ALBERT

Description: Abstract 2742 Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10−5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10−3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10−5, 10−4, 10−3, and 10−2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10−5, 10−4, and 10−3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10−5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 – 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 – 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 – 1.138)) laboratory results (Fig. 1–3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing. Disclosure: Sher: Invivoscribe Technologies Inc: Employment. Miller:Invivoscribe Technologies Inc: Employment. Huang:Invivoscribe Technologies Inc: Employment. Shaw:Invivoscribe Technologies Inc: Employment.

Description: Background: Contemporary treatment schedules for pediatric acute myeloid leukemia (AML) have improved 5 year-overall survival rates up to 70% . These schedules usually consist of 4 to 5 intensive courses mainly based on cytarabine (AraC) and anthracyclines, with allo-HSCT upfront in a subset of patients . Long-term side effects such as late cardiotoxicity caused by high cumulative anthracycline dosages are a major concern. Therefore, the DCOG/BSPHO collaboration adapted the NOPHO 2004 protocol to DB-AML01 to reduce cumulative anthracyclines from 420-450 mg/m² to 300-330 mg/m² and to omit HSCT in CR1. Material: The treatment consisted of 5 chemotherapy courses: first induction course AIET (AraC conventional dose and Idarubicin (Ida) 36 mg/m2); second induction course AM (AraC conventional dose and Mitoxantrone (Mitox) 30 mg/m2) when blasts at day 15 〈 5% or FLA-DNX (high dose AraC and Daunoxome (DNX) 180 mg/m2) when blasts 〉 5% at day 15 or t(8;21) AML; 3 consolidation courses (high dose AraC with Etoposide as course 3 and 5 and in between high dose AraC alone). Cumulative dose of AraC is 43,4 gr/m2 and of anthracyclines 300 mg/m² for patients receiving AIET+AM or 330 mg/m² for patients receiving AIET/FLA-DNX. Results: From 2010 to 2014 a total of 113 children with AML, median age 6.0 yrs (range 0-16), gender 56 males, were included. Second induction course was AM in n=75 and FLA-DNX in n= 33. Death in induction occurred in 4 patients, CR rate was 102/113 (90.3%) and 7 patients were refractory after induction. The later events were death in CR (n=2), relapse in CR (n=40), and secondary malignancies (n=0). HSCT was given to 3 patients in CR1 (physician's decision). Median follow up time is 2.6 years (range (range 1-5). Probability of EFS at 2 years was 60% (95% CI 49-69%) and 2-yr OS was 76 (95% CI 66-83%)77.9%. Especially inv 16 (n=10) and t(8;21) AML (n=16) had an excellent outcome (inv 16:2-yr EFS: 70% (95% CI 32-89%) and 2-yr OS 91% (95% CI 51-99%) resp t(8;21):2-yr EFS: 87% (95% CI 57-97%) and OS: 94% (95% CI 63-99%)), EFS nor OS in other genetic subgroups such as FLT3-ITD and MLL rearrangements were significantly different from the total group. Children with an older age (〉 10 years) (n=34) or high WBC count (〉50.109/l) (n=41) had the poorest 2-yr EFS: 51 (95% CI 33-67%) resp EFS: 40% (95% CI 24-56%), whereas OS was not significantly worse. For the patients with a relapse, 32 achieved a second CR and 4 a partial remission (90%) . In second CR 34 patients received an allo-HSCT. Until now 26 of the 40 patients are still in second CR. Conclusion: The DB-AML01 outcome data suggest that the choice of high cumulative doses of AraC with moderate cumulative doses of anthracyclines up to 300-330 mgr/m² results in a favorable overall survival for particularly those children with t(8;21), inv (16) AML, young age or lower WBC counts. Children with relapsed AML could be rescued with second line treatment and allo-HSCT. These results underscore the relevance of identification of subgroups of pediatric AML patients, based on age, WBC count and t(8;21) or inv 16, curable with "lower risk" treatment arms without allo-HSCT in CR1. Disclosures Kaspers: Janssen-Cilag: Research Funding.

Description: Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which has an incidence of 40 children on average per year in the Netherlands and Belgium combined (population of 28 million). Despite the fact that almost all children achieve complete remission (CR), 30-40% of patients eventually relapse. Since survival after relapse is poor (35-40%), the overall survival (OS) of pediatric AML remains relatively low (~70%) compared to the increasing remission rates. The most effective strategy to improve the dismal outcome of AML patients is thus to prevent relapse. Over the last decades accumulating evidence is suggesting that AML develops in a hierarchical structure; originating from hematopoietic stem cells which are transformed to leukemia initiating cells, also referred to as leukemic stem cells (LSC). LSC possess self-renewal capacity and are more resistant to therapy. Therefore, LSC are supposed to be responsible for outgrowth of both the initial leukemia and the relapse. This holds true for adult AML, where the frequency of LSC at diagnosis has shown to be of importance for clinical outcome. However, while it has been shown that a high number of immature cells (CD34+/CD38-/CD45-/low) associates with an increased risk of relapse in pediatric AML, relatively little is known about the prognostic impact of LSC within this compartment. RAEB-t. Flow-cytometric LSC characterization was performed on either bone marrow (BM) or peripheral blood (PB) from AML patients collected at diagnosis. Purified white blood cells (WBC) were obtained after lysing the red blood cells using lysing solution (Pharm Lyse or FACSLysing, BD Biosciences) and were subsequently incubated with monoclonal antibody combinations and analyzed using an 8-color flow cytometer approach. LSC were defined as CD34+ CD38- cells with aberrant expression of CD123, CD7, CD56 or CD2. Results: Data were available from 103 patients and patient characteristics are listed in Table 1. In this cohort relapse-free survival (RFS) was 59.4%. Fortunately 56.4% of relapsed patients achieved a second CR and OS was 85.4%. LSC-load is defined as % of aberrant CD34+ CD38- cells within the CD34+ compartment. In our pediatric cohort, 17 patients (16.5%) had no expression of CD34 on blast cells at all (CD34null) and consequently no aberrant CD34+ CD38- LSC could be detected. Absence of CD34 has often been associated with good prognosis in literature. In our cohort relapse-rate seemed to follow this trend (4 out of the 17 CD34null patients (23.5%) vs. 34 out of the 83 CD34positive patients (41%)) but did not differ significantly (Plogrank = 0.196). ROC curve analysis showed that a cut-off of 17.2% LSC at diagnosis was associated with the occurrence of developing relapse with a specificity of 92%. Kaplan-Meier survival analysis, as depicted in Figure 1, showed a significant association between a high LSC-load and impaired RFS (34% relapses in LSClow vs. 64% relapses in LSChigh) (Plogrank= 0.027). Univariate analysis showed that next to LSC percentage, FLT3 mutation status and WBC count were significantly associated with RFS. After multivariate adjustment (taking into account cytogenetic risk groups and mutational status) LSC frequency was the only independent predictor of relapse or RFS (HR 2.3, 95% CI 1.1-4.8, p=0.032). Conclusion: Our results confirm data from adults and reveal that the frequency of LSC at diagnosis in pediatric AML patients can distinguish patients more likely to fail current treatment regimens as these patients develop significantly more relapses). Identifying this patient group creates opportunities for more personalized medicine and the development of therapies directed against LSC, sparing normal hematopoietic stem cells. Disclosures Kaspers: Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.

Description: Key Points FLT3-ITD-AR measurement based on RNA, but not DNA, is predictive for survival with a cutoff point of 0.5. FLT3-ITD-AR is associated with an ex vivo response to FLT3 inhibition with gilteritinib.