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In acute myeloid leukemia (AML) many patients experience relapse, despite the achievement of morphological complete remission; therefore, conventional morphologic criteria are currently considered inadequate for assessing the quality of the response after treatment. Quantification of measurable residual disease (MRD) has been established as a strong prognostic marker in AML and patients that test MRD negative have lower relapse rates and better survival than those who test positive. Different techniques, varying in their sensitivity and applicability to patients, are available for the measurement of MRD and their use as a guide for selecting the most optimal post-remission therapy is an area of active investigation. Although still controversial, MRD prognostic value promises to support drug development serving as a surrogate biomarker, potentially useful for accelerating the regulatory approval of new agents. In this review, we will critically examine the methods used to detect MRD and its potential role as a study endpoint.

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.

Few data are available on the treatment with DFX in patients with transfusion dependent Ph- Myeloproliferative Neoplasms in fibrotic phase. Here we report 48MPNpatients and iron overload treated with DFX. Starting DFX dose was 20 mg/Kg in 23 patients, 15 mg/Kg in 20 patientsand 10 mg/Kg in 5 patients. After a median treatment of 27.6 months, 5 patients achieved ferritin500 ng/ml, 11 1000 ng/ml and 3 a reduction50% of basal ferritin with a global response rate of 41%. As to hematological improvement, 9/47 patients (19.1%) showed a persistent rise of Hb1.5 g/dl, with disappearance of transfusion requirement in 6 cases. The median OS from DFX initiation in patients with chelation response was 61.0 months compared to 15.8 months in patients without chelation efficacy. Treatment with DFX is feasible and effective in MPN with iron overload and a hematological improvement can occur in a sizeable rate of patients.

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.

Measurable residual disease (MRD) is an important biomarker in acute myeloid leukemia (AML) that is used for prognostic, predictive, monitoring, and efficacy-response assessments. The European LeukemiaNet (ELN) MRD Working Party evaluated standardization and harmonization of MRD in an ongoing manner and has updated the 2018 ELN MRD recommendations based on significant developments in the field. New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement. Major changes include technical specifications for next-generation sequencing-based MRD testing and integrative assessments of MRD irrespective of technology. Other topics include use of MRD as a prognostic and surrogate end point for drug testing; selection of the technique, material, and appropriate time points for MRD assessment; and clinical implications of MRD assessment. In addition to technical recommendations for flow- and molecular-MRD analysis, we provide MRD thresholds and define MRD response, and detail how MRD results should be reported and combined if several techniques are used. MRD assessment in AML is complex and clinically relevant, and standardized approaches to application, interpretation, technical conduct, and reporting are of critical importance.

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.

Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States.

Response to therapy is affected by the genetic heterogeneity of acute myeloid leukemia (AML) and persistence of leukemic cells below the threshold of morphological complete remission (mCR). Such persistence is called minimal (or measurable) residual disease (MRD). Areas covered: MRD assessment allows early identification of patients who are at high risk of relapse and who should timely receive aggressive therapy (e.g. allogeneic stem cell transplantation) and of those with a good quality mCR in whom an aggressive front-line therapy can be spared, avoiding the harm of excessive treatment toxicity. The most exploited methods to assess MRD are multiparameter flow cytometry (via identification of immunophenotypic aberrancies) or PCR-based assays (via identification of cytogenetic/molecular abnormalities). Expert commentary: A growing body of evidences demonstrates that positive MRD-testing at various time-points throughout the treatment course identifies patients at high risk of relapse. We will focus on the role of MRD as a biomarker to refine risk assessment and to prospectively direct treatment choices in adult with AML.

Background: In ‘real-life', the Nordic score guides ESA use in lower-risk MDS with predicted response rates of 25% or 74%. As new treatments emerge, a more discriminating score is needed. Objectives: To validate existing ESA predictive scores and develop a new score that identifies non-responders. Methods: ESA-treated patients were identified in 3 MDS registries in Italy and Canada (FISM 555, GROM 233, MDS-CAN 208). Clinical and disease-related variables were captured. Nordic, MDS-CAN and IPSS-R-based ESA scores were calculated and documented ESA responses compared. Results: 996 ESA-treated patients were identified. Overall response rate(ORR) was 59%. The database was randomly divided into balanced derivation (n=463) and validation (n=462) cohorts. By multivariate analysis, transfusion independence, erythropoietin level

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).

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.

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.

In the acute myeloid leukemia (AML) setting, research has extensively investigated the existence and relevance of molecular biomarkers, in order to better tailor therapy with newly developed agents and hence improve outcomes and/or save the patient from poorly effective therapies. In particular, in patients with AML, residual disease after therapy does reflect the sum of the contributions of all factors associated with diagnosis and post-diagnosis resistance. The evaluation of minimal/measurable residual disease (MRD) can be considered as a key tool to guide patient’s management and a promising endpoint for clinical trials. In this narrative review, we discuss MRD evaluation as biomarker for tailored therapy in AML patients; we briefly report current evidence on the use of MRD in clinical practice, and comment on the potential ability of MRD in the assessment of the efficacy of new molecules.

We designed a trial in which postremission therapy of young patients with de novo acute myeloid leukemia (AML) was decided combining cytogenetics/genetics and postconsolidation levels of minimal residual disease (MRD). After induction and consolidation, favorable-risk patients (FR) were to receive autologous stem cell transplant (AuSCT) and poor-risk patients (PR) allogeneic stem cell transplant (AlloSCT). Intermediate-risk patients (IR) were to receive AuSCT or AlloSCT depending on the postconsolidation levels of MRD. Three hundred sixty-one of 500 patients (72%) achieved a complete remission, 342/361 completed the consolidation phase and were treatment allocated: 165 (48%) to AlloSCT (122 PR, 43 IR MRD-positive) plus 23 rescued after salvage therapy, for a total of 188 candidates; 150 (44%) to AuSCT (115 FR, 35 IR MRD-negative) plus 27 IR patients (8%) with no leukemia-associated phenotype, for a total of 177 candidates. Overall, 110/177 (62%) and 130/188 (71%) AuSCT or AlloSCT candidates received it, respectively. Two-year overall (OS) and disease-free survival (DFS) of the whole series was 56% and 54%, respectively. Two-year OS and DFS were 74% and 61% in the FR category, 42% and 45% in the PR category, 79% and 61% in the IR MRD-negative category, and 70% and 67% in the IR MRD-positive category. In conclusion, AuSCT may still have a role in FR and IR MRD-negative categories. In the IR MRD-positive category, AlloSCT prolongs OS and DFS to equal those of the FR category. Using all the available sources of stem cells, AlloSCT was delivered to 71% of the candidates.This trial was registered at www.clinicaltrials.gov as #NCT01452646 and EudraCT as #2010-023809-36.

Pulmonary infections (PIs) are a major complication of patients with myelodysplastic syndromes (MDS). We retrospectively evaluated 234 MDS patients treated with azacytidine (AZA). The total number of AZA cycles was 2886 (median 8 cycles per patient). There were 111 episodes of PI (3.8% of AZA cycles) in 81 patients (34.6%). PIs were considered of fungal origin in 27 cases (24.3%), associated to bacteremia in 11 cases (9.9%), to influenza infection in two cases (1.8%) and of unknown origin in the remaining 71 cases (64.0%). Forty-five PI episodes were documented in cycles 1 to 4 of AZA (5.1% of 875 cycles) and the remaining 66 episodes beyond the fourth cycle (3.2% of 2011 cycles) (P = .017). Overall, a fungal PI was documented in 13/875 (1.5%) cycles 1 to 4 and in 13/2011 (0.6%) cycles beyond the fourth cycle (P = .001). A baseline chronic pulmonary disease was significantly associated to a higher risk of severe PIs. In the survival analysis, cases of PI in patients who progressed to acute leukemia (PAL) were excluded, in view of the predominant influence of PAL on the outcome of the patients. A PI unrelated to PAL documented during the first 4 AZA cycles was an independent factor predicting lower survival (OR, 2.13; 95% CI, 1.37-3.33; P = .001). In conclusion, PIs are common in MDS patients receiving AZA, in particular during the first cycles of treatment and are associated with an unfavorable outcome. The results of our study raise the issue of the need of a tailored infection prevention strategy.

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.

Despite the increasing knowledge of the genomic landscape of acute myeloid leukemia (AML), prediction merely based on genetics fails to anticipate outcome, presumably due to the heterogeneous composition of the leukemic clone determining complex interactions between different genetic abnormalities. Therefore, the introduction of a post-treatment biomarker exploring the quality of response to therapy such as assessment of measurable (previously minimal) residual disease (MRD) may lead to refinements of the prognostic assessment in AML. In this view, the European LeukemiaNet has recently endorsed the achievement of a MRD negative morphologic complete remission as a purpose the treatment. Techniques like multiparametric flow cytometry and reverse transcriptase-quantitative polymerase chain reaction have reached a level of sensitivity and specificity that make them ready for introduction in clinical practice. In the present review, we will give an update on the efforts in harmonization and/or standardization of MRD assessment in AML, focusing on the newest acquisitions in the clinical applications of MRD, and considering issues like relationship of MRD with leukemic stem cells or MRD assessment in peripheral blood.

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.

Azacitidine (AZA) prolonged overall survival (OS) in the AZA-AML-001 trial. However, few subjects were randomized to AZA or intensive chemotherapy (IC). The Medical Research Council (MRC) and the Leukemia Research Foundation (LRF) developed a score for older AML patients receiving IC or non-intensive regimens, whereas the E-ALMA study validated a score for survival and response in elderly patients receiving AZA in daily practice. Both identified three groups with different risk estimates. This analysis evaluates the efficacy of frontline AZA in older AML patients (N = 710) unfit for IC from different national registries (E-ALMA + series) stratified by the MRC/LRF risk score. Median OS of patients categorized as good, standard and poor-risk groups by the MRC/LRF score was 13.4 (95% CI, 10.8-16), 12.4 (95% CI, 9.9-14.8), and 8.1 months (95% CI, 7-9.1), respectively (p = .0001). In conclusion, this is the largest retrospective cohort of older AML patients treated with AZA.

The present observational study aimed to compare the efficacy of azacitidine (AZA) and intensive chemotherapy (IC) in elderly patients with untreated acute myeloid leukemia (AML), diagnosed according to WHO criteria. In the two groups, we evaluated complete remission (CR), overall survival (OS), and disease-free survival (DFS). The AZA group included 89 patients; median age was 73 years (range 61–80) and median white blood cell count (WBCc) 2.5 × 109/L (range 0.27–83), 45% of the patients had BM blasts ≥ 30%, and 44 (49%) had a secondary AML (sAML). Karyotype was evaluable in 69 patients: 51 (74%) had intermediate-risk abnormalities and 18 (26%) an unfavorable risk karyotype. IC group consisted of 110 patients who received an induction course with mitoxantrone, cytarabine, and etoposide, followed by two consolidation cycles including idarubicin, cytarabine, and etoposide. Median age was 67 years (range 61–78) and median WBCc 8.0 × 109/L (range 0.69–258); 44 (40%) had a sAML. Karyotype was evaluable in 88 patients, 71 (81%) had intermediate risk, and 17 (19%) unfavorable risk karyotype. To minimize the effects of treatment selection bias, adjustments were made using the propensity-score matching method, which yielded 74 patient pairs. CR rate was significantly higher in IC vs AZA group (73 vs 25%, respectively) (p

Purpose: We evaluated leukemia-associated immunophenotypes (LAIP) and their correlation with fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) gene mutational status in order to contribute a better identification of patients at highest risk of relapse in acute myeloid leukemia (AML). Experimental Design: Bone marrow samples from 132 patients with AML were analyzed by nine-color multiparametric flow cytometry. We confirmed the presence of the mutation in diagnostic samples and in sorted cells by conventional RT-PCR and by patient-specific RQ-PCR. Results: Within the CD34+ cell fraction, we identified a discrete population expressing high levels of the IL3 receptor α-chain (CD123) and MIC-2 (CD99) in combination with the IL2 receptor α-chain (CD25). The presence of this population positively correlated with the internal tandem duplications (ITD) mutation in the FLT3 gene (r = 0.71). Receiver operating characteristics showed that, within the CD34+ cell fraction a percentage of CD123/CD99/CD25+ cells ≥11.7% predicted FLT3–ITD mutations with a specificity and sensitivity of >90%. CD34/CD123/CD99/CD25+ clones were also detectable at presentation in 3 patients with FLT3 wild-type/NPM1+ AML who relapsed with FLT3-ITD/NPM1+ AML. Quantitative real-time PCR designed at relapse for each FLT3-ITD in these three cases confirmed the presence of low copy numbers of the mutation in diagnostic samples. Conclusions: Our results suggest that the CD34/CD25/CD123/CD99+ LAIP is strictly associated with FLT3-ITD–positive cells. Clin Cancer Res; 21(17); 3977–85. ©2015 AACR.

Therapy-related myeloid neoplasms (t-MN) are a complication of cytotoxic treatment for primary tumors and autoimmune diseases. We report data on 277 t-MN patients, recruited between 1999 and 2013 by the Italian Network on Secondary Leukemias (104 retrospectively and 173 prospectively registered). Median age at t-MN diagnosis was 64 years (range, 21-87). Most frequent primary malignancies (PMs) were lymphoproliferative diseases and breast cancer. One hundred and thirty-three patients had received chemotherapy (CHT), 43 patients radiotherapy (RT), and 101 patients combined CHT/RT for PM. Median time between cytotoxic treatment and t-MN was 5.7 years, with t-MN following RT alone associated with significantly longer latency, compared to CHT or combined CHT/RT (mean, 11.2 vs. 7.1 years, P = 0.0005). The addition of topoisomerase-II inhibitors to alkylating agents was associated with shorter latency compared to alkylating agents alone (median, 6 vs. 8.4 years, P = 0.02). Median survival was 14.6 months from t-MN diagnosis, and was significantly longer in patients treated with allogeneic stem cell transplantation. Significant factors for survival at the multivariable analysis included age, adverse karyotype, and degree of anemia. Our data underline the prognostic importance of karyotype and age in t-MN, similar to de novo acute myeloid leukemia. Treatment approaches should not preclude the use of conventional treatments for younger t-MN patients, including allogeneic stem cell transplantation as potentially curative approach.

For many years disappointing results have been generated by many investigations which have utilized a variety of immunologic strategies to enhance the ability of a patient’s immune system to recognize and eliminate malignant cells. However, in recent years, immunotherapy has been used successfully for the treatment of hematologic and solid malignancies. The impressive clinical responses observed in many types of cancer have convinced even the most skeptical clinical oncologists that a patient’s immune system can recognize and reject his tumor if appropriate strategies are implemented. The success immunotherapy is due to the development of at least three therapeutic strategies. They include tumor-associated antigen (TAA)-specific monoclonal antibodies (mAbs), T cell checkpoint blockade, and TAA-specific chimeric antigen receptors (CARs) T cell-based immunotherapy. However, the full realization of the therapeutic potential of these approaches requires the development of strategies to counteract and overcome some limitations. They include the off-target toxicity and mechanisms of cancer immune evasion which obstacle the successful clinical application of mAbs and CAR T cell-based immunotherapies. Thus, we and others have developed the Fc gamma chimeric receptors (FcCRs)-based strategy. Like CARs, FcCRs are composed of an intracellular tail resulting from the fusion of a co-stimulatory molecule with the T cell receptor  chain. In contrast, the extracellular CAR single chain variable fragment (scFv), which recognizes the targeted TAA, has been replaced with the extracellular portion of the FcRIIIA (CD16). FcCR T cells have a few intriguing features. First, given in combination with mAbs, FcCR T cells mediate anti-cancer activity in vitro and in vivo by an antibody-mediated-cellular-cytotoxicity (ADCC) mechanism. Second, CD16-CR T cells can target multiple cancer types provided that TAA-specific mAbs with the appropriate specificity are available. Third, the off-target effect of CD16-CR T cells may be controlled by withdrawing the mAb administration. The goal of this manuscript was three fold. First, we review the current state-of-the-art of pre-clinical CD16-CR T cell technology. Second, we describe its in vitro and in vivo anti-tumor activity. Lastly, we compare the advantages and limitations of the CD16-CR T cell technology with those of CAR T cell methodology.

Acute erythroleukemia (AEL) is a rare disease typically associated with a poor prognosis. The median survival ranges between 3-9 months from initial diagnosis. Hypomethylating agents (HMAs) have been shown to prolong survival in patients with myelodysplastic syndromes (MDS) and AML, but there is limited data of their efficacy in AEL. We collected data from 210 AEL patients treated at 28 international sites. Overall survival (OS) and PFS were estimated using the Kaplan-Meier method and the log-rank test was used for subgroup comparisons. Survival between treatment groups was compared using the Cox proportional hazards regression model. Eighty-eight patients were treated with HMAs, 44 front line, and 122 with intensive chemotherapy (ICT). ICT led to a higher overall response rate (complete or partial) compared to first-line HMA (72% vs. 46.2%, respectively; p, Publication costs were supported by the University of Salzburg.

Summary Iron chelation is controversial in higher risk myelodysplastic syndromes (HR-MDS), outside the allogeneic transplant setting. We conducted a retrospective, multicentre study in 51 patients with transfusion-dependent, intermediate-to-very high risk MDS, according to the revised international prognostic scoring system, treated with the oral iron chelating agent deferasirox (DFX). Thirty-six patients (71%) received azacitidine concomitantly. DFX was given at a median dose of 1000 mg/day (range 375–2500 mg) for a median of 11 months (range 0·4–75). Eight patients (16%) showed grade 2–3 toxicities (renal or gastrointestinal), 4 of whom (8%) required drug interruption. Median ferritin levels decreased from 1709 μg/l at baseline to 1100 μg/l after 12 months of treatment (P = 0·02). Seventeen patients showed abnormal transaminase levels at baseline, which improved or normalized under DFX treatment in eight cases. One patient showed a remarkable haematological improvement. At a median follow up of 35·3 months, median overall survival was 37·5 months. The results of this first survey of DFX in HR-MDS are comparable, in terms of safety and efficacy, with those observed in lower-risk MDS. Though larger, prospective studies are required to demonstrate real clinical benefits, our data suggest that DFX is feasible and might be considered in a selected cohort of HR-MDS patients.

We assessed by flow cytometry minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) given standard-dose (SDAC) and high-dose ARA-C (HDAC) regimens. Of 163 patients enrolled, 130 (median age, 45 years; range, 18-59 years) qualified for analysis, all achieving complete remission after treatment with SDAC (n = 78) or HDAC (n = 52) plus etoposide and daunorubicin. Consolidation consisted of intermediate-dose ARA-C and daunorubicin. MRD negativity was significantly more frequent in the SDAC vs. HDAC arm after both induction (37% vs. 15%, P = 0.007) and consolidation (44% vs. 18%, P = 0.002). Respective median residual leukemic cell counts with SDAC and HDAC use were 1.5 × 10(-3) and 4 × 10(-3) (P = 0.033) after induction and 5.7 × 10(-4) and 2.9 × 10(-3) (P = 0.008) after consolidation. Based on ARA-C schedule and post-consolidation MRD status, the four patient groups (SDAC-MRD(-) , HDAC-MRD(-) , SDAC-MRD(+) , and HDAC-MRD(+) ) displayed 5-year overall survival rates of 60%, 33%, 24%, and 42% (P = 0.007), respectively, with 24%, 35%, 74%, and 48% (P

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.

The Gruppo Romano Mielodisplasie (GROM) conducted a retrospective study in 543 patients with myelodysplastic syndromes (MDS) to evaluate the safety and efficacy of erythropoiesis-stimulating agents (ESAs) in "real-life" clinical practice. The 40.000-UI/week erythropoietin (EPO)-alpha and 30.000-UI/week EPO-beta starting dose were defined "standard," and 80,000 UI/week EPO-alpha and 60.000 UI/week EPO-beta were defined "high." Response was defined according to International Working Group (IWG) 2006 criteria. At ESA's start, median age was 74.2 years (interquartile range (IR) 67.8-79.5) and median hemoglobin was 8.9 g/dl (IR 8.2-9.6). Median time from diagnosis to ESAs start was 3.8 months (IR 0.8-13.2). ESA starting dose was "standard" in 361 patients (66.5 %) and "high" in 182 patients (33.5 %). Erythroid response was observed in 82/185 (44.3 %) transfusion dependent (TD) patients as compared with 226/329 (68.6 %) transfusion independent (TI) ones (p

Background Thrombotic episodes are the major complication in the follow-up of Philadelphia negative Myeloproliferative Neoplasms (MPN), with high morbidity and mortality, as reported in several retrospective studies. At present, however, few prospective data are available on the early incidence of these complications. Methods To address this issue, we report on 1087 patients [M/F 508/579, median age 67.6 years, interquartile range (IQR) 55.2 - 75.9] with newly diagnosed MPN enrolled in the prospective database of our regional cooperative group since January 2011. Of them, 571 (52.5%) had Essential Thrombocythemia (ET), 303 (27.9%) Polycythemia Vera (PV) and 213 (19.6%) Primary Myelofibrosis (PMF). The main clinical features at diagnosis of the whole cohort and according to the different MPNs are reported in the Table 1. Results On the whole, 22 episodes of thrombotic complications were reported in 1087 patients (2.0%) at a median interval from diagnosis of 18.2 months (IQR 7.4 - 29.7): in particular, 15 (68.1%) were arterial (8 cerebral, 2 coronaric, 4 in the lower limbs, 1 splancnic) and 7 (31.9%) venous (5 in the lower limbs and 2 in the upper limbs). As to the incidence of early thrombosis in the different MPNs, they were 13/571 (2.2%) in ET patients, 5/303 (1.6%) in PV patients and 4/213 (1.8%) in PMF patients (p=0.810): median time from diagnosis to thrombotic event was also similar in the 3 MPNs (p=0.311). The 4-year cumulative Thrombosis-Free Survival (TFS) of the whole cohort was 97.3% (95%CI 96.0 - 98.6): there was no difference among the 3 MPNs as to 4-year TFS [96.7% (95%CI 94.8 - 98.6) in ET, 97.8% (95%CI 95.9 - 99.7) in PV and 98.7% (95%CI 96.9 - 100) in PMF, respectively, p=0.668). Several clinical features at diagnosis (age, gender, Hb levels, WBC and PLT counts, spleen enlargement, JAK-2 V617F mutation and previous thrombotic events) were evaluated for a role in predicting thrombotic events: only age (p=0.009) and previous thrombotic events (p=0.009) were significant. Conclusions The incidence of early thrombosis seems low in the first 4 years after diagnosis of MPN based on our prospective database, without any difference among ET, PV and PMF: it is worth of note that only age and previous thrombotic events had a predictive role, thus confirming many retrospective reported data and reinforcing the prognostic value of old scoring system for thrombotic risk in MPN. Table 1 Table 1. Disclosures Breccia: Ariad: Honoraria; Pfizer: Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria. Latagliata:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria.

Objective Azacitidine is the standard of care for higher-risk myelodysplastic syndromes (MDS). We evaluated factors affecting the outcome of azacitidine treatment in 196 ‘real-world’ patients, retrospectively collected by two Italian cooperative groups. Methods The study included 184 MDS and 12 low blast count acute myeloid leukemia (AML). Azacitidine was administered at the standard dose of 75 mg/m2/d for 7 d (SD) in 163 patients and 100 mg/d for 5–7 d in 33 patients. Results After a median of 4.5 azacitidine cycles (range 7–15 cycles), 182 patients were evaluable for response. Nineteen percent achieved complete remission (CR), 17% partial remission (PR), and 21% hematological improvement (HI). The disease was stable or progressive in 29% and 14% of patients, respectively. The probability of response was significantly higher in patients who received the 75 mg/m2/7 d compared with 100 mg through 5–7 d dose (CR/PR/HI: 63 vs. 29%, P = 0.0005). Median overall survival was 17.1 months. Low MDS-CI and achievement of CR/PR/HI were significant predictors of survival in the multivariable analysis. Conclusions Our data show that maximal azacitidine efficacy is associated with the standard dose and with prolonged treatment, beyond 4–6 cycles, with the goal of also improving the ‘quality’ of response. Lower MDS-CI and IPSS-R scores, hematologic response and disease stability, are associated with longer survival. The risk of febrile events is highest during the first treatment cycles and is associated with active disease.

Background CD69 is expressed in several hemopoietic cells and is an early activation marker in chronic lymphocytic leukemia. Chronic lymphocytic leukemia is a clinically heterogeneous disease which needs novel prognostic parameters which can be easily and efficiently managed. Design and Methods We investigated CD69 by flow cytometry in a series of 417 patients affected by chronic lymphocytic leukemia and compared this to other biological and clinical prognosticators. Results CD69 was associated with Rai stages ( P =0.00002), β2-microglobulin ( P =0.0005) and soluble CD23 ( P

Introduction Erythrocytosis is characterized by persistently raised hemoglobin (HB) and hematocrit (Ht) levels. Differential diagnosis includes Polycythemia Vera (PV), secondary, hereditary (HE) and idiopathic erythrocytosis (IE). Recently, Randi et al. (Br J Haematol, 2018) demonstrated the recurrence of HFE single nucleotide variants (SNVs) in patients with IE, postulating a possible link between Hemochromatosis genes and erythrocytosis. The most frequent HFE SNVs are C282Y and H63D, reported at allele frequencies of about 13% and 4% in Caucasian countries. HE has also been associated with mutations in genes members of the complex pathway of "oxygen sensing" (EPOR, VHL, HIF-2a and PHD2). To date, 30 mutations have been described in the PHD2 gene, all localized in the catalytic domain, which impair binding to HIF-2a. The PHD2p.C127S variant is frequently observed in Tibetans with D4E in cis (overall prevalence of this haplotype is 88.6% at altitudes above 3000m), in linkage disequilibrium with other missense mutations (in particular HIF-2a/EPAS1). Surprisingly, the combination of PHD2/HIF-2a variants result in a gain-of-function effect that blunts the hypoxic response, providing a molecular mechanism for the observed protection of Tibetans from erythrocytosis at high altitude. In normoxic conditions and in low-landers, the PHD2p.C127S variant may lead to increased erythropoiesis as reported in the literature. Patients and Methods Twenty-three patients with erythrocytosis (21 males and 2 females with a median age of 56 years, range 18-76, Table 1) negative for JAK2 mutations (both V617F and exon 12 variants), and with a bone marrow histology not suggestive of a myeloproliferative syndrome were studied for secondary causes of erythrocytosis using an appropriate algorithm (EPO levels, chest and abdomen imaging, spirometry, venous p50 of HB, arterial blood gas analysis). Since all tests were negative, HE genes mutation analysis was carried out, using Sanger sequencing of EPOR exon 8, the VHL coding region, PHD2 exon 1-3 and HIF-2a exon 12. HFE SNVs were studied using allele-specific real-time PCR. Results Sequencing of HE genes identified 4 carriers of PHD2 variants in 23 patients (17%). One patient had a novel missense heterozygous mutation (PHD2p.I269N). The study of his kindred showed that his sister and one daughter have HE and both carried the same heterozygous mutation of the propositus. Furthermore, his father had died with a diagnosis of PV in the pre-JAK2 era. In contrast, the patient's brother and the other daughter, both with normal Hb and Hct, had wild-type PHD2. Of note, the propositus and his two daughters were heterozygous for H63D in the HFE gene. The patient's pedigree is illustrated in Figure 1. Interestingly, three further patients tested positive for the PHD2 missense heterozygous variant (PHD2p.C127S) previously reported in the Tibetan population, and whose role in patients with erythrocytosis is still unclear. HFE SNVs real-time PCR revealed 7 carriers of the H63D, 1 of C282Y and 1 of S65C SNV in the HFE gene (about 40% of our cohort). Interestingly, two H63D heterozygous patients were a father and his son. They were all males with a median age of 48 years (range 18-64). The prevalence of HFE SNVs in our cohort of patients with erythrocytosis is higher than expected for H63D and S65C for Caucasians (30% vs 13% for H63D and 4.35 vs 1.5% for S65C), while the allele frequency of C282Y was similar to that of the general population. Finally, the patient with the S65C SNV also had the "Tibetan" PHD2 polymorphism. Conclusions In addition to previously known PHD2 gene alterations, we report here the occurrence in HE patients of a novel PHD2 mutation with an autosomal-dominant inheritance likely involved in disease pathogenesis. The milder phenotypic features of this patient's daughter and sister in terms of erythocytosis, may be explained by the childbearing age and the absence of H63D SNV, respectively. The increased prevalence of HFE SNVs in patients with IE may indicate an effect of impaired iron metabolism on erythropoiesis. Our data show that the inclusion of HFE SNVs and oxygen pathway mutational analysis in the diagnostic algorithm of erythrocytosis may help to better define the genetic basis of erythrocytosis. Further studies -including the analysis of molecules involved in iron storage pathway- in larger IE patient cohorts are warranted in order to clarify the link between HFE gene and IE. Disclosures Voso: Celgene: Research Funding, Speakers Bureau.

A total of 143 adult acute myeloid leukemia (AML) patients with available karyotype (K) and FLT3 gene mutational status were assessed for minimal residual disease (MRD) by flow cytometry. Twenty-two (16%) patients had favorable, 115 (80%) intermediate, and 6 (4%) poor risk K; 19 of 129 (15%) carried FLT3-ITD mutation. Considering postconsolidation MRD status, patients with good/intermediate-risk K who were MRD− had 4-year relapse-free survival (RFS) of 70% and 63%, and overall survival (OS) of 84% and 67%, respectively. Patients with good- and intermediate-risk K who were MRD+ had 4-year RFS of 15% and 17%, and OS of 38% and 23%, respectively (P < .001 for all comparisons). FLT3 wild-type patients achieving an MRD− status, had a better outcome than those who remained MRD+ (4-year RFS, 54% vs 17% P < .001; OS, 60% vs 23%, P = .002). Such an approach redefined cytogenetic/genetic categories in 2 groups: (1) low-risk, including good/intermediate K-MRD− with 4-year RFS and OS of 58% and 73%, respectively; and (2) high risk, including poor-risk K, FLT3-ITD mutated cases, good/intermediate K-MRD+ categories, with RFS and OS of 22% and 17%, respectively (P < .001 for all comparisons). In AML, the integrated evaluation of baseline prognosticators and MRD improves risk-assessment and optimizes postremission therapy.