Abstract
Background
Cervical cancer is the fourth most common genital tract cancer and fourth common cause of death among the causes of neoplasm-related mortality in women worldwide. Squamous cell carcinoma is the most common type and constitutes about 90% of all pathological types of cervical cancer. Cyclin-dependent kinase inhibitor 2A (CDKN2A; p16) is a gene that is located on chromosome 9 that encodes a protein (P16) that inhibits cyclin-dependent kinases 4 and 6 which are inhibitors of retinoblastoma protein; the net result is reactivation of retinoblastoma protein and arrest of cell cycle in G1 phase. So, expression of p16 protein within cancer cell may denote good prognosis. The presence of a soft marker that can detect hidden advanced stages in apparently clinically and radiologically early resectable stages of cervical cancer and can replace life-threatening preoperative lymphadenectomy is of great importance. Is P16 protein which when expressed is associated with good prognosis in other cancer can be this soft marker?
Aim of the study
The aim of this study is to estimate the prevalence of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16) immunohistochemistry positive staining in squamous cell carcinoma of the cervix and to correlate its positivity to clinical and radiological disease stage.
Patients and Methods
An analytical cross-sectional observational prospective and retrospective study was conducted on 60 invasive squamous cell cervical cancer patients from gyne-oncology unit at Al Shatby university hospital after taking a written consent and following approval by Alexandria medical school institutional ethics committee. Inclusion criteria included all patients with invasive squamous cell carcinoma of the cervix prospective and retrospective from January 2019 till June 2022 diagnosed by taking wedge, punch and cone biopsy followed by histopathological examination that confirmed the diagnosis and showed grading and types of squamous cell carcinoma. Clinical examination, vaginal ultrasonographic scanning, computerized axial tomography and magnetic resonance imaging were carried out to all cases, and data were recorded for clinical and radiological staging purposes. All data were collected, coded, tabulated and statistically analyzed to estimate the prevalence of p16 positivity in the study cases and to correlate its positivity with clinical and radiological disease stage.
Result
In relation to prevalence of p16 immunostaining, 34 cases (56.7%) were positive in comparison with 26 cases (43.3%) being negative. Considering correlation between early resectable stage and late nonresectable stage with P16 positive and negative staining, the result showed the following: 32 cases (53.3%) were resectable, 30 cases (88.2%) of them were P16 positive immunostaining compared to 2 cases (7.7%) being P16 negative immunostaining while nonresectable cases were 28 cases (46.7%), 4 cases (11.8%) only were P16 positive compared to 24 cases (92.3%) being P16 immunostaining negative, so most of early resectable stage cases were P16 positive immunostaining and most of late nonresectable stage cases were P16 negative immunostaining. There is a positive significant correlation between early resectable stage in relation to positive P16 immunostaining, and the same was present between late nonresectable stage and negative P16 immunostaining (p = 0.000).
Conclusion
The present study concluded that P16 positive immunostaining prevalence in cervical squamous cell carcinoma was 56.7% and its positive staining is highly correlated with early resectable clinically and radiologically disease stage.
Keywords: P16 immunostaining, Cervical cancer, Squamous cell carcinoma
Introduction
Cervical cancer is the fourth most common genital tract cancer and fourth common cause of death among the causes of neoplasm-related mortality in women worldwide [1]. The incidence of cervical cancer in developed countries was lower than in developing countries due to human papilloma virus vaccination and regular cervical cell cytology {Papanicolaou (Pap) smear} and early intervention in the presence of preinvasive lesion [2, 3]. Squamous cell carcinoma is the most common pathological type and constitutes about 90% of all pathological types of cervical cancer [4]. Nearly all cases of squamous cell cervical carcinoma with its pathological variants and the majority of adenocarcinoma are caused by carcinogenic types of human papilloma virus [5]. Early (E) encoded genes of human papilloma virus as E7 that encodes E7 oncoprotein inhibit retinoblastoma protein by competition with E2F transcription factor for binding with it; the net result is increasing level of E2F transcription factor within the cell [6, 7]. E2F transcription factor stimulates the transfer of cell cycle growth phase 1 to synthetic phase by stimulation of transcription of genes that are needed in DNA replication, and this stimulates oncogenesis [8]. Cyclin-dependent kinase inhibitor 2A (CDKN2A; p16) is a gene that is located on chromosome 9 that encodes a protein (P16) that inhibits cyclin-dependent kinases 4 and 6 which are inhibitors of retinoblastoma protein; the net result is reactivation of retinoblastoma protein and arrest of cell cycle in G1 phase [9]. So expression of p16 protein within cancer cell may denote good prognosis [10]. Surgical management of all resectable cases of cervical cancer that includes early stage till stage 1B2 and some cases with 2A1 stage by radical hysterectomy should be preceded by regional pelvic and lower para-aortic lymphadenectomy followed by histopathological examination to exclude lymph nodes microscopic metastasis [11]. If regional lymph nodes show positive invasion, radical hysterectomy should be avoided and the patients are referred for definitive radio chemotherapy [12]. Lymphadenectomy is surgical procedure that carries a life-threatening risk due to vascular injuries and postoperative deep veins thrombosis [13]. The presence of a soft marker that can detect hidden advanced stages in apparently clinically and radiologically early resectable stages of cervical cancer and can replace life-threatening preoperative lymphadenectomy is of great importance. Is P16 protein which when expressed is associated with good prognosis in other cancer can be this soft marker by its negative staining in immunohistochemistry of the squamous cell cervical cancer tissue? The present study tries to answer this question in squamous cell carcinoma of the cervix by correlating its positivity with clinical and radiological disease stage and to estimate the prevalence in the disease.
Aim of the Study
The aim of this study is to estimate the prevalence of cyclin-dependent kinase inhibitor 2A (CDKN2A; p16) immunohistochemistry positive staining in squamous cell carcinoma of the cervix and to correlate its positivity with clinical and radiological disease stage.
Patients and Methods
An analytical cross-sectional observational prospective and retrospective study was conducted on 60 invasive squamous cell cervical cancer patients from gyne-oncology unit at Al Shatby university hospital after taking a written consent from all cases and following approval by Alexandria medical school institutional ethics committee. Inclusion criteria included all admitted patients to gyne-oncology unit at Al Shatby university hospital with invasive squamous cell carcinoma of the cervix prospective and retrospective from January 2019 till June 2022 diagnosed by taking wedge, punch and cone biopsy followed by histopathological examination that confirmed the diagnosis and showed grading and types of squamous cell carcinoma. All submitted cervical tumors biopsies were assessed on hematoxylin and eosin (H&E)-stained sections for morphologic classification. Squamous cell carcinomas were assessed and graded into well, moderate or poorly differentiated according to degree of keratinization and nuclear atypia. Five-micron sections were cut on positively charged glass slides for cyclin-dependent kinase inhibitor 2A (CDKN2A; p16); immunohistochemistry staining was carried out to all study patients. Anti-P16 primary antibody (#805-4713, mouse monoclonal antibody, ready to use) was used by auto-Stainer Ventana benchmark GX IHC system (ROCHE DIAGNOSTICS, USA). The cases were considered positive when all tumor cells showed strong positivity (block staining). Any focal or patchy staining was considered negative. Clinical examination, vaginal ultrasonographic scanning, computerized axial tomography and magnetic resonance imaging were carried out to all cases, and data were recorded for clinical and radiological staging purposes. All data were collected, coded, tabulated and statistically analyzed to estimate the prevalence of p16 positivity in the study cases and to correlate its positivity with clinical and radiological disease stage using IBM SPSS statistics (Statistical Package for Social Sciences) software version 24.0, IBM Corp., Chicago, USA. Descriptive statistics were carried out for qualitative data as number and percentage. Inferential analyses for independent variables were carried out using Chi-square test for differences between proportions and Fisher’s exact test for variables with small, expected number. The level of significance was taken at p value < 0.05 being statistically significant; otherwise, it is non-significant. The p-value is a statistical measure for the probability that the results observed in a study could have occurred by chance.
Justification of the Sample Size
The required sample size has been estimated using the Med Calc statistical software VAT registration number being BE 0809 344 640. Med Calc software is a corporate member of the American Statistical Association which is a member of the International Association of Statistical Computing. The previous software has been used to conduct a comparative study about the prevalence of P16 immunohistochemistry positive staining and its correlation to clinical and radiological staging of squamous cell carcinoma of the cervix. A previous study carried out by Larsen et al. [14] was used to predict the different measurement in our study. The following equation was used for sample size calculation: n = t2 × p(1 − p)/m2. n = required sample size, t = confidence level at 95% (standard value of 1.96), p = estimated measurements, m = margin of error at 5% (standard value of 0.05). Sample size was calculated to be 60 patients in this study. The estimated sample size is made at assumption of 95% confidence level and 90% power of study.
Result
Histopathological examination of the 60 squamous cell cervical carcinoma of the study cases revealed the following: 22 cases were keratinized squamous cell carcinoma (36.7%), 36 cases were nonkeratinized squamous cell carcinoma (60%), and 2 cases were papillary invasive squamous cell carcinoma (3.3%) as shown in Table 1, Figs. 1a, c and 2. Considering pathological grading of the cases, 7 cases were grade 1 (11.7%), 24 cases were grade 2 (40%), and 29 cases were grade 3 (48.3%) as shown in Table 1 and Fig. 3; regarding operative resectability: 32 cases (53.3%) were resectable in comparison with 28 cases (46.7) being not resectable as shown in Table 1. In relation to the prevalence of p16 immunostaining, 34 cases (56.7%) were positive in comparison with 26 cases (43.3%) being negative as shown in Table 1 and Fig. 1b, d; considering distribution of study cases according to FIGO staging diagnosed clinically and radiologically, the following data were found: 9 cases (15%) were 1B1, 20 cases (33.3%) were 1B2, 3 cases (5%) were 2A1, all the previous mentioned stages were early and operatively resectable and constituted 32 cases out of 60 cases (53.3%) while the remaining 28 cases (46.7%) were late nonresectable stage and included the following: 2 cases (3.3%) were stage 1B3, 1 case (1.7%) was stage 2A2, 11 cases (18.3%) were stage 2B, 2 cases (3.3%) were stage 3B, 7 cases (11.7%) were stage 3C1, 2 cases (3.3%) were stage 3C2, 1 case (1.7%) was 4A, 2 cases (3.3%) were stage 4B as shown in Table 1. Considering correlating early resectable stage and late nonresectable stage with P16 positive and negative staining, the result showed the following: 32 cases (53.3%) were resectable, 30 cases (88.2%) of them were P16 positive immunostaining compared to 2 cases (7.69%) being P16 negative immunostaining while nonresectable cases were 28 cases (46.7%), 4 cases (11.76%) only were P16 positive compared to 24 cases (92.3%) being P16 immunostaining negative, so most of early resectable stage cases were P16 positive immunostaining and most of late nonresectable stage cases were P16 negative immunostaining as shown in Table 2 and Fig. 4A. There is a positive significant correlation between early resectable stage in relation to positive P16 immunostaining, and the same was present between late nonresectable stage and negative P16 immunostaining (p = 0.000) as shown in Table 2 and Fig. 4a. Regarding correlation between FIGO staging (clinical and radiological), there was a highly significant positive correlation between positive P16 staining and early stages as B1, B2 and 2A1 (p = 0.000); on the other hand, the same was present between other advanced stages and negative p16 immunostaining (p = 0.000) as shown in Table 2. In relation to correlation between pathological grading of the study cases and P16 immunostaining, there was there a highly significant positive correlation between positive P16 staining and grades 1 and 2 (p = 0.000); on the other hand, the same was present between grade 3 and negative p16 immunostaining (p = 0.000) as shown in Table 2 and Fig. 5. In relation to specificity, sensitivity, positive predictive value and negative predictive value of positive P16 immunostaining with early resectable stage, area under the ROC curve showed that specificity, sensitivity, positive predictive value and negative predictive value of P16 positive staining with early resectable stage were 92.3%, 88%, 93.8% and 85.7%, respectively, as shown in Fig. 4B and Table 3.
Table 1.
Distribution of studied cases according to different pathological and clinical parameters
| Cases (n = 60) | ||
|---|---|---|
| No | % | |
| Pathologic subtypes | ||
| Keratinized | 22 | 36.7 |
| Non keratinized | 36 | 60 |
| Papillary ` | 2 | 3.3 |
| Pathologic grade | ||
| Grade 1 | 7 | 11.7 |
| Grade 2 | 24 | 40 |
| Grade 3 | 29 | 48.3 |
| Resectability | ||
| Resectable | 32 | 53.3 |
| Non resectable | 28 | 46.7 |
| FIGO stage | ||
|
Early resectable n = 32 (53.3%) | ||
| 1B1 | 9 | 15 |
| 1B2 | 20 | 33.3 |
| 2A1 | 3 | 5 |
|
Late non resectable n = 28 (46.7%) | ||
| 1B3 | 2 | 3.3 |
| 2A2 | 1 | 1.7 |
| 2B | 11 | 18.3 |
| 3B | 2 | 3.3 |
| 3C1 | 7 | 11.7 |
| 3C2 | 2 | 3.3 |
| 4A | 1 | 1.7 |
| 4B | 2 | 3.3 |
| P16 immunostaining: | ||
| Positive | 34 | 56.7 |
| Negative | 26 | 43.3 |
Fig. 1.
a Poorly differentiated keratinized squamous cells carcinoma of the cervix showing invasive nests of malignant squamous cells with focal keratin pearls (arrow) (H&E). b P16 immunostaining of same case showing strong positive staining (block positivity) (IHC). c Nonkeratinizing squamous cell carcinoma showing nests of malignant squamous cells without evident of central keratin in inflamed stroma (H&E). d) The same case showed diffuse strong positive P16 immunostaining (block positivity) (IHC). (×100, scale bar = 200 µm)
Fig. 2.
Pathological subtypes of cervical squamous cell carcinoma of study cases. Nb: KSCC (keratinized squamous cell carcinoma), NKSCC (nonkeratinized squamous cell carcinoma), PSCL (papillary squamous cell carcinoma)
Fig. 3.
Pathological grading types of study cases
Table 2.
Correlation of P16 immunostaining with different pathological and clinical parameters in studied cases
| P16 immunostaining No (%) | Total No (%) | χ2, p value | ||
|---|---|---|---|---|
| Negative | Positive | |||
| Pathologic grade | ||||
| Grade 1 | 0 (0.0) | 7 (100) | 7 (11.7) |
χ2 = 16.397 p = 0.000* |
| Grade 2 | 6 (25) | 18 (75) | 24 (40) | |
| Grade 3 | 20 (68.9) | 9 (31.1) | 29 (48.3) | |
| Resectability | ||||
| Resectable | 2 (7.7%) | 30 (88.2%) | 32 (53.3) |
χ2 = 38.402 p = 0.000* |
| Nonresectable | 24 (92.3%) | 4 (11.8%) | 28 (46.7) | |
| FIGO stage | ||||
|
Early resectable n = 32 (53.3%) | ||||
| 1B1 | 1 (3.8) | 8 (23.5) | 9 (15) |
χ2 = 43.811 p = 0.000* |
| 1B2 | 1 (3.8) | 19 (55.9) | 20 (33.3) | |
| 2A1 | 0 (0) | 3 (8.8) | 3 (5) | |
|
Late nonresectable n = 28 (46.7%) | ||||
| 1B3 | 1 (3.8) | 1 (2.9) | 2 (3.3) | |
| 2A2 | 1 (3.8) | 0 (0) | 1 (1.7) | |
| 2B | 9 (34.6) | 2 (5.9) | 11 (18.3) | |
| 3B | 2 (7.7) | 0 (0) | 2 (3.3) | |
| 3C1 | 7 (26.9) | 0 (0) | 7 (11.7) | |
| 3C2 | 2 (7.7) | 0 (0) | 2 (3.3) | |
| 4A | 0 (0) | 1 (2.9) | 1 (1.7) | |
| 4B | 2 (7.7) | 0 (0) | 2 (3.3) | |
| Total | 26 (43.3) | 34 (56.7) | 60 (100) | – |
χ2 = Pearson Chi-square
*p value is considered significant if < 0.05
Fig. 4.
a Bar chart showing the correlation between P16 immunostaining and resectability of cancer cervix in studied cases. It was highly significant (p = 0.000). b Receiver operating characteristic (ROC) curve showing the P16 immunostaining as a predictor of the resectability of cancer cervix cases. It was able to discriminate resectable and nonresectable cases with 88.2% sensitivity and 92.3% specificity (AUC = 0.903, PPV = 93.8%, NPV = 85.7%)
Fig. 5.
Correlation between pathological grading of the study cases and P16 immunostaining
Table 3.
Area under the curve and Pearson Chi-square test
| Area under the curve | |||
| 0.903 | |||
| Pearson chi-square | Value | Degree of freedom | p value Sig. (2-sided) |
| 38.402 | 1 | 0.000 | |
Sensitivity = 88.0%, specificity = 92.3% PPV = 93.8%, NPV = 85.7%
Pearson Chi-square test showing the P16 positive immunostaining as a predictor of the resectability of cancer cervix cases. It was able to discriminate resectable and nonresectable cases with 88.2% sensitivity and 92.3% specificity (AUC = 0.903, PPV = 93.8%, NPV = 85.7%)
Discussion
Cervical cancer is one of the common gynecological cancers and ranking the fourth among genital tract cancer world widely [15]. The function of the carcinogenic types of human papilloma virus in the pathogenesis of cervical cancer is well known since how long [16]. About 90% or more of cervical cancer cases are related to carcinogenic types of human papilloma virus as types 16 and 18, and the overall mortality rate of cervical cancer is 8% from all cancer death in female [17]. P16 is a protein that is encoded by Cyclin-dependent kinase inhibitor 2A (CDKN2A; p16) gene which is located on chromosome 9 and it functions as tumor suppressor by inhibition of kinase that suppresses retinoblastoma protein allowing its reactivation [18]. P16 positive immunostaining has been used as detector for human papilloma virus infection and marker for viral oncoprotein activity, especially in cases with excellent prognosis and early clinical outcome, as the binding of retinoblastoma protein to early protein 7 of human papilloma virus decreases the free retinoblastoma protein which removes the negative feedback on P16, and the net result is overexpression of P16 which suppresses oncogenic activity, especially in cases with good prognosis [19]. The present study estimates the prevalence of p16 immunohistochemistry positive staining in squamous cell carcinoma of the cervix and correlate its positivity to clinical and radiological disease stage. In relation to prevalence of p16 immunostaining, 34 cases (56.7%) were positive in comparison with 26 cases (43.3%) being negative. In comparison with other study, Sano, T et al. found that positive p16 immunostaining was found in all cases with high-grade cervical intraepithelial neoplasia (CIN) and early invasive cancer except several cases with low-grade CIN [20]. De Wispelaere et al. [21] found that prevalence of P16 positive immunostaining in cervical cancer cases was 94.4% in relation to 56.7% in the present study. The result of the previous 2 study is not matching with the present study, but the explanation is that 28 cases (46%) out of 60 cases were advanced nonresectable late stages so nearly half of cases were advanced nonresectable and this finding not only explains the contradictory between the present study with the previous 2 studies but confirms that P16 positive staining is associated with cases with good clinical outcome. Considering correlation between early resectable stage and late nonresectable stage with P16 positive and negative staining, the present study showed that there is highly significant correlation between early resectable FIGO stage and positive P16 staining (p = 0.000) and the same was present between late nonresectable stage and negative P16 immunostaining (p = 0.000). In relation to specificity, sensitivity, positive predictive value and negative predictive value of positive P16 immunostaining with early resectable stage, area under the ROC curve showed that these parameters were 92.3%, 88%, 93.8% and 85.7%, respectively. In fact, this is an important finding in the present study as it answers the question of using P16 negative staining in cervical squamous cell carcinoma in early clinically and radiologically resectable stage as soft marker to detect advanced hidden late-stage undiagnosed preoperatively so we can prevent faulty operative radical interference and proceed directly to radio chemotherapy. In agreement with the present study, Jiaying Lin et al. [22] found that p16 overexpression is highly associated with no regional lymph nodes metastasis and better overall survival. Nathalie Arians et al. [23] found that P16 positive staining is associated with increased progression-free survival and overall survival in squamous cell carcinoma of the vulva. Masako Ishikawa et al. [24] found that p16 positive immunostaining is associated with favorable prognosis of patients with cervical adenocarcinoma. Regarding sensitivity and specificity, Qin Shi et al. [25] found a comparable result to the present study in high-grade squamous intraepithelial lesion and cervical cancer as their result was 94.6% and 85.4% for specificity and sensitivity, respectively. In relation to correlation between pathological grading of the study cases and P16 immunostaining, the present study showed that there was a highly significant positive correlation between positive P16 staining and grades 1 and 2 (p = 0.000); on the other hand, the same was present between grade 3 and negative p16 immunostaining (p = 0.000). In agreement with last finding, De Wispelaere N, Rico SD et al. found a high association between well-differentiated grade and positive P16 staining. The present study concluded that P16 positive immunostaining in squamous cervical cancer is associated with early resectable stage and negative staining even with early clinically and radiologically stage highly associated with hidden undiagnosed late stage so it may have a role in management by referring these cases to radio chemotherapy directly.
Conclusion
The present study concluded that P16 positive immunostaining prevalence in cervical squamous cell carcinoma was 56.7% and its positive staining is highly correlated with early resectable clinically and radiologically disease stage.
Declarations
Conflict of interest
All the authors declare that they have no conflict of interest.
Consent for publication
Written consent was obtained from each participant.
Ethical Approval
This was an analytical cross-sectional observational Prospective and retrospective study involving collection of patient data who were undergoing standard management protocols and hence there was no direct risk to the participants. This study was approved by Ethics Committee of faculty of medicine, Alexandria university, Egypt, member of ICLAS (International Council for Laboratory Animal Science), (reference number: 0305452).
Informed Consent
Informed consent was obtained from all individual participants included in the study.
Research involving human participants
This study was performed in line with the principles of the Declaration of Helsinki. Ethical approval was provided by Ethics Committee of faculty of medicine, Alexandria university, Egypt, member of ICLAS (International Council for Laboratory Animal Science), (reference number: 0305452).
Footnotes
Dr. Hossam H. El Sokkary is an associate professor, Department of Obstetrics and Gynaecology, Faculty of medicine, Alexandria university, Alexandria, Egypt. Dr. Eman Sheta is an Lecturer, Department of Pathology, Faculty of medicine, Alexandria university, Alexandria, Egypt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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