Original article / research
| Year :
2022 |
Month :
October
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Volume :
11 |
Issue :
4 |
Page :
PO05 - PO08 |
Full Version
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Immunohistochemical Expression of p16 Lesions of the Cervix: A Cross-sectional Study
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Radha Priyadharsini, Bindhuja Joseph, Mega Samly, Santha Sadasivan
1. Junior Resident, Department of Pathology, Sree Mookambika Institute of Medical Sciences, Kulasekharam, Tamil Nadu, India.
2. Associate Professor, Department of Pathology, Sree Mookambika Institute of Medical Sciences, Kulasekharam, Tamil Nadu, India.
3. Assistant Professor, Department of Pathology, Sree Mookambika Institute of Medical Sciences, Kulasekharam, Tamil Nadu, India.
4. Professor, Department of Pathology, Sree Mookambika Institute of Medical Sciences, Kulasekharam, Tamil Nadu, India.
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Correspondence
Address :
Radha Priyadharsini, Bindhuja Joseph, Mega Samly, Santha Sadasivan,
Dr. Bindhuja Joseph,
119, Jose Bhavan, Puthukudiyerrupu, Nagercoil, Tamil Nadu, India.
E-mail: bindhuja85@gmail.com
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| | | ABSTRACT |  | : Introduction: Cervical cancer is the second most malignant cancer that affects women globally. The most common cause for cervical cancer is persistent infection of high risk Human Papillomavirus (HPV) subtypes 16,18. Over-expression of p16 may serve as a surrogate biomarker to confirm HPV infection.
Aim: The present study aimed to study high risk HPV in cervical biopsies using p16 immunostaining for detecting high grade lesions.
Materials and Methods: This cross-sectional study was conducted in the Department of Pathology, Sree Mookambika Institute of Medical Sciences, Tamil Nadu, India, from January 2019 to February 2021 involving all women with cervical biopsies and excluding HPV vaccinated women. Samples were collected in 10% formalin. Specimens were processed and paraffin embedded. The section were stained with Haematoxylin and Eosin (H&E) and immunohistochemical staining p16 and analysed using Statistical Package for the Social Sciences (SPSS) software trial version 20.0 by qualitative analysis test i.e Chi-square test.
Results: The study included 53 cases. Age distribution was between 32-78 years, with majority in 4th to 5th decades. In the study 26 (49.06%) cases were non neoplastic lesions, 16 (30.19%) were premaliganant lesions and the remaining 11 (20.75%) were malignant lesions. The difference in the proportion of HPV diagnosis between age group was statistically significant (p-value=0.003). All the non neoplastic lesions of cervix showed negative p16 staining. Out of 16 premalignant lesions, 15 (93.75%) had low grade Squamous Intraepithelial Lesion (LSIL) and only 1 (6.25%) had high grade Squamous Intraepithelial Lesion (HSIL). Few low grade lesions 7 (100%) had negative staining and all the cervical carcinoma patients showed p16 positivity.
Conclusion: p16 expression was progressively increased with increasing grades of cervical neoplasm. So, p16 may be useful as an adjunct in histological sections to detect HPV in those lesions which can help us to predict the progression of disease. |
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| Keywords
: Cervical lesions, Immunostaining, Squamous cell carcinoma, Squamous intraepithelial lesion |
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| DOI and Others
: DOI: 10.7860/NJLM/2022/55890.2668
Date of Submission: Feb 24, 2022
Date of Peer Review: Apr 04, 2022
Date of Acceptance: May 05, 2022
Date of Publishing: Oct 01, 2022
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. Yes
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Mar 02, 2022
• Manual Googling: May 14, 2022
• iThenticate Software: Jun 20, 2020 (23%)
Etymology: Author Origin |
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INTRODUCTION |
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In developing countries like India, cervical cancer is the most common cancer in women and may constitute upto 25% of all female cancers (1). The most common cause for cervical cancer is persistent infection of high risk Human Papillomavirus (HPV 16,18) (2). It is estimated that the prevalance rate of cervical cancer cases in United States is said to be 20 million high. The link between cervical cancer and genital infections was established by Harold Zur Hausen, a german virologist in the year 1980 (3). The correlation between biological and clinical relevance of dividing cervical carcinoma into HPV associated and independent was identified in the last World Health Organisation (WHO) classification. Diffuse and strong p16 positivity is highly noted in all HPV associated tumors (4),(5). Due to its strong association p16 negative lesions behaves more aggressive than p16 positive lesions.
In HPV related cervical lesions, HPV derived oncoprotein E7 interacts with pRb and inactivates it leading to the disruption of the regulatory pathway Cyclin-Dependent Kinase (CKD)-Rb-E2F. Inactivated pRb passes the cell cycle to the checkpoint G1/S without any inhibition. As a response to this event, an overexpression of p16INK4a occurs (6). Over expression of p16 may serve as a surrogate biomarker of HPV infection which makes it useful in evaluating HPV associated preneoplastic and neoplastic lesions of cervix (7). Studies have shown that expression of p16INK4a is upregulated with the increase in dysplastic cells from LSIL to HSIL and to cervical carcinoma which indirectly indicates degree of malignancy (8). The objective of the study was to investigate histomorphological changes and to study high risk HPV in various cervical lesions of cervix using p16 immunostaining. The Novelty of the study depends on the presence or absence of p16. Absence of p16 is a diagnostic and independent prognostic marker in carcinoma of cervix.
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Material and Methods |
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This cross-sectional study was conducted in the Department of Pathology, Sree Mookambika Institute of Medical Sciences, Tamil Nadu, India over a period of two years from January 2019 to February 2021. A total of 53 cases was studied.
Sample size calculation: Sample size was calculated using the following formula:
N=4pq/d2
P=Prevalence
q=100-p
d=Allowable error (20%)
Prevalence: 65.4% (9)
4×65.4×34.6=9051.36
20% of 65.4=13.08
9051.36/(13.08)2=52.9
Rounded off to 53 samples, these were included in this study involving all women with cervical biopsies from Department of Obstetrics and Gynaecology in this study.
Inclusion criteria: All cases of cervical biopsies received from Department of Obstetrics and Gynaecology for histopathological examination were included in the study.
Exclusion criteria: The HPV vaccinated patients and those on chemoradiotherapy were excluded from the study.
After getting approval from the Institutional Human Ethical Committee (IHEC) No- 26/2019, clinical history and results of relevant investigations done were collected from the patient case files. Punch biopsy from both lips of cervix were received in the Pathology Department in adequate 10% formalin. After a detailed specimen description, sampling was done. Entire cervical biopsies were processed and paraffin embedded. Tissue sections of 4 μm thickness were cut and stained by H&E staining for histopathological study.
Study Procedure
Immunohistochemistry (IHC) procedure: Sections were taken on glass slide coated with adhesive (Poly-L-Lysine) for IHC p16. The positive control used was well-differentiated squamous cell carcinoma with known p16 positivity and the negative control was phosphate buffer solution. The primary antibody used was-p16–INK4(MX007)-monoclonal antibody, and the secondary antibody was Horse Raddish Peroxidase (HRP conjugated antibody). Chest brown colour in nucleus and/or cytoplasm positivity is considered positive.
The slide was incubated for 20 minutes at 60ºC, deparaffinised, dehydrated and then rehydrated. Subject tissues underwent heat epitope retrieval using immune Deoxyribonucleic acid (DNA) retriever citrate buffer, pH 6 by keeping in pressure cooker for one whistle, after cooling keep in tap water, followed by distilled water wash. It was then washed in IHC wash buffer for five minutes. The slide was placed in polydetector peroxidase blocker for 15 minutes. It was washed in IHC wash buffer for three minutes. The sections were incubated with the primary antibody for 30 minutes and washed in IHC wash buffer twice for three minutes. The tissues were covered with polydetector HRP label for 30 minutes and washed in IHC wash buffer thrice for three minutes each.
Deaminobenzidine (DAB) was prepared by adding one drop of polydetector DAB chromogen per mL of polydetector DAB buffer and mix. The tissue was covered with prepared DAB substrate chromogen solution and incubated for 10 minutes. It was then rinsed with deionised water and counter stained with haematoxylin (4-8 dips), then dehydrated, cleared and the slide was mounted with DPX. Positive control (well-differentiated squamous cell carcinoma of cervix) and negative controls were run with each batch of slides.
Two parameters were evaluated in p16 expression:
1. Percentage of p16 positive cells and
2. Reaction intensity of p16 immunostaining.
IHC scoring: Score was obtained by semi-quantitative IHC score (0-3 points) depending on intensity (10):
• 0 points- negative staining
• 1 point- weak staining
• 2 points- moderate staining
• 3 points- strong staining
Grading of IHC: (10)
1. Negative when no cells stained and weak cytoplasmic staining.
2. Positive: Grade 1- positive cells 0-5%
Grade 2-positive cells 6-25%
Grade 3-positive cells >25%
Statistical Analysis
Significance level was decided before starting of study which is found to be 5%. Qualitative analysis (Chi-square test) as the statistical test used for data analysis. A p-value <0.05 was considered significant. The data was collected and entered in Microsoft Excel and analysis was done using Statistical Package for the Social Science (SPSS) software trial version 20.0.
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Results |
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Among the study population, 11 (20.75%) of them were upto 40 years, 21 (39.62%) of them were aged between 41 to 50 years, 12 (22.64%) of them were aged between 51 to 60 and 9 (16.98%) of them were more than 60 years old. Majority of the cases 26 (49.06%) were non neoplastic lesions, 16 (30.19%) were premalignant lesions and 11 (20.75%) were malignant lesions (Table/Fig 1).
Comparison of histopathological diagnosis between age group is shown in (Table/Fig 2). The difference in the proportion of histopathological diagnosis between age group was statistically significant (p-value=0.003).
In patients with non neoplastic lesion, majority of 11 (42.31%) had squamous metaplasia, 9 (34.61%) cases had koilocytosis, 5 (19.23%) had koilocytosis and squamous metaplasia and 1 (3.85%) had tunnel cluster. Out of 16 cases with squamous intraepithelial lesion, 15 (93.75%) had LSIL and only 1 (6.25%) had HSIL. Out of 11 with carcinoma, 9 (81.82%) were non keratinizing squamous cell carcinoma and only one (9.09%) was keratinizing and verrucous squamous cell carcinoma for each respectively.
p16 immunostaining was done on all cervical biopsy specimens. Percentage of p16INK4a expression showed increase in score from non neoplastic to premalignant lesions to malignant lesions (Table/Fig 3). In the present study it was noted that 7 out of 15 cases of LSIL showed negative staining pattern.
Intensity of p16INK4a expression showed increase in score from non neoplastic to premalignant lesions to malignant lesions (Table/Fig 4). LSIL/HSIL intensity and percentage positivity were shown separately in [Table/Fig-5,6] In the present study it was noted that none of the premalignant showed strong reaction intensity for p16 immunostaining (Table/Fig 7). All the malignant lesions showed strong intensity for p16 (Table/Fig 8),(Table/Fig 9),(Table/Fig 10).
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Discussion |
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Among many effective immunomarkers used in diagnosis of cervical lesions, p16 is considered to be a cost-effective and has a higher rate of sensitivity in the detection of cervical tumors. The p16 intensity staining reaction to be considered more superior than other parameters in diagnosing cervical lesions. Although numerous screening methods are available, Papanicolaou test (Pap smear) is the most widely used screening method (11).
The new World Health Organisation (WHO) classification (2020) has included HPV along with histopathologic subtypes of cervical adenocarcinoma (12):
1. Squamous cell carcinoma (HPV-associated, independent), Not Otherwise Specified (NOS)
2. Adenocarcinoma (HPV-associated, independent-NOS, gastric type, clear cell type, mesonephric type), NOS
Screening programmes like liquid based cytology and HPV genotyping are mandatory to detect High Risk (HR) HPV groups. The most common HR-HPV genotypes are HPV52, HPV16, HPV51 and the Low Risk (LR) HPV groups are HPV72, HPV62. International guidelines recommend HPV genotyping to be the effective screening method in case of high risk HPV infection. In earlier days, Pap smear was the auxiliary method followed which is now replaced by Liquid Based Cytology (LBC) due to various interference factors. The Society of Cervical Pathology’s (ASCCP) guidelines have included combination of all the three as a good diagnostic tool in screening high risk cervical cases (13).
The overall age range of the 53 patients was 32-78 years. This is similar to studies done by Kim TH et al., and Poste P et al., in which the age group commonly involved ranges from 24-80 years and 29 to 80 years respectively (14),(15). Majority of malignant lesions of cervix was seen above 50 years of age group, which is similar to study done by Poste P et al., (15). The difference in the proportion of HP diagnosis between age group was statistically significant (p-value=0.003).
In the present study majority of the cervical lesions were non neoplastic (49.06%) followed by premalignant lesions (30.19%) and then malignant lesions (20.75%) This was in contrast to study conducted by Ismail AT et al., which showed majority of their cases were premalignant lesions (16). In another study by Rawat N and Shivapriya R analysed 80 biopsy samples, with benign lesions reported in 17.5%, Cervical Intraepithelial Neoplasia (CIN) 1 in 7.5%, CIN 2 in 7.5%, Squamous Cell Carcinoma (SCC) in 17.5% of the study population (17).
In the present study, among the non neoplastic lesions of cervix 11 (42.31%) cases showed squamous metaplasia in correlation with Bhattacharya S et al., (18) in their study of 686 cases, 344 cases (50.14%) showed squamous metaplasia. This is in contrast to study done by Pallipady A et al., where 78.12% of the cases showed squamous metaplasia (19). Koilocytosis is considered as the histological hallmark of papilloma virus infection. Koilocytosis was diagnosed in nine cases and five cases showed koilocytosis along with squamous metaplasia among all cervical specimens during the study period. This is in correlation with studies done by Pallipady A et al., and Naik R et al., showed 18 out of 2053 cervical biopsies and 3 out of 50 cervical biopsy specimens respectively (19),(20).
The incidence of squamous intraepithelial lesion (cervical dysplasia) was 16 (30.19%) of 53 cases. 15 out of 16 cases had LSIL (CIN 1) and one out of 16 cases had HSIL (CIN 3). This is similar to studies done by Nam EJ et al., where the incidence of low grade cervical lesion, CIN 1 (LSIL) is more when compared to CIN 2 and 3 (HSIL) (21). Carcinoma cervix in the present study was 11 (20.75%) of the total 53 cases and found all the cases were SCC’s. Low incidence of carcinoma cervix was reported by Solapurkar ML, with two cases in a total of 551 cases and high incidence was noted in the study done by Nega B et al., with 2318 (55.7%) of 4155 cases (22),(23).
Immunohistochemistry:
p16INK4a in cervical lesions: Out of 26 non neoplastic lesions including non specific cervicitis, this study showed no expression of p16INK4a. In the study done by Munhoz NG et al., non specific cervicitis showed no expression of p16INK4a and 100% of cases show 6.6% of cells positivity for ki67 (24).
In the present study, out of 15 LSIL slides, seven cases were negative for p16INK4a, six cases were positive for score 1 and two cases were positive for score 2. It was observed that p16 negativity in majority of the LSIL samples. Tan GC et al., in 2010 stated that low grade lesion with p16 negativity may be due to infection with low risk HPV or due to subclinical infection (25). A study by Volgareva G et al., in 2004, observed that some of the preneoplastic and neoplastic lesions of cervix do not express p16 (26). They concluded that absence of p16 expression may be due to p16 mutation, deletion or hypermethylation.
In the present study, one HSIL case showed positivity (100%) for p16INK4a with score 1. In the study done by Lorenzato M et al., (27) 100% of HSIL cases show nuclear and cytoplasmic positivity for p16INK4a marker. In the study done by Reuschenbach M et al., demonstrated positivity in 86.95% of HSIL cases for p16INK4a and 85.45% of cases were positivite for ki67 (28).
In total, 100% (11 out of 11) of the cervical carcinoma patients showed positivity for p16INK4a. All cases of SCC in this study showed an over-expression of p16INK4a. Similar evidence were obtained by the studies done by Volgareva G et al., (26). This reflects the close association of overexpression of p16INK4a in HSIL and SCC with the degree of premalignancy and malignancy and its pivotal role in the progression of malignancy in cervix.
In present study, it was demonstrated that a graded increase was seen in the expression of the protein p16INK4a from non specific cervicitis to LSIL and further to HSIL and invasive squamous cell carcinoma. Thus, expression of p16INK4a protein in cervical biopsies can serve as a specific marker for diagnosing premalignant and malignant lesions.
Limitation(s)
The sample size was low in number however, the attempt for HPV DNA detection studies to validate the utility of p16 for detection of HPV in cervical neoplasm could not be made due to financial constraint.
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Conclusion |
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The p16INK4a was found to show graded increase in score from non specific cervicitis to LSIL and further to HSIL and invasive squamous cell carcinoma. So, p16 may be useful as an adjunct in histological sections to detect HPV in those lesions which can help us to predict the progression of disease. If the association of HPV in SCC it can predict the prognosis of the patient. The detection of p16 can help in categorising high risk HPV cases, which can be further treated.
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| TABLES AND FIGURES | |
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