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Informed treatment decision-making in acute myeloid leukemia (AML) requires a comprehensive evaluation of all clinical and biological features that may affect the outcome with any given type or intensity of therapy [...]

Hypofibrinogenemia (HF) in adult acute lymphoblastic leukemia (ALL) of B lineage is uncommon and mostly associated with asparaginase (ASP) delivery. Since we noticed a significant reduction in fibrinogen (FBG) plasma levels even before the first ASP dose, we aim to assess the levels of FBG during induction treatment and explore if the FBG fall correlated with therapies other than asparaginase and/or specific leukemia biological features. We retrospectively analyzed FBG levels in 115 patients with B-ALL. In 74 (64%) out of 115 patients FBG decline occurred during the steroid prephase. In univariate analysis, such a steroid-related HF was significantly associated with

Several studies in immunocompromised patients, such as those with HIV infection, undergoing cancer chemotherapy or organ transplant, have led to the development of guidelines on the use of prophylaxis to prevent Pneumocystis jirovecii pneumonia (PJP), in these specific conditions. Instead, since the association between PJP and acute myeloid leukaemia (AML) is not clearly defined, the role of prophylaxis in patients with AML is not yet established.We retrospectively analysed 251 consecutive patients with newly diagnosed non-M3-AML, admitted at the Hematology Unit of University Tor Vergata in Rome, during the period 2010-2020. The aim of the study was to evaluate the incidence of PJP among AML patients during their first hospital admission, and to identify subjects at a high risk to develop PJP.Among 251 consecutive patients with non-M3-AML, 67 bronchoalveolar lavages (BAL) were performed. PJP was proven in 11/67 (16.7%) subjects undergoing BAL (11 males, median age 71 years), with an incidence of 4.3%. The most common reason for BAL execution were radiological findings such as ground-glass opacities (6/11, 55%) and atypical patterns like consolidations and nodules (5/11, 45%). One patient died because of PJP after 11 days of trimethoprim/sulfamethoxazole therapy. In multivariate analysis older age and smoking habit were independent factors significantly associated with PJP (p = .021 and 0.017 respectively).We conclude that PJP infection is not uncommon among patients with AML. If intensive chemotherapy is planned, physicians should be aware of this risk and prophylaxis should be considered, particularly in older patients.

Pneumocystis jirovecii pneumonia (PJP) is an opportunistic infection affecting immunocompromised patients. Patients with acute myeloid leukemia (AML) are not considered at high risk of PJP, thus, prophylaxis is not recommended. Between 2010 and 2020 we retrospectively analyzed 251 AML patients. We performed molecular diagnosis and genotyping of Pneumocystis jirovecii in 67 bronchoalveolar lavage samples. Eleven cases of PJP were diagnosed, with a prevalence of 4.3%. Our study confirms that the most widespread genotype in Europe is genotype 1; in our patients, 70% presented with genotype 1 and 30% the genotype 3.

The 2017 version of the European LeukemiaNet (ELN) recommendations, by integrating cytogenetics and mutational status of specific genes, divides patients with acute myeloid leukemia into 3 prognostically distinct risk categories: favorable (ELN2017-FR), intermediate (ELN2017-IR), and adverse (ELN2017-AR). We performed a post hoc analysis of the GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) AML1310 trial to investigate the applicability of the ELN2017 risk stratification to our study population. In this trial, after induction and consolidation, patients in complete remission were to receive an autologous stem cell transplant (auto-SCT) if categorized as favorable risk or an allogeneic stem cell transplant (allo-SCT) if adverse risk. Intermediate-risk patients were to receive auto-SCT or allo-SCT based on the postconsolidation levels of measurable residual disease as measured by using flow cytometry. Risk categorization was originally conducted according to the 2009 National Comprehensive Cancer Network recommendations. Among 500 patients, 445 (89%) were reclassified according to the ELN2017 criteria: ELN2017-FR, 186 (41.8%) of 455; ELN2017-IR, 179 (40.2%) of 445; and ELN2017-AR, 80 (18%) of 455. In 55 patients (11%), ELN2017 was not applicable. Two-year overall survival (OS) was 68.8%, 51.3%, 45.8%, and 42.8% for the ELN2017-FR, ELN2017-IR, ELN2017-not classifiable, and ELN2017-AR groups, respectively (P < .001). When comparing the 2 different transplant strategies in each ELN2017 risk category, a significant benefit of auto-SCT over allo-SCT was observed among ELN2017-FR patients (2-year OS of 83.3% vs 66.7%; P = .0421). The 2 transplant procedures performed almost equally in the ELN2017-IR group (2-year OS of 73.9% vs 70.8%; P = .5552). This post hoc analysis of the GIMEMA AML1310 trial confirms that the ELN2017 classification is able to accurately discriminate patients with different outcomes and who may benefit from different transplant strategies. This trial was registered as EudraCT number 2010-023809-36 and at www.clinicaltrials.gov as #NCT01452646.

Simple Summary Upfront genetics/cytogenetics and minimal measurable disease (MRD) are becoming relevant biomarkers in the process of post-remission transplant allocation in AML. However, until recently the transplantation choice relied on the availability of a fully matched familiar donor, whereas individual patient- and disease-related characteristics played a secondary role in transplant allocation. In this paper we analyzed the evolution of the transplantation policy at our center in a 20-year time interval. At the beginning of our observation patients were submitted to allogeneic transplant, per protocol, mostly if a fully matched family donor was available or to autologous transplant if no fully matched family donor was identified (“donor vs. no donor” strategy) regardless of upfront genetics/cytogenetics or MRD status. Thereafter, persistence of MRD after consolidation cycle was included in the decision-making process for transplant selection. Patients with favorable and intermediate-risk cytogenetic risk were to receive allogeneic or autologous stem cell transplantation if MRD positive or negative, respectively, (“transplant vs. no transplant” strategy). In this cohort, patients with FLT3-ITD or adverse risk karyotype were submitted to allogeneic transplant as well. Abstract Measurable residual disease (MRD) is increasingly employed as a biomarker of quality of complete remission (CR) in intensively treated acute myeloid leukemia (AML) patients. We evaluated if a MRD-driven transplant policy improved outcome as compared to a policy solely relying on a familiar donor availability. High-risk patients (adverse karyotype, FLT3-ITD) received allogeneic hematopoietic cell transplant (alloHCT) whereas for intermediate and low risk ones (CBF-AML and NPM1-mutated), alloHCT or autologous SCT was delivered depending on the post-consolidation measurable residual disease (MRD) status, as assessed by flow cytometry. For comparison, we analyzed a matched historical cohort of patients in whom alloHCT was delivered based on the sole availability of a matched sibling donor. Ten-years overall and disease-free survival were longer in the MRD-driven cohort as compared to the historical cohort (47.7% vs. 28.7%, p = 0.012 and 42.0% vs. 19.5%, p = 0.0003). The favorable impact of this MRD-driven strategy was evident for the intermediate-risk category, particularly for MRD positive patients. In the low-risk category, the significantly lower CIR of the MRD-driven cohort did not translate into a survival advantage. In conclusion, a MRD-driven transplant allocation may play a better role than the one based on the simple donor availability. This approach determines a superior outcome of intermediate-risk patients whereat in low-risk ones a careful evaluation is needed for transplant allocation.

In acute lymphoblastic leukemia, flow cytometry detects more accurately leukemic cells in patients' cerebrospinal fluid compared to conventional cytology. However, the clinical significance of flow cytometry positivity with a negative cytology - occult central nervous system disease - is not clear. In the framework of the national Campus ALL program, we retrospectively evaluated the incidence of occult central nervous system disease and its impact on outcome in 240 adult patients with newly diagnosed acute lymphoblastic leukemia. All cerebrospinal fluid samples were investigated by conventional cytology and flow cytometry. The presence of ≥10 phenotypically abnormal events, forming a cluster, was considered as flow cytometry positivity. No central nervous system involvement was documented in 179 patients, while 18 were positive by conventional morphology and 43 were occult central nervous system disease positive. The relapse rate was significantly lower in central nervous system disease negative patients and the disease-free and overall survival were significantly longer in central nervous system disease negative patients than in those with manifest or occult central nervous system disease positive. In multivariate analysis, the status of manifest and occult central nervous system disease positivity was independently associated with a worse overall survival. In conclusion, we demonstrate that in adult acute lymphoblastic leukemia patients at diagnosis flow cytometry can detect occult central nervous system disease at high sensitivity and that the status of occult central nervous system disease positivity is associated with an adverse outcome. (Clinicaltrials.gov NCT03803670)

Acute myeloid leukemia (AML) with FLT3-ITD mutations (FLT3-ITDmut) remains a therapeutic challenge, with a still high relapse rate, despite targeted treatment with tyrosine kinase inhibitors. In this disease, the CD34/CD123/CD25/CD99+ leukemic precursor cells (LPCs) phenotype predicts for FLT3-ITD-positivity. The aim of this study was to characterize the distribution of FLT3-ITD mutation in different progenitor cell subsets to shed light on the subclonal architecture of FLT3-ITDmut AML. Using high-speed cell sorting, we sequentially purified LPCs and CD34+ progenitors in samples from patients with FLT3-ITDmut AML (n = 12). A higher FLT3-ITDmut load was observed within CD34/CD123/CD25/CD99+ LPCs, as compared to CD34+ progenitors (CD123+/−,CD25−,CD99low/−) (p = 0.0005) and mononuclear cells (MNCs) (p FLT3-ITDmut burden was also observed in LPCs of AML patients with a small FLT3-ITDmut clones at diagnosis. On the contrary, the mutation burden of other myeloid genes was similar in MNCs, highly purified LPCs and/or CD34+ progenitors. Treatment with an anti-CD99 mAb was cytotoxic on LPCs in two patients, whereas there was no effect on CD34+ cells from healthy donors. Our study shows that FLT3-ITD mutations occur early in LPCs, which represent the leukemic reservoir. CD99 may represent a new therapeutic target in FLT3-ITDmut AML.

PURPOSE OF REVIEW: In the present manuscript, we will review the current approaches to investigate measurable residual disease (MRD) and its clinical applications in AML management. RECENT FINDINGS: Over the last decades, several methods have been developed to trace MRD, with flow cytometry and polymerase chain reaction (PCR) being the most reliable. However, new technologies, such as digital PCR and Next-Generation Sequencing are emerging as particularly useful in AML. The 2017 European LeukemiaNet (ELN) recommendations have incorporated MRD assessment to define the response criteria to therapy, and more recently, the ELN MRD Working Party has published guidelines for the use of MRD in clinical practice. SUMMARY: Morphologic complete remission (mCR) after induction therapy, has been consistently shown not only to have a critical prognostic role but also to fail in predicting relapse on an individual basis. Major attempts to improve our prediction capability have been made by measuring the residual levels of leukemic cells that persist in the bone marrow after chemotherapy. This number of cells, also called MRD, harbors in the bone marrow below the threshold of morphology and is responsible for leukemia recurrence. Therefore, the detection of MRD promises to help predict the risk of relapse, allowing a more proper patients' risk-stratification and the use of risk-tailored therapeutic strategy.

Baseline cytogenetic/genetic features have been widely recognized to play a critical prognostic role in acute myeloid leukemia (AML) and have proven useful in designing risk-adapted treatment strategies. Nevertheless, to improve further the outcome of AML patients we are still in need of accurate methods to explore the quality of response and to adequately discriminate patients who are likely to relapse over time from those who are in deep and stable remission. In this view, is it well established that measurement of leukemic cells surviving chemotherapy (called measurable residual disease, MRD) during the course of treatment may be a reliable biomarker in predicting relapse. Detection of MRD relies on highly sensitive techniques, such as quantitative polymerase chain reaction and multiparametric flow cytometry, which, due to their levels of specificity and sensitivity, are increasingly included in the decision-making process of AML treatment. In the present manuscript, we will review the current techniques of MRD investigation and their clinical contribution to AML management.

Background: Although bronchoalveolar lavage (BAL) measurements of galactomannan antigen (GM) seems to be more sensitive than serum testing to detect invasive fungal infection (IFI), a consensus on the most appropriate diagnostic threshold of the BAL GM test is still unclear. Moreover, there is uncertainty as to whether BAL is a safe procedure in patients with hematological malignancies (HM) and thrombocytopenia. Objectives: Based on this background, 102 adult patients with HM and associated thrombocytopenia were retrospectively analyzed with the dual aim of 1) determining whether BAL is a safe and feasible procedure; and, 2) identifying the most appropriate threshold for GM positivity in the diagnosis of IFI. Patients/Methods: each BAL was considered as one case/patient. One hundred twelve BALs were carried out in 102 HM patients: at the time of the BAL, the median platelet count (PLTs) in all patients was 47x10e9/L (1-476), and 31 patients (27%) had PLTs< 20x10e9/L. Results: complications from the BAL were infrequent (3.5%) and mild. No bleeding was reported. The BAL GM cut off of >0.8 was associated with the best diagnostic accuracy (sensitivity 72.97% and specificity 80%). Antifungal treatment of patients with BAL GM >0.8 resulted in a clinical-radiological improvement in 35/41patients (85%). Conclusions: BAL was a safe procedure also in thrombocytopenic patients, permitting an IFI diagnosis not otherwise identifiable using EORTC/MSG criteria. Our data suggest that a BAL GM value of>0.8 represents the most useful cut-off in terms of sensibility and specificity. Further prospective studies on a more significant number of patients are needed to confirm these results.

Background: In Acute Myeloid Leukemia (AML), identification of measurable residual disease (MRD) thresholds with clinical significance is still a matter of debate. For this purpose, multiparametric flow cytometry (MFC) is extensively employed for MRD quantification, due to high sensitivity (down to 1:10-3/10-5 cells) and wide applicability (up to 90% of cases). The identification of 20 clustered residual leukemic cells seems sufficient for the recognition of MRD presence (lower limit of detection [LOD]), whereas a cluster of 50 events may be the minimum threshold for the quantification of a cell population (lower limit of quantitation [LOQ]), provided a sufficient denominator of relevant events (500'000-1'000'000) is acquired. Methods: Using a MFC assay, we assessed the predictive power of a threshold calculated applying the criteria of LOD and LOQ on 261 intensively treated AML patients enrolled in the GIMEMA AML1310 prospective trial.According to the protocol design, patients with a bone marrow residual leukemic cells count (RLCc) equal or above 0.035% of the total no. of mononuclear (MNC) cells qualified as MRDpos,, whileusing LOD and LOQ, we selected the following categories of patients: 1) LODneg if RLCc was below LOD (20x100/total no. of events); 2) LODpos-LOQneg if RLCc was between LOD and LOQ; and 3) LOQpos if RLCc was above LOQ (50x100/total no. of events). Results: The ELN target of 500'000 events was reached in 182/261 (69.6%) patients. Overall, using the predefined AML1310 protocol MRD threshold, 154 (59%) and 107 (41%) were MRDneg and MRDpos, respectively, whereas 74 (28.4%), 43 (16.5%) and 144 (54.4%) patients were classified as LODneg, LODpos-LOQneg and LOQpos, respectively. Two-year overall survival (OS) was 75.4% vs. 79.8% vs. 66.4% for LODneg, LODpos-LOQneg and LODpos, respectively (p=0.1197), and 74.5% vs. 66.4% according to AML1310 protocol 0.035% threshold for MRDneg and MRDpos patients, respectively (p=0.3521). Due to superimposable outcome, LOD-LOQneg and LODpos-LOQneg categories were combined. Accordingly, LODneg/LODpos-LOQneg and LOQpos groups clearly differed in terms of OS (77% vs. 66.4%, p=0.0437) [FIGURE 1A]. Such a figure was challenged in multivariate analysis (p=0.048, HR 0.628, 95% CI 0.396-0.997) that confirmed the independent role of LOD-LOQ approach in influencing OS. To enhance the predictivity of LOD-LOQ estimate, we then focused on samples acquisition of which passed the 500'000 events, according to ELN guidelines. Among 182/261 (69.7%) cases with > 500'000 MNC events as denominator, LODneg/LODpos-LOQneg and LOQpos subgroups were clearly distinct in terms of OS (2-years OS of 83.5% vs. 69.4%, p=0.009). [FIGURE 1B] Similarly, also when selecting those patients (158/261 [60.5%]) whose acquisition passed 500'000 CD45+ events, LODneg/LODpos-LOQneg and LOQpos showed a different behavior with 2-years OS of 86.7% vs. 69.0%, respectively (p=0.004). [FIGURE 1C] Finally, when considering the interaction of the 3 LOD-LOQ categories with possible post-remissional strategies, LODneg/LODpos-LOQneg patients submitted to autologous stem cell transplant showed the best 2-years OS (88.9%) as compared to all the other categories (allogeneic stem cells transplant and no graft-based treatments) (p=0.026). Summary/Conclusion: In conclusion, the use of LOD-LOQ method results in a more sensitive detection of MRD that, in turn, translates in a more accurate recognition of patients with different prognosis. Actually, such an approach allowed to dissect even further the category of patients called MRDneg according to the AML1310 protocol definition, since MRDneg subjects who belonged to a "true negative" LOD-LOQ sub-group [LODneg/LODpos-LOQneg] had a better outcome than the other MRDneg ones. This MRD approach could serve as a useful tool to personalize post-remission strategy in intensively treated AML patients, through selection of high-quality remission patients who may benefit from less intensive post-consolidation therapies. Disclosures Voso: Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Venditti:Novartis: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pfizer: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Amgen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Jazz: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company).

Background: In the 2017 version of the ELN recommendations (ELN2017), a comprehensive evaluation of karyotype and mutational status of specific genes (e.g. FLT3 and NPM1) allows to classify patients (pts) with Acute Myeloid Leukemia (AML) into 3 prognostically distinct risk groups (favorable, intermediate and adverse-risk). Before the publication of the ELN2017 guidelines, the Gruppo Italiano Malattie Ematologiche MAligne (GIMEMA) conducted a prospective trial (AML1310) in which prognostic classification relied on the risk assessment criteria (NCCN2009) at that time available. In this post-hoc analysis, we investigated the applicability of the ELN2017 risk stratification to the AML1310 study population. Methods: After induction and consolidation, pts in complete remission (CR) were to receive autologous stem cell transplant (AuSCT) if categorized as favorable-risk (FR) (CBF-AML, NPM1-mutated) or allogeneic stem cell transplant (ASCT) if adverse-risk (AR) (FLT3-ITD, complex karyotype). Intermediate-risk pts (IR) were to receive AuSCT or ASCT based on the post-consolidation levels of MRD as measured by flow-cytometry. Baseline genetic/cytogenetic, together with RUNX1/RUNX1T1, CBFb/MYH11, NPM1, FLT3 mutational status (including the FLT3 allelic ratio for those positive) were used to retrospectively classify pts according to the ELN2017. Results: All 500 pts, enrolled in the AML1310 trial, were included in the present analysis. Retrospective allocation was feasible in 445/500 (89%) cases and pts lacking crucial information for a proper ELN2017 assignment, defined a control group (ELN2017-NC). Median age was 49 (range 18-61). The re-assignmentaccording to the ELN2017, resulted in 186 pts (41.8%) belonging to the FR category (ELN2017-FR), 179 (40.2%) to the IR (ELN2017-IR) and 80 (18%) to the AR (ELN2017-AR) ones. Moreover, 55 (11%) pts were considered ELN2017-NC. Based on this process of re-assignment, 173 pts were reclassified according to ELN2017: 6 from NCCN FR (1 ELN2017-NC, 4 ELN2017-IR, 1 ELN2017-AR), 54 from NCCN IR (34 ELN2017-NC, 4 ELN2017-IR, 1 ELN2017-AR), and 113 from NCCN AR (20 ELN2017-NC, 38 ELN2017-FR, 55 ELN2017-AR) groups. After 1-2 cycles of induction, 361 (72%) pts obtained CR or CR incomplete (CRi): 163 (88.1%), 114 (65%), 45 (56.2%) and 39 (70%) in the ELN2017-FR, ELN2017-IR, ELN2017-AR and ELN2017-NC groups, respectively (p Summary/Conclusion: In this GIMEMA AML1310 post-hoc analysis, we confirmed that the ELN2017 classification is able to accurately define pts that can benefit from different post-remission strategies. Specifically, AuSCT granted longer survival in FR pts, while for IR pts AuSCT and ASCT performed equally when minimal residual disease was used as a driver for opting between one of the two. In conclusion, ELN classification is a reliable grouping system that, combined with MRD assessment, helps addressing pts to the most appropriate treatment. Such an hypothesis will be prospectively challenged in the next GIMEMA trial AML1819. Disclosures Luppi: Abbvie: Consultancy; Novartis: Consultancy, Speakers Bureau; Gilead Sci: Consultancy, Speakers Bureau; Sanofi: Consultancy; Daiichi-Sankyo: Consultancy; MSD: Consultancy. Voso:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Venditti:Pfizer: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Speakers Bureau; Amgen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Jazz: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMODATIONS, EXPENSES (paid by any for-profit health care company); Novartis: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company).

Introduction: Despite recent progress, the prognosis of acute myeloid leukemia remains poor, mainly in older and in relapsed/refractory patients. Recently, a large number of novel agents have been developed thanks to a better understanding of its pathogenesis. Among these, the potent inhibitor of the isocitrate dehydrogenase-2 (IDH2) mutant protein, enasidenib (formerly AG-221), has demonstrated promising antileukemic activity by targeting IDH2 mutations. Area covered: This review describes the mechanisms of action, the pharmacodynamic and pharmacokinetic properties, the safety, and efficacy of enasidenib. Phase I/II/III clinical trials are also reported and discussed. Expert opinion: Enasidenib is a novel agent able to differentiate leukemic blasts in functional, maturating cells. This drug is characterized by oral bioavailability and good tolerability. As a monotherapy, it demonstrates clinical and laboratorial improvement, in 19.6% and 38.8% of cases respectively. Differentiation syndrome is the most relevant, potentially life-threatening side effect, which physicians must be aware of. The authors believe that the way forwards now is to explore the role of enasidenib as a chemoresistance revertant when associated with chemotherapy, as a 'bridge to transplant' or when associated other novel agents if we wish to maximize its use.

The terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase expressed in acute myeloid leukemias (AMLs), where it may be involved in the generation of NPM1 and FLT3-ITD mutations. We studied the correlations between TdT expression and FLT3-ITD or NPM1 mutations in primary AML samples, and the impact on patients' survival. TdT expression was analyzed in 143 adult AML patients by flow cytometry as percentage of positivity and mean fluorescence intensity (MFI) on blasts. TdT was positive in 49 samples (34.2%), with a median of 48% TdT-positivity (range 7-98) and a median MFI of 2.70 (range 1.23-30.54). FLT3-ITD and NPM1 mutations were present in 24 (16.7%) and 34 (23.7%) cases, respectively. Median TdT expression on blasts was significantly higher in FLT3-ITD+, as compared with FLT3-ITD- AMLs (median 8% vs 0% respectively, p = 0.035). NPM1 mutational status, FLT3-ITD allelic ratio, karyotype, and ELN risk groups, did not correlate with TdT expression or MFI on blasts. TdT + patients had poorer survival as compared to TdT-, but this result was not confirmed by the multivariable analysis, where ELN risk stratification as well as age and type of treatment remained independent prognostic factors for OS. In summary, our results support the possible implication of TdT enzyme in the generation of FLT3-ITD mutations in AML.

Acute myeloid leukemia (AML), with an incidence increasing with age, is the most common acute leukemia in adults. Concurrent comorbidities, mild to severe organ dysfunctions, and low performance status (PS) are frequently found in older patients at the onset, conditioning treatment choice and crucially influencing the outcome. Although anthracyclines plus cytarabine-based chemotherapy, also called “7 + 3” regimen, remains the standard of care in young adults, its use in patients older than 65 years should be reserved to selected cases because of higher incidence of toxicity. These adverse features of AML in the elderly underline the importance of a careful patient assessment at diagnosis as a critical tool in the decision-making process of treatment choice. In this review, we will describe selected recently approved drugs as well as examine prognostic algorithms that may be helpful to assign treatment in elderly patients properly.

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 Conclusions: In our real-life analysis, we confirmed that SIE, SIES, GITMO "operational criteria" represent a powerful tool to discriminate categories of older pts with different outcome.In pts older than 60 years, it was observed a high degree of concordance (>90%) between the "operational criteria" and the actual treatment delivered. However, long-term OS was longer when plotted according to the 3 fitness categories rather than to treatment intensity stratification (Fig.1 A and B). Such a discrepancy can be explained by a higher incidence of toxicity/early mortality in the IC group and also by a suboptimal application of treatment selection criteria. Reproducibility of operational criteria applications in a prospective fashion is currently underway. Disclosures Venditti: Novartis: 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; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. 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.

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 ( Conclusions: In line with the experience of other European groups, we demonstrated that MFC monitoring of LSC is feasible and provides prognostic information when performed at diagnosis and during treatment course. MFC assessment of LSC also offers the opportunity to monitor pts who lack aberrant phenotypes suitable for "standard" MRD investigation. When the 2 approaches - standard "MRD" and LSC assessment - are combined together, the prognosis prediction of AML can be further refined. Finally, LSC assessment can potentially represent an effective tool to monitor the effect of LSC targeting agents. Disclosures Buccisano: Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: 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. Venditti: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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy.

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.

Introduction Persistence of leukemia stem cells (LSCs) in patients with Acute Myeloid Leukemia (AML) achieving complete remission (CR) after chemotherapy leads to disease recurrence and poor outcome. Therefore, the identification of LSCs driving resistance to therapy represents an important challenge. LSCs reside within the CD34+/CD38- cells and it has been recently demonstrated that co-expression of CD99 allows for separation of LSCs from functionally normal hematopoietic stem cells in AML. We showed that the presence of a CD123/CD99/CD25+ population within CD34+ cells strongly correlates with FLT3-ITD-positivity. The aim of this study was to deeply characterize the molecular profile of CD34+/CD123+/CD99+/CD25+ LSCs to shed light on the subclonal architecture of FLT3-ITD+ AML and to track the expansion of mutated clones. Methods Molecular status of FLT3-ITD and NPM1 were investigated at the DNA level in a cohort of 150 de novo AML patients at diagnosis and at relapse, using standard procedures. Clonal evolution of FLT3-ITD mutations was studied at diagnosis in 14 FLT3-ITD mutated AML on sorted bone marrow populations, purified by the Cytoflex High-Speed Cell Sorter using a sequential gating strategy, to separate CD34/CD123/CD99/CD25+ LSCs-, CD34+ precursors (CD123/CD99/CD25-) and T-lymphocytes. Moreover, to characterize the genetic profile of FLT3-ITD-mutated clones, DNA targeted sequencing was performed on 22 cell populations isolated from 7 AML patients, using the Oncomine™ Myeloid Research Assay panel on the Ion Torrent™ S5 sequencer. Results 39 of 150 AML cases were FLT3-ITD+, at a median allelic ratio (AR) of 0.36 (range 0.05-7.9). The FLT3-ITD AR was significantly higher within the rare LSCs compartment as compared to MNC (median AR: 0.78, range 0.42-19.3, vs 0.09, range 0.05-0.6m p1, while the number of LSCs do not seem to expand (Fig 1B). These data confirm that LSCs may persist at rare frequency during progression, still representing the treatment-resistant FLT3-ITD reservoir, driving disease relapse with expansion of more mature, FLT3-ITD positive populations. Aiming at shedding light on the molecular heterogeneity and subclonal structure of AMLs, using targeted NGS we compared the mutational profiles of MNCs to that of highly purified LSCs and/or to the CD34+/CD123-/CD25-/CD99- counterpart (n=7 patients). In 4 cases the same mutation pattern was present in both MNCs and LSCs. In 3 cases we found additional mutations in NRAS, BCOR and KRAS in MNCs, indicating acquisition of other transforming events during maturation (Fig 2A). Furthermore, the variant allele frequency (VAF) of mutations was similar in MNCs and LSCs (p=0.12), while FLT3-ITD and TKD burden was enriched in LSCs (p=0.03) (Fig 2B). Conversely, the NPM1 mutation burden remained almost stable in the different cell subsets (Fig 2C). In conclusion, our study shows that FLT3 mutations occur in early LSCs characterized by the CD34+/CD123+/CD99+/CD25+ immunophenotype, which represent the leukemic reservoir. These data may be the rational for CD99-targeted treatments in FLT3-ITD mutated AML to achieve durable disease eradication. Disclosures Venditti: 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy. Buccisano:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees.

Incidence and effect on outcome of central nervous system (CNS) involvement in adult patients with acute myeloid leukemia (AML) is not clearly defined. To address this issue, 103 consecutive adult patients with newly diagnosed AML, regardless of neurologic symptoms, were submitted to a routine explorative lumbar puncture. Cerebrospinal fluid (CSF) samples were collected from 65 males and 38 females. All 103 CSF samples were examined by conventional cytology (CC) whereas 95 (92%) also by flow cytometry (FCM). At diagnosis, 70 patients (68%) were CNS negative (CNS-), whereas 33 (32%) were CNS positive (CNS+). In 11 of 33 (33%), CNS infiltration was documented either by CC or FCM , in 21 (67%) only by FCM. CNS positivity was significantly associated with a M4-M5 phenotype of the underlying AML (P = .0003) and with high levels of lactate dehydrogenase (P = .006). Overall, 80 of 103 (78%) achieved complete remission with no significant differences between CNS+ and CNS- patients. Five-year disease-free survival and overall survival were found to be shorter in CNS+ patients than in those CNS- (18% vs 50%, P = .006 and 19% vs 46%, P = .02, respectively). In multivariate analysis, CNS status and age were found to affect independently overall survival. In conclusion, the incidence of CNS involvement in adult patients with newly diagnosed AML is higher than expected. Regardless of neurologic symptoms, it should always be searched at diagnosis; CSF samples should routinely be investigated by FCM since a certain proportion of CNS involvements might remain undetected if examination is exclusively CC based.

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.

Introduction. Rituximab has been demonstrated to be effective either as intralesional or as systemic therapy in PCBCL, We report our experience in the treatment of PCBCL with intravenous Rituximab. P atients and Methods From February 1999 to February 2014 we treated 75 patients: 47 Follicle Center Lymphoma, 23 Marginal Zone Lymphoma, 5 Diffuse Large Lymphoma Leg type. The stage was T1 in 38 pts, T2 in 22 pts and T3 in 15 pts. In 24 patient prior treatment included: CHT (11), Radiotherapy (4), Surgery (4) or alpha2Interferon (IFN) (5). Rituximab at dosage of 375 mg/m2 for a minimum of 4 cycles, was administered alone (51 patients) or in association with CHT (13). RT (2) or IFN (3). Results. No patient presented adverse effects during the Rituximab infusion. A reduction of circulating B lymphocytes was observed for 11 months, on the average, without an increased risk of infections. No added toxicity was observed in patients treated with Rituximab plus CHT. Overall response rate was 97,3% (CR 82,6 %, PR 14,6 %). Five–years Overall Survival (OS) was 86,9% with Disease Free Survival of 57%. According to stage OS was in T1 94,3%, in T2 90,5%, in T3 73,6%. (T1+T2 vs T3: p Conclusions. Rituximab is effective and safe in the treatment of PCBCL even in heavily-treated or elderly patients. In our patients only the stage of disease was significant for the prognosis. A higher number of patients are necessary to indicate Rituximab in biological and clinical subsets of patients as a front-line therapy. Disclosures No relevant conflicts of interest to declare.