Intermediate Cytogenetics Research Articles

Genetic Alterations and Their Clinical Implications in Older Patients with Acute Myeloid Leukemia

IntroductionRisk-stratification of patients with acute myeloid leukemia (AML) can not only improve treatment response, but also reduce side effects of the treatment, especially in the elderly. A number of patient-specific and leukemia-associated factors are related to the poor outcome in older patients with AML. However, comprehensive studies regarding the impact of genetic alterations in this group of patients are limited.Methods and MaterialsA total of 500 adult patients with newly diagnosed de novo AML who had enough bone marrow cryopreserved cells for analysis at the National Taiwan University Hospital were enrolled consecutively. We compared the clinico-biological features, cytogenetics and molecular gene mutations between patients aged 60 years or older (n=185) and those younger (<60 years, n=315).ResultAmong older patients, those received standard intensive chemotherapy had a longer overall survival (OS) than those treated with palliative care. Compared with younger patients, the elderly had a higher incidence of poor-risk cytogenetic changes, but a lower frequency of favorable-risk cytogenetics. The median number of molecular gene mutations at diagnosis was higher in the elderly than the younger. Older patients had significantly higher incidences of PTPN11, NPM1, RUNX1, ASXL1, TET2, DNMT3A, and P53 mutations but a lower frequency of WT1 mutations. In multivariate analysis for OS among the elderly who received standard intensive chemotherapy, high WBC >50,000/μL at diagnosis, RUNX1 mutations, DNMT3A mutations, and P53 mutations were independent worse prognostic factors, while the presence of NPM1 mutations in the abcence of FLT3/ITD mutations was an independent good prognostic factor. The frequency of acquiring one or more adverse genetic alterations was much higher in older patients than younger ones. Further, the pattern of gene mutations could divide older patients with intermediate cytogenetics into three groups with significantly different complete remission rates, OS, and disease-free survival.ConclusionOlder AML patients frequently harbored high-risk cytogenetics and gene mutations, and had poorer prognosis. Integration of cytogenetics and molecular alterations could risk-stratify older patients into groups with significant different outcomes. For those patients with poor prognosis under current chemotherapy, novel therapies, such as demethylating agents or other targeted therapies may be indicated. DisclosuresTang:Novartis: Consultancy, Honoraria.

A Clinical Measure of DNA Methylation Predicts Outcome in De Novo AML

Background: Despite advances, the majority of patients (pts) with acute myeloid leukemia (AML) will die of their disease. Current genetics-based risk classification schemes inadequately predict outcome. We sought to determine if a novel methylation-based biomarker could enhance risk stratification in AML.Methods: Using a novel methylation assay (xMELP) validated for the clinical laboratory, we performed DNA methylation assessment at 17 previously identified prognostic loci to calculate a summary methylation statistic (M-score, Wertheim et al. Clin Chem 2015) for 166 pts with de novo AML treated at the University of Pennsylvania (UPENN). Targeted next-generation sequencing (NGS) of 33 hematologic malignancy-associated genes was performed for a 136 pt subset. The association of M-score with other prognostic variables and outcome [complete remission (CR) and overall survival (OS)] was evaluated. Median follow-up was 68.1 mos (range 1.4-150.2) among 38 survivors and 10.5 mos (range 0.1-95.2) among those (n=128) deceased. The optimal M-score for identifying groups with differing OS in the total UPENN cohort defined a binary classifier. The classifier was validated in UPENN subgroups and in a separate ECOG ACRIN E1900 trial cohort.Results: The mean and median M-score for the UPENN cohort was 92.3 (95% confidence interval [CI], 87.4-97.2) and 91.4 (range, 30.8-97.3), respectively; the M-score was not significantly associated with age, gender, specimen type, blast percent, NPM1, or FLT3-ITD. Patients with favorable cytogenetics had a lower mean M-score than those with non-favorable cytogenetics; there was no difference between intermediate and unfavorable cytogenetic groups. Univariate analyses demonstrated that a 10-unit increase in M-score was associated with a 10% increase in the hazard of death (HR 1.1; P<.0001) and a 20% increase in the odds of failing to achieve CR (OR 1.2, P<.0001). In multivariable Cox analysis, higher M-score (HR 1.1, P=.011) and older age (P=.001) were significantly associated with increased hazard of death, while NPM1+/FLT3-ITD-status (P=.031) was associated with decreased hazard of death. In a multivariable logistic analysis, higher M-score (HR, 1.1, P=.034) and older age (P=.007) were associated with increased odds of failing to achieve CR, while favorable cytogenetics (P=.030) was associated with achievement of CR.Based on the maximization of the log-rank statistic, the optimal M-score cutpoint was 86, which defined a binary risk classifier (hazard of death for high vs low M-score: unadjusted HR 2.5, P<.0001; adjusted HR 1.9, P=.003). Median OS was 26.6 vs. 10.6 mos for low and high M-score groups (Figure). The CR rates for low and high M-score groups were 84% and 61%, respectively. The performance of the M-score classifier was confirmed in pts ≤ 60 yrs with intermediate cytogenetics (log-rank P=.001, OS in high vs low M-score groups: 36.4 vs 14.0 mos) and among pts who achieved CR with initial therapy (log-rank P<.00001, OS 43.9 vs 17.2 mos). The M-score classifier identified groups with different outcome regardless of allogeneic transplant.The M-score classifier was validated in the E1900 cohort (n=383). The mean and median M-scores were similar to the UPENN cohort and M-score was significantly associated with OS on multivariable analysis (P<0.0001). The M-score classifier identified E1900 subgroups with different OS (log-rank P<.00001; OS 29.5 vs 12.6 mos), a finding confirmed in the intermediate cytogenetics group, in those aged <50 and ≥50 yrs, and among pts assigned to both standard and high dose daunorubicin. Notably, high dose daunorubicin benefited patients with high M-scores (P=.001), but not those with low M-scores (P=.328).We also evaluated the prognostic value of the M-score in the context of an extended molecular profile. Random forest analysis of the UPENN cohort showed that M-score and age are the most robust predictors of OS, while a subset of recurrent mutations (FLT3-ITD, NPM1, IDH2, TET2, TP53, NRAS, CEBPA) also contribute to prognosis, but to a lesser degree.Conclusion: The M-score and associated classifier represent promising tools for clinical decision-making in AML and deserve further investigation. The prognostic value of the M-score may be superior to mutational analysis. Optimal methods for integration of M-score, clinical and genetic information are being defined. [Display omitted] DisclosuresLoren:Merck: Research Funding. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

Very Poor Long-Term Survival, Also in Contemporary Studies, of Patients with AML Who Relapse after Achieving a First Complete Remission: The ECOG-ACRIN Cancer Research Group Experience

This study was coordinated by the ECOG-ACRIN Cancer Research Group (Robert L. Comis, MD and Mitchell D. Schnall, MD, PhD, Group Co-Chairs) and supported in part by Public Health Service Grants CA180794, CA180820, CA180795, CA180791, CA189859, CA180790, CA180853, and from the National Cancer Institute, National Institutes of Health and the Department of Health and Human Services. Its content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute.Background: Relapse after achieving a complete remission (CR) in AML portends for a poor prognosis and allogeneic transplant after achieving a second remission is the only chance for cure. Patients who undergo a transplant have a 30-40% chance of long term survival. This is a follow-up of a report published 10 years ago (Rowe JM, ASH 2005, abstract 546) and includes contemporary studies and longer follow-up. The study examines the long term overall survival (OS) of AML patients who relapsed after achieving first CR in 9 successive ECOG-ACRIN trials for newly-diagnosed AML patients (E3483, E3489, PC486, E3993, E4995, E1490, E3997, E1900 and E3999) from March 1984 to November 2008.Methods:OS was defined as time from first relapse to death from any cause. Kaplan-Meier estimates were used to estimate the event-time distributions for OS. Multivariate model stratified on protocol and treatment were used to examine whether the following factors are prognostic for OS from relapse: age, gender, cytogenetic risk, ECOG performance status, WBC, platelets, hemoglobin, marrow blasts, peripheral blasts, and duration of CR. All P values were based on 2-sided tests.Results:A total of 3160 patients were enrolled in the 9 studies. The median follow-up on patients still alive was 10.0 years. Among those 3160 patients enrolled, 1864 (58.9%) achieved first CR of which 1086 (58.2%) had documented relapse.The median age at diagnosis of the relapsed patients was 50 (range: 16-84) and 50.6% were males. Fifty percent of the patients had reliable cytogenetic results. Of those, 13.5% had favorable cytogenetics, 55.8% - intermediate and 30.6% had unfavorable baseline results.The median OS from relapse was 0.5 years. The 2- and 5-year OS were 16(±1)% and 10(±1)%, respectively. This is true in even the most contemporary studies (E1900 and E3999) with median OS of 0.6 and 0.4 years, respectively.By age stratification (< or ≥ 55), 5-year OS was 13(±1)% and 5(±1)%, respectively (figure 1). Among patients<55, those with unfavorable cytogenetics had the poorest prognosis (median OS of 0.4 years and 5-year OS of 6(±3)%. Those with favorable and intermediate cytogenetics had similar OS with 0.7 and 0.6 years median OS and 5-year OS of 16(±5)% and 17(±3)%, respectively (figure 2).Multivariate analysis was perfomed on 517 patients who had enough baseline information, including cytogenetics. Factors that were significantly associated with OS from relapse included: age (p<0.001), ECOG performance status (p=0.04), hemoglobin (p=0.03), cytogenetics (p=0.045) at baseline and duration of CR (p<0.001).Conclusions: The short- and long-term OS of AML patients post-relapse is dismal (<10%). Although age<55 and favorable cytogenetics at diagnosis are relatively good prognostic factors, the general survival of even these patients is very poor. Disappointingly, these data are also applicable to the most contemporary studies. Long-term survival was possible only in the minority of patients who survived the relapse, achieved a second CR and then successfully underwent an allogeneic transplant. These data are crucial when considering post-remission strategy, and suggest that offering a therapy most likely to lead to cure in CR1 is the preferred option. [Display omitted] [Display omitted] DisclosuresDouer:Gilead: Consultancy. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; BioLineRx Ltd.: Consultancy; Amgen: Consultancy.

Higher Percentages of Ring Sideroblasts and SF3B1 Mutations in Patients with AML Correlate with Mutations in TP53 and Adverse Cytogenetics, but Have No Independent Impact on Outcome

IntroductionRing sideroblasts (RS) are common findings in myeloid malignancies as MDS or MDS/MPN, especially in RARS-T, and are associated with SF3B1 mutations. However, the incidence of both RS and SF3B1 mutations have not yet been assessed properly in acute myeloid leukemia (AML).AimDetermine the frequency of RS and SF3B1 mutations in 1857 patients with de novo and therapy-related AML (t-AML). Define their impact on survival and association with other frequently mutated genes, as well as with cytogenetic abnormalities.Patients and MethodsFrom a total of 1857 AML patients (excluding those with cytogenetically-defined entities according to WHO), bone marrow assessment revealed 473 (25%) with RS ≥ 1% and thereof 183 (10% of all) with ≥15 RS. Sequencing or melting curve analysis were performed in a subcohort of 340/473 patients for the detection of mutations in: SF3B1, ASXL1, DNMT3A, FLT3- TKD, IDH1 R132, IDH2 R140, IDH2 R172, KRAS, NPM1, NRAS, RUNX1, TET2, FLT3 -ITD and MLL -PTD. These 340 cases were subject to the study. Out of these 340, 141 cases (42%) had RS ≥ 15% and the remaining 199/340 (59%) had RS ≥1 to <15%. The cohort consisted of 303 (89%) de novo AML (FAB: M0 n=18, M1 n=67, M2 n=165, M4 n=30, M5 n=3, M6 n=20) and 37 (11%) t-AML. 148 were female and 192 were male, with median age 74 years, range: 20-93 years. Chromosome banding analysis (assisted by FISH if needed) was performed in all 340 cases.ResultsThe percentage of bone marrow blasts correlated inversely with the percentage of RS present (r 0.213, p<0.001). 136 (40%) patients had a normal karyotype. Intermediate cytogenetics according to MRC criteria were found in 193 (57%), and adverse in 147 (43%). Patients with RS ≥15% more frequently had adverse cytogenetics in comparison to those with RS between 1-14% (54% vs 36%, p=0.001).The frequencies of gene mutations were as follows: TP53 103/331 (31%), RUNX1 84/315 (26%), DNMT3A 86/337 (25%), TET2 68/330 (20%), ASXL1 58/334 (17%), IDH2 R140 53/338 (15%), NPM1 43/340 (12%), SF3B1 34/334 (10%), FLT3 -ITD 33/340 (10%), NRAS 29/340 (8%), IDH1 R132 21/339 (6%), MLL -PTD 22/337 (6%), FLT3 -TKD 18/333 (5%), KRAS codon 12 13/299 (4%), IDH2 R172 13/338 (3%) and KRAS codon 61 3/299 (1%). Moreover, in total 30 variants in 28 patients were identified in DNMT3A, RUNX1, TET2 and TP53, which according to current knowledge cannot be assigned to mutations or SNPs yet.Patients with ≥15% more frequently had TP53 mutations (mut) (44% vs 22%, p<0.001) and less frequently IDH2 R140 mut (11% vs 19%, p=0.094) and MLL -PTD (2% vs 6%, p=0.006). Accordingly, patients with TP53 mutations had higher percentages of RS as compared to those without (28% vs 16%, p<0.001) and patients with IDH2 R140 mut and MLL -PTD, respectively, had lower percentages of RS as compared to those without (15% vs 21%, p=0.043 and 11% vs 21%, p=0.025, respectively). Furthermore, patients with mutations in the following genes had fewer RS than patients with the respective wild-types: ASXL1 (15% vs 21%, p=0.040), FLT3 -ITD (14% vs 21%, p= 0.049), IDH2 R140 (15% vs 21%, p=0.043), MLL -PTD (11% vs 21%, p=0.025), NPM1 (13% vs 21%, p=0.018) and KRAS codon 61 (3% vs 20%, p<0.001). Conversely, patients with mutated SF3B1 had more RS than patients with wild type (27% vs 19%, p=0.054). However, the number of RS did not translate into an increase in the mutational burden of SF3B1.Given the limited degree of overlap between mutations of the four most frequently mutated genes, we hierarchically separated the patients into 5 groups: TP53 mut, ASXL1 mut, NPM1 mut, SF3B1 mut and patients without any of these mutations. Interestingly, the percentage of RS was very similar in the two groups, TP53 mut and SF3B1 mut, and significantly higher as compared to all other groups (TP53 mut/SF3B 1mut: 28% vs ASXL1 mut/NPM1 mut/others 14%, p<0.001).The number of RS did not have an impact on the overall survival (OS) and event free survival (EFS) of patients.Conclusion1. Ring sideroblasts ≥ 15% are present in 10% of de novo and t-AML.2. The blast count correlates inversely with the number of RS.3. Patients with ≥ 15% RS more frequently carry TP53 mutations and adverse cytogenetics.4. Although patients with ≥ 15% RS have worse molecular (TP53 mut) and cytogenetic features, there is no statistically significant impact on survival when compared to patients with <15% RS.5. Patients with mutated ASXL1, FLT3 -ITD, IDH2 R140, MLL -PTD and NPM1 have less RS than wild type patients while those with TP53 or SF3B1 mutations have higher RS. [Display omitted] DisclosuresMartin-Cabrera:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Prognostic Significance and Risk Stratification in Acute Myeloid Leukaemia Patients in the Absence of Successful Cytogenetic Analysis

Cytogenetic analysis is a key tool in risk stratification of patients with acute myeloid leukaemia (AML). Sometimes samples are not sent or metaphase analysis fails (~10% cases). In these situations, a cytogenetic risk score cannot be assigned, preventing risk stratification. It has been suggested that in the absence of cytogenetic data, both missing and failed, patients have a poor outcome (Mederios et al (Br. J. Haematol. 2014 164(2): 245-50); Lazarevic et al (Eur. J. Haematol. 2015 94(5): 419-23)). In this study, we investigated the characteristics and prognostic significance of the provision of samples, and their successful analysis across the MRC UK series of AML trials (AML10 - AML16).Data from 14265 non-APL patients recruited to successive UK AML trials (1988 - 2012) were analysed. Patients were classified based on whether a sample was sent for cytogenetic analysis and the outcome of the analysis. Rates of successful cytogenetic analysis were higher in patients treated on intensive chemotherapy protocols (intensive: 11104/13081 (85%); non-intensive 850/1184 (72%), p<.0001). In intensively treated patients, there was a significant association between increasing age and missing or failed cytogenetic samples (p<.0001). Stratification of intensively treated patients with successful cytogenetics, according to the criteria of Grimwade et al (Blood 2010 116(3):354-65), into favourable (18%), intermediate (62%) and adverse risk (19%) were analysed for outcomes and showed 10 year survival of 66%, 30% and 12%, respectively. Survival at 10 years was 24% when no sample was sent compared to 32% when a sample failed cytogenetic analysis. Survival for patients with failed cytogenetics was not significantly different from those with intermediate risk cytogenetics in either univariate analysis (p=0.10) or after adjustment for age, performance status, white cell count and secondary disease (p=0.06). Outcomes were significantly better than those for adverse risk patients (p<.0001 for both analyses). For those with no sample, survival was significantly worse than for the intermediate risk (p<.0001 in both analyses), although the effect size was not large (HR 1.18 in adjusted analyses), yet significantly better than adverse risk patients (p<.0001, HR 0.57). While outcomes worsened with increasing age, a similar pattern was seen across all age groups (favourable/intermediate/adverse/no sample/failed: age<16 81%/57%/49%/63%/62%; age 16-59 67%/36%/13%/30%/41%; age 60+ 32%/12%/2%/8%/9%).In the most recent trial, AML17 (2009-14) of younger patients, 97% had successful cytogenetic analysis, compared to 86% in AML10-15. Here too, there was no suggestion of poor outcomes for those patients with no cytogenetic result. These results indicate that cytogenetic analysis is possible in the vast majority of younger patients, and suggest that absence of cytogenetic information is not in itself an adverse prognostic factor. This poor prognosis identified in other studies may be due to selection bias in the decision to send samples. The experience of AML17 demonstrates that, when cytogenetic analysis is required to guide treatment decisions, if results can be provided in real time, high levels of compliance can be achieved, not only in sending, but also in the quality of samples. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase I/IB Study of Azacitidine and Hedgehog Pathway Inhibition in Myeloid Malignancies

Background: Hypomethylating agents (HMA) such as 5-Azacitidine (Aza) have overall responses rate of ~35-50% in acute myeloid Leukemia (AML) and higher risk myelodysplastic syndrome (MDS) patients (pts). However, eventually all pts develop resistance on therapy. To improve upfront response rates and to address HMA failure, we performed genome-scale RNAi screens targeting genes on chromosomes 5 and 7, recurrent cytogenetic abnormalities in AML/MDS. These screens identified Hedgehog pathway (Hh) genes as potentially targetable vulnerabilities with Aza. Combination experiments with smoothened (SMO) inhibitors (SMO is a central activating gene within the Hh pathway) showed pre-clinical synergy. These and additional published data support the Hh pathway as a targetable vulnerability in AML, and provide a rational for a trial.Methods: A phase I/Ib protocol of the oral SMO inhibitor LDE225 (Sonidegib) in combination with Aza in untreated and relapsed/refractory (rel/ref) AML, chronic myelomonocytic leukemia (CMML), MDS, or MDS/myeloproliferative neoplasm overlap pts. Objectives of the phase I part are to determine safety, toxicity and MTD and for the phase Ib part to assess the preliminary overall efficacy. Sonidegib was started at dose level (DL) 0 at 400mg oral daily continuously and Aza at the approved doses (75mg/m2 x 7 days or days 5-2-2) with dose adjustment per label. A classical 3+3 design was used and DLT (CTCAE v4.0) defined as > grade 3 CPK elevation or hepatic or non-hematologic adverse events (AEs) lasting > 7 days or any grade 4 event, except for hematologic toxicities. DLT was assessed after 42 days when steady-state concentrations of Sonidegib were reached. Pharmacokinetic assessments were not performed for this study.Results:Patients and Dose Levels: Between 5/22/2014 and the data cut-off date (7/20/2015), 36 pts were enrolled. Median age was 72 y (range 52-85 y) and 64% were male. Most AML pts (23 of 24) had intermediate or unfavorable cytogenetics and most MDS/CMML pts were in the int.- to high-/very high-risk group (IPSS-R). Six pts were treated at DL-0 of Sonidegib 400mg/day and one DLT of CPK elevation was observed. Another patient had grade 4 neutropenia (which at the time was included in the DLT definition, later removed). Due to overall tolerance of the combination at DL 0 and investigator assessment, despite only 1/6 pts having a formal DLT, DL -1 at Sonidegib 200mg was explored at which 0/3 pts (1 pt non-evaluable) had DLT and the phase Ib portion was opened at DL -1. To date a total of 24 AML (13 newly dx, 11 rel/ref), 11 MDS/CMML (5 newly dx, 6 rel/ref ) and 1 myelofibrosis pt have been enrolled.Safety and Tolerability: An initial safety and efficacy assessment for 25 of the 29 pts treated on the phase Ib (n=26) + the MTD cohort of 200 mg (n=3) and for who at least 2 cycles of treatment follow up was available, showed that all pts experienced at least one grade-3 or 4 hematologic AE with a lower rate in the untreated vs. treated cohort. Other, non-hematologic grade-3 and 4 AEs respectively were seen in 24 and 28% of patients-most commonly hyponatremia and fatigue, known AEs for this drug class. Treatment delays occurred in 28% of pts and Aza or Sonidegib dose reductions in 16%. 23 (79%) pts are alive and 6 have died (21%), with one death while pt was actively treated but was deemed unrelated; 11 pts remain on study, with the remainder showing disease progression (n=3) or taken-off study for other reasons (n=3 each for AEs and did not want further treatment; n=4 other).Efficacy: As of 7/20/15 with a median treatment duration of 2 cycles, 2/5 untreated MDS/CMML pts achieved CR/CRi; 1 HMA rel/ref AML had CR; 1 untreated AML pt each had PR and MLFS. 90% of pts have not progressed. Updated results will be presented at the ASH 2015 meeting; by then all 29 pts will have completed a minimum of 4-6 cycles on study and full efficacy and AE result on all pts will be reported. To date the OS at 6 mo is 77% (95% CI: 44-97%) with a median PFS of 4 mo (range: 2-NR). Many pts in the untreated cohorts have just enrolled.Conclusion: Hh pathway inhibition with the clinically well tolerated SMO inhibitor Sonidegib in combination with Aza in MDS and AML pts is well tolerated with mostly hematologic AEs as expected with early signs of clinical activity in untreated and HMA-failure pts. Full efficacy results of 29 pts and updated safety data will be reported for this first combination trial of a SMO inhibitor in combination with a HMA in MDS and AML pts. DisclosuresMesa:Gilead: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Incyte Corporation: Research Funding; Genentech: Research Funding; Pfizer: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; CTI Biopharma: Research Funding. Al-Kali:Novartis: Research Funding.

Feasibility of a Symptom-Adapted Physical Activity Intervention during Induction Chemotherapy for Older Adults with Acute Myeloid Leukemia (AML)

Background: Poor treatment tolerance contributes to suboptimal outcomes for many older adults with acute myeloid leukemia (AML). Treatment-associated physical deconditioning during induction may limit therapeutic options and impair quality of life. Interventions to enhance physical function during therapy may improve treatment tolerance and benefit.Objective: To test the feasibility of conducting a symptom-adapted inpatient physical activity (PA) intervention among older adults receiving intensive induction chemotherapy for AML.Methods: A single institution randomized controlled pilot study (N=70) was conducted from October 2012-July 2015. Eligibility included age ≥60 years, newly diagnosed AML, and receipt of induction chemotherapy. Exclusion criteria included medical contraindication to PA at the time of enrollment, cognitive impairment, and/or receipt of low intensity therapy. Participants randomized to the intervention were offered a PA session five days per week tailored daily to symptoms and health status during the induction hospitalization. Session options included: 1) Standard (ward-based), walking + balance training + resistance exercises; 2) Intermediate (room-based), upper-body ergometer + balance training + resistance exercises; and 3) Low-intensity (bed-based), upper-body ergometer + resistance exercises. Counseling sessions to establish PA goals and trouble-shoot barriers were conducted weekly during hospitalization. Phone counseling to reinforce PA goals continued monthly post-hospitalization during follow-up (up to 6 months). The control arm received usual care. Assessment of physical function (self-report and objective), mood, symptoms, and quality of life was done at baseline, 3 months and 6 months with weekly physical function testing while hospitalized. The primary outcome was feasibility defined as recruitment (≥60%), adherence (>75%, average 3 sessions/week), and retention (85% follow-up for eligible participants).Results: There were 97 eligible patients of which 70 enrolled (recruitment rate 72%). The study sample was 70% male, mean age was 72.1 years (Standard Deviation [SD] 6.3), mean Hematopoietic Cell Transplantation Comorbidity index score was 2.0 (SD 1.8), mean hemoglobin 9.3 (SD 1.6), mean white cell count 17 (SD 33.7), and 93% had adequate ECOG performance status (0-2). The majority had intermediate (61%) or poor (33%) risk cytogenetics. Most common induction regimens included anthracycline+cytarabine (80%) and clofarabine (15.7%). A total of 732 PA sessions were offered during the course of the study. Patients were deemed medically ineligible to participate in 13% of these sessions. Of eligible sessions, the participation rate was 80%. Of weeks with at least one eligible day, the average number of weekly sessions conducted per participant was 3.0 (SD 1.6). Overall mean number of sessions/participant was 14.5 (SD 9.4). Among the 35 participants randomized to the intervention 74% completed a program evaluation. Most reported that they liked the program (88%), found it helpful (88%) and planned to continue physical activity post discharge (69%). The activities rated to be most helpful were: combination of balance + resistance exercises + walking (31%), resistance exercises alone (23%) and balance exercises alone (15%). Retention was 96% among evaluable participants (survived at least to the 3 month follow-up assessment, N=53).Conclusions: Delivery of a symptom-adapted inpatient PA intervention to older adults receiving intensive induction chemotherapy for AML is feasible and well received by participants. Next steps include estimation of the effect size of the intervention on physical function, symptoms, and quality of life. DisclosuresPardee:Celgene: Speakers Bureau; Novartis: Speakers Bureau.

Molecular Predictors of Outcome in Patients with MDS and AML Following MDS after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: The landscape of molecular aberrations in patients with myelodysplastic syndromes (MDS) has been well characterized and has identified ASXL1, BCOR, CUX1, IDH1, IDH2, SRSF2, RUNX1, U2AF1, TP53 and others as negative prognostic markers for overall survival (OS). We comprehensively investigated the prognostic impact of genetic aberrations in the context of allogeneic hematopoietic stem cell transplantation (alloHSCT) in a large cohort of patients with high risk MDS or secondary AML following MDS (sAML). Patients and Methods: 308 Patients with a diagnosis of MDS (47.4%) or sAML (52.6%) who received an alloHSCT at four German university medical centers and for whom genomic DNA was available at a time with active disease before transplantation were evaluated for the presence of mutations in 54 genes by Illumina high-throughput sequencing. Results: At least one mutation was identified in 82% of our patients with a median number of 2 mutations per patient. The most frequently mutated genes were ASXL1 (24.4%), DNMT3A, (23.1%), RUNX1 (16.9%), TET2 (16.9%), STAG2 (12%), TP53 (11.7%), and SRSF2 (11%) in agreement with previous reports. Mutation frequencies were similar between MDS and sAML patients for all mutated genes except SRSF2, TET2 and WT1, which were more frequently mutated in sAML. We grouped the mutated genes into functional classes and found that patients most frequently had mutations in modifiers of DNA methylation (42.2%), followed by chromatin modifiers (41.5%), splicing genes (31.1%), transcription factors (30.8%), signal transducers (28.8%) and tumor suppressors (14.6%). Mean variant allele frequencies were highest in modifiers of DNA methylation (33.2%), while signal transducers had the lowest allele frequency (20.4%). We next assessed the prognostic impact of gene classes and individual genes on outcome of patients after alloHSCT. Median follow up from transplantation was 4.15 years. Median patient age at time of HSCT was 58 years (range 19-75). 76 patients (25%) werein complete remission and 232 patients (75%) had active disease before transplantation. Low, intermediate, and high risk cytogenetics according to IPSS were found in 116 (38%), 59 (19%), and 115 (37%) patients, respectively. Matched and mismatched related donor HSCT was performed in 71 and 4 patients, respectively (23.1 and 1.3%), and matched and mismatched unrelated donor HSCT in 171 and 62 patients, respectively (55.5 and 20.1%). The six functional gene classes had no prognostic impact on survival, cumulative incidence of relapse and non-relapse mortality. We therefore evaluated the prognostic impact of individual gene mutations, of aberrations of chromosomes 3, 5, 7, 8, 17, 20 or a complex karyotype, and of transplant characteristics on OS. Parameters with significant impact on OS in univariate analysis were included in a multivariate cox proportional hazards model. Significant predictors of OS in multivariate analysis were mutations in PTPN11 (HR 3.1, present in 2.3% of pts.), IDH2 (HR 2.6, present in 4.2%), PHF6 (HR 2.2, present in 4.9%), NRAS (HR 1.8, present in 7.5%), presence of a complex karyotype (HR 1.8, present in 16.6%), transplantation from haploidentical donor (HR 5.5), RAEB/sAML not in complete remission before transplantation compared to untreated RA/RARS or RAEB/sAML and treated RAEB/sAML in remission (HR 2.0), GvHD prophylaxis other than calcineurin inhibitor with methotrexate or mycophenolate mofetil (HR 1.7) and female sex of the donor (HR 1.7). TP53 mutations lost their unfavorable prognostic impact when complex karyotype was added to the multivariate model. Competing risk analysis for cumulative incidence of relapse and non-relapse mortality showed that IDH2 and NRAS mutations and a complex karyotype were significantly associated with higher risk for relapse while PTPN11 and PHF6 mutations predicted for a higher incidence of non-relapse mortality. Importantly, a negative prognostic impact of ASXL1, BCOR, CUX1, IDH1, SRSF2, RUNX1 and U2AF1 seen previously in MDS patients not undergoing alloHSCT was not found in the transplant setting, suggesting that alloHSCT may overcome the unfavorable effect of these mutations. Conclusion: By extensive genetic characterisation of 308 MDS or sAML patients undergoing alloHSCT we identified mutations in IDH2, NRAS and complex karyotype as predictors of relapse and reduced OS and provide a matrix to refine risk prediction for allogeneic HSCT. Disclosures Heuser: Karyopharm: Research Funding. Platzbecker:Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Thiede:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AgenDix GmBH: Equity Ownership.

Increased p53 Expression in Patients with Therapy-Related MDS/AML Is Common and May be Associated with a Poor Prognosis

Therapy-related myelodysplastic syndromes and acute myeloid leukaemia (t-MDS/AML) occur as a late complication of cytotoxic chemotherapy and/or radiation for neoplastic and non-neoplastic disorders and are a major cause of non-relapse mortality. The prognosis of t-MDS/AML is poor because of an increased incidence of adverse cytogenetic features and relative resistance/intolerance to conventional therapies. TP53 is a tumour suppressor and cell cycle regulatory protein. Mutations in the TP53 gene are known to be associated with adverse outcomes in a variety of cancers including haematological malignancies.We examined p53 expression using immunohistochemical staining, as a surrogate marker for TP53 mutation, in marrow biopsies of 39 patients with t-MDS/AML from a single institution and correlated p53 expression with survival. Formalin fixed, paraffin embedded marrow trephine samples at diagnosis of t-MDS/AML were stained with DO-7 mouse p53 monoclonal antibody. Positive expression was defined as per Modified Quick Score. Of 39 patients, 35 had t-MDS and 4 had t-AML. 51% of patients had marrows at diagnosis that showed p53 positivity and 49% were negative. In a control group of 47 MDS patients in our centre, 22% were p53 positive and 78% were p53 negative. Of the t-MDS/AML group, age at diagnosis and gender distribution was similar in p53 positive and negative patients. Median latency (time from diagnosis of primary malignancy to diagnosis of t-MDS/AML) was similar between p53 positive and negative patients (66 vs. 52 months, p=0.51). A similar distribution was noted for the type of primary tumours (haematological vs. solid tumour) among the two groups, but all patients with more than one prior malignancy were noted to be p53 positive (N=4) whereas all patients who developed t-MDS after cytotoxic therapy for non-neoplastic disorders were p53 negative(N=3). There was no difference in p53 expression based on the type of primary therapy received (chemotherapy and/or radiotherapy) but a greater proportion (71%) of patients who received combined chemotherapy and radiation was p53 positive. A higher proportion of p53 negative patients had lower risk cytogenetics and normal karyotype whereas in p53 positive group there was lower incidence of good risk and a higher proportion to intermediate risk cytogenetics. However, equal numbers of patients with higher risk cytogenetics were found between p53 positive and negative groups. These findings were again reflected by IPSS-R risk groups (Table 1). Median overall survival was 10.5 months in p53 positive group compared to 22.5 months in p53 negative patients, p= 0.208. (Figure 1)In conclusion, our study demonstrated that a higher proportion of patients with t-MDS/AML were positive for p53 than in de novo MDS which may be related to poor outcomes in this group. p53 positive t-MDS/AML patients tend to have lower incidence of favourable karyotypes and showed poor survival compared to their counterparts who were p53 negative. Analysis of p53 expression by immunohistochemistry is a readily accessible, cost-effective method of assessment without the need for expensive gene sequencing and is a clinically useful prognostic tool. It may be particularly useful in patients with t-MDS/AML.Table 1p53 expression in Therapy-related Myeloid Neoplasmsp53 Positive (N=20, 51%)p53 Negative (N=19, 49%)Primary TumourHaematological (22) Solid Tumour (10) Autoimmune disease (3) More than one disorder (4)45% 60% 0 100%55% 40% 100% 0Primary TherapyChemotherapy alone (27) Radiotherapy alone (5) Combined therapy (7)44% 60% 71%56% 40% 29%Cytogenetic RisksLow (very good, good) (14) Intermediate (8) High (poor, very poor) (12)29% 75% 50%71% 25% 50%IPSS-RLower (very low, low)(12) Intermediate (4) Higher (high, very high) (16)33% 75% 50%67% 25% 50% [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Discordance Between Morphologic and Cytogenetic Findings Following Induction Chemotherapy for Acute Myeloid Leukemia Is Prevalent in Patients with Bone Marrow Blasts >2% and Is Associated with Poor Clinical Outcomes

BACKGROUND: Following induction chemotherapy for Acute Myeloid Leukemia (AML), patients in morphologic complete remission (mCR) with persistent cytogenetic abnormalities (PCA) have poor outcomes relative to those achieving a mCR without a PCA. Whether these patients have similar outcomes to those failing to achieve a mCR remains unknown. Furthermore, the reasons for the discordance between morphologic and cytogenetic findings have not been well described. To address these questions we conducted a retrospective review of AML patients treated at our centre.METHODS: All non-Acute Promyelocytic Leukemia AML patients from 2004 - 2014 were eligible for inclusion however patients with normal cytogenetics at diagnosis, those not evaluated for remission and those lacking post-induction cytogenetics were excluded. mCR was determined by the attending hematopathologists after review of the bone marrow aspirate (BMA), the bone marrow trephine biopsy (BMTB) with or without immunohistochemistry and/or flow cytometry for the detection of residual disease particularly in equivocal cases.RESULTS: Study criteria were met in 111 patients. Median age was 50 years (17.6 - 77) and 63% were males. Median white blood count at diagnosis was 5.5 x 109/L (0.4 - 307) and median BMB% was 56% (11 - 96%). Most (84%) had primary AML. Cytogenetics were favorable in 28%, intermediate in 41%, and unfavorable in 31% of the patients. Chemotherapy was anthracycline based in 88% and fludarabine based in 12% of patients.Post-induction, 94 patients (85%) had mCR (78 CR + 16 CR with incomplete count recovery, CRi) following induction, 18 (19%) of whom had a PCA and thus discordance between morphologic and cytogenetic findings. There was no difference in the rate of discordance in those age ≤60 vs. age >60 (18.8 vs. 20%, p=1.0), in those with secondary AML vs. primary AML (36 vs. 16%, p=0.13) or in those treated with an anthracycline vs. fludarabine regimen (21 vs. 9%, p=0.68). Discordance was 0%, 28% and 27% in those with favorable, intermediate and unfavorable risk cytogenetics, respectively (p=0.002). A CRi was associated with higher rates of discordance relative to those in CR (50 vs. 12.8%, p=0.002).When the effects of the quality of the BMA and BMTB on the rates of discordance was evaluated, higher rates of discordance were seen in patients with dilute BMA vs. those with non-dilute aspirates (75 vs. 16.7%, p=0.02) and in those with hypocellular BMTB vs. those with cellular/hypercellular/packed BMTB (43.8 vs. 14.7%, p=0.016). Patients with BMB% of >2% had higher rates of discordance vs. those with BMB% ≤2% (46 vs. 9%, p=0.0001)No difference in discordance rates was observed between patients whose BMA were aparticulate/pauciparticulate vs. those with particulate samples (33.3 vs. 16.5%, p=0.15), in those whose BMA samples were hypocellular vs. those that were cellular/hypercellular (20 vs. 16.4%, p=0.67) and in those who had suboptimal BMTB vs. those who had adequate vs. good/excellent samples (25 vs. 18 vs. 20%, p=0.89). In multivariate analysis only the BMB% >2% was associated with high rates of discordance (p=0.0002, odds ratio = 26) between morphologic and cytogenetic findings.The outcomes of patients in mCR with a PCA (n=18) were compared with those failing to achieve a mCR (n=17). The two groups had similar characteristics although patients in mCR with a PCA had a lower median BMB% post-induction vs. those not in a mCR (3 vs. 6%, p=0.004). Of the 18 patients in mCR with a PCA, 10 (56%) underwent an allogeneic stem cell transplant (SCT) while 8 of the 17 (47%) patients not in mCR, after additional chemotherapy, went onto a SCT. Rate of relapse following SCT was similar for those with mCR with PCA vs. those not in mCR (62.5 vs. 33.3%, p=0.35) as was time to relapse (651 vs. 1973 days, p=0.56, Fig 1A). The median overall survival was also similar for patients with mCR and PCA relative to those not in mCR (542 vs. 670 days, p=0.88, Fig 1B).Conclusions: Our results strongly reinforce the need to evaluate for persistent karyotype abnormalities post induction particularly amongst patients with BMB% >2%. Further prospective studies with larger cohorts are necessary to confirm our findings. [Display omitted] DisclosuresSandhu:Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Outcomes of Down-Syndrome Associated Acute Lymphoblastic Leukemia in Adults: A Single Center Retrospective Review

Background:Children and adults with Down Syndrome (DS) have an approximately 20-fold increased risk of developing acute lymphoblastic leukemia, which in the majority of cases is of the B-cell precursor (BCP) type, and have significantly inferior outcomes due to both higher relapse rates and increased treatment-related mortality.During treatment for ALL, children with DS are at a higher risk of complications such as prolonged neutropenia, severe mucositis and longer hospitalizations. In view of the significant barriers to successful treatment for pediatric DS-ALL and the absence of outcome data for DS-ALL in adults, we retrospectively reviewed the outcomes of adult patients with DS-ALL treated at our center.Methods:All patients greater than 18 years of age diagnosed with DS-ALL who were followed at Princess Margaret Cancer Center/University Health Network (PMH/UHN) between January 1, 2000 and June 30, 2014 were identified using the institutional leukemia database.Diagnosis of ALL was established by standard FAB WHO Criteria using multiparameter flow cytometry for immunophenotyping according to ISCN guidelines. Treatment of adult DS patients with de novo ALL and those with relapsed ALL who had not previously received intensive asaparaginas therapy, were treated according to a modified Dana Farber Cancer Institute (DFCI) ALL regimen; reduced doses of methotrexate were used due to the increased risk of mucositis reported in children with DS. This regimen includes an at least four fold higher total dose of E. coli derived asparaginase compared to other adult regimens.Results:Seven adult patients with DS-ALL were treated at our center from 2000 to 2014. Four of these were diagnosed with de novo DS-ALL (after age of 18 years) and three patients developed a late isolated bone marrow relapse of DS-ALL as adults after previous treatment for childhood DS-ALL. Approximately half of our patients had favourable cytogenetics and half had intermediate risk cytogenetics . Treatment was not altered based on cytogenetics.The median age of 4 patients, with de novo adult DS-ALL, was 26 years (range 21-42 years). 75% achieved a CR after initial induction; one patient died during induction from sepsis. Two of the four de novo adult DS-ALL patients relapsed after CR1 durations of 11 and 35 mon.. One patient received palliative chemotherapy. At the time of last follow up, 3 of these patients have died. The one remaining patient is alive in continuous CR at 41 mos.The three adult DS patients with relapsed ALL had a median age of 14 years (7-15 years) at diagnosis of primary DS-ALL and 29 years (21-36) at the diagnosis of relapse. Two of these received DFCI as re-induction while the third received Hyper-CVAD. Despite achieving an initial remission all patients with relapsed DS-ALL died , two from subsequent relapsed and one from an invasive aspergillosis. One patient relapsed while on therapy at 4.7 months. The other patient relapsed while off therapy at 15.2 months.The overall and relapsed-free survival of adult patients with DS and de novo ALL at 3 years was 50% and 33.3%, respectively, and thus markedly inferior to results of a similarly treated population of adults (aged 18-35 years) without DS ( 3-year OS 83%, 3-year RFS 77%) at our center.Conclusions:The results of our series of adult patients with DS and ALL suggest that the barriers to successful treatment of ALL in adults with DS are similar to those observed in children. Although the rarity of adult ALL in general, and that of adult DS-ALL in particular, limits sample size and conclusions, this report is to our knowledge the first describing survival outcomes of adults with DS and ALL and highlights that treatment of primary adult DS-ALL is feasible but significantly less successful compared to adults without DS. As in children, subsequent relapse and treatment-related mortality impacting outcome are equally problematic. DisclosuresGupta:Incyte: Honoraria, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Outcomes of Mixed Phenotype Leukemia, Not Otherwise Specified (NOS), in Adults: A Single Centre Retrospective Review from 2000 to 2014

Introduction:Acute leukemias of ambiguous lineage (AUL), biphenotypic leukemias (BAL) and mixed phenotypic leukemias (MPAL) are a heterogeneous group of rare, poorly characterized leukemias with unfavorable outcomes. AUL, BAL and MPALs are believed to constitute 2-5% of all leukemias. The optimal treatment regime for this group of leukemias is currently unknown.The primary objective of this study is to determine the complete remission rate (CR) and overall survival (OS) among adult patients with mixed phenotypic leukemia, not otherwise specified (NOS), as defined by the WHO 2008 classification criteria.Methods:Patients with mixed phenotype acute leukemia (MPAL) older than 17 years of age who were treated at Princess Margaret Cancer Centre from January 1, 2000 to June 30, 2014 were identified through the Leukemia Database at Princess Margaret Cancer Registry. Pathology review of the original diagnostic bone marrow was completed to ensure patients met the WHO 2008 criteria for MPAL, not otherwise specified (NOS). Patients with MLL+, BCR-ABL+, PDGRFA+ were excluded from analysis.Results:Fourteen patients were identified and confirmed by pathology review. Eleven (79%) were male, 3 (29%) were female. The median age was 57 years (range: 18-84). Eight (57%) had extramedullary involvement at presentation. Six (42%) had poor risk cytogenetics, whereas 8 (57%) had intermediate risk cytogenetics at presentation as per the MRC criteria. Two patients (14%) had p53 mutations/loss of chromosome 17. Four patients (42%) had monosomy 7 karyotype. Six cases (43%) were myeloid/B, seven cases (50%) were myeloid/T and one (7%) was myeloid/T/B type. The median WBC at presentation was 10.8 x 109/L (2.3 x 109/L-106 x 109/L).Five patients were treated upfront with daunorubicin and cytarabine (3&7) with 1 of these patients achieved a CR with this upfront regime. Five patients were treated upfront with the Dana Farber Consortium Protocol (DFCI) and 4 of these patients achieved a CR with this upfront regime. Two patients were treated upfront with HyperCVAD, and 1 achieved a CR. Two patients were treated palliatively. One received azacytidine as palliative treatment and achieved a CR with a duration of 5 months. Ten (71%) of these patients achieved a CR. Three of the primary nonresponders achieved a CR with an alternative induction regime. The median CR1 duration was 5.5 mos (range, 2-40 mos).Overall median survival was 13 mos (95% CI, 8 mos-27 mos). The OS at 1 year and 2 years was 56% (95% CI, 29%- 82%) and 32% (95% CI, 6%-57%), respectively.Four patients (29%) underwent an allogeneic hematopoietic stem cell transplant. At last follow up, 13 of the 14 patients have died, 11 from relapsed leukemia and 2 from sepsis post HCT. One patient died of relapsed leukemia post HCT.Conclusions:This retrospective review confirms the high risk nature and poor prognosis of MPAL NOS. Our limited cohort showed a male predominance, a high rate of extramedullary involvement at presentation, and a higher rate of CR with DFCI as the initial remission induction regime. HCT or other forms of maintenance therapy should be considered in CR1 in eligible patients due to the high- risk nature of MPAL NOS. DisclosuresGupta:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding.

DC/Aml Fusion Cell Vaccination Administered to AML Patients Who Achieve a Complete Remission Potently Expands Leukemia Reactive T Cells and Is Associated with Durable Remissions

We have developed an autologous whole cell leukemia vaccine, whereby patient derived leukemic blasts are fused to ex-vivo generated dendritic cells. In this way, a broad array of leukemia-associated antigens are presented in the context of DC mediated co-stimulation, resulting in the expansion of a polyclonal anti-leukemia immune response. We are conducting a clinical trial in which AML patients who are not candidates for allogeneic transplantation undergo vaccination with DC/AML fusion cells after achieving a complete remission. 56 patients underwent collection of AML cells for vaccine generation from either a bone marrow aspirate (N=38), 20cc of peripheral blood (N=15) or a leukapheresis product (N=3) at presentation with newly diagnosed (N=55) or first relapsed (N=1) AML. The mean yield of AML cells was 692 x 106 cells with a mean viability of 99%. Patients who did not achieve remission or who underwent allogeneic transplant in first remission came off study and did not complete vaccine generation. Eligible patients achieving CR who did not proceed to allogeneic transplant (N=21) underwent leukapheresis for DC generation and vaccine preparation. Adherent peripheral blood mononuclear cells were cultured in the presence of GM-CSF and IL-4 for 5-7 days, and exposed to TNFα for 48-72 hours to generate mature DCs. Fusion cells were generated by co-culture of DCs with AML cells in the presence of 50% polyethylene glycol and identified as cells co-expressing antigens that were unique to the DC and tumor population. Mean fusion efficiency and viability was 42% and 87%, respectively. Vaccination with DC/leukemia fusion cells was initiated within 12 weeks from count recovery following the final cycle of post-remission therapy. 16 patients (median age 60) have received at least two monthly vaccinations at a dose of 5x106 fusion cells. Remission induction therapy consisted of 7+3 in 13 patients, 7+3 followed by 5+2 re-induction in 1 patient, MEC in one patient, and decitabine in 1 patient. 9 patients had intermediate risk cytogenetics, 2 patients had good risk cytogenetics, and 5 patients had poor cytogenetic risk disease. Vaccination was well tolerated, and importantly, was not associated with clinically significant auto-immunity. Possibly related adverse events were transient and of grade 1-2 intensity, including vaccine site reactions, pruritis, arthalgias, myalgias, eosinophilia, leukopenia, thrombocytopenia. Biopsy of vaccine site reactions demonstrated a dense infiltrate of CD4 and CD8 T cells consistent with recruitment of reactive T cell populations to the vaccine bed. To date, 12 of 16 evaluable patients remain in remission (75%), with a median follow up of 45 months. Notably, no patient has relapsed later than 1 year from completing chemotherapy and the patient who was treated in CR2 remains in remission with 51 months of follow up, after having relapsed within a year of CR1. Vaccination resulted in the potent induction of leukemia specific immunity as measured by a mean fold increase of 4.9 CD8 T cells expressing IFNγ in response to ex vivo exposure to autologous leukemia cell lysates (n=14). In a subset of patients who are HLA2.1+, vaccination resulted in the expansion of T cells recognizing MUC1 (4.4 fold increase), WT1 (2.7 fold increase), NY-ESO (3.8 fold increase) tumor antigens by pentamer analysis. In conclusion, DC/AML fusion cell vaccination administered to patients who achieve complete remission results in the potent expansion of leukemia reactive T cells, and durable remissions. A second cohort is planned in which patients will be treated with DC/AML fusion cell vaccination in conjunction with immune checkpoint blockade. DisclosuresStone:Karyopharm: Consultancy; Roche/Genetech: Consultancy; Merck: Consultancy; Novartis: Research Funding; Celgene: Consultancy; Pfizer: Consultancy; Agios: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Amgen: Consultancy; Abbvie: Consultancy; Celator: Consultancy; AROG: Consultancy; Juno: Consultancy. Steensma:Celgene: Consultancy; Amgen: Consultancy; Incyte: Consultancy; Onconova: Consultancy. DeAngelo:Incyte: Consultancy; Celgene: Consultancy; Agios: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Bristol Myers Squibb: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

Final Results from a Phase 2 Study of Pracinostat in Combination with Azacitidine in Elderly Patients with Acute Myeloid Leukemia (AML)

Background: Combination studies with histone deacetylase (HDAC) inhibitors plus hypomethylating agents (HMA) have suggested beneficial clinical activity in higher risk MDS and AML, though exceptions have also been reported. Pracinostat is a potent oral HDAC inhibitor selective for class I, II and IV isoforms. A pilot phase Ib study of pracinostat in combination with azacitidine (AZA) in higher risk MDS demonstrated a complete response (CR)/CR with incomplete blood count recovery (CRi) rate of 89% (Proc ASH:3821, 2012). Preliminary data on 33 patients from a multi-center, open-label, single-arm Phase II study of Pracinostat in combination with AZA in elderly AML also reported a high CR/CRi rate (ASH 2014). Herein we report the latest survival and response results for this study.Methods: Eligibility includes previously untreated AML (≥20% bone marrow blasts), age ≥65 years, unsuitable for intensive therapy due to co-morbidities and/or AML related features, and intermediate or high-risk cytogenetics. Study therapy includes pracinostat, 60 mg p.o. 3 alternate days/week for 3 weeks plus AZA, 75 mg/m2) days 1-7 or days 1-5 and 8-9 either s.c. or i.v. with cycles repeated every 28 days until progressive disease, lack of response or intolerance. The primary endpoint is CR+CRi+ morphologic leukemia free state (MLFS) per IWG criteria. Response assessments occur at the end of cycle 1 or 2 then every other cycle or when clinically indicated. A Simon 2-stage statistical design is utilized with the following assumptions: null=0.10, alternate=0.25, a=0.10, power=0.90. Stage 1 n=27 and total stage 2 n=40. Secondary endpoints include overall response rate (ORR; CR+CRi+MLFS+partial response [PR]+PRi), duration of response and overall survival.Results: Between Dec 2013 and Dec 2014, 50 patients from 15 study sites were enrolled. At this time, 50 are evaluable for efficacy. Baseline disease characteristics for all patients include: median age 75 (range 66-84); 32 de novo AML, 13 evolved from AHD, 5 were treatment-related; 28 intermediate-risk and 20 high-risk cytogenetics and 2 unknown; baseline bone marrow blast counts ranged from 20% to 89% with a median of 40%. Thirty-one patients (62%) continue to be followed for survival (range: 8.5 to 18.5 months). Median overall survival has not been reached in the overall study population and neither in patients with high-risk cytogenics or those with AML secondary to MDS or prior anti-cancer therapy. The 1-year overall survival estimate is 60%. The primary endpoint of CR +CRi +MLFS has been observed in 27/50 evaluable patients (54%) to date, including 21/50 (42%) CR. The 60-day all-cause mortality rate is 10% (5/50). Treatment emergent adverse events (TEAEs) Grade ≥3 seen in >5% of patients: febrile neutropenia 30%; thrombocytopenia 22%; neutropenia 10%; cellulitis 10%; anemia 8%; fatigue 8%; sepsis 6%, and pancytopenia 6%. TEAE's leading to study therapy discontinuation: peripheral motor neuropathy (1), parainfluenza (1), atrial fibrillation/prolonged QTc (1), subdural hematoma after a fall (1), and sepsis (3).Conclusions: Pracinostat plus AZA produces a high rate of durable responses in this AML population. Median overall survival has not been reached; 1-year overall survival is estimated at 60%. Final response data and overall survival estimates will be presented at the meeting. DisclosuresOff Label Use: Azacitidine is not approved for use in acute myelogenous leukemia.. Khaled:Sequenom: Research Funding. Arellano:Cephalon Oncology: Research Funding. Butler:MEI Pharma, Inc.: Employment. Ashby:MEI Pharma, Inc.: Employment. Medeiros:Celgene: Honoraria, Research Funding; Agios Pharmaceuticals: Honoraria.

Improved Outcomes of Low-Dose Cytarabine Regimen By Increased Dose and Duration of Cytarabine in Combination with Oral Etoposide for Elderly Acute Myeloid Leukemia

Background: For elderly patients unfit for intensive chemotherapy in acute myeloid leukemia (AML), low-dose cytarabine (LDAC; 20 mg SQ BID for 10 days) still remains to be the standard treatment, despite its unsatisfactory complete response (CR) rate of 18% and median overall survival (OS) of < 6 months (Burnett, 2007). Recently, there have been huge efforts to develop more effective and less-toxic therapies, such as decitabine, azacitidine, clofarabine, or gemtuzumab ozogamicin, but their benefits were not concrete, even though they were compared to the classical LDAC. To improve outcomes of the classical LDAC, we modified it by giving a higher dose of cytarabine for an extended duration in combination with oral etoposide. Herein, we present the results.Methods: Between 2002 and 2014, 93 consecutive older (≥ 60 years) patients with AML, who were unfit for intensive chemotherapy, received 1st cycle of modified LDAC (mLDAC) regimen consisting of cytarabine (20 mg/m2 SQ BID) and oral etoposide (50 mg PO BID) for 14 days. Thereafter, they received additional subsequent cycles (for a maximum of 7 cycles) for 10 days every 6 to 8 weeks. We retrospectively analyzed their overall response (OR), disease-free survival (DFS), and overall survival (OS) rates. In this analysis, OR was defined as CR plus CR with incomplete platelet recovery (CRp) or blood count recovery (CRi).Results: The median age of patients in our cohort, including 69 (74.2%) with poor performance status (ECOG ≥ 2), 15 (16.1%) with AML with myelodysplastic-related changes or secondary AML, and 13 (14.0%) with poor cytogenetic risk, was 68 years (range, 60-83). The median number of mLDAC regimen cycles which they received was 2 (range, 1-8). Clinically relevant toxicities of grade III-IV including nausea/vomiting, diarrhea, hyperbilirubinemia and neutropenic fever were observed in 4 (4.3%) patients, 6 (6.5%), 3 (3.2%), and 42 (45.2%), respectively, which were comparable with those of classical LDAC (Burnett, 2007). The early mortality rates at 30 and 60 days were 11.8% and 15.0%, respectively. The OR was observed in 45 (48.4%) patients, including 34 (36.6%) CR, 7 (7.5%) CRp, and 4 (4.3%) CRi, within two cycles of mLDAC. With median follow-up duration of 26.1 months, the median DFS and OS were 6.2 and 15.8 months, respectively. For patients who achieved OR, they were 14.5 and 36.9 months, respectively. The OR of patients who had poor cytogenetic risk was not significantly different compared to others (57.1%, 46.2%, and 38.5% for favorable, intermediate, and poor cytogenetics, respectively; P=0.50). However, they showed significantly shorter median DFS (9.8, 6.6, and 5.1 months, respectively; P=0.01) and OS (NR, 1.4, and 5.1 months, respectively; P=0.01) with significantly shorter OR duration (30.6, 19.1, and 8.6 months, respectively; P=0.01). Between 2009 and 2014, among 17 patients treated with hypomethylating agents (HMA; 14 decitabine and 3 azacitidine), 1 CR and 3 partial response were achieved with a median survival of 5.5 months, and 5 patients after HMA treatment failure received subsequent mLDAC, and 3 achieved additional CR (n=2) and CRp (n=1).Conclusions: These results suggest that the outcomes of classical LDAC in elderly patients with AML can be improved by modifying it, with improved response and survival rates without increasing toxicities, even in patients with poor cytogenetics. Additionally, mLDAC could induce clinical responses in patients with HMA failure. Our mLDAC regimen may become another therapeutic option with emerging novel agents for elderly patients with AML, and these should be confirmed by large randomized trials. DisclosuresNo relevant conflicts of interest to declare.

Myelodysplastic Syndromes in Adolescent Young Adults (AYA)

Introduction:There has been little improvement in cancer survival of adolescent and young adult (AYA) patients, ages 18-39, possibly reflecting different disease biology in this subgroup. Myelodysplastic syndrome (MDS) is mainly a disease of the elderly. The characteristics, outcomes and response to treatment are not well described among AYA population.Patients and Methods:Retrospective review of patients from the Moffitt Cancer Center MDS database. We compared baseline characteristics and outcomes of AYA population to older patients. Descriptive statistics were used for baseline characteristics. Chi-square test was used for categorical variables, and t-test for continuous variables comparison. Kaplan-Meier estimates were used for overall survival (OS), and cox regression method for multivariable analysis.Results:We identified 51 AYA and 1,897 older MDS patients. Table-1 summarizes baseline characteristics. More females and Hispanics were noted in AYA group. The AYA patients had higher risk disease, more circulating myeloblasts and more hypoplastic MDS. Autoimmune disorders were more prevalent in older patients.The median OS was 47 months (mo) in the AYA group versus 40 mo in the older group (p 0.26). The median OS was 47 mo versus 56 months in lower risk (low and intermediate-1(int-1)) IPSS MDS AYA group and older group respectively (p 0.46). In the higher risk IPSS group (int-2 and high), median OS was 82 mo in AYA group compared to 17 mo in older group (p 0.001). Thirty individuals were transplanted in the AYA versus 241 in the older group. The median OS for transplanted patients was 55 mo in the AYA group and 46 mo in the older (p 0.4). Whereas, in the non-transplanted patients median survival was 31 months for AYA and 39 months for the older group (p 0.9). The rate of AML transformation was 37% versus 28% in AYA and older group respectively (p 0.17). No difference in use or response to hypomethylating agents was observed. Lenalidomide therapy was seldom used in younger patients.In AYAs, poor karyotype was the only variable strongly associated with worse outcome. Fifteen patients had poor risk karyotype. The median OS was 47 months, not reached and 29 months among patients with good, intermediate and poor risk cytogenetics, respectively (p 0.035)Conclusion:MDS is rare and tends to be more aggressive in the AYA population. The karyotype was the most important prognostic factor. The differences in underlying disease biology should be further explored. Allogeneic stem cell transplant offered younger patients best outcomes.Table 1Baseline characteristics of AYA and Older PatientCharacteristicAYA (18-39) (N= 51)Older Patients (> 39 years old) (N=1,897)P valueGenderFemale25 (49%)655 (34.5%)0.025RaceWhite Black Hispanic Other34 (66.7%) 2 (3.9%) 13 (25.5%) 2 (3.9%)1,736 (91.5%) 47 (2.5%) 57 (3%) 41 (2.2%)0.000t-MDSYes10 (19.6%)359 (18.9%)0.902WHO SubtypeRA RARS RCMD Deletion 5q RAEB-1 RAEB-2 AML CML MDS-U MDS/MPN3 (5.9%) 1 (2%) 22 (43.1%) 0 (0%) 11 (21.6%) 10 (19.6%) 0 (0%) 0 (0%) 1 (2%) 2 (3.9%)196 (10.4%) 151 (8%) 583 (30.8%) 51 (2.7%) 372 (19.7%) 336 (17.7%) 1 (0.1%) 60 (3.2%) 44 (2.3%) 94 (5%)0.188IPSSLower risk Higher risk28 (59.6%) 19 (40.4%)1,264 (68.2%) 590 (31.8%)IPSS-RVery low/low Intermediate High/very high13 (30.1%) 14 (32.6%) 16 (37.3%)826 (45.3%) 394 (21.6%) 602 (33%)Hypoplastic BMYes11 (23.4%)178 (9.8%)0.009LGL cloneYes0 (0%)159 (8.4%)0.033Autoimmune diseaseYes8 (15.7%)500 (26.4%)0.055KaryotypeGood Intermediate Poor24 (50%) 9 (18.8%) 15 (31.3%)1120 (60.4%) 300 (16.2%) 434 (23.4%)0.324Peripheral BlastsYes14 (29.8%)246 (13.2%)0.003RBC TransfusionDependent36 (70.6%)1274 (67.3%)0.372 DisclosuresShah:Seattle Genetics: Research Funding; Rosetta Genomics: Other: Grant support; Acetylon: Other: Advisory board; Plexus Communications: Honoraria; Pharmacyclics: Speakers Bureau; Spectrum: Other: Advisory board, Speakers Bureau; Bayer: Honoraria; Celgene: Other: Advisory board, Speakers Bureau; DeBartolo Institute for personalized medicine: Other: Grant support. Lancet:Pfizer: Consultancy; Kalo-Bios: Consultancy; Boehringer-Ingelheim: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy, Research Funding. List:Celgene Corporation: Honoraria, Research Funding. Komrokji:Celgene: Consultancy, Research Funding; Incite: Consultancy; Novartis: Speakers Bureau; GSK: Research Funding.

North American Leukemia, Intergroup Phase III Randomized Trial of Single Agent Clofarabine As Induction and Post-Remission Therapy, and Decitabine As Maintenance Therapy in Newly-Diagnosed Acute Myeloid Leukemia in Older Adults (Age ≥60 Years): A Trial of the ECOG-ACRIN Cancer Research Group (E2906)

▪Background: Induction therapy with daunorubicin (Dauno) & cytarabine (Ara-C) [DA] has been the standard of care for eligible older adults (age ≥ 60 years) with newly diagnosed acute myeloid leukemia (AML) for over 2 decades. Single agent Clofarabine (CLO) induction & consolidation (Consol.) therapy has demonstrated important clinical activity in this age group in large phase II studies. Lower induction mortality (IM) & similar reported complete remission rate (CR) & overall survival (OS), as well as notable activity in those with higher risk disease features [including unfavorable cytogenetics, therapy-related AML (t-AML) & prior antecedent hematologic disorder (AHD)] raises the possibility that a non-Ara-C-based regimen could achieve similar or superior OS with lower toxicity.Methods: We performed a randomized United States Intergroup Phase III trial of single agent CLO [30mg/m2 x 5 days induction; 20 mg/m2 re-induction (if indicated) & 2 cycles Consol.] vs. standard DA therapy [Dauno 60mg/m2 D1-3 & Ara-C 100mg/m2 D1-7 induction x 1-2 cycles; 2 cycles Consol. with Ara-C (1.5g/m2 Q12hrs D1-6 age 60-69; once daily if age 70+)] in patients (pts) age ≥ 60 yrs with newly diagnosed AML. Patients with serum creatinine >1.0 (or GFR <60 mL/min) and those with AML-M3 and ECOG performance status >3 (PS>2 if age 70+ yrs) were excluded. Randomization was stratified by age (60-69 vs. 70+), t-AML, & AHD. Pts with HLA-matched donor were eligible for allogeneic transplantation (AlloHCT) after induction, and those completing Consol. were eligible for randomization #2 (R#2) to maintenance decitabine [20mg/m2 x 3D, monthly x 1 year] versus observation. With a target accrual of 747, E2906 was powered to determine non-inferiority [and possible superiority] of CLO vs. standard DA, and primary endpoint was OS. A weighted statistical analysis was performed to account for confounding impact of R#2. AlloHCT patients were censored at transplant in this analysis. Responses & cytogenetics were confirmed centrally and OS & CR rates were monitored by an independent Data Safety Monitoring Committee (DSMC) at pre-specified time points.Results: As of Feb 23, 2015, 727 pts were randomized. Median age was 68 years (range 60-86); 57% were male, and 38% were age ≥70 yrs. Treatment arms are well balanced for all baselineclinical & AML characteristics, & 30% had unfavorable cytogenetics. Of 659 with complete treatment information reported, 30.4% on DA vs. 40.1% on CLO received 2 cycles of induction (p=0.006).Median follow-up of surviving patients is 7.6 months.Table 1shows early treatment results (CR, toxicity) for the 686 pts randomized as of Dec 23, 2014 (2 months prior to study end, & excluding 90 with ongoing response evaluation).DACLOp-valueCR/CRi43.8%42.8%p=0.8730-day mortality8.5%7.9%p=0.8960-day mortality14.9%13.1%p=0.58Gr 4-5 Non-Heme Tox.Induction27%19%p=0.02Gr 4-5 Non-Heme Tox.Consol.20%7%p=0.001374 pts have died (174, DA; 200, CLO) & significantly inferior OS was observed for CLO vs. DA [Hazard Ratio (HR) 1.41 (95% CI 1.12-1.78)] (Fig. 1). Planned subgroup analyses were performed (Table 2) demonstrating significant differences in OS after CLO for patients age 60-69 yrs, without AHD, & with intermediate risk cytogenetics; but not for those with Unfav. Cytogen. (Fig. 2) or t-AML. Based on the primary weighted analysis, DSMC recommended suspension of new accrual to E2906 on Feb 23, 2015 & all active patients on CLO were transitioned to DA Arm. Table 2NHR CLO/Standard (95% CI)*Weighted OS7271.41 (1.12-1.78)Unweighted OS7271.23 (1.00-1.50)Age 60-694491.48 (1.10-1.99)Age 70+2781.34 (0.93-1.93)Intermed. Risk Cytogen.3781.77 (1.27-2.47)Unfav. Risk Cytogen.2160.96 (0.65-1.43)No AHD6041.46 (1.13-1.89)AHD1231.22 (0.74-2.00)De novo AML6271.52 (1.18-1.96)Therapy-related AML1000.94 (0.54-1.61)Conclusions: Despite similar CR & IM, OS after single agent CLO is inferior to standard DA therapy for pts age ≥60 years with newly diagnosed AML who are fit for intensive therapy, and DA remains the standard of care. However no difference in OS was observed after CLO in some pre-specified high risk AML subgroups. R#2 & AlloHCT arms continue in E2906 for pts already enrolled. Embedded prospective minimal residual disease study at CR is being performed to identify pts at higher risk after CLO & DA. [Display omitted] [Display omitted] DisclosuresOff Label Use: Use of clofarabine in AML, and maintenance therapy with decitabine in AML. Claxton:Medimmune: Research Funding; BMS: Consultancy; Astellas: Research Funding; Cyclacel: Research Funding; Merck: Research Funding; Ambit: Research Funding. Levine:Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy. Altman:Seattle Genetics: Consultancy; BMS: Consultancy; Spectrum: Consultancy; Astellas: Consultancy; Ariad: Consultancy; Novartis: Consultancy. Al-Kali:Novartis: Research Funding.

Impact of GvHD and Other Patient-, Disease-, Donor and Transplantation-Related Factors on 5 Year Relapse after Unrelated Cord Blood Transplantation for Children with Acute Lymphoblastic Leukemia in Remission

Relapse remains a major barrier to the long-term overall survival (OS) of pediatric patients with acute lymphoblastic leukemia (ALL). Allogeneic HSCT is a potentially curative option for these patients and its mechanism relies on graft-versus-leukemia (GvL) and delivery of high-dose chemotherapy. However, the mechanism and degree of GvL in curing ALL is not fully understood. Unrelated cord blood (CB) is an attractive donor option for these patients due to rapid procurement and lenient HLA matching without increased graft-versus-host disease (GvHD). Despite less GvHD, relapse is not increased when compared to other donor sources. Therefore, through collaboration between Eurocord, PDWP-EBMT and Duke University, we performed a retrospective analysis to identify risk factors associated with relapse, and the role of GvHD (as time dependent co-variate) in preventing relapse after unrelated CB transplantation (CBT) for children with ALL.We analyzed 640 children (<18 years (y)) with ALL in complete remission (CR) who received a single-unit CBT in either first (n=257, 40%) or second (n=383, 60%) CR from 2000-2012. Most patients were diagnosed with B-cell (79%) or T-cell (18%) lineage ALL and had no CNS involvement (92%). Cytogenetic data was available for 411 patients (CR1=171, CR2=240), and 37% were considered intermediate risk and 36% poor risk. Pre-transplant, 49% of patients were CMV seropositive. All patients received myeloablative regimens and most received anti-thymocyte globulin (88%). Regimens included total body irradiation (TBI; 69%) with either cyclophosphamide (Cy)+Fludarabine (Flu; 10%) or other agents (59%), Busulfan (Bu)+ Cy (16%), or Thiotepa+Bu+Flu (8%). Most patients received HLA mismatched CBs at 1 (50%) or 2+ (34%) loci. GvHD prophylaxis included cyclosporine with either steroids (72%) or mycophenolate mofetil (21%), or other agents (6%).Considering CR1 patients only, the median age at CBT was 5.3 y. Most were diagnosed with B-cell ALL (75%) and approximately half of those with known cytogenetics were considered poor risk. The median duration from diagnosis to CBT was 6.7 months and median follow up was 47 months. Estimated OS and LFS at 5 y for CR1 patients was 59% and 52%, respectively. Improved OS and LFS were associated with TBI-containing regimens and younger age at time of CBT (Table 1). The CI of relapse at 5 y was 23% for CR1 patients. In a multivariate analysis (MVA) the presence of acute GvHD (grade II-IV) and TBI-containing regimens were both protective of relapse (Table 1). No impact of GvHD was observed on OS, LFS or NRM.In CR2 patients, the median age at CBT was 6.9 y. Disease was primarily B-lineage ALL (82%) with intermediate or poor risk cytogenetics in 40% and 28% of patients, respectively. Median time from diagnosis to CBT was 36 months and median follow up was 54 months. Estimated OS and LFS in CR2 patients were 46% and 44%, respectively. In MVA, younger age and longer duration from diagnosis to CBT (>30 months) was associated with improved OS and LFS (Table 2). Conversely, the use of ATG was associated with lower OS.In CR2 patients, the CI of relapse was 28%. In MVA, longer duration from diagnosis to CBT (>30 months) and receiving TBI were both associated with less relapse. Importantly, receiving a fully matched HLA CB graft was a strong risk factor for increased relapse compared to mismatched CB recipients. Acute GvHD (II-IV) in this cohort was associated with higher mortality but not with relapse.In this large retrospective collaborative study, we investigated the impact of GvHD as a marker of GvL in pediatric ALL patients after CBT. In CR1 patients, acute GvHD and use of TBI were protective of relapse. However in CR2 patients, use of TBI, longer duration from diagnosis to CBT and HLA mismatched grafts were associated with decreased incidence of relapse but not with the presence of GvHD. These results showed that the impact of GvHD as a GvL marker is more evident in CR1 patients than CR2. Whereas, the impact of HLA mismatch CB on relapse is more important in CR2 patients. Decreasing immunosuppression more rapidly for CR1 patients or avoiding HLA identical CB for CR2 patients should be further investigated. In both cohorts, TBI has shown to be an important protective factor for relapse. [Display omitted] [Display omitted] DisclosuresBader:Riemser: Other: Institutional grants; Neovii: Other: Institutional grants; Medac: Other: Institutional grants; Amgen: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy.

Integration of Minimal Residual Disease with Other Patient Risk Factors Identifies a Population with Very Poor Overall Survival in Pediatric ALL: Results from the UKALL 2003 Trial

Background:Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies.Methods:The UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS (<75% at 5 years). MRD was considered positive at a level ≥0.005% and low risk if <0.005% unless otherwise stated.Results:Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003).Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p<0.0001) whilst high WCC in T-ALL had little effect on survival, even at WCC >200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively).Older age (≥10yr) was also associated with poorer outcomes compared to those <10yr (5yr OS 95.8 vs. 87.4%, p<0.0001). Integration with MRD results further separated these groups but did not identify a very high risk subgroup. However, combining older age with a higher EOI MRD cut-off of 1% successfully identified a subgroup (n=96) with a very poor 5yr OS of 51.3% (vs. 91.4% in those with MRD<1%, p<0.0001). Interestingly, this was independent of cytogenetic risk group.Conclusions:Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. DisclosuresNo relevant conflicts of interest to declare.

Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: Implications for leukemia therapy.

Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI-1 in AML and the effects of a novel small molecule selective inhibitor of BMI-1, PTC-209. BMI-1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse-phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI-1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI-1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC-209 reduced protein level of BMI-1 and its downstream target mono-ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase-3 cleavage, BAX activation, and phosphatidylserine externalization. PTC-209 induced apoptosis in patient-derived CD34(+)CD38(low/-) AML cells and, less prominently, in CD34(-) differentiated AML cells. BMI-1 reduction by PTC-209 directly correlated with apoptosis induction in CD34(+) primary AML cells (r = 0.71, P = 0.022). However, basal BMI-1 expression was not a determinant of AML sensitivity. BMI-1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.