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Updated: Sep 23, 2025
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
RNA-Based Targeted Gene Sequencing Improves the Diagnostic Yield of Mutant Detection in Chronic Myeloid Leukemia
Naranie Shanmuganathan1, Carol Wadham2, Daniel Thomson2
1Department of Haematology, Royal Adelaide Hospital and SA Pathology, Adelaide, South Australia, Australia; Department of Genetics and Molecular Pathology, Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia; Precision Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, South Australia, Australia; Clinical and Health Sciences, University of South Australia, Adelaide, South Australia, Australia; Adelaide Medical School, University of Adelaide, Adelaide, South Australia, Australia.
Abstract:
Mutation detection is increasingly used for the management of hematological malignancies. Prior whole transcriptome and whole exome sequencing studies using total RNA and DNA identified diverse mutation types in cancer-related genes associated with treatment failure in patients with chronic myeloid leukemia. Variants included single-nucleotide variants and small insertions/deletions, plus fusion transcripts and partial or whole gene deletions. The hypothesis that all of these mutation types could be detected by a single cost-effective hybridization capture next-generation sequencing method using total RNA was assessed. A method was developed that targeted 130 genes relevant for myeloid and lymphoid leukemia. Retrospective samples with 121 precharacterized variants were tested using total RNA and/or DNA. Concordance of detection of precharacterized variants using RNA or DNA was 96%, whereas the enhanced sensitivity identified additional variants. Comparison between 24 matched DNA and RNA samples demonstrated 95.3% of 170 variants detectable using DNA were detected using RNA, including all but one variant predicted to activate nonsense-mediated decay. RNA identified an additional 10 variants, including fusion transcripts. Furthermore, the true effect of splice variants on RNA splicing was only evident using RNA. In conclusion, capture sequencing using total RNA alone is suitable for detecting a range of variants relevant in chronic myeloid leukemia and may be more broadly applied to other hematological malignancies where diverse variant types define risk groups.
Insights
Total RNA sequencing effectively detects diverse mutations in chronic myeloid leukemia, including gene deletions and fusion transcripts. This cost-effective method aids in managing hematological malignancies and identifying patient risk groups.
Area of Science:
- Genomics
- Molecular Biology
- Hematology
Background:
- Mutation detection is crucial for managing hematological malignancies, with previous studies identifying various mutation types in chronic myeloid leukemia (CML) linked to treatment failure.
- Diverse variants, including single-nucleotide variants, small insertions/deletions, fusion transcripts, and gene deletions, have been identified.
Purpose of the Study:
- To assess if a single, cost-effective hybridization capture next-generation sequencing method using total RNA can detect all mutation types relevant to hematological malignancies.
- To develop and validate a method targeting 130 genes critical for myeloid and lymphoid leukemia.
Main Methods:
- A hybridization capture next-generation sequencing method was developed targeting 130 leukemia-relevant genes.
- Retrospective samples with 121 precharacterized variants were analyzed using total RNA and/or DNA.
- Matched DNA and RNA samples from 24 patients were compared for variant detection.
Main Results:
- The RNA-based method demonstrated 96% concordance with precharacterized variants detected by RNA or DNA, with enhanced sensitivity identifying additional variants.
- Comparison of matched DNA and RNA samples showed 95.3% of DNA-detectable variants were identified by RNA, including most variants affecting nonsense-mediated decay.
- RNA sequencing uniquely identified fusion transcripts and revealed the true impact of splice variants on RNA splicing.
Conclusions:
- Capture sequencing using total RNA alone is effective for detecting a wide spectrum of variants in chronic myeloid leukemia.
- This approach shows potential for broader application in other hematological malignancies for risk stratification based on diverse variant types.
