; range, 1.8-27 years). The two groups were similar in terms of disease and demographic characteristics. Patients in the RD group received pre transplant cytoreduction/immunosuppression with hydroxyurea, azathioprine from day -45 pretransplant, and fludarabine from day -16 through day -12. Their conditioning regimen consisted of weight based targeted i.v. Busulfan (Bu), thiotepa (TT) (10 mg/kg), Cyclophosphamide (CY) (200 mg/kg), and Thymoglobulin 10 mg/kg. The conditioning regimen in the MSD group consisted of BuCY200 ±TT10 for class 2 patients and BuCY90-160 preceded by preconditioning cytoreduction/immunosuppression for class 3 patients. All patients received cyclosporine, methylprednisolone, and methotrexate as GVHD prophylaxis. Most patients in both groups were heavily transfused before transplantation and had moderately severe iron overload and liver fibrosis. In the RD group 11 of 16 donors were HLA-phenotypically identical (parents eight, cousin two, and uncle one), and 5 were one-antigen mismatched (siblings three, mother or aunt one). Results In total, 16 patients in the RD and 58 (88%) in the MSD group had sustained engraftment. The median times to an ANC〉0.5 x 109/L or to a platelet count 〉20 x 109/L were similar in both groups. Graft rejection occurred in 8 MSD group patients but in none of the RD group patients. The cumulative incidence of rejection was 12% (95% CI, 6–21%) in the MSD group and 0% in the RD group, which was not statistically significant (P = 0.15). The cumulative incidence of acute GVHD (grades 2–4) in the RD group (19%; 95% CI, 4–41%) was less than that in the MSD group (36%; 95% CI, 24–48%), but the difference was not statistically significant (P = 0.21). Two patients in the RD group and six in the MSD group had chronic extensive GVHD. At present, all patients are off immunosuppressive medication. The cumulative incidence of transplant-related mortality (TRM) at 100 days and 1 year was 0% and 6% (95%CI 1-26%) in the RD and 8% and 8% (95% CI, 3–16%) in the MSD group, respectively (P = 0.77). At the time of survival analysis, 15 patients (94%) in the RD group and 61 patients in the MSD group (92%) were alive, with median follow-up durations of 72 months (range, 17–93 months) and 80 months (range, 25–107 months), respectively. The probabilities of overall survival were 94% (95% CI, 63–99%) for the RD group and 92% (95% CI, 83–97%) for the MSD group (P = 0.83). The respective probabilities of thalassemia-free survival were 94% (95% CI, 63–99%) and 82% (95% CI, 70–89%), with no statistically significant difference (P = 0.24). Despite the preexisting disease and treatment-related organ damage this intensified preparative regimen was well tolerated as no significant toxicity was observed. The treatment-related toxicities were similar for the two group of patients, and none of the patients experienced grade 4 toxicity. Conclusion The novel treatment protocol Pc 26.1 effectively and safely prevented graft rejection and ensured a high thalassemia-free survival rate in patients who received BMT from related donors who were not HLA-matched siblings. Our data show that thalassemia patients treated with Pc26.1 and receiving RD transplant have similar outcomes (rates of OS, TFS, GVHD, and TRM) to recipients of MSD transplantation. These findings are significant because they expand the availability of the treatment to more patients. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Key Points A novel approach to BMT for thalassemia using related phenotypically matched or 1-antigen–mismatched donors improved transplant outcomes. BMT from phenotypically matched or 1-antigen–mismatched donors is associated with a high thalassemia-free survival rate (94%).

Beschreibung: In chronic lymphocytic leukemia (CLL) the distribution and prognostic impact of genetic and molecular markers has been assessed on retrospective series of patients in different phases of the disease. Our aim was to assess the distribution and clinical significance of a comprehensive panel of clinical and biologic parameters prospectively evaluated at presentation in all young patients diagnosed with CLL at our Institution, taking advantage of the fact that in Italy individuals with a lymphocytosis are referred to the hematologist for the diagnostic work-up. From November 2002 to June 2008, 105 young CLL patients (20% cells) and del(11q) (cut off 〉10% cells) was 2% and 7%, respectively. Patients with del(17p) or del(11q) exclusively showed unmutated IgVH and ZAP-70+, and were mostly CD38+. p53 mutations were present in 4 cases, 3 with unmutated IgVH and del(17p) and 1 with mutated IgVH and no del(17p). In univariate analysis, the following variables resulted associated to a short TFI: advanced stage Binet B/C and Rai I/IV (