ALBERT

Description: Introduction Azacytidine(AZA) is the current standard of care for patients with high-risk myelodysplastic syndrome (MDS) in Europe. AZA has shown a survival advantage when compared with conventional therapies and has also shown activity in IPSS lower-risk patients. However, about 40% of patients do not respond and most patients show a loss of response within 2 years. Treatment options for MDS patients who progressed while on, failed to respond to, or became intolerant tohypomethylatingagents are scarce and it has been shown that overall survival (OS) is extremely short. Objectives of this study were to describe in a cohort of real life MDS patients treated with AZA, the reasons to discontinue treatment, and to evaluate their clinical outcomes. Methods We present here the results of a real life study from a large group ofnon selectedpatients recorded in the MDS registry of FondazioneItalianaSindromiMielodisplastiche(FISM). Only patients treated with AZA from January 2009 to June 2014 were considered for the analysis. All types of conventional published schedules of AZA were allowed. The primary end point was the evaluation of OS from start of AZA treatment to the date of death from any cause. Secondary end points were clinical response, cause of discontinuation, salvage treatments and OS from discontinuation of the drug. Results Between January 2009 to June 2014 1799 newly diagnosed MDS patients were enrolled , and 420 received AZA; 271 patients received AZA as 1st line treatment, 115 patients as 2nd line treatment, 34 as a line ≥3rd. Median age was 73 years (IQR: 67-77); 261 patients (62%) were male. WHO diagnosis was RA or RARS (n=27, 6%), RCMD with or without RS (n=62, 15%) AREB-1 (n=126, 30%), AREB 2 (n=190, 45%), other subtypes (n=15, 4%). At start of AZA therapy IPSS score was low in 11 (3%), int-1 in 80 (19%), int-2 in 143 (34%), high in 54 patients (13%), and not available in 132 patients (31%). Forty-three (47%) low and int-1 risk MDS patients had a transfusion-dependent anemia. Patients received a median of 7 courses of treatment (range 3-12). Twenty-four patients (6%) received concomitant erythropoietin therapy. OS at 1 year from beginning of AZA therapy was 73% for the whole cohort (420 pts)(95%CI: 0.69-0.78), and median OS was 23 months, 25 months for patients with IPSS lower-risk MDS and 21 months for those with IPSS higher risk MDS (log-rank test: 0.72). OS after discontinuation of AZA was 8 months. Clinical response was reported according to IWG criteria only in 288/420 patients (69%); 94 patients (33%) achieved a hematological response, that was complete in 35 patients (12%) and partial in 59 (20%), 78 patients (27%) had stable disease while 116 (40%) patients did not respond. Response was achieved after a median of 6 cycles (IQR: 4-11), in both lower and higher risk MDS patients. After a median follow up of 16 months (IQR:7-35) in 97 patients (23%) AZA therapy was still ongoing and in 323 has been discontinued (77%). Interruption of treatment was due to loss of response/worsening of hematological parameters in 24 patients who achieved complete or partial response (7%) and in 20 patients with stable disease (6%). AML evolution was the cause for interruption in 105 cases (32%), death in 28 (9%), toxicity or poor compliance in only 26 (8%), while clinical decision of the treating hematologist determined interruption in 22 cases (7%). In additional 98 patients AZA was discontinued early for reasons not reported by the treating physician (30%). Of the 323 patients who discontinued AZA 10 (3%) were managed with intensive AML-like chemotherapy, 17 (5%) underwent an allogeneic HSCT, 18 (6%) received low-dose chemotherapy, 42 (14%) other treatments and 236 patients (73%) received only transfusions and other supportive therapy. Conclusions Our data confirm that AZA therapy is effective for MDS patients, both with higher and lower IPSS risk disease. Response rate is consistent with what previously reported and median OS is 23 months. The interesting observation is that at 16 months, 77% of patients had discontinued treatment, either for progression or loss of response (45% of cases) and only in 8% of cases for reported toxicity. As there are few treatment options after AZA interruption, it is important to establish the reasons other than progression yielding to a stop in therapy, in order to avoid too early discontinuation and loss of survival advantage. Disclosures Finelli: Celgene: Research Funding; Celgene: Other: Speaker fees; Novartis: Other: Speaker fees. Angelucci:Novartis oncology, celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Santini:Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Astex: Consultancy; Amgen: Consultancy; Onconova: Consultancy.

Description: Abstract 2810 Azacitidine is a pyrimidine nucleoside analogue of cytidine and it is at present the standard treatment for myelodysplastic syndromes (MDS). A large number of studies performed to evaluate the mechanism of resistance/response to nucleoside chemotherapeutics like cytosine arabinoside or gemcitabine, have demonstrated the importance of the levels of expression of the enzymes involved in their metabolism. Despite the high clinical interest in azacitidine, little is known about its intracellular metabolism and scanty data are available in literature. Recently, differences in the expression of these enzymes has been reported in cell lines differentially sensitive to hypomethylating agents. Clinical responses to azacitidine are heterogeneous, mainly due to clinical characteristics of MDS patients, so there is a need to identify molecular markers that could predict and/or monitor the efficacy of this therapy. A better understanding of the biological mechanisms involved in the activation and DNA uptake of azacitidine is therefore necessary to possibly identify responsive MDS patients. Objectives: To measure the expression levels of the 5 enzymes involved in azacitidine metabolism and to correlate their gene expression with the clinical response to the drug. To test possible causes of differences in gene expression. Material and Methods: DNA and RNA was extracted from mononuclear cells of 39 IPSS high risk MDS patients treated with at least 6 cycles of azacitidine 75mg/m2/7 days every 28 days. Of them, 14 patients were responders and 25 non-responders to azacitidine according to IWG 2006 criteria. Gene expression was assessed with quantitative PCR, using an ABI GeneAmp® 5700. The specific oligonucleotides and TaqMan® probes of every selected gene will be acquired among the Assay-on-Demand® Gene Expression Products (Applied Biosystems). Promoter methylation of UCK1 was evaluated by methylation specific PCR using specific primers. The UCK1 coding region was sequenced by Sanger method. Results: Gene expression of hENT1, UCK1, DCK, RNR1 and RNR2 was compared between the groups of responders and non-responders. Individually, none of the genes was differentially expressed in responders versus non-responders. Nevertheless, a trend for lower expression in non-responders was shown for the UCK1 gene (median responders: 12.4 versus median non responders: 10.7; P = 0.095). In order to verify whether the presence of aberrant methylation could impact in UCK1 gene expression, we measured the methylation status of a putative CpG island in the UCK1 gene promoter region. The CpG island was unmethylated in all the samples analyzed, including responders and non-responder cases, as well as healthy donors. Moreover, to verify whether the difference in gene expression could be due to nucleotide variants in the gene sequence, we sequenced the UCK1 sequence. We found one polymorphic locus (rs2296957) in exon 6 and additional five intronic SNPs (rs3904060 and rs7867616, Intron 1; rs2296956 and rs189473964, Intron 3; rs150900763, 5' UTR) at the intron/exon boundaries. The presence of an individual genotype at these loci was not associated with therapy response and UCK1 gene expression. Conclusions: Our results show that absence of clinical response to azacitidine could be partly correlated with lower expression of UCK1. This is most likely causing a scarce azacitidine monophosphorylation by UCK1 in non-responders patients. The methylation status of the UCK1 gene promoter is not affecting gene expression. In addition, the genetic polymorphisms found in the UCK1 sequence do not associate with therapy response nor with gene expression. Further studies on larger series of MDS cases are needed to confirm these results which could help driving therapeutic decisions. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1761 Background: Mutation(s) of the JAK2 gene (V617F) has been described in a significant proportion of Philadelphia negative MPN patients and its detection is now a cornerstone in the diagnostic algorithm. The method most frequently used for measuring the distribution of cell populations is based on JAK2 sequencing and Q-PCR. Therefore the chance of distinguishing the JAK2 wild type or mutated population at the single-cell level still represents a challenge. The aim of the study was to developed a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of JAK2-mutation thus improving both the diagnostic resolution and the study of clonal prevalence. Methods: We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring the mutated (V617F) or the wild type form of JAK2. CD34+ cells were enriched from 24 PV patients (5 of them were selected for the absence of JAK2V617F), 13 PMF (10 with the mutation and 3 JAK2 wild type) and 6 ET patients (2 of them were wild type). In addition 20 BM samples were collected from healthy donors and used as control. Patients were a priori found to be either positive or negative for the JAK2V617F mutation by standard sequencing and by Q-PCR. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the human JAK2 sequence surrounding the nucleotide at position 1849, which is responsible for the V617F substitution (JAK2V617F/PNA). Slides were analyzed by fluorescence confocal microscopy. Results: The analysis revealed that among JAK2V617F PV patients the distribution pattern is fairly similar to that reported by Scott and colleagues in 2006 analyzing JAK2V617F in colonies. We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 40% to 100% in PV patients, from 15% to 80% in ET and from 25% to 100% in PMF. These findings are in agreement with previous data reporting that a variable proportion of progenitors from patients affected by JAK2V617F positive PV are capable of generating JAK2V617F negative colonies. In addition these data indicate that fluorescinated JAK2V617F/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of JAK2V617Fpositive cells collected from 3 JAK2 wild type subjects defined by sequencing and by Q-PCR, we identified a small percentage of cells positive for the JAK2V617F/PNA staining. However, this apply only to patients with PV but not to PMF and ET patients. Quantitatively, this percentage did not exceed 3% of the CD34+ cell population indicating a high level of sensitivity of the procedure since the PNA/FISH technique may detect JAK2V617F-positive cells within a CD34+ population isolated from patients reported as JAK2V617Fnegative by standard approaches. Importantly, the lack of positivity detected in CD34 positive cells from 20 healthy subjects demonstrates a high specificity of this method. Conclusions: JAK2V617F/PNA-FISH method displays high specificity and reliability in discriminating cell subpopulations harbouring the JAK2V617F mutation. In addition, it allows to analyze the CD34+ population at the single cell level, avoiding the time consuming analysis of hematopoietic colonies. In addition, this approach allows to monitor longitudinally the evolution of a defined cell population over time in MPNs and the characterization of the CD34+ compartment in patients with MPNs which still represents a challenging issue. Disclosures: No relevant conflicts of interest to declare.

Description: In acute myeloid leukemia (AML), blasts lose their ability to differentiate into mature cells and to undergo apoptosis. Accordingly, a proapoptotic and differentiating therapy (arsenic and retinoic acid) has dramatically improved survival in acute promyelocytic leukemia; however, a similar combination therapy is not available for other AML subtypes. In 2016, inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme of the pyrimidine biosynthesis, was found to induce differentiation in several AML models; for in vivo studies, brequinar (BRQ) was utilized. Starting from BRQ and applying a scaffold-hopping replacement, we have recently developed a new DHODH inhibitor, Meds433, which could induce differentiation at a 1-log lower concentration compared to BRQ (Sainas, J Med Chem 2018). Here we characterize Meds433 with in vitro and in vivo experiments, showing that it has a significant pro-apoptotic effect in several AML cell lines, which is at least partially independent from the differentiating effect. Analyzing the kinetic of differentiation induced by Meds433 on U937 and THP1 cell lines, and comparing the data with the number of viable cells, we noticed that cells started to die before the differentiation effect could be significant. Hence, we decided to investigate the proapoptotic effect of Meds433, treating several AML (U937, THP1, OCI-AML3, NB4) and non-AML (CEM, P3j, peripheral blood mononuclear cells-PBMC) cell lines with Meds433, and analyzing the expression of Annexin V and propidium in flow cytometry. Experiments demonstrated that Meds433 had a significant pro-apoptotic effect on several AML cell lines (Fig.1), but not on non-AML cell lines. The apoptotic rate increased with the time of exposure (3 vs 6 days), allowing to obtain a good apoptotic rate also in OCI-AML3, the most resistant cell line in our hands. As for the differentiation experiments, Meds433 could induce apoptosis at a 1-log inferior concentration compared to BRQ. More interestingly, in NB4 cells, a strong apoptotic effect was not associated with any differentiating feature, indicating that DHODH inhibition can induce apoptosis directly. As NB4 is a promyelocytic cell line, we also compared the effects of ATRA (all-trans retinoic acid), Meds433 and their combination. ATRA alone was found to induce strong differentiation and mild apoptosis on NB4 cells, while Meds433 alone could induce exclusively apoptosis; finally, the combination of ATRA and Meds433 increased both the rate of differentiating and apoptotic cells compared to ATRA only. We next tried to further characterize the apoptotic effect. When experiments were performed in the presence of uridine, a downstream product of DHODH in the pyrimidine biosynthesis, the apoptotic effect was totally abrogated. This phenomenon was already observed in the differentiation experiments (Sainas, J Med Chem 2018), and it confirms that both differentiation and apoptosis are indeed caused by the pyrimidine depletion rather than off-target mechanisms. Moreover, when experiments were performed in hypoxic conditions, the rate of apoptosis did not change, suggesting that Meds433 could work in the bone marrow hypoxic niche of (leukemic) stem cells. Further analyzing the effect of Meds433 on non-AML cells, we evaluated the maturation of T-lymphocytes, from naïve to TEMRA (T effector memory RA), finding no influence at all. Finally, preliminary results from in vivo experiments show that i) Meds433 is not toxic on Balb/c mice after 5 weeks of intraperitoneal administration; ii) the half-life is limited to 4-6 hours and iii) Meds433 has a good antileukemic activity (approximately 50% reduction of the tumor volume compared with control, after an 18-day treatment of THP1-xenograft in NSG mice). In conclusion, our work demonstrates that: i) DHODH inhibition can induce apoptosis in AML cells both directly and as a result of differentiation, partially depending on the cell line; ii) Meds433 is a novel, promising antileukemic drug, with limited toxicity, which could be active on a wide variety of AML. Since continuous exposure to the drug is fundamental in the pyrimidine starvation strategy, we are currently optimizing Meds433 pharmacokinetic profile in order to maximize the in vivo antileukemic activity. Disclosures Saglio: BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy.

Description: Introduction Multiple myeloma (MM) remains an incurable malignancy despite important recent advances in treatments. Neo-vascularization entails a crucial aspect of interactions between neoplastic plasma cells (PCs) and their microenvironment. Without it, MM would be unable to grow and progress, and would probably regress to a low-mass steady-state comparable to monoclonal gammopathy of undetermined significance (MGUS). To overcome drug resistance and improve clinical response to novel therapeutic approaches halting both PC growth and the increased bone marrow (BM) microvascular density are needed. In this setting, monoclonal antibodies against MM-specific cell surface antigens represent a promising therapeutic approach, which is however hampered by a lack of appropriate membrane target structures expressed across all MM cells. The Eph receptors, a large family of receptor tyrosine kinases, have been implicated in many processes involved in malignancy, including alteration of the tumour microenvironment, and in angiogenesis, in both of which EphA3 likely plays an active role. Interestingly, the over-expression of EphA3 is sufficient to confer tumorigenic potential, although probably further mechanisms can occur to abnormally activate the receptor. A first-in-class engineered IgG1 antibody targeting the EphA3 was developed and it is now under phase I clinical trials in USA and Australia for the treatment of EphA3 over-expressing hematological myeloid malignancies refractory to conventional treatment. Methods We investigated the EphA3 role in MM patients in order to define whether it may represent a potential new molecular target for a novel therapeutic approach with a specific anti EphA3 monoclonal antibody. The EphA3 expression was studied through a comparative proteomic analysis between BM endothelial cells (ECs) of patients with MM (MMECs) or with MGUS (MGECs), of control subjects (normal ECs). Moreover, the effects of anti EphA3 antibody in MM were studied in vitro and in vivo in a MM xenograft mouse model. After written informed consent, BM aspirates were collected from 26 MM and 6MGUS patients. Normal ECs were derived from 5 BM aspirates of subjects with anemia due to iron or vitamin B12 deficiency. We analyzed both mRNA and protein levels of EphA3 in normal ECs, MGECs and MMECs and in MM cell lines by absolute RT-PCR and by WB coupled to immunofluorescence and FACS analysis respectively. Immunoistochemistry was also performed on MM BM biopsies. The biological effects of EphA3 targeting were studied in vitro silencing (siRNA) the EphA3 mRNA in MMECs and using the anti EphA3 antibody testing them in series of in vitro functional assays including viability, apoptosis, adhesion, migration, wound healing and angiogenesis tests. We further examined the inhibitory capacity of anti-EphA3 Ab on tumor growth in SCID mice bearing MM tumor cell xenografts. Finally, we assessed morphology, vessel density, and apoptosis of excised xenotransplanted tumors. Results Briefly, our data showed that EphA3 mRNA and protein levels are progressively increased from ECs to MGECs, reaching the highest values in MMECs. EphA3 stained intensely and diffusely MM microvessels and PC in MM BM biopsies. The EphA3siRNA MMECs revealed a protein level reduction of approximately 80% when compared to the control. We not detected viability or apoptotic defects, whereas in vitro adhesion, migration and angiogenesis inhibition was evident when compared to the not silenced counterpart. The anti EphA3 antibody inhibited MMECs migration and reduced in vitro MM angiogenesis. In particular, tumour masses developed in xenograft mice treated with anti-EphA3 Abs were smaller in size and showed foci of ischemic-hemorrhagic necrosis, in association with a significant (P 〈 0.05) reduction in the number of intact tumor microvessels. The proliferative activity was not significantly different from that observed in tumors from untreated or control isotype treated mice, while the apoptotic index was significantly (P 〈 0.05) increased in comparison with tumors from both groups of mice. Conclusions In this study we have characterized the role of the EphA3in MM patients, providing in vitro and in vivo experimental evidences that support the possibility of using EphA3 as a new molecular target for MM. Disclosures: No relevant conflicts of interest to declare.

Description: The introduction of different generations of tyrosine kinase inhibitors (TKIs) significantly improved outcome and survival rate in chronic myeloid leukemia (CML) patients. However, long-term use of TKIs is concomitant with many side effects that affect the quality of life in patients. Approximately half of CML patients achieve deep molecular response (DMR), this makes them suitable candidates to discontinue the TKI therapy in a controlled condition, and about half of them will remain in treatment free remission (TFR) after discontinuation. It has been shown that a small population of leukemia stem cells (LSCs) as the residual disease burden is present at diagnosis, during the treatment, and in patients who are in TFR. While CML LSCs have many features in common with HSCs, they express specific markers such as CD25, CD26, IL1-RAP, etc., which can be used for the diagnosis and targeting. Protection by the bone marrow microenvironment and activity of signaling pathways such as WNT/β catenin, Hedgehog, PI3K, JAK/STAT in CML LSCs in a BCR-ABL dependent and independent manner guarantee their survival and elimination of these cells solely using TKIs seems ineffective. Herein we designed a pegylated liposomal nanocarrier conjugated with a specific antibody against CD26 (Begelomab, ADIENNE, Lugano, Switzerland). Then we loaded this immunoliposome with venetoclax, a BCL2 inhibitor, to eliminate CML LSCs selectively and to spare normal HSCs. First, we measured the expression of CD26 in the bone marrow and peripheral blood samples of newly diagnosed patients. We had a high expression of CD26 in CD34+/CD38- of both PB and BM, and a low expression on CD34+/CD38+ (progenitors) cells. Also, the expression of this marker in resistant patients to TKIs was visible while it was absent in normal stem cells. After the synthesis of the liposome, we conjugated Begelomab to the liposome. Then, we tested the selectivity of the designed system in different positive and negative cells. Our designed immunoliposome showed a strong selectivity toward CD26 positive cells. We also tested the selectivity on CML primary cells; in particular, we sorted newly diagnosed CML samples based on CD34+/CD38-/CD26- for HSCs and CD34+/CD38-/CD26+ for LSCs. Based on the confocal and flow cytometry analysis, our designed immunoliposome selectively targets LSCs and spares HSCs. Then we loaded this immunoliposome with venetoclax, and we treated CD26 positive and negative cells with this system. Based on our preliminary results, this immunoliposome loaded venetoclax specifically induced apoptosis in CD26+ cells, with higher activity compared to free venetoclax at the same dose. However, more analysis will be performed to confirm the selectivity of this system. Based on the obtained results, CD26 in newly diagnosed CML patients is expressed by CML LSCs and is a suitable option for diagnosis and targeting. Our preliminary data strongly suggest that we can selectively target CML LSCs. The main advantage of this system is its precision to hit the target. So we expect that after the drug release, the LSCs will be eliminated without any side effects on normal cells. Liposomes are suitable carriers because of their biocompatibility, self-assembly, large drug payload, and minimal toxicity. This strategy may help us to increase the number of patients attaining and maintaining TFR without relapsing. Disclosures Saglio: Ariad: Research Funding; Pfizer: Research Funding; Incyte: Research Funding; Roche: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Research Funding.