Circulating Tumor DNA in Primary and Secondary Liver Cancers: A Comprehensive Review

Abstract

Similar to many other cancer types, liver malignancies pose the common challenges of late detection of primary tumors and recurrences. Liquid biopsies, which assess the presence of circulating tumor DNA, have emerged as a novel, non-invasive clinical tool for diagnostic and surveillance purposes. This review represents an introductory and comprehensive overview of the current circulating tumor DNA (ctDNA) literature relevant to primary and secondary liver malignancies. Herein, we highlight key findings, landmark discoveries, challenges, and future directions.

Graphical abstract

Keywords

circulating tumor DNA / ctDNA / liquid biopsy / liver cancer / liver malignancy

1. Introduction

Liver-related malignancies comprise the sixth most common cause of cancer and third leading cause of cancer death [1]. Primary liver cancers include hepatocellular carcinoma (HCC) (85–90% of cases) and cholangiocarcinoma (CCA) (10–15% of cases) [1]. Secondary liver malignancies include metastases arising from the lung, colorectum, pancreas, stomach, breast, and cecum [2]. Approximately 5% of cancer patients present with synchronous liver metastases, with the most common primary site being breast cancer for young females and colorectal cancer for young males [3]. For older patients, lung, pancreatic, and colorectal cancers are the most common primary sites, although a greater percentage of primary esophageal, stomach, small intestine, melanoma, and bladder cancers start to emerge with age [3].

Common to all liver malignancies is a worse prognosis associated with late stage of disease. The one-year survival of patients with liver metastases from any primary cancer is lower (15.1%) than those with non-hepatic metastases (24%) [3]. Advanced disease is multi-factorial due to limited sensitivity and specificity of biomarkers, more aggressive tumor biology, and lack of early, specific symptoms [4]. Current methods of liver cancer detection include radiologic imaging, tissue sampling, and traditional serum biomarkers [5]. However, radiologic imaging is limited in patients with small nodules and early microscopic lesions [6]. Tissue sampling requires invasive biopsy procedures and may yield insufficient tissue for diagnosis and high false negative rates [7, 8]. Traditional serum biomarkers, such as alpha-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA) are limited in their sensitivity and specificity [9, 10].

To address these limitations, circulating tumor DNA (ctDNA) has become of interest due to its non-invasive nature through liquid biopsy and its ability to provide insight into the nature of individual patient tumor biology [11]. One meta-analysis revealed a high overall sensitivity (72.2%) and specificity (82.3%) along with diagnostic odds ratio (18.53) and area under the curve (AUC) (0.88) for ctDNA in HCC, displaying its potential as a diagnostic marker [12]. This tool, now FDA-approved for monitoring minimal residual disease for colorectal liver metastasis, holds significant potential as a surveillance and prognostic tool for liver malignancies. In terms of prognosis, patients with detectable ctDNA following curative-intent local therapy for colorectal cancer with liver metastasis have been shown to have significantly higher potential for recurrence and shorter overall survival (OS) compared to those without detectable ctDNA [13].

This comprehensive narrative review serves as an introduction to ctDNA and the landscape of ctDNA literature within primary and secondary liver cancers. First, the concept of ctDNA as a biomarker is introduced, followed by summaries of key studies on clinical utility and mutational profiles by cancer subtype. Lastly, insight into challenges and limitations of current studies involving ctDNA is provided, along with commentary on future directions for research and clinical applications. Detailed descriptions of the methodologies are not included in the present review due to heterogeneity in ctDNA detection methods; however, readers are encouraged to refer to cited materials for further information.

2. Circulating Tumor DNA (ctDNA)

ctDNA refers to genomic material released by tumor cells into the patient’s systemic circulation. An early study in 1977 showed the presence of cell-free DNA from peripheral blood of cancer patients, but further characterization was limited by technology at the time [14]. Recent advancements in genetic amplification technology have allowed deeper investigation into this genomic material, identifying single-nucleotide changes [15], methylation patterns [16, 17, 18], and viral sequences [19] derived from or reflective of the original tumor.

ctDNA is thought to originate from several sources, including apoptotic or necrotic tumor cells, live tumor cells, and circulating tumor cells (Fig. 1) [20, 21]. Due to its relatively short half-life of up to two hours, ctDNA reflects the tumor biology in a dynamic fashion [22]. Methods of detection include droplet digital PCR (ddPCR), personalized amplicon-based NGS, personalized hybridization-based next-generation sequencing (NGS), whole-genome sequencing, exome sequencing, or array comparative genomic hybridization (Figs. 2,3) [23, 24]. Detection and analytic methods can be performed in tumor-informed, which decreases risk of false positive results, or tumor-uninformed fashions, which can allow for detection of clonal evolution and tumor resistance.

3. Primary Liver Malignancies

3.1 Hepatocellular Carcinoma (HCC)

HCC is the sixth most common cancer worldwide and third leading cause of cancer-related deaths [1]. The standard diagnostic method consists of radiologic imaging (e.g., computer tomography (CT), magnetic resonance imaging (MRI)) and detection of elevated alpha-fetal protein (AFP) levels. Despite a high specificity for early HCC, AFP has limited and variable sensitivity [25, 26]. In contrast, ctDNA has a reported sensitivity of 100% (42/42) and specificity of 97.4% (75/77) for detecting recurrence in patients with HCC following surgery and adjuvant therapy [27]. Recently published clinical trial results also show the ability of a liquid biopsy-based DNA methylation signature to detect HCC with 84.5% sensitivity, 95% specificity, and 0.94 AUC [18]. Further information regarding this trial, along with other completed and recruiting clinical trials for primary and secondary liver malignancies, is summarized in Table 1 (Ref. [18, 28, 29, 30, 31]) and Table 2 respectively.

Several studies have explored the clinical utility of ctDNA in HCC. In 2020, Wang et al. [32] found a higher cancer detection rate using pre-operative plasma ctDNA (70.4%, n = 57/81) with a panel of 4 mutations (TP53 (c.747G>T), TRET (c.1-124C>T), CTNNB1 (c.121A>G) and CTNNB1 (c.133T>C)) compared to a detection rate of 56.8% (n = 46/81) with AFP. In addition, pre-operative detection of plasma ctDNA was associated with larger tumor size, multiple tumor lesions, microvascular invasion, and advanced Barcelona Clinic Liver Cancer (BCLC) stage, which are also associated with worse clinical prognosis [32]. Pre-operative ctDNA was also independently associated with disease-free survival (DFS) and overall survival (OS) on multivariate analysis. Another study featuring 26 operable HCC cases found that patients with at least one detectable mutation on postoperative plasma ctDNA had worse DFS than those without (17.5 months vs. 6.7 months, hazard ratio (HR) = 7.655, p 0.0001), and post-operative ctDNA status was an independent risk factor for recurrence (HR = 10.293, p 0.0001) [33]. Additionally, TERT promoter mutations on plasma ctDNA were identified as a factor for poor overall survival among 130 patients undergoing systemic chemotherapy or transcatheter arterial chemoembolization (HR = 1.94; 95% confidence interval [CI], 1.18–3.24; p 0.01) [34]. High serum cfDNA levels are also an independent prognostic factor for worse OS (HR: 3.4, 95% CI: 1.5–7.6, p = 0.004) and increased recurrence in distant organs (HR: 4.5, 95% CI: 1.3–14.9, p = 0.014) for patients with hepatitis C virus-related HCC who underwent curative intent hepatectomy [35]. Additionally, serum cfDNA levels were significantly different in patients who had HCV-related HCC compared to patients with HCV only (115.9 \pm 98.3 vs 34.4 \pm 40.4 ng mL^-1 respectively, p 0.0001) [35].

Characteristics provided by ctDNA testing, such as extent of tumor mutational burden (TMB), have also been shown to correlate with survival. Wehrle et al. (2024) [36] demonstrated an association between TMB on post-operative ctDNA and shorter recurrence-free survival (RFS) in 48 patients with HCC following surgical resection. With immunotherapy emerging as a treatment option for HCC as suggested by the Imbrave50 trial results, this may suggest a role for adjuvant immunotherapy in this patient population. Similarly, preliminary data from Marron et al. (2023) [37] shows the ability of post-operative ctDNA in detecting disease relapse and shorter recurrence-free survival for patients undergoing neoadjuvant and adjuvant cemiplimab (anti-PD-1) and surgical resection.

For patients with unresectable HCC, ctDNA also plays a role in predicting treatment response. Preliminary results from a personalized, tumor-informed assay show a prolonged PFS in patients whose ctDNA became undetectable following treatment [38]. In another study, 3/4 (75%) patients undergoing immunotherapy and locoregional therapy changed from detectable to non-detectable TMB on their ctDNA following curative-intent hepatectomy [39]. CtDNA can also provide insight into the mutational landscape driving resistance to systemic therapies in advanced HCC. For example, von Felden et al. (2021) [40] conducted targeted sequencing of 25 genes and ddPCR of the TERT promoter on plasma ctDNA, and found that patients with mutations in the PI3K/MTOR pathway exhibited significantly shorter PFS (2.1 vs. 3.7 months, p 0.0001) following treatment with tyrosine kinase inhibitors compared to those without such pathway-specific mutations. For these 77 HCC, the most frequently mutated genes on ctDNA were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (6% each) [40].

Other studies have used ctDNA to investigate mutational pathways driving HCC tumorigenesis. For example, Ikeda et al. (2018) [41] performed ctDNA and tissue NGS testing on 26 patients with HCC and identified common mutations of TP53 (50%), CTNNB1 (100%), and ARID1A (90%). An et al. (2019) [33] assessed the presence of nine presumptive driver genes for HCC (TP53, AXIN1, CTNNB1, CDKN2A, ARIN1A, ARID2, SMARCA4, KEAP1 and NFE2L2) and found 37 driver events in 88.5% cases (23/26). On an epigenetic level, aberrant promoter methylation of tumor suppressor genes p16, GSTP1, and RASSF1A have been noted in both the plasma and tumor tissue of patients with HCC [42, 43, 44, 45, 46]. More specifically, p16 methylation was noted in 73% (16/22) HCC tissues and 81% (13/16) HCC plasma/serum samples, while being absent in the plasma/serum of healthy patients and patients with chronic hepatitis/cirrhosis [43]. Hypermethylation of GSTP1, which encodes glutathione S-transferase, was observed in 88.5% (23/26) of HCC tumor tissue and 50% (16/32) patients with HCC, while none of the normal peripheral blood mononuclear cell samples from healthy patients (n = 12) had aberrant GSTP1 methylation [45]. For RASSF1A, aberrant promoter methylation was detected in 92.5% (37/40) of HCC tissues and 42.5% (17/40) of paired plasma, while being associated with HCC tumor size of at least 4 centimeters (p = 0.035) [46]. Hypomethylation of serum LINE-1 has also been shown to be a significant and independent prognostic marker of overall survival for patients with HCC [47]. Additionally, the average level of serum LINE-1 hypomethylation differed significantly among patients who were healthy and those with HCC, cirrhosis, or hepatitis B virus [47]. Thus, specific genetic and epigenetic changes within HCC tumors may be detectable peripherally from patient plasma/serum, establishing another method of clinical diagnosis.

Finally, we hypothesize potential utility of ctDNA in transplant selection criteria. Studies have demonstrated generally equivalent outcomes across the many currently described selection criteria, indicating the current spectrum of biomarkers are ineffective at advancing our discriminatory capability [48, 49, 50]. In contrast, ctDNA status has been shown to be associated with shorter RFS and higher recurrence rate based on serial ctDNA testing [51]. In a study by Huang et al. (2024) [51] featuring 74 patients undergoing liver transplant for cancer, patients with plasma ctDNA detected postoperatively had a shorter RFS of 17.2 months (vs. 19.2 months, p = 0.010) and higher recurrence rate (46.2% vs. 21.3%, p 0.0001) compared to those without detectable ctDNA postoperatively. Significantly, dynamic changes in ctDNA could also predict disease progression prior to changes in levels of traditional tumor biomarkers. In the same study, increase in ctDNA levels occurred prior to changes in AFP and DCP (des-gamma-carboxy prothrombin) for one patient, in whom recurrent liver tumor lesions were subsequently found on MRI [51]. Initial data are promising in this sense, and we hypothesize that addition of ctDNA to morphologic characteristics may improve our ability to stratify pre-transplant oncologic risk.

3.2 Cholangiocarcinoma (CCA)

CCA accounts for 10–15% of primary liver cancers. In the U.S., Australia, and Europe, CCA affects 0.3–3.5 individuals per 100,000 people, but can also reach incidences of 85 cases per 100,000 people in areas such as northeastern Thailand, where liver fluke infection is more common [52]. 5-year overall survival rates for CCA are only about 15–20%, even after curative-intent surgery and adjuvant therapy [53]. Timely diagnosis of CCA is challenging due to limited samples obtained during biopsy, equivocal results of diagnostic testing and radiologic imaging, and lack of specific tumor markers [54]. Therefore, many patients have advanced or systemic disease at time of diagnosis, precluding surgical resection, which is considered the gold-standard therapy [55]. CA19-9 is a traditional serum biomarker used in diagnosing and monitoring CCA. However, its poor sensitivity and specificity, high false positive rate, and unreliable nature in patients with benign conditions including primary sclerosing cholangitis render it a suboptimal biomarker [56, 57].

In terms of clinical utility, ctDNA has been shown to predict survival in patients with CCA. A study by Uson Junior et al. (2022) [58] featured patients with metastatic intrahepatic CCA, extrahepatic CCA, and gallbladder cancer who underwent ctDNA testing prior to initiation of first-line treatment with platinum-based chemotherapy. After adjusting for cancer subtype, metastatic site, largest tumor size, age, sex, and CA19-9 levels, each 1% increase in ctDNA level was associated with HR of 13.1 in OS [58]. Additionally, when stratifying dominant clonal allele frequency (DCAF) from ctDNA by quartile (ctDNA \le0.6%, 0.6%–3%, 3%–10%, and \ge10%), there was a significant association with PFS (p = 0.014) and OS (0.001) [58]. Wintachai et al. (2021) [59] have also shown the diagnostic value of ctDNA in CCA. In particular, they identified cut-off values of ctDNA of 0.2175 and 0.3388 ng/uL to distinguish CCA from healthy patients and those with benign biliary diseases at high sensitivity (88.7, 82.3%) and specificity (96.7%, 57.6%). Plasma cfDNA had the highest AUC in discriminating CCA from patients with benign biliary disease (AUC: 0.72, 95% CI: 0.61–0.83) compared to CA19-9 (AUC: 0.59, 95% CI: 0.46–0.71) and CEA (AUC: 0.50, 95% CI: 0.36–0.65), showing superior diagnostic efficacy of ctDNA [59].

More recently, preliminary results from the STAMP (adjuvant gemcitabine plus cisplatin (GemCis) versus capecitabine (CAP) in node-positive extrahepatic cholangiocarcinoma (CCA)) trial show feasibility of ctDNA monitoring prior to and during adjuvant chemotherapy, with improved RFS in patients who remained ctDNA negative on adjuvant chemotherapy relative to patients who were ctDNA positive [60]. The utility of tumor-informed ctDNA testing-based minimal residual disease detection in CCA was also recently exemplified in a case study published by Yu et al. [61]. In this case, tumor-informed ctDNA testing identified high levels of microsatellite instability and tumor mutational burden, leading to early treatment with pembrolizumab and a DFS within the study follow up period of two years.

Given the wide genetic heterogeneity of CCA, investigation of tumor mutational profiles with ctDNA is valuable. One multi-institutional study of 1671 patients with advanced biliary tract cancer showed fibroblast growth factor receptor 2 (FGFR2) fusions, isocitrate dehydrogenase 1 (IDH1) mutations, and BRAF V600E mutations to be clonal alterations, likely representing early oncogenic drivers [62]. A smaller scale study of 71 patients with CCA showed alterations in TP53 (38%), KRAS (28%), and PIK3CA (14%) as the most common [63]. Another study surveying the mutational landscape of biliary tract cancers using ctDNA among 124 patients identified TP53 and KRAS as the most common alterations in all subtypes of disease, followed by FGFR2 for the intrahepatic subtype, ARID1A for the extrahepatic subtype, and CDK6, APC, and SMAD4 for the gallbladder subtype [64]. Interestingly, the spectrum of detectable alterations on ctDNA can vary based on age, as patients with early-onset biliary tract cancer (or less than 50 years of age) were shown to have higher rates of FGFR2 fusions or single-nucleotide variations (21%) compared to those greater than 50 years of age (2%, p = 0.2). Conversely, older patients had higher rates of TP53 mutations (67%) compared to early-onset cancer patients (35%, p = 0.6) [64].

Matching systemic therapy regimens based on ctDNA molecular testing may lead to improved treatment outcomes. For example, a study featuring 80 patients who underwent systemic treatment for biliary tract cancers showed significantly prolonged PFS (HR = 0.60 [0.37–0.99], p = 0.047) and higher rates of disease control (61% vs. 35%, p = 0.04) for patients whose therapy were molecularly matched to ctDNA and/or tissue-DNA genomic profiling compared to those with unmatched regimens [63]. Following targeted inhibitor treatment, serial ctDNA measurements can also provide insight into mechanisms of acquired resistance. For example, Varghese et al. (2021) [65] and Goyal et al. (2017) [66] have shown acquisition of new mutations for patients with metastatic iCCA on FGFR-targeting treatments, while Cleary et al. (2022) [67] has shown identification of secondary IDH1 and acquired IDH2 mutations following treatment with ivosidenib. Given that resistance may occur due to clonal evolution as well, single site biopsy results may not be reliable in capturing polyclonal states as ctDNA.

4. Secondary Liver Malignancies

Colorectal Liver Metastases (CRLM)

Colorectal cancer (CRC) is the third most lethal cancer worldwide and the most common cause of liver metastasis for young males [1, 3]. About 30–50% of patients with CRC experience liver metastasis, with a 10-year survival of only 5% [68]. Traditional biomarkers, such as carcinoembryonic antigen (CEA), are limited by low sensitivity and specificity in detecting CRLM [69]. In contrast, ctDNA has been shown to have high sensitivity rates across all stages of CRC [70].

Recently, the landmark DYNAMIC trial showed that ctDNA-guided management was noninferior to standard management for adjuvant therapy following curative-intent surgery for stage II CRC with respect to two and five-year RFS (Table 1) [28, 71, 72]. For example, five-year RFS was 88% for the ctDNA-guided group, which was similar to 87% in the standard management group (difference 1.1%, 95% CI: –5.8%–8%) [72]. Five-year OS was also similar between the two groups (93.8% vs. 93.3% for ctDNA vs. standard, HR: 1.05; 95% CI: 0.47–2.37, p = 0.887) [72]. Long-term follow-up of these trials showed the significance of ctDNA clearance at time of adjuvant therapy completion, as patients with ctDNA clearance had a much higher RFS (85.2%) compared to those with ctDNA persistence (20%) (HR: 15.4, 95% CI: 3.91–61.0, p 0.001) [73].

Similarly, a study featuring 48 patients with CRLM with paired pre- and post-hepatectomy ctDNA showed that negativity of ctDNA following hepatectomy, whether ctDNA+/– or ctDNA–/–, is associated with improved RFS compared to ctDNA+/+, after adjusting for prehepatectomy chemotherapy, synchronous disease, and presence of 2+ CRLM (ctDNA+/–: HR = 0.21, 95% CI 0.08–0.53; ctDNA–/–: HR = 0.21, 95% CI 0.08–0.56) [74]. These findings have led to a single-institution, risk-stratified, prospective trial evaluating ctDNA-directed chemotherapy for patients with following hepatectomy for CRLM (NCT05062317) (Table 3). Likewise, another study by Wehrle et al. (2023) [75] showed an association with positive postoperative ctDNA and increased likelihood of disease recurrence (p = 0.090). A study by Bolhuis et al. (2021) [76] showed early evidence for this relationship—detectable postoperative ctDNA was associated with shorter median RFS (4.8 vs. 12.1 months) and lack of response on pathology. Overall, a recent systematic review and meta-analysis by Wullaert et al. (2023) [13] summarized supporting evidence for ctDNA as a prognostic marker, as the presence of ctDNA following surgery had a hazard ratio of 3.12 (2.27–4.28, 95% CI) for recurrence and 5.04 (2.53–10.04, 95% CI) for overall survival.

There are ongoing studies assessing selection criteria for liver transplant for colorectal liver metastasis based on novel pre-transplant protocols, including suggestions that ctDNA may help assess disease burden prior to transplantation [77, 78, 79]. This suggestion has not yet been validated but is of interest in guiding patient selection in this relatively novel disease approach.

In terms of mutational landscape, ctDNA can also provide insight into mutations or genetic alterations driving CRLM. In the same study of 51 CRLM patients by Wehrle et al. (2023) [75], the most common mutations detected on ctDNA in the were TP53 (57%), APC (53%), KRAS, (37%) and EGFR (24%). Another study by Shi et al. (2022) [80] similarly found KRAS, APC, and TP53 to be the most commonly altered genes among 41 patients with metastatic CRC. Such patterns are in line with mutational profile analyses conducted on tissue for CRLM [81, 82]. Additionally, alterations on ctDNA have been shown to be associated with therapeutic response, as patients with low-KRAS mutational burden had improved response rates, PFS, and OS within the Shi et al. (2022) study [80]. On an epigenetic level, methylation status of certain gene promoters have also been used for diagnostic, prognostic, and monitoring purposes. A few examples of commercially available tests that detect altered promoter methylation patterns include the Epi proColon (SEPT9) [83], ColoDefense (SEPT9, SDC2) [84], SpecColon (SFRP2, SDC2) [85], and TriMeth (C9orf50, KCNQ5, CLIP4) [86]. Compared to standard diagnostic tumor biomarkers (e.g., CEA, CA19-9), methylation markers SEPT9, DCC, BOLL, and SFRP2 were shown to have a stronger correlation with tumor volume and operability [87].

5. Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid tumor with poor prognosis and high recurrence rates. It is the sixth leading cause of cancer mortality in both sexes, accounting for 5% of all cancer deaths globally [1]. A significant challenge in improving outcomes in PDAC is early detection of primary tumors and metastases. One study investigating ctDNA via ddPCR found higher rates of ctDNA detection in patients with occult metastases (41% vs. 14.6%, p = 0.001) compared to patients without occult metastases [88]. In fact, ctDNA was determined to be an independent predictor of occult metastases (OR: 3.113, p = 0.039) with a sensitivity of 66.7% and specificity of 81.6% [88].

In terms of survival, ctDNA has been found to be independently associated with worse OS and PFS on multivariable analysis of 104 patients with advanced pancreatic cancer and liver metastasis (HR = 3.1, 95% CI = 1.9–5.0, p 0.0001; HR 2.6, 95% CI = 1.7–4.0, p 0.0001, respectively) [89]. Additionally, detection of ctDNA correlates with increased number of liver lesions, presence of lung and/or peritoneal metastases, tumor burden, and CA19-9 levels [89]. PDAC with liver metastasis has been associated with increased rates of KRAS mutations (78%) and higher median maximum variant allele frequency (VAF) (1.9%) compared to other metastatic sites, indicating a more aggressive tumor biology [90]. In addition, somatic copy number alterations of ctDNA seems to be unique to PDAC patients with liver metastasis [91]. Overall, for pancreatic cancer generally, detection of KRAS-mutated ctDNA has been shown to feasibly predict early progression of pancreatic cancer [92], while somatic mutation burden on ctDNA can also predict treatment response to first-line chemotherapy [93].

For clinical management, ctDNA-guided treatment is now being explored following upfront resection of PDAC in the AGITG DYNAMIC-Pancreas trial. Preliminary results show association of ctDNA with earlier recurrence, as patients with positive ctDNA 5 weeks following tumor resection had a lower median RFS compared to ctDNA negative patients (13 vs. 22 months, HR: 0.52, p = 0.003) [94].

Regarding mutational landscape, ctDNA whole exome sequencing has been used to identify unique molecular profiles in patients with aggressive pancreatic cancer and those with liver metastasis. In particular, enrichment of somatic mutations in KRAS, LAMA1, FGFR1, and IFF01 in tumor cells and mutations pertaining to the adaptive immune response (HLA-H, HLA-DRB1, TRBV6-7) have been noted on ctDNA for pancreatic cancer with liver metastasis [95]. Concurrent KRAS copy number gains and somatic mutations on ctDNA have also been associated with extremely poor overall survival for patient with PDAC and metastatic liver lesions [96].

6. Lung Cancer

Lung cancer is the most frequently diagnosed cancer and primary cause of cancer-related deaths worldwide (18.7%) [1]. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases [97]. Approximately 15% of patients with NSCLC have metastasis to the liver, which is associated with the worst prognosis and resistance to targeted therapy against epidermal growth factor receptor (EGFR) [98, 99]. According to the National Cancer Comprehensive Network (NCCN) guidelines, ctDNA testing is warranted for patients with advanced NSCLC who are deemed medically unfit for invasive tissue sampling, have insufficient tissue sample for analysis, or uncertain timing of tissue acquisition. Sensitivity and specificity of ctDNA for NSCLC are 70–94% and 90% respectively [100, 101, 102].

Noninvasive versus Invasive Lung Evaluation (NSCLC) features the most extensive collection of ctDNA related studies. In the NILE trial, ctDNA was shown to have a 100% positive predictive value and greater than 98.2% concordance with tissue for FDA-approved targets (e.g., EGFR, ALK, ROS1, BRAF) in 34 patients with ctDNA testing prior to treatment of metastatic NSCLC [103]. Additionally, ctDNA was shown to be noninferior compared to standard-of-care tissue genotyping for identifying guideline-recommended biomarkers while decreasing median turnaround time (9 vs. 15 days, p 0.0001) compared to tissue [103]. The ACCELERATE trial further underscored the ability of ctDNA to decrease result turn-around time, as patients with advanced NSCLC and ctDNA testing had a lower median time from referral to treatment initiation compared to those undergoing tissue genotyping (39 days (interquartile range (IQR), 27–52) vs. 62 (IQR, 44–82, p 0.001) [104]. When assessed in a larger multicohort study through the Blood First Assay Screening Trial (BFAST), ctDNA guided treatment of patients with ALK mutations had a high objective response rate (87.4% [78.5–93.5] by investigator, 92% [84.1–96.7] by independent facility) [105]. One recent nonrandomized controlled trial demonstrated feasibility for using ctDNA in de-escalating tyrosine kinase inhibitor treatment following local consolidative therapy in advanced NSCLC patients while attaining full remission, thereby minimizing unnecessary treatment toxicity [106].

A few studies have investigated the use of ctDNA specifically in the context of liver metastasis for lung cancer. One study noted that concordance of therapeutically targetable mutations on ctDNA with tissue-based genotyping results was highest for patients with liver metastases (100%, 13/13) compared to patients with metastatic disease to other sites (46.2% concordance), indicating promising potential for the clinical applicability of ctDNA-guided treatment for this patient population [107]. When surveying the mutational landscape of 115 patients with NSCLC and liver metastasis, Zhao et al. (2024) [108] identified TP53 and EGFR as the most frequently altered genes on ctDNA. This is consistent with findings from Jiang et al. (2021) [109], who found TP53 and EGFR to be the most commonly mutated genes for lung adenocarcinoma with liver metastasis. Interestingly, this group also noted higher similarity in mutational and copy number between paired primary lesions and metastases in patients with liver metastases compared to those with brain metastases, indicating a more linear progression model for hepatic metastatic lesion development [109]. Another study by Lam et al. (2021) [110] found that visceral metastasis (hepatic, adrenal, renal, or splenic) was associated with increased ctDNA VAF and greater tumor burden. Their discussion highlights a hypothesis regarding decreased clearance of ctDNA in hepatic metastasis specifically, potentially leading to high ctDNA VAF [110].

7. Breast Cancer

Breast cancer is the most common malignancy in female patients globally, with the liver being third most common site of metastasis [1, 111]. Several studies have explored the utility of ctDNA in the context of breast cancer at various stages of treatment. For example, a study following 283 patients with a tumor-informed approach found that ctDNA positivity was a notable adverse prognostic factor for distant RFS for both triple negative (TNBC) and hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative cancer subtypes [112]. Interestingly, ctDNA concentration was found to be strongly associated with pathologic complete response and residual cancer burden in TNBC specifically [112]. The I-SPY2 trial results also support the use of ctDNA as a tool for guiding de-escalation of therapy, as early ctDNA clearance at 3 weeks following treatment initiation predicted good outcomes [113]. Another multicenter trial (plasmaMATCH) showed a high sensitivity of digital PCR ctDNA testing (93%) and high concordance rate with targeted sequencing (96–99%) [114]. Among 1044 patients, 533 (51.1%) were identified to have potentially targetable mutations (e.g., PIK3CA, ESR1, HER2, AKT1, PTEN). Additionally, patients who underwent ctDNA guided treatment (neratininb for HER2 mutant and capivasertib for AKT1 mutation) were found to have comparable durable responses to those who received treatments based on tissue testing [114].

For liver metastasis specifically, one multicenter study featuring 223 metastatic breast cancer patients (NCT05079074), of which 30.9% (72/233) had liver metastasis, demonstrated prolonged PFS for patients with no alterations detected on pre-treatment ctDNA compared to patients with at least 1–2 or 3–4 alterations on ctDNA (6.63 vs. 5.70 vs. 4.90 months respectively, p 0.05) [115]. Another study including 58 patients with metastatic breast cancer showed that copy number changes of ctDNA was significantly correlated with the presence of hepatic metastases (p = 0.002), suggesting a high rate of variant mutant DNA in the circulation of patients with metastatic disease [116].

Evaluation of ctDNA has also been used to study tumoral genetics and resistance patterns in breast cancer. One study surveying the ctDNA mutational landscape in Chinese women with breast cancer found the most prevalent mutated genes to be PIK3CA (24%), TP53 (43%), and ERBB2 (14%) [117]. They also identified significant associations between cfDNA yield and cancer stage (p = 0.033, r = 0.9) [117]. A proof-of-concept study featuring a ER+/HER2+ patient with mixed invasive ductal-lobular carcinoma showed the ability of ctDNA to capture mutations present in the primary tumor and/or liver metastasis, while primary tumor biopsy sites failed to reliably identify all mutations in the metastasis [118]. Another study evaluating paired plasma and tissue samples from 40 HR+ early-stage breast cancer patients found a broader mutation spectrum with ctDNA compared to tissue DNA, while maintaining dependable assessments of microsatellite instability, tumor mutational burden, loss of heterogeneity, and homologous recombination deficiency [119]. Significantly, ctDNA was able to detect mutations in ESR1 early—potentially identifying patients who are at risk for resistance to endocrine therapy—along with mutations in DNA damage response and proliferative signaling pathways [118]. For example, one patient who had tumor recurrence and liver metastasis had mutations in PIK3CA, ESR1, and TP53 [119]. A separate study with whole genome sequencing from ctDNA in two HR+/HER2- breast cancer patients with liver metastasis identified similar driver mutations (e.g., PIK3CA, ESR1) and convergent evolution for drug resistant mutants following endocrine therapy [120].

8. Challenges & Future Directions

Several challenges remain for the use of ctDNA in clinical practice. Firstly, concordance between ctDNA and tissue-based DNA have shown varying rates based on cancer and platform type [29]. When possible, exploration of paired tissue sequencing may provide insight into the presence of tumor-based mutations, while also elucidating ctDNA specific mutations. Additionally, the differences in sequencing methods among commercial and research platforms for ctDNA detection and sequencing may impede comprehensive interpretation of results. Furthermore, certain somatic alterations, such as detection of fusions, are still not as accurately captured on ctDNA compared to primary tissue biopsy sequencing [121]. In the clinic, timing and administration of ctDNA testing may be logistically difficult to obtain both pre- and post-operative testing results for each patient. Furthermore, managing cost of the commercially available assays is necessary to ensure equitable access to cancer care. Given the expanding nature of this field, initial studies have featured smaller sample sizes for feasibility testing and retrospective experimental designs, leading to limitations in drawing reliable conclusions. Thus, increased recruitment, prospective studies, and incorporation into clinical trials may improve our knowledge and understanding of ctDNA and its results. Additionally, although advantages of ctDNA testing include its non-invasive nature, rapid result times, and potential for treatment de-escalation, the impact of liquid biopsy with respect to patient quality-of-life remains to be explored and will likely require investigation based on specific cancer type.

Despite these challenges, the future clinical applications of ctDNA are promising. For diagnostic purposes, ctDNA may be helpful in cancer types for which adequate tissue sampling is difficult to obtain. As prospective studies evolve for primary liver malignancies, ctDNA can be used to select neoadjuvant therapy regimens for high-risk patients and guide de-escalation of therapy by monitoring minimal residual disease. Such changes can significantly impact patient care by decreasing time to treatment, minimizing unnecessary toxicity, and tailoring targeted therapy. ctDNA-guided de-escalation of adjuvant therapy or post-treatment surveillance can also reduce overall cost of care [122, 123]. Additionally, ctDNA may identify unique, novel mutations for targeted therapy development and allow for serial monitoring of clonal evolution of tumors and development of resistance mechanisms in a dynamic manner [66, 124]. Particularly for immunotherapy, several parameters (e.g., microsatellite instability, high tumor mutational burden) are identifiable from ctDNA and can provide personalized predictive value for the benefit of immune checkpoint inhibitors, as shown in the KEYNOTE 158, MYSTIC phase III, and OAK (atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer) clinical trials [125, 126, 127, 128]. On a larger scale, with a refined understanding of ctDNA results, liquid biopsy may even be considered for screening purposes, although associated ethical and cost-related challenges may arise.

For research, several avenues remain to be explored for the use of ctDNA within liver cancer. In addition to delineating mutational profiles on ctDNA, epigenomic analysis of tumor-specific methylation patterns has been preliminarily explored and warrants further investigation [129]. As highlighted above, the utility of ctDNA within patients receiving liver transplant for liver cancer also requires further investigation to ensure unwanted sources of cfDNA are not introduced by the donor organ during data acquisition and interpretation. Combining the use of ctDNA with established serum tumor biomarkers and other liver-specific factors is also an open area for investigation for clinical research for patients with cancer and those undergoing liver transplant. On a mechanistic level, research into the exact origins and biologic basis of circulating tumor cells and ctDNA may be helpful in elucidating the mismatch between ctDNA mutational profiles and tumor tissue profiles. The sensitivity and specificity of ctDNA in early-stage malignancies, including primary liver cancers, remains limited due to smaller amounts of ctDNA shedding [22, 130]. For example, large-scale ctDNA profiling of 236 HCC patients with staging data revealed a lower sensitivity for ctDNA detection for stage I-III cancers (68%, 95% CI: 62.6%–73.4%) compared to stage IV cancers (86.3%, 95% CI: 83.6%–89%) [130]. Although strides are being made to detect alterations in ctDNA for early stage cancers [22, 101, 131], further research is needed to optimize detection of localized cancers at such timepoints for diagnostic purposes. Lastly, for studies evaluating ctDNA in other primary cancer types (e.g., colorectal, pancreas, lung, breast), including subgroup analyses based on metastatic site is crucial to broadening our knowledge of the mutational landscape and tumor biology in secondary liver malignancies.

9. Conclusion

Circulating tumor DNA has several advantages for liver malignancies in the modern era, such as its non-invasive nature, quick turnaround time with results, detection of actionable mutations, and monitoring of minimal residual disease. Information generated from this tool can guide treatment decisions, such as de-escalation of therapy, initiation of targeted therapy, or treatment switches, thereby facilitating precision oncology. Barriers and challenges to consider when implementing ctDNA testing in the clinical setting include evaluating concordance with patient tumor tissue, deciding on the use of tumor-informed versus uninformed approaches, and timing of pre- and post-treatment ctDNA. As liquid biopsy platforms evolve, ctDNA will become a clinically significant tool in guiding neoadjuvant and adjuvant therapy agents and timelines, while shifting diagnostic and surveillance guidelines. Future studies involving liver cancers should include larger scale studies with paired tissue sampling, while prospective clinical trials can consider integrating serial ctDNA measurements into treatment arms and subgroup analyses based on metastatic site to evaluate its full utility.

References

[1] Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a Cancer Journal for Clinicians. 2024; 74: 229–263. https://doi.org/10.3322/caac.21834.

[2] Wang ZG, He ZY, Chen YY, Gao H, Du XL. Incidence and survival outcomes of secondary liver cancer: a Surveillance Epidemiology and End Results database analysis. Translational Cancer Research. 2021; 10: 1273–1283. https://doi.org/10.21037/tcr-20-3319.

[3] Horn SR, Stoltzfus KC, Lehrer EJ, Dawson LA, Tchelebi L, Gusani NJ, et al. Epidemiology of liver metastases. Cancer Epidemiology. 2020; 67: 101760. https://doi.org/10.1016/j.canep.2020.101760.

[4] Nault JC, Cheng AL, Sangro B, Llovet JM. Milestones in the pathogenesis and management of primary liver cancer. Journal of Hepatology. 2020; 72: 209–214. https://doi.org/10.1016/j.jhep.2019.11.006.

[5] Marrero JA, Kulik LM, Sirlin CB, Zhu AX, Finn RS, Abecassis MM, et al. Diagnosis, Staging, and Management of Hepatocellular Carcinoma: 2018 Practice Guidance by the American Association for the Study of Liver Diseases. Hepatology (Baltimore, Md.). 2018; 68: 723–750. https://doi.org/10.1002/hep.29913.

[6] Roberts LR, Sirlin CB, Zaiem F, Almasri J, Prokop LJ, Heimbach JK, et al. Imaging for the diagnosis of hepatocellular carcinoma: A systematic review and meta-analysis. Hepatology (Baltimore, Md.). 2018; 67: 401–421. https://doi.org/10.1002/hep.29487.

[7] Kopystecka A, Patryn R, Leśniewska M, Budzyńska J, Kozioł I. The Use of ctDNA in the Diagnosis and Monitoring of Hepatocellular Carcinoma-Literature Review. International Journal of Molecular Sciences. 2023; 24: 9342. https://doi.org/10.3390/ijms24119342.

[8] Renzulli M, Pecorelli A, Brandi N, Brocchi S, Tovoli F, Granito A, et al. The Feasibility of Liver Biopsy for Undefined Nodules in Patients under Surveillance for Hepatocellular Carcinoma: Is Biopsy Really a Useful Tool? Journal of Clinical Medicine. 2022; 11: 4399. https://doi.org/10.3390/jcm11154399.

[9] Sekiguchi M, Matsuda T. Limited usefulness of serum carcinoembryonic antigen and carbohydrate antigen 19-9 levels for gastrointestinal and whole-body cancer screening. Scientific Reports. 2020; 10: 18202. https://doi.org/10.1038/s41598-020-75319-8.

[10] Hanif H, Ali MJ, Susheela AT, Khan IW, Luna-Cuadros MA, Khan MM, et al. Update on the applications and limitations of alpha-fetoprotein for hepatocellular carcinoma. World Journal of Gastroenterology. 2022; 28: 216–229. https://doi.org/10.3748/wjg.v28.i2.216.

[11] Tivey A, Church M, Rothwell D, Dive C, Cook N. Circulating tumour DNA - looking beyond the blood. Nature Reviews. Clinical Oncology. 2022; 19: 600–612. https://doi.org/10.1038/s41571-022-00660-y.

[12] Zhang Z, Chen P, Xie H, Cao P. Using circulating tumor DNA as a novel biomarker to screen and diagnose hepatocellular carcinoma: A systematic review and meta-analysis. Cancer Medicine. 2020; 9: 1349–1364. https://doi.org/10.1002/cam4.2799.

[13] Wullaert L, van Rees JM, Martens JWM, Verheul HMW, Grünhagen DJ, Wilting SM, et al. Circulating Tumour DNA as Biomarker for Colorectal Liver Metastases: A Systematic Review and Meta-Analysis. Cells. 2023; 12: 2520. https://doi.org/10.3390/cells12212520.

[14] Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Research. 1977; 37: 646–650.

[15] Wang Z, Li L, Kuang Y, Yao J, Xu F, Chen Y. Simultaneous quantification of multiple single nucleotide variants in PIK3CA ctDNA using mass-tagged LCR probe sets. Talanta. 2023; 258: 124426. https://doi.org/10.1016/j.talanta.2023.124426.

[16] Chen X, Gole J, Gore A, He Q, Lu M, Min J, et al. Non-invasive early detection of cancer four years before conventional diagnosis using a blood test. Nature Communications. 2020; 11: 3475. https://doi.org/10.1038/s41467-020-17316-z.

[17] Cai J, Chen L, Zhang Z, Zhang X, Lu X, Liu W, et al. Genome-wide mapping of 5-hydroxymethylcytosines in circulating cell-free DNA as a non-invasive approach for early detection of hepatocellular carcinoma. Gut. 2019; 68: 2195–2205. https://doi.org/10.1136/gutjnl-2019-318882.

[18] Cheishvili D, Wong C, Karim MM, Kibria MG, Jahan N, Das PC, et al. A high-throughput test enables specific detection of hepatocellular carcinoma. Nature Communications. 2023; 14: 3306. https://doi.org/10.1038/s41467-023-39055-7.

[19] Han K, Leung EW, Zou J, Wang TT, Yu C, Zhao Z, et al. Viral Genome Sequencing for Ultrasensitive Detection of Circulating Tumor DNA. International Journal of Radiation Oncology, Biology, Physics. 2019; 105: S177. https://doi.org/10.1016/j.ijrobp.2019.06.214.

[20] Stroun M, Lyautey J, Lederrey C, Olson-Sand A, Anker P. About the possible origin and mechanism of circulating DNA apoptosis and active DNA release. Clinica Chimica Acta; International Journal of Clinical Chemistry. 2001; 313: 139–142. https://doi.org/10.1016/s0009-8981(01)00665-9.

[21] Stejskal P, Goodarzi H, Srovnal J, Hajdúch M, van ‘t Veer LJ, Magbanua MJM. Circulating tumor nucleic acids: biology, release mechanisms, and clinical relevance. Molecular Cancer. 2023; 22: 15. https://doi.org/10.1186/s12943-022-01710-w.

[22] Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of circulating tumor DNA in early- and late-stage human malignancies. Science Translational Medicine. 2014; 6: 224ra24. https://doi.org/10.1126/scitranslmed.3007094.

[23] Banys-Paluchowski M, Fehm TN, Grimm-Glang D, Rody A, Krawczyk N. Liquid Biopsy in Metastatic Breast Cancer: Current Role of Circulating Tumor Cells and Circulating Tumor DNA. Oncology Research and Treatment. 2022; 45: 4–11. https://doi.org/10.1159/000520561.

[24] Lee MS, Kaseb AO, Pant S. The Emerging Role of Circulating Tumor DNA in Non-Colorectal Gastrointestinal Cancers. Clinical Cancer Research: an Official Journal of the American Association for Cancer Research. 2023; 29: 3267–3274. https://doi.org/10.1158/1078-0432.CCR-22-3626.

[25] Parikh ND, Mehta AS, Singal AG, Block T, Marrero JA, Lok AS. Biomarkers for the Early Detection of Hepatocellular Carcinoma. Cancer Epidemiology, Biomarkers & Prevention: a Publication of the American Association for Cancer Research, Cosponsored by the American Society of Preventive Oncology. 2020; 29: 2495–2503. https://doi.org/10.1158/1055-9965.EPI-20-0005.

[26] Chaiteerakij R, Addissie BD, Roberts LR. Update on biomarkers of hepatocellular carcinoma. Clinical Gastroenterology and Hepatology: the Official Clinical Practice Journal of the American Gastroenterological Association. 2015; 13: 237–245. https://doi.org/10.1016/j.cgh.2013.10.038.

[27] Cai Z, Chen G, Zeng Y, Dong X, Li Z, Huang Y, et al. Comprehensive Liquid Profiling of Circulating Tumor DNA and Protein Biomarkers in Long-Term Follow-Up Patients with Hepatocellular Carcinoma. Clinical Cancer Research: an Official Journal of the American Association for Cancer Research. 2019; 25: 5284–5294. https://doi.org/10.1158/1078-0432.CCR-18-3477.

[28] Tie J, Cohen JD, Lahouel K, Lo SN, Wang Y, Kosmider S, et al. Circulating Tumor DNA Analysis Guiding Adjuvant Therapy in Stage II Colon Cancer. The New England Journal of Medicine. 2022; 386: 2261–2272. https://doi.org/10.1056/NEJMoa2200075.

[29] Narayan RR, Goldman DA, Gonen M, Reichel J, Huberman KH, Raj S, et al. Peripheral Circulating Tumor DNA Detection Predicts Poor Outcomes After Liver Resection for Metastatic Colorectal Cancer. Annals of Surgical Oncology. 2019; 26: 1824–1832. https://doi.org/10.1245/s10434-019-07201-5.

[30] Hsu C, Chang YF, Yen CJ, Xu YW, Dong M, Tong YZ. Combination of GT90001 and nivolumab in patients with advanced hepatocellular carcinoma: a multicenter, single-arm, phase 1b/2 study. BMC Medicine. 2023; 21: 395. https://doi.org/10.1186/s12916-023-03098-w.

[31] Portelinha A, Thompson S, Smith RA, Da Silva Ferreira M, Asgari Z, Knezevic A, et al. ASN007 is a selective ERK1/2 inhibitor with preferential activity against RAS-and RAF-mutant tumors. Cell Reports. Medicine. 2021; 2: 100350. https://doi.org/10.1016/j.xcrm.2021.100350.

[32] Wang J, Huang A, Wang YP, Yin Y, Fu PY, Zhang X, et al. Circulating tumor DNA correlates with microvascular invasion and predicts tumor recurrence of hepatocellular carcinoma. Annals of Translational Medicine. 2020; 8: 237. https://doi.org/10.21037/atm.2019.12.154.

[33] An Y, Guan Y, Xu Y, Han Y, Wu C, Bao C, et al. The diagnostic and prognostic usage of circulating tumor DNA in operable hepatocellular carcinoma. American Journal of Translational Research. 2019; 11: 6462–6474.

[34] Hirai M, Kinugasa H, Nouso K, Yamamoto S, Terasawa H, Onishi Y, et al. Prediction of the prognosis of advanced hepatocellular carcinoma by TERT promoter mutations in circulating tumor DNA. Journal of Gastroenterology and Hepatology. 2021; 36: 1118–1125. https://doi.org/10.1111/jgh.15227.

[35] Tokuhisa Y, Iizuka N, Sakaida I, Moribe T, Fujita N, Miura T, et al. Circulating cell-free DNA as a predictive marker for distant metastasis of hepatitis C virus-related hepatocellular carcinoma. British Journal of Cancer. 2007; 97: 1399–1403. https://doi.org/10.1038/sj.bjc.6604034.

[36] Wehrle CJ, Hong H, Kamath S, Schlegel A, Fujiki M, Hashimoto K, et al. Tumor Mutational Burden From Circulating Tumor DNA Predicts Recurrence of Hepatocellular Carcinoma After Resection: An Emerging Biomarker for Surveillance. Annals of Surgery. 2024; 280: 504–513. https://doi.org/10.1097/SLA.0000000000006386.

[37] Marron TU, Brennan L, Hamon P, Fiel MI, Ward SC, Zhu YO, et al. Abstract 2178: Circulating tumor DNA (ctDNA) correlates closely with tumor necrosis and relapse-free survival (RFS) in hepatocellular carcinoma (HCC) patients treated with perioperative cemiplimab. Cancer Research. 2023; 83: 2178. https://doi.org/10.1158/1538-7445.AM2023-2178.

[38] Hsu CH, Lu S, Abbas A, Guan Y, Zhu AX, Aleshin A, et al. Longitudinal and personalized detection of circulating tumor DNA (ctDNA) for monitoring efficacy of atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma (HCC). Journal of Clinical Oncology. 2020; 38: 3531–3531. https://doi.org/10.1200/JCO.2020.38.15_suppl.353.

[39] Raj R, Wehrle CJ, Aykun N, Stitzel H, Ma WW, Krishnamurthi S, et al. Immunotherapy Plus Locoregional Therapy Leading to Curative-Intent Hepatectomy in HCC: Proof of Concept Producing Durable Survival Benefits Detectable with Liquid Biopsy. Cancers. 2023; 15: 5220. https://doi.org/10.3390/cancers15215220.

[40] von Felden J, Craig AJ, Garcia-Lezana T, Labgaa I, Haber PK, D’Avola D, et al. Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma. Oncogene. 2021; 40: 140–151. https://doi.org/10.1038/s41388-020-01519-1.

[41] Ikeda S, Tsigelny IF, Skjevik ÅA, Kono Y, Mendler M, Kuo A, et al. Next-Generation Sequencing of Circulating Tumor DNA Reveals Frequent Alterations in Advanced Hepatocellular Carcinoma. The Oncologist. 2018; 23: 586–593. https://doi.org/10.1634/theoncologist.2017-0479.

[42] Matsuda Y, Ichida T, Matsuzawa J, Sugimura K, Asakura H. p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology. 1999; 116: 394–400. https://doi.org/10.1016/s0016-5085(99)70137-x.

[43] Wong IH, Lo YM, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Research. 1999; 59: 71–73.

[44] Tchou JC, Lin X, Freije D, Isaacs WB, Brooks JD, Rashid A, et al. GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas. International Journal of Oncology. 2000; 16: 663–676. https://doi.org/10.3892/ijo.16.4.663.

[45] Wang J, Qin Y, Li B, Sun Z, Yang B. Detection of aberrant promoter methylation of GSTP1 in the tumor and serum of Chinese human primary hepatocellular carcinoma patients. Clinical Biochemistry. 2006; 39: 344–348. https://doi.org/10.1016/j.clinbiochem.2006.01.008.

[46] Yeo W, Wong N, Wong WL, Lai PBS, Zhong S, Johnson PJ. High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma. Liver International: Official Journal of the International Association for the Study of the Liver. 2005; 25: 266–272. https://doi.org/10.1111/j.1478-3231.2005.01084.x.

[47] Tangkijvanich P, Hourpai N, Rattanatanyong P, Wisedopas N, Mahachai V, Mutirangura A. Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma. Clinica Chimica Acta; International Journal of Clinical Chemistry. 2007; 379: 127–133. https://doi.org/10.1016/j.cca.2006.12.029.

[48] Wehrle CJ, Kusakabe J, Akabane M, Maspero M, Zervos B, Modaresi Esfeh J, et al. Expanding Selection Criteria in Deceased Donor Liver Transplantation for Hepatocellular Carcinoma: Long-term Follow-up of a National Registry and 2 Transplant Centers. Transplantation. 2024. https://doi.org/10.1097/TP.0000000000005097. (online ahead of print)

[49] Fares S, Wehrle CJ, Hong H, Sun K, Jiao C, Zhang M, et al. Emerging and Clinically Accepted Biomarkers for Hepatocellular Carcinoma. Cancers. 2024; 16: 1453. https://doi.org/10.3390/cancers16081453.

[50] Wehrle CJ, Raj R, Maspero M, Satish S, Eghtesad B, Pita A, et al. Risk assessment in liver transplantation for hepatocellular carcinoma: long-term follow-up of a two-centre experience. International Journal of Surgery (London, England). 2024; 110: 2818–2831. https://doi.org/10.1097/JS9.0000000000001104.

[51] Huang A, Guo DZ, Zhang X, Sun Y, Zhang SY, Zhang X, et al. Serial circulating tumor DNA profiling predicts tumor recurrence after liver transplantation for liver cancer. Hepatology International. 2024; 18: 254–264. https://doi.org/10.1007/s12072-023-10594-x.

[52] Vogel A, Bridgewater J, Edeline J, Kelley RK, Klümpen HJ, Malka D, et al. Biliary tract cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up. Annals of Oncology: Official Journal of the European Society for Medical Oncology. 2023; 34: 127–140. https://doi.org/10.1016/j.annonc.2022.10.506.

[53] Mavros MN, Economopoulos KP, Alexiou VG, Pawlik TM. Treatment and Prognosis for Patients With Intrahepatic Cholangiocarcinoma: Systematic Review and Meta-analysis. JAMA Surgery. 2014; 149: 565–574. https://doi.org/10.1001/jamasurg.2013.5137.

[54] Banales JM, Marin JJG, Lamarca A, Rodrigues PM, Khan SA, Roberts LR, et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nature Reviews. Gastroenterology & Hepatology. 2020; 17: 557–588. https://doi.org/10.1038/s41575-020-0310-z.

[55] Ruff SM, Pawlik TM. Clinical management of intrahepatic cholangiocarcinoma: surgical approaches and systemic therapies. Frontiers in Oncology. 2024; 14: 1321683. https://doi.org/10.3389/fonc.2024.1321683.

[56] Arechederra M, Rullán M, Amat I, Oyon D, Zabalza L, Elizalde M, et al. Next-generation sequencing of bile cell-free DNA for the early detection of patients with malignant biliary strictures. Gut. 2022; 71: 1141–1151. https://doi.org/10.1136/gutjnl-2021-325178.

[57] Choi WJ, Ivanics T, Gravely A, Gallinger S, Sapisochin G, O’Kane GM. Optimizing Circulating Tumour DNA Use in the Perioperative Setting for Intrahepatic Cholangiocarcinoma: Diagnosis, Screening, Minimal Residual Disease Detection and Treatment Response Monitoring. Annals of Surgical Oncology. 2023; 30: 3849–3863. https://doi.org/10.1245/s10434-023-13126-x.

[58] Uson Junior PLS, Majeed U, Yin J, Botrus G, Sonbol MB, Ahn DH, et al. Cell-Free Tumor DNA Dominant Clone Allele Frequency Is Associated With Poor Outcomes in Advanced Biliary Cancers Treated With Platinum-Based Chemotherapy. JCO Precision Oncology. 2022; 6: e2100274. https://doi.org/10.1200/PO.21.00274.

[59] Wintachai P, Lim JQ, Techasen A, Lert-Itthiporn W, Kongpetch S, Loilome W, et al. Diagnostic and Prognostic Value of Circulating Cell-Free DNA for Cholangiocarcinoma. Diagnostics (Basel, Switzerland). 2021; 11: 999. https://doi.org/10.3390/diagnostics11060999.

[60] Yoo C, Laliotis G, Jeong H, Jeong JH, Kim KP, Lee S, et al. Utility of circulating tumor DNA (ctDNA) as a predictive biomarker for disease monitoring in patients (pts) with cholangiocarcinoma (CCA) before and during adjuvant chemotherapy (ACT): Sub-analysis of the randomized phase 2 STAMP trial. Journal of Clinical Oncology. 2023; 41: 4123–4123. https://doi.org/10.1200/JCO.2023.41.16_suppl.4123.

[61] Yu J, Avriett TA, Ray CM, Kim RD. Circulating tumor DNA analysis guiding adjuvant treatment in resected stage III cholangiocarcinoma: a case report. Journal of Gastrointestinal Oncology. 2024; 15: 485–490. https://doi.org/10.21037/jgo-23-815.

[62] Berchuck JE, Facchinetti F, DiToro DF, Baiev I, Majeed U, Reyes S, et al. The clinical landscape of cell-free DNA alterations in 1671 patients with advanced biliary tract cancer. Annals of Oncology: Official Journal of the European Society for Medical Oncology. 2022; 33: 1269–1283. https://doi.org/10.1016/j.annonc.2022.09.150.

[63] Okamura R, Kurzrock R, Mallory RJ, Fanta PT, Burgoyne AM, Clary BM, et al. Comprehensive genomic landscape and precision therapeutic approach in biliary tract cancers. International Journal of Cancer. 2021; 148: 702–712. https://doi.org/10.1002/ijc.33230.

[64] Mody K, Kasi PM, Yang J, Surapaneni PK, Bekaii-Saab T, Ahn DH, et al. Circulating Tumor DNA Profiling of Advanced Biliary Tract Cancers. JCO Precision Oncology. 2019; 3: 1–9. https://doi.org/10.1200/PO.18.00324.

[65] Varghese AM, Patel J, Janjigian YY, Meng F, Selcuklu SD, Iyer G, et al. Noninvasive Detection of Polyclonal Acquired Resistance to FGFR Inhibition in Patients With Cholangiocarcinoma Harboring FGFR2 Alterations. JCO Precision Oncology. 2021; 5: PO.20.00178. https://doi.org/10.1200/PO.20.00178.

[66] Goyal L, Saha SK, Liu LY, Siravegna G, Leshchiner I, Ahronian LG, et al. Polyclonal Secondary FGFR2 Mutations Drive Acquired Resistance to FGFR Inhibition in Patients with FGFR2 Fusion-Positive Cholangiocarcinoma. Cancer Discovery. 2017; 7: 252–263. https://doi.org/10.1158/2159-8290.CD-16-1000.

[67] Cleary JM, Rouaisnel B, Daina A, Raghavan S, Roller LA, Huffman BM, et al. Secondary IDH1 resistance mutations and oncogenic IDH2 mutations cause acquired resistance to ivosidenib in cholangiocarcinoma. NPJ Precision Oncology. 2022; 6: 61. https://doi.org/10.1038/s41698-022-00304-5.

[68] Zeineddine FA, Zeineddine MA, Yousef A, Gu Y, Chowdhury S, Dasari A, et al. Survival improvement for patients with metastatic colorectal cancer over twenty years. NPJ Precision Oncology. 2023; 7: 16. https://doi.org/10.1038/s41698-023-00353-4.

[69] Nyström H, Tavelin B, Björklund M, Naredi P, Sund M. Improved tumour marker sensitivity in detecting colorectal liver metastases by combined type IV collagen and CEA measurement. Tumour Biology: the Journal of the International Society for Oncodevelopmental Biology and Medicine. 2015; 36: 9839–9847. https://doi.org/10.1007/s13277-015-3729-z.

[70] Bessa X, Vidal J, Balboa JC, Márquez C, Duenwald S, He Y, et al. High accuracy of a blood ctDNA-based multimodal test to detect colorectal cancer. Annals of Oncology: Official Journal of the European Society for Medical Oncology. 2023; 34: 1187–1193. https://doi.org/10.1016/j.annonc.2023.09.3113.

[71] Tie J, Cohen JD, Wang Y, Christie M, Simons K, Lee M, et al. Circulating Tumor DNA Analyses as Markers of Recurrence Risk and Benefit of Adjuvant Therapy for Stage III Colon Cancer. JAMA Oncology. 2019; 5: 1710–1717. https://doi.org/10.1001/jamaoncol.2019.3616.

[72] Tie J, Wang Y, Lo SN, Lahouel K, Cohen JD, Wong R, et al. Circulating tumor DNA analysis guiding adjuvant therapy in stage II colon cancer: Overall survival and updated 5-year results from the randomized DYNAMIC trial. Journal of Clinical Oncology. 2024; 42: 108–108. https://doi.org/10.1200/JCO.2024.42.16_suppl.108.

[73] Oh DY, He AR, Qin S, Chen LT, Okusaka T, Vogel A, et al. 78P Updated overall survival (OS) from the phase III TOPAZ-1 study of durvalumab (D) or placebo (PBO) plus gemcitabine and cisplatin (+ GC) in patients (pts) with advanced biliary tract cancer (BTC). Annals of Oncology. 2022; 33: S1462–S1463. https://doi.org/10.1016/j.annonc.2022.10.114.

[74] Newhook TE, Overman MJ, Chun YS, Dasari A, Tzeng CWD, Cao HST, et al. Prospective Study of Perioperative Circulating Tumor DNA Dynamics in Patients Undergoing Hepatectomy for Colorectal Liver Metastases. Annals of Surgery. 2023; 277: 813–820. https://doi.org/10.1097/SLA.0000000000005461.

[75] Wehrle CJ, Raj R, Aykun N, Orabi D, Stackhouse K, Chang J, et al. Circulating Tumor DNA in Colorectal Cancer Liver Metastasis: Analysis of Patients Receiving Liver Resection and Transplant. JCO Clinical Cancer Informatics. 2023; 7: e2300111. https://doi.org/10.1200/CCI.23.00111.

[76] Bolhuis K, van ‘t Erve I, Mijnals C, Delis-Van Diemen PM, Huiskens J, Komurcu A, et al. Postoperative circulating tumour DNA is associated with pathologic response and recurrence-free survival after resection of colorectal cancer liver metastases. EBioMedicine. 2021; 70: 103498. https://doi.org/10.1016/j.ebiom.2021.103498.

[77] Line PD, Dueland S. Transplantation for colorectal liver metastasis. Current Opinion in Organ Transplantation. 2024; 29: 23–29. https://doi.org/10.1097/MOT.0000000000001126.

[78] Wehrle CJ, Fujiki M, Schlegel A, Uysal M, Sobotka A, Whitsett Linganna M, et al. Intensive locoregional therapy before liver transplantation for colorectal cancer liver metastasis: A novel pretransplant protocol. Liver Transplantation: Official Publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society. 2024; 30: 1238–1249. https://doi.org/10.1097/LVT.0000000000000417.

[79] Wehrle CJ, Fujiki M, Schlegel A, Whitsett Linganna M, Pita A, Kim JK, et al. Update to ‘A Contemporary Systematic Review on Liver Transplantation for Unresectable Liver Metastasis of Colorectal Cancer’. Annals of Surgical Oncology. 2024; 31: 697–700. https://doi.org/10.1245/s10434-023-14611-z.

[80] Shi M, Yuan H, Ji J, Zhang S, Li Q, Chen Y, et al. Mutational landscape of circulating tumor DNA identifies distinct molecular features associated with therapeutic response in patients with metastatic colorectal cancer. Therapeutic Advances in Medical Oncology. 2022; 14: 17588359211070643. https://doi.org/10.1177/17588359211070643.

[81] Yang S, Qian L, Li Z, Li Y, Bai J, Zheng B, et al. Integrated Multi-Omics Landscape of Liver Metastases. Gastroenterology. 2023; 164: 407–423.e17. https://doi.org/10.1053/j.gastro.2022.11.029.

[82] Lee CS, Song IH, Lee A, Kang J, Lee YS, Lee IK, et al. Enhancing the landscape of colorectal cancer using targeted deep sequencing. Scientific Reports. 2021; 11: 8154. https://doi.org/10.1038/s41598-021-87486-3.

[83] Lofton-Day C, Model F, Devos T, Tetzner R, Distler J, Schuster M, et al. DNA methylation biomarkers for blood-based colorectal cancer screening. Clinical Chemistry. 2008; 54: 414–423. https://doi.org/10.1373/clinchem.2007.095992.

[84] Zhao G, Li H, Yang Z, Wang Z, Xu M, Xiong S, et al. Multiplex methylated DNA testing in plasma with high sensitivity and specificity for colorectal cancer screening. Cancer Medicine. 2019; 8: 5619–5628. https://doi.org/10.1002/cam4.2475.

[85] Zhao G, Ma Y, Li H, Li S, Zhu Y, Liu X, et al. A novel plasma based early colorectal cancer screening assay base on methylated SDC2 and SFRP2. Clinica Chimica Acta; International Journal of Clinical Chemistry. 2020; 503: 84–89. https://doi.org/10.1016/j.cca.2020.01.010.

[86] Jensen SØ Øgaard N, Ørntoft MBW, Rasmussen MH, Bramsen JB, Kristensen H, et al. Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study. Clinical Epigenetics. 2019; 11: 158. https://doi.org/10.1186/s13148-019-0757-3.

[87] Bhangu JS, Beer A, Mittlböck M, Tamandl D, Pulverer W, Schönthaler S, et al. Circulating Free Methylated Tumor DNA Markers for Sensitive Assessment of Tumor Burden and Early Response Monitoring in Patients Receiving Systemic Chemotherapy for Colorectal Cancer Liver Metastasis. Annals of Surgery. 2018; 268: 894–902. https://doi.org/10.1097/SLA.0000000000002901.

[88] Hata T, Mizuma M, Iseki M, Takadate T, Ishida M, Nakagawa K, et al. Circulating tumor DNA as a predictive marker for occult metastases in pancreatic cancer patients with radiographically non-metastatic disease. Journal of Hepato-biliary-pancreatic Sciences. 2021; 28: 648–658. https://doi.org/10.1002/jhbp.993.

[89] Uesato Y, Sasahira N, Ozaka M, Sasaki T, Takatsuki M, Zembutsu H. Evaluation of circulating tumor DNA as a biomarker in pancreatic cancer with liver metastasis. PloS One. 2020; 15: e0235623. https://doi.org/10.1371/journal.pone.0235623.

[90] Umemoto K, Sunakawa Y, Ueno M, Furukawa M, Mizuno N, Sudo K, et al. Clinical significance of circulating-tumour DNA analysis by metastatic sites in pancreatic cancer. British Journal of Cancer. 2023; 128: 1603–1608. https://doi.org/10.1038/s41416-023-02189-y.

[91] Wei T, Zhang J, Li J, Chen Q, Zhi X, Tao W, et al. Genome-wide profiling of circulating tumor DNA depicts landscape of copy number alterations in pancreatic cancer with liver metastasis. Molecular Oncology. 2020; 14: 1966–1977. https://doi.org/10.1002/1878-0261.12757.

[92] Watanabe F, Suzuki K, Aizawa H, Endo Y, Takayama Y, Kakizawa N, et al. Circulating tumor DNA in molecular assessment feasibly predicts early progression of pancreatic cancer that cannot be identified via initial imaging. Scientific Reports. 2023; 13: 4809. https://doi.org/10.1038/s41598-023-31051-7.

[93] Huang L, Lv Y, Guan S, Yan H, Han L, Wang Z, et al. High somatic mutations in circulating tumor DNA predict response of metastatic pancreatic ductal adenocarcinoma to first-line nab-paclitaxel plus S-1: prospective study. Journal of Translational Medicine. 2024; 22: 184. https://doi.org/10.1186/s12967-024-04989-z.

[94] Lee B, Tie J, Wang Y, Cohen JD, Shapiro JD, Wong R, et al. The potential role of serial circulating tumor DNA (ctDNA) testing after upfront surgery to guide adjuvant chemotherapy for early stage pancreatic cancer: The AGITG DYNAMIC-Pancreas trial. Journal of Clinical Oncology. 2024; 42: 107–107. https://doi.org/10.1200/JCO.2024.42.16_suppl.107.

[95] Huerta M, Martín-Arana J, Gimeno-Valiente F, Carbonell-Asins JA, García-Micó B, Martínez-Castedo B, et al. ctDNA whole exome sequencing in pancreatic ductal adenocarcinoma unveils organ-dependent metastatic mechanisms and identifies actionable alterations in fast progressing patients. Translational Research: the Journal of Laboratory and Clinical Medicine. 2024; 271: 105–115. https://doi.org/10.1016/j.trsl.2024.05.003.

[96] Sivapalan L, Thorn GJ, Gadaleta E, Kocher HM, Ross-Adams H, Chelala C. Longitudinal profiling of circulating tumour DNA for tracking tumour dynamics in pancreatic cancer. BMC Cancer. 2022; 22: 369. https://doi.org/10.1186/s12885-022-09387-6.

[97] Choi MG, Choi CM, Lee DH, Kim SW, Yoon S, Kim WS, et al. Different prognostic implications of hepatic metastasis according to front-line treatment in non-small cell lung cancer: a real-world retrospective study. Translational Lung Cancer Research. 2021; 10: 2551–2561. https://doi.org/10.21037/tlcr-21-206.

[98] Ren Y, Dai C, Zheng H, Zhou F, She Y, Jiang G, et al. Prognostic effect of liver metastasis in lung cancer patients with distant metastasis. Oncotarget. 2016; 7: 53245–53253. https://doi.org/10.18632/oncotarget.10644.

[99] Jiang T, Cheng R, Zhang G, Su C, Zhao C, Li X, et al. Characterization of Liver Metastasis and Its Effect on Targeted Therapy in EGFR-mutant NSCLC: A Multicenter Study. Clinical Lung Cancer. 2017; 18: 631–639.e2. https://doi.org/10.1016/j.cllc.2017.04.015.

[100] Chaudhuri AA, Chabon JJ, Lovejoy AF, Newman AM, Stehr H, Azad TD, et al. Early Detection of Molecular Residual Disease in Localized Lung Cancer by Circulating Tumor DNA Profiling. Cancer Discovery. 2017; 7: 1394–1403. https://doi.org/10.1158/2159-8290.CD-17-0716.

[101] Abbosh C, Birkbak NJ, Wilson GA, Jamal-Hanjani M, Constantin T, Salari R, et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017; 545: 446–451. https://doi.org/10.1038/nature22364.

[102] Mulshine JL, Makuch RW, Johnston-Early A, Matthews MJ, Carney DN, Ihde DC, et al. Diagnosis and significance of liver metastases in small cell carcinoma of the lung. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 1984; 2: 733–741. https://doi.org/10.1200/JCO.1984.2.7.733.

[103] Leighl NB, Page RD, Raymond VM, Daniel DB, Divers SG, Reckamp KL, et al. Clinical Utility of Comprehensive Cell-free DNA Analysis to Identify Genomic Biomarkers in Patients with Newly Diagnosed Metastatic Non-small Cell Lung Cancer. Clinical Cancer Research: an Official Journal of the American Association for Cancer Research. 2019; 25: 4691–4700. https://doi.org/10.1158/1078-0432.CCR-19-0624.

[104] García-Pardo M, Czarnecka-Kujawa K, Law JH, Salvarrey AM, Fernandes R, Fan ZJ, et al. Association of Circulating Tumor DNA Testing Before Tissue Diagnosis With Time to Treatment Among Patients With Suspected Advanced Lung Cancer: The ACCELERATE Nonrandomized Clinical Trial. JAMA Network Open. 2023; 6: e2325332. https://doi.org/10.1001/jamanetworkopen.2023.25332.

[105] Dziadziuszko R, Mok T, Peters S, Han JY, Alatorre-Alexander J, Leighl N, et al. Blood First Assay Screening Trial (BFAST) in Treatment-Naive Advanced or Metastatic NSCLC: Initial Results of the Phase 2 ALK-Positive Cohort. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2021; 16: 2040–2050. https://doi.org/10.1016/j.jtho.2021.07.008.

[106] Dong S, Wang Z, Zhang JT, Yan B, Zhang C, Gao X, et al. Circulating Tumor DNA-Guided De-Escalation Targeted Therapy for Advanced Non-Small Cell Lung Cancer: A Nonrandomized Controlled Trial. JAMA Oncology. 2024; 10: 932–940. https://doi.org/10.1001/jamaoncol.2024.1779.

[107] Aggarwal C, Thompson JC, Black TA, Katz SI, Fan R, Yee SS, et al. Clinical Implications of Plasma-Based Genotyping With the Delivery of Personalized Therapy in Metastatic Non-Small Cell Lung Cancer. JAMA Oncology. 2019; 5: 173–180. https://doi.org/10.1001/jamaoncol.2018.4305.

[108] Zhao J, Zhong J, Chen Y, Chen Z, Yin H, He Y, et al. Molecular features of NSCLC patients with liver metastasis. Therapeutic Advances in Medical Oncology. 2024; 16: 17588359241275421. https://doi.org/10.1177/17588359241275421.

[109] Jiang T, Fang Z, Tang S, Cheng R, Li Y, Ren S, et al. Mutational Landscape and Evolutionary Pattern of Liver and Brain Metastasis in Lung Adenocarcinoma. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2021; 16: 237–249. https://doi.org/10.1016/j.jtho.2020.10.128.

[110] Lam VK, Zhang J, Wu CC, Tran HT, Li L, Diao L, et al. Genotype-Specific Differences in Circulating Tumor DNA Levels in Advanced NSCLC. Journal of Thoracic Oncology: Official Publication of the International Association for the Study of Lung Cancer. 2021; 16: 601–609. https://doi.org/10.1016/j.jtho.2020.12.011.

[111] Liu C, Mohan SC, Wei J, Seki E, Liu M, Basho R, et al. Breast cancer liver metastasis: Pathogenesis and clinical implications. Frontiers in Oncology. 2022; 12: 1043771. https://doi.org/10.3389/fonc.2022.1043771.

[112] Shaw JA, Page K, Wren E, de Bruin EC, Kalashnikova E, Hastings R, et al. Serial Postoperative Circulating Tumor DNA Assessment Has Strong Prognostic Value During Long-Term Follow-Up in Patients With Breast Cancer. JCO Precision Oncology. 2024; 8: e2300456. https://doi.org/10.1200/PO.23.00456.

[113] Magbanua MJM, Brown Swigart L, Ahmed Z, Sayaman RW, Renner D, Kalashnikova E, et al. Clinical significance and biology of circulating tumor DNA in high-risk early-stage HER2-negative breast cancer receiving neoadjuvant chemotherapy. Cancer Cell. 2023; 41: 1091–1102.e4. https://doi.org/10.1016/j.ccell.2023.04.008.

[114] Turner NC, Kingston B, Kilburn LS, Kernaghan S, Wardley AM, Macpherson IR, et al. Circulating tumour DNA analysis to direct therapy in advanced breast cancer (plasmaMATCH): a multicentre, multicohort, phase 2a, platform trial. The Lancet. Oncology. 2020; 21: 1296–1308. https://doi.org/10.1016/S1470-2045(20)30444-7.

[115] Liu B, Hu Z, Ran J, Xie N, Tian C, Tang Y, et al. The circulating tumor DNA (ctDNA) alteration level predicts therapeutic response in metastatic breast cancer: Novel prognostic indexes based on ctDNA. Breast (Edinburgh, Scotland). 2022; 65: 116–123. https://doi.org/10.1016/j.breast.2022.07.010.

[116] Heidary M, Auer M, Ulz P, Heitzer E, Petru E, Gasch C, et al. The dynamic range of circulating tumor DNA in metastatic breast cancer. Breast Cancer Research: BCR. 2014; 16: 421. https://doi.org/10.1186/s13058-014-0421-y.

[117] Wang Y, Lin L, Li L, Wen J, Chi Y, Hao R, et al. Genetic landscape of breast cancer and mutation tracking with circulating tumor DNA in Chinese women. Aging. 2021; 13: 11860–11876. https://doi.org/10.18632/aging.202888.

[118] De Mattos-Arruda L, Weigelt B, Cortes J, Won HH, Ng CKY, Nuciforo P, et al. Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle. Annals of Oncology: Official Journal of the European Society for Medical Oncology. 2014; 25: 1729–1735. https://doi.org/10.1093/annonc/mdu239.

[119] Bharde A, Nadagouda S, Dongare M, Hariramani K, Basavalingegowda M, Haldar S, et al. ctDNA-based liquid biopsy reveals wider mutational profile with therapy resistance and metastasis susceptibility signatures in early-stage breast cancer patients. The Journal of Liquid Biopsy. 2025; 7: 100284. https://doi.org/10.1016/j.jlb.2024.100284.

[120] Cresswell GD, Nichol D, Spiteri I, Tari H, Zapata L, Heide T, et al. Mapping the breast cancer metastatic cascade onto ctDNA using genetic and epigenetic clonal tracking. Nature Communications. 2020; 11: 1446. https://doi.org/10.1038/s41467-020-15047-9.

[121] Israel MA, Danziger N, McGregor KA, Murugesan K, Gjoerup O, Sokol ES, et al. Comparative Genomic Analysis of Intrahepatic Cholangiocarcinoma: Biopsy Type, Ancestry, and Testing Patterns. The Oncologist. 2021; 26: 787–796. https://doi.org/10.1002/onco.13844.

[122] Li Y, Heer AK, Sloane HS, Edelstein DL, Tie J, Gibbs P, et al. Budget Impact Analysis of Circulating Tumor DNA Testing for Colon Cancer in Commercial Health and Medicare Advantage Plans. JAMA Health Forum. 2024; 5: e241270. https://doi.org/10.1001/jamahealthforum.2024.1270.

[123] Kowalchuk RO, Kamdem Talom BC, Van Abel KM, Ma DM, Waddle MR, Routman DM. Estimated Cost of Circulating Tumor DNA for Posttreatment Surveillance of Human Papillomavirus-Associated Oropharyngeal Cancer. JAMA Network Open. 2022; 5: e2144783. https://doi.org/10.1001/jamanetworkopen.2021.44783.

[124] Nong J, Gong Y, Guan Y, Yi X, Yi Y, Chang L, et al. Circulating tumor DNA analysis depicts subclonal architecture and genomic evolution of small cell lung cancer. Nature Communications. 2018; 9: 3114. https://doi.org/10.1038/s41467-018-05327-w.

[125] Kelley RK, Ueno M, Yoo C, Finn RS, Furuse J, Ren Z, et al. Pembrolizumab in combination with gemcitabine and cisplatin compared with gemcitabine and cisplatin alone for patients with advanced biliary tract cancer (KEYNOTE-966): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet (London, England). 2023; 401: 1853–1865. https://doi.org/10.1016/S0140-6736(23)00727-4.

[126] Marabelle A, Le DT, Ascierto PA, Di Giacomo AM, De Jesus-Acosta A, Delord JP, et al. Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study. Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 2020; 38: 1–10. https://doi.org/10.1200/JCO.19.02105.

[127] Rizvi NA, Cho BC, Reinmuth N, Lee KH, Luft A, Ahn MJ, et al. Durvalumab With or Without Tremelimumab vs Standard Chemotherapy in First-line Treatment of Metastatic Non-Small Cell Lung Cancer: The MYSTIC Phase 3 Randomized Clinical Trial. JAMA Oncology. 2020; 6: 661–674. https://doi.org/10.1001/jamaoncol.2020.0237.

[128] Rittmeyer A, Barlesi F, Waterkamp D, Park K, Ciardiello F, von Pawel J, et al. Atezolizumab versus docetaxel in patients with previously treated non-small-cell lung cancer (OAK): a phase 3, open-label, multicentre randomised controlled trial. Lancet (London, England). 2017; 389: 255–265. https://doi.org/10.1016/S0140-6736(16)32517-X.

[129] Slater S, Bryant A, Aresu M, Begum R, Chen HC, Peckitt C, et al. Tissue-Free Liquid Biopsies Combining Genomic and Methylation Signals for Minimal Residual Disease Detection in Patients with Early Colorectal Cancer from the UK TRACC Part B Study. Clinical Cancer Research: an Official Journal of the American Association for Cancer Research. 2024; 30: 3459–3469. https://doi.org/10.1158/1078-0432.CCR-24-0226.

[130] Zhang Y, Yao Y, Xu Y, Li L, Gong Y, Zhang K, et al. Pan-cancer circulating tumor DNA detection in over 10,000 Chinese patients. Nature Communications. 2021; 12: 11. https://doi.org/10.1038/s41467-020-20162-8.

[131] Phallen J, Sausen M, Adleff V, Leal A, Hruban C, White J, et al. Direct detection of early-stage cancers using circulating tumor DNA. Science Translational Medicine. 2017; 9: eaan2415. https://doi.org/10.1126/scitranslmed.aan2415.