P53 GENE: MUTATION AND IMMUNOHISTOCHEMICAL ANALYSIS IN PATIENTS WITH INVASIVE DUCTAL CARCINOMA OF BREAST

The p53 tumor suppressor gene is the most commonly mutated gene in cancer. In breast cancer, the presence of p53 gene alterations has been associate d with worse prognosis. This study was attempted to associate p53 gene mutations with its protein expre ssion in North Eastern Indian population. We used single-stranded conformation polymorphism to screen samples for mutations in five conserved regions, exons 4, 5, 6, 7 and 8, of the p53 gene. Mutations were confirmed by direct DNA sequencing. Samples we re also analyzed for expression of p53 immunohistochem ically. We found two critical mutations in the exon 4. A well known missense mutation at codon 72 (pro to arg) with a frequency of 47% was found which was significantly correlated with the immunohistochemic al analysis of p53 protein in such patients. A nove l nonsense mutation at codon 107 which leads to stop codon was also found. Although the occurrence of th is mutation was very less, we did not find expression of p53 protein immunohistochemicaly. We support tha mutation in p53 gene can be exploited as a prognost ic marker for the early diagnosis of breast cancer, although more clinical and epidemiological data is required to establish this claim.


INTRODUCTION
In the light of the existing literature breast cancer is the most prevalent cancer among women and affects approximately 1 million women worldwide each year comprising 18% of all female cancers (Baum, 2002). Several epidemiological studies have documented extensively for the incidence of breast cancer in Indian scenario (Badwe et al., 1990;Jussawalla et al., 1980;Kapoor et al., 1993). One in fifty eight women are affected by breast cancer in the age group of 30-70 years and are mainly seen in the urban areas. The well established epidemiological risk factors for the development of the breast cancer are age at diagnosis, family history, parity, age at the menarche and menopause, diet, socioeconomic status, history of exposure to radiation and use of oral contraceptive pills (Helmrich et al., 2009;Dupont and Page, 1985).
In the last decade several lines of evidence have correlated a significant association between the polymorphism of p53 gene and development of breast cancer (Lacroix et al., 2006). p53 plays a key role in Science Publications AJBB mediating cell response to various stresses, mainly by inducing or repressing a number of genes involved in cell cycle arrest, senescence, apoptosis, DNA repair and angiogenesis. The qualitative and quantitative activity of p53 depends on its integrity (mutation status), its amount and its specific post-translational modifications induced by the activation of the different stress-induced signaling pathways (Lacroix et al., 2006). This leads to variable patterns of association between p53 and a number of other co-regulatory proteins, of which some may be tissue-or cell type-specific. Several association studies between the p53 expression, polymorphism and development of breast cancer have been carried out in various geographical regions of India like Bombay, Eastern Rajasthan, Aligarh, Bangalore, Jammu, Surat and Chennai (Jussawalla et al., 1998;Kapoor et al., 1993;Sharma et al., 1994;Hussain et al., 1994;Nandakumar et al., 1995;Gadhia et al., 1995;Juneja et al., 1990;Lin et al., 2001;Nayak and Das, 1997;1999a;1999b;Nayak et al., 1996;Kannan et al., 2000;Keasri et al., 1997), In continuation, the present study was designed to explore three specific issues of p53 in the subjects suffering from Invasive Ductal Carcinoma (IDC) of breast among middle North-Eastern Indian population: (a) Does the exonic region of p53 contain any sequence aberration? (b). Is there any correlation between the mutational analysis of p53 with clinico-pathological data of the subjects? (c) Is there any correlation between the p53 mutation (if any) with expression status of p53 protein?

Subject Selection
A total of 62 (58 females and 4 males) fresh excision biopsies were obtained from the patients of Invasive Ductal Carcinoma with the mean age of 53.35±9.29 years (aged between 33 to 75). All the patients included in the present study were recruited from in patient department of Cancer Research Institute, North Eastern Railways, Lehertara, Varanasi, India, between January 2009 to March 2010 with their prior informed consent and their detailed history was obtained. Patients receiving chemotherapy and radiotherapy were excluded from the study. Study was approved by the institutional ethical committee.

Sample Collection
Tumor biopsies were collected directly from the surgical operation theater of the department of oncology in chilled phosphate-buffered saline from the patients who underwent mastectomy. Each freshly microdissected tumor was divided into two equal portions by use of a disposable scalpel. Slices were taken from the outer, viable, cellular region of one half for later DNA analysis and sequencing. Corresponding slices from the other half taken in 10% formalin were prepared for histopathological examination. The part of the tumor that was frozen was stored at -80°C until analysis. The adjacent tissue biopsy from each individual was taken as control.

DNA Isolation
Cellular DNA from collected breast tumor biopsies was isolated using standard proteinase K digestion, phenol-chloroform extraction and ethanol precipitation method routinely being used in our laboratory (Sambrook and Russell, 2001). The quality and concentration of DNA was measured on an ethidium bromide-stained 1% agarose gel and by standard spectrophotometric methods.

Single-Strand Conformation Polymorphism Analysis
Single-Strand Conformation Polymorphism (SSCP) analysis was performed as described by Michaelides et al. (1995). Depending on the PCR product concentration, 4±1.2 mL of the PCR product was added to the same volume of denaturing solution containing 800 µL formamide, 100 µL of 1% Bromophenol Blue, 100 µL of 1% xylene cyanol, 2 µL of 0.5 M EDTA and 1 µL of 10 M NaOH per 1 mL solution. Samples were denatured at 93°C for 5 min and transferred immediately to ice to prevent renaturation of DNA. Each sample was loaded onto a 10% polyacrylamide gel with 0.5 mm thickness. Electrophoresis was performed in 1X TBE at 4°C and at 19 mA for 5 to 7 h depending on the size of the PCR product. Each PCR product was electrophoresed at least three times to increase precision and to prevent false positive results. Bands were visualized after silver staining of gel, as described (Bourn et al., 1994).

Sequencing
Purified PCR products of the samples showing mobility shift on SSCP analysis and randomly chosen samples were sent for sequencing. Direct DNA sequencing using an automated ABI 3100 Genetic Analyzer (Bangalore Genei) using ABI's AmpliTaq FS dye terminator cycle sanger's sequencing chemistry.

Sample Processing for Histology and Analysis
Paraffin-embedded tumor sections on poly-L-lysine coated slides were dewaxed in xylene and rehydrated in ethanol and distilled water. Antigen retrieval in a microwave oven at 750W (two cycles for 10 minutes each) enhanced p53 antigen accessibility to antibodies. The expression of Estrogen Receptor (ER), Progesterone Receptor (PR) and HER2 (Human Epidermal growth factor Receptor-2) were also checked using specific antibodies. Positive p53 IHC staining was seen in the nucleus of cancer cells. IHC-positive samples were subclassified with regard to immunostaining intensity and extent according to graded scales that ranged from 1 to 3. Allred's method was used for the scoring of p53, ER and PR and for HER2 expression, Hercep Test; DAKO was used (Allred et al., 1990;Dako and Hoffman, 2002). Super sensitive polymer-HRP detection system and primary antibodies p53, ER, PR and HER2 from Biogenex, CA, USA were used. All slides were viewed and judged microscopically by the pathologist.

Statistical Analysis
Analysis was performed with the Statistical Package for Social Science (SPSS) windows version 16. Chisquare test was used to compare the frequencies.
Statistical significance was considered when p≤0.05.

RESULTS
Clinico-pathological features of the recruited patients are presented in Table 2. Most of the patients at diagnosis were either in tumor stage II (75.1%) or in stage III (24.1%). All patients underwent mastectomy.
Sequencing of amplified p53 exons (4 to 8) from all the recruited subjects were carried out to confirm the mobility shift bands detected on SSCP gel. Out of 62 patients, we found 29 subjects with p53 mutations. Table  3 summarizes the age wise frequency of patients with and without p53 mutations. We found 2 critical mutations in the exon 4 at codon positions 72 and 107 resulting in the missense and nonsense mutations, respectively (Fig. 1) as compared to the corresponding controls. Table 4 represents a summary of p53 gene mutations in breast cancer patients. No sequence aberration was found in exons 5, 6, 7 and 8 in the patients with respect to their corresponding control biopsies. The sequencing results were further confirmed by Single-strand conformation polymorphism analysis also (Fig. 2). The nonsense mutation at codon 107 occurred along with the missense one at codon 72. To search for suitable mutations of p53 gene PCR-SSCP analysis was used as a screening technique.
Out of 62 biopsies from patients, 27 were found to be p53 positive, further immunohistochemical analysis revealed a significant higher expression of p53 protein in the breast cancer biopsies as compared to their corresponding control biopsies (Fig. 3). A total of 51 (82.0%) tumors were found negative for ER, 54 (87.0%) tumors were found negative for PR and 44 (71.0%) were found positive for HER2 membrane staining. Interestingly we did not find the expression of p53 in the 2 patients having nonsense mutation. Table 6 summarizes the age wise frequency of patients with p53 expression. Correlation of menstrual status with p53 expression and HER2 expression was found to be significant, their p values being 0.031 and <0.001, respectively.           Tumor size was found to be significantly associated with ER negativity (p = 0.015), histological grade was found to be significantly associated with p53 expression (p = 0.018) and ALN involvement was also found to be significantly associated with p53 expression (0.025).

DISCUSSION
p53, a well established tumor suppressor gene has the centre of attraction for the researchers in the field of cancer biology for its ability to regulate several fundamental cellular processes like cell cycle arrest, senescence, apoptosis, DNA repair and angiogenesis (Lacroix et al., 2006). The status of p53 in human breast cancer has been the subject of intensive investigation (Gasco et al., 2002;Anne-Lise, 2003). The presence of mutation, with or without accompanying Loss of Heterozygosity (LOH), was an early finding in cell lines and primary breast cancers, establishing p53 as a bona-fide tumor suppressor gene in the breast. Epidemiological studies have documented extensively the incidence of breast cancer in the western population (Ursin et al., 1994) but the available information in India is limited. India is a vast sub-continent with a huge population of multiracial society with widely varying cultures and several ethnic groups. Different aspects of breast cancer involving epidemiology, risk factors and susceptibility have been dealt in various communities and regions. Some association studies between the p53 expression, polymorphism and development of breast cancer have been carried out in various geographical regions of India like Bombay, Eastern Rajasthan, Aligarh, Bangalore, Jammu, Surat, Chennai (Jussawalla et al., 1998;Kapoor et al., 1993;Sharma et al., 1994;Hussain et al., 1994;Nandakumar et al., 1995;Gadhia et al., 1995;Juneja et al., 1990;Lin et al., 2001;Nayak and Das, 1997;1999a;1999b;Nayak et al., 1996;Kannan et al., 2000;Keasri et al., 1997). The present study is an extension of these Indian studies for the geographically distinct middle north eastern population in order to explore the sequence aberration in p53 gene and its protein expression as well as correlation with demographic and clinical findings.
Our study comprised of 62 subjects with confirmed diagnosis of IDC of breast. Association of the p53 mutation was found significant with gender postmenopausal status, histological grade and ALN involvement which was consistent with existing data in the literature (Giordano et al., 2004;Cutler et al., 2009;Madigan et al., 1995). Our sequencing data revealed p53 gene mutations in 29 (47%) tumor biopsies from different individuals. We observed two critical mutations in the exonic region 4 of p53 ( Fig. 2  and 3). The missense mutation at codon 72 (pro to Arg) has been shown to have varying ethnic and geographical distribution (Chosdol et al., 2002;Ihsan et al., 2011;Kalemi et al., 2005). The frequency of this mutation in our breast cancer population was found to be around 50%. It has already been reported that the p53 Arg homozygous genotype could be a potential genetic risk factor for cancer (Buyru et al., 2003;Papadakis et al., 2000). Further our mutation data was correlated with the immunohistochemical analysis of significant high expression of p53 protein (Fig. 4). In healthy humans, the p53 protein is continually produced and degraded in the cell. The degradation of the p53 protein is associated with MDM2 binding. In a negative feedback loop, MDM2 is itself induced by the p53 protein (Haupt et al., 1997). However, mutant p53 protein often does not induce MDM2 and is thus able to accumulate at very high concentrations (Peng et al., 2001) and hence detectable by IHC. We reported another novel nonsense mutation at codon 107 which leads to the stop codon. Although the occurrence of this mutation is very less, but interestingly we did not find the expression the p53 protein by histological analysis which supports the translation of truncated p53 protein and ultimately leads to protein degradation (Heck et al., 2010).

CONCLUSION
Our data thus indicate that p53 gene can be exploited as a prognostic marker for the early diagnosis of breast cancer, although more clinical and epidemiological data across the Indian and Asian geographical regions is required to establish this claim.

ACKNOWLEDGMENT
We are indebted to all the patients and their family members who participated in this study. We would like to thank the nurses and the O.T. staff for helping us during the sample collection from the mastectomized tissues.