Abstract: Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p 〈 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p 〈 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.

Abstract: In older patients with chronic myelomonocytic leukaemia (CMML) and limited life expectancy due to age and or comorbidities, it is particularly important to consider the risk of transformation for individualised treatment decisions. There is limited information on potential differences between younger and older CMML patients regarding the cumulative risk of transformation as well as haematological, molecular and biologic characteristics. We analysed data from the Austrian Biodatabase for CMML (ABCMML) to compare these parameters in 518 CMML patients. Categorisation of patients into 3 age‐related groups: 〈 60 years, 60‐79 years and ≥80 years, showed a significantly lower risk of transformation at higher age by competing risk analysis, with a 4‐year risk of 39%, 23% and 13%, respectively ( P   〈  .0001). The lower probability of transformation was associated with a lower percentage of blast cells in the peripheral blood (PB) of older patients. Furthermore, we provide a simple score based on age, PB blasts and platelet counts that allowed us to define subgroups of CMML patients with a different cumulative transformation risk, including a low‐risk group with a transformation risk of only 5%. Our findings may facilitate reasonable treatment decisions in elderly patients with CMML.

Abstract: Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK 1 . Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children.

Abstract: The classical BCR-ABL1-negative myeloproliferative neoplasms (MPN) are characterized by over-production of myeloid cells, disease-related mutations in certain driver-genes (JAK2, CALR, MPL) and an increased risk to transform to secondary acute myeloid leukemia (sAML). Although considered stem cell-derived neoplasms, little is known about the phenotype and functional properties of disease-initiating neoplastic stem cells (NSC) in MPN and sAML. Recent data suggest that MPN NSC reside in a CD34+ fraction of the malignant clone. Therefore, these cells are considered most critical target populations to be examined for expression of molecular and immunological targets with the aim to develop improved or even curative NSC-eliminating therapies, such as antibody-based or CAR-T cell approaches. Using a panel of monoclonal antibodies (n=40) and multicolor flow cytometry, we established the immunological phenotype and target expression profiles of putative CD34+/CD38─ NSC and CD34+/CD38+ progenitor cells in patients with polycythemia vera (PV, n=18), essential thrombocythemia (ET, n=29), primary myelofibrosis (PMF, n=38) and post-MPN sAML (n=11). In almost all patients, the putative MPN stem cells expressed the stem cell invasion receptors Hermes (CD44) and ADGRE5 (CD97), C1qR1 (CD93), the migration/adhesion receptor MIC2 (CD99), and the stem cell antigen AC133 (CD133). Contrasting normal stem cells, MPN NCS and sAML stem cells failed to express Thy-1 (CD90). Among the cytokine receptors tested, MPN NSC invariably displayed the TGFßR-related antigen endoglin (CD105), TPOR (CD110), SCFR KIT (CD117), IL-3RA (CD123), CXCR4 (CD184) and IGF-1R (CD221). NSC expressed particularly high levels of KIT and low levels of TPOR and IGF-1R. The IL-2RA (CD25) was identified on NSC in most patients with PMF and sAML, and in a few with ET, but not in patients with PV. Similarly, the GM-CSFR (CD116) was found to be expressed on NSC in most patients with PMF, a few with ET and no with PV. MPN NSC did not exhibit substantial amounts of M-CSFR (CD115), IL-3RB (CD131), FLT3 (CD135), NGFR (CD271) VEGFR-2 KDR (CD309), EPOR, MET or OSMRB. The CD34+/CD38+ MPN progenitor cells displayed a similar profile of cytokine receptors. In addition, MPN and sAML progenitor cells expressed IL-1RAP and CLL-1 in most donors examined. We next examined the expression of various immunological targets and resistance-mediating immune checkpoint antigens on NSC and MPN progenitor cells. In all MPN patients and all sAML patients tested, NSC were found to express substantial amounts of Siglec-3 (CD33) and low levels of Campath-1 (CD52) and MDR-1 (CD243). In addition, MPN NSC and sAML stem cells invariably displayed the "don't eat" me checkpoint IAP (CD47) and the classical checkpoint PD-L1 (CD274). Exposure to interferon-gamma (200 U/ml, 24 hours) resulted in an upregulation of PD-L1 on NSC. In a subset of patients, MPN NSC expressed low levels of HB15 (CD83). In contrast, MPN NSC and sAML stem cells failed to express B7-1 (CD80), B7-2 (CD86), PD-L2 (CD273) and PD1 (CD279). MPN progenitor cells and sAML progenitors expressed an identical profile of cell surface targets and checkpoint antigens. Finally, we confirmed the disease-initiating capacity of MPN stem- and progenitor cells (CD34+ cells) using primary PMF cells in xenotransplantation experiments employing NSGS mice expressing human interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF). After 28 weeks post injection, engraftment of human CD45+ cells in the bone marrow of NSGS mice was found in 15/15 mice injected with bulk mononuclear cells (MNC) containing CD34+ cells and in 0/15 NSGS mice injected with MNC depleted of CD34+ cells. Together, MPN NSC reside in a CD34+ fraction of the malignant clone and display a unique phenotype, including cytokine receptors, immune checkpoint molecules and other target antigens. The phenotypic characterization of neoplastic stem cells should facilitate their enrichment and the development of NSC-eradicating treatment concepts in MPN. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding.

Abstract: Background In myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML) achievement of complete remission (CR) is a prerequisite for potential cure. In AML, CR/CR with incomplete recovery (CRi) is deemed the major outcome associated with improved overall survival (OS); patients (pts) without CR/CRi are considered non-responders, and hematologic improvement (HI) without (assessment of) bone marrow (BM) blast clearance is considered treatment (trt) failure. Achievement of CR may not be necessary for prolonged OS in pts treated with azacitidine (AZA) (Pleyer L, Annals Hematol 2014, 1825; Schuh AC, ASH 2015, P575). Outside of clinical trials, BM evaluations (BME) are only performed in ~50% of pts (Dinmohamed, Leuk Res 2015, 177) when either response or progression are obvious from peripheral blood (PB) values, or when pts are unable or unwilling to have BME. Aims To assess of the impact of response type on AZA trt outcomes in multivariate adjusted analyses (MVA). Methods 1441 pts included in the Austrian Azacitidine Registry were analyzed (NCT01595295). Data cut-off was 1 July 21. Marrow response was assessed for MDS/CMML and AML at each BME; HI was assessed on day 1 of each AZA cycle (Döhner H, Blood 2017, 424; Cheson BD, Blood 2006, 419; Pleyer L, ASH 2019, P3821); peripheral blood complete remission (PB-CR) was defined as hemoglobin ³11 g/dL, platelet count ≥100 G/L, neutrophil count ³1.0 G/L, white blood cell count & lt;15 G/L, PB blasts =0%, and no transfusions. Response types were calculated from electronic case report form data. To identify which response type achieved by which AZA cycle had the highest impact on time-to-event endpoints, likelihood ratios (LR) of the Cox-regression model for OS or time to next treatment (TTNT) were calculated using the respective response types as covariates. Baseline characteristics with univariate p & lt;0·10 (n=23) for association with OS were included in the multivariate regression. After stepwise selection n=17 variables remained and were used for MVA. Assign Data Management and Biostatistics GmbH performed statistical analyses with SAS® 9.4. Results In total, 521, 135, and 785 pts had MDS, CMML and AML. Median year of initial diagnosis was 2012, median time to AZA start was 3·0 (IQR 1·0-13·2) months (mo), median follow-up time from AZA start was 10.6 (IQR 4·0-21·1) mo, 894 pts received AZA as first line trt, median age at AZA start was 73 (range 23-99) years. In total, 13956 AZA cycles were applied, median duration of AZA trt was 5·0 (IQR 1·9-12·1) mo, median AZA dose was 875 (IQR 700-1000) mg/cycle, AZA was applied for a median of 7 (IQR 5-7) days. Median time to best response was 3·7 (IQR 2·0-5·9) months. Early mortality was 5.5% within 30 days. During AZA trt 1225 BM evaluations (BME) were performed in 697 (48·4%) of pts. Of these, 204 achieved CR/CRi. Irrespective of BME, 622 (43%) of 1441 pts achieved an HI and 264 (18·3%) of 1441 pts achieved a PB-CR. Pts achieving CR had longer adjusted OS (23·7 vs 19·7 mo, p=0·0227; HR=0·621 [0·413-0·936]) and TTNT (19·4 vs 15·7 mo, p=0·0262; HR=0·644 [0·436-0·949] ) than pts achieving CRi. Among pts achieving CR, those additionally achieving PB-CR had longer adjusted OS (24·8 vs 16·3; p=0·0040; HR=0·256 [0·101-0·647]; Fig 1A) and TTNT (21·2 vs 11·0; p=0·0005; HR=0·219 [0·094-0·513] ; Fig 1B) than those who did not. Among pts not achieving CR, those additionally achieving PB-CR had longer adjusted OS (20·8 vs 14·1; p & lt;0·0001; HR=0·510 [0·397-0·657]; Fig 1A) and TTNT (17·7 vs 10·9; p & lt;0·0001; HR=0·485 [0·357-0·589]; Fig 1B) than those who did not. Among all pts, irrespective of BM blast count, achievement of PB-CR resulted in longer adjusted OS (21·7 vs 10·0 mo; p & lt;·0001; HR 0·363; Fig 1C) and TTNT (18.5 vs 7.8 mo; p & lt;0.0001; HR=0.346; Fig 1D) and provided added value to CR and CR/CRi. Among all response types and after MVA, the highest prognostic impact on both OS and TTNT was observed when achieving PB-CR or CR/CRi by cycle 9 or 10 (Fig 2A-B). Conclusions Above data indicate that achievement of PB-CR is a strong predictor of OS and TTNT that provides additional information to current response criteria. Inclusion of PB-CR in updated response criteria of pts with MDS, CMML or AML receiving non-intensive trt should be considered. The greatest advantage of PB-CR is that it can be easily, nearly painlessly and quickly assessed. Inclusion of PB-CR as an endpoint in clinical trials would be desirable for validation of these results. Figure 1 Figure 1. Disclosures Pleyer: AbbVie, BMS, Novartis: Honoraria, Other: Travel Sport. Pfeilstocker: BMS: Honoraria. Stauder: Celgene/BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Heibl: BMS: Honoraria. Sill: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees. Hartmann: Celgene, Amgene, Janssen, AbbVie: Honoraria. Petzer: Kite-Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Saegen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene-BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees. Geissler: BMS: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Leisch: Honoraria from BMS, Celgene, Gilead, Takeda and Novartis; Travel support: Celgene and Novartis: Honoraria, Other: Travel support. Melchardt: Abbvie, Celgene, Novartis: Honoraria. Zebisch: Novartis: Consultancy; AbbVie: Consultancy; Celgene: Consultancy, Honoraria. Machherndl-Spandl: AbbVie, Celgene, BMS, Pfizer: Honoraria. Wolf: Roche: Honoraria, Research Funding; MSD: Honoraria, Research Funding; BMS-Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Takeda: Honoraria; Gilead: Honoraria; Incyte: Honoraria; GEMOAB: Honoraria. Greil: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Daiichi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Other: Travel, Accommodations, Expenses, Research Funding; Sandoz: Honoraria, Research Funding. OffLabel Disclosure: Azacitidine is approved for all types of MDS and CMML as well as low blast count AML by FDA, but not for all subtypes of MDS and CMML by EMA.

Abstract: Febrile children below 3 months have a higher risk of serious bacterial infections, which often leads to extensive diagnostics and treatment. There is practice variation in management due to differences in guidelines and their usage and adherence. We aimed to assess whether management in febrile children below 3 months attending European Emergency Departments (EDs) was according to the guidelines for fever. This study is part of the MOFICHE study, which is an observational multicenter study including routine data of febrile children (0–18 years) attending twelve EDs in eight European countries. In febrile children below 3 months (excluding bronchiolitis), we analyzed actual management compared to the guidelines for fever. Ten EDs applied the (adapted) NICE guideline, and two EDs applied local guidelines. Management included diagnostic tests, antibiotic treatment, and admission. We included 913 children with a median age of 1.7 months (IQR 1.0–2.3). Management per ED varied as follows: use of diagnostic tests 14–83%, antibiotic treatment 23–54%, admission 34–86%. Adherence to the guideline was 43% (374/868) for blood cultures, 29% (144/491) for lumbar punctures, 55% (270/492) for antibiotic prescriptions, and 67% (573/859) for admission. Full adherence to these four management components occurred in 15% (132/868, range 0–38%), partial adherence occurred in 56% (484/868, range 35–77%). Conclusion : There is large practice variation in management. The guideline adherence was limited, but highest for admission which implies a cautious approach. Future studies should focus on guideline revision including new biomarkers in order to optimize management in young febrile children. What is Known: •  Febrile children below 3 months have a higher risk of serious bacterial infections, which often leads to extensive diagnostics and treatment. • There is practice variation in management of young febrile children due to differences in guidelines and their usage and adherence. What is New: •  Full guideline adherence is limited, whereas partial guideline adherence is moderate in febrile children below 3 months across Europe. •  Guideline revision including new biomarkers is needed to improve management in young febrile children.