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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 3  |  Issue : 1  |  Page : 20-24

Correlation of immunohistochemical expression of p16, Ki-67, and p53 with histopathological diagnosis of noninvasive cervical lesions: A multicenter study from South East Nigeria


Department of Histopathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria

Date of Submission18-Oct-2021
Date of Decision05-Dec-2021
Date of Acceptance19-Dec-2021
Date of Web Publication24-Mar-2022

Correspondence Address:
Dr. Chinedu Onwuka Ndukwe
Department of Histopathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jascp.jascp_26_21

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  Abstract 


Background: There is a high burden of cervical cancer in our environment. Most patients present late when the prognosis is guarded. Hence, accurate diagnosis of preinvasive lesions from cervical biopsies is important for clinical decisions and patient management. The aim of the study is to correlate the expression of p16INK4a, p53, and Ki-67 with histopathological diagnosis of noninvasive cervical lesions. Materials and Methods: The paraffin blocks of all cervical biopsies (excluding cases histologically diagnosed as invasive lesions) seen in two histopathology laboratories in Nnewi, Southeast Nigeria, over a 10-year period (2011–2020) were retrieved from the archives of both facilities. The cases were subjected to immunohistochemistry using p16INK4a, Ki-67, and p53 monoclonal antibodies. Results: There were 23 normal/reactive (45.1%), 6 low-grade squamous intraepithelial lesion (11.8%), and 22 hIL (43.1%). There is a very strong positive correlation between p16INK4a expression and the histopathological diagnosis (Spearman's correlation = 0.98). There is a strong positive correlation between Ki-67 expression and the histopathological diagnosis (Spearman's correlation = 0.70). There is a weak positive correlation between p53 expression and histopathological diagnosis (Spearman's correlation coefficient = 0.40). Conclusion: p16INK4a shows the best correlation with histopathological diagnosis of noninvasive cervical lesions and may be a very useful adjunct to H and E for diagnosing preinvasive cervical lesions. However, p53 correlates poorly with histopathologically diagnosed noninvasive cervical lesions and therefore may not be diagnostically useful.

Keywords: Cervical intraepithelial neoplasia, cervix, Ki-67, Nigeria, p16, p53


How to cite this article:
Ogbu CC, Ndukwe CO, Chiemeka ME, Madubuike KC, Ukah CO. Correlation of immunohistochemical expression of p16, Ki-67, and p53 with histopathological diagnosis of noninvasive cervical lesions: A multicenter study from South East Nigeria. J Appl Sci Clin Pract 2022;3:20-4

How to cite this URL:
Ogbu CC, Ndukwe CO, Chiemeka ME, Madubuike KC, Ukah CO. Correlation of immunohistochemical expression of p16, Ki-67, and p53 with histopathological diagnosis of noninvasive cervical lesions: A multicenter study from South East Nigeria. J Appl Sci Clin Pract [serial online] 2022 [cited 2022 May 17];3:20-4. Available from: http://www.jascp.com/text.asp?2022/3/1/20/340881




  Introduction Top


Cervical cancer is the fourth most common malignancy in women after breast cancer, colon cancer, and endometrial cancer worldwide.[1] It is the most common type of cancer among females in the majority of developing countries including sub-Saharan Africa.[1] In Nigeria, it is the second most common female cancer after breast cancer with age-standardized incidence rate of 34.5 cases/100,000 women in 2010.[2] The age-standardized rate of cervical cancer in Nnewi is 13.2 cases/100,000 females.[3]

Human papillomavirus (HPV) plays an etiologic role in cervical carcinogenesis and is detectable in preinvasive and invasive cervical epithelial neoplasms.[4] Virtually all cases of cervical precancer and cancer are associated with high-risk HPV (hr-HPV).[5] The most common hr-HPV subtypes include types 16 and 18, which account for approximately 70% of HPV species detected in cervical cancer.[6]

Cervical cancer emerges from cervical intraepithelial neoplasia (CIN).[7] CIN can be treated effectively to prevent progression to cervical cancer.[8] On the basis of morphology alone, it can be difficult to distinguish between squamous metaplasia coupled with hyperplasia (SMH) and CIN I and between CIN I and CIN II-III.[9] Moreover, small biopsy size, tangential sectioning, thermal artifact, coexistent inflammatory or reactive lesions, and application of subjective criteria all increase the difficulty for the diagnosis of CIN.[9] Therefore, objective diagnostic methods in addition to histology are required to accurately diagnose CIN in histological specimens.[6] Immunohistochemistry is an ancillary technique that is helpful in equivocal cases. A panel of monoclonal antibodies, used as surrogate markers, is now available, and these include, p16INK4a, p53, and Ki-67.[10]

The aim of this study is to perform an immunohistochemical review of all cervical biopsies (excluding cases confirmed as invasive lesions) in Nnewi over a 10-year period and to correlate the expression of p16INK4a, p53, and Ki-67 with histopathological diagnosis.


  Materials and Methods Top


The study locations

Nnamdi Azikiwe University Teaching Hospital (NAUTH) in Nnewi is the 440-bed hospital that serves as the teaching hospital for Nnamdi Azikiwe University, Awka. Pathocon Specialist Clinic and Research Institute is a private histopathology laboratory also located in Nnewi. Both facilities serve mainly Nnewi and environs. Nnewi is the second-largest city in Anambra state. It is an industrial and commercial city with a population of 391,227 people as at 2006.

Inclusion criteria

All cervical biopsies (excluding cases histologically diagnosed as invasive lesions) with well-preserved blocks, adequate clinical data, and tissue left for sectioning.

Exclusion criteria

Poorly preserved tissue blocks such as those that are matted together, partially eaten by rodents or infested by mould. Also excluded are tissue blocks without adequate tissue left for sectioning, cases with missing blocks, and cases with incomplete data like age and indication for biopsy.

Study design

This is a retrospective study of the immunohistochemical expression of p16INK4a, Ki-67, and p53 over a 10-year period (January 2011 to December 2020). Formalin-fixed, paraffin-embedded tissue blocks of all cervical biopsies (excluding cases histologically diagnosed as invasive lesions) that met the inclusion criteria in both facilities were selected for the study.

Procedure

Formalin-fixed, paraffin-embedded tissue blocks of all cervical biopsies excluding cases histologically diagnosed as invasive lesions in both facilities from January 2011 to December 2020 were retrieved from the archives. Fresh sections were cut and stained with hematoxylin and eosin. These were reviewed for diagnostic consistency. The cases were immunostained for p16INK4a, Ki-67, and p53 antigens through the indirect biotin-streptavidin method. All sections were deparaffinized in xylene, rehydrated in graded alcohols, washed in phosphate-buffered saline buffer, boiled in ethylenediaminetetraacetic acid solution (pH 8.0, 100°C, 2.5 min) for antigen retrieval, and cooled down. The slides were stained. The primary antibodies were as follows: p16INK4a (DB Biotech clone R19-D) at a dilution of 1:80, Ki-67 (Ventana clone 30-9) at a dilution of 1:200, and p53 (Ventana clone Bp53-11) at a dilution of 1:100.

Interpretation of immunohistochemistry staining results

p16INK4a, Ki-67, and p53 were expressed in the nucleus. p16 was adjudged positive when there was diffuse staining in the nuclei and/or cytoplasm of basal or parabasal cells. To determine the grade of p16INK4a expression, a four-tiered semi-quantitative classes were used to describe the percentage of positively stained tumor cells as follows: negative (below 5%), weak positive (+1, 5%–25%), moderate positive (+2, 26%–50%), and intense positive (+3, >50%). To determine the grade of Ki-67 expression, nuclei of 1000 epithelial cells located across the whole epithelial layer were examined. Ki-67 index was defined as the percentage of Ki-67 positive cells: negative, +1, +2, and +3 were given when the Ki-67 index is below 5%, 5%–25%, 26%–50%, and >50%, respectively. To determine the grade of p53 immunoreactivity, a 5-tiered scoring system was used (0–4 depending on percentage of immunoreactive cells). p53 immunoreactivity of <5% was scored negative; 5%–24% was scored + 1; 25%–49% was scored + 2; 50%–75% was scored + 3; and 75%–100% was scored + 4. Positive controls included tissue sections known to express antigens under investigations.

Data analysis

Data were analyzed using simple descriptive statistics such as Chi-square test to measure association with level of significance of <0.05. This analysis was done using SPSS Statistics for Windows, Version 23.0. IBM Corp. Armonk, NY, USA.

Ethical consideration

Ethical approval for the study with registration number NAUTH/CS/66/VOL 8/130 was obtained from the Ethics Committee of NAUTH.


  Results Top


Fifty-one (51) cases were enrolled in this study (30 cases from PATHOCON and 21 cases from NAUTH). The cases were classified as follows: 23 reactive/normal (45.1%), 6 low-grade squamous intraepithelial lesion (LSIL) (11.8%), and 22 hIL (43.1%).

Demographic characteristics

The mean age of the patients was 47.9 ± 10.2 years for normal/reactive lesions, 48.3 ± 9.4 years for LSIL, and 51.9 ± 11.7 years for high-grade squamous intraepithelial lesions (HSIL). Both normal/reactive lesions and LSIL had a modal age group of 41–50 years, whereas the modal age group for HSIL was a decade later (51–60 years).

Correlation of expression of p16INK4a, p53, and ki-67 with histopathological diagnosis

p16 showed > 50% positivity in 18/22 (81.8%) of HSIL cases and 26%–50% positivity in 4/22 (18.2%) of HSIL cases. Furthermore, p16 showed 5%–25% positivity in 6/6 (100%) of LSIL cases, whereas all normal/reactive cervical lesions were negative for p16 (P < 0.01) [Table 1] and [Figure 1] and [Figure 2].
Figure 1: Low-grade squamous intraepithelial lesion with dysplasia limited to lower third of the epithelium (a) H and E stain (b) Ki-67 stain (c) p16 stain

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Figure 2: High-grade squamous intraepithelial lesions with full-thickness dysplasia (a) H and E stain (b) Ki-67 stain (c) p16 stain

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Table 1: p16INKa × final diagnosis crosstabulation

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Ki-67 was positive in only 2/23 (8.7%) of reactive lesions, but 2/6 (33.3%) of LSIL, and 18/22 (81.8%) of HSIL (P < 0.01). In addition, none of the reactive lesions, one (16.7%) of LSIL cases, and 10/22 (45.5%) of HSIL cases showed a Ki-67 labeling index >25% [Table 2] and [Figure 1] and [Figure 2].
Table 2: Ki-67 × final diagnosis crosstabulation

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p53 was positive in 6/22 (27.3%) of HSIL cases but completely negative in both LSIL and reactive lesions [Table 3]. There was no statistically significant difference in p53 expression across the three groups (P = 0.176).
Table 3: p53 × final diagnosis crosstabulation

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There is a very strong positive correlation between p16INK4a expression and the histopathological diagnosis (Spearman's correlation coefficient = 0.98), a strong positive correlation between Ki-67 expression and the histopathological diagnosis (Spearman's correlation coefficient = 0.70), and a weak positive correlation between p53 expression and the histopathological diagnosis (Spearman's correlation coefficient = 0.40). Furthermore, there is a strong positive correlation between p16INK4a immunoreactivity and Ki-67 immunoreactivity (Spearman's correlation coefficient = 0.68), whereas there is a weak positive correlation between p16INK4a immunoreactivity and p53 immunoreactivity (Spearman's correlation coefficient = 0.34).


  Discussion Top


p16INK4a has been extensively investigated. It labels cervical dysplastic squamous and glandular cells with a sensitivity of 99.9%, and a specificity of 100%, both immunohistochemically and immunocytochemically.[11] It has also been shown to be able to separate low- and high-grade lesions, and identify histologically or cytologically classified low-grade lesions that are susceptible to cancer progression.[11]

There was a very strong positive correlation between p16INK4a expression and the histopathological diagnosis in this study. Similarly, a study in Kuala Lumpur (Malaysia) showed that p16INK4a expression correlates with grade of CIN.[12] A total of 201 cases of cervical neoplasms were subjected to immunohistochemical analysis (129 cases of CIN and 72 cases of squamous cell carcinoma).[12] p16INK4a expression in CINI, CINII, and CINIII was 25.4%, 42.9%, and 95.9%, respectively. Approximately 99% (98.6%) of cases diagnosed as squamous cell carcinoma expressed p16INK4a.[12] There was a linear relationship between p16INK4a expression and grade of CIN.[12] Furthermore, a study in Shanghai (China) showed that the expression of p16INK4a exhibited a significantly higher rate in the CIN I group than in the SMH group (SMH; P < 0.0001) and the CIN II-III group (P = 0.005).[13] Another study done in Anderson Cancer Center (Houston, Texas) showed that the positivity of p16INK4a increased significantly with the severity of cervical lesions.[14]

The correlation between Ki-67 expression and histopathological diagnosis is lower than that of p16INK4a. This is very similar to a study done by Ungureanu et al. in Romania.[15] Ki-67 is an antigen that corresponds to a nuclear nonhistone protein, expressed by cells in the proliferative phases G1, G2, M, and S. On its own, it reflects the cell turnover rate only. It is usually associated with the other markers.[16] This makes its use less attractive outside of a research context. If used, it should be used in conjunction with other markers, such as p16INK4a.[17]

There is a strong positive correlation between p16INK4a immunoreactivity and Ki-67 immunoreactivity in this study. A study in Goyang (Korea) showed that CIN grade was positively related to the expression of Ki-67 and p16INK4a.[18] A total of 31 CIN specimens (CINI, 12; CINII, 6; CINII, 13) obtained by colposcopy were subjected to immunohistochemical analysis using p16INK4a and Ki-67.[18] The expression of p16INK4a (P < 0.001) and Ki-67 (P = 0.003) was positively associated with the grade of CIN.[18] There was also a positive correlation between expression of p16INK4a and Ki-67.[18]

The sensitivity and specificity of p16INK4a for diagnosis of HSIL are 90% and 85%, respectively.[19] The sensitivity and specificity of Ki-67 for diagnosis of HSIL are 89% and 87%, respectively.[19] These values increase by 5% when used together.[19]

There is a weak correlation between p53 expression and histopathological diagnosis in this study. This is in keeping with work done by Wu et al. in Shanghai (China).[13] However, this is at variance with works done by Vecchione et al. and Herbsleb et al. in Rome (Italy) and Copenhagen (Denmark), respectively.[20],[21] So far, p53 has received less attention than p16INK4a in cervical pathology. p53 mutation, resulting in the inactivation of the wild-type p53, is the most frequent genetic event in cancer. Wild-type p53 gene product is an important tumor suppressor, apoptosis inducer, and cell-cycle controller. Its mutation may occur during cervical carcinogenesis, through downstream mutagenesis. The mutated p53 leads to a malignant phenotype. The prevalence of p53 mutation in cervical cancer is <10%. This makes it a less suitable surrogate marker.[22]

From the foregoing, we recommend p16 immunohistochemistry for resolving diagnostic dilemmas involving preinvasive cervical lesions. This will go a long way in rendering the appropriate diagnosis and also increase the quality of patients' care. This will also go a long way in reducing the burden of cervical cancer in our environment. Furthermore, it will assist in making the diagnosis at an early and preinvasive stage when chances of cure are very high.

This study is limited mainly by its relatively small sample size. This may be attributed to the significant number of cases excluded from the study due to inadequate clinical data and failed antigen retrieval. Furthermore, there is lack of molecular genotyping facility to confirm the genotype of the HPV in cases immunohistochemically categorized as squamous intraepithelial lesions.

Nonetheless, from this study, we can conclude that immunohistochemistry using p16INK4a shows good correlation with histopathological diagnosis of noninvasive cervical lesions and may be a very useful adjunct to H and E for diagnosing preinvasive cervical lesions especially challenging cases. Comparatively, in this study, we found that p53 may not be very useful in the diagnosis of preinvasive lesions as it shows a poor correlation with histopathological diagnosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Bray F, Ferlay J, Laversanne M, Brewster DH, Gombe Mbalawa C, Kohler B, et al. Cancer Incidence in Five Continents: Inclusion criteria, highlights from Volume X and the global status of cancer registration. Int J Cancer. 2015 Nov 1;137(9):2060-71. doi: 10.1002/ijc.29670. PMID: 26135522.  Back to cited text no. 1
    
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Ukah CO, Nwofor AM. Cancer incidence in south-east Nigeria : A report from Nnewi Cancer Registry. Orient J Med 2017;29:48-55.  Back to cited text no. 3
    
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Vuyst HD, Ndirangu G, Moodley M, Tenet V, Estambale B, Meijer CJ, et al. Prevalence of human papillomavirus in women with invasive cervical carcinoma by HIV status in Kenya and South Africa. Int J Cancer 2012;131:949-55.  Back to cited text no. 5
    
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Bauer HM, Ault K. Human papillomavirus: Current prevalence and future protection. Sex Transm Dis 2006;33:509-11.  Back to cited text no. 6
    
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Zhong P, Li J, Gu Y, Liu Y, Wang A, Sun Y, et al. P16 and Ki-67 expression improves the diagnostic accuracy of cervical lesions but not predict persistent high risk human papillomavirus infection with CIN1. Int J Clin Exp Pathol 2015;8:2979-86.  Back to cited text no. 8
    
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Mulvany NJ, Allen DG, Wilson SM. Diagnostic utility of p16INK4a: A reappraisal of its use in cervical biopsies. Pathology 2008;40:335-44.  Back to cited text no. 11
    
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13.
Wu J, Li XJ, Zhu W, Liu XP. Detection and pathological value of papillomavirus DNA and p16INK4A and p53 protein expression in cervical intraepithelial neoplasia. Oncol Lett 2014;7:738-44.  Back to cited text no. 13
    
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Guo M, Baruch AC, Silva EG, Jan YJ, Lin E, Sneige N, et al. Efficacy of p16INK4a and ProExC immunostaining in detection of high-grade cervical intraepithelial neoplasia and cervical carcinoma. Am J Clin Pathol 2011;135:212-20.  Back to cited text no. 14
    
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Ungureanu C, Teleman S, Socolov D, Anton G, Mihailovici MS. Evaluation of p16INK4a and ki-67 proteins expression in cervical intraepithelial neoplasia and their clinical correlation. Rev Med Chir 2010;114:823-8.  Back to cited text no. 15
    
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Vasilescu F, Ceauşu M, Tănase C, Stănculescu R, Vlădescu T, Ceauşu Z. P53, p63 and Ki-67 assessment in HPV-induced cervical neoplasia. Rom J Morphol Embryol 2009;50:357-61.  Back to cited text no. 16
    
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Nierkerk DV, Guillaud M, Matisic J, Benedet JL, Freeberg JA, Follen M, et al. p16 and MIB1 improve the sensitivity and specificity of the diagnosis of high grade squamous intraepithelial lesion: Methodical issues in a report of 447 biopsies with consensus diagnosis and HPV HCII testing. J Gynaecol Oncol 2007;107:S233-40.  Back to cited text no. 19
    
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Vecchione A, Cermele C, Giovagnoli MR, Valli C, Alimandi M, Carico E, et al. p53 expression and genetic evidence for viral infection in intraepithelial neoplasia of the uterine cervix. Gynecol Oncol 1994;55:343-8.  Back to cited text no. 20
    
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Herbsleb M, Knudsen UB, Orntoft TF, Bichel P, Norrild B, Knudsen A, et al. Telomerase activity, MIB-1, PCNA, HPV 16 and p53 as diagnostic markers for cervical intraepithelial neoplasia. APMIS 2001;109:607-17.  Back to cited text no. 21
    
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Senba M, Buziba N, Mori N, Wada A, Irie S, Toriyama K. Detection of human papillomavirus and cellular regulators p16INK4a, p53, and NF-kappaB in penile cancer cases in Kenya. Acta Virol 2009;53:43-8.  Back to cited text no. 22
    


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