Expression Analysis of Esophageal Squamous Cell Carcinoma in Khyber Pakhtunkhwa and Afghan Patients Using Wt1 and Cox-2 Biomarkers

Esophageal cancer (EC) is the major global health concern that ranks seventh among the common types of cancers and is the sixth most fatal disease worldwide. Esophageal squamous cell carcinoma (ESCC) is the most common major histologic subtype of EC in regions of Greater Iran with high prevalence, more than 140/100,000 population. In Pakistan it was ranked fourth overall, third in males and fifth in females. Molecular carcinogenesis of the disease has not been explored in depth so far leading to lack of effective chemotherapeutic approach and adverse prognosis. Material and methods: This study was carried out in Pakistani and Afghan patients of ESCC, living in Peshawar, Khyber Pakhtunkhwa (KP) province of Pakistan. We investigated the risk factors and molecular abnormalities of ESCC. For molecular analysis formalin fixed paraffin embedded tissue (FFPE) of ESCC and paired healthy esophageal mucosa were used. The study’s objective was to investigate the expression profile of COX-2 and WT1 biomarkers at protein level using immunohistochemistry (IHC). Also, Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) was performed to identify expression profile of COX-2 and WT1 at mRNA level by RNA extraction and cDNA synthesis in ESCC cases. The DNA isolated from ESCC samples were sequenced to find the spectrum of mutation relevant to COX-2 and WT1 gene. The immunohistochemistry and qRT-PCR data were analyzed using SPSS version 20. ANOVA test was used to determine the variations in means of gene expression in normal esophageal mucosa, carcinoma in situ and tumor tissue of esophagus. Furthermore, Pearson’s Chi square test was applied to assess the correlation of Clinico Pathological variables. For RT-PCR analysis paired sample t test was used. Results: High expression of WT1 and COX-2 was observed in 38% and 67% of ESCC cases, respectively, when compared to paired normal mucosa by immunohistochemistry. Similarly, for in carcinoma in situ (CIS), high expression was 36% of WT1and 79% in COX-2. Naswar users constituted 67% of ESCC patients, among them high expression of WT1 and COX-2 was observed in 42% and 65% patients, respectively. High relative fold difference of mRNA in ESCC samples was noticed in both WT1 (1.092) and COX-2 (1.2500) when compared to paired normal esophageal mucosa (1.0000) by qRT-PCR. When comparing the expression of WT1 by IHC with the relative fold difference by qRT-PCR, correlation between protein and mRNA expression was found in negative, weakly positive and strongly positive ESCC cases. Synonymous mutation A 903 G was detected at exon 7 in WT1 gene in malignant and normal samples of ESCC patient, without any change in amino acid. Conclusion: Naswar use is more common in patients of ESCC in the KP province of Pakistan and may be a risk factor in ESCC and highlights the need for improved public health practices and awareness to reduce the addiction to tobacco and snuffing. qRT-PCR is a fast tissue and powerful technique to identify low level of mRNA for quantification of gene expression levels. Few studies have addressed the issue of gene expression by qRT PCR in FFPE tissue which is often the only sample for research in routine practice. In this study increased relative expression of WT1 and COX-2 gene in ESCC patients was found by IHC and qRT-PCR techniques in FFPE tissue samples. Overexpression of COX-2 and WT1 is positively associated with ESCC.Our results may provide the evidence for immunotherapy targeting of the WT1 protein in ESCC cases expressing WT1 while its anti-oligomers may be the part of molecular targeted therapy in these individuals. The results of this study suggest that inhibitors of COX-2 may potentially be beneficial for preventive and therapeutic measures with an adjuvant therapy in high risk ESCC patients. Further studies on a larger scale are needed to confirm the prognostic and predictive value of COX-2 and WT1 in this high risk population. Raising public awareness of these cancers and improved knowledge of symptoms could help to identify earlier stage tumors and consequently improve the prognosis of these cancers, particularly in more deprived localities