ALBERT

Description: Abstract 5146 INTRODUTION: Most hereditary hemochromatosis (HH) patients are homozygous for the p.C282Y mutation in the HFE gene. But rare HFE variants have been shown to be associated with HH. In addition, four main types of non-HFE HH are caused by mutations in the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1) genes. The main aim of this study was to screen for HFE, HJV, HAMP, TFR2 and SLC40A1 mutations and to investigate their relationship with HH. MATERIAL E METHODS: Fifty-one Brazilian patients with primary iron overload (transferrin saturation 〉 50% in females and 60% in males) were eligible. Subsequent bidirectional sequencing for each exon of HFE, HJV, HAMP, TFR2 and SLC40A1 genes was performed. The effect of HFE p.V256I novel mutations on protein structure was analyzed by in silico molecular dynamic and free energy calculations. RESULTS: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, the p.S65C mutation was found in heterozygosis with p.H63D in two patients and the homozygous genotype for the p.H63D was found in two patients. One novel mutation (p.V256I) was indentified in heterozygosis with the p.H63D mutation. In silico modeling analysis of protein behavior suggested that the p.V256I mutation does not reduce the binding affinity between HFE and β2-microglobulin (β2M) in the same way the p.C282Y mutation does compared with the native HFE protein. Sequencing HJV revealed one patient presenting Juvenile hemochromatosis (JH) (homozygous genotype for the HJV p.G320V mutation); two patients carrying heterozygous genotype for the p.E302K mutation; and one patient with heterozygosis p.A310G polymorphism. Sequencing HAMP revealed one patient carrying p.P48G novel mutation in the heterozygous form. Three and five non-pathogenic polymorphisms were observed in the TFR2 and SLC40A1 genes, respectively. Sequencing SLC40A1 also identified one patient with homozygous genotype for the p.R561G described mutation; and one patient with homozygous genotype for the p.G204S novel mutation. CONCLUSION: The HFE p.C282Y in homozygosis or in heterozygosis with p.H63D was the most frequent mutation associated with HH in our sample population. The novel HFE p.V256I mutation could not be implicated in the molecular basis of the HH phenotype. Two described mutations, HJV p.E302K and SLC40A1 p.R561G, could have functional consequences according to previously studies contributing to HH phenotype. Two novel mutations, HAMP p.R48G and SLC40A1 p.G204S, may be implicated with iron overload in these patients, but further studies are need to explain their impact on proteins. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4702 INTRODUCTION The allogeneic hematopoietic stem cell transplantation remains the only curative option for certain hematological malignancies. The use of reduced intensity conditioning (RIC) has diminished the transplant-related mortality, making it possible to consider this therapeutic option in elderly patients with associated morbidity. The graft versus host disease (GVHD) is a late-onset complication in this type of transplant which still remains one of the major causes of morbidity and mortality. Hematopoietic chimerism (HQ) levels related to relapse or GVHD in RIC allogeneic transplantation (allo-RIC) have not been established. We have studied the relationship between HQ and the incidence of GVHD and relapse, using real time-PCR for chimerism quantification. PATIENTS AND METHODS We evaluated 16 patients with hematological malignancies (11 AML, 3 ALL, 1 CLL, 1 plasma cell leukemia) undergoing allo-RIC in the last three years. The median follow up of this series was 310 days (range 86–958 days), with a median age of 53 years (range 26–64). A sibling donor was found in 11 cases (69%) and fourteen donors (88%) were HLA identical. Bone marrow was the source of progenitors in 12 cases, peripheral blood in 3 and umbilical cord in one. The conditioning regimen was: fludarabine + busulphan (44%), fludarabine + busulphan + timoglobulin (23%), fludarabine + melphalan (13%) and others (21%). GVHD prophylaxis was performed with cyclosporine A (CsA) + methotrexate (44%), CsA + mycophenolate (31%) and tacrolimus + syrolimus (19%). The HQ was studied in peripheral blood DNA at the time of granulocyte engraftment, and later 30, 45 and 60 days after stem cells infusion. Chimerism was expressed as a percentage of residual host cells. Quantitative real-time PCR amplification of null alleles or insertion/deletion polymorphisms was used for HQ analysis (Jimenez et al, 2005; Leukemia 2005; 19:336-43). Chimerism was expressed as a percentage of residual host cells. Chi-square test was employed to evaluate qLas variables cualitativas se analizaron mediante chi-cuadrado y se usaron test no paramétricos para las cuantitativas. ualitative variables and non-parametric tests for quantitative variables. El análisis multivariante fue realizado mediante regresión de Cox. The Kaplan-Meier model with log-rank test comparisons were applied for survival analysis. RESULTS The median time of engraftment was 16 days (12-27) for granulocytes and 18 days (10-69) for platelets. Ten patients (63%) developed acute GVHD (aGVHD), eight of them Grade II-IV. The incidence of late-onset aGVHD was 38%, with a mean time to appearance of 117 days (90-190). Six patients (38%) developed chronic GVHD (cGVHD), and in four cases a previous late-onset aGVHD had been recorded. In our series, disease relapse was observed in 6 patients (37.5%), whereas 7 patients died (3 infections, 2 progressive disease, 1 aGVHD and one bleeding). No relapse was found in the group of patient who developed cGVHD compared with 60% relapses in those without cGVHD (Fisher's exact test, p = 0.026). However, overall survival did not differ significantly in both groups. Median HQ at the time of granulocyte engraftment was 2,65% (0.07 to 55), on day +30 was 1.4% (0.01-12), on day +45 was 1.0% (0.01-9) and on day +60 1.7% (0.01-15). HQ showed significantly lower levels in those patients who did not relapsed either on day +30 (0.4% vs 4.0%, p= 0.016), on day +45 (0.35% vs 5.5%, p= 0.04), and on day +60 (0.4% vs 4.5%, p= 0.04). With respect to cGVHD, we observed a trend to lower HQ levels in those patients who developed cGVHD compared with those who did not develop it on day +30 (0.25% vs 1.9%, p= 0.056) and +45 (0.26% vs 1.7%, p= 0.066), but these results were at the limit of statistical significance. CONCLUSIONS Our data show that HQ levels may help us to identify a group of patients whith higher risk of relapse also in allo-RIC. The monitoring of HQ may also be useful to predict patients who will develop cGVHD after SCT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4832 Background Myeloproliferatives neoplasms (MPN) are clonal stem cell disorders characterized by proliferation of one or more of the myeloid lineages, associated with genetic abnormalities that include translocations or point mutations of genes that encode cytoplasmic or tyrosin quinase (TK) receptor proteins. This produces an abnormal constitutively activation of signal transduction pathways, leading to an unregulated proliferation. According to the WHO criteria, MPN are classified into BCR-ABL+/Philadelfia Ph+: chronic myeloid leukemia (CML) and MPN BCR-ABL fusion-/Ph-. A single acquired point mutation, JAK2V617F, has been described in 95% of Polycytemia vera (PV), in 50 % of essencial thrombocythemia (ET) and idiopathic myelofivrosis (IMF), and generally absent in MPN Ph+. In the last years, it has been described the co-ocurrence of both BCR-ABL and V617F mutation in few cases of CML patients. We report here the rare and concomitant ocurrence of JAK2V617F mutation with BCR-ABL translocation at presentation in atypical CML. Methods Blood samples from six patients with clinical suspicion of MPN diagnosis, were referred to our laboratory to cytogenetic studies and molecular analysis of BCR-ABL fusion gene expression by conventional RT-PCR and JAK2V617F status mutation by ASO-PCR in three of them. All patients showed a slightly elevated white blood cells level (7200-25900), trombocythosis (700 -1036 platelets) and small splenomegaly. Three patients, after CML diagnosis, recieved Imatinib therapy and were monitored by quantitative BCR-ABL real time PCR. Due to persistent thrombocytosis, slightly elevated white blood cells level and small splenomegaly; JAK2 status was analized in these blood specimens, and later retrospectively in the stored initial diagnosis samples. Results BCR-ABL rearrangement (b3a2 isoform) and JAK2V617F mutation were identified in all 6 patients at diagnosis. Three cases showed lack of Ph chromosome, 1 patient showed 15 % Ph+ metaphases and in the remained two patients no data was available. Quantitative PCR for BCR-ABL expression performed in 3 patients during follow-up (8-12 months) showed BCR-ABL/BCR ratio 〈 = 0.0018 % (scored according to the International Scale) and the presence of JAK2V617F mutation. Retrospective assessment of stored bood samples showed that JaK mutation was already present at the time of the diagnosis of CML. Conclusions The coexistence of both genetic defects, BCR-ABL fusion gene and JAK2V617F mutation in NMP patients is a rare and uncommon feature. We found 6 MPN patients hourboring both genetics features in blood dignosis samples. In three cases JAK2V617F mutation was detected in MPN patients BCR-ABL positive after the remission induction with Imatinib. The rapid remission of BCR-ABL transcript, after a short period of Imatinib treatment, led us to think that BCR-ABL fusion gene expression was present in a low burden at diagnosis. The complete reduction of BCR-ABL rearrangement, after the imatinib therapy, and the persistence of JAK 2 mutation suggests two possible mechanisms for this double genetic alteration: 1) a haematopoyetic cell subclone with a pre-existing JAK2V617F acquires the BCR-ABL fusion gene, which confers a selective advantage to double mutant progenitor; 2) two clons, one of them having BCR-ABL rearrangement, and the other one the JAK2V617F mutation (biclonal origin). These cases intend to contribute to the discussion about the onset of the molecular alterations, and their correlation with the differente phenotypes and clinical management. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4737 Objective: Microscopic examination of peripheral blood cells is an important diagnostic tool. We evaluated the CellaVision DM96 (CellaVision AB, Lund, Sweden), an automated image analysis system for digital peripheral blood cell analysis, comparing the results with direct manual microscopy. The system obtains digital images of the blood cells at high magnification and these images are analyzed using a neural network based on a large database of cells. Material and Methods: We analyzed 234 PB films stained with May-Grünwald-Giemsa from patients of the Hospital Clínic of Barcelona. Leukocyte values were from 1.12 to 282 × 109/L (Advia 2120, Siemens Healthcare Diagnostics SL). 177 of the PB films were from patients with hematological diseases: lymphoid neoplasias: 83, acute leukemias: 52, chronic myeloproliferative diseases: 20, myelodisplastic syndromes: 18, paroxysmal nocturnal hemoglobinuria: 2, hemoglobin S: 1 and thrombotic thrombocytopenic purpura: 1. Manual differentials were performed using standard microscopy. After the microscopic analysis the slides were loaded into the CellaVision DM96 obtaining digital images of preclassified cells, which were verified or corrected when necessary by the hematologist (DM96POST). WBC differentials were abnormal in 120 cases. Statistical analysis was performed using correlation (Pearson) and concordance (Lin) tests. Results: Correlation coefficients between results obtained from the CellaVision DM96 preclassification and by conventional direct microscopy were excellent for segmented neutrophils, lymphocytes, monocytes and blasts (r〉0.87

Description: Abstract 4839 Introduction. Chronic myeloid leukemia (CML) is characterized by the Philadelphia chromosome (Ph) observed in more than 90% of patients with CML as a result of t(9;22)(q34;q11), leading to the formation of chimeric gene BCR/ABL encoding for proteins with abnormal tyrosine kinase activity. Cytogenetic variants of Ph chromosome can be identifed in 5 to 10% of CML patients, involving additional chromosomes other than 9 and 22. To explain the formation of variant translocations one-step, two-step and multi-step mechanisms have been proposed. Rarely, the variant Ph chromosome results from a BCR insertion on the ABL region and form a BCR/ABL fusion gene, generally mapping to 9q34, instead of the usual location at 22q11. In very few variant Ph cases, the insertion of the BCR/ABL product in a third chromosome was demonstrated. Case Report 28 year-old man, with bilateral central scotoma and gingivorragia. Physical examination: Grade 4 splenomegaly. Peripheral blood count showed hemoglobin concentration 11.5 g/dl, platelet count: 300.000/mm3, and white blood cell count 590.000/mm3. Blood smear: myelemia exhibiting 30% of myeloid blasts. Bone marrow biopsy: panmyelosis showing 20% of myeloid blasts. Cytogenetic analysis by G-banding performed in peripheral blood verified the following karyotype: 46, XY, t(9;22;10)(q34;q11;q24)[20] The analysis of the BCR-ABL fusion gene according to standard protocols detected the presence of the b3a2 isoform. Fluorescence in situ hybridization (FISH) studies using dual color dual fusion probes in metaphases showed a signal pattern 1F2G1R. The fusion signal mapped to 10q24, the red signal to 9q34, and the normal green signal to chromosome 22, while a second low intensity green signal mapped to the Ph chromosome. No signal was observed in der(9). Interphase FISH analysis in nuclei (n=200) presented the same signal pattern. Instead of using whole chromosome probes for 9 and 22, we hybridised probes used to detect DiGiorge syndrome. These probes detect gene control ARSA (spectrum green) localized at 22q13 and Tuple1 at 22q11 (spectrum orange). Two signals, green and orange were identified in normal chromosome 22. Ph chromosome showed the orange signal, whereas the green signal mapped to der(10). Discussion. The localization of the hybrid BCR/ABL gene on chromosomes other than 22q is a rare event wich can only be detected by FISH techniques. When these unusual translocation occurs, the hypothesis most often put forward is that several consecutive chromosome rearrangements have taken place. In the present case the interpretation of karyotypes, FISH data and molecular evidence lead to the following hypothesis: Insertion of the BCR sequence from chromosome 22 to chromosome 9 may have ocurred, producing a BCR/ABL fusion in der(9). The Ph chromosome detected by G-banding showed a different green fluorescence intensity in the metaphase FISH signal pattern with BCR/ABL dual color dual fusion probes, as a result of an insertion on chromosome 9. This first event was followed by the translocation between the derivative 9 and chromosome 10, being the final localization of the BCR/ABL gene in 10q24. FISH analysis using a DiGeorge syndrome probe, supports the hypothesis of a multistep mechanism underlying insertion and translocations events in the present case. The relocation of BCR/ABL fusion sequence on sites other than chromosme 22q11 represent a rare type of variant Ph translocation. At least 21 cases described in the literature, showed fusion gene BCR/ABL located at 9q24. Only 12 patients with variant Ph were reported bearing BCR/ABL on a third chromosome. All of them involved a masked Ph chromosome. To our best knowledge this is the first report showing a variant Ph chromosome detected by G-banding in a CML patient due to a BCR insertion on ABL sequences and exhibiting the fusion signal in a third chromosome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2070 Background and Aim. Patients with transfusion-dependent hemoglobinopathies, as thalassemia major (TM) and sickle/β-thalassemia (S/βThal), have an high risk to be infected by hepatitis C virus (HCV), developing chronic hepatitis C (CHC). Current standard-of-care therapy for CHC consists of pegylated interferon-α 2 and ribavirin (PEG-IFN- α/RBV). A genome-wide association study (GWAs) identified a single nucleotide polymorphism (SNP), on chromosome 19, 3 kilobases (kb) upstream of the IL28B gene (IL28B -3kb C〉T). This SNP is associated to the natural clearance of HCV-RNA. The IL28B -3kb C〉T SNP is in strong linkage disequilibrium with a non-synonymous variant in the exon2 of IL28B gene (213A〉G, K70R). Because of in these patients the PEG-IFN-α/RBV treatment of chronic HCV infection could be complicated by severe side adverse events, i.e. ribavirin-associated hemolysis, it is noteworthy to predict the spontaneous C virus clearance by investigate these two IL28B DNA variants. Therefore, in a cohort of 42 patients with hemoglobinopathies, it was analyzed the two mentioned DNA variants to evaluate their allele frequencies and to eventually correlate them to the C virus clearance. Patients and Methods. Forty two patients with hemoglobinopathies, not-treated with PEG- IFN-α/RBV, were included in this study. Molecular analysi:. Genomic DNAs were extracted from peripheral blood mononuclear cells. DNA variants, IL28B -3kb C〉T e K70R, were investigated by direct genomic sequencing (CEQ880-Beckman), endonuclease digestion and reverse dot-blot hybridization. PCR primers, specific for the investigated DNA regions, were designed in our laboratory on the basis of IL28B gene map (GENATLAS) and FASTA SNP sequences. According to literature, the genotype IL28B -3kbCC/K70RAA was considered most frequently associated with the spontaneous HCV-RNA clearance, whereas the genotypes IL28B 3kbTT/K70R GG and -3kbCT/K70R AG were considered most frequently associated with a persistent infection. Result: Allele frequencies of -3kbC〉T polymorphism were 63,1% for the favourable C allele and 36,9% for the T allele. Allele frequencies for the K70R variant were 63.1% for the A allele and 36,9% for the G allele. Twenty of 42 studied patients (47,6%) showed a spontaneous HCV-RNA clearance: 15/20 (75%) had IL28B genotype -3kbCC/K70R AA, 5/20 (25%) had IL28B genotype -3kbCT/K70R AG. Twenty two of 42 studied patients (52,4%) showed HCV-RNA persistence: 13/22 (59,1%) showed viral genotype 1b and IL28B genotype -3kbCT/K70R AG; 4/22 (18,2%) showed viral genotype 1b and IL28B genotype -3kbCC/K70R AA; 4/22 (18,2%) showed IL28B genotype -3kbTT/K70R GG (3 had viral genotype 2a2c and one viral genotype 1b; one case had viral genotype 2a2c and was homozygous for the favourable -3kb C allele but it was heterozygous for the K70R mutation (Table 1). Conclusion: It seems that IL28B genotype has a marked impact on natural clearance of HCV-RNA: 15/20 (75%) not treated patients who showed a spontaneous HCV-RNA clearance had an IL28B genotype promoting the viral defence (IL28B-3kbCC/K70R AA) (Table 1). These data are in agreement with Thomas et al (Thomas et al. Nature 461,798-801-2009) who showed a -3kb CC genotype in the 60% of patients who clear HCV. Therefore, they may suggest that waiting and watch approach should be advised in patients with hemoglobinopathy and favourable IL28B genotype. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3932 ITK (Interluekin-2 Inducible Tyrosine Kinase) is a member of the TEC family of intracellular protein tyrosine kinases. ITK is highly expressed in T cells and NK cells, with expression detected in mast cells. ITK plays a key role in several aspects of T cell biology, including T cell development, differentiation, migration, proliferation and activation. The function of ITK in immunity and allergy is well documented. T cells from ITK knock out mice show several developmental and functional defects, including defective signal transduction, altered CD4+ to CD8+ T cells ratios, reduced Th2 lineage differentiation, diminished IL4 and IL2 production and reduced T cell proliferation. Importantly ITK deficient mice fail to mount an immune response to infection and show reduced allergic asthma reactions. In contrast to its well described role in immune function, ITK's function in cancer biology is still emerging. Recent studies had reported enhanced ITK expression and activation of the ITK pathway in several types of leukemias and lymphomas. In addition, the dependence of T cell malignancies on an ITK-regulated pathway, namely the IL2/IL2R (CD25) pathway, has also been observed. Taken together, this information indicates that ITK is a therapeutic target, with applicability in leukemias and lymphomas. MannKind scientists have developed a series of selective small molecule ITK inhibitors, including the orally available tool compound described within, and evaluated their activity in enzyme, cell-based and in vivo studies. In cellular assays, the compounds showed significant inhibition of the T cell-receptor mediated activation of the ITK pathways and related downstream cytokine production. In addition to inhibiting the phosphorylation of ITK and its downstream mediator, PLCg, our tool compounds inhibited the production of IL2 and expression of CD25 in a dose dependent manner. Importantly, our compound regulated the in vitro growth of tumor T cells but not that of unrelated control cells. In vivo studies revealed that the tool compounds inhibited the growth and progression of patient derived ATL tumors in a xenograft pre-clinical model, and prolonged the survival of treated mice in a dose dependent manner, in addition to regulating cytokine production in vivo. In summary, our team has identified ITK selective compounds with demonstrated on-target and anti-tumor activity in vitro and preclinical T cell tumor models, and validated this pathway relative to T cell malignancies. This effort provides a platform for further compound optimization and evaluation for hematologic malignancies. Disclosures: Faris: MannKind Corp: Employment. Malyankar:MannKind Corp: Employment. Zeng:MannKind Corp: Employment. Kertesz:Mannkind Corporation: Employment, Equity Ownership. Vuga:MannKind Corp.: Employment. Rosario:MannKind Corp: Employment. Bot:MannKind Corp: Employment.

Description: Abstract 4239 Paroxysmal Nocturnal Hemoglobinuria (PNH) is associated with clonal expansion of stem cells with an acquired somatic PIG-A mutation. The PIG-A gene is essential for the biosynthesis of glycosylphosphatidylinositol (GPI), and mature blood cells derived from the PNH stem cell clone exhibit a loss of all proteins that require this structure for attachment to the cell surface, notably the complement inhibitors CD55 and CD59. The loss of these proteins on the surface of red cells is responsible for hemolysis, and thrombosis may be the consequence of the loss of these proteins from the surface of platelets: both hemolysis and thrombosis can be attenuated by anti-complement therapy. Thrombosis can occur, however, despite anticomplement therapy and despite anticoagulation and has been historically the most important determinant of death in patients with PNH. Some patients will present with thrombosis and some patients will not be candidates for anticoagulation or anticomplement therapy: therefore treatment of thrombosis remains an important part of the management of PNH patients. Thrombolysis with tissue plasminogen activator (tPA) in PNH has been reported in small series or case reports, generally with encouraging outcomes. Here we report what we believe to be the largest series on the outcome of the use of tPA. Of 38 patients with PNH who had at least one thrombotic event, 13 were thought to have had a thrombus sufficiently recent to be amenable to fibrinolysis; of these, 4 patients were regarded as ineligible on account of active hemorrhage or high risk of hemorrhage. Of the 9 eligible patients who received tPA, all of whom had potentially life-threatening thromboses, 3 also required tPA on subsequent hospitalizations, and the results of a total of 15 hospitalizations during which tPA infusions were given are reported here. tPA was given in the ICU by systemic infusion through a peripheral vein at a dose of 1 mg/kg delivered over 24 hours, with anticoagulants withheld temporarily during this time. Response was monitored by follow-up imaging, and most patients required several 24 hour infusions. Platelets were given for thrombocytopenia and FFP was given to reverse oral anticoagulation or when low circulating plasminogen was documented. On all 15 occasions a radiologically documented response was obtained, including reversal of thrombosis in hepatic veins, portal veins, the IVC, cerebral dural venous sinuses, and an intrahepatic portocaval shunt. Among the 15 courses of tPA, serious hemorrhagic complications developed in 3 cases. At last follow-up visit, of the 9 patients treated, 3 have expired, one patient (who has been non-compliant with post treatment anticoagulation and anticomplement therapy) was in good clinical condition despite extensive residual occlusions, and 5 others were in good to excellent condition in terms of clinical and radiological outcome. The only patient in whom tPA may have contributed to a fatal outcome also had complications of ‘heparin induced thrombocytopenia with thrombosis’ (HITT), which we diagnosed in a milder form in 3 additional patients. The other two fatalities were associated with bowel edema (probably due to progressive small vessel thrombosis) in one case, and a progressive concurrent myeloproliferative disorder associated with a JAK2 mutation in the other case. On the other hand, we feel tPA must be credited as having been immediately life-saving in 2 patients who had been moribund with Budd-Chiari syndrome, and in one who had impending renal failure associated with an IVC thrombosis. Given the high incidence of HITT, we favor the use of direct thrombin inhibitors or fondaparinux rather than heparin products in patients with PNH. Given the high mortality and morbidity associated with thrombosis in PNH patients, and given the excellent radiographic responses, we conclude that, in spite of the risk of hemorrhage, thrombolysis is strongly indicated to reverse intra-abdominal and intracranial thromboses. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1973 Background. Hematological and extra-hematological adverse events associated to Hydroxyurea (HU) treatment in patients with chronic myeloproliferative neoplasms (MPN) are object of particular interest in the Ph-negative MPN since they can significantly limit the HU use. Objective. To evaluate the extra-hematological adverse events associated to HU treayment in a large cohort of Essential Thrombocythemia (ET) patients. Material and Methods. One thousand and seventy-five out of the 2005 ET patients registered in the RIT were treated with HU and are the object of this report. The patients, 641 females (59.6%) and 434 males (40.4%), diagnosed according to the PVSG or WHO criteria in 54 hematological centers of the RIT, started treatment with HU as first (92%) or second (8%) line, at median age of 65 years.The mean duration of HU treatment was 3.3 years. The HU treatment was withdrawn in 221 (20.5%) patients after a mean of 3.0 years.The administered dose of HU was 0.25–3.0 g/day (median 1), and a mean cumulative dose was 1113 g. The extra-hematological adverse events (EHAEs) observed during the HU treatment were distinguished in HU related AEs (HU-EHAEs), ET related AEs (ET-EHAEs) and HU or ET unrelated AEs (U-EHAEs).Results. During the HU treatment (3587 pt-y) 378 EHAEs were reported in 207 (19.3%) patients, being the HU-EHAEs 244 (6.8/100 pt-y) in 170 (15.8%) patients. In detail, the HU-EHAEs were: dermatological in 108 (48.3%) cases (38 hyperpigmentation, 26 leg ulcers, 22 maculo-papular rash, 10 lichenoid eruptions, 5 skin cancer, 4 alopecia); gastro-intestinal in 80 (32.8%) cases (37 nausea/vomiting, 30 diarrhea, 13 gastro-intestinal intolerance); systemic in 35 (14.3%) cases (28 fever, 7 fatigue); neurological in 19 (7.8%) cases (headache); miscellanea in 2 (0.8%) cases. Conclusion. This preliminary analysis in 1075 ET patients of the Registro Italiano Trombocitemia (RIT) treated with HU shows that the extra-hematological adverse events referred to the HU (HU-EHAEs) occurred with not negligible rate (6.8/100 pt-y) and need to be object of attention in the management of ET patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1782 Background: Chemo immunotherapy (R-CHOP) has improved outcomes of both GC (germinal center) and ABC (activated B-cell) subtypes of DLBCL. However outcomes in DLBCL patients treated with R-CHOP with ABC subtype (vs. GC) and/or with poor-risk features (High IPI, high Ki-67) remain inferior. These patients might benefit from more dose-intensive or high-dose therapy approaches. In our practice at The John Theurer Cancer Center, we have employed a risk-adaptive strategy with R-HCVAD to treat patients with DLBCL with aggressive features. Methods: Utilizing Kaplan Meier (KM) survival and Cox regression analyses, we conducted a retrospective cohort study to describe the outcome of patients treated with R-HCVAD in the 1st-line setting with the following high-risk features: high Ki-67 (MIB-1), high IPI, multiple extra-nodal (EN) sites, bulky disease or immunohistochemistry (IHC) staining patterns (GC vs. non GC by Hans’ model). The primary study endpoints were PFS and OS. The proportional hazards assumption was met for this analysis. Results: 45 patients (median age 57, range 34–71 yrs) with newly diagnosed DLBCL treated with R-HCVAD (median 6 cycles, range 1–8) were available for this analysis, representing 1010 total months of follow up at-risk. Baseline characteristics included: stage III-IV (90%), IPI ≥ 3 (52%), median Ki-67 (80%, range 10–100%), median EN sites (2), non-GC subtype (34%), bone marrow (BM) involvement (38%), EBER positive (14%), HIV negative (100%). With 17 months (range 9–64 months) median follow up, median OS and PFS (graph) are not yet reached. 2-yr PFS and OS were 80% (95% CI 61–91%) and 78% (95% CI 61–88%) respectively. In Cox regression analysis, survival outcomes were not adversely affected by: patient age 〉 60 (HR .8, p=.18), LDH 〉 ULN (HR 2.3, p=.3), non-GC IHC pattern (HR .5, p=.5), BM involvement (HR 1.9, p=.4), Ki-67 ≥ 80% (HR 1.7, p=.6) or EN sites ≥ 2 (HR 4.7, p=.15). Conclusions: This analysis represents the largest reported cohort of DLBCL patients treated with R-HCVAD. These data suggest that R-HCVAD can overcome traditional poor-risk features such as high IPI, high Ki-67 and non-GC IHC pattern. Future work will focus on identifying molecular markers for failure in DLBCL patients treated with dose-intensive regimens. A randomized trial comparing R-HCVAD to R-CHOP in selected high-risk patients is warranted. Disclosures: No relevant conflicts of interest to declare.