Expression of P16 biomarker in squamous cell carcinoma of uterine cervix and its association with clinico-pathological parameters: A cross-sectional study

Use your smartphone to scan this QR code and download this article ABSTRACT Introduction: Cervical cancer is the most common cancer among females. P16 is the surrogate marker for cervical carcinoma. This study aimed to evaluate the association of P16 marker with clinic-pathological parameters in squamous cell carcinoma of uterine cervix. Methods: This was a cross-sectional study. Histological confirmed cases of squamous cell carcinoma (SCC) of cervix were considered. All cases were evaluated for IHC P16 expression as per lower anogenital squamous terminology (LAST) criteria and correlated with clinico-pathological parameters. The data was analyzed by SPSS software version 22. Results: Out of 75 cases, P16 biomarker expression was block positive, ambiguous and negative in 67 (89.3%), 5 (6.6%), and 3 (4%) cases, respectively. There was a significant association between P16 expression and age (p = 0.005). All cases between 30-59 years of age showed block positivity. There was no significant association between P16 expression and age at marriage (p = 0.951), age at menopause (p = 0.311), parity (p = 0.554), clinical symptoms/signs, stage of disease (p = 0.28), or histopathological grade (p = 0.877). Maximumexpression was seen between 40-44 years. Moreover, all cases having 1 & 2 parity showed block positivity and all stage I cases showed block positivity. Conclusion: P16 biomarker was significantly expressed in cervical cancers of the relatively younger age group and those with early stage of disease.


INTRODUCTION
Cervical cancer is the third most common cancer in women worldwide with a global prevalence of 11.7%, and accounts as the 5 th most common cause of cancerrelated deaths. Its annual estimated global incidence is 500,000 cases, with India accounting for approximately 100,000 cases [1][2][3] . Cervical cancer is the second common cancer in underdeveloped countries among females 1 . The Human Papilloma Virus (HPV) is a proven etiological factor 1,3,4 . The age range for cervical cancer is reported to be 27-80 years with mean age of 54.2 years; the maximum cases have been noted between 41-60 years of age 1 . The most common symptom is vaginal bleeding. Many times the colposcopic appearance is non-specific 2 . Histologically, squamous cell carcinoma is common, and classified as non-keratinizing or keratinizing variants 1 . Histomorphological diagnosis of cervical biopsy can result in under-or over-treatment and low diagnostic agreement rates 5 . Use of P16 as an adjuvant biomarker has improved diagnostic agreement 6 . P16 expression indicates infection with high risk HPV (HR-HPV) and integration of viral genome with host genome 4 . Different pathologists use different criteria and threshold for interpretation of P16 expression 7,8 . Lower anogenital squamous terminology (LAST) criteria defines P16 immunoreactivity as block positive, ambiguous or negative, based on the consideration of P16 expression in the nucleus with or without cytoplasmic staining. The LAST project has been cosponsored by the American Society for Colposcopy and Cervical Pathology (ASCCP) and the College of American Pathologists (CAP). LAST gives standard guidelines for the precise utility of P16 biomarker, decreases interobserver's variation, and increases accuracy 8,9 . In this study, we have evaluated the association of immunohistochemistry (IHC) P16 biomarker expression and clinico-pathological parameters in our patient population.

MATERIALS -METHODS
This was a cross-sectional study from October 2017 to September 2018. Ethical clearance was obtained from the Institutional Ethics Committee before the start of the study. A total of 75 clinically suspected and Cite this article : R Kalyani, CV Raghuveer, SR Sheela. Expression of P16 biomarker in squamous cell carcinoma of uterine cervix and its association with clinico-pathological parameters: A crosssectional study. Biomed. Res. Ther.; 7(9):3954-3961.
histopathologically confirmed cases of SCC of cervix, without radiotherapy or chemotherapy, was considered for the study. The case details such as hospital number, biopsy number, age, presenting complaints, menstrual history, personal history, past history, family history, per-abdominal examination findings, perspeculum examination findings, per-vaginal examination findings, and stage was taken from hospital records. Staging was done per the FIGO staging 10 . Paraffin blocks and the corresponding slides were retrieved from the Department of Pathology. The slides were screened by two pathologists and histological typing was conducted. Histologically, the cases were classified as keratinizing or non-keratinizing types. Keratinizing squamous cell carcinomas was further classified as well-differentiated (WDSCC), moderately differentiated (MDSCC) and poorly differentiated (PDSCC). The non-keratinizing squamous cell carcinomas were further classified as small cell type (NKSCSCC) or large cell type (NKLCSCC) 10 . Tissue sections were cut from paraffin blocks and evaluated by IHC for P16 marker using a mouse monoclonal anti-p16 INK4 clone G175-405 as primary antibody (Biogenex, USA); tissue from all 75 cases were evaluated with positive and negative controls. The IHC procedure was carried out per the manufacturer's instructions. The P16 expression on the tissue sections was classified as block positivity, ambiguous staining, or negative per the LAST criteria. "Block" pattern staining corresponded to strong, continuous, nuclear positivity-with or without cytoplasmic staining extending from basal layers upwards for at least 1/3 rd thickness of the epithelium (basal & parabasal layers)-which can be further graded as 1/3 rd , 2/3 rd , more than 2/3 rd , and laterally over a significant distance with diffuse staining of > 25% of cells. "Ambiguous" staining corresponded to strong, basal, diffuse and continuous staining (involving only lower 1/3 rd without upward extension) or weak, diffuse and discontinuous staining (involving at least 2/3 rd of the epithelium), or strong, focal and discontinuous staining (located at any level of the epithelium). "Negative" staining meant a total absence or weak, focal and discontinuous, or only cytoplasmic staining 9 . All data were entered in Microsoft Excel, and the data analyzed using SPSS version 22. The categorical data were presented as frequency and proportions. Continuous data were presented as mean, standard deviation and confidence intervals. Significance of difference between the groups was estimated using standard 't' test and chi-square test; p value < 0.05 was considered as statistically significant.

RESULTS
Seventy-five cases of SCC of cervix were considered for the study. The ages of the cases ranged from 30-80 years with a mean of 54.3 ± 12.0. Table 1 shows the age distribution of the cases. Postmenopausal cases and pre-or peri-menopausal cases accounted for 74.7% and 25.3%, respectively. The majority of women had attained menopause between 40-57 years of age with a mean of 46.3 ± 3.9 years. Age range at marriage was 12-23 years with a mean of 15.7 ± 2.1 years; maximum cases were at 15 years (26.6%) followed by 14 years (16%) of age. Parity ranged from 1-11 with a mean of 3.6 ± 1.6; the cases had a maximum parity of ≥ 5 (28%) followed by 4 (26.6%). Clinical presentations of the cases are shown in Table 2, where bleeding per vagina was the common symptom. Per-speculum findings of cases are shown in Table 3, with growth and bleeding as the most common findings. Per-vaginal findings of cases are shown in Table 4. Stages of the disease of the cases are shown in Table 5, with the maximum number of cases in stage III (40%). The histological grade of disease in the cases are shown in Table 6, with keratinizing SCC as the maximum (90.6%) compared to non-keratinizing type (9.2%). Among the keratinizing SCC, the maximum cases were WDSCC (56%) followed by MDSCC (21.3%). Out of 75 cases, P16 expression was block positive, ambiguous or negative in 67 (89.3%), 5 (6.6%), and 3 (4%) cases, respectively. Block positivity was maximal in pre-& peri-menopausal women (94.7%) compared to post-menopausal women (87.5%); the differences were not statistically significant (p = 0.55) ( Table 7). A statistically significant association was observed between age of cases and P16 expression     (p = 0.005) ( Table 8). All cases (100%) between 30-59 years of age showed block positivity. There was no significant association between age at marriage of cases and P16 expression (p = 0.951) ( Table 9). The p16 expression was maximum among females with age of marriage between 15 and 18 years. There was no statistically significant association between age at menopause of cases and P16 expression (p = 0.311) ( Table 10). Among the post-menopausal women, P16 expression was maximal between 40-44 years. There was no significant association (p = 0.554) between parity and P16 expression. However, all cases (100%) with parity one and two showed block positive P16 expressions (Table 11).
There was no significant association between P16 expression and clinical presentation (p = 0.135), or perspeculum examination findings (p = 0.217), or pervaginal examination findings (p = 0.982). There was no significant association between P16 expression and stage of the disease (p = 0.28) (

DISCUSSION
Cervical cancer is one of the most common cancers in women worldwide, especially in women from developing countries. It is the most common cause of cancer-related deaths in females. The prevalence of cervical cancer in the Southeast part of Karnataka (India) at a tertiary health care center is 17% of the total cancers of females 11  Pap smear test as a screening test has helped decrease the incidence of cervical cancer by 75%, especially in developed countries. However, it has some limitations such as low sensitivity, false negative results and low reproducibility. To overcome this, the P16 biomarker has been used as an alternative and has emerged as a surrogate marker for in-situ as well as advanced cervical cancer. The P16 biomarker test has better reproducibility. The P16 gene undergoes alterations like amplification following HPV infection and integration of viral genome with host genome. P16 may also undergo mutations 14,15 . Normally, the expression of P16 increases with age which results in a decrease of the renewal activity of stem cells. In conditions of inhibition / low P16 expression, there will be high expression of cancer stem cells which results in increased ability of self-renewal of cancer stem cells 16 16,20 . In the present study, the cases were not followed up for prognosis. P16 is also an indicator for radiosensitivity. Thus, due to its anti-cancer activity, P16 can be exploited for development of targeted chemotherapy in cervical cancer 16 .
The limitation of the present study was that we did not follow up with the cases to assess their prognosis. However, P16 block positivity was high in young females and was statistically significant. P16 expression was maximal in pre-and peri-menopausal females, post-menopausal females between 40-44 years of age, females with one/two parity and in stage I disease, although these were not statistically significant.
The study can be conducted in a larger population to further confirm the above findings.

CONCLUSION
Significant p16 biomarker expression was observed in cervical patients of younger age and early stage of the disease. Therefore, P16 biomarker may have a beneficial use in screening or early diagnosis leading to better prognosis of SCC of cervix. This findings from the study can also be used as a concept for targeted therapy as P16 protein has anti-cancer properties.

AVAILABILITY OF DATA AND MATERIALS
Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE
This study was conducted in accordance with the amended Declaration of Helsinki. The institutional review board (The Institutional Ethics Committee of Sri Devaraj Urs Medical College, Tamaka, Kolar) approved the study, and all participants provided written informed consent.