Jae Myoung Noh and Yeon Jeong Kim contributed equally to this work.
We investigated the feasibility of using an anatomically localized, target-enriched liquid biopsy (TLB) in mouse models of lung cancer.
After irradiating xenograft mouse with human lung cancer cell lines, H1299 (NRAS proto-oncogene, GTPase [
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.
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.
Tumors are spatially heterogenous and temporally dynamic, with differently evolving genetic clones responsible for disease progression over time. Next-generation sequencing technology can capture intratumor genomic heterogeneity through deep-sequencing of multiple biopsies taken at different times and regions. Characterizing complex intratumor heterogeneity has yielded important insights for therapeutic target selection and drug development. However, characterizing intratumor heterogeneity at a sufficient resolution remains difficult, primarily because of the high risk caused by multiple and repetitive biopsies.
Recently, analyzing circulating (cell-free) tumor DNA (ctDNA) has been recognized as a powerful, real-time approach to comprehensive genetic profiling of temporally dynamic diseases. Because it removes the need for repeated invasive procedures [
There is unfortunately no practical way to control or modulate the release of cell-free DNA (cfDNA) in a region of interest, but its release from normal cells and tumor cells into blood is at least partly understood. Because cfDNA is known to be released upon cell death via apoptosis or necrosis, we hypothesized that the induction of tumor cell death in an anatomically defined region would facilitate ctDNA release and thus enable a liquid biopsy enriched with ctDNA released from the target region. To test that hypothesis, we used radiotherapy (RT) as a modality to induce tumor cell death because RT can be delivered to a well-defined anatomical target region. From the perspective of radiation oncology, personalized radiotherapeutic approaches based on biomarkers are now under development, and ctDNA could be a biomarker for radiation response, the detection of minimal/molecular residual disease (MRD), and the early detection of tumor recurrence [
In this study, we investigated whether RT could enhance the detection sensitivity of ctDNA in mouse models of lung cancer, and we determined the feasibility of anatomically localized, target-enriched liquid biopsy (TLB) using a double-xenograft mouse model. In addition, we evaluated cancer tissue transcripts from animal models to find changes in the cell composition of the tumor microenvironment that we hoped would allow us to discover the mechanism by which ctDNA release was modulated. Furthermore, increases in ctDNA levels were examined in longitudinal plasma samples from lung cancer patients before, during, and after RT.
BALB/c mice (male, 5–6 weeks old) were purchased from Orient Bio (Seongnam, Korea).
Four types of human non–small cell lung cancer (NSCLC) cell lines, H460, H1299 (Ras mutant), H1975, and HCC827 (epidermal growth factor receptor [
NSCLC cells, 1×106 cells in a 100 μL suspension of Matrigel Basement Membrane Matrix (Corning, Tewksbury, MA), were subcutaneously injected into the hind legs of mice. The double-xenograft model used H1299, H1975, and HCC827 cells. Different mutant types of cancer cells were inoculated into the hind legs of each mouse. Tumor volumes were measured with calipers every 3 days and calculated according to the following formula: volume=DShort2×DLong/2. In both the single and two-tumor models, irradiation was delivered to the tumor-bearing legs of the mice in the irradiation groups using 6-MV photon beams from a linear accelerator (Varian Medical System, Palo Alto, CA) when the tumor volume reached 500 mm3 or 1,000 mm3. During irradiation, the mice were anesthetized by an intraperitoneal injection of tiletamine+zolazepam (50 mg/kg) and xylazine (10 mg/kg), under the approval of the Ministry of Food and Drug Safety. At certain times after irradiation, we sacrificed the mice and harvested their plasma and tumor tissues.
Immunohistochemistry (formalin-fixed paraffin-embedded sections) analysis of tumor tissues sections labeling F4/80 at 1/50 dilution (BM8, eBioscience, San Diego, CA). Heat mediated antigen retrieval citrate buffer. Anti-rat IgG, horseradish peroxidase conjugated (Dako, Glostrup, Denmark) was used as the secondary antibody. Hematoxylin was used as a counterstain.
Circulating DNA was extracted from plasma using a QIAamp Circulating Nucleic Acid Kit (Qiagen, Santa Clara, CA). Genomic DNA (gDNA) was isolated from blood samples using a QIAamp DNA Mini Kit (Qiagen). An AllPrep DNA/RNA Mini Kit (Qiagen) was used to purify gDNA and mRNA from tissues. DNA/RNA concentrations and purity were quantified using a Nanodrop 8000 UV-Vis spectrometer (Thermo Fisher Scientific) and a Picogreen fluorescence assay on a Qubit 2.0 fluorometer (Life Technologies, Waltham, MA). The fragment size distribution was measured using a 2200 TapeStation Instrument (Agilent Technologies, Santa Clara, CA).
To quantify isolated cfDNA, the long interspersed nuclear element-1 (LINE-1) locus was amplified by real-time polymerase chain reaction (PCR) using SYBR Green (Exiqon) according to the manufacturer’s protocols. The LINE-1 locus region was amplified by the following pair of PCR primers: hLINE1: 5′-TCA CTC AAA GCC GCT CAA CTA C-3′, 5′-TCT GCC TTC ATT TCG TTA TGT ACC-3′ and mLine1: 5′-GGA GGG ACA TTT CAT TCT CAT CA-3′, 5′-GCT GCT CTT GTA TTT GGA GCA TAG A-3′. The Ct (threshold cycle) values of the target genes were determined using LightCycler 480 software (Roche, Branchburg, NJ).
To validate the mutations in the NSCLC cell lines, droplet digital PCR (ddPCR) was performed using a QX200TM Droplet Digital PCR System (Bio-Rad, Hercules, CA) according to the manufacturer’s guidelines. The TaqMan ddPCR Liquid Biopsy Assays for NRAS p.Q61K and EGFR p.E746–A750del (Hs000000079_rm and Hs000000027_rm, Life Technologies) were used. The ddPCR data were analyzed using QuantaSoft software (Bio-Rad).
Purified gDNA was sonicated (7 minutes, 0.5% duty, intensity of 0.1, and 50 cycles/burst) into 150–200 bp fragments using a Covaris S2 (Covaris Inc., Woburn, MA). The tumor biopsy sample libraries were constructed using a SureSelect XT reagent kit, HSQ (Agilent Technologies) according to the manufacturer’s instructions. The peripheral blood lymphocytes and plasma DNA libraries were created using a KAPA Hyper Prep Kit (Kapa Biosystems, Woburn, MA). Briefly, after completing end repair and A-tailing according to the manufacturer’s protocol, we performed adaptor ligation at 4°C overnight using a pre-indexed PentAdapter (PentaBase ApS, Soendersoe, Denmark). Hybrid selection was performed using customized baits that targeted ~117 kb of the human genome, including exons from 38 cancer-specific genes (
All liquid biopsy data were aligned to the hg19 reference using BWA-mem (v0.7.5) and analyzed as previously reported [
First, all bases were subjected to Phred quality filtering using a threshold Q of 30, and only positions where total depths were above 500× were considered for variant identification. The error suppression method using unique molecular identifiers was carried out to select highly confident reads supporting a non-reference. Non-reference alleles present at a frequency greater than 1% in the matched germline DNA were removed. Otherwise, non-reference alleles were subjected to the binomial test to determine if a non-reference allele was significantly more abundant in plasma DNA than the matched germline DNA (Bonferroni adjusted p-value < 0.01). To minimize false-positives due to cross-contamination among multiplexed samples, we also excluded non-reference alleles if they were found as germline single nucleotide polymorphisms in other samples processed in a capture reaction or the same lane of a sequencing flow cell. Variant candidates with a high strand bias (90% if supporting reads ≥ 20; Fisher exact test, p-value < 0.1 if supporting reads < 20) were removed. Next, we performed a Z-test to identify variants that were present at a significantly higher frequency than the corresponding background errors in the normal samples (Bonferroni adjusted p-value < 0.05). We further applied the following threshold as previously reported [
All statistical data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). All data were evaluated using analysis of variance (ANOVA) with the Bonferroni
To assess the clinical feasibility of TLB in lung cancer patients, we launched a prospective study for NSCLC patients undergoing definitive RT. The first protocol (IRB 2017-09-120) included patients with stage I–III NSCLC according to the 8th American Joint Committee on Cancer staging manual and pathologic confirmation who were inoperable because of their medical condition. They were recommended to receive definitive RT, and dose-fractionations were determined based on the tumor size, location, and stage. Approximately 10 mL of whole blood sample was obtained at simulation for RT planning. During RT, blood samples were drawn after 1, 2, and 3 days and 1 week, followed by weekly sampling. Posttreatment samples were drawn at 1 month, followed by 3-month intervals for 2 years with chest computed tomography. The second protocol (IRB 2018-05-155) included clinically diagnosed lung cancer patients who failed to have a pathologic diagnosis or for whom diagnostic procedures were significantly risky. All other procedures were the same as in the first protocol.
First, we examined if RT is an adequate modality to promote cfDNA release from a targeted tumor tissue using mouse xenograft model as schemed in
To determine the relationship between tumor burden and ctDNA in the xenograft mouse models, we established mouse subcutaneous tumor models using various NSCLC cell lines. The xenograft models showed significant differences in tumor growth according to the various NSCLC cell lines (
ctDNA could be measured using the amount of hLINE-1 or variation in the injected cells at tumor volumes of more than 400 mm2, confirming that ctDNA reflects the tumor burden.
The hLINE-1 concentration increased significantly after irradiation in all animal models except the H1975 tumor model (
In addition to hLINE-1 quantitative PCR, we performed a ddPCR assay to quantify DNA fragments carrying tumor-specific mutations and thereby double check the elevated ctDNA levels upon irradiation. In H1299 tumor-bearing mice, the
Based on the increased level of ctDNA after irradiation, we hypothesized that radiation aimed at a particular tumor region would locally facilitate ctDNA release from the irradiated region and thus enable TLB. TLB was anticipated not only to improve sensitivity of ctDNA but also to overcome tumor heterogeneity. To emulate intratumor genetic heterogeneity, we established two-tumor model mice bearing H1299 and HCC827 tumors, one in each hind leg.
To investigate the hypothesis, we irradiated one tumor site in two-tumor model mice. When irradiation was delivered to the H1299 tumor,
To investigate the mechanism by which the tumor micro-environment modulates releases of cell-free DNA from cancer cells into blood vessels, we performed transcriptome analysis of xenograft tumor tissues comprising tumor cells and infiltrated normal cells. We obtained both tumor tissues in the two-tumor mouse model and generated next-generation sequencing-based RNA sequencing (RNA-seq) data.
As a result, a mean of 19,144,381 human and 2,806,448 mouse reads were uniquely mapped to a concatenated human and mouse genome, indicating that 13.3% of transcripts were originated from host cells (
Because recent studies showed that radiation, particularly high-dose, hypo-fractionated administration [
Taken together, our data indicated that the secretion of ctDNA following RT was not only caused directly by cancer cells but also indirectly related to changes in the cell composition of the tumor microenvironment.
We launched a prospective study for patients undergoing definitive RT with or without a histologic diagnosis as schemed in
In total, we analyzed ctDNA in longitudinal blood samples from 11 patients (
During the past few decades, the emergence of precision medicine and personalized cancer treatment based on genetic analyses of tumor tissue has resulted in more specific treatment options. The shifting paradigm toward serially monitoring tumor molecular characteristics to more precisely guide therapy provides an opportunity for new diagnostic studies to determine the best way to acquire this information [
In this study, we demonstrated that RT temporarily amplifies the release ctDNA in lung cancer mouse models. We also demonstrated the feasibility of TLB in a double-xenograft mouse model. Preliminary results from our clinical study also demonstrated a temporary increase in ctDNA following RT. In addition, monitoring of tumor response might be also feasible (
Although the plasma volumes from mouse xenograft models were small, we reliably used them to quantify ctDNA with both the hLINE-1 assay and digital PCR. At several doses, the concentration of ctDNA and cfDNA increased within 24 hours of irradiation except the H1975 tumor model. Different kinetics of cfDNA release might be affected by a complex interplay between apoptosis, necrosis, and senescence, as described by Rostami et al. [
Similar to patients with lung cancer, the total concentration of cfDNA in mice is known to increase as the disease progresses [
In addition, target-specific amplification of ctDNA levels in the two-tumor mouse model suggests that the diagnostic value of liquid biopsy could be improved by using irradiation as a local stimulus. Although liquid biopsy itself cannot identify a specific site or represent intratumoral heterogeneity, local irradiation could make it target-specific. Therefore, we could apply this concept clinically to patients who need repeated liquid biopsies for tumor foci resistant to previous treatments. A future clinical study should assess the feasibility of TLB for patients with advanced disease who are receiving RT to treat a metastatic tumor.
Why did we use RT for ctDNA amplification? Other local stimulation methods might be used, such as high-intensity, focused ultrasound, but the lung environment is quite different from other solid organs. X-rays can be delivered anywhere precisely, and they are widely used to treat lung cancer. Clinically, lung cancer has various mutations that produce resistance to treatment, and serial liquid biopsy during systemic anticancer therapy is needed to identify response or progression [
Compared with previous studies on monitoring ctDNA during RT or chemoradiotherapy, the current study has some limitations (
When we analyzed bulk RNA-seq data, the mouse gene expression profiles differed significantly between cell lines. This observation raised the possibility that ctDNA levels were partly influenced by specific interactions with the host system. Because radiation not only induces the death of tumor cells but also modulates the tumor microenvironment, the increase in ctDNA after irradiation might be related to changes in immune cell proportions or activities. The results of our bulk RNA-seq deconvolution analysis are consistent with the previous observation that irradiation modulates immune cells, such as macrophages and NK cell proportions [
In conclusion, we used irradiation and high-throughput techniques to temporarily amplify ctDNA release in both animal models and lung cancer patients to decipher the heterogeneity and distinct molecular signatures of a specific anatomical tumor site. TLB could have clinical utility for diagnosis and identifying relevant mutations in tumor foci resistant to previous treatments, and allow for serial liquid biopsies to monitor treatment response and to detect MRD during lung cancer treatment.
Supplementary materials are available at Cancer Research and Treatment website (
The animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (20170706001, 20180109002) of Samsung Biomedical Research Institute before the investigational use of any animals. Before beginning the study, we received approval from our Institutional Review Board (IRB; 2017-09-120, 2018-05-155) and written informed consent was provided before enrollment.
Conceived and designed the analysis: Noh JM, Lee HY, Choi C, Pyo H, Park WY.
Collected the data: Ahn WG, Park JH.
Contributed data or analysis tools: Kim YJ, Ahn WG, Lee T, Park D.
Performed the analysis: Kim YJ, Lee T.
Wrote the paper: Noh JM, Kim YJ, Park D.
Funding acquisition: Lee HY.
Study supervision: Pyo H, Park WY.
Technical support: Park D.
Conflict of interest relevant to this article was not reported.
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (NRF-2016R1A2B4013046, NRF-2017M2A2A7A02018568, NRF-2017M2-A2A7A02018569) and by the Korea Health Technology R&D Project through the Korean Health Industry Development Institute, funded by the Ministry of Health & Welfare (HI17C0086).
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 [
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 (
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,
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.
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.
Summary of previous studies on ctDNA during radiotherapy
Study (year) | Cancer type | No. of patients | Treatment | Implication |
---|---|---|---|---|
Chaudhuri et al. (2017) [ |
Lung cancer | 40 | RT/CRT Surgery | ctDNA analysis can identify posttreatment MRD |
Moding et al. (2020) [ |
Lung cancer | 65 | CCRT±ICI | Consolidation ICI was beneficial in patients with MRD |
Lv et al. (2019) [ |
Nasopharyngeal cancer | 673 | CRT | Persistent circulating EBV DNA was adverse prognostic factor |
Chera et al. (2019) [ |
Oropharyngeal cancer | 103 | CRT | Rapid clearance profile of plasma HPV DNA predicts likelihood of disease control |
Current study | Lung cancer | 11 | RT alone | Temporary increase of ctDNA within 72 hours after initiation of RT |
CCRT, concurrent chemoradiation therapy; CRT, chemoradiotherapy; ctDNA, circulating (cell-free) tumor DNA; EBV, Epstein-Barr virus; HPV, human papillomavirus; ICI, immune check point inhibition; MRD, molecular residual disease; RT, radiotherapy.