Application of circulating tumor DNA liquid biopsy in nasopharyngeal carcinoma: A case report and review of literature

Abstract

BACKGROUND

Circulating tumor DNA (ctDNA)-based liquid biopsy has been found to be effective for the detection of minimal residual disease and the evaluation of prognostic risk in various solid tumors, with good sensitivity and specificity for identifying patients at high risk of recurrence. However, use of its results as a biomarker for guiding the treatment and predicting the prognosis of nasopharyngeal carcinoma (NPC) has not been reported.

CASE SUMMARY

In this case study of a patient with stage IVb NPC, we utilized ctDNA as an independent biomarker to guide treatment. Chemotherapy was administered in the early stages of the disease, and local intensity-modulated radiation therapy was added when the patient tested positive for ctDNA, while radiation therapy was stopped and the patient was observed when the ctDNA test was negative. During the follow-up period, ctDNA signals became positive before tumor progression and became negative again at the end of treatment. We also explored the potential of ctDNA in combination with Epstein–Barr virus (EBV) DNA status to predict the prognosis of NPC patients, as well as the criteria for selecting genetic mutations and the testing cycle for ctDNA analysis.

CONCLUSION

The results of ctDNA-based liquid biopsy can serve as an independent biomarker, either independently or in conjunction with EBV DNA status, to guide the treatment and predict the prognosis of NPC.

Key Words: Nasopharyngeal cancer; Radiation therapy; Circulating tumor DNA; Epstein–Barr virus; Minor residual disease; Guide treatment; Predicting prognosis; Case report

Core Tip: The results of circulating tumor DNA (ctDNA)-based minimal residual disease testing have been used as a biomarker for some solid tumors, but their use as a biomarker to guide the treatment and predict the prognosis of nasopharyngeal carcinoma (NPC) has not yet been reported. We report a patient who tested positive for ctDNA and negative for Epstein‒Barr virus DNA. During the follow-up period, ctDNA signals became positive before tumor progression and became negative again at the end of treatment. This study represents a breakthrough in the use of biomarkers to assess the prognosis of NPC.

INTRODUCTION

Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates from the epithelium of the nasopharynx. It has a unique geographic and ethnic distribution and is prevalent in East Asia and Southeast Asia[1]. In 2020, there were approximately 133000 newly confirmed cases of NPC worldwide[2]. Currently, the treatment of NPC is guided mainly by the tumor–node–metastasis classification, which is a one-size-fits-all approach[3]. While this method is generally effective, overtreatment or insufficient treatment can still occur. Unfortunately, more than three-quarters of NPC patients seek medical attention late in tumor progression, by which time they have already developed locally advanced or metastatic disease. Approximately 10% of patients have distant metastasis at the initial diagnosis, and 15% to 34.4% of patients eventually develop metastases after treatment[4]. Previous studies have reported differences in survival rates among NPC patients with distant metastases, with a median survival time and 2-year survival rates ranging from falling within the range of 9 to 15.6 months and from 15.0% to 34.4%, respectively[5-8]. Recurrence of NPC is difficult to detect early, making it challenging to perform subsequent curative rescue treatment. Therefore, the development of reliable and cost-effective biomarkers for screening, early detection, improved prognostic prediction, customized treatment stratification, and disease monitoring is crucial. Evidence suggests that residual Epstein-Barr virus (EBV) DNA after treatment can serve as a biomarker that can be used to assess the prognosis of NPC[9]. However, the use of plasma EBV DNA as a screening tool for NPC presents a challenge with respect to balancing sensitivity and specificity. This method lacks standardization, with varying minimum detection limits across institutions leading to different false-negative rates and recommended cutoff values[10]. Notably, false -negatives and false- positives are not uncommon. Additionally, Chan et al[11] reported that age, ambient temperature, and smoking status are factors that can affect the detection of plasma EBV DNA, with the likelihood of positivity for EBV DNA being 1.59 times greater in smokers than in nonsmokers. In epidemic areas, plasma EBV DNA failed as a biomarker in 17.2%-29.3% of NPC patients during their initial diagnosis[12,13]. While plasma EBV DNA is currently the most valuable biomarker for NPC, it is not an ideal target because of its limitations. If plasma EBV -DNA alone is used as a population screening tool, 60.0%, 23.0%, 14.5%, and 5.0% of stage I, II, III, and IVA NPC cases, respectively, may be missed[14]. Therefore, a new biomarker is necessary to overcome the current limitations of EBV DNA-based monitoring methods. Minimal residual disease (MRD) refers to the presence of an unknown number of tumors in cancer patients even after treatment. In the case of solid tumors such as NPC, MRD indicates that malignant cells still exist in the patient's body after a specific treatment regime, without causing any clinical symptoms or detectable signs of disease. These cells cannot be identified through conventional screening methods or through examination of their cellular morphology[15]. The detection of MRD has been proven effective for the prediction of recurrence and the development of risk-adaptive treatment strategies for many solid tumors, offering numerous advantages. Monitoring MRD with circulating tumor DNA (ctDNA) allows the identification of high-risk patients and can be used to guide further treatment strategies[16]. Liquid biopsy and the detection of ctDNA in plasma provide genetic information specific to the tumor and valuable insight into the tumor genome map, facilitating the personalization of treatment decisions and the monitoring of treatment responses[16]. Liquid biopsy also offers a noninvasive alternative to tissue biopsy for obtaining cancer-related genomes and other information[17]. Liquid biopsy and the detection and analysis of ctDNA can be used for various clinical applications, such as early screening for cancer, monitoring disease, evaluating the efficacy of treatment, and detecting drug resistance. For example, changes in ctDNA levels during treatment can be used to evaluate treatment effectiveness, allowing treatment plans to be adjusted in a timely manner, and the detection of drug-resistant mutations can support the development of subsequent treatment strategies[18]. However, there is currently limited research on the relationship between ctDNA-based liquid biopsy and NPC prognosis[19,20]. We present a rare case of an NPC patient who was tested negative for EBV DNA but positive for ctDNA and presented with axillary lymph node metastasis. We describe the patient's clinical course and compare it with evidence from the published literature.

CASE PRESENTATION

Chief complaints

A male patient, aged 39 years, presented at the First Affiliated Hospital of China Medical University (Shenyang, China) on July 19, 2022, with palpable masses in the right neck.

History of present illness

An ultrasound examination conducted in January did not reveal any clear cause, and inflammation was initially suspected. The patient was treated orally with cephalosporin, which did not provide significant relief. The painless nature of the mass caused the patient to initially ignore it. However, in July 2022, the patient experienced an enlarged mass in the right side of his neck, accompanied by palpable masses in the right clavicle. A positron emission tomography/computed tomography (PET/CT) scan was carried out on July 5, 2022, at Liaoning Health Industry Group Fushun Mining Group General Hospital (Fushun, China), revealing multiple enlarged lymph nodes in the bilateral walls of the oropharynx, bilateral parapharyngeal spaces, carotid artery spaces, posterior cervical spaces, upper and lower clavicle areas, mediastinum, and bilateral hila of the lungs. The scan also revealed increased glucose metabolism and thickening of the soft tissue in the posterior wall of the nasopharynx (Figure 1). As a result, the patient was referred to our hospital for further treatment.

Figure 1 Positron emission tomography/computed tomography findings (July 5, 2022). A: (Head and neck) Thickening of soft tissue in the posterior wall of the nasopharynx, accompanied by a slight increase in glucose metabolism; Multiple enlarged lymph nodes in the bilateral walls of the oropharynx and bilateral parapharyngeal spaces, accompanied by increased glucose metabolism; B: (Pulmonary hilum and mediastinum) Multiple enlarged lymph nodes in the mediastinum and bilateral hilum, accompanied by increased glucose metabolism.

History of past illness

The patient displayed satisfactory overall health, with regular sleep patterns and diet and no reported history of hypertension, coronary heart disease, or diabetes.

Personal and family history

The patient had a 20-year history of smoking, with daily consumption of 40 cigarettes. The patient had quit smoking 4 months prior and reported occasional alcohol consumption. Furthermore, there was no indication of a family history of tumors.

Physical examination

On physical examination, multiple enlarged lymph nodes were detected in the right neck and clavicle region, which had a hard texture and limited mobility. The patient then underwent a nasopharyngoscopic procedure, during which a smooth tumor was found in the posterior wall of the right nasopharynx, right tubal torus, and right pharyngeal recess. The neoplasm presented a disorganized vascular pattern near the midline (Figure 2).

Figure 2 Electronic endoscope images (July 9, 2022). A: Right pharyngeal recess, tubal torus; B: Nasopharyngeal posterior wall. The nonsmooth new organism is located on the posterior wall of the right nasopharynx, on the right round pillow, and in the pharyngeal recess. The surface of the new organism presents a vascular disorder like appearance.

Laboratory examinations

Pathologic examination was subsequently performed, and the immunohistochemical results revealed a nonkeratinizing squamous carcinoma (Figure 3). Importantly, the patient tested IgG positive and IgM negative for EBV and had DNA levels below the limit of detection 5000 copies/mL). The patient subsequently underwent genetic testing of ctDNA from a liquid biopsy via ATG-Seq, performed by Nanjing Geneseeq Technology, Inc. (International Patent No. US9963694B2), which detected a positive MRD (ctDNA) status with an equivalent of 475.9 hGE/mL (Figure 4). The tumor-specific mutation was a missense mutation in exon 9 of p.L230R of BAP1 (BRCA1-associated protein 1), a tumor suppressor gene that is involved in DNA repair mechanisms, particularly the repair of double-strand breaks[21]. Reduced or absent BAP1 expression is associated with reduced overall and disease-free survival in NPC patients[21].

Figure 3 Pathology. Two grayish-white to yellowish rice-grain-sized masses were obtained from the nasopharynx of the patient. Immunohistochemical analysis revealed the following results: Cytokeratin (+), tumor Protein p53 (wild-type), tumor Protein p63(+), marker of Proliferation Ki-67 (30%+), tumor Necrosis Factor Receptor Superfamily Member 8 (scattered+), ΔNp63 Isoform of tumor Protein p63 (P40) (+), Cytokeratin 7 (-), and Cytokeratin 5/6 (+). Hematoxylin and eosin staining; magnification, ×100.

Figure 4 Table of patient's disease progression during treatment and line graph of the abundance of circulating tumor DNA -specific tumor mutations in liquid biopsies as a function of treatment time point. EBV: Epstein–Barr virus.

Imaging examinations

On July 27, 2022, nasopharyngeal magnetic resonance imaging (MRI) revealed thickening of the posterior wall of the nasopharyngeal parietal region, partial necrosis, and fusion in the bilateral neck and retropharyngeal groups (Figure 5A). Multiple enlarged lymph nodes were also detected. Lung CT on August 27, 2022 revealed multiple enlarged bilateral hilar and mediastinal lymph nodes, indicating multiple metastases (Figure 5B).

Figure 5 Nasopharynx magnetic resonance imaging (T1+C) and lung computed tomography findings of the nasopharynx. A: Nasopharynx magnetic resonance imaging (MRI) (July 27, 2022) reveals multiple enlarged lymph nodes in both neck and retropharyngeal groups, with enhancement, partial necrosis, and fusion; B: Lung computed tomography (CT) (August 27, 2022) reveals multiple enlarged lymph nodes in the right neck, bilateral hilum, and mediastinum; C: Nasopharynx MRI (November 03, 2022) reveals that the range of lesions is slightly smaller than that in July; D: Lung CT (December 23, 2022) reveals that bilateral hilar and mediastinal lymph nodes are enlarged; E: Nasopharyngeal MRI (March 8, 2023) reveals that the range of lesions was significantly reduced compared to the October film; F: Lung CT (March 8, 2023) reveals that the range of lesions is significantly reduced compared to the November film; G: Nasopharyngeal MRI (July 5, 2023) reveals swelling and resolution of lymph nodes, diffuse swelling of submandibular soft tissue, slight thickening of the left side of the mucosa in the nasopharynx, and a more uniform signal; H: Lung CT (July 5, 2023) reveals lymph node enlargement subside.

FINAL DIAGNOSIS

On the basis of the patient's clinical presentation, imaging studies, and pathological examination, the final diagnosis was confirmed to be stage IVb nasopharyngeal squamous carcinoma (T1N3M1) according to the AJCC staging system (8^th edition).

TREATMENT

Between August 6, 2022, and November 1, 2022, the patient underwent four cycles of GP chemotherapy and toripalimab immunotherapy. Follow-up MRI and CT scans were performed promptly. After the second cycle of treatment, a nasal MRI showed slight regression of the tumor, but the treatment outcome did not meet expectations. An additional dose of cetuximab-targeted therapy was administered, but two cycles later, a rash developed on the patient’s face, upper chest, and back. This patient was treated with minocycline and hydrocortisone ointment and gradually recovered one month after treatment with cetuximab was stopped.

At the end of treatment, MRI of the nasopharynx revealed slight regression of the tumor (Figure 5C), and ctDNA test results revealed a decrease in ctDNA haploid genome equivalents to 154.2 hGE/mL (initial haploid genome equivalents were 475.9 hGE/mL), whereas BAP1 abundance increased. Repeat lung CT revealed enlarged lymph nodes (Figure 5D), and ultrasound suggested thyroid nodules and lymph nodes of varying grades. From November 24, 2022, to February 3, 2023, local intensity-modulated radiotherapy was administered (Figure 6), which resulted in tumor regression, as observed via fiber-optic laryngoscopy after 20 radiotherapy sessions. CT of the lungs on the following day revealed significant shrinkage of the lymph nodes.

Figure 6 Delineation of the target area for local intensity modulated radiotherapy plan. A: Lung target area: GTVnd2 = 2 Gy × 33f, CTV = 1.82 Gy × 33f; B: Nasopharynx target area: GTV = 2.12 Gy × 33f, CTV = 1.82 Gy × 33f; C: Neck Lymph Nodes: GTVnd1 = 2.12 Gy × 33f, CTV = 1.82 Gy × 33f. CTV: Clinical target volume.

OUTCOME AND FOLLOW-UP

The patient’s MRD was measured again one month after the end of radiotherapy, which revealed that the patient had become negative for MRD. A follow-up MRI of the nasopharynx revealed slight thickening and irregularities in the posterior wall and parietal region of the nasopharynx. However, compared with the parameters on November 11, 2022, the width was slightly decreased, and there was a significant reduction in the extent of the lesions in multiple lymph nodes on both sides (Figure 5E). Additionally, a CT scan of the lungs revealed a further reduction in lymph node size, indicating that the treatment was effective (Figure 5F). A study by Yu-Pei Chen showed that the addition of metronomic adjuvant capecitabine to radiotherapy significantly improved the failure-free survival rate of nasopharyngeal cancer patients[22]. We asked our patient to take capecitabine and to have regular follow-ups. A review of MRI of the nasopharynx and CT of the lungs five months after the completion of radiotherapy revealed that the primary site and lymph nodes had recovered well (Figure 5G and H). However, in January 2024, the patient underwent another MRD measurement, which revealed that ctDNA haploid genome equivalents had increased from undetectable to 154.0 hGE/mL and that the abundance of BAP1 had increased. We suspected that the patient had signs of recurrence. A repeat lung CT revealed slightly enlarged mediastinal lymph nodes (Figure 7A). He was treated with TP-regimen chemotherapy combined with reslizumab from March 26, 2024, to September 13, 2024. We continued to check ctDNA during treatment and found that ctDNA haploid genome equivalents decreased on May 29, 2024, after the third chemotherapy cycle. Repeat CT revealed tiny nodules in the left lung, and the bilateral hilar and mediastinal lymph nodes were enlarged but smaller than before (Figure 7B). At the end of the last chemotherapy cycle on September 13, the ctDNA test results decreased again. Repeat CT revealed that the lymph nodes were smaller than before (Figure 7C). Nasopharyngeal MRI did not reveal significant changes during the treatment period. We asked him to continue oral capecitabine combined with toliparibizumab and to undergo regular follow-up.

Figure 7 Computed tomography of the patient's lungs during the second pre-treatment period. A: (March 2024) reveals slightly enlarged mediastinal lymph nodes; B: (June 17, 2024) reveals that Left lung micronodule, bilateral hilar and mediastinal lymph nodes enlarged but smaller than before; C: (September 7, 2024) reveals that Lymph nodes are smaller than before.

DISCUSSION

ctDNA testing has become an adjunctive test for the diagnosis and treatment of many solid tumors[23]. However, its value in NPC has not yet been reported. ctDNA is a promising marker for detecting MRD. ctDNA can be used to detect the progression of breast cancer up to 5 months before imaging evidence appears[24]. With respect to the detection of Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, ctDNA tends to be more sensitive and specific than circulating tumor cells (CTCs), another biomarker used in MRD testing. For example, in a study comparing CTC and ctDNA methods for the detection of KRAS mutations in patients with suspected lung cancer, ctDNA detection was found to be more accurate[25]. Another study revealed that ctDNA detection had a higher detection rate (97%) than either CTC (87%) or the protein marker CA 15-3 (78%) in patients with somatic genomic changes[26]. Over the past few decades, numerous methods based on different technologies have been developed for detecting MRD using ctDNA (Table 1).

Table 1 Minimal residual disease detection methods based on circulating tumor DNA.

MRD detection methods based on ctDNA and their advantages and disadvantages	Ref.
PCR-based	[28]
Hybridization capture-based NGS	[29]
PCR amplicon-based NGS	[30]
whole-genome sequencing	[31]

PCR: Polymerase chain reaction; NGS: Next-generation sequencing; MRD: Minimal residual disease; ctDNA: Circulating tumor DNA.

In this case, the patient tested negative for EBV, and we used a ctDNA liquid biopsy to guide the patient's treatment and predict his prognosis. ctDNA levels are a well-known biomarker that can be used to predict the prognosis of solid tumors, but research is still lacking on its relevance in patients with NPC, whether as a replacement or an adjunct measure to EBV DNA. Nevertheless, several articles have revealed the potential usefulness of ctDNA in both directions[27-30]. In a recent study by You et al[31], plasma-derived ctDNA and EBV DNA were analyzed and evaluated in patients with NPC receiving triple therapy. The goal was to correlate the clinical outcomes with molecular biomarkers. The results showed that ctDNA could indicate prognosis more precisely than could EBV DNA. These findings showed that ctDNA could be used as a prognostic biomarker alternative when EBV DNA is negative and that ctDNA has better specificity and accuracy than does EBV DNA[31].

In a prospective study, continuous liquid biopsy monitoring via targeted capture sequencing was conducted in 21 patients with metastatic NPC. The mutation load analysis covered 1366 tumor-related genes with customized probes. This study compared the results of liquid biopsy with those of plasma EBV DNA levels and PET/CT imaging to evaluate treatment response and patient prognosis[32]. The results showed that in metastatic NPC, MRD can play a role in evaluating treatment response and prognosis, even in patients with clear or negative plasma EBV DNA test results[32]. However, research on the use of ctDNA-based liquid biopsy assays to guide therapy is still in its infancy. Ghimire B documented a case study of an NPC patient who underwent multiple ctDNA tests during chemotherapy and immunotherapy. The results of Ghimire B’s study revealed that ctDNA levels exhibited consistent changes in tandem with the imaging results. Specifically, ctDNA levels increased during disease progression and decreased when treatment was effective. These findings indicate that ctDNA results have the potential to accurately reflect disease status and treatment response in NPC patients[20]. Although ctDNA-based MRD detection in NPC still faces several challenges with respect to detection technology, various studies[20,31-34] have demonstrated that MRD is a promising biomarker for predicting the prognosis of NPC and guiding its treatment. ctDNA has been established as a reliable prognostic biomarker for many other solid tumors, and its potential for use in conjunction with EBV for the prediction of NPC prognosis and the guidance of treatment remains equally attractive. Furthermore, ctDNA levels may serve as an independent biomarker in specific situations, such as in patients who are negative for EBV. These findings highlight the potential of ctDNA detection as a valuable tool in the treatment of NPC. BAP1 mutations were detected in the liquid biopsies of our patient. Notably, BAP1 mutations detected in liquid biopsies were also detected in histological biopsies, confirming the consistency of liquid and tissue biopsies. In a study by Perkail et al[35], thymic tumor mice with BAP1 knockout presented significant DNA damage and increased apoptosis in their tumors after radiation exposure. Our patient's BAP1 mutation abundance was transiently elevated after the first treatment, whereas the number of ctDNA haploid genome equivalents decreased. A possible reason for this is that after radiotherapy, tumors release less ctDNA into the bloodstream owing to shrinkage of the tumor, but tumors with BAP1 mutations are made to release a greater proportion of it into the bloodstream due to their heightened radiosensitivity. When selecting ctDNA mutations to guide treatment and prognosis prediction in NPC patients, several key criteria should be considered. First, the mutation abundance detected in the patient is a critical factor, as higher mutation abundance typically indicates a more significant proportion in tumor mutations, potentially carrying greater clinical relevance. Second, the specificity of the mutation to the tumor is another important criterion, as tumor-specific mutations can accurately reflect the presence and progression of the disease, increasing the sensitivity and specificity of detection. Additionally, the stability and prevalence of the mutation should not be overlooked, as mutations that are stable and widely present may offer stronger prognostic value. These considerations collectively contribute to the selection of ctDNA mutations that are most informative for clinical decision-making in NPC. In this study, we selected the missense mutation p.L230R in exon 9 of the BAP1 gene as a ctDNA biomarker for several key reasons. First, BAP1 mutations are tumor specific, enabling accurate reflection of tumor presence and progression, which enhances their clinical utility. Second, the mutation exhibited a relatively high detection abundance in the patient, suggesting greater clinical significance. Finally, the p.L230R mutation demonstrated excellent stability across multiple detection time points, consistently being identified in subsequent tests. This stability further reinforces its reliability as a biomarker for prognosis and treatment monitoring in NPC patients. BAP1 mutations are frequently observed in various tumor types, including uveal melanoma, malignant pleural mesothelioma, and clear cell renal cell carcinoma[33]. However, research on BAP1 in NPC remains limited. Previous studies have shown that BAP1 functions as a tumor suppressor by regulating chromatin structure, gene expression, DNA repair, and cell cycle progression[36-40]. Dysregulation of these functions may contribute to tumorigenesis and progression. Although only the BAP1 p.L230R mutation was selected in this study, our findings provide preliminary evidence for its potential value as a ctDNA biomarker in NPC. Furthermore, the broad role of BAP1 in oncology and its association with radiotherapy efficacy suggest that further studies in larger patient cohorts are warranted to validate its clinical utility in NPC. We established multiple time points for ctDNA monitoring to comprehensively evaluate treatment efficacy and patient prognosis. Specifically, the baseline ctDNA level prior to treatment served as a reference for subsequent comparisons. Following the initial four cycles of GP chemotherapy combined with toripalimab immunotherapy, ctDNA levels were measured to assess the preliminary treatment response and guide further therapeutic decisions. Postradiotherapy ctDNA monitoring, conducted one month after treatment and during follow-up, was intended to evaluate the efficacy of radiotherapy and predict patient outcomes. At the 11-month follow-up after radiotherapy, ctDNA analysis not only provided insights into long-term treatment efficacy but also served as a reference for subsequent chemotherapy. ctDNA levels were also measured after three cycles of chemotherapy to assess the treatment response promptly and allow the therapeutic regimen to be adjusted accordingly. Finally, ctDNA levels at the end of chemotherapy were used to evaluate overall treatment efficacy and predict patient prognosis. This comprehensive monitoring approach aligns with the treatment timeline and demonstrates clinical feasibility. Since only one patient was included in this study, confirmation of the universal applicability of MRD still depends on evidence from subsequent studies, but the detection performance of ATG-Seq ctDNA liquid biopsy has been fully validated in clinical practice[41-43].

CONCLUSION

ctDNA-based liquid biopsy can not only serve as an independent tool in the field of NPC but also be combined with the detection of EBV to predict patient prognosis and guide treatment.