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| ORIGINAL ARTICLE |
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| Year : 2021 | Volume
: 48
| Issue : 3 | Page : 179-185 |
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Epidemiological aspects of preinvasive cervical lesions and the role of p16INK4a immunohistochemistry in their diagnosis: Experience from a tertiary hospital in South-east Nigeria
Christian C Ogbu, Chinedu O Ndukwe, Michael E Chiemeka, Kingsley C Madubuike, Cornelius O Ukah
Department of Histopathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria
| Date of Submission | 28-Apr-2021 |
| Date of Acceptance | 02-Jul-2021 |
| Date of Web Publication | 28-Dec-2021 |
Correspondence Address:
Dr. Chinedu O Ndukwe
Department of Histopathology, Nnamdi Azikiwe University Teaching Hospital, Nnewi
Nigeria
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/jss.jss_39_21
Introduction: There is a high burden of cervical cancer in our environment. Most patients present late when the prognosis is guarded. Hence, accurate diagnosis of preinvasive lesions from cervical biopsies is important for clinical decision and patient management. Therefore, the use of immunohistochemistry in most difficult cases is paramount and justifies the study. The aim of the study is to evaluate the expression and diagnostic role of p16INK4a Immunohistochemistry in preinvasive cervical lesions. Materials and Methods: The paraffin blocks of all histologically diagnosed preinvasive and normal/reactive lesions of the cervix seen in two histopathology laboratories in Nnewi: Nnamdi Azikiwe University Teaching Hospital and Pathocon Specialist Clinic and Research Institute over a 10-year retrospective period (2011–2020) were retrieved from the archives. Fresh h and e slides were produced and reviewed for diagnostic consistency and a consensus diagnosis rendered for each case. The cases were subjected to immunohistochemistry using p16INK4a monoclonal antibodies. Results: A total of 402 cervical biopsies were seen in the records of both facilities. Ninety of them were noninvasive lesions (62 preinvasive lesions and 28 normal/reactive lesions). Thus, the prevalence of preinvasive lesions in this study was 15.4% (62/402). However, 51 cases met our inclusion criteria. The consensus H and E diagnosis consists of 15 normal/reactive, 12 low squamous intraepithelial lesion (LSIL), and 24 hIL. The diagnosis using immunohistochemistry consisted of 23 normal/reactive (45.1%), 6 LSIL (11.8%), and 22 hIL (43.1%). The age range of the patients was 34–80 years. The mean and median ages were 49.7 and 47 years respectively. The peak ages for LSIL and high squamous intraepithelial lesion (HSIL) were 41–60 years and 51–60 years, respectively. Only 21.5% (ca in situ-3.9% and squamous intraepithelial lesion-17.6) of the patients were biopsied on account of preinvasive cervical lesions. The sensitivity and specificity of p16INK4a in the diagnosis of HSIL were 75% and 85%, respectively. There was a significant statistical difference between the diagnosis before and after the application of p16INK4a (P < 0.001). Conclusion: There is a high prevalence of preinvasive lesions in our environment. Majority of the preinvasive lesions were incidental findings. Immunohistochemistry using p16INK4a is a very useful adjunct to H and E for diagnosing preinvasive cervical lesions, especially in challenging cases.
Keywords: Cervix, cervical intraepithelial neoplasia, high squamous intraepithelial lesion, low squamous intraepithelial lesion, Nigeria, p16
How to cite this article:
Ogbu CC, Ndukwe CO, Chiemeka ME, Madubuike KC, Ukah CO. Epidemiological aspects of preinvasive cervical lesions and the role of p16INK4a immunohistochemistry in their diagnosis: Experience from a tertiary hospital in South-east Nigeria. J Sci Soc 2021;48:179-85 |
How to cite this URL:
Ogbu CC, Ndukwe CO, Chiemeka ME, Madubuike KC, Ukah CO. Epidemiological aspects of preinvasive cervical lesions and the role of p16INK4a immunohistochemistry in their diagnosis: Experience from a tertiary hospital in South-east Nigeria. J Sci Soc [serial online] 2021 [cited 2022 May 25];48:179-85. Available from: https://www.jscisociety.com/text.asp?2021/48/3/179/333845 |
| Introduction | |  |
Cervical intraepithelial neoplasia (CIN) is a precursor to invasive squamous cell carcinoma.[1] CIN is classified as CIN I, CIN II, and CIN III. CIN II frequently progresses to CIN III or invasive cancer, and CIN III to invasive cancer, but the progression rate from CIN I to a higher grade lesion or to cancer is low (9% from CIN I to CIN III and 1% to invasive cancer).[2],[3],[4]
The age-standardized rate of cervical cancer in Nnewi is 13.2 cases/100,000 females.[5] However, in Nigeria, it is 34.5 cases/100,000 women.[6]
The grading of CIN is based on the proportion of the epithelium occupied by the neoplastic, undifferentiated, and basaloid cells.[7] They are semi-quantitatively classified into 3 categories: CIN I-the neoplastic and basaloid cells occupy the lower third of the epithelium; CIN II – the neoplastic and basaloid cells occupy the lower third to two-thirds of the epithelium; CIN III – the neoplastic and basaloid cells occupy two-thirds to the full thickness of the epithelium.[7]
Studies done in Thailand, Colombia, Spain, and the US highlighted some of the risk factors implicated in cervical carcinogeneses like human papillomavirus (HPV) infection, illiteracy, multiple sexual partners, sexually transmitted infections, oral contraceptives, and smoking.[8],[9],[10]
The indications for cervical biopsies include: An abnormality seen during the pelvic examination, abnormal pap smear, a positive test for HPV, as part of colposcopy (colposcopy-guided cervical biopsy), to diagnose and treat precancerous and cancerous lesions in the cervix, to diagnose and treat cervical polyps, to diagnose and treat genital warts, and in women exposed to diethylstilbestrol in utero.[11]
Cytological and histological diagnosis of cervical biopsies that are often considered as the “gold standards” for cervical screening programs can be significantly hampered by intra-and inter-observer variability.[12],[13] It is difficult to distinguish CIN I reliably from nonneoplastic lesions, and CIN I from CIN II/III, resulting in either overtreatment or undertreatment.[14],[15] Therefore, accurate diagnosis of cervical lesions is important for clinicians' decisions and patients' treatment.[1]
Immunohistochemistry is an ancillary technique that is helpful in equivocal cases. A panel of monoclonal antibodies, used as surrogate markers, is now available, and these include CD40, p16INK4a, p53, and Ki-67.[16]
p16INK4a has been extensively investigated. It labels cervical dysplastic squamous and glandular cells with a sensitivity of 99.9%, and a specificity of 100%, both immunohistochemically and immunocytochemically. It has also been shown to be able to separate low- and high-grade lesions, and identify histologically or cytologically classified low-grade lesions that are susceptible to cancer progression.[17],[18]
The aim of this study is to carry out an immunohistochemical review of all cervical biopsies (excluding cases confirmed as invasive lesions) in Nnewi over a 10-year retrospective period using p16INK4a. It also seeks to correlate the expression of p16INK4a with histopathological diagnosis and to determine if there is a significant difference between the diagnosis before and after the application of p16INK4a.
| Materials and Methods | | |
The study locations
Nnamdi Azikiwe University Teaching Hospital (NAUTH) is a 440-bed hospital. Pathocon Specialist Clinic and Research Institute is a private histopathology laboratory. Both facilities are located in Nnewi and serve mainly Nnewi and environs. Nnewi is the second-largest city in Anambra state with a population of 391,227 people as at the 2006 census.[19] It is an industrial and commercial city.
Study design
This is a retrospective study of immunohistochemical expression of p16INK4a over 10 years (January 2011 to December 2020).
Inclusion criteria
All cervical biopsies histologically diagnosed as either preinvasive or normal/reactive lesions with well-preserved blocks, adequate clinical data, and tissue left for sectioning were included in the study.
Exclusion criteria
Poorly preserved tissue blocks such as those that are matted together, partially eaten by rodents or infested by mold were excluded from the study. Also excluded are tissue blocks without adequate tissue left for sectioning, cases with missing blocks, and cases with incomplete data like age and indication for biopsy.
Ethical consideration
Ethical approval for the study has been obtained from the Ethics Committee of NAUTH.
Procedure
Formalin-fixed and paraffin-embedded tissue blocks of cases histologically diagnosed as either preinvasive or normal/reactive cervical lesions in both facilities from January 2011 to December 2020 were retrieved from the archives. Those blocks that met our inclusion criteria were eventually enrolled in the study. Fresh sections were cut and stained with hematoxylin and eosin. These were reviewed for diagnostic consistency and a consensus diagnosis rendered for each case. The cases were immunostained for p16INK4a antigen through the indirect biotin streptavidin method. All sections were deparaffinized in xylene, rehydrated in graded alcohols, washed in PBS buffer, boiled in EDTA solution (pH 8.0, 100°C, 2.5 min) for antigen retrieval, and cooled down. The slides were stained. The primary antibody was p16INK4a (DB Biotech clone R19-D) at a dilution of 1:80.
Interpretation of immunohistochemistry staining results
p16INK4a was considered positive when there was continuous staining of the cells of the basal and parabasal cell layers of the cervical squamous epithelium, with or without staining of cells of intermediate or intermediate and superficial layers [Figure 1]. It was considered negative when there was no staining or only focal staining defined as staining of isolated cells or small clusters of cells (i.e., noncontinuous staining) [Figure 2]. Positive controls included tissue sections known to express the antigen under investigation. | Figure 1: Upper panel: High squamous intraepithelial lesion (H and E, ×40); lower panel: High squamous intraepithelial lesion with strong and diffuse staining (block positivity) of both the nuclei and cytoplasm by p16INK4a (×200)
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 | Figure 2: Upper panel: Low squamous intraepithelial lesion (H and E, ×40); Lower panel: Low squamous intraepithelial lesion with focal staining of the nuclei by p16INK4a (×100)
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Data analysis
Data were analyzed using simple descriptive statistics such as the Chi-square test to measure association with a level of significance of <0.05. This analysis was performed using the SPSS Statistics for Windows, Version 22.0. IBM Corp.Armonk, NY, USA.
| Results | | |
A total of 402 cervical biopsies were seen in the records of both facilities for the 10 years under review (2011–2020). Ninety of them are noninvasive lesions (62 preinvasive lesions and 28 normal/reactive lesions). Thus, the prevalence of preinvasive lesions in this study was 15.4% (62/402). Forty-nine (49) and 41 cervical biopsies from NAUTH and PATHOCON, respectively. Thirty-nine cases were excluded for various reasons such as inadequate clinical data on the request forms, missing tissue blocks, and poor preservation of the blocks, thereby making antigen retrieval impossible in the course of immunohistochemistry.
Fifty-one cases were eventually enrolled in this study (30 cases from PATHOCON and 21 cases from NAUTH). They consist of 15 low squamous intraepithelial lesion (LSIL), 18 hIL (7 CIN II, 9 CIN III, 2 carcinoma in situ [ca in situ]), and 18 normal/reactive (15 normal, 3 chronic-nonspecific cervicitis). LSIL, high squamous intraepithelial lesion (HSIL), and normal/reactive of 29.4%, 35.3%, and 35.3%, respectively.
The cases were jointly reviewed by the authors for diagnostic consistency and a consensus diagnosis adopted for those with discrepant diagnoses leading to reclassification as follows: 15 normal/reactive (13 normal and 2 chronic-nonspecific cervicitis), 12 LSIL and 24 hIL (9 CIN II, 10 CIN III and 5 ca in situ). Therefore, the consensus H and E diagnosis consisted of 15 normal/reactive (29.4%), 12 LSIL (23.5%) and 24 hIL (47%).
The inter-observer agreement between the original and reviewed histopathological diagnosis was good (Cohen's κ = 0.63). The sensitivity and specificity of the original histologic diagnosis for HSIL were 62.5% and 66.7%, respectively.
Following immunohistochemistry using p16INK4a, the cases were subsequently re-classified as follows: 23 reactive/normal (45.1%), 6 LSIL (11.8%), and 22 hIL (43.1%) [Table 1]. | Table 1: Frequency table and age demographics of the final diagnosis after p16 immunohistochemistry
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Demographic characteristics
The age range of the patients was 34–80 years with mean and median of 49.7 and 47 years, respectively [Table 1].
The age range of the patients with the diagnosis of HSIL was 36–80 years. The mean age was 51.9 years and the median age was 50.5 years [Table 1]. There was a sharp increase in the incidence of HSIL from the age group 31–40 years and a peak in the age group 51–60 years, followed by a sharp decline in the age group 61–70 years and another peak in the age group 71–80 years [Figure 1].
The age range of patients with the diagnosis of LSIL was 37–63 years. The mean age was 48.3 years and the median age was 46.5 years [Table 1]. There was a sharp increase in the incidence of LSIL from the age group 31–40 years and a peak in the 41–50 years age group. Followed by drastic decline and a plateau in the age groups 51–60 years and 61–70 years [Figure 3].
The age range of patients with the diagnosis of normal/reactive was 34–76 years. The mean age was 47.9 years and the median age was 47 years [Table 1].
Indications for biopsy (clinical diagnosis)
Leiomyoma had the highest frequency of 17, followed by ca cervix (cancer of the cervix) and squamous intraepithelial lesion (SIL) having frequencies of 11 and 9, respectively. Both malignant ovarian tumor and abnormal uterine bleeding had the same frequencies of three. ca in situ and endometrial hyperplasia also had the same frequencies of two. Retained products of conception, chronic nonspecific cervicitis, cervical polyp and endometrial cancer (endometrial ca) each had the least and same frequency of one [Table 2].
Determination of significant difference between the diagnosis before and after application of p16INK4a
There was a significant statistical difference between the H and E diagnosis and the diagnosis after the application of p16INK4a (P < 0.05).
The sensitivity and specificity of p16INK4a in the diagnosis of HSIL were 75% and 85%, respectively [Table 3]. | Table 3: Consensus diagnosis and diagnosis with p16INK4a cross tabulation for sensitivity and specificity
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| Discussion | | |
Preinvasive lesions of the cervix are fairly common in our environment. However, there are few published local studies on the prevalence of histopathologically diagnosed preinvasive cervical lesions in Nigeria. Studies done in Zaria, Abakaliki and Lagos reported prevalence of 1.4%, 15.1%, and 8.9%, respectively (a range of 1.4%–15.1%).[20],[21],[22] In this study, the prevalence was 15.4% which is slightly higher than earlier quoted works. This is because a large proportion of our sample size was from a private histopathology laboratory patronized mainly by general practitioners who send tissues for histology only when there is a high index of suspicion.
Other studies done in neighboring African countries; rural Gambia, Yaoundé in Cameroon, and Kigali in Rwanda reported prevalence of 7%, 3.9%, and 5.9%, respectively, which are within the range of the prevalence in our environment.[23],[24],[25]
In other parts of the world, the prevalence ranges from 0.1% to 0.8%.[26],[27] This can be explained by the fact that these countries have better health infrastructures, higher literacy rate, better economic indices, availability and affordability of HPV vaccines, and affordable health services. Many Nigerian women are illiterates due to their preference for the training of the male child. Furthermore, many of them are dependent on their partners or spouses (full-time housewives) economically and otherwise. Many of them do not have health insurance and cannot afford the rarely available HPV vaccines; hence the higher prevalence rate in our environment.[28] Furthermore, polygamous marriage is very common in our environment. This habit increases the risk of contracting high-risk HPV from a partner with multiple sexual partners.
Demographic characteristics
The age range of the patients in this study was 34–80 years. This is comparable with the age range of the patients in the studies done in Lagos and Ibadan (age range of 19–87 years and 23–80 years), respectively.[22],[29] The mean age of the patients was 49.7 ± 10.7 years. This is very similar to the study done in Lagos (mean age of 49 ± 13.0 years).[22] However, this is at variance with studies done in Zaria, Abakaliki, and Ibadan that reported mean age of 37.6 years, 43.6 ± 6.3 years, and 46.3 years, respectively.[20],[21],[29] A similar study done in neighboring Yaoundé in Cameroon (West Africa) reported an age range of 27–55 years which is at variance with this study and earlier quoted studies done in Lagos and Ibadan.[24] Another study done in Kigali in Rwanda (East Africa) reported a much lower mean age of 37 years.[25]
The peak age of HSIL in this study was 51–60 years. This is a decade later than the peak age for preinvasive lesions reported in the studies done in Zaria, Lagos and Ibadan.[22],[29],[30] This is also later than the peak age for similar studies done in neighboring Gambia and Cameroon (West Africa) that reported 35–44 years and 40–44 years respectively.[23],[24]
The peak age for LSIL in this study was 41–50 years. This is similar to the studies on preinvasive lesions done in Zaria, Lagos, and Ibadan.[22],[29],[30] It is also similar to the study done in Cameroon-a neighboring West African country.[29] In view of this, screening should be commenced at least a decade earlier. Furthermore, vaccination should be commenced as early as possible. According to the Centre for Disease Control recommendations immunization should be commenced at 11 or 12 years in both boys and girls or earlier, and the recommended doses should be completed.[31]
Indications for biopsy (clinical diagnosis)
21.5% (ca in situ-3.9% and SIL-17.6) of the patients were biopsied on account of preinvasive cervical lesions. This is about 30.6% of the preinvasive lesions diagnosed using H and E. This is not surprising because most preinvasive cervical lesions are asymptomatic. They are mainly discovered during cervical screening.[32] Majority of the preinvasive cervical lesions diagnosed using H and E were incidental findings probably because there is no government policy on cervical screening in women.
Determination of significant difference between the diagnosis before and after application of p16INK4a
There is a significant statistical difference between the diagnosis before and after the application of p16INK4a in this study. This is in agreement with works done by Horn et al. and Gurrola-Díaz et al. in Germany and Mexico, respectively.[33],[34]
The sensitivity and specificity of p16INK4a for diagnosis of HSIL in this study were 75% and 85%, respectively. A lot of studies have validated the utility of this biomarker in the diagnosis of preinvasive cervical lesions. It has high sensitivity and specificity for diagnosing HSIL.
Our findings are very similar to a study done in Virginia (USA) by Galgano et al. that reported a specificity of 82.8%.[13] However, this sensitivity is lower than the 86.7% that Galgano et al. reported. Furthermore, our findings are at variance with what was reported by Nierkerk et al. in Houston (Texas). They reported sensitivity and specificity of 90% and 85%, respectively.[35]
Recommendations
In view of the high prevalence of preinvasive lesions in our environment, there should be a well-articulated government policy on cervical screening for women. There should be universal health coverage for the citizens. Vaccines should be readily made available free of charge for the population at risk or at least heavily subsidized. This will go a long way in reducing the overall burden of cervical cancer and its precursor lesion: SIL.
There should be routine use of p16INK4a immunohistochemistry for difficult or equivocal preinvasive cervical lesions. This will go a long way in rendering the appropriate diagnosis and also increase the quality of patients' care. This will also go a long way in reducing the burden of cervical cancer in our environment. Furthermore, it will assist in making the diagnosis at an early and preinvasive stage when chances of cure are very high.
Government should allocate more funds to cancer research especially cervical cancer and its precursor lesion: SIL since it is a leading cause of cancer deaths amongst women in our environment. This will shed more light on the biology of cervical cancer and its precursor lesion and also expand the body of knowledge presently in existence.
| Conclusion | | |
There is a high prevalence of preinvasive cervical lesions in our environment. Majority of the preinvasive cervical lesions diagnosed using H and E were incidental findings probably because there is no government policy on cervical screening in women. Immunohistochemistry using p16INK4a is a very useful adjunct to H and E for diagnosing preinvasive cervical lesions, especially in challenging cases.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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