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Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whose translation is enhanced by cytoplasmic localization of METTL3, independently from its catalytic activity. In conclusion, our results point to METTL3 as a novel relevant oncogene in CML and as a promising therapeutic target for TKI resistant CML.

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: 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.

In adult patients with acute lymphoblastic leukemia (ALL), Central Nervous System (CNS) involvement is associated with a very poor prognosis. The diagnostic assessment of this condition relies on the use of neuroradiology, conventional cytology (CC) and flow cytometry (FCM). Among these approaches, which is the gold standard it is still a matter of debate. Neuroradiology and CC have a limited sensitivity with a higher rate of false negative results. FCM demonstrated a superior sensitivity over CC, particularly when low levels of CNS infiltrating cells are present. Although prospective studies of large series of patients are still awaited, a positive finding by FCM appears to anticipate an adverse outcome even if CC shows no infiltration. Current strategies for adult ALL CNS-directed prophylaxis or therapy involve systemic and intrathecal chemotherapy and radiation therapy. Actually, early and frequent intrathecal injection of cytostatic combined with systemic chemotherapy is the most effective strategy to reduce the frequency of CNS involvement. In patients with CNS overt ALL, at diagnosis or upon relapse, allogenic hematopoietic stem cell transplantation might be considered. This review will discuss risk factors, diagnostic techniques for identification of CNS infiltration and modalities of prophylaxis and therapy to manage it.