Liquid biopsy in HPV-associated cervical cancer
Cervical cancer and HPV
Cervical cancer is one of the most frequently diagnosed cancer types in women worldwide. According to 2020 statistics, it is the fourth leading cause of cancer death , which is mainly due to the high incidence rate in developing countries, where the disease is often diagnosed at a later stage.
Almost all cervical cancer cases are preceded by HPV infection. HPV, or human papillomavirus is a circular, non-enveloped dsDNA virus. The Papillomaviridae family is greatly heterogeneous; more than a hundred subtypes have been described. Papillomaviruses can be categorized by infection site (mucosal or cutaneous), genera (alpha, beta, gamma, mu), or carcinogenic risk . A subgroup of HPV viruses has been identified as specifically carcinogenic to humans, the so-called high-risk HPV types, comprising 14 HPV viruses, namely HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 . These 14 high-risk HPV types are associated with 98% of cervical cancer cases, among them, 70% are caused by HPV 16 or HPV 18 infection . HPV infection may lead to other types of cancers primarily in the urogenital tract (for example vaginal, vulval, anal, and penile cancers) or in the upper respiratory tract (oropharyngeal carcinomas) .
With adequate screening programs, a high number of cervical cancer cases and mortality can be prevented. Thanks to more widely available testing programs, incidence and mortality rates have been declining in the past few decades in most areas of the world . Only persistent infection (defined as longer than two years) with high-risk HPV leads to progressive cervix changes, ultimately, cancer formation; although it is important to mention that not every HPV infection will cause malignancies , in fact, the vast majority of these infections resolve on its own . Testing procedures, therefore, can identify malignancies in the early stages. Other factors should also be taken into account during testing procedures, such as the patient’s age: under 25 years, HPV infections usually clear on their own . Vaccination against oncogenic HPV types is also deemed effective in preventing cervical cancer cases .
HPV infection targets squamous epithelial cells, more specifically the basal cell layer. Here the virus induces the expression of viral genes that promote viral replication. These so-called early genes (E6 and E7) later cause the carcinogenic effect of the virus. These viral genes integrate into the host cell’s genome, then inhibit the expression of important cell cycle genes (p53 and Rb protein-coding genes), which leads to uncontrolled proliferation .
Liquid biopsy screening
Although cytology testing has largely improved the incidence and mortality decline of cervical cancer, there is still a huge need for non-invasive and highly specific methods for diagnosis and monitoring the risk of recurrent metastatic disease. Liquid biopsy techniques could help solve these problems. Liquid biopsy is a form of non-invasive test that detects biomarkers in bodily fluids such as whole blood, serum, plasma, urine, saliva, and others. The non-invasive nature of liquid biopsies allows continuous recurring testing, monitoring of diseases, and treatment response with minimal discomfort to the patient. The most commonly used sample is blood, from which it is possible to analyze, for example, circulating tumor cells, cell-free nucleic acids, or exosomes. The huge advancement of molecular detection techniques in the last few years has led to the possibility of studying small amounts of cell-free nucleic acids released from tumor cells . Once a cancer diagnosis has been established by the clinical examination of the cervix, a sensitive blood marker could improve the patient’s surveillance as part of a biomarker panel .
Circulating tumor cells (CTCs)
CTCs originate from the primary tumor site and enter the bloodstream, potentially giving rise to distant metastases. They appear in the blood in a relatively small number compared to white blood cells therefore their isolation and identification is a difficult process. CTC collection could represent a non-invasive method for real-time tumor heterogeneity investigation, prognosis prediction, and treatment response monitoring.
In cervical cancer, the CTC isolation strategies rely on the cells’ morphological properties and identification of HPV oncogenes and epithelial markers. Despite numerous attempts, CTC identification and quantitation are still limited in cervical cancer, compared to solid tumor biopsies. This is due to the lack of specific tumor markers and the generally low number of detectable CTCs in blood .
Some research groups applied nucleic acid-based detection methods for CTCs: specific mRNA amplification appeared to be more useful than ctDNA detection. mRNAs are extremely degradable, therefore accurately reflect the presence of viable cells; whereas the more stable ctDNA could also be derived from necrotic or apoptotic cells .
When performing in vitro experiments, Cell viability is inevitable for culturing the isolated cells. For this purpose, size-based isolation systems are used, which may be able to isolate whole viable cells from the samples. Immunofluorescence-based detection techniques negatively affect the viability of cells. After size-based isolation and culturing, molecular analysis is achievable and more reliable, for example, qPCR tests help proper characterization of possible metastasis-causing CTCs .
Circulating cell-free DNA (cfDNA)
Cancer cells shed circulating tumor DNA (ctDNA) into the bloodstream, although the exact mechanism is still unclear . The amount of ctDNA in blood depends on several factors (for example tumor burden) and takes up only a fraction of cfDNA. Therefore, highly sensitive detection methods are needed for clinical practice.
Multiple studies found that ctDNA levels are significantly higher in cervical cancer patients versus healthy controls and are associated with tumor stage.
ctDNA can contain specific mutations that are associated with prognostic value, even gene fusions. Specific mutation detection requires sensitive methods, such as qPCR, dPCR, or NGS. Based on several research studies, specific ctDNA detection could aid in assessing response to treatment or simply characterizing the tumor cells’ genetic makeup .
As most cervical cancer cases are caused by HPV infection, another line of research is investigating the applicability of circulating HPV DNA as a biomarker , . Cheung et al.  applied dPCR for HPV DNA detection in blood samples and found that patients with a high viral load are at increased risk for disease recurrence and death in 5 years. Kang et al.  also utilized dPCR to detect HPV DNA in cancer patients’ blood and found that the presence of HPV DNA was able to differentiate between patients and controls and appeared to be a good marker of immunotherapy response. Interestingly, the HPV genotype identified in blood samples matched the type that was found in the primary tumor tissue.
Circulating RNAs have been widely investigated in cervical cancer: RNA present in the blood can originate from tumor cell necrosis, apoptosis, or even active release from the tumor cells .
First, researchers investigated circulating mRNAs. They found some promising mRNAs, whose levels were significantly higher in cancer patients compared to controls (e.g., EGFR and Bmi-1 ). HPV E6 mRNA was also isolated from cancer patients , showing a significant correlation with advanced disease. However, observations such as the degradation of extracellular mRNAs by blood-based ribonucleases and potential contamination by intracellular mRNAs limited the use of circulating mRNA as a viable biomarker.
Researchers also investigated circulating non-coding RNAs, such as long non-coding RNAs (lncRNA) and microRNAs (miRNA). Non-coding RNAs have important roles as epigenetic modulators in several cellular pathways. lncRNAs control tumorigenesis and metastasis processes and a few types were identified as potential biomarkers . miRNAs act as either oncogenic or tumor-suppressing molecules, hence they are often deregulated in cancer patients. A meta-analysis study found several differentially expressed miRNAs, most of which target key oncogenic pathways . Other groups identified miR-20a upregulated, and another found a panel of five miRNAs that differentiate cancer patients from controls. Some additional miRNAs were found to provide prognostic information, for example, miR-218 and miR-196a; and some emerging evidence suggests miR-486-5p to be a putative therapeutic target .
In addition to circulating miRNAs, recent research focuses more on miRNAs packed within exosomes. Exosomes can be easily detected in nearly any bodily fluid, and their importance is in participating in the crosstalk between cancer cells and their environment. Researchers used dPCR and RNA sequencing techniques, and so far, the most interesting finding is exosomal miR-125a-5p, differentiating between controls and patients and associated with clinical parameters .
Liquid biopsy approaches emerge as promising diagnostic and prognostic testing methods for cervical cancer patients, although several limitations hinder their routine clinical application. As for CTC quantification and analysis, the recent results in terms of prognostic applications are promising, but the identification methods are variable and not standardized yet. Circulating tumor DNA profiling also suggests promising perspectives in clinical utilization, yet few studies investigated it. The most recent evidence supports the use of circulating RNAs as markers for disease progression – however, further standardized research on larger sample sizes is needed. Most likely, a multi-analyte approach would be ideal for routine clinical approaches.
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