Contributions
Abstract: PB1654
Type: Publication Only
Background
With frequency of 11-30% the NRAS mutations represent one of the most frequently reported mutations in patients with de novo acute myeloid leukemia (AML). Point mutations of the gene are heterozygous and exclusively affect glycine hotspot codons 12, 13 (exon 2) and 61 (exon 3). In some AML patients more than one NRAS mutation can be identified. The exact number of patients with multiple point mutations is unclear as well as it is not known whether the multiple point mutations are localised on the same allele of one clone or are carried by separate clones.
Aims
To describe the frequency and type of NRAS mutations and to analyse the polyclonality of mutations in selected patients.
Methods
NGS libraries were prepared from peripheral blood samples from the time of diagnosis of 258 consecutive de novo AML patients (median age 54 years; range 19-72) by ClearSeq AML panel (Agilent Technologies) and sequenced on NextSeq machines (Illumina). NRAS positive samples with a variant allele frequency (VAF) ≥ 1% according to NGS were verified by NRAS StripAssay (ViennaLab). The clonal background of multiple point NRAS mutations was analysed using TA Cloning Kit (Thermo Fisher Scientific). Sanger sequencing was used to analyse bacterial clones.
Results
In the studied cohort of AML patients the presence of NRAS mutation was identified in 58/258 (22.5%) patients analysed by NGS method. NRAS positivity was confirmed in all samples (58/58, 100%) by hybridization strips. One mutation was detected in 42/58 patients (72.4%), in remaining 16/58 (27.6%) patients multiple point mutations were revealed. Two point mutations were detected in 12/58 (20.7%), 3 mutations in 3/58 (5.2%) and 4 mutations in 1/58 (1.7%) patients. In total, 13 different gene aberrations were identified. The most frequent mutations were G12D (23/58 patients; 39.7%), G12S (14/58; 24.1%) and G13D (12/58; 20.7%). The polyclonality of NRAS mutations was analysed in two patients. First patient carried three mutations (G12D, G12S, G13D) in two different codons, second patient carried two mutations (G13D, Q61H) in two different exons. Analysis of 111 bacterial clones of the first patient and 112 clones of the second patient did not reveal any double-locus mutants with two mutations in the same allele. Each of the identified mutations was separate in different alleles indicating the presence of more clones.
Conclusion
More than one fifth of AML patients carry somatic missense mutation in NRAS gene and approximately a quarter of them has multiple point mutations. Our results show that each of the multiple point mutations is present separately in the allele and thus likely forms independent clone.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Clonality, mutation analysis
Abstract: PB1654
Type: Publication Only
Background
With frequency of 11-30% the NRAS mutations represent one of the most frequently reported mutations in patients with de novo acute myeloid leukemia (AML). Point mutations of the gene are heterozygous and exclusively affect glycine hotspot codons 12, 13 (exon 2) and 61 (exon 3). In some AML patients more than one NRAS mutation can be identified. The exact number of patients with multiple point mutations is unclear as well as it is not known whether the multiple point mutations are localised on the same allele of one clone or are carried by separate clones.
Aims
To describe the frequency and type of NRAS mutations and to analyse the polyclonality of mutations in selected patients.
Methods
NGS libraries were prepared from peripheral blood samples from the time of diagnosis of 258 consecutive de novo AML patients (median age 54 years; range 19-72) by ClearSeq AML panel (Agilent Technologies) and sequenced on NextSeq machines (Illumina). NRAS positive samples with a variant allele frequency (VAF) ≥ 1% according to NGS were verified by NRAS StripAssay (ViennaLab). The clonal background of multiple point NRAS mutations was analysed using TA Cloning Kit (Thermo Fisher Scientific). Sanger sequencing was used to analyse bacterial clones.
Results
In the studied cohort of AML patients the presence of NRAS mutation was identified in 58/258 (22.5%) patients analysed by NGS method. NRAS positivity was confirmed in all samples (58/58, 100%) by hybridization strips. One mutation was detected in 42/58 patients (72.4%), in remaining 16/58 (27.6%) patients multiple point mutations were revealed. Two point mutations were detected in 12/58 (20.7%), 3 mutations in 3/58 (5.2%) and 4 mutations in 1/58 (1.7%) patients. In total, 13 different gene aberrations were identified. The most frequent mutations were G12D (23/58 patients; 39.7%), G12S (14/58; 24.1%) and G13D (12/58; 20.7%). The polyclonality of NRAS mutations was analysed in two patients. First patient carried three mutations (G12D, G12S, G13D) in two different codons, second patient carried two mutations (G13D, Q61H) in two different exons. Analysis of 111 bacterial clones of the first patient and 112 clones of the second patient did not reveal any double-locus mutants with two mutations in the same allele. Each of the identified mutations was separate in different alleles indicating the presence of more clones.
Conclusion
More than one fifth of AML patients carry somatic missense mutation in NRAS gene and approximately a quarter of them has multiple point mutations. Our results show that each of the multiple point mutations is present separately in the allele and thus likely forms independent clone.
Session topic: 3. Acute myeloid leukemia - Biology & Translational Research
Keyword(s): Acute Myeloid Leukemia, Clonality, mutation analysis