Double CEBPA Mutations Are Not Associated With Favorable Clinical Outcome In Pediatric AML: A Report From The Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4942-4942
Author(s):  
Hidemasa Matsuo ◽  
Mio Kajihara ◽  
Daisuke Tomizawa ◽  
Tomoyuki Watanabe ◽  
Akiko Moriya Saito ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
Study Group ◽  
Prognostic Impact ◽  
Pediatric Leukemia ◽  
Favorable Prognosis ◽  
Pediatric Aml ◽  
Cebpa Mutations ◽  
Lymphoma Study Group

Abstract Background CCAAT/enhancer binding protein alpha (CEBPA) is a transcription factor that coordinates cellular differentiation. Mutations in the CEBPA genes are found in about 10% of patients with AML and are associated with favorable prognosis. However, recent data suggests that the favorable prognosis is restricted only to the patients with double CEBPA mutations and normal karyotype. These data have large implications for risk-stratified therapy and require confirmation. In this study, we investigated CEBPA mutation status and clinical outcome of the pediatric AML patients treated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 study. Patients & Methods AML-05 study, registered at http://www.umin.ac.jp/ctr/as UMIN000000511, is a nation-wide multi-institutional study for children (age <18 years) with de novo AML enrolled from 11/1/2006 to 12/31/2010. Cases with acute promyelocytic leukemia or Down syndrome were excluded. Patients were stratified by the specific cytogenetic characters and morphological treatment response into 3 risk groups; CBF-AML patients to the low risk (LR) group, those with either unfavorable cytogenetics (-7, 5q-, t(16;21)(p11;q22), Ph1, FLT3-ITD) or poor induction responders to the high risk (HR) group, and the rest to the intermediate risk (IR) group. In this trial, morphology was prospectively diagnosed by a central review system. Cytogenetic tests were carried out in regional laboratories, but the reports were reviewed centrally. FLT3-internal tandem duplication (ITD) was examined for all patients. Using the surplus RNA from the AML-05 study, CEBPA gene mutation was analyzed by RT-PCR and direct sequencing. Results Of the 317 evaluable patients, 73 patients (23.0%) had normal karyotype, 42 patients (13.2%) were FLT3-ITD positive, and 19 patients (6.0%) had both normal karyotype and FLT3-ITD. Among the 54 cases with normal karyotype and negative FLT3-ITD, 16 patients (29.6%) had a single CEBPA mutation and 17 patients (31.5%) had double or more (hereafter described as Double) CEBPA mutations. Between the CEBPA wild type (WT) and Double mutated patients, there were no statistically significant differences in relapse-free survival (RFS) (53.9% vs. 71.1%, P=0.27) nor overall survival (OS) (68.9% vs. 64.7%, P=0.57). An in-frame insertion of 6bp (ACCCGC) in CEBPAtrans-activator (TAD2) domain, resulting in a His-Pro duplication (HP196-197 ins), was detected in 131 patients (41.3% of all patients) in the current study, a considerably high incidence. However, this insertion was previously reported as polymorphism in adult AML (Leukemia 2008). When categorizing this insertion cases as CEBPA WT, 24 patients were CEBPA mutated among the 54 cases with normal karyotype and negative FLT3-ITD; 11 patients (20.4%) with single CEBPA mutation and 13 patients (24.1%) with Double CEBPA mutations. Although not statistically significant, there was a tendency of higher RFS (83.3% vs. 55.5%, P=0.20) and OS (79.1% vs. 63.3%, P=0.39) in patients with Double CEBPA mutations versus WT patients. Conclusions The current study is the first Japanese nation-wide investigation of the clinical significance of CEBPA mutations in pediatric AML. Our results suggest that CEBPA mutations have no prognostic impact on children with AML. Disclosures: No relevant conflicts of interest to declare.

Effectiveness of Intrathecal Liposomal Cytarabine in Treatment of Childhood Hematopoietic Malignancies – Experience of Polish Pediatric Leukemia/Lymphoma Study Group

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4240-4240
Author(s):  
Tomasz Szczepanski ◽  
Lidia Kajdas ◽  
Aneta Pobudejska-Pieniazek ◽  
Ninela Irga ◽  
Maciej Niedzwiecki ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Study Group ◽  
Liposomal Cytarabine ◽  
Pediatric Leukemia ◽  
Cns Disease ◽  
Hematopoietic Malignancies ◽  
Lymphoma Study Group ◽  
Hodgkin's Lymphomas

Abstract Abstract 4240 Introduction: Liposomal Cytarabine for intrathecal administration is characterized by prolonged activity and better penetration to central nervous system (CNS). This makes it promising medicine for treating children with hematopoietic malignancies relapsing in CNS or refractory CNS disease. The purpose of the Study: The study aimed at retrospective evaluation of the effectiveness of liposomal Cytarabine (Depocyte®) administrated intrathecally as a part of the treatment of hematopoietic malignancies in Polish children. Patients and methods: The study group consisted of 23 patients, 11 boys and 12 girls, treated in the centers of Polish Pediatric Leukemia/Lymphoma Study Group, including 18 patients with acute lymphoblastic leukemia (ALL), 3 patients with acute myeloid leukemia (AML) and two children with high grade Non-Hodgkin’s Lymphomas (NHL). The median age of the children was 10.8 years (range: 1.3 to 18 years). Liposomal cytarabine treatment was administered on compassionate basis to 20 children with relapsed acute leukemia / NHL, a single child with secondary leukemia, one patient with severe neurotoxicity after intrathecal Methotrexate during front-line treatment and in one child with large granulocytic sarcoma, penetrating into CNS. Thirteen patients received liposomal cytarabine dosage of 50 mg, while the remaining 10 children were exposed to the doses of 25–35 mg, all in association with prophylactic dexamethasone administration. The number of liposomal Cytarabine injections ranged from 1 to 11, mean 5 doses per patient. Results: The clearance of CNS disease was achieved in 15 of 23 patients (65%). Eight children were alive during the follow-up procedure, including 3 patients in complete remission after treatment completion. Grade IV neurotoxicity was observed in five children, which might be also partly related to CNS malignancy. Another side effects occurred in 4 patients, including headache, vertigo, paresthesias and seizures. Conclusion: Liposomal cytarabine administered intrathecally is effective treatment for CNS disease in children with relapsed acute leukemia/NHL with acceptable toxicity profile. Disclosures: No relevant conflicts of interest to declare.

scholarly journals FLT3, NPM1 and CEBPA Mutations in 195 Children with Acute Myeloid Leukemia in Argentina

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5318-5318
Author(s):  
Cristina N. Alonso ◽  
Patricia L. Rubio ◽  
Adriana Medina ◽  
Silvia Eandi Eberle ◽  
Andrea Bernasconi ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Normal Karyotype ◽  
Prognostic Impact ◽  
Sequencing Analysis ◽  
Rt Pcr ◽  
Genetic Abnormalities ◽  
Childhood Aml ◽  
The Mean ◽  
Cebpa Mutations

Abstract Background: Mutations of FLT3, NPM1 and CEBPA are found in 25 to 35% of adult-AML. These mutations correlate with outcome, especially in AML with normal karyotype. There are few reports concerning the incidence and prognostic significance of these mutations in childhood-AML and there is no data from Argentina. Objectives: To describe the prevalence of FLT3, NPM1 and CEBPA mutations and to analyze the prognostic impact in the outcome in our setting. Methods: Samples from 195 children treated with AML protocols were retrospectively analyzed. The mean age at diagnosis was 6.8 [0.0-17.9] years, including 65 patients younger than 2 years of age. FAB subtypes were M2: 18%, M3: 15%, M4: 12%, M5: 34%, M6: 3%, M7: 10%, while 16 cases (8%) disclosed an ambiguous lineage immunophenotype. Genetic abnormalities of AML cases were characterized by cytogenetic analysis (97%) and/or RT-PCR for AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF4, MLL-AF9, MLL-ENL and MLL-AF10 fusion transcripts (95%). The distribution of the genetic abnormalities was: AML1-ETO: 11%, PML-RARA: 15%, CBFB-MYH11: 6%, MLL/11q23: 23%, other abnormalities: 25% and normal karyotype: 16%. Detection of NPM1 and CEBPA mutations was performed by Gene-scanning; FLT3-ITD and FLT3-TKD were studied by RT-PCR and RFLP respectively. Positive cases were further characterized by sequencing analysis. Results: The prevalences of the studied mutations were: FLT3-ITD: 10.3%, FLT3-TKD: 8.2%, NPM1mut: 4.6% and CEBPAmut: 2.1%. Within the group of AML with normal karyotype the incidences were: FLT3-ITD: 12.5%, FLT3-TKD: 6.3%, NPM1mut: 25.0% and CEBPAmut: 12.5%. The mean age for each subgroup was: FLT3-ITD: 14 years, FLT3-TKD: 9 years, NPM1mut: 12 years and CEBPAmut: 12 years. Simultaneous presence of FLT3-ITD and NPM1 mutations was detected in 2 cases while 1 patient disclosed both FLT3-TKD and CEBPAmut. FLT3-ITD and FLT3-TKD showed significant association with the presence of PML-RARA (p<0.00001 and p=0.055 respectively). Eight out of nine patients with NPM1mut and 4/4 patients with CEBPAmut were AML with normal karyotype. The FAB subtypes more frequently observed for each subgroup were: FLT3-ITDmut: M3 (n:10/20; p<0.00001), FLT3-TKDmut: M5 (n:8/16; p=n.s.), NPM1mut: M2 (n:4/9; p=0.062) and CEBPAmut: M2 (n:3/4; p=0.019). The mean ages of patients with FLT3-ITDmut, NPM1mut and CEBPAmut were significantly higher (p<0.00001, p=0.006 and p=0.033, respectively). FLT3-TKD was the only mutation detected in 5/45 (11%) of patients younger than 1 year of age. The five-years leukemia-free survival probabilities (pLFS) and standard error (SE) were: Total AML: 49 (4)%, FLT3-ITDmut:68 (12)%, FLT3-TKDmut:46 (17)%, NPM1mut: 75 (15)%, CEBPAmut: 100 (0)% and NPM1mut/CEBPAmut/FLT3-ITDneg: 83 (15)% (p<0.00001). The pLFS (SE) of patients with normal karyotype and FLT3-ITDneg and NPM1mut or CEBPAmut was 88 (12)% (p=0.066). Conclusions: This is the first report of the frequencies of FLT3, NPM1 and CEBPA mutations in childhood AML in our country. The incidences of NPM1mut and CEBPAmut were significantly higher in AML with normal karyotype. Our data confirm the favorable prognosis of AML with NPM1mut/FLT3-ITDneg and CEBPAmut/FLT3-ITDneg genotypes, especially in cases with normal karyotype. The present results support the notion that this group should be considered as a new AML subset with better outcome. This group of AML patients with better outcome could be included in the standard risk group, thus avoiding intensive treatments and related toxicity. Disclosures No relevant conflicts of interest to declare.

The Prognostic Effect of High WT1 Gene Expression in Pediatric AML Depends on WT1 SNP rs16754 Status: A Report From the Children's Oncology Group (COG)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1444-1444
Author(s):  
Phoenix A. Ho ◽  
Todd A. Alonzo ◽  
Robert B. Gerbing ◽  
Julia Kuhn ◽  
Jessica A. Pollard ◽  
...  
Keyword(s):  
Clinical Outcome ◽  
Conflicts Of Interest ◽  
Small Sample ◽  
Survival Outcomes ◽  
Study Cohort ◽  
Prognostic Impact ◽  
Expression Levels ◽  
Molecular Abnormalities ◽  
Pediatric Study ◽  
Pediatric Aml

Abstract Abstract 1444 Background: WT1 is aberrantly over-expressed in the blast cells of most AML patients. Several adult AML groups have reported poor outcome in association with high WT1 expression. In contrast, in previously the largest pediatric study, COG investigators found no association between diagnostic WT1 expression and survival in 155 patients treated on the legacy POG-9421 trial. We recently demonstrated that the WT1 synonymous SNP rs16754 correlates with favorable outcome in childhood AML; further, SNP+ patients had higher median WT1 expression. In the present study we examine the clinical correlates of diagnostic WT1 expression within a contemporary COG trial, accounting for SNP rs16754 genotypes. Methods: WT1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 225 patients enrolled on the pediatric trial COG-AAML03P1. Expression levels were normalized against pooled normal bone marrow (NBM) controls. Direct sequencing of WT1 exon 7 was performed to determine SNP rs16754 genotype. WT1 expression was correlated with disease characteristics, SNP status, and clinical outcome. Results: WT1 expression varied over 4 log folds across the study cohort (range: 0.00 to 3148.27 fold NBM, median: 75.91). In 29 (13%) patients, WT1 expression was undetectable. These patients had distinct biologic characteristics compared to the remainder of the cohort, as they were predominantly FAB class M5 (56%, p<0.001) and the majority (52%, p<0.001) had 11q23 rearrangements. This group was also younger (median age 5.1 years), and rarely harbored favorable cytogenetics (Inv(16): 1 patient; t(8;21): 1 patient) or molecular abnormalities (FLT3/ ITD: 1 patient; NPM1- mutated: 1 patient; no patients with mutated CEBPA or WT1). Survival outcomes for this group did not differ significantly from the study cohort at large. WT1 expression higher than NBM was detected in 183/196 of the remaining patients. We grouped patients into WT1 expression quartiles (Q1-Q4) for comparison of clinical characteristics and outcome. FLT3/ ITD (23% vs. 9%, p=0.017) and WT1 mutations (22% vs. 4%, p<0.001) occurred more frequently in those within the highest quartile (Q4; WT1 expression >302.97 fold NBM) compared to the lower 3 quartiles. Infant patients were also less common in Q4 (p=0.010). WT1 SNP rs16754 was equally distributed among quartile groups. Survival outcomes did not differ significantly between the quartiles, confirming that diagnostic WT1 expression has no prognostic impact in contemporarily-treated pediatric AML patients, when all patients were considered. However, given our recent findings regarding the clinical relevance of the WT1 SNP, we examined the prognostic impact of WT1 expression in SNP+ and SNP- subgroups. Median WT1 expression for SNP+ and SNP- patients was 149.76 (range: 0 – 2104.44) vs. 74.40 (range: 0 – 3148.27) respectively (p=0.299). When restricting analysis to SNP rs16754 negative patients (n=150), those in the highest quartile had significantly worse 3-year overall survival (OS) from study entry (51% vs. 72%, p=0.034) and event-free survival (EFS, 35% vs. 54%, p=0.033) than those in lower quartiles. When comparing SNP- patients with higher vs. lower than median WT1 expression (Q3 + Q4 vs. Q1 + Q2), patients with higher expression again had inferior EFS (39% vs 59%, p=0.008) with a corresponding relapse rate from CR of 45% vs. 25% (p=0.011). Notably, similar comparisons in WT1 SNP+ patients with high vs. low WT1 expression did not demonstrate significant outcome differences. Surprisingly, SNP+ patients within the lowest expression quartile had the poorest outcome, while those in the highest quartile had no relapses from CR. Conclusion: WT1 expression levels vary across cyto-molecular subgroups of AML. WT1 expression was not prognostic when all AML patients were considered; however, stratifying patients by SNP rs16754 genotype revealed that high WT1 expression was significantly associated with poor clinical outcome in SNP- patients. Conversely, although the small sample size of SNP+ patients precluded significant comparisons, increasing WT1 expression trended towards improved outcome in the SNP+ group. The clinical and biologic significance of WT1 over-expression appears to differ depending on the presence or absence of SNP rs16754. WT1 expression must be considered in the context of SNP status, to determine which patients would most benefit from novel WT1 -targeted therapies. Disclosures: No relevant conflicts of interest to declare.

Prognostic Significance of CXCR4 Overexpression in Pediatric Acute Myeloid Leukemia with Low-Risk: A Report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3814-3814
Author(s):  
Hidemasa Matsuo ◽  
Naomi Nakamura ◽  
Daisuke Tomizawa ◽  
Akiko M. Saito ◽  
Nobutaka Kiyokawa ◽  
...  
Keyword(s):  
Prognostic Factor ◽  
Myeloid Leukemia ◽  
Adverse Outcome ◽  
Prognostic Significance ◽  
Study Group ◽  
Pediatric Leukemia ◽  
Adverse Prognostic Factor ◽  
Significant Difference ◽  
Pediatric Aml ◽  
Lymphoma Study Group

Abstract Background: The outcome of acute myeloid leukemia (AML) has been improved by advanced risk-stratified chemotherapy based on prognostic factors. However, the long-term survival rate remains 60-70% and further investigation is needed. Core binding factor (CBF)-AML, characterized by t(8;21) or inv(16)/t(16;16), is the most frequent subtype in pediatric AML. Although CBF-AML is generally classified as a low-risk (LR) group, approximately 30% of the patients relapsed within 3 years in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial. This finding suggests that the LR patients had other risk factors that could account for their adverse outcome. CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor that helps leukemia cells adhere to bone marrow stromal cells that affect the cell survival and proliferation. There are several reports that CXCR4 overexpression is an adverse prognostic factor in adult AML; however, few studies have examined their significance in pediatric AML. Here, we retrospectively examined whether CXCR4 overexpression can be an adverse prognostic factor in pediatric AML with LR. Patients and Methods: From 2006 to 2010, 485 de novo pediatric AML patients aged <18 years were registered in the AML-05 trial. Overall, 42 patients were excluded, mainly because of misdiagnosis. The risk groups were defined as follows: all of the CBF-AML patients with complete remission (CR) after the initial induction course without FLT3 -ITD were included in the LR group, while patients with -7, 5q-, t(16;21)(p11;q22), Ph1, or non-CR after the initial induction course or with FLT3-ITD were included in the high-risk (HR) group. Patients with no LR or HR features were in the intermediate-risk. CXCR4 mRNA expression was analyzed by quantitative RT-PCR using diagnostic bone marrow samples. CXCR4 expression levels were dichotomized based on the average expression level. Results and Discussion: A total of 248 samples were available for this study. No significant differences in the clinical outcomes were observed between these 248 patients and the 195 patients who did not have CXCR4 data (EFS, P =0.55; OS, P =0.87). CXCR4 overexpression (CXCR4 +) was present in 81 patients (32.7%). CXCR4 + was not correlated with age, sex, and WBC count. CBF-AML was similarly distributed between CXCR4 + and CXCR4 - patients. The frequency of FLT3 -ITD in CXCR4 + patients was significantly higher in CXCR4 - patients (P =0.049), while there were no significant differences in that of KIT and CEBPA mutation (P =0.08 and 0.71, respectively). In total (n=248), no significant differences were observed between CXCR4 + and CXCR4 - patients in 3-year OS (68.6% vs. 75.1%, P =0.42) and 3-year EFS (65.6% vs. 58.9%, P =0.27). However, in the LR group (n=93), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (79.2% vs. 98.3%, P =0.008), whereas no significant difference was observed in 3-year EFS (83.3% vs. 69.6%, P =0.33). We have revealed that KIT mutations are adverse prognostic factors in t(8;21) pediatric AML in our recent study (Leukemia, 2015). In t(8;21) AML without KIT mutations (n=44), a significant difference was observed between CXCR4 + and CXCR4 - patients in 3-year OS (76.1% vs. 100.0%, P =0.01), whereas no significant difference was observed in 3-year EFS (77.8% vs. 74.2%, P =0.85). Multivariate Cox regression analysis, including CXCR4 +, KIT mutation, and age identified CXCR4 + as the only prognostic factor predicting poor OS in the cohort of AML with LR (hazard ratio, 11.01; P =0.01), but not EFS (hazard ratio, 0.54; P =0.21). The results suggest that CXCR4 + is associated with an adverse prognosis in pediatric AML with LR. The adverse outcome was restricted in OS, not in EFS, suggesting that CXCR4 + may be associated with the poor prognosis after recurrence, although the underlying mechanism remains unknown. Furthermore, it is noteworthy that the OS of LR patients with CXCR4 - was quite high, which should be confirmed in larger studies. Conclusions: We identified the adverse prognostic significance of CXCR4 overexpression in pediatric AML with LR. The adverse prognosis may be restricted after recurrence. Further studies of the underlying biology are required. There are several promising CXCR4 antagonists including AMD3100, therefore, our findings will help to develop better therapeutic approaches in pediatric AML with LR. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adverse prognostic impact of KIT mutations in childhood CBF-AML: the results of the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial

Leukemia ◽  
2015 ◽  
Vol 29 (12) ◽  
pp. 2438-2441 ◽  
Author(s):  
M Tokumasu ◽  
C Murata ◽  
A Shimada ◽  
K Ohki ◽  
Y Hayashi ◽  
...  
Keyword(s):  
Study Group ◽  
Prognostic Impact ◽  
Pediatric Leukemia ◽  
Lymphoma Study Group

scholarly journals The Detection of Minor Clones with Somatic KIT D816V Mutations Using Droplet Digital PCR in Pediatric De Novo AML: AML-05 Trial from the Japanese Pediatric Leukemia/Lymphoma Study Group

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1419-1419
Author(s):  
Koji Sasaki ◽  
Yuri Uchiyama ◽  
Junji Ikeda ◽  
Masahiro Yoshitomi ◽  
Yuko Shimosato-Wada ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Gene Mutations ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Study Group ◽  
Pediatric Leukemia ◽  
The Mean ◽  
Pediatric Aml ◽  
Lymphoma Study Group ◽  
Kit D816v

Introduction Recent medical advances and development of comprehensive genetic understanding dramatically improve the clinical outcome of whole pediatric cancers, particularly in pediatric acute lymphoblastic lymphoma. However, approximately 50% of patients have disease relapse, and overall survival (OS) of pediatric acute myeloid leukemia (AML) is less than 70% as of the major therapeutic challenges. AML is caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. AML with RUNX1-RUNX1T1 gene fusions are generally classified as a low-risk group and resulted in favorable prognosis. However approximately 30% of the patients relapsed within 3 years. Conversely, KIT mutations were found in approximately 30% of AML cases with RUNX1-RUNX1T1 and thought to be a risk factor for relapse, particularly when occurring in D816V within KIT exon 17. Recently, droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, has been used to determine low-prevalence somatic mutations that were not detectable using Sanger sequencing. It shows the possibility that there are some of Pediatric AML cases which were not detected minor clones with somatic KIT mutation by using ordinary PCR. In this study, we explored KIT D816V mutations including the cases which are not detected by Sanger sequencing and found the prognosis of them by using Japanese pediatric AML cases. Methods We reanalyzed somatic KIT mutations (p.D816V) in the DNA extracted from 335 pediatric AML patients with RUNX1-RUNX1T1 who participated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial using ddPCR . In this trial, we conducted the tests as follows,: PCR mixture containing 10 μL 2x ddPCR Supermix for probes, 900 nM target-specific PCR primers, and 250 nM mutant-specific (FAM) and wild-type-specific (HEX) probes.20 µL of PCR mixture and 70 μL Droplet generation oil were mixed, and droplet generation was performed using a Bio-Rad QX100 Droplet Generator. The droplet emulsion was thermally cycled in the following conditions: denaturing at 95 °C for 10 min, 40 cycles of PCR at 94 °C for 30 s and at 57 °C for 2 min, and a final extension at 98 °C for 10 min. PCR amplification in the droplets was confirmed using Bio-Rad QX200 Droplet Reader. ThresholdThe threshold was determined by comparing the non-template ddPCR results. All the data were evaluated above the threshold. We also performed targeted gene mutation analysis of KIT in all patients using Sanger sequencing. Results We identified 24 KIT D816V mutations (7.2%) in the 335 pediatric AML. Variant allele frequency (VAF) was 0.1%-46.9%.It is noteworthy that 12 out of 24 KIT D816V mutations were undetected in our previous study using Sanger sequencing. Fourteen out of these 24 patients were AML with RUNX1-RUNX1T1, 5 cases with inv(16), and 5 cases with other alterations. Ten of the 14 RUNX1-RUNX1T1 (71%) patients were newly identified using ddPCR. Six of these 14 RUNX1-RUNX1T1 patients had relapsed, and D816V mutations were only detected using ddPCR in 4 of these 6 relapsed cases. The mean VAF of KIT D816V was 3.8% (0.1%-13.4%) in the 10 undetected patients with RUNX1-RUNX1T1. Two of the 5 patients with inv(16) were newly identified, and 1 had relapsed. All 5 cases with other alterations were already identified using Sanger sequencing. The mean VAF of KIT D816V in the 3 patients with inv(16) was 44.8% (42.7%-46.9%), detected using Sanger sequencing, whereas the mean VAF of KIT D816V was 8.6% (6.5% and 10.7%) in the undetected patients with inv(16). The mean VAF of KIT D816V with other alterations was 28.1% (16.2%-42.9%). Conclusion We identified 12 KIT D816V mutations using ddPCR that were undetected using Sanger sequencing. ddPCR may be useful for detecting accurate frequencies of mutations that were undetected using Sanger sequencing. Potential co-existing gene mutations may contribute different significance of leukemogenesis and relapse. Disclosures Ito: Asahi Kasei Pharma: Consultancy, Other: Grants; Astellas Pharma: Consultancy, Other: Grants; Teijin: Other: Grants; Novartis Pharma: Consultancy; Zenyaku Kogyo Co. Ltd: Consultancy; Chugai Pharma: Consultancy, Other: Grants; Japan Blood Products Organization: Other: Grants; Pfizer inc.: Other: Grants.

Outcome of Adolescent and Young Adults with Acute Myeloid Leukemia Treated with Pediatric Protocols: A Report from the 3 Japanese Cooperative Studies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 374-374 ◽  
Author(s):  
Daisuke Tomizawa ◽  
Tomoyuki Watanabe ◽  
Ryoji Hanada ◽  
Keizo Horibe ◽  
Yasuo Horikoshi ◽  
...  
Keyword(s):  
Young Adults ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
National Study ◽  
Study Group ◽  
Pediatric Leukemia ◽  
Cancer Study ◽  
Cancer Study Group ◽  
Adolescent And Young Adults ◽  
Pediatric Aml

Abstract BACKGROUND: There have been conflicting results from several large pediatric AML collaborative groups comparing survival of adolescent and young adults (AYAs) to that of younger patients with AML. We conducted a retrospective analysis using data from 3 Japanese pediatric AML studies; AML99 (2000-02), AML9805 (1998-2002), and AML-05 (2006-10), conducted by the Japanese Childhood AML Cooperative Study (consisted of Tokyo Children’s Cancer Study Group (TCCSG), Japan Association of Childhood Leukemia Study (JACLS), and Kyushu Yamaguchi Children’s Cancer Study Group (KYCCSG)), the Japanese Childhood Cancer and Leukemia Study Group (CCLSG), and the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG, the first Japanese national study group), respectively. PATIENTS & METHODS: Among the 782 patients with de novo AML (excluding acute promyelocytic leukemia and Down syndrome), 44 were AYAs (age ≥15 years), 574 were age 2 to 14 years, and 164 were infants (age <2 years) at diagnosis. Because of the underlying differences in biological characteristics, further analysis on event-free survival (EFS), overall survival (OS), cumulative incidence of relapse (RR) and treatment-related mortality (TRM) were compared between AYAs and patients 2 to 14 years old. RESULTS: 29.5% (13/44) of AYAs were t(8;21) (vs. 37.1% of 2 to 14 years old, P=0.315), 38.6% (17/44) were normal karyotype (vs. 22.0%, P=0.011), and 16.2% of the tested cases (6/37) were FLT3-ITD positive (vs. 9.4%, P=0.554). Complete remission (CR) rate after 2 induction courses was similar between AYAs and patients 2 to 14 years old: 88.6% vs. 88.8% (P=0.965). Five-year EFS rate was also similar between the two cohorts: 55.2% vs. 59.2% (P=0.578). However, 5-year OS rate was significantly poorer in AYAs: 54.7% vs 73.8% (P=0.005). Total 19 events were observed among the 44 AYA patients; 4 failures to achieve CR, 12 relapses, and 3 deaths. Fourteen out of the 16 patients with non-CR or relapse eventually died and 8 deaths were treatment-related. In fact, 5-year RR was similar between the 2 age groups (32.7% vs. 30.1%, P=0.819), but TRM was significantly higher in AYAs (33.2% vs. 12.4%, P=0.001). In multivariate analysis, using Cox proportional hazard regression model, age ≥15 years old at diagnosis, absence of low risk cytogenetics (t(8;21) or inv(16)), and no remission after initial induction course were associated with both poorer OS and higher TRM, while high white cell count at diagnosis (≥ 100,000/μL) and presence of high risk cytogenetics (-7, 5q-, t(16;21)(p11:q22)/FUS-ERG, Philadelphia chromosome, and/or FLT3-ITD) were not siginificant. CONCLUSIONS: AYAs with AML had inferior OS, but not EFS, due to higher incidence of TRM especially after failure of initial treatment. Disclosures No relevant conflicts of interest to declare.

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