Histopathology, Immunohistochemistry and Molecular Study of Tumor Protein 53 Gene Mutations in Esophageal Cancer among Sudanese Patients

Abstract

The protein product of the normal tumor protein 53 (TP53) gene performs an essential function in cell cycle control and tumor suppression, and the mutation of a TP53 gene is an essential step in the development of many cancers. Despite the reported association of TP53 gene mutations with many human cancers, the comprehensive computational analysis of single nucleotide polymorphisms (SNPs) is of paramount importance in predicting functional and structural impacts of specific SNPs. The aim of this study was to detect TP53 gene mutations in exon 5-8 in Sudanese patients with esophageal cancer (EC). In this study 204 esophageal cancer specimens were collected from different hospitals in Khartoum state, and investigated using hematoxylin and eosin (H&E) stain for histopathologic classification of EC, in addition to immunohistochemistry (IHC) stain to detect p53 accumulation, TP53 gene alterations were investigated using Deoxyribonucleic acid (DNA) extraction from the same specimens followed by polymerase chain reaction (PCR) and finally DNA sequencing were screened in 50 formalin fixed paraffin embedded (FFPE) specimen due to DNA scarcity or absence in part of the specimens and degradation of the DNA in the rest of them. Computational analysis was performed using different algorithms to screen for deleterious single nucleotide polymorphisms (SNPs). In the 204 specimens used, their patients age ranged from 18-93 years, mean 60 years; those who were <60 years of age were classified as the young group which comprises 72/204 (35.3%) and classified the other as the elderly group 132/204 (64.7%). Males were 76 (37.3%) and females were 128 (62.7%). Histopathologically, the EC specimens were classified as squamous cell carcinoma (SCC; n= 170), Adenocarcinoma (AC; n= 21) and undifferentiated carcinoma (n= 13). IHC positive samples were 56 (27.5%) and negative samples were 148 (72.5%), the study found there is a significant relationship between sex and histopathologic diagnosis with SCC being more prevalent in females, AC and UDC in males. The 50 EC specimens used for detection of TP53 gene alterations comprised 43 SCC and 7 AC. The results also demonstrated that there are synonymous SNPs (sSNPs) (n= 4) and non-synonymous SNPs (nsSNPs) (n= 6) in the TP53 gene that may be deleterious to p53 structure and function. The 50 specimens selected for sequencing, their age range from 20-93 years, with (<60 years) group comprising 20/50 (40%) and (>60 years) group was 30/50 (60%), also 33(66%) of them were female and 17 (34%) were males and 43 (86%) Were diagnosed as squamous cell carcinoma (SCC) and 7 (14%) as adenocarcinoma (AC), Additionally, TP53 gene alterations were found in 20/50 (40%) of samples. Six out of ten of TP53 gene alterations occurred in exon 5, two in each of exons 6 and 8. Only one SNP in position E298Q was predicted to have a neutral effect and other SNPs were predicted to be disease related according to MutationTaster software. A total of 15/40 (37.5%) of SCC samples were found to be altered, 12/15 (80%) of them exist in exon 5 and 1/15 (6.7%) in exon 6, whereas AC showed a higher rate of alterations 4/7 (57.1%) with 100% involvement of exon 5, and no involvement of exon 6 and 8. TP53 gene alterations were detected in 20 (40%) of the 50 EC cases investigated. Five out of 13 (38.5%) of SCC cases that stained positive for p53 protein, showed TP53 alterations, only one (14.3%) of seven AC samples was positive for p53 protein without associated genetic alterations and 4/7 (57.1%) showed TP53 mutations only. Immunohistochemical stain For P53 was positive in 14/50 (28%) of EC samples; 13 (92.9%) of them were SSC cases and one (7.1%) AC. Five out of forty three (11.6%) of SSC and none of AC cases showed both immunohistochemical positive stain and alterations of TP53 gene while 8/43 (18.6%) of SSC and 1/7 (14.3%) of AC samples showed immunohistochemical positive stain but no TP53 gene mutations were present. Finally 15/50 (30%) of EC; 11 (73.3%) SSC and 4 (26.7 %) AC, showed immunohistochemical negative stain and positive TP53 gene alterations. Statistically there is no correlation or significant association between TP53 gene alteration and sex, age of patients or tumor type. The study concluded that alterations of exon 5 in TP53 gene of EC patients were the most frequent. Genomic results have identified a higher TP53 mutation rate in esophageal AC in contrast to SCC. Also Immunohistochemical patterns of p53 were not significantly related to mutational analysis results in the cases examined. Mutation analysis is recommended to be done on the entire length of the gene using fresh tissue to complete the whole picture in future studies.