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Idle time at work is a phase of involuntary downtime during which employees experience that they cannot carry out their work tasks. In contrast to breaks, interruptions, procrastination, or withdrawal behavior, employees cannot work because of the absence of work-related tasks. Based on action regulation theory, we develop an integrative conceptual model on the antecedents and consequences of the subjective experience of idle time. We propose that work constraints (i.e., regulation problems) have negative effects on occupational well-being and task performance, and that these effects are mediated by subjective idle time. The strength of these effects is further assumed to be influenced by individuals’ use of proactive (i.e., prevention) and adaptive (i.e., coping) strategies. Results of a supplemental qualitative study, for which we interviewed 20 employees from different occupations, provided preliminary support for the propositions. Finally, we develop theory on how individual, situational, and organizational characteristics may influence the proposed effects on and of idle time. Overall, this conceptual development paper contributes to a better theoretical understanding of idle time at work by extending its definition and applying action regulation theory to this practically important phenomenon.

Prognostic impact of the European LeukemiaNet standardized reporting system in older AML patients receiving stem cell transplantation after non-myeloablative conditioning

In Leipzig ist es gelungen, uber ein Modellprojekt eine „Koordinierungsstelle kommunale Gesundheit“ innerhalb der Stadtverwaltung fest zu verankern, durch die Gesundheitsforderung als kommunale Querschnittsaufgabe sowie ein von Krankenkassen poolfinanzierter Fonds zur Forderung von Stadtteilprojekten etabliert wurde.

In acute myeloid leukemia (AML), leukemia-initiating cells exist within the CD34+/CD38- cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38- cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft-versus-leukemia effects. Percentage of bone marrow CD34+/CD38- cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%-89% of all mononuclear cells). A high CD34+/CD38- cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38- cell burden had shorter relapse-free and overall survival which may be mediated by residual leukemia-initiating cells in the CD34+/CD38- cell population, escaping the graft-versus-leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38- cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia-initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.

7042 Background: Surface antigen expression evaluation is part of the standard work-up at acute myeloid leukemia (AML) diagnosis. The biological & prognostic implications of surface antigen expression patterns in normal karyotype (NK) AML patients (pts) remain unknown. Methods: The diagnostic antigen expression patterns of mononuclear cells in bone marrow (BM) of 111 NK-AML pts were assessed using a standard flow cytometric panel. At diagnosis common AML gene mutations (mut) & expression levels were analyzed. Pts received stem cell transplantation (SCT, 98% allogeneic, 2% autologous; median age 63 years [y, range 26-74y]) after induction therapy at our institution. Median follow up was 3.3y. With R’s gplot package unsupervised hierarchical clustering of surface antigens was performed & revealed 4 distinct clusters. Results: Pts in cluster 1 (n = 36) had higher expression of immature, in cluster 2 (n = 31) of thrombocytic/T-cell/erythroid, in cluster 3 (n = 24) of monocytic & in cluster 4 (n = 20) of myeloid surface antigens. All 4 clusters associated with distinct clinical & molecular features. At diagnosis, compared to all others, pts in cluster 1 had a higher CD34+/CD38- cell burden ( P< .001), higher blood blasts ( P< .03) & BM blasts ( P< .06) by trend. They had less NPM1 mut ( P< .001) & DNMT3A mut ( P= .02), were more likely to be EVI1 positive ( P= .03) & had higher EZH2 ( P= .02), RUNX1 ( P= .009), BAALC ( P< .001), ERG ( P= .02) & MN1 ( P< .001) expression. Compared to all others, pts in cluster 1 had a higher cumulative incidence of relapse (CIR, P= .002, at 1y 41% vs 15%) & shorter event-free survival (EFS, P= .02, at 1y 50% vs 69%). In multivariate analysis, cluster 1 pts had a significantly higher CIR (Hazard Ratio [HR] 5.4, P= .01) after adjustment for FLT3-ITD & shorter EFS (HR 2.1, P= .02) after adjustment for FLT3-ITD, age & disease status at SCT. Conclusions: Pts in cluster 1 had high expression of immature surface antigens (eg CD34, CD117, CD13), genes involved in stem cell renewal & worse outcome. Our data indicate a relationship between easily accessible surface antigen expression patterns at diagnosis, molecular disease features & aggressiveness of the NK-AML phenotype.

Expression levels of miR-29 family members (i.e. miR-29a, miR-29b, & miR-29c) are deregulated in various neoplastic diseases, including acute myeloid leukemia (AML), known to affect DNA-methylation profiles by targeting epigenetic modifiers, & have been shown to be important for normal hematopoietic stem cell function. Mir-29 is organized in two distinctively regulated bi-cistronic clusters: the miR-29a/b-1 cluster & the miR-29b-2/c cluster. Here we evaluated the biological associations & clinical impact of the differential expression of pre-miR-29a/b-1 & pre-miR-29b-2/c clusters in AML. We analysed121 AML patients (pts) (median age 63 years [y], range 37-75 y) who have been consolidated with hematopoietic stem cell transplantation following non-myeloablative conditioning (nma-HCT; Fludarabin 30 mg/m2 on day -4 till -2 & 2 Gy total body irradiation) between 2000 & 2014 with pretreatment bone marrow material (BM) available. Disease status at nma-HCT was first (CR1 62%) or second complete remission (CR2 18%) or CR with incomplete peripheral recovery (CRi 20%). The mutation status (mut) of the ASXL1, CEBPA, DNMT3A IDH1, IDH2, NPM1, & TP53 gene & the FLT3-ITD & EVI1 expressionstatusas well as common surface marker expressions were assessed at diagnosis. European LeukemiaNet (ELN) classification was favorable (25%), intermediate-I (23%), intermediate-II (21%), adverse (27%) or unknown (4%). Pretreatment pre-miR-29a/b-1 & pre-miR-29b-2/c clusters expressionin bone marrow (BM)was measured by quantitative reverse transcription polymerase chain reaction & normalized to 18S. The median normalized gene expression defined high & low pre-miR-29a/b-1 & pre-miR-29b-2/c clusterexpressers. Median follow-up was 4.4y for pts alive. At diagnosis a high pre-miR-29a/b-1 expression did not associate with clinical characteristics. High pre-miR-29a/b-1 expressers were less likely to be TP53 mut (p=.01). Pts with high pre-miR-29b-2/c expression at diagnosis had higher BM blast counts (p=.01), were more likely to have a normal cytogenetics (CN, p=.03) & were less likely to be TP53 (p=.004) or ASXL1 mutated (p=.03). When we combined the expression status information of the two miR-29 clusters we found that AML blasts of pts with high expression of both clusters were less likely to be CD34 (p=.05) or CD117 (p=.04) positive & more likely to be CD11b positive (p=.05). These pts more often had CN-AML (p=.04) & better ELN genetic risk (p=.03). High expressers of both miR-29 clusters were also more likely to be DNMT3A mut (p=.01) & less likely to be EVI1 positive (p=.007). Noteworthy, none of the pts with high expression of both clusters had a TP53 (p=.16) or ASXL1 mutation (p=.08). Pts with a high expression of both miR-29 clustershad a significant longer relapse free survival (RFS, p=.01, Figure 1a) & overall survival (OS, p=.03) compared to pts with low expression of one or both miR-29 clusters. In conclusion, high expression of pre-miR-29a/b-1 & pre-miR-29b-2/c associated with different clinical & genetic characteristic at AML diagnosis. High expressers of both clusters were more often DNMT3A mutated, a gene targeted by miR-29. Furthermore, none of these patients harbored TP53 mutations, a gene known to be indirectly activated by miR-29 family members. These findings provide new insights into the miR-29 associated AML biology, which may contribute to the observed impact on AML pts outcomes. While we observed a trend for better survival for each miR-29 cluster, pts with high expression of the pre-miR-29a/b-1 & the pre-miR-29b-2/c clusterhad significantly longer RFS & OS. Figure 1 Figure 1. Disclosures Poenisch: Mundipharma: Research Funding.

In recent years expression levels of several genes & microRNAs (miR) were identified as strong prognostic markers, capable to refine AML risk stratification. So far technical difficulties, including the limitations of established methods for comparable, absolute quantification & the lack of defined cut points prevented translation of these findings into clinical practice. Innovative digital droplet PCR (ddPCR) is a novel technique with high sensitivity, specificity that allows absolute quantification - without the need for standard curves - & promises better inter-laboratory comparability. In AML pts, high miR-155 expression levels associate with the presence of FLT3-ITD & independently predict inferior outcome. Here, for the first time we applied ddPCR for absolute quantification of pre-miR-155 to define an absolute cut point to discriminate between high & lowexpressers, which was then validated in a second set of AML pts. We analyzed a homogeneous test set of 71 AML pts treated between January 2000 & June 2012 in our institution. All pts received cytarabinebased induction therapies & were consolidated with allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablativeconditioning (NMA; consisting of fludarabine[30mg / m² at days -4 to -2] & 2Gy total body irradiation [day 0]). At NMA-HCT ptswere in first (n=43; 60.6%) or second complete remission (CR; n=16; 22.5%) or CR with incomplete recovery (n=12; 16.9%). At diagnosis, cytogenetics & mutation status of the NPM1, CEBPA, IDH1, IDH2 & DNMT3A gene & presence of FLT3-ITD or FLT3-TKD mutation were assessed. The expression of the pre-miR-155 stem loop was measured using an EvaGreen-based ddPCR assay & normalized to the absolute copy numbers of ABL1. The R Package OptimalCutpointswas used to determine a cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies to discriminate between high (n =29; 40.8%) & low (n =42; 59.2 %) miR-155 expressers. High miR-155 expressers, more often had a FLT3-ITD (p=.039) & less frequently a mutation in the FLT3-TKD (p=.010). No significant association was found for other clinical or biological markers at diagnosis. In the test set, pts with more than 1.104 copies pre-miR-155 per 100 ABL1 copies at diagnosis had a significant shorter event-free survival (EFS; p=.03, figure 1A) & overall survival (OS; p=.009, figure 1B). To validate these findings, we used a second set of 71 pts (median age 63.4y [range 37.1 to 74.7]) with a median follow-up of 3.7y for pts alive that all received cytarabinebased induction therapies & NMA-HCT as consolidation. The ptsin the validation set also did not differ significantly in the analyzed clinical or molecular characteristics (i.e. white blood count, hemoglobin, platelets, blasts in bone marrow or peripheral blood at diagnosis, remission status at HCT [CR1 vs. CR2 vs. CRi], ELN genetic group, mutational status of FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 & presence of FLT3-ITD). Using the determined cut point of 1.104 copies pre-miR-155 / 100 ABL1 copies in the test set, patients in the validation set were divided in 39 patients (54.9%) with a high miR-155 expression & 32 (45.1%) with a low miR-155 expression. Pts with high miR-155 expression in the validation set had shorter EFS (p=.11, figure 2A) by trend & a significant shorter OS (p=.05, figure 2B). In conclusion, ddPCRis a novel, feasible method that allows absolute quantification of miRexpression. We defined an absolute cut point of 1.104 copies pre-miR-155 per 100 ABL1 copies for the prognosticator miR-155 in AML without the need for standard curves. Pts with pre-miR-155 expression above the cut point had a significant shorter EFS & OS. Remarkably, using a second clinically comparable set, we were able to validate our test set findings. Future studies are planned to confirm the clinical impact of pre-miR-155 expression levels at diagnosis, as well as the identified absolute pre-miR-155 / ABL1 copy number cut point to distinguish high from low miR-155 expressers. Figure 1 Test Set Figure 1. Test Set Figure 2 Validation Test Figure 2. Validation Test Disclosures Poenisch: Mundipharma: Research Funding.

Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC (Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALC copy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR. We identified 82 AML pts with PB in first (60%) or second CR (23%) or CRi (17%) up to 28 days prior to HSCT available. Median age at HSCT was 63.9 (range 50.8-76.2) years (y). All pts received non-myeloablative (NMA) conditioning (fludarabine 3x30 mg & 2 Gy total body irradiation). At diagnosis, mutation status (mut) of the NPM1, CEBPA, IDH1, IDH2,& DNMT3A gene & presence of FLT3-ITD or FLT3-TKD were assessed. In pre-HSCT PB, absolute quantification of BAALC copy numbers was performed by ddPCR & results were normalized to ABL1 copy numbers.Additionally, absolute BAALC copy numbers wereassessedin PB of healthy controls (n=7) with a median age of 62.7 (range 39.6-82.0) y. Pts were grouped according to the European LeukemiaNet (ELN) classification in 21% favorable, 23% intermediate-I, 24% intermediate-II, 23% adverse & 9% unknown. Pts & healthy control were evenly matched in age (P=1) & sex (P=1). BAALC/ABL1 copy numbers did not differ between AML pts at HSCT (median 0.03 [range 0.01-2.48]) & the healthy controls (median 0.04 [range 0.03-0.10], P=.34, Figure 1). A cut-off point of 0.14absolute BAALC/ABL1 copies was determined using the R package 'OptimalCutpoints' & used to define pts with high (26%) & low (74%) pre-HSCT BAALC/ABL1 copy numbers. The copy number at this cut-off point was higher than the two-fold standard deviation over the median of the healthy controls (0.10 BAALC/ABL1). Pts with high & low pre-HSCT BAALC/ABL1 copy numbers did not differ significantly in pre-treatment characteristics (i.e. hemoglobin, white blood count, platelets, blasts in bone marrow or PB, ELN genetic group, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 mut) or remission status at HSCT (CR1 vs. CR2 vs. CRi). However, pts with high pre-HSCT BAALC/ABL1 copy numbers had a significantly higher cumulative incidence of relapse (CIR, P=.02, Figure 2a) & shorter overall survival (OS, P=.02, Figure 2b). High pre-HSCT BAALC/ABL1 copy numbers especially impacted on CIR when we restricted our analysis to pts with normal cytogenetics (P=.003). In multivariate analysis for the entire cohort, high pre-HSCT BAALC/ABL1 copy numbers retained the prognostic impact on CIR (Hazard Ratio [HR] 3.6, Confidence Interval [CI] 1.6-8.2, P=.002) after adjustment for disease status at HSCT (P=.006) & the prognostic impact on OS (HR 2.2, CI 1.1-4.3, P=.02). In conclusion, ddPCR is a feasible method for absolute quantification of BAALC copy numbers in PB, which may indicate residual disease burden in AML pts. High PB BAALC/ABL1 copy numbers (>0.14) in AML pts in hematologic CR at HSCT associated with higher CIR & shorter OS in univariate & multivariate models. AML pts with high PB BAALC/ABL1 copy numbers at HSCT should be closely monitored for relapse in the post-transplant period. In the future prospective studies will be required to validate the absolute PB BAALC/ABL1 copy number cut-off point & to evaluate whether AML pts with high BAALC/ABL1 copy numbersmight benefit from additional treatment before HSCT. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Poenisch: Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

Introduction: The transcription factor ZBTB7A regulates early differentiation of hematopoietic progenitors & has been associated with oncogenic as well as oncosuppressive functions. While it was shown that ectopic overexpression of Zbtb7a in immature lymphocytes leads to the development of an aggressive T-cell lymphoblastic leukemia, high ZBTB7A expression in cytogenetically normal acute myeloid leukemia (CN-AML) is associated with improved outcomes. Furthermore, recently a leukemogenic cooperation between RUNX1/RUNX1T1 & ZBTB7A mutations in t(8;21)-associated AML was suggested. Here, we further evaluated the complex role of ZBTB7A expression in hematopoietic malignancies by assessing its potential prognostic impact in AML pts undergoing hematopoietic stem cell transplantation (HSCT) after non-myeloablative conditioning (NMA). Methods: We analyzed bone marrow (BM) at diagnosis of 140 pts (median age 63 years [y], range 37-75y) treated at our institution between 2000 & 2015. All pts received NMA conditioning (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) followed by HSCT in complete remission with (CR; n=111; 79.3%) or without peripheral hematological recovery (CRi; n=29; 20.7%). Median follow-up for pts alive was 3.5y. Our cohort included pts with CN-AML (n=62, 44.3%), complex karyotype (n=17; P=12.1%) & other cytogenetic abnormalities (n=61; 43.6%). At diagnosis mutations in the genes CEBPA, DNMT3A, IDH1, IDH2, NPM1 & the presence of FLT3-ITD were determined. In diagnostic BM cytogenetics were analyzed using standard techniques for banding & fluorescence in-situ hybridization & the expression of common surface markers was analyzed using flow cytometry. The expression of ZBTB7A was assessed using quantitative RT-PCR & normalized to ABL1 as internal control. As a cut-off the third quartile of normalized gene expression was identified to group high & low ZBTB7A expressers. Results: At diagnosis pts with a high ZBTB7A expression more often had a complex karyotype (P=.02) & were less likely to have core-binding factor AML by trend (P=.18). Additionally, high ZBTB7A levels associated with significantly fewer blasts in peripheral blood (P=.008) & BM (P=.02). The BM mononuclear cells in high ZBTB7A expressers were to a smaller extent positive for myeloid markers (CD38 P=.03; CD33 P=.11; CD13 P=.13) & exhibited a higher percentage of erythroid (Glycophorin A P=.03) as well as monocytic (CD11b P=.04; CD14 P=.01) surface markers. We did not find any statistical associations between ZBTB7A levels & the mutation status of NPM1, CEBPA, IDH1, DNMT3A or the presence of FLT3-ITD. Yet, there was a trend for more IDH2 mutations in the group of high ZBTB7A expressers (P=.18). At diagnosis a high expression of ZBTB7A associated with a significantly higher cumulative incidence of relapse (CIR; P=.002, Figure 1A). This finding also translated into a significantly shorter overall survival (OS; P=.01; Figure 1B) for AML pts with high ZBTB7A levels at diagnosis. When we restricted our analyses to CN-AML, high ZBTB7A expression remained a negative prognostic factor by trend (CIR P=.16; OS P=.11). Conclusion: Expression of ZBTB7A associated with distinct biological features & surface marker pattern in AML. This underlines the results of recent studies which identified ZBTB7A as a novel player in leukemogenesis. However, our findings are in contrast with the previously shown favorable prognostic impact of high ZBTB7A levels in a CN-AML cohort mainly treated with chemotherapy. In contract all pts included in our studies were consolidated with NMA-HSCT. Since this treatment regimen is mainly based on the graft versus leukemia effect a high ZBTB7A expression could potentially interfere with the immunological recognition of the AML blasts resulting in a reduced response to NMA-HSCT. Consequently, future functional studies & clinical trials should aim at further characterize the complex role of ZBTB7A in AML. Figure Figure. Disclosures Poenisch: Mundipharma: Research Funding.

Introduction: Despite intensive research most patients with acute myeloid leukemia (AML) still have a dismal prognosis. The transcription factor RUNX1 is a master regulator of myeloid differentiation and in AML its function is often disrupted by chromosomal translocations or mutations. These genetic alterations impact on leukemogenesis, disease progression and prognosis of AML patients. The expression of fusion proteins involving RUNX1 has been shown to exert a dominant-negative effect over wild-type RUNX1 leading to impaired myeloid differentiation and enhanced proliferation, while loss of RUNX1 may impact negatively on the frequency and self-renewal capacities of long-term hematopoietic stem cells. However, it remains unclear if differential expression of RUNX1 affects the AML phenotype. Here we analyzed the prognostic value of RUNX1 expression levels in AML patients undergoing allogeneic hematopoietic stem cell transplantation (HCT) after non-myeloablative conditioning (NMA). Patients & Methods: We analyzed 132 patients (median age 64 years [y], range 38-75y) with diagnostic bone marrow material available who received NMA-HCT (3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation on day 0 followed by infusion of granulocyte-colony stimulating factor mobilized peripheral blood stem cells) at our institution between 2000 and 2012. Donors were human leukocyte (HLA)-matched related (15%) or HLA-matched (61%) or mismatched (24%) unrelated. 63 (48%) patients had a normal karyotype. Presence of FLT3 -ITD or FLT3 -TKD, mutational status of IDH1, IDH2, CEBPA, NPM1 and DNMT3A as well as expression levels of mir-9, mir-181a, BAALC, ERG and MN1 were determined at diagnosis. European LeukemiaNet cytogenetic classification was: 26% favorable, 27% intermediate-I, 20% intermediate-II and 27% adverse. RUNX1 expression at diagnosis was determined by qRT-PCR and normalized to 18S as internal standard. The median cut was used to define highand low RUNX1 expressers. Median follow-up was 3.9y for patients alive. Results: High RUNX1 expressers were more likely to have de novo AML (73% vs. 52%; P =.019) and higher % blasts in peripheral blood (median 38% vs. 19%; P =.004) and bone marrow (median 68% vs. 50%; P =.002) at diagnosis. Patients with high RUNX1 expression more often had IDH1 mutations by trend (P =.061) while there was no difference in IDH2 mutation frequency (P =.32). High RUNX1 expressers showed significantly higher ERG (P Conclusion: Our results revealed that high expression of the hematopoietic master regulator R UNX1 at diagnosis independently associated with survival in AML patients with normal karyotype receiving NMA-HCT. High RUNX1 expression associated with distinct clinical and molecular markers. Assessing pretreatment RUNX1 levels may help to refine risk stratification in AML patients undergoing NMA-HCT. Figure 1. Figure 1. Disclosures Franke: Novartis: Other: Travel Costs; MSD: Other: Travel Costs; BMS: Honoraria. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction: Minimal residual disease (MRD) monitoring after achievement of hematologic complete remission (CR) in patients (pts) with acute myeloid leukemia (AML) aids in identifying pts at risk of relapse. Evaluating MRD markers for clinical practice is crucial for improving outcomes in AML. Since mutations in the gene encoding Nucleophosmin (NPM1 mut) occur early in leukemogenesis & are relatively stable during disease course, they represent suitable markers for MRD monitoring. Approximately 30% of AML pts harbor NPM1 mut of which approximately 80% are type A. Some studies already demonstrated the feasibility to use NPM1 mut as MRD markers in AML pts and showed NPM1 mut MRD positivity at different time-points during disease course to be predictive for AML relapse. Hematopoietic stem cell transplantation (HCT) is a consolidating therapy option offering potential cure to AML pts. Here, we tested the feasibility to use NPM1 mut type A as MRD marker in pts receiving HCT applying digital droplet polymerase chain reaction (ddPCR) methodology. This novel technique offers high sensitivity, specificity & absolute quantification without the need of standard curves, promising inter-laboratory comparability. At the same time cost effectiveness may be superior to standard qualitative real-time PCR making ddPCR a promising new application. Methods: We analyzed 134 pts (median age 64 years [y], range 38-76y) who received HCT after non-myeloablative conditioning (NMA, 3x30mg/m2 Fludarabine on days -4 to -1 & 2Gy total body irradiation) in CR in our institution between 2000 & 2013. Donors were human leukocyte antigen (HLA)-matched related (n=23; 17.1%) or HLA-matched (n=79; 63.7%) or mismatched (≥1 antigen; n=32; 23.9%) unrelated. Median follow-up was 4.3y for pts alive. NPM1 mut status was assessed in diagnostic bone marrow (BM) samples by Sanger sequencing of the mutation hot spot in exon 12. All pts with NPM1 mut type A & available BM samples at the time of NMA-HCT (within 28 days before HCT) were analyzed by ddPCR. cDNA was applied to a duplex assay measuring NPM1 wild type (wt) & mutation type A simultaneously using fluorescent labeled probes. The mutation burden, defined as % mut type A copies/wt copies ratio, was normalized to ABL. Samples with NPM1 mut type A burden >0.01% were defined as MRD positive (MRD+) & samples with mutation burden ≤0.01% or Results: We identified 42 AML pts (24.8%) with NPM1 mut of whom 24 pts had a type A mutation & BM samples at the time of NMA-HCT available. In this set 16 pts (66.7%) were classified to have favorable risk according to European LeukemiaNet (ELN) classification, 7 pts intermediate-I (29.2%) & 1 patient intermediate-II (4.1%) risk. 8 of the 16 pts harbored a FLT3 -ITD. In 4 pts NPM1 mut type A MRD was detectable at the time of NMA-HCT of which 3 pts experienced relapse. The fourth patient died 3 months after NMA-HCT due to transplantation-related complications & no further MRD time-points were available for analysis. In our cohort all clinical relapses occurred within 6 months after HCT. Two pts relapsed without detectable NPM1 mut type A at NMA-HCT. For these two pts there were no follow-up samples for further MRD monitoring after NMA-HCT available. MRD+ status at the time of NMA-HCT associated with a significantly lower cumulative incidence of relapse (P=0.001, Figure 1A) & shorter overall survival (P=0.003, Figure 1B). Conclusion: Our data demonstrate that ddPCR is a feasible method to determine the NPM1 mut type A burden by absolute quantification. Additionally, we showed the applicability of NPM1 mut type A as MRD marker in AML pts at the time of NMA-HCT. 3 out of 5 pts with clinical relapse were MRD+ at NMA-HCT. Assessing the NPM1 mut type A MRD status at the time of NMA-HCT & possibly at later time-points after HCT by ddPCR might help to identify pts at high risk of relapse. Our results strengthen the observation to use NPM1 mut for MRD monitoring. Consequently, we aim at expanding the applied ddPCR methodology to other NPM1 mut types & testing the validity in the context of clinical studies at various time points before & after NMA-HCT. Figure 1. Figure 1. Disclosures Franke: Novartis: Other: Travel Costs; MSD: Other: Travel Costs; BMS: Honoraria. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Characterisation of antigen expression patterns is part of the standard diagnostic work-up in acute myeloid leukemia (AML). But the biological & clinical implication of such antigen expression patterns have not been studied extensively & remain unclear in AML patients (pts) undergoing allogeneic stem cell transplantation (SCT). We analyzed the diagnostic antigen expression patterns of 162 AML pts (median age 64.7 years [y, range 46.6-76.2y]) with available data who received allogeneic peripheral blood SCT after non-myeloablative conditioning (NMA-SCT) between 2001 & 2013 at our institution. Conditioning regimen was fludarabine 3x30mg/m2 & 2Gy total body irradiation. Donors were human leukocyte antigen (HLA) matched related (12%) or HLA matched (59%) or mismatched unrelated (29%). Mutation (mut) status of the NPM1, CEBPA, IDH1, IDH2 & DNMT3A gene, presence of FLT3 -ITD & FLT3-TKD & the expression status of BAALC, ERG, MN1, EVI1, miR-9 & miR-181a at diagnosis were accessed. Pts were grouped according to the European LeukemiaNet (ELN) genetic classification in 22% favorable (fav), 24% intermediate-I (int-I), 21% intermediate-II (int-II) & 32% adverse (adv). Median follow up was 3.2y. To assess antigen expression patterns at diagnosis for all pts, flow cytometric analysis utilizing a standard panel (CD2, CD7, CD11b, CD13, CD14, CD15, CD33, CD34, CD38, CD45, CD56, CD61, CD64, CD65, CD117 & Glycophorin A) of mononuclear cells in bone marrow (BM) was performed. Using R's gplot package we performed unsupervised hierarchical clustering of the antigen expression which revealed 4 subgroups with distinct antigen expression patterns (Figure 1). At diagnosis, pts grouped in cluster 1 (n=19) had higher white blood count (WBC, P=.004) & lower peripheral blood (PB) blast count (P =.03) & were more likely to have de novo AML (P =.05). They were also less likely to have trisomy 8 (P=.08) by trend & more likely to have normal karyotype (KT, P=.05), to have ELN fav risk (P =.04), to be NPM1 mut (P =.002) & to be DNMT3A mut by trend (P=.08) & had lower miR-181a (P=.04), lower BAALC (P Pts grouped in cluster 2 (n=35) had higher WBC (P Pts grouped in cluster 3 (n=59) had lower WBC (P Pts grouped in cluster 4 (n=49) had lower WBC (P=.03), higher PB blasts (P=.007) & BM blasts (P For the entire set of pts, belonging to one of the antigen expression clusters did not impact on outcome. However, when the ELN groups were regarded separately, within the ELN fav group, cluster 1 pts had a significantly shorter event free survival (EFS, P=.04, Figure 2A) & within the ELN int-I group, cluster 3 pts had a trend for better (P=.096) & cluster 4 pts for worse EFS (P=.087). In conclusion, the antigen expression patterns at diagnosis obtained by unsupervised cluster analysis associated with distinct biological & clinical features (Figure 2B): NPM1 mut were enriched in clusters 1 & 2. Cluster 1 was characterized by ELN fav risk, normal KT, de novo disease & lower BAALC, ERG, MN1 & miR-181a expression. Cluster 2 was characterized by a high incidence of FLT3-ITD. We found more pts with ELN adv risk, monosomal KT, secondary AML & low miR-9 expression in cluster 3 & higher miR-9 as well as lower BAALC, ERG & MN1 expression levels in cluster 4. Even though we did not observe a prognostic impact of the antigen expression patterns in the entire cohort, the patterns may help to refine the ELN risk classification for AML pts undergoing SCT. Assessing the diagnostic antigen expression patterns provides information on disease biology, clinical parameters and potentially disease aggressiveness in AML. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Franke: BMS: Honoraria; MSD: Other: Travel Costs; Novartis: Other: Travel Costs. Niederwieser:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

In retrospective studies, CALR mutation is found to be a favorable prognostic variable in myelofibrosis (MF) compared with JAK2, or MPL mutations. With the availability of the JAK1/JAK2 inhibitor ruxolitinib (RUX) for treatment of MF, it is not yet known whether response varies across mutational subgroups and whether the prognostic implication of the driver mutations in terms of survival (OS) changes under RUX. We studied the prevalence and prognostic weight of clinical parameters in the IPSS and DIPSS-plus and the impact of RUX on OS in the context of mutation status. Patient and Methods: As of November 2009, RUX for treatment of splenomegaly and/or constitutional symptoms became an option at the University of Leipzig. All patients (pt) with MF seen since then were included (n=127; f=43%, median age 58 years) with the exception of pt with MPL mut+ because of small number. Screening for the JAK2V617F and CALR mutations was performed as previously published. Cytogenetic-risk categorization was conducted as published (Caramazza et al. Leukemia 2011). Results: JAK2 mut+ (group A; 60.6%) constituted the largest group, followed by CALR mut+ (group B; 19.7%), and “triple-negative” (group C; 19.7%). The median duration of MF was 22 months with no difference in the 3 groups. Higher-risk IPSS was present in the majority [Int-II 34.4%; high-risk 42.4%]. Constitutional symptoms were present across all groups, although more frequent in group A which was also characterized by prominent splenomegaly (Table). Group B pt were younger with low WBC in contrast to goup C pt who were older, anemic, with high WBC and low platelets, unfavorable cytogenetics and high-risk IPSS. Hb < 100 g/L (54%), and transfusion-dependency (32%) were frequently present in group B and was similar to group A. RUX was given to 72 (57%) pt [graoup A (66%), group B (56%), group C (29%)] with a median exposure of 8 months. The response of 80% was not influenced by the mutation status, cytogenetics, or IPSS variables. Leukemic transformation was documented in 22 pt [14 (64%) pt had no RUX exposure] after a median of 18 months after diagnosis. The incidence was highest in group C (36%) and lowest in group B (4%) (p=0.004). Unfavorable cytogenetics were 75%, and 27% in group C and A respectively. After a median follow-up of 31 months, OS at 3-years was 82% and worst in group C (72%) vs 80% for group A (p=0.05) vs 96% in goup B (p=0.003). In univariate analysis, non-mutated status (p=0.02), Int-2/high-risk IPSS (p=0.06), anemia (p=0.03), transfusion dependency (p=0.04), and platelets < 100 x109/L (p=0.06) but not unfavorable cytogenetics were associated with an inferior OS. In multivariate analysis, advanced IPSS only (p=0.04) but not the mutation status (p=0.4) retained its negative impact on OS. Generally, in group A pt treated with RUX (n=50), OS was only marginally inferior to group B (p=0.08). In pt with int-2/high-risk IPSS, OS in group A with RUX (n=41) was similar to that of group B (p=0.4). Conclusions: The mutation status in MF bestows distinct clinical phenotypes and has crucial prognostic and therapeutic implications. Patients with non-mutated MF had the worst prognosis, unfavourable cytogenetics, and the highest rate of leukemic transformation. Although the IPSS retains its value, the prognostic power of certain factors such as anemia and constitutional symptoms in CALR-mutated pt needs to be re-evaluated. Response to ruxolitinib seems to be independent of the mutation status. More importantly, a JAK1/JAK2 inhibition appears to be capable of attenuating the prognostic implication of the different mutation profiles by improving the less-favorable survival associated with non-CALR-mutated MF through a disease-modifying effect. The full potential of a sustained JAK1/JAK2 inhibition in modifying survival in the context of the various mutational profiles needs to be further studied in a larger cohort of patients. Table Clinical variables in patients with different driver mutations Variable JAK2 mut+ CALR mut+ Triple-negative p N (%) 77 (60.6) 25 (19.7) 25 (19.7) Median age (years) 59 51 61 0.02 Constitutional symptoms (%) 69 52 46 0.08 Median palpable spleen (cm) 10 4 3 0.001 Int-2/high-risk IPSS (%) 79 52 96 0.01 WBC > 25 x109/L (%) 21 11.5 44 0.008 Median peripheral blasts (%) 1 0.5 2 ns Median Hb (g/L) 106 99 86 0.008 Hb < 100 g/L (%) 42 54 80 0.03 Transfusion dependency (%) 33 32 67 0.009 Platelets < 100 x109/L (%) 30 8 52 0.04 Unfavorable cytogenetics (%) 31 14 48 0.06 Disclosures Al-Ali: Novartis: Honoraria, Research Funding. Jaekel:Novartis: Honoraria. Lange:Novartis: Honoraria. Roskos:Novartis: Honoraria. Niederwieser:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction: Despite progress in understanding acute myeloid leukemia (AML) biology, most patients (pts) still have a poor prognosis. MicroRNAs (miR) have emerged as important players in AML biology. High expression of miR-181a has been associated with improved outcomes in AML pts. Mature miR-181a originates from two precursor molecules pri-miR-181a-1 & pri-miR-181a-2 which have been associated with distinct biological features & are derived from chromosome 1q32.1 & 9q33.3, respectively. Allogeneic stem cell transplantation (HCT) offers a potential curative treatment option for AML pts. With the introduction of reduced intensity conditioning (RIC) regimens, this procedure also became available for older or comorbid individuals. Here, we measured pri-miR-181a-1 & pri-miR-181a-2 and analyzed their impact on survival in RIC-HCT treated AML pts. Patients and Methods: 139 AML pts (median age 64 years [y], range 22-75y) who received RIC-HCT between 2000 and 2012 with pretreatment material available were included in our analysis. The conditioning regimens were based on Fludarabine 30mg/m2 on day -4 till -1 followed by 2 Gy total body irradiation. Disease status at RIC-HCT was first (n=79, 57%) or second complete remission (CR; n=23, 16%) or more advanced disease (n=37, 27%). Donors were human leukocyte antigen (HLA) matched related (n=22, 16%) or HLA matched (n=83, 60%) or mismatched (n=34, 24%) unrelated. The mutation status of the NPM1 & CEBPA genes & the FLT3-ITD status were assessed at diagnosis. Pts were grouped according to the European LeukemiaNet (ELN) classification in favorable (26%), intermediate-I (26%), intermediate-II (21%) or adverse (27%). Pri-miR-181a-1 & pri-miR-181a-2 expressions were measured by RT-PCR, normalized to 18S & the median normalized gene expression was used to define high & low expressers. Median follow up was 4.3y for pts alive. Results: The expression of both precursor molecules correlated well (Pearson Correlation 0.78), but 23% had a discordant expression status. At diagnosis pts with high pri-miR-181a-1 expression were younger at time of RIC-HCT (P=.01), had higher % peripheral blood (P=.05) & bone marrow (P=.04) blasts & by trend fewer NPM1 mutations (P=.054). Pts with high pri-miR-181a-2 expression had higher white blood count (WBC, P=.04). Combining the expression status information for both precursors, pts with high pri-miR-181a-1 and/or high pri-miR-181a-2 expression were younger at time of RIC-HCT (P=.04).Pts with high pri-miR-181a-1 and/or high pri-miR-181a-2 expression had longer overall survival (OS; P=.004, Figure 1) & event-free survival (EFS; P=.002). Interestingly a strong impact on outcome was observed in the ELN favorable group (OS: P=.001 & EFS: P=.008, Figure 1) and in the ELN intermediate II group (OS: P Conclusion: In conclusion, pri-miR-181a-1 & pri-miR-181a-2 expression correlated well, but were not fully concordant & associated with distinct clinical characteristics. The expression status of pri-miR-181a-1 & pri-miR-181a-2 at diagnosis is a strong independent prognostic factor for AML pts undergoing RIC-HCT. The prognostic impact was strongest in the ELN favorable and intermediate II groups. Pri-miR-181a-1 & pri-miR-181a-2 expression levels may contribute to improved risk stratification for AML pts undergoing RIC-HCT, refining the ELN favorable and intermediate II group. Furthermore, pretherapeutic miR elevation either pharmacologically or by miR-replacement therapies may improve outcomes in AML pts undergoing RIC-HCT. Figure 1 Figure 1. Disclosures Lange: Novartis: Consultancy, Honoraria, Research Funding.

In acute myeloid leukemia (AML) high expression of the transcription factor ERG (ETS related gene) is associated with dismal outcome. The mechanisms that regulate differential ERG expression remain to be fully elucidated. MicroRNAs (miRs), small RNAs that are able to regulate gene expression, have emerged as important players in AML. We hypothesized that part of the differential expression of ERG is mediated by miRs. In silico prediction tools identified three putative miR-9 binding sites (BS) in the 3'-untranslated region (UTR) of ERG. First, we determined the expression levels of ERG & miR-9 in eight leukemia cell lines (i.e. KG1a, K562, THP-1, MV4-11, EOL1, NB4, OCI-AML3 & ME1) & found an inverse correlation between ERG & miR-9 expression (rank correlation -0.90). The cell line KG1a had the highest ERG & low miR-9 expression, and was therefore used for miR-9 overexpression experiments. In these cells miR-9 overexpression decreased ERG expression at the mRNA level to 82±7% (P=.079) & at the protein level to 72±14% (P=.005) after 12 hours (h) compared to empty vector control transfected cells. Next, we tested the activity of the three putative miR-9 BS in the 3'-UTR of ERG using luciferase assays. 12 h after cotransfection of HEK-293T cells with a miR-9 overexpression vector & an appropriate luciferase vector containing two of the putative BS (BS1 & BS2) from the 3'-UTR of ERG, we found the luciferase activity reduced to 52±4% (P=.023). Mutation experiments showed BS1, but not BS2 to be essential for this activity. The insertion of BS3 into the luciferase vector had no effect on reporter gene expression. Thus miR-9 most likely regulates expression of the transcription factor ERG by directly binding to BS1 in its 3'-UTR. To test if a differential expression of miR-9 is also of functional significance in AML, we first analyzed its impact on cell proliferation. Overexpression of miR-9 led to decreased proliferation rates in KG1a cells compared to control vector treated cells. After 5 days, the relative cell count was 133±21% vs. 241±67% in the miR-9 overexpressing cells compared to empty vector expressing cells, respectively. Next, we determined if this difference was based on a higher apoptosis rate. An Annexin V staining revealed no significant difference between the apoptotic threshold of miR-9 overexpressing (21%) and empty vector cells (21%) after 24 h. However, a subsequent cell cycle analysis demonstrated a higher percentage of miR-9 overexpressing cells in the G2/M phase, (39±2%) compared to the empty vector control treated cells (31±3%) after 24 h (P=.084), indicating that the cell cycle is slowed or stopped during cell division. Since miR-9 targets the poor prognostic marker ERG & higher miR-9 expression led to decreased proliferation & reduced cycling in AML cells in vitro we speculated that the differential miR-9 expression would also impact the outcome of AML patients (pts). Mature miR-9 is derived from three precursor molecules of which pre-miR-9-1 & pre-miR-9-3 are known to be expressed in hematopoietic cells. We assessed the pre-miR-9-1 expression of bone marrow by real-time PCR in 131 AML pts (median age 64 [range 22 – 75] years) with favorable (n=4, 3%), intermediate (n=90, 69%), adverse (n=33, 25%), or unknown (n=4, 3%) cytogenetic risk (according to the Medical Research Council [MRC] Classification) who received a RIC-HCT. The median follow-up was 4 years. The pre-miR-9-1 expression levels were normalized to ABL to define high & low pre-miR-9-1 expressers by the median expression of all AML pts. At diagnosis, high pre-miR-9-1 expresser status associated with a lower white blood count (P=.065) and lower % of peripheral blasts (P=.108) by trend. Furthermore, pts with high pre-miR-9-1 expression were more likely to be NPM1 wild-type (P=.047) & FLT3-ITD negative (P=.020). Pts with high pre-miR-9-1 had a lower probability of relapse (P=.048). In conclusion, miR-9 targets & regulates expression of the poor outcome predictor ERG. Overexpression of miR-9 led to decreased proliferation and a pause in AML cell cycling. Furthermore, high pre-miR-9-1 expression associated with reduced relapse rates in AML. Thus a pharmacologically induced expression of miR-9 in AML blasts may improve outcomes of AML pts. Disclosures No relevant conflicts of interest to declare.

Background: Most acute myeloid leukemia (AML) patients (pts) stilldo not achieve long-term survival. Better risk stratification and novel therapeutic avenues are needed to improve pts outcomes. The expression of microRNAs has been demonstrated to be altered in AML & miR-based therapies are entering clinical trials. MiR-320a, maps to chromosome 8p21.3 & is known to play a role in several tumors; e.g. higher miR-320a expression suppresses the progression of colorectal cancer. In AML miR-320a has been shown to inhibit cell proliferation, likely by targeting the transferrin receptor 1. Objective: The objective of this study was to investigate whether a differential expression of pri-miR-320a associated with outcome in AML pts. Methods: We assessed the expression levels of pri-miR-320a, a precursor molecule of mature miR-320a. The pri-miR-320a expression levels in 129 AML pts were assessed by quantitative reverse transcription polymerase chain reaction & normalized to a housekeeping gene (18S). The 75th percentile was chosen as a cut-off discriminating between high & low pri-miR-320a expressers. We analyzed 129 AML pts (median age at HCT 64 years [y]; range 22–74 y) who received reduced intensity conditioning (RIC; Fludarabine 30mg/m^2 at day -4 to -2 & 2 Gy total body irradiation at day 0)-hematopoietic cell transplantation (HCT) at the University of Leipzig, with pretreatment bone marrow available. The median follow-up was 4.5 y for pts alive. European LeukemiaNet (ELN) genetic classification was: favorable (n=33; 25.6%), intermediate I (n=33; 25.6%), intermediate II (n=29; 22.5%) or adverse (n=30; 23.3%). The pts were also characterized for FLT3-ITD status, CEBPA, IDH1, IDH2 and NPM1 mutations. Results: At diagnosis high pri-miR-320a expression associated by trend with lower hemoglobin levels (P=.094), lower white blood cell counts (P=.079) & lower peripheral blast counts (P=.096). High pri-miR-320a expressers less frequently had NPM1 (P=.038) or CEBPA (P=.025) mutations. Interestingly, pri-miR-320a expression was significantly lower in pts with trisomy 8 (P=.018) compared to non-trisomy 8 pts & in the ELN intermediate II group none of the trisomy 8 pts was in the high pri-miR-320a expressing group. Analysis of our AML pts, showed a significant association between pri-miR-320a expression status & clinical outcome. In the entire group of pts high pri-miR-320a expressers had a longer overall survival (OS; P=.086; Figure 1) by trend & a significantly longer event-free survival (EFS; P=.032). The strongest impact of pri-miR-320a was found in the ELN intermediate II group, where high pri-miR-320a expression was associated by trend with longer OS (P=.059; Figure 1) & a significantly longer EFS (P=.034). In multivariate analysis, the prognostic impact of pri-miR-320a expression status was confirmed in the entire group of pts (OS: P Conclusion: High expression of pri-miR-320a associates with distinct clinical & molecular features. Interestingly, in AML pts with trisomy 8 pri-miR-320a that maps to chromosome 8 has a significantly lower expression, indicating a possible leukemogenic link to trisomy 8 associated AML. High pri-miR-320a expression significantly associates with better outcome especially in the ELN intermediate II group. These results suggest that pri-miR-320a could be used as a marker to refine current AML risk stratifications. Increasing miR-320a, by e.g. miR-replacement therapies, might improve outcomes of AML pts. Figure 1: Figure 1:. OS (A) and EFS (B) in AML pts according to pri-miRNA-320a expression status, OS (C) and EFS (D) in AML pts in the ELN intermediate II group according to pri-miRNA-320a expression status Disclosures No relevant conflicts of interest to declare.

The presence of BCR-ABL kinase domain mutations below the detection limit of sanger sequencing (low level mutations, LLM) predicts the outcome of subsequent therapy in patients with imatinib resistance (Parker et al. JCO 2011, Blood 2012). We have evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of deep molecular response (MR4) after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). We have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment and correlated the mutation status (LLM vs. no LLM) with the primary endpoint of the ENEST1st trial, MR4at 18 months. Additionally, we evaluated MMR at 18 months. Seventy eight ENEST1st study patients (pts) provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+purity was subsequently determined by fluorescent activated cell sorting (FACS). Aliquots of 105 CD34+ were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to 50% were available for BCR-ABL amplification. Amplification was successful from 41 (52%) of these CD34+samples. A total of 205 L-PCR assays of CD34+ cells identified 29 (14%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) from 21/41 pts (51%). The median quantitative level of mutant alleles in CD34+ cells was 0.13 (range 0.06–0.54) BCR-ABLmutant/ BCR-ABLunmutated. One pt was taken off the study because of adverse events before the 3 months evaluation, 2 pts between 3 and 6 months and 1 after the 6 months evaluation (9.7%). Two pts were screened for the trial and did not fulfil the inclusion criteria (4.9%). One pt lost complete hematological response after 6 months and was switched to a different TKI and one pt had a persistent suboptimal response and switched TKI after 12 months on NI (4.9%). These two pts were considered as non-optimal responders at all time points after the change of treatment. All patients except one had typical transcripts (b2/a2 or b3/a2). One pt had an atypical transcript and was not available for MR4 assessment. At 18 months, 31 of 41 patients were evaluable for BCR-ABL levels. Thirteen out of 31 (42%) pts had MR4 or higher at 18 months (6 pts in the LLM group). Twenty six (84%) pts had MMR (10 in the LLM group), while five pts did not reach MMR at 18 months (4 pts with LLM). No statistically significant differences in the proportion of patients achieving MR4 or MMR could be detected between the two groups. In conclusion, T315I, Y253H, E255K or F359V mutations in CD34 selected cells below the detection limit of sanger sequencing do not predict the achievement of MR4 or higher in patients on nilotinib in the ENEST1st study. Therefore, screening for low level BCR-ABL mutations at diagnosis of CML is currently not recommended. Disclosures Giles: Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; MSD: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Research Funding.

Droplet Digital PCR (ddPCR) is based on the separation of a standard PCR reaction into many thousand single droplets, each of which is tested independently for the presence of the target. This has the potential to generate an absolute read out that is largely tolerant of variations in PCR efficiency, reducing the requirement for internal standardisation. The ddPCR method should therefore be suitable for monitoring even very low BCR-ABL levels in CML patients with a high degree of reliability and sensitivity. We compared standardized quantitative (Q) RT-PCR with ddPCR results in patients with BCR-ABL e13/e14/a2 positive chronic myeloid leukemia and deep molecular remission (MR). 439 cDNA samples from CML patients with e13 or e14/a2 BCR-ABL fusion genes were received for routine monitoring in our laboratory, which is accredited for the detection of molecular response (MR) down to 4.5 by the European Treatment Outcome Study (EUTOS) collaboration. QRT-PCR was performed using the WHO certified ERM-AD623 BCR-ABL standard and showed molecular response (MR) 3, 4, or at least 4.5 in 78, 129 and 232 samples, respectively. For comparison, the same cDNA samples were analysed by ddPCR using a QX200 Droplet Digital PCR System (BIO-RAD). In line with the manufacturer's recommendations, samples yielding a minimum of 3 positive droplets from the 10-15.000 routinely analysed were scored as positive, although the effects of reducing this cut-off value were also investigated. The median total ABL copies in 5µl cDNA were in close agreement between the 2 methods with 37769 (range 5032 to 470057) by QRT-PCR and 37810 (range 4200 to 110100) by ddPCR. The congruence at the level of single samples was relatively low, as may be expected at limiting target levels, with 104 samples scoring positive by both methods, 65 by QRT-PCR only and 84 by ddPCR only (table 1). Where both methods scored positive, the calculated BCR-ABL/ABL ratios were comparable with a median of 0.011 for QRT-PCR (range 0.0001-0.1) and 0.013 for ddPCR (range 0.0037-0.12). Further analysis of the data following pre-categorization of the samples into MR3, MR4 or MR4.5 based on the standardised QRT-PCR procedure revealed a higher number of samples scoring positive by ddPCR but not QRT-PCR at the lower BCR-ABL/ABL ratios, with 75 samples registered positive by ddPCR only, compared to 8 by QRT-PCR only in the MR4.5 category. This suggests a possible advantage of ddPCR at very low target levels. Further reducing the cut-off value to 2 positive droplets results in out hands in a small (1%) incidence of positivity in the negative controls, but leads to a marked increase in the frequency of CML samples detected positive by ddPCR only. Conclusions: 1) Although QRT-PCR and ddPCR yield similar overall results throughout all categories of molecular response, there is relatively low congruence at the level of single samples. It is likely that stochastic variation in pipetting the low numbers of target molecules involved contributes significantly to the variability between individual assays. 2) Using the recommended cut-off of 3 positive droplets, ddPCR scored more positive samples than did QRT-PCR at low BCR-ABL/ABL ratios. 3) Given the low numbers of target molecules present, the recommended use of a 3 droplet cut-off may limit the sensitivity of the ddPCR method. For these applications, it will therefore be important to define and reduce the source of background in the ddPCR procedure to a reproducible minimum. Overall, our results suggest that ddPCR may have the potential for more sensitive quantitation at low transcript levels then the current standard QRT-PCR. | | ddPCR Cut-off 3 positive droplets | ddPCR Cut off 2 positive droplets | | ----------------------------- | ----------------------------------------- | ----------------------------------------- | ------------------ | | | QRT-PCR only | QRT-PCR + ddPCR | ddPCR only | QRT-PCR only | QRT-PCR + ddPCR | ddPCR only | | All (n=439) | 65 | 104 | 84 | 44 | 125 | 149 | | MR3* (n=78) | 23 | 55 | | 14 | 64 | | | MR4* (n=129) | 34 | 33 | 9 | 26 | 41 | 21 | | MR4.5* (n=232) | 8 | 16 | 75 | 4 | 20 | 128 | Table 1: Comparison of BCR-ABL positive and negative samples by QRT-PCR and ddPCR. *MR groups defined on the basis of standard QRT-PCR results. Disclosures Lange: Novartis: Consultancy, Honoraria, Research Funding.

Abstract 1671 The presence of BCR-ABL kinase domain mutations below the detection limit of conventional screening techniques (low level mutations, LLM) predicts outcome of subsequent therapy in patients with imatinib resistance (Parker et. al JCO 2011 and Blood 2012). We have further evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of complete molecular responses after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Here, we have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment in comparison to total white cells (TWBC). Sixty nine ENEST1st study patients with CP CML provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+ purity was subsequently determined by fluorescent activated cell sorting (FACS). The results were compared to those derived from stored TWBC from 23 of the same patients and a further 16 patients at diagnosis. Aliquots of 105 CD34+ or at least 106 TWBC were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to 50% were available for BCR-ABL amplification. Amplification was successful from 36 (52%) of these CD34+ samples and from 38 of the 39 (97%) TWBC samples. A total of 180 L-PCR assays of CD34+ cells identified 29 (16%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) in CD34+ cells from 21/36 patients (58%). In comparison, 190 assays of TWBC identified 10 (5%) mutations (T315Ix3, Y253Hx6, E255Vx1, p=0.0005) in 8/38 patients (21%, p=0.001 Fishers exact test). Significantly more T315I (33%) and E255K (22%) mutations were observed in CD34+ cells than in TWBC (8%, p=0.007 and 0% p= 0.003 respectively). The quantitative levels of all mutant alleles were median 0.135 (range 0.06–0.535) and 0.1 (range 0.04-0, 25) BCR-ABLmutant/ BCR-ABLunmutated for mutations in CD34+ cells and TWBC, respectively and were not significantly different. Where both CD34+ and TWBC were available from the same patient (n=23), 11 patients showed a total of 18 mutations in the CD34+ fraction but only one of these mutations was confirmed in TWBC. One additional mutation was detectable in the TWBC. The remaining 12 patients with no detectable mutation in the CD34+ fraction showed 3 mutations (2x Y253H, T315I) in 2 patients in TWBC only. In conclusion, LLM with either no (T315I) or intermediate (Y253H, E255K/V, F359V) sensitivity to nilotinib are detectable in CP CML patients at a frequency of 21% in the TWBC but with a significantly higher frequency of 58% in the enriched CD34+ progenitor cell compartment. Longterm patient follow up on the ENEST1st and ENESTobserve studies will allow analysis of the relationship between LLM and clinical outcomes on nilotinib. Disclosures: Hochhaus: Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange: BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.