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Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Formalin-fixed paraffin-embedded sample conditions for deep next generation sequencing
Masayuki Nagahashi1, Yoshifumi Shimada1, Hiroshi Ichikawa1
1Division of Digestive and General Surgery, Niigata University Graduate School of Medical and Dental Sciences, Niigata City, Niigata, Japan.
Introduction:
Precision medicine is only possible in oncology practice if targetable genes in fragmented DNA, such as DNA from formalin-fixed paraffin-embedded (FFPE) samples, can be sequenced using next generation sequencing (NGS). The aim of this study was to examine the quality and quantity of DNA from FFPE cancerous tissue samples from surgically resected and biopsy specimens.
Methods:
DNA was extracted from unstained FFPE tissue sections prepared from surgically resected specimens of breast, colorectal and gastric cancer, and biopsy specimens of breast cancer. A total quantity of DNA ≥60 ng from a sample was considered adequate for NGS. The DNA quality was assessed by Q-ratios, with a Q-ratio >0.1 considered sufficient for NGS.
Results:
The Q-ratio for DNA from FFPE tissue processed with neutral-buffered formalin was significantly better than that processed with unbuffered formalin. All Q-ratios for DNA from breast, colorectal and gastric cancer samples indicated DNA levels sufficient for NGS. DNA extracted from gastric cancer FFPE samples prepared within the last 7 years is suitable for NGS analysis, whereas those older than 7 years may not be suitable. Our data suggested that adequate amounts of DNA can be extracted from FFPE samples, not only of surgically resected tissue but also of biopsy specimens.
Conclusions:
The type of formalin used for fixation and the time since FFPE sample preparation affect DNA quality. Sufficient amounts of DNA can be extracted from FFPE samples of both surgically resected and biopsy tissue, thus expanding the potential diagnostic uses of NGS in a clinical setting.
Insights
High-quality DNA suitable for next-generation sequencing (NGS) can be extracted from formalin-fixed paraffin-embedded (FFPE) cancerous tissue. This study confirms FFPE samples, including biopsies, yield sufficient DNA for precision medicine applications in oncology.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Precision medicine in oncology relies on sequencing targetable genes from fragmented DNA.
- Formalin-fixed paraffin-embedded (FFPE) samples are a common source of DNA but present challenges for sequencing.
- Next-generation sequencing (NGS) requires high-quality and sufficient quantity DNA for accurate analysis.
Purpose of the Study:
- To evaluate the quality and quantity of DNA extracted from FFPE cancerous tissue.
- To determine the suitability of DNA from FFPE samples for NGS applications in clinical oncology.
Main Methods:
- DNA was extracted from FFPE tissue sections of breast, colorectal, and gastric cancers (surgical and biopsy specimens).
- DNA quantity was assessed, with ≥60 ng considered adequate for NGS.
- DNA quality was determined using Q-ratios, with >0.1 deemed sufficient for NGS.
Main Results:
- Neutral-buffered formalin resulted in significantly better DNA Q-ratios compared to unbuffered formalin.
- All tested FFPE samples yielded sufficient DNA quantity and quality for NGS.
- Gastric cancer FFPE samples prepared within 7 years were suitable for NGS; older samples may not be.
Conclusions:
- Formalin type and sample age impact FFPE DNA quality for NGS.
- Adequate DNA can be extracted from both surgical and biopsy FFPE specimens.
- These findings support the expanded use of NGS in clinical oncology settings for FFPE samples.
