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The immunohistochemical biomarker B-cell lymphoma-2 expression in malignant and premalignant lesions of the uterine cervix and its association with human papillomavirus infection


1 Department of Obstetrics and Gynecology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
2 Department of Patholog, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
3 Department of Microbiology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India
4 Department of Obstetrics and Gynecology, All India Institute of Medical Sciences, Bhubaneswar, Odisha, India
5 Department of Pathology, All India Institute of Medical Sciences, Raipur, Chhattisgarh, India

Date of Submission27-Mar-2020
Date of Decision31-May-2020
Date of Acceptance04-Sep-2020
Date of Web Publication20-Aug-2021

Correspondence Address:
Prasanta Kumar Nayak,
Qtr. No 302, Block C, Jainam Planet, Tatibandh, Raipur, Chhattisgarh
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_388_20

 > Abstract 


Introduction: Cervical cancer is one of the leading causes of cancer deaths among women. It results due to human papillomavirus (HPV) infection. Cervical intraepithelial neoplasia (CIN) is the preinvasive condition of cervical cancer. Various objective immunohistochemical (IHC) markers have been studied for cervical cancer. This study is aimed at studying the expression of B-cell lymphoma-2 (Bcl-2) IHC marker among preinvasive and invasive lesions of cervical cancer and its association with HPV infection.
Methodology: This prospective study was conducted over a period of 1 year in a tertiary care hospital in central India, included 73 women suffering from CIN and cancer cervix. The expression of Bcl-2 and the presence of HPV genotypes were studied.
Results: Out of 73 patients, 34 had cancer cervix, out of which 15 (44%) had Bcl-2 positivity, 24 had CIN 1, out of which 13 (54%) had Bcl-2 positivity, 10 had CIN 2, out of which 4 (40%) had Bcl-2 positivity and 5 had CIN 3, out of which 3 (60%) had Bcl-2 positivity. No significant difference was found in Bcl-2 positivity among CIN-1, CIN-2, CIN-3, and cancer cervix cases with a Chi-square value of 1.116 and P = 0.77. HPV positivity was found in 41 (56%) out of 73 patients where HPV 16 subtype was the most common (31.5%), followed by HPV 18 (13.7%). No significant association between HPV positivity and Bcl-2 positivity was found with P = 0.34.
Conclusion: Bcl-2 IHC seems to have variable expression among CIN cases. Although its expression is low among invasive cancer cases when compared with preinvasive lesions, the difference is not significant. Similarly, no significant association was found between Bcl-2 expression and HPV infections.

Keywords: B-cell lymphoma-2, cancer cervix, human papillomavirus



How to cite this URL:
Nayak PK, Hussain N, Negi S, Agrawal S, Bagde N, Mitra S, Singh V. The immunohistochemical biomarker B-cell lymphoma-2 expression in malignant and premalignant lesions of the uterine cervix and its association with human papillomavirus infection. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=324164




 > Introduction Top


Cervical cancer is the fourth most common cancer of females in the world, which is a serious threat to the health of women.[1] On the other hand, one-fifth of the world's burden of cervical cancer is from India.[2] The current WHO findings indicate that every year, 132,082 women are diagnosed with cervical cancer and 74,118 die from this disease in India alone.[3] Cervical intraepithelial neoplasia (CIN) is a premalignant (dysplastic) condition which is characterized by abnormal cellular proliferation, maturation, and nuclear atypia. If it is not treated, CIN may regress to normal or progress to invasive cancer. Approximately one-third to one-half of cases of CIN 1 and CIN II regress without treatment. Even cases of CIN III have been observed to regress spontaneously. Regression to normal condition is inversely proportional to the severity of the premalignant lesion. However, it is not possible at present to predict which cases of CIN will progress and which will persist or regress. The histopathological finding is considered as the “Gold standard” for the diagnosis of CIN lesions.[4] The intraepithelial distribution, density, and nature (typical or atypical) of mitotic figures are the diagnostic criteria used to grade dysplasia, and at the same time, it is used to distinguish high-grade dysplasia from potential histological mimics such as transitional metaplasia, atrophy, or immature squamous metaplasia. However, a considerable interobserver variability in the grading of CIN is seen. Therefore, an objective biomarker will be useful in the identification of truly dysplastic cells and in predicting disease progression. On the other hand, it is known that apoptosis plays an important role in preventing the potential abnormal proliferation of cells.[5],[6] The different protein families those involved in the process of apoptosis have been identified, such as B-cell lymphoma-2 (Bcl-2), Bcl xL (Bcl-xL), and cellular inhibitor of apoptosis protein 1.[7] Out of these, Bcl-2 is an important regulator of apoptosis. The regulation of apoptosis by Bcl-2 is implemented through the mitochondrial pathway.[8] The main biological function of Bcl-2 protein is its prolonging the cell life and increasing the resistance to many kinds of apoptosis stimulating factors, and hence, it prevents apoptotic cell death.[9] It is also found that overexpression of Bcl-2 can block p53-mediated G1 arrest.[10] The possible mechanism is that Bcl-2 protein inhibits the increase of intracellular calcium concentration and oxidation damage by blocking cell nuclear transfer. The occurrence of cervical cancer is not only related to the mutation of oncogene and the inactivation of tumor suppressor genes but also closely related to the imbalance of cell proliferation and apoptosis. Inhibition of apoptosis or the proliferation out of control is involved in the formation of tumor.[11] Bcl-2 overexpression is present in premalignant and malignant lesions of the cervix. In histological sections of CIN and carcinoma in tissue biopsy specimens, the biomarkers like Bcl-2 show a clear-cut crisp staining in different compartments of the epithelium. It is also found that the alteration of Bcl-2 expression is a relatively early event in cervical tumorigenesis. Hence, the expression of Bcl-2 in premalignant cells of the cervix can be used as a prognostic marker. Studies have also shown that Bcl-2 positivity confers a better 5-year survival rate.[12] Human papillomavirus (HPV) is the causative agent of cancer cervix. More than 200 types of HPV have so far been identified on deoxyribonucleic acid (DNA) sequence analysis. Approximately, 80%–90% of cervical carcinomas contain DNA sequences of specific HPV subtypes. HPV DNA testing is the preferred cervical screening method for women, as HPV is the proven causative agent of cervical carcinoma. The HPV life cycle and molecular events leading to cellular transformation have not been completely elucidated, but it has provided insight into potential biomarkers that can be used as adjunctive tests to improve the diagnostic accuracy of cervical lesions as well as identify those patients at risk for progression to cancer. Numerous biomarkers have been studied in CIN and carcinoma of the cervix. Not many studies have established a correlation between HPV infection and the overexpression of the commonly used biomarker such as Bcl-2. This study was undertaken to study the significance of the expression of this biomarker and their correlation with HPV infection in premalignant and malignant lesions of the cervix.


 > Methodology Top


This prospective study was undertaken in a tertiary care center in central India over a period of 1 year from July 2018 to June 2019 after taking approval from the institute ethical committee. A total of 73 women attending the gynecology outpatient department of our hospital who were already diagnosed to have either CIN or cancer cervix from cervical biopsy during their previous visits in our hospital were included in this study after informed consent. A detailed history was elicited pertaining to complaints such as persistent vaginal discharge, metrorrhagia, postcoital bleeding, intermenstrual bleeding, and postmenopausal bleeding. A colposcopy-guided biopsy was taken from the cervix which was used for histopathological study and immunohistochemical (IHC) study for the detection of Bcl-2 expression. The IHC antigen is retrieved in citrate buffer in a microwave oven. Hydrogen peroxide (3%) was used for blocking of endogenous peroxidase and then incubation with primary mouse monoclonal antibody against Bcl-2 protein linking with rabbit antimouse secondary antibody was performed. Followed by it, enzyme labeling with streptavidin-biotin and counterstaining with hematoxylin was done. Positive and negative controls were used with each batch of slides. B and T cells of the mantle zone of tonsillar lymphoid tissue were used as positive control and the germinal centers of tonsillar lymphoid tissue were used as negative control. The categorization of the cervical lesions as CIN 1, 2, 3, and invasive cancer was done by studying the H and E stained slides under the microscope. Cytoplasmic staining of Bcl-2 was examined by studying the immunostained slides. The proportion of positive staining cells was expressed as a percentage and the intensity of staining was expressed as 0, 1, 2, or 3. A case was taken as positive if more than 10% cells showed cytoplasmic reactivity, then it was considered as Bcl-2 positive. Staining intensity of epithelium and stroma was described as 0, 1, 2, and 3 for no staining, weak staining, moderate staining, and intense staining, respectively. [Figure 1] shows the microscopic picture of a Bcl-2 positive slide and [Figure 2] shows the microscopic picture of a Bcl-2 negative slide.
Figure 1: Microscopic picture showing B-cell lymphoma-2 positive at ×1840

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Figure 2: Microscopic picture showing B-cell lymphoma-2 negative at ×1940

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Sampling for the HPV study was done by taking a cytobrush smear from the cervical canal. A total of 14 high risk (HR)-HPV genotypes, namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 were detected by tagging oligonucleotide cleavage and extension mediated Anyplex HR-HPV genotyping assay on CFX 96 real-time polymerase chain reaction (PCR) machine (Bio-Rad, USA). The L gene was targeted to individually identify 14 h-HPV in the given cervical scrape samples. The human beta-globin gene was taken as the internal control. Principally, it is a multiplex PCR reaction which works on template-based melting temperature instead of the current probe-based melting temperature and distinguishes multiple targets in a single channel in a real-time PCR reaction (Lee, 2012). The approximate time from the processing of clinical samples and genotype analysis was around 6 h. The detection limit obtained in the assay was 50 HPV copies/reaction.


 > Results Top


In this study, a total of 73 patients were evaluated whose demographic distributions are mentioned in [Table 1]. The patients between 25 and 73 years of age groups with a mean age of 45.9 years were included in this study. Majority of our patients (60%) belonged to the reproductive age group. Majority of our patients (52%) had a marriage before the age of 19 years, and also majority of our patients (62%) were from rural areas. Out of 73 patients, 72 were parous. As it is shown in [Table 2], out of 73 patients, 34 had cancer cervix, out of which 15 (44%) had Bcl-2 positivity, 24 had CIN 1, out of which 13 (54%) had Bcl-2 positivity, 10 had CIN 2, out of which 4 (40%) had Bcl-2 positivity and 5 had CIN 3, out of which 3 (60%) had Bcl-2 positivity. Hence, Bcl-2 positivity was found more among preinvasive cases taken together (CIN = 51%) in comparison to invasive (cancer = 44%) cases. As it is shown in [Table 3], no significant difference was found in Bcl-2 positivity among CIN-1, CIN-2, CIN-3, and cancer cervix cases with a Chi-square value of 1.116 and P = 0.77. As it is shown in [Table 4], HPV positivity was found in 41 (56%) out of 73 patients where HPV 16 subtype was the most common (31.5%), followed by HPV 18 (13.7%). No significant association between HPV positivity and BCL-2 positivity was found with P = 0.34 [Table 5]. Similarly, no significant association was found between Bcl-2 positivity and individual HPV genotypes with P = 0.43 [Table 6].
Table 1: Demographic distribution

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Table 2: Bcl-2 positivity in cervical intraepithelial neoplasia and cervical cancer

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Table 3: Association between Bcl-2 positivity and cervical intraepithelial neoplasia, cervical cancer

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Table 4: Distribution of human papillomavirus types

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Table 5: Association between Bcl-2 positivity and human papillomavirus positivity

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Table 6: Association between Bcl-2 positivity and human papillomavirus genotype

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 > Discussion Top


As it is described, 60% of our patients belong to the reproductive age group. Majority of our patients (52%) had a marriage before the age of 19 years and majority of them (62%) were from the rural area. It is a fact that in rural India, the girls get married at a very young age, and hence, the development of cervical cancer is seen in premenopausal age groups as it is shown in our study results.

In the present study, Bcl-2 positivity was found to be variable among different types of CIN without any statistically significant difference (CIN 1 – 54%; CIN 2 – 40%; and CIN 3 – 60%). Many of the previously reported studies including a recently published study by Kamaraddi et al. have reported that the Bcl-2 positivity increases as the grade of CIN increases.[10],[13],[14],[15],[16],[17] However, few studies such as Saegusa et al. and Grace et al. have reported a statistically significant difference in the trend.[16],[18] At the same time, some studies have also reported a decreasing trend for the same.[19],[20] The study by Shukla et al. shows that the Bcl-2 expression reduced with increasing grade of CIN, but the difference was not significant (P = 0.373).[20]

In our study, the expression of Bcl-2 is lower in invasive cervical cancer (44%), compared with preinvasive lesions (51%). A study reported from China in 2018, comparing the Bcl-2 expression in cervical cancer (44.4%) and normal cervical tissue (57.1%), also did not show any significant difference.[7] For the relationship between Bcl-2 and the prognosis of cervical cancer, Ozalp et al. and Jain et al. have found that Bcl-2, as a single variable, has no effect on the survival rate of patients with cervical cancer.[21],[22] However, it is believed that the patients with high-positive Bcl-2 protein expression have low degree of malignancy, high survival rate, and good prognosis.[23] The present study has not investigated the correlation of Bcl-2 protein expression and prognosis of cervical cancer, which should be further studied.

Our laboratory has used multiplex PCR reaction technique which can detect 14 HR HPV genotypes. In our study, HPV positivity was found in 41 (56%) out of 73 patients where HPV 16 subtype was the most common (31.5%), followed by HPV 18 (13.7%), HPV 39 was 6.9% and HPV 33 was 4%. HPV 16 and 18 combined together were detected among 45.2% of cases. Similar HPV positivity ranging between 35% and 55% was obtained using in situ hybridization by Al-Saleh et al., Choudhury and Singh, and Jeffers et al.[24],[25],[26] On the other hand, a study by Dellas et al. and Menon et al. found a much higher incidence of HPV infection (75%–100%).[27],[28] Other authors using PCR, restriction fragment length polymorphism, or hybrid capture II found higher positivity rates varying between 70% and 95% for HPV 16 and HPV 18 infection in cases of CIN and carcinoma cervix.[16],[18],[29] The higher rates have been reported because of the use of more sensitive methods of HPV detection. Low HPV positivity in our study can be attributed to sampling error, since we have used the cytobrush only to collect the cervical sample whereas various studies have used cervical tissue for the same purpose. We did not find any significant statistical correlation between HPV infection and Bcl-2 expression, which is similar to findings by Shukla et al. and contrary to the findings of Saegusa et al. and Grace et al.[16],[18],[20]

The limitation of the present study is that we have not investigated the correlation of Bcl-2 protein expression and prognosis of cervical cancer.


 > Conclusion Top


Bcl-2 expression is found among preinvasive and invasive conditions of cancer cervix, but its expression is more in preinvasive diseases as compared to invasive diseases. Some studies have shown an increasing trend of Bcl-2 expression as the disease advances from low-grade preinvasive lesions to high-grade ones; some studies have shown a decreasing trend of Bcl-2 expression for the same, but the present study has shown a variable expression. Hence, further studies taking large sample size required to establish the facts. Similarly, although HPV infection is present among majority of the cases of preinvasive and invasive lesions, no significant association was found between Bcl-2 positivity and HPV positivity.

Acknowledgment

The authors acknowledge Dr. Veena Yaduvansi, Dr. Pavan B.C, and Dr. Zameer Lone.

Financial support and sponsorship

The intramural project was sponsored by the All India Institute of medical sciences, Raipur, India.

Conflicts of interest

There are no conflicts of interest.



 
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[PUBMED]  [Full text]  


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