Cancer Res Treat.  2024 Apr;56(2):484-501. 10.4143/crt.2023.712.

Revolutionizing Non–Small Cell Lung Cancer Diagnosis: Ultra-High-Sensitive ctDNA Analysis for Detecting Hotspot Mutations with Long-term Stored Plasma

• Lee JY
• Jeon S
• Jun HR
• Sung CO
• Jang SJ
• Choi CM
• Chun SM

• KMID: 2554338
• DOI: http://doi.org/10.4143/crt.2023.712

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.

Keyword

Cell-free nucleic acids; Circulating tumor DNA; Ultra-high sensitive lung version 1; Semi-quantitation; Non-small cell lung carcinoma

Figure

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  Fig. 1. Performance of the ultra-high-sensitivity lung version 1 (ULV1) panel. (A) Diagrams illustrating the principles of ultrahigh sensitive polymerase chain reaction (PCR) applied to obtain Enriched variant allelic frequency (VAF) in the ULV1 panel compared with conventional PCR. (B) Direct comparison of ULV1 panel and conventional iPLEX sensitivity using serially diluted H1975 gDNA demonstrated stronger intensity of mutant signal with ULV1 panel (red arrow) compared to iPLEX in the same sample. Wild signal intensity indicated with black arrow. (C) Amplification efficiency and sensitivity of ULV1 panel. Assays had different cutoff values depending on multiplexing amplification efficiency. Red dashed line indicates the mean cutoff value. Blue and green dotted lines indicate the acceptable cutoff ranges. (D) Amplification curve shape of enriched VAF versus initial VAF. Initial and enriched VAFs of positive samples shown on the x- and y-axis, respectively. All assays showed enriched VAF saturation over 1% initial VAF.

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  Fig. 2. Validation of the ultra-high-sensitivity lung version 1 (ULV1) panel using 104 patient samples. (A) Mutational profiling and concordance between tumor tissue and matched plasma in 104 patients. The heatmap shows mutational concordance between tissue and plasma samples of each patient and between plasma methods. (B) Association of cell-free DNA (cfDNA) concentration with advanced cancer stage. (C) Comparison of epidermal growth factor receptor (EGFR) variant allelic frequency (VAF) between stages. High VAF of cfDNA is associated with advanced cancer. (D) Concordance rate comparison of EGFR hotspot mutations in tissue and matched plasma. Gray bars represent tissue EGFR hotspot mutations. Red and blue dotted lines show the concordance rate of EGFR hotspot mutations identified by ULV1 and CT-ULTRA compared to tissue. FFPE, formalin-fixed paraffin-embedded; ns, not significant; *p < 0.05, ***p < 0.001.

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  Fig. 3. Feasibility of semi-quantitative analysis. (A) Semi-quantitative detection range. The linear relation between the initial variant allelic frequency (VAF) and the enriched VAF identified by positive samples was in the range of 0.025%-1%. (B) Amplification curve shape between the CT-ULTRA VAF and the enriched VAF in patient samples. The CT-ULTRA VAF and the enriched VAF in each patient sample are shown on the x-axis and y-axis as a percentage. The saturation of enriched VAF was shown in patient samples with CT-ULTRA VAF of more than 1%. (C) Correlation between initial VAF and expected VAF obtained by CT-ULTRA and ultra-high-sensitivity lung version 1 (ULV1), respectively. Expected VAF for each epidermal growth factor receptor (EGFR) mutation was measured using the respective equations shown in Fig. 3A.

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  Fig. 4. Distribution of plasma storage duration between stages in the cohort. Plasma samples from patients with advanced cancer showed that the plasma storage time was relatively short.

Reference

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