Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.
Lab Invest
; 104(1): 100280, 2024 01.
Article
em En
| MEDLINE
| ID: mdl-38345263
ABSTRACT
Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Artefatos
Tipo de estudo:
Guideline
/
Prognostic_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article