ALBERT

Description: The absolute content of CD34+ cells in the peripheral blood of 84 patients with myelofibrosis with myeloid metaplasia (MMM) and 23 patients with other Philadelphia-negative (Ph−) chronic myeloproliferative disorders (CMDs) was investigated. In MMM, the median absolute number of circulating CD34+cells was consistently high (91.6 × 106/L; range, 0-2460 × 106/L). Receiver operating characteristic curve analysis showed that 15 × 106/L as a decision criterion for CD34+cells produced an almost complete discrimination between MMM patients out of therapy and other Ph− CMDs (positive predictive value, 98.4%; negative predictive value, 85.0%). MMM patients with higher numbers of CD34+ cells had a significantly longer disease duration (P = .019) and higher spleen volume index (P = .014), liver volume (P = .000), percentage of circulating immature myeloid cells (P = .020), and percentage of myeloid blasts (P = .000). When CD34+ cells were correlated with the use of Dupriez risk stratification, CD34+ cells increased significantly from low-risk (median, 68.1 × 106/L) to intermediate-risk (median, 112.8 × 106/L) and high-risk patients (median 666.1 × 106/L) (F = 4.95;P = .009). When CD34+ cells were correlated with a severity score on the basis of both myeloproliferative and myelodepletive characteristics of the disease, only the myeloproliferation index was significantly associated with CD34+ cell level (F = 5.7;P = .000). Overall survival and interval to blast transformation from the time of CD34+ cell analysis were significantly shorter in patients with more than 300 × 106/L CD34+ cells (P = .005 and .0005, respectively). In conclusion, the absolute number of CD34+ circulating cells allows MMM to be distinguished from other Ph− CMDs; it is strongly associated with the extent of myeloproliferation and predicts evolution toward blast transformation.

Description: Background. Primary myelofibrosis (PMF) is a clonal, neoplastic disorder of the hematopoietic stem cell, categorized among the Ph-negative myeloproliferative neoplasms (MPNs). Available evidence indicates that enhanced activation of JAK-STAT pathway is the main molecular mechanism of myeloproliferation due to somatic mutations in JAK2, CALR or MPL genes. However, imbalanced immune responses that lead to chronic inflammation has been hypothesized to play an important role in the pathogenesis of the disease (Hasselbalch HC et al. Leuk Res 2013;37:214-20). Study design. In the attempt to explain the mechanism of immune dysregulation, and to explore how it could influences the disease phenotype, we examined the frequency of circulating CD4+CD25++CD127low/-FOXP3+ regulatory T cells (Tregs), as measured by cytofluorimetric technique, in 193 consecutive patients with PMF and 16 healthy controls (HCs). Results. The percentage of Treg cells between PMF patients and HCs was significantly different [median, 0.87% (range 0% to 10.2%) vs. 2.09% (range 0.54% to 6.5%); Mann-Whitney U test, P

Description: Abstract 2141 Poster Board II-118 Background: CD133+ (Prominin-1 positive) is a 5-transmembrane glycoprotein that identifies immature progenitor stem cells. Immature hematopoietic stem cells retain the possibility to give origin to tissues different from hematopoietic cell lines (transdifferentiation). Material and methods: In this preliminary study we investigated the possibility to mobilize, collect, immunoselect and reinfuse autologous CD133+ immature stem cells in liver cancer patients. This approach was adopted to obtain, in a short time, an adequate volume increase of the disease free liver thus extending the resectability criteria of the liver, with the final goal to prolong survival. We enrolled 4 patients with a large liver cancer and no chance of resection. The mobilization protocol consisted in: G-CSF administration (10 μg/kg/day ) for 3-5 days, peripheral blood stem cell (PBSC) monitoring starting from the 3rd day, leukapheresis (LKF) collection processing 3 blood volumes when CD133+ cells〉15 μL. Patients were monitored during mobilization, collection and post collection phase for clinical status, blood pressure and bleeding. Positive CD133+ immunoselection (Miltenyi Clinimacs) was performed on LKF product after overnight storage. Quality controls on positive fraction consisted in viability and purity of CD133+ cells by cytofluorimetric analysis and clonogenic assays. Microbial tests were performed on the negative fraction. After LKF, patients underwent right portal embolization and infusion of CD133+ cells into the opposite portal vein by a percutaneous access. Evaluation of liver regeneration was performed 30 days after stem cell infusion by spiral CT and galactose clearance. Liver resection was carried out if liver regeneration reached 30-40%. Results: Stem cell mobilization, LKF content and immunoselected cells are detailed in tab 1. No relevant side effects were observed. We obtained an efficient stem cell mobilization in all patients enrolled. No bacterial or fungal contamination was observed in cells infused. Results about liver regeneration and patients' follow up are detailed in table 2. Conclusions: Our approach to liver regeneration was feasible and safe with no relevant side effects. We observed an efficient stem cell mobilization comparable to healthy donors also in liver cancer patients. The infusion of CD133+ cells allowed a significant hepatic tissue regeneration in all patients. Controlled clinical trials are needed to confirm our preliminary results. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2829 Background: The somatically acquired Janus Kinase 2 (JAK2) mutation (V617F) is borne by approximately 60% of patients with primary myelofibrosis (PMF). The mutation increases JAK2 kinase activity, with the potential to affect phenotype and clinical outcome of mutated subjects. Accordingly, PMF V617F mutants result associated with higher white-blood cell counts, higher propensity to develop large splenomegaly, and are less likely to require transfusion during follow-up. At variance, different results have been obtained as far as the influence of JAK2 V617F mutation on the major disease outcomes, such as blast transformation (BT) and overall survival (OS). In this work we considered our cohort of patients with PMF-fibrotic type consecutively registered from 1990 and prospectively followed, who reached a median follow-up of more than 4 years and we used statistical analysis to account for competing risks. With this cohort of patients we strove to provide a more informative population and appropriate analysis for studying the influence of JAK2 V617F mutation on BT and OS in PMF. Patients and Methods: In 462 PMF –fibrotic type patients (bone marrow [BM] fibrosis grade 〉0) we computed the incidence of BT and death in the framework of Cox regression analysis and of Fine and Gray competing risks analysis for BT. Results: At the Cox regression analysis, having either a wild-type (wt) or a homozygous JAK2 V617F genotype were factors for BT (HR, 1.98 and 2.04, respectively, with respect to the heterozygous genotype), but not for OS. At the competing risks regression analysis, the risk for BT in wt and homozygous V617F patients increased with respect to Cox analysis, giving a sHR of 2.17 and 2.12, respectively. Correcting the results for the variables that could have influence on BT, JAK2 V617F wt and homozygous genotypes remained independently associated with BT. In a validation cohort of 133 independent cases with PMF-prefibrotic type (BM fibrosis grade =0), the BT predictive model including JAK2 V617F genotype, older age, and lower hemoglobin retained high discriminant capacity (C statistics, 0.70; 95% CI, 0.47 to 0.92). A total of 147 patients were genotyped with a quantitative assay for JAK2 V617F mutation in granulocyte DNA collected within 6 months from diagnosis, provided that no treatment had been delivered in the meantime. A total of 97 patients were JAK2 V617F mutated, giving an overall frequency of 65.9%. Comparing the four groups, the time to BT, death for any cause, and death for BT among the 4 quartiles were not significantly different (P=0.5). However, there was a shorter time to BT in the upper quartile compared with lower quartiles (P= 0.04). Conclusion: The accumulation of mutated alleles in the JAK2 V617F clone or the selective acquisition of a proliferative advantage in the wt clone are two relevant routes to BT in PMF. The influence of these results on treatment decisions with anti-JAK2 agents should be tested. Disclosures: No relevant conflicts of interest to declare.

Description: Myelofibrosis with Myeloid Metaplasia (MMM) is characterized by an abnormal constitutive mobilization of CD34+ stem/progenitor cells. The SDF-1alpha axis chemoactracts hematopoietic stem and progenitor cells and is thought to play a crucial role in the homing/mobilization of these cells from the bone marrow, interacting with its unique receptor CXCR4. CD26 (dipeptidylpeptidase IV/DPPIV) is an extracellular peptidase, present both as membrane-bound and as a catalitically active soluble form in plasma. It has the ability to cleave SDF-1alpha at its position-2 proline inducing significant changes in its receptor binding and/or functional activation, thus inhibiting normal SDF-1alpha induced migration. In this study we evaluated the expression of CD26/DPPIV in patients with MMM (N=63) and its functional activity (N=8). CD26/DPPIV cell surface expression was measured by flow cytometry on peripheral blood mononuclear cells (PBMNC) or on immunoselected circulating CD34 + cells. The CD26/DPPIV activity was measured in 96-well microplates using the chromogenic substrate gly-pro-p-nitoanilide. The proteolytic activity was determined by measurements of the amount of nitoanilide (pNA) formed in the supernatant at 405 nm. Absorbance was measured at 405 nm and the picomoles of pNA formed were calculated by comparison with a pNA standard curve. The CD26 expression on the membrane of PBMNC of patients with MMM was comparable to that found on PBMNC of normal controls (median 29.5 %, range 0.72–61.1; median 33.1 %, range 20.3–39.2, respectively). However, the percentage of circulating CD34+CD26+ cells was significantly reduced (P

Description: Introduction: At moment PBSC collections can be performed using semiautomated or automated cell separator devices. The collection with semiautomated methods implies an augmented working load for the dedicated personnel and is strongly influenced by the operator. On the contrary, the automated methods offer the advantages of a diminuished working load for the dedicated personnel and an high standardization of the collection procedure. Herein we report our experience on 60 PBSC collections employing the new automated COM.TEC Fresenius autoMNC program that provides the possibility to predict the total number of CD34+ cells collected basing on the CD34+ cell count (x μL) pre-leukapheresis (LKF) collection in peripheral blood. Materials and Methods: 39 patients affected with various onchohematological diseases and10 healty donors were mobilized with chemotherapy + G-CSF or G-CSF alone, respectively, and subsequently underwent LKF collection for auto or allotransplant. According to our internal protocol 60 LKF collections were performed starting with a CD34+ cell count in peripheral blood at least of 20/μL. Net weight of the final LKF product and its CD34+ cell content were evaluated at the end of each PBSC collection procedure and then compared to the expected data calculated by the cell separator device. Moreover a post collection peripheral blood Plt count was evaluated for each patient/donor. Results: The mean starting WBC count was 25.86x103/μL (range: 4–82.3), Plt count was 151.38x103/μL (20–395), CD34+ cells was 96.63/μL (20–332). The mean WBC and CD34+ cells in the LKF collection were 224.78x103/μL (20.71–425.3) and 565.45x106 (59.3–1609.3), respectively. The mean volume of the LKF collection was 237.28 ml (120–503). The mean estimated CD34+ cell content was 498.37x106 while the real mean CD34+ LKF cell content was 623.32x106. The mean CD34+ cell collection efficiency was 91% (66–126). Finally, the mean post procedure Plt count in patient/donor was 77.91x103/μL (12–164). Conclusions: The automatized PBSC collection with the new program COM.TEC Fresenius autoMNC demonstrated a very high CD34+ cell collection efficiency. Moreover the possibility to predict the CD34+ cell yield permits an optimal management of the LKF collection, reducing the number of procedures per patient/donor. The difference observed between the mean estimated CD34+ cells and the real CD34+ cell content may be due to the intra-procedure stem cell mobilization phenomenon. Finally, this new automatized collection system demonstrated to limit the collection related thrombocytopenia either in patient or in donor.

Description: Introduction: Platelet concentrates (PC) collected by apheresis are effective in supporting deeply thrombocytopenic patients. The reduced risk of multiple allogeneic exposure and transmissible infectious diseases together with the high WBC depletion and diminished transfusion reactions are the main advantages offered by PC transfusion. At moment, the availability of several synthetic solutions for platelets storage permits to prepare hyperconcentrate(dry) apheresis platelets with the advantage of reducing febrile non-hemolytic transfusion reactions and, in low body weight patients the citrate toxicity, without the necessity of further manipulations. The aim of this study was to test the quality of 20 dry platelets (DP) in comparison to 20 standard plateletpheresis (SP) concentrates. Materials and methods: A total of 40 apheresis procedures were performed by the single-needle Cobe Trima separation device (Gambro BCT, Lakewood, CO, USA) collecting either DP or SP concentrates. Within 1h after collection, the bag containing DP was added with the appropriate amount (70% of DP final volume) of synthetic solution for platelets storage (SSP, MacoPharma). Both DP and SP concentrates were stored at room temperature with gentle agitation for 4 days. For both concentrates, platelet yield was calculated and in vitro studies of membrane glycoproteins expression and aggregation at day +1 and day +4 were carried out. Results: The comparison between 20 DP and 20 SP concentrates in terms of ability to aggregate in vitro and membrane glycoproteins expression at day +1 and day +4 of storage is reported in table A and B respectively. Conclusions: The in vitro tests documented a major activation of dry platelets. In particular, the ability to aggregate was reduced in the 20 DP concentrates analised and this phenomenon was more evident at day +4 of storage. The alteration of membrane glycoproteins expression (markers of storage lesion) confirms the lower in vitro quality of DP concentrates. The effectiveness of this new blood component in vivo should be evaluated in a controlled clinical trial. Table A. At collection Day +1 Day +4 SP DP SP DP SP DP Collagen μg/ml 4 93 88 97 82 80 53 ADP 10 μM 32 11 24 16 16 5 Ristocetin 1.5 mg/ml 91 97 77 81 71 52 Collagen 10 μg/ml + Adrenaline 10 μM 98 95 93 94 93 80 ADP 10 μM + Adrenaline 10 μM 87 72 76 61 57 30 Table B. At collection Day +1 Day +4 SP DP SP DP SP DP GPIb alfa (MFI) 5.06 5.75 6.31 6.13 5.26 4.54 GPIIb-IIIa (MFI) 35.47 36.71 34.61 37.7 31.76 40.9 GP IV (MFI) 11.49 11.4 11.67 10.28 11.65 11.38 GP 53 24.23 27.11 21.74 25.91 21.46 31.84 GMP 140 21.79 29.29 22.65 30.38 20.58 34.89