Noninvasive biomarkers in thymic epithelial tumors: a systematic review of cfDNA/ctDNA detection, molecular profiling, and organoid-based monitoring

Abstract

Background

Thymic epithelial tumors (TETs), including thymomas and thymic carcinomas, are rare malignancies with limited treatment options and no established biomarkers for surveillance. Circulating cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) provide a non-invasive method for understanding tumor biology, detecting minimal residual disease (MRD), and possibly identifying recurrence. While this approach has added to the management of other solid tumors, its role in TETs remains poorly defined. The objective of this review was to evaluate the feasibility, molecular insights, and clinical utility of cfDNA and ctDNA for diagnosis, molecular profiling, and recurrence monitoring in TETs.

Methods

This systematic review summarizes the current evidence on cfDNA and ctDNA in TETs. Studies were identifies through systematic searches of PubMed, Embase, Web of Science, MEDLINE, Cochrane Library, and American Society of Clinical Oncology (ASCO) meeting abstracts from inception through July 2025. Eligible studies reported cfDNA or ctDNA analysis in patients with histologically confirmed thymoma or thymic carcinoma, and excluded reviews, commentaries, abstracts without full text, and non-blood based liquid biopsy studies. Data extraction included patient characteristics, assay platforms, mutational findings, and clinical applications. Data were synthesized narratively due to methodological heterogeneity. No formal risk of bias assessment was performed because of the small number of included studies.

Results

Six studies involving 289 patients met inclusion criteria. ctDNA detection was feasible across all studies, with detection rates ranging from 46% to 80%. Recurrent alterations included TP53, CDKN2A/B, KIT, and other variants. Liquid biopsy enabled genomic profiling at diagnosis and dynamic monitoring during treatment. Notably, several studies have suggested that disease recurrence may be detectable through liquid biopsy prior to the appearance of radiographic changes on conventional imaging. Despite these promising observations, evidence remains limited by small sample size, variability in assay methods, and short follow up duration.

Conclusions

Liquid biopsy approaches based on cfDNA and ctDNA have shown applicability in TETs and provide clinically relevant molecular information in settings where tissue-based analysis is limited. Tumor informed ctDNA strategies show particular promise for postoperative monitoring and longitudinal disease assessment, whereas broader clinical adoption remains investigational. Further prospective, multicenter studies are needed to establish standardized workflows and clarify the role of liquid biopsy across diagnostic, therapeutic, and surveillance contexts in TETs.

Highlight box.

Key findings

• Six studies evaluating circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), and circulating tumor cell (CTC) derived organoids in thymic epithelial tumors (TETs) demonstrate the feasibility of liquid biopsy across analytic platforms.

• ctDNA identifies recurrent, clinically meaningful genomic alterations, including TP53, CDKN2A/B, KIT, EGFR, PTEN, BRCA2, and PIK3CA, with moderate concordance to paired tumor tissue.

• ctDNA detected molecular recurrence earlier than radiographic imaging in select patients.

• CTC derived organoids provide a complementary functional platform for drug sensitivity testing, supporting integration of genomic and phenotypic profiling in TETs.

What is known and what is new?

• Liquid biopsy using cfDNA and ctDNA is well established for molecular profiling and minimal residual disease assessment in several solid tumors; however, evidence in TETs has remained limited due to disease rarity and small, heterogeneous studies.

• This review provides the first comprehensive synthesis of ctDNA, cfDNA, and CTC-derived organoid data in TETs, demonstrating that liquid biopsy can capture disease biology, identify actionable alterations, and may enable earlier detection of recurrence in addition to conventional surveillance.

What is the implication, and what should change now?

• Liquid biopsy has strong potential to complement or partially replace tissue biopsy when tissue is limited or unobtainable.

• Earlier recurrence detection via ctDNA could refine surveillance strategies and support more personalized clinical decision making.

• Larger, prospective, multicenter studies with standardized ctDNA assays are needed before routine clinical adoption in TETs can be recommended.

Introduction

Thymic epithelial tumors (TETs) are rare neoplasms that originate in the anterior mediastinum and include thymomas, thymic carcinomas, and thymic neuroendocrine tumors. Their rarity has limited representation in large-scale genomic databases and surgical resection remains the cornerstone of management. Despite generally indolent behavior, thymomas can recur after surgery and thymic carcinomas carry a poorer prognosis with limited systemic treatment options (1). Furthermore, current surveillance relies heavily on cross-sectional imaging, which may delay detection of molecular relapse and expose patients to cumulative radiation (2). In contemporary series, thymoma recurrence after resection occurs in about 6–13% of patients over 5–9 years, influenced strongly by stage, histology, and margin status. Stage I disease recurs in ~1–6% of cases, compared to ~27–30% in stage III and up to 50–100% in stage IV. Low-risk World Health Organization (WHO) subtypes (A/AB/B1) have minimal recurrence, while higher-risk subtypes (B2/B3) have recurrence rates approaching 20% despite complete resection (3). Incomplete resection can increase recurrence risk to over 60% (1,4,5).

TETs are uniquely challenging due to unpredictable oncological outcomes, frequent association with immunological dysregulation, and preserved thymopoietic activity. Although progress is limited by a lack of large phase II and III clinical and therapeutic development, our understanding of molecular profiles of newly diagnosed and unresectable/metastatic/recurrent TETs have expanded (6). Recurrent gene mutations in both thymoma and thymic carcinoma have been identified, and pathogenetic mechanisms linking TETs with autoimmune diseases have been increasingly understood. Early clinical data suggest potential benefit of targeted therapies and immune checkpoints inhibitors (ICIs), however their oncogenic potential and long term use remains unclear (7,8).

Circulating tumor DNA (ctDNA), consisting of short DNA fragments shed from tumor cells into the bloodstream, has emerged as a powerful tool for real-time tumor genotyping, detection of minimal residual disease (MRD), and early recurrence monitoring. It is important to distinguish ctDNA from cell-free DNA (cfDNA), a broader term that includes all DNA fragments circulating in plasma, including those released from normal, non-cancerous cells. Baseline cfDNA is present in healthy individuals and rises with nonmalignant stressors such as surgery, inflammation, or exercise, thereby diluting tumor-derived fragments. Since ctDNA typically represents less than 1% of total cfDNA, its detection requires highly sensitive tumor-informed or tumor-agnostic assays capable of identifying cancer-specific variants against background cfDNA (9). Importantly, due to its short half-life (approximately 2 hours), ctDNA is thought to provide real-time molecular changes in the tumor (10).

Across multiple tumor types, ctDNA has demonstrated clinical utility. In non-small cell lung cancer (NSCLC) ctDNA, it is guideline endorsed for identifying driver mutations and resistance profiling when tissue is limited (11). In breast cancer, ctDNA is prognostic and valuable for monitoring resistance tracking, though its use in routine practice is still investigational (11,12). In colorectal cancer, ctDNA guides adjuvant therapy decisions and provides prognostic information, though survival benefit remains unproven (13). Despite these advances, the role of ctDNA and cfDNA in rare thoracic tumors such as TETs remains poorly defined. This review synthesizes current evidence on their application in TETs and identifies key areas for future investigation. Given these gaps, we conducted a systematic review to evaluate the feasibility, molecular characteristics, and clinical utility of cfDNA and ctDNA in TETs. We present this article in accordance with the PRISMA reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1-2467/rc).

Methods

Literature identification and eligibility criteria

A systematic literature review was performed to identify published studies evaluating ctDNA and cfDNA in TETs, including thymomas and thymic carcinomas. A comprehensive literature search was conducted in PubMed, Embase, Web of Science, MEDLINE, Cochrane Library, and the American Society of Clinical Oncology (ASCO) Meeting Abstracts database. The search included all records from database inception to July 2025. Reference lists from relevant reviews and meta-analyses were also screened to identify additional studies.

The final PubMed search string combined Medical Subject Headings (MeSH) and free text terms: “thymic epithelial tumors” OR “thymoma” OR “thymic carcinoma” AND “ctDNA” OR “circulating tumor DNA” OR “cell-free DNA” OR “liquid biopsy”. The Embase strategy used the terms: ‘thymic epithelial tumor’ OR ‘thymoma’ OR ‘thymic carcinoma’ AND ‘ctDNA’ OR ‘circulating tumor DNA’ OR ‘cell-free DNA’ OR ‘liquid biopsy’. The search was restricted to human studies published in English.

To capture unpublished or recently presented clinical data not yet indexed in PubMed or Embase, the ASCO abstracts and proceedings database was searched using the keywords “thymic epithelial tumor” and “circulating tumor DNA” (and synonyms). Where available, corresponding peer-reviewed manuscripts were retrieved for eligible ASCO abstracts.

Full-text clinical research articles were included if they evaluated ctDNA in patients with histologically confirmed TETs and linked ctDNA findings to clinical features (e.g., histology, stage, metastatic status) or patient outcomes (e.g., recurrence, progression-free survival, MRD). Eligible study designs included prospective and retrospective studies as well as as observational cohort analyses. Studies assessing tumor-related genomic alterations in cfDNA were also included.

Exclusion criteria were: non-English language publications, abstracts, editorials, conference abstracts without sufficient data, commentaries and study protocols. Studies focused exclusively on non-blood-based specimens or cfDNA/ctDNA quantification without correlation to tumor characteristics or outcomes were excluded.

Titles and abstracts were independently screened by two reviewers. Full texts of potentially eligible studies were then assessed in detail. Discrepancies were resolved by consensus or, if needed, adjunction by a third reviewer. Reference lists from relevant reviews and meta-analysis articles were also manually screened to identify additional eligible studies. The search yielded 311 records after duplicate removal. Titles and abstracts were screened, and 23 articles were selected for full-text review. Of these, six met all inclusion criteria and were included in the final synthesis. Common reasons for exclusion included lack of ctDNA/cfDNA analysis, absence of correlation with clinical features or outcomes, non-human studies, and conference abstracts without sufficient data. The selection process is summarized in Figure 1, following PRISMA guidelines.

Figure 1.

PRISMA flow diagram of study selection. Seven studies were included in the review (5 from databases, 2 from websites) after screening 311 records and excluding studies that were duplicates, animal-based, not using ctDNA/cfDNA, or not specific to TETs. ASCO, American Society of Clinical Oncology; cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; TETs, thymic epithelial tumors.

No formal GRADE (Grading of Recommendations, Assessment, Development and Evaluation) assessment was performed due to the small number and heterogeneity of included studies. Overall confidence in the body of evidence was judged to be low to moderate, reflecting the limited sample sizes, retrospective design of most studies, and methodological variability across cfDNA/ctDNA assays. Despite these limitations, consistent detection of tumor-derived variants across independent studies supports a reproducible signal of feasibility.

Data collection and extraction

A standardized data extraction form (Excel, Microsoft Corporation) was used to capture the following information from each eligible study:

• ❖ Study characteristics: authors, year of publication, study design, study period, location, and setting (research vs. clinical).

• ❖ Population characteristics: number of patients, histologic subtype (thymoma, thymic carcinoma, other), stage, and metastatic status.

• ❖ cfDNA/ctDNA details: assay platform, sample source (plasma, serum), timing of collection (e.g., pre-treatment, post-surgery, surveillance), and analytic approach (quantitative vs. genomic profiling).

• ❖ Endpoints and outcomes: detection rate, mutational profile, tumor mutational burden (TMB), microsatellite instability (MSI) status, correlation with imaging or pathology, recurrence MRD detection, and turnaround time.

• ❖ Key findings and study limitations.

Data extraction was performed by one reviewer and independently cross-checked by a second reviewer for accuracy and completeness. A formal risk of bias assessment was not performed due to the small number of included.

Classification of cfDNA and ctDNA utilization

The framework for classifying cfDNA/ctDNA use was adapted from prior literature (12) and applied to TETs. Studies were categorized based on the primary aim of DNA testing, the timing of sample acquisition, and the reported outcomes:

1. Diagnosis—cfDNA/ctDNA for early detection or diagnostic confirmation.

2. Prognostication—cfDNA/ctDNA for baseline molecular profiling, risk stratification, or correlation with disease aggressiveness.

3. Monitoring—cfDNA/ctDNA for surveillance, MRD detection, treatment response assessment, or early identification of recurrence/progression using serial sampling.

Categories were not mutually exclusive; studies meeting multiple criteria were classified in all relevant domains.

Data synthesis

Given the rarity of TETs and the heterogeneity of study design, patient populations, assay platforms, and endpoints, results were synthesized descriptively. Extracted data were organized into structured tables and complementary figures to facilitate comparison across studies. Table 1 summarizes study design, patient cohort, platform, timing of sample collection, and key findings, while Figure 1 illustrates the PRISMA selection flow. Figure 2 provides a schematic overview and pooled mutation frequencies, and Figure 3 displays a heatmap of recurrent genomic alterations. This descriptive and visual approach was used to highlight methodological diversity, clinical applications, feasibility, and limitations among included studies. No effect measures were calculated because no quantitative synthesis or meta-analysis was performed.

Table 1. Summary of published studies evaluating circulating cfDNA and ctDNA in TETs.

Study No.	Author	Design	N	Platform	Timing	Key findings	Recurrence monitoring
1	Ottaviano et al. [2020]	Prospective	26 advanced TETs + 6 resected	QIAamp + PCR	Pre-therapy and post resection	cfDNA levels: carcinoma > thymoma > controls; higher in metastasis; but no correlation with stage	No
2	Tagliamento et al. [2024]	Prospective multi-cohort	46 TETs	FoundationOne® CDx on FFPE tumor tissue vs. FoundationOne® Liquid CDx on ctDNA from plasma	Baseline	Alterations in 48%; TP53, CDKN2A/B, MTAP, KIT, PTEN. 50% detection in plasma vs. 46% in tissue	No
3	Dada et al. [2021]	Retrospective	157 TETs	Guardant360	Advanced disease	Cohort mostly thymic carcinoma; 80% had ≥1 mutation; TP53 55%, KIT 13%, EGFR 12%, BRCA2 11%, PIK3CA 10%, ARID1A 10%, ATM 10%, KRAS 9%, APC 9%, BRAF 9%	No
4	Ardeshir-Larijani et al. [2023]	Retrospective	49 advanced or metastatic TETs; cfDNA analysis on 24	FoundationOne® Liquid CDx (Illumina NovaSeq 6000)	Advanced disease	Advanced thymomas: GTF2I/HRAS; metastatic: TP53. Primary TC: CYLD; metastatic TC: TP53/DNA repair	No
5	Lagarde et al. [2022]	Case report	1 NET of the thymus	QIAamp + NGS	Pre/post-op (3 timepoint)	MEN1 ctDNA dropped post-op; recurrence detected prior to imaging	Yes
6	Wu et al. [2023]	Observational	TET patients	CTC-derived organoids	Baseline	Demonstrated feasibility of using CTCs to generate avatars	No

The table presents study design, sample size, assay platform, timing of blood collection, key findings, and whether recurrence monitoring was performed. APC, adenomatous polyposis coli; ATM, ataxia-telangiectasia mutated; BRAF, v-raf murine sarcoma viral oncogene homolog B1; CDx, companion diagnostic; cfDNA, cell-free DNA; CTC, circulating tumor cell; ctDNA, circulating tumor DNA; CYLD, cylindromatosis; EGFR, epidermal growth factor receptor; FFPE, formalin fixed paraffin embedded; MEN1, multiple endocrine neoplasia type 1; NET, neuroendocrine tumor; NGS, next-generation sequencing; PCR, polymerase chain reaction; TC, thymic carcinoma; TETs, thymic epithelial tumors.

Figure 2.

Schematic overview, study design, and mutational profiles from cfDNA/ctDNA studies in thymic epithelial tumors. (A) Schematic illustration of circulating cfDNA and ctDNA released into the bloodstream from TETs, highlighting their role as minimally invasive biomarkers for genomic profiling. (B) Characteristics of published studies evaluating cfDNA/ctDNA in thymic malignancies. Designs included 4 prospective cohorts, 2 retrospective cohorts, and 1 case report. Platforms were predominantly NGS-based (n=4), with fewer PCR-based (n=1) and experimental/other approaches (n=2), reflecting methodological heterogeneity across the literature. (C) Weighted average frequencies of recurrent genomic alterations detected across cfDNA/ctDNA studies. The most prevalent alteration was TP53 (48.6%), followed by CDKN2A (30.5%), EGFR (10.1%), BRCA2 (9.5%), PIK3CA (8.6%), ARID1A (8.6%), ATM (8.2%), and KRAS (7.9%). cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; NGS, next-generation sequencing; PCR, polymerase chain reaction; TETs, thymic epithelial tumors.

Figure 3.

Heatmap of recurrent genomic alterations across three cfDNA/ctDNA cohorts of TETs. Study 2 (Tagliamento et al., n=46) included FFPE tumor samples analyzed with FoundationOne^® CDx and plasma samples analyzed with FoundationOne^® Liquid CDx; mutation frequencies were approximated from Fig. S2 provided in the original publication. Study 3 (Dada, n=157) reflects cfDNA profiling using the Guardant360 targeted NGS panel. Study 4 (Ardeshir-Larijani et al., n=49) represents advanced or metastatic TETs profiled with FoundationOne^® Liquid CDx on the Illumina NovaSeq 6000 platform, with matched tissue sequenced using FoundationOne^® CDx when available. Percentages indicate the proportion of patients with alterations in each gene, and color intensity is scaled to mutation frequency. Blank cells indicate genes not reported for that study. cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; FFPE, formalin fixed paraffin embedded; NGS, next-generation sequencing; TETs, thymic epithelial tumors.

To explore potential sources of heterogeneity, studies were qualitatively compared based on their design (prospective vs. retrospective), patient population (thymoma vs. thymic carcinoma), stage distribution, cfDNA/ctDNA assay type [polymerase chain reaction (PCR)-based vs. next-generation sequencing (NGS)], and clinical endpoints assessed (diagnostic, prognostic, or surveillance applications). Differences in detection rates and mutational spectra were interpreted in light of these factors to identify methodological and biological contributors to variability. Quantitative analyses of heterogeneity were not performed due to the limited number of studies and lack of standardized outcome measures across datasets.

No sensitivity analyses were performed due to the small number of studies and lack of standardized outcome measures. Reporting bias was not formally assessed due to the small number of eligible studies and the lack of comparable reported outcomes across included datasets. Certainty of evidence (e.g., GRADE) was not evaluated because methodological heterogeneity and limited sample sizes precluded meaningful assessment. No protocol was prepared for this review.

Results

Overview of included

The six included studies comprised prospective and retrospective cohorts and one case report, representing a total of 289 patients with TETs. Study characteristics, including author, year, study design, cohort size, assay platform, timing of sample collection, and main objectives, are summarized in Table 1. Figure 2 provides a schematic overview of included studies and pooled mutation frequencies.

Detection feasibility and profiling

Detection of ctDNA in TETs has gained increasing feasibility and clinical relevance, evolving from simple quantification of circulating cfDNA to comprehensive genomic profiling. Early prospective work by Ottaviano et al. (14) (Study 1) established that plasma cfDNA levels are significantly elevated in thymic carcinomas compared to thymomas, and that metastatic disease is associated with higher cfDNA concentrations. Importantly, while cfDNA levels correlated with tumor histology and metastatic status, they did not demonstrate a clear relationship with traditional staging metrics such as Masaoka-Koga or tumor-node-metastasis (TNM) classification or tumor burden assessed by RECIST criteria. Among 26 patients with advanced TET (aTET), 6 with completely resected TET (crTET), and 10 healthy controls, median cfDNA levels were marked higher in aTET patients (11.4 ng/µL in thymomas and 25.6 ng/µL in thymic carcinomas) compared with controls (3.3 ng/µL), while crTET patients did not differ from controls. cfDNA levels did not correlate with overall clinical stage or tumor burden but were significantly higher in patients with metastatic disease (M1a/M1b) compared to non-metastatic cases. This foundational study confirmed that circulating nucleic acids are both detectable and quantifiable in TET patients, laying the groundwork for future investigations of ctDNA as a noninvasive biomarker.

Building on these findings, the European multicenter EORTC-SPECTA Arcagen study by Tagliamento et al. (15) (Study 2) applied a broad targeted NGS panel to paired tissue and plasma samples from patients with thymomas and thymic carcinomas. Molecular alterations were detected in 48% of patients, with recurrent mutations identified in key genes such as TP53, CDKN2A/B, PTEN, and KIT. Detection rates were comparable between plasma and tissue samples (50% and 46%, respectively).The rapid median turnaround time of just eight days highlighted the potential clinical utility of ctDNA testing as a complementary tool to tissue-based diagnostics, particularly when tissue acquisition is limited or delayed. This study also provided valuable insights into the mutational landscape of TETs as captured in ctDNA, reinforcing its relevance in precision oncology and molecular profiling.

Further supporting the value of plasma-based genomic profiling, Dada et al. (Study3) retrospectively evaluated ctDNA in advanced TETs using the Guardant360 commercial assay (16). Their abstract echoed the mutational patterns observed in the Arcagen cohort, underscoring the reproducibility of ctDNA assays across platforms. In this study, 66% of patients had thymic carcinoma and 34% had thymoma, with a median age of 60 years; 59% were male. Somatic alterations were identified in 80% of patients, most frequently involving TP53 (55%), KIT (13%), EGFR (12%), BRCA2 (11%), and PIK3CA (10%). Mutations occurred more often in thymic carcinoma than thymoma, though this difference was not statistically significant due to limited sample size.

Another study by Ardeshir-Larijani et al. (Study 4) evaluated cfDNA in both advanced and metastatic TETs (17). Advanced non-metastatic thymomas were characterized predominantly by GTF2I (49%) and HRAS (8%) mutations, which were largely absent in metastatic thymomas. Instead, metastatic thymomas showed frequent TP53 alterations (31%). These differences between non metastatic and metastatic thymomas are strongly associated with tumor histotype, as GTF2I and HRAS mutations occur virtually exclusively in type A and type AB thymomas, whereas TP53 mutations are commonly observed in B2 and B3 thymomas. Primary thymic carcinomas harbored CYLD mutations (27%) with fewer TP53 changes (18%), whereas metastatic thymic carcinomas showed high TP53 mutation rates (36%) along with alterations in DNA repair (ATM, CHD1, ARID1A) and cell-cycle regulators (CDKN2A/B). Pathway analysis demonstrated that metastatic TETs were enriched for TP53/DNA repair, EGFR/RAS, and PI3K/mTOR alterations, while primary thymomas were more strongly associated with immune-related pathways.

Collectively, these studies demonstrate that both cfDNA and ctDNA are feasible and informative biomarkers in TETs, providing quantitative measures of disease burden as well as detailed molecular characterization. Early work emphasized differences in cfDNA levels by histology and metastasis, while subsequent studies showed that ctDNA profiling can identify actionable mutations with a turnaround time compatible to clinical decision-making. The consistency of mutational profiles across research and commercial platforms further supports the potential of ctDNA as a minimally invasive surrogate for tumor tissue, particularly when biopsies are challenging or risky. The frequency of recurrent genomic alterations across three independent cohorts is summarized in Figure 3.

Nonetheless, important limitations remain. Plasma detection rates can be lower than in tissue, likely due to tumor shedding dynamics and the relatively low tumor burden seen in some TET patients. Small cohorts and the rarity of TETs also limit generalizability. Future research should focus on improving assay sensitivity, incorporating longitudinal monitoring, and defining prognostic and predictive value of specific genomic alterations detected in plasma.

Considerable heterogeneity was observed among the included studies, largely attributable to differences in study design (prospective vs. retrospective), cohort composition (thymoma vs. thymic carcinoma), disease stage, and cfDNA/ctDNA assay methodology (quantitative PCR vs. targeted or broad NGS panels). Detection rates ranged from approximately 46% to 80%, reflecting variations in tumor burden, ctDNA shedding dynamics, and plasma DNA input volumes. Timing of sample collection, such as pre-treatment, post-resection, or during surveillance, also contributed to differences in ctDNA detection and mutation profiles. These methodological and biological factors collectively account for much of the variability observed across study results.

Surveillance and recurrence monitoring

The use of ctDNA for surveillance and recurrence monitoring is gaining attention as a promising biomarker across multiple tumor types. Unlike circulating cfDNA, which included DNA fragments from both malignant and nonmalignant calls, ctDNA originates from tumor cells and carries tumor-specific alterations, enabling more precise detection of MRD and early recurrence.

The potential was highlighted by a case report from Lagarde et al. (Study 5), describing a patient with a thymic neuroendocrine tumor in the context of multiple endocrine neoplasia type 1 (MEN1) mosaicism (18). Following surgical resection, ctDNA sequencing revealed a marked postoperative decline in the allelic frequency of the MEN1 mutation, consistent with effective tumor removal. Notably, ctDNA detected recurrence nearly 1 month before the onset of clinical symptoms or radiological findings, underscoring its value as a real-time, minimally invasive tool for postoperative monitoring. This dynamic assessment of tumor burden highlights how ctDNA could provide earlier indication of relapse compared to conventional methods. Incorporating ctDNA into the postoperative TET surveillance could refine follow-up paradigms by enabling earlier recurrence detection and supporting more personalized management strategies. This approach may allow clinicians to intervene sooner, potentially improving long-term outcomes. However, validation through larger prospective studies is needed to define standardized clinical thresholds and determine optimal integration with imaging. Given the heterogeneity of TET subtypes—including thymomas, thymic carcinomas, and thymic neuroendocrine tumors—further research is required to evaluate the performance of ctDNA across these diverse subtypes.

Alternative liquid biopsy approaches

Although most studies have focused on cfDNA or ctDNA, Wu et al. (Study 6) demonstrated the feasibility of generating patient-derived organoids from circulating tumor cells (CTCs), highlighting an alternative liquid biopsy approach that may complement ctDNA in precision medicine strategies for TETs (19).

Tumor organoids are three-dimensional cellular structures derived from patient tumors or cancer stem cells that replicate the histopathological, genetic, and phenotypic characteristics of the original tumor. Organoid technology was first described in 2009, when adult stem cells were shown to grow in a three dimensional culture system to form self-organizing structures that resemble native tissue. In cancer research, tumor organoids are generated by enzymatic dissociation of tumor specimens into single cells or small cell clusters, which are subsequently embedded in a basement membrane matrix (e.g., Matrigel) and cultured in defined media, rich in growth factors that support self-renewal (20,21).

Tumor organoids preserve tumor heterogeneity and microenvironmental interactions and can be indefinitely expanded in vitro using aforementioned specialized media and extracellular matrices (21,22).

Organoids are increasingly used in translational cancer research for drug screening, evaluation of treatment response, and investigation of tumor-stromal and immune interactions. Compared to traditional cell lines, they more closely mimic in vivo tumor architecture, supporting applications in personalized medicine and preclinical therapeutic modeling (22).

Such organoid models have already been described for colorectal, pancreatic, breast, prostate, liver, stomach, brain, bladder, and lung cancers (23,24). Their extension to TETs through CTC-derived organoids underscores the potential of alternative liquid biopsy strategies to broaden research and clinical applications in this rare tumor type.

Wu’s observational study enrolled 12 patients with pathologically confirmed thymic malignancies between August 2018 and January 2023. Tumor staging followed American Joint Committee on Cancer (AJCC) guidelines, and clinical response was assessed using RECIST 1.1 at ~3 months before, at the time of, and ~3 months after liquid biopsy. Peripheral blood (20 mL) was collected from patients ≥2 weeks post-chemotherapy, and CTCs were isolated using Ficoll-Paque centrifugation and RosetteSep enrichment. CTCs were seeded on a binary colloidal crystal substrate and cultured in platelet lysate-based medium with growth factors for 3 weeks. Organoid expansion was monitored by microscopy, and adenosine triphosphate (ATP)-based bioluminescence quantified viable cells for drug testing. Drug sensitivity was classified as positive (E+) or negative (E−) based on predefined cell viability thresholds. Results then correlated with patient clinical response over retrospective and prospective 3-month intervals.

Organoid cultures were successfully established from 16 of 21 blood samples (76.2%), providing a feasible platform for evaluating drug sensitivity testing. Drug sensitivity profiles of the CTC-derived organoids showed high concordance with clinical responses: sensitivity (C+E+) was 100% (9/9) and specificity (C−E−) was 60% (3/5), with a statistically significant correlation (P=0.03).

CTC-derived organoids preserve the molecular and phenotypic characteristics of the original tumor, enabling detailed study of tumor biology and therapeutic responses in a controlled ex vivo setting. This model allows for high-throughput drug screening, assessment of treatment efficacy, and investigation of mechanisms of drug resistance, offering insights into tumor adaptability and strategies to overcome therapeutic challenges.

Integrating CTC-derived organoid models with ctDNA analysis could enhance TET monitoring by combining genomic insights with functional assessments of treatment responses. Together, these complementary approaches may advance precision oncology in TETs, supporting more comprehensive and personalized treatment strategies aimed at improving patient outcomes.

Discussion

TETs are rare anterior mediastinal neoplasms, including thymomas, thymic carcinomas, and thymic neuroendocrine tumors. Management has historically centered on surgical resection. Although thymomas often behave indolently, recurrence occurs in 6–13% of patients within 5–9 years, particularly in advanced-stage disease, aggressive histologic subtypes, or following incomplete resection. Thymic carcinomas, in contrast, are associated with poorer prognosis, limited systemic treatment options, and surveillance strategies that rely largely on imaging—methods that may delay detection of molecular relapse and expose patients to cumulative radiation.

This review synthesized findings from six studies examining the feasibility and clinical utility of ctDNA, cfDNA, and other liquid biopsy modalities in TETs. Evidence spans prospective and retrospective cohorts, a case report, and a feasibility study of CTC-derived organoids. Collectively, these highlight both the promise and current limitations of liquid biopsy in this rare group of thoracic malignancies. cfDNA represents fragmented DNA in the bloodstream, while ctDNA—its tumor derived subset—harbors tumor-specific genetic and epigenetic alterations (25). ctDNA reflects the tumor’s molecular landscape, providing a non-invasive alternative when tissue biopsy is limited.

Worthy of mention for clinical applicability is the difference between tumor naïve/agnostic ctDNA and tumor informed/specific ctDNA (26). Tumor informed ctDNA uses prior analysis of a patient’s tumor to create a personalized set of mutations that are then tracked in the blood, whereas tumor naïve/agnostic approaches analyze plasma using a fixed gene panel without prior knowledge of the tumor’s specific mutations. The former achieve much higher sensitivity, allowing detection of very low levels of ctDNA and making them particularly well suited for MRD monitoring and early detection of recurrence (27). In contrast, tumor naïve/agnostic evaluations are easier to implement (not requiring tumor tissue) but have the trade-off of lower sensitivity (28). Tumor-agnostic ctDNA strategies are for now preferred in advanced or metastatic tumors when tissue is unavailable or rapid molecular profiling is needed to guide treatment selection, whereas tumor-informed strategies are better suited for postoperative MRD detection and longitudinal surveillance following resection.

For example, in colorectal and other solid tumors, ctDNA has shown utility in precision oncology, including real-time assessment of tumor burden, early recurrence detection, and identification of actionable mutations (25). ctDNA dynamics also correlate with treatment response and disease progression (29). In breast cancer, ctDNA can detect MRD following surgery or systemic therapy, highlighting its potential across disease stages (30).

In the context of thymic epithelial neoplasms, early prospective studies showed cfDNA levels are higher in thymic carcinoma compared with thymoma or healthy controls, with metastatic disease showing the highest concentrations. However, cfDNA levels did not consistently correlate with tumor stage or RECIST-defined burden. Targeted NGS of paired plasma and tissue confirmed the feasibility of ctDNA detection, identifying genomic alterations in approximately half of patients and demonstrating concordance between plasma and tumor tissue. Retrospective analyses further reinforced these findings, with recurrent somatic mutations across subtypes, including TP53, KIT, EGFR, BRCA2, PIK3CA, and others, reflecting both the genomic heterogeneity of TETs and ctDNA’s ability to capture subtype-specific biology. Notably, MTAP deletion was reported in one study but not observed in others. This discrepancy may reflect differences in sequencing panel design, particularly with respect to detection of copy number loss, as well as technical variability in identifying homozygous deletions in plasma compared with tissue-based assay. Alternatively, this finding may reflect true biological heterogeneity within TETs, as MTAP loss has been associated with specific disease contexts.

Moreover, ctDNA has shown potential for early detection of relapse, as described in a case report where recurrence was identified one month before radiographic progression. Functional approaches such as CTC-derived organoids add a complementary dimension, enabling ex vivo assessment of tumor biology and therapeutic sensitivity.

Despite these uses, ctDNA and cfDNA assays face technical and biological challenges. Low ctDNA abundance in early-stage disease, preanalytical variability, and the need for highly sensitive assays necessitate rigorous validation and standardization (31). Consensus guidelines emphasize strict quality control, reference materials, and analytical validation to ensure reproducibility (32).

In addition to these technical and biological constraints, substantial heterogeneity was evident across studies in both methodology and study population. Variability in assay platforms, timing of sample collection, and tumor histology likely contributed to differences in ctDNA detection rates and mutation spectra. Such heterogeneity complicates cross-study comparisons and underscores the need for standardized protocols for plasma collection, sequencing, and reporting criteria to enable reproducible and clinically meaningful interpretation of ctDNA findings in TETs.

Taken together, the available evidence suggests that ctDNA and cfDNA are among the most clinically mature liquid biopsy modalities, with ctDNA—particularly tumor informed approaches—showing the greatest potential for clinical implementation and increasing integration into routine cancer care for genotyping advanced disease, monitoring MRD, and assessing treatment response. Several commercial assays have received regulatory clearance or breakthrough designations reflecting this progress. Tempus xF is a 105 gene hybrid capture NGS assay designed primarily for comprehensive genomic profiling to identify actionable mutations with published data supporting concordance with tissue genomics in various tumors, reflecting progress toward clinical application (33). Both tumor naïve/agnostic and tumor informed ctDNA analysis are available. The Signatera™ assay (Natera) is a tumor informed ctDNA MRD test customized from each patient’s tumor exome or genome and is now use in clinical practice for surveillance following curative therapy in multiple malignancies (34,35).

While both approaches illustrate the growing clinical maturation of ctDNA platforms, widespread adoption for routine surveillance in TETs remains limited outside of evidence generating contexts. Nevertheless, a growing literature now focuses on the practical considerations of ctDNA use in clinical practice (11).

This review has methodological limitations. The search was restricted to English-language publications and did not include gray literature or conference proceedings beyond ASCO, which may have introduced selection bias. The review was not prospectively registered, and no formal risk of bias or certainty assessment tool was applied. Additionally, because of the small number of heterogeneous studies, quantitative synthesis and meta-analysis were not feasible, and findings were summarized descriptively.

Overall, these studies suggest that cfDNA and ctDNA are feasible and informative biomarkers in TETs, providing both quantitative measures of tumor burden and detailed genomic profiling. However, key limitations persist, including technical constraints, low abundance in some patients, biological confounders such as clonal hematopoiesis, and limited prospective validation for surveillance or recurrence detection. Future research should focus on larger, multicenter cohorts with longitudinal follow-up to establish standardized protocols, validate clinical utility, and integrate liquid biopsy into routine care for patients with TETs.

Conclusions

Based on the current body of evidence, liquid biopsy approaches in TETs are feasible and provide meaningful molecular insight, though their clinical application remains exploratory. Both prospective and retrospective studies demonstrate that cfDNA/ctDNA can capture the genomic heterogeneity of thymomas and thymic carcinomas, offering a minimally invasive alternative when tissue acquisition is limited. A single case report suggests that ctDNA can detect recurrence earlier compared to conventional imaging. However, prospective validation of this concept is still lacking. Complementary modalities such as CTC-derived organoids further expand the scope of liquid biopsy by enabling functional testing, though these remain investigational. Future research should prioritize larger, multicenter studies with standardized methodologies to define the prognostic and predictive value of tumor specific and/or naïve ctDNA, and to establish its role within diagnostic, therapeutic, and surveillance strategies for TETs

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