ALBERT

Description: Introduction:Acute Myeloid Leukemia (AML) predominantly affects the older population, with a median age at diagnosis of 67 years (yrs). In the last decades, Overall Survival (OS) has not changed meaningfully for these patients (pts). This worse outcome is explained by the poor-risk biological profile of the disease but also by the scarce propensity in administering curative treatments in this age category. Despite this general attitude, there is consensus that age alone should not be representative of the functional profile of older pts and that making decisions based on the sole age parameter can compromise possible therapeutic attempts. Therefore, a panel of experts from SIE (Italian Society of Hematology),SIES (Italian Society of Experimental Hematology) and GITMO (Italian Group for Bone Marrow Transplantation) summarized a list of operational criteria to be used in the process of treatment allocation, identifying 3 fitness categories of patients to address to differentiated strategies: 1)Fit pts (FP), eligible to intensive chemotherapy (IC) with the aim to achieve complete remission (CR); 2)Unfit pts (UP), eligible to non-intensive chemotherapy (NIC) with the aim to prolong survival; 3)Frail pts (FP) for whom, since the natural course of disease cannot be altered, supportive therapy (ST) is the best option in the attempt to preserve an acceptable quality of life (Ferrara et. al, Leukemia 2013). Aim: We retrospectively applied the operational SIE, SIES, GITMO criteria to a series of 180 consecutive non-APL AML pts diagnosed at our institution from 2013 to 2018 to investigate (1) the degree of concordance between the "operational criteria derived categories" and the actual treatment received; (2) the impact of this evaluation on long-term OS. Methods: We analyzed 180 consecutive pts with AML (median age 66 yrs, range 21-91) diagnosed at our institution from January 2013 to December 2018. We mainly focused on 125 pts older than 60 yrs (median 70 yrs, range 61-91). For the purpose of comparison, 55 younger pts, submitted to IC (51/55, 93%), were also analyzed. Results: SIE, SIES, GITMO operational criteria were retrospectively applied through medical files backtracking. One-hundred-48 out of 181 pts were stratified according to ELN 2010 as follow: 24 (16%) low risk, 59 (40%) intermediate-I, 25 (17%) intermediate-II and 40 (27%) high risk. This risk stratification did not differ between younger and older pts, suggesting that risk distribution may not be always an age-related factor. Overall, according to physician choice, 98 pts were submitted to IC, 40 to NIC, 42 to ST (54%, 22%, 24%, respectively). When focusing on pts older than 60 yrs, 47/125 were submitted to IC, 39/125 to NIC, 39/125 to ST (38% vs. 31% vs. 31%, respectively). A high concordance between treatment actually given and the one derived from the application of the "operational criteria" (165/180, 92%) was observed. The highest concordance was observed for the association of ST with FrP (39/40, 98%), whereas the associations IC with FP (96/107, 90%) and NIC with UP (30/33, 91%) showed a lower level of concordance. Overall, by applying the "operational criteria" the 3 categories differed significantly in terms of OS: 15,3 mos (range 0,4-78) for FP, 8,6 mos (range 2,2-47,9) for UP and 1 mos (range 0,1-29,9) for FrP, respectively (p

Description: Introduction: Despite the recent advances in chemotherapy regimens, relapse still substantially affects prognosis of intensively treated adult acute myeloid leukemia (AML) patients. There is growing evidence that a residual populations of leukemic cells may survive chemotherapy and outgrow, eventually causing relapse. These chemo-resistant cells are particularly abundant in the fraction of leukemic stem cells (LSC), which are endowed with pronounced self-renewal properties allowing to initiate and maintain leukemic clone. These cells can be detected, by high sensitivity multiparametric flow-cytometry (MFC), in the CD34+/CD38- fraction of the leukemic populations and can be distinguished from normal hematopoietic stem cells by the expression of specific markers. In recent clinical trials, LSC have been demonstrated to represent a biomarker of poor prognosis when detected at diagnosis but also during treatment course. Moreover, the combined estimate of measurable residual disease (MRD) and LSC refines the prognostic assessment as determined by the sole application of MRD detection. Aim: We analyzed a series of patients (pts) treated in the context of GIMEMA trials, in whom the LSC frequency was assessed by MFC at diagnosis. Pts with measurable levels of LSC were tested again after the consolidation cycle. At the same timepoint "standard" MRD was also determined. The purpose of the study was to demonstrate a correlation between LSC burden at baseline and prognosis in terms of overall (OS) and disease-free survival (DFS). Furthermore, we wanted to investigate the relationship between LSC and "standard" MRD persistence (〉0.035%) after consolidation, and possible correlation with outcome. Methods: LSC were evaluated by MFC as described elsewhere (Terwijn, PLoS 2014). LSC were quantified exploiting the expression of the C-type lectin-like molecule-1 (CLL1) and applying a sequential gating strategy that contained the CD34+/CD38- population. Pts were defined as LSC negative (LSCneg) in case of zero LSC count, LSClow or LSChigh when LSC were 〉00.03%, respectively. After consolidation, any level 〉0 was considered as a LSC persistence. Methods of analysis and thresholds were set according to previous publications (Zeijlemaker, Leukemia 2019). Results: We analyzed 130 pts with de novo AML, in whom LSC determination was available at the baseline. Fifty-nine (45,4%) pts were LSCneg, 49 (37,7%) LSClow, 22 (16,9%) LSChigh. We did not observe any correlation between baseline LSC level and genetic/cytogenetic risk at diagnosis. There was not a significant difference in terms of OS duration according to the 3 LSC levels, however, pts who were LSChigh had the shortest OS (36-month estimate OS of 71.5% vs. 65.4 % vs 52.4 % for the LSCneg, LSClow and LSChigh categories respectively; p=0.21). A statistically significant difference, regardless of the belonging to the LSClow or LSChigh category was observed when we focus on the subgroup of 30 pts with intermediate-risk AML, with a 36-month estimate OS of 76% vs. 77.8% vs 25% for the LSCneg, LSClowand LSChigh categories respectively (p=0.023) (Figure 1A). In 19 patients, LSC persistence was assessed at the post-consolidation time-point. Nine LSChigh pts who failed to eradicate residual LSC at this timepoint had a worse outcome as compared to those belonging to the same category but achieving a LSC clearance or those who were LSClow (36-month OS of 62.5% vs. 59.2% vs. 66.7% vs. 25% for the LSClow converted into LSCneg, LSClow not converted into LSCneg, LSChigh converted into LSCneg and LSChigh not converted into LSCneg categories, respectively; P=0.062) (Figure 1B). In 27 pts LSC and "standard" MRD determination was available. LSC persistence determined a worse 3-years OS both in MRD negative (66.7% vs 85.7%, p=0.44) and MRD positive pts (

Description: Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to 〉 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; 〉0.1%-1%; 〉1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p 0.1% and 〉 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories 0.1%-1%, 〉1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories 0.1%-1%, 〉1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD 〉 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD 〉1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Background Routine diagnostic lumbar puncture is not recommended in adult patients with acute myeloid leukemia (AML) without Central Nervous System (CNS) symptoms and little is known about the incidence of CNS involvement and its impact on survival in these patients. Furthermore, several studies have demonstrated that flow cytometry (FCM) is superior to conventional cytology (CC) for detection of CNS involvement in lymphoproliferative disorders but the role of this approach for the investigation of cerebrospinal fluid (CSF) in AML is unknown. Design and Methods The aims of our study were 1) to determine the incidence of occult/manifest CNS disease in a homogenous series of AML patients; 2) to correlate CNS disease with clinico-biologic parameters; 3) to examine the impact of CNS involvement on outcome. CSF samples were collected from 98 newly diagnosed AML patients, 62 males and 36 females, median age 53 years (range 18-75). Sixty-five and 33 patients aged 60 years, respectively.Seventy-one patients received standard and 22 high-dose-ARA-C-based regimens, 5 supportive care. All of 98 CSF samples were examined by CC whereas 90 (91%) also by FCM. CC positivity was defined as unequivocal morphologic evidence of leukemic blast in CSF and/or white blood cells count (WBCc) ≥ 5/µl with less than 10 erythrocytes/µl. A cluster of at least 10 phenotypically abnormal events was regarded as a proof of FCM positivity. Results Sixty-seven patients were CNS negative (CNS-) while thirty-one (31%) were CNS positive (CNS+). Among the last, 10 (10%) were positive on both CC and FCM (manifest CNS+) and 21 (21%) only on FCM (occult CNS+). There was an equal male/female distribution among CNS- and CNS+ patients, as well as median age (52 years, range, 20-71, vs 56 years, range, 18-75, p=NS) and WBCc(27.5 x109/L, range, 1,20-223 x109/L, vs. 11,6 x109/L, range, 0,70-315 x109/L, p= NS) were similar in both groups. Instead, higher levels of lactate dehydrogenase (LDH) were observed among CNS+ than CNS- patients (p=. 01). Forty-seven patients (48%) had monoblastic/monocytic or myelomonocytic AML and belonging to one of these categories was significantly associated with a condition of CNS positivity (55% vs 45%, P = 002). Cytogenetic/genetic data were available in 82/98 (84%). Twenty-for patients (29%), 33 (39%), 12 (14%) and 12 (14%), belonged to the category of favorable, intermediate-I, intermediate-II, and adverse karyotype, respectively. Cytogenetic/genetic characteristics did not differed significantly between CNS+ and CNS- patients. Overall, response rate was 70%, with complete remission rate being not statistically different between CNS+ and CNS- patients (69% vs 81% p= NS). Five-year DFS and OS were found to be significantly shorter in occult or manifest CNS+ patients than in those CNS- (23% vs 50% p= .03 and 19% vs 46%, p=.02, respectively)(Figure 1A and 1B). The prognostic variables achieving a statistical significance in univariate analysis (CNS status, age , WBCc, favorable vs adverse karyotype) were challenged in a multivariate model to determine to what extent they affected treatment outcome. In multivariate analysis, CNS positivity was found to be independently and significantly associated with a shorter duration of DFS.(p=.03 HR= 0.46). Age 〉50 years was found to be the only independent prognostic factor affecting OS (p=.01 HR= 2.26). Conclusion Our data suggest that incidence of CNS involvement in newly diagnosed AML pts is higher than expected. Regardless of neurologic symptoms, manifest and occult CNS positivity should always be sought at diagnosis since it may affect outcome and influence therapeutic decision. Further prospective studies on larger series are warranted to confirm this data. Figure 1 DFS and OS based on CNS status Figure 1. DFS and OS based on CNS status Disclosures Lo Coco: Teva: Consultancy, Honoraria, Speakers Bureau; Lundbeck: Honoraria, Speakers Bureau; Novartis: Consultancy; Baxalta: Consultancy; Pfizer: Consultancy.

Description: Clonal haematopoiesis of indeterminate potential (CHIP) is defined by the detection of somatic mutations in genes recurrently mutated in myeloid neoplasms (MNs), in the blood of healthy individuals with normal blood values and lack of morphological evidence of MN. Recent studies have highlighted the potential association between CHIP and the development of MN, in particular therapy-related MN (t-MN) in patients with lymphoma treated with chemotherapy and/or autologous stem-cell transplantation. In the present study, we investigated whether the presence of CHIP is associated with a higher risk for the development of t-MN in patients with chronic lymphocytic leukemia (CLL) treated with chemo(immuno)therapy, including fludarabine and cyclophosphamide combinations. To this end, we studied 9 patients with CLL who developed a t-MN [acute myeloid leukemia (AML): n=6, myelodysplastic syndrome (MDS): n=3] after the administration of chemo(immuno)therapy (FCR: n=7, other, n=2), with available samples collected both before CLL treatment and at diagnosis of t-MN. The median interval between the two samples was 26 months (range: 9-38months). NGS was performed on DNA extracted from bone marrow mononuclear cells (MNCs) at t-MN diagnosis, using the Trusight Myeloid Sequencing Panel (n=4) and the Archer VariantPlex Myeloid kit (n=5). Backtracking of the variants detected at the t-MN phase was performed by NGS of DNA extracted from peripheral blood MNCs (n=8) or CD19+ selected cells (n=1) in the samples from the CLL phase. In case no variants were detected in the t-MN phase, targeted digital droplet PCR (ddPCR) was also performed in paired CLL samples to confirm the presence of the variants. Moreover, using the Trusight Myeloid Sequencing Panel, we evaluated the prevalence of CHIP in a population cohort of 285 patients with CLL at the time of diagnosis. The variant allele frequency (VAF) cut-off for the detection of the variants was set to 5%. Variants were reported if meeting the following criteria: (i) located within an exonic or splicing region; (ii) be non-synonymous; (iii) not listed in the gnomAD database, if not also recurrently reported in Cosmic v85. Overall, 16 variants were detected in 7/9 cases analyzed at the time of t-MN [NRAS (n=4), DNMT3A (n=3), TET2 (n=2), EH2 (n=2), TP53 (n=2), KRAS (n=1), U2AF1 (n=1) and SF3B1 (n=1)], while no variants were detected in 2 t-MN samples. In 6/7 cases with detectable variants at t-MN diagnosis, the same variants were present at the CLL phase with either lower (n=4) or similar (n=2) VAF. Overall, CHIP was detectable in 6/9 (66.7%) CLL patients who later developed a t-MN. Among the untreated CLL patients, 45 CHIP-related variants were detected in 35/285 cases (12%) as 7 patients harbored more than one variant. The median VAF was 12.7% (5.1-58.6%) with 27/45 (60%) having a VAF