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Mitochondrial carnitine/acylcarnitine carrier (CAC) is a member of the mitochondrial carrier (MC) family and imports acylcarnitine into the mitochondrial matrix in exchange for carnitine, playing a pivotal role in carnitine shuttle, crucial for fatty acid oxidation. The crystallized structure of CAC has not been solved yet, however, the availability of several in vitro/in silico studies, also based on the crystallized structures of the ADP/ATP carrier in the cytosolic-conformation and in the matrix-conformation, has made possible to confirm the hypothesis of the single-binding centered-gated pore mechanism for all the members of the MC family. In addition, our recent bioinformatics analyses allowed quantifying in silico the importance of protein residues of MC substrate binding region, of those involved in the formation of the matrix and cytosolic gates, and of those belonging to the Pro/Gly (PG) levels, proposed to be crucial for the tilting/kinking/bending of the six MC transmembrane helices, funneling the substrate translocation pathway. Here we present a combined in silico/in vitro analysis employed for investigating the role played by a group of 6 proline residues and 6 glycine residues, highly conserved in CAC, belonging to MC PG-levels. Residues of the PG-levels surround the similarly located MC common substrate binding region, and were proposed to lead conformational changes and substrate translocation, following substrate binding. For our analysis, we employed 3D molecular modeling approaches, alanine scanning site-directed mutagenesis and in vitro transport assays. Our analysis reveals that P130 (H3), G268 (H6) and G220 (H5), mutated in alanine, affect severely CAC transport activity (mutant catalytic efficiency lower than 5 % compared to the wild type CAC), most likely due to their major role in triggering CAC conformational changes, following carnitine binding. Notably, P30A (H1) and G121A (H3) CAC mutants, increase the carnitine uptake up to 217 % and 112 %, respectively, compared to the wild type CAC.

Mitochondria and mitochondrial proteins represent a group of promising pharmacological-target candidates in the search of new molecular targets and drugs to counteract the onset of hypertension and more in general cardiovascular diseases (CVDs). Indeed, several mitochondrial pathways result impaired in CVDs, showing ATP depletion and ROS production as common traits of cardiac tissue degeneration. Thus, targeting mitochondrial dysfunction in cardiomyocytes can represent a successful strategy to prevent heart failure. In this context, the identification of new pharmacological targets among mitochondrial proteins paves the way for the design of new selective drugs. Thanks to the advances in omics approaches, to a greater availability of mitochondrial crystallized protein structures and to the development of new computational approaches for protein 3D-modelling and drug-design, it is now possible to investigate in detail impaired mitochondrial pathways in CVDs. Furthermore, it is possible to design new powerful drugs able to hit the selected pharmacological targets in a highly selective way to rescue mitochondrial dysfunction and prevent cardiac tissue degeneration. The role of mitochondrial dysfunction in the onset of CVDs appears increasingly evident, as reflected by the impairment of proteins involved in lipid peroxidation, mitochondrial dynamics, respiratory chain complexes, and membrane polarization maintenance in CVD patients. Conversely, little is known about proteins responsible for the cross-talk between mitochondria and cytoplasm in cardiomyocytes. Mitochondrial transporters of the SLC25A family, in particular, are responsible for the translocation of nucleotides (e.g., ATP), amino acids (e.g., aspartate, glutamate, ornithine), organic acids (e.g. malate and 2-oxoglutarate), and other cofactors (e.g., inorganic phosphate, NAD

COVID-19 is a severe infectious complication in patients with underlying medical conditions such as having undergone hematopoietic stem cell transplantation (HCT). This prospective survey reports outcome on 272 COVID-19 patients from 19 countries having undergone allogeneic (n = 175) or autologous (n = 97) HCT reported to the EBMT registry or to the GETH. All patients had the diagnosis of SARS-CoV-2 documented by PCR. Patients were included in this analysis if COVID-19 diagnosis was before April 10, 2020. The overall survival was estimate by using the Kaplan Meier methods, considering the death due to any cause as an event and the time from COVID-19 infection to the latest follow-up as survival time; difference between groups were tested by the log-rank test. Univariate and multivariate risk factor analysis for overall survival were performed with the Cox regression model. The median age was 54.4 years (1.0 - 80.3) for allogeneic and 60.9 years (7.7 - 73.4) for autologous HCT patients. 20 patients were children (< 18 years of age; median age 11.3 (1.0 - 16.9)). The median time from HCT to diagnosis of COVID-19 was 13.7 months (0.2 - 254.3) in allogeneic and 25.0 months (-0.9 - 350.3) in autologous recipients. Lower respiratory tract disease (LRTD) developed in 84.8% and 21.5% were admitted to an intensive care unit (ICU). At the time of analysis, 68/238 (28.6%) patients had died (47/155 allogeneic patients; 21/83 autologous patients). No follow-up had been received on 34 patients. The median time from infection to death was 19 days (0-102). Five patients were reported to have other primary causes of death than COVID-19. Of the patients reported to be alive, the median follow-up was 44 days. 144 (84.7%) patients (93 allogeneic; 51 autologous) had virologic resolution of the COVID-19 infection having at least one negative PCR. 26 patients were alive and known to be still COVID-19 positive (15 allogeneic; 11 autologous). For 34 patients the resolution status was unknown. Factors influencing the likelihood of resolution in multivariate analysis were underlying diagnosis (p=.01) and longer time from transplant to diagnosis of COVID-19 (p=.035). Overall survival at 6 weeks from COVID-19 diagnosis was 76.8% and 83.8% in allogeneic and autologous HCT recipients (p =ns), respectively (figure 1). Children (n=20) tended to do better with a 6-week survival of 95.0% although the difference was not significantly different (p =.12). In multivariate analysis of the total population older age (HR 1.26; 95% CI 1.05 - 1.51; p = .01) increased the risk and better performance status decreased the risk for fatal outcome (HR 0.79; 95% CI 0.69 - 0.90; p = .0003). The same factors had significant impact on overall survival in allogeneic HCT recipients (age HR 1.28; 95% CI 1.05 - 1.55; p=.01; performance status HR 0.79; 95% CI 0.68 - 0.92); p=.002) while only age impacted survival among autologous HCT patients (data not shown). Other transplant factors such as underlying diagnosis, time from HCT to diagnosis of COVID-19, graft-vs-host disease, or ongoing immunosuppression did not have a significant impact on overall survival. We conclude that HCT patients are at an increased risk compared to the general population to develop LRTD, require admission to ICU, and have increased mortality in COVID-19. Figure 1 Disclosures Duarte: Incyte Corporation: Other: Has received speaker and advisor fees. Kwon:Jazz: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria. Mielke:Novartis: Honoraria, Other: received via my institution, Speakers Bureau; Celgene/BMS: Honoraria, Other: received via my institution , Speakers Bureau; Bellicum: Honoraria, Other: received via my institution, Speakers Bureau; Kite/Gilead: Honoraria, Other: received via my institution , Speakers Bureau; Miltenyi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: received via my institution , Speakers Bureau; KIADIS Pharma: Honoraria, Other: received via my institution , Speakers Bureau; DNA Prime: Honoraria, Other: received via my institution , Speakers Bureau. López Jiménez:MSD: Speakers Bureau; Roche: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Abbvie: Research Funding, Speakers Bureau; Gilead: Research Funding, Speakers Bureau.

ADP/ATP carriers (AACs) are mitochondrial transport proteins playing a strategic role in maintaining the respiratory chain activity, fueling the cell with ATP, and also regulating mitochondrial apoptosis. To understand if AACs might represent a new molecular target for cancer treatment, we evaluated AAC expression levels in cancer/normal tissue pairs available on the Tissue Cancer Genome Atlas database (TCGA), observing that AACs are dysregulated in most of the available samples. It was observed that at least two AACs showed a significant differential expression in all the available kidney cancer/normal tissue pairs. Thus, we investigated AAC expression in the corresponding kidney non-cancer (HK2)/cancer (RCC-Shaw and CaKi-1) cell lines, grown in complete medium or serum starvation, for investigating how metabolic alteration induced by different growth conditions might influence AAC expression and resistance to mitochondrial apoptosis initiators, such as &ldquo, staurosporine&rdquo, or the AAC highly selective inhibitor &ldquo, carboxyatractyloside&rdquo, Our analyses showed that AAC2 and AAC3 transcripts are more expressed than AAC1 in all the investigated kidney cell lines grown in complete medium, whereas serum starvation causes an increase of at least two AAC transcripts in kidney cancer cell lines compared to non-cancer cells. However, the total AAC protein content is decreased in the investigated cancer cell lines, above all in the serum-free medium. The observed decrease in AAC protein content might be responsible for the decrease of OXPHOS activity and for the observed lowered sensitivity to mitochondrial apoptosis induced by staurosporine or carboxyatractyloside. Notably, the cumulative probability of the survival of kidney cancer patients seriously decreases with the decrease of AAC1 expression in KIRC and KIRP tissues making AAC1 a possible new biomarker of metabolic remodeling and survival in kidney cancers.

Background Haploidentical allogeneic hematopoietic cell transplantation (haplo-HCT) with post-transplant cyclophosphamide (PTCy) has greatly expanded potential donor options for patients (pts) without a fully HLA-matched donor. However, an HLA-mismatched unrelated donor (UD) or cord blood transplantation (CBT) are valid options for pts who do not have an haploidentical-related or a fully HLA-matched donor. The incorporation of PTCy with mismatched UD allo-HCT is associated with reduced risk of non-relapse mortality (NRM) and graft-versus-host disease (GvHD) (Jorge et al., BBMT, 2018). No previous study has compared outcomes of these two graft sources in contemporary era. In this study, we compared the outcomes of CBT versus (vs) single antigen-mismatched (HLA 9/10) UD allo-HCT with post-transplant cyclophosphamide (PTCy) in pts with acute myeloid leukemia (AML). Methods Pts with AML who underwent a first CBT without PTCy (N=902) or allo-HCT from a 9/10 UD with PTCy (N=280) between 2010 to 2019 were selected from The European Society of Blood and Marrow Transplantation (EBMT) registry. We excluded pts who had ex vivo T cell depletion. The median pt age was comparable between CBT and UD cohort [51 years (y) vs. 52 y, p=0.09]. The recipients of CBT had a longer median follow up (47 vs. 19 months, p Results In the univariate analysis, day +180 incidence of grade II-IV acute GvHD was 36% vs. 32% (p=0.07) and grade III-IV acute GvHD was 15% vs. 11% (p=0.16) for CBT and UD cohorts, respectively. The 2-y incidence of total chronic GvHD was 26% vs. 32% (p=0.20) and extensive chronic GvHD was 12% vs. 12% (p=0.83), respectively. CBT was associated with higher incidence of graft failure (11% vs. 4%, p In the MV analysis (Table 1), CBT was associated with higher risk of grade II-IV acute GvHD [hazard ratio (HR)=1.32, 95% confidence interval (CI):1-1.74,p Conclusions In this large registry-based observational study, CBT was associated with inferior LFS, OS and GRFS due to higher NRM and RI compared to 9/10 UD allo-HCT with PTCy. In the absence of a fully matched or haplo donor, 9/10 UD allo-HCT with PTCy may be preferred over CBT in pts with AML. A prospective study is needed to validate our findings. Disclosures Dholaria: Poseida: Research Funding; Angiocrine: Research Funding; Takeda: Research Funding; J&J: Research Funding; bms: Research Funding. Labopin:Jazz Pharmaceuticals: Honoraria. Blaise:Jazz Pharmaceuticals: Honoraria. Chevallier:Incyte Corporation: Honoraria. Mohty:BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Stemline: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau.

In this review we discuss the structure and functions of the aspartate/glutamate carriers (AGC1-aralar and AGC2-citrin). Those proteins supply the aspartate synthesized within mitochondrial matrix to the cytosol in exchange for glutamate and a proton. A structure of an AGC carrier is not available yet but comparative 3D models were proposed. Moreover, transport assays performed by using the recombinant AGC1 and AGC2, reconstituted into liposome vesicles, allowed to explore the kinetics of those carriers and to reveal their specific transport properties. AGCs participate to a wide range of cellular functions, as the control of mitochondrial respiration, calcium signaling and antioxydant defenses. AGC1 might also play peculiar tissue-specific functions, as it was found to participate to cell-to-cell metabolic symbiosis in the retina. On the other hand, AGC1 is involved in the glutamate-mediated excitotoxicity in neurons and AGC gene or protein alterations were discovered in rare human diseases. Accordingly, a mice model of AGC1 gene knock-out presented with growth delay and generalized tremor, with myelinisation defects. More recently, AGC was proposed to play a crucial role in tumor metabolism as observed from metabolomic studies showing that the asparate exported from the mitochondrion by AGC1 is employed in the regeneration of cytosolic glutathione. Therefore, given the central role of AGCs in cell metabolism and human pathology, drug screening are now being developed to identify pharmacological modulators of those carriers. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

Flavine adenine dinucleotide (FAD) dependent glucose methanol choline oxidoreductase (GMC oxidoreductase) is the terminal key enzyme of the patulin biosynthetic pathway. GMC oxidoreductase catalyzes the oxidative ring closure of (E)-ascladiol to patulin. Currently, no protein involved in the patulin biosynthesis in Penicillium expansum has been experimentally characterized or solved by X-ray diffraction. Consequently, nothing is known about P. expansum GMC oxidoreductase substrate-binding site and mode of action. In the present investigation, a 3D comparative model for P. expansum GMC oxidoreductase has been described. Furthermore, a multistep computational approach was used to identify P. expansum GMC oxidoreductase residues involved in the FAD binding and in substrate recognition. Notably, the obtained 3D comparative model of P. expansum GMC oxidoreductase was used for performing a virtual screening of a chemical/drug library, which allowed to predict new GMC oxidoreductase high affinity ligands to be tested in in vitro/in vivo assays. In vitro assays performed in presence of 6-hydroxycoumarin and meticrane, among the highly affinity predicted binders, confirmed a dose-dependent inhibition (17&ndash, 81%) of patulin production by 6-hydroxycoumarin (10 &micro, M&ndash, 1 mM concentration range), whereas the approved drug meticrane inhibited patulin production by 43% already at 10 &micro, M. Furthermore, 6-hydroxycoumarin and meticrane caused a 60 and 41% reduction of patulin production, respectively, in vivo on apples at 100 &micro, g/wound.

Background: Ovarian cancer is the second most common gynecologic malignancy, accounting for approximately 220,000 deaths annually worldwide. Despite radical surgery and initial high response rates to platinum- and taxane-based chemotherapy, most patients experience a relapse, with a median progression-free survival of only 18 months. Overall survival is approximately 30% at 5 years from the diagnosis. In comparison, patients out from breast cancer are more than 80 % after ten years from the disease discovery. In spite of a large number of published fundamental and applied research, and clinical trials, novel therapies are urgently needed to improve outcomes of the ovarian cancer. The success of new drugs development in ovarian cancer will strongly depend on both fully genomic disease characterization and, then, availability of biomarkers able to identify women likely to benefit from a given new therapy. Methods: In this review, the focus is given to describe how complex is the diseases under the simple name of ovarian cancer, in terms of cell tumor types, histotypes, subtypes, and specific gene mutation or differently expressed in the tumor with respect the healthy ovary. The first- and second-line pharmacological treatment clinically used over the last fifty years are also described. Noteworthy achievements in vitro and in vivo tested new drugs are also summarized. Recent literature related to up to date ovarian cancer knowledge, its detection by biomarkers and chemotherapy was searched from several articles on Pubmed, Google Scholar, MEDLINE and various Governmental Agencies till April 2019. Results: The papers referenced by this review allow a deep analysis of status of the art in the classification of the several types of ovarian cancer, the present knowledge of diagnosis based on biomarkers and imaging techniques, and the therapies developed over the past five decades. Conclusion: This review aims at stimulating more multi-disciplinary efforts to identify a panel of novel and more specific biomarkers to be used to screen patients for a very early diagnosis, to have prognosis and therapy efficacy indications. The desired final goal would be to have available tools allowing to reduce the recurrence rate, increase both the disease progression free interval and of course the overall survival at five years from the diagnosis that today is still very low.

Haem–copper oxidases are the terminal enzymes in both prokaryotic and eukaryotic respiratory chains. They catalyse the reduction of dioxygen to water and convert redox energy into a transmembrane electrochemical proton gradient during their catalytic activity. Haem–copper oxidases show substantial structure similarity, but spectroscopic and biochemical analyses indicate that these enzymes contain diverse prosthetic groups and use different substrates (i.e. cytochrome c or quinol). Owing to difficulties in membrane protein crystallization, there are no definitive structural data about the quinol oxidase physiological substrate-binding site(s). In the present paper, we propose an atomic structure model for the menaquinol:O2 oxidoreductase of Bacillus subtilis (QOx.aa3). Furthermore, a multistep computational approach is used to predict residues involved in the menaquinol/menaquinone binding within B. subtilis QOx.aa3 as well as those involved in quinol/quinone binding within Escherichia coli QOx.bo3. Two specific sequence motifs, R70GGXDX4RXQX3PX3FX[D/N/E/Q]X2HYNE97 and G159GSPX2GWX2Y169 (B. subtilis numbering), were highlighted within QOx from Bacillales. Specific residues within the first and the second sequence motif participate in the high- and low-affinity substrate-binding sites respectively. Using comparative analysis, two analogous motifs, R71GFXDX4RXQX8[Y/F]XPPHHYDQ101 and G163EFX3GWX2Y173 (E. coli numbering) were proposed to be involved in Enterobacteriales/Rhodobacterales/Rhodospirillales QOx high- and low-affinity quinol-derivative-binding sites. Results and models are discussed in the context of the literature.

CoA is an essential cofactor that holds a central role in cell metabolism. Although its biosynthetic pathway is conserved across the three domains of life, the subcellular localization of the eukaryotic biosynthetic enzymes and the mechanism behind the cytosolic and mitochondrial CoA pools compartmentalization are still under debate. In humans, the transport of CoA across the inner mitochondrial membrane has been ascribed to two related genes, SLC25A16 and SLC25A42 whereas in D. melanogaster genome only one gene is present, CG4241, phylogenetically closer to SLC25A42. CG4241 encodes two alternatively spliced isoforms, dPCoAC-A and dPCoAC-B. Both isoforms were expressed in Escherichia coli, but only dPCoAC-A was successfully reconstituted into liposomes, where transported dPCoA and, to a lesser extent, ADP and dADP but not CoA, which was a powerful competitive inhibitor. The expression of both isoforms in a Saccharomyces cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact.

Background Treatment of patients developing steroid-refractory acute GvHD (SR-aGvHD) after allogeneic hematopoietic cell transplantation (HCT) remains an unmet clinical need and a major therapeutic challenge. Mesenchymal stromal cells (MSC) induce immunosuppression and reduce the inflammatory status initiated by aGvHD. We performed an interim analysis of a multicenter, prospective, non-randomized, phase I/II study, planned to test the safety and activity of human umbilical cord (UC), third-party MSC given sequentially after Pentatostatin for the treatment of SR-aGvHD grade III-IV as well as the overlap syndrome (EUDRACT n. 2012-000582-21), NCT02032446). Patients and Methods Third-party UC MSC were produced under GMP conditions as previously described (Capelli C et al Cytotherapy, 2011, 13, 786-801). The treatment schedule was based on the sequential administration of Pentostatin, given iv at dose of 1 mg/m^2 for 3 consecutive days, followed by 3 MSC infusions given at weekly intervals. Between October 2013 and May 2018 (range 4.6 years) we enrolled 27 allogeneic HCT adult recipients (22 male) with SR-aGvHD (n 25) or overlap syndrome (n 2), median age 47 years (range 19-62) transplanted from HLA-identical sibling donors (6; 22%), haploidentical donors (2; 7%), unrelated donors (18; 67%) or cord blood (1; 4%). Target organ involvement included skin in 11 cases (44%), gastrointestinal tract (GI) in 23 (92%) and liver in 12 (48%). Nine patients had grade III (33%), 16 patients grade IV (60%) and 2 patients had severe overlap syndrome (7%). Most of patients exhibited multiple organ involvement (n 16, 64%). Patients started MSC therapy at median of 22 days (10-2101) after the onset of aGvHD or overlap syndrome. Results The infusions of UC MSC-infusion were always well tolerated without any immediate or late toxic event. Response to MSC therapy, by day 30 after the final MSC dose, was evaluable in 16 of 27 patients: 7 achieved CR (26%), 6 PR (22%) and 3 NR (11%). Ten patients died before reaching the evaluation time of response: 8 patients for aGvHD and 2 for haematological disease. One patient was lost to follow up after 9 days from the last UC-MSC infusion. Remarkably, the subgroup of patients with single organ involvement (8 GI, 1 liver) were all evaluable showing 6 patients in overall response (67%, 4 RC and 2 PR). No response was observed in 2 cases of severe overlap syndrome. Patients achieving CR had improved longer term OS vs patients in PR/NR (80% vs 50% at 1 year). For patients with grade III and IV SR-aGvHD, CR rates at day 30 were 67% and 31% respectively. During tapering of steroid therapy 4 out of 7 patients that obtained CR had aGvHD recurrence, at median time of 87 days. We observed relapse of underlying malignancy in 5 patients and all died with active disease. At last follow-up 8 out of 27 patients were alive with no GVHD in 5 of them; 14 patients died from TRM: aGvHD or overlap syndrome itself (n=10), infections (n= 2), cGvHD (n= 2). Two patients with uncontrolled GvHD had clinical signs of Transplant Associated Microangiopathy (TAM). A total of 17 reported AE and 12 SAE were registered. In 23 out of 27 patients infectious complications were observed, in most cases virus related (n.25; CMV followed by BK virus and EBV), and fungal infections (3 aspergillus pneumoniae). Blood-stream documented infections (5) and pneumoniae (5) were also observed. Conclusions The infusion of cord blood derived MSC proved safe in all cases. As expected in patients with steroid refractory aGvHD, a high rate of infectious complications were observed. When considering that most patients (60 %) had grade IV aGvHD, the response rate and the OS are promising and this warrants not only the completion of this Phase I/II study but also planning a future pivotal trial. Disclosures No relevant conflicts of interest to declare.

In the study of the protein folding problem with ab initio methods, the protein backbone can be built on some periodic lattices. Any vertex of these lattices can be occupied by a "ball," which can represent the mass center of an amino acid in a simplified coarse-grained model of the protein. The backbone, at a coarse-grained level, can be constituted of a No Reverse Self Avoiding Walk, which cannot intersect itself and cannot go back on itself. There is still much debate between those who use lattices to simplify the study of the protein folding problem and those preferring to work by using an off-lattice approach. Lattices can help to identify the protein tertiary structure in a computational less-expensive way, than off-lattice approaches that have to consider a potentially infinite number of possible structures. However, the use of a lattice, constituted of insufficiently accurate direction vectors, constrains the predictive ability of the model. The aim of this study is to perform a systematic screening of 7 known classic and 11 newly proposed lattices in terms of predictive power. The crystal structures of 42 different proteins (14 mainly alpha helical, 14 mainly beta sheet and 14 mixed structure proteins) were compared to the most accurate simulated models for each lattice. This strategy defines a scale of fitness for all the analyzed lattices and demonstrates that an increase in the coordination number and in the degrees of freedom is necessary but not sufficient to reach the best result. Instead, the introduction of a good set of direction vectors, as developed and tested in this study, strongly increases the lattice performance.

Alternaria brown spot is one of the most important diseases of tangerines and their hybrids worldwide. Recently, outbreaks in Mediterranean areas related to susceptible cultivars, refocused attention on the disease. Twenty representatives were selected from a collection of 180 isolates of Alternaria spp. from citrus leaves and fruit. They were characterized along with reference strains of Alternaria spp. Micro- and macroscopic characteristics separated most Alternaria isolates into six morphotypes referable to A. alternata (5) and A. arborescens (1). Phylogenetic analyses, based on endopolygalacturonase (endopg) and internal transcribed spacer (ITS), confirmed this finding. Moreover, a five-gene phylogeny including two anonymous genomics regions (OPA 1-3 and OPA 2-1), and the beta-tubulin gene (ß-tub), produced a further clustering of A. alternata into three clades. This analysis suggested the existence of intra-species molecular variability. Investigated isolates showed different levels of virulence on leaves and fruit. In particular, the pathogenicity on fruit seemed to be correlated with the tissue of isolation and the clade. The toxigenic behavior of Alternaria isolates was also investigated, with tenuazonic acid (TeA) being the most abundant mycotoxin (0.2-20 mg/L). Isolates also synthesized the mycotoxins alternariol (AOH), its derivate alternariol monomethyl ether (AME), and altenuene (ALT), although to a lesser extent. AME production significantly varied among the six morphotypes. The expression of pksJ/pksH, biosynthetic genes of AOH/AME, was not correlated with actual toxin production, but it was significantly different between the two genotypes and among the four clades. Finally, ten isolates proved to express the biosynthetic genes of ACTT1 phytotoxin, and thus to be included in the Alternaria pathotype tangerine. A significant correlation between pathogenicity on leaves and ACTT1 gene expression was recorded. The latter was significantly dependent on geographical origin. The widespread occurrence of Alternaria spp. on citrus fruit and their ability to produce mycotoxins might represent a serious concern for producers and consumers.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 μM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 μM and 2.1 μM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Therapeutic mAbs have high efficacy in treating TNFalpha-related immunological diseases such as ankylosing spondylitis, psoriatic-rheumatoid arthritis, and skin diseases. Three mAbs are available:E...

Mitochondrial and nuclear DNAs contribute to encode the whole mitochondrial protein complement. The two genomes possess highly divergent features and properties, but the forces influencing their evolution, even if different, require strong coordination. The gene content of mitochondrial genome in all Metazoa is in a frozen state with only few exceptions and thus mitochondrial genome plasticity especially concerns some molecular features, i.e. base composition, codon usage, evolutionary rates. In contrast the high plasticity of nuclear genomes is particularly evident at the macroscopic level, since its redundancy represents the main feature able to introduce genetic material for evolutionary innovations. In this context, genes involved in oxidative phosphorylation (OXPHOS) represent a classical example of the different evolutionary behaviour of mitochondrial and nuclear genomes. The simple DNA sequence of Cytochrome c oxidase I (encoded by the mitochondrial genome) seems to be able to distinguish intra- and inter-species relations between organisms (DNA Barcode). Some OXPHOS subunits (cytochrome c, subunit c of ATP synthase and MLRQ) are encoded by several nuclear duplicated genes which still represent the trace of an ancient segmental/genome duplication event at the origin of vertebrates.

Introduction: Patients with disease relapse after allogeneic transplantation (alloHSCT) have a poor prognosis. Donor lymphocyte infusion (DLI) is one of the main clinical options to salvage patients in relapse after transplant. Cytokine Induced Killer (CIK) cells are in vitro activated and expanded T lymphocytes which have acquired NK like cytotoxicity as well as CD56 expression. CIK cells have shown Graft versus Leukemia (GvL) activity with little GvHD and therefore may represent an ideal candidate to treat post-transplant relapse. We report the final results of a phase II multicenter pilot study, whose objective was to evaluate the safety and efficacy of sequential administration of donor derived unmanipulated DLI and CIK cells in patients with recurrent hematologic cancers after alloHSCT. Methods Seventy-four patients relapsed after alloHSCT, performed using either a matched related (N=42) or unrelated donor (n= 32), were enrolled in the study. This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, and approved by the Agenzia Italiana del Farmaco (AIFA). The trial was registered as (EUDRACT number 2008-003185-26, ClinicalTrial.gov : NCT01186809). Results Among the 74 patients (including 16 children and 58 adults) enrolled into this study (median age 45, range 1-67), 20 had a diagnosis of ALL (27%), 41 of AML (55%), 4 of MM (5%), 3 of HD (4%), 2 of NHL (3%) and 4 of MPN (5%). All patients relapsed after matched allogeneic transplants (32 unrelated and 42 sibling), of whom 44 (59%) suffered from a hematological, 4 (5%) from a cytogenetic and 26 (35%) from a molecular relapse. The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with increasing numbers of CIK cells, in groups of three patients per dose level. Since dose limiting toxicity (DLT) was never observed (acute GVHD of grade III or more), the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to all patients. Ten patients died for disease progression, 1 patient developed aGVHD (grade I, skin only) and 1 withdrawn for medical decision before or during the DLI treatment and could not proceed to the planned subsequent CIK administration. Sixty-two patients received at least one infusion of CIK cells, of whom 43 patients (61%) completed the cell therapy program, while 3 patients are still under treatment. The study flow is outlined in Figure 1. As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 24 (32%) patients (4 during the DLI), no response was observed in 18 (24%) patients, a stable disease in 1 patient (1%), a complete remission in 21 (28%) and a partial remission in 6 (8%), for an overall response rate of 36%. In 4 patients clinical response could not be evaluated (3 patients still in treatment and 1 withdrawn from the protocol). Acute GVHD was observed in a total of 11 patients (15%): grade 1 (n=4), 2 (n=2) and 3-4 (n=5). During follow up, chronic GVHD was observed in 8 patients (11 %) (3 mild, 4 moderate and 1 severe). By univariate analysis, progression free survival (PFS) and overall survival (OS) were significantly associated (p Conclusion Our study shows that administration of CIK cells is feasible in patients with recurrent hematologic cancer after alloHSCT with a relatively low toxicity in terms of GvHD. Particularly in the setting of the molecularly relapsed patients, long-term survival can be achieved. In future studies, we are planning to test CIK cells in preventing post-transplantation relapse in high risk AML. Finally, CIK cells may represent an innovative platform to transduce chimeric antigen receptors in allogeneic T cells with a reduced risk to induce GvHD. Disclosures Biondi: Cellgene: Other: Advisory Board; BMS: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board.

Background The p53 gene family consists of the three genes p53, p63 and p73, which have polyhedral non-overlapping functions in pivotal cellular processes such as DNA synthesis and repair, growth arrest, apoptosis, genome stability, angiogenesis, development and differentiation. These genes encode sequence-specific nuclear transcription factors that recognise the same responsive element (RE) in their target genes. Their inactivation or aberrant expression may determine tumour progression or developmental disease. The discovery of several protein isoforms with antagonistic roles, which are produced by the expression of different promoters and alternative splicing, widened the complexity of the scenario of the transcriptional network of the p53 family members. Therefore, the identification of the genes transactivated by p53 family members is crucial to understand the specific role for each gene in cell cycle regulation. We have combined a genome-wide computational search of p53 family REs and microarray analysis to identify new direct target genes. The huge amount of biological data produced has generated a critical need for bioinformatic tools able to manage and integrate such data and facilitate their retrieval and analysis. Description We have developed the p53FamTaG database (p53 FAMily TArget Genes), a modular relational database, which contains p53 family direct target genes selected in the human genome searching for the presence of the REs and the expression profile of these target genes obtained by microarray experiments. p53FamTaG database also contains annotations of publicly available databases and links to other experimental data. The genome-wide computational search of the REs was performed using PatSearch, a pattern-matching program implemented in the DNAfan tool. These data were integrated with the microarray results we produced from the overexpression of different isoforms of p53, p63 and p73 stably transfected in isogenic cell lines, allowing the comparative study of the transcriptional activity of all the proteins in the same cellular background. p53FamTaG database is available free at http://www2.ba.itb.cnr.it/p53FamTaG/ Conclusion p53FamTaG represents a unique integrated resource of human direct p53 family target genes that is extensively annotated and provides the users with an efficient query/retrieval system which displays the results of our microarray experiments and allows the export of RE sequences. The database was developed for supporting and integrating high-throughput in silico and experimental analyses and represents an important reference source of knowledge for research groups involved in the field of oncogenesis, apoptosis and cell cycle regulation.

Introduction Cytokine Induced Killer (CIK) cells are memory T lymphocytes which have acquired CD56 expression and Natural Killer (NK) like unrestricted cytotoxicity, following in vitro activation by anti CD3 OKT3 and IFNg and subsequent expansion with IL-2. CIK cells have demonstrated in vitro and in vivo anti tumor activity, direct intratumor homing following iv. administration and, more importantly, a very reduced Graft Versus Host (GVH) activity, in several experimental allogeneic models. Indeed we and others have demonstrated very limited GVH activity in preliminary phase I studies, with donor derived (matched) CIK cells in patients with different hematological neoplasms, previously treated by allogeneic Hematopoietic Stem Cells (HSC) transplantations and subsequently relapsed of diseases 1. Methods To better delineate the toxicity profile, as well as the potential anti tumor efficacy, of donor derived CIK cells, we prospectively studied 48 patients relapsed after allogeneic stem cell transplantation performed using either a matched related (N=28) or unrelated donor (n= 20, including 1 haplo). This phase II multicenter study was authorized by Istituto Superiore di Sanità, as for Advanced Therapeutic Medicinal Product (ATMP) regulations, approved by the Agenzia Italiana del Farmaco and (AIFA). The trial was registered as (EUDRACT n 2008-003185-26, ClinicalTrial.gov: NCT01186809) Results In this interim analysis, forty-eight patients (including childrens and adults) have been so far enrolled into this study protocol. The median age was 48 (range 6-67) and a diagnosis of ALL (n=9, 20%), AML (n=29, 60%), MM (n=5, 8%), HD (n=3, 6%), MPN (n=2, 4%), NHL (n=1, 2%). Reasons for being enrolled into study was a hematologic relapse in 36 (75%) or a molecular relapse in 12 (25%). The therapeutic strategy consisted of two infusions of unmanipulated DLI (each of 1 x 106/kg cells) at 3 weeks interval, followed by three infusions of donor derived CIK cells given at 3 weeks interval. The first 12 patients were treated with growing numbers of CIK cells, in groups of three patients per dose level. Since DLT was never observed (acute GVHD of grade III or more) the highest dose planned (5 x 106/kg, 5 x 106/kg and 10 x 106/kg) was then administered to subsequent consecutive 36 patients. 4 patients died for disease progression and 1 patient developed aGVHD (grade I, skin only) during the DLI treatment and could not proceed to the planned subsequent CIK administration. Of the 43 patients who eventually received at least one infusion of CIK cells, 15 patients did not complete the program, 9 for disease progression and death, 3 for insurgence of grade II aGVHD (skin only in 2 cases, skin and gut in 1 case), 1 for hemolytic anemia, 1 for insufficient cell supply and 1 for medical decision. Overall, 28 patients received the complete cell therapy planned (58%). Overall, of the 48 patients enrolled, 5 (10%) suffered from aGVHD (1 grade I, 3 grade II, 1 grade III). During follow up, chronic GVHD was observed in 7 patients (14 %) (3 mild and 4 moderate). As per protocol, clinical response was determined 100 days after the last CIK administration and the study was analyzed on an intent to treat basis. An early death occurred in 13 (27%) patients (4 during the DLI), before the clinical response could be evaluated. A CR was observed in 9 (19 %) and a PR in 7 (14%) for an overall response rate of 16 (33%). No response was observed in 19 (39%). At 2 and 4 years, the event free survival of the 48 patients is 22% and 18%, while the overall survival is of 37% and 34%, respectively. For the small group of patients who achieved a complete response, the disease free survival is of 64% at 2 years and 51% at 4 years. By univariate analysis, survival was significantly associated to the type of relapse (molecular) (p 0.0081) since at 2 and 4 years it was and 24% and 27% vs. 71% and 71 % for patients enrolled for a hematologic or a molecular relapses, respectively. By multivariate analysis, the type of relapse remained the only significant predictor of survival (0.0160 p value). Conclusion This study shows the feasibility of CIK preparation and administration as well as the relatively low toxicity of the program (10% aGVHD grades I-III) in spite of the fact that 20 patients received cells from matched unrelated donors. Finally, the study offers the suggestion that CIK cells may be efficacious to treat post-transplant relapse. 1 Introna M. et al, Haematologica, 2007, 92, 7, 948. Figure 1. Figure 1. Disclosures Introna: roche: Research Funding. Rambaldi:Roche: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Research Funding; Pierre Fabre: Honoraria.

Background With the advent of imatinib and the other tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, the outcomes of Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) improved substantially. Nonetheless, allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1) remains the consolidation therapy of choice in Ph+ ALL. Evidence is emerging that post-transplant relapse is influenced by the persistence of minimal residual disease (MRD), with an inferior outcome of patients undergoing transplantation with measurable level of MRD (Sramkova L et al, Pediatr Blood Cancer 2007; Bar M et al. Leuk Res Treatment 2014). Considering that a deeper molecular response can probably be achieved with innovative targeted therapies, such as second and third-generation TKIs or immunotherapy, an accurate evaluation of MRD values before alloSCT may be very relevant. Aim of the study. To evaluate the predictive relevance of MRD levels before transplant in Ph+ALL patients in CR1 on the probabilities of (i) overall survival (OS), (ii) relapse incidence (CIR) and (iii) leukemia free survival (LFS) Patients and methods. One hundred and six adult patients (median age 41.2, range 19-62) with newly diagnosed Ph+ ALL (as determined by cytogenetic or molecular analysis) were enrolled into 2 prospective NILG protocols (09/00 ClinicalTrial.gov Identifier: NCT00358072 and 10/07 ClinicalTrial.gov Identifier: NCT00358072) and were treated with chemotherapy and imatinib. One hundred (94%) achieved CR1, of whom 72 patients underwent an alloSCT in CR1 and are the subject of this report. MRD was determined by quantitative polymerase chain reaction (RQ-PCR) according to validated methods. Results. Among the 72 patients undergoing alloSCT, MRD status before transplant was available for 65 patients (90%). Twenty-four patients (37%) achieved a complete molecular response (BCR-ABL/ABL For the whole patient cohort (n=106), the median follow-up was 2.8 years (range 0.06-11.8), with a 5 years OS of 41%. The OS of patients receiving alloSCT was 50%. The MRD negativity at time of conditioning was associated with a significant benefit in terms of risk of relapse with a CIR of 8% compared to 39% of patients with MRD positivity (p=0.007) (Figure 1A). Nonetheless, the LFS and OS probability were not significant different in MRD- compared to MRD+ patients (58% vs 41%, p=0.17 and 58% vs 49%, p=0.55, respectively) (Figure1B), likely due to the effective post-relapse treatment with TKIs and/or DLI. The cumulative incidence of non relapse mortality was similar in MRD- compared to that of MRD+ group (33% vs 20%, p=0.22). Conclusions. Our results confirm that patients undergoing alloSCT with measurable levels of MRD show a significant increase risk of relapse after transplant. These results highlight the importance of achieving a complete molecular remission before transplant that should be considered an essential prerequisite for successful alloSCT. Table 1. Patients' characteristics according to MRD group Characteristics MRD negative (N=24) MRD positive (N=41) P Age years , median (range) 45.0 (21.4-58.2) 42.7 (18.5-62.4) 0.95 Male sex (%) 16 (67) 15 (37) 0.01 WBC, X 109/L, median (range) 27.7 (0.9-350.0) 12.0 (1.1-680.0) 0.12 Hemoglobin, g/dL, median (range) 11.4 (5.4-14.6) 9.0 (3.7-16.5) 0.02 Platelets, X 109/L, median (range) 41.0 (4.0-336.0) 34.0 (3.0-325.0) 0.44 LDH, U/L median (range) 1231 (353-8104) 715 (65-6194) 0.12 Conditioning regimen (%)Reduced intensity Myeloablative 4 (17)20 (83) 7 (17)34 (83) 1.00 Donor type (%)SiblingUnrelated 13 (54)11 (46) 18 (44)23 (56) 0.73 Graft type (%)Bone marrow Peripheral blood Cord blood 3 (12)20 (83)1 (4) 9 (22)31 (76)1 (2) 0.91 Figure 1. CIR and LFS according to MRD group Figure 1. CIR and LFS according to MRD group Disclosures No relevant conflicts of interest to declare.

Background The combination of a myeloablative dose of intravenous (iv) busulfan with cyclophosphamide is the standard preparative regimen for allogeneic hematopoietic stem cell transplantation (HSCT) in acute myeloid leukemia (AML) patients. However, in patients older than 40 years, this conditioning can be associated to high non relapse mortality (NRM). A similar myeloablative dose of busulfan combined to fludarabine was found associated to a lower NRM in older AML (Alatrash, BBMT 2011). Patients and study design The Gruppo Italiano Trapianto Midollo Osseo (GITMO) conducted a Phase III, randomized, multicenter, trial to compare the standard myeloablative combination of iv busulfan (Busilvex®, Pierre Fabre, Boulogne, France) at a dose of 0.8 mg/kg/6h over two hours infusion for 4 consecutive days (16 doses), for a total dose of 12.8 mg/kg, in combination with cyclophosphamide at the dose of 60 mg/kg/day for 2 consecutive days for a total dose of 120 mg/kg (BUCY2 arm) or fludarabine at the dose of 40 mg/m2/day for 4 consecutive days, for a total dose of 160 mg/m2 (BUFLU arm). Eligible were patients with a diagnosis of AML in 1st or 2nd complete remission (CR) with an age ≥40 and ≤ 65 years, and the availability of an HLA compatible sibling or unrelated donor as defined by molecular high-resolution typing (4 digits) of the HLA gene loci class I (HLA- A, B, and C) and class II (DRB1). Excluded were patients with a t(15;17) or PML/RARα positive APL or with a t(8;21)(q22;q22) or an inv(16) or t(16;16)(p13;q22) positive AML in 1st CR. The GvHD prophylaxis was based on conventional Cyclosporine A and Methotrexate. In case of unrelated donors, anti Thymocyte Globulin (Thymoglobuline®, Sanofi-Aventis) was given at a total dose of 5 mg/kg (or 7.5 mg/kg, in case of HLA acceptable disparity) (one antigen/allele disparity in class I, or one allele disparity in class II). The primary study end-point was the one-year NRM using an intent-to-treat analysis. The required sample size was calculated assuming that the one-year NRM would have been halved (from 25% to 12.5%) in the BUFLU arm. The cumulative incidence of NRM was estimated by considering relapse as a competing event. All outcomes were evaluated from the date of transplantation. The study was approved by the Institutional Review Boards of each center. Results From July 2008 to February 2013, 25 centers in Italy and 1 in Israel, enrolled 245 patients who were randomly assigned to BUCY2 (n=121) or BUFLU (n=124), stratified according to donor type and remission (1st vs. 2nd or more). The main clinical features (balanced between the randomization arms) were as follows: the median age was 50 years, 209 patients (85%) were in 1st and 36 (15%) in 2ndCR and the ELN risk subgroups were good (11%), intermediate-1 (46%), intermediate-2 (20%) and adverse (23%). The donor was a sibling related (n= 112, 46%) or matched unrelated (n= 133, 54%) while the stem cell graft was the peripheral blood (PB, n= 168, 69%) or the bone marrow (BM, n= 77, 31%). The overall survival rate in the BUCY2 and BUFLU arm was 71% vs. 78% at 1 year, 65% vs. 62 % at 2 years and 56% vs. 57% at 5 years, respectively (P=ns). A non-significant lower incidence of relapse was documented in the BUCY2 vs. the BUFLU arm being 20.7% vs. 24.2% at 1 year, 25.6% vs. 29% at 2 years and 28.9 vs. 32.3 at 5 years, respectively. On the contrary, at 1 year, the overall NRM in the BUCY2 arm was 17.4% vs. 7.3% in the BUFLU (Gray Test P=0.02). At 2 years and throughout the study, the same significantly different NRM was observed between study arms being respectively 18.2% vs. 8.9% and 19% vs. 9.7% (Gray Test P=0.03) (Figure 1). Causes of NRM in the BUCY2/BUFLU arms were: infections 8/6, organ failures 9/0, GvHD 5/3, hemorrhage 1/1, others 0/2. All in all, at 1, 2 and 5 years the leukemia free survival of the BUCY2 and BUFLU arm was similar being 62% vs. 69%, 56% vs. 62% and 50% vs. 56%, respectively (P=ns) (Figure 2). The number of patients with grade III-IV acute GvHD was higher in the BUCY2 arm (P= 0.02). There were no significant between-group differences in the incidence of chronic GvHD. Conclusion In AML patients older than 40 years, the reduced toxicity conditioning with iv BUFLU significantly reduced the NRM compared to BUCY2. The increased incidence of leukemia relapse in the BUFLU arm was not associated with a detrimental effect on overall and leukemia free survival. (Funded by a grant from the Agenzia Italiana per il Farmaco (AIFA), ClinicalTrial.gov Identifier: NCT1191957). Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Rambaldi: Pierre Fabre Pharma: Consultancy.

Abstract 2733 Introduction: The best treatment of peripheral T-cell lymphomas (PTCLs) currently remains a matter of debate. Some retrospective study and few phase II trials have shown that first line high dose therapy (HDT) with autologous stem cell transplant (SCT) may apparently offer better results. However, no comparative trials are currently available. Material and Methods: We retrospectively evaluated the outcome of previously untreated patients with PTCL, excluding mycosis fungoides and Sézary syndrome, referred to our Center between 1996 and 2012. An in-house histologic revision was performed to confirm the diagnosis according to the latest WHO criteria. From this analysis we excluded patients with diagnosis of ALK positive anaplastic large cell lymphoma (ALCL). We stratify patients according to treatment. Conventional therapy (CT) consisted of CHOP, CHOEP or MACOP(-B). From early 90s at our Center was adopted the strategy to consolidate response to initial treatment with either autologous or allogeneic SCT, whenever possible, in patients aged Results: The specific diagnosis of the 124 patients evaluated was PTCL-not otherwise specified (n=70), angioimmunoblastic T-cell lymphoma (n=21), ALCL ALK negative (n=19), other subtypes (n=14). At diagnosis, 43 patients were planned to receive HDT plus SCT but only 26 (60%) eventually received it (22 autologous, 4 allogeneic) while the remaining experienced early death (8 patients), progression (8 patients) or mobilization failure (1 patient). The median age of this first cohort was 47.9 years (range 23–63), that also showed an advanced Ann-Arbor stage (III-IV) or an int-high/high IPI in 77% and 58% of the cases, respectively. The rate of complete remission (CR) was 57% with 21% of patients dying during treatment (5 patients not evaluable for response, 3 responders and 1 with progressive disease). Eighty-one patients were treated according to a CT strategy. The median age of this latter group was 66.7 years (25–85), an advanced stage (III-IV) or an int-high/high IPI was present in 73% and 61%, respectively. The CR rate was 57% with 19% of the patient dying during treatment (11 patients not evaluable for response, 2 responders and 1 with progressive disease). With a median follow up of 1.63 years (0–25) and 82 deaths, by intention to treat analysis the 5-years overall survival (OS) was 43% and 32% for HDT and CT (p=.90, figure 1), respectively, while the 5-years OS of those patients eventually receiving SCT was 64%. Irrespectively from the adopted treatment strategy, patients who achieved a CR showed a similar 5-years OS and disease free survival that were 57% and 52% in the CT group and 80% and 64% in the HDT cohort (p=.43 and p=.44), respectively. Of the 54 patients with relapsed or refractory disease, 9 underwent salvage SCT (4 autologous, 5 allogeneic) with 3 patients achieving a sustained remission. Conclusions: The overall clinical outcome of most PTCL patients remains unsatisfactory, with a large fraction of patients not responding to front-line treatment. The advantage of a post-remission consolidation with SCT has to be confirmed by appropriate ad hoc designed clinical studies. On the contrary, allogeneic SCT, despite remaining a potentially curative treatment option for these patients, unfortunately has a limited applicability due to the frequent advanced age and comorbidities as well as to the difficulty in finding a donor. Disclosures: No relevant conflicts of interest to declare.

Abstract 657 Background: Cytokine Induced Killer (CIK) cells have been previously shown by us and others to possess non restricted, NK-like anti-tumoral cytotoxicity in vitro and in vivo, with little graft-versus host disease (GVHD), in vitro and in several animal models. We have previously performed a phase I study with donor derived CIK cells in 11 adult hematologic patients and shown that the infusions of CIK cells (median 12.4 × 106/kg, range 7.2 to 87.4) were well tolerated and no acute or late infusion related reactions were recorded. Acute GVHD (grade I and II) was observed in 4 patients and progressed into extensive chronic GVHD in two cases (Introna et al, Haematologica 92:952, 2007). Methods: This study is an open-labeled, multicenter, exploratory phase IIA study to evaluate the safety (dose-finding) and efficacy of a sequential administration of donor derived unmanipulated lymphocytes (DLI) plus in vitro expanded CIK cells to patients with hematologic malignancies relapsing after related or unrelated allogeneic hematopoietic stem cells transplantation. The protocol was formally approved by the italian competent national authority (Agenzia Italiana del Farmaco, AIFA) on 14/04/2009. Two infusions of unmanipulated DLI (1×106/kg each) were given with a minimum interval of 3 weeks. Three infusions of donor CIK cells were then administered according to a dose escalating program, starting 3 weeks after the second DLI. CIK administrations were separated by 3 weeks intervals. Up to 4 combinations of dose escalating levels were provided in sequential order until the maximal tolerated dose (MTD) was reached. Indeed the first triplet of patients was supposed to receive CIK cells at the doses of 1×106/kg, 1×106/kg and 5×106/kg, the second 1×106/kg, 5×106/kg, 5×106/kg, the third 1×106/kg, 5×106/kg,10×106/kg, the last triplet 5×106/kg, 5×106/kg, 10×106/kg. In case of grade II or more severe acute GVHD, the next scheduled infusion was planned to be suspended. Only grade IV acute GVHD was considered the dose limiting toxicity (DLT). Once identified the MTD, this same dose will be administered up to 24 patients in a two-stage Simon's design. Results: So far 16 patients have been enrolled and 12 are evaluable. No DLT (grade IV acute GVHD) was observed at any dose. One patient suffered from grade III skin and gut acute GVHD in the group which received the highest dose and the therapy was stopped after the first administration of CIK cells (5×106/kg), while another patient showed grade I skin acute GVHD in this same group, but completed the 3 administrations program. So far, 7 patients have received the highest dose of CIK cells and they all completed the schedule without toxicity except one case. As per protocol, the best clinical response was evaluated 100 days after the end of the last CIK administration. We observed 3 CR (3 AML), 5 PR (3 AML, 1 MM and 1 HD) and 4 NR (2 AML, 1 NHL and 1 MM) in the 12 evaluable patients. Of the 12 evaluable patients, 5 required additional therapy at the end of the cell therapy program because of NR (3 patients) or to improve the PR (2 patients). Four patients died after a mean 63 days (3 due to relapse and 1 for infection), 6 patients are in PR and 2 patients remain in CR. Conclusions: These preliminary observations suggest that the sequential infusion of DLI and CIK cells is feasible with relatively minor toxicity, the MTD has not been achieved and the triplet of three CIK administrations of 5×106/kg, 5×106/kg and 10×106/kg will be further evaluated in 17 additional patients. This cell therapy program confirms the antineoplastic activity of this cell therapy schedule in some patients relapsing after allogeneic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 682 Background The treatment outcome of adult patients with Philadelphia positive Acute Lymphoblastic Leukemia (Ph+ ALL) remains remarkably unfavorable and this is why patients under the age of 60 are usually considered good candidates for allogeneic hematopoietic stem cell transplantation (alloHSCT). The availability of Imatinib and other tyrosine kinase inhibitors (TKI) like Dasatinib and Nilotinib, seems to be changing rapidly the clinical outcome of Ph+ ALL since these drugs may reduce early relapse, increase the clinical response and the proportion of patients to whom the transplant can be offered. In addition, the strict and accurate evaluation of minimal residual disease by quantitative polymerase chain reaction (RQ-PCR) is crucial to guide the post transplant treatment including the use of TKI and donor lymphocytes infusions. Nonetheless, the complications of the transplant (engraftment, immune reconstitution, graft versus host disease, GVHD) may still offset the benefit of the procedure in many patients, the elderly and those with comorbidity, in particular. Aim of the study To evaluate the role of Imatinib and molecular monitoring of minimal residual disease on the clinical outcome of adult Ph+ ALL patients undergoing or not alloHSCT. Patients One-hundred consecutive, untreated adult patients (median age 46, range 19–66) with Ph+ ALL (as determined by cytogenetic or molecular analysis) enrolled into Northern Italy Leukemia Group (NILG) protocol 09/00 (ClinicalTrial.gov Identifier: NCT00358072) are the object of this study. The protocol was approved by the institutional review board of all participating institutions and amended in February 2003, when imatinib became available and added to each chemotherapy course at 600 mg/d. Sixty-five patients received Imatinib during induction/consolidation (IM+ group, Bassan et al.: Journal of Clinical Oncology, 2010). Fifty-eight patients received alloHSCT from a sibling (n= 33), unrelated (n= 24) or haplo (n= 1) donor, while 42 received a conventional treatment (chemotherapy, n= 33 or an autologous transplant, n= 9) (with or without Imatinib). The median age of patients receiving or not an alloHSCT was 40 and 54 years, respectively. The conditioning regimen to alloHSCT was myeloablative in 49 and reduced intensity in 9. The stem cell source was the bone marrow (BM) in 20 and the peripheral blood (PB) in 38. Results The addition of Imatinib to chemotherapy during induction/consolidation increased the proportion of patients who achieved CR (93% vs 80%, p= 0.05) and who had the opportunity to undergo alloHSCT (66% vs 43%, p= 0.02). With a median follow-up of 1.5 years, the overall survival (OS) at 5 years was 35% for the whole patient cohort (n= 100), 39% in the IM+ group and 23% in the IM-, respectively (p=0.007). At 5 years, the OS of patients receiving alloHSCT was 46% vs 21% of the others (p= 0.0001) (Figure 1). The transplant related mortality at 4 years was 23%, no matter whether Imatinib was given or not any time during treatment. The cumulative incidence of relapse (CIR) of patients undergoing alloHSCT was 32% in the IM+ vs 57% in the IM- group. Interestingly, the CIR of alloHSCT patients was 19% for patients who proved MRD negative at time of conditioning vs 51% for those bearing any positive MRD level in the bone marrow or peripheral blood (p= 0.04). Accordingly, the disease free survival probability was 67% of MRD negative vs 42% of MRD positive patients (p=0.06). Conclusions This study shows that the combined use of Imatinib and chemotherapy during induction/consolidation (1) improves the long term clinical outcome of all patients with Ph+ ALL, (2) reduces the relapse probability of patients undergoing alloHSCT and (3) is associated with a better disease free and overall survival after transplantation. Finally, alloHSCT either from a sibling or unrelated donor remains the best post remissional treatment of Ph+ adult ALL patients despite a still remarkable transplant toxicity. Disclosures: No relevant conflicts of interest to declare.

Background Very recently, encouraging results indicate that third party human mesenchymal stromal cells (hMSCs) are a rapidly available therapeutic tool for the treatment of severe (grade III–IV), steroid resistant, acute graft versus host disease (aGVHD). In the clinical experience published so far, hMSCs have been expanded in Fetal Bovine Serum (FBS), which may constitute a problem for its antigenicity and as a possible vehicle of animal pathogens. We have established a highly efficient protocol for the in vitro expansion, under strict GMP compliance, of bone marrow derived hMSCs using human platelets lysate (PL) in place of FBS (Capelli C. et al.: BMT, 2007). In this study, upon Ethical Committee approval and patient’s informed consent, hMSCs were administered on a compassionate basis for the treatment of refractory GVHD. Methods hMSCs were prepared from washouts of bags and filters, left over at the end of the standard filtration procedures of the bone marrow harvests from third party HLA mismatched healthy donors. Cells were grown in the presence of DMEM with 5% PL obtained from the Blood Bank of our Hospitals. In a short period of time (10–33 days), low density seeding of unmanipulated cells (100–200/cm2), obtained from 7 bone marrow harvests allowed to prepare large quantities of hMSCs (median 115×106, range: 67–375), with only one in vitro passage. Twenty-three frozen bags of hMSCs (each containing approximately 1×106/kg of recipient body weight) have been quarantined until the completion of quality tests, including viability, phenotype, absence of detectable bacteria, fungi, mycoplasma or endotoxin, according to European Pharmacopea guidelines. Differentiation to osteogenic and chondrogenic cells as well as the immunosuppressive potential of these cells was confirmed when tested in mixed lymphocyte reaction (MLR). Q banding and clonogenic assays were performed for each batch and never showed abnormalities of karyotype or autonomous growth in vitro. Results Two adult and 4 pediatric patients were treated for aGVHD (grade II–IV) and 2 adults for extensive chronic GVHD (cGVHD) between January and July 2008, using 12 hMSCs bags that had completed quarantine. Before hMSCs, second or third line treatments had been given to patients with aGVHD, including Etanercept (n= 5), Mycophenolate Mofetil (MMF, n= 4) and Extracorporeal Photopheresis (ECP, n= 3), Rituximab (1 patient). Patients with cGVHD were previously treated with ECP and MMF (n= 2), Imatinib (n= 1) and Etanercept (n= 1). Each infusion contained a median dose of 1×106/kg (range, 0.7–1.2×106) hMSCs. For patients with aGVHD, a single infusion was performed in 4 pediatric patients while 1 and 3 infusions were performed in 2 adult patients. The 2 patients with cGVHD received 1 and 4 infusions, respectively. All infusions were very well tolerated with no immediate or late adverse events according to WHO common criteria. Among pediatric patients with aGVHD, 3 complete and 1 partial responses were registered and all patients are alive and in complete hematologic remission. A complete response was observed in 1 adult with grade III cutaneous aGVHD although the patient rapidly relapsed and died of leukemia progression. No response was observed in the other adult patient who died of progressive grade IV gut and liver aGVHD. The 2 adult patients with cGVHD had both a partial response and are alive. Conclusions These data show that large numbers of third party hMSCs can be expanded in vitro with PL containing medium and stored for immediate use in patients with GVHD. Moreover, the clinical results and the toxicity profile confirm those reported with hMSCs expanded in FBS containing media.

Cytokine induced killer cells (CIK) are T/NK cells with demonstrated anti-tumoral activity but lack of GVHD reactivity. They are expanded in vitro after stimulation of PBMC with OKT3, IFN-γ and rhIL-2. This phase I study was designed to test the safety and feasibility of repeated infusions of in vitro expanded donor derived CIK cells given to patients relapsed after allogeneic HSCT. The mean number of starting total nucleated cells was 707 × 106 (range 58–1500 × 106). After a median 22 days of culture, a mean percentage of 51% CD3+CD56+ cells (range 40–71%) was obtained corresponding to an absolute mean number of 1421×106 total CIK cells (range 422–2470 × 106). Eleven patients with AML (n=4), HD (n=3), CMML (n=1), pre-B ALL (n=1) and MDS (n=2), all relapsed after sibling (6) or matched unrelated donor (5) HSCT, entered this study. Before CIK administration, 7 patients had received one or more additional salvage treatments including chemotherapy (5), radiotherapy (1) and unmanipulated DLI (6) without any significant tumor response. The median number of CIK infusions was 2 (range 1–7) and the median number of total CIK cells was 14.5 ×106/kg (7.2–51). The infusions were well tolerated and no acute or late infusion-related reactions were registered. Acute GVHD (grade I and II) was observed in 4 patients 30 days after the last CIK infusion, which progressed into extensive chronic GVHD in 2 cases. In 6 patients, no significant clinical response could be registered so that disease progression and death occurred rapidly. In contrast, 5 patients achieved measurable responses: a patient with MDS, who had been treated with CIK cells alone, showed a hematologic improvement but subsequently progressed and died. One patient with HD received local radiotherapy and 7 CIK infusions (total of 51×106/kg CIK cells) which allowed the achievement of a good PR. After almost 1 year (300 days), he progressed and chemotherapy was given with achievement of a very good PR. A second patient with HD received one DLI at day 516 and 1 CIK infusion (12.2×106/kg) at day 537. At day 572, chemotherapy was initiated due to the persistence of disease. At the end of chemotherapy he received 3 additional CIK infusions for a total of 34×106/kg from days 711–752, without signs of aGVHD and is presently in CR at more than 780 days. One patient with CMML had been treated with DLI on day 102 and with a total of 38×106/kg CIK cells, given in four infusions from days 137–530, because of the appearance of mixed chimerism. Full chimerism was achieved 42 days after the first CIK infusion. The patient remains in CR at day 576. A second MDS patient, who had not achieved any significant response after five DLI (days 411–559), obtained a complete hematologic, cytogenetic and molecular remission after a single CIK infusion (7.6 ×106/kg) given at day 603 and remains in CR at day 680. This study shows that the production of allogeneic CIK cells is feasible, their infusion is generally safe and may induce clinical remission in patients relapsing after HSCT.

A procedure to obtain a controlled-release microencapsulated anti-inflammatory drug based on a solvent evaporation method is described. The present method makes use of ethylcellulose as the polymer and methylene chloride as solvent. The evaporation of solvent is controlled by means of an air stream. Variations in the preparative procedure and their effects on capsule dimensions and permeabilities were studied. The release behavior of the drug is determined, and two different diffusion constants are also determined: 7.0 X 10(-10) cm2/s and 1.2 X 10(-10) cm2/s, corresponding to low and high release time. Based on these results it is proposed that these microcapsules have a nonhomogeneous polymeric wall, and are more porous in the outer surface. This model might be applicable to the microcapsules obtained by means of the solvent evaporation method.

Glutaminolysis is known to correlate with ovarian cancer aggressiveness and invasion. However, how this affects the tumor microenvironment is elusive. Here, we show that ovarian cancer cells become addicted to extracellular glutamine when silenced for glutamine synthetase (GS), similar to naturally occurring GS‐low, glutaminolysis‐high ovarian cancer cells. Glutamine addiction elicits a crosstalk mechanism whereby cancer cells release N‐acetylaspartate (NAA) which, through the inhibition of the NMDA receptor, and synergistically with IL‐10, enforces GS expression in macrophages. In turn, GS‐high macrophages acquire M2‐like, tumorigenic features. Supporting this in␣vitro model, in silico data and the analysis of ascitic fluid isolated from ovarian cancer patients prove that an M2‐like macrophage phenotype, IL‐10 release, and NAA levels positively correlate with disease stage. Our study uncovers the unprecedented role of glutamine metabolism in modulating macrophage polarization in highly invasive ovarian cancer and highlights the anti‐inflammatory, protumoral function of NAA., This study reveals a crosstalk between ovarian cancer cells and tumor associated macrophages. Glutamine addicted cancer cells release the signaling metabolite N‐acetylaspartate (NAA), which in turn polarizes macrophages towards a GS‐high, M2‐like state.