|Year : 2013 | Volume
| Issue : 3 | Page : 200-205
Immunohistochemical expression of MIB-1 and PCNA in precancerous and cancerous lesions of uterine cervix
Madhu Mati Goel1, Anju Mehrotra2
1 Department of Pathology, Chhatrapati Shahuji Maharaj Medical University, Lucknow- 226 003, Uttar Pradesh, India
2 Department of Pharmacology and Therapeutics, Chhatrapati Shahuji Maharaj Medical University, Lucknow- 226 003, Uttar Pradesh, India
|Date of Web Publication||23-Sep-2013|
Department of Pharmacology and Therapeutics, Chhatrapati Shahuji Maharaj Medical University, Lucknow- 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background and Objective: The present study was done to analyze the immunoexpression of diagnostic markers (MIB-1: molecular immunology borstel and PCNA: proliferating cell nuclear antigen) in grading cervical intraepithelial lesion (CIN) and squamous cell carcinoma (SCC) in cervix. Setting and Design: Total 150 cervical biopsies were divided into four groups respectively; Group I-Normal (n = 32), Group II- CIN (n = 60), Group III- SCC (n = 44), Group IV- CA cervix (n = 14) respectively. Materials and Methods: These biopsies were stained with monoclonal antibodies by streptavidin--biotin method. Mean labeling index was calculated and grading was performed using the I--III scoring system. Statistical Analysis: Findings were correlated with age and menopausal status. Statistical analysis was done by using student sample't' test and analysis of variance (ANOVA) by SPSS 10 package. Results: MIB-1 immunostaining was positive in 112/150 (74.6%) cases and PCNA in 118 /150 (78.6%) cases. Labeling indices showed linear progression from normal to CIN to SCC to cancer lesion. Few cases of low-grade CIN lesion had high proliferative index. A significant positive correlation was found between age and PCNA and MIB-1 values (P < 0.05) when comparison was made for all the cases. Conclusion: These markers may be useful in identifying low-grade CIN lesion with high proliferative index. These cases should be kept for follow up studies so that proper intervention can be taken at an early stage. This method is simple and cost effective and can easily be done in formaline-fixed paraffin embedded tissues in a clinical laboratory for grading CIN and SCC lesions in cervix.
Keywords: Cervical cancer, cervical intraepithelial lesion, MIB-1, PCNA
|How to cite this article:|
Goel MM, Mehrotra A. Immunohistochemical expression of MIB-1 and PCNA in precancerous and cancerous lesions of uterine cervix. Indian J Cancer 2013;50:200-5
| » Introduction|| |
In developing countries, cervical cancer is a major cause of death in women. In India, cervical cancer is common in the females between 15 and 44 years of age group. It is the first most common malignancy among females. India accounts for one fifth of the world's burden of cervical cancer.  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. 
Different methods were identified for an early detection of cervical cancer. Pap smear More Details test is a time-consuming method and sensitive with limited reproducibility and many times showed high rate of false-positive and false-negative results.  Moreover, colposcopic biopsy gives the patient to unnecessary surgical intervention.
Histopathological evaluation is known as "Gold standard" for the diagnosis of squamous intraepithelial lesions (SIL) and cervical intraepithelial neoplasia (CIN) lesions.  Reactive/reparative epithelial changes, immature squamous metaplasia, and atrophy are well-recognized mimics of HSIL and frequently cause problems in histological interpretation. Moreover, the morphological criteria assessed do not provide information about the further development of these lesions, which may be the meaning of regression or progression to invasive disease.  Therefore, additional diagnostic and prognostic markers for the detection of precursors lesions and cervical cancers are required, which could save the patients from surgical intervention and the high screening cost. There are reports that have highlighted the usefulness of PCNA and MIB-1 in SIL diagnosis and prognosis. ,
MIB-1, an antigen expressed in the nuclei of proliferating cells and detected with the MIB-1 antibody. MIB-1 is a monoclonal antibody raised against a recombinant part of the MIB-1 antigen.  It is a sensitive biological indicator of progression in cervical intraepithelial neoplasia (CIN) lesions. 
Proliferating cell nuclear antigen (PCNA) has been described as a non-nuclear histone protein that appears in the nucleus during the late G1 phase, increases during S phase and declines during the G2 and M phase.  In India, only sporadic reports are published in relation to the role of MIB-1 and PCNA expression in non-neoplastic and neoplastic lesions of cervical cancer. Therefore, it is interesting to study the immunohistochemical expression of MIB-1 and PCNA in different grades of CIN lesions and invasive squamous cell carcinoma and its correlation with age and menopausal status of the patients.
| » Materials and Methods|| |
Total 150 tissue biopsies were collected consecutively from Histopathology laboratory of the Postgraduate Department of Pathology, during the period of 2007--2010. The hospital medical ethics committee approved study and informed formal consent was taken from each patient. Histopathological examination of biopsies for grading and typing of lesions was done. Samples were divided into four groups respectively; Group I: Normal (n = 32), Group II: CIN (n = 60), Group III: SCC (n = 44), Group IV: Ca cervix (n = 14), respectively. Tissue sections were selected where sufficient material was present in the block and clinical data was available. From each block 3--4 μm thick multiple sections were cut. One section was stained with hematoxylin-eosin (HE) staining for observing histological typing and rest of the sections were kept for immmunostaining (MIB-1 and PCNA). Primary antibodies MIB-1 (Code No. N1633) and PCNA (Code No. N1529) were purchased from Dakota Cytomatin Ltd and B sap universal kit (Code No. 37101) from Span Diagnostics Ltd. Immunostaining was done by streptavidin--biotin method.
Positive control for MIB-1 staining: A histological section of gall bladder adenocarcinoma was used as positive control with each batch of staining.
Positive control for PCNA staining: A histological section of reactive lymph node was used as positive control with each batch of staining.
Negative control: For negative control 1% nonimmune serum was used in place of primary antibodies, with rest of the steps same as for the positive control.
Interpretation of MIB-1 and PCNA labeling index
MIB-1 and PCNA labeling index was calculated from area showing positive cells per 100 cervical epithelial cells under X400 magnification in triplicate and mean was calculated. Positive nuclei were expressed as a percentage of total nuclei counted. 
| » Results|| |
In normal group immunohistochemical, staining showed negative staining for PCNA and MIB-1. Brown color staining was confined to the nuclei in all positive cases [Figure 1] and [Figure 2]. All cases were divided into different age groups and menopausal status. A detail of Clinicopathological relationship was shown in [Table 1]. Maximum cases of CIN lesion were present in the age group of 31--50. Premenopausal group showed 72% cases while 28% women were of postmenopausal group.
|Figure 1: Squamous cell carcinoma is showing positive staining for PCNA (×100)|
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|Figure 2: Squamous cell carcinoma is showing positive staining for MIB-1 (×400)|
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MIB-1 immunoexpression was confined to the basal layer in the normal cervical epithelium. Out of these 150 cases, 112 (74.6%) cases were positive with MIB-1 and 118 (78.6%) cases for PCNA. As we moved from the normal to the carcinoma group, the LI increased with increasing severity of intraepithelial neoplasia to the carcinoma group. Mean labeling index was maximum in carcinoma group with MIB-1 (37.69) and PCNA (40.21).Significant difference was seen (P = 0.000) for all the groups [Table 2]. The mean values for both MIB-1 and PCNA present the following pattern
Normal < CIN < SCC < CA
When intergroup comparison was performed the p value was found to be insignificant, (P = 0.18, 0.05) for both the markers in CA group [Table 3]. In order to compare the difference analysis of variance was performed in all the groups [Table 4]. Variance was maximum in SCC group with both the markers. [Table 5] depicts the correlation between age and PCNA and MIB-1. When comparison was made for all the cases, a significant positive correlation was seen between age and PCNA and MIB-1 values (P < 0.05). However, when correlations were made in individual groups, no significant correlation was found in any group. Comparison of mean PCNA and MIB-1 labeling index was done with menopausal status among different groups. However, on making comparison in the individual groups, there was no significant difference in mean PCNA and MIB-1 values between pre and postmenopausal status. Therefore, menopausal status does not play a significant role in determining the MIB-1 and PCNA values.
|Table 3: Comparison of PCNA and MIB-1 among different histological groups|
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|Table 4: Analysis of variance for cervical biopsies for different histological groups|
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| » Discussion|| |
Immunohistochemical assessment of the proliferative activity is very helpful to establish the diagnosis of preneoplastic lesions of uterine cervix and represents an important parameter in the prognostic evaluation of the patients. 
In normal cervical epithelium MIB-1 antigen, labeled cells were exclusively found in the parabasal and basal layers.  PCNA immunostained cells were mainly present in the basal layer in control cases but enhanced in layers containing dysplastic cells.  We observed intense immunostaining in full thickness of epithelium of CIN III lesion and carcinoma group.
In our findings, labeling indices of MIB-1 and PCNA increased from dysplasia to carcinoma group. Our results supported the findings of Maeda MY observed that the labeling indices of PCNA and MIB-1 immunostaining increased with increasing grades of cervical lesions although PCNALI was greater than MIB-1 LI.  The reason for higher reactivity may be explained that half-life of PCNA exceeded 20 hours which could result in some staining of nuclei in the G0 phase in the basal cell. 
MIB-1 may be helpful in differentiating between CIN III and SCC cases in routinely stained histological sections.  It is a valuable marker for cervical carcinoma diagnosis, grade, and clinical course.  Bulten et al. found the percentage of MIB-1 labeled cells in CIN III was 2.5 times higher when compared with CIN I and comparable and even higher stain in cases of CIN II/III to invasive lesions. MIB-1 immunostaining was positive in the dysplastic epithelial layers and increased with lesion grade.  MIB-1 immunoexpression was present in all SIL cases proportional to the severity of lesions and cell proliferation index was greater the degree of lesions was increased. In LSIL, proliferation index was below 20% and in cases where atypical mitoses were present. 
Looi ML observed that MIB-1 index was higher in high grade CIN and SCC lesions as compared to normal cervix.  Wang et al. observed negative PCNA expression in normal and inflammatory cases of cervix but increased expression in CIN (63.2%) and SCC (100%) groups respectively (P < 0.01, P < 0.05). They also reported that PCNA might be valuable clinical marker to predict the progression of cervical neoplasia. 
Pahuja et al. also reported statistically significant difference between preinvasive and invasive squamous epithelial lesions of cervix.  PCNA index may be a predictive indicator for the prognosis of patients with squamous cell carcinoma of the cervix treated with radiation therapy alone. These markers may be helpful in the identification of those patients whose CIN lesion will progress and require treatment to be distinguished from those, whose lesion will stay static or regress.  Our results also showed that these markers might be helpful in low-grade CIN lesion showing high proliferative index. These cases should be kept for follow-up studies so proper intervention can be given to these patients.
A significant difference was found between MIB-1 labeling indices in young and older patients of cervical carcinomas, suggesting a biologic aggressiveness of age related cervical carcinomas.  In our study, MIB-1 and PCNA was found to be independent markers of proliferation irrespective of age and menopausal status. In another study, MIB-1 immunostaining had no relationship between growth fraction of tumor cell and age.  In contradictory, Cole reported that MIB-1 expression could be used in determining the aggressiveness of CIN lesions. 
This marker in the tissue section can be used as an adjunct to diagnose preneoplastic and neoplastic lesions in the cervix.  The use of specific biomarkers of dysplasia in conjunction with histological procedures could greatly improve the accuracy, precision and sensitivity of cervical screening program. In nutshell, it was concluded that MIB-1 and PCNA markers might be of greater importance in those cases, which are seen low grade in histology sections but have a high proliferative index. This will put the low-grade CIN lesions in higher grade thus indicating the utility of proliferative markers in decision making for intervention. Since MIB-1 does not stain the nuclei in G0 phase of growth, so this would be a preferable marker. This method is simple and cost effective in comparison to HPVDNA test and can easily be done in formaline fixed paraffin embedded tissues in a clinical laboratory for grading CIN and SCC lesions in cervix.
| » Acknowledgement|| |
Indian Council of Medical Research, New Delhi, financially supported this work.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]