Search

Background: FL is an indolent yet incurable disease characterized by recurrent relapses. Pts with R/R FL often have a poor prognosis and limited treatment options, particularly those who have progression of disease within 24 months of frontline treatment (POD24) or are refractory to multiple agent classes. Glofitamab is a T-cell-engaging, CD20xCD3 bispecific, full-length, 2:1 format antibody with bivalent binding to CD20 (B cells) and monovalent binding to CD3 (T cells). Glofitamab monotherapy with obinutuzumab pretreatment (Hutchings, et al. JCO 2021), or combined with obinutuzumab (Morschhauser et al, Blood 2019; NCT03075696), has shown efficacy and manageable safety in heavily pretreated R/R NHL. Here, we present updated results of glofitamab with three different step-up dosing (SUD) regimens as monotherapy (mono) or combined with obinutuzumab (combo) in R/R FL. Methods: Obinutuzumab (1000mg) was given 7 days prior to the first dose of glofitamab. For the 3 mono cohorts, intravenous glofitamab SUD was given on Days (D) 1 and 8 of Cycle (C) 1; then at target dose on C2, or as SUD on C1D1, C1D8, C2D1 and target dose on C3D1. For the combo cohort, glofitamab SUD was given on D1 and D8 of C1, then at target dose combined with obinutuzumab 1000mg from C2D1 and onwards (every 21 days for up to 12 cycles). Response rates were based on the Lugano criteria (Cheson et al. J Clin Oncol 2014). Results: As of May 18, 2021, 53 pts received glofitamab mono SUD (2.5/10/16mg, n=3; 2.5/10/30mg, n=21; 0.5/2.5/10/30mg, n=29) and 19 pts received glofitamab combo SUD (2.5/10/30mg). All pts had FL Grade (Gr) 1-3A (FLIPI 1 high risk score: mono, 28 [53%] pts; combo, 11 [58%] pts). Median age was 64 years (range 33-83) in mono cohorts and 61 years (range 41-78) in the combo cohort; median number of prior therapies was 3 (range 1-12) and 2 (range 1-5), respectively. Twenty-eight (53%) pts in the mono cohorts and 8 (42%) pts in the combo cohort were refractory to last therapy; 16 (30%) and 7 (37%), respectively, were refractory to prior CD20 and alkylating therapy (double refractory). A total of 19 (36%) pts in the mono cohorts and 10 (53%) pts in the combo cohort had experienced POD24. In the mono cohorts, overall response rate (ORR) was 81% (n=43) and complete metabolic response rate (CMR) was 70% (n=37), with 72% (n=21) CMR in the 0.5/2.5/10/30mg cohort and 67% CMR in both the 2.5/10/16mg (n=2) and 2.5/10/30mg (n=14) cohorts. In the combo cohort, ORR was 100% (n=19) and CMR was 73.7% (n=14). In the mono cohorts 87% (32/37) pts were in CMR, while in the combo cohort 71% (10/14) pts were in CMR. Median follow-up of CMR ranged from 0-14 months (mono) and 0-5 months (combo), and at this cut-off the follow-up is insufficient to assess CMR duration. High CMR rates were observed in high-risk pts (Table). The most common adverse event (AE) was cytokine release syndrome (CRS): 66% and 79% in the mono and combo cohorts, respectively. CRS rates by mono cohort were as follows: 2.5/10/16mg cohort, 100% (3/3); 2.5/10/30mg cohort, 76% (16/21); 0.5/2.5/10/30mg cohort, 55% (16/29). With mono, CRS events (Lee et al. ASTCT 2019) were mostly Gr 1 (47.2%) and 2 (17.0%); 1 pt had a Gr 3 event (2.5/10/16mg cohort). With combo, 52.6% had Gr 1 CRS and 26.3% had Gr 2 CRS. There were no Gr 3 CRS events in the combo cohort and no Gr 4 or 5 CRS with either regimen. Of pts with CRS, tocilizumab was used to treat CRS in 22.9% pts in mono cohorts and 33.3% pts in the combo cohort. All CRS events were manageable and had resolved at data cut-off. Neurologic AEs were seen in 26 pts (16 mono, 10 combo; 36%); all Gr 1 (n=17) or Gr 2 (n=9). No ICANS-like events related to glofitamab were reported. Other common AEs were infusion related reactions and pyrexia (events separate from CRS; both 28%) and neutropenia (26%) with mono, and neutropenia (58%), anemia (37%) and thrombocytopenia (32%) with combo. Conclusions: Glofitamab SUD administered as monotherapy or in combination with obinutuzumab achieved high response rates in pts with heavily pretreated R/R FL, including high-risk subgroups. Response rates were comparable to those reported for CAR-T in R/R FL. The safety profile of glofitamab was manageable; CRS events were mostly low grade and occurred mainly in C1 and C2. Considering the short duration of follow-up, additional follow-up is required to further assess the safety and efficacy of glofitamab in this heterogeneously treated population. Further analyses will be presented. Figure 1 Figure 1. Disclosures Morschhauser: BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy; Genmab: Membership on an entity's Board of Directors or advisory committees; AstraZenenca: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Servier: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Carlo-Stella: AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Research Funding; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; Janssen Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Membership on an entity's Board of Directors or advisory committees. Dickinson: Celgene: Research Funding; Takeda: Research Funding; Gilead Sciences: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Honoraria; Roche: Consultancy, Honoraria, Other: travel, accommodation, expenses, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau. Phillips: ADCT, BeiGene, Bristol Myers Squibb, Cardinal Health, Incyte, Karyopharm, Morphosys, Pharmacyclics, Seattle Genetics: Consultancy; AstraZeneca: Consultancy; BMS: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Incyte: Consultancy, Other: received travel expenses from Incyte, Research Funding. Houot: Bristol-Myers Squibb: Honoraria; CHU Rennes: Current Employment; Roche: Honoraria; Kite: Honoraria; Gilead: Honoraria; MSD: Honoraria; Celgene: Honoraria; Jsnssen: Honoraria; Novartis: Honoraria. Haioun: Celgene: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Miltenyi: Honoraria, Research Funding; Servier: Honoraria, Research Funding. Corradini: AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Honoraria; Incyte: Consultancy; Amgen; Takeda; AbbVie: Consultancy, Honoraria, Other: Travel and accommodations; Novartis; Gilead; Celgene: Consultancy, Other: Travel and accommodations; BMS: Other: Travel and accommodation; Sanofi: Consultancy, Honoraria; KiowaKirin; Incyte; Daiichi Sankyo; Janssen; F. Hoffman-La Roche; Kite; Servier: Consultancy; Novartis, Janssen, Celgene, BMS, Takeda, Gilead/Kite, Amgen, AbbVie: Other: travel and accomodations; AbbVie, ADC Theraputics, Amgen, Celgene, Daiichi Sankyo, Gilead/Kite, GSK, Incyte, Janssen, KyowaKirin, Nerviano Medical Science, Novartis, Roche, Sanofi, Takeda: Consultancy. Hutchings: Novartis: Research Funding; Janssen: Honoraria, Research Funding; Incyte: Research Funding; Genentech: Honoraria, Research Funding; Celgene: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Genmab: Consultancy, Honoraria, Research Funding. Sureda: GSK: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Other: Support for attending meetings and/or travel; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MSD: Consultancy, Honoraria, Speakers Bureau; BMS/Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support for attending meetings and/or travel, Research Funding, Speakers Bureau; Bluebird: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Consultancy. Martínez-López: Janssen, BMS, Novartis, Incyte, Roche, GSK, Pfizer: Consultancy; Roche, Novartis, Incyte, Astellas, BMS: Research Funding. Wrobel: Janssen: Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; BeiGene: Honoraria, Speakers Bureau. Lundberg: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Mulvihill: F. Hoffmann-La Roche Ltd: Current Employment, Ended employment in the past 24 months. Perez-Callejo: F. Hoffmann-La Roche Ltd: Current Employment, Current equity holder in publicly-traded company. Relf: Harpoon Therapeutics: Divested equity in a private or publicly-traded company in the past 24 months; F-Star Therapeutics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Roche Pharmaceutical Ltd: Current Employment, Current equity holder in publicly-traded company. Panchal: F. Hoffmann-La Roche Ltd: Current Employment. Humphrey: Roche: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Bachy: Kite, a Gilead Company: Honoraria; Novartis: Honoraria; Daiishi: Research Funding; Roche: Consultancy; Takeda: Consultancy; Incyte: Consultancy. OffLabel Disclosure: Glofitamab is a full-length, humanized, immunoglobulin G1 bispecific antibody with a 2:1 molecular format that facilitates bivalent binding to CD20 on B-cells, and monovalent binding to CD3 on T-cells. Glofitamab redirects T cells to engage and eliminate malignant B cells. Glofitamab is an investigational agent. Obinutuzumab (Gazyva) is a CD20-directed cytolytic antibody indicated: in combination with chlorambucil, for the treatment of pts with previously untreated CLL; in combination with bendamustine followed by obinutuzumab monotherapy, for the treatment of pts with FL who relapsed after, or are refractory to, a rituximab-containing regimen; in combination with chemo followed by obinutuzumab monotherapy in pts achieving at least a PR, for the treatment of adult pts with previously untreated stage II bulky, III or IV FL

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy worldwide. A sizable fraction of these patients ended up succumbing to this fatal disease, so the standard of care after the second-line chemotherapy and/or autologous stem cell transplants (ASCT) remains to be improved. Polatuzumab vedotin-piiq (PoV), an anti-CD79b antibody-drug conjugate, gained accelerated approval by the U.S. Food and Drug Administration in June, 2019. The phase II randomized controlled study (GO29365) showed PoV in combination with bendamustine and rituximab (BR) conferred an additional 22% complete response rate for relapsed/refractory DLBCL, compared with BR alone (40% vs. 18%) . Interestingly, how PoV fares when combined with other salvage therapy remains as an open question. Method DLBCL patients who developed progressive diseases after at least 2 prior lines of therapy including R-CHOP and not previously treated with BR were enrolled into this study between Nov. 2018 and Apr. 2020 at the National Taiwan University Hospital. PoV was given at 1.8 mg/kg in combination with salvage chemotherapies based on clinicians' choices. The compassionate use of PoV was approved by the ethics committee, and clinical data were retrospectively analyzed. Results Thirty-two patients were enrolled in this study. According to the Hans' criteria of immunohistochemical subtypes, 13 patients were germinal center B cell-like (GCB), 17 were non-GCB, and 2 patients were unclassifiable. The median age was 62.5 years (range 20-82). At diagnosis, 26 (81%) patients had stage III or IV diseases, 22 (68.8%) had extranodal involvement, and 24 (75%) had the IPI scores equaled to 3 or higher. The median of failed prior lines of treatment was 4 (range 2-11), including 7 (21.9%) relapsing from ASCT and 16 (50%) were of primary refractory disease. Regarding combination therapies, 20 (62.5%) received BR, 5 (15.6%) had rituximab, 5 (15.6%) had rituximab/carmustine-containing regimens, and rituximab/gemcitabine-based ones were given to 4 (12.5%) patients. The median of cycles of PoV was 4 (range 1-7). Notably, the combinatory use of PoV and salvage chemotherapy helped transition 5 patients to ASCT and 4 to allogeneic SCT. The median overall survival (OS) was 8.9 months (95% confidence interval 6.2-not estimable) with the median follow-up of 11.4 months (range 0.3-20.7). The overall response rate (ORR) was 45.2%, including 29.1% complete response (CR) and 16.1% partial response (PR). The patients achieving CR or PR after PoV had better overall survival (OS) than those who did not (median OS: not reached (NR) vs. 5.6 months, p=0.0001, Figure A). The ORR for patients failing prior 2, 3, 4, and ≥5 lines of treatment were 50% (3/6), 40% (2/5), 50% (4/8), and 42% (5/12), respectively. The patients failing prior 4 lines of treatment had better OS than those with failed prior 5 or more lines (NR vs. 6.47 months). Non-GCB patients had better ORR (69.2% vs. 30.8%) and OS (NR vs. 6.23 months) than GCB ones did. Intriguingly, the patients received SCT had better OS compared with the others (NR vs. 6.23 months, p=0.011, Figure B). As for grade 3 & 4 hematological adverse effects, 16 (50%) patients developed anemia, 20 (62.5%) had thrombocytopenia, and 18 (56.3%) were neutropenic. Grade 5 toxicities included pneumonia (2 patients, 6.3%), fungemia (1, 3.1%), intracranial hemorrhage (1, 3.1%), and other infections (2, 6.3%). Conclusion As the third-line or above treatment for DLBCL in this study, the efficacy and safety associated with PoV-based therapies not only were encouraging but also helped bridge to transplants. The application of this monoclonal antibody-drug conjugate in future clinical trials for DLBCL is expected. Figure Disclosures Tsai: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Harvester: Honoraria; Janssen: Honoraria; Kirin: Honoraria; Takeda: Honoraria; BMS: Honoraria; Astellas: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ko:Roche: Honoraria. Cheng:Roche: Honoraria. Huang:MundiPharma: Honoraria; Chugai: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Bristol Meyer & Squibb: Honoraria.

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.

DNMT3A mutations are associated with poor prognosis in acute myeloid leukemia (AML), but the stability of this mutation during the clinical course remains unclear. In the present study of 500 patients with de novo AML, DNMT3A mutations were identified in 14% of total patients and in 22.9% of AML patients with normal karyotype. DNMT3A mutations were positively associated with older age, higher WBC and platelet counts, intermediate-risk and normal cytogenetics, FLT3 internal tandem duplication, and NPM1, PTPN11, and IDH2 mutations, but were negatively associated with CEBPA mutations. Multivariate analysis demonstrated that the DNMT3A mutation was an independent poor prognostic factor for overall survival and relapse-free survival in total patients and also in normokaryotype group. A scoring system incorporating the DNMT3A mutation and 8 other prognostic factors, including age, WBC count, cytogenetics, and gene mutations, into survival analysis was very useful in stratifying AML patients into different prognostic groups (P < .001). Sequential study of 138 patients during the clinical course showed that DNMT3A mutations were stable during AML evolution. In conclusion, DNMT3A mutations are associated with distinct clinical and biologic features and poor prognosis in de novo AML patients. Furthermore, the DNMT3A mutation may be a potential biomarker for monitoring of minimal residual disease.

The impact of WT1 mutations in acute myeloid leukemia (AML) is not completely settled. We aimed to determine the clinical implication of WT1 mutation in 470 de novo non-M3 AML patients and its stability during the clinical course. WT1 mutations were identified in 6.8% of total patients and 8.3% of younger patients with normal karyotype (CN-AML). The WT1 mutation was closely associated with younger age (P < .001), French-American-British M6 subtype (P = .006), and t(7;11)(p15;p15) (P = .003). Multivariate analysis demonstrated that the WT1 mutation was an independent poor prognostic factor for overall survival and relapse-free survival among total patients and the CN-AML group. A scoring system incorporating WT1 mutation, NPM1/FLT3-ITD, CEBPA mutations, and age into survival analysis proved to be very useful to stratify CN-AML patients into different prognostic groups (P < .001). Sequential analyses were performed on 133 patients. WT1 mutations disappeared at complete remission in all WT1-mutated patients studied. At relapse, 3 of the 16 WT1-mutated patients who had paired samples lost the mutation and 2 acquired additional mutations, whereas 3 of 110 WT1-wild patients acquired novel mutations. In conclusion, WT1 mutations are correlated with poor prognosis in AML patients. The mutation status may be changed in some patients during AML progression.

Somatic mutation of the AML1/RUNX1(RUNX1) gene is seen in acute myeloid leukemia (AML) M0 subtype and in AML transformed from myelodysplastic syndrome, but the impact of this gene mutation on survival in AML patients remains unclear. In this study, we sought to determine the clinical implications of RUNX1 mutations in 470 adult patients with de novo non-M3 AML. Sixty-three distinct RUNX1 mutations were identified in 62 persons (13.2%); 32 were in N-terminal and 31, C-terminal. The RUNX1 mutation was closely associated with male sex, older age, lower lactic dehydrogenase value, French-American-British M0/M1 subtypes, and expression of HLA-DR and CD34, but inversely correlated with CD33, CD15, CD19, and CD56 expression. Furthermore, the mutation was positively associated with MLL/PTD but negatively associated with CEBPA and NPM1 mutations. AML patients with RUNX1 mutations had a significantly lower complete remission rate and shorter disease-free and overall survival than those without the mutation. Multivariate analysis demonstrated that RUNX1 mutation was an independent poor prognostic factor for overall survival. Sequential analysis in 133 patients revealed that none acquired novel RUNX1 mutations during clinical courses. Our findings provide evidence that RUNX1 mutations are associated with distinct biologic and clinical characteristics and poor prognosis in patients with de novo AML.

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.

Alterations of galectin-3 expression are often seen in cancers and may contribute to tumorigenesis, cancer progression, and metastasis. The studies concerning clinical implications of galectin-3 expression in patients with acute myeloid leukemia (AML) are scarce. We investigated the expression of LGALS3, the gene encoding galectin-3, in the bone marrow (BM) mononuclear cells from an original cohort comprising 280 adults with primary non-acute promyelocytic leukemia. Higher LGALS3 expression was closely associated with older age, French-American-British M4/M5 subtypes, CD14 expression on leukemic cells, and PTPN11 mutation, but negatively correlated with CEBPA mutation and FLT3-ITD. Compared with patients with lower LGALS3 expression, those with higher expression had lower complete remission rates, higher primary refractory rates, and shorter overall survival. This result was validated in an independent validation cohort. A scoring system incorporating higher LGALS3 expression and 8 other risk factors, including age, white blood cell count, cytogenetics, and gene mutations, into survival analysis proved to be very useful to stratify patients with AML into different prognostic groups (P < .001). In conclusion, BM LGALS3 expression may serve as a new biomarker to predict clinical outcome in patients with AML, and galectin-3 may serve as a potential therapeutic target in those patients with higher expression of this protein.

Introduction GATA-binding factor 2 (GATA2) contributes to the regulation of hematopoietic stem cell proliferation and differentiation. Mutations of GATA2 are identified in AML patients, but their clinical and biological correlations in AML remain to be delineated. Methods and Materials We determined GATA2 mutations by Sanger sequencing and next generation sequencing in consecutively enrolled 766 newly diagnosed de novo AML patients who had cryopreserved cells for analysis. The expression of GATA2 was also analyzed by microarray in 335 patients. We then looked for the clinical and biological correlations of the expression levels and mutation status of GATA2. Computational network analysis of GATA2 mutations was conducted by Ingenuity Pathway Analysis (IPA) and Gene set enrichment analysis (GSEA). Result GATA2 mutations were detected in 58 patients (7.5%) and were associated with FAB M1 subtype, and intermediate-risk cytogenetics, but negatively associated with M4 subtype and favorable-risk cytogenetics. There was no difference in other clinical parameters such as age, hemogram and LDH levels between the patients with and without GATA2 mutations. GATA2 mutations were closely associated with CEBPA double mutations (44.8% vs. 10.2%, P With a median follow-up time of 25 months (ranges 0 to 160), patients with GATA2 mutations had a trend of better overall survival (OS) than those without GATA2 mutations (median 40.7 vs. 24.9 months P=0.123). Among patients with CEBPA double mutations, there was also a trend toward better OS in patients concomitant with GATA2 mutations (P=0.199). By comparing the mRNA expression profiles between patients with and without GATA2 mutations, we found GATA2 expression levels were higher in those with GATA2 mutations (P=0.003). GATA2 mutations were also associated with a significant change in genes related to cell proliferation and differentiation by IPA. We also performed GSEA analysis to identify modest functional changes related to GATA2 mutation status. Leukemogenesis-related genes were significantly enriched in the GATA2-mutated subgroup (nominal P value= 0.006; Normalized enrichment score= 1.62), whereas gene signatures associated with myeloid differentiation, apoptosis, leukemia cell death, CEBPA and WT1 pathways were enriched in GATA2 -wild type patients. Conclusion GATA2 mutations, which are commonly heterozygous point mutations between amino acids 315 to 361, occur in 7.5% of AML patients and are associated with certain clinical features, a trend of better treatment outcome, and higher GATA2 expression. The GATA2 mutation-associated expression signatures suggest the effects on leukemogenesis by GATA2 mutations. Figure 1. Figure 1. Disclosures Tang: Novartis: Consultancy, Honoraria.

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.

The studies concerning clinical implications of TET2 mutation in patients with primary acute myeloid leukemia (AML) are scarce. We analyzed TET2 mutation in 486 adult patients with primary AML. TET2 mutation occurred in 13.2% of our patients and was closely associated with older age, higher white blood cell and blast counts, lower platelet numbers, normal karyotype, intermediate-risk cytogenetics, isolated trisomy 8, NPM1 mutation, and ASXL1 mutation but mutually exclusive with IDH mutation. TET2 mutation is an unfavorable prognostic factor in patients with intermediate-risk cytogenetics, and its negative impact was further enhanced when the mutation was combined with FLT3-ITD, NPM1-wild, or unfavorable genotypes (other than NPM1+/FLT3-ITD− or CEBPA+). A scoring system integrating TET2 mutation with FLT3-ITD, NPM1, and CEBPA mutations could well separate AML patients with intermediate-risk cytogenetics into 4 groups with different prognoses (P < .0001). Sequential analysis revealed that TET2 mutation detected at diagnosis was frequently lost at relapse; rarely, the mutation was acquired at relapse in those without TET2 mutation at diagnosis. In conclusion, TET2 mutation is associated with poor prognosis in AML patients with intermediate-risk cytogenetics, especially when it is combined with other adverse molecular markers. TET2 mutation appeared to be unstable during disease evolution.

The incidence of chronic lymphocytic leukemia (CLL) in Taiwan is markedly lower than that in Western countries, but we have seen a drastically increasing trend. We explored this distinct incidence trend of CLL for Taiwanese. The epidemiologic data of CLL for Taiwanese and Caucasian Americans during 1986 to 2005 were obtained from the Taiwan National Cancer Registry and Surveillance, Epidemiology, and End Results Program, respectively. The individual effects of time period and birth cohort on the incidence trends were analyzed using an age-period-cohort model. Although there was a weak period effect corresponding to the increased applications of immunophenotyping in 1991 to 1995 in Taiwan, evidences suggested that the age-adjusted incidence rate of CLL for Taiwanese was continuously increasing during the 20-year period while that for Caucasian Americans remained steady. In addition, a much stronger birth-cohort effect was identified for Taiwanese but not for Caucasian Americans. This effect corresponded to the westernization of lifestyle in Taiwan since 1960. We conclude that, in addition to the ethnic difference of incidence, there is distinct increasing incidence trend of CLL in Taiwan. The strong birth-cohort effect underlying this increasing trend indicates that lifestyles and environmental factors may play a role in the development of CLL for Taiwanese.

Mutations in the additional sex comb-like 1 (ASXL1) gene were recently shown in various myeloid malignancies, but they have not been comprehensively investigated in acute myeloid leukemia (AML). In this study, we analyzed ASXL1 mutations in exon 12 in 501 adults with de novo AML. ASXL1 mutations were detected in 54 patients (10.8%), 8.9% among those with normal karyotype and 12.9% among those with abnormal cytogenetics. The mutation was closely associated with older age, male sex, isolated trisomy 8, RUNX1 mutation, and expression of human leukocyte antigen–DR and CD34, but inversely associated with t(15;17), complex cytogenetics, FLT3–internal tandem duplication, NPM1 mutations, WT1 mutations, and expression of CD33 and CD15. Patients with ASXL1 mutations had a shorter overall survival than patients without, but the mutation was not an independent adverse prognostic factor in multivariate analysis. Sequential analyses showed that the original ASXL1 mutations were lost at relapse and/or refractory status in 2 of the 6 relapsed ASXL1-mutated patients studied, whereas 2 of the 109 ASXL1-wild patients acquired a novel ASXL1 mutation at relapse. In conclusion, AML bearing ASXL1 mutations showed distinct clinical and biological features. The ASXL1 mutation status can change during disease evolution in a few patients.

Mutations of nicotinamide adenine dinucleotide phosphate-dependent isocitrate dehydrogenase gene (IDH1) have been identified in patients with gliomas. Recent genome-wide screening also revealed IDH1 mutation as a recurrent event in acute myeloid leukemia (AML), but its clinical implications in AML are largely unknown. We analyzed 493 adult Chinese AML patients in Taiwan and found 27 patients (5.5%) harboring this mutation. IDH1 mutation was strongly associated with normal karyotype (8.4%, P = .002), isolated monosomy 8 (P = .043), NPM1 mutation (P < .001), and French-American-British M1 subtype (P < .001), but inversely associated with French-American-British M4 subtype (P = .030) and expression of HLA-DR, CD13, and CD14 (P = .002, .003, and .038, respectively). There was no impact of this mutation on patient survival. Sequential analysis of IDH1 mutation was performed in 130 patients during follow-ups. None of the 112 patients without IDH1 mutation at diagnosis acquired this mutation at relapse. In all 18 IDH1-mutated patients studied, the mutation disappeared in complete remission; the same mutation reappeared in all 11 samples obtained at relapse. We conclude that IDH1 is associated with distinct clinical and biologic characteristics and seems to be very stable during disease evolution.

Background Heritable mutation in GATA2, a zinc-finger (ZF) transcription factor crucial for hematopoietic stem cell proliferation and differentiation, was identified in the families with myelodysplastic syndrome related acute myeloid leukemia (MDS-AML). Recently, GATA2 mutations were found to be strongly associated with CEBPA double mutations (CEBPAdouble-mut) in AML, which define a distinct genetic entity of AML. However, the stability of the mutation during the clinical course remains unclear. In this study, we evaluated the clinical and biological features of AML with GATA2 mutation and analyze its sequential changes during the clinical follow-ups. Materials and Methods Mutation analysis of GATA2 exons 3-7 was performed by polymerase chain reaction and direct sequencing in 192 de novo AML patients. The association between GATA2 mutations and clinical features, chromosomal changes and genetic mutations were analyzed. Furthermore, sequential analyses of GATA2 mutation during the clinical course were performed in 375 samples from 138 patients. Results GATA2 mutations were seen in 27.4%, 6.7% and 1% of patients with CEBPAdouble-mut, CEBPA single mutation and CEBPA-wild type, respectively. Fourteen different missense GATA2 mutations, which were all clustered in the highly conserved N-terminal ZF-1 domain, were identified in 20 patients, most commonly A318V (n=4), followed by L321F (n=3) and A318T (n=2). GATA2 mutations were detected much more frequently in younger patients, and were closely associated with FAB M1 subtype, intermediate-risk cytogenetics, and expression of HLA-DR, CD7, CD15 or CD34 on leukemic cells. In contrast, GATA2 mutations were negatively associated with FAB M4 subtype and favorable-risk cytogenetics. A thorough mutation screening in 166 patients, including 15 GATA2-mutated ones, was performed to investigate the association between mutations of GATA2 and 17 other genes. The most common additional genetic aberrations in 15 GATA2-mutated patients at initial diagnosis were CEBPA mutation (n=14), followed by TET2 mutation (n=3) and NRAS mutation (n=2). Patients with GATA2 mutations had a significantly higher incidence of CEBPA mutation than those with GATA2-wild type (93.3% versus 34.4%, P Of the 154 AML patients undergoing conventional intensive induction chemotherapy, 124 (81%) patients achieved a complete remission (CR). Patients with GATA2 mutation had a higher CR rate and a trend of lower relapse rate compared with those with GATA2 wild-type (CR rate: 95% versus. 78.5%; relapse rate: 21% versus. 44.8%). With a median follow-up of 24.3 months (ranges, 0.1 to 150), patients with GATA2 mutation had significantly better overall survival (OS) and relapse-free survival (RFS) than those without GATA2 mutation (P=0.010 and P=0.033, respectively). Interestingly, among the subgroup of CEBPAdouble-mut patients, GATA2-mutated patients still had a trend of better OS and RFS than GATA2-wild type (median, not reached versus. 61 months, P=0.068 and not reached versus. 17 months, P=0.066, respectively). Multivariate analysis demonstrated that GATA2 mutation was an independent favorable prognostic factor for OS irrespective of age, white blood cell counts, cytogenetics and NPM1/FLT-ITD (HR: 0.0133, 95% CI: 0.018-0.978, P=0.047). In the serial studies of GATA2 mutations in 375 samples from 138 patients, including 14 GATA2-mutated and 124 GATA2-wild patients, all 14 GATA2-mutated patients lost the original mutations when first CR was achieved. Among them, four patients relapsed; two regained the original mutations and the other two lost them. On the other hand, two of the 124 GATA2-wild patients acquired a novel GATA2 mutation at first relapse and lost it at second CR. Interestingly, one of them regained the same GATA2 mutation at the second relapse. Conclusion AML patients with GATA2 mutations presented distinct clinical and biological features and a favorable prognosis in the total cohort. In the subgroup of patients with CEBPAdouble-mut, those with GATA2 mutations also had a trend of better OS and RFS compared with GATA2-wild patients. Sequential studies showed GATA2 mutation was not stable during disease evolution; it might be lost or acquired at disease progression, implying it was a second hit in the leukemogenesis of AML with CEBPA mutation. Disclosures: No relevant conflicts of interest to declare.

Abstract 3799 Background and Purpose Mutations of the DNMT3A gene, which encodes the enzyme DNA methyltransferase 3A, were identified in patients with myeloid malignancies and are associated with poor prognosis in primary AML patients. However, the clinical and prognostic implications of these mutations in myelodysplastic syndrome (MDS) remain to be determined. Methods and Materials A total of 328 de novo MDS patients diagnosed according to French-American-British (FAB) criteria at the National Taiwan University Hospital who had cryopreserved bone marrow cells for study were recruited into mutational analyses. Mutations in DNMT3A gene at exon 2–23 were analyzed by polymerase chain reaction and direct sequencing. The results were correlated with clinical features, cytogenetics, gene mutations and treatment outcomes. Results Among the 328 patients, 115 patients (35.0%) had refractory anemia (RA), 19 (5.8%) had RA with ring sideroblasts (RARS), 122 (37.2%) had RA with excess blasts (RAEB), 35 (10.7%) had RAEB in transformation (RAEBT), and 37 (11.3 %) had chronic myelomonocytic leukemia (CMMoL). DNMT3A mutations at 20 different positions were identified in 33 patients, including thirteen missense mutations, two nonsense mutations and five frame-shift mutations. Among these 33 patients, 31 had single mutation of DNMT3A, and the other 2 patients had double mutations. The most common mutation was R882H (n = 8), followed by R882C (n = 7), Y735C (n=2), and R720H (n=2). All other mutations were detected in only one patient each. Totally, DNMT3A mutations were identified in 33 (10.1%) of 328 patients diagnosed according to the FAB classification and in 25 (9.8%) of 256 diagnosed according to 2008 WHO classification. DNMT3A-mutated patients were older (median age, 74 years vs. 66 years, P=0.048) and had higher platelet counts at diagnosis than DNMT3A-wild patients (median, 123.5×103/μL vs. 73 ×103/μL, P=0.016). According to FAB classification, patients with RARS had the highest incidence (26.3%) of DNMT3A mutations, followed by RAEBT (14.3%), RAEB (11.5%), and CMMoL (8.1%), whereas those with RA had the lowest incidence (5.2%, P=0.035). Chromosome data were available in 308 patients (93.9%) at diagnosis and clonal chromosomal abnormalities were detected in 155 patients (50.3%). There was no difference in the distribution of 2008 WHO classification, karyotype and international prognostic scoring system (IPSS) between patients with and without DNMT3A mutations. To investigate the association of gene mutations in the pathogenesis of MDS, a mutational screening of 10 other genes was also performed. Among the 33 patients with DNMT3A mutations, 16 patients (48.5%) showed additional molecular abnormalities at diagnosis, including seven with concurrent IDH1/IDH2 mutations, seven ASXL1 mutations, five AML1/RUNX1 mutations, two MLL-PTD, two RAS mutations and one JAK2 mutation. Eight of these 16 patients (50%) had two other concurrent mutations, and the others had one additional mutation. It's clear that DNMT3A mutation was closely interacted with IDH mutation in MDS (IDH mutation occurring in 21.2% of DNMT3A-mutated patients vs. 3.4% in DNMT3A-wild ones, P=0.001). With a median follow-up of 57.6 months (range, 0.1–250.7 months), there was no significant difference in overall survival (OS) between patients with and without DNMT3A mutation by either FAB or 2008 WHO classifications (median, 22.5 months vs. 30.9 months, P=0.669 and 25.2 months vs. 34.9 months, P=0.538, respectively) as well as in the rate of acute transformation. However, among the subgroup of patients with RA by FAB classification or refractory cytopenia with unilineage dysplasia by 2008 WHO classification, DNMT3A-mutated patients had significantly shorter OS than DNMT3A-wild patients (median, 28.3 months vs. 39.8 months, P Conclusion Our findings provided evidence that DNMT3A mutations could be detected in a substantial portion of de novo MDS patients. DNMT3A mutations are associated with distinct clinical and biological features and poor prognosis in selected groups of patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 409 Background: DNMT3A mutations are associated with poor prognosis in acute myeloid leukemia (AML), but the stability of this mutation during the clinical course remains unclear. Materials and Methods: Mutation analysis of DNMT3A exons 2–23 was performed by polymerase chain reaction and direct sequencing in 506 de novo AML patients. Their interaction with clinical parameters, chromosomal abnormalities and genetic mutations were analysed. Results: DNMT3A mutations were identified in 14% of total patients and 22.9% of patients with normal karyotype (CN-AML). 30 different kinds of DNMT3A mutations were identified in 70 patients. Twelve were missense mutations, eight were nonsense mutations, nine were frame-shift mutations and one, in-frame mutation. The most common mutation was R882H (26 patients), followed by R882C (15 patients), R882S (3 patients), R736H (3 patients) and R320X (2 patients). DNMT3A mutations were closely associated with older age, higher white blood cell (WBC) and platelet counts at diagnosis, FAB M4/M5 subtype, intermediate-risk and normal cytogenetics. Among the 70 patients with DNMT3A mutations, 68 (97.1%) showed additional molecular abnormalities at diagnosis. The most common associated molecular event was NPM1 mutation (38 cases), followed by FLT3-ITD (30 cases), IDH2 mutation (16 cases) and FLT3-TKD (9 cases). Patients with DNMT3A mutations had significantly higher incidences of NPM1 mutation, FLT3-ITD, IDH2 and PTPN11 mutations than those with DNMT3A-wild type (54.3% vs. 15.3%, P With a median follow-up of 55 months (ranges, 1.0 to 160), patients with DNMT3A mutation had significantly poorer overall survival (OS) and relapse-free survival (RFS) than those without DNMT3A mutation (median, 14.5 months vs. 38 months, P =0.013, and medium, 7.5 months vs. 15 months, P=0.012, respectively). In the subgroup of 130 younger patients (less than 60 years) with CN-AML, the differences between patients with and without DNMT3A mutation in OS (median, 15.5 months vs. not reached, P= 0.018) and RFS (median, 6 months vs. 21 months, P=0.004) were still significant. Multivariate analysis demonstrated that DNMT3A mutation was an independent poor prognostic factor for OS and RFS among total patients (HR 2.218, 95% CI 1.333–3.692, P=0.002 and HR 2.898, 95% CI 1.673–5.022, P DNMT3A mutations were serially studied in 316 samples from 138 patients, including 35 patients with distinct DNMT3A mutations and 103 patients without mutation at diagnosis. Among the 34 patients with DNMT3A mutations who had ever obtained a CR and had available samples for study, 29 lost the original mutation at remission status, but five retained it; all these five patients relapsed finally within a median of 3.5 months and died of disease progression, suggesting presence of leukemic cells. In the 13 patients who had available samples for serial study at relapse, all patients regained the original mutations, including mutant clone was found by TA cloning in one patient. Among the 103 patients who had no DNMT3A mutation at diagnosis, none acquired DNMT3A mutation at relapse, while karyotypic evolution was noted at relapse in 39% of them. Conclusion: DNMT3A mutations are associated with distinct clinical and biological features and poor prognosis in de novo AML patients. Furthermore, the mutation may be a potential biomarker for monitoring of minimal residual disease. 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.

Abstract 4378 Chronic lymphocytic leukemia (CLL) is a prevalent leukemia in western countries. However, the incidence of CLL in Taiwan, is markedly lower but shows an increasing trends. We explored the incidence of CLL in Taiwan during the past two decades and compared it with Caucasian Americans. The epidemiologic data of CLL (1979-2003) in Taiwan was obtained from Taiwan Cancer Registry, and the corresponding data of Caucasian Americans was from the Surveillance, Epidemiology, and End Results Program (SEER). From 1979-2003, the age-adjusted annual incidence (with WHO 2000 standard population) of CLL in Taiwan increased from 0.08 to 0.36 per 100,000 people with about 4.5 folds increment, while that in Caucasian Americans was much higher but remained in steady state in this period (2.6 to 3.1 per 100,000 people). Age-specific incidence rate of CLL plotted by calendar year at diagnosis revealed a stronger period effect in Taiwanese (Figure 1A). The estimation of age-specific annual percentage changes (AAPC) of incidences revealed that, in every age group, the annual increase of the overall incidence throughout the past 2 decades was much higher for Taiwanese compared with that for Caucasian Americans (Figure 1B). Age-specific incidence rates plotted by birth cohort showed that the CLL incidence was higher for later birth cohorts for every age group in Taiwanese but not in Caucasian Americans (Figure 1C). The individual effects of time period and birth cohort on the trends of incidence of CLL in both populations were analyzed using the age-period-cohort analysis for comparison. These analyses showed a much stronger birth-cohort effect on the incidence trend of CLL in Taiwanese than in Caucasian Americans. This strong birth-cohort effect corresponded to the Westernization of lifestyle in Taiwan since 1960. This finding suggests that, in addition to the racial differences of genetic backgrounds that may be responsible for the differences in the CLL incidence, the environmental effects also play some roles. We also analyzed the cytogenetic abnormalities (CA) by conventional cytogenetics (CG) and fluorescence in situ hybridization (FISH) and assessed their clinical significance in Taiwanese patients who were diagnosed and treated at National Taiwan University Hospital. Thirty-nine patients (45.9%) out of 85 patients possessed CG-CA and 58 (69.9%) out of 83 patients had FISH-CA. FISH was more sensitive to detect CA than CG. Those who had neither CG-CA nor FISH-GA had excellent overall survival (OS). Patients with hypodiploidy or complex CG-CA had poorer prognosis than others (p=0.043 and 0.037, respectively). Among those specific chromosome changes detected by CG, trisomy 12 was the most common CA (14.1% among 85 patients). Detection of 17p- by CG was independently associated with poor prognosis. By FISH, 13q- was the most common CA (45.8% among 83 patients), followed by trisomy 12 (20.5%); the frequencies of these two abnormalities were similar to those reported in western countries. Both FISH-CA of 17p- and 11q- were significantly independent factors for poor prognosis. Trisomy 3 was found in five patients; all were of Binet A stage but had a poor prognosis, suggesting that CLL with trisomy 3 may be a distinct disease entity. From these data we conclude that the incidence of CLL in Taiwan was dramatically increased in recent years and was associated with a birth-cohort effect. The CA of CLL in Taiwanese patients are similar to those in Western countries, indicating that although the incidence of CLL is lower in Taiwan than in the West, the pathogenesis of this disease in these two areas is similar. The finding from combined CG and FISH analysis can help predict prognosis. The clinical significance of trisomy 3 in CLL needs to be further investigated. Disclosures: No relevant conflicts of interest to declare.

Abstract 1564 Poster Board I-587 Somatic mutation of AML1/RUNX1 (RUNX1) gene is seen in acute myeloid leukemia (AML) M0 subtype and AML transformed from myelodysplastic syndrome, but the impact of this gene mutation on survival in AML patients remains unclear. In this study, we sought to determine the clinical implications of RUNX1 mutations in 470 adult patients with de novo non-M3 AML. Sixty-three distinct RUNX1 mutations were identified in 62 individuals (13.2%); 32 were in N-terminal and 31, C-terminal. The RUNX1 mutation was closely associated with male gender, older age, lower LDH value, FAB M0/M1 subtypes and expression of HLA-DR and CD34, but inversely correlated with CD33, CD15, CD19 and CD56 expression. Furthermore, the mutation was positively associated with MLL/PTD (P=0.0061), but negatively associated with CEBPA (P=0.0057) and NPM1 mutations (P=0.0001). AML patients with RUNX1 mutations had a significantly lower complete remission rate, shorter disease-free and overall survival than those without the mutation (P=0.0087, P Disclosures No relevant conflicts of interest to declare.

The suppressor of cytokine signaling-1 (SOCS1) protein is a tumor suppressor. Hypermethylation of SOCS1, resulting in transcriptional silencing, is suggested to play an important role in the development of cancers. We sought to characterize SOCS1 methylation in primary myelodysplastic syndrome (MDS) and to clarify its clinical implications. We analyzed the methylation status of SOCS1 by methylation specific polymerase chain reaction in 114 patients with primary MDS and performed serial studies in 29 of them. SOCS1 methylation occurred in 54 patients (47.4%), more frequently in patients with high-risk subtypes of MDS than in those with low-risk ones (52.6% vs. 25.8%, p = 0.011). SOCS1 methylation was closely associated with N-RAS gene mutation (p = 0.010) and inversely associated with good-risk karyotype (p = 0.021). With a median follow-up of 17 months (range, 1 to 231 months), two patients, who did not have SOCS1 methylation at diagnosis, acquired it during disease progression. SOCS1 methylation disappeared after hematopoietic stem cell transplantation in two patients who had it initially. The patients with SOCS1 methylation had a higher cumulative risk of leukemic transformation than the others (55.8% vs. 27.7% at 3 years, p = 0.004). This difference remained significant within the subgroup of patients with high-risk subtypes of MDS (67.3% vs 45.1% at 3 years, p = 0.045). This is the first report to demonstrate the clinical relevance of SOCS1 methylation in MDS. It may play an important role in the pathogenesis of MDS, especially among patients with high-risk subtypes. Correlation of SOCS1 methylation with clinical characteristics, cytogenetics and RAS mutations Total(n=114) SOCS1 methylated(n=54) SOCS1 unmethylated(n=60) p value Age 64(7~68) 64(7~84) 65(19~86) 0.231 Sex(M/F) 81/33 36/18 45/15 0.327 WBC(1000/ml) 4675(220~227200) 4710(440~227200) 4640(1570~87420) 0.913 Hemoglobin(g/dl) 8.5(3.9~14.4) 8.9(4.4~12.9) 7.8(3.9~14.4) 0.348 Platelet(1000/ml) 88.5(2~607) 102(2~400) 74(3~607) 0.453 BM blast(%) 5.8(0~54.8) 7.2(0~54.8) 4.4(0~25.4) 0.033 Cytogenetics 0.021 Poor 19 13(68.4%) 6(31.6%) -7/7q- 11 8(72.7%) 3(27.3%) complex 10 6(60%) 4(40%) Intermediate 15 10(66.7%) 5(33.3%) Good 70 27(38.6%) 43(61.4%) FAB subtypes 0.011 Low-risk 31 8(25.2%) 23(74.2%) RA 21 4(19.0%) 17(81.0) RARS 10 4(40%) 6(60%) High-Risk 78 41(52.6%) 37(47.4%) RAEB 33 20(60.6%) 13(39.4%) RAEB-T 20 11(55.0%) 9(45.0%) CMML 25 10(40.0%) 15(60.0%) AML 5 5(100%) 0 IPSS 0.008 Low-risk 57 21(36.8%) 36(63.2%) Low 21 4(19.0%) 17(81.0%) Int-1 10 4(40.0%) 6(60.0%) High-risk 46 29(63.0%) 17(37.0%) Int-2 33 29(63.0%) 17(37.0%) High 20 11(55.0%) 9(45.0%) N-RAS 0.010 Mutated 11 9(81.8%) 2(18.2%) Wild 96 38(39.6%) 58(60.4%) K-RAS 1.000 Mutated 4 2(50%) 2(50%) Wild 101 44(43.6%) 57(56.4%) Cumulative risk of leukemic transformation in MDS patients. A. Comparison for all patients with and without SOCS1 methylation. B. Comparison for patients of high-risk subtypes of MDS (RAEB, RAEB-T, CMML) with and without SOCS1 methylation Cumulative risk of leukemic transformation in MDS patients. A. Comparison for all patients with and without SOCS1 methylation. B. Comparison for patients of high-risk subtypes of MDS (RAEB, RAEB-T, CMML) with and without SOCS1 methylation

Purpose: In patients of acute myeloid leukemia (AML) with mutations in Nucleophosmin (NPM), minimal residual disease (MRD) can be quantified by real-time polymerase chain reaction (qPCR). However, the validity of MRD detection by qPCR on a long-term basis and its prognostic significance remain undetermined. Patients and Methods: We quantified NPM mutants by qPCR in genomic DNA of 202 bone marrow samples serially collected during a median follow-up of 21.9 months (range 2.5 to 204) from 40 AML patients bearing this mutation. Results: With a relatively homogeneous number of mutants at diagnosis, induction chemotherapy resulted in a median of 2.78 logs of reduction (p Conclusion: Quantification of MRD by qPCR on genomic DNA in AML bearing NPM mutation predicts outcomes of this group of patients.