Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples

Abstract

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Suitability of melanoma FFPE samples for NGS libraries: time and quality thresholds for downstream molecular tests

“…Despite the tested use of FFPE tissue in NGS [6], further studies are required to check whether archival FFPE samples are suitable for NGS [7,8]. A recent study found that FFPE samples stored for more than 7 years were not worth being included in NGS studies [9].…”

Formalin-fixed paraffin-embedded sample conditions for deep next generation sequencing

“…Over-fixation has been reported to cause excessive crosslinking, which interferes with the extraction of nucleic acids and proteins for NGS analysis. 18 On the other hand, under-fixation can lead to the degradation of nucleic acid and protein, or a change in gene expression in poorly perfused areas where the formalin has not reached. 18 Furthermore, fixation with formalin needs to be performed immediately after removal of the specimen to avoid ischemic changes in the tissue.…”

“…18 On the other hand, under-fixation can lead to the degradation of nucleic acid and protein, or a change in gene expression in poorly perfused areas where the formalin has not reached. 18 Furthermore, fixation with formalin needs to be performed immediately after removal of the specimen to avoid ischemic changes in the tissue. 18 Here, we have shown that high-quality DNA can be obtained as long as an appropriate preservation procedure is performed following a standard protocol with NBF.…”

“…18 Furthermore, fixation with formalin needs to be performed immediately after removal of the specimen to avoid ischemic changes in the tissue. 18 Here, we have shown that high-quality DNA can be obtained as long as an appropriate preservation procedure is performed following a standard protocol with NBF.…”

“…17 Recently, it has been reported that the quality of FFPE samples varies depending on how surgical specimens have been prepared and preserved. 18 Therefore, it is extremely important that the next generation of surgeons who will be harvesting samples for NGS know how to appropriately handle samples for FFPE processing. Furthermore, it is important to clarify how much DNA can be harvested from a minimally sized sample, because often only a small amount of tissue is available in clinical settings such as from a biopsy or after neoadjuvant therapy.…”