Contributions
Abstract: PB1688
Type: Publication Only
Background
The genetic characterization of pediatric acute myeloid leukemia (AML) is an important outcome predictor. Genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of AML. High-Throughput Sequencing (HTS) provides a unique insight into mechanisms of disease initiation, risk stratification and treatment and multi-gene mutation panel is a part of diagnostic routine testing by now.
Aims
To perform expanded prospective molecular diagnosis in children with AML mainly concerning comprehensive risk assessment and stratification and to improve our understanding of mutational and clonal complexity in each patient because of the morphological and molecular heterogeneity of AML.
Methods
Genetic analyses of bone marrow (BM) samples from pediatric patients with newly diagnosed de novo AML recruited into the Russian National Prospective AML Study between September 2016 and February 2018 were performed by a combination of fragment analysis, Sanger sequencing for genes involved in risk stratification, and HTS with Human Myeloid Neoplasms Panel (HMNP) (Qiagen, Germany) for analysis of 141 genes most commonly mutated in myeloid neoplasm. Library was constructed for sequencing on a MiSeq using v3 chemistry with 2 x 150 bp read length.
Results
Diagnostic samples from 146 (75 male and 71 female) pediatric patients with de novo AML were analyzed. Age at diagnosis ranged from 0 to 17,6 years (median 6,6). An average depth of 978x was achieved. The total read fragments number was about 2 358 019 generated reads per sample with primer found on-target ~ 95.7%. Coding and splice site regions of FLT3, NPM1, KIT, CEBPA genes were fully covered, GATA1 gene had almost full coverage with the exception of exon 4. All SNV mutations in risk stratification genes were confirmed by Sanger sequencing. However indel variants such as FLT3-ITD (18 cases, size range - from 20 to 115) we accurately identified in all cases just only by fragment analysis, HTS analysis algorithms could only find the smallest of them - 20 and 22 bp.
Overall, 15% (22/146) of analyzed patients had normal karyotype, they showed molecular mutations in risk-stratification genes: 1(4,6%) patient had isolated FLT3-ITD, 4 (18%) had isolated NPM1mut, in 5 (22,7%) cases combination of NPM1mut+FLT3mut was observed and only 1 (4,6%) patient had biCEBPA. The numerous mutations were identified in additional genes: RUNX1, NRAS, KRAS, PTPN11, WT1, IDH1, IDH2, DNMT3A, ASXL1, ASXL2, STAG2. A highest mutational rate and their variable cooperation were observed in patients with normal karyotype and t(8;21). In 16 patients with t(8;21) KIT mutations were found in 10 (62,5%). AML with t(8;21) displayed also elevated mutational diversity: NRAS, RUNX1, ASXL2, EZH2, STAG2, NOTCH1, CSF3R, KDM6A, TP53, RAD21, JAK3, U2AF2.
Conclusion
Our results reflect the profile of pediatric AML cases in Russia. In our study the identification of subgroups defined by molecular aberrations, according to national diagnostic recommendations for specific treatment and assessment of molecular heterogeneity were performed. There were significant differences in gene mutations among morphological, cytogenetic and age groups. The nonrandom mutational cooperations were observed.
Identification of genetic subgroups is important for the molecular epidemiology and biology of AML worldwide.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, mutation analysis, Pediatric, Somatic mutation
Abstract: PB1688
Type: Publication Only
Background
The genetic characterization of pediatric acute myeloid leukemia (AML) is an important outcome predictor. Genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of AML. High-Throughput Sequencing (HTS) provides a unique insight into mechanisms of disease initiation, risk stratification and treatment and multi-gene mutation panel is a part of diagnostic routine testing by now.
Aims
To perform expanded prospective molecular diagnosis in children with AML mainly concerning comprehensive risk assessment and stratification and to improve our understanding of mutational and clonal complexity in each patient because of the morphological and molecular heterogeneity of AML.
Methods
Genetic analyses of bone marrow (BM) samples from pediatric patients with newly diagnosed de novo AML recruited into the Russian National Prospective AML Study between September 2016 and February 2018 were performed by a combination of fragment analysis, Sanger sequencing for genes involved in risk stratification, and HTS with Human Myeloid Neoplasms Panel (HMNP) (Qiagen, Germany) for analysis of 141 genes most commonly mutated in myeloid neoplasm. Library was constructed for sequencing on a MiSeq using v3 chemistry with 2 x 150 bp read length.
Results
Diagnostic samples from 146 (75 male and 71 female) pediatric patients with de novo AML were analyzed. Age at diagnosis ranged from 0 to 17,6 years (median 6,6). An average depth of 978x was achieved. The total read fragments number was about 2 358 019 generated reads per sample with primer found on-target ~ 95.7%. Coding and splice site regions of FLT3, NPM1, KIT, CEBPA genes were fully covered, GATA1 gene had almost full coverage with the exception of exon 4. All SNV mutations in risk stratification genes were confirmed by Sanger sequencing. However indel variants such as FLT3-ITD (18 cases, size range - from 20 to 115) we accurately identified in all cases just only by fragment analysis, HTS analysis algorithms could only find the smallest of them - 20 and 22 bp.
Overall, 15% (22/146) of analyzed patients had normal karyotype, they showed molecular mutations in risk-stratification genes: 1(4,6%) patient had isolated FLT3-ITD, 4 (18%) had isolated NPM1mut, in 5 (22,7%) cases combination of NPM1mut+FLT3mut was observed and only 1 (4,6%) patient had biCEBPA. The numerous mutations were identified in additional genes: RUNX1, NRAS, KRAS, PTPN11, WT1, IDH1, IDH2, DNMT3A, ASXL1, ASXL2, STAG2. A highest mutational rate and their variable cooperation were observed in patients with normal karyotype and t(8;21). In 16 patients with t(8;21) KIT mutations were found in 10 (62,5%). AML with t(8;21) displayed also elevated mutational diversity: NRAS, RUNX1, ASXL2, EZH2, STAG2, NOTCH1, CSF3R, KDM6A, TP53, RAD21, JAK3, U2AF2.
Conclusion
Our results reflect the profile of pediatric AML cases in Russia. In our study the identification of subgroups defined by molecular aberrations, according to national diagnostic recommendations for specific treatment and assessment of molecular heterogeneity were performed. There were significant differences in gene mutations among morphological, cytogenetic and age groups. The nonrandom mutational cooperations were observed.
Identification of genetic subgroups is important for the molecular epidemiology and biology of AML worldwide.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, mutation analysis, Pediatric, Somatic mutation