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Angiogenic factors play an essential role in normal and pathologic angiogenesis, but their clinical role in acute myeloid leukemia (AML) remains unclear. We investigated the expression of Ang-1, Ang-2, Tie2, VEGF-A, and VEGF-C genes in bone marrow (BM) mononuclear cells by real-time quantitative PCR (RQ-PCR) in a cohort of 126 patients with newly diagnosed de novo AML and normal marrow donors. Here we show that high pre-treatment levels of Ang-2 in the BM indicate an unfavorable prognosis in AML. Only karyotype (hazard ratio 2.19, 95% CI 1.25-3.42, P=0.005) and expression of Ang-2 (hazard ratio 2.05, 95% CI 1.20-3.52, P=0.009), but not other angiogenic factors, were independent prognostic factors for overall survival by multivariate analysis. The prognostic significance of Ang-2 expression was more obvious in the subgroup of patients with intermediate-risk cytogenetics. Subgroup analysis showed that Ang-2 expression had prognostic impact on patients with low (but not high) Ang-1 or Tie2 levels, and on patients with high (but not low) VEGF-A or VEGF-C levels.

The growth of myeloma cells depends on bone marrow (BM) stroma consisting of stromal cells, secreted cytokines and the extracellular matrix (ECM). Decorin, a small leucine-rich proteoglycan in the ECM, is a signaling ligand and native anti-tumor agent. However, the role of decorin in patients with myeloma is not clear. We evaluated the correlation between the decorin levels measured by enzyme-linked immunosorbent assay in BM plasma from 121 patients with newly diagnosed myeloma based on their clinical features and treatment response. The median decorin levels in the patients and the normal control group were 12.31 ng/mL [standard deviation (SD), 7.50 ng/mL; range, 2.45 to 44.46 ng/mL] and 10.31 ng/mL (SD, 2.42 ng/mL; range, 4.85-15.14 ng/mL), respectively (P < 0.001). Using 15.15 ng/mL as a cut-off, 46 patients (38%) exhibited higher decorin levels (H-DCN), whereas the other patients exhibited normal to lower decorin levels (NL-DCN). Except for the median age, which was significantly younger in the H-DCN than in the NL-DCN group (60.6 ± 14.0 vs. 65.8 ± 12.2 years, respectively; P = 0.034), there were no differences between the two groups. However, in 79 patients who had received novel agent-based induction, the overall response rate was significantly better in the H-DCN than in the NL-DCN (97 vs. 63%, respectively; P < 0.001), as was the depth of responses (P = 0.008), which were not observed in those who had received chemotherapeutic agents alone. Progression-free survival (PFS) was significantly longer in H-DCN than NL-DCN (not reached vs. 19.5 mo, respectively; P = 0.0003). Multivariate analyses indicated that H-DCN, as a significantly independent factor, was associated with better treatment response (odds ratio, 20.014; 95% CI, 2.187-183.150; P = 0.008) and longer PFS (hazard ratio, 0.135; 95% CI, 0.051-0.361; P < 0.001). These findings disclose the potential role of decorin in myeloma and provide a basis for further study on possible synergistic anti-myeloma effects between decorin and the novel agents that target BM stroma.

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, was reported to over-express in multiple cancers, including gastric, lung, breast, and prostate cancers. It has been shown that the enzymatic activities of its extracellular metalloprotease domain mediate ErbB signaling pathway and promote breast cancer proliferation and migration. Nevertheless, in non-small cell lung cancer (NSCLC), the role of ADAM15 and its correlation to patient prognosis is still not well understood. Moreover, unlike in breast cancer, the enzymatic activities of the ADAM15 extracellular domain appeared to be independent of ErbB signaling and lung cancer proliferation, indicating an alternative mechanism of ADAM15 in promoting lung cancer progression. Here, we demonstrated that ADAM15 was over-expressed in primary NSCLC tissues compared to the adjacent normal tissues. Patients with ADAM15 high-expressing lung tumors had shorter survival times. Furthermore, we examined different isoforms of ADAM15 and discovered that, isoform 6 (i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, was significantly upregulated in primary lung cancer tissues. Comparison of i6 and isoform 1 (i1), which has no cytoplasmic SH3 binding motifs, revealed that the cytoplastmic domain is essential for lung cancer proliferation, as well as aggressiveness in an in vitro lung cancer migration model. We also showed that ADAM15 i6, but not i1, mediated ErbB signaling pathway through physical interaction with growth factor receptor-bound protein 2 (Grb2), and activation of Src homolog 2 domain containing (Shc). Most importantly, overexpression of ADAM15 i6 promoted NSCLC cell growth in a xenograft mouse model while knockdown of i6 resulted in tumor shrinkage. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will not only shed light on the molecular mechanisms of ADAM proteins, but also facilitate development of novel therapy in NSCLC. Citation Format: Hsin-Han Hou. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) proliferation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2015-4948

The TP53 mutation is frequently detected in acute myeloid leukemia (AML) patients with complex karyotype (CK), but the stability of this mutation during the clinical course remains unclear. In this study, TP53 mutations were identified in 7% of 500 patients with de novo AML and 58.8% of patients with CK. TP53 mutations were closely associated with older age, lower white blood cell (WBC) and platelet counts, FAB M6 subtype, unfavorable-risk cytogenetics and CK, but negatively associated with NPM1 mutation, FLT3/ITD and DNMT3A mutation. Multivariate analysis demonstrated that TP53 mutation was an independent poor prognostic factor for overall survival and disease-free survival among the total cohort and the subgroup of patients with CK. A scoring system incorporating TP53 mutation and nine other prognostic factors, including age, WBC counts, cytogenetics and gene mutations, into survival analysis proved to be very useful to stratify AML patients. Sequential study of 420 samples showed that TP53 mutations were stable during AML evolution, whereas the mutation was acquired only in 1 of the 126 TP53 wild-type patients when therapy-related AML originated from different clone emerged. In conclusion, TP53 mutations are associated with distinct clinic-biological features and poor prognosis in de novo AML patients and are rather stable during disease progression.

Squared M-ary quadrature amplitude modulation (QAM) constellations have been widely adopted for modern high-data-rate digital communications. Nevertheless, carrier recovery required by demodulation of 128-QAM signals is almost a formidable task for practical receiver design. In this paper, two types of modified 128-QAM constellations are proposed allowing utilization of a low-complexity carrier recovery mechanism in the receiver. With the proposed constellations, perfect recovery of a frequency offset up to plusmn2.5% of baud rate is guaranteed using a conventional carrier recovery method at the expense of slight Es/N0 and PAPR increment compared with the conventional 128-QAM constellations

Mutations in a variety of genes have been identified in MDS patients. Among them, mutations of additional sex combs-like 1 (ASXL1), found in 15-20% of MDS patients, have been identified as an independent poor prognostic factor. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced an MDS-like disease in mice after 1~2 years by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27 (Inoue et al. J Clin Invest. 2013). Given that ASXL1 mutations have been shown to be related to high-risk MDS or leukemic transformation, it is not clear how ASXL1-mutated MDS clones can transform into advanced MDS or AML. First, we examined genetic alterations in 368 WHO-defined MDS patients; ASXL1 mutations were detected in 64 of them (17.39%). Intriguingly, the patients with ASXL1 mutations had a significantly higher incidence of the concurrent SET binding protein 1 (SETBP1) mutation than those with the wild-type ASXL1 (6 out of 64, 9.38% vs. 2 out of 304, 0.66%, P=0.0005). Moreover, among ASXL1-mutated MDS patients, those harboring SETBP1 mutations had a higher incidence of leukemic transformation than those without (P=0.042), and MDS patients with both mutations had a significantly shorter overall survival compared to those without SETBP1 mutations (median, 10.5 vs. 22.5 months, P=0.046). In addition, we demonstrated that most SETBP1 mutations, such as D868N, occur in the PEST domain of the SKI homology region, preventing ubiquitination and subsequent proteasomal degradation. These results prompted us to investigate whether SETBP1 mutations play a critical role in the leukemic transformation of ASXL1-mutated MDS cells. In in vitro experiments, the expression of SETBP1-D868N enhanced myeloid colony formation of ASXL1-MT-transduced LSK cells, augmenting ASXL1-MT-induced differentiation blocking of 32Dcl3 cells. Of note, SETBP1-D868N collaborated with ASXL1-MT to induce AML after a short latency (median survival, 73 days) in a murine BMT model, while all mice expressing either ASXL1-MT or SETBP1-D868N survived for 6 months after transplantation (P To clarify the molecular mechanism leading to leukemic transformation, we first investigated the Pp2a-Akt pathway because SETBP1 protein has been shown to interact with SET oncoprotein, resulting in Pp2a phosphorylation and subsequent inhibition. Consistent with previous reports using overexpression systems of SETBP1 wild type protein (SETBP1-WT), BM cells of leukemic mice displayed phosphorylated Pp2a and Akt compared to those of the control mice. Administration of FTY720, a Pp2a activator, efficiently repressed the growth rate in vitro and slightly improved the survival of serially transplanted mice. Next, using RNA-seq and GSEA, we demonstrated that SETBP1-D868N enriched hematopoietic stem cell-related genes and posterior Hoxa genes. Chromatin immnoprecipitation assay showed that both SETBP1-WT and SETBP1-D868N interacted with the promoter regions of Hoxa9 and Hoxa10, raising the possibility that a gain-of-function mutant of SETBP1 enhances transcription of these genes, directly or indirectly. Moreover, GSEA indicated global repression of the TGF-β signaling pathway and reciprocal upregulation of the Myc pathway in leukemic mice. In conclusion, our data provide evidence for the role of SETBP1 mutations in leukemic transformation and suggest the resulting deregulated pathways as potential therapeutic targets to prevent disease progression in MDS. Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding.

Introduction Acute myeloid leukemia (AML) is a highly heterogeneous disease with various cytogenetic and molecular abnormalities, some of which bear prognostic significance. Expression levels of some single microRNAs are influential for prognosis, but a system integrating several together and considering the weight of each should be more powerful. We sought to define a simple microRNA signature to predict prognosis in the patients. Method A cohort of 195 de novo AML patients who had cryopreserved marrow cells for study were subjected to global microRNA analysis. Analysis for overall survival (OS) was based on the 138 patients who received standard intensive chemotherapy (NTUH cohort). The AML cohort from the Cancer Genome Atlas (TCGA), which contains publically available data of microRNA and clinical information, serves as a validation cohort. To build a risk scoring system based on the global microRNA expression, we first analyzed the association between OS and the expression levels of individual microRNAs using univariate Cox proportional hazards regression model. MicroRNAs whose expression levels harbored top significance on OS (univariate Cox P < 0.005) were then analyzed with the multivariate Cox model. These microRNAs with independent survival significance (multivariate Cox P < 0.1) were selected to generate a risk scoring system, in which the expression of component microRNAs went through another round of multivariate Cox regression test to get beta values as weights for each microRNA. We also analyzed the global mRNA expression profiles from ours and TCGA cohort to sort out the physiologic pathways associated with high/low scores. Results Eleven microRNAs were significantly associated with OS by univariate Cox analysis (P < 0.005). After introducing the expression of these microRNAs into a multivariate Cox model, we identified high expression of hsa-miR-9-5p and hsa-miR-155-5p were independently associated with poor OS, while that of hsa-miR-203 had a trend of association with favorable OS. We constructed a risk scoring system: Risk = 0.4908 [Zhsa-miR-9-5p] + 0.2243 [Zhsa-miR-155-5p] - 0.7187 [Zhsa-miR-203], where the weights of microRNAs are beta values from multivariate Cox analysis and ZmicroRNA means the levels of specific microRNA expression after z-transformation. AML patients with higher scores had significantly shorter OS compared with those with lower scores in ours (median 13.5 months vs. not reached, P < 0.0001, Figure 1) as well as in the validation TCGA AML cohort (median 12.2 vs 26.4 months, P = 0.008, Figure 2). When restricting the analysis in our patients with a normal karyotype, the OS of patients with higher scores still fared worse (median 17.0 months vs. not reached, P = 0.006). Moreover, high score appeared to be an unfavorable prognostic factor independent of age, white cell count, cytogenetics, and gene mutation in both ours (HR=2.079, 95% CI 1.407-3.073, p Conclusion We present a simple and user-friendly 3-microRNA signature as a powerful prognostic factor for AML through multiple rounds of statistical analyses on our cohort and further validation by another independent patient group. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Development of the eye is morphologically similar among vertebrates, indicating that the underlying mechanism regulating the process may have been highly conserved during evolution. Herein we analyzed the promoter of the human betaB1-crytallin gene in zebrafish by transgenic experiments. To delineate the evolutionarily conserved regulatory elements, we performed serial deletion assays in the promoter region. The results demonstrated that the -90/+61-bp upstream proximal promoter region is sufficient to confer lens-tissue specificity to the human betaB1-crystallin gene in transgenic zebrafish. Through phylogenetic sequence comparisons and an electrophoretic mobility shift assay (EMSA), a highly conserved cis-element of a six-base pair sequence TG(A/C)TGA, the consensus sequence for the Maf protein binding site, within the proximal promoter region was revealed. Further, a site-mutational assay showed that this element is crucial for promoter activity. These data suggest that the fundamental transcriptional regulatory mechanism of the betaB1-crystallin gene has been well conserved between humans and zebrafish, and plausibly among all vertebrates, during evolution.

Lung cancer is the leading cause of cancer-related deaths in worldwide, including in Taiwan. Lung cancer can typically be grouped into two large categories: small-cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which account for 15% and 85% of lung cancers, respectively. Despite the continuing endeavors by scientists and clinicians, the cure rate remains low and there is urgent to develop a novel target for NSCLC therapy. The a disintegrin and metalloprotease (ADAM)-15 is a transmembrane protease mediating a wide variety of activities including proteolysis, adhesion, cell fusion and signaling. The expression of ADAM-15 is significantly up-regulated in NSCLC compared to normal tissue. However, the mechanisms of ADAM-15-modulated tumorigenesis in NSCLC are remained unclear. The external enzymetic activity of ADAM15 doesn’t involve in the common pathways in tumoregenesis of NSCLC, such as HER3-EGFR signaling. ADAM15 isoforms are different in cytoplasmic tails, which may drive different biological behavior through intracellular signaling. Therefore, we hypothesized that the diverse cytosolic domain of ADAM15 isoforms plays a crucial role in tumoregenesis of NSCLC. In this study, using real-time quantitative PCR measuring the mRNA expression of 22 ADAMs in NSCLC, we identified a correlation of higher expression of ADAM15 in NSCLC of non-smokers (p=0.002, compared with ex- and current smokers) and in adenocarcinoma (p=0.038, compared with squamous cell ca). Comparing to non-invasive CL1-0 cells and matched normal tissue, CL1-5 cells and NSCLC tissue expressed more the longest ADAM-15 isoform with the largest amount of Src homology (SH)3-binding domain. The association ADAM-15 with SH3-motif containing protein, growth factor receptor-bound (Grb) 2, promoted the ability of proliferation in NSCLC in vitro and in vivo. In conclusion, we suggested that the cytosolic domain of ADAM-15 promotes proliferation of NSCLC via enhance of Grb2-associated signaling pathways, and the inhibition of ADAM-15 may be a potential therapeutic target for NSCLC. Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintegrin and metalloprotease (ADAM)-15 promotes proliferation of non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3317. doi:10.1158/1538-7445.AM2014-3317

Recently, mutations of the additional sex comb-like 1 (ASXL1) gene were identified in patients with myelodysplastic syndrome (MDS), but the interaction of this mutation with other genetic alterations and its dynamic changes during disease progression remain to be determined. In this study, ASXL1 mutations were identified in 106 (22.7%) of the 466 patients with primary MDS based on the French-American-British (FAB) classification and 62 (17.1%) of the 362 patients based on the World Health Organization (WHO) classification. ASXL1 mutation was closely associated with trisomy 8 and mutations of RUNX1, EZH2, IDH, NRAS, JAK2, SETBP1 and SRSF2, but was negatively associated with SF3B1 mutation. Most ASXL1-mutated patients (85%) had concurrent other gene mutations at diagnosis. ASXL1 mutation was an independent poor prognostic factor for survival. Sequential studies showed that the original ASXL1 mutation remained unchanged at disease progression in all 32 ASXL1-mutated patients but were frequently accompanied with acquisition of mutations of other genes, including RUNX1, NRAS, KRAS, SF3B1, SETBP1 and chromosomal evolution. On the other side, among the 80 ASXL1-wild patients, only one acquired ASXL1 mutation at leukemia transformation. In conclusion, ASXL1 mutations in association with other genetic alterations may have a role in the development of MDS but contribute little to disease progression.

Background Ruxolitinib is a potent JAK1/JAK2 inhibitor that has demonstrated rapid and durable reductions in splenomegaly, improved MF-related symptoms and quality of life (QoL), and prolonged survival in 2 phase 3 studies comparing ruxolitinib with placebo (COMFORT-I) and best available therapy (COMFORT-II). However, no clinical trial in pts with MF had been conducted in Asian countries, and only a limited number of Asian pts or healthy volunteers had been enrolled in any ruxolitinib study. Methods This study was an open-label phase 2 study evaluating ruxolitinib in Asian pts with PMF, PPV-MF, or PET-MF who had palpable splenomegaly ≥ 5 cm below the costal margin and intermediate-2– or high-risk MF by the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) criteria. Pts received starting doses of ruxolitinib 15 or 20 mg twice daily (bid) based on baseline platelet count (100-200 or > 200 × 109/L, respectively); dose adjustments balancing safety and efficacy were allowed to titrate each pt to their most appropriate dose. The primary endpoint was met if the proportion of pts achieving ≥ 35% reduction in spleen volume from baseline at week 24 was ≥ 27.5% as measured by MRI/CT. Symptomatic response was assessed as a secondary endpoint using the 7-day modified MF Symptom Assessment Form (MFSAF) v2.0 total symptom score (TSS) and European Organisation for Research and Treatment of Cancer QoL Questionnaire Core 30 (EORTC QLQ-C30). The study was conducted in China (n = 63), Japan (n = 30), Korea (n = 17), and Taiwan (n = 10). The data cutoff date for this analysis was 7 June 2013. Results Overall, 120 pts were enrolled (PMF, n = 80; PPV-MF, n = 21; PET-MF, n = 19), and their baseline characteristics were as follows: median age, 61 years (range, 25-80 years); 51.7% female; 69.2% intermediate-2 and 30.8% high risk by IWG-MRT criteria; median palpable spleen size, 15 cm (range, 5-45 cm); median spleen volume, 2159 cm3; 55.8% of pts had prior exposure to hydroxyurea. The median follow-up was 8.44 months; 22.5% of pts discontinued treatment, primarily for adverse events (AEs; 9.2%) and disease progression (7.5%). The median duration of treatment was 8.44 months (range, 0.5-21.7 months), and the median daily dose was 20.64 mg/day in the 15 mg bid group (n = 46) and 36.11 mg/day in the 20 mg bid group (n = 74). All pts were evaluable for achievement of the primary endpoint, 101 pts remained on study and were evaluable at week 24, and 96 pts had nonzero scores on the MFSAF-TSS and were evaluable for a reduction from baseline. Most pts who had assessments at week 24 (91% [92/101]) had a reduction from baseline in spleen volume (Figure). The study met the primary endpoint, with 31.7% (38/120) of all pts achieving ≥ 35% reduction from baseline at week 24. Overall, 38.3% (46/120) of pts achieved ≥ 35% reduction from baseline in spleen volume at any time on study. As measured by the 7-day MFSAF, 49% (47/96) of pts achieved ≥ 50% reduction from baseline in TSS (median reduction, 47.2%). Pts experienced an improvement from baseline at week 24 in EORTC global health status/QoL (mean change, 5.2). The most common nonhematologic AEs (≥ 10%) regardless of relationship to study medication included diarrhea (25.8%), upper respiratory tract infection (17.5%), ALT level increased (15.0%), pyrexia (15.0%), AST level increased (13.3%), cough (11.7%), herpes zoster infection (11.7%), nasopharyngitis (10.8%), constipation (10.0%), gamma-glutamyl transferase level increased (10.0%), and headache (10.0%), and most were grade 1/2. Serious AEs were reported for 24.2% of pts, and 65.8% of all pts had grade 3/4 AEs. The most common new or worsening laboratory abnormalities were low hemoglobin (all grade 3, 55.7%), low lymphocyte (grade 3/4, 19.5%), low platelet (grade 3/4, 15.3%), and low ANC (grade 3/4, 7.6%) levels. AEs observed in this study were consistent with those observed in the 2 large phase 3 COMFORT studies. Six pts (5%) died on treatment or within 30 days of discontinuation. Summary/conclusions Findings from this study demonstrated that ruxolitinib was relatively well tolerated in Asian pts with MF and provided substantial reductions in splenomegaly and modest improvements in MF-associated symptoms. The AEs observed with ruxolitinib treatment in this study are consistent with those observed in the large phase 3 COMFORT studies, and there were no new AEs associated with ruxolitinib in Asian pts with MF. Disclosures: Okamoto: Novartis: Honoraria, Research Funding. Sirulnik:Novartis: Employment. Ruiz:Novartis: Employment. Amagasaki:Novartis: Employment. Ito:Novartis: Employment. Akashi:Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Introduction Homeodomain-only protein homeobox (HOPX) gene encodes a homeodomain protein without DNA-binding domains and functions as an adaptor protein to mediate transcriptional repression. HOPX is known to regulate cardiac development through interaction with serum responsive factor (SRF), thereby inhibiting SRF-dependent transcription either by interference of its DNA binding or recruitment of histone deacetylase. Recent studies have suggested that HOPX acts as a tumor suppressor gene since loss of its expression has been reported in several solid cancers. However the biological effects of HOPX in hematopoietic malignancies have not been explored. In this study, we aim to see the clinical and biological relevance of HOPX expression in acute myeloid leukemia (AML). Methods and materials We performed global mRNA arrays for 181 newly diagnosed AML patients in the National Taiwan University Hospital. There are also detailed demographic, clinical and genetic data for these patients. The HOPX gene expression levels extracted from the array data were analyzed for its clinical and biological relevance. We also tried to validate our findings by analyzing the public databases of AML. Results The 181patients were divided into two groups based on the median level of HOPX expression on the arrays. Patients with higher expression had higher platelet counts than those with lower expression (median, 59000/μL vs. 35000/μL, P=0.008). Higher HOPX expression was negatively associated with favorable karyotypes including t(8;21) and t(15;17). To investigate the association of gene mutations with HOPX expression in AML, we also performed a mutational screening for 17 genes. We found that patients with higher HOPX expression had significantly higher incidence of RUNX1 mutation (23.1% vs. 4.4%, P With a median follow-up of 33 months, patients with higher HOPX expression had shorter overall survival (OS) compared with patients with lower HOPX expression (median, 17 months vs. not reached, P=0.004). In multivariate analyses, higher expression of HOPX still represented a poor prognostic factor for OS independent of age, white blood cell counts, karyotype, NPM1 mutation, FLT3-ITD, CEBPAdouble mutation and RUNX1 mutation (P=0.021). Considering the hypothetical role of HOPX as a tumor suppressor in solid tumors, it seems surprising to see the negative impact on OS of high HOPX expression in AML. To be more assuring, we analyzed HOPX expression in another two large independent AML patient cohorts, TCGA and GSE12417_GPL96. We obtained the same results that higher HOPX expression was an unfavorable prognostic factor. Conclusions Higher expression of HOPX in AML patients was correlated with higher platelet counts and mutations in RUNX1 and IDH2, but negatively associated with favorable karyotypes such as t(8;21) and t(15;17) and CEBPA mutation or FLT3-TKD. Higher expression of HOPX appeared to be an independent unfavorable prognostic factor in our cohort, and its negative prognostic impact could be validated in another 2 large independent cohorts of AML. Further studies are needed to explore the biological significance of this gene expression in AML. Disclosures: No relevant conflicts of interest to declare.

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.

Chronic pulmonary obstructive disease (COPD) is the fourth leading cause of death worldwide, however, the pathogenic factors and mechanisms are not fully understood. Pulmonary emphysema is one of the major components of COPD and is thought to result from oxidative stress, chronic inflammation, protease–antiprotease imbalance and lung epithelial (LE) cell apoptosis. In our previous studies, COPD patients were noted to have higher levels of placenta growth factor (PlGF) in serum and bronchoalveolar lavage fluid than controls. In addition, transgenic mice overexpressing PlGF developed pulmonary emphysema and exposure to PlGF in LE cells induced apoptosis. Furthermore, intratracheal instillation of porcine pancreatic elastase (PPE) on to PlGF wild type mice induced emphysema, but not in PlGF knockout mice. Therefore, we hypothesized that PPE generates pulmonary emphysema through the upregulation of PlGF expression in LE cells. The elevation of PlGF then leads to LE cell apoptosis. In the present study, we investigated whether PPE induces PlGF expression, whether PlGF induces apoptosis and whether the downstream mechanisms of PlGF are related to LE cell apoptosis. We found that PPE increased PlGF secretion and expression both in vivo and in vitro. Moreover, PlGF-induced LE cell apoptosis and PPE-induced emphysema in the mice were mediated by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways. Given these findings, we suggest that the increase in PlGF and PlGF-induced JNK and p38 MAPK pathways contribute to PPE-induced LE cell apoptosis and emphysema. Regulatory control of PlGF and agents against its downstream signals may be potential therapeutic targets for COPD.

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 2697 Purpose: Although the clinical and biological features of Isocitrate dehydrogenase (IDH) mutations in acute myeloid leukemia (AML) have been characterized, its stability and in vivo sufficiency of the mutation alone for leukemogenesis remain uninvestigated. Patients and Methods: Mutations of IDH and other clinically relevant genes were analyzed in the bone marrow from 446 adult patients with de novo non-M3 AML. IDH2 mutations were examined serially in 140 patients at diagnosis and after chemotherapy. Results: Among the 446 adults with de novo non-M3 AML, IDH2 R172, R140, and IDH1 R132 mutations occurred at a frequency of 2.9%, 9.2%, and 6.1%, respectively. IDH2 mutation was associated with higher platelet counts (p=0.046), intermediate-risk (p=0.002) or normal karyotype (p=0.023), and isolated +8 (p=0.014), but was inversely correlated with expression of HLA-DR (p=0.002), CD34 (p=0.039), CD15 (p=0.003), CD7 (p=0.010), and CD56 (p=0.048), and was mutually exclusive with WT1 mutation (p=0.037) and core-binding factor translocations (p=0.001). All these correlations became stronger when IDH1 and IDH2 mutations were considered together, suggesting similarity of biological roles between these 2 mutations. However, IDH2 but not IDH1 mutation conferred a better prognosis (Fig 1), especially in those with normal karyotype or intermediate cytogenetics (median overall survival: not reached vs. 58 months, p=0.044 and not reached vs. 19 months, p=0.027 for normal and intermediate karyotype group, respectively). Importantly, IDH2 but not IDH1 mutation was an independent favorable prognostic factor (HR: 0.332, 95% CI: 0.159–0.694; p=0.003). Patients with IDH2−/FLT3-ITD+ genotype had especially worse prognosis (median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD− group: 12 months vs. not reached; p=0.003; median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD+ or IDH2−/FLT3-ITD− group : 12 months vs. 35 months; p Conclusion: IDH2 mutation is a stable marker during disease evolution and confers favorable prognosis. FLT3-ITD combined with wild type IDH2 exerted synergistic negative impact on survival. IDH2 mutation alone is insufficient for leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1569 Poster Board I-593 Acute myeloid leukemia (AML) is a clonal disorder characterized by acquired genetic abnormalities in hematopoietic progenitors. CCAAT/enhancer binding protein α (C/EBPα) is a basic leucine zipper transcription factor that regulates differentiation-dependent genes during granulocyte differentiation. CEBPA mutations have been observed in several human malignancies. Epigenetic modification of the distal CEBPA promoter region (-1422 to -896) has been reported to result in down-regulation of CEBPA expression in lung cancer, pancreatic cancer, and head and neck squamous cell carcinoma. Apart from solid tumors, epigenetic modification of CEBPA in AML has also been reported. Chim et al. reported aberrant CEBPA core promoter methylation in a small proportion (2.85%) of patients with AML. Wouters et al. identified a subgroup of AML patients (1.4%) in which the CEBPA gene was silenced in association with CEBPA core promoter methylation. Hackanson et al. observed CEBPA methylation in the distal promoter region in 51% of selected AML patients. Due to the selection of differing patient populations and CEBPA promoter regions for analysis in past studies, the clinical significance of CEBPA methylation in AML remains unclear. To evaluate the CEBPA methylation status of patients with AML, 193 patients with de novo AML were evaluated for CEBPA methylation by bisulfite sequencing and MSP methods. A substantial proportion of patients were noted to have some degree of heterogeneous methylation in the distal region (-1423 to -1121), but not in the proximal region (-1121 to -896) or in the core region (-141 to +103), of the CEBPA promoter. Only one patient was noted to have broad methylation from the distal region to the core of the CEBPA promoter, and extending into exon1 (-25 to +103). This patient was a 61-year-old female with M1 subtype AML, a normal karyotype and an absence of CEBPA, NPM1 or FLT3 mutations. We further correlated the CEBPA methylation levels with CEBPA transcript levels in leukemic cells from 12 patients with AML and found a negative correlation (coefficient of correlation = -0.722, P = 0.017). To clarify the correlation between CEBPA methylation and clinical features in AML, quantitative MassARRAY analyses for CEBPA methylation were performed. Among the 193 patients studied, methylation levels ranged from 0.073 to 0.971 with a median value of 0.229, compared to normal bone marrow cells (NBM) with levels ranging from 0.207 to 0.269 (n=5, median: 0.218) and mature leukocytes (NPB) which ranged from 0.144 to 0.201 (n=5, median 0.161). Using complete linkage clustering, we divided the patients into two groups: one with high CEBPA methylation levels (≥ 0.55, n = 28), and the other with low methylation levels (< 0.55, n = 165). Clinical and laboratory features of these two groups are explored. Gene alteration analysis revealed NPM1 mutation was mutually exclusive with CEBPA methylation (P = 0.004), so was similar trend in CEBPA mutation. Of the 28 patients with high CEBPA methylation, only one patient had a CEBPA mutation. immunophenotyping work revealed that leukemic blasts from patients with high CEBPA methylation were predominately negative for HLA-DR (P = 0.005) and CD56 (P = 0.015) expression. Of the 140 patients receiving standard induction therapy, 110 (79.3%) patients achieved CR. With a median follow-up of 17.5 months, patients with high CEBPA methylation demonstrated a greater probability of survival (median >60 months vs. 20 months, P = 0.020) than those with low CEBPA methylation. In addition, in the subgroup of AML patients without favorable karyotypes as well as NPM1 and CEBPA mutations, patients with high CEBPA methylation (n = 14) still had better five-year survival (median 34 months vs. 11 months, P = 0.048) than those with low CEBPA methylation. Intriguingly, among the cytogenetically normal AML patients, except those with CEBPA and NPM1 mutations, the five-year survival of patients with high CEBPA methylation (n=8) was remarkably better than those with low CEBPA methylation (n=19; median >60 months vs. 11 months, P = 0.002). These results suggest that, in addition to current cytogenetic and molecular markers, CEBPA methylation status could emerge as a prognostic factor for survival advantage in AML patients. Disclosures No relevant conflicts of interest to declare.

Abstract 3373 Poster Board III-261 Purpose Several gene mutations had been found to have clinical implications in patients with acute myeloid leukemia (AML), especially in those with normal karyotype. However, the role of such gene mutations for AML patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT) was unclear and inconclusive. We retrospectively evaluated the prognostic impact of 8 gene mutations in adult AML patients undergoing allo-HSCT. Materials & Methods From 1995 to 2007, a total of 463 consecutive adult patients with de novo non-M3 AML had comprehensive gene mutation analyses at the National Taiwan University Hospital. Three hundred and twenty five patients who received conventional induction chemotherapy were enrolled in this study. Those who received only low dose chemotherapy or palliative treatment were excluded. The genetic alterations analyzed included NPM1, FLT3/ITD, FLT3/TKD, CEBPA, AML1/RUNX1, RAS, MLL/PTD, and WT1. The clinical implication of these genetic alterations in the patients receiving allo-HSCT was analyzed, and the result was compared with that in patients without allo-HSCT. Results The clinical characteristics in the patients receiving allo-HSCT (n=100) and those without (n=225) were similar with the exception of age, being younger in the former group (35.4 years vs. 49.5 years p 45 years), higher initial WBC count (WBC>50K/μL), elevated LDH level, unfavorable karyotype, FLT3/ITD, mutations of AML1/RUNX1 were significantly associated with poorer overall survival (OS) in patients not receiving allo-HSCT; While NPM1mut/FLT3ITDneg and CEBPA mutations served as significantly good prognostic indicators. In multivariate analysis, age, WBC count, karyotype, FLT3/ITD, AML1/RUNX1, CEBPA and NPM1mut/FLT3ITDneg remained to be independent prognostic factors in non-allo-HSCT patients. However, in patients receiving allo-HSCT, only unfavorable karyotype and disease status (refractory or remission) at the time of transplantation were associated with poorer OS both in univariate and multivariate analyses. The similar prognostic impact of FLT3/ITD, CEBPA, AML1/RUNX1 and NPM1 on OS was not seen in patients receiving allo-HSCT. Furthermore, in contrast to its poor prognostic impact in non-allo-HSCT patients, mutation of AML1/RUNX1 was a significant good prognostic factor for relapse free survival (p=0.046), although not for OS, in allo-HSCT group. Conclusion FLT3/ITD, mutations of AML1/RUNX1, CEBPA and NPM1 have great prognostic implication for OS in AML patients not receiving allo-HSCT. However, their impact on OS is ameliorated in patients receiving allo-HSCT. The results need to be confirmed by further studies on more patients. 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.

Purpose: Acute myeloid leukemia (AML) with t(7;11)(p15;p15), which results in fusion of NUP98 and HOXA9 genes, is rarely seen, especially in Western countries. Till now only few studies about this entity have been reported in literature, and they usually contained limited number of patients and did not have detailed characterization. This study evaluated the clinical and biological features of this group of patients from a large cohort. Methods: From 1994 to 2007, we had comprehensive studies on a total of 536 AML patients. We compared the clinical features and genetic mutations of NPM1, FLT3/ITD, FLT3/TKD, AML1, K-RAS, N-RAS, CEBPA, MLL/PTD, JAK2, PTPN11, and WT1 between patients with and without this translocation. We also characterized the fusion points in patients with this chromosomal abnormality and devised a quantitative real-time PCR to specifically quantify the leukemia cells bearing the genetic fusion. Results: Among the 536 patients, 11 patients (2%) were found to have t(7;11)(p15;p15). Comparing with the others, we found that AML patients bearing this translocation were younger (p=0.044), female predominant (p=0.010), and having a trend of higher lactate dehydrogenase (LDH) level (p=0.071). This group of patients had worse overall survival (OS) (median 13.5 vs. 36.5 month, p=0.005), relapse free survival (RFS) (median 6 vs. 15.5 months, p Conclusion: AML with NUP98-HOXA9 fusion has a characteristic profile in both clinical and biological aspects, and should be regarded as a poor prognostic group.

An increasing number of epigenetic deregulation was reported to be associated with acute myeloid leukemia (AML). Hypermethylation of several genes is associated with hematologic malignancies, such as the calcitonin gene, p15INK4B, CEBPA, SOCS1, DAPK, p21Cip/Waf1, the ER gene, SDC4, MDR. MicroRNAs (miRNAs) are endogenous nonconding small RNAs that inhibit the translation of genes whose 3′-UTR are targeted. Many miRNA genes, including let-7a-3, are located in CpG islands, suggesting possible epigenetic regulation of their expression. The let-7a-3 gene has been reported to be heavily methylated in normal human placenta, brain, bone marrow, colon, skin, and lung tissue but unmethylated in some lung cancer samples. In this communication, we used bisulfite sequencing and real-time reverse transcription-PCR to analyze methylation status in 33 CpG sites of the let-7a-3 gene and miRNA expression of let-7a in four myeloid cell lines, HEL, HL60, K562 and U937. All of them showed hypermethylation (93%, 80%, 71% and 90%, respectively) in let-7a-3 promoter region (Figure 1) and displayed little let-7a miRNA expression. To evaluate the significance of let-7a-3 methylation status in patients with AML, 90 patients with de novo AML were evaluated for let-7a-3 methylation status by using combined bisulfite restriction analysis (COBRA) and confirmation with bisulfite sequencing. Most AML patients were noted to have hypermethylation in the promoter region of let-7a-3 gene. We divided these patients into three groups: demethylated (n=6), hypomethylated (n=11) and hypermethylated (n=73). Correlation of let-7a-3 methylation with biological and clinical features was evaluated. We found that the methylation status was not associated with patient age at diagnosis, sex, FAB classification and cytogenetic change. Further sequential study revealed that no association was found between let-7a-3 methylation and disease status at either complete remission or relapse stage. We previously demonstrated that 25% AML patients showed hypermethylation in distant region of CEBPA promoter and the methylation status correlated with disease status. In this study, we found that up to 81% AML patients showed let-7a-3 methylation. In the meanwhile, further analysis revealed inverse association between CEBPA and let-7a-3 methylation (p=0.007). It has be widely known that CpG islands hypermethylation of tumor-suppressor genes is a common alteration in cancer cells, leading to the transcriptional inactivation of these genes and normal cellular malfunctions. At the same time, it is also known that the genome of the cancer cell undergoes global hypomethylation at repetitive sequences, leading to genome fragility and tumorigenesis. CEBPA is widely known to be tumor suppressor gene, and let-7a-3 was previously identified as with oncogenic function in lung adenocarcinomas. Consistent to the results shown in lung adenocarcinomas, our results indicate that the miRNA let-7a might play an oncogenic function in AML. Figure 1 let-7a-3 methylation status in 33 CpG sites of promoter region in four myeloid cell lines, HEL, HL60, K562 and U937. The nucleotide numbering are according to the DNA sequence from NT_011523. Figure 1. let-7a-3 methylation status in 33 CpG sites of promoter region in four myeloid cell lines, HEL, HL60, K562 and U937. The nucleotide numbering are according to the DNA sequence from NT_011523.

The development of acute myeloid leukemia (AML) is a multistep process. Gilliland and colleagues proposed a two hit theory of leukemogenesis that requires collaboration of at least two classes of gene mutations. The Class I gene mutations activate the signal transduction pathway and confer proliferation and survival advantage to hematopoietic cells. The Class II gene mutations affect transcriptional activators or coactivators and serve to impair cell differentiation. In this study, comprehensive analyses of a panel of gene mutations, their interactions and associations with antigen expression of leukemia cells were performed in 324 patients with primary AML, including 275 adults and 49 children(≤18years). The gene mutations included FLT3/ ITD (78 cases, 24.1%), FLT/ TKD (24 cases, 7.4%), NPM(63 cases, 19.4%), CEBPA(45 cases, 13.9%), NRAS (39 cases, 12%), AML1 (31 cases, 9.6%), PTPN11 (14 cases, 4.3%), MLL/PTD(13 cases, 4%), KIT(10 cases, 3.1%), KRAS (8 cases, 2.5%), and JAK2 (3 cases, 0.9%). In addition, 33 patients had t(8;21), 24 had t(15;17), 9 had inv(16) and 13 had 11q23 translocations. Totally, the Class I gene mutations were detected in 155 patients (47.8%), and Class II gene mutations, in 228 patients (70.4%). Most Class II mutation was associated with a distinct immunophenotype of leukemic cells, such as CEBPA mutation: HLADR(+)CD7(+)CD15(+)CD19(−)CD34(+) (p Table I. Interaction of Class I and Class II gene mutations

The development of acute myeloid leukemia (AML) is a multistep process. Recently, somatic mutations of PTPN11, the gene encoding non-receptor protein tyrosine phosphatase SHP-2, were observed in some hematological malignancies. However, the characteristics of adult AML with PTPN11 mutations have not been comprehensively studied, and the specific genetic alteration cooperative with a PTPN11 mutation in the leukemogenesis remains largely unknown. In this study, PTPN11 mutation and its association with other gene aberrations were analysed for 272 adult patients with de novo AML. Missense mutations were identified in 14 individuals (5.1%). The PTPN11 mutation was closely associated with old age (P=0.036), FAB M4/M5 subtypes (P=0.049), CD14 expression (P=0.026), a normal karyotype (P=0.044) and NPM1 mutation (P=0.037), but negatively associated with FLT3/ITD (P = 0.025). In addition, four patients revealed simultaneous mutations of PTPN11 and AML1 (n=3) or MLL (n=1). We observed a shorter overall survival for patients with PTPN11 mutation than those without amongst NPM1-wild patients (P=0.001), but not amongst NPM1-mutated patients (P=0.738). Our findings provide evidence that AML patients with PTPN11 mutations had some distinct biological and clinical characteristics, and the mutation may cooperate with other gene alterations to lead to AML in a subset of patients.

Cereblon (CRBN) is essential for the anti-myeloma (MM) activity of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide. However, the clinical implications of CRBN in MM patients are unclear. Using immunohistochemical (IHC) staining on paraffin-embedded bone marrow sections, the expression of CRBN protein in myeloma cells (MCs) was assessed in 40 relapsed/refractory MM (RRMM) patients who received lenalidomide/dexamethasone (LD) and 45 and 22 newly diagnosed MM (NDMM) patients who received thalidomide/dexamethasone (TD) and melphalan/bortezomib/prednisolone (MVP), respectively. IHC staining were scored on a scale representing the diffuseness and intensity of positive-staining MCs (range, 0-8) and a score ≥4.5 was used for CRBN positivity (CRBN(+)) on a cut-point analysis of all possible scores and response of TD and LD. Compared to CRBN(+) NDMM patients, CRBN(-) NDMM patients had more international staging system (ISS) III (26 vs. 61 %, respectively; P = 0.006). In the LD and TD cohorts, the response rate (RR) was higher in CRBN(+) patients than CRBN(-) patients (LD 79 vs. 33 %, respectively; P = 0.005) (TD 75 vs. 29 %, respectively; P = 0.005); however, this trend was not observed in the MVP cohort. In the LD and TD cohorts, the positive and negative prediction value of CRBN(+) for treatment response was 79 and 67 % and 75 and 71 %, respectively. Multivariate analysis showed that CRBN(+) was a significant factor associated with superior RR for LD and TD. The data suggest that expression of CRBN protein in MCs assessed using the IHC is a feasible approach to predict the response of IMiDs in MM patients.

Introduction Suppressor of cytokine signaling1 (SOCS1) protein, which encodes a member of the signal transducers and activators of transcription (STATs)-induced inhibitors, takes part in a negative regulation of cytokine signaling. The mechanism of SOCS1 in tumor carcinogenesis is complex and remains to be defined. Till now, there have been no studies concerning the prognostic implication of SOCS1 expression in acute myeloid leukemia (AML). Methods and Materials A total of 223 adult patients with newly diagnosed de novo AML who had enough cryopreserved cells for analysis at the National Taiwan University Hospital were enrolled consecutively. SOCS1 expression in bone marrow (BM) mononuclear cells was analyzed by quantitative real-time polymerase chain reaction. The results were correlated with FAB subtypes, clinical features, cytogenetics, other genetic alterations, and clinical outcome. Result The median value of SOCS1 expression was used as the cut-off value to divide patients into lower- and higher-expression groups. Higher SOCS1 expression was closely associated with older age (P=0.032) but inversely related to FAB M1 subtype and t(8;21)(q22;q22). There was no difference in other clinical parameters, including sex, hemoglobin level, white blood cell (WBC) counts, blast counts, and lactate dehydrogenase level between the two groups. Compared to patients with lower SOCS1 expression, those with higher expression had higher incidence of CD7 and CD34 expression on leukemic cells. To investigate the interactions of SOCS1 expression and other genetic alterations in the pathogenesis of AML, a complete mutational screening of 17 genes was performed. Higher SOCS1 expression was closely associated with NPM1 mutation and DNMT3A mutation (33% vs. 14.4%, P=0.002 and 20.9% vs. 10.8%, P=0.044, respectively), but negatively associated with CEBPA mutation (5.4% vs. 18.9%, P=0.002). Of the 154 AML patients receiving conventional intensive induction chemotherapy, 112 (72.7%) patients achieved complete remission (CR). The patients with higher SOCS1 expression had a lower probability of achieving CR than those with lower SOCS1 expression (62.9% vs. 81%, P=0.001). With a median follow-up time of 37 months (ranges, 0 to 160), patients with higher SOCS1 expression had poorer overall survival (OS) than those with lower SOCS1 expression (median 20 months vs. not reached, P=0.004). The same was also true among the patients with intermediate-risk cytogenetics and normal karyotype. In multivariate analysis, higher SOCS1 expression was an independent poor prognostic factor for OS in total cohort (relative risk, RR 1.947, 95% CI 1.081-3.508, P=0.026) irrespective of age, WBC, cytogenetics, NPM1/FLT3-ITD and CEBPA mutation. In the 77 cytogenetically-normal patients, higher SOCS1 expression was still an independent poor prognostic factor (RR 2.410, 95% CI 1.012-5.738, P=0.047). Interestingly, a scoring system incorporating SOCS1 expression and six other risk factors, including age, WBC, karyotype, FLT3/ITD, and mutations of NPM1 and CEBPA, into survival analysis was proved to be very useful to stratify AML patients into different risk groups (P =0.002). Conclusion AML patients with higher SOCS1 expression had distinct clinic-biologic features and poorer outcome. BM SOCS1 expression may serve as a new biomarker to risk stratify the patients. Disclosures No relevant conflicts of interest to declare.

Address: 1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, 2Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, 3Institute of Pharmacology and Toxicology, School of Medicine, Tzu Chi University, Hualien, Taiwan, 4Department of Pediatrics, Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan, 5Department of Animal Science and Technology, National Taiwan University, Taiwan and 6Graduate Institute of Medical Sciences and Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan