Original article
Correlation between p53 mutations and HPV in bilharzial bladder cancer

https://doi.org/10.1016/S1078-1439(03)00014-0Get rights and content

Abstract

Alterations of the p53 tumor suppressor gene are the most common genetic changes detected in human cancers as well as in papillary and invasive bladder cancer. Several studies have demonstrated an association between HPV infection and urological malignancies. In the present work, the p53 gene status was studied together with the frequency of HPV in 99 cases of Bilharzial bladder cancer [BBC] in Egypt and both were correlated to the clinicopathological features of the patients. SSCP and sequencing were used to screen the p53 gene for mutations at exons 4–10 and IHC was performed to detect protein overexpression. PCR was used for detection and typing of HPV-DNA in tumor samples. p53 mutations were detected in 33.3% of the studied cases whereas protein overexpression was detected in 35.6% of the cases. The highest concordance rate was observed in cases harboring mutations at exon 4 [87.5%]. Bilharzial infestation was obvious in 72.2% of the cases that showed mutations. Exon 8 showed the highest rate of mutation [32%] followed by exons 4 and 5 [22% each]. The commonest mutational event was G:C transversion [15/50] especially at CpG dinucleotides. A mutational hot spot was detected at exon 4, codons 72-73. HPV-DNA was detected in 48.97% of the cases the majority of which [64.6%] were of type 16. Significant correlation was found between p53 mutation and the pathological stage as well as p53 overexpression and tumor grade. Our results demonstrate that the mutational spectrum in BBC is different from that of bladder cancer in Western countries in many aspects and suggest an etiological role of HPV in this type of neoplasm. However, both HPV infection and p53 gene abnormalities may contribute to Bilharzial bladder carcinogenesis in an independent way.

Introduction

Bilharzial bladder cancer (BBC) is the commonest neoplasm in Egypt, accounting for more than 30.3% of all cancer cases diagnosed at the NCI, Cairo [1]. Various mechanisms have been proposed to account for the association between infection with Schistosoma hematobium and the development of bladder cancer. Some investigators [2] suggest that the organism may potentiate the mutagenic action of some environmental carcinogens through its chronic irritation of the urothelial lining.

To date, most genetic studies on bladder cancer have focused on transitional cell carcinoma (TCC) and little is known about the genetic alterations that occur in BBC [3], [4], [5]. However, it has been shown in several studies that, not only the clinicopathological characteristics of BBC are different from those of TCC of the Western countries [6] but also the genetic abnormalities detected in them are not the same, for example, incidence, type, and distribution of p53 mutations [7].

In BBC, data on p53 mutations are still conflicting, ranging from 35% to 86% [4], [8], [9], [10]. However, these data suggest a role for the p53 gene in BBC development or progression. A growing list of p53-regulated target genes has been identified through which p53 is involved in pathways of cell cycle control, angiogenesis, apoptosis, and DNA repair [11]. In addition to specific and nonspecific DNA-binding, several cellular and viral proteins capable of binding p53 and inactivating its function have also been described including the HPV viral oncoproteins [12].

Some investigators [13] reported the presence of HPV structural antigens in 10% to 30% of bladder tumors using in situ hybridization (ISH) or southern blotting. Others detected HPV-DNA in a higher proportion of bladder tumors (50%) using the PCR technique [14]. HPV-DNA 6,16 and 18 were detected in 23.3% of 30 cases of Egyptian BBC cases where ultrastructural examination revealed the presence of intracellular inclusion bodies 150 to 250 nm, suggestive of HPV in tumor cells [15].

However, the significance of this association, the true incidence of viral association and the exact mechanism(s) through which the virus may induce cancer has to be substantiated yet. One proposed mechanism is the binding of HPV/E6 protein to p53 which alters the regulatory function of p53 on cellular proliferation. It has been shown that binding of E6 protein encoded by high-risk HPV types result in rapid degradation of the p53 protein through the ubiquitin-directed pathway. Furthermore, E6 can abrogate p53-mediated transcriptional repression following the binding of E6 to p53 even without p53 degradation [16]. Recently, it has been shown that, HPV-E6 inhibits DNA repair along both the transcribed (transcription-coupled nucleotide excision repair) and nontranscribed (global excision repair) DNA strands [17].

Considering the numerous contradictory reports on the presence of HPV in BBC and the role of p53 gene mutations in the pathogenesis of this type of neoplasms, we analyzed 99 cases of BBC for the presence of p53 gene mutations using SSCP and sequencing technique. Immunohistochemistry (IHC) was also performed to assess the degree of p53 protein expression. The results of sequencing were correlated to those of the IHC. We also correlated the prevalence of HPV in tumor samples with p53 gene mutation detected by both techniques. Data regarding p53 gene mutations and HPV incidence in tumors was also correlated with the clinicopathologic and the epidemiological characteristics of the patients.

Section snippets

Patients

A total of 101 consecutive BBC patients who attended the National Cancer Institute, Cairo during the years 1996 through 1997 were evaluated for p53 mutation, protein overexpression as well as for the incidence and typing of HPV. Bilharzial infestation was confirmed either by the detection of S. hematobium ova in hematoxylin and eosin-stained sections obtained from tumor samples or their adjacent tissue or by the presence of definite history or clinical manifestations of bilharziasis. The

Results

Out of the 101 cases collected, 99 BBC cases were analyzed for the presence of p53 gene mutations by IHC and SSCP-sequencing techniques as well as for the presence and typing of HPV. The remaining 2 cases were diagnosed as rhabdomyosarcoma. The studied cases included 49 cases of SCC, 36 TCC, 7 adenocarcinoma, 5 TCC with squamous differentiation, and 2 cases of undifferentiated carcinoma. Histopathologic examination of tumor samples or their adjacent tissues revealed bilharziasis in 77/94 cases

Discussion

Alterations of the p53 are the most common genetic changes detected in human cancers. We investigated the relation between p53 mutation, nuclear overexpression and HPV infection in 99 cases of BBC. In the present study, p53 nuclear overexpression was demonstrated in 36.6% whereas mutations were recorded in 33.3% of the cases. Our 35.6% incidence of positive p53 immunostaining is comparable to those of Bochner et al. [23], Esring et al. [24], and Pycha et al. [5] who detected nuclear

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