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TP53 gene mutations are common in Myelodysplastic Syndromes (MDS) with del5q and have a clinical and prognostic significance. Next Generation Sequencing (NGS) is an accurate, but expensive, technique, and not commonly available. Immunohistochemistry (IHC) for TP53 expression has been recently used as a surrogate to assess TP53 mutations. To compare the concordance between TP53 expression in IHC and TP53 mutations by NGS, 30 cases with MDS harbouring a del5q abnormality were evaluated. Overall, 10/30 patients (33.3%) had TP53 mutations by NGS, while 16/29 (55.1%) had TP53 overexpression in IHC. TP53 expression by IHC had a 70% sensitivity to identify patients with TP53 mutation by NGS, but its specificity was low (52.6%, kappa = 0.198; P = 0.24). In addition, ROC curve analyses showed that the overall diagnostic value (accuracy) of TP53 expression in IHC to identify patients with TP53 mutation by NGS was 68% in the whole study sample and 67% in patients with isolated del5q-. In both cases, the areas under the curves did not attain the statistical significance (P = 0.11 and P = 0.29, respectively). Based on the ROC curve, the cut-off of 2.3% TP53 expression in IHC was shown to be the best cut-off to identify TP53 mutations: using this cut-off, the agreement between TP53 expression and TP53 mutation by NGS reached statistical significance (kappa = 0.42; P = 0.023). In conclusion, the agreement between TP53 expression in IHC and TP53 mutation analysis by NGS is rather unsatisfactory in MDS patients with del5q at the standard cut-off. Thus, the IHC technique cannot be considered a valid alternative to NGS evaluation of TP53 mutational status in these patients.

Mature adipocytes have shown dynamic plasticity to be converted into fibroblast-like and lipid-free cells. After the dedifferentiation process, these cells re-entered the cell cycle and acquired a high proliferation potential, becoming a valid source of stem cells. However, many aspects of the cellular biosafety about dedifferentiated fat cells remained unclear. This study aimed to elucidate their potential susceptibility to malignant transformation and to ascertain the safety of these cells for clinical use. To evaluate the genomic stability of dedifferentiated adipocytes, telomere length, hTERT gene transcription, the capacity of these cells to grow in an anchorage-independent manner and the presence of DNA damage by single cell gel electrophoresis assay were studied. Spontaneous chromosomal alterations were excluded by cytogenetic analysis and the expression level of c-myc and p53, tumor associated genes, were assessed, evaluating also p53 loss of function mutations. Despite the high proliferation capacity of dedifferentiated adipocytes, these cells showed stable telomere length compared with mature adipocytes, no hTERT transcriptions and consequently no telomerase activity, suggesting that both transformation and senescence were avoided. A constant expression level of c-myc and p53, the inability of dedifferentiated adipocytes to grow in an anchorage-independent manner, the absence of DNA damage suggested the safety of these cells. Moreover, a normal karyotype was preserved throughout the dedifferentiation process. Data in vivo showed that dedifferentiated adipocytes analyzed for tumorigenicity did not develop tumors. In conclusion, our data indicated that dedifferentiated adipocytes could be a relatively easily accessible resource for cell therapy and regenerative medicine.

To detect factors associated with quality of life (QOL) of patients with myelodysplastic syndrome (MDS) and to compare the MDS patients’ self-assessed QOL with that perceived by their physicians. In an observational, non-interventional, prospective, multicentre study, QOL was evaluated in 148 patients with newly diagnosed low- and intermediate-risk IPSS MDS. QOL measures (QOL-E v.2, LASA and EQ-5D) and patient-related candidate determinants of QOL were assessed for up to 18 months. Patients' QOL scores were compared with those obtained by appointed hematologists’ assessment and with ECOG performance status (PS). Fatigue was not prevalent at diagnosis, though physical QOL and energy levels were low. Transfusion-dependent patients had worse QOL scores. In multivariate analysis, Hb levels and comorbidities were a major determinant of QOL. Physicians’ perception of patients’ well-being correlated with patients’ QOL. Physicians underestimated the impact of disturbances on patients’ QOL, mainly in the MDS-specific components. ECOG PS did not discriminate patients according to QOL status. In conclusion, the association of anemia with QOL is confirmed, while co-morbidities emerge as an independent predictor of QOL in MDS. Fatigue is not a major concern. ECOG PS is not a valuable surrogate of patient’s QOL, thus highlighting that physician’s judgment of patient’s well-being must not substitute patient-reported outcomes. Appropriate questionnaires should be used to assess MDS patients’ QOL in order to improve communication and therapeutic choice.

Abstract 3822 Poster Board III-758 BACKGROUND The heterogeneous nature of myelodysplastic syndromes (MDS), age- and disease-related factors (complications and progression to acute leukemia) are associated with the complexity of quality of Life (QoL). Patients are offered mainly non-curative or experimental drugs and/or supportive care. The exploration of QoL in MDS is a prerequisite for adequate therapeutic choice. METHODS We designed an observational study in MDS patients with IPSS risk score ≤2 to evaluate determinants of QoL and its correlates. Clinical and laboratory data were collected up to 18 months and QoL instruments (QOL-E v.2, LASA scale, and EQ-5D) were completed by patients and physicians (both blind to each other's responses) at baseline, months 1, 3, 6, 12, and 18. After diagnosis, treatment was assigned based on investigators' judgment. RESULTS Of 148 patients enrolled (mean age 72 years, 56% males), 115 (78%) patients were anemic at diagnosis and 38 (26%) had already received transfusions. Mean (± SD) Hb was 10.3 ± 2.1, ANC 2.3 ± 1.9×103/μL and PLT count 155 ± 118×103/μL. Charlson's Comorbidity Score was > 1 in 33 (22%). Physical and functional QoL, health, energy, activity and general states were generally poor ( CONCLUSIONS QoL, together with prognostic scores, should guide therapeutic choice in MDS. Avid research in MDS tends to focus on younger and fit patients to offer more aggressive or novel therapies with the aim of increasing survival. However, patients of advanced age with comorbidities complicated with severe anemia and transfusion-dependence represent a fragile category with particularly poor QoL that actually require special therapeutic attention to favor transfusion-independence and possibly an hemoglobin response. Disclosures: No relevant conflicts of interest to declare.

Introduction The “classical” IPSS, based on cytogenetics, marrow blast percentage, and number of cytopenias, has played a major role in prognosis assessment in MDS. The recently published revised IPSS (IPSS-R), refines the original IPSS prognostic value (Greenberg et al, Blood 2012). However, its prognostic value for response to ESA has not been assessed. We analyzed it retrospectively in 456 IPSS Low-/Int-1-risk MDS patients (pts) treated with ESA in France, Germany and Italy. Results Those 456 pts had serum EPO < 500mU/ml and Hb≤10g/dl, and had received ESA (EPO alfa or beta 40000-60000 IU/week, or darbepoietin 150 - 300µg/week) for at least 12 weeks. In addition to IPSS-R parameters, age, sex, serum EPO level, serum ferritin (SF), and RBC transfusion requirement before ESA onset were assessed for response to ESA (based on IWG 2006 criteria), and overall survival (OS) from ESA onset. Characteristics of the 456 pts at ESA onset are listed in Table 1. 71% of the pts had never received RBC transfusions, with a median Hb level of 9.3g/dl (range 7.0-10) , and 29% pts had received at least 4 RBC concentrates/8wks before ESA onset (with a maximum of 12). Median SF was 357ng/ml and serum EPO 60mU/ml (range 6-483). IPSS was low in 55% and intermediate-1 in 45% pts. IPSS-R was very low in 15%, low in 61%, intermediate in 19% and high in 4% of the pts. 303 (61%) pts had an erythroid response (ER), including 72% and 52% of low and int-1 risk pts respectively (p=0.001). Using IPSS-R, 85%, 68%, 48% and 31% of pts had ER in the very low, low, intermediate and high risk group respectively (p Other prognostic factors of ER, in univariate analysis, included individual IPSS-R parameters analyzed according to IPSS-R thresholds (Hb level, platelet count, ANC, marrow blast %, cytogenetics) serum EPO level, SF (variables tested as continuous variables) and previous RBC-transfusions. In multivariate analysis, IPSS-R, serum EPO, and SF remained significantly associated with ER (p Applying 1 point to each of the following unfavorable variables of ER: serum EPO >200mU/ml (=1), SF >350 ng/ml (=1), IPSS-R (very low=0, low=1, intermediate=2 and high=3), yielded a score ranging from 0 to 4, with ER rates of 85%, 80%, 64%, 40% and 20% respectively. As expected, IPSS-R also had strong prognostic value for OS (not shown). Conclusion In this patient cohort with overall favorable prognostic factors of response to ESA according to the Nordic score (ie serum EPO =3 (using IPSS-R, serum EPO and SF) proved useful to identify pts with low response to ESA, who also have worse OS and may require alternative treatments (Kelaidi et al, Leukemia 2013). Disclosures: No relevant conflicts of interest to declare.

Shwachman-Diamond syndrome (SDS) is one of the most commonly inherited bone marrow failure syndromes (IBMFS). In SDS, bone marrow is hypocellular, with marked neutropenia. Moreover, SDS patients have a high risk of developing myelodysplastic syndrome (MDS), which in turn increases the risk of acute myeloid leukemia (AML) from an early age. Most SDS patients are heterozygous for the c.183-184TA>CT (K62X) SBDS nonsense mutation. Fortunately, a plethora of translational read-through inducing drugs (TRIDs) have been developed and tested for several rare inherited diseases due to nonsense mutations so far. The authors previously demonstrated that ataluren (PTC124) can restore full-length SBDS protein expression in bone marrow stem cells isolated from SDS patients carrying the nonsense mutation K62X. In this study, the authors evaluated the effect of a panel of ataluren analogues in restoring SBDS protein resynthesis and function both in hematological and non-hematological SDS cells. Besides confirming that ataluren can efficiently induce SBDS protein re-expression in SDS cells, the authors found that another analogue, namely NV848, can restore full-length SBDS protein synthesis as well, showing very low toxicity in zebrafish. Furthermore, NV848 can improve myeloid differentiation in bone marrow hematopoietic progenitors, enhancing neutrophil maturation and reducing the number of dysplastic granulocytes in vitro. Therefore, these findings broaden the possibilities of developing novel therapeutic options in terms of nonsense mutation suppression for SDS. Eventually, this study may act as a proof of concept for the development of similar approaches for other IBMFS caused by nonsense mutations.

Background aims Adipose tissue is a critical organ that plays a major role in energy balance regulation and the immune response through intricate signals. Methods We report on the inter-relation between mature adipocytes and lymphocytes in terms of adipocyte-derived T-cell chemo-attractants and adipocyte metabolic effects on lymphocytes. Results During the culture time, mature adipocytes changed their structural and functional properties into de-differentiated cells. Isolated mature adipocytes expressed significantly higher levels of CIITA , major histocompatibility complex II (human leukocyte antigen [HLA]-DR) and costimulatory signal molecule CD80 compared with adipocytes after the de-differentiation process. Moreover, human leukocyte antigen–G, which may prevent the immune responses of mesenchymal stromal cells, was expressed at lower level in mature adipocytes compared with de-differentiated adipocytes. In line with these molecular data, functional results showed different immunoregulatory properties between adipocytes before and after the de-differentiation process. Mature adipocytes stimulated the proliferation of total lymphocytes and immunoselected cell populations CD3+, CD4+ and CD8+ in a direct contact-dependent way that involved the major histocompatibility complex I and II pathways. Moreover, adipocytes secreted potential chemo-attractant factors, but data showed that adipocyte-derived culture medium was not sufficient to activate lymphocyte proliferation, suggesting that a direct contact between adipocytes and immune cells was needed. However, specific mature adipocyte cytokines enhanced lymphocyte proliferation in a mixed lymphocyte reaction. Conclusions In conclusion, cross-talk occurs between adipocytes and lymphocytes within adipose tissue involving T-cell chemo-attraction by mature adipocytes. Our findings, together with current observations in the field, provide a rationale to identify adipocyte-lymphocyte cross-talk that instigates adipose inflammation.

Summary Iron chelation is controversial in higher risk myelodysplastic syndromes (HR-MDS), outside the allogeneic transplant setting. We conducted a retrospective, multicentre study in 51 patients with transfusion-dependent, intermediate-to-very high risk MDS, according to the revised international prognostic scoring system, treated with the oral iron chelating agent deferasirox (DFX). Thirty-six patients (71%) received azacitidine concomitantly. DFX was given at a median dose of 1000 mg/day (range 375–2500 mg) for a median of 11 months (range 0·4–75). Eight patients (16%) showed grade 2–3 toxicities (renal or gastrointestinal), 4 of whom (8%) required drug interruption. Median ferritin levels decreased from 1709 μg/l at baseline to 1100 μg/l after 12 months of treatment (P = 0·02). Seventeen patients showed abnormal transaminase levels at baseline, which improved or normalized under DFX treatment in eight cases. One patient showed a remarkable haematological improvement. At a median follow up of 35·3 months, median overall survival was 37·5 months. The results of this first survey of DFX in HR-MDS are comparable, in terms of safety and efficacy, with those observed in lower-risk MDS. Though larger, prospective studies are required to demonstrate real clinical benefits, our data suggest that DFX is feasible and might be considered in a selected cohort of HR-MDS patients.

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here, we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes, suggesting that these cells could be committed to the neural lineage. After neural induction, NeuroD1, Sox1, Double Cortin, and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties, indicating the inability of these cells to differentiate into mature neurons. In contrast, the differentiated cells expressed a high level of CLDN11, as demonstrated using molecular method, and stained positively for the glial cell markers CLDN11 and GFAP, as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production, which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion, our data demonstrate morphological, molecular, and immunocytochemical evidence of initial neural differentiation of mature adipocytes, committing to a glial lineage.