Introduction

Cervical cancer is the third most common cancer in women worldwide with a global prevalence of 11.7%, and accounts as the 5 most common cause of cancer-related deaths. Its annual estimated global incidence is 500,000 cases, with India accounting for approximately 100,000 cases1, 2, 3. Cervical cancer is the second common cancer in underdeveloped countries among females1. The Human Papilloma Virus (HPV) is a proven etiological factor1, 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 age1. The most common symptom is vaginal bleeding. Many times the colposcopic appearance is non-specific2. Histologically, squamous cell carcinoma is common, and classified as non-keratinizing or keratinizing variants1.

Histomorphological diagnosis of cervical biopsy can result in under- or over-treatment and low diagnostic agreement rates5. Use of P16 as an adjuvant biomarker has improved diagnostic agreement6. P16 expression indicates infection with high risk HPV (HR-HPV) and integration of viral genome with host genome4. Different pathologists use different criteria and threshold for interpretation of P16 expression7, 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 co-sponsored 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 accuracy8, 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 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, per-speculum examination findings, per-vaginal examination findings, and stage was taken from hospital records. Staging was done per the FIGO staging10. 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 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 thickness of the epithelium (basal & parabasal layers)- which can be further graded as 1/3, 2/3, more than 2/3, 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 without upward extension) or weak, diffuse and discontinuous staining (involving at least 2/3 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 staining9.

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 per-speculum examination findings (p = 0.217), or per-vaginal examination findings (p = 0.982). There was no significant association between P16 expression and stage of the disease (p = 0.28) (Table 12). However, all stage I cases (100%) showed block positive P16 expression. There was no significant association between histopathological grade and P16 expression (p = 0.877) (Table 13). However, most of the cases of WDSCC (88.0%) and MDSCC (93.7%) showed block positive P16 expression, though there was no statistical significance.

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 females11. The prevalence reported at Bangalore by the Kidwai Memorial Institute of Oncology between 2004 – 2005 was 15.9%12. The National Cancer Registry Program newsletter has reported the incidence to be 6.2% — 22.6% between 2001 - 2011 in India and 21.1% in Bangalore13.

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 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 mutations14, 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 cells16. The sensitivity, specificity and accuracy of P16 biomarker to diagnose SCC of cervix is 96.0%, 88.2% and 91.7%, respectively17.

In the present study, IHC P16 expression per the LAST classification showed block positivity in 67 cases (89.3%), ambiguity in 5 cases (6.6%) and negativity in 3 cases (4%). In a study by Sarwath H ., block positivity was seen in 92.2% of cases and negativity in 7.8% of cases, with sensitivity, specificity, PPV and NPV of 79.2%, 46%, 83.9% and 27.2%, respectively. The absence of P16 expression in SCC of cervix may be due to absence of HPV infection, improper IHC technique, mutations in promoter region, epigenetic mechanisms and hypermethylation1. Stoler MH . classified P16 expression as diffuse, focal and negative per the LAST criteria and found that P16 expression was diffuse in 100% of invasive cancers6. Amaro-Filho has reported P16 expression as diffuse, focal and negative in 85.5%, 9.9% and 4.6% cases, respectively, in SCC of cervix; the data were statistically significant (p < 0.001)18.

In the present study, there was a statistically significant association between age and P16 expression, where all cases (100%) between 30-59 years showed block positivity. Pre- and peri-menopausal women showed maximal (94.73%) block positivity over post-menopausal women; the data was not statistically significant. Among the post-menopausal women, women between 40-44 years showed maximal (93.75%) block positivity, although this data was not statistically significant. Sarwath . stated that there was a significant correlation between P16 expression and age group between 41–60 years. This was thought to be due to active transforming precancerous lesions in younger age group women. Hence, P16 is an appropriate surrogate marker for use in early screening of cervical cancer1.

In the present study, parity and P16 expression did not show a statistically significant association. However, all cases (100%) with parity one and two showed block positivity. There was no statistically significant association between stage / histological grade of the disease and P16 expression. However, all stage I cases (100%) showed block positivity and a majority of cases of WDSCC (88.0%) and MDSCC (93.7%) showed block positivity. Fu HC . in their study stated that P16 expression was not found to have an association with tumor stage, tumor size, histological grade, vascular invasion, CEA levels, SCC Ag levels, or non-squamous cell carcinoma. The independent prognostic factors for cervical cancer regarding disease-free survival (DFS) is high-grade SCC, non-SCC and low P16 expression16. Sarwath . stated that P16 expression did not correlate with tumor grade and size of the tumor1. Huangfu M . in their study reported that auto-antibodies against P16 protein (tumor-associated antigen) are released in cases of cervical cancer, and they are found to be at the highest levels in serum of stage I cervical cancer patients, with sensitivity of > 90% and specificity of 20.3%. Hence, P16 auto-antibody can be used as one of the parameters for early diagnosis and assessment of prognosis19.

Cervical cancer with P16 expression has better prognosis16, 20. High P16 expression in cervical cancer is reported to have high five-year overall survival and DFS, both of which were statistically significant. Five-year overall survival in high and low P16 expression was 62.0% and 35.2%, respectively. DFS in high and low P16 expression was 60.0% and 31.2%, respectively16, 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 cancer16.

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.

Abbreviations

Ag: Antigen

ASCCP: American Society for Colposcopy and Cervical Pathology

CAP: College of American Pathologists

CEA: Carcinoembryonic Antigen

DFS: Disease Free Survival

FIGO: International Federation of Gynaecology and Obstetrics

HPV: Human Papilloma Virus

HR-HPV: High Risk Human Papilloma Virus

IHC: Immunohistochemistry

LAST: Lower Anogenital Squamous Terminology

MDSCC: Moderately Differentiated Squamous Cell Carcinoma

NKLCSCC: Non-Keratinizing Large Cell Squamous Cell Carcinomas

NKSCSCC: Non-Keratinizing Small Cell Squamous Cell Carcinomas

PDSCC: Poorly Differentiated Squamous Cell Carcinoma

SCC: Squamous Cell Carcinoma

WDSCC: Well Differentiated Squamous Cell Carcinoma

Acknowledgments

Nil

Author’s contributions

Kalyani Raju: Concept, Study design, data collection, literature search, statistical analysis, manuscript writing, manuscript editing. Raghuveer CV: Concept, Manuscript editing, manuscript review. Sheela SR: Data collection, manuscript editing.

Funding

Nil

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.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.