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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 2  |  Page : 377-381

Immunohistochemical evaluation of prime molecules in cervical lesions towards assessment of malignant potentiality


1 Department of Medical Science and Technology, Indian Institute of Technology, Kharagpur; Department of Materials Engineering, Indian Institute of Science, Bangalore, Karnataka, India
2 Department of Pathology, Calcutta National Medical College, Kolkata, India
3 Department of Radiology, Apollo Gleanengles Hospital, Kolkata, India
4 Department of Pathology, Midnapore Medical College and Hospital, Midnapore, West Bengal, India
5 Department of Medical Science and Technology, Indian Institute of Technology, Kharagpur, India

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Lopamudra Das
Department of Materials Engineering, Biomaterials and Tissue Engineering Lab, Indian Institute of Science, Bangalore - 560 012, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.158029

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 > Abstract 


Objective: A comparative immunohistochemical evaluation of p63, CD105, and E-cadherin expression pattern in histopathologically confirmed normal cervical epithelium (NCM), dysplastic cervical epithelium (DYS) and squamous cell carcinoma (SCC) of uterine cervix towards assessing malignant potentiality of the precancerous condition.
Materials and Methods: The biopsies from cervical mucosa (normal, dysplasia, and cancer) were studied by routine hematoxylin and eosin (H and E) and by immunohistochemistry for p63, E-cadherin, and CD105 expression. The expressions of these molecules were assessed in a semiquantitative way by (i) counting p63 cell population and distribution, (ii) intensity scoring of E-cadherin along the expression path, and (iii) measuring CD105 expression density.
Result: p63+ cells were highest in carcinomas followed by dysplasia and normal. An abrupt increase in CD105 expression was observed through change of normal to dysplasia and cancer. A decrease in membranous E-cadherin expression was noticed in the transformation from normal to precancer and cancers.
Conclusion: The malignant potential of the dysplastic conditions is likely to be correlated with upregulation in p63 and CD105 expression and a simultaneous downregulation of membranous E-cadherin.

Keywords: CD105, cervical lesion, E-cadherin, immunohistochemistry, malignant potentiality, p63


How to cite this article:
Das L, Naskar S, Sarkar T, Maiti AK, Das S, Chatterjee J. Immunohistochemical evaluation of prime molecules in cervical lesions towards assessment of malignant potentiality. J Can Res Ther 2018;14:377-81

How to cite this URL:
Das L, Naskar S, Sarkar T, Maiti AK, Das S, Chatterjee J. Immunohistochemical evaluation of prime molecules in cervical lesions towards assessment of malignant potentiality. J Can Res Ther [serial online] 2018 [cited 2019 Nov 14];14:377-81. Available from: http://www.cancerjournal.net/text.asp?2018/14/2/377/158029




 > Introduction Top


At present high incidence rate of cervical cancer worldwide is a major health concern. Mortality due to this fatal disease occurs at an alarming rate in developing countries, and India is no exception.[1] The cause of high mortality rate may be linked to late diagnosis of the dysplasia and its malignant potentiality. Fallacy in the existing ambiguous diagnostic technique is a prime reason for late diagnosis. Hence, to address the existing diagnostic lacunae, especially in the event of carcinogenesis at an early stage, an integrated approach may be developed by drawing logical correlation between findings of histopathology with that of the molecular pathology attributes.

Cervical cancer is headed by an asymptomatic condition of precursor lesions called cervical intraepithelial neoplasia (CIN) or dysplasia/precancer.[2] This stage is characterized by proliferative, metabolic, and atypical changes in epithelial cells; their structural association; and connective tissue organization.[3] Hyperplasia is usually observed in both dysplasia and cancerous progression.[4] Indeed, numerous epithelial and subepithelial changes occur during initiation of cervical carcinogenesis and its malignant transformation.[5]

In the context of understanding malignant potentiality of cervical dysplasia and its progression to cancer, the study of prime molecular expressions associated with cervical epithelial stratification and progressive maturation, epithelial cell–cell adhesion, migratory phenotype development, etc., are considered crucial.[6]

p63, the master regulator of the epithelia exhibits vital role in development, proliferation, differentiation, maintenance, and maturation of stratified squamous epithelium viz. oral mucosa, skin, and cervical mucosa.[7] As a transcriptional regulator, p63 plays crucial role in epithelial stem cell regeneration and skin renewal.[8],[9] Strong expression of p63 occurs in basal keratinocytes.[10] Recent researches have shown that p63 support the viability and maintenance of basal epithelial and cancer cells, and promotes squamous differentiation.[11],[12]

The epithelial cells are tightly bound to each other at the cellular junctions. In this process, adherens junction plays a crucial role in regulating epithelial cell–cell adhesion. E-cadherin is one of the important components of the adherens junction. E-cadherin is a calcium-dependent cell-surface glycoprotein located on the chromosome 16q-22 and encoded by CDH1 gene. E-cadherin acts as an adhesion molecule between epithelial cells. It is also engaged in the signal transduction controlling various cellular events like polarity, differentiation, growth, and cell migration.[13] E-cadherin has been reported as a marker for assessment of epithelial cell–cell adhesiveness to differentiate normal from neoplastic tissues.[14],[15] Loss of membranous E-cadherin expression has been correlated with dysplasia progression, and hence considered as a hallmark of EMT.[16]

Endoglin, known as CD105, is coded by the gene located on chromosome 9q34. It is a transmembrane glycoprotein and restricted to angiogenic endothelial cells in adult. It acts as a receptor for transforming growth factor (TGF-β) superfamily. Under hypoxia and in the absence of TGF-β1, CD-105 acts as an apoptosis inhibitor of vascular endothelial cells.[17] Thus, hypoxia is reported to induce CD-105 expression which antagonizes the growth inhibitory effect of TGF-β1.[18] The expression level of CD-105 may be clinicopathologically correlated with decreased survival, neoangiogenesis, and metastasis.[18] Thus, this molecule is reported to be useful as theranostic target of malignancy also.

The current study evaluated the immunohistochemical attributes for the expression status of prime genes of cervical mucosa like p63, E-cadherin, and CD105 in normal, dysplasia, and cancerous cervical biopsies towards developing an approach to assess their malignant potentiality.


 > Materials and Methods Top


Study samples

A total of 44 samples were collected, of which 15 were from histologically confirmed dysplasia, that is, CIN stage 2 (dysplastic cervical epithelium (DYS); n = 15), 12 of normal cervical mucosa (NCM; n = 12), and 17 were from squamous cell carcinoma (SCC; n = 17) of uterine cervix. Normal cervical mucosal samples were collected from patients undergoing total hysterectomy due to abnormalities in uterus or ovary. All the above samples were collected under the informed consent of patients and ethical clearance. The samples were collected from women with mean age of the volunteers being 54.6 years. The study was conducted in full accordance with the ethical principles and guidelines of Medical Council of India including the World Medical Association's Declaration of Helsinki.

Tissue processing

The biopsy specimens were fixed in 10% phosphate-buffered formalin (pH 7.2–7.4) and then processed for obtaining 4 μm thick paraffin sections, which were then subsequently placed on albumin and poly-L-lysine (Cat. No. P8920, Sigma Aldrich, St Louis, Missouri, USA) coated glass slides for histological (hematoxylin and eosin (H and E)) and immunohistochemical (p63, E-cadherin, and CD105) studies, respectively.

H and E staining

The tissue sections were deparaffinized and rehydrated for staining with H and E. The sections were dehydrated following staining through upgrades of alcohol and mounted.[19]

Immunohistochemistry

Tissue sections were baked and subsequently deparaffinized and hydrated for antigen retrieval in a 10 mM citrate buffer (pH 6.0) using EZ-Retriever System V.2 (BioGenex, San Ramon, California, USA) and immunostained using the kit (i.e., Super Sensitive Polymer-HRP IHC Detection System, Cat. No. QD400-60K, BioGenex). Sections were incubated with primary antibodies (antihuman p63 clone 4A4, Cat. No. AM418-5M; E-cadherin, clone EP700Y, Cat. No. ab40772, Abcam, Cambridge, UK; and anti-CD105 clone 4G11, Cat. No. AM441-5M, BioGenex). p63 and CD105 antibodies were 'ready to use', whereas a dilution of 1:500 was used for E-cadherin. Primary binding of antibody was visualized using a horseradish peroxidase conjugated secondary antibody using the chromogen 3,39-diaminobenzidine and counterstained with hematoxylin. Appropriate controls were put up to validate the experiments.

Microscopic image grabbing

The images were grabbed digitally at 1388 × 1040 pixels by a Zeiss Observer. Z1 microscope (Carl Zeiss, Germany) under ×10 (numerical aperture (NA) 0.25, with final magnification ×100) and ×20 (NA 0.8, with final magnification ×200X) objectives with their respective resolutions being 0.63 and 0.31 mm.

Semiquantitative assessment of immunohistochemical features

Towards making the immunohistochemical features more quantitative in respect to their expressional intensities and spatial distribution in the target tissue, targeted molecular expressions were semiquantified in respect to their grey scale intensity values by using AxioVision Software 4.7.2 version, Carl Zeiss, Germany as well as by application of visual intensity scoring scale (0–10).

2.6.1 p63 expression

Under ×20 objective, the p63+ and p63 epithelial cells were counted per microscopic field. The field selection was random for each sample. In each study, class 30 fields were assessed. The intensity of p63 expressions were also evaluated for the epithelia of NCM, DYS, and SCC based on their grey values and tested statistically for significant changes.

2.6.2 E-cadherin expression

Using the software AxioVision (version 4.7.2, Carl Zeiss, Germany) and specific expression evaluation framework as shown in [Figure 1] (Adapted from[20]), E-cadherin expression was assessed at 50 randomly chosen reference points in the photomicrographs with final magnification of ×100 and grabbed under ×10 objective of each study group with respect to (i) the distance between basement membrane and E-cadherin expression initiation point (P1); (ii) the ratio (ET/EI) between epithelial thickness (ET) and the thickness displaying E-cadherin (EI); and (iii) E-cadherin intensity variation along the epithelial expression path, as per intensity scoring scale (i.e., 0–10), across three equidistant study points: P1, P2, and P3 [Figure 1]. E-cadherin expression intensity scores at different study points, that is, P1, P2, and P3 were analyzed, using analysis of variance (ANOVA).
Figure 1: Schematic diagram to show the methodology used to generate data on intensity of E-cadherin expression in normal cervical mucosa (NCM), precancer/dysplasia (DYS) and cancer (SCC). P1 signifies the expression initiation point and P3 the termination of expression. ET = Total epithelium thickness, EI = Epithelial thickness expressing E-cadherin in different tissues, P1, P2, and P3 = Equidistant points considered for intensity scoring along the E-cadherin expression path, SCC = Squamous cell carcinoma

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2.6.3 CD105 expression

CD105 positive microvessels were counted per microscopic field under ×20 objective, on chromogenic images. The field selection was random for each sample. In each study class 30 fields were assessed and data were tested statistically.

Statistical analysis

An independent sample t-test was applied for comparing the p63 data. E-cadherin expression intensity scores at different study points (P1, P2, and P3) were analyzed using ANOVA. The CD105 data were statistically analyzed using independent sample t-test.


 > Results Top


p63 expression in cervical epithelium

The chromogenic immunohistochemical (IHC) studies depicted nuclear reactivity of p63 proteins in the epithelial cells of uterine cervix in normal, dysplasia, and cancer as shown in [Figure 2]a,[Figure 2]b,[Figure 2]c. In normal cervical epithelium (NCM), p63 positive nuclei were only present in the basal region [Figure 2]a, but in dysplasia such nuclei was also observed in the upper layers also [Figure 2]b; and in case of cancer [Figure 2]c the positive nuclei extended throughout the whole epithelium [Figure 3]. Furthermore, on transformation of the normal cervical mucosa to premalignant or malignant one, the percentage of p63 positive nuclei increased significantly as represented by [Table 1].
Figure 2: (a-c) Microphotographs (magnification, ×200) showing immunohistochemical (chromogenic) expression of p63 in uterine cervix; (a) normal cervical mucosa, (b) dysplasia, and (c) cancer. P63 positive nuclei are marked by ''

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Figure 3: Grayscale intensity distribution of p63 positive nucleus in layers of cervical epithelium in normal, dysplasia and cancer

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Table 1: p63 expression in normal, dysplasia, and cancerous cervical epithelium

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E-cadherin expression in cervical epithelium

The chromogenic microphotographs in [Figure 4]a,[Figure 4]b,[Figure 4]c represented the expression pattern of E-cadherin in the epithelium of the uterine cervix under normal, dysplasia, and cancerous conditions. Membranous expression of this molecule occurred in normal condition [Figure 4]a. A loss of membranous E-cadherin along with simultaneous gain in its cytoplasmic form occurred in dysplasia [Figure 4]b. Further loss in the membranous expression of E-cadherin was observed in cancer [Figure 4]c.
Figure 4: (a-c) Microphotographs (magnification, ×400) showing immunohistochemical expression of E-cadherin in uterine cervix; (a) normal, (b) dysplasia, and (c) cancer

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Analysis of E-cadherin with respect to their membranous expressions showed a gradual decrease in the intensity through dysplasia and cancer [Table 2]. Membranous decline in E-cadherin was found throughout the epithelia in the order of NCM to DYS to SCC [Table 2].
Table 2: ANOVA of changes in expression status (grey scale intensity) of E-cadherin in normal, precancer (dysplastic), and cancerous cervical epithelium

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CD105 expression in cervical epithelium

CD105 was not expressed in normal condition [Figure 5]a and faintly its expression was noted in cervical dysplasia [Figure 5]b, while prominent expression of this molecule was observed in cancer [Figure 5]c. [Table 3] showed the percentage of CD105 positive vasculature in the different cervical tissues studied here.
Figure 5: (a-c) Microphotographs (magnification, ×100) showing immunohistochemical expression of CD105; (a) normal, (b) dysplasia, and (c) cancer

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Table 3: Assessment of % of CD105+ in normal, dysplasia, and cancerous cervix

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 > Discussion Top


The assessment of malignant potential of cervical dysplasia is generally performed by histopathological evaluation. However, this approach suffers from subjectivity and inadequacy of the available data, especially in assessing its progression towards malignancy. In general, carcinoma of uterine cervix is headed by an asymptomatic condition or dysplasia/precancer characterized by proliferative, metabolic, and atypical changes in epithelial cells, their structural association, and connective tissue organization.[3] Numerous epithelial and subepithelial changes occur during initiation of cervical carcinogenesis and its malignant transformation.[5]

The enhanced expression (intensity and number density) of epithelial master regulator, the p63 proteins[11] throughout the cervical epithelium in dysplasia, and cancerous conditions [Figure 2] and [Figure 3] and [Table 1] and [Table 4], having impacts on cell proliferation and carcinogenesis, indicated increased cellular proliferation and impaired epithelial progressive maturation and stratification during initiation and advancement of cervical dysplasia.[7],[8]
Table 4: Immunohistochemical observations on alteration of p63 expression in normal, dysplasia, and cancerous cervix

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Expressional deregulation of another crucial epithelial molecule E-cadherin [Figure 4] and [Table 2] has also been observed in cervical dysplasia and cancer. This molecule is vitally associated with maintaining cellular adhesion, integrity, as well as cancer suppression.[13],[21] Decrease in membranous and increase in cytoplasmic expression of this molecule pointed out the loss of cervical epithelial structural integrity and cell–cell adhesion, migratory phenotype development, and increased metastatic potentiality as reported in other cancer also.[13],[14],[15],[16],[22] These findings were corroborative with the relevant observation on p63 in the context of impairment in epithelial progressive maturation and progression of the epithelium towards malignancy. Interestingly, the appearance of tumorigenic vasculature specific and hypoxia induced CD105 expressions [Figure 5] and [Table 3] in cervical dysplasia and cancer demonstrated the hypoxic conditions in these disease conditions.[18],[23],[24],[25],[26],[27],[28] Especially CD105 expressions depicted increase in tumorigenic vasculature which had strong pathobiological connotation with cervical epithelial structural and molecular pathology attributes related to progression of carcinogenesis as well as plausible impact on reorganization of the subepithelial connective tissue components.

The present findings conclude that p63 was able to define the malignant potentiality in case of dysplasia, but E-cadherin and CD105 were more potent in describing the same. An increase in expression of p63 was noted with the advancement of the disease along with concurrent loss of membranous E-cadherin and increase in CD105 expression. These indicated the occurrence of remarkable molecular changes in the cervical mucosa during the progression of dysplasia. A sharp increase in expression of CD105 in dysplasia with a similar rise in p63+ nuclei also indicated a possible association between these genes in the biological events relating to malignant potentiality. The study demonstrated the comparative expression of p63, E-cadherin, and CD-105 in normal, dysplasia, and cancer depicting the loss in progressive epithelial stratification, loss of cellular adhesion, and increased tumorigenic angiogenesis. The role of the above biomarkers associated with the development of dysplasia or precancer has been explained in this study. However, further in-depth analysis is required to justify use of these molecules as diagnostic biomarkers.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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[PUBMED]  [Full text]  
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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