ZRSR2 Mutations Research Articles

A Personalized Prediction Model for Outcomes after Allogeneic Hematopoietic Stem Cell Transplant in Patients with Myelodysplastic Syndromes: On Behalf of the CIBMTR Chronic Leukemia Committee

▪BackgroundAllogeneic hematopoietic cell transplantation (HCT) remains the only potentially curative option for myelodysplastic syndromes (MDS). Genetic alterations have an impact on outcomes after HCT in MDS (Lindsley C, et al, NEJM 2017). Given the significant risks of transplant-related mortality and relapse, identifying patients (pts) who may or may not benefit from HCT is clinically important.We explored how the incorporation of genomic / clinical data using machine learning algorithms can create a personalized prediction model that can predict outcomes after HCT in MDS pts.MethodMDS patients enrolled in the Center for International Blood and Marrow Transplant Research (CIBMTR) Registry were considered for inclusion. Pts were excluded only if blasts were >/= 20% or if they had diagnosis of CMML or MDS/MPN overlap. A panel of 127 gene mutations was performed on samples from peripheral blood prior to HCT. OS was measured from the time of transplant to death or last follow up. Relapse was measured from the time of transplant to time of relapse or last follow up. A random survival forest (RSF) algorithm was used to build the model, in which clinical and molecular variables are randomly selected for inclusion in determining survival and relapse probability, thereby avoiding some of the limitations of traditional Cox step-wise regression in accounting for variable interactions. The accuracy of the proposed model was assessed by concordance (c-) index.ResultsA total of 1514 MDS pts included and randomly divided into training (1059 pts) and validation (455 pts) cohorts. The median age of the entire cohort was 59 years (range 0.4 - 77). Donors were 8/8 HLA-matched unrelated donors (n = 862, 57%), HLA-identical siblings (n = 181, 12%), < 8/8 HLA-matched unrelated donors (n = 296, 20%), or umbilical cord blood (n = 174, 11%). Conditioning regimens included: myeloablative (n=789, 52%), reduced-intensity (n=582, 38%), nonmyeloablative (n=130, 9%), and missing data (n=13, 1%). Cytogenetic analysis by IPSS-R criteria: 579 (38%) very good/good, 269 (18%) intermediate, 287 (19%) poor, 125 (8%) very poor, missing 254 (17%). Median number of mutations/sample was 2 (range, 0-15). The most commonly mutated genes were: ASXL1 (20%), TP53 (19%), DNMT3A (15%), TET2 (12%), RUNX1 (11%), SF3B1 (9%), U2AF1 (8%), and SRSF2 (6%).Variable importance analysis (the most important variables that contributed to the outcome) and multiple backward elimination analysis (identifying the least number of variables that can provide the least error rate) identified the following variables prior to HCT that impacted OS: age, TP53 mutation, absolute neutrophils counts (ANC), cytogenetics per IPSS-R, Karnofsky performance status (KPS), conditioning regimen, donor age, WBC, hemoglobin, diagnosis of therapy-related MDS (t-MDS), peripheral blast percentage, mutations in RAS pathway, JAK2 mutation, number of mutations, ZRSR2, and CUX1 mutations. Variables prior to HCT that affected the cause-specific hazard of relapse included: TP53 mutation, conditioning regimen, cytogenetics per IPSS-R, hemoglobin, disease status prior to HCT, receiving hypomethylating agent vs chemotherapy prior to HCT, number of mutations, age, year of transplant, IPSSR, WBC, KPS, mutations in RAS pathway, in-vivo T cell depletion, ANC, peripheral blast percentage, t-MDS, monosomal karyotype status, number or prior treatments, ASXL1 mutation, bone marrow blast percentage, and PPM1D mutation.To ease the translation of this tool into the clinic, a web application was built where clinical and mutational variables can be entered and the survival probability at 6,12, and 24 months will be computed, Figure 1.The model was valid for OS when it was applied to the validation cohort (c-index of the model in the training cohort was .6, and validation cohort .61.Conclusions:We built a personalized prediction model that uses clinical, molecular, and transplant-related data and can predict the probability of survival in MDS pts that are specific for a given pt. The new model identified several clinical and molecular variables that impacted OS and the cause-specific hazard of relapse. The OS probability at different time points may aid physicians and pts in their decision prior to HCT. [Display omitted] DisclosuresNazha:MEI: Consultancy. Majhail:Incyte: Honoraria; Atara: Honoraria; Anthem, Inc.: Consultancy.

Therapy-Related Myeloid Neoplasms (T-MN) and Primary MDS (PMDS) Patients with Very Low (VL) or Low (L) IPSS-R Score Share Clinical and Biological Characteristics and Have Similar Outcome

▪Introduction: Myeloid neoplasms occurring after exposure to chemo- and/or radiotherapy, termed Therapy-related myeloid neoplasms (T-MN), are considered poor prognosis. This perception creates an inherent bias in treatment decision, resulting in either over- (allo-HSCT) or under-treatment (palliation) and exclusion from most clinical trials. The optimal T-MN treatment paradigm is unknown, partly because its mutational architecture is not well defined. Few small studies show mutations in only 50-60% (Ok, Leuk Res 2015; Lindsley, Blood 2015) of T-MN (compared to 80-90% in PMDS). This study compares the mutational architecture of T-MN and PMDS from the South Australian Myelodysplastic Syndrome registry.Methods: Demographic, clinical and laboratory data including cytogenetic profiles of 129 T-MN (95 T-MDS; 34 T-AML [≥20% blasts]) and 108 PMDS patients were analysed. Targeted Massively Parallel Sequencing of a custom panel of 43 myeloid neoplasms associated genes (all coding regions) was performed on diagnosis bone marrow samples. Mutations with VAF ≥3% were selected for further assessment. Overall survival (OS) was calculated from date of diagnosis to date of last follow-up or death and adjusted using time varying covariate to account for disease modifying therapy (DMT) exposure.Results: Compared to PMDS, T-MN patients were younger at diagnosis (71.1 vs 75.3 years, p<0.001), had higher frequency of IPSS-R High and Very High (VH) scores (39.5 vs 20.3%, p<0.001), poor risk cytogenetic abnormalities (del7/7q [18.4 vs 6.5%, p=0.0099], complex [28.8 vs 8.3%; p<0.0001] or monosomal karyotype [26.4 vs 4.6%; p<0.0001]), translocations (39.2 vs 10.2%; p<0.0001), and marker chromosomes (20.8 vs 1.9%; p<0.0001) and poor overall survival (10.6 vs 31.4 months, p<0.0001). Mutation frequency in our T-MN cohort was substantially higher than previously published frequency of 50-60% in t-MN and comparable to PMDS (93 vs 92.6%). The most common mutations were TP53 in T-MN and spliceosomal complex in PMDS. Within the spliceosomal complex, SF3B1 (25.9% vs. 6.2%; p<0.0001) and U2AF1 mutations (7.4% vs. 1.6%; p=0.04) were detected at higher frequency in PMDS compared to T-MN, while there was no significant difference in the SRSF2 (21.3% vs 15.5%) or ZRSR2 (5.6 vs 2.3%) mutations. Importantly, SRSF2 mutations were the most frequent spliceosome mutations in T-MN, in contrast to SF3B1 in PMDS.Although the OS was poorer for T-MN than PMDS (10.6 vs 31.4 mo, p<0.0001, Figure 1) as a group, the characteristics of T-MN and PMDS were different when segregated according to Revised International Prognostic Scoring System (IPSS-R) score. T-MN with intermediate IPSS-R score had poorer OS compared to PMDS (13.6 vs 35 mo, p=0.01). T-MN with High or VH IPSS-R scores had poorer OS than PMDS (8.8 vs 12.8 mo) but was not significant (p=0.09) due to application of DMT in a significant subset of patients. Interestingly, survival of T-MN with VL or Low risk was similar to PMDS (63.8 vs 45.3 mo, p=0.9, Figure 2). Hence, we compared the clinical and genetic characteristics of PMDS (n=52) and T-MN (n=22) patients with VL or Low IPSS-R score (Table 1). There was no significant difference between their demographics and MDS subtypes. A significant majority of patients had normal karyotype; no poor-risk chromosomal abnormalities were seen in remainder. The mutation profile of both groups was also remarkably similar. The median number of mutations and frequency of mutated genes were not different.As expected, VL or Low risk T-MN patients were different from T-MN with intermediate, High or VH IPSS-R score (n=74) who showed higher frequency of high-risk karyotypes and poorer OS. Additionally, the mutation profile was also different between the two groups; TP53 mutations were less common while TET2, SF3B1, IDH2 mutations were significantly more frequent in low-risk T-MN.Conclusions: In our cohort, somatic mutations were seen in 93% of T-MN which is higher than published literature. TP53 and spliceosomal mutations were commonest in T-MN and PMDS, respectively. Although, the T-MN survival is poorer than PMDS, the subgroup with IPSS-R Very Low or Low risk mirrors clinical and genetic characteristics of PMDS and has similar outcome. Such patients should be managed at par with PMDS counterparts and, importantly, should not be excluded from appropriate clinical trials based on pre-MDS chemotherapy or radiotherapy exposure. [Display omitted] DisclosuresHiwase:Novartis: Research Funding; Celgene: Research Funding.

The prognostic significance of global aberrant alternative splicing in patients with myelodysplastic syndrome

Aberrant alternative splicing (AS) is a hallmark of cancer development. However, there are limited data regarding its clinical implications in myelodysplastic syndrome (MDS). In this study, we performed an in-depth analysis of global AS in 176 primary MDS patients with 20 normal marrow transplant donors as reference. We found that 26.9% of the expressed genes genome-wide were aberrantly spliced in MDS patients compared with normal donors. These aberrant AS genes were related to pathways involved in cell proliferation, cell adhesion and protein degradation. A higher degree of global aberrant AS was associated with male gender and U2AF1 mutation, and predicted shorter overall survival and time to leukemic change. Moreover, it was an independent unfavorable prognostic factor irrespective of age, revised international prognostic scoring system (IPSS-R) risk, and mutations in SRSF2, ZRSR2, ASXL1, TP53, and EZH2. With LASSO-Cox regression method, we constructed a simple prognosis prediction model composed of 13 aberrant AS genes, and demonstrated that it could well stratify MDS patients into distinct risk groups. To our knowledge, this is the first report demonstrating significant prognostic impacts of aberrant splicing on MDS patients. Further prospective studies in larger cohorts are needed to confirm our observations.

Sustained clonal hematopoiesis by HLA-lacking hematopoietic stem cells without driver mutations in aplastic anemia

AbstractClonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA− HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA− HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA− HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA− granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA− granulocytes revealed somatic mutations (DNMT3A, n = 2; TET2, ZRSR2, and CBL, n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA− HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA− granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow–fluorescence in situ hybridization analyses of sorted HLA+ and HLA− granulocytes showed no telomere attrition in HLA− granulocytes. The findings suggest that HLA− HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.

IntEREst: intron-exon retention estimator

BackgroundIn-depth study of the intron retention levels of transcripts provide insights on the mechanisms regulating pre-mRNA splicing efficiency. Additionally, detailed analysis of retained introns can link these introns to post-transcriptional regulation or identify aberrant splicing events in human diseases.ResultsWe present IntEREst, Intron–Exon Retention Estimator, an R package that supports rigorous analysis of non-annotated intron retention events (in addition to the ones annotated by RefSeq or similar databases), and support intra-sample in addition to inter-sample comparisons. It accepts binary sequence alignment/map (.bam) files as input and determines genome-wide estimates of intron retention or exon-exon junction levels. Moreover, it includes functions for comparing subsets of user-defined introns (e.g. U12-type vs U2-type) and its plotting functions allow visualization of the distribution of the retention levels of the introns. Statistical methods are adapted from the DESeq2, edgeR and DEXSeq R packages to extract the significantly more or less retained introns. Analyses can be performed either sequentially (on single core) or in parallel (on multiple cores). We used IntEREst to investigate the U12- and U2-type intron retention in human and plant RNAseq dataset with defects in the U12-dependent spliceosome due to mutations in the ZRSR2 component of this spliceosome. Additionally, we compared the retained introns discovered by IntEREst with that of other methods and studies.ConclusionIntEREst is an R package for Intron retention and exon-exon junction levels analysis of RNA-seq data. Both the human and plant analyses show that the U12-type introns are retained at higher level compared to the U2-type introns already in the control samples, but the retention is exacerbated in patient or plant samples carrying a mutated ZRSR2 gene. Intron retention events caused by ZRSR2 mutation that we discovered using IntEREst (DESeq2 based function) show considerable overlap with the retained introns discovered by other methods (e.g. IRFinder and edgeR based function of IntEREst). Our results indicate that increase in both the number of biological replicates and the depth of sequencing library promote the discovery of retained introns, but the effect of library size gradually decreases with more than 35 million reads mapped to the introns.

Splicing factor mutations in myelodysplastic syndromes

Splicing factor mutations represent a novel class of driver mutations in myelodysplastic syndromes, where four genes, including SF3B1, SRSF2, U2AF1, and ZRSR2, are most frequently affected. SF3B1 and SRSF2 mutations show prominent specificity to the syndrome subtypes characterized by increased ring sideroblasts and chronic myelomonocytic leukemia, respectively. These mutations are suspected to be involved in the pathogenesis of the above mentioned syndromes most likely via abnormal RNA splicing. However, the precise mechanism and target genes have not been fully understood. Splicing alterations induced by these mutations are extensively studied using RNA sequencing. SF3B1 mutations caused misrecognition of 3' splice sites. Target genes included those involved in mitochondrial iron metabolism or heme biosynthesis, such as ABCB7 and PPOX, suggesting a role in the abnormal erythropoiesis associated with increased ring sideroblasts. By contrast, SRSF2 and U2AF1 mutations were mainly associated with alternative exon usage in hundreds of genes. The targets included genes of which mutations are definitive or putative drivers in myeloid malignancies. Protein structure modeling and experiments with cell lines and mouse models supported these findings. ZRSR2 mutations were associated with the retention of U12-type introns, and this is consistent with the known role of ZRSR2 as an essential component of the U12-type spliceosome. Further studies are warranted to determine the biological effects of splicing alterations.

Refractory macrocytic anemias in patients with clonal hematopoietic disorders and isolated mutations of the spliceosome gene ZRSR2

Although mutations in RNA splicing genes occur frequently in patients with clonal cytopenias of unknown significance (CCUS) and myelodysplastic syndromes (MDS), very often additional common myeloid gene driver mutations are present at diagnosis. Thus, the clinical significance of isolated mutations in the most commonly mutated RNA splicing genes remains unknown. Here we report five unusual patients with an isolated mutation causing a loss of function of ZRSR2, a protein required for recognition of a functional 3′ splice site. Two of the patients had a diagnosis of CCUS and three patients had an MDS disorder characterized by low risk features and absence of complex cytogenetic abnor-malities. Notably, all five cases were characterized predominantly by macrocytic anemia. In addition, one CCUS patient followed for more than 15 years with a transfusion dependent macrocytic anemia was found to have an inactivating ZRSR2 mutation with an allele frequency of >60%. We conclude that the common clinical features of patients with an isolated mutation of ZRSR2 are a macrocytic anemia without leukopenia, thrombocytopenia or an increase in marrow blast percentage. At least in some cases, the presence of an isolated ZRSR2 mutation can accompany a dominant hematopoietic clone with a low risk for transformation to frank dysplasia or acute leukemia.

H3B-8800-G0001-101: A first in human phase I study of a splicing modulator in patients with advanced myeloid malignancies.

TPS7075 Background: Dysregulated mRNA splicing is important in tumorigenesis and in resistance to cancer therapy. Somatic heterozygous mutations in core spliceosome genes (e.g. SF3B1, SRSF2, U2AF1) have been reported at high frequencies in patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML). These mutations confer a change of function resulting in aberrant mRNA splicing that, in preclinical models, results in defects in hematopoietic cell development and myelodysplasia. Recurrent mutations in the spliceosome of patients with malignancies suggests importance in disease pathogenesis. Cells bearing splicing mutations depend on wild-type spliceosome function, suggesting the spliceosome as a therapeutic target. In vitro data indicate preferential induction of apoptosis (measured by caspase 3/7 activation) in SF3B1-mutant cells following treatment with the SF3B1 modulator H3B-8800. H3B-8800 inhibits growth in human AML cell lines, including those with mutations in U2AF1, SRSF2 or SF3B1. Oral administration of H3B-8800 modulates splicing and induces antitumor activity in xenograft leukemia models expressing mutant core spliceosome components. Methods: This study explores the safety of H3B-8800 in patients with myeloid cancers. Dose escalation (Cohort A) follows a 3+3 design with a starting dose of 1 mg daily for 5 consecutive days every 14 days in a 28 day cycle. Cohort A is open to patients with MDS, AML or CMML, irrespective of spliceosome mutations. In parallel to dose escalation, up to 6 patients with mutations of interest may be enrolled at doses determined to be safe in Cohort A (Cohort B). After determining the recommended phase 2 dose, 4 expansion cohorts will enroll patients with: (1) International Prognostic Scoring System (IPSS) low/int-1 risk MDS with SF3B1 mutations, (2) IPSS low/intermediate risk-1 MDS with mutations in SRSF2, U2AF1, or ZRSR2, (3) high/intermediate risk-2 MDS or AML, and (4) CMML; 3 and 4 having mutations in SF3B1, SRSF2, U2AF1, or ZRSR2. The first cohort enrolled 3 patients and the trial is currently enrolling patients at the second dose level. Clinical trial information: NCT02841540.

Aberrant splicing in maize rough endosperm3 reveals a conserved role for U12 splicing in eukaryotic multicellular development

RNA splicing of U12-type introns functions in human cell differentiation, but it is not known whether this class of introns has a similar role in plants. The maize ROUGH ENDOSPERM3 (RGH3) protein is orthologous to the human splicing factor, ZRSR2. ZRSR2 mutations are associated with myelodysplastic syndrome (MDS) and cause U12 splicing defects. Maize rgh3 mutants have aberrant endosperm cell differentiation and proliferation. We found that most U12-type introns are retained or misspliced in rgh3 Genes affected in rgh3 and ZRSR2 mutants identify cell cycle and protein glycosylation as common pathways disrupted. Transcripts with retained U12-type introns can be found in polysomes, suggesting that splicing efficiency can alter protein isoforms. The rgh3 mutant protein disrupts colocalization with a known ZRSR2-interacting protein, U2AF2. These results indicate conserved function for RGH3/ZRSR2 in U12 splicing and a deeply conserved role for the minor spliceosome to promote cell differentiation from stem cells to terminal fates.

Exploration of the role of gene mutations in myelodysplastic syndromes through a sequencing design involving a small number of target genes

Novel sequencing designs are necessary to supplement the recognized knowledge of myelodysplastic syndrome (MDS)-related genomic alterations. In this study, we sequenced 28 target genes in 320 Chinese MDS patients but obtained 77.2% of recall factors and 82.8% of genetic abnormalities (including karyotype abnormalities). In addition to known relationships among mutations, some specific chromosomal abnormalities were found to link to specific gene mutations. Trisomy 8 tended to be linked to U2AF1 and ZRSR2 mutations, and 20q- exhibited higher SRSF2/WT1 and U2AF1 mutation frequency. Chromosome 7 involvement accounted for up to 50% of RUNX1 mutations and 37.5% of SETBP1 mutations. Patients carrying a complex karyotype were prone to present TP53 mutations (36.1%). However, individuals with normal karyotypes rarely possessed mutations in the TP53, RUNX1 and U2AF1. Moreover, DNMT3A, TP53, SRSF2, STAG2, ROBO1/2 and WT1 predicted poor survival and high AML transformation. By integrating these predictors into international prognostic scoring system (IPSS) or revised IPSS, we built a set of mutation-based prognostic risk models. These models could layer different degrees of risk in patients more satisfactorily. In summary, this sequencing design was able to detect a number of gene mutations and could be used to stratify patients with varied prognostic risk.

Dynamics of clonal evolution in myelodysplastic syndromes.

To elucidate differential roles of mutations in myelodysplastic syndromes (MDS), we investigated clonal dynamics using whole-exome and/or targeted sequencing of 699 patients, of whom 122 were analyzed longitudinally. Including the results from previous reports, we assessed a total of 2,250 patients for mutational enrichment patterns. During progression, the number of mutations, their diversity and clone sizes increased, with alterations frequently present in dominant clones with or without their sweeping previous clones. Enriched in secondary acute myeloid leukemia (sAML; in comparison to high-risk MDS), FLT3, PTPN11, WT1, IDH1, NPM1, IDH2 and NRAS mutations (type 1) tended to be newly acquired, and were associated with faster sAML progression and a shorter overall survival time. Significantly enriched in high-risk MDS (in comparison to low-risk MDS), TP53, GATA2, KRAS, RUNX1, STAG2, ASXL1, ZRSR2 and TET2 mutations (type 2) had a weaker impact on sAML progression and overall survival than type-1 mutations. The distinct roles of type-1 and type-2 mutations suggest their potential utility in disease monitoring.

Clonal Evolution of Gene Mutations in AML Transformation from Patients with De Novo Myelodysplastic Syndromes Carrying Spliceosome Mutations: An Analysis of 71 Paired Samples

Background and Purpose: The molecular pathogenesis of progression of myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (sAML) remains incompletely understood. We studied genemutations involving spliceosome in the transformation of MDS to sAML by comparing matched paired MDS/sAML bone marrow samples to determine the roles of SRSF2, U2AF1 SF3B1, and ZRSR2 mutations in the evolution of MDS to sAML.Patients and Methods: One hundred and forty-nine de novo MDS patients (2 RCUD, 5 RARS, 27 RCMD, 52 RAEB-1, and 63 RAEB-2) were examined forspliceosome mutations at initial diagnosis and 93 patients progressed to sAML with a median follow-up of 16.4 months. Seventy one of them had paired MDS/sAML bone marrow samples available for comparative analyses. Mutational analyses of spliceosome were performed by pyrosequencing with a detection sensitivity of 5% for U2AF1 (exons 2 and 6) and SRSF2 (P95 of exon 1) mutations and by direct sequencing for SF3B1 (exons13-16) and ZRSR2 (whole coding exons 1-11) mutations. Additional 29 gene mutations known to involve in myeloid neoplasms were also analyzed by direct sequencing or next-generation sequencing (NGS, Ion Torrent PGM) followed by Sanger sequencing validation; NGS was mainly used for mutation detection of epigenetic regulators and cohesin complex. The allele burden of targeted genes was determined by pyrosequencing and/or NGS.Results: The frequencies of U2AF1, SRSF2, SF3B1, and ZRSR2 mutations in the 149 MDS patients were 14.8% (22/149), 12.8% (19/149), 12.1% (17/141), and 7.5% (10/133), respectively. Together, spliceosome mutations occurred in 51.5% of MDS patients at initial diagnosis and were mutually exclusive. All the 5 RARS patients had SF3B1 mutations. Co-existed mutations with epigenetic regulators were detected in 36 patients (52.9%), with RUNX1 in 13 (19.1%), with cohesin complex including STAG2, SMC3, RAD21,or SMC1A, in 13 (19.1%), with signaling pathways including RAS, PTPN11, JAK2, or FLT3-TKD in 4 (5.9%), BCOR in 2, and one each with CEBPa and SETBP1. There were no differences in blood counts, percentage of blasts in bone marrow and blood, WHO subtype, cytogenetic risk group, and IPSS-R between spliceosome-mutated and -unmutated patients. Of the patients carrying spliceosome mutations, only SRSF2 mutation had prognostic impact on predicting a higher risk of sAML transformation (P = 0.025) and sAML-free survival (median 10.8 months, 95% CI 5.1-16.5 months) compared to SRSF2-unmutated patients (median 17 months, 95% CI 8.5-25.5 months, P = 0.050). Of the 71 paired MDS/sAML samples, 37 (52.1%) had spliceosome mutations at diagnosis; the mutational status and patterns remained unchanged in all the 37 matched sAML samples but allele burden was apparently increased in the sAML samples with SRSF2 (P = 0.017) or SF3B1 (P = 0.015) mutations. In addition, one ZRSR2 mutant clone and two SRSF2 mutant clones evolved during sAML progression. Acquisition of other mutated genes was found in 37 spliceosome-mutated patients at sAML phases, including RUNX1 in 5, N-RAS in 5, CEBPa in 4, K-RAS in 3, FLT3-ITD in 3, and one each with ASXL1, TET2, STAG2, WT1, PTPN11, CBL, FLT3-TKD, and C-FMS. Notably, of the 3 patients acquiring spliceosome mutations, none gained other mutated genes during sAML transformation.Clonal expansion of other mutated genes were observed in 4 cases with RUNX1, in 2 with TET2, and one each with N-RAS, CEBPa, ASXL1, SMC1A, and STAG2.Conclusions: Our results showed that spliceosome mutations occurred in more than half of de novo MDS patients at initial diagnosis. Clonal expansion, evolution, or unchanged allele burden of spliceosome mutations might occur during sAML transformation with frequent acquisition of additional mutated genes. SRSF2 mutation predicted a higher risk and more rapid sAML transformation. (Grants support: NHRI-EX103-10003NI, MOHW103-TD-B-111-09 and CMRPG3D1532) DisclosuresNo relevant conflicts of interest to declare.

Amplicon Based Panel Targeted Resequencing Identified ZRSR2 As a Potential New Favorable Marker in Pediatric AML

Introduction:Acute myeloid leukemia (AML) is one of the most threatening malignancies in children and adolescents. The accumulation of mutations in leukemia stem cells (LSC) is believed to lead to the development of leukemia. Cyto- and molecular genetics already identified several aberrations which are relevant in leukemogenesis, prognosis and therapy. Nevertheless, the molecular landscape and clonal evolution of AML and its clinical relevance, especially for pediatric patients, is not yet well described. Next Generation Sequencing (NGS) as an emerging sequencing technology provides the possibility to generate sequence data of high quality and detect genetic aberrations in a minimum of time. The aim of this study was to apply amplicon based panel targeted resequencing by using the TruSight Myeloid Panel (Illumina) to identify variants in 54 genes.MethodsPatients:In total 150 samples derived from pediatric patients diagnosed with AML at the time of initial diagnosis or relapse were analysed regarding their mutational profile. All patients treated according to the AML-BFM therapy protocols (n=103) were chosen to determine the potential impact in prognosis.Sequencing and analysis of variantsSequencing with the TruSight Myeloid panel (Illumina) was performed on a MiseqDX. The sequencing panel is designed to identify somatic mutations associated with myeloid malignancies in 54 genes. To define variants, we excluded intronic, synonymous and variants with an allele frequency below 5% and a read depth below 50 reads. False positive variants were excluded by including healthy donors and reference samples.Variants were detected and analysed using Variant studio (Illumina) and Sophia DDM (Sophia Genetics). Almost all variants were detectable in both software, although great insertions and deletions were detectable only by Sophia DDM.ResultsIn the cohort of 150 patients, we detected 408 mutations in the 54 genes included in the panel (fig. 1). 26% of the patients showed more than 4, 24% 3, 24% 2, 17% 1 and 9% of the patients showed 0 mutations. Four and more mutations occurred mostly in AML FAB M1 (n=17) and patients with a complex karyotype (n=6). Treatment related AML show less mutations compared to primary AML.Within the group of patients treated according the 1st line AML-BFM protocol (n=103), CEBPA, FLT3, KIT, NRAS, KRAS, NPM1 or WT1 mutations did not have prognostic relevance. Interestingly, we were able to detect mutations in ZRSR2 in 21 patients in total (SNVs in 6, InDels in 9 and splice acceptor variants in 6 patients). 15 patients were part of the group of patients who were treated according to the 1st line AML-BFM protocol. ZRSR2 encodes an essential splicing factor and the encoded protein associates with the U2 auxiliary factor heterodimer and may play a role in network interactions during spliceosome assembly [RefSeq 2008]. The presence of a ZRSR2 mutation seems to be associated with better EFS and lower cumulative incidence of relapse, respectively (fig.2). Even if patients with favourable cytogenetics were excluded, patients with mutated ZRSR2 might have better EFS (fig.2).Conclusions:Amplicon based panel targeted resequencing with the TruSight Myeloid panel provides the possibility to detect mutations in 54 genes associated to myeloid malignancies within 3 days. This will enable a faster and possible more precise characterisation of pediatric AML, either for risk group stratification or the addition of more specific treatment options. Due to the limited number of patients, the results concerning the prognostic relevance of ZRSR2 need to be confirmed in a larger patient group. [Display omitted] [Display omitted] [Display omitted] DisclosuresReinhardt:Jazz Pharma: Other: Travel Accomodation; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

The Mutational Patterns Associated with Cytogenetic Subsets of De Novo Acute Myeloid Leukemia (AML): A Study of 1603 Adult Patients (Pts)

The classification of AML is based on the morphology of leukemic blasts, their immunophenotypes, and the presence of specific chromosomal abnormalities and selected gene mutations. It is widely acknowledged that some gene mutations are enriched in certain cytogenetic subsets, in which they may serve as therapeutic targets. We created a comprehensive oncoprint of mutations associated with centrally reviewed cytogenetic findings in 1603 adults (1080 aged <60 years, and 523 aged ≥60 years) with de novo AML (excluding acute promyelocytic leukemia) who were treated on CALGB/ALLIANCE protocols. The pts were classified into 5 cytogenetic subgroups: (1) cytogenetically normal (CN); (2) with complex karyotype (CK); (3) with t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22), i.e., core-binding factor (CBF)-AML; (4) with balanced rearrangements other than t(8;21) and inv(16)/t(16;16); (5) with unbalanced chromosomal abnormalities in non-complex karyotype.Using a targeted next-generation sequencing panel, we analyzed the mutational status of 80 cancer- and leukemia-associated genes. The mutated genes were assigned to 9 previously described by the Cancer Genome Atlas Research Network (N Engl J Med. 2013;368:2059) functional groups: chromatin remodeling, cohesin complex, kinases, methylation-related, NPM1, RAS pathway, spliceosome, transcription factors, and tumor suppressors. The mutational oncoprint revealed major differences in gene mutation frequencies among the 5 major cytogenetic groups.CN-AML pts had a broad mutational spectrum, involving all functional groups. Mutations in methylation-related genes (61% of pts), NPM1 (57%), and kinases (46%) dominated the mutation pattern.TP53 mutations were most common in pts with CK (38%). The second most common mutation, TET2, was detected only half as often (17%). The mutation patterns associated with "typical" (containing abnormalities resulting in loss of chromosome material from 5q, 7q and/or 17p), and "atypical" (without the aforementioned abnormalities) CKs differed. TP53 mutations were present in 52% of pts with typical but only in 5% of pts with atypical CKs (P<0.001). Conversely, the latter pts harbored mutations in another tumor suppressor gene, PHF6, more often (15% vs. 2%, P=0.03). Furthermore, atypical CKs had a broader spectrum of recurrent mutations, with 6 mutations occurring in ≥10% of pts (IDH2, NPM1, PHF6, TET2 and ZRSR2 mutations and FLT3-TKD), compared with only 2 genes, TP53 and TET2, mutated in ≥10% of pts with typical CKs, suggesting major biological differences between typical and atypical CKs.CBF-AML pts with had few detectable mutations, with a median of 1 (range, 0-5) versus 3 mutations (range, 0-9) detected in the remaining AML pts (P<0.001).In AML with balanced rearrangements, AML pts with MLL-rearrangements often had mutations in RAS pathway genes, whereas they rarely harbored mutations in other functional groups compared with pts with non-MLL-rearranged balanced translocations [MLL-rearranged vs. non-MLL-rearranged: chromatin remodeling, 9% vs. 26%; methylation-related, 12% vs. 23%; spliceosome, 12% vs. 24%]. Some cytogenetic subgroups with particular recurrent chromosome abnormalities presented with specific molecular features, supporting claims that they represent separate AML entities. For instance, 51% of pts with t(6;11)(q27;q23) harbored KRAS mutations, compared with only 3% of all remaining pts. Among t(9;22)(q34;q11.2)-positive pts, 47% had mutations in RUNX1,and 27% mutations in ZRSR2. Pts with inv(3)(q21q26.2/t(3;3)(q21q26.2) had frequent mutations in SF3B1 and BCOR (both 38%).Pts with gains or losses of chromosome material in non-complex karyotypes (unbalanced chromosomal abnormalities) were characterized by high mutation frequencies in methylation-related (55%) and spliceosome genes (34%). Strikingly, spliceosome mutations were enriched in pts with chromosome gains, with almost half of these pts harboring one or more mutations. Except for pts with sole +11, who most often harbored U2AF1 mutations (43%), SRSF2 was the most frequently mutated spliceosome gene in pts with unbalanced chromosomal abnormalities.Our comprehensive depiction of frequencies of single gene mutations and functional groups in recurrent cytogenetic subsets may be useful in guiding mutation testing in AML pts, and result in more focused application of targeted therapy of AML. DisclosuresWang:Immunogen: Research Funding; Incyte: Speakers Bureau. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Novartis: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Agios: Consultancy; Juno Therapeutics: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; ONO: Consultancy; Celator: Consultancy; Amgen: Consultancy; Jansen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Roche: Consultancy.

A 6-Lncrna Scoring System for Prognostication of Adult Myelodysplastic Syndromes

Background:Myelodysplastic syndromes (MDS) are highly heterogeneous regarding pathogenesis and clinical outcome. Traditionally, the revised international prognostic scoring system (IPSS-R)incorporating clinical features and cytogenetic abnormalities has been the standard for prognostication, while in recent years, recurrent somatic mutations are becoming increasingly important for this purpose. Additionally, gene expression profiling of coding genes and microRNAs has also emerged as a powerful tool to separate patients into distinct prognostic categories. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without protein-coding capacity. lncRNAs not only participate in normal hematopoiesis, but perturbation of their expressions may contribute to development of acute leukemia. However, the impact of lncRNA expression profiling on the prognosis of MDS patients has not yet been explored to date.Aims:This study was aimed to evaluate lncRNAs expression profiling in MDS and to find out lncRNAs whose expression levels were associated with clinical outcomes. A scoring system was constructed to better risk-stratify MDS patients. We also tried to seek clues to the functionality of these lncRNAs.Materials & Methods:By using the Affymetrix Human Transcriptome Array 2.0 platform, we obtained the global expression profiles of 24120 lncRNAs in 176 adult patients with de novo MDS diagnosed according to the 2008 WHO classification. Through mathematical modeling, we identified six lncRNAs whose expression levels were significantly associated with overall survival (OS). We then constructed a risk scoring system with the weighted sum of each of these six lncRNAs, and evaluated the correlation of the scores with clinical features,cytogenetic abnormalities, gene mutations, andtreatment outcomesof the MDS patients.The reliability of our lncRNA scoring system was assured based on the coefficient of variance obtained from a permutation test, and further validated by using a five-fold cross-validation, in which 80% of the patients were used as the training samples to develop the scoring model, whose prediction performances were evaluated by using the rest of the 20% of the patients. Analysis of mutations in 21 genes was performed by conventional Sanger sequencing technique.Results:Higher lncRNA scores were positively associated with refractory anemia with excess of blasts (RAEB)-1 or RAEB-2 subtypes, complex cytogenetic changes, and IPSS-R high and very high risks, but inversely associated with RCUD, RARS, RCMD subtypes, a normal karyotype, and IPSS-R low risk. Patients with higher lncRNA scores had more frequent RUNX1, ASXL1, TP53, EZH2, SRSF2 and ZRSR2 mutations, shorter OS (median 14.0 months vs. 77.3 months, P<0.001, Figure 1), and higher five-year leukemic transformation rate (72.0% vs. 17.9%, P<0.001), compared with those with lower lncRNA scores. The predictive power of this 6-lncRNA model was validated by a five-fold cross validation procedure. In multivariable analyses, a higher lncRNA score remained an independent unfavorable risk factor for OS (RR 2.428, 95% CI 1.751-3.367, P<0.001) after adjusting for other clinical and molecular variables. Moreover, the survival difference between higher-score and lower-score patients remained statistically significant in both IPSS-R higher-risk (high and very high risks) and lower-risk (very low, low and intermediate risks) subgroups (Figure 2). We also correlated our lncRNA score with mRNA expression profiling, and identified that higher lncRNA scores were associated with pathways related to acute myeloid leukemia and hematopoiesis.Conclusion:Our integrated 6-lncRNA risk scoring system provides distinct insights into the clinical and biological implications of lncRNAs expression in de novo MDS patients. The scoring system represents an independent prognostic factor for survival and leukemic transformation. It may assist improving risk stratification of newly-diagnosed MDS patients. Further prospective trials are necessary to confirm the findings of our study. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Characterization of MDS Harboring TET2 Mutations and/or TET2 Deletions

Background: TET2 mutations and deletions have been reported in MDS. A detailed analysis of the prognostic impact of TET2 deletions and their association to TET2 mutations is lacking. Aim: To characterize MDS with TET2 mutations (mut) and/or TET2 deletions (del) with respect to accompanying cytogenetic and molecular genetic abnormalities and their impact on prognosis. Patients and Methods: First 788 unselected MDS cases (cohort A) were evaluated. As in this cohort only 8 cases with TET2 deletion were detected, further MDS were screened for TET2 deletions. In total 77 MDS harboring a TET2 deletion were identified and included in cohort B. Both cohorts were analyzed by chromosome banding analysis, FISH, genomic arrays and mutation analysis of TET2. Cases from cohort A were also analyzed for mutations in ASXL1, ATM, BCOR, BRCC3, CBL, CTCF, DNMT3A, ETV6, EZH2, FBXW7, IDH1, IDH2, JAK2, KRAS, LAMB4, MPL, NCOR1, NCR2, NF1, NRAS, PHF6, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SMC3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Results: In cohort A 248 cases (31%) with TET2mut were identified. TET2del and a normal karyotype were more frequent in MDS with TET2mut as compared to those with TET2 wild-type (wt) (3% vs 1%, p=0.006; 89% vs 78%, p&lt;0.001). SF3B1 and ASXL1 were frequently mutated in both TET2mut and TET2wt MDS (32% and 34%, 22% and 18%, respectively). In MDS with TET2mut compared to MDS with TET2wt the following genes were less frequently mutated: ATM (0.5% vs 3%, p=0.05), DNMT3A (9% vs 15%, p=0.02), ETV6 (0.5% vs 3%, p=0.03), IDH1 (0.5% vs 3%, p=0.02), IDH2 (1% vs 5%, p=0.002), TP53 (2% vs 7%, p=0.004), U2AF1 (4% vs 9%, p=0.04), while the following genes were more frequently mutated: CBL (6% vs 2%, p=0.01), EZH2 (8% vs 2%, p&lt;0.001), SRSF2 (27% vs 12%, p&lt;0.001), and ZRSR2 (15% vs 3%, p&lt;0.001). Overall spliceosome genes were more frequently mutated in TET2mut than in TET2wt MDS (77% vs 56%, p&lt;0.001). In the total cohort A neither TET2mut nor TET2del had an impact on overall survival (OS). In TET2mut MDS and TET2wt MDS SF3B1mut were associated with favorable outcome, while TP53mut were associated with shorter OS in both subsets (table 1). However in TET2mut MDS mutations in RUNX1 (p&lt;0.0001), CBL (p=0.001), and U2AF1 (p=0.03) were independently associated with shorter OS, while in TET2wt MDS mutations in KRAS (p=0.03), EZH2 (p=0.02), NRAS (p=0.02), SRSF2 (p=0.007), IDH2 (p=0.05), and ASXL1 (p=0.01) were independently associated with shorter OS. In cohort B 40/77 (52%) MDS with TET2del also harbored a TET2mut. The 4q deletion encompassing the TET2 gene was &lt; 10 MB in size and thus cytogenetically cryptic in 77% of cases with TET2mut, while the TET2 deletion was cryptic in only 24% of cases without TET2mut. A normal karyotype was present in 37 cases (48%), a complex karyotype in 29 (38%) and other abnormalities in 11 cases (14%). TET2mut were frequent in cases with a normal karyotype (68% vs aberrant karyotype: 32%, p&lt;0.001) and were rare in cases with a complex karyotype (13%). Relating the mutation load of TET2mut to the proportion of cells with TET2del as determined by FISH revealed in 60% of cases that both TET2 alterations were present in the main clone, while in 23% of cases the TET2mut was present in a subclone only and in 17% the TET2del was observed in a subclone only. In the subset of patients with TET2del in a subclone only, 83% showed a normal karyotype and none a complex karyotype, while in the subset of cases with TET2mut in a subclone only, 43% showed a normal and 29% a complex karyotype. In the total cohort B the presence of a TET2mut in addition to the TET2del had no prognostic impact, while the presence of a complex karyotype was associated with shorter OS (RR: 8.0, p=0.004). Conclusions: 1) TET2 deletions are rare in TET2 mutated MDS (3%). 2) TET2 mutations are frequent in MDS with TET2 deletion (52%). 3) TET2 mutations are highly correlated to a normal karyotype and are rare in complex karyotype. 3) Neither TET2 mutations nor TET2 deletions have a prognostic impact in MDS. 4) In TET2 mutated MDS mutations in RUNX1, TP53, CBL, and U2AF1 have the strongest negative independent impact on OS, which in TET2 wild-type MDS is the case for mutations in TP53, KRAS, EZH2, NRAS, SRSF2, IDH2 and ASXL1. Table The relative risk of parameters significantly (p&lt;0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Table. The relative risk of parameters significantly (p&lt;0.05) associated with overall survival are depicted in TET2 mutated and TET2 wild-type MDS Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Stengel:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

MDS with Deletions in the Long Arm of Chromosome 11 Are Associated with a High Frequency of SF3B1 Mutations

Background: According to the revised International Prognostic Scoring System (IPSS-R), patients with MDS and deletions in the long arm of chromosome 11 (del(11q)) as sole abnormality are categorized as very good prognosis, even better than patients with a normal karyotype. Molecular data on this rare MDS subset is still limited. Aims: Comprehensive cytogenetic and molecular genetic characterization of MDS patients with del(11q) with and without additional aberrations and evaluation of prognosis. Patient cohorts and methods: Within 9225 unselected cases with de novo MDS, 33 cases with isolated del(11q) (del(11q) sole) and 23 cases with del(11q) accompanied by additional chromosomal aberrations (del(11q) other) were detected. All 56 cases were investigated using chromosome banding analysis (CBA) and interphase FISH. For 46 patients (33 of del(11)q sole, 13 of del(11q) other) material was available for investigation by genomic arrays (SurePrint G3 ISCA CGH+SNP Microarray, Agilent, Waldbronn, Germany). In 44 cases amplicon sequencing was performed to detect mutations in ASXL1, CALR, CBL, CSF3R, CSNK1A1, DNMT3A, ETNK1, ETV6, EZH2, FLT3-TKD GATA1, GATA2, IDH1, IDH2, JAK, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SRSF2, TET2, TP53, U2AF1 and ZRSR2. Variants of unknown significance were excluded from statistical analysis. The cohort comprised 23 male and 33 female patients, median age was 75 years (range: 47-89 years). Results: 33/9925 cases with del(11q) sole were found by CBA (0.4%). In the 23 del(11q) other cases additional aberrations included del(5q) (12/23 cases, 52%), del(20q) (3/23 cases, 13%), del(12p) (2/23 cases, 9%) and +8 (2/23 cases, 9%) as recurrent abnormalities. In 6/23 cases (26%), a complex karyotype (&gt;3 aberrations) was observed. In 13/33 cases with del(11q) sole by CBA, array CGH identified additional chromosomal aberrations (all non-recurrent, submicroscopic). In the total cohort analyzed by array CGH, the size of del(11q) varied between 12 - 58 Mb with a median of 39 Mb. In more detail, in cases with del(11q) sole the size ranged from 12 - 58 Mb (median: 38 Mb), in cases with del(11q) other from 21 - 56 Mb (median: 40 Mb). While the minimal deleted region for cases with del(11q) other was identified between genomic position 107 251 381 - 120 088 150, for cases with del(11q) sole no common minimal deleted region was determined due to the strong variation between the respective deleted regions. Mutation analyses revealed a high frequency of SF3B1 mutations in the total cohort (47%). Mutation frequencies &gt;10% were also detected for ASXL1 (17%), SRSF2 (13%), TET2 (13%) and U2AF1 (10%). The median number of mutations was 1 (range 0-4) without differences between both subgroups. However, the high frequency of SF3B1 mutations was even more prominent when only cases with del(11q) sole were analyzed (56% vs. 23% in del(11q) other). Moreover, mutations in U2AF1 were exclusively found in cases with del(11q) sole (14% vs. 0%). By contrast, mutations in ASXL1 were more frequent in del(11q) other (23% vs. 14%). However, these correlations were not found to be statistically significant due to the low number of cases. Comparison of the SF3B1 mutation loads with the proportion of cells with del(11q) observed by FISH revealed that in most cases, del(11q) is found in the main clone. Ring sideroblasts (≥15%) were detected in 11/45 cases (24%), 10 of them harbored an SF3B1 mutation. The minimal deleted region of del(11q) for SF3B1 mutated cases was identified between 107 789 141 - 119 437 544. In the total cohort, the overall survival (OS) at 5 years was 91% and is thus in line with the very good prognosis as defined by the IPSS-R. A lower OS at 5 years was observed for patients with del(11q) other compared to del(11q) sole, although it was not found to be statistically significant (94% vs. 85%). Conclusion: In 0.4% of MDS patients a del(11q) sole was observed. This incidence is in line with data used for calculation of the IPSS-R (0.7% in Schanz et al., 2012). Especially in cases with del(11q) sole, but also in del(11q) other, a high frequency of SF3B1 mutations was observed, which might explain in part the very good prognosis as stated in the IPSS-R and also observed in the present study. Moreover, these patients were found to show a low number of other molecular mutations. The role of U2AF1 mutations, which were exclusively detected in cases with del(11q) sole, needs further investigation. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

The Clinical and Biological Effects of Expression of Dedicator of Cytokinesis 1 (DOCK1) in Myelodysplastic Syndrome

Background: DOCK1 is an archetype member of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors and functions as an RAC activator.DOCK1 is involved in diverse biological processes including cytoskeleton rearrangement, cell motility and phagocytosis. Expression of DOCK1 is ubiquitous except in hematopoietic cells. Several studies highlighted the functional implications of DOCK1 in migratory and invasive properties of cancer cells, yet the roles of DOCK1 in hematological malignancies are not known.Materials and Methods: In this study, we focused on myelodysplastic syndrome (MDS) according to the diagnostic criteria of 2008 WHO classification, a clinically heterogeneous myeloid cancer whose pathogenesis remains enigmatic. We employed Affymetrix Human Transcriptome Array 2.0 to quantify the global expression patterns of the marrow mononuclear cells (MNC) in 243 newly diagnosed MDS patient recruited in the National Taiwan University Hospital (NTUH). We specifically analyzed the clinical, genetic, and prognostic significance of DOCK1 expression in various stages of MDS.Results: Our data showed that higher expression of DOCK1 conferred an unfavorable prognosis in MDS patients. Increased expression levels of DOCK1 was associated with higher-risk MDS defined by either revised international scoring system (IPSS-R) or WHO classification. Notably, DOCK1 expression levels in lower-risk MDS such as RA, RARS, RCMD, RCMD-RS, in which the marrow blast percentages were not increased, were still significantly higher than normal marrow (Figure 1), implying that the higher DOCK1 expression was independent of blast numbers in the samples, at least in these subgroups of patients. Among the 51 patients documented with leukemic transformation, 36 patients (70.6%) had higher DOCK1 expression compared with 70 out of 168 patients (41.7%) without transformation (P < 0.001). Regarding the genetic alterations, mutations in RUNX1, EZH2, SETBP1, ZRSR2, ASXL1 and TP53 were more frequently seen in the patients with higher DOCK1 expression, whilst SF3B1 mutation showed the contrary pattern. Remarkably, we demonstrated step-wise decreasing DOCK1 expression in the marrow MNC from higher-risk MDS, lower-risk MDS, to normal marrow, suggesting an impact of DOCK1 in pathogenesis in MDS. With a median follow up of 37.4 months, higher expression of DOCK1 was associated with shorter overall survival (OS) (median 13.7 months vs. 69.9 months) and shorter interval to leukemic transformation (median 23.6 months vs. not reached) after a follow up of 20.0 months (both P < 0.001) (Figure 3A and 3B). Independent analysis of DOCK1 expression showed higher level in the very poor cytogenetic subgroup in MDS (P < 0.001). In the multivariate analysis, higher DOCK1 expression proved to be a strong unfavorable prognostic factor for OS and leukemic transformation (P = 0.005 and P = 0.001, respectively) independent of other unfavorable factors including age, karyotype, and mutations of ASXL1, RUNX1, EZH2 and TP53.Conclusion: To our knowledge, our study is the first to show the clinical, genetic, prognostic, and biological association of DOCK1 expression in MDS patients. The step-wise increasing expression levels of DOCK1 in MNC from normal marrow, lower-risk MDS to higher-risk MDS patients highlight the hitherto unappreciated role of DOCK1 in the pathogenesis of MDS. Further studies on large and prospective cohorts are necessary to confirm our observations. Animal studies are needed to elucidate the in vivo roles of DOCK1 in MDS. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Driver Mutations in Patients with Lower-Risk Myelodysplastic Syndromes Classified By the MD Anderson Lower-Risk Prognostic Scoring System

▪INTRODUCTION: Patients with lower-risk myelodysplastic syndromes (MDS) as defined by the International Prognostic Scoring System (IPSS) are generally considered to have favorable prognosis. The MD Anderson Lower-risk Prognostic Scoring system (MDA LR-PSS) is a validated prognostic model that defines a subset of patients with shorter than expected survival. Presence of EZH2mutations has been reported to independently impact survival of these patients. Further evaluation and validation of the addition of mutation data to this prognostic model is needed.METHODS: We conducted whole exome sequencing of 74 previously untreated patients with Low or Int-1 IPSS. Exome capture hybrid was performed using Agilent SureSelect All Exon V4. Sequencing was performed with Illumina HiSeq 2000 and aligned to the hg19 human genome reference. Common virtual normal in house pool was used for somatic variant calling. Generalized linear models were used to study association of overall response (OR), complete response (CR) and risk factors. Response was defined following 2006 IWG criteria. The Kaplan-Meier produce limit method was used to estimate the median overall survival (OS) and leukemia-free survival (LFS).RESULTS: Patient characteristics are summarized in Table 1. Seventeen (23%) patients had CMML and 57 (77%) had MDS. Seventy (95%) patients had evaluable cytogenetics. A total of 46 (66%) patients had normal karyotype with 4 (6%) patients having complex karyotype. Twenty-three (31%) patients were classified as Low risk by IPSS and 51 (69%) as Int-1. Twelve (16%) patients were classified as Category 1 by the MDA LR-PSS, 38 (51%) as Category 2 and 24 (32%) as Category 3. A total of 148 driver mutations in 27 genes were detected. Median number of driver mutations per patient was 2 (range 0-5). Mutations in TET2, SRSF2, ASXL1, SF3B1 and ZRSR2 were the most frequently detected mutations present in >10% cases. Frequency of identified mutations is shown in Figure 1A. The median follow up was 26.5 months (range 3-102 months). Median OS was 43.2months (95% CI 35.04-50.46). Survival by MDA LR-PSS category is shown in Figure 1B. A total of 6 patients had transformation to acute myeloid leukemia with a median time to transformation of 22.4 months (95% CI 0.00-45.28). By univariate analysis, mutations in BCOR (HR 5.49, 9% CI 1.24-24.29, p=0.011), RUNX1 (HR 2.8, 95% CI 1.11-7.09, p=0.023) and STAG2 (HR 3.32, 95% CI 1.12-9.82, p=0.022) predicted for shorter OS. When analyzing for LFS, mutations in EZH2 (HR 18.61, 95% CI 2.99-115.92, p<0.001) and U2AF1 (HR 5.08, 95% CI 0.92-28.06, p=0.038) predicted for shorter LFS. By multivariate analysis combining the identified driver mutations with prognostic relevance and the MDA LR-PSS category, presence of MDA LR-PSS Category 3 (HR 5.98, 95%CI 1.28-28, p=0.023), RUNX1 mutation (HR 4.18, 95%CI 1.36-12.81, p=0.012) and STAG2 mutation (HR 3.7, 95%CI 1.02-13.47, p=0.047) retained there prognostic significance. Based on these results we designed a new model with scoring being based on HR for each given variable: MDA LR-PSS Category 3 2 points, RUNX1mut 1 point and STAG2mut 1 point. Patients were classified into three categories: Low, High or Very high with significantly different survival outcomes (Figure 1C). Based on this new model, patients with Category 2 MDA LR-PSS with either STAG2 or RUNX1 mutations had similar outcomes to those with Category 3 and no mutations (p=0.747). Patients with either Category 1 or Category 2 by MDA LR-PSS without STAG2 or RUNX1 mutations had similar OS (p=0.306) and represented the population with most favorable outcomes (p<0.001). Finally, patients with Category 3 with STAG2 or RUNX1 mutations had the worse outcomes in terms of survival (p<0.001).CONCLUSION: Similar to previous studies, our data suggests integration of mutation data into the MDA LR-PSS can improve our ability to predict outcomes of patients with lower-risk MDS. Mutations in STAG2 and RUNX1 can help identify a subset of patients with worse than expected outcomes as predicted by the MDA LR-PSS. [Display omitted] [Display omitted] DisclosuresJabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Ravandi:Seattle Genetics: Consultancy, Honoraria, Research Funding; BMS: Research Funding. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding. DiNardo:Daiichi Sankyo: Other: advisory board, Research Funding; Abbvie: Research Funding; Novartis: Other: advisory board, Research Funding; Celgene: Research Funding; Agios: Other: advisory board, Research Funding. Daver:Otsuka: Consultancy, Honoraria; Kiromic: Research Funding; Ariad: Research Funding; BMS: Research Funding; Sunesis: Consultancy, Research Funding; Karyopharm: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Kantarjian:Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Pfizer Inc: Research Funding; Delta-Fly Pharma: Research Funding; Novartis: Research Funding.

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Is Highly BCL-2 Dependent and Sensitive to Venetoclax

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a particularly aggressive hematologic malignancy with median survival of 80% bone marrow blasts. His BPDCN carried the mutations ASXL1 Y581fs*, ASXL1 E553fs*, GNB1 K57E, IDH2 R140W, and NRAS G12D, and expressed high levels of BCL-2 protein in bone marrow and skin. BH3 profiling of a skin tumor biopsy revealed marked BCL-2 dependence and dynamic BH3 response to venetoclax (4 hr delta priming 55.6%). We treated him using a regimen recently FDA-approved for chronic lymphocytic leukemia (CLL) consisting of weekly dose escalation (20 -> 50 -> 100 -> 200 mg), to a target dose of 400 mg daily. At the time of this writing, he had reached 200 mg without significant toxicity, including no evidence of tumor lysis syndrome. His skin disease has responded remarkably (Figure), with the first response evident within 10 days. Our data suggests that BPDCN is highly sensitive to BCL-2 inhibition, which could provide an urgently needed new treatment for patients with this disease. We propose that BCL-2 inhibition should undergo expedited clinical evaluation in BPDCN. In addition, this case offers an example of precision cancer medicine by functional rather than genetic means. Disclosures Davids:Infinity: Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Gilead: Honoraria; Janssen: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria, Research Funding; TG Therapeutics: Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Stone:ONO: Consultancy; Novartis: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Celator: Consultancy; Abbvie: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Agios: Consultancy; Celgene: Consultancy, Membership on an entity9s Board of Directors or advisory committees; Karyopharm: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; Juno Therapeutics: Consultancy; Merck: Consultancy; Sunesis Pharmaceuticals: Consultancy; Xenetic Biosciences: Consultancy. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding. Letai:AbbVie: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding. Lane:N-of-1: Consultancy; Stemline Therapeutics: Research Funding.