Abstract: Abstract 4851 Background Nucleoside 5-Azacitidine (5-Aza) administered at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates, reduces progression to acute myeloid leukaemia (AML) and increases overall survival in the high risk MDS patients. Aim The use of 5-Aza in the earlier phases of MDS could reduce the proliferative advantage of MDS clone and favours the regrowth of normal hematopoiesis. In the setting of low-risk MDS patients lower doses of 5-Aza could be enough to induce hematologic responses. We attempted to use an alternative schedule, 75 mg/mq subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses, to evaluate its efficacy and tolerability in low risk MDS patients. Pharmacogenomic studies (gene expression profile and single nucleotide polymorphism analysis), cytokine network and phosphatidylinositol-phospholipase C – β1 (PI-PLC - β1) expression, before and after 5-Aza treatment, were planned to identify new biological markers to predict the response. Methods From September 2008 to August 2009, 25 patients were enrolled into the study. According to WHO criteria 12 patients had refractory anemia (RA), 5 patients refractory cytopenia with multilineage dysplasia (RCMD), 7 patients refractory anemia with excess blasts-1 (RAEB-1) and 1 patient refractory anemia with ringed sideroblasts (RARS). All patients were classified as Low Risk (IPSS score 0-1). Age at diagnosis ranged between 56 and 84 years. All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results 9/25 (36%) patients completed at least 4 courses of therapy and only 6/25 (24%) patients finished the treatment plan (8 courses). Five patients obtained an hematologic improvement (3 erythroid response and 2 neutrophil response) whereas 4 patients maintained a stable disease. The others 16 patients are ongoing. The drug was very well tolerated. WHO grade III hematologic toxicity consisted in neutropenia (1 case) and thrombocytopenia (1 case) was observed in only two patients, but it was transitory and no delay of treatment was necessary. Conclusion Our preliminary results show that the 5-Aza five days/monthly schedule is very well tolerated and it appears to induce hematologic improvements as well as the seven days/monthly schedule, at least in the low-risk MDS setting. Biological studies will be useful to elucidate the genetic bases of sensititvity or resistance to 5-AZA and to optimise the therapy.Acknowledgments: this work was supported by MIUR 2008. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction. The association of Azacitidine (AZA) and Lenalidomide (LEN), either administered concurrently (Sekeres, 2010; 2012; 2017), or sequentially (Platzbecker, 2013; Di Nardo 2015; Mittelman 2016; Narayan 2016) has proven effective in Myelodysplastic Syndromes (MDS), however the optimum dose and schedule remains unknown. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in higher-risk MDS pts. Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the IWG criteria (Cheson, 2006). Secondary endpoints: a) rate of CR; b) duration of responses; c) overall survival (OS). Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS with IPSS risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles. For responder patients the same treatment was continued until disease progression or unacceptable toxicity. Results. From March 2013, 44 pts (27 males), median age: 72 (48-83 yrs) were enrolled, from 13 hematologic Centers. At baseline, IPSS risk was: Intermediate-2: 31 pts; High: 9 pts; not determined (N.D.) (because of lack of cytogenetic data): 2 pts. (all with RAEB-2). In 2 pts IPSS risk was Intermediate-1, but they were enrolled because of severe thrombocytopenia and neutropenia, respectively. IPSS-R risk was: intermediate: 8 pts; High: 16 pts; Very-High: 18 pts; N.D.: 2 pts. In 5 pts (11.4%) del(5q) was present (additional cytogenetic alterations: 1 in 1 pt, and 〉 1 in 4 pts , respectively). 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. Treatment was given for a median of 8.5 (1-52) cycles; in ARM 1: 9 (1-51) cycles; in ARM 2: 8 (1-52) cycles, respectively. Median follow-up: 15 (2-54) months; 47 (37-54) months for survivors. 10/44 pts (22.7%) did not complete at least 6 cycles of treatment for causes other than disease progression (6 pts for adverse events, 2 pts for consent withdrawal and 2 pts for medical decision), and were not considered evaluable for response. Among the 34/44 pts (77.3%) evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. Intention-to-treat (ITT) analysis: ORR: 59.1%. First response was observed after a median of 2 (1-8) cycles. The Best Response achieved was: CR: 8 pts (23.5%) (ITT: 18.1%), PR: 1 pt (2.9%) (ITT: 2.2%), mCR: 3 pts (8.8%) (ITT: 6.8%), HI: 8 pts (23.5%) (ITT: 18.1%), mCR+HI: 6 pts (17.6%) (ITT: 13.6%). The remaining 8 pts showed either Stable Disease (SD) (6 pts, 17.6%) or Disease Progression (DP) (2 pts, 5.9%). Among the 27 pts (21 evaluable for response) with an abnormal karyotype at baseline, ORR was 66.7% (ITT: 51.8%) and 4 pts achieved complete cytogenetic response. Median duration of hematologic response: 10.5 months. 34 pts (77,3%) died , and 17 pts (38.6%) showed progression to AML. Grade 〉 2 non haematological toxicity: 54.5%. Median OS: 15 months. No significant differences between the 2 arms were observed, in terms of ORR (ARM 1: 76.5%, ITT: 61.9%; ARM 2: 76.5%, ITT: 56.5%), CR rate (ARM 1: 17.6%, ITT: 14.3%; ARM 2: 29.4%, ITT: 21.7%), grade 〉 2 non haematological toxicity (ARM 1: 66.7%; ARM 2: 43.5%), AML incidence (ARM 1: 28.6%; ARM 2: 47.8%) and OS (ARM 1: 14 months; ARM 2: 16 months), but the median response duration was significantly longer in ARM 2 (18 months) as compared to ARM 1 (6 months) (p=0.0459). At the time of last analysis, 5/44 (11.4%) patients, 1/21 (4.8%) in ARM 1, and 4/23 (17.4%) in ARM 2, were still maintaining the haematological response, and were still in treatment, after 54, 54, 52, 51 and 37 months, and after 52, 51, 33, 48 and 35 cycles of therapy, respectively. The changes observed during treatment in mutational status of inositide-specific genes and microRNA expression profiling were related to clinical outcome, predicting a negative response to therapy. Conclusions. Our results confirm the efficacy of both AZA+LEN treatment regimens in higher-risk MDS pts, in terms of ORR and OS, although pts treated with the sequential regimen showed a significantly longer duration of haematological response. Disclosures Finelli: Celgene: Other: speaker fees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: speaker fees; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fees. Candoni:Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Merck SD: Honoraria, Speakers Bureau. Gobbi:Novartis: Consultancy; Janssen: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfister: Membership on an entity's Board of Directors or advisory committees. Rigolin:Gilead: Research Funding. Cavo:GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Abstract: Connective tissue growth factor (CTGF/CCN2), a cysteine-rich protein of the CCN (Cyr61, CTGF, Nov) family of genes, emerged from a microarray screen of genes expressed by human rhabdomyosarcoma cells. Rhabdomyosarcoma is a soft tissue sarcoma of childhood deriving from skeletal muscle cells. In this study, we investigated the role of CTGF in rhabdomyosarcoma. Human rhabdomyosarcoma cells of the embryonal (RD/12, RD/18, CCA) and the alveolar histotype (RMZ-RC2, SJ-RH4, SJ-RH30), rhabdomyosarcoma tumor specimens, and normal skeletal muscle cells expressed CTGF. To determine the function of CTGF, we treated rhabdomyosarcoma cells with a CTGF antisense oligonucleotide or with a CTGF small interfering RNA (siRNA). Both treatments inhibited rhabdomyosarcoma cell growth, suggesting the existence of a new autocrine loop based on CTGF. CTGF antisense oligonucleotide-mediated growth inhibition was specifically due to a significant increase in apoptosis, whereas cell proliferation was unchanged. CTGF antisense oligonucleotide induced a strong decrease in the level of myogenic differentiation of rhabdomyosarcoma cells, whereas the addition of recombinant CTGF significantly increased the proportion of myosin-positive cells. CTGF emerges as a survival and differentiation factor and could be a new therapeutic target in human rhabdomyosarcoma.

Abstract: Purpose: This phase II prospective study aimed to evaluate the efficacy and safety of 5-days azacytidine (5d-AZA) in patients with low-risk myelodysplastic syndromes (MDS). Second, single-nucleotide polymorphism (SNP) genetic profile and phosphoinositide-phospholipase C (PI-PLC) β1 levels were studied to evaluate possible biologic markers able to predict the hematologic response. Experimental Design: The study tested a lower intensity schedule of azacytidine. The treatment plan consisted of 75 mg/sqm/d subcutaneous administered for 5 days every 28 days, for a total of 8 cycles. Results: Thirty-two patients were enrolled in the study. The overall response rate was 47% (15 of 32) on intention-to-treat and 58% (15 of 26) for patients completing the treatment program. In this latter group, 5 (19%) achieved complete remission (CR) and 10 (38%) had hematologic improvement, according to the International Working Group (IWG) criteria. Three patients have maintained their hematologic improvement after 37, 34, and 33 months without other treatments. Moreover, 21 and 2 of 26 cases completing 8 cycles were transfusion-dependent for red blood cells and platelets at baseline, respectively. Of these, 7 (33%) and 2 (100%) became transfusion-independent at the end of the treatment program, respectively. Grade 3–4 neutropenia occurred in 28% of patients and 4 patients died early due to infections or hemorrhage. SNP results were not significantly correlated to the clinical outcome, whereas PI-PLCβ1 level anticipated either positive or negative clinical responses. Conclusions: 5d-AZA is safe and effective in a proportion of patients with low-risk MDS. PI-PLCβ1 gene expression is a reliable and dynamic marker of response that can be useful to optimize azacytidine therapy. Clin Cancer Res; 19(12); 3297–308. ©2013 AACR.

Abstract: Abstract 4635 Nuclear inositide signalling pathways are involved in the MDS progression to AML. Indeed, in the last few years our group demonstrated not only that MDS cells can show alterations on PI-PLCbeta1 and Akt pathways, but also that Akt is inversely correlated with PI-PLCbeta1, therefore affecting MDS cell survival and differentiation. Lenalidomide has proven effectiveness in 70–80% low-risk MDS cases with del(5q), resulting in transfusion-independence with a rise in hemoglobin levels, suppression of the 5q clone, and improvement of bone marrow morphologic features. In particular, in del(5q) MDS, Lenalidomide probably acts by directly suppressing the dysplastic clone, while in non-del(5q) it might enhance an effective erythropoiesis, possibly via activation of the EPO signalling, which in turn is associated with PI-PLCgamma1 pathways. However, the exact molecular mechanisms underlying the effect of Lenalidomide in MDS cells are still not completely clarified. Interestingly, Lenalidomide might inhibit the phosphatase PP2A, whose gene is located in the common deleted region and which usually targets Akt. Indeed, Akt-dependent pathways are critical in low-risk MDS cells, which display a marked apoptosis and a low proliferation rate. In this study we examined four MDS patients treated with Lenalidomide, and compared them with four low-risk MDS patients (IPSS: Low or Int-1) who only received best supportive care. In our study, all of the patients treated with Lenalidomide were affected by del(5q) low-risk MDS (IPSS: Low or Int-1), with transfusion-dependent anemia, and had only received supportive care before undergoing Lenalidomide treatment. Clinically, all of the patients responded to Lenalidomide: one patient reached Complete Remission, whilst the other three patients showed erythroid Hematologic Improvement. In contrast, all of the patients who were treated only with best supportive care maintained a Stable Disease. As for the molecular effects of Lenalidomide on lipid signalling pathways, we analyzed the expression of critical molecules involved in both cell proliferation and differentiation, that is PI-PLCbeta1 and its downstream target Cyclin D3, as well as PI-PLCgamma1, which is linked with EPO signalling. Ongoing analyses are also trying to examine the effect of Lenalidomide on Akt phosphorylation and Globin genes, which are specifically associated with erythropoiesis. So far, our results indicate that, in our responder patients, both PI-PLCbeta1 and Cyclin D3 are not significantly affected by Lenalidomide, whereas PI-PLCgamma1 is specifically induced, as compared with both healthy subjects and low-risk MDS patients treated with supportive care. Overall, these findings hint at a specific activation of PI-PLCgamma1 signalling following Lenalidomide treatment, and possibly pave the way to larger investigations aiming to better understand the role of these pathways in the mechanism of action of Lenalidomide in del(5q) MDS. Disclosures: No relevant conflicts of interest to declare.

Abstract: Abstract 233 Azacitidine, a DNA methyltransferase inhibitor currently used for the treatment of higher-risk myelodysplastic syndromes (MDS) patients, was shown to delay the evolution into acute myeloid leukemia (AML) and prolong overall survival (Fenaux P et al, Lancet Oncol 2009). In addition, azacitidine has recently been shown to potentially be a feasible and effective treatment even for patients with lower-risk MDS (Musto P et al, Cancer 2010). Lipid signalling pathways are involved in many important biological processes, such as cell growth, differentiation and apoptosis and play a role in the progression of MDS towards AML (Follo MY et al, J Cell Biochem 2010). Moreover, we recently demonstrated that phosphoinositide-phospholipase C beta1 (PI-PLCbeta1) promoter gene is hyper-methylated in higher-risk MDS and that azacitidine treatment can induce an increase in the level of PI-PLCbeta1 splicing variants as well as a down-regulation of activated Akt (Follo MY et al, Leukemia 2008; Follo MY et al, PNAS 2009). In fact, responding patients showed an increase in PI-PLCbeta1 expression in correlation with the therapeutic response, whereas their PI-PLCbeta1 promoter methylation was reduced. Furthermore, the decrease of promoter methylation anticipated the hematologic response, since the variations in PI-PLCbeta1 gene expression were observed prior to the clinical outcome. Stemming from these data, we further investigated the role of inositide signalling pathways during the epigenetic therapy, focusing on the effect of azacitidine on lipid signal transduction pathways in lower-risk MDS patients. The study included 25 patients (IPSS risk: low or intermediate-1) treated with azacitidine (75mg/m2 subcutaneous daily for 5 consecutive days every 28 days, for a total of 8 courses). For each patient we followed the effect of azacitidine in correlation to both PI-PLCbeta1 promoter methylation and gene expression, as well as the molecular profile of key molecules involved in the regulation of methylation processes, such as histone deacetylases (HDACs), methyl-CpG binding domain proteins (MBDs), and transcription factors correlated to hematopoietic stem cell differentiation and proliferation. Our results show that 8/25 (34%) of our lower-risk MDS patients, showing hematologic improvements after azacitidine therapy, had a significant increase in PI-PLCbeta1 expression, as compared with the amount of the pre-treatment period, thus confirming the involvement of this molecule in the response to demethylating agents. As for the remaining patients, mainly showing a stable disease, we observed slight increases or almost constant levels of PI-PLCbeta1 expression. Moreover, ongoing analyses are trying to disclose whether lower-risk MDS patients responding to azacitidine show a specific molecular epigenetic profile during the regulation of methylation processes. Taken together, our data suggest a correlation between azacitidine treatment and PI-PLCbeta1 signalling even in lower-risk MDS, thus hinting at a role for PI-PLCbeta1 in the evaluation of patients likely to respond to azacitidine and paving the way for the development of innovative therapeutic strategies in lower-risk MDS patients. Disclosures: No relevant conflicts of interest to declare.