The Role of p16/Ki-67 Immunostaining, hTERC Amplification and Fibronectin in Predicting Cervical Cancer Progression: A Systematic Review

Simple Summary Human papillomaviruses (HPV) are common sexually transmitted infections and they are responsible for cervical cancer (CC), as well as for several other anogenital cancers. CC is the fourth leading cause of death in women with cancer, although it could be preventable by enforcement of optimal screening programs. The Pap smear is the standard screening test for CC and precancerous lesions, and a combination of Pap smear and HPV testing is generally recommended as a triage step before colposcopy. However, these tests cannot predict lesion progression, which is why several adjunctive biomarkers have been studied. Our aim was to summarize current scientific data on the role of these biomarkers, with a view to determining which biomarkers could help to more accurately establish the need for colposcopy and at the same time, to limit the number of unnecessary colposcopy referrals. Abstract Human papillomaviruses (HPVs) are common sexually transmitted infectious agents responsible for several anogenital and head and neck cancers. Cervical cancer (CC) is the fourth leading cause of death in women with cancer. The progression of a persistent HPV infection to cancer takes 15–20 years and can be preventable through screening. Cervical cytology (Pap smear) is the standard screening test for CC and precancerous lesions. For ASC-US and ASC-H lesions, a combination of Pap smear and HR-HPV analysis is recommended as a triage step before colposcopy. However, these tests cannot predict progression to CC. For this purpose, we summarized current scientific data on the role of p16/Ki-67 immunohistostaining, telomerase and fibronectin in predicting progression to CC. p16 and p16/Ki-67 dual staining (DS) were more specific than HR-HPV DNA testing for the detection of CIN2+/CIN3+ in women with ASC-US and LSIL. Similarly, hTERC FISH analysis significantly improved the specificity and positive predictive value of HPV DNA testing in differentiating CIN2+ from CIN2 cytological samples. In conclusion, p16 IHC, p16/Ki-67 DS and hTERC FISH amplification are all valid adjunctive biomarkers which significantly increase the sensitivity and specificity of cervical dysplasia diagnosis, especially when combined with HPV DNA testing. However, considering the global socioeconomic background, we can postulate that p16 and p16/ Ki-67 IHC can be used as a next step after positive cytology for ASC-US or LSIL specimens in low-income countries, instead of HPV DNA testing. Alternatively, if HPV DNA testing is covered by insurance, p16 or p16/Ki-67 DS and HPV DNA co-testing can be performed. In middle- and high-income countries, hTERC amplification can be performed as an adjunctive test to HPV DNA testing in women with ASC-US and LSIL.

The main role of p16/Ki-67 IHC in the triage of HPV-positive women is to distinguish between those with underlying high-and low-grade cervical lesions, which aids in determining the necessity for immediate colposcopy referrals [20]. It is cost-effective, highly reproducible and has a relatively low technical complexity [13], which makes it easily accessible and widely used.
Telomerase up-regulation is known to arrest cellular apoptosis, thus having a central role in malignant proliferation [21,22]. Moreover, the E6/E7 oncogene encoding the HPV proto-oncoprotein can up-regulate telomerase activity by human telomerase RNA component (hTERC) gene amplification. Studies have shown an important correlation between HR-HPV infection and hTERC up-regulation in CC progression [23][24][25][26]. Telomerase activity as a prognostic biomarker in CC has been demonstrated through numerous studies and it is generally recommended as an ancillary biomarker in CC screening, after cytology and HPV DNA detection.
Fibronectin (FN1) is a glycoprotein component of the extracellular matrix that plays an important part in cell growth, cell adhesion and differentiation [27]. A few studies have discussed its potential role in different malignancies such as hepatocellular, renal, gastrointestinal, head and neck cancers [28,29]. We further discuss the literature published so far.

Study Selection
We conducted a systematic review of the literature following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. We searched the PubMed database for studies published between 2011 and 2022 using the term cervical cancer in combination with the following terms: telomerase, fibronectin, p16, ki-67, HPV. The last search was run on 25th March 2022. There was no limit to study design.

Data Extraction
Two investigators independently selected relevant articles according to predefined inclusion and exclusion criteria, as described above. Disagreements were resolved by discussion, with a prior arrangement that any unsettled discrepancy would be determined by a third author.

Inclusion Criteria
Eligibility was restricted to studies in which p16, ki-67, telomerase and fibronectin positivity were correlated with histopathologic modifications in cervical specimens classified according to the Bethesda system. The relationship between the grade of cervical dysplasia and mentioned markers was analyzed. Only articles in English were selected. Only studies in which telomerase activity and HPV detection were performed by genomic amplification techniques and not by staining procedures were included. Other potentially relevant articles were identified by manually checking the references of the articles included.

Exclusion Criteria
We excluded the following studies: those where the number of patients was either not specified or expressed as different age frequencies; those where the main inclusion criterion was only HPV-positive patients; those where different comparisons were drawn, either between the sensitivity and specificity of various detection methods, or between selfsampled specimens and samples collected by healthcare professionals. Additionally, studies in which p16 and ki-67 staining were assessed only according to the level of expression and not as either positive or negative specimens were excluded, given the great heterogeneity of histopathological assessment techniques and grading systems [30,31].

Data Synthesis and Statistical Analysis
Pertinent data were selected in the form of: number of biopsy specimens analyzed, number of specimens for each category of the Bethesda classification system, number of HPV-positive specimens, HPV types detected, number of p16-, Ki-67-, telomerase-and fibronectin-positive specimens.

Limitations
The limitations of this review lie in study heterogeneity, which is reflected in the different scoring systems used for cervical modifications, for HPV detection, and for p16/ ki-67 staining positivity. In order to limit bias in reporting, we objectively summarized relevant data from the literature in Tables 1-4. We only included studies where a definitive histopathologic diagnosis was provided and cervical dysplasia was classified according to either of the Bethesda systems [16,17]. Non-neoplastic lesions (NNL) included any type of modification, including inflammation (cervicitis), infection and atypical metaplasia, which was mentioned in just one study [32]. Cervical cancer (CC) was ascribed to both squamous cell carcinoma, either in situ or invasive, and adenocarcinoma, considering that most studies included both types of cervical cancer under this common nomenclature.

Results
A total of 853 records were initially identified in the literature search, of which 34 were duplicates and 728 did not meet the inclusion criteria, thus being further excluded ( Figure 1). A total of 20,877 biopsy specimens were investigated, of which there were 7174 for p16 IHC, 745 for Ki-67 IHC, 5329 for p16/ ki-67 dual staining (DS) and 9084 for telomerase up-regulation.

Data Synthesis and Statistical Analysis
Pertinent data were selected in the form of: number of biopsy specimens analyzed, number of specimens for each category of the Bethesda classification system, number of HPV-positive specimens, HPV types detected, number of p16-, Ki-67-, telomerase-and fibronectin-positive specimens.

Limitations
The limitations of this review lie in study heterogeneity, which is reflected in the different scoring systems used for cervical modifications, for HPV detection, and for p16/ ki-67 staining positivity. In order to limit bias in reporting, we objectively summarized relevant data from the literature in Tables 1-4. We only included studies where a definitive histopathologic diagnosis was provided and cervical dysplasia was classified according to either of the Bethesda systems [16,17]. Non-neoplastic lesions (NNL) included any type of modification, including inflammation (cervicitis), infection and atypical metaplasia, which was mentioned in just one study [32]. Cervical cancer (CC) was ascribed to both squamous cell carcinoma, either in situ or invasive, and adenocarcinoma, considering that most studies included both types of cervical cancer under this common nomenclature.

Fibronectin
Zhou et al. [84] performed a comparative study assessing the levels of FN1 expression in 94 paired patients with CC by quantitative real-time polymerase chain reaction (qRT-PCR). They found significantly higher FN1 levels in cervical cancer tissues than in adjacent normal tissues. Furthermore, higher FN1 expression was correlated with poor prognosis.

Discussion
Currently, cervical cytology (Pap smear) is the standard screening test for CC and precancerous lesions [11]. For ASC-US and ASC-H lesions, a combination of Pap smear and HR-HPV analysis is generally recommended as a triage step before colposcopy [66]. However, these tests have low applicability: Pap smear can only identify abnormal cell morphologies and most HPV infections are self-limited, thus neither test has predictive value [22].
Despite being considered transitory, low-grade lesions, a critically large number of ASC-US and LSIL specimens had underlying CIN2 and CIN3 morphologic changes, which carry a high risk for malignant transformation [18]. Consequently, adjunctive biomarkers have been investigated in order to increase the accuracy of CC screening and to guide selection of the most appropriate treatment option.

P16/Ki-67 staining
p16inka (p16) is a cyclin-dependent kinase inhibitor involved in the normal cycle of somatic cells and acts as a tumor suppressor [44]. p16 overexpression is associated with keeping the retinoblastoma protein (Rbp) in an unphosphorylated state which deaccelerates cell cycle progression from G1 to S phase [85,86]. Viral oncogenes E6 and E7 are known to be drivers of proliferation, promoting and maintaining the malignant growth of cervical cells in the process of high-risk HPV-linked carcinogenesis [13,87]. p16 protein is considered a surrogate biomarker for the transforming activity of high-risk HPV and it can be detected via IHC staining of cytology or histology specimens [88,89]. p16-positivity is defined as strong and diffuse staining, meaning nuclear and/or nuclear plus cytoplasmic expression affecting the basal and para-basal cell layers and extending to the surface of the squamous epithelium on histological sections [90].
Ki-67 is a non-histone cell cycle progression antigen expressed only during the active phases of the cell cycle (G1, S, G2 and mitosis) and it is described as a biomarker for determining the growth fraction of a tumor [91]. According to IHC studies, Ki-67 is normally expressed in the basal and para-basal layers of the epithelium, whereas highgrade CIN lesions containing abnormally proliferating cells appear as increased Ki-67 staining in all layers of the squamous epithelium [19]. Isolated expression of p16 or Ki-67 within a cell may be considered physiologic, whereas simultaneous positive staining of the two biomarkers is linked with cell cycle dysregulation associated with a transformative high-risk HPV infection [13]. Co-expression of p16 and the cell cycle progression biomarker Ki-67 in one cell allows for the unequivocal identification of HPV-transformed epithelial cells and can be detected via dual immunostaining (DS) of p16/Ki-67 [92].
Additionally, p16/Ki-67 DS positivity was strongly associated with HR-HPV persistence and the presence of CIN2+ lesions [57]. One study found that p16/Ki-67 DS had sensitivity and specificity rates of 93.2% and 46.1%, respectively, for CIN3+ detection and these increased to 97.2% and 60.0% in women older than 30 years; for women with HR-HPV-positive ASC-US and LSIL, sensitivity and specificity rates were as high as 90.6% and 48.6%, respectively, which might make p16/Ki-67 DS a potent biomarker for LSIL triage [22]. Additionally, Ma et al. [36] showed that p16 immunostaining had significantly higher specificity and accuracy in predicting high-grade CIN and CC in ASC-US and LSIL specimens, as compared with HR-HPV DNA testing.
In a prospective, cross-sectional study on 599 patients, Liu et al. [65] compared the clinical performance of Pap smear, HPV DNA testing and p16/Ki-67 DS for the detection of CIN2+/VAIN2+. They found that for women who tested positive for HR-HPV and had a Pap smear ≥ ASC-US, DS reduced the number of unnecessary colposcopy referrals from 274 to 181. Additionally, DS identified four high-grade lesions that had initially negative colposcopy-guided biopsy results.
A recent meta-analysis evaluating the accuracy of p16 and p16/Ki-67 DS versus HR-HPV testing for the detection of CIN2+/CIN3+ in women with ASC-US and LSIL found that p16 staining and p16/Ki-67 DS were more specific than HR-HPV DNA testing, whereas p16 staining was less sensitive and DS has similar sensitivity [93].
Throughout the studies, however, sensitivity (Se) rates remained above 90%, whereas specificity (Sp) rates were below 50% [22], which indicates a high risk of unnecessary biopsy referrals. p16 IHC had significantly higher specificity and accuracy rates in predicting high-grade CIN and CC in ASC-US and LSIL specimens, as compared with HR-HPV DNA testing [36]. Additionally, p16 and HR-HPV co-testing had Se = 89.58% and Sp = 72.73% [33]. However, no studies analyzing the combined Se and Sp rates of cytology, HPV DNA testing and DS have been performed. On the other hand, p16 IHC was shown to be a significant negative predictor of survival [44], whereas HPV-positive CC had better survival rates [45][46][47]. Finally, according to The Lower Anogenital Squamous Terminology (LAST), p16 IHC is recommended for distinguishing between H-SIL and benign lesions mimicking precancerous lesions (immature squamous metaplasia, atrophy, repair changes and tangentially sectioned specimens) and also for the assessment of morphologically equivocal cases interpreted as L-SIL versus H-SIL [94].
Hence, given the current literature, it can be postulated that DS can be used ancillary to, or instead of HPV DNA detection, for women with ASC-US and LSIL. Additionally, p16 IHC can be used as a negative survival predictor for women with CC [44].

Telomerase
Telomerase is a ribonucleoprotein enzyme complex that adds 50-TTAGGG-30 repeats to the chromosomal ends known as telomeres, which play an important part in maintaining chromosomal stability during DNA replication [21,23,24]. Human telomerase consists of three subunits: one RNA component (hTERC), which functions as a template for DNA replication; one of unknown function (TP1) and the human telomerase reverse transcriptase (hTERT) [95,96]. hTERC gene expression is consistent with telomerase activity and it is generally expressed in many normal tissues [24]. However, telomerase up-regulation can reflect a malignant process as it stops cellular apoptosis, consequently leading to tumorigenesis [21,22]. The majority of studies have demonstrated the importance of increased telomerase activity as a prognostic marker in CC, its level being positively correlated with viral load, clinical stage, tumor size and lymph node metastases [96]. The activity of telomerase might be a potential method for the differential diagnosis between low-grade and highgrade precancerous cervical neoplasia, reaching Se and Sp rates of over 90% [21,23,97,98]. HR-HPV positivity and increased hTERC activity have been linked to more aggressive CC and might have an important role in future screening algorithms [23][24][25].
Furthermore, it has been suggested that hTERC amplification be used as a triage test, ancillary to HPV DNA in ASC-US and LSIL cytological samples, as a predictor of progression to more severe cervical neoplasia [21]. Studies have shown that increased telomerase activity detected by FISH analysis increased with the degree of cervical dysplasia [21]. In addition, hTERC FISH analysis significantly improved the specificity and positive predictive value of HPV DNA testing in differentiating CIN2+ from CIN2 cytological samples [25,79]. Currently, the determination of telomerase activity is not used in routine screening tests, but most authors have proposed that this method become part of future screening tests for cervical dysplasia [24,77,80,96].
Moreover, the combination of cytology, HPV DNA testing and hTERC amplification reached Se and Sp levels as high as 100% and 98.11%, respectively [68,71]. This makes hTERC an important adjunctive biomarker for CC screening and it can be recommended as an ancillary test to cytology and HPV DNA detection in women with ASC-US and LSIL lesions.

Fibronectin
Fibronectin is an extracellular matrix glycoprotein that plays a major role in cell differentiation, growth and migration. Furthermore, it is involved in the processes of wound healing and embryonic development, as well as oncogenic transformation. The highest levels of fibronectin expression were detected in colorectal, renal and esophageal cancers and were associated with poor prognosis [84]. Few studies have shown a significantly higher expression of fibronectin in cervical cancer tissues compared with adjacent normal tissues, but further evidence is lacking [84,99]. Consequently, the role of fibronectin as a prognostic marker in patients with CC requires additional investigation and might have potential diagnostic and therapeutic implications.

Challenges and Future Scope
CC screening and HPV vaccination campaigns are the pillars of CC prevention. However, given the financial, political and educational differences worldwide, strategies for CC prevention cannot be implemented homogenously. Access to medical care, information campaigns and health financing influence the addressability of CC screening and the treatment options. Hence, there is continuous research for more reliable and accessible biomarkers that can be used irrespective of the socioeconomic background of each country.

Conclusions
Currently, cervical cytology and HR-HPV analysis are the well-known and widely accepted screening tests for CC and precancerous lesions. However, they cannot be used to predict lesion progression to high-risk intraepithelial neoplasia. ASC-US and LSIL specimens can have underlying CIN2 and CIN3 morphologic changes, which carry a high risk for CC progression, which emphasizes the need for adjunctive biomarkers with predictive value. p16 IHC had significantly higher specificity and accuracy rates than HPV DNA testing in predicting high-grade cervical dysplasia and CC in ASC-US and LSIL specimens. Thus, p16 IHC can be used as an alternative to HPV DNA testing in low-income countries for women with ASC-US and LSIL cytology. However, p16 and HPV DNA co-testing have better sensitivity and specificity rates (Se = 89.58% and Sp = 72.73%), which lowers the number of unnecessary colposcopy referrals, but each case should be investigated according to financial availability. Additionally, p16 can be used as a negative survival predictor for women with CC.
The combination of cytology, HPV DNA testing and hTERC FISH amplification reached sensitivity and specificity levels as high as 100% and 98.11%, respectively, which make hTERC an important, although expensive, adjunctive biomarker for CC screening. It can be recommended as an ancillary test to cytology and HPV DNA detection in women with ASC-US and LSIL lesions, in medium-and high-income countries.
Author Contributions: S.T.V. and N.N. designed the study, prepared the material, statistically processed and analyzed the data and developed and edited the manuscript; C.D., B.G. and M.A.H. developed the methodology; C.C.U. and S.G.T. coordinated and monitored the study activities and critically reviewed the manuscript; F.F.R. and Z.K. reviewed the material used for the study and critically reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.