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Introduction Recent advances in the discovery of the genomic landscape in AML prompts necessity to re-examine the 2017 European LeukemiaNet (ELN) recommendation. In this study we aimed to validate the usefulness of 2017 ELN risk stratification in a large Taiwan cohort with special focus on the prognostic relevance of FLT3-ITD allelic ratio and its interaction with other mutations. Methods We retrospectively included 1040 de novo non-M3 AML patients. AML was risk-stratified according to the 2017 ELN recommendation. 739 (71.1%) patients who received standard chemotherapy were included for survival analysis. Allogeneic hematopoietic stem cell transplantation (HSCT) was performed in 293 (39.6%) patients. Mutational analyses of fifteen genes, including CEBPA, NPM1, FLT3, RUNX1, ASXL1, TP53, splicing factors (SF), such as SRSF2, U2AF1, and SF3B1, as well as KIT, NRAS, KRAS, DNMT3A, TET2, and WT1 were performed. FLT3-ITD/wild allelic ratios were calculated as the ratio of the area under the curve by fragment analysis. High FLT3-ITD allelic ratio (FLT3-ITDhigh) was defined as ³ 0.5 and low allelic ratio (FLT3-ITDlow) defined as < 0.5. Results According to the 2017 ELN risk classification, favorable, intermediate and adverse categories comprised 34.6%, 29.2% and 36.2% patients, respectively. NPM1 mutations and FLT3-ITD, the most common mutations in this cohort, were detected in 217 (20.9%) and 216 (20.8%) patients, respectively, with a significant association between each other. The median value of the FLT3-ITD/wild ratio was 0.68 without difference between NPM1-mutated and NPM1-wild group. Of note, patients with FLT3-ITDhigh had higher WBC count and LDH level than those with FLT3-ITDlow. Overall, the CR rate and relapse rate were 74.2% and 54.7%, respectively and 5-year overall survival (OS) was 43.2±1.9%. The CR rate (92.3%) was higher in the 2017 ELN favorable risk group than in the intermediate (73.0%) and adverse groups (52.0%, P As to the prognostic impact of FLT3-ITD, we showed that FLT3-ITD patients had significantly lower CR rate, higher relapse rate, reduced DFS and OS than those without. There was a strikingly difference in treatment response between the low and high FLT3-ITD allelic ratio groups: CR rate (80.7% vs. 63.6%, P=0.0319), relapse rate (56.5% vs. 66.2%, P=0.329), DFS (14.2 vs. 4.6 months P=0.011) and OS (24.0 vs. 11.9 months, P=0.048). Interestingly, patients with FLT3-ITDhigh had a better OS if they received allogeneic HSCT than those who did not. Among the 2017 ELN favorable-risk category, we found that patients with mutated NPM1 and FLT3-ITDlow had significantly shorter OS (median, not reached vs. 31.6 months, P=0.003, Figure. 1A) and a trend of shorter DFS (median 14.9 months vs. 93.9 months, P=0.089, Figure. 1B) compared to other ELN favorable subgroups. To find the cause of the difference, we investigated the concurrent mutations in the patients with mutated NPM1 and FLT3-ITDlow. 46.2% of them had concurrent poor-risk mutations, such as ASXL1, RUNX1, TP53, WT1, TET2, DNMT3A, and SF mutations. Similarly, among the 2017 ELN intermediate-risk category, patients with mutated NPM1 and FLT3-ITDhigh had more unfavorable outcomes compared to those with wild-type NPM1 and without FLT3-ITD (DFS, median 3.7 vs. 11.6 months, P=0.028 and OS, median, 11.4 vs. 26.5 months, P=0.067). Presence of concurrent poor-risk mutations were also identified in 72.9% of these patients. Based on these findings, we postulated that concomitant poor-risk genetic alterations at least partially affected the prognosis of FLT3/ITD patients. In the cohort of FLT3-ITD patients, patients harboring poor-risk mutations had shorter DFS and OS than those without (P=0.028 and P=0.031, respectively). Further, co-occurrence of FLT3-ITDhigh and poor-risk mutations that predicted a worst outcome, seemed to define a highly adverse prognostic group. Conclusions We showed that ELN 2017 risk classification could well stratify AML patients in Taiwan. The prognostic relevance of FLT3-ITD may further depend on the presence or absence of co-occurring poor-risk genetic alterations, which seemed to add an adverse effect in patients with FLT3-ITD. These observations warrant confirmation in other prospective and large-scale studies. Disclosures Ko: Roche: Research Funding; GNT Biotech & Medicals Crop.: Research Funding; Abbevie: Research Funding; Mumdipharma Taiwan: Consultancy.

Recurrent somatic mutation of SRSF2, one of the RNA splicing machinery genes, has been identified in a substantial proportion of patients with myelodysplastic syndrome (MDS). However, the clinical and biologic characteristics of MDS with this mutation remain to be addressed. In this study, 34 (14.6%) of the 233 MDS patients were found to have SRSF2 mutation. SRSF2 mutation was closely associated with male sex (P = .001) and older age (P < .001). It occurred concurrently with at least 1 additional mutation in 29 patients (85.3%) and was closely associated with RUNX1, IDH2, and ASXL1 mutations (P = .004, P < .001, and P < .001, respectively). Patients with SRSF2 mutation had an inferior overall survival (P = .010), especially in the lower risk patients. Further exploration showed that the prognostic impact of SRSF2 mutation might be attributed to its close association with old age. Sequential analyses in 173 samples from 66 patients showed that all SRSF2-mutated patients retained their original mutations, whereas none of the SRSF2-wild patients acquired a novel mutation during disease evolution. In conclusion, SRSF2 mutation is associated with distinct clinical and biologic features in MDS patients. It is stable during the clinical course and may play little role in disease progression.

Introduction Acute myeloid leukemia (AML) with hyperleukocytosis (HL), commonly defined as white blood cell (WBC) counts >100,000/uL, are intuitively thought as a unique group with dismal prognosis. However, comprehensive studies regarding the clinical characteristics, genetic alterations in this group of patients are limited, and the role of hematopoietic stem cell transplantation (HSCT) is controversial. Method A cohort of 755 de novo AML patients diagnosed from 1994 to 2011 who had cryopreserved cells for analysis were enrolled. The mutation status of 18 genes was determined by Sanger sequencing and/or next generation sequencing (NGS). We compared cytogenetics and relevant mutations in these genes between AML patients with and without HL, and exposed their prognostic implications. Results The median age was 54 (range 15-94). 101 (13.4%) patients had HL. HL was associated with younger age (median age 47 vs. 54, P=0.022), higher peripheral blast percentage (77.6% vs. 39%, P Survival analysis was performed on the 525 patients who received standard intensive chemotherapy; among whom 69 patients had HL. The HL patients had lower complete remission (CR) rates compared to those without (63.8% vs. 78.2%, P=0.009). Further, the HL patients had significantly poorer overall survival (OS) and disease-free survival (DFS) than those without (median 24 months vs. not reached (NR), P=0.042; 6.5 vs. 11.8 months, P=0.005, respectively, Figure 1). Older age, poor-risk cytogenetics, RUNX1, TP53 and WT1 mutations were other poor prognostic factors for OS (median, 18.1 months vs. NR, P Conclusion The HL patients represented 13.4% of our AML cohort and showed distinct clinic features and genetic alterations compared to those without HL. HL was an independent poor prognosis factor irrespective of cytogenetics change or mutation status, and the HL patients may potentially benefit from HSCT. Figure 1 The Kaplan-Meier survival curves for OS (A) and DFS (B) stratified by hyperleukocytosis (HL) or not in the 525 AML patients who received standard intensive chemotherapy (A) (B) Figure 1. The Kaplan-Meier survival curves for OS (A) and DFS (B) stratified by hyperleukocytosis (HL) or not in the 525 AML patients who received standard intensive chemotherapy. / (A). / (B) Figure 2 The Kaplan-Meier survival curves for OS stratified by HSCT or not in the 69 HL patients who received standard intensive chemotherapy Figure 2. The Kaplan-Meier survival curves for OS stratified by HSCT or not in the 69 HL patients who received standard intensive chemotherapy Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Background Aberrant patterns of alternative splicing (AS) are reported in acute myeloid leukemia and other malignances, but have not yet been investigated in myelodysplastic syndrome (MDS). In addition, mutations in spliceosome genes are especially common in MDS. Therefore, it would be highly interesting to analyze the clinical and biological significance of aberrant AS in MDS. Materials and Methods A total of 243 newly diagnosed MDS patients, including 78 with refractory anemia (RA), 22 with RA with ring sideroblasts(RARS), 76 with RA with excess blasts (RAEB), 24 with RAEB in transformation (RAEBT), and 43 with chronic myelomonocyticleukemia (CMMoL), who had complete clinical and genetic information were enrolled. We also included 20 healthy donors of hematopoietic stem cell transplantation for comparison. We extracted RNA from mononuclear cells (MNC) isolated from the bone marrow (BM), followed by hybridization on the microarrays of AffymetrixHuman Transcriptome Array 2.0, which contained > 6 million distinct probes covering coding, non-coding transcripts, and the exon-exon junctions among 67539 genes (44710 coding genesand 22829 noncoding genes). This comprehensive coverage of exons and junctions facilitated investigation of the splicing patterns of the genes. The aberrant AS was analyzed with the splicing index (SI) method using the default setting of AffymetrixTranscriptome Analysis Console version 3.0 software. The SI of a probe in a gene was derived from the ratio between MDS patients and normal controls in terms of the probe intensity divided by the gene expression level, ie. [intensityof probe A in gene A /gene A expression level]MDS/[intensity of probe A in gene A /gene A expression level]normal control. SI had to be less than -2 or more than 2 to be defined as an aberrant AS event. Results Totally 52730 of 67539 genes (78.1%) on the array were expressed in BM from both MDS patients and normal donor controls. Approximately 17565 of the 52730 expressed genes (33.3%) were differentially spliced between the MDS and normal marrow MNC (P < 0.05). As a whole, 88.9% of these aberrant spliced events were mapped to coding genome and 11.1% to noncoding regions. These aberrant AS exons fell into five common patterns, including exon skipping (57.8%), alternative donor sites (18.1%), alternative acceptor sites (17.8%), intron retention (5.6%) and mutually exclusive exons (0.7%). In average, when compared with the normal marrow MNC, there were 6.4 (4.8-12.0) more aberrant AS events in each AS gene (ASG) in the marrow MNC of 243 MDS patients.More aberrant AS events per ASG implied a more complex aberrant AS pattern.The numbers of aberrant AS events per ASG were not significantly associated with MDS subtypes and frequencies of spliceosome gene mutations. However, with a median follow up of 48.4 months, the patients with more aberrant AS events per ASG had a trend of shorter overall survival (OS) (median 20.1 vs 29.6 months, P=0.074) and a trend of shorter time to acute leukemic transformation (P=0.212) than the others (Fig.1). Further subgroup analysis showed a significantly shorter OS in the patients with more aberrant AS events per ASG than those with less events among RAEB patients (median OS: 10.8 months vs 16.9 months, P=0.007, Fig.2A). In addition, RA patients with more aberrant AS events per ASG had shorter time to acute leukemic transformation than RA patients with fewer aberrant AS events per ASG (P=0.006, Fig.2B). Among the patients without detectable mutations in spliceosome genes or genes related to epigenetic modification, those with more aberrant AS events per ASG also had a shorter OS than the others (median OS: 27.9 months vs not reached, P=0.013, Fig.3). Multivariate analysis in our cohort of 243 MDS patients revealed that higher aberrant AS events per ASG was an unfavorable prognostic factor for OS (P=0.016) independent of age, IPSS-R risk score, and mutations of SF3B1, ASXL1 and TP53. Conclusion To our knowledge, we are the first to present the prognostic impact of aberrant AS pattern in MDS patients. Large prospective cohorts are needed to confirm our observations. Disclosures No relevant conflicts of interest to declare.

Introduction Risk-stratification of patients with acute myeloid leukemia (AML) can not only improve treatment response, but also reduce side effects of the treatment, especially in the elderly. A number of patient-specific and leukemia-associated factors are related to the poor outcome in older patients with AML. However, comprehensive studies regarding the impact of genetic alterations in this group of patients are limited. Methods and Materials A total of 500 adult patients with newly diagnosed de novo AML who had enough bone marrow cryopreserved cells for analysis at the National Taiwan University Hospital were enrolled consecutively. We compared the clinico-biological features, cytogenetics and molecular gene mutations between patients aged 60 years or older (n=185) and those younger ( Result Among older patients, those received standard intensive chemotherapy had a longer overall survival (OS) than those treated with palliative care. Compared with younger patients, the elderly had a higher incidence of poor-risk cytogenetic changes, but a lower frequency of favorable-risk cytogenetics. The median number of molecular gene mutations at diagnosis was higher in the elderly than the younger. Older patients had significantly higher incidences of PTPN11, NPM1, RUNX1, ASXL1, TET2, DNMT3A, and P53 mutations but a lower frequency of WT1 mutations. In multivariate analysis for OS among the elderly who received standard intensive chemotherapy, high WBC >50,000/μL at diagnosis, RUNX1 mutations, DNMT3A mutations, and P53 mutations were independent worse prognostic factors, while the presence of NPM1 mutations in the abcence of FLT3/ITD mutations was an independent good prognostic factor. The frequency of acquiring one or more adverse genetic alterations was much higher in older patients than younger ones. Further, the pattern of gene mutations could divide older patients with intermediate cytogenetics into three groups with significantly different complete remission rates, OS, and disease-free survival. Conclusion Older AML patients frequently harbored high-risk cytogenetics and gene mutations, and had poorer prognosis. Integration of cytogenetics and molecular alterations could risk-stratify older patients into groups with significant different outcomes. For those patients with poor prognosis under current chemotherapy, novel therapies, such as demethylating agents or other targeted therapies may be indicated. Disclosures Tang: Novartis: Consultancy, Honoraria.

Abstract 4490 Purpose Allogeneic hematopoietic stem cell transplantation (allo-SCT) may rescue patients with acute myeloid and lymphoblastic leukemia (AML/ALL) even in high risk disease. Little has been investigated about prognostic impact of pre- and post-transplant residual disease (RD) status, hematogones (HG) level and natural killer (NK) cells level in the adult patients with acute leukemia. Materials & Methods We retrospectively studied 45 consecutive adult patients, including 31 AML and 14 ALL, receiving allo-SCT at National Taiwan University Hospital. Based on disease status at transplant, 18 patients were in standard risk; 16 in intermediate risk; and 11 in high risk disease. 28 patients underwent myeloablative conditioning and 17 received reduced-intensity SCT. Bone marrow samples were obtained both before conditioning therapy (pre-SCT) and at hemogram recovery after transplantation (post-SCT). RD, HG level and NK cells level were evaluated by multidimensional flow cytometry. Leukemia-free survival (LFS) was estimated by using Kaplan-Meier method. Results Pre-SCT RD-negative (RD-) was shown in 26 patients and RD-positive (RD+) in 19 patients; whereas post-SCT RD- was found in 39 patients and RD+ in 6 patients. Level of pre-SCT RD status ranges from 0–90% and level of post-SCT RD status ranges from 0–3%. Estimated one-year LFS were 71.8% in pre-SCT RD- compared to 0% in pre-SCT RD+ group (P=0.008, Fig. 1). In post-SCT flow cytometric analysis, one-year LFS were 33.2% in post-SCT RD- versus 25.0% in post-SCT RD+ group (P=0.002, Fig. 2). After combining pre- and post-SCT RD status, three groups of patients were identified; pre-SCT RD−/post-SCT RD- (n=26), pre-SCT RD+/post-SCT RD- (n=13), and pre-SCT RD+/post-SCT RD+ (n=6). The classification further stratify the patients into different groups with distinct outcome (P=0.003, Fig. 3). We also found the median HG 0.2% (range 0–12%) and 0.1% (range 0–2.1%) and median NK cells 1.8% (range 0.2–20%) and 1.8% (0.02–15.5%) pre-SCT and post-SCT, respectively. The patients with higher NK cells, either pre-SCT or post-SCT, had longer LFS (P Conclusion Our data suggest both pre- and post-SCT RD status and NK cells level could predict the transplant outcome. Further large-scale studies may be needed to confirm this point. Disclosures: No relevant conflicts of interest to declare.

Abstract 3018 Background and Purposes Epstein-Barr virus (EBV)-associated PTLD is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Independent risk factors include use of ATG, acute GVHD, CMV antigenemia, T-depleted graft, and unrelated donor in our previous study. Quantitative real-time polymerase chain reaction (Q-PCR) was developed for early detection and intervention of EBV reactivation to prevent PTLD-related mortality. Patients and Methods Between Apr 2004 and Oct 2010, EBV viral load in plasma was monitored by Q-PCR in 222 HSCT patients (total 2945 samples) in NTUH. EBV reactivation was defined as > 500 copies/mL in two consecutive assays or > 10-fold elevation than baseline level. Results EBV reactivation occurred in 50 (22%) patients. The cumulated incidence of EBV reactivation was 28% at 1-year and 32% at 2-year. Median time to EBV reactivation was 40 days (ranges, 26–406) after SCT and median peak EBV-viral load, 10888 copies/ml (ranges, 948–3×107). The risk of EBV reactivation was significantly higher in patients receiving ATG (52% vs. 13%, p< 0.001), use of TBI (58% vs. 26%, p Conclusions Monitoring of EBV viral load is a sensitive and useful tool in the surveillance of EBV-reactivation. Frequent monitoring in high-risk patients is important to prevent occurrence of EBV-PTLD. Disclosures: No relevant conflicts of interest to declare.