1. Introduction

In the structure of morbidity and mortality, cervical cancer (CC) is one of the most common types of carcinomas among women in all countries of the world, in fourth place after breast cancer, colorectal cancer, and lung cancer [1]. According to the data of the International Agency for Research on Cancer, in 2018, the number of patients with cervical cancer was 570,000, an increase of 7.8% over the past decade, and the number of deaths from this disease amounted to 311,000 ^[1][2][1,2]. In 2020, 604,100 new cases of cervical cancer were detected around the world, and the number of deaths was 341,800 ^[1][2][3][4][1,2,3,4]. Therefore, despite the recommended screening programs for cervical cancer, morbidity and mortality rates from this oncopathology have been steadily growing in recent years, both worldwide and within the Russian Federation ^[4][5][6][7][4,5,6,7]. Indicators of later diagnosis of cervical cancer in stages III-IV of the disease in Russia have also reached high numbers—they made up 32.6% in 2018 [4]. Currently, the oncology, oncobiology, and oncomorphology of cervical cancer are characterized by the accumulation of new information; various molecular biological research methods aimed at studying the mechanisms of cervical carcinogenesis are being tested and applied; targeted anticancer drugs and biomarkers which are useful for the diagnosis of various stages of the tumor process in cervical cancer are being sought ^{[8][9][10][11][12][13][14][15][16][17][18][19]}[8,9,10,11,12,13,14,15,16,17,18,19]. The search for reliable genetic, molecular and immunohistochemical markers for early diagnosis of precancerous and neoplastic processes in the cervix is also an important task of current interest in modern oncology. Biomarkers in oncology include certain genes, DNA and RNA molecules, proteins, enzymes, antigens, and other cellular and biological products that can be detected at various stages of carcinogenesis, under the influence of therapy, and are described in many modern reviews and articles concerning carcinogenesis, precancerous lesions, and cervical cancer ( Table 1 ).

According to the type of biomolecules and methods of their detection, genomic, transcriptomic, and metabolic factors (immunohistochemical, biochemical and others) are distinguished. Biomarkers can be divided into diagnostic markers and prognostic and predictive clinical factors ( Figure 1 ).

Figure 1. Types of biomarkers for investigation of precancerous lesions and cervical carcinoma.

Diagnostic markers make diagnosis possible; clinical biomarkers include the following groups: (1) prognostic ones, which allow one to predict the course of the disease and survival without antitumor treatment; these are factors associated with tumor proliferation, differentiation, angiogenesis, invasion, or metastasis; (2) predictive ones, which allow prediction of the clinical effect, the relapse-free period, survival, sensitivity to treatment, and the toxicity of the planned treatment; they are associated with molecular targets for targeted drugs and a cascade of intracellular signals in a tumor cell; (3) there are factors that can simultaneously have both prognostic and predictive properties [82].

2. Mechanisms of Cervical Carcinogenesis

A complex genomic study of cervical cancer identified new mutated genes and gene amplifications in endometrioid-like cancers, consisting mainly of HPV-negative tumors with a high frequency of the mutations KRAS, ARID1A, and PTEN; it established that in more than 70% of cases of cervical cancer, genomic changes take place in one or both of the PI3K/MAPK and TGFβ signaling pathways [16]. These studies support the existence of various molecular subtypes of cervical cancer [16].

According to different research, in HPV infection and cervical cancer there are various epigenetic changes and DNA methylation processes, and the participation of microRNAs in these processes has been described ^{[18][19][65][83][84][85][86][87]}[18,19,65,83,85,86,87,88]. At present, many of these processes have been studied insufficiently, and their interpretation is ambiguous, so the molecular mechanisms underlying these pathological changes are being investigated ^[11][14][84][11,14,85]. Recently, Wang et al. (2021) described the epigenetic regulation of the p16 tumor suppressor by miR 29a, which can justify the search for miRNA-based treatment options for cervical cancer ^[84][85]. It has been found that during the development of carcinomas, including various stages of cervical cancer, numerous epigenetic changes, such as hyper- and hypomethylation of DNA and tumor suppressor genes and modification of histones associated with the promoter regions of regulatory genes are observed. The results of meta-analysis confirm the hypermethylation of p16INK4a as an epigenetic marker for the progression of carcinogenesis in cervical cancer [65]. Expression of tumor suppressor genes, oncogenes, and growth factors is regulated by many histone-modifying enzymes, such as deacetylases and histone acetyltransferases; changes in the expression of histone modifiers also lead to epigenetic control of gene expression [16]. For example, it was found that DNA methylation is much higher in CIN2 and CIN3 compared to CIN1 [18]; the DNA methylation test is characterized by high specificity and sensitivity as compared to the ASCUS + cytological study and HPV16/18 genotyping [18]. Understanding the role of epigenetic changes in cervical carcinogenesis will help to develop effective methods for the detection and treatment of cervical cancer ^[8][18][84][8,18,85].

At the moment, the possible role of circular RNAs (circRNAs) in oncogenesis in the development of carcinomas of the organs of the female reproductive system is being investigated. It is believed that in cancer of the ovaries, endometrium, cervix, and breast carcinoma, circRNAs have prognostic, diagnostic, and therapeutic potential ^{[85][86][87][88]}[86,87,88,89]. Circular RNAs are a new class of non-coding RNAs. In several types of cancer, abnormal expression of these biomolecules happens, for example, in virus-associated malignant neoplasms—anogenital carcinoma associated with HPV, oropharyngeal and oral cancer ^[88][89]. It has been shown that circRNAs are involved in oncogenesis, cancer progression, and drug resistance, and some of them are useful diagnostic and prognostic markers.

In cervical cancer, aberrant expression of miRNA, circRNA, and IncRNA has been revealed, making a new contribution to the understanding of the molecular mechanisms of oncogenesis, and these molecules may turn out to be potential biomarkers for oncological screening and monitoring of cancer recurrence ^[86][87][88][87,88,89]. Long non-coding RNAs (LncRNAs) (>200 nucleotides) are non-coding transcripts, and some of them have been associated with the development, progression, and metastasis of certain neoplasia, such as leukemia and breast, colon, and liver cancers. LncRNA can play a role at the transcriptional and post-transcriptional levels, contributing to the development of various diseases, drug resistance, signal modulation, DNA repair, the cell cycle, and apoptosis. Patterns of aberrant expression of some LncRNAs have been found in cervical carcinoma cells and in precancerous lesions of the cervix; nevertheless, molecular mechanisms and biological effects associated with LncRNA require further research ^[89][90]. The role of long non-coding RNAs associated with HPV and cervical cancer, which can play a role in the processes of cell proliferation, migration, invasion, and apoptosis, is being investigated. LncRNAs can be seen as candidates for prognostic and predictive markers; however, molecular mechanisms involving LncRNAs, ncRNAs, and mRNAs have not been elucidated yet and are subject to further clarification ^[89][90]. It is known that approximately 20–40% of high-grade intraepithelial neoplasms regress spontaneously; however, an increase in the 3q chromosomal region, in which the human telomerase RNA gene is localized at 3q26, correlates with the degree of disease in CIN and cervical cancer lesions. In a study of an increase in the 3q26 gene as a genetic marker for the prognosis of neoplasia using in situ hybridization, a connection with the prognosis was established; the absence of an increase in this DNA region during a diagnostic biopsy may indicate a high probability of regression of the disease [20].

3. Biomarkers of HPV Infection and Carcinogenesis in Cervical Cancer

Potential diagnostic and prognostic biomarkers in most neoplastic lesions of the cervix are based on molecular mechanisms associated with HPV infection ^[10][11][13][10,11,13]. Clinically significant markers of HPV-mediated oncogenesis are used in the following methods: determination of virus by PCR methods, assessment of viral load, detection of E6/E7 mRNA, p16INK4a methylation, p53, Rb and p16INK4a, other cell cycle biomarkers, proliferation and apoptosis, determined by immunohistochemistry ^{[11][21][22][27]}[11,21,22,27]. The most widely used and well-studied biomarkers are HPV DNA in cervical epithelial cells and the immunohistochemically detectable expression of the p16INK4a protein, which is directly related to HPV oncogenic risk and Ki-67, which is a proliferation marker ^[10][11][12][10,11,12]. The p16 protein takes part in the regulation of the cell cycle and is an inhibitor of the activity of the cyclin-dependent kinase CDK4/6; normally its concentration in the cell is extremely low. Overexpression of p16 indicates active expression of the HPV E7 viral oncogene and inactivation of the retinoblastoma gene by the E7 virus protein; this is observed in malignant neoplasms caused by HPV of high oncogenic risk ^[10][11][12][10,11,12]. From 10% to 25% of cervical carcinomas are adenocarcinomas of various histotypes; high-risk HPV DNA is found in 94% of lesions in situ, 85% of adenosquamous carcinomas, and 76% of adenocarcinomas [23]. HPV DNA is most often found in endocervical adenocarcinoma of the usual type (90%), less often in serous (30%), clear cell (27%) and endometrioid carcinoma (13%). A surrogate marker of HPV is overexpression of p16, detected immunohistochemically; p16 is a sensitive and specific marker not only in the diagnosis of squamous cell neoplasms and squamous cell carcinoma, but also in identifying several histotypes of glandular neoplasms of the cervix [23].

HPV infection can lead to aneuploidy, chromosomal aberrations, and DNA hypermethylation. Potential biomarkers in cervical neoplasia are methylation factors and chromosomal abnormalities in cervical carcinoma detected by fluorescence in situ hybridization (FISH), in particular in the region of the telomerase RNA gene (TERC) located in the 3q26 region [20]. It has been established that E6 proteins of oncogenic HPV promote transcription of telomerase reverse transcriptase (TERT), which restores DNA at the telomeric end of chromosomes and therefore increases the cell division limit [11]. Increases of chromosome 3q, which contains the telomerase sequence (TERC), and chromosome 5p, which contains the TERT gene, are associated with intraepithelial neoplasia, which is a useful marker for detecting progressive lesions ^[8][11][15][8,11,15]. Telomerase is an enzyme which with an increase of its activity, compensates for the shortening of telomeres during sequential replications, which is observed during cell division; an increase in telomerase activity is characteristic of stem and tumor cells, and telomerase activity is not detected in most somatic cells [15]. It was found that the telomerase RNA gene in the 3q26 region is found in CIN lesions and cervical carcinoma and correlates with the degree of the disease. It was demonstrated by fluorescent in situ hybridization that patients without an increase in 3q26 showed regression of the disease, i.e., the lack of 3q26 increase on diagnostic biopsy can potentially be used to identify high-grade CIN lesions with a high probability of disease regression, preferably as part of a wider biomarker panel [20]. The authors suppose that 3q26 FISH may help select women with low-grade cervical lesions (LSIL) who do not need immediate colposcopy and patients with severe lesions (HSIL) who do not need immediate treatment. Such approach can help to reduce treatment costs and reduce the side effects of surgery treatment; however, further studies on larger sample groups are needed to confirm this hypothesis [20].

Serum biomarkers play an important role in the monitoring of many malignant neoplasms, including cervical cancer ^[24][25][24,25]. A well-known biomarker is the squamous cell carcinoma antigen SCC-Ag, which was discovered in 1977. The squamous cell carcinoma antigen (SCCa) is the most widely used and most reliable tumor marker for squamous cell carcinomas [24]. During the analysis of the literature devoted to the study of the level of SCCa in the blood serum as a predictor of cervical cancer metastases to lymph nodes, it was found that SCCa can serve as a reliable prognostic factor; the meta-analysis confirmed the value of the predicting property of SCCa, even though the predictive value was moderate. Elevated SCCa is clinically relevant information in addition to CT scan or magnetic resonance imaging (MRI) supporting the need for laparoscopic evaluation of the pelvic and/or para-aortic lymph nodes [24]. In a meta-analysis of the association of serum SCC-Ag with relapse and mortality in patients with squamous cell carcinoma of the cervix, it was found (in 61 articles) that SCC-Ag in serum was invariably associated with relapse and mortality in newly diagnosed cervical cancer; this marker may be useful for monitoring disease progression in patients with cervical cancer [25].

Despite the huge progress that has been made in the screening and management of women with HPV-associated cervical pathology, there is still a need for clinically reliable biomarkers to further improve the screening, diagnosis, and prognosis of cervical carcinomas ^{[8][10][21][22]}[8,10,21,22]. An important role in clinical pathology and oncologic morphology is given to immunohistochemical methods aimed at solving diagnostic problems and searching for new prognostic and predictive factors. Taking into account the role of individual biomolecules in the cell during the development of physiological and pathological processes, the following categories of immunohistochemical biomarkers are distinguished –markers of the cell cycle, proliferation, apoptosis, adhesion, invasion, angiogenesis, microenvironment, immune factors, and others—which are currently being tested for verification of “cancer stem cells” [56].

4. Biomarkers of Cancer Stem Cells in Cervical Carcinomas

In recent years, the concepts of tumor (cancer) stem cells (CSCs) and tumor microenvironments (niches) have been suggested and developed ^[57][90][57,91]; various biomarkers of tumor stem cells, including immunohistochemical ones, are being investigated. According to the modern concept, the CSC population is a small fraction of the total quantity of tumor cells, characterized by the expression of certain surface markers that allow it to be identified and isolated, support the growth of a heterogeneous population of tumor cells, form a separate pool of cells, be verified by biological and physicochemical methods, and have an unlimited capability of self-renewal and differentiation in various directions with the formation of a heterogeneous population of tumor cells consisting of different clones, and it is characterized by high resistance to therapy [57]. Resistance to therapy may occur due to the selective expression of some members of the multidrug resistance transporter family, an increase in the expression of anti-apoptotic molecules, an increased ability to repair DNA, or activation of stem cell-specific survival signals (pro-survival signaling), namely Notch, Hedgehog (Hh), Wnt, JAK/STAT, and others [57].

An important role in the structure of the tumor is assigned to the CSC microenvironment, the so-called “niches”. Niches are certain anatomical compartments in tissue surrounding stem cells which regulate the participation of these cells in the formation, maintenance, and repair of tissue ^[48][57][48,57]. The microenvironment includes such structures and components as the extracellular matrix, mesenchymal and endothelial cells, cells of the immune system, adhesion molecules, growth factors, cytokines and their receptors, and blood vessels [57]. The tumor microenvironment is represented by a stroma with cells of various types: fibroblasts, tumor-associated fibroblasts, myofibroblasts, smooth muscle cells, endotheliocytes, pericytes, neutrophilic and eosinophilic leukocytes, basophils, mast cells, T- and B-lymphocytes, macrophages, and dysplasia ^[90][91]. According to Zibirov and Mozerov (2018), understanding the mechanisms of interaction between tumor cells and the microenvironment is promising for increasing the effectiveness of treatment [49].

Currently, there is a significant number of publications dedicated to different problems associated with CSCs of various tumors. The tasks of verifying CSCs by detecting cell markers (antigens) by immunohistochemical methods are urgent. A large number of experimental works and reviews are devoted to the identification of surface antigens characteristic of CSCs in various tumors, including cervical cancer, and the list of such markers is constantly growing ^{[48][56][58][59][60][61][62][63][64]}[48,56,58,59,60,61,62,63,64]. Recently, it is believed that cervical cancer develops from stem cells of the transformation zone upon infection with HPV of high oncogenic risk, where the transformation zone is a niche for cells with a unique expression profile and embryonic characteristics [48]. Modern data on CSCs in cervical cancer are, however, incomplete. It has been established that one of the markers of cervical CSC is Nanog, a factor that is expressed mainly in the internal cell mass of blastocysts; its expression is significantly expressed in early embryonic stem cells and decreased in the processes of differentiation. Nanog plays an important role in the regulation of pluripotent cells in embryogenesis, and recently it was found to be closely associated with oncogenesis; it is found in cancer of the stomach and breast, glioblastoma, lung cancer, and various human sarcomas ^[48][58][48,58]. There is evidence of a pronounced expression of Nanog in the cervix with CIN-II and -III and in cervical carcinoma cells, in contrast to weak expression in CIN I and in normal epithelium [58]. Ye et al. (2008), in an immunohistochemical study of the cervix of 235 patients, showed that the expression of Nanog, nucleostemin (NS) and musashi1 (Msi1) was significantly higher in cervical cancer compared to CIN, and in CIN compared to normal cervical epithelium; the results give evidence that Nanog, NS, and Msi1 may be involved in the carcinogenesis and progression of cervical cancer [58]. Recent studies have demonstrated the expression of CK 17 and CK19 on CSCs; however, further research is required to understand the possible role of CK19 in CSCs, as the study of cervical CSCs is currently at an early stage, so there are no internationally recognized markers of the cells [48].

There is evidence that Sox2-positive SiHa and C33A cells, as compared to Sox2-negative cells, demonstrate more pronounced abilities for self-renewal, differentiation, and tumor formation [62]. In addition, Liu et al. (2014) found that Sox2-positive SiHa and C33A cells expressed higher levels of several genes associated with SCSs, i.e., cells expressing endogenous Sox2 are CSCs in cervical carcinomas [62]. In a review publication devoted to CSCs in cervical cancer, the following factors are included in the group of cancer stem cell markers: ABCG2, ALDH1, CD133, CD49f, OCT4, OPN, and SOX2 [63]. Nevertheless, at present, many issues related to cervical CSCs have not been sufficiently studied, so the specificity of such a general CSC biomarker as CD44, which is used in the diagnosis of many tumors in cervical cancer, is not sufficiently established. The proto-oncogene c-Kit (protein tyrosine kinase KIT or CD117), a transmembrane cytokine receptor, is phosphorylated and activated by binding to a KIT ligand called stem cell factor, which is a marker of SCSs in ovarian and endometrial cancers and osteosarcoma; however, the proposal that c-Kit is a marker of CSCs in cervical cancer is not yet proved. NANOG is a transcription factor that plays an important role in maintaining the pluripotency of CSCs and regulation of proliferation and asymmetric division; it is used widely as a marker of CSCs that regulate renewal and tumorigenesis in many tumors [63]. Therefore, stem cells play a key role in the physiology of the female reproductive system; however, according to Lopez et al. (2013), this field is still in its infancy—definitive markers need to be identified for more selective isolation and enrichment of stem cells, and further research is needed to assess clinical characteristics, prognosis, survival, and population characteristics; these studies will improve the understanding of carcinogenesis in ovarian and uterine cancer and cervical carcinoma and may be useful in the treatment of these neoplasms [64].