Papillomaviruses are widely distributed in mammals and are species-specific [1]. Human papillomavirus (HPV) cannot be cultivated in tissue cultures or in common experimental animals [1]. They are members of the Papillomaviridae family, have no coating, measure from 50 to 55 nm in diameter, and have an icosahedral capsid of 72 capsomers [2]. Of the approximately 200 genotypes of HPV, subdivided into 14 species, about 40 can infect the epithelial cells (skin or mucous membranes) of the anogenital regions and other areas [3]. Differentiation into types is made based on the characteristics of the L1 protein [4]. HPV was the first virus to be recognized as responsible for cervical cancer (CC). According to the degree of association with invasive tumors, HPV genotypes have been subdivided into: high oncogenic risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68), related with an increased risk of developing CC [5]; low oncogenic risk (6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81, 89) associated with no disease most commonly or with benign epithelial lesions (such as anogenital and oropharyngeal warts) [6]; and, finally, HPV with an “undetermined risk” (3, 7, 10, 27, 28, 29, 30, 32, 34, 55, 57, 62, 67, 69, 71, 74, 77, 83, 84, 85, 86, 87, 90, 91) include those whose oncogenicity has not yet been fully defined [7]. It has been established that about 99.8% of CC have a high-risk HPV DeoxyriboNucleic Acid (DNA) sequence, particularly HPV 16 and 18, found in about 70% of invasive carcinomas [8]. The prevalence of infection is very high (70% of sexually active female patients over 25 years old), and most infections tend to regress spontaneously, with or without manifestations of dysplasia; only in some cases can HPV infection become persistent [9]. Data from the scientific literature show that in female patients over 30 years old, persistent high-risk HPV infections play a critical role in predicting the risk of developing CC [8,9,10]. The risk of developing in histopathologic high-grade cervical intraepithelial lesion HSIL (CIN2 and CIN3) or invasive CC is estimated to be much higher in women with persistent high-risk HPV infection, being 11 times higher in the 30–44 age group, 35-fold higher between 45 and 54 years, and 49-fold in those over 50 years of age [11]. The ability of HPV viruses, especially those at high risk, to integrate into infected cells, and to orchestrate a gene expression program that allows the transcription of oncogenic proteins (E6, E7), promotes carcinogenicity [12]. Cervical intraepithelial lesions (CIN) can regress spontaneously, or progress to invasive neoplasia in different percentages depending on their severity. More specifically, histopathologic low-grade cervical intraepithelial lesion LSIL (CIN1) tends to regress spontaneously, particularly in young patients. Ostor and coworkers [13] report that CIN1 subsides spontaneously in 60% of cases, persists in 30%, and can progress in 10% of cases. On the other hand, CIN2 regresses spontaneously in 40%, persists in another 40% of cases, and can progress in 20% of cases; finally, CIN3 can regress in 33% of cases and progress in more than 12% of cases.

The natural history of CC is typically characterized by the progression, over the years, of non-invasive HPV-related precancerous lesions to invasive carcinoma [7,8,9,10,11,12,13]. Cytology (Pap-Test) and human papillomavirus detection (HPV-DNA) are two screening tests whose purpose is to detect CC or precancerous changes at an early stage. Before the development of the HPV-DNA test, the Pap-Test alone was performed every 3 years in women after the onset of sexual activity or in any case from 25 years of age [14]. Today, according to guidelines from different countries, HPV-DNA tests are used for women over 30 or over 25, and a Pap-Test is only done if it gives a positive result [15,16,17,18,19,20]. In fact, globally the recommended method of primary CC screening is the HPV-DNA test, independently of resource settings [21], due to its sensitivity compared to the Pap-Test, even in the presence of a lower specificity especially for the identification of CIN2 and CIN3 lesions. HPV testing is recommended by the World Health Organization [14] and other guidelines [15,22] even with respect to the Pap-Test which is now considered a secondary test [14]. In fact, the Pap-Test even if it has allowed to tangibly reduce the incidence of invasive carcinoma, however, presenting a variability between different operators, it can lead to the diagnosis of false negatives as well as cases of invasive CC are also reported in the literature in women regularly investigated with the Pap-Test [23]. In view of the crucial role of persistent infection of hr-HPV, the focus has shifted to the use of HPV-DNA testing as a screening test [24] so that access to treatment is increasingly targeted and timely. According to the ASCO guidelines, if the HPV-DNA test results positive, genotyping for HPV 16/18 (with or without HPV 45) and/or Pap-Test are also indicated [21]. In the event of a positive or abnormal result, the HPV-DNA test procedure involves colposcopy and related biopsy [21]. Conversely, in discordant results between the HPV test and cytological examination, it is recommended to repeat the HPV-DNA test one year later, then repeat the test at 12–24 months in case of negativity or colposcopy in women who tested positive [21]. Finally, in cases of CIN2 histological diagnosis, patients should be offered a surgical solution followed by targeted follow-up over time [21]. According to data from the randomized study published by Ronco et al. [25], HPV-research-based screening is more effective than the Pap-Test in preventing CC in women aged 25–60, because it allows an earlier identification of high-grade persistent lesions. In fact, the execution of the HPV-DNA test is useful in stratifying the population according to the degree of risk: a negative test indicates a low risk of developing CC, and so in these controls can be made at longer intervals. Although with differences between the various settings, the HPV-DNA test should be started from the age of 30 in the general female population, regular screening being done with the HPV test validated every 5–10 years, versus 25 years of age in women living with HIV, who should be screened more frequently, every 3–5 years. CC is the fourth most common malignancy among women worldwide, accounting for approximately 7% of all female cancers [26,27]. As reported in the literature, most of the diagnoses of CC can be associated with the presence of HPV infection and, in some studies, these associations can reach levels comparable to almost all cases [28]. Among the high-risk HPV genotypes, variant 16 has the highest affinity for neoplastic progression with over 50% of cases, followed by variant 18 which occurs in 20% of cases; this association tends to vanish in the remaining high-risk genotype up to 5% and even less [29]. Conversely, there does not appear to be a significant difference between the HPV status and the histotype of the carcinomas except for squamous-cell carcinoma (SCC) which is unlikely to be HPV-negative [30], as well as in mixed adeno-squamous form where the HPV positivity may reach up to 86% [31] of cases and also in the vast majority of Adenocarcinoma in situ (AIS) [30]. Vice versa, the prevalence of HPV among adenocarcinoma (AD) types can vary and, according to the International Endocervical Adenocarcinoma Criteria and Classification, ADs are divided in two categories: HPV-associated (HPVA) and not HPV-associated (NHPVA), with well-defined characteristics due to histology HPVA shows more mitotic activity or apoptotic figures than NHPVA. If focal or equivocal HPVA features are visible at ×200, a tumor can be classified as a “limited HPVA” and provisionally diagnosed as NHPVA AD [32]. To this histotype belong different variants such as mucinous (that can show aspects of HPVA and NHPVA), gastric (prevalent NHPVA type), endometrioid and serous carcinomas (extraordinarily rare). NHPVA comprehends histological variants [32] such as gastric, clear cell, serous, endometrioid, or mesonephric carcinomas that notoriously tend to be HPV negative [33] compared to histotypes presenting glandular/villo-glandular/intestinal aspects that appear to have a higher percentage of HPV positivity.

The study of viral and cellular biomarkers that could be useful for identification of specific stages of cervical intraepithelial lesions related to hrHPV infection is closely linked to the biology of HPV and the different stages from infection through intraepithelial lesion, and, if this is not properly treated, to invasive CC. After hrHPV infection, the infected cervical epithelium begins to proliferate, leading to the transformation into CIN1, 2, and 3 [34,35,36,37]. Progression from a transient to a transformation HPV infection is characterized by a sharp increase in HPV mRNA E6/E7 and protein expression [34,37]. With this in mind, several authors have assessed how the detection of mRNA transcripts belonging to E6/E7 proteins could be helpful for identifying cervical precancers [36,37,38,39,40,41,42]. From these works, we can deduce a good sensitivity and specificity of these tests; there are two platforms used for the study of this biomarker (PreTect. Proofer and APTIMA GenProbe). The p16INK4a protein is a cyclin-dependent kinase inhibitor that plays a key role in cell cycle regulation and is upregulated when E7 is overexpressed as in HPV infections, representing an ideal biomarker to define the nature of cervical lesions [43,44], especially if combined in a single test together with Ki-67 [45,46] which is generally used in immunohistochemistry to evaluate the cell proliferation index [47]. Double-stained cytology p16/Ki-67 (approved by FDA on 3 October 2020 [48]) is a qualitative immunocytochemical assay intended for the simultaneous detection of proteins P16INK4a (clone E6H4) and Ki-67 (clone 274-11AC3V1) in cervical specimens of women aged 25–65 years with positive HPV test (high oncogenic risk); it is also indicated for patients aged between 30 and 65 who have to postpone colposcopy or have other risk factors regardless of the result of the HPV test [48]. It is highly expressed in almost 100% of cases of CIN2, CIN3, squamous CC, but is rarely found in benign forms; it is highly expressed in 100% of AIS cases. Several studies have highlighted the ability of the p16 test to identify the neoplastic transformation of cervical cells infected with human papillomavirus, being able to predict with greater precision the underlying cervical intraepithelial neoplasia of grade 3 or worse [49,50]. Specifically, the ATHENA study, on a group of 7727 patients, showed that p16/Ki-67 dual-stained cytology was significantly (p < 0.0001) more sensitive than Pap-Test (74.9% vs. 51.9%) for the triage of HPV-positive women and that specificity was comparable between the two methods [50]. In studies directed by Bergeron and Petry [46,51], as well as by Wright and coworkers, the authors described a p16/Ki-67 dual-stained cytology, that either alone or combined with HPV16/18 genotyping, represents a promising approach as a sensitive and efficient triage for colposcopy of HPV-positive women when primary HPV screening is utilized [50]. These studies led the way in using p16/ki-67 as a reliable tool for risk stratification of HPV-positive women with cervical lesions, reporting elevated sensitivity values similar to those of the HPV test [51,52,53,54,55]. Furthermore, other authors have shown that p16/ki-67 had a higher specificity than the HPV test [45,53,54,55]. These data reinforce the diagnostic value of p16/Ki-67, which represents a useful tool in avoiding further diagnostic investigations such as unnecessary colposcopies given its ability to more precisely identify female patients at increased CIN2 risk. In the field of research into serum biomarkers for the early detection of CC, the activation of Macrophage-Colony Stimulating Factor (M-CSF) and Vascular Endothelial Growth Factor (VEGF) is likely involved in the pathogenesis and spread of CC. In particular, M-CSF is overexpressed in the CC lines compared to CIN and the blocking of the M-CSF receptor determines both a growth arrest and an increase in intratumoral apoptosis [56,57]. In various papers, Lawicki and coworkers [38,58], using the immunosorbent assay (ELAISA), evaluated the plasma levels of M-CSF as compared to commonly accepted tumor markers, such as CA 125 and SCC-Ag in CC patients before surgery and in healthy subjects. The M-CSF plasma level was significantly higher, as also CA 125 and SCC-Ag, in CC patients. The diagnostic sensitivity of M-CSF was higher than stem cell factor, CA 125, and SCC-Ag (25%, 30%, 40%, respectively). The diagnostic specificity was high and equal for all tested cytokines and CA 125 (92%). Positive and negative predictive values were higher for all tested parameters but highest for M-CSF (83% and 69.7%, respectively) [38]. More recent studies [59,60,61,62] confirmed that cytokine members of hematopoietic growth factors may have a diagnostic potential in CC. Zajkowska evaluated serological markers against CA 125 and SCC-Ag in 100 CC patients with chemiluminescent microparticle immunoassay and showed statistically significant M-CSF values from all parameters tested in the CC cohort compared to the control groups [59]. Similar results have also been described by Lubowicka et al. who investigated M-CSF, matrix metalloproteinase-2, and its inhibitor beyond CA 125 and SCC-Ag in 89 CC patients and in 50 healthy women (aged 22–61 years) reporting for M-CSF the highest specificity (86%) in the CC group [60]. Moreover, the association of these different markers increased in specificity, as observed in the combination between matrix metalloproteinase-2 and CA 125 in different CC stages [60]. While median levels of M-CSF and VEGF, as well as CA 125 and SCC-Ag are shown to be significantly different in women with CC, this relationship does not seem to be specific to SCC alone; in fact, the plasma levels of M-CSF and VEGF are also higher in AD than in the control group and, moreover, no significant differences were observed between SCC and AD [61]. Sidorkiewicz reported 81% sensitivity and 74% specificity for VEGF in the SCC group and 86% and 76% in the AD group [61]. Although the results of these studies are encouraging and suggest a diagnostic utility and a possible clinical applicability of serum biomarkers in patients with CC, to date they are not yet sufficiently studied, and more confirmations would be useful for their wider use than p16/Ki67 testing and mRNA detection.