Molecular Taxonomy of Myelodysplastic Syndromes and its Clinical Implications.

Myelodysplastic syndromes/neoplasms (MDS) are clonal hematologic disorders characterized by morphologic abnormalities of myeloid cells and peripheral cytopenias. While genetic abnormalities underlie the pathogenesis of these disorders and their heterogeneity, current classifications of MDS rely predominantly on morphology. We performed genomic profiling of 3,233 patients with MDS or related disorders to delineate molecular subtypes and define their clinical implications. Gene mutations, copy-number alterations (CNAs), and copy-neutral loss of heterozygosity (cnLOH) were derived from targeted sequencing of a 152-gene panel, with abnormalities identified in 91, 43, and 11% of patients, respectively. We characterized 16 molecular groups, encompassing 86% of patients, using information from 21 genes, 6 cytogenetic events, and LOH at the TP53 and TET2 loci. Two residual groups defined by negative findings (molecularly not-otherwise specified, absence of recurrent drivers) comprised 14% of patients. The groups varied in size from 0.5% to 14% of patients and were associated with distinct clinical phenotypes and outcomes. The median bone marrow blast percentage across groups ranged from 1.5 to 10%, and the median overall survival from 0.9 to 8.2 years. We validated 5 well-characterized entities, added further evidence to support 3 previously reported subsets, and described 8 novel groups. The prognostic influence of bone marrow blasts depended on the genetic subtypes. Within genetic subgroups, therapy-related MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN) had comparable clinical and outcome profiles to primary MDS. In conclusion, genetically-derived subgroups of MDS are clinically relevant and may inform future classification schemas and translational therapeutic research.

Molecular and clinical presentation of UBA1-mutated myelodysplastic syndromes.

Mutations in UBA1, which are disease-defining for VEXAS syndrome, have been reported in patients diagnosed with myelodysplastic syndromes (MDS). Here, we define the prevalence and clinical associations of UBA1 mutations in a representative cohort of patients with MDS. Digital droplet PCR profiling of a selected cohort of 375 male patients lacking MDS disease-defining mutations or established WHO disease classification identified 28 patients (7%) with UBA1 p.M41T/V/L mutations. Using targeted sequencing of UBA1 in a representative MDS cohort (n=2,027), we identified an additional 27 variants in 26 patients (1%), which we classified as likely/pathogenic (n=12) and unknown significance (n=15). Among the total 40 patients with likely/pathogenic variants (2%), all were male and 63% were classified by WHO2016 as MDS-MLD/SLD. Patients had a median of one additional myeloid gene mutation, often in TET2 (n=12), DNMT3A (n=10), ASXL1 (n=3), or SF3B1 (n=3). Retrospective clinical review where possible showed that 83% (28/34) UBA1-mutant cases had VEXAS-associated diagnoses or inflammatory clinical presentation. The prevalence of UBA1-mutations in MDS patients argues for systematic screening for UBA1 in the management of MDS.

Overlapping features of therapy-related and de novo NPM1-mutated AML.

NPM 1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of "therapy-related" NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity.

Oral azacitidine prolongs survival of patients with AML in remission independently of measurable residual disease status.

Measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy is predictive of early relapse and poor survival. Postremission maintenance therapy that prolongs MRD negativity or converts MRD+ patients to MRD- status may delay or prevent relapse and improve overall survival (OS). In the phase 3 QUAZAR AML-001 trial, oral azacitidine (oral-AZA; formerly CC-486), a hypomethylating agent, significantly prolonged OS and relapse-free survival (RFS) compared with placebo in patients aged ≥55 years with AML in first remission after intensive chemotherapy who were not candidates for hematopoietic stem cell transplantation. In this trial, MRD (≥0.1% leukemic cells in bone marrow) was assessed by multiparameter flow cytometry in serial samples collected at baseline and on day 1 of every 3 cycles. As expected, baseline MRD status was significantly associated with both OS and RFS. Multivariate analyses showed oral-AZA significantly improved OS and RFS vs placebo independent of baseline MRD status. Oral-AZA treatment also extended the duration of MRD negativity by 6 months vs placebo and resulted in a higher rate of conversion from MRD+ at baseline to MRD- during treatment: 37% vs 19%, respectively. In the oral-AZA arm, 24% of MRD responders achieved MRD negativity >6 months after treatment initiation. Although presence or absence of MRD was a strong prognostic indicator of OS and RFS, there were added survival benefits with oral-AZA maintenance therapy compared with placebo, independent of patients' MRD status at baseline. Registered at clinicaltrials.gov as #NCT01757535.

Diagnostic capabilities, clinical features, and longitudinal UBA1 clonal dynamics of a nationwide VEXAS cohort.

VEXAS is a prototypic hemato-inflammatory disease combining rheumatologic and hematologic disorders in a molecularly defined nosological entity. In this nationwide study, we aimed at screenshotting the current diagnostic capabilities and clinical-genomic features of VEXAS, and tracked UBA1 longitudinal clonal dynamics upon different therapeutics, including allogeneic hematopoietic cell transplant. We leveraged a collaboration between the Italian Society of Experimental Hematology and of Rheumatology and disseminated a national survey to collect clinical and molecular patient information. Overall, 13/29 centers performed UBA1 genomic testing locally, including Sanger sequencing (46%), next-generation sequencing (23%), droplet digital polymerase chain reaction (8%), or combination (23%). A total of 41 male patients were identified, majority (51%) with threonine substitutions at Met41 hotspot, followed by valine and leucine (27% and 8%). Median age at VEXAS diagnosis was 67 years. All patients displayed anemia (median hemoglobin 9.1 g/dL), with macrocytosis. Bone marrow vacuoles were observed in most cases (89%). The most common rheumatologic association was polychondritis (49%). A concomitant myelodysplastic neoplasm/syndrome (MDS) was diagnosed in 71% of patients (n = 28), chiefly exhibiting lower Revised International Prognostic Scoring System risk profiles. Karyotype was normal in all patients, except three MDS cases showing -Y, t(12;16)(q13;q24), and +8. The most frequently mutated gene was DNMT3A (n = 10), followed by TET2 (n = 3). At last follow-up, five patients died and two patients progressed to acute leukemia. Longitudinal UBA1 clonal dynamics demonstrated mutational clearance following transplant. We collected a nationwide interdisciplinary VEXAS patient cohort, characterized by heterogeneous rheumatologic manifestations and treatments used. MDS was diagnosed in 71% of cases. Patients exhibited various longitudinal UBA1 clonal dynamics.

Luspatercept in Patients with Lower-Risk Myelodysplastic Syndromes.

BACKGROUND: Patients with anemia and lower-risk myelodysplastic syndromes in whom erythropoiesis-stimulating agent therapy is not effective generally become dependent on red-cell transfusions. Luspatercept, a recombinant fusion protein that binds transforming growth factor ß superfamily ligands to reduce SMAD2 and SMAD3 signaling, showed promising results in a phase 2 study. METHODS: In a double-blind, placebo-controlled, phase 3 trial, we randomly assigned patients with very-low-risk, low-risk, or intermediate-risk myelodysplastic syndromes (defined according to the Revised International Prognostic Scoring System) with ring sideroblasts who had been receiving regular red-cell transfusions to receive either luspatercept (at a dose of 1.0 up to 1.75 mg per kilogram of body weight) or placebo, administered subcutaneously every 3 weeks. The primary end point was transfusion independence for 8 weeks or longer during weeks 1 through 24, and the key secondary end point was transfusion independence for 12 weeks or longer, assessed during both weeks 1 through 24 and weeks 1 through 48. RESULTS: Of the 229 patients enrolled, 153 were randomly assigned to receive luspatercept and 76 to receive placebo; the baseline characteristics of the patients were balanced. Transfusion independence for 8 weeks or longer was observed in 38% of the patients in the luspatercept group, as compared with 13% of those in the placebo group (P<0.001). A higher percentage of patients in the luspatercept group than in the placebo group met the key secondary end point (28% vs. 8% for weeks 1 through 24, and 33% vs. 12% for weeks 1 through 48; P<0.001 for both comparisons). The most common luspatercept-associated adverse events (of any grade) included fatigue, diarrhea, asthenia, nausea, and dizziness. The incidence of adverse events decreased over time. CONCLUSIONS: Luspatercept reduced the severity of anemia in patients with lower-risk myelodysplastic syndromes with ring sideroblasts who had been receiving regular red-cell transfusions and who had disease that was refractory to or unlikely to respond to erythropoiesis-stimulating agents or who had discontinued such agents owing to an adverse event. (Funded by Celgene and Acceleron Pharma; MEDALIST ClinicalTrials.gov number, NCT02631070; EudraCT number, 2015-003454-41.).

What's new in the pathogenesis and treatment of therapy-related myeloid neoplasms.

Therapy-related myeloid neoplasms (t-MNs) include diseases onsetting in patients treated with chemo- and/or radiotherapy for a primary cancer, or an autoimmune disorder. Genomic variants, in particular, in familial cancer genes, may play a predisposing role. Recent advances in deep sequencing techniques have shed light on the pathogenesis of t-MNs, identifying clonal hematopoiesis of indeterminate potential (CHIP) as a frequent first step in the multihit model of t-MNs. CHIP is often detectable prior to any cytotoxic treatment, probably setting the fertile genomic background for secondary leukemogenesis. The evolution pattern toward t-MNs is then a complex process, shaped by the type of cancer therapy, the aging process, and the individual exposures, that favor additional hits, such as the acquisition of TP53 mutations and unfavorable karyotype abnormalities. The pathogenesis of t-MNs differs from MN associated with environmental exposure. Indeed, the genetic aberration patterns of MN developing in atomic bomb survivors show few mutations in classical DNA methylation genes, and a high prevalence of 11q and ATM alterations, together with TP53 mutations. Survival in t-MNs is poor. In addition to the biology of t-MNs, the patient's previous disease history and the remission status at t-MN diagnosis are significant factors contributing to unfavorable outcome. New drugs active in secondary leukemias include CPX-351, or venetoclax in combination with hypomethylating agents, monoclonal antibodies as magrolimab, or targeted drugs against pathogenic mutations. Allogeneic stem cell transplantation remains the best currently available therapeutic option with curative intent for fit patients with unfavorable genetic profiles.

Myeloid lncRNA LOUP mediates opposing regulatory effects of RUNX1 and RUNX1-ETO in t(8;21) AML.

The mechanism underlying cell type-specific gene induction conferred by ubiquitous transcription factors as well as disruptions caused by their chimeric derivatives in leukemia is not well understood. Here, we investigate whether RNAs coordinate with transcription factors to drive myeloid gene transcription. In an integrated genome-wide approach surveying for gene loci exhibiting concurrent RNA and DNA interactions with the broadly expressed Runt-related transcription factor 1 (RUNX1), we identified the long noncoding RNA (lncRNA) originating from the upstream regulatory element of PU.1 (LOUP). This myeloid-specific and polyadenylated lncRNA induces myeloid differentiation and inhibits cell growth, acting as a transcriptional inducer of the myeloid master regulator PU.1. Mechanistically, LOUP recruits RUNX1 to both the PU.1 enhancer and the promoter, leading to the formation of an active chromatin loop. In t(8;21) acute myeloid leukemia (AML), wherein RUNX1 is fused to ETO, the resulting oncogenic fusion protein, RUNX1-ETO, limits chromatin accessibility at the LOUP locus, causing inhibition of LOUP and PU.1 expression. These findings highlight the important role of the interplay between cell-type-specific RNAs and transcription factors, as well as their oncogenic derivatives in modulating lineage-gene activation and raise the possibility that RNA regulators of transcription factors represent alternative targets for therapeutic development.

Towards a personalized preventive strategy of Herpes zoster infection in patients with hematologic diseases or hematopoietic stem cell transplant recipients: a position paper from an ad hoc Italian expert panel.

The identification of patients at high risk of herpes zoster (HZ) requiring a prevention strategy with antiviral prophylaxis and anti-HZ vaccine is a clinically relevant issue in patients with immunological impairment. Absence of trials comparing vaccination to pharmacological prophylaxis or defining their sequential use makes the optimal prevention strategy uncertain. This article presents the results of group discussion among an ad hoc constituted panel of experts aimed to review the literature regarding antiviral prophylaxis and vaccine efficacy and safety in populations with malignant and non-malignant hematological diseases, and submitted to hematopoietic stem cell transplantation. The panel used the consensus methodology and proposed solutions for prevention strategy producing advice for the management of the most relevant unmet clinical needs. Such a comprehensive overview aims to support at the practice of HZ pharmacological and vaccine prevention and informing the design and the need of implementation of new studies in the field.

Reliability of flow-cytometry in diagnosis and prognostic stratification of myelodysplastic syndromes: correlations with morphology and mutational profile.

Diagnosis and prognostic stratification of myelodysplastic syndromes (MDS) have been complemented by new techniques, including flow cytometry and NGS. To analyze the relationship between molecular and cytofluorimetric data, we enrolled in this retrospective study, 145 patients, including 106 diagnosed with MDS and 39 controls. At disease onset, immunophenotypic (IF), cytogenetic tests, and cytomorphological (CM) examination on bone marrow were carried out in all patients, while NGS was performed in 58 cases. Ogata score presented a specificity of 100% and a sensitivity of 59%. The detection of at least two phenotypic aberrancies in Ogata negative patients increased the sensitivity to 83% and specificity to 87%. Correlations were identified between IF aberrancies and mutations, including positive Ogata>2 and mutations in SRSF2 (p=0.035), CD15 and U2AF1 (0.032), CD56 and DNMT3A (p=0.042), and CD38 and TP53 (p=0.026). In multivariate analysis, U2AF1 mutations, associated with del(20q) and/or abnormalities of chromosome 7 (group 4 as defined by the EuroMDS score), significantly correlated with an inferior overall survival (p=0.019). These parameters and Ogata score>2 also showed a significant correlation with inferior event-free survival (p=0.023 and p=0.041, respectively). Both CM and FC features correlated with prognosis and mutational patterns. In an integrated MDS work-up, these tools may guide indications for mutational screening for optimal risk stratification.

Correlation analysis between auto-immunological and mutational profiles in myelodysplastic syndromes.

OBJECTIVE AND DESIGN: Systemic-Inflammatory-Autoimmune-Diseases (SIAD) is increasingly considered in Myelodysplastic-Syndromes (MDS). In this line, we evaluated the MDS auto-immunological profile, correlating it to the mutational landscape, trying to identify a molecular-genetic trigger agent related to SIAD. METHODS AND MATERIALS: Eighty-one MDS were enrolled and t-NGS was performed. Anti-Nuclear-Antibodies (ANA) were tested, and ANA-antigenic-specificity was characterized by ANA-profile, ENA-screen, anti-dsDNA. Non-Hematological-Patients (NHP) and Healthy-Donors (HD) were used as controls. RESULTS: At clinically relevant cut-off (≥ 1:160), ANA was significantly more frequent in MDS, while ANA-antigenic-specificity showed a low association rate. ANA ≥ 1:160-positive MDS showed a mutational landscape similar to ANA-negative/ANA < 1:160 MDS. No significant correlations between mutational and immunological profiles were found and UBA1 mutations, related to VEXAS, were absent. CONCLUSIONS: Although ANA-positivity was found to be increased in MDS, the low ANA-antigenic-specificity suggests that autoantibodies didn't recognize autoimmune-pathognomonic antigens. The lack of relationship between genetic profile and ANA-positivity, suggests that MDS genetic variants may not be the direct cause of SIAD.

Acute Myeloid Leukemia with NPM1 Mutation and Disseminated Leukemia Cutis: Achievement of Molecular Complete Remission by Venetoclax/Azacitidine Combination in a Very Old Patient.

We describe a case of acute myeloid leukemia with NPM1 mutation and disseminated leukemia cutis in a very old patient, who achieved a long-lasting response to the azacitidine/venetoclax combination with molecular complete remission, given the potential value of this rarely observed clinical outcome.

Clonal haematopoiesis as a risk factor for therapy-related myeloid neoplasms in patients with chronic lymphocytic leukaemia treated with chemo-(immuno)therapy.

Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p = 0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.

Molecular dissection of a hyper-aggressive CBFB-MYH11/FLT3-ITD-positive acute myeloid leukemia.

Acute Myeloid Leukaemia (AML) is a haematological malignancy showing a hypervariable landscape of clinical outcomes and phenotypic differences, explainable by heterogeneity at the cellular and molecular level. Among the most common genomic alterations, CBFB-MYH11 rearrangement and FLT3-ITD gene mutations, have opposite clinical significance and are unfrequently associated. We present here a Molecular Case Report in which these two events co-exist an ultra-aggressive phenotype resulting in death in 4 days from hospital admittance. Somatic and germline Whole Exome Sequencing analysis was performed to uncover other putative driver mutations, de-novo genomic structural events or germline clusters increasing cancer insurgence. Only three mutations in LTK, BCAS2 and LGAS9 were found, unlikely causative of the exhibited phenotype, prompting to additional investigation of the rare CBFB-MYH11/ FLT3-ITD scenario.

Impact of NPM1/FLT3-ITD genotypes defined by the 2017 European LeukemiaNet in patients with acute myeloid leukemia.

Patients with acute myeloid leukemia (AML) harboring FLT3 internal tandem duplications (ITDs) have poor outcomes, in particular AML with a high (≥0.5) mutant/wild-type allelic ratio (AR). The 2017 European LeukemiaNet (ELN) recommendations defined 4 distinct FLT3-ITD genotypes based on the ITD AR and the NPM1 mutational status. In this retrospective exploratory study, we investigated the prognostic and predictive impact of the NPM1/FLT3-ITD genotypes categorized according to the 2017 ELN risk groups in patients randomized within the RATIFY trial, which evaluated the addition of midostaurin to standard chemotherapy. The 4 NPM1/FLT3-ITD genotypes differed significantly with regard to clinical and concurrent genetic features. Complete ELN risk categorization could be done in 318 of 549 trial patients with FLT3-ITD AML. Significant factors for response after 1 or 2 induction cycles were ELN risk group and white blood cell (WBC) counts; treatment with midostaurin had no influence. Overall survival (OS) differed significantly among ELN risk groups, with estimated 5-year OS probabilities of 0.63, 0.43, and 0.33 for favorable-, intermediate-, and adverse-risk groups, respectively (P < .001). A multivariate Cox model for OS using allogeneic hematopoietic cell transplantation (HCT) in first complete remission as a time-dependent variable revealed treatment with midostaurin, allogeneic HCT, ELN favorable-risk group, and lower WBC counts as significant favorable factors. In this model, there was a consistent beneficial effect of midostaurin across ELN risk groups.

Clinical relevance of an objective flow cytometry approach based on limit of detection and limit of quantification for measurable residual disease assessment in acute myeloid leukemia. A post-hoc analysis of the GIMEMA AML1310 trial.

Using a multiparametric flow cytometry assay, we assessed the predictive power of a threshold calculated applying the criteria of limit of detection (LOD) and limit of quantitation (LOQ) in adult patients with acute myeloid leukemia. This was a post-hoc analysis of 261 patients enrolled in the GIMEMA AML1310 prospective trial. According to the protocol design, using the predefined measurable residual disease (MRD) threshold of 0.035% bone marrow residual leukemic cells (RLC) calculated on mononuclear cells, 154 (59%) of the 261 patients were negative (MRD <0.035%) and 107 (41%) were positive (MRD ≥0.035%). Using LOD and LOQ, we selected the following categories of patients: (i) LODneg if RLC were below the LOD (74; 28.4%); (ii) LODpos-LOQneg if RLC were between the LOD and LOQ (43; 16.5%); and (iii) LOQpos if RLC were above the LOQ (144; 54.4%). Two-year overall survival of these three categories of patients was 75.4%, 79.8% and 66.4%, respectively (P=0.1197). Given their superimposable outcomes, the LODneg and LODpos-LOQneg categories were combined. Two-year overall survival of LODneg/LODpos-LOQneg patients was 77.0% versus 66.4% of LOQpos individuals (P=0.043). This figure was challenged in univariate analysis (P=0.046, hazard ratio=1.6, 95% confidence interval: 1.01-2.54) which confirmed the independent role of the LOD-LOQ approach in determining overall survival. In the AML1310 protocol, using the threshold of 0.035%, 2-year overall survival of patients with MRD <0.035% and MRD ≥0.035% was 74.5% versus 66.4%, respectively (P=0.3521). In conclusion, the use of the LOD-LOQ method results in more sensitive detection of MRD that, in turn, translates into a more accurate recognition of patients with different outcomes.

Azacitidine to Consolidate and Deepen the Therapeutic Response Achieved by Intensive Induction Treatment in a Young Patient Affected by NPM1mut-AML Who Has Become Ineligible for High-Dose Consolidation.

Acute myeloid leukemia (AML) is the most common leukemia in adults. In spite of the most recent discoveries about the molecular landscape of this disease, the treatment of elderly and unfit young patients continues to be a great challenge. The hypomethylating agents (HMA) still represent an effective therapeutic option for these categories, especially for the low-risk subgroups. We report the case of a young patient with NPM1mut-AML who underwent a first cycle of intensive induction treatment, achieving a complete remission, but suffered from a serious life-threatening neurologic toxicity. Due to the ineligibility to further lines of intensive chemotherapy, we decided to consolidate the response with azacitidine, administered according to the regular schedule. The minimal residual disease (MRD), monitored through the NPM1 mutation at diagnosis, progressively decreased and became undetectable after 36 cycles of hypomethylating therapy. After 1 year from discontinuation of azacitidine, MRD remains undetectable. Therefore, HMA might still represent a feasible and effective option for patients with low-risk AML, especially when the standard chemotherapy is not indicated, or as maintenance therapy in nontransplantable patients.

Early intracranial haemorrhages in acute promyelocytic leukaemia: analysis of neuroradiological and clinico-biological parameters.

Acute promyelocytic leukaemia (APL) represents a modern success of precision medicine. However, fatalities occurring within the first 30 days of induction treatment, in particular intracranial haemorrhage (ICH), remain the main causes of death. We studied the clinico-biological characteristics of 13 patients with APL who experienced ICH. Compared to 85 patients without this complication, patients with ICH were older and more frequently had high-risk APL. Moreover, positivity for the 'swirl' sign at neuroradiological imaging and hydrocephalus were predictors of a fatal outcome, together with lower fibrinogen, prolonged international normalized ratio (INR) and higher lactate dehydrogenase levels.

Poly(ADP-Ribose) Polymerase Inhibitors for Arsenic Trioxide-Resistant Acute Promyelocytic Leukemia: Synergistic In Vitro Antitumor Effects with Hypomethylating Agents or High-Dose Vitamin C.

Arsenic trioxide (ATO) is an anticancer agent used for the treatment ofacute promyelocytic leukemia (APL). However, 5%-10% of patients fail to respond or experience disease relapse. Based on poly(ADP-ribose) polymerase (PARP) 1 involvement in the processing of DNA demethylation, here we have tested the in vitro susceptibility of ATO-resistant clones (derived from the human APL cell line NB4) to PARP inhibitors (PARPi) in combination with hypomethylating agents (azacitidine and decitabine) or high-dose vitamin C (ascorbate), which induces 5-hydroxymethylcytosine (5hmC)-mediated DNA demethylation. ATO-sensitive and -resistant APL cell clones were generated and initially analyzed for their susceptibility to five clinically used PARPi (olaparib, niraparib, rucaparib, veliparib, and talazoparib). The obtained PARPi IC50 values were far below (olaparib and niraparib), within the range (talazoparib), or above (rucaparib and veliparib) the C max reported in patients, likely as a result of differences in the mechanisms of their cytotoxic activity. ATO-resistant APL cells were also susceptible to clinically relevant concentrations of azacitidine and decitabine and to high-dose ascorbate. Interestingly, the combination of these agents with olaparib, niraparib, or talazoparib resulted in synergistic antitumor activity. In combination with ascorbate, PARPi increased the ascorbate-mediated induction of 5hmC, which likely resulted in stalled DNA repair and cytotoxicity. Talazoparib was the most effective PARPi in synergizing with ascorbate, in accordance with its marked ability to trap PARP1 at damaged DNA. These findings suggest that ATO and PARPi have nonoverlapping resistance mechanisms and support further investigation on PARPi combination with hypomethylating agents or high-dose ascorbate for relapsed/ATO-refractory APL, especially in frail patients. SIGNIFICANCE STATEMENT: This study found that poly(ADP-ribose) inhibitors (PARPi) show activity as single agents against human acute promyelocytic leukemia cells resistant to arsenic trioxide at clinically relevant concentrations. Furthermore, PARPi enhance the in vitro efficacy of azacitidine, decitabine, and high-dose vitamin C, all agents that alter DNA methylation. In combination with vitamin C, PARPi increase the levels of 5-hydroxymethylcytosine, likely as a result of altered processing of the oxidized intermediates associated with DNA demethylation.

GIMEMA AML1310 trial of risk-adapted, MRD-directed therapy for young adults with newly diagnosed acute myeloid leukemia.

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.