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Research Article | Volume 14 Issue: 2 (March-April, 2024) | Pages 1108 - 1112
Unravelling the Role of P16 in Cervical lesions in a Tertiary Care Centre – A Retrospective Study
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1
Assistant Professor, Department of Pathology, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology,Visakhapatnam, Andhra Pradesh, India.
2
Associate Professor, Department of Pathology, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology, Visakhapatnam, Andhra Pradesh, India.
3
Associate Professor, Department of Pathology, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology, Visakhapatnam, Andhra Pradesh, India.
4
Professor, Department of Pathology, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology, Visakhapatnam, Andhra Pradesh, India.
Under a Creative Commons license
Open Access
PMID : 16359053
Received
Feb. 21, 2024
Revised
March 13, 2024
Accepted
April 18, 2024
Published
April 17, 2024
Abstract

Introduction: Carcinoma cervix is the most common cancer and  the invasive stage of cervical cancer is preceded by pre-invasive phases and all the phases can be picked up and diagnosed by Pap smear. p16INK4 is a tumour suppressor protein which serves as a surrogate marker for the oncogenic activities of Human Papilloma virus  and its overexpression is well established in cervical intraepithelial neoplasia (CIN) and invasive cancers.

Aim: To evaluate the pattern of cervical cytology at a tertiary care hospital and to correlate the findings with histopathological diagnosis  and to determine the importance of  p16INK4a as a diagnostic marker of HPV by IHC staining method.

Materials and Methods: This is a two year  retrospective  study done on Pap smears reported between April 2021 to  May 2023 in the Department of Pathology , Gayatri Vidya Parishad Institute of  Healthcare and Medical Technology, Visakhapatnam .Frequency and percentage statistics was used to present the results.

Results: A total of 844 Pap smears  were included in the study, out of which 52 were cytologically abnormal. Biopsy was done on 42 cases, out of which 33 cases showed concordance which included18 LSIL, 8 HSIL , 5 squamous cell carcinoma and 2 cases of adenocarcinoma , among them  p16INK4a was found to be positive in 6 cases of LSIL, 5 cases of HSIL and  4 cases of SCC and 2 cases of adenocarcinoma.

Conclusion: Pap smear is a sensitive and cost effective screening modality for premalignant and malignant lesions of cervix and expression of  p16 as IHC marker has the potential to accredit the lesions with an increased risk of progression to high-grade lesions.

Keywords
INTRODUCTION

Carcinoma cervix is the most common cancer and also a common cause of cancer related deaths, particularly in developing countries like India.The incidence of the disease increases in 30 – 34 years and summits at 55 – 65 years with a median age of 38 years.The invasive stage of carcinoma cervix is preceded by pre-invasive phases and all the phases can be picked up and diagnosed by Pap smear as there are cellular alterations in the cervix(1). A long pre-invasive state, availability of screening programs and effective treatment of pre-invasive lesion makes it a preventable disease(2). Pap smear is a simple, safe, non-invasive, cost effective screening tool and cytology provides excellent nuclear details, making early nuclear abnormalities easier to appreciate for detection of precancerous, cancerous and noncancerous changes in the cervix. In biopsy a portion of squamocolumnar junction is sampled and it remains the gold standard for the confirmatory diagnosis ofcervical lesions. p16INK4 is a tumour suppressor protein which serves as a surrogate marker for the oncogenic activities of Human Papilloma virus and its overexpression is well established in cervical intraepithelial neoplasia (CIN) and invasive cancers(3). This study was designed to evaluate the pattern of cervical cytology at a tertiary care hospital and to correlate the findings with histopathological diagnosis and to determine the importance of p16INK4a as a diagnostic marker of HPV by IHC staining method

MATERIAL AND METHODS:

This is a two year retrospective study done on Pap smears reported between April 2021 to May 2023 in the Department of Pathology ,Gayatri Vidya Parishad Institute of Healthcare and Medical Technology, Visakhapatnam . Ethical clearance was obtained to carry out the study.Detailed clinical history including socioeconomic status, parity and complaints like vaginal discharge, post coital bleeding, post menopausal bleeding, pain in lower abdomen, intermenstrual bleeding was taken.Total 844 females were screened by Pap smear for various cervical lesions. The Pap smears were taken with Ayer’s spatula on a clean glass slide and fixed immediately in 95% alcohol. Staining of the slides was performed by means of the conventional Pap staining and reported according to Bethesda System 2014.
Inclusion criteria:
1. Age group from 21 – 75 years irrespective of marital status and parity
2. All symptomatic women
Exclusion criteria:
1. Pregnant women
2. Patients previously diagnosed with active lesions.
3. All inadequate PAP smears according to Bethesda system of reporting.

Methodology
The epithelial cell abnormalities , ASCUS (Atypical Squamous Cells of Undetermined Significance), LSIL (Low grade Squamous Intraepithelial Lesion), HSIL (High grade Squamous Intraepithelial Lesion), ASC-H (AtypicalSquamous cells, cannot exclude HSIL) , AIS (Adenocarcinoma in situ) and SCC (Squamous cellcarcinoma) were registered in cytology smears and corresponding cervix biopsies sent for histopathological examination. The cervical biopsies were fixed in 10% neutral buffered formalin, processed routinely, embedded in paraffin blocks and stained with haematoxylin and eosin (H and E)stain. The concordance and discordance in cytology and histopathology reports were noted.
33 Cases were selected for IHC to detect the intensity of HPV by immunohistochemistry method. For Immunohis to chemistry antigen retrieval was done by using Tris buffer. The sections were stained using peroxidase- antiperoxidase technique and were incubated with primary monoclonal antibody- p16 and secondary antibody for one hour and 30 minutes respectively. Diamino Benzidine (DAB) chromogen and Haematoxylin counter stain was used and covers lipped. The p16 immunostaining was considered positive when the nucleus and cytoplasm stain in at least 1% of tumor cells. The percentage of positive cells was evaluated in the highest expression area and based on the intensity of staining score given as 0-3. Grade 0 was considered negative, while Grade 1 to Grade 3 were considered positive for p16 staining.

RESULTS:

total of 844 Pap smears received in Department of Pathology during the period from April 2021 to May 2023, Gayatri Vidya Parishad Institute of Healthcare and Medical Technology were included in the study . Age of patients ranged from 21 to 75 years. Most of the patients belong to 3rd and 4th decade and were predominantly Para 2 (52% )followed by Para 3 (30 %).
45% of patients were asymptomatic and the remaining presented with complaints of white discharge and lower abdomen pain followed by menorrhagia, postcoital bleeding and postmenopausal bleeding.
The Pap smear findings revealed 757 cases of Negative for Intraepithelial lesion or malignancy (NILM), 37 specific pathogenic organisms and 50 cases of Epithelial cell abnormalities(ECA) . Out of the 50 cases reported, 25 were Low grade squamous intraepithelial lesion, 11 High grade squamous intraepithelial lesion , 6 Atypical squamous cells of undetermined significance , 4 ASC -H , 2 Adenocarcinoma in situ,followed by 2 cases of Squamous cell carcinoma (SCC) (Table 1) .
For the atypical Pap smear cases , corresponding biopsy was taken in 42 cases and predominant lesion was LSIL(18 cases) followed by 8cases of HSIL , 5 cases of squamous cell carcinoma and 2 cases of Adenocarcinoma. (Table 2) . Immunohistochemistry was done on all 33 positive histopathology sections, out of which 20 showed positivity for HPV in the present study (Table 3)

 

DISCUSSION

Cervical cancer is one of the health burdens in many developing countries and in the past decade the role of human papilloma virus in the pathogenesis of cervical cancer has been well acknowledged. Cervical neoplasia has a long period of pre-invasive stage which is usually asymptomatic that can be screened by Pap smear, appropriately managed and prevent the progression to be an invasive cervical cancer. The protein p16INK4a is a cellular protein involved in cell cycle regulation at G1-S interphase , and its expression in normal cells is at a very low level and is almost undetectable by immunohistochemistry. Physiological expression of p16 is seen in cells undergoing squamous metaplasia during transdifferentiation process whereas strong expression of p16 is noted in dysplatic cells due to the functional inactivation of Rb by HPV E7 protein which results in an accumulation of p16 protein(4) The objective of the present study was to analyze the results of the expression of p16 in low-grade and high-grade squamous intraepithelial lesion, squamous cell carcinoma and adenocarcinoma of the cervix in order to help draw a distinction between low risk and high risk patients with cervical lesions. In the present study age of patients ranged from 21 to 75 years, most of the women were multiparous and belong to 4th and 5th decade accounting for 33.3% and 23.1% of the population respectively. These findings are comparable to the study by Meenakshi et al , Kalyankar et al and Thobbi VA et al (5,6,7) .
In the present study most of the cases were categorized as Negative for intraepithelial malignancy (NILM) contributing to 89.6% cases and Epithelial cell abnormalities accounted for 5.9 % cases which was in accordance with the study conducted by Bamanikar et al and Amritha sinha et al (2,8).
In this study LSIL, HSIL , ASCUS , ASC-H, AIS and frank malignancy cases were 25(2.9%), 11 (1.3%), 6 (0.7%) , 4(0.5%) 2 (0.2%) and 2 (0.2%) respectivelywhich was similar to the study done by Bamanikar et al (7) who reported ASCUS, LSIL, HSIL and frank malignancy in 2.98%, 1.19%, 0.66% and 0.95% respectively. However Gandavaram et al study accounted for maximum number of ASCUS cases in their study compared to other cervical lesions.
The present study included 6 ASCUS cases on cytology, cervix biopsy was done on four cases, two reported as chronic non specific cervicitis and other two LSIL on histopathology. 3 cases out of 4 ASC-H cases were diagnosed as 2 HSIL and 1 Chronic non specific cervicitis with squamous metaplasia on histopathology . Out of 22 cases of LSIL sent for HPE , 6 were reported as non neoplastic, 13 LSIL and 3 HSIL respectively. The false positive cases of ASCUS, ASC-H and LSIL on PAP smear were due to misinterpretation of regenerative changes on cytological smears.
Out of 9 cases of HSIL sent for HPE, 2 LSIL, 4 HSIL and 3 SCC were diagnosed on HPE. 2 cases of AIS and SCC on pap smear were confirmed as Adenocarcinoma and SCC on histopathological examination. The cytology histopathology correlation observed in the present study was 78.5 % which was comparable to the study done by Atla et al (83.3%), Patil et al ( 82.1%) and Joshi et al ( 80.0%) (10,11,12).
The present study included 18 cases of LSIL, 8 cases of HSIL , 5 cases of squamous cell carcinoma and 2 cases of adenocarcinoma , among them p16INK4a was found to be positive in 7 cases of LSIL, 5 cases of HSIL and 4 cases of SCC and 2 cases of Adenocarcinoma. The percentage of overall P16 positivity in the present study was % which correlated with the study of Atla et al, Patil et al and Joshi et al. The high percentage of p16 negativity (61.1%) in LSIL can be attributed to either low risk HPV infection or low viral load. 71.5% HSIL and 90% of invasive carcinomas show P16 overexpression which highlights the prime association between HPV and cervical cancer ( Fig 1). Among p16 positive cases, the study demonstrated the p16 expression was directly related to the increasing grades of CIN which were similar to the study by Bharadwaj et al and Umar et al ( 3, 13)

CONCLUSION

Pap smear is sensitive and cost effective screening modality for premalignant and malignant lesions of cervix. IHC should always be used as an adjunct to morphological examination in cervical lesions and expression of p16 has the potential to accredit the lesions with an increased risk of progression to high-grade lesions. Immunohisto chemistry along with molecular investigations will play an increasingly important role in the field of diagnostic uterine cervical pathology.

REFERENCES

1.Kumar S D, Rajeswari K, Krishna R, Meenakshisundaram K. A retrospective study of pap smear and cervical biopsy correlation in abnormal cervical cytology cases. IP Arch Cytol Histopathology Res 2021;6(2):87-90.
2. Amrita Sinha, Santosh Upadhyaya Kafle, Mrinalini Singh, Neeta Kafle.,Variation of Cervical Cytology by Pap Smear and its Correlation with Cervical Biopsy in Patients Attending Birat Medical College Teaching Hospital, Morang, Nepal. BJHS 2021;6(3)16. 1615 - 1620. 3. Vedula B , Rama Reddy B V, Rajani M, Sree Ramulu Naidu R, Srikanth Reddy K, Expression of p16 in cervical premalignant and malignant lesions- IHC study. Indian J Pathol Oncol 2020;7(3):404-407. 4. Izadi-Mood N, Asadi K, Shojaei H, Sarmadi S, Ahmadi SA, Sani S, Chelavi LH. Potential diagnostic value of P16 expression in premalignant and malignant cervical lesions. J Res Med Sci. 2012 May;17(5):428-33. PMID: 23626605; PMCID: PMC3634266. .
5. VenkateshM, GopalanU. A comparative study of Pap smear and colposcopy guided biopsy in the evaluation of unhealthy cervix.Int J Reprod Contracept Obstet Gynecol2020;9:2305-9.
6. 6.Kalyankar VY, Kalyankar BV, Gadappa SN, Kute S. Colposcopic evaluation of unhealthy cervix and it’s correlation with Papanicolau smear in cervical cancer screening. Int J Reprod Contracept Obstet Gynecol. 2017;6:4959-65.
7. 7.Thobbi VA, Khan F. Cervical cytology by Pap smear in reproductive population. Int J Reprod Contracept Obstet Gynecol. 2018;7:1988-92
8. Bamanikar SA, Baravkar D, Chandanwale S, Dharwadkar A, Paranjape S. Study of cervical cytology and its correlation with clinical and histopathological findings. Clin Cancer Investig J 2016;5:403-8. DOI:10.4103/2278-0513.197869.
9. Gandavaram J, Pamulapati BR. Correlation of pap smear and colposcopic findings in relation to histopathological findings among women attending a tertiary care hospital: a two year study . Int J Reprod Contracept Obstet Gynecol.2019;8(6):2163-8.
10. Atla B, Uma P, Shamili M, Kumar S. Cytological patterns of cervical pap smears with histopathological correlation. Int J Res Med Sci. 2015;3:1911–6. doi:10.18203/2320-6012.ijrms20150300.

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