cfDNA refer to short DNA fragments with an average length of 120 to 160 bp that are present outside cells. ctDNA is the subset of cfDNA that are derived from tumor cells following apoptosis and/or necrosis and are usually <145 bp in length. ctDNA is highly diluted in plasma, accounting for approximately 0.1%-10% of the total cfDNA and its concentration is impacted by clearance and degradation by nuclease activity [6,7]. While tumor tissue biopsy is the gold standard in establishing the diagnosis of NSCLC, diagnostic rates vary by region and biopsy technique, but it is reported that over 40% of patients in the United States required repeat biopsies before diagnosis [8]. Furthermore, the failure rate of molecular characterization has been reported in approximately 30% of cases [9]. ctDNA can thus be an used as an alternative tool in the molecular genotyping of patients with NSCLC, especially in situations where tissue or time is limited [10,11].

Liquid biopsy testing of tumor DNA has some advantages over tissue-based DNA testing including less invasive, shorter turn around time and lower sample isolation costs. A liquid biopsy enables the assessment of quantitative changes in ctDNA which could be used for real time monitoring of therapeutic effects. In addition, detection of genetic mutations, changes in copy number and chromosomal rearrangement have the potential to be applied to detect resistance mechanisms or treatment efficacy [12,13]. On the other hand, an important limitation of liquid biopsy is the inability to assess the histology of the tumor and the risk of false negative results due to either insensitive ctDNA assays or inadequate ctDNA levels in circulation (Table 1).

ctDNA levels are influenced by the degree of shedding and clearance. Factors associated with shedding include tumor burden with early-stage disease are less likely to shed ctDNA, metabolic activity, histologic type and grade, lymph node involvement, tumor microenvironment, and location of metastasis [14]. Factors associated with ctDNA clearance includes soluble blood enzymes such as DNase I, protease factor VII-activating protease (FSAP) and factor H as well clearance by organs such as liver, spleen and kidneys [15]. The advantages and disadvantages of tissue biopsy and liquid biopsy are summarized in Table 1. Multiple applications exist for ctDNA including molecular genotyping of disease, screening, detection of measurable disease, treatment selection, treatment monitoring and the molecular characterization of resistance (Figure 1).

Although driver mutation-based therapy is widely used in NSCLC, complete and timely tissue genotyping is difficult, and few patients have been examined for all eight genomic biomarkers recommended by professional guidelines (National Comprehensive Cancer Network [NCCN] Guidelines 2024) [16]. Molecular genotyping using ctDNA could overcome the tissue sampling barrier in advanced NSCLC.

The detection of plasma ctDNA in advanced NSCLC is prognostic and is associated with metabolic tumor volume, more frequent in extrapulmonary disease (74%) than intrapulmonary disease (40%) [17]. In the Noninvasive vs. Invasive Lung Evaluation (NILE) study, a prospective trial that compared the utility of plasma next generation sequencing (NGS) using Guardant360 versus tissue genotyping in patients with advanced NSCLC, the Guardant360 assay had a concordance of >98.2% and 100% positive predictive value versus tissue testing. In addition, the median turnaround time (TAT) for cfDNA was significantly faster than for tissue (9 days vs. 15 days, p<0.0001) [18]. In a prospective observational study comparing tissue genotyping with ctDNA genotyping, NGS results were significantly better for ctDNA at the first visit after biopsy for diagnosis (85% vs. 9%, p<0.01), especially in cases with specific driver gene mutations significantly shorter time to treatment initiation in cases (10 days vs. 19 days, p<0.01) [19].

Another important trial on this topic is the Blood First Assay Screening Trial, a Phase II/III umbrella trial. This study was designed to evaluate plasma-based NGS for the detection of actionable genetic alterations in patients with advanced NSCLC and to examine the efficacy of targeted and immunotherapy-based therapies using plasma results alone. Confirmed objective response rate (ORR) was 87.4% for alectinib in the anaplastic lymphoma kinase (ALK) cohort, better than existing prospective trials [20]. The ROS1 cohort also showed response rates and progression-free survival (PFS) comparable to tissue-based assessments [21].

Another potential application of ctDNA in advanced NSCLC is the evaluation of blood-based tumor mutation burden (bTMB). Tumor mutation burden (TMB), defined as the total number of non-synonymous somatic mutations in the coding region of a cancer genome, is a predictive biomarker of immune checkpoint inhibitor (ICI) in a range of solid tumours [22,23] and exome or large panel sequencing allow for the quantification of TMB. More recently, the U.S. Food and Drug Administration (FDA) approved pembrolizumab for the treatment of cancer patients with TMB >10 mutations per megabase (mut/Mb) [24] FDA. FDA approves pembrolizumab for adults and children with TMB-H solid tumors in 2020 [25]. A retrospective study of two large randomized trials, bTMB was a predictive biomarker for PFS in patients treated with atezolizumab [26]. In a single arm prospective study of first line atezolizumab (Blood First Assay Screening Trial [BFAST] study), investigator-assessed PFS in the bTMB ≥ 16 versus bTMB < 16 mut/Mb groups were not statistically significant but bTMB ≥16 was associated with higher ORR [27]. In the BFAST study, Cohort C compared first line atezolizumab versus platinum doublet chemotherapy in patients with a high TMB (>16 mut/Mb). Unfortunately the study did not meet its primary endpoint of investigator-assessed PFS (hazard ratio [HR], 0.77; 95% confidence interval [CI], 0.59 to 1.00; p=0.05). A major limitation of this study was programmed death-ligand 1 (PD-L1) expression was unknown [28]. The Evaluation of Blood tumor mutation burden for improved efficacy of atezolizumab (BUDDY) study showed that high cfDNA concentration at C0 (cutoff: 8.6 ng/mL) and cycle 4/baseline bTMB ratio >1 was significantly associated with PFS in atezolizumab treatment after platinum combination therapy [29]. Future research is required to define the optimal bTMB cutoff and standardization of assay technologies [30,31].

The characterization of resistance mutations with plasma ctDNA genotyping has been investigated extensively. In the AZD9291 (Osimertinib) Versus Platinum-Based Doublet-Chemotherapy in Locally Advanced or Metastatic Non-Small Cell Lung Cancer (AURA3) study, of the 564 patients for whom both plasma and tumor tissue was performed on the Cobas-based epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) post-progression screening, 41% were ctDNA T790M positive and 64% were tissue EGFR T790M positive, for a ctDNA/tissue concordance rate of 61% [32]. Compared to the EGFR sensitive mutations L858R and exon19 deletion, the ctDNA-tissue concordance is lower for EGFR T790M. While sensitive mutations are clonal mutations and thus relatively homogeneous within the tumor, resistance genes such as T790M are subclonal mutations, and thus greater tumor heterogeneity is assumed to be the cause of the tissue-ctDNA discrepancy.

In the AZD9291 Versus Gefitinib or Erlotinib in Patients With Locally Advanced or Metastatic Non-small Cell Lung Cancer (FLAURA) study, 109 patients who were included in the osimertinib arm and had EGFR mutations detected by ctDNA at baseline had mesenchymal epithelial transition (MET) amplification at the time of resistance (amplification [n=17, 16%] and the EGFR C797S mutation [n=7, 6%]) was more frequent at the time of resistance [33]. Chinese Thoracic Oncology Group (CTONG1509), a phase III trial comparing erlotinib with erlotinib plus bevacizumab, also showed 57% (33/58) patients with known resistance mechanisms including EGFR T790M, oncogenic fusion, amplification, mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) changes, and cell cycle gene changes at progression of disease [34]. Similarly, ctDNA has successfully detected resistance mutations after osimertinib as a second line [32,35,36].

The molecular mechanisms of resistance by ctDNA genotyping have also been described in patients after targeted therapy for BRAF V600E mutations, ALK rearrangement, MET exon14 skipping mutations, and ROS1 fusions [37-40].

The utility of ctDNA to monitor for plasma EGFR T790M acquired resistance in patients treated with a 1st- or 2nd- generation EGFR-TKIs. The authors found the median time of detectable plasma EGFR T790M was 2.2 months (range, 0.8 to 6) before clinical progressive disease [41].

This intriguing finding posits the question: does the early detection of acquired EGFR T790M resistance mutations improve outcomes with a change in therapeutic agent? To test this proposition, the Osimertinib Treatment on EGFR T790M Plasma Positive NSCLC Patients (APPLE) trial was designed to evaluate the feasibility of longitudinal plasma EGFR T790M monitoring performed intensively using the Cobas EGFR Mutation Test v2 and the best sequencing strategy from gefitinib to osimertinib (molecular progression by T790M positivity in plasma test or radiographic progression by Response Evaluation Criteria in Solid Tumors [RECIST] 1.1 criteria). In the trial arm B, which switches to osimertinib if T790M mutation is detected in plasma ctDNA, 17% of patients (eight of 47) switched to it before radiographic progression. However, overall survival (OS) was comparable between the group that switched early to osimertinib based on ctDNA detection and the group that switched after disease progression was confirmed by RECIST 1.1 criteria [42].

It is well established clearance of ctDNA has been shown to be a prognostic factor in multiple clinical trials, including the FLAURA trial of osimertinib versus first generation EGFR-TKI for EGFR mutations [36], the A Study Of Lorlatinib Versus Crizotinib In First Line Treatment Of Patients With ALK-Positive NSCLC (CROWN) trial of lorlatinib versus crizotinib for ALK fusions [43], the Phase 1/2 Study of MRTX849 in Patients With Cancer Having a KRAS G12C Mutation (KRYSTAL-1) trial of adaglasib for KRAS G12C mutation [44], and the BRAF-targeted therapies (BRAF-TT) trial of targeted therapy against BRAF V600E mutation [37]. More recent phase III trials have reported on the association between ctDNA clearance status and outcomes. In the A Study of Amivantamab and Lazertinib Combination Therapy Versus Osimertinib in Locally Advanced or Metastatic Non-Small Cell Lung Cancer (MARIPOSA) trial examining the efficacy of 1^st-line amivantamab plus lazertinib versus osimertinib in patients with EGFR mutated NSCLC, 78% of patients had available droplet digital polymerase chain reaction (ddPCR) testing for ctDNA at baseline. In the patients with ctDNA detected at baseline, the PFS for the group of amivantamab plus lazertinib and the group of osimertinib were 20.3 and 14.8 months, respectively (HR, 0.68), while, in the patients with no detectable ctDNA, the PFS for the group of amivantamab plus lazertinib and the group of osimertinib were 27.7 and 21.9 months, respectively (HR, 0.72). In addition, the therapeutic efficacies of amivantamab plus lazertinib were greater than that of osimertinib, regardless of ctDNA clearance at 9 weeks [45]. In an exploratory analysis of the FLAURA2 trial comparing the clinical efficacies of osimertinib plus chemotherapy to osimertinib, a better PFS for the group of osimertinib plus chemotherapy was observed in patients with positive ctDNA. Regardless of ctDNA clearance, the addition of chemotherapy to osimertinib provided a PFS advantage over osimertinib alone [46].

To evaluate the role of treatment escalation based on plasma ctDNA status, a number of trials are ongoing. In a randomized phase II trial by Memorial Sloan Kettering Cancer Center (NCT04410796) where patients with advanced EGFR NSCLC without ctDNA clearance after one cycle of osimertinib are randomized to carboplatin and pemetrexed plus osimertinib versus continuation of osimertinib. In another ongoing study, patients with plasma EGFR mutations detected after one cycle of lazertinib are randomized to pemetrexed, carboplatin and lazertinib versus lazertinib alone (Lazertinib and Chemotherapy Combination in EGFR-mutant NSCLC Patients Without ctDNA Clearance After lead-in Lazertinib Monotherapy [CHAMELEON] trial, NCT06020989) in which chemotherapy is combined with lazertinib based plasma EGFR mutation status [47]. Furmonertinib Monotherapy and Combination Therapy in Advanced EGFR Mutant NSCLC With Uncleared ctDNA (FOCUS-C) is a randomized phase II where patients without ctDNA clearance with furmonertinib are randomized to furmonertinib or furmonertinib plus chemotherapy (pemetrexed and carboplatin) or furmonertinib plus chemotherapy plus bevacizumab (NCT05334277).

In advanced stage NSCLC, oligometastatic disease (OMD) has limited number of metastatic lesions (usually up to 5). The addition of local treatment is expected to prolong survival, but there is no clear consensus on its definition [48-50]. In 2020, European Society for Radiotherapy and Oncology/European Organisation for Research and Treatment of Cancer (ESTRO/EORTC) classified OMD into synchronous, metachronous, induced, persistence, and progression [51].

In a prospective study of ctDNA in patients with OMD, patients with ctDNA detected before local ablative radiotherapy had an mPFS of 5.4 months, significantly shorter than the 8.4 months for ctDNA negative patients (HR, 1.57; 95% CI, 1.15 to 2.13; p=0.004) [52], consistent with the notion detectable ctDNA is a prognostic factor.

More recently, a nonrandomized study was conducted to assess the feasibility of adaptive TKI de-escalation guided by ctDNA to achieve complete remission after local consolidative therapy (LCT) in patients with advanced NSCLC. The median PFS was 18.4 months and the authors concluded an adaptive de-escalation TKI approach was feasible in those with no lesions after LCT with undetectable ctDNA [53]. While these results are promising, potential challenges include the presence of ctDNA non-shedders and the use of a highly sensitive ctDNA assay is crucial. Furthermore, a randomized trial is required to confirm the validity of a de-escalation treatment strategy.

Minimal Residual Disease (MRD) is a micro-tumor that remains in the body after or during cancer treatment. MRD was originally applied in hematologic malignancies and is a prognostic factor in acute leukemias and plays an important role in the choice of appropriate treatment. Quantitative polymerase chain reaction and multiparameter flow cytometry are typical methods used to detect MRD. In solid tumors, emerging data suggests blood-based ctDNA analysis is effective in detecting MRD [54-56]. Several prospective observational studies have reported MRD is associated with increased recurrence.

In a landmark analysis of plasma samples taken within 120 days of surgery detected ctDNA in 25% of patients, including 49% of all patients who experienced clinical recurrence [57]. In a prospective observational study of 261 patients with resected stage I to III NSCLC, longitudinal undetectable MRD was associated with decreased disease recurrence with a negative predictive value of 96.8%. These results suggests a potentially cured patient population in those with longitudinal undetectable MRD. Conversely, the positive predictive value of longitudinal detectable MRD was 89.1%, with a median lead time of 3.4 months [58].

In patients with locally advanced unresectable stage III NSCLC, the success of the A Global Study to Assess the Effects of MEDI4736 Following Concurrent Chemoradiation in Patients With Stage III Unresectable Non-Small Cell Lung Cancer (PACIFIC) trial led to the approval of durvalumab consolidation therapy after chemoradiotherapy (CRT). The 5-year PFS rate was 19.0% in the BSC group and 33.1% in the durvalumab group, and the 5-year survival rate was 33.4% in the BSC group compared to 42.9% in the durvalumab group [65].

In a retrospective study of ctDNA in patients NSCLC patients treated with CRT with or without consolidation ICI, cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis of 218 samples from 65 patients undergoing CRT showed that patients with undetectable ctDNA after CRT had longer DFS, regardless of whether they had been treated with ICI. Of note, one MRD negative case died of G5 pneumonitis. Among patients with positive MRD, those who received consolidation ICI treatment had significantly longer DFS than those who did not. Early ctDNA responses identified patients who would benefit particularly from ICI treatment [66]. These results, albeit retrospective in nature, suggests consolidation ICI improves outcomes for NSCLC patients with MRD and ctDNA analysis may be useful in the personalization of consolidation therapy. Prospective randomized studies are required to confirm if personalization of consolidation ICI based on MRD status can be applied.

In the LAURA trial, osimertinib therapy after CRT prolonged DFS and OS in EGFR mutated locally advanced unresectable stage III NSCLC [67]. The length of treatment in adjuvant therapy is longer than in the advanced stage, and there is concern side effect including quality of life. Therefore, MRD may have a promising role in EGFR -positive early-stage NSCLC by (1) replacing or complementing pathologic stage as a criterion for selection of adjuvant TKI extension therapy; (2) reducing treatment and economic/resource burden; (3) improving prognosis; and (4) reducing frequency of imaging diagnosis.

Randomized trials of lung cancer screening with low-dose computed tomography (LDCT) in selected high risk patient population has been shown to be effective in reducing lung cancer mortality [68,69]. Blood-based biomarkers may potential advantages over LDCT including ease of convenience, high-throughput, lower costs and lack of exposure for participants to ionizing radiation. However several challenges using ctDNA as a screening modality. First, the sensitivity may be inadequate due to limited ctDNA shedding or assay sensitivity. Plasma ctDNA mutations may be non-malignant or premalignant and their tissue of origin is often unknown. To overcome these barriers, screening in combination with positron emission tomography-computed tomography, protein biomarkers, methylation patterns, miRNAs, etc. is being considered.

In a study comparing the mutational profile of cfDNA in either apparent heathy subjects and patients with stage I or II colorectal, breast, lung, or ovarian cancer, somatic mutations were detected in the plasma of 71%, 59%, 59%, and 68%, respectively and sensitivity increased with stage of disease. However, approximately 16% of healthy controls had mutations in genes associated with clonal hematopoiesis of indeterminate potential [70].

The Circulating Cell-Free Genome Atlas Study (CCGA; NCT02889978) is a prospective, case-control, observational study that examined whether a blood-based multiple cancer early detection test combining targeted methylation of ctDNA and machine learning could detect cancer signals across multiple cancer types and predict cancer signal origin by detecting cancer signals across multiple cancer types. The sensitivity and specificity for all carcinomas were 51.5% and 99.5%, respectively, with a sensitivity of 75% for lung cancer For NSCLC, sensitivity increased with stage, with 21.9% for stage I, 79.5% for stage II, 90.7% for stage III, and 95.2% for stage IV [71]. The Cancer SEEK trial investigated the role of screening with mutated DNA and eight standard biomarkers in cfDNA and reported a sensitivity of 60% for resectable lung cancer [72]. In another study integrating genomic features with machine learning, termed 'lung cancer likelihood in plasma' (Lung-CLiP), this approach could discriminate early-stage lung cancer patients from risk-matched controls with a specificity of 96% [73].

Collectively, ctDNA assays have a high specificity for lung cancer but sensitivity is limited, especially for early-stage disease. Further prospective evaluation of blood-based biomarkers are ongoing to confirm its utility in lung cancer screening.