Missing link: Viral RNA signatures in circulating exosomes as early diagnostic biomarkers for gastrointestinal cancers

Abstract

This editorial highlights the critical role of viral RNA signatures encapsulated within circulating exosomes as a potential missing link in the early diagnosis of gastrointestinal (GI) cancers. Current diagnostic methods for virus-associated GI malignancies often fall short in detecting infections at subclinical or pre-cancerous stages. We propose that viral RNA-loaded exosomes, by offering stable, specific, and non-invasive biomarkers, can bridge this gap and revolutionize early detection compared to conventional approaches. As highlighted by Zhang et al in their recent review, viral infections, such as hepatitis B and C viruses, Epstein-Barr virus, and human papillomavirus, are well-established contributors to the pathogenesis of various GI malignancies. However, current diagnostic methods often underperform in detecting these infections at subclinical or pre-cancerous stages. We highlight the shared points between virology, exosome biology, and oncology, reinforcing the importance of viral RNA-loaded exosomes as a “missing link” in the early detection of virus-associated GI cancers. We also discuss current challenges, translational opportunities, and the requirements for clinical validation of these promising biomarkers.

Key Words: Exosomes; Viral RNA; Liquid biopsy; Gastrointestinal cancer; Early detection; Epstein-Barr virus; Hepatitis B virus; Hepatitis C virus; Biomarkers; Noncoding RNA

Core Tip: Early and accurate diagnostic markers are essential for managing gastrointestinal (GI) cancers, as their prognosis is linked to the stage of detection. This editorial emphasizes the potential of virus-enriched exosomes as a novel, non-invasive diagnostic tool for early detection and monitoring of GI cancers, particularly those associated with oncogenic viruses like Epstein-Barr virus and hepatitis B virus. The viral RNA signatures within these circulating exosomes serve as a crucial missing link, providing specific, and non-invasive biomarkers that can not only aid in distinguishing malignant from benign conditions but also offer insights into tumor progression and potential therapeutic targets.

INTRODUCTION

Gastrointestinal (GI) cancers, including malignancies of the stomach, colorectum, liver, esophagus, and pancreas, represent a significant global health burden, accounting for approximately 35% of all cancer-related mortalities[1]. The often insidious onset and non-specific symptoms of these cancers frequently lead to diagnosis at advanced stages, severely limiting therapeutic options and contributing to undesirable survival rates[2]. Consequently, the development of highly sensitive, specific, and non-invasive diagnostic tools for early detection and prognostic assessment is fundamental to improving patient outcomes[3]. Liquid biopsy, a cutting-edge diagnostic approach, has demonstrated potential as a transformative method for the early detection of premalignant lesions and subclinical cancers, facilitating prompt intervention and predicting tumor recurrence[4].

In this context, the recent publication by Zhang et al[5], published in the World Journal of Gastrointestinal Oncology, provides a comprehensive and perceptive study of exosomes as novel biomarkers in the diagnosis of GI cancers. Zhang et al[5] precisely defined the composition of exosomes, their cargo sorting mechanisms, and presented convincing clinical evidence supporting their diagnostic utility across various GI malignancies[5]. Their report substantiates the significant potential of circulating exosomes, which are enriched with tumor-derived proteins, lipids, microRNAs (miRNAs), and other RNAs, as indicators of tumorigenesis, tumor progression, and treatment response[6]. Although their work broadly covers the general diagnostic and prognostic value of exosomal components, a critical dimension that justifies further emphasis is the role of virus-derived signatures encapsulated within these extracellular vesicles[5].

This editorial aims to highlight this specific aspect, proposing that viral RNA-loaded exosomes represent a crucial “missing link” in the early detection and management of virus-associated GI cancers.

THE EXOSOMAL CONNECTION: VIRAL HIJACKING OF INTERCELLULAR COMMUNICATION

Oncogenic viruses, including Epstein-Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human papillomavirus (HPV), are well-established contributors to the pathogenesis of various GI malignancies[7]. These viruses utilize sophisticated mechanisms to manipulate host cellular processes, ultimately promoting carcinogenesis[8]. A particularly insidious strategy involves the subversion of the exosomal pathway, where viral components, including genetic material, are packaged into these extracellular vesicles. These virus-laden exosomes act as crucial vehicles for paracrine signaling, disseminating oncogenic signals and influencing the tumor microenvironment throughout the body[9].

EBV and gastric cancer

EBV is a leading etiological agent in a subset of gastric cancers (EBVaGC)[10]. During latent infection, EBV-infected gastric cells actively release exosomes containing viral noncoding RNAs, such as EBV-encoded small RNAs (EBERs) and specific miRNAs [such as miR-Bam HI fragment A rightward transcript (BARTs)][11]. These exosomal viral RNAs can profoundly influence host gene expression, often leading to the suppression of tumor suppressor genes and the promotion of cell proliferation and survival[12]. These exosomal viral RNAs can profoundly influence host gene expression, often leading to the suppression of tumor suppressor genes and the promotion of cell proliferation and survival. Accordingly, EBV-encoded miRNAs carried by exosomes can directly target and downregulate key components of immune signaling pathways, such as specific interferon-stimulated genes, thereby fostering an immunosuppressive tumor microenvironment and contributing to immune evasion[13]. The exosomal transfer of these viral components contributes to the switch between EBV’s latent and lytic cycles, facilitates viral persistence, and can enhance EBV transmission from infected B cells to epithelial cells, expanding the oncogenic reservoir[14]. Furthermore, viral proteins such as Epstein-Barr nuclear antigen 1 and Latent membrane protein 2A (LMP2A), frequently encapsulated within exosomes, modulate various cellular pathways intrinsically linked to tumorigenesis and immune evasion[15]. Studies have indicated that EBV-positive gastric cancer patients exhibit exosomes enriched with EBERs and LMP2A, which are correlated with poorer clinical outcomes[16]. The potential of these virus-enriched exosomes as non-invasive biomarkers and even therapeutic targets in EBVaGC is substantial, especially regarding the observed overexpression of programmed cell death ligand 1 in EBVaGC and its responsiveness to immunotherapy[17].

HBV and HCV in hepatocellular carcinoma

HBV and HCV are principal drivers of hepatocellular carcinoma. Infected hepatocytes release exosomes containing viral nucleic acids (such as Hepatitis B virus X protein RNA from HBV) and proteins[18]. These exosomal viral components can induce chronic inflammation, promote fibrosis, and directly or indirectly contribute to cellular transformation[19]. The systemic dissemination of these virus-laden exosomes can establish a pro-tumorigenic microenvironment in distant sites, potentially facilitating metastasis even before the primary tumor is clinically detectable[20].

HPV and anal/colorectal cancers

HPV is well-established as a major etiologic agent in anal cancers and its role in colorectal cancers is increasingly recognized, albeit with ongoing research and debate regarding its precise contribution to carcinogenesis. Exosomes derived from HPV-infected cells have been shown to encapsulate viral oncoproteins such as E6 and E7, along with viral RNAs. The exosomal transfer of these viral components plays a role in modulating host immune responses, particularly by influencing antigen presentation pathways, and can promote uncontrolled cell proliferation in recipient cells[21,22]. Therefore, the detection of these distinct viral signatures within circulating exosomes presents a promising non-invasive strategy for the early identification and monitoring of HPV-associated GI malignancies, including those of the anal canal and potentially a subset of colorectal cancers[23].

EXOSOMES AS A DIAGNOSTIC FRONTIER FOR VIRUS-ASSOCIATED GI CANCERS

The unique biological properties of exosomes, as comprehensively reviewed by Zhang et al[5], makes them promising candidates for cancer diagnosis, particularly for the early detection of virus-associated GI tumors. Their constitutional advantages, amplified by the presence of specific viral cargo, position them as promising diagnostic tools compared to conventional methods (Table 1).

Table 1 Exosomal viral RNA biomarkers in gastrointestinal cancers.

Virus type	Exosomal marker(s)	Associated GI cancer(s)	Diagnostic utility/clinical relevance
Epstein-Barr virus	Epstein-Barr virus-encoded small RNAs (EBERs), specific microRNAs (miR-BARTs, miR-BART7-3p, BART13-3p), viral proteins (EBNA1, LMP2A)	Gastric cancer (EBVaGC), nasopharyngeal carcinoma	Exosomal viral RNAs influence host gene expression, promoting cell proliferation and survival. EBERs and LMP2A in exosomes correlate with poorer outcomes in EBVaGC. miR-BART7-3p is a highly specific biomarker for gastric cancer. BART13-3p distinguishes cancerous from benign EBV infections in nasopharyngeal carcinoma
Hepatitis B virus	Viral nucleic acids (e.g., HBV DNA, HBV RNA, HBx RNA), viral proteins (e.g., HBsAg, HBx protein), miRNAs (e.g., miR-212)	Hepatocellular carcinoma	Exosomal viral components induce chronic inflammation, fibrosis, and cellular transformation. Their systemic dissemination can facilitate metastasis. Exosomes from infected cells contain HBV DNA, RNA, and proteins. Exosomal miR-212 is upregulated in HBV-hepatocellular carcinoma
Hepatitis C virus	Viral nucleic acids (e.g., HCV RNA, replication-competent viral RNA), viral proteins (e.g., HCV core protein, E2 envelope protein), miRNAs (e.g., miR-122)	Hepatocellular carcinoma	Exosomal viral components induce chronic inflammation, fibrosis, and cellular transformation. Exosomes from infected patients contain HCV RNA, mediating viral receptor-independent transmission. HCV RNA in exosomes can associate with Ago2, HSP90, and miR-122
Human papillomavirus	Viral oncoproteins (E6 and E7), viral RNAs	Anal cancers, colorectal cancers (to a lesser extent)	Exosomal transfer contributes to immune evasion by modulating antigen presentation and promotes cell proliferation. Detecting these signatures offers a non-invasive tool for identifying HPV-associated GI malignancies

EBERs: Epstein-Barr virus-encoded small RNAs; BART: Bam HI fragment A rightward transcript; EBNA1: Epstein-Barr nuclear antigen 1; LMP2A: Latent membrane protein 2A; HBV: Hepatitis B virus; HBx: Hepatitis B virus X protein; HBsAg: Hepatitis B surface antigen; miRNA: MicroRNA; HCV: Hepatitis C virus; EBVaGC: Epstein-Barr virus is a leading etiological agent in a subset of gastric cancers; Ago2: Argonaute 2; HSP90: Heat shock protein 90; HPV: Human papillomavirus; GI: Gastrointestinal.

One of the most significant advantages of exosomes is their remarkable stability in biological fluids. Encapsulated within a protective lipid bilayer membrane, exosomal cargo, including viral DNA, mRNA, and miRNAs, is protected from enzymatic degradation by circulating nucleases[24]. This intrinsic stability ensures that viral RNA signatures remain intact and detectable, making exosomes ideal for non-invasive liquid biopsy applications, such as blood tests, a point also mentioned by Zhang et al[5] and Zeng et al[25].

The presence of specific viral RNA signatures within exosomes offers a high level of diagnostic specificity, distinct from general tumor markers. Unlike general tumor markers, these viral components are directly linked to the oncogenic virus, allowing for precise identification of virus-associated cancers[26]. For instance, EBV’s miR-BART7-3p is overexpressed in gastric cancer exosomes, providing a highly specific biomarker[27]. Similarly, in nasopharyngeal cancer, exosomal BART13-3p has demonstrated efficacy in distinguishing cancerous conditions from benign EBV infections[28]. This high specificity, coupled with the ability to detect even low-abundance viral cargo, translates into enhanced diagnostic sensitivity, crucial for early detection when tumor burden is minimal.

The accessibility of exosomes in various bodily fluids, including blood, urine, and saliva, facilitates non-invasive sample collection. This eliminates the need for invasive and often painful tissue biopsies, improving patient compliance and enabling serial monitoring of disease progression and treatment response. This non-invasive approach aligns perfectly with the liquid biopsy paradigm championed by Zhang et al[5] as a revolutionary method for early detection.

CHALLENGES AND FUTURE DIRECTIONS

Despite the interesting scientific evidence demonstrating the potential of exosomal biomarkers, particularly those carrying viral RNA signatures, their widespread translation into routine clinical applications may face several challenges. As Zhang et al[5] detailed in their review, these hurdles are complex and require coordinated efforts to overcome.

Standardization of exosome isolation and analysis

The lack of standardized and universally adopted techniques for exosome isolation and purification remains a major issue. Different isolation methods, such as ultracentrifugation, ultrafiltration, or microfluidic technologies, can yield different subpopulations of extracellular vesicles with varying compositions of miRNAs, proteins, and sizes. Such heterogeneity can impact the accuracy and reproducibility of exosome-based diagnoses, making the purity and consistent yield of exosomes a primary concern[29,30]. For viral RNA analysis, this challenge is amplified by the potentially low abundance of viral transcripts within the exosomal cargo, demanding highly efficient and sensitive isolation methods[31]. The development of standardized operating procedures and robust data-handling methods is required. Current clinical guidelines for liquid biopsy, while evolving, still lack uniform and strong evidence, leading to considerable variability across studies that propose exosomes as biomarkers[32]. This inconsistency in purification methods and detection techniques results in substantial heterogeneity in assay quality, complicating comparative analyses and hindering the establishment of reliable diagnostic benchmarks. Variations in sensitivity and specificity due to different detection platforms further underlines the need for detailed standardization[33].

Different isolation methods, such as ultracentrifugation, ultrafiltration, or microfluidic technologies, can yield different subpopulations of extracellular vesicles with varying compositions of miRNAs, proteins, and sizes. This heterogeneity can directly impact the accuracy and reproducibility of exosome-based diagnoses, especially for viral RNA analysis where low abundance demands highly efficient and sensitive isolation methods[34]. While specific comparative data on viral RNA yield across all techniques is still emerging, ongoing research is actively addressing these methodological challenges, with various academic and commercial entities developing advanced exosome-based diagnostics.

Need for robust clinical validation

Comprehensive validation through large-scale, prospective clinical studies is a crucial, yet often neglected, aspect of the clinical application of exosomes. As Zhang et al[5] correctly point out, many current studies suffer from insufficient sample sizes, limited data, and short validation periods, prohibiting defined conclusions regarding the clinical utility of exosomal biomarkers. Furthermore, the predominant focus on specificity and sensitivity often overshadows other vital aspects required for comprehensive clinical testing, such as consistency, reproducibility, accuracy, established reference ranges, and minimum detection limits. For viral RNA-loaded exosomes, well-designed clinical trials are essential to demonstrate their incremental value over existing diagnostic tools and to define their precise role in various clinical settings[35].

Technological innovations and future prospects

Continued innovation in microfluidics and other advanced separation techniques is necessary to achieve high-purity, high-yield exosome isolation from diverse biological fluids, even with low viral loads[36]. Concurrently, the development of ultra-sensitive detection platforms, such as advanced nucleic acid amplification methods or next-generation sequencing, tailored for low-abundance viral RNA within exosomes, is critical[37].

A deeper understanding of the exosomal cargo profiles in various virus-associated GI cancers would facilitate the discovery of novel and highly specific viral RNA biomarkers that can differentiate between benign and malignant conditions, and even predict disease progression or therapeutic response. Moreover, large-scale, multi-center clinical trials are needed to validate the diagnostic and prognostic utility of exosomal viral RNA signatures. Combining exosomal viral RNA analysis with other omics data (exosomal proteins, host miRNAs, circulating free DNA) could provide a more holistic and accurate diagnostic picture, enhancing sensitivity and specificity. Furthermore, the real-world implementation of exosomal viral RNA biomarkers will necessitate addressing practical considerations such as the cost-effectiveness of exosomal profiling technologies and ensuring accessibility in low- and middle-income countries. Overcoming these barriers will be essential for equitable global adoption of these diagnostic tools.

CONCLUSION

The persistent challenge of late diagnosis in GI cancers significantly contributes to their high mortality rates. While the review by Zhang et al[5] provides a valuable overview of the transformative potential of exosomes as emerging tumor markers due to their stability, non-invasive accessibility, and diverse bioactive cargo, the specific contribution of virus-derived signatures within these vesicles represents an underexplored yet highly promising frontier. The integration of exosomal viral RNA signatures into liquid biopsy platforms holds strong promise, potentially serving as first-tier screening tools in high-risk populations or as complementary diagnostics to improve the sensitivity and specificity of existing methods. The ability of oncogenic viruses to package their genetic material into exosomes offers a unique and highly specific avenue for early cancer detection. These viral RNA-loaded exosomes serve as a missing link, providing stable, specific, and non-invasive biomarkers that can reflect early oncogenic processes and immune responses in virus-associated GI cancers. By optimizing these unique viral signatures, we can potentially achieve earlier and more precise diagnoses, distinguish between malignant and benign conditions, and gain better understanding of tumor progression and therapeutic targets. Despite the technical challenges in exosome isolation, standardization, and the need for extensive clinical validation, the integration of exosomal viral RNA signatures into liquid biopsy platforms holds strong promise. Converting these obstacles into tangible clinical applications will be crucial for revolutionizing the early detection and management of virus-associated GI cancers, ultimately leading to improved patient outcomes in the near future.