ALBERT

Description: Introduction With standard intensive induction regimens, up to 80% of Acute Myeloid leukemia (AML) patients can achieve complete remission (CR). Several evidences demonstrated that the persistence of detectable disease (MRD) assessed with highly sensitive techniques such as Multicolor-Flow-Cytometry (MFC) and PCR based molecular analysis, retains a prognostic value among patients achieving morphological remission (Walter RB, 2015; Araki D et al, 2016, Zhou Y et al, 2016). The aim of the present study was to retrospectively evaluate the prognostic impact of MRD in a cohort of uniformly treated AML patients. One hundred and ten consecutive AML patients who had been treated in our center between January 2004 and December 2014 were retrospectively analyzed. All patients had received a fludarabine-containing induction (FLAI-5) and received second cycle and further consolidation therapy according to our published strategy (Guolo F, 2016). Median age was 47 years (range 17-61). Median follow up was 59 months. Patients features are summarized in Table I. MRD assessment was performed through 4-colour MFC analysis (MFC-MRD)and through WT1-gene expression analysis, as previously described (Guolo F, 2016). Three different MRD time-points (TP)were considered: TP1, after induction I; TP2, after induction II; TP3, after consolidation therapy for patients who did not undergo HSCT and at HSCT for patients who underwent HSCT. Relapse-free survival (RFS) was calculated from the time of diagnosis until last follow-up or documented leukemic relapse. CR rate after 1st and 2ndinduction was 82.7 and 85.5%, respectively, whereas 30 and 60 days mortality was 6.4% and 8.2%, respectively. Overall, patients showed MRD reduction from TP1 to TP2. Detailed MRD negativity rates are provided in table II. MRD clearance probability was significantly influenced only by ELN risk group and Karyotype (p

Description: Background: Acute Myeloid Leukemia (AML) is an incurable disease characterized by a highly unstable genome, resulting in large-scale changes at diagnosis, as well as further evolution contributing to disease progression. However, the mechanisms whereby tumor cells adapt to genomic instability are largely unknown, but recent observations have correlated these abnormalities with dysfunctional DNA damage repair (DDR) machinery. SIRT6 is an important regulator of cellular stress response and genomic integrity. Here we investigated the role of this NAD+ -dependent deacetylase in regulating ongoing DNA damage observed in AML patients. Methods: SIRT6 mRNA level was determined by RT-qPCR in AML patients (n=100) diagnosed at the Hematology Department of University of Genoa (Italy), compared with normal bone marrow derived CD34+ cells (n=5). Correlation studies with clinical and molecular characteristics of these patients were also performed. A panel of different AML cell lines and primary cells, both sensitive and resistant to conventional and novel anti-AML therapies, was used in the study. The anti-leukemic effect of DNA-damaging agents (DDAs) including idarubicin, Ara-C and fludarabine was evaluated in presence of SIRT6 depletion/inhibition by CTG assay and Annexin-V/propidium iodide staining. Mechanistic studies were performed with Western-blotting, lentivirus-mediated shRNAs and immunofluorescence assay. Analysis of DNA DSB repair was done using chromosomally integrated reporter constructs, followed by cytometer analysis. Results: AML patients were grouped into lower and higher SIRT6 expressers according to its median mRNA level. Patients with lower expression had a higher incidence of FLT3-ITD (p=0.034, Wilcoxon signed rank test). No significant association was observed with respect to mutations of NPM1, nor with WT1 and BAALC expression. SIRT6 expression correlated also with adverse clinical outcome in term of event free and overall survival (p=0.035 and p=0.025, respectively; unpaired t test). Based on these data, we evaluated SIRT6 role in biology of AML. We found higher SIRT6 protein level in AML cell lines carrying FLT3-ITD mutation (MOLM-14 and MV 4-11) compared to cell lines harboring other mutations (OCI-AML3, THP-1, KG, NB4, HL60, Nomo1 and U937). Targeting SIRT6 by specific shRNAs weakly reduced AML cell survival compared with control-scrambled cells, by impairing DNA repair efficiency. Indeed, a restricted effect of SIRT6 impairment on DNA damage proteins (H2AX, RAD51, 53BP1, RPA32) was measured. We next examined the therapeutic relevance of SIRT6 inhibition in AML by testing effects of its depletion in combination with genotoxic agents. Remarkably, SIRT6 depletion conferred increased sensitivity of AML cells to idarubicin, Ara-C and Fludarabine. Overall, enhancing genotoxic stress while concomitantly blocking DNA double-strand breaks (DSBs) repair response, may represents an innovative strategy to increase chemosensitivity of AML cells. Further mechanistic studies revealed that SIRT6 acts as a genome guardian in leukemia cells by binding DNA damage sites and activating DNA-PKcs and CtIP by deacetylation, which in turn promotes DNA repair. Conclusion: Genomic instability is present in all hematologic malignancies including AML. Strategies aimed to shift the balance towards high DNA damage and reduced DNA repair by SIRT6 inhibition can decrease AML growth and may benefit patients with otherwise unfavorable outcomes. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND AND AIMS Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for patients with high-risk acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict relapse risk after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are the most common tools to evaluate MRD. We recently reported that combining WT1 expression and MFC for MRD detection after induction therapy strongly impacts on relapse risk in AML. The aim of this study was to analyze the role of pre-BMT MRD assessment as predictor for the post-transplant relapse risk. MATERIALS AND METHODS We retrospectively analyzed the outcome of 253 consecutive AML patients receiving allo-BMT. Pre-BMT marrow samples were analysed for WT1 expression and MFC as MRD evaluation . Median age at transplant was 45 years. Disease phase was CR1 in 161, CR2 in 63, and CR3 in 29 patients. One hundred eighty-two received myeloablative conditioning, whereas 71 patients received reduced intensity conditioning. Median follow-up was 59 months (95% CI 46.2 - 71.8 months). Relapse-free survival (RFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall Survival (OS) was calculated from the time of transplantation until death by any cause or last follow-up. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells/105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for abnormal WT1 expression. RESULTS Relapse occurred in 81 patients (32%). Three-year estimate of RFS was 63.7% (median not reached). The probability of relapse was significantly affected by disease status (first or subsequent CR, p

Description: Background and aims Myelodisplastic syndromes (MDS) are a group of hemopoietic disorder characterized by an impaired blood cell production, morphologic dysplasia and peripheral cytopenias; they are the most common hematologic neoplastic disorder and its diagnosis relays on morphologic evaluation, associated to a karyotypic assay. In order to predict the outcome of patients affected from these disorders, knowing that the order of survival can be extremely variable, several prognostic index were developed such as International Prognostic Score Sistem (IPSS) or the most usefull WHO-Prognostic Score Sistem (WPSS). On the contrary of acute leukemia, these disorders have not a biomolecular profile evalutation of intrinsic markers able to stratify patients in different prognostic risk groups. The aim of our study is to assess the risk of leukemic evolution, in MDS patients, on the basis of the levels expression of WT1 and BAALC at disease diagnosis, and to evaluate the leukemia free survival (LFS) at 6-12-24-36 months of follow up, among the different risk category according to IPSS and irrespectively of treatment. Materials and method In five years we analized 102 patients with a diagnosis of MDS divided according to the WHO classification such as follows: 38 AR, 1 AR with del(q5), 21 aREB-1, 23 AREB-2, 3 chronic myelomonocitic leukemia, 1 RARS, 1 MDS, 11 RCMD, 1 5q- syndrome, 2 suspected MDS. According to IPSS 58 belonged to the low risk category, 21 to the intermediate-1, 23 to the intermediate-2/high risk. Cytogenetic assay showed 20 people with an abnormal karyotype, 8 of them fallen into the high risk class and 12 into the intemediate risk. Low risk and intermediate-1 patients were treated only with supportive care; high risk patients were treated with hypomethylating agents. Iron chelation were used when necessary. Lenalidomide was used in the only case of 5 q- syndrome Samples of bone marrow were analized with Real-Time quantitave PCR and levels of WT1 and BAALC expression were determinated. Molecular datas were analized with X-square Test and a significant association was recorded between overexpression of the genes evaluated (WT1 higher than 100 copy numbers and BAALC higher than 1000 copy numbers) and the probability of develop acute myeloid leukemia ( AML ). Results Nine out of 102 patients showed an isolated WT1 hyperexpression (3 of them developed an AML ), in 15 cases we reported an isolated BAALC overexpression (3 of them developed an AML ), while 13 out 18 patients ( 72% ) with combined WT1 and BAALC overexpression developed AML within an average time of 6 months; instead only 5% of patients, which expressed low levels of WT1 and BAALC, developed AML within the interval of observation. In particulary a combined high expression of WT1 and BAALC were strongly associated with an high risk to develop leukemia and a short LFS, especially in INT-1 subset. After that we calculated the LFS, divided for the risk category at 6-12-24-36 months of follow up. Patients with combined overexpression of WT1 and BAALC showed a LFS of 40% at 6 months of follow up and 0% at 24 months. Conclusion MDS have a great variable survival, and the current approach to these diseases relays on morphological evaluation, karyotypic assay and need of transfusional support; gene expression could be a promising system to predict the prognosis in these patients. Analysis of gene expression, which belong to AML evaluation, allows to divide patients in several risk groups; furthermore is not the single gene evaluation that is more predictable but a combined assay. With this method, which seems to be more realiable than IPSS, we could find that a great percentage of patients with levels of WT1〉100 and BAALC 〉1000, indipendently from karyotypic status and treatment, developed AML and have a shorter LFS than the population with WT1

Description: Backgrounds and aims Prevention and prompt treatment of invasive fungal infections (IFI) in acute myeloid leukemia (AML) patients can improve overall survival not only by reducing infection-related mortality but also allowing to comply induction regimens on time. The aim of the study was to estimate efficacy and feasibility of primary antifungal prophylaxis with posaconazole (PSZ) in patients affected by acute myeloid leukemia receiving front-line chemotherapy and to optimize our clinical practice. Materials and methods From January 2013 to May 2014, 28 AML patients undergoing intensive chemotherapy and potentially eligible for bone marrow transplantation in our institute received PSZ prophylaxis for IFI. All patients received a fludarabine, cytarabine and idarubicin containing regimen as first line treatment. We performed a retrospective analysis to evaluate the efficacy and feasibility of PSZ prophylaxis; to detect factors affecting drug exposure we analyzed each period of hospitalization as a single independent event. PSZ was given at the standard dose of 200 mg for 3 times/day, concurrently with a fat snack or with at least 100 ml of an acidic drink. Because of unpredictable absorption rates PSZ serum levels (TDM) were assessed routinely according to a validated high-performance liquid chromato-graphic (HPLC) method as described. A comparison with an historical cohort with similar features who had received fluconazole (FLC) prophylaxis was made. The use of empirical or targeted antifungal therapies and the incidence of IFI were compared. Results and discussion PSZ showed a good tolerability profile with no serious adverse events clearly related to prophylaxis occurring. A median number of 2 TDM for each period of hospitalization was performed (range 2-5). The achievement of a plasmatic PSZ concentration 〉 0,7 mcg/mL is considered optimal for prophylaxis efficacy; in 30/47 (64%) episodes of hospitalization and treatment, with at least two TDM, the threshold PSZ serum concentration was reached, with stable plasmatic levels. Median PSZ plasmatic value at first assessment was 0.89 mcg/mL (range 0.1-3.3). Table 1 summarizes patients features and factors that might affect PSZ plasma concentrations. The strongest negative factors affecting PSZ absorption are the discontinuation of prophylaxis and the concomitant assumption of proton pump inhibitors since their negative impact is shown both in univariate and multivariate analysis. No proven IFI were observed in our cohort with only one probable IFI occurring in a patient with refractory disease who did not reach adequate serum PSZ concentration. Table 2 summarizes the comparison with our historical cohort. The risk of experiencing IFI (proven or probable) during AML treatment is significantly higher in the FLC cohort (HR: 9.488, CI: 1,404 - 64,122). Moreover, the use of targeted or empirical antifungal therapies had been significantly higher in FLC cohort (HR: 2.7, CI: 1,212 - 6,050). Our clinical experience confirms the utility and cost-effectiveness of primary prophylaxis with PSZ in AML patients receiving intensive treatment. Table 1: Factors affecting PSZ serum concentration Reached plasmatic concentration threshold (%) p(univariate) p(multivariate) All Hospitalizations 30/47 (64) - - Sex Male 17/23 (74) 0.227 - Female 13/24 (54) Disease Status Active Disease 13/27 (57) 0.014 0.156 Complete Response 17/20 (85) Mucositis None or Grade 1 25/32 (78) 0.008 0.228 Grade 〉=2 5/15 (33) Age 45 years 18/26 (69) Concomitant PPI No 28/36 (78) 0.001 0.000 Yes 2/11 (18) Concomitant Ranitidine No 26/42 (62) 0.640 - No 4/5 (80) Concomitant Levofloxacine prophylaxis Yes 27/39 (69) 0.118 0.042 No 3/8 (38) Prophylaxis Discontinuation Never 27/36 (75) 0.009 0.003 At least for 2 dd 3/11 (27) Infectious Complications None 8/11 (73) 0.722 - At least one episode 22/36 (61) PSZ Assumption with Fat snack 5/11 (46) 0.153 0.150 Acidic drink 25/35 (71) Table 2: Historical comparison FCZ PSZ p All hospitalizations 54 47 - Median age (range) 47 (17-72) 47 (19-68) 0.560 Median ANC

Description: Background: Allogeneic bone marrow transplantation (BMT) offers the greatest chance of cure for most patients affected by acute myeloid leukemia (AML). Persistence of disease or high levels of pre BMT minimal residual disease (MRD) have been reported to predict disease relapse after BMT. WT1 expression levels and multicolor flow cytometry (MFC) are widely used as markers of MRD. We recently reported that combined evaluation of MRD by WT1 and MFC after induction therapy can predict relapse risk in AML patients. Aims: The aim of the present study was to apply the same MRD assessment in pre BMT setting to evaluate its reliability in predicting relapse. Methods: We retrospectively analyzed BMT outcome of 66 AML patients with both WT1-based and MFC-based MRD evaluation on bone marrow samples before transplant. Median age at transplant was 44 years. Forty-one were transplanted in first and twenty-five in second or subsequent complete remission. Induction therapies included fludarabine-containing regimens or standard ara-C and daunorubicin schedule (3+7). Median follow-up was 44 months (range 0-119 months); pre-transplantation evaluations were performed at a median of one month before transplant (range 1-3). Disease-free survival (DFS) was calculated from the time of transplantation until last follow-up or documented leukemic relapse. Overall survival was calculated from the time of transplantation to the last follow-up or death for any cause. All causes of death not directly due to relapse or progression of leukemia were considered as non-relapse mortality. A positive MFC MRD was defined by the presence of no less than 25 clustered leukemic cells /105 total events (threshold of 2.5x10-4 residual leukemic cells) at four-color flow-cytometry. Real-time PCR for WT1 was performed on DNA Engine 2 (Opticon®, MJResearch®). WT1 copy number/Abl copy number 500x104 was used as cut-off value for high WT1 expression. Results: Twenty-five relapses (37.9%) were observed. Median DFS was 31 months. Our analysis shows that the probability of relapse was significantly influenced only by disease status (first or subsequent CR) and MRD status at transplantation. Specifically, MFC-MRD was the strongest predictor of longer disease free survival (p

Description: BACKGROUND AND AIMS In patients with myelodysplastic syndromes (MDS) several validated prognostic scores, such as IPSS and R-IPSS, are available to assess the risk of AML progression and predict overall survival (OS) as well as leukemia-free survival (LFS). A number of molecular aberrations can be identified in MDS. However, differently from AML, none of the current prognostic indexes takes into account molecular profile at diagnosis. WT1 expression has often been evaluated in acute leukemias and MDS. High WT1 expression levels on bone marrow at diagnosis have been reported to identify MDS patients who are at high risk of progression to AML. BAALC (Brain And Acute Leukemia Cytoplasmic) hyper-expression has been associated with a poor prognosis in AML patients, whereas its prognostic value in MDS is not yet clearly defined. The aim of our study was to determine if combined assessment of WT1 and BAALC expression levels at diagnosis could be predictive of leukemic evolution. MATERIALS AND METHODS We selected 86 patients with available WT1 and BAALC expression levels on BM samples at diagnosis. According to IPSS score, 22 patient were considered low-risk, 27 intermediate-1 and 28 intermediate-2 or high risk. Patients underwent different treatment schedules including supportive care, erythropoietin, hypomethylating and immunomodulating agents, according to their risk group. Median follow-up was 36 months (range 4 -121 months). Leukemia-free survival (LFS) was calculated from the diagnosis until last follow-up or documented leukemic progression as defined in literature. LFS was estimated using the Kaplan–Meier method. All Real-Time PCR were performed on DNA Engine 2 (Opticon®, MJ Research®). WT1 copy number/Abl copy number 1000x104 was used as cut-off value for high WT1 expression, a level of 1000x104 BAALC copy number/Abl copy number was set as cut-off for BAALC hyper-expression. RESULTS After a median time of 32 months, 43 patients died. The main cause of death was leukemic evolution (accounting for 31/43 deaths, 72%), other causes were cardiovascular events and infections (data not shown). The risk of death by any cause was significantly affected by leukemic evolution, diagnosis according to WHO classification and molecular expression profile at diagnosis. Multivariate analysis showed that leukemic evolution was an independent predictor of death (p

Description: Background Blastic plasmacytoid dendritic cell neoplasm (BPDCN) are a very rare group of diseases included by WHO 2016 classification among the myeloid neoplasms and usually display an aggressive course with dismal outcome. BPDCN are characterized by a recurrent phenotype (CD45low/CD34-/CD56+/CD4+/CD123+), in the absence of other lineage differentiation markers. Our group recently reported that a subset of patients diagnosed with AML with NPM1-mutation carrying co-expression of CD123, CD56 and CD4, a "BPDCN-like" phenotype, showed poor prognosis. The aim of the present study was to evaluate the incidence and the prognostic impact of BPDCN-like phenotype in a wider cohort of cytogenetically normal AML patients, irrespectively of NPM1-mutational status. Methods We retrospectively evaluated a cohort of 83 younger (age

Description: Background: The presence of FLT3 "internal tandem duplication" (FLT3-ITD) mutation is associated with poor prognosis in acute myeloid leukemia (AML). However, the concomitant presence of NPM1 mutation (NPM1-mut) partially overcomes the negative prognostic impact of FLT3-ITD, which is also modulated by FLT3-ITD/wild-type allelic ratio. NPM1 and FLT3 mutational status assessment is strongly recommended for risk stratificationat diagnosis by the last European Leukemia Net (ELN) guidelines. Aims: To investigate the efficacy of an intensive fludarabine-containing induction regimen (FLAI) for fit, de novo AML patients according to NPM1 and FLT3-ITD mutational status. Methods: One-hundred and sixteen consecutive AML patients, treated in 3 Hematology Italian centers from January 2008 to January 2018, were included in this analysis. Twenty five patients showed isolated FLT3-ITD, 39 concomitant FLT3-ITD and NPM1-mutation and 52 isolated NPM-1-mutation.Median age was 52 yrs (range: 18-65 years). All patients received fludarabine, high dose cytarabine-based induction (fludarabine 30 mg/sqm and ARA-C 2g/sqm ondays 1 to 5 plus idarubicin 10 mg/sqm on days 1-3-5). For patients achieving CR fludarabine was omitted on II induction course and idarubicin dose was increased to 12 mg/sqm. Before2017 patients with isolated FLT3-ITD mutation were scheduled to receive allogeneic bone marrow transplantation (BMT) in first complete remission regardless of allelic burden whereas after 2017 only patients with high allelic burden received BMT in 1st CR. Minimal residual disease was evaluated on marrow samples using multicolor flow-cytometry (MFC)or NPM1 expression levels. Negative MFC-MRD wasdefined by the presence of less than 25 clustered leukemic cells/105 total events (threshold of 0.025% residual leukemic cells, Minetto et. al, BJH 2019). NPM1mutation (NPM1-A, B and D) was measured using Muta Quant Kit Ipsogen from Qiagen. FLT3-ITD allelic burden was available in31/64 of FLT3-ITD patients. Results: Overall 60-days mortality was 4%. After two induction cycles, 101 patients achieved CR (87%). Thirty-three/101 (33%) CR patients underwent BMT in first CR. After a median follow up of 61 months, 3-year overall survival (OS) was 56.8% (median not reached). In univariate analysis OS duration was favorably affected by age

Description: BACKROUND: Hodgkin Lymphoma (HL) is characterized by a high degree of response to chemotherapy and an overall favorable outcome in responding patients. Despite the efficacy of first line therapy, however, about 30% of patients affected by HL eventually relapse or are refractory (R/R) to first or second line therapy followed by autologous hemopoietic stem cell transplantation (ASCT). Recently, the clinical application of immune checkpoint inhibitors (CI), in particular the PD-1 targeting antibody Nivolumab, has dramatically improved the prognosis of patients affected by advanced phase solid tumours. In HL, Nivolumab has shown good activity in the difficult setting of patients relapsing after ASCT; however, complete response (CR) rate was less than 20%. Many studies in the solid tumours field have shown that the efficacy of CI is strictly related to the host degree of immune competence, which is greatly impaired in heavily pre-treated HL patients who received ASCT. Therefore, there is a strong rationale in enhancing the activity of the CI with the co-infusion of autologous lymphocytes (ALI), that have been collected in the early phase of therapy, and that are functionally activated. AIMS OF THE STUDY: Primary endpoint of this prospective trial was the evaluation of the efficacy of ALI in combination with pre-emptive CI administration early post ASCT in patients affected by R/R HL. Secondary pre-clinical endpoint was the study of the peripheral blood lymphocyte subpopulation, pre and post adoptive immune cell therapy and CI administration. METHODS: HL patients under the age of 60 with active R/R disease who have already failed at least two chemotherapy lines and Brentuximab (BV) were eligible for the trial. All enrolled patient underwent early lymphocyte apheresis, with a target cell dose of 5x107 CD3+/kg. All patients then received ASCT with FEAM conditioning. After ASCT, the first ALI was delivered 7 days after engraftment. ALI dosing was incremental, one logarithm at each step, starting from 1x104/kg in the first infusion to a maximum of 1x107/kg in the fourth and last infusion. Each ALI was followed after 48 hours by the administration of Nivolumab 240 mg flat dose. The second ALI dose was administered at 14 days after the first one. The third and the fourth were given every 21 days. Lymphocyte subpopulations were extensively studied on peripheral blood samples before and after each ALI and each Nivolumab administration, by 8-colours flow cytometry. Clinical response was evaluated 21 days after completion of the fourth ALI + Nivolumab. RESULTS: Six R/R HD patient have completed treatment so far and one is currently being treated. All patients failed to achieve CR with chemotherapy and then progressed during BV therapy. PET scan before ASCT showed progressing disease in all patients, with multiple-extra nodal involvement in 5 of them. All patients achieved complete hematological engraftment after a median of 10 days (8-12) from ASCT. Overall, infusion of ALI resulted in significantly higher lymphocyte counts at day 90, as compared to HL patients receiving the same conditioning without ALI (p