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Background:
Systematic Review

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

by
Septimiu Toader Voidăzan
1,
Caterina Dianzani
2,
Mădălina Aurelia Husariu
3,
Bíborka Geréd
3,
Sabin Gligore Turdean
4,
Cosmina Cristina Uzun
5,
Zsolt Kovacs
5,
Florin Francisc Rozsnyai
6 and
Nicoleta Neagu
1,3,*
1
Department of Epidemiology, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Târgu Mureş, 540139 Târgu Mureş, Romania
2
Plastic and Reconstructive Surgery Unit, Campus Biomedico University of Rome, 00128 Rome, Italy
3
Dermatology Clinic, Mureș County Hospital, 540136 Târgu Mureș, Romania
4
Department of Pathology, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Târgu Mureş, 540139 Târgu Mureș, Romania
5
Department of Biochemistry, Environmental Chemistry, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Târgu Mureş, 540139 Târgu Mureș, Romania
6
Department of Obstetrics Gynecology, George Emil Palade University of Medicine, Pharmacy, Science and Technology of Târgu Mureş, 540139 Târgu Mureș, Romania
*
Author to whom correspondence should be addressed.
Biology 2022, 11(7), 956; https://doi.org/10.3390/biology11070956
Submission received: 16 May 2022 / Revised: 11 June 2022 / Accepted: 21 June 2022 / Published: 23 June 2022
(This article belongs to the Special Issue Infection, Inflammation and Cancer)
Browse Figure
Versions Notes

Abstract

:

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.

1. Introduction

Human papillomaviruses (HPVs) are common sexually transmitted infectious agents described as non-enveloped, double-stranded, circular DNA viruses belonging to the Papovaviridae family [1]. Approximately 90% of HPV infections are transient and become undetectable in 1–2 years. However, persistent infections with oncogenic HPV types have been associated with the progression of the disease [2,3]. According to epidemiological data, 12 mucosal alpha HPVs are categorized as high-risk HPV (HR-HPV) types and are responsible for several anogenital and head and neck cancers [4]. HPV16 and 18 are the most carcinogenic types: HPV16 has been associated with 50–60% of cervical cancers (CCs), HPV18 with 10–15% of CCs and the remaining HR-HPV types have been implicated in 25–40% of CCs [5,6].
CC is the fourth most frequently diagnosed cancer worldwide [7] and according to the WHO it is the fourth leading cause of death in women with cancer, with an estimated annual incidence of 604,000 cases and 342,000 deaths reported worldwide in 2020 [8]. The progression of a persistent HPV infection to cancer usually takes 15–20 years and it is preventable by the optimal application of secondary prevention programs [9]. CC screening is recommended to be initiated at the age of 21 years via cytology every three years or, for women aged 30–65 years, cytology in combination with HR-HPV testing every five years. Screening can be discontinued in women with a hysterectomy or women older than 65 years who have a history of regular screening with negative results [10,11,12].
Cytology-based screening, also known as the Papanicolau smear (Pap smear) test, was first introduced in 1940 by Georgios Papanicolau as a CC screening tool. Conventionally, microscopic evaluation is performed on cervical cells obtained from cervical scraping after fixing them on a glass slide. Another cytology-based screening method is liquid-based cytology (LBC), by which cervical cells are suspended in a liquid medium and then filtered and transferred onto a monolayer for microscopic evaluation [11,13,14]. Both methods have shown similar sensitivity, specificity, positive predictive value, negative predictive value and accuracy for the detection of cervical intraepithelial neoplasia (CIN) 2 or higher [15].
Cytological findings have been classified according to the Bethesda system [16], which was updated in 2014 [17] and includes the following categories: atypical squamous cells of undetermined significance (ASC-US); atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H); low-grade squamous intraepithelial lesion (LSIL—corresponding to mild dysplasia/ CIN 1); high-grade squamous intraepithelial lesion (HSIL—corresponding to moderate or severe dysplasia, CIS; CIN 2 or CIN 3) and squamous cell carcinoma (SCC) [17]. ASC-US and LSIL are generally considered transient lesions of the cervical epithelium, although an important proportion of women with ASC-US and LSIL have underlying CIN2 or 3 and an increased risk for developing CC [18]. Rigurous triage of women with ASCUS or LSIL is warranted for early diagnosis and treatment of CIN2 or 3 lesions, as well as for minimizing unnecessary biopsies, especially in young women who wish to conceive. The Pap smear test is currently used as a first step in the CC screening method and more recently, HR-HPV co-testing has been integrated into cervical cancer screening guidelines [19]. Despite existing protocols, CC maintains high incidence and mortality rates, which is why several adjunctive biomarkers and their role in accurately predicting progression to CC have been studied. For this purpose, we have summarized current scientific data on the role of p16/Ki-67 immunohistostaining (IHC), telomerase and fibronectin biomarkers.
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.

2. Materials and Methods

2.1. 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.

2.2. 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.

2.3. 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.

2.4. 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 self-sampled 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].

2.5. 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.

2.6. 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.

3. 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.
  • p16 staining
Nineteen studies had relevant data regarding p16 IHC, totaling 7174 biopsy specimens: 1375 NNL, 1857 CIN1, 2 CIN 1/2, 1923 CIN2, 43 CIN2/3, 1664 CIN3, 310 CC. p16 was positive in 3813/7069 biopsy specimens: 2.54% NNL, 15.02% CIN1, 0.05% CIN1/2, 35.79% CIN2, 0.91% CIN2/3, 38.18% CIN3, 6.74% CC. HPV genotyping was positive in 4486/6335 biopsy specimens: 5.84% NNL, 19.30% CIN1, 0.02% CIN1/2, 35.95% CIN2, 0.53% CIN2/3, 32.56% CIN3, 4.34% CC (Table 1).
The majority of the studies demonstrated a directly proportional increase in the likelihood of p16 positive staining and the severity of cervical dysplasia [6,33,34,35,36,37,38,39,40,41,42,43]. Similarly, the number of HPV-positive specimens increased with the degree of intraepithelial lesion [6,33,34,35,36,37,43]. Moreover, higher sensitivity and specificity rates were demonstrated for the combination of HR-HPV detection and p16 IHC in the early diagnosis of cervical lesions, as compared with either test alone: p16 sensitivity (Se) = 95.83% and specificity (SP) = 65.34%, HR-HPV Se = 91.67% and Sp = 53.4%, combination p16 and HR-HPV testing Se = 89.58% and Sp = 72.73% [33].
Additionally, Alhamlan et al. [44] found that p16 IHC was a significant negative predictor of survival. In a retrospective, cross-sectional study conducted on 315 cervical biopsy specimens collected from women aged 23–95 years old who were also PCR-tested for HPV L1 protein, p16 overexpression correlated with poorer survival rates (multivariate Cox regression, hazard ratio, 3.2; 95% CI, 1.1–8.8). Conversely, a multivariate Cox regression analysis showed that HPV-positive cervical cancer (CC) had better survival rates, whereas HPV-negative CC was linked to significantly worse disease-free survival [36]. Similar findings were reported in the literature [45,46,47].
Table
Table 1. p16 IHC in cervical tissue biopsy specimens.
Table 1. p16 IHC in cervical tissue biopsy specimens.
Reference, YearNumber of Biopsy SpecimensHPV Detection and Correlation with Biopsy Resultsp16 Positive IHC and Correlation with Biopsy Results
Alhamlan et al., 2021 [44] 315, of which: 96/315, of which:111/212, of which:
82 NNL6/82 NNL2/54 NNL
54 CIN16/54 CIN19/37 CIN1
16 CIN23/16 CIN27/10 CIN2
45 CIN317/45 CIN320/25 CIN3
118 CC64/118 CC73/84 CC
Castle et al., 2019 [6]4010, of which: 283 NNL3172/40102520/4010, of which:
934 CIN159/283 NNL21/283 NNL
1512 CIN2507/934 CIN1248/934 CIN1
1208 CIN3 1386/1512 CIN21087/1512 CIN2
73 CC1154/1208 CIN31095/1208 CIN3
66/73 CC, of which:69/73 CC
1283/3172 HPV16:
9/283 NNL
67/934 CIN1
506/1512 CIN2
658/1208 CIN3
43/73 CC
242/3172 HPV 18/45:
7/283 NNL
49/934 CIN1
111/1512 CIN2
65/1208 CIN3
10/73 CC
1357/3172 OHR-HPV:
28/283 NNL
270/934 CIN1
659/1512 CIN2
390/1208 CIN3
10/73 CC
213/3172 IR-HPV:
11/283 NNL
82/934 CIN1
85/1512 CIN2
34/1208 CIN3
2/73 CC
76/3172 LR-HPV:
4/283 NNL
39/934 CIN1
25/1512 CIN2
7/1208 CIN3
1/73 CC
Haltas et al., 2012 [48]64, of which:N/A37/64, of which:
8 NNL 0/8 NNL
26 CIN1 12/26 CIN1
19 CIN2 15/19 CIN2
8 CIN3 7/8 CIN3
3 CC3/3 CC
Huang et al., 2011 [33]272, of which:170/272, of which: (HR-HPV)153/272, of which:
82 NNL19/82 NNL 14/82 NNL
94 CIN163/94 CIN1 47/94 CIN1
41 CIN235/41 CIN2 37/41 CIN2
28 CIN327/28 CIN3 28/28 CIN3
27 CC26/27 CC27/27 CC
Indarti et al., 2013 [34]30, of which:14/30, of which:17/30
11 CIN1 0/11 CIN10/11 CIN1
9 CIN2 5/9 CIN27/9 CIN2
10 CIN39/10 CIN310/10 CIN3
Liao et al., 2013 [35]463, of which:248/463160/463
187 NNL29/187 NNL5/187 NNL
171 CIN1124/171 CIN173/171 CIN1
53 CIN245/53 CIN240/53 CIN2
49 CIN347/43 CIN339/49 CIN3
3 CC3/3 CC3/3 CC
Ma et al., 2011 [36]131, of which:88/131 HR-HPV, of which:49/131
79 NNL43/79 NNL10/79 NNL
26 CIN121/26 CIN116/26 CIN1
23 CIN2/321/23 CIN2/320/23 CIN2/3
3 CC3/3 CC3/3 CC
Pabuccu et al., 2017 [49]27, of which:N/A13/27
14 NNL1/14 NNL
5 CIN15/5 CIN1
8 CIN2/37/8 CIN2/3
Pacchiarotti et al., 2014 [50]577, of which: N/A193/577, of which:
312 NNL 6/312 NNL
159 CIN1 91/159 CIN1
39 CIN2 36/39 CIN2
58 CIN3 53/58 CIN3
9 CC 7/9 CC
Sarma et al., 2017 [51]110, of which:N/A60/110, of which:
25 NNL2/25 NNL
25 CIN18/25 CIN1
21 CIN211/21 CIN2
12 CIN312/12 CIN3
27 CC27/27 CC
Tsoumpou et al., 2011 [52]126, of which:64/126, of which:28/126, of which:
12 NNL28/78 NNL/CIN18/78 NNL/CIN1
66 CIN136/48 CIN2/320/48 CIN2/3
36 CIN2
12 CIN3
Valasoulis et al., 2013 [37]200, of which: 133/200 HPV:53/200, of which:
23 NNL6/23 NNL2/23 NNL
79 CIN141/79 CIN112/79 CIN1
50 CIN241/50 CIN217/50 CIN2
48 CIN345/48 CIN322/48 CIN3
118/200 HR-HPV:
5/23 NNL
30/79 CIN1
38/50 CIN2
45/48 CIN3
60/200 HPV16/18:
0/23 NNL
14/79 CIN1
17/50 CIN2
29/48 CIN3
van Baars et al., 2015 [39]104, of which:90/104, of which:76/104, of which:
25 NNL13/25 NNL 0/25 NNL
11 CIN111/11 CIN1 8/11 CIN1
23 CIN223/23 CIN2 23/23 CIN2
45 CIN343/45 CIN345/45 CIN3
  • p16/Ki-67 DS
Seventeen studies had relevant data regarding p16/Ki-67 DS, totaling 5329 biopsy specimens: 2704 NNL, 936 CIN1, 2 CIN1/2, 655 CIN2, 12 CIN2/3, 810 CIN3, 210 CC. p16/Ki-67 DS was positive in 2327/5300 biopsy specimens: 20.24% NNL, 15.68% CIN1, 22.04% CIN2, 0.34% CIN2/3, 30.46% CIN3, 8.51% CC. HPV genotyping was positive in 2376/4883 biopsy specimens: 28.78% NNL, 17.97% CIN1, 0.04% CIN1/2, 18.01% CIN2, 0.37% CIN2/3, 24.41% CIN3, 7.53% CC.
p16/Ki-67 IHC has been used most frequently throughout the studies. Similar to p16 and Ki-67 IHC alone, an increase in the number of DS-positive biopsy specimens was correlated with a more severe histological diagnosis [32,41,42,43,53,54,55,56,57,58,59,60,61,62,63] and with HPV DNA positivity [41,42,43,58,59,63] (Table 2).
Table
Table 2. p16/Ki-67 DS in cervical tissue biopsy specimens.
Table 2. p16/Ki-67 DS in cervical tissue biopsy specimens.
Reference, YearNumber of Biopsy SpecimensHPV Detection and Correlation with Biopsy Resultsp16/Ki67 Positive IHC and Correlation with Biopsy Results
Celewicz et al., 2018 [53]43, of which:NA30/43, of which:
17 NNL 9/17 NNL
5 CIN12/5 CIN1
10 CIN29/10 CIN2
8 CIN37/8 CIN3
3 CC3/3 CC
Diouf et al., 2020 [54]69, of which:30/38, of which:32/46, of which:
30 NNL 1/7 NNL1/7 NNL
14 CIN1 4/6 CIN16/14 CIN1
3 CIN2 6/6 CIN2/36/6 CIN2/3
3 CIN3 19/19 CC19/19 CC
19 CC
Donà et al., 2012 [64]113, of which:95/10762/107, of which:
14 NNL5/13 NNL0/13 NNL
35 CIN131/33 CIN113/33 CIN1
24 CIN223/24 CIN217/24 CIN2
37 CIN336/37 CIN3/CC32/37 CIN3/CC
3 CC
84/107 HR-HPV
3/13 NNL
25/33 CIN1
20/24 CIN2
36/37 CIN3/CC
11/107 O-HPV
2/13 NNL
6/33 CIN1
3/24 CIN2
0/37 CIN3/CC
El-Zein et al., 2020 [55] 492, of which:321/492, of which:279/492, of which:
134 NNL47/134 NNL41/134 NNL
130 CIN169/130 CIN154/130 CIN1
99 CIN286/99 CIN272/99 CIN2
121 CIN3111/121 CIN3105/121 CIN3
8 CC8/8 CC7/8 CC
119/492 HPV16:
7/134 NNL
17/130 CIN
37/99 CIN2
55/121 CIN3
3/8 CC
26/492 HPV18:
6/134 NNL
4/130 CIN1
5/99 CIN2
5/121 CIN3
6/8 CC
139/492 HPV16/18:
12/134 NNL
20/130 CIN1
41/99 CIN2
58/121 CIN3
8/8 CC
235/492 OHR-HPV:
41/134 NNL
63/130 CIN1
58/99 CIN2
70/121 CIN3
3/8 CC
321/492 ANY HR-HPV:
47/134 NNL
69/130 CIN1
86/99 CIN2
111/121 CIN3
8/8 CC
Frega et al., 2019 [56]78, of which:73/78, of which:74/78, of which:
53 CIN250/53 CIN250/53 CIN2
25 CIN323/25 CIN324/25 CIN3
Liu et al., 2020 [65]305, of which:N/A165/305, of which:
90 NNL3/90 NNL
48 CIN18/48 CIN1
35 CIN226/35 CIN2
117 CIN3113/117 CIN3
15 ICC15/15 CC
Ngugi et al., 2015 [57]22, of which:21/22 HR-HPV, of which:8/22, of which:
12 NNL11/12 NNL1/12 NNL
2 CIN12/2 CIN10/2 CIN1
2 CIN22/2 CIN21/2 CIN2
6 CIN36/6 CIN36/6 CIN3
Waldstrøm et al., 2013 [59]226, of which:174/226, of which:154/226, of which:
42 NNL28/42 NNL23/42 NNL
97 CIN166/97 CIN154/97 CIN1
41 CIN236/41 CIN233/41 CIN2
45 CIN343/45 CIN343/45 CIN3
1 CC1/1 CC1/1 CC
Wentzensen et al., 2012 [32]623, of which:171/623 HPV16, of which:371/623, of which:
137 NNL24/137 NNL42/137 NNL
228 CIN131/228 CIN1106/228 CIN1
169 CIN260/169 CIN2140/169 CIN2
83 CIN353/83 CIN377/83 CIN3
6 CC3/6 CC6/6 CC
Yu et al., 2016 [60]1290, of which:463/1290, of which:427/1290, of which:
996 NNL204/996 NNL183/996 NNL
63 CIN1 41/63 CIN134/63 CIN1
42 CIN2 40/42 CIN234/42 CIN2
119 CIN3 111/119 CIN3111/119 CIN3
70 CC 67/70 CC65/70 CC
Yu et al., 2016 [61]701, of which:173/701, of which:149/701, of which:
640 NNL126/640 NNL111/640 NNL
46 CIN132/46 CIN126/46 CIN1
11 CIN211/11 CIN28/11 CIN2
4 CIN34/4 CIN34/4 CIN3
Zhang et al., 2019 [62]537, of which:294/537, of which:234/537, of which:
298 NNL76/298 NNL39/298 NNL
29 CIN18/29 CIN10/29 CIN
49 CIN245/49 CIN238/49 CIN2
111 CIN3106/111 CIN399/111 CIN3
50 CC49/50 CC48/50 CC
168/537 HPV16/18
23/298 NNL
8/29 CIN
16/49 CIN2
80/111 CIN3
41/50 CC
168/537 O-HPV
59/298 NNL
10/29 CIN
34/49 CIN2
50/111 CIN3
15/50 CC
Zhu et al., 2019 [63]300, of which:256/300, of which:96/300, of which:
138 NNL103/138 NILM3/138 NILM
108 CIN1100/108 CIN140/108 CIN1
29 CIN228/29 CIN228/29 CIN2
22 CIN322/22 CIN322/22 CIN3
3 CC3/3 CC3/3 CC
  • Ki-67 staining
Six studies had data regarding Ki-67 IHC alone, totaling 745 biopsy specimens, of which: 243 NNL, 196 CIN1, 2 CIN1/2, 104 CIN2, 12 CIN2/3, 141 CIN3, 47 CC. Ki-67 was positive in 384/654 biopsy specimens: 17.18% NNL, 18.48% CIN1, 0.26% CIN1/2, 22.39% CIN2, 2.60% CIN2/3, 27.60% CIN3, 13.54% CC. HPV genotyping was positive in 411/684 specimens: 21.16% NNL, 22.62% CIN1, 0.24% CIN1/2, 18.24% CIN2, 0.73% CIN2/3, 28.95% CIN3, 8.02% CC.
Ki-67 was generally expressed in combination with p16 IHC, as DS positivity. Where data were available, a direct proportionality relation between Ki-67 expression alone and the severity of intraepithelial lesion was demonstrated [40,41,42,43,66], as well as between Ki-67 expression and HPV DNA positivity [40,41,42,43] (Table 3).
Table
Table 3. p16, Ki-67 and DS IHC in cervical tissue biopsy specimens.
Table 3. p16, Ki-67 and DS IHC in cervical tissue biopsy specimens.
Reference YearNumber of Biopsy SpecimensHPV Detection and Correlation with Biopsy Resultsp16 Positive IHC and Correlation with Biopsy ResultsKI-67 Positive IHCDS Positive IHC and Correlation with Biopsy Results
and Correlation with Biopsy Results
Chang et al., 2014 [40]143, of which: 70/143, of which: 31/141, of which:29/124 of which:NA
77 NNL23/77 NNL5/75 NNL2 /69 NNL
33 CIN21/33 CIN13/33 CIN12/27 CIN1
6 CIN24/6 CIN24/6 CIN24/5 CIN2
22 CIN318/22 CIN315/21 CIN317/19 CIN3
5 CC4/5 CC4/5 CC4/4 CC
Gatta et al., 2011 [67]72, of which: 9/72, of which:41/72, of which:N/ANA
10 NNL (controls)0/10 NNL0/10 NNL
32 CIN18/32 CIN111/32 CIN1
10 CIN21/10 CIN210/10 CIN2
10 CIN30/10 CIN310/10 CIN3
10 CC0/10 CC10/10 CC
Jackson et al., 2012 [43]97, of which: 17/36, of which:14/97, of which:25//97, of which:13/97, of which:
39 NNL4/9 NNL1/39 NNL14/39 NNL1/39 NNL
46 CIN110/24 CIN15/46 CIN111/46 CIN14/46 CIN1
12 CIN2/33/3 CIN2/38/12 CIN2/310/12 CIN2/38/12 CIN2/3
Koo et al., 2013 [41]70, of which: 36/70 HR-HPV:50/70, of which:48/70, of which:43/70, of which:
27 NNL9/27 NNL15/27 NNL16/27 NNL4/27 NNL
6 CIN12/6 CIN1 2/6 CIN12/6 CIN14/6 CIN1
20 CIN214/20 CIN216/20 CIN214/20 CIN218/20 CIN2
17 CIN311/17 CIN317/17 CIN316/17 CIN317/17 CIN3
of which:
18/36 HPV 16/18:
3/9 NNL
0/2 CIN1
7/14 CIN2
8/11 CIN3
Li et al., 2019 [42]350, of which: 271/350, of which:197/350, of which:276/350, of which:185/350
84 NNL49/84 NNL
77 CIN150/77 CIN19/84 NNL41/84 NNL8/84 NNL
68 CIN256/68 CIN222/77 CIN156/77 CIN117/77 CIN1
89 CIN387/89 CIN355/68 CIN260/68 CIN250/68 CIN2
32 CC29/32 CC80/89 CIN387/89 CIN379/89 CIN3
31/32 CC32/32 CC31/32 CC
271/350, of which:
141/350 HPV16
16/350 HPV 18
16/350 HPV 31
21/350 HPV 33
13/350 HPV 35
13/350 HPV 39
3/350 HPV 45
16/350 HPV 51
56/350 HPV 52
10/350 HPV 56
61/350 HPV 58
8/350 HPV 59
11/350 HPV 68
Toll et al., 2014 [66]13, of which:8/13, of which:10/13, of which:6/13, of which:5/13, of which:
6 NNL2/6 NNL4/6 NNL3/6 NNL2/6 NNL
2 CIN12/2 CIN11/2 CIN10/2 CIN10/2 CIN1
2 CIN1/21/2 CIN1/22/2 CIN1/21/2 CIN1/21/2 CIN1/2
3 CIN33/3 CIN33/3 CIN32/3 CIN32/3 CIN3
  • Telomerase
Seventeen studies had data regarding telomerase up-regulation detected via fluorescence in situ hybridization (FISH) of hTERC amplification, totaling 9084 biopsy specimens: 1998 NNL, 2423 CIN1, 65 CIN1/2, 1617 CIN2, 120 CIN2/3, 1832 CIN3, 1029 CC. Telomerase was detected in 4337/9084 biopsy specimens: 4.28% NNL, 12.12% CIN1, 0.94% CIN1/2, 24.53% CIN2, 1.86% CIN2/3, 34.67% CIN3, 22.09% CC. HPV genotyping was positive in 2872/ 3937 biopsy specimens: 12.74% NNL, 23.39% CIN1, 1.11% CIN1/2, 17.79% CIN2, 2.09% CIN2/3, 25.52% CIN3, 11.90% CC (Table 4).
Throughout the studies, telomerase activity increased with the severity of cervical dysplasia [67,68,69,70,71,72,73,74,75,76,77,78,79,80]. Furthermore, significant differences in telomerase activity levels between L-SIL versus H-SIL, L-SIL versus CC and H-SIL versus CC, with higher activity levels in the more advanced groups, were demonstrated [22,81,82]. Similarly, He et al. [69] showed significant differences in the frequency of genomic amplification of hTERC between NNL and CIN2/CIN3/SCC, between CIN1 and CIN2/CIN3/SCC, as well as between CIN2 and SCC lesions. Additionally, Chen et al. [68], further demonstrated the superiority in terms of sensitivity and specificity of hTERC and HPV DNA co-testing when compared with hTERC amplification testing alone, for cervical cancer screening: hTERC Se = 90.0% and SP = 89.6%, HPV DNA Se = 100% and Sp = 44.0%, combination hTERC and HPV DNA Se = 90.0% and Sp = 92.2% [33].
Table
Table 4. hTERC up-regulation in cervical tissue biopsy specimens.
Table 4. hTERC up-regulation in cervical tissue biopsy specimens.
Reference, YearNumber of Biopsy SpecimensHPV Detection and Correlation with Biopsy ResultshTERC up-Regulation and Correlation with Biopsy Results
Chen et al., 2012 [68]243, of which:158/243, of which:55/243, of which:
NNL = 164NNL = 84/164NNL = 15/164
CIN1 = 29CIN1 = 24/29CIN1 = 5/29
CIN2 = 21CIN2 = 21/21CIN2 = 6/21
CIN3 = 22CIN3 = 22/22CIN3 = 22/22
CC = 7CC = 7/7CC = 7/7
He et al., 2012 [69]175, of which:N/A86/175, of which:
NNL = 24NNL = 0/24
CIN1 = 34CIN1 = 5/34
CIN2 = 36CIN2 = 18/36
CIN3 = 33CIN3 = 23/33
CC = 48CC = 40/48
He et al., 2020 [70]135, of which:97/135109/135
CIN 1/2 = 65CIN1/2 = 32/65CIN 1/2 = 41/65
CIN3 = 39CIN3 = 35/39CIN3 = 37/39
CC = 31CC = 30/31CC = 31/31
Ji et al., 2019 [71]213, of which:103/213 64/213, of which:
NNL = 15975 HR, 28 LR, of which:NNL = 29/159
CIN1 = 31NNL = 41 HR, 25 LR/159CIN1 = 18/31
CIN2 = 14CIN1 = 16 HR, 2 LR/31CIN2 = 9/14
CIN3 = 7CIN2 = 10 HR, 1 LR/14CIN3 = 6/7
CC = 2CIN3 = 6 HR/7CC = 2/2
CC = 2 HR/2
Jiang et al., 2010 [72]6726, of which: 1752/2313, of which:3250/6726, of which:
NNL = 1257NNL = 156/385NNL = 124/1257
CIN1 = 2054CIN1 = 560/794CIN1 = 428/2054
CIN2 = 1387CIN2 = 406/461CIN2 = 952/1387
CIN3 = 1410CIN3 = 490/522CIN3 = 1162/1410
CC = 618CC = 140/151CC = 584/618
Jin et al., 2011 [73]130, of which:N/A46/130, of which:
NNL = 52NNL = 2/52
CIN1 = 33CIN1 = 6/33
CIN2 = 9CIN2 = 6/9
CIN3 = 26CIN3 = 22/26
CC = 10CC = 10/10
Koeneman et al., 2019 [83]19, of which:19/1915/19, of which:
CIN2 = 3CIN 2 = 3/3
CIN3 = 16CIN 3 = 12/16
Kudela et al., 2018 [74] 111, of which:90/111, of which:58/111, of which:
NNL = 27NNL = 14/27NNL = 1/27
CIN1 = 15CIN1 = 7/15CIN1 = 4/15
CIN2 = 24CIN2 = 24/24CIN2 = 11/24
CIN3 = 25CIN3/CIS = 25/25CIN3 = 21/25
CC = 20CC = 20/20CC = 20/20
Kuglik et al., 2015 [75]74, of which:64/74, of which:23/74, of which:
NNL = 12NNL = 10/12NNL = 3/12
CIN1 = 6CIN1 = 3/6CIN1 = 1/6
CIN2 = 6CIN2 = 3/6CIN2 = 3/6
CIN3 = 12CIN3 = 10/12CIN3 = 7/12
CC = 38CC = 34/38CC = 33/38
Li et al., 2014 [77]171, of which:N/A67/171, of which:
NNL = 64NNL = 6/64
CIN1 = 26CIN1 = 6/26
CIN2 = 29CIN2 = 15/29
CIN3 = 36CIN3 = 26/36
CC = 16CC = 14/16
Liu et al., 2012 [23] 114, of which:77/11451/114
NNL = 27NNL = 0/26
CIN1 = 26CIN1 = 4/19
CIN2 = 16CIN2 = 6/12
CIN3 = 24CIN 3 = 22/27
CC = 21CC = 19/19
Liu et al., 2019 [24]150, of which:108/150, of which:64/150
NNL = 32NNL = 10/32NNL = 4/32
CIN1 = 38CIN1 = 25/38CIN1 = 13/38
CIN2/3 = 66CIN2/3 = 60/66CIN2/3 = 35/66
CC = 14CC = 13/14CC = 12/14
Xiang et al., 2012 [21] 92, of which:N/A62/92
NNL = 20 NNL = 0/20
CIN3 = 14CIN3 = 12/14
CC = 58CC = 50/58
Yin et al., 2012 [78] 166, of which:N/A101/166
NNL = 40NNL = 0/40
CIN1 = 27CIN1 = 12/27
CIN2/3 = 54CIN2/3 = 46/54
CC = 45CC = 43/45
Zappacosta et al., 2015 [25]54, of which:52/5420/54, of which:
NNL = 8NNL = 0/8
CIN1 = 26CIN1 = 6/26
CIN2 = 9CIN2 = 6/9
CIN3 = 11CIN3 = 8/11
Zheng et al., 2013 [79]373, of which:267/373, of which:192/373, of which:
NNL = 89NNL = 26/89NNL = 0/89
CIN1 = 36CIN1 = 19/36CIN1 = 5/36
CIN2 = 43CIN2 = 32/43CIN2 = 18/43
CIN3 = 129CIN3 = 119/129CIN3 = 101/129
CC = 76CC = 71/76CC = 68/76
Zhu et al., 2018 [80]138, of which:85/138, of which:74/138, of which:
NNL = 23NNL = 4/23NNL = 2/23
CIN1 = 42CIN1 = 16/42CIN1 = 13/42
CIN2 = 20CIN2 = 14/20CIN2 = 11/20
CIN3 = 28CIN3 = 26/28CIN3 = 23/28
CC = 25CC = 25/25CC = 25/25
  • 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.

4. 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 high-grade 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 high-grade 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.

5. 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.

6. 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 pro-cessed 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.

Funding

This research was funded by George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania, grant number 615/14/17.01.2019, and the publication fee is supported by George Emil Palade University of Medicine, Pharmacy, Science, and Technology of Targu Mures, Romania.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

This work was supported by the University of Medicine, Pharmacy, Science and Technology of Târgu Mureș Research Grant number 615/14/17.01.2019.

Conflicts of Interest

The authors declare no conflict of interest.

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Biology 11 00956 g001 550
Figure 1. Literature search and article selection.
Figure 1. Literature search and article selection.
Biology 11 00956 g001
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Voidăzan, S.T.; Dianzani, C.; Husariu, M.A.; Geréd, B.; Turdean, S.G.; Uzun, C.C.; Kovacs, Z.; Rozsnyai, F.F.; Neagu, N. The Role of p16/Ki-67 Immunostaining, hTERC Amplification and Fibronectin in Predicting Cervical Cancer Progression: A Systematic Review. Biology 2022, 11, 956. https://doi.org/10.3390/biology11070956

AMA Style

Voidăzan ST, Dianzani C, Husariu MA, Geréd B, Turdean SG, Uzun CC, Kovacs Z, Rozsnyai FF, Neagu N. The Role of p16/Ki-67 Immunostaining, hTERC Amplification and Fibronectin in Predicting Cervical Cancer Progression: A Systematic Review. Biology. 2022; 11(7):956. https://doi.org/10.3390/biology11070956

Chicago/Turabian Style

Voidăzan, Septimiu Toader, Caterina Dianzani, Mădălina Aurelia Husariu, Bíborka Geréd, Sabin Gligore Turdean, Cosmina Cristina Uzun, Zsolt Kovacs, Florin Francisc Rozsnyai, and Nicoleta Neagu. 2022. "The Role of p16/Ki-67 Immunostaining, hTERC Amplification and Fibronectin in Predicting Cervical Cancer Progression: A Systematic Review" Biology 11, no. 7: 956. https://doi.org/10.3390/biology11070956

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