Revolutionizing Non–Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
Purpose Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non–small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). Materials and Methods Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. Results The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). Conclusion Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.
Abstract
Purpose Circulating cell-free DNA (cfDNA) has great potential in clinical oncology. The prognostic and predictive values of cfDNA in non–small cell lung cancer (NSCLC) have been reported, with epidermal growth factor receptor (EGFR), KRAS, and BRAF mutations in tumor-derived cfDNAs acting as biomarkers during the early stages of tumor progression and recurrence. However, extremely low tumor-derived DNA rates hinder cfDNA application. We developed an ultra-high-sensitivity lung version 1 (ULV1) panel targeting BRAF, KRAS, and EGFR hotspot mutations using small amounts of cfDNA, allowing for semi-quantitative analysis with excellent limit-of-detection (0.05%). Materials and Methods Mutation analysis was performed on cfDNAs extracted from the plasma of 104 patients with NSCLC by using the ULV1 panel and targeted next-generation sequencing (CT-ULTRA), followed by comparison analysis of mutation patterns previously screened using matched tumor tissue DNA. Results The ULV1 panel demonstrated robust selective amplification of mutant alleles, enabling the detection of mutations with a high degree of analytical sensitivity (limit-of-detection, 0.025%-0.1%) and specificity (87.9%-100%). Applying ULV1 to NSCLC cfDNA revealed 51.1% (23/45) samples with EGFR mutations, increasing with tumor stage: 8.33% (stage I) to 78.26% (stage IV). Semi-quantitative analysis proved effective for low-mutation-fraction clinical samples. Comparative analysis with PANAMutyper EGFR exhibited substantial concordance (κ=0.84). Conclusion Good detection sensitivity (~80%) was observed despite the limited volume (1 mL) and long-term storage (12-50 months) of plasma used and is expected to increase with high cfDNA inputs. Thus, the ULV1 panel is a fast and cost-effective method for early diagnosis, treatment selection, and clinical follow-up of patients with NSCLC.
참고문헌 (19)
- Lynch TJ, "Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib", N Engl J Med, 350, pp.2129~2139 (2004)
- Yuan M, "The emerging treatment landscape of targeted therapy in non-small-cell lung cancer", Signal Transduct Target Ther, 4, pp.61~ (2019)
- Wang S, "Third-generation inhibitors targeting EGFR T790M mutation in advanced non-small cell lung cancer", J Hematol Oncol, 9, pp.34~ (2016)
- Wang S, "EGFR C797S mutation mediates resistance to third-generation inhibitors in T790M-positive non-small cell lung cancer", J Hematol Oncol, 9, pp.59~ (2016)
- Taus A, "Dynamics of EGFR mutation load in plasma for prediction of treatment response and disease progression in patients with EGFR-mutant lung adenocarcinoma", Clin Lung Cancer, 19, pp.387~394 (2018)
- Zhang JT, "Longitudinal undetectable molecular residual disease defines potentially cured population in localized non-small cell lung cancer", Cancer Discov, 12, pp.1690~1701 (2022)
- Russano M, "Liquid biopsy and tumor heterogeneity in metastatic solid tumors : the potentiality of blood samples", J Exp Clin Cancer Res, 39, pp.95~ (2020)
- Bronkhorst AJ, "The emerging role of cell-free DNA as a molecular marker for cancer management", Biomol Detect Quantif, 17, pp.100087~ (2019)
- Yi X, "The feasibility of using mutation detection in ctDNA to assess tumor dynamics", Int J Cancer, 140, pp.2642~2647 (2017)
- Corcoran RB, "Application of cell-free DNA analysis to cancer treatment", N Engl J Med, 379, pp.1754~1765 (2018)
- Shin SJ, "Feasibility of multiplexed gene mutation detection in plasma samples of colorectal cancer patients by mass spectrometric genotyping", PLoS One, 12, pp.e0176340~ (2017)
- Elazezy M, "Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management", Comput Struct Biotechnol J, 16, pp.370~378 (2018)
- Sozzi G, "Effects of prolonged storage of whole plasma or isolated plasma DNA on the results of circulating DNA quantification assays", J Natl Cancer Inst, 97, pp.1848~1850 (2005)
- Jahangiri L, "Assessing the concordance of genomic alterations between circulating-free DNA and tumour tissue in cancer patients", Cancers(Basel), 11, pp.1938~ (2019)
- Guo Q, "Heterogeneous mutation pattern in tumor tissue and circulating tumor DNA warrants parallel NGS panel testing", Mol Cancer, 17, pp.131~ (2018)
- Jiang J, "Concordance of genomic alterations by next-generation sequencing in tumor tissue versus cell-free DNA in stage I-IV non-small cell lung cancer", J Mol Diagn, 22, pp.228~235 (2020)
- Bettegowda C, "Detection of circulating tumor DNA in early-and latestage human malignancies", Sci Transl Med, 6, pp.224ra24~ (2014)
- Kujala J, "Circulating cell-free DNA reflects the clonal evolution of breast cancer tumors", Cancers(Basel), 14, pp.1332~ (2022)
- Kujala J, "High mutation burden of circulating cellfree DNA in early-stage breast cancer patients is associated with a poor relapse-free survival", Cancer Med, 9, pp.5922~5931 (2020)