ALBERT

Description: Background and Aims: Myelodysplastic syndromes (MDS) are mainly diagnosed in the elderly with an increasing burden on healthcare systems. As a consequence, population-based studies are important in order to estimate the evolution of this emerging disease in different countries. Objectives: The objective of this study was to provide trends ofannual case frequency, morphological subtypes, incidence, mortality and survival of patients diagnosed with MDS in Switzerland between 2001 and 2012. Methods: A retrospective, population-based, epidemiological study was carried out on MDS cases reported to the Swiss Cantonal Cancer Registries and aggregated by the National Institute for Epidemiology and Cancer Registration. The Swiss Federal Statistical Office provided mid-year population estimates and cause of death statistics. Due to changes in the WHO classification of MDS, data was stratified for two time periods 2001-2007 and 2008-2012, respectively. 56 million person-years (py) were observed, covering 60%-65% of the Swiss population, during a time period of 12 years. Results: 2138 MDS cases were reported with a median age of 77 years (range of means: 75-78 years). The estimated annual case frequency increased from 263 to 316 (+20%) but the overall age-standardized (adjusted) incidence-rate did not change between the time periods (Table 1). A substantial increase in incidence was only visible for men aged 80-84 (+57.7%), men aged 85+ (+29.3%) and women aged 85+ (+13.4%). With respect to mortality rates, a 10% decline was observed for men aged 85+. Incidence and mortality were very low below the age of 60 years but the rates steeply increased thereafter (Figure 1a). Irrespective of time period, incidence- and mortality-rates were almost twice as high among men compared to women (Figure 1b). Classification in MDS subtypes was poor and improved only modestly from 20% to 39% with a higher awareness for diagnosis of higher-risk diseases. Relative survival at 5 years (RS at 5y) for all patients was 37% in 2008-2012 with better survival for younger patients 〈 65 years (61%) compared to older patients 〉 65 years (24-37%). No differences in survival could be observed between the two time periods (Figure 2). Conclusions: In this study we provide the first population-based, epidemiologic data from MDS patients in Switzerland. The analysis showed a 20% increase of annual incidence mainly due to population aging and not explained by increase in age-specific risk. This observation will impact on the future prevalence of the disease and its burden on healthcare systems.The age-specific incidence-ratesin patients 〉 75 years increased markedlyconsistent with the general increase of cancer-incidence in the elderly population. An increased diagnostic awareness of higher-risk disease seems to shift the population-based data for MDS sub-classification. We observed that younger patients without classified MDS subtypes have a similar survival like lower-risk disease, indicating that lower-risk MDS is underreported. Unsurprisingly, our data showed that younger patients have a better survival than elderly patients. This is most likelyrelated to higher frequency of lower-risk diseases in younger patients and their eligibility for allogeneic HSCT. However,the lack of a survival benefit observed in elderly patients on population-based level, after introduction of hypomethylating agents as standard treatment for transplant ineligible patients, is intriguing. The underlying reasons require further health-service research investigations. Relevance: The currently available data from CCRs in Switzerland is insufficient for detailed health service research on MDS patients, since important data is lacking on treatment, side effects and outcomes. A new cancer registration law with mandatory notification of cancer cases will be implemented in Switzerland by 2018. Moreover, the Swiss MDS Study Group has launched a Swiss MDS Registry that has started recruitment in 2016. Both initiatives will be of great value to improve data collection in order to foster future health service research of MDS patients in Switzerland with international collaborators. Table 1 Incidence and Mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Table 1. Incidence and Mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 1 Incidence and mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 1. Incidence and mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 2 Age-specific relative survival of MDS patients diagnosed in 2001-2007 and 2008-2012 Figure 2. Age-specific relative survival of MDS patients diagnosed in 2001-2007 and 2008-2012 Disclosures Bonadies: Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ruefer:Novartis: Consultancy; Celgene: Consultancy, Research Funding. Gerber:Celgene: Consultancy. Benz:Celgene: Consultancy. Lehmann:Novartis: Consultancy.

Description: BACKGROUND: Symptom burden in essential thrombocythemia (ET) and polycythemia vera (PV) is severe even among individuals with low risk disease (Blood 2012. 12;123(24):3803-10). New therapies exist which alleviate the severe symptom burden and reduce splenomegaly in ET and PV patients (N Engl J Med 2015; 372:426-435). This analysis is the first to date to evaluate thresholds at which symptom-based treatment can be considered for ET and PV patients who are intolerant or resistant to hydroxyurea (HU). METHODS: Patient demographics, symptom burden, and disease traits were collected from ET and PV patients at a single time point during therapy. The MPN-10 total symptom score (TSS, JCO 2012;30(33)4098-103) was utilized to assess symptom burden. Symptom criteria models were determined as previously described among a population of MF patients (Scherber et. al. EHA 2016: a2250). Cutoffs were then evaluated in a cohort of ET and PV patients to assess for utility as a symptom model among this population. RESULTS: Demographics and symptom burden: 838 PV and 867 ET patients with previous hydroxyurea therapy were included in this analysis. Patients were of mean age (54.9 years ET, 64.0 years PV) and gender (69.2% female ET, 55.7% female). Mean disease duration was 6.0 years for ET and 7.3 years for PV.Among ET and PV patients, 15.0% and 24.2% had prior thrombosis respectively. In evaluating prognostic risk, ET patients tended to be low (45.5%) or intermediate risk (42.9%) with only a minority of patient meeting criteria forhigh risk disease (11.6%). Laboratory findings: ET patients had a mean platelet value of 598.7x 109/L(SD=283.4). Among PV patients, mean hematocrit was 45.8% (SD=8.1) and 42.6% of patients had a hematocrit of greater than 45%. White blood cell count was normal between the two groups (ET mean 8.3 x 109/L, PV mean 9.0 x 109/L). Symptoms: Mean worst symptom severity was 6.4 out of 10 (SD=2.7). Among ET patients, worst symptom was most frequently fatigue (32.7%, mean 5.0/10, SD=3.1, overall prevalence 88%) followed by night sweats (13.6%, mean 2.0/10, SD=3.0, overall prevalence 53%) and concentration difficulties (8.6%, mean 3.1/10, SD=3.0, overall prevalence 68%). For PV, worst individual symptom items were most frequently fatigue (29.2%, mean 5.2/10, SD=3.0, overall prevalence 91%), pruritus (14.1%, mean 3.2/10, SD=3.2, overall prevalence 69%), and night sweats (12.8%, mean 2.5/10, SD=3.0, overall prevalence 57%). Cutoff Scoring: 47.0% of ET patients fit criteria for TSSgreater than to equal to 20; 59.0%% had a single itemgreater than 5; and 45.7% had both a TSS greater than or equal to 20 and a single item greater than 5. Among PV participants, 54.5% had aTSS greater than to equal to 20; 66.1% had a single itemgreater than 5; and 51.5% had both a TSS greater than or equal to 20 and a single item greater than 5. Each scoring method was significantly associated with individual item scores (Table 1). Prognostic scoring was not significantly associated with any of the symptom cutoffs evaluated. Correlations: Among ET patients, a prior history of thrombosis was significantly associated with having a worst symptom item greater than 5 (p=0.043). ET patients with lower hemoglobin were significantly more likely to meet criteria for a MPN-10 score greater than or equal to 20 or to meet combined criteria for a MPN-10 greater than or equal to 20 and single worst item greater than 5 (for both p=0.01 or less). For PV, lower hematocrit levels were significantly associated with having an individual worst symptom score of greater than 5 (44.9% versus 46.7%, p=0.0376). CONCLUSION: Assessment of ET and PV symptoms, now measurable through standardized and practical instruments such as the MPN-10, is an integral part of determining therapeutic impact of newer therapies in both clinical practice and trial settings. In our modeling, patients with severe symptom burden profiles are well represented by utilizing cutoff criteria including aworst individual symptom item of greater than 5 out of 10, an MPN-10 score of greater than or equal to 20, or combined criteria of both cutoffs. These cutoffs can be considered when determiningwhich HU intolerant or resistant patients would most benefit fromsymptom orientedtreatment. Disclosures Kiladjian: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding. Schouten:Novartis: Consultancy; Sanofi: Consultancy. Etienne:BMS: Speakers Bureau; ARIAD: Speakers Bureau; Pfizer: Speakers Bureau; novartis: Consultancy, Speakers Bureau. Harrison:Incyte Corporation: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Radia:Pfizer: Honoraria; Novartis: Honoraria. Cervantes:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mesa:Promedior: Research Funding; Celgene: Research Funding; CTI: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy.

Description: Key Points IDH1 promotes leukemogenesis in vivo in cooperation with HoxA9. Pharmacologic inhibition of mutant IDH1 efficiently inhibits AML cells of IDH1-mutated patients but not of normal CD34+ bone marrow cells.

Description: Dyskeratosis congenita (DC) is an inherited bone marrow failure (BMF) syndrome caused by defects in telomere biology genes. BMF occurs in approximately 80% of patients by 30 years of age and, in addition to pulmonary failure and malignancies, remains a major contributor to mortality. Hematopoietic cell transplantation (HCT) cures BMF in DC patients but is associated with a high incidence of graft failure and transplant-related mortality. Radiation and/or alkylating agents (e.g. cyclophosphamide, busulfan, melphalan) used in conditioning may have a role in the poor outcomes in DC patients following HCT. Recently, reduced-intensity conditioning approaches have improved short-term outcomes, but concerns remain about the long-term effects of DNA damaging agents in these regimens, given the multisystem disease and lifelong predisposition to malignancy conferred by telomere dysfunction. Radiation and alkylating agents are mainstays of allogeneic HCT preparative regimens: their highly effective myeloablative and immunosuppressive properties provide a niche for donor hematopoietic progenitors and decrease graft rejection. Based on a rationale that presumes (1) niche availability in DC patients with BMF, and (2) an intrinsic replicative defect in DC hematopoietic cells, we hypothesized that engraftment would be feasible in DC patients using a regimen of immunosuppressive therapy alone. Here, we report that in a single-institution prospective study, 4 consecutive patients with DC have undergone successful HCT using a radiation- and alkylator-free preparative regimen. Patients received alemtuzumab and fludarabine, followed by bone marrow transplantation (BMT) from unrelated donors. Cyclosporine A and mycophenolate mofetil were used for graft versus host disease (GVHD) prophylaxis. The primary outcome measures were neutrophil engraftment at day +30 and overall survival at day +100. Secondary outcome measures included acute toxicities and rates of graft failure, infections, and GVHD. The first two patients were 18 month-old twins with TINF2 mutations and transfusion-dependent BMF who received a BMT from a fully HLA-matched unrelated donor. The third patient was an 18 year-old female with CTC1 mutations and transfusion-dependent BMF, who underwent a B-mismatched unrelated donor BMT. The fourth patient, a 22 year-old male with clinical DC and short telomeres, had significant pulmonary disease (diffusing capacity of lung for carbon monoxide 28% of predicted) and BMF, and underwent a fully HLA-matched unrelated donor BMT. All 4 patients engrafted neutrophils by day +30, and are alive and well with follow-up ranging from 6 months (1 patient) to 2 years (3 patients). Platelet transfusion-independence occured between day +4 to +18, and red cell transfusion-independence occured between day 0 to +90. No patients required transfusions thereafter. All patients showed full donor myeloid chimerism by day +60. 3 of 4 patients showed full donor lymphoid chimerism by year 2; the fourth patient has high and increasing mixed donor lymphoid chimerism (60%) at day +180. There were no significant, unexpected toxicities, bacterial or fungal bloodstream or tissue infections. All patients were monitored weekly by serum PCR for CMV, EBV, adenovirus and HHV6 until day +100; only one patient showed CMV reactivation which was controlled with pre-emptive ganciclovir therapy. There was no acute GVHD. Only one patient developed chronic GVHD with limited skin involvement controlled with topical steroids. These results provide promising early data for engraftment and a favorable short-term toxicity profile using a radiation- and alkylator-free conditioning regimen in HCT for BMF due to DC. This approach could spare DC patients the acceleration of non-hematologic complications and malignancies, which has been attributed to HCT, and improve long-term survival. The limited toxicity of the alemtuzumab/fludarabine regimen may also allow patients with severe disease-related co-morbidities such as pulmonary dysfunction to safely undergo HCT. To our knowledge, this is the first prospective HCT study to achieve full donor myeloid engraftment in a series of patients following conditioning without radiation or alkylating agents. Disclosures Off Label Use: alemtuzumab - conditioning for bone marrow transplantation fludarabine - conditioning for bone marrow transplantation cyclosporine A - graft versus host disease prophylaxis in bone marrow transplantation mycophenolate mofetil - graft versus host disease prophylaxis in bone marrow transplantation.

Description: Background: Severe hepatic veno-occlusive disease (VOD, also referred to as sinusoidal obstruction syndrome) with associated multi-organ failure (MOF) is a life-threatening complication of hematopoietic stem cell transplant (HSCT) and has an associated mortality rate 〉80%. Defibrotide has been shown to have a protective effect on injured endothelium and to restore the thrombo-fibrinolytic balance. In severe VOD, defibrotide has improved complete response (CR) rates and survival at Day +100 post HSCT compared with historical controls, and has also been shown to have a favorable safety profile. In the EU, defibrotide is now approved for the treatment of severe hepatic VOD in HSCT therapy. It is indicated in adults, adolescents, children, and infants 〉1 month of age. In the USA, there are currently no approved therapies for this complication; however, defibrotide has been made available since 2007 through an expanded access protocol directed treatment IND (T-IND). The aim of the T-IND is to gather additional data on safety and efficacy of defibrotide in a broader patient population, including those with severe VOD/MOF post HSCT, non-severe VOD post HSCT, and VOD following chemotherapy in the non-HSCT setting. This is the largest prospective evaluation of defibrotide for the treatment of VOD. Here we provide an update on the safety of defibrotide from this ongoing study. Methods: The original T-IND protocol required patients to have a diagnosis of VOD by Baltimore criteria (total bilirubin ≥2.0 mg/dL with ≥2 of the following: hepatomegaly, ascites, or 5% weight gain) with MOF (either renal and/or pulmonary failure) following HSCT; the study was amended to allow inclusion of patients with non-severe VOD (defined as no MOF) occurring either post-HSCT or post-chemotherapy. Key exclusion criteria include clinically significant bleeding or the need for 〉1 vasopressor. Defibrotide was given as a 2-hour infusion at 6.25 mg/kg IV every 6 hours (25 mg/kg/d) with a recommended minimum treatment duration of 21 days. Results: The current interim safety analysis is based on 612 patients enrolled between December 2007 and December 2013 (including 99 in 2013) for whom safety data is available and who received ≥1 dose of defibrotide. Across the USA, over the course of the study approximately 86 centers were active to enroll patients. Median patient age was 12 years (range 18 to 65 years, and 1.2% aged 〉65 years. Patients were primarily male (55.8%) and predominantly white (65.6%). Overall, 454 patients (74.2%) reported ≥1 treatment emergent adverse events (AEs). Of these, 138 patients (22.5%) had AEs that investigators assessed as related (possibly, probably, or definitely) to study medication. Related AEs in 〉2.0% were pulmonary hemorrhage (4.7%), gastrointestinal hemorrhage (3.6%), epistaxis (3.1%), and hypotension (2.8%). Serious AEs (SAEs) were reported by 368 patients (60.1%). The majority of SAEs were assessed as not related to study treatment; 82 patients (13.4%) had an SAE at least possibly related to study treatment, most commonly pulmonary hemorrhage (3.9%) and gastrointestinal hemorrhage (2.9%). AEs leading to death occurred in 254 patients (41.5%); these AEs were deemed by the investigators to be possibly related to study medication in only 17 patients (2.8%). Previously reported efficacy data at D +100 in 425 patients evaluable for outcome have shown survival of 55% (by Kaplan-Meier estimate) for patients following HSCT, and survival of 62% (by Kaplan-Meier estimate) in 45 patients following chemotherapy (without HSCT), respectively. Conclusions: Defibrotide therapy in patients with VOD was generally well tolerated in this population, with manageable toxicity, and promising results seen in terms of response and survival. Safety results from prior studies, which have also been associated with a low incidence of defibrotide-associated toxicities, have proven very consistent with the favorable tolerability profile seen in this largest experience to date. Enrollment to the T-IND study continues; updated results will be presented at the meeting. Support:Jazz Pharmaceuticals Off-Label Use Defibrotide is an investigational treatment for hepatic veno-occlusive disease in the United States. Disclosures Richardson: Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals Inc.: Research Funding. Off Label Use: Defibrotide is an investigational treatment for veno-occlusive disease in the United States.. Antin:Dana-Farber Cancer Institute: Employment; Tempera: Consultancy; Enlivex: Consultancy. Lehmann:Dana Farber/Boston Children's Hospital: Employment. Bandiera:Gentium S.p.A.: Employment. Hume:Jazz Pharmaceuticals Inc.: Employment. Hannah:Jazz Pharmaceuticals: Consultancy. Nejadnik:Jazz Pharmaceuticals: Employment. Study Group:Gentium S.p.A.: Employment.

Description: Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 24 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Median age was 59 (range, 37–70) years. The series included 8 females and 16 males. Prior to allo-SCT, all patients were heavily pretreated with a median of 6 chemotherapy cycles including autologous and allo-SCT in all patients. Also, prior to lenalidomide, salvage treatment included donor lymphocyte infusions (DLI) in 18 pts,, thalidomide in 11 pts and bortezomib in 13 pts. Lenalidomide was given at 15mg (n=4) or 25 mg (n=20) orally once daily on day 1–21 every 28 days and in 20 patients in combination with dexamethason.. No prophylactic anticoagulation was used. The median number of completed cycles was 5 (range 2–17). Myelotoxicity according NCI criteria was the primarily and major encountered side effect (leukopenia: 4% grade 4, 21% grade 3, 17% grade 2, thrombopenia: 17% grade 3, 29% grade 2) and led to dose reduction in 54% of the patients. Infectious complications were observed in 50%. Non-hematological toxicity consisted of cramps (n=9), fatique (n=5) and constipation (n=2). Thrombembolic complications (cerebral infarction) were observed in one patient, who received concomitant corticosteroid treatment for acute graft-vs.-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 3 patients (one grade 2, and 2 grade 1), with one case occurring shortly after an additional DLI.. Objective remission was achieved in 66% of the patients (CR: 8%, VGPR: 8%, PR: 50%) and stable disease (SD) in 13% of the patients, while in 21% progressive disease was noted. Prior treatment with thalidomide or with bortezomib did not influenced the rate of CR/PR. Surprisingly, patients with del 13q14 achieved a higher CR/PR rate than those without del 13q14 (p=0.02). The median time to progression was 9.7 months (95% CI: 7.5–11.9) and the median overall survival was 19.9 months (95% CI: 17.3–22.5). Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in the majority of patients. The optimal dose of lenalidomide after allo-SCT has to be investigated.

Description: Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved 〉60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107 and 2.5 x 107 cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105 cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

Description: Acute myeloid leukemia (AML) is characterized by impaired myeloid differentiation of hematopoietic progenitors, causing uncontrolled proliferation and accumulation of immature myeloid cells in the bone marrow. Rearrangements of the mixed lineage leukemia (MLL) gene are common aberrations in acute leukemia and occur in over 70% in childhood leukemia and 5-10% in leukemia of adults. MLL rearrangements encode a fusion oncogenic H3K4 methytransferase protein, which is sufficient to transform hematopoietic cells and give rise to an aggressive subtype of AML. Leukemia where the MLL fusion oncogene is expressed is characterized by dismal prognosis and 30-60% of 5-years overall survival rate. The current standard treatment for AML is chemotherapy and in certain cases bone marrow transplantation. However, chemotherapy causes severe side effects on normal cells and an increased risk of relapse. Consequently, discovery of novel drug targets with better efficacy and low toxicity are needed to improve treatment of AML. In this study, we aimed to identify genes that are required for growth of AML cells and that encode proteins that potentially could be used as therapeutic targets. To do this, we performed high-throughput RNAi screening covering all annotated human genes and the homologous genes in mice, using barcoded lentiviral-based shRNA vectors. Stable loss-of-function screening was done in three AML cell lines (two human and one murine AML cell lines) as well as in a non-transformed hematopoietic control cell line. The candidate genes were selected based on that shRNA-mediated knockdown caused at least a 5-fold growth inhibition of leukemic cells and that the individual candidates were targeted by multiple shRNAs. The chromodomain Helicase DNA binding protein 4 (CHD4), a chromatin remodeler ATPase, displayed the most significant effect in reduced AML cell proliferation upon inhibition among the overlapping candidate genes in all three AML cell lines. CHD4 is a main subunit of the Nucleosome Remodeling Deacetylase (NuRD) complex and has been associated with epigenetic transcriptional repression. A recent study has shown that inhibition of CHD4 sensitized AML cells to genotoxic drugs by chromatin relaxation, which increases rate of double-stranded break (DSB) in leukemic cells. To verify whether CHD4 is exclusively essential for AML with MLL rearrangements, we inhibited CHD4 expression with two independent shRNAs in various AML cell lines with and without MLL translocations. In vitro monitoring of growth and viability indicated that knockdown of CHD4 efficiently suppressed growth in all tested cell lines, suggesting that CHD4 is required in general for growth of leukemic cells. To test the effect of CHD4 inhibition in normal hematopoiesis, we pursued knockdown of CHD4 and monitored effects in hematopoiesis using colony formation assays of human CD34+ cells. The results demonstrated that CHD4 knockdown had minor effects in colony formation as well as growth and survival of normal hematopoietic cells. Furthermore, to explore whether inhibition of CHD4 can prevent AML tumor growth and disease progression in vivo, we have generated a mouse model for AML. By transplanting AML cells transduced with shRNA against CHD4 into recipient mice, we showed that shRNA-mediated targeting of CHD4 not only significantly prolonged survival of AML transplanted mice but also in some cases completely rescued some mice from development of the disease. Collectively, these data suggested that CHD4 is required for AML maintenance in vivo. Next, to determine whether suppression of CHD4 can inhibit cell growth of different subpopulations and subtypes of AML, we performed loss of function studies of CHD4 on patient-derived AML cells ex vivo. Loss of CHD4 expression significantly decreased the frequency of leukemic initiating cells in different subtypes AML patient samples. In further in vivo studies using a xeno-tranplantation model for AML, we demonstrated that shRNA-mediated inhibition of CHD4 significantly reduced the frequency of leukemic cells in the marrow 6 weeks after transplantation. Taken together our results demonstrated the critical and selective role of CHD4 in propagation of patient-derived AML cells as well as in disease progression in mouse models for AML. We believe that CHD4 represents a novel potential therapeutic target that can be used to battle AML. Disclosures No relevant conflicts of interest to declare.

Description: Introduction : TP53 mutated (TP53m) MDS and AML have very poor outcome irrespective of the treatment received, including 40% responses (20% CR) with azacitidine (AZA) with short response duration and a median overall survival (OS) of about 8 months (Bejar, Blood 2014). APR-246 is a prodrug spontaneously converted to methylene quinuclidinone (MQ), a Michael acceptor that binds covalently to cysteines in mutant p53, leading to protein reconformation that reactivates its pro apoptotic and cell cycle arrest functions. The combination of AZA and APR 246 showed promising results in a phase Ib study in TP53m MDS (Sallman, ASH 2018). We report interim results of a GFM phase 2 study of AZA+ APR-246 in TP53m MDS and AML, conducted in parallel with a similar US MDS consortium study. Patients and Methods : The study included hypomethylating agent (HMA) naïve and not previously allografted intermediate, high or very high IPSS-R TP53m MDS and AML adult patients. Patients received APR-246 4500 mg IV /d (6 hour infusions) (days 1-4) followed by AZA 75 mg/m²/d (days 4-10) in 28 day cycles. Response (primary endpoint, assessed by IWG 2006 for MDS and ELN criteria for AML) was evaluated after 3 and 6 cycles in the intent to treat (ITT) population, ie all patients who had received any protocol treatment, and in patients who had at least a blood and bone marrow evaluation after cycle 3 (evaluable population). Allo-SCT, when possible, was proposed after 3 to 6 cycles, and treatment with reduced APR 246 and AZA doses could be continued after allo-SCT. Results : 53 patients were enrolled between Sept 2018 and July 2019 in 7 GFM centers, with a median age of 73 years (range 44-87), and M/F: 28/25. 34 patients had MDS (including 74% very high IPSS-R) and 19 had AML. IPSS-R cytogenetic risk was very poor in 30/34 MDS, and unfavorable in 18/19 AML, complex in 89% of the patients. Median baseline mutated TP53 VAF was 21% (range 3-76). Nineteen of the 53 patients had been included at least 7 months before date of analysis (25 July 2019), had received protocol treatment and were thus potentially evaluable for response after 6 treatment cycles (ITT population). One of them died after only one cycle from an unrelated cause (cerebral ischemic stroke), and 2 during the third cycle (from bleeding and sepsis, respectively). In the remaining 16 patients (evaluable population per protocol), the response rate was 75% including 9 (56%) CR, 3 (19%) marrow CR or stable disease with hematological improvement (HI), and 4 treatment resistance. In the ITT population, the response rate was 63%, including 47% CR, and 16% stable or marrow CR+ HI. Among CR patients, complete cytogenetic CR and negative NGS for TP53 mutation (VAF cutoff of 2%) were achieved in 7/9 (78%) and 8/8 (100%), respectively. So far, 1 patient has undergone allo-SCT. All 53 patients had received at least one treatment cycle, and no increased myelosuppression, compared with AZA alone, was apparent. Treatment related AEs observed in ≥ 20% of patients were febrile neutropenia in 19 (36%) and neurological AEs in 21 (40%) of the patients. The latter, reviewed with a neurological team, were mainly grade 1 or 2 and consisted of ataxia (n=13), sometimes associated with cognitive impairment (n=4), suggesting a cerebellar origin. Other patients experienced acute confusion (n=4), isolated dizziness (n=3) and facial paresthesia (n=1). Neurological AEs reached grade III in 3 cases (1 acute confusion, 2 ataxia). Occurrence of neurological AEs was correlated with lower glomerular filtration rate at treatment onset (p