Alternative protocols of tissue fixation dramatically reduces the impact of DNA artifacts, unraveling the interpretation of clinical Comprehensive Genomic Profiling

Abstract

ABSTRACTFormalin-fixed-paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing (NGS) 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 (CGP) analysis is required. 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: i) neutral buffered formalin (NBF); ii) acid-deprived formalin fixation (ADF); iii) pre-cooled ADF (coldADF); iv) Glyoxal Acid-Free (GAF). DNA extraction, fragmentation analysis and sequencing by using two large NGS panels (OCAv3, TSO500) followed. We comprehensively analyzed library and sequencing QCs, and the quality of sequencing results.Libraries fromcoldADF samples showed significantly longer reads than the others with both panels. ADF andcoldADF 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 TSO500 results, which also highlighted the best performance in terms of total region sequenced for ADF andcoldADF samples. NBF samples had a significantly smaller region sequenced, displayed a significantly lower number of evaluable MS loci and a significant increase in SNVs compared to other protocols. The mutational Signature 1 (aging-, FFPE artifact-related) showed the highest (37%) and the lowest (17%) values in NBF andcoldADF samples, respectively.Most of identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: four discordant results involved NBF samples.In conclusion, acid-deprived fixatives guarantee the highest DNA preservation/sequencing performance, suggesting possible implementation of even more complex molecular profiling on tissue samples.