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IMMUNOHISTOCHEMICAL EXPRESSION OF WILM'S TUMOR (WT1) MARKER IN HISTOLOGICAL GRADES OF ORAL SQUAMOUS CELL CARCINOMA.

Byline: DANIAL QASIM BUTT, FAROOQ AHMAD CHAUDHARY, NAVEED MAZHAR BHATTI, DANIAL KHALID, AAMIR RAFIQ and ZULEIKHA MALIK

Key Words: Well differentiated Oral Squamous Cell Carcinoma, Moderately Differentiated, Poorly Differentiated.

INTRODUCTION

Squamous cell carcinoma (SCC) considerably occupies the highest percentage among the heterogenous group of malignant cancers. It is the most common type of cancer in South Asian Countries like India, Srilanka, Pakistan and Bangladesh and contributes nearly onefourth of all new cases of cancer.1 OSCC is involved in ninety percent of all the malignancies.2 They initiate through rapid uncontrolled division of cancer epithelial cells and once matured gets infiltrated into the adjacent structures, or can disseminate to other organs (secondary metastasis). Most commonly affected sites are lip, tongue, buccal mucosa and floor of the mouth.

The OSCC predominantly arises from the non-keratinized mucosa, with the most common sites involve are buccal mucosa accounting for 50% of all the oral cavity cancers, followed by tongue which accounts for 20% and lower alveolus 13%.3 This disease is characterized clinically by signs and symptoms (ulcerated, exophytic growth). The early onset is painless as it becomes aggressive, it becomes painful and eventually bleeds if it traumatized and become secondarily infected. The prognosis and therapy strictly depend on the histological grades and correlated to rapid diagnosis.

Wilm's Tumor 1 (WT1) is a tumor suppressor gene located on the chromosome 11p13 that have been detected in variable neoplasms such as mesotheliomas and primary peritoneal serous and ovarian carcinomas.4

WT1 gene contains 10 exons and spans around 50kb approximately generating 3 kb mRNA. The carboxyl terminal portion having four zinger motifs forming a domain for DNA binding and regulates the early growth response gene 1 family i.e. EGR1 and EGR2 proteins, indicative of its role as transcription factor.5

24 different isoforms are generated from alternative RNA splicing and RNA editing, it depends on the target site where WT1 is binding. WT1 down regulates the EGFR6, PAX 2 and IGF-2.7 The addition of 68 amino acid amino terminal portion has a slight effect on the transcription.8 In comparison with the shorter versions of amino terminal, it increases activation potential.9

Spliced exon 5 has the repressing effect of WT1 on some promoters10. WT1 17AA isoform has shown its role in cell division and cell survival. Its mutation causes neoplasm of the kidney in 1 of 10,000 infants. Alteration of WT1 leads to Wilm's tumor and Denys-Drash syndrome, presented both in sporadic and hereditary forms. The WT1 protein has been documented for its binding capability with the cellular components e.g. p5311. Previous studies on breast cancer have provided the fact of Wilm's tumor oncogenic role12.WT1 overexpression has been documented in variety of cancers like urinary tract cancers, cancer of male genital organs, malignant melanomas and pancreatic cancers.

Many reported evidences reveal the role of WT1 in cancer cells proliferation and at present a cancer therapy in the form of WT1 peptide based immunotherapy has been suggested as a capable method of dealing with the cancers13. The purpose of the study was to demonstrate the immunoexpression of WT1 in the histological grades of OSCC which can be beneficial in using WT1 peptide based immunotherapy in treating different patients of OSCC.

METHODOLOGY

The Descriptive study was carried out in the Department of Histopathology, Armed Forces Institute of Pathology (AFIP, Rawalpindi), Ethical approval was taken from the respective institute. The sample size was 100 cases was calculated by WHO calculator13 (n= Z2 PQ/ d2, where n = desired sample size, Z = standard normal deviate, corresponding to 95% confidence level, P = proportion in the target population estimated to have a particular characteristic, Q =1-P = proportion in the target population not having the particular characteristics) and d= degree of accuracy required, Z2 =3.8416, P=7% (0.07) , Q=1-P, d2=0.0025). The sampling technique was Non Probability convenience sampling. Freshly diagnosed cases or archival tissue blocks of OSCC of all the grades were recruited from different hospitals. All age groups, both genders involving the oral cavity diagnosed at AFIP Rawalpindi were included.

Poorly fixed specimen, necrosed or autolysed tissue samples and tumors with scanty tissue were excluded and previously treated cases of OSCC were also excluded. Quantitative variables were age and gender while the Qualitative variables were histological grades, immunohistochemical (IHC) expression of WT1 and Immunohistochemical score. Percentage of positively reactive tumor cells was measured as 75%. Similar grading scales have previously been used in the immunohistochemical expression studies of WT114,15. The expression pattern was organized into high expression and low expression groups and their relationship were correlated with histological grade of differentiation16.

Tissues were prepared for microscopic examination. The tissue processing was start with the fixation of the surgical specimen or biopsy using buffered formalin (10%). All the representative sections from the surgical specimen were taken and processed in labeled plastic cassettes with perforated walls. These were then placed in automatic tissue processor Tissue Tek VIP-5 processor. The processing involved dehydration using increasing strengths of alcohol, clearing in which the alcohol was replaced by xylene at 380C, followed by impregnation of the tissue with molten paraffin wax at temperature of 52-550C. Casting or blocking was then carried out using Tissue Tek Embedding Console system, filling metal moulds with fresh molten wax and was allowed to cool. Finally the cooled blocks were trimmed and very thin sections (3-5 microns) were cut using Accu Cut Rotary Microtome SRM 200-1.

The sections were picked up on clean and labeled frosted glass slides and were stained in Varistain Multiproy slide stainer. The immunohistochemistry was then applied using the indirect method, It is a two-step method with an unlabeled primary antibody (first layer) reacting with tissue antigen followed by application of secondary labelled antibody (second layer) reacting with primary antibody. This method has adequate sensitivity and second layer antibody can be labeled with a fluorescent dye or enzyme.

The information collected in the form of variables on specially designed data collection proforma was analyzed using SPSS version 17.0. Descriptive statistics were used to describe the data. Mean and standard deviation was used to describe numeric variables like age and gender. Categorical variables were analyzed by calculating frequency and percentages. Immunomarker result and their association with the histological grade with the help of Chi square test was carried out. P value significance was set at < 0.05.

RESULTS

The results are made on one hundred cases of OSCC of different histological grades. There were fifty (53%) males and forty seven (47%) females, with a male to female ratio of 1.12:1. Mean age was 57.11 years with SD +-14.6. Sixty eight (68%) were WD-OSCC, with twenty one (21%) were MD-OSCC and eleven (11%) were PD-OSCC. The tongue was found in fifty seven cases (57%), followed by buccal mucosa thirty three cases (33%), lower lip six cases (6%), lower jaw two cases (2%), maxillary gingiva one case (1%) and hard palate one case (1%). WT1 was found weak or absent in the basal layer and in the infiltrating nest of cells. IHC of WT1 in OSCC with the percentage of stained cells were investigated in all of the histological grades of OSCC.

TABLE 01: IMMUNOEXPRESSION OF WT1 PERCENTAGE OF STAINED CELLS IN WD-OSCC

Percentage of###Intensity

stained cells###Low###High

0###14###-

75###-###4

TABLE 2: IMMUNOEXPRESSION OF WT1 PERCENTAGE OF STAINED CELLS IN MD-OSCC

Percentage of###Intensity

stained cells###Low###High

0###12###-

75###-###-

TABLE 3: IMMUNOEXPRESSION OF WT1 PERCENTAGE OF STAINED CELLS IN PD-OSCC

Percentage of###Intensity

stained cells###Low###High

0###06###-

75###-###-

TABLE 4: RELATION OF WT1 IMMUNOEXPRESSION IN HISTOLOGICAL GRADES OF OSCC

Tumor###WT1 Expression###P Value

Differen-###Low###High

tiation

WD - OSCC###45###23

MD - OSCC###20###02###0.024*

PD - OSCC###09###01

WD-OSCC

Fifty four cases showed the positivity out of sixty eight cases of WD-OSCC, with fourteen cases (20.5%) showed 0 reactivity, sixteen cases (23.5%) showing < 5 % of stained cell with and twenty cases (29.05%) of 5-50%, nineteen cases (27.9%) showed and four cases (5.8%) (Figure 1 and 2) were above the 75% stained cells (as shown in the table no.1)

MD-OSCC

Four cases (19%) showed the positivity out of twenty one cases of MD-OSCC, with seventeen cases (80.9%) showed 0 reactivity, two cases (9.5%) showing < 5 % of stained cell with and one case (4.7%) of 5-25% each with 1+ nuclear reactivity, while only one case (4.7%) showed 51-75% of stained cells (as shown in the table no.2).

PD-OSCC

Five cases (45.4%) showed the positivity out of eleven cases of PD-OSCC, with six cases (54.5%) showed 0 reactivity, two cases (18%) showing < 5 % of stained cells with and one case (9%) of 5-50%, two cases (18.18%) showed 51-75% of stained cells (as shown in the Table no.3).

79.4% (54 cases) of positivity in WD-OSCC (68 cases), 19% (4 cases) positivity in MD-OSCC (21 cases) and 45.4% (5 cases) positivity in PD-OSCC (11 cases).

The expression pattern was organized into two groups; high expression and low expression and their relationship was correlated with histological grade of differentiation (as shown in Table no.4).

WT1 immunoexpression was found correlated with the histological differentiation of OSCC and with the age parameter (P value 0.004), However no significant correlation was found with the gender and tumor site (P value 0.8 and 0.6 respectively).

DISCUSSION

In normal epithelium, there is continuous renewal of cells by the mitotic division at the basal cell layer. These cell migrate to the surface of the epithelium to replace the cells that are shed. In WD-OSCC, same process occurs where the cancer nests proliferate and infilterate from the basal layer into the underlying tissue. WT1 was observed expressed in the cancer nests suggesting it role in neoplastic proliferation of cancer nests.17 Wilm's tumor 1 (WT1) is a tumor suppressor gene responsible for a kidney neoplasm in children. In oral SCC, the alliance of WT1 protein and Ribonucleoprotein particles (RNPs) suggested its involvement in RNA metabolism by the shuttling of WT1 protein between nucleus and cytoplasm.18

Oji et al.19 in a study of esophageal squamous cell carcinoma observed the overexpression of WT1 protein in esophageal proliferating tumor nests in the underlying connective tissue infiltrating from the basal layer of the epithelium. The expression was weak to moderate at the basal cell layer. The study suggested significant correlation with PD-OSCC as more expression was observed when the tumor cells differentiated from well differentiated to poorly differentiated tumor cells. Similar expression of WT1 in basal cell layers and infiltrating tumor nests were observed in our study. WT1 overexpression was observed more in the WD-OSCC in this study. Mikami et al.13 investigated the role of WT1 protein in OSCC and found positive expression of WT1 in well differentiated tumor cells and suggested its role in tumor cell proliferation. Similarly Fattahi et al.20 observed the same phenomenon and suggested its strong role in tumorigenesis.

Our results also showed the positivity of WT1 in each of the histological grade of OSCC suggesting the tumorigenesis role. More detailed studies should be carried out worldwide of IHC expression of WT1 in OSCC. Previous studies have proposed its role in tumorigenesis and its detection in various cancers such as leukemia, breast cancers, sarcomas21,22 and lung cancers [13]. These cancers expressed WT1 and were treated with WT1 antisense oligomers. Tsuboi et al.23 used WT1 peptide-based immunotherapy, in which anti WT1 vaccinations caused increase in the WT1-specific cytotoxic-T lymphocytes which reduced the leukemic blast cells in patients with leukemia. More studies are needed to establish conclusive role of WT1 expression in OSCC and immunotherapy as treatment in treating patients of OSCC.

CONCLUSION

WT1 expression results suggested a strong role in tumorigenesis, which is consistent with other studies. New WT1 peptide based oligomers (vaccinations) are being used in immunotherapy for the direct tumor cell killing and in future may be tried on the cases of oral squamous cell carcinoma.

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Publication:Pakistan Oral and Dental Journal
Article Type:Report
Date:Dec 31, 2018
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