ALBERT

Description: Introduction Splicing factor (SF) mutations represent a novel class of driver mutations in myelodysplastic syndromes (MDS), where SF3B1 and SRSF2 are most frequently affected. Although abnormal RNA splicing is thought to play a central role in the pathogenic mechanism of mutated SFs, little is known about exact gene targets whose abnormal splicing is responsible for the pathogenesis of MDS. Methods We enrolled a total of 480 patients with MDS, for whom complete clinical and pathological data were available. RNA sequencing was performed for bone marrow mononuclear cells (BM/MNCs) and/or CD34+ cells from 215 MDS patients. Observed splicing junctions were compared between samples with and without each SF mutation. In SF-mutated cases, NMD could cause severe degradation of abnormal transcripts and obscure the effect of SF-mutants. To sensitively detect abnormal transcripts otherwise degraded by nonsense-mediated RNA decay (NMD), analysis was also performed on BM/MNCs from 7 patients and CD34+ bone marrow cells from 3 patients with or without inhibition of NMD by cycloheximide (CHX). Common mutations and copy number variations were also investigated using targeted sequencing. Results SF3B1 and SRSF2 mutations were associated with distinct clinical phenotypes and outcomes. SF3B1-mutated cases typically showed isolated erythroid dysplasia and high proportion of ring sideroblasts, whereas SRSF2 mutations correlated with a significantly higher incidence of myeloid and megakaryocyte dysplasia (P

Description: Background Intensive efforts of genome sequencing studies during the past decade identified 〉100 driver genes recurrently mutated in one or more subtypes of myeloid neoplasms, which collectively account for the pathogenesis of 〉90% of the cases. However, approximately 10% of the cases have no alterations in known drivers and their pathogenesis is still unclear. A possible explanation might be the presence of alterations in non-coding regions that are not detected by conventional exome/panel sequencing; mutations and complex structural variations (SVs) affecting these regions have been shown to deregulate expression of relevant genes in a variety of solid cancers. Unfortunately, however, no large studies have ever been performed, in which a large cohort of myeloid malignancies were analyzed using whole genome sequencing (WGS) in an attempt to identify a full spectrum of non-coding alterations, even though its efficacy have been demonstrated in many solid cancers. In this study, we performed WGS in a large cohort of pan-myeloid cancers, in which both coding and non-coding lesions were comprehensively analyzed. Patients and methods A total of 338 cases of myeloid malignancies, including 212 with MDS, 70 with AML, 17 with MDS/MPN, 23 with t-AML/MDS, and 16 with MPN were analyzed with WGS, of which 173 were also analyzed by transcriptome sequencing. Tumor samples were obtained from patients' bone marrow (N=269) or peripheral blood (N=69), while normal controls were derived from buccal smear (N=263) or peripheral T cells (N=75). Sequencing of target panel of 86 genes were performed for all samples. Sequencing data were processed using in-house pipelines, which were optimized for detection of complex structural variations (SVs) and abnormalities in non-coding sequences. Results WGS identified a median of 586,612 single nucleotide variants (SNVs) and 124,863 short indels per genome. NMF-based decomposition of the variants disclosed three major mutational signatures, which were characterized by age-related C〉T transitions at CpG sites (Sig. A), C〉T transitions at CpT sites (Sig. B), and T〉C transitions at ApTpN context (Sig. C). Among these, Sig. C showed a prominent strand bias and corresponds to COSMIC signature 16, which has recently been implicated in alcohol drinking. Significant clustering of SNVs and short indels were interrogated across the genome divided into different window sizes (1Kbp, 10Kbp, 100Kbp) or confining the targets to coding exons and known regulatory regions, such as promoters, enhancers/super enhances, and DNase I hypersensitive sites. Recapitulating previous findings, SNVs in the coding exons were significantly enriched in known drivers, including TP53, TET2, ASXL1, DNMT3A, SF3B1, RUNX1, EZH2, and STAG2. We detected significant enrichment of SNVs in CpG islands, and promoters/enhancers. We also detected a total of 8,242 SVs with a median of 15 SVs/sample, which is more prevalent than expected from conventional karyotype analysis. Focal clusters of complex rearrangements compatible with chromothripsis were found in 8 cases, of which 7 carried biallelic TP53 alterations. NMF-based signature analysis of SVs revealed that large (〉1Mb) deletions, inversions, and tandem duplications and translocations are clustered together and were strongly associated with TP53 mutations, while smaller deletions and tandem duplications, but not inversions, constitute another cluster. As expected, FLT3-ITD (N=15) and MLL-PTD (N=12) were among the most frequent SVs. Unexpectedly, in addition to known SVs associated with t(8;21) (RUNX1-RUNX1T1) (N=6) and t(3;21) (RUNX1-MECOM) (n=1) as well as non-synonymous SNVs within the coding exons (N=30), we detected frequent non-coding alterations affecting RUNX1, including SVs (N=15) and SNVs around splicing acceptor sites (N=5), suggesting that RUNX1 was affected by multiple mechanism, where as many as 38% of RUNX1 lesions were explained by non-coding alterations. Other recurrent targets of non-coding lesions included ASXL1, NF1, and ETV6. Conclusions WGS was successfully used to reveal a comprehensive registry of genetic alterations in pan-myeloid cancers. Non-coding alterations affecting known driver genes were more common than expected, suggesting the importance of detecting non-coding abnormalities in diagnostic sequencing. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Usuki:Mochida Pharmaceutical: Speakers Bureau; Astellas Pharma Inc.: Research Funding; Sanofi K.K.: Research Funding; GlaxoSmithKline K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Daiichi Sankyo: Research Funding; Celgene Corporation: Research Funding, Speakers Bureau; SymBio Pharmaceuticals Limited.: Research Funding; Shire Japan: Research Funding; Janssen Pharmaceutical K.K: Research Funding; Boehringer-Ingelheim Japan: Research Funding; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Pfizer Japan: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau; MSD K.K.: Speakers Bureau. Chiba:Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding. Miyawaki:Otsuka Pharmaceutical Co., Ltd.: Consultancy; Novartis Pharma KK: Consultancy; Astellas Pharma Inc.: Consultancy.

Description: Introduction: Since its implementation in 2012, the IPSS-R (Greenberg et al., 2012), defines the latest standard in risk stratification of patients with Myelodysplastic Syndromes (MDS). However, the prognostic impact of rare abnormalities remains unclear as yet because the number of these aberrations was too low to allow a valid statistical analysis. Hence, rare abnormalities were coalesced in one group in the IPSS and IPSS-R and, due to the unknown prognosis of these abnormalities, classified as prognostically intermediate. The main goal of the study presented here was to analyse the type, frequency and prognosis of rare single abnormalities in a large cohort of patients with primary, untreated MDS and integrate them in the existing IPSS-R in order to refine its predictive power. Methods: The data set analyzed was derived from the IPSS-R database and extended by additional data from European centers. In total, 7245 patients with primary, untreated MDS were included. Of these, we identified 410 (6%) pts. with rare single abnormalities. An aberration was defined as rare when it occurred in less than 10 patients in the cytogenetic scoring system that was the basis for the IPSS-R (Schanz et al., 2012). Additionally, further cytogenetic abnormalities not considered in this score were detected. A specific cytogenetic subgroup was defined as having at least n=5 cases with the same abnormality. Survival analyses was performed in cytogenetic subgroups with a minimum number of n=10 exclusively. The participating centers (in numerical order) were: Spain (n=110; 26.8%), MD Anderson Cancer Center (69; 16.8%), Düsseldorf (44; 10.8%), IMRAW (41; 10.0%), France (32; 7.8%), Pavia (21; 5.1%), Vienna (19; 4.6%), Japan (13; 3.2%), Vienna Medical University (12; 2.9%) Italy (11; 2.7%), Cleveland (10; 2.4%), Dundee (9; 2.2%), Brazil (8; 2.0%), Netherlands (5; 1.2%), Czech (2, 0.5%), Freiburg (1; 0.2%), Innsbruck (1; 0.2%), Sweden (1; 0.2%), and Russia (1; 0.2%). The median overall- (OS) and AML-free survival (AFS) was calculated for any specific cytogenetic subgroup. Results: Rare single abnormalities detected were: der(1;7)(n=24; 5.9%), partial or total monosomy 13 (22; 5.4%), partial or total monosomy 9 (22; 5.4%), +21 (20; 4.9%), +mar (14; 3.4%), del(3p) (12; 2.9%), total or partial monosomy 21 (11; 2.7%), total or partial monosomy X (11; 2.7%), total or partial monosomy 18 (10; 2.4%), +1/+1q (10; 2.4%), del(17p) (9; 2.2%), total or partial monosomy 14 (9; 2.2%), total or partial monosomy 16 (9; 2.2%), total or partial monosomy 6 (9; 2.2%), total or partial monosomy 1 (8; 2.0%), t(11q23;varia) (7; 1.7%), total or partial monosomy 19 (6; 1.5%), +11/+11q (6; 1.5%), +13 (6; 1.5%), +14 (6; 1.5%), del(5p) (5; 1.2%), total or partial monosomy 2 (5; 1.2%), +15 (5; 1.2%) and +X (5; 1.2%) The remaining 159 patients (38.7%) showed very rare abnormalities occurring in less than 5 patients each. The median overall survival as well as the AML-free survival in each category will be presented in detail. Furthermore, a multivariate model including all relevant confounders and a proposal to integrate these abnormalities in the cytogenetic module of the IPSS-R will be suggested. Conclusions: In order to overcome the problem of their extremely low frequency, knowledge about rare single abnormalities in MDS can only be gained by large, international cooperative projects. The present study was performed to identify and comprehensively analyze rare abnormalities occurring in MDS, uninfluenced by therapy or additional abnormalities. The results will lead to a further refinement of the cytogenetic prognostic classification in patients with MDS. The study was supported by a grant from the European Leukemia Net (ELN) Disclosures Schanz: Novartis: Honoraria, Other: Travel Grant; Celgene: Honoraria, Research Funding; Alexion: Other: Travel Grant; Lilly: Other: Travel Grant. Sole:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fenaux:Amgen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Ohyashiki:Kyowa Kirin KK: Honoraria; Novartis Pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jansen Pharma KK: Honoraria, Research Funding, Speakers Bureau; Chugai Pharna KK: Research Funding; Bristol Meyer Squib KK: Research Funding; Taiho Yakuhin KK: Research Funding; Asahikasei: Research Funding; Teijin Pharma KK: Research Funding; Alexion Pharma KK: Research Funding; Asteras: Research Funding; Shinbaio Pharma KK: Honoraria; Toyama Kagaku KK: Speakers Bureau; MSD KK: Honoraria; Nippo Shinyaku KK: Speakers Bureau; Sumitomo Dainippon: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Somatic mutations of RNA splicing machinery were identified in myelodysplastic syndromes (MDS), and a strong association was found between SF3B1 mutation and disease phenotype with ring sideroblasts. Recent studies showed that this mutation identifies a distinct group of MDS with ring sideroblasts, characterized by isolated erythroid dysplasia with a high degree of expanded but ineffective erythropoiesis, resulting in inappropriately low hepcidin levels. In this work we analyzed the relationship between SF3B1 mutation, body iron status, hepcidin level and liver and cardiac iron overload detected with MRI T2* in patients with MDS. We studied 34 patients with MDS diagnosed according to WHO criteria 2008 at the Department of Hematology, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy and at Ospedale Galliera, Genova, Italy (8 patients with RA or RCMD, 20 with RARS or RCMD-RS, 6 with RAEB-1 or -2). Serum hepcidin-25 measurements were performed by a validated Mass Spectrometry based method (SELDI-TOF MS). SF3B1 mutations were analyzed on DNA from circulating granulocytes on an Illumina HiSeq. Magnetic Resonance Imaging (MRI) examinations were performed with a validated scanner using a gradient echo T2* technique. Somatic mutations of SF3B1 were detected in 19 of 34 patients, with a median variant allele frequency of 0.40 (range 0.12-0.54). Ten of 19 SF3B1-mutated and 10 of 15 SF3B1 wild-type patients (67%) were RBC transfusion-dependent (median number of RBC transfusions 32, range 7-490). Variable hepcidin levels were found in the patients studied (median value 13.24 nM, range 3.25-75.56, versus 29.19 nM, range 3.26-66.15, in SF3B1-mutated and SF3B1 wild-type patients, respectively). We calculated the hepcidin to ferritin ratio, as a measure of adequacy of hepcidin levels relative to body iron stores, which was inversely related to the SF3B1 mutation (median value 0.015 nmol/mcg, range 0.004-0.035, versus 0.035, range 0.002-0.17, in SF3B1-mutated and SF3B1 wild-type patients, respectively, P=.04). In a multivariable regression model adjusted for RBC transfusion requirement, the hepcidin to ferritin ratio was independently associated with SF3B1 mutation (P=.042), indicating a blunted hepcidin response to iron overload in SF3B1-mutated patients. We then investigated the relationship between SF3B1 mutation status and parenchymal iron overload estimated by liver and cardiac MRI. Median hepatic T2* in the study population was 7 ms, ranging from 1.49 to 30.08. Focusing the analysis on patients with RBC transfusion-dependency, a higher prevalence of hepatic iron overload (defined as MRI T2* values less than 6.3 ms) was observed in SF3B1-mutated compared with SF3B1 wild-type patients (9/10 versus 5/10 respectively). In a multivariable regression analysis adjusted for RBC transfusion history, SF3B1 mutation was an independent predictor of hepatic T2* value (P=.038). When the analysis was limited to patients without RBC transfusion need, two SF3B1-mutated patients showed a liver iron overload, associated with mild increased in AST and ALT values. Cardiac iron overload defined as MRI T2* values less than 10 ms was detected in two out of 10 patients receiving more than 50 RBC units, both of them carrying SF3B1 mutation. In conclusion, our study shows that MDS patients carrying a somatic mutation of SF3B1 have inappropriately low hepcidin levels, resulting in parenchymal iron loading due to excessive iron absorption and reticuloendothelial release. These results suggest that SF3B1 mutation may be associated with liver iron overload even in untransfused patients, as observed in congenital iron loading anemias. These results may be relevant for decision-making concerning treatment of transfusion iron overload in MDS patients. Disclosures No relevant conflicts of interest to declare.

Description: Background The outcome of lower-risk MDS patients with red blood cell transfusions (RBCT) dependency is inferior to that of RBCT independent patients, but whether the intensity of RBCT is important for prognosis is unknown. The EUMDS Registry is a non-interventional, observational longitudinal study enrolling patients with lower-risk MDS from 142 sites in 17 countries as described elsewhere (1). The EUMDS registry has accrued 1,902 patients as of July 21, 2015. We hypothesized that RBCT intensity is an independent prognostic factor for survival. Methods We first assessed the impact of RBCT intensity in the first year post-diagnosis (1yrPD) on progression-free survival among the 1034 patients who survived at least 1yrPD and had potential for a further year of follow-up. Secondly, we developed a longitudinal model of platelet counts throughout follow-up for 1660 patients in the registry with potential for at least one year follow-up. Results Among the 1034 patients, 323 patients had died: 67 after progression to higher-risk MDS/AML and 256 without progression. A further 41 surviving patients had progressed to AML. The overall 5-year survival was 52%. In a proportional hazards regression model (Table), the risk of death or progression increased in a non-linear fashion with age at diagnosis (p

Description: The diagnostic approach to unexplained cytopenia is hampered by the poor specificity of dysplastic changes that may complicate the distinction between myeloid neoplasms (MN) and non-malignant cytopenias. In the last years, several somatic mutations were identified in MN; however, the diagnostic value of mutation analysis needs to be defined. In this study, we performed a mutation screening in a prospective cohort of patients with unexplained cytopenia undergoing a comprehensive diagnostic work-up, with the aim to estimate the predictive value of somatic mutations. This study included two cohorts: a learning cohort that consisted of 683 consecutive patients investigated for unexplained cytopenia at the Policlinico San Matteo & University of Pavia, Italy, between 2003 and 2015; and a validation cohort, including 190 patients referred as second opinion for suspected MDS. A set of 42 genes was analyzed on DNA from peripheral blood granulocytes using Illumina HiSeq (Illumina Inc., CA, USA). The diagnosis of patients in the learning cohort was MN in 409 cases (233 MDS, 86 MDS/MPN, 35 MPN; 55 AML), other cytopenia in 120 cases, whereas in 154 patients a provisional diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) was adopted. After a median follow-up of 22 months (range 3-136), 38 patients in this category developed a MN (ICUS-MN). The most frequently mutated genes were TET2 (171/683, 25%), ASXL1 (15%), SRSF2 (14%), SF3B1 (11%), DNMT3A (10%), RUNX1 (9%). Significantly higher number of mutations per subject and variant allele frequency (VAF) were observed in MN (n=2, range 0-9; VAF=0.39, 0.03-0.57) compared with ICUS (n=0, 0-7; VAF=0.31, 0.03-0.51) or other cytopenia (n=0, 0-2; VAF=0.06, 0.03-0.44) (P

Description: Background Data on causes of death (COD) in patients with lower-risk (LR-MDS) is limited and sometimes conflicting. In contrast to higher-risk MDS, many LR-MDS patients die from conditions associated with advanced age, not directly associated with the underlying disease. Infections and cardiovascular disorders (CVD) have been reported as frequent COD in LR-MDS, but whether the incidence is higher than in age-matched population, is not known. The EUMDS Registry has been collecting prospective observational data on LR-MDS since 2008. The comprehensive clinical and laboratory data provides a unique chance to assess the impact of LR-MDS on survival either by causes related to MDS or indirectly related to MDS by aggravation of co-morbidities. Objectives To assess the impact of MDS and associated co-morbidities on COD in patients with LR-MDS and to evaluate the COD in the whole group and across participating countries. Methods: We evaluated clinical and laboratory data of LR-MDS patients registered in EUMDS registry from 2008 to 2018. Data were obtained by 145 centers from 16 European countries and Israel. MDS related causes of death were defined as infection, bleeding, MDS progression and AML transformation. Overall survival(OS) and relative survival(RS) were estimated using the Stata program 'strel' with age, sex and country specific background obtained from national life tables for the CONCORD program. RS is a standard approach used to take into account competing causes of death by adjusting for the age and sex specific mortality in the general population, estimating the excess mortality in these patients compared to that seen in the general population of each country. Results Overall data on 2235 LR-MDS patients was available in the EUMDS registry. Of these, 822 (36,7%) patients had died at the time of analysis. Median age was 77 years and 65% of the patients were male. Nearly half of them (46.9%) were diagnosed as IPPS low risk. The MDS-Comorbidity Index was low, intermediate and high in 55.7%, 37.5% and 6.8% of patients respectively. The most common COD were those considered as related to MDS 41.7% (Table 1). Deaths due to cardiovascular and pulmonary diseases were reported in 10.1% and 4.9% respectively. Other reasons (e.g. liver, renal failure, second malignancy) were found in 18.2%. In 25% of patients, the precise reason of death remained unknown. The proportion of MDS related COD were different between participating countries with lower rates in Germany (30%), France (31.3%) and higher in Portugal (55%), Greece (55.2%) and Romania (63.1%). Median follow-up was 2.1 years (0.1-10 years). Five-year overall survival in the whole cohort was 47.1% (95% CI: 44.1%-49.9%) and 5-year relative survival (attributed to MDS/AML only) was 59.1% (95% CI:55.4%-62.5%)(Figure 1). One year overall and relative survival was 90.4% (95% CI:89.1%-91.6%) and 94.4% (95% CI:93%-95.5%) respectively. Conclusions: MDS- related complications are the most common causes of death in LR-MDS patients. Comparison of overall and relative survival supports that observation and indicates that excess mortality in LR-MDS patients can be mainly explained by MDS/AML related causes. Interestingly, the strongest influence of MDS/AML attributable deaths was observed during the first year from diagnosis. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Roche: Honoraria; Otsuka: Honoraria, Research Funding. Stauder:Teva: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Germing:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria, Research Funding. de Witte:Novartis: Research Funding; Celgene: Honoraria, Research Funding; Amgen: Consultancy, Research Funding. Smith:Jazz Pharmaceuticals: Research Funding; Johnson & Johnson: Research Funding; Gilead Sciences: Consultancy; Novartis: Research Funding.

Description: Splicing factors are the most frequently mutated genes in myeloid neoplasms with myelodysplasia, including myelodysplastic syndromes (MDS), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) and acute myeloid leukemia with myelodysplasia-related changes (AML-MDS). SF3B1 mutations was shown to be strongly associated with disease phenotype with ring sideroblasts, and SF3B1-mutant MDS has been proposed as a distinct disease subtype (Malcovati et al. Blood 2015). Conversely, SRSF2 has been found to be mutated in chronic myelomonocytic leukemia (CMML), and to be associated with multilineage dysplasia and excess blasts in MDS, as well as with clinico-pathological features of secondary AML (Linsdley et al. Blood 2015). In this study, we carried out a comprehensive mutation analysis in a well-annotated prospective cohort of 551 patients with myeloid neoplasms with myelodysplasia, and focused on clinical and molecular features of SRSF2-mutated patients with the aim to characterize patterns of co-occurring mutations and to identify genotype-phenotype correlations. A core panel of 54 genes recurrently mutated in myeloid neoplasms was analyzed using an Illumina HiSeq (Illumina Inc., CA, USA). Multivariable survival analyses were performed by means of Cox proportional hazards regression accounting for left censoring of the observation at the time of mutation assessment. Unsupervised hierarchical clustering analyses were performed in order to identify homogeneous groups of patients with respect to the variables of interest. Overall, 102 patients carrying SRSF2-mutation were identified, including 37 MDS (2 MDS with single lineage dysplasia, 9 with multilineage dysplasia, 6 with ring sideroblasts, 20 with excess blasts), 40 MDS/MPN (35 CMML, 5 MDS/MPN unclassified) and 25 AML-MDS. The median number of co-mutated genes was 2 (range 0 to 5); SRSF2 mutation was the unique mutation detected in 11 cases (5 AML-MDS, 4 MDS/MPN and 3 MDS). The most frequently co-mutated genes were TET2 (31/102 cases, 30%), ASXL1 (28%), IDH1/IDH2 (24%), RUNX1 (21%), STAG2 (14%) and CBL (10%). A significant association was found between co-mutation of TET2 and SRSF2 and myeloid neoplasm with monocytosis (P

Description: Key Points Risk assessment is crucial in patients with CMML because survival may range from a few months to several years. Integrating clinical features, morphology, and genetic lesions significantly improves risk stratification in CMML.