Eun-Hye Hur

C3435T polymorphism of the MDR1 gene is not associated with P-glycoprotein function of leukemic blasts and clinical outcome in patients with acute myeloid leukemia

We investigated the association between the MDR1 C3435T polymorphism and P-glycoprotein function of leukemic blasts as well as clinical outcomes in 200 patients with AML, excluding the M3 subtype. The CC, CT and TT genotype frequencies of the C3435T polymorphism among patients were 71, 93 and 36, respectively. The C3435T polymorphism genotypes did not have influence on the P-glycoprotein function of leukemic blasts. Complete remission rates and overall, relapse-free and event-free survival rates were not significantly different among the C3435T polymorphism genotypes. In conclusion, the MDR1 C3435T polymorphism does not appear to have significant clinical implications in AML.

Somatic mutations predict outcomes of hypomethylating therapy in patients with myelodysplastic syndrome.

Although hypomethylating therapy (HMT) is the first line therapy in higher-risk myelodysplastic syndromes (MDS), predicting response to HMT remains an unresolved issue. We aimed to identify mutations associated with response to HMT and survival in MDS. A total of 107 Korean patients with MDS who underwent HMT (57 responders and 50 non-responders) were enrolled. Targeted deep sequencing (median depth of coverage 1,623X) was performed for 26 candidate MDS genes. In multivariate analysis, no mutation was significantly associated with response to HMT, but a lower hemoglobin level (<10g/dL, OR 3.56, 95% CI 1.22-10.33) and low platelet count (<50,000/μL, OR 2.49, 95% CI 1.05-5.93) were independent markers of poor response to HMT. In the subgroup analysis by type of HMT agents, U2AF1 mutation was significantly associated with non-response to azacitidine, which was consistent in multivariate analysis (OR 14.96, 95% CI 1.67-134.18). Regarding overall survival, mutations in DNMT1 (P=0.031), DNMT3A (P=0.006), RAS (P=0.043), and TP53 (P=0.008), and two clinical variables (male-gender, P=0.002; IPSS-R H/VH, P=0.026) were independent predicting factors of poor prognosis. For AML-free survival, mutations in DNMT3A (P<0.001), RAS (P=0.001), and TP53 (P=0.047), and two clinical variables (male-gender, P=0.024; IPSS-R H/VH, P=0.005) were independent predicting factors of poor prognosis. By combining these mutations and clinical predictors, we developed a quantitative scoring model for response to azacitidine, overall- and AML-free survival. Response to azacitidine and survival rates became worse significantly with increasing risk-scores. This scoring model can make prognosis prediction more reliable and clinically applicable.

Clinical significance of GSTM1 and GSTT1 polymorphisms in younger patients with acute myeloid leukemia of intermediate-risk cytogenetics

We investigated the association between GSTM1 or GSTT1 polymorphisms and clinical outcomes in 133 younger patients with AML of intermediate-risk cytogenetics. Clinical outcomes were not significantly different among the GSTM1 polymorphism genotypes, whereas cumulative incidence of relapse (CIR) was significantly lower and event-free survival (EFS) was significantly higher in patients with the GSTT1-present genotype compared with those with the GSTT1-null genotype (CIR at 5 year, 28.9% vs. 44.6%, P=0.018; EFS at 5 year, 51.4% vs. 34.1%, P=0.029). Our results suggest that GSTT1 gene polymorphism has significant clinical implications in younger patients with AML of intermediate-risk cytogenetics.

Prospective Randomized Comparison of Idarubicin and High-Dose Daunorubicin in Induction Chemotherapy for Newly Diagnosed Acute Myeloid Leukemia

Purpose We compared two induction regimens, idarubicin (12 mg/m2/d for 3 days) versus high-dose daunorubicin (90 mg/m2/d for 3 days), in young adults with newly diagnosed acute myeloid leukemia (AML). Patients and Methods A total of 299 patients (149 randomly assigned to cytarabine plus idarubicin [AI] and 150 assigned to cytarabine plus high-dose daunorubicin [AD]) were analyzed. All patients received cytarabine (200 mg/m2/d for 7 days). Results Complete remission (CR) was induced in 232 patients (77.6%), with no difference in CR rates between the AI and AD arms (80.5% v 74.7%, respectively; P = .224). At a median follow-up time of 34.9 months, survival and relapse rates did not differ between the AI and AD arms (4-year overall survival, 51.1% v 54.7%, respectively; P = .756; cumulative incidence of relapse, 35.2% v 25.1%, respectively; P = .194; event-free survival, 45.5% v 50.8%, respectively; P = .772). Toxicity profiles were also similar in the two arms. Interestingly, overall and event-free survival times of patients with FLT3 internal tandem duplication (ITD) mutation were significantly different (AI v AD: median overall survival, 15.5 months v not reached, respectively; P = .030; event-free survival, 11.9 months v not reached, respectively; P = .028). Conclusion This phase III trial comparing idarubicin with high-dose daunorubicin did not find significant differences in CR rates, relapse, and survival. Significant interaction between the treatment arm and the FLT3-ITD mutation was found, and high-dose daunorubicin was more effective than idarubicin in patients with FLT3-ITD mutation.

Expression and prognostic significance of microRNAs in Korean patients with myelodysplastic syndrome

Background/Aims Various alterations of microRNA (miRNA) expression have been reported in myelodysplastic syndrome (MDS). We aimed to investigate the unique patterns and prognostic significance of miRNA expression in Korean patients with MDS. Methods Bone marrow mononuclear cells were collected from eight healthy controls and 26 patients with MDS, and miRNAs were isolated and assessed via quantitative real-time polymerase chain reaction for selected miRNAs, including miR-21, miR-124a, miR-126, miR-146b-5p, miR-155, miR-182, miR-200c, miR-342-5p, miR-708, and Let-7a. Results MiR-124a, miR-155, miR-182, miR-200c, miR-342-5p, and Let-7a were significantly underexpressed in patients with MDS, compared to healthy controls. MiR-21, miR-126, 146b-5p, and miR-155 transcript levels were significantly lower in international prognostic scoring system lower (low and intermediate-1) risk MDS than in higher (intermediate-2 and high) risk MDS. Higher expression levels of miR-126 and miR-155 correlated with significantly shorter overall survival and leukemia-free survival. Higher miR-124a expression also tended to be related to shorter survivals. Conclusions Although our study was limited by the relatively small number of patients included, we identified several miRNAs associated with pathogenesis, leukemic transformation, and prognosis in MDS.

Comparison of anthracyclines used for induction chemotherapy in patients with FLT3 -ITD-mutated acute myeloid leukemia

This retrospective analysis compared anthracyclines (as part of an induction regimen) in 128 newly diagnosed FLT3-ITD-mutated AML patients. Induction regimens comprised high-dose daunorubicin (HD-DN; 90 mg/m2/d × 3d; n = 44), standard-dose daunorubicin (SD-DN; 45 mg/m2/d × 3d; n = 51), or idarubicin (IDA; 12 mg/m2/d × 3d; n = 33) in combination with cytarabine (100-200 mg/m2/d × 7d). Fifty-three patients showing persistent leukemia on interim bone marrow examination received a second course of induction chemotherapy comprising 2 days of daunorubicin (45 mg/m2/d) or IDA (8 or 12 mg/m2/d) in addition to 5 days of cytarabine. Complete remission (CR) rates were 77.3%, 56.9%, and 69.7% for HD-DN, SD-DN, and IDA, respectively (P = 0.101; HD-DN vs. SD-DN, P = 0.036; HD-DN vs. IDA, P = 0.453; IDA vs. SD-DN, P = 0.237). The HD-DN showed higher overall survival (OS) and event-free survival (EFS) than SD-DN and IDA: the differences between HD-DN and SD-DN (P = 0.009 for OS and P = 0.010 for EFS) were statistically significant. Results of in vitro studies using FLT3-ITD-mutated cell lines supported these findings. In conclusion, HD-DN improved the CR rate, OS, and EFS of FLT3-ITD-mutated AML patients. HD-DN also tended to yield better outcomes than IDA, though the difference was not significant. The superiority of HD-DN over IDA should be confirmed in future studies.

TP53 mutation in allogeneic hematopoietic cell transplantation for de novo myelodysplastic syndrome

We investigated the prognostic role of somatic mutations in allogeneic hematopoietic cell transplantation (HCT) for de novo myelodysplastic syndrome (MDS). We performed targeted deep sequencing analysis of 26 genes on bone marrow samples obtained within 6 weeks before HCT from 202 patients with de novo MDS. Overall, 76% of patients carried one or more somatic mutations, and TP53 mutation was present in 23 patients (11.4%). Overall survival (OS) at 5 years was 63.6%, cumulative incidence of relapse (CIR) was 18.6%, event-free survival (EFS) was 58.5%, and non-relapse mortality (NRM) was 22.9%. TP53 mutation was an independent risk factor for lower OS (41% vs. 67%; P =  0.001), higher CIR (49% vs. 15%; P =  0.001), and lower EFS (38% vs. 61%; P =  0.005), but not for NRM (13% vs. 24%). N-RAS mutation was an independent risk factor for higher CIR (HR, 5.91; P =  0.008). TP53 mutation did not have significant interactions with conditioning intensity or the occurrence of graft-versus-host disease with regard to post-transplant outcomes. In conclusion, TP53 mutation was significantly associated with poor outcomes after HCT for patients with de novo MDS, mainly due to a higher incidence of disease relapse.

Establishment and characterization of hypomethylating agent-resistant cell lines, MOLM/AZA-1 and MOLM/DEC-5

Two hypomethylating agents (HMAs), azacitidine and decitabine, have demonstrated clinical activities in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML); however, potential problems include development of acquired resistance. HMA-resistant patients have very poor prognosis and this cohort of patients constitutes an important area of research. To understand the mechanisms underlying HMA-resistance and to overcome it, we established an azacitidine-resistant cell line, MOLM/AZA-1 and a decitabine-resistant cell line, MOLM/DEC-5 using MOLM-13. For cytogenetic characterization, we performed microarray-based comparative genomic hybridization (array-CGH), which identified a total of 15 copy number alterations (CNAs). Among these CNAs, eight regions in HMA-resistant cell lines showed CNA patterns distinct from the parental MOLM-13 genome. Single nucleotide polymorphism (SNP) microarray was also performed to obtain a more reliable interpretation of the identified CNAs, and all HMA-resistance-specific CNAs except one detected by array-CGH were successfully validated. In addition to CNAs, copy neutral loss of heterozygosity and mosaic loss events were identified in HMA-resistant cell lines. In our resistant cell lines, MDR-1 was not overexpressed, while DNMT3b was upregulated. Azacitidine and decitabine did not inhibit DNMT1, DNMT3a, or DNMT3b in both HMA-resistant cell lines, while they inhibited the enzymes in parental MOLM-13. We also developed mouse xenograft models using MOLM/AZA-1 and MOLM/DEC-5. Our in vitro and in vivo models of HMA-resistant cell lines will provide clues for the elucidation of molecular mechanisms related to the development of resistance to HMA and tools for the application of novel therapeutics for AML and MDS.

Induction of immunoglobulin transcription factor 2 and resistance to MEK inhibitor in melanoma cells

Primary or acquired resistance to MEK inhibitors has been a barrier to successful treatment with MEK inhibitors in many tumors. In this study, we analyzed genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines to identify candidate genes whose expression changes are associated with responses to a MEK inhibitor, selumetinib (AZD6244). Of 62 identified differentially expressed genes, we selected Immunoglobulin Transcription Factor 2, also known as transcription factor 4 as a potential drug resistance marker for further analysis. This was because the ITF-2 expression increase in resistant cell lines was relatively high and a previous study has suggested that ITF-2 functions as an oncogene in human colon cancers. We also established an AZD6244 resistant cell line (M14/AZD-3) from an AZD6244 sensitive M14 cell line. The expression of the ITF-2 was elevated both in primary AZD6244 resistant cell line, LOX-IMVI and acquired resistant cell line, M14/AZD-3. Targeted silencing of ITF-2 by siRNA significantly enhanced susceptibility to AZD6244 in resistant cells. Wnt/β-catenin pathway was activated through direct interaction of p-ERK and GSK3β. Our results suggest that up-regulation of the ITF-2 gene expression is associated with cellular resistance to MEK inhibitors, and activation of Wnt signaling pathway through interaction of p-ERK and GSK3β seems to be a mechanism for increase of ITF-2.

Abstract 5455: Resistance to a MEK inhibitor (AZD6244): Its association with increased expression of transcription factor 4

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The important role of Ras/Raf/MEK/ERK pathway in carcinogenesis has led to clinical development of MEK inhibitors for treatment of various cancers. Although recent studies have demonstrated impressive antitumor activities of the agents, many tumors show intrinsic and acquired resistance to MEK inhibitors. We tried to find biomarkers that were associated with intrinsic or acquired resistance to a MEK inhibitor (AZD6244) by public microarray data acquisition and development of AZD6244-resistance cell lines. First, we analyzed a set of genome-wide gene expression profiling data from 6 sensitive and 6 resistant cell lines of various tissue origins to identify candidate genes whose expression changes are associated with resistant or sensitive responses to AZD6244. Of 62 differentially expressed genes, we selected Transcription Factor 4 (TCF4) gene as a potential drug resistance marker for further analysis because degree of increased expression of TCF4 in resistance cell lines was relatively high and a previous study suggests that TCF4 functions as an oncogene in human colon cancers. The mRNA levels of TCF4 in AZD6244 resistant cell lines were significantly elevated than those in sensitive cell lines. The siRNA was used for the targeted silencing of TCF4 to elucidate the role of increased expression of TCF4 in intrinsic resistance to AZD6244. TCF4 siRNA significantly enhanced susceptibility of a AZD6244-resistant cell line, LOXIMVI, to AZD6244. Next, we established AZD6244-resistant cell lines, using originally AZD6244-senstive cell lines, M14 and COLO-205. The half maximal inhibitory concentration (IC50) values for AZD6244 in two acquired resistant cell lines were 34 fold and 200 fold higher than those of their parent cell lines, respectively. Protein levels of TCF4 in the acquired resistant cell lines were higher than those in the sensitive parent cell lines. Our study results suggest that increase expression of TCF4 may be associated with both intrinsic and acquired resistance to MEK inhibitors. Citation Format: Bon-Kwan Goo, Eun-Hye Hur, Yunsuk Choi, Sung-Doo Kim, Jung Jin Hwang, Choung-Soo Kim, Kyun Seop Bae, Jene Choi, Suk Young Cho, Sang-Hwa Yang, Je-Hwan Lee. Resistance to a MEK inhibitor (AZD6244): Its association with increased expression of transcription factor 4. [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 5455. doi:10.1158/1538-7445.AM2014-5455