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Published on: April 11, 2016
Impact of the quality of resected thyroid cancer tissue sample on next-generation sequencing testing
Kanako C Hatanaka1, Kenichi Nakamura2, Ryohei Katoh3
1Center for Development of Advanced Diagnostics, Hokkaido University Hospital, Sapporo, Japan.
Abstract:
Activating rearranged during transfection (RET) proto-oncogene alterations can be identified using next-generation sequencing (NGS) of tumor DNA/RNA. We assessed factors associated with NGS (Oncomine Dx Target Test [ODxTT]) success for resected thyroid cancer (TC) specimens, including sample age, processing conditions, and DNA/RNA quality. TC samples were from three Japanese hospitals, with sample age <1-<10 years, fixative 10%/15% neutralized buffered formalin (NBF), and fixation time ≤48 h/>48 h-≤72 h. NGS success rate was defined as the percentage of samples returning validated NGS results (RET fusion-positive/negative [RNA] or RET mutation-positive/negative [DNA], detected using ODxTT). DNA/RNA quality was assessed with indexes based on electrophoresis (DNA/RNA integrity number, DV200 ) and quantitative polymerase chain reaction (DNA/RNA integrity score [ddCq/ΔCq]). NGS success rate (N = 202) was 90%/93% (DNA/RNA) overall, 98%-100% (DNA and RNA) for samples <3 years old, and 91% (DNA and RNA) for samples ≥3-<5 years old fixed in 10% NBF for ≤48 h. Multivariate logistic regression analysis identified ddCq and ΔCq as significant predictors of DNA and RNA NGS success rates, respectively. Quality assessment of nucleic acid extracted from archival tissue samples is important for achieving high NGS success rates in clinical practice, especially for samples ≥3 years old.
Insights
Next-generation sequencing (NGS) for thyroid cancer (TC) relies on nucleic acid quality. Older samples (≥3 years) require careful assessment to ensure high NGS success rates for detecting rearranged during transfection (RET) alterations.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- Activating rearranged during transfection (RET) proto-oncogene alterations are crucial in thyroid cancer (TC) pathogenesis.
- Next-generation sequencing (NGS) is a key method for identifying these RET alterations in tumor DNA/RNA.
- Assessing factors influencing NGS success is vital for reliable clinical diagnostics.
Purpose of the Study:
- To evaluate factors affecting the success rate of NGS for detecting RET alterations in resected TC specimens.
- To determine the impact of sample age, fixation conditions, and nucleic acid quality on NGS outcomes.
- To establish quality assessment criteria for archival TC samples undergoing NGS.
Main Methods:
- Analysis of 202 TC samples from three Japanese hospitals with varying storage durations (<1 to <10 years) and fixation parameters.
- Next-generation sequencing (NGS) performed using the Oncomine Dx Target Test (ODxTT) for DNA and RNA.
- Assessment of DNA/RNA quality using integrity numbers (DV200) and integrity scores (ddCq/ΔCq).
Main Results:
- Overall NGS success rates were 90% for DNA and 93% for RNA.
- Samples <3 years old showed high success rates (98%-100%), while samples ≥3-<5 years old had 91% success.
- Nucleic acid integrity scores (ddCq for DNA, ΔCq for RNA) were significant predictors of NGS success.
Conclusions:
- Nucleic acid quality assessment is critical for achieving high NGS success rates in clinical settings.
- Particular attention to quality is needed for archival TC samples aged 3 years or older.
- Validated NGS results for RET alterations depend on robust sample handling and quality control.
