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Introduction Dasatinib, a second-generation tyrosine kinase inhibitor (TKI), has been administered at a fixed starting dose of 100 mg once daily for patients with chronic myeloid leukemia in chronic phase (CP-CML). However, the relationships between TKI exposure, and safety and efficacy responses suggest that such fixed dosing may not be optimal for many Asian patients. In this study, the relationships between the starting dose of dasatinib adjusted for BW (Dose/BW), and dose limiting toxicities (DLTs) and molecular responses (MRs) were evaluated to develop an initial dosing strategy of dasatinib. Methods The safety and efficacy responses of dasatinib therapy were explored from the clinical data obtained in patients newly diagnosed with CP-CML at seventeen hospitals in South Korea. The safety response was assessed as the occurrence of first DLTs by 36 months that required an interruption in dasatinib therapy. The efficacy responses were evaluated as the achievement of MR at 3 months (MR1), 6 months (MR2), 12 months (major molecular response, MMR) and 24 months (deep molecular response, MR4.5). The rates and patterns of DLT occurrence and MR achievement by 36 months were examined using a Kaplan-Meier method. A logistic regression method was used to determine the effect of the Dose/BW of dasatinib on the occurrence of first DLTs or the achievement of MRs. A chi-square test for trend was used to assess the trend of DLT occurrence and MR achievement in patient subgroups divided into quartiles (Q1 to Q4) based on Dose/BW. Results The clinical data were obtained from a total of 102 Asian patients with CP-CML whose median BW was 64.0 kg (range, 37.8 to 106.0 kg) and Dose/BW 1.60 mg/kg (1.00 to 2.80 mg/kg). Safety. The most frequent DLTs were thrombocytopenia (46%), pericardial or pleural effusion (31%) and anemia (7%); the median time of occurrence for the DLTs were 29 days (range, 7 to 417 days), 336 days (7 to 681 days) and 35 days (10 to 140 days), respectively. The rate of DLT occurred first increased rapidly by four months of starting dasatinib therapy and then steadily by 28 months until reaching approximately 56% in the Kaplan-Meier curve. Patients with higher Dose/BW had a greater risk of DLT occurrence by 36 months as determined using a logistic regression (logit [P] = 1.58 × [Dose/BW] - 2.27, p = 0.03). As Dose/BW increases from Q1 to Q4 (median Dose/BW; 1.23, 1.45, 1.65 and 2.00 mg/kg, respectively), the rate of DLTs rises from 43.5% (Q1) to 66.7% (Q4) with a statistically significant trend of increment (p = 0.03). The median dose that would produce the lowest rate of DLTs is 78.7 mg as calculated by multiplying the median Dose/BW of Q1 (1.23 mg/kg) by the median BW of patients (64.0 kg). Efficacy. The achievement rates of MMR and MR4.5 increased continuously and reached 82% and 43%, respectively, by 36 months of starting dasatinib therapy. The Dose/BW of dasatinib did not affect the rates of MR achievements based on a logistic regression analysis. Even though Dose/BW increased, the achievement rates of MR1, MR2 and MMR demonstrated neither an increasing nor a decreasing trend from Q1 to Q4. Conclusion The higher the starting Dose/BW of dasatinib, the greater is the risk of DLT occurrence without improving the potential for MR achievement. Therefore, in order to minimize the risk of DLTs without compromising MRs, a lower starting dose of dasatinib 80 mg once daily is suggested for Asian patients with CP-CML especially with lighter BW. Disclosures Kim: ILYANG: Consultancy, Honoraria, Research Funding; Takeda: Research Funding; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sun Pharma.: Research Funding.

Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders (P 5 .007). Frequently mutated genes at diagnosis were ASXL1, IKZF1, and RUNX1. The methyltransferase SETD1B was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders (P 5 .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including ABL1 kinase domain mutations in 58%. However, ABL1 mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated ABL1 mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach. Refereed/Peer-reviewed

Introduction: Cytogenetically normal acute myeloid leukemia (CN-AML) accounts for 40 to 50 percent of all AML and shows significant heterogeneity in clinical outcomes. Despite the advancements in the molecular pathophysiology of CN-AML, clinical outcome of this leukemia remains to be elucidated. Prohibitin (PHB) were typically known to have the potential act as a tumor suppressor, anti-proliferative regulator of cell-cycle in normal cells. However, our previous study discovered that high expression of PHB in AML cells was involved in escaping apoptosis process. In this study, we investigated the clinical significance of expression levels of PHB1 and PHB2 genes in patients with CN-AML. Methods: We developed quantitative reverse transcription PCR (qRT-PCR) detection system for mRNA expression status for PHB1 and PHB2.The expression levels was analyzed in 69 CN-AML patients. Total RNA was extracted from bone marrow samples at the diagnosis using InvitrogenRNAqueous Total RNA Isolation Kit (Thermo Fisher Scientific, USA). The concentration of isolated total RNA was measured by Micro UV-VIS spectrophotometer (Malcom, Japan). Quantification of mRNA of PHBs were performed in CFX96 Touch Real-Time PCR system (BIO-RAD, USA) using our laboratory-developed, customized primers and probes.The gene expression levels werenormalized compared to those of ABL1 gene.Survival analysis was performed compared with other prognostic factors: age, NPM1 mutation, and FLT3-ITD. FLT3-ITD was tested by PCR & fragment analysis (LeukoStrat FLT3 Mutation kit; Invivoscribe, USA) and NPM1 mutation was tested using qRT-PCR kit (ipsogen NPM1 MutaQuant; Qiagen, Germany). All statistical computations and graphical process were performed using R version 3.5.1. A P value ≤ 0.05 (for 95% confidence interval) was considered statistically significant. Results: The median age of enrolled patients was 68 (ranged 23-85) at the diagnosis. The ratio of male and female was 1:0.86 (37:32). There was no significant difference in age, sex, WBC count in peripheral blood, blast percentage of peripheral blood and bone marrow, the achievement rate of complete remission, and the enforcement of stem cell transplant according to expression status of PHB1 and PHB2. Using 0.61 as cutoff, PHB1 high-expressed group showed superior overall survival (OS) (47.7% vs 15.1%; P = 0.014) and low hazard ratio (0.43 [95% CI: 0.215-0.861]; P = 0.017) compared to PHB1 low-expressed group. In PHB2, using a cutoff 2.39, high-expressed group showed as inferior OS (17.5% vs 40.0%; P = 0.16) and high hazard ratio (1.93 [95% CI:0.739-5.042]; P = 0.18) compared to low expressed group. Conclusions: PHB1 expression status was associated with favorable clinical outcome. In contrast, those of PHB2 was related with poor prognosis in CN-AML. Disclosures No relevant conflicts of interest to declare.

Introduction: Fusion transcript is a chimeric RNA encoded by a fusion gene or by two different genes by subsequent trans-splicing. Detection of fusion transcripts is an integral part of routine diagnostics of hematological malignancies. However, most of previous analytical methods couldn't detect all fusion transcripts in leukemia. In this study, we developed accurate fusion transcript detection methood using whole transcriptome sequencing, fusion gene detection software and expression analysis. Methods: RNA sequencing (RNA-seq) for whole transcriptome was performed in 11 patients with hematological malignancies (4 AML, 2 APL, 2 ALL, and 3 CML) having fusion transcripts detected by multiplex RT-PCR (HemaVision, DNA Diagnostic, Risskov, Denmark). Library were prepared with 1 ug of total RNA for each sample by TruSeq mRNA Sample Prep kit (Illumina, San Diego, USA). The libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantificatoin kits for Illumina Sequecing platforms) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, Santa Clara, USA). Indexed libraries were then sequenced using the HiSeq2500 platform (Illumina). The data obtained from the sequencing was analyzed using STAR-Fusion (v1.2.0). Novel fusion transcripts were confirmed by conventional sequencing. Results: Using STAR-Fusion, average number of fusion candidates per sample was 949.8 (range, 286-1752). To exclude false positive results and obtain true positive results, we developed the following filtering algorithm. First filtering criterion is to have more than 5 junction reads, the second is to detect more than one number of spanning reads, and the third criterion is to be in-frame fusion, which type of fusion can actually synthesize intact protein. Fusion candidates remaining after applying the above three filtering criteria were 1-3 per sample. All known fusion transcripts (PML--RARA, RUNX--RUNX1T1, CBFB--MYH11, KMT2A--MLLT3, BCR--ABL1, DEK--NUP214, ETV6--RUNX1) by multiplex RT-PCR were also detected in RNA-seq. In addition, 10 novel fusion transcripts (IGKV4-1--IGKC, IGLV1-47--IGLC2, HBA2--HBB, DEFA3--MBNL1, HBB--HBA2, MPO--HBA2, HBS1L--AHI1, HBB--HBA2, IGKV4-1--IGKC, SS18L1--ADRM1) were detected and among them, 6 fusions were confirmed by conventional sequencing. Conclusions: Whole transcriptome sequencing and optimized filtering algorithms successfully detected all known fusion transcripts and various novel fusions. Disclosures No relevant conflicts of interest to declare.

Introduction: As chronic myeloid leukemia (CML) patents are generally diagnosed at old age and live longer by active use of BCR-ABL1 tyrosine kinase inhibitors (TKIs), the occurrence of other malignancy (OM) is becoming a critical issue as a long-term comorbidity. An increased rate of OM has been reported in myeloproliferative disorders and long-term TKI treatment may induce OM in CML. To explore exact prevalence and characteristics of OM, we reviewed medical records of CML patients and compared with those of age-matched Korean population. Methods: The medical records of 1,469 CML patients who diagnosed between January 2000 and December 2014 were reviewed using Korean data-set of Asia CML Registry (ACR). With a cut-off date of July 2016, age-standardized prevalence rates (A-SPR) of OM (except benign tumors and other leukemias) were analyzed and compared with that of general population in Korea Central Cancer Registry (KCCR). In addition, we analyzed cumulative incidence rate of OM and various risk factors. Results: The median duration of follow-up was 84 (1-197) months, and 96 CML patients had at least one OM. Forty three patients had a history of OM before a median 69 (1-161) months of CML diagnosis and 53 patients developed OM after a median 53 (range; 0.2-172) months of CML diagnosis. The OM included 32 thyroid cancers, 19 colorectal cancers, 16 stomach cancers, 9 breast cancers, 4 gynecological cancers (3 cervical cancers and 1 uterine endometrial cancers), 3 lymphoma (2 non-Hodgkin's lymphoma and 1 Hodgkin's lymphoma), 3 biliary cancer, 3 skin cancers, 3 prostate cancers, 2 lung cancer, 2 tongue cancer, 2 liver cancer, 2 esophageal cancer, 1 pancreatic cancer, and 1 bladder cancer.A-SPR of OM was 1.7 times higher in CML patients. Hodgkin's lymphoma (8.7 times), thyroid cancer (2.6 times), biliary cancer (2.6 times), colorectal cancer (2 times), non-Hodgkin's lymphoma (1.8 times), cervical cancer (1.8 times), and breast cancer (1.6 times) had a higher A-SPR. On the other hands, skin cancer (3.3 times), lung cancer (2 times), and liver cancer (2 times) were lower than that of general population. With 53 patients who had OM after CML diagnosis, we analyzed the cumulative incidence. The risk of OM was increased over the follow-up period (2.7% at 7 years) Univariate analysis revealed that patients who were more than 37 years old at CML diagnosis (4.3% vs. 0.4%, p Conclusion: Although comorbid malignancies did not significantly affect CML survival, poor survival in advanced stages and the high risk of other cancers warn the need of systematic screening in long-term CML survivors. In addition, the specific cancer types with a significantly higher A-SPR should be considered for further studies including genetic mechanisms. Disclosures Kim: ILYANG: Consultancy, Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Backgroud: Recent reports showing that imatinib (IM) discontinuation can be employed in patients who had enough IM therapy and undetectable molecular residual disease (UMRD) durations prior to IM discontinuation result in treatment-free remission (TFR) as a new therapeutic goal in chronic phase chronic myeloid leukemia (CP CML). Although 50-70% of patients experienced molecular relapse by several TFR studies, the most of patients resumed molecular responses (MR) following restart of IM. Through the Korean multicenter prospective study (Korean Imatinib Discontinuation Study; KID Study), we have identified predictors for sustained UMRD and explored molecular kinetics after the first IM discontinuation. In patients regaining durable UMRD with IM resumption, we tried second IM discontinuation and compared molecular kinetics between the first IM stop and second IM stop. Methods: CP CML patients who were treated with IM for more than 3 years and had undetectable levels of BCR-ABL1 transcripts determined by quantitative reverse transcriptase polymerase chain reaction (PCR) for at least 2 years were eligible for KID study and in cases of MMR loss after 2 consecutive assessments, IM treatment was re-introduced. After IM resumption for MMR loss, the molecular response was evaluated every month until MMR was re-achieved and every 3 months thereafter. The second stop was permitted in the patients who were in second UMRD for at least 2 years. Results: By the data cut-off date of 31 July 2015, among 90 non-transplant UMRD patients with at least 12 months of follow-up, 37 patients lost MMR in 2 consecutive analyses and resumed IM. Among them, 9 patients (5 men and 4 women) with a median age of 54 years (range, 35-59 years) entered into a second IM discontinuation after maintaining UMRD at least 2 years. Prior to first discontinuation, the median duration of IM therapy was 76.8 months (range, 38.5-129.0 months) and the duration of sustained UMRD was 30.1 months (range, 24.4-64.5 months). After first attempt of IM discontinuation, they relapsed after a median duration of 3.7 months (range, 1.9-20.8 months) and re-achieved UMRD at a median of 5.5 months (range, 1.8-9.4 months) after IM resumption. After sustaining a second UMRD for a median of 25.7 months, IM therapy discontinued for a second time. After a median follow-up of 37.4 months (range, 19.7-58.5 months) after second IM discontinuation, 7/9 patients (78%) and 4/9 patients (44%) lost UMRD and MMR, respectively. Among three patients who lost UMRD but not MMR, one patient showed fluctuation of BCR-ABL1 transcript under the level of 0.1% on IS for 19.5 months and two patients has not yet been followed up after a first detection of UMRD loss. Four patients who experienced second relapse (MMR loss) after a median 2.9 months (range, 2.7-3.9 months), which was similar to those of the first IM discontinuation [median 3.75 (range, 1.9-3.9 months)]. The patients who lost MMR were retreated with IM for a median of 13.6 months (range, 0.8-18.6 months); three patients re-achieved MMR at 3.5, 5.5, and 11.5 months, respectively and one re-achieved UMRD at 7.2 months. Conclusion: Our data demonstrated that a second attempt might be possible and the median time to MMR loss after second discontinuation was similar to those of the first discontinuation. But the molecular kinetics after second IM resumption needs longer follow-up with more patients. Further studies on the predictors to select patients for a trial of second TFR and novel strategies such as intermittent therapy will be warranted. Disclosures No relevant conflicts of interest to declare.

Background: Prohibitin (PHB) is a highly conserved protein, widely expressed in multiple cellular compartments such as mitochondria and nucleus. Recent studies have demonstrated that PHB played an important role in the regulation of mitochondrial function, cell proliferation and development. Our proteome analysis has revealed PHB was highly expressed in primary AML cells compared with normal cells. This finding prompted us to investigate whether PHB could serve as a potential diagnostic, prognostic, and therapeutic target in AML. Patients and Methods: PHB2 protein expression was assessed in 133 newly diagnosed normal karyotype AML (NK-AML) patients by using immunohistochemical staining (IHCS) on paraffin-embedded bone marrow sections. The degree of PHB2 protein expression was scored as 0-8 as a sum of a diffuseness score (0-5) and an intensity score (0-3). Overall survival (OS) and event-free survival (EFS) was analyzed according to PHB2 expression score. Also, we developed quantitative reverse transcriptase PCR (qRT-PCR) method to measure mRNA expression of PHB1 and PHB2 genes for the optimal algorithms for the discrimination of AML cells from the normal blood or hematopoietic cells. Because accumulation of beta-catenin in the nucleus has been identified in leukemic cells, we investigated the effect of beta-catenin accumulation on PHB expression. We monitored the expression levels of PHB with lithium chloride treatment, which blocks GSK-3beta activity and ultimately affects beta-catenin accumulation. For the development of anti-leukemic drug candidates targeting PHB in AML cells, we synthesized two potent chemical substances that can alkylate PHB: cyclohexylphenyl-chloroethyl urea (CCEU) and iodophenyl-chloroethyl urea (ICEU). We evaluated their therapeutic effects in the primary AML and AML cell lines. Results: In the PHB2 IHCS, using the cutoff as positivity score 4.0, PHB2 positive patients showed inferior OS (23.7% vs 50.0%; P = 0.026) and high hazard ratio (1.808; P = 0.028) compared to PHB2 negative patients (figure 1). Using the same cutoff, PHB2 positive patients showed inferior EFS (20.7% vs 47.1%; P= 0.024) and high hazard ratio (1.795; P = 0.026) (figure 2). In the qRT-PCR assay of PHB1 and PHB2, quantitative expression (ddCt) of PHB1 gene was higher in the control group: 0.31-28.32 (4.40+/-5.80; Mean+/-SD) versus 0.22-6.52 (1.83+/-1.56) in patient group. Meanwhile, PHB2 gene expression was higher in the NK-AML patient group: 0.15-3.49 (0.80+/-0.47) versus 0.00-0.46 (0.16+/-0.13) in control group. Using linear discrimination analysis, we derived a linear equation for discrimination between normal and AML cells. Estimating the prevalence of AML as 57.2/1,000,000, the sensitivity of newly developed algorithm was 93.6%, specificity was 76.9%, negative predictive value was 99.9%, and positive predictive value was 2.28% (figure 3). PHB protein expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This beta-catenin-mediated activation of PHB expression was independent of c-MYC activation, a product of Wnt signaling. These data indicated that PHB was a direct target of beta-catenin and the increased level of PHB in leukemic cells can be regulated by Wnt signaling. Using CCEU and ICEU, time and dose dependent manner of proliferation suppression was observed in primary AML and AML cell lines. Notable morphological transformation of AML cells was observed when treated with 10-100 umol of CCEU and ICEU for 24 hours. The half maximal inhibitory concentration (IC50) was 25 umol for most AML cells. Conclusions: Overexpression of PHB2 was associated with poor prognosis in NK-AML. Newly developed discrimination algorithm using qRT-PCR showed the possibility as a rapid diagnostic tool for leukemic cell identification. Our functional study suggested that the elevated level of PHB in leukemic cells was the result of Wnt signals which were up-regulated in AML cells. Finally, this study developed novel alkylating chemotherapeutics targeting PHB for selective killing of leukemic cells. PHB2 expression status can provide clinically valuable information for the diagnosis, prediction of the prognosis, detection of minimal residual disease, and therapeutic targeting of NK-AML. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures No relevant conflicts of interest to declare.

Recently, a striking example of the effects of acquired somatic mutations in splicing factors such as SF3B1, U2AF1, and SRSF2 has been described. The DNA sequences from abnormal blood cells of patients with hematologic malignancies has shown that a high proportion of these diseases are associated with somatic mutations of spliceosomal proteins. However, little is known about the aberrant splicing pathways in multiple myeloma (MM) patients. Therefore, this study investigated the presence and prognostic implication of splicing gene aberration in MM patients. Also, we planned functional experiments in cultured normal and Hela cell line to determine the effects of the common spliceosome mutations on cellular growth rate, cell count, and apoptosis. In total, 87 MM patients and 100 healthy controls were examined for somatic mutations in SF3B1, U2AF1, and SRSF2 by direct sequencing. Also, the clinical and laboratory characteristics of MM patients were reviewed. Regarding to the initial findings in recent reports, this study expressed the wild-type and the mutant (Q157P) U2AF1 in Hela cells and examined their proliferation. MM patients did not show mutation in SF3B1, U2AF1,and SRSF2 splicing genes. However, the patients displayed 39198T>C or 39198T>T/C polymorphism (70.1 %) in exon 18 of SF3B1, 8345T>T/G polymorphism (13.8 %) in exon 2 of U2AF1, and 5399C>T or 5399C>C/T polymorphism (100.0 %) in exon 1 of SRSF2 (Fig. 1). In the entire cohort, the number of patients with one polymorphism, two polymorphisms, and three polymorphisms was counted up to 24.1 %, 67.8 %, and 8.0 %, respectively. Meanwhile, in 100 normal controls, polymorphism of SF3B1 exon 18 and U2AF1 exon 2 was counted up to 82.0 % and 10.0 %, respectively. Free light chain (FLC) kappa/lambda (K/L) ratio and chromosome analysis showed significant difference according to polymorphism status (p=0.002) while other clinical characteristics did not. The patient with polymorphisms in both SF3B1 and U2AF1 gene had unfavorable overall survival (p =0.067) and disease-free survival (p=0.001), compared to patients without polymorphism. Our results show no recurrent SF3B1 and U2AF1 mutations in MM patients. Instead, polymorphisms were found in SF3B1 or U2AF1, which were significantly implicated in worse prognosis. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: The efficacy of second-generation tyrosine kinase inhibitors (2G-TKI) as frontline therapy for newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP) has been established. 2G-TKI treated patients achieved faster and deeper molecular responses compared with imatinib (IM) treated patients. Because CML patients currently have multiple treatment options, their impact on long-term outcomes needs to be determined and each agent should be chosen with a consideration of distinct toxicity profile and patient's comorbidities. The aim of this study is to provide a comprehensive analysis of the outcome of CML patients treated with different 2G-TKIs and to explore contributing factors on the outcome, as additional information to guide clinical decisions on selecting a frontline therapy. Methods: 237 new CP CML patients who were treated with frontline 2G TKIs were analyzed. Molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). Main study objectives were to compare event-free survival (EFS), failure-free survival (FFS), and progression-free survival (PFS) to different 2G-TKIs as frontline therapy. FFS and EFS was measured from the date of treatment start until death, progression to AP or BP, or ELN failure on treatment (FFS), and until death, progression to AP or BP, ELN failure on treatment, or treatment discontinuation for any cause (except treatment-free remission) (EFS), respectively, whichever came first. PFS included progression to AP or BP as well as death resulting from any reason and collected on patients who were treated with other TKIs after frontline 2G-TKI discontinuation. Results: From Oct 2007 to Jul 2015, a total of 237 patients were treated with nilotinib (NIL; n = 69), dasatinib (DAS; n = 113), and radotinib (RAD; n = 55). The median age was 43 years (range, 18-81). The percentages of patients with low, intermediate and high Sokal risk scores were 35%, 38% and 27%, respectively. With a median follow-up of 25.1 (NIL; range, 0.2-94.6), 13.8 (DAS; range, 2.0-86.8), and 36.8 (RAD; range, 9.9-48.2) months, 180 (75.9%) patients continue on the therapy and 57 (24.1%) patients were permanently discontinued the first 2G TKI due to intolerance (9 NIL, 11 DAS, 16 RAD), ELN failure (3 NIL, 7 DAS, 3 RAD), progression (2 NIL, 1 DAS), and others (2 NIL, 3 DAS). The probability of 2-year (y) EFS, FFS, and PFS were 72.6%, 89.4%, and 94.2%, respectively. There was no significant difference between treatment groups (NIL vs DAS vs RAD): 2-y EFS (78.9% vs 69.3% vs 66.1%, P = 0.363), 2-y FFS (89.4% vs 85.6% vs 92.7%, P = 0.487), and 2-y PFS (95.4% vs 88.8% vs 98.2%, P = 0.420). Among 209 patients with an available qRT-PCR data at 3 months, patients achieving BCR-ABL1 ≤ 10% (n = 182, 87.1%) had a better outcomes in terms of 2-y EFS (80.2% vs 44.2%, P10%. In the 195 patients with an available 6-month data, achievement of 6-month early molecular response (BCR-ABL1 ≤1% at 6 months) was associated with a higher 2-y EFS (82.0% vs 61.8%, P = 0.002) and 2-y FFS (96.9% vs 69.0, P Conclusions: Our data showed the outcome of CML patients treated with different 2G-TKI as frontline therapy, which showed no difference of EFS, FFS, and PFS. However, the kind of intolerance resulting in treatment discontinuation was significantly different, suggesting that distinct toxicity profile should be a consideration when selecting a therapy. The 3-month and 6-month EMRs also provided prognostic information in patients treated with frontline 2G-TKI. Additively, the predictive impact of age and Sokal risk was observed. However, further investigations in a larger patient population with longer follow-up are needed. Disclosures No relevant conflicts of interest to declare.

Background: Second-generation BCR-ABL tyrosine kinase inhibitors (2nd generation TKIs) including nilotinib (NIL) and radotinib (RAD) are approved for the treatment of newly diagnosed and other TKI failed chronic myeloid leukemia (CML). Uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) gene promoter polymorphism, distinct number of TA repeats in promoter, has been reported for an association with hyperbilirubinemia on nilotinib therapy (Singer et al. Leukemia (2007) 21, 2311–2315). However, the distribution of UGT1A1 (TA)7 repeat polymorphism differs greatly between Caucasians and Japanese, namely, the frequency of UGT1A1(TA)7 repeat polymorphism is high in Caucasians, whereas it is low in Asians (Beutler et al. Proc. Natl. Acad. Sci. USA 95 (1998) 95, 8170–8174 ). Aims: The aim of this study was to investigate the association between UGT1A1 gene promoter polymorphism and hyperbilirubinemia in Korean CML patients treated with NIL and RAD as a frontline therapy. Methods: A total of 88 newly diagnosed chronic phase CML patients who treated with frontline NIL (n=39) and RAD (n=49) was screened for UGT1A1 promoter polymorphism genotype analysis. We used the High Pure PCR Template Preparation Kit (Roche, Germany) to prepare genomic DNA from whole blood and genotyped by direct sequencing of the 253- to 255-bp fragments produced by PCR. Results: The (TA)6/(TA)6 homozygote and (TA)6/(TA)7 heterozygote were seen in all genotyped population and frequency of (TA)6/(TA)6 homozygote was 79.5% (70/88) in our patients. (TA)6/(TA)6 homozygote predominated with 81.6% of the alleles in the RAD group and 76.9% in the NIL group. Relative riskfor each genotype presented in Table 1, with hyperbilirubinemia defined as CTCAE grade 2 or greater observed at any post-baseline point. The relative risks for 6/6 genotype versus 6/7 genotype was 3.5 with 95% CIs of (0.6, 18.9) in the RAD group compared with 8.1 (1.5, 43.8) in the NIL group and NIL group showed significantly high association with UGT1A1 gene promoter polymorphism (P Conclusions: This finding suggests that UGT1A1 gene promoter polymorphism may be an important determinant of hyperbilirubinemia in CML patients with NIL therapy, but not in RAD. However other mechanisms should be explored in patients who have significant hyperbilirubinemia with RAD therapy. Updated data with longer follow-up duration will be presented in the meeting. Table 1. Relative risk calculations for prevalence of hyperbilirubinemia Radotinib Nilotinib Max grade=,,=2 Total Max grade=,,=2 Total (TA)6/(TA)6 20 20 40 (TA)6/(TA)6 26 4 30 50.00% 50.00% 65.00% 10.00% (TA)6/(TA)7 2 7 9 (TA)6/(TA)7 4 5 9 22.22% 77.78% 44.44% 55.56% Total 22 27 49 Total 30 9 39 Relative risk=3.5, 95% CI of (0.6, 18.9) Relative risk=8.1, 95% CI of (1.5, 43.8) Abbreviation: CI, confidence interval. Disclosures No relevant conflicts of interest to declare.

Background. In chronic myeloid leukemia (CML), achievement of optimal responses by time point has improved long-term outcomes. In IRIS study, patients who achieved major molecular response (MMR) at 18 months had event-free survival (EFS) benefit, compared to those who achieved complete cytogenetic response (CCyR) without MMR. However, the best treatment for these patients is still not confirmed. By the previous studies, sustaining standard-dose of imatinib (IM) is expected to yield less than 20 percent of additive MMR. In this study, we investigated the efficacy of nilotinib (NIL) versus high-dose IM versus sustaining standard-dose IM for CCyR patients with suboptimal molecular response to frontline IM therapy. Methods. Early chronic phase (CP) CML patients who have achieved CCyR but no MMR after at least 18 months and up to 24 months ( 18 to 24 months) on first-line IM therapy at a daily dose of 400 mg were divided into 3 treatment groups; NIL 400mg BID (800 mg/day; group 1) vs IM 400 mg BID (800 mg/day; group 2) vs IM 400mg QD (400mg/day; group 3). Group 1 and 2 patients were selected in RE-NICE multicenter study and group 3 patients were selected with the same inclusion criteria of RE-NICE. The efficacy endpoints are MMR rate by 12 months and MMR rate and undetectable molecular residual disease (UMRD) rates by 36 months. Safety profiles of each group were compared. Patients showing lack of response (lack of complete hematologic response (CHR) at 6 months, increasing WBC, no major cytogenetic response (MCyR) at 24 months), loss of response (loss of CHR or MCyR) or intolerance to treatment were allowed to switch to other treatment. Results. With a data cut-off date of 17 Jul 2014, a total of 83 patients were evaluated; 29 patients in NIL group (group 1), 29 patients in high-dose IM group (group 2) and 25 patients in standard-dose IM group (group 3). With a median follow-up of 36 months (range, 1-63), all patients in group 1 remained in nilotinib treatment, 17 patients in group 2 switched to NIL 400mg BID due to intolerance (n=4) and lack of response (no MMR; n=13). In group 3, with a median follow-up of 71 months (range, 6-132), 15 patients switched to other treatment due to intolerance (n=5) and lack of response (no MMR; n=10). Up to now, all patients in three groups have maintained CCyR without progression or resistance. 10 in 29 (35%), 8 in 29 (28%) and 5 in 25 (20%) patients achieved MMR by 12 months, and 20 in 29 (69%), 15 in 29 (51%) and 11 in 25 (44%) patients achieved MMR by 36 months in group 1, group 2 and group 3 respectively. Overall, 3 patients in group 1 (3/29, 10%) achieved confirmed UMRD. Overall 3 years probability of MMR was significantly higher in group 1 than the other two groups (67.8% vs 41.0% vs 40.4%, group 1, 2, 3 respectively, group 1 vs 2, P=0.089, group 1 vs 3, P=0.035, group 2 vs 3, P=0.614). Compare to other groups, the patients in group 2 showed higher toxicities, such as leukopenia, anemia, thrombocytopenia, edema, fatigue, dyspnea and hypophosphatemia. Conclusions. Nilotinib 400mg twice daily treatment showed better efficacy than high-dose or same standard-dose imatinib for the treatment of patients who have suboptimal molecular response to initial standard-dose imatinib. Additionally, a switch to nilotinib in suboptimal molecular responder to imatinib would also be preferable option in terms of tolerability. Updated data with longer follow-up duration will be presented in the meeting. Disclosures No relevant conflicts of interest to declare.

Abstract Background: The first BCR-ABL1 tyrosine kinase inhibitor (TKI), imatinibmesylate (IM), has become a first-line therapy for chronic phase (CP) chronic myeloid leukemia (CML). However, approximately one third of IM-treated patients discontinue therapy due to an inadequate response or adverse event. More potent second or third generation TKIs such as nilotinib, dasatinib, radotinib, bosutinib and ponatinib have developed and these agents have shown high rates of hematologic and cytogenetic responses after failure of IM therapy. Although the new European Leukemia Net (ELN) recommendation serves provisionally the definitions of the response to second-line therapy, early molecular milestones which are associated with the best long-term are not confirmed. The aim of this study was to identify 3-month molecular milestone for predicting long-term survival to second-line therapy using second or third generation TKIs in CP CML patients who showed a failure or warning to IM. Methods: Between March 2005 and January 2014, 198 CP CML patients with failure or warning to IM (defined by 2013 ELN recommendation) had been treated with nilotinib, dasatinib, radotinib, bosutinib or ponatinib as a second-line therapy. Among them, 180 patients had available molecular data at 3 months from the initiation of second-line therapy. Based on receiver operating characteristic (ROC) curveanalysis, the predictive cutoffs of BCL-ABL1 transcripts at 3 months for progression-free survival (PFS), and overall survival (OS) were evaluated. OS included any death regardless of causes, and PFS included progression to AP or BP as well as death resulting from any reason. OS and PFS were also collected on patients who were treated with other TKIs after failure of second-line TKI therapy. Results: A total of 180 patients were treated with second-line TKI, dasatinib (n=66), nilotinib (n=61), radotinib (n=44), bosutinib (n=7), and ponatinib (n=2). 119 men and 61 women were included and their median age was 42 years (range, 15-75). Using a ROC curve analysis, BCR-ABL1 transcript level 7.93% on the international scale, at 3 months were predictive cutoffs for both PFS and OS. The median follow-up for survivors since the start of second-line TKI was 78.73 months (range, 6.3-114.0 months). 104 patients continue on therapy and 76 patients were permanently discontinued due to intolerance (n=38), failure (n=20), progression (n=14), and others (n=4). The7-year PFS and OS were 82.6% and 85.3%, respectively. The patients with transcript levels below 7.93% at 3 months had significantly better 7-year PFS (95.1% vs. 60.4%; P < 0.001) and OS (96.3% vs. 67.9%; P < 0.001). After adjusting for potential factors affecting PFS and OS in univariate analyses, multivariate analyses showed that BCR-ABL1 transcript of 7.93% at 3 months was the independent predictor for PFS (RR of 8.37, P < 0.001) and OS (RR of 13.53, P = 0.001). In addition, increasing age (RR of 1.05, P = 0.023) and presence of BCR-ABL1 kinase domain abnormalities (KDA) at baseline (RR of 5.83, P = 0.007) were associated with a lower OS. Conclusions: Our data showed BCR-ABL1 transcript of 7.93% at 3 months was an early independent predictor for long-term survival to second-line TKIs in IM-resistant CP CML. It implies that the patients who failed an achievement of reduction of BCR-ABL1transcripts to this level may require more precise monitoring on second-line therapy, allowing early clinical intervention using other third-line TKI therapy or allografting. Disclosures No relevant conflicts of interest to declare.

Background Approximately 50% of CP CML patients achieve undetectable molecular residual disease (UMRD) at 6-7 years of first-line imatinib (IM) therapy. Although imatinib therapy is effective in chronic myeloid leukemia (CML) patients and a substantial portion of patients achieve UMRD with prolonged IM therapy, up to 10^7 leukemic cells can still be present in the absence of detectable BCR-ABL1 in RQ-PCR assay due to the sensitivity limit of current RQ-PCR technology. The recent several reports to assess whether IM can be discontinued in CML patients have shown that IM discontinuation can be employed based on clinical study in patients who had enough IM therapy and UMRD durations prior to IM discontinuation. In our previous report, the 12-month probability of sustained UMRD of 48 patients was 80.8% (78.4% in 37 patients with at least 12 months follow-up), a higher rate than that reported by the ‘STIM’ (Stop Imatinib) trial. To identify predictors for safer, successful IM discontinuation and to explore additional contributing factors for sustained molecular responses (MRs), this multicenter prospective Korean Imatinib Discontinuation Study (KIDS) is on-going. Methods Patients who were treated with IM for more than 3 years and whose BCR-ABL1 was undetectable in RQ-PCR for at least 2 years were enrolled in this study. After discontinuation, molecular response was monitored using RQ-PCR assay every month up to 6 month follow-up, every 2 months up to 12 month follow-up, and every 3 months thereafter. In case of relapse, defined as loss of MMR on 2 consecutive assessments, IM therapy was re-introduced and molecular response after resumption was observed every month using RQ-PCR. Our primary objectives were to evaluate the probability of persistent UMRD at 12 month follow-up after discontinuation, and to measure the duration of persistent UMRD after discontinuation. The secondary objective was to evaluate the probability of major molecular response (MMR) loss and the time taken to lose MMR after discontinuation. Results Of the 148 patients who were screened for KIDS, 30 patients have failed because of positive result of RQ-PCR tested in the central laboratory (N= 21, 70%), failure to meet inclusion criteria (N=7, 23%) and informed consents withdrawal (N=2, 7%). As of data cut-off date of 2 July 2014, a total of 115 patients (66 females, 49 males) who were diagnosed between 20 Mar 1996 and 12 Jul 2012 were enrolled on KIDS, with a median age of 44 years (range, 19 – 77), the percentages of patients with low, intermediate and high Sokal risk scores were 35%, 25% and 17%, respectively with unknown Sokal risk scores in 23%. And the median time on IM therapy and the median duration of sustained UMRD prior to discontinuation were 84 months (range, 32 – 149) and 44 months (range, 22 – 131), respectively. With a median follow-up of 15 months (range, 0.2 – 45.4), 28 patients loss MMR and the probability of sustained MMR was 73.5 ± 4.3%. The 12-month probability of sustained MMR was 67.0 ± 5.2%. Among 21 patients who lost UMRD without confirmed MMR loss, 10 spontaneously re-achieved UMRD, 2 fluctuated BCR-ABL1 transcript under MMR and 9 continuously increase BCR-ABL1 transcript. All of 28 patients who confirmed MMR loss were re-treated with IM for a median of 9.9 months (range, 0.7 – 34.2 months), 22 patients re-achieved MMR at a median of 4.1 months (range, 0.5 - 6.5 months) after resuming IM therapy and 14 of these patients re-achieved UMRD at a median of 6.7 months (range, 3.3 - 13.3 months). One patient, who lost MMR at 7.6 months after KIDS enrollment, resuming IM for 26.7 months and sustained UMRD for 23.9 months, currently discontinues IM therapy (KIDS2) with the follow up of 6.5 months. Univariate analysis showed that IM duration and UMRD duration before treatment discontinuation had a higher 12-month probability of sustained MMR. Conclusions Based on results, IM discontinuation with resuming after MMR loss can be applied safely. In particular, the rate of screening failure due to positive result of RQ-PCR tested in the central laboratory implied that a strict PCR sensitivity criterion is important for safer, successful IM discontinuation. Overall, both UMRD and IM duration on treatment were the most important predictors for successful IM-off. Further studies on underlying mechanisms about immunological control, minimal residual leukemia and stem cell biology during TKI-off study should be conducted. Disclosures No relevant conflicts of interest to declare.

[Graphic][1] Background: To explore a real incidence of dasatinib-induced pulmonary arterial hypertension (D-PAH) in clinical practice, we investigated 82 imatinib or other 2G tyrosine kinase inhibitor (TKI)-failed chronic myeloid leukemia (CML) patients who received dasatinib as a second-line therapy. Methods: Routine chest X-ray and Doppler echocardiography were regularly evaluated in all patients and additional tests were performed if dyspnea developed on treatment. Results: Median age at the time of starting dasatinib was 48 (16-82) years. Of 82 patients, 8 patients (9.8%) showed an elevation of right ventricular systolic pressure (RVSP>35mmHg) by Doppler echocardiography. Among them, 7 patients (8.5%) were considered D-PAH with a median dasatinib treatment duration of 32.6 (10.3-108.7) months. They underwent follow-up Doppler echocardiography median 5 (2-9) times. Five patients showed severe D-PAH (RVSP >70mmHg), 1 was moderate (RVSP 46mmHg), and 1 was mild (RVSP 41mmHg). Advanced studies such as pulmonary angiographic catheterization (patient 1 and 2) or pulmonary arterial computed tomography (patient 3 and 4) were performed for confirming D-PAH or ruling out PAH due to pulmonary vascular abnormality. Six patients had bilateral pleural effusion and 1 had unilateral pleural effusion. With sildenafil (n=5) + dose reduction (n=1) + switch to other TKI (n=6), all of patients improved dyspnea, and RVSP level was completely resolved in 3 patients. In addition, previous nilotinib therapy and concomitant pleural effusion were significant contributing factors for D-PAH. Conclusion: Regardless of complete resolution of pleural effusion, a patient with sustained dyspnea on dasatinib treatment should be carefully evaluated by Doppler echocardiography and a regular monitoring will be needed for early intervention. | Cohort | Age at PAH diagnosis (year) | Sex | Treatment duration before dasatinib (month) | Previous therapy for CML | Duration between initiation of dasatinib and diagnosis of PAH (month) | Daily mean dose of dasatinib (mg/d) | Duration between diagnosis of D-PAH and last follow up (month) | Treatment of D-PAH | Switch to other TKI | Outcome | | ------ | --------------------------- | --- | ------------------------------------------- | ------------------------------------------------------- | --------------------------------------------------------------------- | ----------------------------------- | -------------------------------------------------------------- | ------------------------------------ | ----------------------- | -------- | | 1 | 53 | M | 54.4 | Interferon, Hydroxyurea, Imatinib, nilotinib | 26.4 | 123 | 73.5 | Sildenafil | nilotinib and ponatinib | partial | | 2 | 50 | M | 36.6 | Interferon, Hydroxyurea, Imatinib, dasatinib, nilotinib | 50.3 | 112 | 55.2 | Sildenafil | nilotinib and radotinib | partial | | 3 | 37 | F | 31.7 | Imatinib, nilotinib | 21.7 | 88 | 39.7 | Sildenafil Dose de-escalation | radotinib | partial | | 4 | 45 | M | 70.9 | Hydroxyurea, Imatinib | 69.8 | 101 | 35.2 | Sildenafil Dose de-escalation | ponatinib | complete | | 5 | 59 | F | 107.4 | Interferon, Hydroxyurea, Imatinib | 83.6 | 92 | 14.3 | none | radotinib | partial | | 6 | 46 | F | 12.6 | Imatinib | 29.1 | 76 | 13.0 | Steroid, Sildenafil | radotinib | complete | | 7 | 38 | F | 30.2 | Imatinib | 33.1 | 98 | 10.2 | Dose reduction | NA | complete | Abstract 5535. Table 1. Characteristics of patients with dasatinib-induced PAH Disclosures No relevant conflicts of interest to declare. [1]: /embed/inline-graphic-2.gif

Introduction Recently, recurrent somatic SET-binding protein 1 (SETBP1) mutations were found in atypical chronic myeloid leukemia (aCML) and other related myeloid neoplasms. According to reports so far, SETBP1 mutations occur in 9% of myelodysplastic/myeloproliferative neoplasms (MDS/MPN), especially in high frequency (24∼30%) of aCML. SETBP1 mutations were associated with worse prognosis and higher white blood cell (WBC) counts. Most of the reports came from western countries and there was a need to further study its clinicopathological impacts in East Asian patients because of paucity of reports. Therefore, this study investigated the prevalence and clinical implications of SETBP1 mutations in MDS/MPN patients at a single medical center in South Korea. Patients and methods We analyzed a cohort of 34 MDS/MPN patients (10 aCML, 7 CMML-1, 9 CMML-2, 5 JMML, 3 MDS/MPN unclassifiable) who were diagnosed and treated in Chonnam National University Hwasun Hospital (Hwasun, Korea) from October 2004 to June 2013. The mononuclear cells from bone marrow of the patients were separated and the total DNA was extracted by commercial kit (QIAGEN, Hilden, Germany). PCR and sequencing reaction were performed by targeting the hot spot (exon 4, codon 778-979) of the SETBP1 gene. The PCR mixture consisted of 50 to 100 ng of total DNA, 20 pmol of each forward (5'-CCACTTTCAACACAGTTAGGTG-3') and reverse (5'-TCTCGTGGTAGAAGGTGTAACTC-3') primer, 0.4 mM of each dNTP, 5 μL 10X F-taq reaction buffer, 5 U of DNA Polymerase (Solgent, Daejeon, Korea) and H2O in a final reaction volume of 50 μL. Direct sequencing was performed using the ABI Prism 3130XL Genetic Analyzer with the BigDye Terminator v3.1 Ready Reaction Kit (Applied Biosystems). Clinical information about patients was obtained from our electronic medical record database. All statistical computations were performed using PASW 18.0 (SPSS Inc., Chicago, Illinois, USA). Results In this analysis, 4 (11.7%) of 34 MDS/MPN patients showed SETBP1 mutations. 3 of them were aCML patients and 1 was CMML-2 patient. The frequencies in aCML and CMML-2 were 30% and 11.1%, respectively. All of the aCML patients with SETBP1 mutation showed mutation encoding c.2898G>A (p.Asp868Asn) and the CMML-2 patient displayed c.2903C>T synonymous mutation (Ser869).The mutated SETBP1 patients showed a tendency of higher mean WBC counts, lower mean hemoglobin, lower mean platelet counts and lower mean BM blasts percentage than the wild-type patients, but they were not statistically significant. One of the mutated SETBP1 patients showed a i(17)(q10) cytogenetic abnormality. We found no statistical difference in overall survival (OS) between mutated SETBP1 patients and wild-type patients. Conclusions Alteration of SETBP1 gene was a common genetic event in aCML with an impact as a diagnostic marker for MDS/MPN. Disclosures: No relevant conflicts of interest to declare.

Background Recent studies have demonstrated that measurements of BCR-ABL1 transcript levels at 3 and 6 months were able to identify high-risk patients treated with IM. However, the value of early molecular response has not been fully defined. New European Leukemia Net (ELN) recommendations concluded that a single measurement of BCR-ABL1 transcripts level after 3 months of treatment is not sufficient to define failure necessitating a change of treatment. The aim of this study was to identify predictive factors for an achievement of 3-month EMR. For the purpose, we explored contributing factors including trough plasma concentrations of IM, precise IM dose schedule. Additionally, in the same population, prognostic implications of 3- month EMR were analyzed. Methods Between December 2009 and June 2012, 102 patients with newly diagnosed CP CML were enrolled. They started IM (400 mg/day) therapy without prior treatment except hydroxyurea or anagrelide and molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). All qRT-PCR tests were performed in a single laboratory (Cancer Research Institute, The Catholic University of Korea, Seoul, Korea). Measurements of IM plasma concentrations were performed on day 29. Results A total of 102 newly diagnosed CP CML patients (including 61 men and 41 women) were analyzed. With a median age of 41 years (range, 18-75 years), the distribution of low, intermediate and high Sokal risk scores were 42%, 42% and 16%, respectively. All patients, except one patient who showed less than complete hematologic response, were evaluable for molecular analyses at 3 months. Day 29 trough IM level data were available from 99 patients. The median trough concentrations of IM were 1,252 (range, 439-3,491) and cut-off IM levels for Q1 and Q4 quartiles were 958 and 1767 ng/mL on day 29, respectively. Univariate analyses revealed that age of ≥ 40 years (P = 0.046), high Sokal risk (P = 0.066), high Euro risk (P = 0.004), increased platelet count (P = 0.028), increased blast percentage (0.023), and large spleen size (P = 0.046) were potential predictive factors for no achievement of 3-month EMR. In addition, plasma IM trough level of ≤ Q1 quartile on day 29 was associated with no achievement of 3-month EMR. After adjusting for factors affecting achievement of 3-month EMR on univariate analyses, multivariate analyses showed that large spleen size (RR of 0.79, P = 0.030) was predictor for no achievement of 3-month EMR and patients in ≤ Q1 of plasma IM trough level on day 29 had a lower 3-month EMR, compared with those in Q2-4 (RR of 15.61, P = 0.005). To evaluate the prognostic value of 3-month EMR in our cohort, we analyzed CCyR at 6 months, MMR at 12 months, and the 3-year CI of CCyR, MMR, and UMRD. In addition, the 3-year EFS, FFS, and PFS were also assessed. In patients with BCR-ABL1 ≤10% at 3 months, significantly higher rates of CCyR at 6 months (79% vs 13%, P < 0.001) and MMR at 12 months (54% vs 10%, P = 0.014) were observed, compared with that of patients with BCR-ABL1 >10%. They also had significantly better 3-year CI of CCyR (100% vs 63.0%, P Conclusions This study analyzed various factors, such as baseline biological characteristics, adherence to IM, IM dose intensity, pharmacokinetics. It provides predictors for 3-month EMR and re-confirmed the prognostic significance of 3-month EMR. The considering of IM plasma concentrations for 3-month EMR should be needed in the clinical decision of changing therapy at this time point. Further clinical investigations in a larger patient population with longer follow-up are needed. Disclosures: No relevant conflicts of interest to declare.

Introduction Recent randomized trial (DASISION trial), compared clinical outcomes between dasatinib and imatinib in newly diagnosed (chronic myeloid leukemia) CML patients. Results showed that dasatinib had faster and deeper responses compared with imatinib, and thus dasatinib became to be considered as a first-line therapy in CML patients. However, the long-term administration of dasatinib has been reported to have a risk of pleural effusion and dasatinib-related (pulmonary arterial hypertension) PAH. So, we have prospectively evaluated for emergence of PAH in all CML patients treated with dasatinib in our institute. Materials and methods Between May 2005 and July 2013, a total of 89 CP CML patients on dasatinib treatment were tested with laboratory studies, Brain natriuretic peptide (BNP), echocardiography (echo) in the Catholic University of Korea, Seoul, and baseline functional capacity (NYHA or WHO functional class) was assessed. PAH was defined as mean pulmonary artery pressure (mPAP) of >25mmHg at rest, with wedge pressure < 15mmHg, or Rt. Ventricular systolic pressure (RVSP) of 40 mmHg on echocardiography. And patients who had abnormal RVSP and/or symptoms (New York heart association-NYHA class 3, 4) were performed with additional studies such as pulmonary angiographic catheterization or pulmonary arterial computed tomography. Results So far, 62 patients (70%) of total 89 were evaluated with echocardiography (46 male, 16 female). The median age was 48 year old (range, 22∼72). The median duration of disease was 8.5 years (range, 0.8∼16.1). The median mean daily dosage of dasatinib was 102mg (range, 73∼140mg). The duration of dasatinib treatment was 34.6 months (range, 0.5∼99.6). 8 of 66 patients (12.1%) showed abnormal echocardiographic findings as increased right ventricular systolic pressure or symptom. All of 8 patients had treated with dasatinib as second line, and had exertional dyspnea (2 patients on class 2, and 5 patients on class 3 of NYHA). 5 patients of these showed abnormal BNP levels. The mean RVSP in screened patients was 65.2 mmHg (range, 40∼108mmHg), 2 of 8 patients were confirmed diagnosis with overt PAH on as pulmonary angiographic catheterization, and right ventricular hypertrophy on pulmonary angiographic computed tomography. Clinical, functional, or hemodynamic improvements were observed within 5 months of dasatinib discontinuation and then, in 5 patients the treatment was switched to radotinib, and another 3 patients received reduced dosage of dasatinib. Conclusion Although the lowest estimate of incident PAH occurring in patients exposed to dasatinib was 0.45% in France group. our preliminary data show more higher incidence rates (8 of 66 patients, 12.1%) in Korea. So, we suggest that the long-term dasatinib treatment for CML requires careful attention to cardiopulmonary adverse effects. and routine cardiovascular and pulmonary evaluation on regular basis is strongly recommended before and during treatment with dasatinib. Disclosures: No relevant conflicts of interest to declare.

Background BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients through impaired binding of TKI to the target site. Recent studies have reported that multiple mutations detected in 2-9% of patients with imatinib (IM)-resistant CML were associated with poor response rate and survival outcomes. However, biological characteristics and dynamics of multiple low-level mutations are still not assessed with a quantitative serial follow-up data in the same populations. Aims The aim of this study was to investigate biological characteristics and dynamics of low-level mutations in the serial samples from the patients carrying multiple mutations using subcloning and sequencing. Methods Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKIs including imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib (BOS), radotinib (RAD) or ponatinib (PON) at Seoul St Mary’s Hospital using direct sequencing and allele specific oligonucleotide-polymerase chain reaction (ASO-PCR). Among them, 31 patients carried ≥ 2 BCR-ABL1 kinase domain mutations. We analyzed 137 samples from these 31 patients using subcloning and sequencing (in total, 2737 colonies were sequenced). By cloning and sequencing, two or more missense mutations present in the same clone were defined as compound mutation and co-existence of single missense mutations in the separated clones was defined as polyclonal mutation. Co-existence of single missense mutation and compound mutation harboring two or more missense mutations in the same clone was defined as mixed mutation. Missense mutations detected by direct sequencing are defined as predominant mutations. Missense mutations detected by cloning and sequencing but not by direct sequencing are defined as low-level mutations. Results In a total of 2737 colonies from 137 samples, 1596 (58%) colonies harbored ≥ 2 missense mutations with a median 2 (range, 2 – 7) mutations, and 905 (33%) colonies with a single mutation and 236 (9%) colonies with wild type were observed. In 2737 colonies, 692 different low-level mutations were detected by cloning and sequencing but not by direct sequencing. Among them, M244V, G250E, Y253H, E279K, T315I, F317L, M351T, E355A, F359I, and F359V were detected by direct sequencing in the followed-up samples, and the others remain undetectable by direct sequencing. To address whether these low-level mutations were distinct in the patients with TKI resistance, we applied the cloning and sequencing to samples from 3 healthy controls and 3 patients with optimal response to IM. 38 different mutations were detected healthy controls. 52 different mutations were detected in optimal responders. S349P, N374S, E450G, and S485P were detected in both healthy controls and optimal responders. All mutations detected in optimal responders except 3 missense mutations (V335A, F382V, and A395S) were detected as low-level mutations in the 31 patients’ cohort with TKI resistance. Of 38 different mutations detected in healthy control, 31 mutations except 7 missense mutations (Y264H, M343R, A350V, E373G, G298R, E462K, and T495K) were also detected as low-level mutations in the 31 patients’ cohort. Conclusions We showed basic characteristics and dynamics of low-level mutations by subcloning and sequencing. Some of the low-level mutant clones harboring previously known (clinically common) TKI resistant mutations changes to the predominant in the followed-up samples. Except them, most low-level mutant clones were not detected repeatly and did not increase gradually in the serial samples, implying that they may not have clinical significance. Disclosures: No relevant conflicts of interest to declare.

Background Approximately 50% of CP CML patients achieve undetectable molecular residual disease (UMRD) at 6-7 years of first-line imatinib (IM) therapy. Although imatinib therapy is effective in chronic myeloid leukemia (CML) patients and a substantial portion of patients achieve UMRD with prolonged IM therapy, up to 10^7 leukemic cells can still be present in the absence of detectable BCR-ABL1 in RQ-PCR assay due to the sensitivity limit of current RQ-PCR technology. The recent several reports to assess whether IM can be discontinued in CML patients have shown that IM discontinuation can be employed based on clinical study in patients who had enough IM therapy and UMRD durations prior to IM discontinuation. In our previous report, the 12-month probability of sustained UMRD of 48 patients was 80.8% (78.4% in 37 patients with at least 12 months follow-up), a higher rate than that reported by the ‘STIM’ (Stop Imatinib) trial. To identify predictors for safer, successful IM discontinuation and to explore additional contributing factors for sustained molecular responses (MRs), this multicenter prospective Korean Imatinib Discontinuation Study (KIDS) is on-going. Methods Patients who were treated with IM for more than 3 years and whose BCR-ABL1 was undetectable in RQ-PCR for at least 2 years were enrolled in this study. After discontinuation, molecular response was monitored using RQ-PCR assay every month up to 6 month follow-up, every 2 months up to 12 month follow-up, and every 3 months thereafter. In case of relapse, defined as loss of MMR on 2 consecutive assessments, IM therapy was re-introduced and molecular response after resumption was observed every month using RQ-PCR. Our primary objectives were to evaluate the probability of persistent UMRD at 12 month follow-up after discontinuation, and to measure the duration of persistent UMRD after discontinuation. The secondary objective was to evaluate the probability of major molecular response (MMR) loss and the time taken to lose MMR after discontinuation. Results Of the 100 patients who were screened for KIDS, 22 patients have failed because of positive result of RQ-PCR tested in the central laboratory (N= 15, 69%), failure to meet inclusion criteria (N=4, 18%) and informed consents withdrawal (N=3, 13%).As of data cut-off date of 10 July 2013, a total of 78 patients (42 females, 36 males) who were diagnosed between 26 Feb 1998 and 11 Dec 2009 were enrolled on KIDS, with a median age of 45 years (range, 19 – 82), the percentages of patients with low, intermediate and high Sokal risk scores were 37%, 29% and 13%, respectively with unknown Sokal risk scores in 18%. And the median time on IM therapy and the median duration of sustained UMRD were 85 months (range, 38 – 141) and 44 months (range, 23 – 114), respectively, prior to discontinuation. With a median follow-up of 14 months (range, 0.3 – 33.5), the 12-month probability of sustained MMR and UMRD were 78.5% ± 5.3% and 78.5% ± 5.1%, respectively. All patients with MMR loss were in non-transplant group, whereas none of the 21 patients in the transplant group experienced MMR loss. The probability of sustained MMR and UMRD in non-transplant group were 67.3% ± 7.7% and 68.5% ± 7.0%, respectively. Nine of 13 patients who lost MMR were resumed IM with a median time of 15 months (range, 0.1 - 22.3 months). All 9 patients, who resumed IM, re-achieved MMR at a median of 3 months (range, 1.4 - 4.7 months) after resuming IM therapy and re-achieved UMRD at a median of 7.5 months (range, 3.3 - 13.3 months). Univariate analysis of factors affecting loss of MMR showed that IM therapy duration and UMRD duration before treatment discontinuation had a higher 12-month probability of sustained MMR. Conclusions Our updated data shows lower relapse rate after discontinuation compared to previous discontinuation studies. In particular, the rate of screening failure due to positive result of RQ-PCR tested in the central laboratory implied that strict PCR sensitivity criteria is important for safer, successful IM discontinuation. Disclosures: No relevant conflicts of interest to declare.

Background Recent studies have demonstrated that early molecular response (EMR) is predictive for long-term outcomes. However, the value of EMR has not been fully defined. Recently, European Leukemia Net (ELN) recommended that BCR-ABL1 ≤ 10%, and/or Ph+ Methods CP CML patients who were newly diagnosed and receiving 400mg IM once daily with no prior treatment were eligible for this study. Molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). All qRT-PCR tests were performed in a single laboratory (Cancer Research Institute, The Catholic University of Korea, Seoul, Korea). Pharmacokinetics data of IM, drug adherence, and dose intensity as well as baseline biological characteristics were included as variables affecting the achievement of 6-month EMR. Results A total of 102 patients (including 61 men and 41 women) were enrolled. One patient changed IM treatment to second-generation TKI due to less than complete hematologic response before 3 months. At the time between 3 and 6 months, 9 patient were discontinued permanently from IM treatment due to progression (n = 1), ELN failure (n = 3), and intolerance (n = 5). Ninety-two patients’ molecular responses were analyzed at 6 months. Day 29 trough IM level data were available from 99 patients and trough IM level data on the end of cycle 6 were available from 84 patients. Univariate analyses revealed that age of ≥ 40 years (P = 0.061), male sex (P = 0.042), b3a2 transcript type (P = 0.008), intermediate (P = 0.007) and high Sokal risk (P = 0.013), increased leukocyte count (P = 0.018), increased blast percentage (0.028), large splenic size (P = 0.020), and mean daily dose by 6 months of In the current study, patients with BCR-ABL1 ≤1% at 6 months had a better MMR rates at 12 months (63% vs 10%, P1% at 6 months showed a trend for lower 3-year PFS, compared with those who achieved ≤1% (100% vs 94.1%, P = 0.063). Conclusions In this study, we re-confirmed the prognostic significance of 6-month EMR and found that b2a2 transcript type, early decline of BCR-ABL1 transcript, mean daily dose by 6 months (≥350 mg/day), and Sokal risk were associated of the achievement of 6-month EMR. These predictive factors for 6-month EMR should be considered in the clinical decision of changing therapy at this time point. Further clinical investigations in a larger patient population with longer follow-up are needed. Disclosures: No relevant conflicts of interest to declare.

Introduction Since the introduction of imatinib (IM), it has been used as a standard frontline therapy for newly diagnosed chronic phase (CP) chronic myeloid leukemia (CML). Its efficacy and durable response have been proved by several clinical studies. However, several prior studies suggested that there may be differences of clinical outcomes according to age groups, and the effect of age on clinical outcomes remains uncertain. The purpose of this study was to evaluate the safety and efficacy of IM in elderly patients by the comparison between age groups (younger patients < 60 years vs. elderly patients ≥ 60 years). Methods Between January 2001 and March 2013, 798 patients with newly diagnosed CP CML were analyzed. They started IM therapy without prior treatment for leukemia except for hydroxyurea or anagrelide within 6 months of diagnosis. Routine chromosomal analyses were performed using standard banding techniques with bone marrow aspirates and molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). The main end points of this study included the comparisons of IM intolerance, response for IM therapy and survival between two age groups. Results A total of 798 newly diagnosed CP CML patients (including 476 men and 322 women) were analyzed. The patients were divided into the following two age subgroups: younger patients < 60 years (n = 698) vs. elderly patients ≥ 60 years (n = 100). Median age was 38 years (range, 5-59) and 66 years (range, 60-86) for each group. In the characteristics for each age group, older age group had a higher Sokal risk score (P< 0.001) and EURO score (P< 0.001). In contrast, EUTOS score was similar (P = 0.157). IM treatment duration of younger and elderly patients were 37.7 and 22.5 months, respectively (P Conclusions The incidence of IM intolerance was higher in elderly patients and IM treatment duration of elderly patients was significantly shorter than it of younger patients. The elderly patients showed low CHR, CCyR and MMR compared with younger patients. Accordingly, an effective treatment strategy with tolerable side effect profiles for elderly patients should be considered. For these reasons, upfront use of second-generation TKIs in elderly patients may be beneficial. Disclosures: No relevant conflicts of interest to declare.

Background In chronic myeloid leukemia (CML), achievement of optimal responses by time point has improved long-term outcomes. In IRIS study, patients who achieved major molecular response (MMR) at 18 months had event-free survival (EFS) benefit, compared to those who achieved complete cytogenetic response (CCyR) without MMR. However, the best treatment for these patients is still not confirmed. By the previous studies, sustaining standard-dose of imatinib (IM) is expected to yield less than 20 percent of additive MMR. In this study, we investigated the efficacy of switching to nilotinib (NIL) versus high-dose IM versus standard-dose IM for patients with suboptimal molecular response to IM as first-line therapy. Methods Early chronic phase (CP) CML patients who have achieved CCyR but no MMR after at least 18 months and up to 24 months ( 18 to 24 months) on first-line IM therapy at a daily dose of 400 mg were enrolled in RE-NICE study, and informed consents were obtained from all patients. In NIL arm, patients received 400 mg BID (800 mg/day) and in high-dose IM arm, patients received 800 mg/day administrated as 400 mg BID. Primary endpoint is to evaluate the cumulative MMR rates by 12 months, and secondary endpoints are to evaluate the cumulative MMR, MR4.0 and undetectable molecular residual disease (UMRD) rates during further 24 month follow-up. The efficacy of switching to NIL or high-dose IM were compared with that of the patients who maintained standard-dose of IM. Patients with standard-dose IM were selected with the same inclusion criteria and maintained standard-dose IM after enrollment period. To compare the efficacy among three groups, MMR rate, MR4.0 and undetectable molecular residual disease (UMRD) rates by 12 months were analyzed. Safety profiles also be assessed. Patients showing lack of response (lack of complete hematologic response (CHR) at 6 months, increasing WBC, no major cytogenetic response (MCyR) at 24 months), loss of response (loss of CHR or MCyR) or severe intolerance to treatment were allowed to crossover to the alternative treatment. Results With a data cut-off date of 15 Jul 2013, a total of 52 patients were randomized into NIL arm (n = 26) or high-dose IM arm (n = 26) and 16 patients were included in standard-dose IM group. With a median follow-up of 21 months (range, 1-36) in NIL arm and high-dose IM arm, all patients have maintained CCyR without progression to advanced disease, and progressive decrease in BCR-ABL1 transcript levels was observed in all patients. With a median follow-up of 12 months (range, 1-60), all patients in standard-dose IM group have maintained CCyR without progression to advanced disease. Cumulative incidence (CI) of MMR by 12 months showed no significant difference between NIL arm and high-dose IM arm (41.1% vs 28.8, P = 0.334). Only in NIL arm, 2 in 26 (8%) achieved confirmed MR4.0 and UMRD. By 12 months, 10 in 26 (39%), 7 in 26 (27%) and 3 in 16 (19%) patients achieved MMR, in NIL arm, high-dose IM arm and standard-dose IM group respectively. Overall, the patients treated with high-dose IM showed toxicities more frequently, such as leucopenia, anemia, Thrombocytopenia, edema, fatigue, dyspnea and decreased phosphate. In addition, 14 patients in high-dose IM arm have cross-over to NIL treatment due to lack of response (n=11) and intolerance (n=3), and the median duration of NIL treatment was 23 months (range, 2-36 months). Among them, 6 (43%) patients have achieved MMR with a median NIL treatment duration of 12 months (range, 0-18). Conclusions These results demonstrate that early switching to NIL or dose escalation of IM could be recommended, considering the results of standard dose of IM in suboptimal molecular responders. When the tolerability of treatment was considered for switching to NIL or high-dose IM, NIL may be preferred. Through further clinical investigation on a large patient population and longer period observation, the efficacy and safety of early intervention of suboptimal molecular response using NIL or dose escalation of IM will be needed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 1677 Background: BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL1 kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. Methods: Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKI including imatinib, nilotinib, dasatinib, bosutinib, radotinib or ponatinib at Seoul St Mary's Hospital using direct sequencing and ASO-PCR. Among them, 31 patients showed multiple mutations. We analyzed serial samples from the 31 patients using subcloning and sequencing to investigate whether the multiple mutations are on same clone (defined as compound clone), separated clones (defined as multiple clone) or co-existent clones (defined as mixed clone) and characterize its clinical relevance and dynamics. Results: Status of the patients with multiple mutations is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 31 patients. Cloning of cDNA region corresponding to BCR-ABL1 KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 65% patients (20 out of 31) existed compound mutation which means the individual mutant types are located on the same BCR-ABL1 molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL1 KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL1 mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL1 mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL1 gene in patients with point mutation are relatively unstable. Among 20 patients with compound mutation, 9 patients were available for serial timepoint samples under same TKI therapy. In all nine patients (100%), portion of compound clone was increased as treatment went on. With a median follow-up 53.3 months (range, 0–113.2 months), of 31 patients with multiple mutation, 7 patients remained alive; 4 of 11 (36%) in the multiple clone group vs 3 of 20 (15%) in the mixed clone group (P = 0.066). Conclusion: Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutations as treatment went on. Patients with compound clone showed poor outcomes compared with multiple clone group in our cohort, further investigation on a large patient cohort will be needed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 2794 Background The main difference of recently developed new prognostic classification, EUTOS score, from the conventionally used Sokal and Hasford scores is the simplicity of adopting only 2 clinical parameters, basophil counts and size of spleen, to discriminate high vs. low risks in chronic myeloid leukemia (CML). Concerns may rise from possible inter-observer gap when measuring the size of spleen. While there is currently controversial debate on its prognostic power, we present our single center results of comparing EUTOS score with other two scores. Patients and Methods Among 380 adult patients, consecutively registered as early chronic phase (CP) CML at Seoul St. Mary's Hospital (Jan. 2004 – Apr. 2010), who received imatinib (IM) as frontline therapy, 255 were evaluable for analysis: subjects were strictly limited to those with documented clinical parameters measured at very early time of diagnosis by a single doctor, before any cytoreduction therapies. The median age was 42 years (range, 19–77) with a slight male predominance (57%). Cytogenetic and molecular responses were calculated at specific time points and cumulative rates of response were compared according to risk scores. Event-free survival (EFS) was analyzed by time from start of IM to the time of events, defined by death from any cause, primary or secondary resistance to IM, progression to advanced phases, intolerance to IM and switch to second line tyrosine kinase inhibitor (TKI) or allografting. Response assessment followed current European Leukemia Net recommendation. Results Median time from diagnosis to IM start was 14 days (range, 1–183). After a median follow-up duration of 56.6 months (range, 13.0–102.2) in survivors, there were 10 deaths and 3 patients with disease progression to accelerated phase of blastic crisis. Sixty-four patients had switched IM to second TKI due to either IM resistance or intolerance. Overall comparisons are described in Table 1. Of 42 EUTOS high risk patients, only 25 high risk patients by Sokal and 15 by Hasford were included. Almost 90% of either low or intermediate (INT) risk patients by all risk models achieved complete cytogenetic response (CCyR) at 12 months after IM, whereas high risk groups had lower rates, especially in EUTOS high risk (57%, p = Conclusion By simply comparing the p-values, our data may implicate that high risk group discriminated by the EUTOS score is predicted with more unfavorable outcomes in achieving CCyR, MMR and survival after IM frontline therapy. Furthermore, the lack of statistically significant difference between outcomes of low and INT risk groups by conventional risk models may challenge the need for future classification of INT from low risk in this era of TKI. Disclosures: No relevant conflicts of interest to declare.

Abstract 3775 Background. In chronic myeloid leukemia (CML), achievement of optimal responses by time point has improved long-term outcomes. In contrast, several clinical studies investigating the clinical implications of suboptimal response showed that patients with suboptimal responses tend to have poor long-term outcomes. In IRIS study, patients who achieved major molecular response (MMR) at 18 months had event-free survival (EFS) benefit, compared to those who achieved complete cytogenetic response (CCyR) without MMR. However, the best treatment for these patients is still not confirmed. By the previous studies, sustaining standard-dose of imatinib (IM) is expected to yield less than 20 percent of additive MMR. In this prospective study, we investigated whether switching to nilotinib (NIL) or high-dose IM may be more effective for patients with suboptimal molecular response to IM as first-line therapy. Methods. Early chronic phase (CP) CML patients who have achieved CCyR but no MMR after at least 18 months and up to 24 months (≤ 18 to ≥24 months) on first-line IM therapy at a daily dose of 400 mg were enrolled in this clinical trial, and informed consents were obtained from all patients. In NIL arm, patients received oral dose of 400 mg BID (800 mg/day) and in high-dose IM arm, patients received 800 mg/day administrated as 400 mg BID. Primary endpoint is to evaluate the cumulative MMR rates by 12 months, and secondary endpoints are to evaluate the cumulative MMR, MR4.0 and undetectable molecular residual disease (UMRD) rates during further 24 month follow-up. Safety profiles will also be assessed. Patients showing lack of response (lack of complete hematologic response (CHR) at 6 months, increasing WBC, no major cytogenetic response (MCyR) at 24 months), loss of response (loss of CHR or MCyR) or severe intolerance to treatment were allowed to crossover to the alternative treatment. Results. With a data cut-off date of 10 Jul 2012, a total of 43 patients were randomized into NIL arm (n = 22) or high-dose IM arm (n = 21). With a median follow-up of 15 months (range, 1–36), all patients have maintained CCyR without progression to advanced disease, and progressive decrease in BCR-ABL1 transcript levels was observed in all patients. Cumulative incidence (CI) of MMR by 12 months showed no significant difference between NIL arm and high-dose IM arm (37.8 ± 11.9% vs 34.8 ± 10.6%, P = 0.789). In NIL arm, 3 in 22 (14%) and 2 in 22 (9%) patients achieved MR4.0 and UMRD, respectively, and in high-dose IM arm, 1 in 21 (5%) patients achieved MR4.0. Overall, the patients treated with high-dose IM showed toxicities more frequently, such as fatigue, dyspnea and decreased phosphate. In addition, 10 patients in high-dose IM arm have cross-over to NIL treatment due to lack of response (n=9) and intolerance (n=1), and the median duration of NIL treatment was 14 months (range, 7–26 months). Among them, 5 (50%) patients have achieved MMR with a median NIL treatment duration of 12 months (range, 3–18). Conclusions. These results demonstrate that early switching to NIL or dose escalation of IM could be recommended, considering the results of standard dose of IM in suboptimal molecular responders. When the tolerability of treatment was considered for switching to NIL or high-dose IM, NIL may be preferred. Through further clinical investigation on a large patient population and longer period observation, the efficacy and safety of early intervention of suboptimal molecular response using NIL or dose escalation of IM will be needed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 2763 Approximately 50% of CP CML patients achieve complete molecular response (CMR) at 6–7 years of first-line imatinib therapy. Although imatinib therapy is effective in CML patients and a substantial portion of patients achieve CMR with prolonged imatinib therapy, up to 10^7 leukemic cells can still be present in the absence of detectable BCR-ABL in RQ-PCR assay due to the sensitivity limit of current RQ-PCR technology. The recent data from STIM (Stop Imatinib) trial showed that the probability of persistent CMR at 12 month follow-up after imatinib discontinuation was 41%, and the conclusion was that imatinib can be safely discontinued, at least in some patients with persistent CMR. However, it is still not clearly defined whether discontinuation of imatinib therapy can be safely employed in patients with sustained CMR. In our prospective study, we examined if imatinib therapy can be safely discontinued in CML patients with sustained CMR4.5 according to strict PCR sensitivity criteria, and CMR4.5 was defined as undetectable BCR-ABL using RQ-PCR assay with at least 4.5-log sensitivity. CML patients who were treated with imatinib for more than 3 years and whose BCR-ABL was undetectable in RQ-PCR for at least 2 years were enrolled in this study. Our primary objectives were to evaluate the probability of persistent CMR4.5 at 12 month follow-up after discontinuation, and to measure the duration of persistent CMR4.5 after discontinuation. The secondary objective was to evaluate the probability of major molecular response (MMR) loss and the time taken to lose MMR at 12 month follow-up after discontinuation. In patients with loss of MMR, the probability of re-achieving MMR/CMR4.5 and the time taken to re-achieve MMR/CMR4.5 after imatinib resumption were also evaluated. After discontinuation, molecular response was monitored using RQ-PCR assay every month up to 6 month follow-up, every 2 months up to 12 month follow-up, and every 3 months thereafter. Digital PCR methodology with higher sensitivity compared to RQ-PCR assay was also applied before discontinuation and every year after discontinuation for more accurate estimation of BCR-ABL transcript levels. In case of relapse, defined as loss of MMR on 2 consecutive assessments, imatinib therapy was re-introduced and molecular response after resumption was observed using both RQ-PCR and digital PCR assays. As of data cut-off date of 15 Jul 2011, 20 patients (13 females, 7 males) who were diagnosed in Seoul St. Mary's Hospital between 20 Mar 1996 and 25 Apr 2005 were enrolled in this study with a median follow-up of 7 months (range, 2–9), and informed consents were obtained from all patients prior to participation. With a median age of 44 years (range, 25–67), the percentages of patients with low, intermediate and high Sokal risk scores were 30%, 30% and 15%, respectively with unknown Sokal risk scores in 25%. Ten patients (50%) received SCT and/or interferon therapy prior to imatinib therapy, while 10 patients (50%) received first-line imatinib therapy. The median time on imatinib therapy and the median duration of sustained CMR4.5 were 91 months (range, 40–112) and 60 months (range, 23–104), respectively, prior to discontinuation. Since discontinuation of imatinib therapy, all of 20 patients remained off therapy at the last follow-up with persistent CMR4.5 in 18 patients (90%) and loss of CMR in 2 patients (10%). Although loss of CMR was observed in 2 patients, both patients have not resumed imatinib therapy as MMR was maintained at the last follow-up. Our preliminary data show lower relapse rate after discontinuation compared to previous discontinuation studies. Strict PCR sensitivity criteria should be employed to assess the accurate measurement of BCR-ABL transcript levels prior to discontinuation, and then it might be possible to safely stop imatinib therapy in CML patients with stable CMR4.5. Through further clinical investigation on a large patient population and longer period of observation, more concrete conclusion can be made regarding the outcome of imatinib discontinuation. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.

Abstract 2765 In CML, achievement of major molecular response (MMR) is a significant prognostic factor as it has been shown to be associated with longer duration of complete cytogenetic response (CCyR) and long-term progression-free survival. In IRIS study, patients who achieved both CCyR and MMR showed higher progression-free survival rates, compared to those who had CCyR without MMR. Higher doses of imatinib are expected to yield higher CCyR and MMR rates, compared to standard dose of imatinib, and second-generation tyrosine kinase inhibitor, nilotinib also produces high CCyR and MMR rates in patients with CP CML who are resistant to imatinib. In this prospective study, the efficacy of nilotinib and high-dose imatinib was investigated in suboptimal molecular responders who received standard-dose imatinib as first-line therapy. Early CP CML patients who have achieved CCyR but no MMR after at least 18 months and up to 24 months (≥ 18 to ≤ 24 months) on first-line imatinib therapy at a daily dose of 400 mg were enrolled in this clinical trial, and informed consents were obtained from all patients prior to participation. In nilotinib arm, patients received oral dose of 400 mg BID (800 mg/day), and patients received 800 mg/day administrated as 400 mg BID in imatinib dose-escalation arm. To assess the drug efficacy, cytogenetics and RQ-PCR analysis were performed at regular intervals, and baseline mutational analysis was conducted for every patient with subsequent mutational analyses performed in patients demonstrating either lack of response or disease progression. Primary endpoint is to evaluate the cumulative MMR rates by 12 months, and secondary endpoints are to evaluate the cumulative CMR rates and time to and duration of MMR and CMR during further 24 month follow-up. Progression-free survival and safety profiles will also be assessed as secondary endpoints. Patients showing lack of response (lack of complete hematologic response (CHR) at 6 months, increasing WBC, no major cytogenetic response (MCyR) at 24 months), loss of response (loss of CHR or MCyR) or severe intolerance to treatment were allowed to crossover to the alternative treatment arm. With a data cut-off date of 18 Jul 2011, a total of 30 patients were randomized into nilotinib arm (n =13) or imatinib arm (n = 17), and 6 patients have crossed-over to nilotinib arm due to lack of response. With a median follow-up of 11 months (range, 0.2–28 mos), all patients have maintained CCyR without progression to advanced disease, and progressive decrease in BCR-ABL transcript levels was observed in all patients. Cumulative MMR rates at 20 months were significantly higher in nilotinib arm compared to imatinib dose-escalation arm (59.00% vs. 27.40%, P = 0.047), and patients treated with nilotinib also showed faster molecular response rates, with 5 patients achieving MMR within 3 months of nilotinib therapy. At the last follow-up, 7/13 (53.85%) and 2/11 (18.18%) patients achieved MMR in nilotinib arm and in high-dose imatinib arm, respectively, with 1 patient in nilotinib arm achieving 4-log reduction of BCR-ABL transcripts. Although toxicity was observed more frequently in imatinib dose-escalation arm, all patients currently maintain the initial dose (except 1 patient who interrupted imatinib therapy due to neurosurgical operation), and based on the toxicity data, no additional or serious adverse events were developed except for pre-existing toxicities before randomization. These preliminary results demonstrate that early intervention using nilotinib or dose escalation of imatinib could be recommended in suboptimal molecular responders, with nilotinib being more preferable. Through further clinical investigation on a large patient population and longer period of observation, efficacy and safety of early intervention of suboptimal molecular response using nilotinib or dose escalation of imatinib will be assessed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: Woodman: Novartis: Employment, Equity Ownership. Szczudlo:Novartis: Employment, Equity Ownership. Kim:Novartis: Employment.

Abstract 3441 Resistance to imatinib can occur in chronic myeloid leukemia (CML). Mutations in BCR-ABL kinase domain have been known as the clinically most relevant mechanisms of imatinib resistance. Several studies have shown that 40~80% of the imatinib resistant patients have BCR-ABL kinase domain mutations. However, there has not been much information about mutation status in CML patients with suboptimal response to imatinib. With samples from Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) study which investigates efficacy of imatinib 400 mg daily and 800 mg daily with Philadelphia positive CML CP patients, and relationship between various responses and emergence of mutation was investigated by both standard and highly sensitive mutation assays. As a clinical correlative study (CCS) program with samples from TOPS study, we analyzed BCR-ABL mutation from 51 patients with optimal response, suboptimal response or treatment failure to imatinib in order to investigate their mutation status at different time points including diagnosis, 6 months, 12 months and 18 months. In addition, 53 non-clinical trial patients from Seoul St. Mary's hospital were involved in this analysis. All patients were in CP. Suboptimal responders and treatment failures were selected based on European Leukemia Net (ELN) 2009 guideline. CML patients' samples from the TOPS study and Seoul St. Mary's hospital were collected at particular time points after initiation of imatinib treatment and stored as cryopreserved cells or isolated RNAs. Mutations in BCR-ABL kinase domain were analyzed using direct sequencing and allele-specific oligonucleotide (ASO)-PCR (for Y253H, Y253F, G250E, E255K, E255V, T315I, F359V, and M351T). We performed mutation analysis with total 164 samples collected from 104 patients at different time points including diagnosis, 6 months, 12 months and 18 months after initiation of imatinib treatment. Serially collected samples at all time points were not available for all the patients; samples from 31 patients were available at diagnosis, 41 patients at 6 months, 58 patients at 12 months and 34 patients at 18 months (Table 1). We found ten BCR-ABL kinase domain point mutations including G250E, Q252H, Y253H, T315I, F317L, E355G, F359V, F359I and D444Y in 13 patients. In addition, we also found other mutations including 35 base pair insertion between exon 8 and exon 9 of ABL, and deletion of exon 7 of ABL in other 10 patients. No mutation was found from the patients' samples collected at diagnosis. At 6 months, mutation was found 5% (1 of 21), 18% (2 of 11) and 22% (2 of 9) patients in optimal response, suboptimal response and treatment failure group, respectively. ASO-PCR revealed that one patient in optimal response group had T315I. The same mutation status of the patient maintained at 12 months and the patients showed treatment failure at 12 months. At 12 months, mutation portion was 0% (0 of 15), 13% (2 of 15) and 25% (7 of 28) in optimal response, suboptimal response and treatment failure group, respectively. At 18 months, 36% (5 of 14) of suboptimal molecular responders who achieved CCyR, but no MMR showed mutation, and 62% (8 of 13) of failure group who showed less than CCyR had mutations. No particular difference in mutation frequency was found between 400mg group and 800mg group. Patients with suboptimal response or treatment failure showed much higher chance of BCR-ABL point mutation, 35 base pair insertion or exon 7 deletion in comparison with optimal responders, suggesting that mutation screening is important for patients with suboptimal response as well as treatment failure on the basis of ELN guideline. Treatment failure who achieved less than CCyR at 18 months and suboptimal responders who achieved CCyR, but no MMR at 18 months were highly recommended for mutation screening based on these data. Highly sensitive ASO-PCR provided early detection of point mutation in BCR-ABL kinase domain. However, clinical relevance of low level mutant clone, 35 base pair insertion and exon 7 deletion require long-term follow up for better understanding. Table 1. Response Diagnosis 6 months 12 months 18 months Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Optimal response 0 31 1 (5%) 21 0 (0%) 15 0 (0%) 7 Suboptimal response 2 (18%) 11 2 (13%) 15 5 (36%) 14 Failure 2 (22%) 9 7 (25%) 28 8 (62%) 13 Total Patients 31 41 58 34 Disclosures: Pane: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hochhaus:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Abstract 3443 BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD. We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available. Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable. Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Table 1. Patients Tx at mutation detection (mg) Compound type Compound % 1 Nilotinib400 G250E+T315I 6.7 G250E+D444G 33.3 T315I+D444G 6.7 2 Nilotinib400 M244V+T315I 95.0 3 Dasatinib100 Y253H+T315I 95.0 4 Dasatinib140 T315I+E459K 55.6 5 Dasatinib200 T315I+M351T 66.7 6 Dasatinib100 NCM Dasatinib80 NCM Dasatinib100 M244V+F359V 16.7 7 Bosutinib500 NCM 8 Dasatinib140 T315I+F359C 35.3 9 Imatinib400 E255K+T315I 5.6 10 Dasatinib80 E255V+T315I 90.0 11 Imatinib800 E255K+T315I 10.5 12 Nilotinib800 E255K+T315I 12.5 13 Dasatinib100 F311I+T315I 35.0 F311I+F317Lb 10.0 Imatinib400 F311I+T315I 10.0 F311I+F317La 15.0 F311I+F317Lb 55.0 14 Nilotinib800 Y253H+F359I 5.6 15 Bosutinib500 V299L+E459K 95.0 Nilotinib400 + Dasatinib100 V299L+F359I 5.0 V299L+E459K 55.0 V299L+F317La+E459K 15.0 V299L+F359I+E459K 15.0 V299L+F317La+F359I+E459K 5.0 16 Imatinib600 NCM 17 Imatinib400 NCM NCM: no compound mutation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1240 Use of reliable predictable factors in newly diagnosed chronic phase (CP) CML patients could ensure the most appropriate selection of therapy in individual patients, and the Sokal score which was developed in the pre-interferon era still retains prognostic values in patients treated with imatinib. In addition to the Sokal score, patients with a high BCR-ABL transcript level at diagnosis might respond differently to therapy. However, to our knowledge, there has been no distinct study to investigate the association between BCR-ABL transcript level at diagnosis and the response to therapy. It has been generally agreed that baseline BCR-ABL transcript levels are not relevant to predict the response to therapy, and the use of international scale (IS) is also based on the concept in which 100%IS is defined as the standardized baseline and 0.1%IS corresponds to major molecular response (MMR) in all CML patients. In this study, cytogenetic and molecular responses to the therapy was investigated in patients with high BCR-ABL transcript levels at diagnosis and patients with low BCR-ABL transcript levels at diagnosis to assess if baseline BCR-ABL transcript levels could be the reliable predictable factor in CP CML patients. Between May 2001 and Aug 2008, total 756 CML patients were treated with imatinib in St. Mary's Hospital of the Catholic University of Korea, and among them, 113 CP patients have been treated with imatinib at a dose of 400 mg/day for more than 6 months without interferon or stem cell transplantation prior to imatinib therapy. BM or PB samples were obtained at regular intervals from diagnosis for hematologic response (HR), cytogenetic response (CyR) and molecular response (MR) monitoring. For additional statistical analysis, 102 patients with Sokal scores available at the time of diagnosis were divided into 3 groups: low, intermediate and high Sokal groups. The median baseline BCR-ABL transcript level for 113 patients was 103.79%(IS), and the median BCR-ABL transcript levels were compared between 57 patients in a group of high baseline BCR-ABL levels and 56 patients in a group with low BCR-ABL levels by 3, 6, 12 and 18 months of imatinib therapy (Table 1). No difference was observed when the median values at month 3 and month 6 were considered, but from month 12 to month 18 after initiation of imatinib, the median BCR-ABL levels in patients with high baseline BCR-ABL levels was at least twice as high as median BCR-ABL levels in patients with low baseline BCR-ABL levels. However, differences cannot be considered to be significant due to the small number of patients under analysis. Regarding the CCyR rate by 3, 6, 12 and 18 months of imatinib therapy, no significant difference was observed between 2 groups of high and low baseline BCR-ABL levels, and there was also no statistically significant difference in CCyR and MMR rates depending on the baseline BCR-ABL levels even after grouping the patients according to Sokal scores.Table 1:Median BCR-ABL levels over time under imatinib treatment depending on the baseline BCR-ABL transcript levels.BaselineBCR-ABLtranscriptlevels (IS%)Month 3Month 6Month 12Month 18> 103.79n25534625median3.900.850.330.20range0.74 - 38.870 - 77.840 - 26.660 - 3.00< 103.79n36513122median4.110.950.150.07range0 - 183.300.001 - 41.980 - 8.040 - 2.51 In this study, no evidence of the correlation of the baseline BCR-ABL levels and the response to therapy was observed, and BCR-ABL level at diagnosis does not seem to give additional prognostic information to predict the response to therapy. Thus, the value of baseline BCR-ABL levels is still questionable to be used to identify patients who require higher dose of imatinib or more potent tyrosine kinase inhibitors, and further study with larger cohort and longer follow-up would be necessary to confirm whether baseline BCR-ABL levels are relevant to predict the response to therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1726 With prolonged imatinib therapy, BCR-ABL transcript levels measured by RQ-PCR assay show a progressive reduction, and some patients achieve complete molecular remission (CMR), which is defined as sustained undetectable BCR-ABL using RQ-PCR assay with a sensitivity of at least 4.5-log below the standardized baseline. However, due to the sensitivity limit of the current RQ-PCR technology, PCR negativity should not be considered as cure as more than 106 leukemic cells can still remain in the absence of detectable BCR-ABL transcripts by RQ-PCR. Although the numbers of patients with undetectable BCR-ABL are increasing with prolonged imatinib therapy and also with advent of more potent novel tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib and bosutinib, currently there is no methodology to further classify the patients in CMR. In this study, digital PCR (dPCR) assay was applied for measurement of BCR-ABL transcript levels in patients with CMR to assess if more sensitive detection methodology can be implemented for molecular monitoring in CML. Between May 2001 and Aug 2008, total 757 CML patients were treated with imatinib in St. Mary's Hospital of the Catholic University of Korea, and 192 chronic phase (CP) CML patients have been under imatinib therapy for more than 2 years. Among them, 36 patients achieved PCR negativity at least once during imatinib treatment, and serial PB samples collected from the patients who have maintained CMR for at least 3 years in RQ-PCR were screened by dPCR. In dPCR assay, each sample was partitioned into hundreds to tens of thousands of reaction chambers, and this sample partitioning enables detecting extremely low copy numbers that would normally be undetectable by conventional RQ-PCR platforms. Using the BioMark Real-Time PCR System (Fluidigm) and 12.765 Digital Array (Fluidigm) in dPCR assay, only 1 liquid-transfer step is required to automatically partition each of 12 samples into 765 reaction chambers of approximately 4.6 ul (6 nl × 765), and pre-amplification step was performed prior to dPCR assay to improve the sensitivity. Regarding the detection limit, whereas down to 10-5 of cell line dilutions and down to 10-4 of patient sample dilutions were detectable using conventional RQ-PCR, dPCR showed 2–3 log improvement in the detection sensitivity limit by detecting down to 10-7 of cell line dilutions and patient sample dilutions. In all the patient samples collected at the first time point of PCR negativity, positive BCR-ABL signals were detected in dPCR assay, and gradual decrease in the number of positive signals were observed in the serial samples collected on yearly basis after achieving CMR. PB samples collected from 5 healthy individuals were also screened to confirm the significance of positive results in dPCR, and no amplification was detected in all of 5 samples. This study shows that the patients in CMR who is currently categorized based on the data derived from conventional RQ-PCR assay could be classified further using more sensitive methodology such as dPCR assay. In the previous studies on imatinib discontinuation, the selection criteria of candidates was solely based on the duration of PCR negativity prior to discontinuation, and conventional RQ-PCR was employed to measure PCR negativity. The fact that the absolute number of residual leukemia clones could not be measured under the detection limits of conventional RQ-PCR might have resulted in relapse in more than half of the patients, and it reflects that firm conclusions cannot be drawn about if a patient could safely discontinue the therapy solely based on conventional RQ-PCR. It might be necessary to have more sensitive assays which will allow further classification of patients who could be candidates for imatinib discontinuation without relapse. This study shows the potential of highly sensitive PCR approach for molecular monitoring, and dPCR examined here can be extended to expand our understanding of molecular profiles in CML patients and to correlate to clinical significance. Detection and quantitation of low copy numbers may help to more precisely follow-up the course of disease and thereby to more accurately tailor personalized therapeutic choices. Disclosures: No relevant conflicts of interest to declare.

Abstract 4696 SET-NUP214 rearrangement at chromosome 9q34 is an extremely rare recurrent cryptic deletion resulting from del(9)(q34.11q34.13) mainly found in T-cell acute lymphoblastic leukemia (T-ALL). A multiplex RT-PCR system (HemaVision; DNA Technology) covering 28 leukemic fusion transcripts was applied to 271 bone marrow (BM) samples obtained from acute leukemia patients at initial diagnosis to investigate new pathophysiologic role of SET-NUP214 fusion gene. Out of 271 samples, we identified three cases (1.1%) harboring SET-NUP214 rearrangement – each case of adult T-ALL, pediatric acute myeloid leukemia (AML) from chronic myeloid leukemia (CML) and adult de novo AML with minimal differentiation (M0). Adult T-ALL occurred in 38-year-old man presented with dizziness and hepatomegaly. Conventional cytogenetic study disclosed normal karyotype and existence of SET-NUP214 fusion gene generated by del(9)(q34.11q34.13) was confirmed by RT-PCR, FISH and direct sequencing. Hematological remission was achieved after treatment of induction chemotherapy but during 7 months follow-up, SET-NUP214 fusion gene was steadily detected by RT-PCR. 13-year-old female with AML M0 converted from CML after treatment of imatinib for 2 months was the second case. Leukemic cells showed negative myeloperoxidase (MPO) and sudan-black B (SBB) staining, positive reaction against CD13, CD33, CD117 and MPO monoclonal antibodies by flowcytometry-based immunophenotyping. Major BCR-ABL1 rearrangement and SET-NUP214 fusion genes were simultaneously detected by RT-PCR, FISH and direct sequencing. She died at 38 days after diagnosis during the chemotherapy. The fusion protein contributed to the leukemogenesis by promoting expression of HOXA cluster genes. Last case was a de novo AML M0 in 39-year-old male. Cytochemical stains of leukemic cells on MPO and SBB were negative and immunophenotyping studies were positive for CD34, HLA-DR, CD13 and CD33. SET-NUP214 rearrangement was detected by multiplex RT-PCR. After treatment of induction and consolidation chemotherapy, he received allogeneous sibling stem cell transplantation (SCT), then complete chimerism was achieved. In clonclusion, SET-NUP214 rearrangement is associated with the development of not only T-ALL, but also primary and secondary AML via aberrant expression of HOXA cluster genes. Disclosures: No relevant conflicts of interest to declare.

Abstract 4568 Background Acute myeloid leukemias (AML) are maintained by a subpopulation of leukemia initiating cell that can regenerate themselves as well as give rise to more differentiated and less proliferative cells that constitute the bulk of the disease. Recent studies discovered both the nature of leukemia stem cells and their origin. Immunophenotype of AML stem cell (ASC) is known as CD34+CD38– leukemic cells. Yet, despite their critical importance, much remains to be learned about the frequency, proportion and dividing activity of ASC in a single cell levels. To address these issues, the current study investigated the frequency, proportion according to AML FAB subtypes and single cell plating efficiency of ASC from bone marrow (BM) at the initial diagnosis. Materials and methods The proportion and frequency of ASC were examined on BM samples of twenty AML patients using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). Growth and proliferation capacities of single normal hematopoietic stem cells and LSC were determined using plating efficiency (number of the wells in which more than two cells grew / total number of cells in 96-well plate culture x 100). Individual single cells were cultured in 96-well plates with each well containing 100 uL of serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF for five days. Results The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of remaining other subtypes was 13.6±16.2%. CD34 percentage in BM at the initial diagnosis closely correlated with the proportion of ASC. ASC proportion was statistically significant different between patient's group with more than 20% of CD34 and those with less than 20% (P=0.003). Single cell plating efficiency of ASC was markedly reduced compared with non-ASC (CD34+CD38+ leukemic cell population) (7.2 ± 4.7% in ASC vs. 12.6 ± 7.6% in non-ASC). Conclusion ASC proportion and frequency in BM samples were very diverse according to FAB subtypes. This study directly proved lower dividing activity of single ASC, and also implicated strategies for single ASC cloning to develop new therapeutics targeting ASC. Disclosures: No relevant conflicts of interest to declare.

Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.

Dasatinib, as a 2nd generation tyrosine kinase inhibitor, is an effective Bcr-Abl kinase inhibitor for chronic myeloid leukemia (CML) with more than 300-fold and 20-fold potency than imatinib and nilotinib, respectively. Through a series of phase I and II clinical trials, dasatinib has demonstrated durable efficacy in CML patients with resistance, suboptimal response, or intolerance to imatinib. However, some adverse effects from dasatinib therapy have been reported in several studies. Among them, pleural effusion is one of the most common adverse effects, and was reported in 15 to 30% of patients. We investigated the development of pleural effusion from 64 Korean patients registered in a single center for a phase II study which was approved by the institutional review committee and carried out in accordance with the Declaration of Helsinki with informed consent provided from patients. All the patients are under dasatinib therapy after failure of imatinib treatment (22 patients with intolerance and 44 patients with resistance). Median age was 44 years old (range, 18 to 65) and median duration of dasatinib therapy was 29 months (range, 0.5 to 41). The patients were in different phases including 30 in chronic phase (CP) and 30 in accelerated phase (AP) when they started dasatinib treatment. We found that 23 patients (36%) experienced pleural effusion of any grade (grade 1/2 in 22, grade 3/4 in 1) at least one time. Among the 23 patients, 14 (61%) patients experienced recurrent pleural effusion, and two of them showed a change in grade from grade 1 to 2. The median time to the first occurrence of pleural effusion was 18 weeks (range, 1 to 132), developing within the first 6 months and 12 months of treatment in 13 patients (57%) and in 15 patient (65%), respectively, while the adverse effect occurred even after 28 months (2.3 years) of treatment in 3 patients (13%). Age, gender, previous interferon a treatment, duration of imatinib treatment, and duration of dasatinib treatment were not significantly different between patients without pleural effusion and patients developed pleural effusion. However, disease phase when dasatinib therapy started showed significant difference (P=0.002) in the development of pleural effusion. Patients in AP showed higher cumulative % of pleural effusion in comparison with those in CP (16/34, 47% vs. 7/30, 23%). Daily dose amount and dose schedule also gave an influence on occurrence of pleural effusion. We used 4 different categories in dose amount and dose schedule; 100 mg QD (n=3), 140 mg QD (n=32), 50 mg BID (n=7) and 70 mg BID (n=22). Frequency of pleural effusion was higher in the patients treated with BID schedule than in QD schedule (56% vs. 22%, P=0.005), and also higher in the patients treated with 140 mg per day than in 100 mg per day, but not significant (39% vs. 20%, P=0.23). Among 4 categories, 70 mg BID showed relatively higher % of pleural effusion (64%, 14/22, P=0.006). To see whether dose amount or dose schedule can influence on efficacy, we investigate cytogenetic responses from patients. Portion of complete cytogenetic response (CCyR) was not significantly different in 100 mg doge group and 140 mg dose group (67% vs. 64%). Time to achieving CCyR showed no significant difference (P=0.21) between 100 mg dose group (median 3 months, range; 2 to 17) and 140 mg dose group (median 6 month, range; 1 to 18). In addition, dose schedule did not make any significant difference in portion of achieving CCyR between QD group and BID group (60% vs. 59%). Based on the observed characteristics of pleural effusion and analysis of cytogenetic response in this study, lower dose (100 mg) administration by QD can be proposed for dasatinib therapy to reduce any possible occurrence of pleural effusion. Figure Figure

Diagnosis of chronic myeloid leukemia (CML) is based on detection of the BCR-ABL gene or Philadelphia chromosome, and the BCR-ABL tyrosine kinase inhibitor imatinib has been the standard therapy for CML patients. Although imatinib therapy is effective in CML, it is still unclear whether imatinib can be safely discontinued without relapse. This study was designed to investigate the outcome of 26 CML patients after discontinuation of imatinib and to determine whether intermittent imatinib therapy can be employed in CML patients. Between May 2001 and Jun 2007, 555 patients have been treated with imatinib in St Mary’s Hospital of the Catholic University of Korea, and 26 patients discontinued imatinib when they achieved either complete cytogenetic response (CCyR) or complete molecular response (CMR). These 26 patients were diagnosed as Philadelphia positive (Ph+) CML between November 1995 and May 2002, and 22 patients were in chronic phase (CP) and 4 patients were in accelerated phase (AP) at diagnosis. The median age was 35 years (22–56), and 12 patients (46%) were female and 14 (54%) were male. Among 26 patients, 7 received interferon prior to imatinib therapy and 7 underwent SCT. Five patients received both interferon and SCT before imatinib therapy, and the remaining 7 patients received the imatinib as a front line therapy. Imatinib was started at oral dose of 400mg and 600mg daily for patients in CP and AP, respectively, and when they achieved CCyR or CMR, imatinib was discontinued after informed consent of the patient. In case of cytogenetic or molecular relapse, patients in all phases were retreated with imatinib at 400mg daily. Bone marrow (BM) or peripheral blood (PB) samples were obtained at regular intervals from diagnosis for hematologic response (HR), cytogenetic response (CyR) and molecular response (MR) monitorings. Eleven patients discontinued imatinib when they achieved CCyR, and 15 patients discontinued imatinib after achieving CMR. After the median duration of 7 month (4–48) observation without imatinib therapy, hematologic, cytogenetic and molecular relapses occurred in 4, 7 and 10 patients, respectively, and imatinib at oral dose of 400mg daily was reintroduced to all patients except 2 who continued to remain in CMR after imatinib discontinuation. Except 1 patient who expired and 2 patients who are in persistent molecular remission, all of 23 patients are maintaining the best response achieved after imatinib resumption with a median duration of 38 months (16–58). In conclusion, although imatinib cannot be discontinued completely, intermittent therapy can be considered for the treatment of CML patients. Intermittent imatinib treatment should not be restricted to CP patients who achieve CMR, and AP patients or patients with CCyR also can be considered for intermittent imatinib treatment. We will continue the follow-up of the patients enrolled in this study, and long-term study of intermittent imatinib treatment with expanded pool of patients will enable us to determine the accurate consequences of discontinuation of imatinib and intermittent imatinib treatment.

Background: An acquired somatic mutation in the JAK2 gene (V617F) could present the primary causative lesion in BCR-ABL-negative chronic myeloproliferative disorders (CMPD). However, some of the clinical and genetic data implied that the pathophysiological role of JAK2 V617F mutation in the CMPD would be more complex. Quantitative assessment of JAK2 V617F mutation has shown substantial heterogeneity in the genomic DNA. Recently, it has been reported that allelic variation in gene expression is common in the human genome. To test the hypothesis that JAK2 V617F mutant allele could be increased in cDNA, we examined JAK2 V617F mutation status in the genomic DNA and cDNA from a total of 78 patients with BCR-ABL-negative CMPD. Patients and Methods: Enrolled patients with BCR-ABL-negative CMPD in this study comprised 42 cases of essential thrombocythemia (ET), 26 polycythemia vera (PV), 7 idiopathic myelofibrosis (MF) and 3 unclassifiable (UC) CMPD. A 364-bp PCR product containing JAK2 V617F mutation was bidirectionally sequenced from total BM cells. A quantitative real time PCR-based allelic discrimination assay and pyrosequencing (Pyrosequencer PSQ96) were developed for quantitative analysis of JAK2 V617F mutation status. Homozygous JAK2 V617F mutation was defined if the mutant peak was more than 50% of total peak area. Results: The proportion of mutant alleles ranged from 36% to 100% in real-time PCR and pyrosequencing analysis. Patients with MF had higher percentages of JAK2 mutant alleles than patients with ET (MF > PV > ET). The prevalence of homozygous V617F mutations was significantly higher in PV patients (73%) than in patients with ET (17%). Allelic expression imbalance of heterozygous JAK2 mutation was common in patients with PV and ET. Interestingly, allelic expression imbalance in patients with MF was not remarkably impaired, but preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with PV and ET. Conclusion: Allelic imbalance in the gene expression of JAK2 V617F mutant could provide the underlying mechanisms to elucidate phenotypic variation in BCR-ABL negative CMPD.