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.

Keywords: DNA amplifiability; DNA extraction; formalin-fixed and paraffin-embedded tumor tissue (FFPE); next-generation sequencing (NGS); pre-analytics.

Figure 1

(A) DNA yield (ng/mm² tissue) from different labs. QIAamp FFPE kit (QIAGEN Cat# 56404, Hilden, Germany) was used by Labs A, C, D. RecoverAll (ThermoFisher Cat# AM1975, Waltham, MA, USA) was used by Lab B; (B) DNA amplifiability from different labs. Amplifiability values have been converted to the same log 2 scale (C_t value). Values across labs are not comparable due to lack of common reference DNA. Values from the same lab correlate with the quality of FFPE.

Figure 2

Amplifiability of DNA extracted by different labs measured by Asuragen Quantitative Functional Index (QFI) (A) and RNase P (B) assays. S12 from Lab A was run out and quality control (QC) data is not available. Missing data for S11 is due to poor FFPE block quality and high fat content of the breast tissue. CRC: colorectal cancer.