Cancer Res Treat.  2022 Jan;54(1):40-53. 10.4143/crt.2021.151.

Targeted Liquid Biopsy Using Irradiation to Facilitate the Release of Cell-Free DNA from a Spatially Aimed Tumor Tissue

• Noh JM
• Kim YJ
• Lee HY
• Choi C
• Ahn WG
• Lee T
• Pyo H
• Park JH
• Park D
• Park WY

• KMID: 2524586
• DOI: http://doi.org/10.4143/crt.2021.151

Abstract

Purpose
We investigated the feasibility of using an anatomically localized, target-enriched liquid biopsy (TLB) in mouse models of lung cancer.
Materials and Methods
After irradiating xenograft mouse with human lung cancer cell lines, H1299 (NRAS proto-oncogene, GTPase [NRAS] Q61K) and HCC827 (epidermal growth factor receptor [EGFR] E746-750del), circulating (cell-free) tumor DNA (ctDNA) levels were monitored with quantitative polymerase chain reaction on human long interspersed nuclear element-1 and cell line-specific mutations. We checked dose-dependency at 6, 12, or 18 Gy to each tumor-bearing mouse leg using 6-MV photon beams. We also analyzed ctDNA of lung cancer patients by LiquidSCAN, a targeted deep sequencing to validated the clinical performances of TLB method.
Results
Irradiation could enhance the detection sensitivity of NRAS Q61K in the plasma sample of H1299-xenograft mouse to 4.5- fold. While cell-free DNA (cfDNA) level was not changed at 6 Gy, ctDNA level was increased upon irradiation. Using double-xenograft mouse with H1299 and HCC827, ctDNA polymerase chain reaction analysis with local irradiation in each region could specify mutation type matched to transplanted cell types, proposing an anatomically localized, TLB. Furthermore, when we performed targeted deep sequencing of cfDNA to monitor ctDNA level in 11 patients with lung cancer who underwent radiotherapy, the average ctDNA level was increased within a week after the start of radiotherapy.
Conclusion
TLB using irradiation could temporarily amplify ctDNA release in xenograft mouse and lung cancer patients, which enables us to develop theragnostic method for cancer patients with accurate ctDNA detection.

Keyword

Radiotherapy; Cell-free DNA; Liquid biopsy; Lung neoplasms

Figure

• 

  Fig. 1 Schematic view of the study design. In xenograft mouse model using human lung cancer lines; H460, H1299 (ras-mutant), H1975 and HCC827 (epidermal growth factor receptor [EGFR]-mutant), we estimated the effect of irradiation on circulating (cell-free) tumor DNA (ctDNA) level using human long interspersed nuclear element-1 quantitative polymerase chain reaction (qPCR) and mutation-specific droplet digital polymerase chain reaction (ddPCR). Irradiation of 6, 12, and 18 Gy was delivered on the tumor-bearing leg using 6-MV photon beams. Two-tumor xenograft model was developed inoculating H1299 and HCC827 to different legs. Then, prospective study for patients undergoing definitive radiotherapy (RT) with or without histologic diagnosis were launched, where targeted deep sequencing was performed to analyze ctDNA. NSCLC, non–small cell lung cancer.

• 

  Fig. 2 Circulating human long interspersed nuclear element-1 (hLINE-1) DNA and tumor growth of various tumor models. (A) The concentration of plasma hLINE-1 correlated with the NRAS proto-oncogene, GTPase (NRAS) Q61K mutation in the H1299 xenograft model and the epidermal growth factor receptor (EGFR) E746-A750del mutation in the HCC827 xenograft model. (B) The tumor growth rate and concentration of plasma hLINE1 according to tumor volumes. Data represent means±standard error of mean (n=5).

• 

  Fig. 3 Target-specific liquid biopsy using irradiation in xenograft mouse model. (A) The concentration of plasma human long interspersed nuclear element-1 (hLINE-1) in various tumor models before and after irradiation. (B) Three different doses (6, 12, and 18 Gy) and time points after irradiation (6, 18, and 24 hours) were examined. (C) Tumor-specific mutations, NRAS mutation in the H1299 tumor model and EGFR mutation in the HCC827 tumor model, increased after irradiation in tumor-bearing mice. Data represent means±stasndard error of mean (SEM) (n=5).(D) When irradiating either the H1299 or HCC827-bearing leg in the two-tumor model, an increase in the target-specific mutation was observed. Data represent means±SEM (n=5). cfDNA, cell-free DNA; ctDNA, circulating (cell-free) tumor DNA; EGFR, epidermal growth factor receptor; NRAS, NRAS proto-oncogene, GTPase; RT, radiotherapy. *p < 0.05 compared with non-irradiation groups.

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  Fig. 4 Modulation of circulating (cell-free) tumor DNA (ctDNA) release by the tumor microenvironment. RNA sequencing (RNA-seq) transcriptome analysis of both tumor tissues in the two-tumor mouse model was performed. (A) Sequenced reads were mapped separately to the human and mouse genomes to delineate tumor (human) and host (mouse) gene expression. (B) Principal component analysis plots of the RNA-seq data show the characteristics of samples according to gene expression levels. Each dot indicates a sample. RT, radiotherapy. (C) Heat map of the transcriptome analysis for host genes correlated with ctDNA levels. Analysis of the varying cell-type proportions in the bulk data using a deconvolution method from the host gene. (D) Changes in cell fractions after irradiation. (E) Correlation between the amount of ctDNA and the cell-type proportions by deconvolution in individual xenograft two-tumor model mice. NK, natural killer. (F) Immunohistochemical analysis of paraffin-embedded tumor tissues using F4/80. F4/80 is a cell surface protein and known marker of mouse macrophage populations.

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  Fig. 5 Circulating (cell-free) tumor DNA (ctDNA) analysis after irradiation in patients with lung cancer. The ctDNA levels estimated by targeted deep sequencing are plotted on the left y-axis for patients with non–small cell lung cancer before and during radiotherapy. (A) Increase in ctDNA after radiotherapy for a patient with squamous cell carcinoma of the lung. (B) An increase was also observed in a patient with clinically diagnosed lung cancer without a histologic diagnosis. (C) Relative ctDNA levels of 11 patients with non–small cell lung cancer during radiotherapy (mean±standard error of mean). (D) Frequency of patients with increasing ctDNA levels over time after radiation therapy. Responders, patients with an increase in their ctDNA levels; non-responders, patients without an increase in their ctDNA levels. cfDNA, cell-free DNA.

Reference

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