ALBERT

Description: Abstract 4020 A retrospective analysis of influence of different clinical and laboratory parameters on disease outcome was performed in a cohort of 43 patients with advanced myelodysplasia (MDS)(RAEB 〉 10% blasts + RAEB-T according to the FAB classification) who underwent allogeneic stem cell transplantation (SCT) in our institute within past 20 years; 21 patients were transplanted with 〈 10% of bone marrow (BM) blasts after 1 or 2 courses of induction followed by 1 or 2 courses of consolidation chemotherapy (Group A), 22 patients were transplanted with 〉 10% BM blasts either prior treated with combination chemotherapy or transplanted up-front with intensified conditioning (Group B). Median survival of all transplanted patients was 35,5 months (+/− 53,9 months) with a significant difference between Group A and B (57,5+/−62,3 months v.s. 18,0 +/−36,7 months, p=0.017). Estimated 3 year and 10 year survival for all patients were 53,5% and 41,9%, respectively. Estimated 3 and 10 year survival also significantly differed between Group A and B (71,4% and 57,1% for Group A and 36,4% and 27,3% for Group B). Complete remission (CR) rate was 44,2%, 18,6% patients relapsed (14,3% in Group A and 22,7% in Group B). No difference in overall survival was observed between patients with 〉 10% BM blasts transplanted either after chemotherapy or up-front (median survival: 26,8+/− 41,4 v.s. 18,0+/−33,8 months, respectively, p=0.65). Univariate analysis using Kaplan-Meier curves and log-rank2 test revealed as significant variables affecting overall survival: achievement of CR (p=0.007), achievement of 〈 10% BM blasts prior SCT (p=0.011), SCT performed 〈 4 months after diagnosis (p=0.031) and absence of relapse (p=0.046). Independent variables for determining overall survival (identified by Cox regression multivariate analysis) were: SCT performed 〈 4 months after dg. (p=0.003,χ2= 8,798), achievement of CR (p=0.011,χ2= 6,457), and age 〈 50 years (p=0.044,χ2= 4,053). None independent variable determining occurrence of relapse was found. Neither the percentage of BM blasts at the time of dg. and initial transfusion dependency, nor the donor origin (related or unrelated) and number of consolidation courses affected survival. Conclusions: combination chemotherapy leading to a rapid clearance of BM blasts below 10% followed by immediate SCT represented the best treatment option for younger patients with MDS with 〉 10% BM blasts. Patients transplanted with 〉 10% BM blasts at the time of conditioning had significantly inferior outcome either transplanted after previous chemotherapy or up-front with intensified conditioning. In this subset of patients, a possible benefit of addition of hypomethylating agents to the treatment schedules prior SCT should be studied. The study was supported by scientific programme MZCR 00023736. Disclosures: No relevant conflicts of interest to declare.

Description: The influence of different clinical and laboratory parameters and various therapeutic approaches on the survival was retrospectively studied in a group of 142 adult patients with advanced forms of primary MDS: RAEB with 〉 10% blasts or RAEB-T. Univariate statistical analysis was performed using Kaplan-Meier curves and log-rank2 test. Independent variables for determining survival were studied using proportional hazards regression multivariate analysis. Median survival, estimated 1 year (ES1y) and 3 year (ES3y) survival of patients stratified according to different treatment modalities are shown in the table. Median survival and estimated 1 and 3 year survival of patients stratified according to different treatment approaches. treatment Nr.of patients median survival (months) ES1y (%) ES3y (%) allo SCT (+/− chemotherapy) 31 37,5 90,3 58,0 chemotherapy + auto SCT 6 21,5 100 33,3 combination chemotherapy 47 8,0 40,4 4,3 low-dose chemotherapy 18 5,5 22,2 11,1 demethylating agents 3 11,5 33,3 NE oral single agent chemoth. 6 2,8 0 0 supportive care only 31 3,0 3,2 0 Allogeneic stem cell transplantation (SCT) regardless to the number of bone marrow blasts at the time of conditioning or to whether the patients received combination chemotherapy prior SCT or not was the most significant parameter affecting survival in univariate analysis (χ2=46,3, P

Description: No definitive consensus whether to treat or not to treat patients with advanced MDS with combination chemotherapy before SCT has been obtained yet. Between 1998 and 2003, fifty patients with primary MDS underwent allogeneic SCT from HLA-matched related (31 cases) or unrelated donors (19 cases). Twenty-five patients with less advanced disease (RA and RAEB with 10% of blasts or RAEB-T) were pretreated with combination chemotherapy (anthracycline + standard dose cytosine arabinoside [ARA-C] +/− etoposide for induction, intermediate or high dose ARA-C +/− anthracycline for 1 or 2 consolidation courses) followed by myeloablative conditioning in all but one patient; ten patients were transplanted immediately after myeloablative preparative regimen (Bu+Cy, Cy+TBI or Flu+TBI) intensified by idarubicin(21mg/m2x2) as initial therapy. Complete remission (CR) rate after combination chemotherapy was 72% (11 patients); 3 patients (19%) achieved partial remission (PR), 1 patient had resistant disease. Median age, median survival, 3 year DFS, transplant related mortality (TRM) and relapse rate in different subgroups of patients according to the FAB criteria are shown in the Table. No significant difference in relapse rate was observed between patients pretreated with chemotherapy and those transplanted directly after intensified conditioning; nevertheless, median survival and 3 year DFS were 31,5 months and 54% in pretreated patients compared to 6,8 months and 40% in non-pretreated patients. Five of the six patients who relapsed after SCT were transplanted either in PR or with resistant disease after chemotherapy (3 patients) or with the initial number of bone marrow blasts ranging from 21 to 24% before immediate SCT (2 patients); 1 patient relapsed after SCT with reduced conditioning performed in the first CR. On the other hand, seven out of eight patients with advanced disease surviving more than 3 years without signs of the disease had low or intermediate-1 score according to IPSS at the time of SCT. We conclude, that the reduction of bone marrow blasts using combination chemotherapy prior to SCT may benefit patients with advanced MDS and may contribute to prolonged DFS after transplantation. Results of SCT in different groups classified according to the FAB criteria FAB subtype Nr.of patients Median age (years) Median survival (years) 3 years DFS (%) TRM (%) relapse rate (%) RA 16 38,0 22,3 69 25 0 RAEB ≤ 10% blasts 9 43,0 4,0 22 50 11 RA EB〉 10% blasts + RAEB-T-pretreated 15 34,0 31,5 54 27 26 RAEB 〉 10% blasts + RAEB-T-no pretreatment 10 44,5 6,8 40 40 20

Description: AIM OF THE STUDY, PATIENTS AND METHODS : The data obtained from a long-term follow-up for a period of 30 years (1988-2017) were analyzed in a group of 529 patients with primary MDS and factors affecting prolonged survival were detected using different statistical methods including Kaplan Maier test and multivariate analysis. RESULTS : The results confirmed usefulness of both IPSS and IPSS-R (median survival in months for risk groups : very low - 73.7, low - 40.0, intermediate - 27.0, high - 9.0, very high - 3.5). In a subgroup of 249 patients with less advanced disease without excess of blasts, allogeneic SCT represented the most favouring treatment approach leading to estimated 10 years survival in 49.1% of patients. However, when compared to patients treated by supportive care only, a benefit in overall survival for SCT did not become significant before 5 years of follow-up (estimated 5 years survival /e5yS/: 43.3% for supportive care vs. 54.7 % for SCT). Only 2 (3.8%) out of 53 transplanted patients died later than after 5 years follow-up in comparison to 31 (20.8%) out of 149 patients on supportive care; 22 of them died on complications not directly related to MDS and a late disease progression (between 6 and 26 years after diagnosis of MDS) was observed in 9 patients. Transplantation related mortality was 30.2%, SCT at the time of disease progression (〉5% of bone marrow /BM/ blasts) and prolonged (〉3 months) administration of corticosteroids prior to SCT were independent adverse prognostic factors for SCT outcome (P

Description: Background: Efficiency of CAR T cell based therapies against cancer is often limited by a poor survival of CAR T following recognition of tumor target cells. Interaction of CAR T with target cells induces their rapid differentiation into late memory subtypes (Teff) which lack expression of CD27, CD28, CD62L and CCR7. Although these terminally differentiated T cells are highly cytotoxic, their in vivo engraftment capacity is lower which thus reduces their in vivo survival and enables only temporary antitumor effects. It is generally believed that CAR T cells with early memory phenotypes (Tscm + Tcm) would provide stronger antitumor effects due to better survival in vivo. Recently, we have developed a transposon-based protocol of clinical-grade CAR19 T manufacture (Otahal et al, Cytotherapy 2018) which uses a combination of cytokines IL-4, IL-7 and IL-21 which strongly enhance the generation of CAR T cells with Tscm/Tcm phenotypes. Methods: We have thoroughly studied the effects of IL-21 on the survival, differentiation status and the expression of major immunoinhibitory receptors using CAR T cells specific to antigens CD19 and PSMA. After the co-culture of CAR T with their tumor target cells, the phenotypes were analyzed by multi-color flow cytometry, together with the assessment of effector functions and proliferation. We have compared the outcomes of signaling initiated by IL-21 on the fate of CAR T during this co-culture with the effects initiated by IL-2. Results: We have found out that IL-21 is a strong regulator of CAR T memory differentiation initiated by recognition of tumor target cells. IL-21 supported expansion of CAR T with Tscm/Tcm phenotypes and inhibited their terminal differentiation into CD45RA+/- CD62L neg, CD27 neg, CD28 neg late memory subtypes (i.e. Teff and Tem). Additionally, IL-21 suppressed up-regulation of inhibitory receptors PD-1 and TIGIT by CAR T cells. Both IL-21 and IL-2 were indispensable to maintain proliferation of CAR T following their activation via the recognition of tumor target cells however, IL-2 induced a rapid differentiation of CAR T into late memory subtypes and resulted in significantly lower expansion than CAR T cells co-cultivated with tumor cells in the presence of IL-21. Conclusions: Our data strongly suggest that the in vivo functions of CAR T cells can be significantly boosted by omitting the use of IL-2 during production because IL-2 drives CAR T towards their terminal effector differentiation state that reduces their ability to form long-lived memory cells. We are currently developing CAR T with transgenically expressed IL-21 and we are preparing a clinical testing of CAR T manufactured according to this protocol in patients diagnosed with relapsed-refractory B-ALL and B-NHL. Supported by grants NV15-34498A and Primus/MED/34, MH CZ - DRO (Institute of hematology and blood transfusion, IN - 00023736) and by gifts from Heřmanský foundation. Disclosures No relevant conflicts of interest to declare.

Description: Background: Recently we have published results of pilot study on CML patients demonstrating fast development of hyperinsulinaemia, peripheral insulin resistance, hypoadiponectinaemia and hypercholesterolemia during nilotinib therapy. Aims: To analyze results from follow up multicenter study “ENIGMA 2” with the aim to confirm or to exclude results from the pilot study, as well as to analyze whether these abnormalities are detected in control groups of patients treated with other TKIs - imatinib and dasatinib. Methods: Patients received intensive laboratory workup before the start of TKI and after 3 month of therapy. This included fasting insulin, glucose, adiponectin and lipid serum concentration, HbA1c and oral glucose tolerance test. Patients with TKI treatment interruption for 〉2 weeks and/or dose reduction for 〉 25% were excluded. Results: Between 2/2011-6/2014 in 5 centers 37 CML patients initiated therapy with nilotinib, 18 with imatinib and 8 with dasatinib. After 3 months patients treated with nilotinib developed significant hypersinulinaemia and hyperglycaemia as result of fast development of peripheral insulin resistance. This was proved by significant increase in HOMA-2 index during 3 months of nilotinib therapy (mean – 1.4 vs. 1.8; p = 0.0023). Moreover, we have proved significant decrease of adiponectin (major insulin sensitizer) concentration as well as significant increase in total and LDL cholesterol concentration after 3 month of nilotinib treatment. Details are presented in Table. Contrary – none of these abnormalities were detected in the control group of patients treated with imatinib and dasatinib, including any change in insulin resistance measured by HOMA-2 index (means – 0.9 vs. 1.3; p = 0.1046 and 1.1 vs. 1.1; p = 0.9255). Moreover, administration of imatinib (and probably also dasatinib, however only limited data are available at this moment) leads to increase of adiponectin concentration, which serves as major insulin sensitizer in peripheral tissues. Conclusions: Our study proved fast development of peripheral insulin resistance already during the first 3 months of nilotinib therapy as underlying cause of glucose and secondary also lipid metabolism impairment during this treatment. Moreover, this was not proved for patients treated with imatinib and dasatinb and significant increase in adiponectin concentration during imatinib (and probably dasatinib) therapy could at least partly explain observed amelioration of diabetes 2 during its administration described in some studies Supported by the CELL – the Czech Leukemia Study Group – for life Table. NILOTINIB THERAPY (n=37) Start Month 3 p mean (range) mean (range) Fasting glucose [mmol/l] 5.3 (4.5-6.7) 5.7 (4.6-8.2)

Description: Background: Core binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) and is associated with a favorable outcome, particularly if treated with repetitive cycles of high-dose cytarabine as post-remission therapy. Long-time 10-year overall survival (OS) rate was reported of 58% in FLT3-ITD negative patients (pts; Allen et al. Leukemia 2013). Nevertheless, 30-40% CBF-AML pts experience relapse. FLT3-ITD mutations occur in roughly 5-10% of adult CBF-AML. However, their prognostic relevance is still controversial. Aims: To characterize CBF-AML with FLT3-ITD and compare outcomes according to their genetic background. Methods: We retrospectively studied 65 AML pts with CBF-AML and FLT3-ITD (median age at diagnosis, 54 years; range, 22-81 years) diagnosed between 1996 and 2018 within seven study groups/institutions of the US and Europe. Results: Thirty-two (49%) of the 65 pts harbored t(8;21). Median white blood cell and platelet counts at diagnosis of patients with t(8;21) and inv(16) were 18.3/nl (range, 1.8-202/nl) and 31/nl (range, 7-372/nl), respectively. AML diagnoses were de novo in 61 (94%) and therapy-related in 4 (6%) of the pts. Thirty (46%) pts were female. Cytogenetic analysis revealed additional abnormalities (abn) in 38 (58%) pts, most frequently loss of X or Y (n=13; n=12 associated with t(8;21)), complex karyotype (≥3 abn; n=12; n=7 occurring in t(8;21)), trisomy 22 (n=7, all associated with inv(16)) or trisomy 8 (overall n=6, n=5 occurring in inv(16)). Four pts were positive for both mutations, FLT3-ITD as well as FLT3-TKD. Median ITD allelic ratio were 0.44 (range, 0.003-50) and median ITD size 60 bp (range, 3-120 bp). Three older pts (median age, 75.5 years) were treated with either azacitidine + sorafenib, azacitidine + venetoclax or with etoposide + tipifarnib. All three patients receiving non-intensive therapy died within one year and were excluded from further analysis. Complete remission (CR) after anthracycline-based induction therapy was achieved in 98% (n=61/62) of patients fit for intensive treatment including two pts treated with 7+3 ± midostaurin within the RATIFY trial. One patient died during induction. Fifteen (24%) pts underwent allogeneic hematopoietic cell transplantation. Of those, 10 pts were transplanted in 1st and 5 pts in 2nd CR. Median follow-up for the entire cohort was 4.43 years (95%-CI, 3.35-8.97 years). Median and 4-year relapse-free survival (RFS) rates were 3.41 years (95%-CI, 1.26 years - not reached) and 44.9% (95%-CI, 32.9-61.4%). Median and 4-year overall survival rates (OS) were 4.48 years (95%-CI, 2.26 years - not reached) and 51.8% (95%-CI, 39.6.2-67.9%). Neither type of CBF-AML (p=0.60), nor additional chromosomal abn (p=0.80), nor presence of a complex karyotype (p=0.50) had a prognostic impact on OS. Higher age (≥60 years) was an in trend negative prognostic factor on RFS and OS (p=0.07, each). High allelic ratio (≥0.5) had no impact on RFS (p=0.3), but in trend on OS (p=0.10). Conclusions: Despite a high remission rate pts with FLT3-ITD had an inferior outcome as compared to previously published data on CBF-AML without FLT3-ITD. Thus, CBF-AML with FLT3-ITD should not be classified within the low-risk category. CBF pts with FLT3-ITD warrants further study and should be included in FLT3-inhibitor trials. Disclosures Brunner: Astra Zeneca: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Forty Seven Inc: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Novak:Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel,Accommodations; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel,Accommodations; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Stoelzel:Neovii: Other: Travel funding; Shire: Consultancy, Other: Travel funding; JAZZ Pharmaceuticals: Consultancy. Thiede:Daiichi Sankyo: Honoraria; AgenDix GmbH: Employment, Equity Ownership; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Diaceutics: Membership on an entity's Board of Directors or advisory committees. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Levis:Agios: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Description: Abstract 2495 Background. The intracellular concentration of imatinib (IM) in patients with chronic myeloid leukemia (CML) is supposed to be influenced by the expression of its main cellular transporters, hOCT1 (influx) and ABCB1 (efflux). The assessment of the genes expression may be potentially important in clinical practice to effectively manage the therapy. Our recent results showed the necessity to cautiously interpret results from gene expression measurements of both transporters as their detected mRNA levels are affected by the proportion of different cell types in the sample analyzed (Racil et al. 2010 Am J Hem 85:525, Racil et al. 2011 Leuk & Lymph 52:331). Aims. In this report, we aimed to comprehensively assess differences in the expression of hOCT1 and ABCB1 in total leukocytes of peripheral blood (PB) in relation to the blood cell lineage in CML patients with different responses to IM. Methods. Kruskall Wallis's and Dunn's multiple comparison tests were applied to calculate differences in transcript levels of hOCT1 and ABCB1 in patients at diagnosis (Dg=43), in major molecular response (MMR=27), complete molecular response (BCR-ABL log 4.5 reduction; CMR4.5 =15), therapy failure (TF=13), accelerated phase (AP=12) and in 75 healthy controls. CMR4.5 is defined here as either a detectable disease ≤0.0032% BCR-ABLIS or as undetectable by nested PCR. TF is defined as non CCgR achievement. Additionally, we used the Spearman's correlation test to investigate relationship between expressions and percentage of immature cells and neutrophils in patients with non-physiological blood count (Dg and AP). In patients with normal blood count (CMR4.5, MMR, TF), we calculated correlation with BCR-ABL transcript level. Finally, we performed in vitro experiments with BCR-ABL negative (SKM-1, MOLM-13) and positive cell lines (K562, MOLM-7) treated with IM to study its effect on ABCB1 expression. Results. We found a significantly lower expression of hOCT1 and ABCB1 at Dg (P

Description: Abstract 4485 Purpose Thanks to the development of knowledge in the field of molecular biology, the great progress has been done in risk stratification of patients with acute myeloid leukemia (AML) at diagnosis, in recent years. Based on the recommendations of international expert groups there were identified the patients who may benefit from the allogeneic stem cell transplantation (allo-SCT) as a consolidation of first complete remission (CR). In the absence of an universal marker for minimal residual disease (MRD) measurements, there is still little information about the importance of MRD prior to allo-SCT. Our department has a very good experience with quantitative monitoring of WT1 gene expression as a marker of MRD during treatment of AML. The aim was to retrospectively evaluate the significance of MRD in patients indicated for allo-SCT in 1.CR. Patients and methods Overall 35 patients (pts) in the first morphological CR were transplanted from April 2005 - July 2011. Median age was 46 years (range; 20–63), mens 14, women 21, three good risk, intermediate risk 23, high risk 7 (NA 3). A total of 19 pts achieved CR after second induction (salvage), 11 pts were in 1st iCR. Induction 3+7 was given to 31 pts (4x other), as consolidation has been used HIDAC in 28 pts (7x other). As the graft, peripheral blood stem cells were used in 27 pts, bone marrow in 8 pts. The donor was identical sibling in 15 pts (1x mismatched sibling), matched unrelated donor (MUD) in 10 pts and mismatched UD in 9 pts. Conditioning regimen was myeloablative in 29 pts, reduced-intensity in 6 pts. Median follow-up was 18 months (range; 2–56). The expression of WT1 gene was measured by real-time polymerase chain reaction in peripheral blood according to the European Leukemia Net recommendations. The WT1 expression was related to the expression of a reference gene and the results were calculated with a number of WT1 copies related to 104 copies of ABL gene. The upper limit of normal WT1 expression was set as 50 copies of WT1 to 104 copies of ABL. Before allo-SCT, 25 pts were WT1-negative, ten pts were WT1-positive. Results When comparing the two groups according the MRD status, there was not significant difference in terms of age, risk groups, first induction failure, number of iCR, induction or consolidation type. Also, type of graft, conditioning regimen, or HSCT-CI was not significantly different. The group of WT1-positive pts had more unrelated donors, more aGVHD and shorter follow-up. In terms of cGVHD, the groups were comparable. When comparing the overall survival (OS) and cumulative relapse incidence (RI) of the entire group in terms of: risk group, first induction failure, iCR, consolidations number and incidence of aGVHD, we found no significant difference. Pts with cGVHD had a better OS, lower RI with comparable non-relapse mortality (NRM). In contrast, the MRD status measured by WT1 gene expression appears as clearly significant factor. The outcome of WT1-positive pts is significantly worse in terms of OS (55% vs 83% at 3 years, p = 0.03), RI (50% vs 11% at 3 years, p = 0.008), and there is a trend toward higher NRM (23% vs 5% in 3 years, p = 0.08). Conclusion Our results show that MRD status measured by WT1 gene expression in patients with AML in 1.CR significantly affects their future prognosis. Opportunities to influence the unfavorable prognosis of MRD-positive patients may be more intensive pre-transplantation therapy or earlier immunomodulatory intervention after allo-SCT (pre-emptive DLI). The larger prospective studies are necessary to confirm this hypothesis. The study was supported by scientific project MZ 00023736 granted by the Ministry of Health, Czech Republic. Disclosures: No relevant conflicts of interest to declare.