RESUMO
BACKGROUND: Clinical specimens undergoing diagnostic molecular pathology testing are fixed in formalin due to the necessity for detailed morphological assessment. However, formalin fixation can cause major issues with molecular testing, as it causes DNA damage such as fragmentation and non-reproducible sequencing artefacts after PCR amplification. In the context of massively parallel sequencing (MPS), distinguishing true low frequency variants from sequencing artefacts remains challenging. The prevalence of formalin-induced DNA damage and its impact on molecular testing and clinical genomics remains poorly understood. METHODS: The Cancer 2015 study is a population-based cancer cohort used to assess the feasibility of mutational screening using MPS in cancer patients from Victoria, Australia. While blocks were formalin-fixed and paraffin-embedded in different anatomical pathology laboratories, they were centrally extracted for DNA utilising the same protocol, and run through the same MPS platform (Illumina TruSeq Amplicon Cancer Panel). The sequencing artefacts in the 1-10% and the 10-25% allele frequency ranges were assessed in 488 formalin-fixed tumours from the pilot phase of the Cancer 2015 cohort. All blocks were less than 2.5 years of age (mean 93 days). RESULTS: Consistent with the signature of DNA damage due to formalin fixation, many formalin-fixed samples displayed disproportionate levels of C>T/G>A changes in the 1-10% allele frequency range. Artefacts were less apparent in the 10-25% allele frequency range. Significantly, changes were inversely correlated with coverage indicating high levels of sequencing artefacts were associated with samples with low amounts of available amplifiable template due to fragmentation. The degree of fragmentation and sequencing artefacts differed between blocks sourced from different anatomical pathology laboratories. In a limited validation of potentially actionable low frequency mutations, a NRAS G12D mutation in a melanoma was shown to be a false positive. CONCLUSIONS: These findings indicate that DNA damage following formalin fixation remains a major challenge in laboratories working with MPS. Methodologies that assess, minimise or remove formalin-induced DNA damaged templates as part of MPS protocols will aid in the interpretation of genomic results leading to better patient outcomes.
Assuntos
Artefatos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Neoplasias/genética , Fixação de Tecidos , Linhagem Celular Tumoral , Fragmentação do DNA , Reações Falso-Positivas , Formaldeído , Humanos , Inclusão em Parafina , Reação em Cadeia da Polimerase , Estudos Prospectivos , Análise de Sequência de DNA , Moldes Genéticos , Uracila-DNA Glicosidase/metabolismoAssuntos
Análise Mutacional de DNA/métodos , Leucemia Mieloide Aguda/genética , Mutação , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Domínio Catalítico/genética , Éxons/genética , Feminino , Humanos , Lactente , Recém-Nascido , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Mutação/fisiologia , Desnaturação de Ácido Nucleico/genética , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Fatores de Tempo , Tirosina Quinase 3 Semelhante a fms/análise , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
BACKGROUND: Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45-60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples. RESULTS: We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. CONCLUSION: HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases.
Assuntos
Testes Genéticos/métodos , Mutação , Proteínas Nucleares/genética , Tirosina Quinase 3 Semelhante a fms/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Duplicação Gênica , Testes Genéticos/economia , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Fatores de Tempo , Adulto JovemRESUMO
The point mutation 1849 (GT) V617F in the JAK2 gene occurs at high frequency in several chronic myeloproliferative diseases. Although a number of V617F mutation detection methods have been described, few are readily implemented in a diagnostic setting. We developed a simple and sensitive allelespecific competitive blocker polymerase chain reaction (ACB-PCR) assay to detect the V617F mutation. DNA was extracted from peripheral whole blood samples of 26 patients with chronic myeloproliferative disease. The ACB-PCR limit of detection was 1%. All positive samples detected by sequencing were detected by ACB-PCR. In 3 patients with essential thrombocythemia, the V617F mutation was readily detected by ACB-PCR but was near the detection limit of sequencing, confirming that ACB-PCR is more effective at detecting V617F when the mutant cell population is low. Detection of the monomorphic JAK2 V617F mutation using the ACB-PCR assay is easy to perform, rapid, sensitive, and cost-effective, which are key features of an ideal diagnostic method.