Postgraduate Training Course in Reproductive Health 2004

Phenotypic Features with p53 Alterations Related to HPV
and Prognostic Evaluation in Cervical Cancer

Dr. Xin Lu
Ob & Gyn Hospital at Fudan University, Shanghai

See also presentation

Abstract

Background: Cervical cancer is one of the most common tumors affecting women worldwide. The multiple genetic alterations involved in the stepwise development of this malignancy. There is evidence that human papillomavirus (HPV) were found have a causal relationship with cervical cancer and its precursors. The tumor suppressor gene p53 have been shown to be associated with the prognosis for certain tumors. The interaction between HPV E6 protein and p53 was identified in vitro studies.
Objectives: To evaluate the prevalence of p53 alterations related to HPV infection, such as p53 mutation, LOH of p 53, p53 polymorphism; To evaluate the prognostic significance of p53 alterations in cervical cancer.
Methods: Studies were identified by a MEDLINE search and all relevant articles were retrieved from 1991 to March 2004. Some data from the World Health Organization (WHO) and p53 database of the International Agency for Research on Cancer (IARC) were also cited. All references not match the selective criteria were excluded.
Results: The prevalence of p53 mutations is rare event in cervical cancer. The correlation between p53 mutations and HPV or prognosis is controversy. LOH of p53 is more common found in cervical cancer and related with the prognosis of this disease. There is no significant correlation between p53 polymorphism and the development of cervical cancer.
Discussion: The significance of various p53 alterations with prognosis in cervical cancer are discussed. The p53 mutations were found not common in cervical cancer. LOH of p53 may contribute to the progression of this malignancy. p53 mutations and p53 polymorphism failed to be an independent prognostic factor in predicting the outcome of patients with cervical cancer. Further epidemiological surveys should be undertaken in larger populations and in different geographical regions.

Introduction

1. Introduction of cervical cancer

Cervical cancer is one of the most common and aggressive malignancy in female. Worldwide about 500 000 women acquire the disease annually and 75% are from developing countries (1,2). Cervical cancer has leading mortality rate in the world, every year around 300 000 women die of this disease. Although the etiology of cervical cancer has not been clear elucidated, but the causal relation between human papillomavirus (HPV) and the cervical cancer was well documented [3-6]. It was reported that around 95% of cervical cancers contain different types of HPV. To date, over 120 types of the HPV have been described (7). The World Health Organization (WHO), International Agency for Research on Cancer (IARC) has classified the HPV into three groups: “carcinogenic (HPV types 16 and 18); probably carcinogenic (HPV types 31 and 33) and possibly carcinogenic (other HPV types except 6 and 11) (6, 7). On the one hand, epidemiological studies had identified the major risk factors of cervical cancer. The population with HPV infection, multiple sex partners, smoking, oral contraceptives as well as family history have higher risk of cervical cancer (7-10). On the other hand, in addition to social-environmental and infectious factors, numerous molecular biologic studies have demonstrated that molecular genetic factors were also involved in the carcinogenesis of cervical cancer. These factors are cell cycle regulator genes, tumor suppressor genes, including p53 alteration (11-13).

2. Introduction of p53 gene

2.1 p53 gene structure and function
The first p53 gene mutation arising in a human cancer was described by Baker (14). It is estimated that p53 mutaitons are the most frequent genetic events in human cancers. During the past 25 years, p53 mutations are found in all human cancers, account for more than 50% of the cases (15,16). The p53 gene is located on chromosome 17p and is composed of 11 exons which encoding for 393 amino acids. It is highly conserved in vertebrates. p53 gene contains three domains: DNA binding domain, transactivation domain and oligomerization domain (Fig. 1). The transcriptional domain at the N-terminus contains a binding site for the product of MDM2 gene. The DNA binding domain is sequence-specific and contains the binding sites for SV40 large T-antigen. The C-terminal region contains a domain necessary for p53 oligomerization, one primary and two secondary nuclear localization signal sequences, mediating non-specific DNA binding. Wild type p53 is an important transcription factor and coupling many functions. It was well-known that wild type p53 can acting as a tumor suppressor, apoptosis inducer, and a protein able to arrest cell cycle. p53 plays a critical role in the cell cycle regulation mechanisms and cell proliferation control, and its inactivation is considered a key event in human carcinogenesis (17).

2.2 p53 mutation in human cancer
p53 mutations are the main cause which contribute to its inactivation. For most human cancers, p53 mutations are mis-sense mutations within the coding sequence of the gene (Fig 1). About 90% of the mutations reported to be clustered between exon 4 and 10 and are localized in DNA binding domain of the p53 gene (15,16). The correlations between distinct mutants and functional changes are well-established during past two decades. Mis-sense mutations result in loss of p53 tumor suppress function by changing sequence-specific transactivation activity (18). However, gain of oncogenic function by different pathways which are controlled by this transcription factor was also seen (19).

3. Introduction of p53 and HPV

Certain HPV serotypes have been associated with cervical cancers. Among HPV types, the most frequent one is HPV-16 (3-5). Previous studies have shown that in cervix, HPV-16 DNA is present in an episomal form in asymptomatic infections and benign lesions. While in malignant lesions it is frequently integrated into the host cell genome. The frequency of cervical cancer specimens that carry HPV-16 genome in integrated form ranges between 30-50% (20). The products of the E6 and E7 Open Reading Frames (ORFs) of HPV-16 genome are responsible of the immortalising effects on transformed epithelial cells. In vitro studies have demonstrated that the major transforming E6 and E7 activities include the targeted degradation of p53 and transcriptional induction of the cellular telomerase enzyme by E6 and the inactivation of the cellular retinoblastoma protein pRB by E7 (21, 22).

4. The prognostic factors of cervical cancer

The clinical and pathologic prognostic factors in cervical cancer are well defined, such as tumor histologic type, low grade differentiation, parametrial infiltration, lymph node metastases. A large study demonstrated that HPV 16 genotype may play an important role in the assessing progress of cervical cancer patients (23). In the literature, the alteration of mutated p53 leads to malignant phenotype, and the interaction between HPV and p53 pathways may contribute to cervical carcinogenesis. To better understand the role of p53 alterations related to HPV infection, and to predict the prognosis in cervical cancer, mutation, LOH of p 53 as well as p53 polymorphism will be reviewed.

Objectives

The objectives of this study are as follows:

• To analyse data about of p53 mutation in cervical cancer
• To investigate the correlation of p53 mutation and HPV infection in cervical carcinoma
• To evaluate whether p53 can be a prognostic factor in this malignancy
• To examine LOH of p53 and p53 polymorphism

Materials and Methods

Studies were identified by a MEDLINE search and all relevant articles from 1991 to March 2004 were retrieved. Searching the reference lists of retrieved full-text articles and abstracts, some relevant journals were obtained by hand searching. Data from the World Health Organization (WHO) and p53 database of the International Agency for Research on Cancer (IARC) were reviewed.

About p53 database

In 1997, a remarkable p53 database had been generated by IARC. The p53 mutation database contains all publications related on p53 gene alterations in human tumors cell lines and human cancers. It was compiled from the published literature and made available through electronic media. The database is now maintained at the IARC and is updated twice a year (24,25) and is available online. Today, there were over 18,585 mutations reported in the IARC database of human p53 tumor mutations (26,27). This database also provides an important information on the environmental factors and biological processes that are important causes of human gene aberrations (28).

The MEDLINE database was searched by using the following key words (MeSH): Cervical cancer/carcinoma, HPV, p53 mutation, LOH , p53 polymorphism
References related to the following topics were reviewed: Etiological factors for cervical cancer, the structure and function of p53 gene, interaction between p53 gene and HPV E6 protein, the incidence of p53 mutation in cervical cancer, correlation of p53 mutation with pathologic factors and prognosis, LOH of p53 and p53 polymorphism in cervical cancer, methods commonly used to detect p53 alterations.

So far, there are three methods used to detect mutations of p53 gene: immuno-histochemistry (IHC) to detect and localize the mutated protein expressed in cancer cells, Single-Strand Conformation Polymorphism (SSCP) to direct sequencing of the p53 gene and PCR based methods to analysis LOH of p53 which is located in 17p13 and polymorphism in p53. Each method has its own limitations in terms of specificity to detect p53 mutations. One important feature of mutated p53 protein is to increase stability and its accumulation in the nucleus of neoplastic cells. Positive immuno-staining is usually indicative of abnormalities of the p53 gene and its product, but it is highly dependent on the type of p53 mutation (29, 30).

Study Selection

The articles excluded were: 1). Technical papers without original data. 2). Letters and those that did not address cervical cancer or p53 mutation. 3). Those data only contain result from cell line study. 4). Paper that did not measure HPV and p53 mutation as the reference standard. The final sample for critical appraisal consisted of 103 studies. All articles were obtained from Medline, IARC p53 database as well as WHO library.

Results

1. Mutations of p53 in cervical cancer

1.1 Prevalence of p53 mutations and cervical cancer
The worldwide distribution of human cancer countries is shown in Fig 2.
Compared to other type of cancers, p53 mutations were found not common in cervical cancer (25).

Table 1. Prevalence of p53 mutation in cervical cancer based on gene sequencing. (SSCP : Single-Strand Conformation Polymorphism ; DDGE :Denaturing . Gradient Gel Electrophoresis ; CDGE : Constant Denaturant Gel Electrophoresis ; Yeast : Yeast Function Study).

According the data based on gene sequencing, from 19 studies, 13 studies showed the prevalence of p53 mutations less than 10% in cervical cancer (Table 1). Among them, only one study (43) showed high p53 mutation rate (42%). In two study, there is no significant difference between p53 mutations and histological type of cervical cancer(32,34).

1.2 Pattern of p53 mutations in cervical cancer
The complied data in the IARC p53 mutation database was used to analyze the pattern of p53 mutations in cervical cancer. Fig 3 shows the pattern and codon distribution of p53 mutations in cervical cancer. The codon distribution in cervical cancer shows similar hotspots of mutations as in all other cancers. The global analysis indicates that the p53 gene mutation pattern in cervical cancer does not show features that significantly distinguish it from most other human cancers.

1.3 Association of HPV infection and p53 mutations in cervical cancer
Data from in vitro experiments showed that HPV E6 protein involved in p53 degradation. There are several groups have studied the correlation between p53 mutations and HPV infection in cervical cancer patients (40, 43, 50-52). The correlation between p53 mutation and HPV infection is controversy. Ishikawa et al showed the p53 mutation was related with HPV infection (50). In contrast, Helland reported that a significantly higher p53 mutation frequency was found in HPV-negative cervical cancers (43). And the rest studies had shown that there is no relationship between p53 mutations and HPV infection in cervical cancer patients (40, 41, 47).

Table 2. Association between p53 mutation and HPV infection in cervical cancer (p53 data based on SSCP)

2. The cervical cancer prognosis and p53 mutations

During past two decades, the major p53 alterations were found in cervical cancers and their precursors. These include p53 expression, mutations, LOH, as well as polymorphism in p53. This review analyzed the impact of these various p53 abnormalities on the cervical cancer patient prognosis. Overall, the IHC data showed that the correlation of p53 over expression with prognosis is discrepancy (53-66). In addition, because the p53 mutations are not common event in this malignancy, the evaluation of p53 mutations on the prognosis of cervical cancer remains to be defined (67).

2.1 p53 expression and cervical cancer prognosis based on IHC studies
p53 gene mutation was found correlated with prognosis in a wide variety of malignancies, especially in lung cancer and breast cancer (67). In this review, although the prevalence of p53 prevalence based on IHC is much higher than that based on gene sequencing, however the two groups (related and unrelated prognosis) based on the IHC data showed no differences on prevalence of p53 (46% vs 47.8%) (Table 3).

Table 3. p53 expression and prognosis in cervical cancer (IHC)

2.2 P53 expression and cervical cancer prognosis based on SSCP studies
Because the p53 mutations are not common event in this malignancy and the perspective study using gene sequencing limited the investigation, so there are only two studies analysed the correlation between p53 mutations based on SSCP and prognosis of cervical cancer patients (50,52). ISHIKAWA et al. has studied 52 patients with squamous-cell carcinomas (SCC) who received radiation therapy alone and investigated the effects of HPV infection, p53 status, and other parameters on clinical outcome by univariate analysis. They found that the p53 mutation had a significant correlation with local tumor recurrence, but no obvious correlation between HPV infections with any clinical outcome for cervical cancer patients (50). Milde-Langosch K et al. has reported 51 cervical cancers and 40 vulvar SCC for the presence of HPV and mutant p53. In this study, p53 mutations were found in 7.8% in cervical cancer and showed no relation with prognosis of the disease (52).

Table 4. p53 Mutation and Prognosis in Cervical Cancer (SSCP)

2.3 p53 mutation and radiotherapy (RT) in cervical cancer
Cervical cancer is the most common cancer in women. Radiotherapy is the first choice of treatment in those cases at late clinical stages. Even though, the five-year survival rate remains low in those cases (68). It is well known that the presence of mutant p53 is related to radio resistance in a variety of tumor types (69,70). In cervical cancer, the results are controversy. Five of eight reports demonstrated that the presence of p53 was significant associated with the radio resistant of radiotherapy (71-75) (Table 5). However, the rest three studies shown no relationship between p53 and radio resistant of radiotherapy (76-78). We also reviewed whether or not there is a correlation between p53 alteration and prognosis related radiotherapy. Only two studies reported that p53 expression has impact on prognosis in patients undergoing definitive radiotherapy for cervical cancers (74, 75). They found that patients with immuno-histologically mutant p53-negative tumors had a clear survival advantage over patients with immuno-histologically mutant p53-positive carcinomas. However, another investigation found that p53 expression did not influence survival in patients with primary cervical cancers that were treated with radiotherapy (72).

Table 5. p53 alteration related with radiotherapy in cervical cancer (Prevalence of p53 based on IHC data; RT : radiotherapy ; ND: not detected)

3. LOH of TP 53 in cervical cancer

3.1 Prevalence of LOH of p53 in cervical cancer

Cytogenetic analysis of cervical cancers has shown that chromosomes 1, 3, 11, and 17 are commonly abnormal (79). Chromosome 17 alterations are found in more cancers than those of any other chromosome, and frequently involve the p53 gene on 17p13. Most of studies showed that numerical abnormalities of chromosome 17 were found in cervical cancers (80-91) (Table 6). There is no correlation between LOH of p53 and HPV status in cervical cancers (87).

Table 6. Prevalence of LOH of p53 in cervical cancers (LOH: Loss of heterozygosity; ND: not detected)

References	Cases	Prevalence of LOH	LOH and prognosis
Atkin NB, et al. 1990 (80)	43	17	ND
Kinoshita M, et al.1994 (81)	11	36.4	ND
Busby-Earle RM, et al.1994 (82)	20	15	ND
Mitra AB, et al. 1994 (83)	17	41.2	ND
Park SY et al. 1995 (84)	26	40	ND
Wistuba I et al. 1996 (85)	12	50	ND
Mullokandov MR, et al. 1996 (86)	38	15	ND
Kim JW et al. 1997 (87)	55	5.5	ND
Southern SA, et al. 1997 (88)	25	36	ND
Kersemaekers AMF, 1998 (89)	64	38	ND
Helland A et al. 2000 (90)	79	18	related
Harima Y et al. 2001 (91)	65	33.8	related

3.2 LOH of p53 and prognosis in cervical cancer
The clinical course of the cervical cancer is highly complex, and genetic factors underlying carcinogenesis are poorly understood. Loss on chromosomes 17p13.1 and p53 inactivation emerged as the disease progressed and were closely associated with each other. There are some reports showing that LOH of p53 related with the progression of certain tumor types (26). In cervical cancer, two studies also found that the LOH of p53 related with the clinical stages of tumors (90, 91) (Table 6).

4. p53 polymorphism in cervical cancer

4.1 Prevalence of p53 polymorphism in cervical cancer
Single nucleotide polymorphism (SNP) is a frequent form of single base pair variations in genome, which occur once in every 1200-1500 base pairs by the processes of deletion, addition, and substitution. These base pair variations make each person's sequence unique. Thus the different susceptibility manifested in each individual to a specific disease may also explained by genetic factors in some cancers (92). It was known that the tumor suppressor gene p53 has a SNP in codon 72. Polymorphism occurring in p53 codon 72 sequence causes CGC to change to CCC at exon 4, changing the end product arginine to proline.

Table 7. Prevalence of p53 polymorphism in cervical cancer (Cxca: cervical cancer; Arg: arginine; Pro: proline; ND: not detected; NS: not significant)

In cervical cancer, most of studies showed that no relationship exists between p53 polymorphism and cervical cancer (93-98) (Table 7). However, there are studies showing that an over-expression of homozygous p53Arg in cervix cancer compared to heterozygous or homozygous p53Pro, suggesting that individuals homozygous for p53Arg genetically susceptible to cervical cancers (99-101). Recently, Koushik et al. summarized all data related to p53 polymorphism and cervical cancer in a meta-analysis (102). The meta-analysis has shown that there is no correlation between p53 polymorphism and cervical cancer.

4.2 P53 polymorphism and HPV prognosis in cervical cancer
In this review, there are four studies showing that there is no significant difference between p53 polymorphism and HPV of cervical cancer patients (94-97). However, an India group have found that the frequencies of the three p53 genotypes Arg/Arg, Arg/Pro and Pro/Pro in the HPV-positive tumour samples were 0.34, 0.57 and 0.09 in comparison with frequencies of 0.18, 0.44 and 0.38 for HPV-negative tumours. There is a significant difference in the allelic frequency of p53 Arg/Arg in high-risk HPV-infected cervical carcinoma cases (0.34) and HPV-negative carcinomas (0.18) (103).

4.3 P53 polymorphism and prognosis in cervical cancer
A prospective cohort study was done to evaluation p53 codon 72 polymorphism in predictive the outcome of cervical cancer with other factors (94). Among 39 patients with HPV-16 positive cervical cancer, there was no difference between the lymph nodal metastases and p53 codon 72 polymorphism (Arg/Arg 54.5% vs 67.9%, Pro/Pro 0 vs 7.1%, Arg/Pro 45.5% vs 21.4%). The p53 codon 72 genotype did not influence the disease-free survival significantly (94). Similarly, another two studies also showed that there is no significant difference between p53 polymorphism and prognosis of cervical cancer patients (96,98).

Discussion

The development of cervical cancer correlates with some risk factors (7-11). There is evidence showing that certain human papillomavirus types, such as HPV-16 and HPV-18 are the main cause of cervical cancer (3-5). HPV-16 and HPV-18 encode two major oncoproteins, E6 and E7. The E6 protein binds to the cellular tumour-suppressor protein p53 and directs its degradation through the ubiquitin pathway and the E7 protein binds to and inactivates the cellular tumour-suppressor protein Rb (21, 22). Although inactivation of these two tumour-suppressor proteins by HPV is probably important for tumour development. So far, no genetic factors have been conclusively identified that might an infected individual to develop cervical carcinoma. p53 mutations were found in many human tumours. However, according the data based on gene sequencing, from 21 studies, 14 studies showed the prevalence of p53 mutations less than 10% in cervical cancer (Table 1). Among them, only one study (43) showed high p53 mutation rate (42%). The result may be effected by selected samples. Because the p53 mutations are not common event in this malignancy, the perspective study using gene sequencing limited the investigation. It is controversy to use IHC to determine the prognosis. Although p53 immuno-histochemical analysis may be incomplete as a prognostic indicator, but the relationship to p53 mutation by sequence analysis is still not well defined. It has been shown that p53 over expression may result from either the short half-life of the wild type due to protein folding, over-production due to repair, or may be caused by technical factors. The evaluation of p53 over expression by immuno staining without molecular analysis may not give the exact informations. In this review, all studies on prognosis were based on IHC data. There is a need of large number of control study to compare differences between whether or not based on IHC and SSCP, further to confirm whether or not the p53 mutation related with prognosis as well as response to radio resistant of radiotherapy. If there is a correlation, It can be concluded that the assessment of the p53 status could aid in the selection of patients for different treatment strategies. The clinical course of the cervical cancer is highly complex, and genetic factors underlying carcinogenesis are poorly understood. Loss on chromosomes 17p13.1 and p53 inactivation emerged as the disease progressed and were closely associated with each other. There are some reports showing that LOH of p53 related with the progression of certain tumor types (26). In this review, only two studies found that the LOH of p53 related with the clinical stages of tumors (90, 91). In addition, the role of p53 polymorphism in cervical cancer remains to be elucidated (67). Although previous study had demonstrated that homozygous for p53Arg more likely to develop HPV-associated cervical cancer than individuals having one or more p53Pro alleles, suggesting that p53Arg may represent a risk factor for HPV-associated tumorgenesis (99). However, recently a meta-analysis on this issue was done by Koushik A et al. (102).They had shown that there is no evidence that p53 polymorphism is a risk factor in cervical cancer. In summary, the p53 mutations were found not common in cervical cancer. LOH of p53 may contribute to the progression of this malignancy. p53 polymorphism failed to be a risk factor in predicting the outcome of patients with cervical cancer. Understanding the behaviour of p53 alterations, and analysing it thoroughly for each patient could allow us to develop sound correlations between p53 status and patient outcome. Epidemiological surveys should be undertaken in larger populations and in different geographical regions to determine the role of various p53 aberrations in HPV-associated diseases and whether the p53 phenotype affects the persistence of HPV infection and the development of cervical cancer.

Conclusions

In this review, several phenotypic features with p53 alterations related to HPV are summarized and discussed. The tumor suppressor gene p53 mutations were found not common in cervical cancer compared with other cancer types. From the literatures, the correlation between p53 mutation and HPV infection is controversy. LOH of p53 has also been found in cervical cancers from several studies. LOH of p53 also related to the progression of this malignancy, but not related with HPV status. The p53 polymorphism failed to be a individual risk factor in predicting the outcome of patients with cervical cancer. In order to determine the role of various p53 aberrations in the development of cervical cancer, further epidemiological surveys should be undertaken in larger populations and in different geographical regions.

Acknowledgements

I do wish to thank Geneva Foundation for Medical Education and Research organizing this wonderful program.

Thanks IAMANEH for supporting me to join this course.

I also thanks all teachers for giving us wonderful lessons on methodology and advanced reproductive health.

Finally I am very grateful to my tutor Dr. Anis Feki, for helping me with this review correction.

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Figure 1:

Fig 2. Worldwide distribution of cancers and p53 mutations.