Abstract: Background and Objective: According to the Saudi cancer registry reports, breast cancer is the first cause of cancer-related death in women and the eleventh cancer incidence in male of Saudi Arabia. Phosphatase and tensin homolog and tumor protein 53 PTEN and TP53 mutations in codons are relatively common in increasing prevalence of the most types of cancer incidence as tumor suppresser genes. The primary objective of this study was to investigate the mutations in PTEN and TP53 in both male and female patients with breast cancer in Saudi Arabia and the role of these mutations on the protein expression of PTEN and TP53 antibodies using immunohistochemical method. Materials and Methods: The present study encompasses 342 Saudi breast cancer patients that were carrier of either PTEN or TP53 gene mutations. The analysis was conducted using sanger sequence and immunohistochemical staining for protein expression. Results: The frequency of PTEN mutation in male and female was 22.0 and 78.0%, respectively. Similarly, the frequency of TP53 mutation among male and female was 19.2 and 80.8%, respectively. Seven distinct mutations were identified in PTEN and TP53 gene. The PTEN and TP53 mutations involving transitions, transversions and additions or deletions were identified in the present investigation. Inactivation of PTEN and overexpression of p53 by mutation has been detected in number of breast cancer samples as screened through immunohistochemical staining. Breast cancer samples were tested with anti-PTEN monoclonal antibody 6H2.1 and anti-p53 monoclonal antibody DO 7 for estimation of immunohistochemical expression. Conclusion: Overall 26.8% of the analyzed breast cancer tumors displayed loss of PTEN expression. The TP53 over expression was observed in 65.7% of the tumors.
INTRODUCTION
In the last few decades genetic testing for mutations in cancer susceptibility genes has been highly emphasized. However, owing to the expensive testing procedure and technical limitations, most of the mutations still remain unidentified or poorly understood. Several studies have linked genetic predispositions of cancer with mutations of tumor suppressor genes. Of the several genes, genetic testing of inherited mutations in PTEN and TP53 tumor suppressor genes have been extensively carried out to explore the detail mechanism underlying its role in tumorigenesis and tumor progression.
The PTEN (phosphatase and tensin homolog deleted on chromosome 10) and TP53 (in chromosome 17) are the most frequently mutated tumor suppressor genes have been implicated in the pathogenesis of breast cancer1. The PTEN encodes for a dual specific phosphatase protein. The TP53 gene encodes a 53-kd transcription factor that mediates tumor suppressor activity. These genes are known to regulate each other’s activity at both transcription as well as translational level2. Though inherited mutations of TP53 and PTEN are reported to be infrequent they are often found associated with early onset of breast cancer. The tumour suppressor activity of PTEN is mediated by protein phosphatase activity and lipid phosphatase activity. Lipid phosphatase activity is manifested by dephosphorylation of 3-phosphoinositide products (PIP3) of the phosphatidylinositol-3-kinase (PI3K), antagonizing AKT (also known as protein kinase B, PKB) signaling. In normal condition PTEN negatively regulates PI3K pathway responsible for cell proliferation, survival and increase in cell size. When PTEN is mutated AKT is activated leading to initiation of tumorigenesis3.
The tumor suppressor activity of TP53 is mediated by protein p53 through many auto-regulatory pathways with the involvement of several modulator proteins. The p53 protein is a multifunctional transcription factor and regulates either by inducing or repressing genes involved in regulation of cell cycle, DNA repair, differentiation, senescence and apoptosis4,5. Mutations in the TP53 tumor suppressor gene have been reported in15-30% of breast cancer patients6 and are thought to be associated with poor clinical prognosis7. Mutations alter p53 activity by activating several stress-induced regulatory pathways.
A comprehensive knowledge of PTEN and TP53 mutations in breast cancer is still insufficient in Saudi population. Present study addresses the mutation frequency of PTEN and TP53 in breast carcinoma tissues among Saudi male and female patients with breast cancer and to determine the level of gene expression patterns using immunohistochemistry method for archived formalin-fixed paraffin-embedded blocks and histomorphological portraits.
MATERIALS AND METHODS
Ethics statement: The current study was approved by the deanship of scientific research of Princess Nourah Bint Abdul Rahman University. Informed consent was taken from all of the breast cancer patients before collection of tumor samples from them.
Study population and samples collection: A total 342 samples of malignant breast tumors diagnosed between November, 2017 to January, 2018 were collected during surgeries from King Fahad Medical City (KFMC), Riyadh, Saudi Arabia. Only Saudi patients with breast cancer diagnosed within the age of 40 years were included in the study. Approximately 7-8 mm of breast tumors’ biopsies were immersed immediately in 10% buffered formalin in labeled tubes for one hour then these fixed biopsies were embedded in paraffin wax. All the specimens used in the study were coded and the patient’s confidentiality was preserved in accordance with the guidelines for studies of human subjects.
DNA extraction: The DNAs were isolated from formalin fixed, paraffin embedded (FFPE) breast cancer tumor tissues (malignant) of 342 samples and 20 randomly selected non tumor tissues (benign) using Qiagen DNA isolation kit (Cat No./ID: 69506, Quigen, Hilden, Germany) according to the manufacturer’s recommendations. The ratio of DNA concentration and quality were estimated by using NanoDrop Spectrophotometer (Thermo Scientific, USA).
Targeted sequencing of PTEN and TP53 mutations: Mutational analysis for all coding exons of the PTEN and TP53 genes were assessed in 342 breast cancer samples (212 samples of female patients and 130 samples of male patients) through heteroduplex analysis8 or single-stranded conformation analysis9.
PCR and sanger sequencing for detection of PTEN and P53 mutations: Conventional Sanger sequencing was performed for the entire coding regions of PTEN and TP53 from 342 FFPE blocks. Primer 3 software was used to design the primers for all coding exons of PTEN and TP53 genes, the sequences of primers for PTEN and TP53 are provided in Table 1, respectively.
| Table 1: | PTEN and TP53 primers sequence |
| Table 2: | Anti-p53 and anti-PTEN antibodies used for immunohistochemistry profile |
MM-mouse monoclonal, PC-pressure cooker | |
The 50 ng of tumor DNA was used for each PCR analysis. Sequencing reactions were performed using BigDye® terminator v1.1 Cycle Sequencing kit for each purified PCR product. The products of sequencing reaction were purified using MontageTM SEQ96 Sequencing Reaction kit, followed by electrophoresis through Applied Biosystems 3130 Genetic Analyzer. Mutation peak reaching 20% height of the normal peak were estimated.
Tumor tissue preparation and histopathological examination: The histological features of malignant breast tissues were stained with hematoxylin and eosin stains (H and E). Different lesions from malignant tumor’s from male and female breast tissues (10 each) were selected randomly from the stained section. Similarly, 10 benign breast tumour tissues were selected as controls. The H and E sections were arrayed based on standard procedures10. The histological types investigated in the study were infiltrative ductal carcinoma, lobular ductal carcinoma and in situ ductal carcinoma.
Immunohistochemical stating for PTEN and TP53 antibodies: Immunohistochemical studies were performed in 114 breast cancer samples (41 with PTEN mutation and 73 with TP53 mutation) obtained from paraffin-embedded blocks. 10 benign breast tumour tissues were selected as controls. The 3 μm sections cut from paraffin wax blocks were mounted in saline-glass slides and air-dried overnight at 37°C, deparafinized in xylene and then rehydrated in graded alcohol (100 and 70%). Slides were incubated in 3% H2O2/methanol for 10 min, followed by washing in phosphate-buffered saline (PBS). The antigen retrieval procedure is mentioned in Table 2. The sections were incubated overnight in primary antibodies at 4°C. After washing in PBS, sections were incubated with biotinylated secondary antibody, followed by the streptavidin-biotin-peroxidase complex reagent. Immunostaining was visualized with 4,6-diamidino-2-phenylindole (DAPI). Negative control slides were obtained by omitting the primary antibody. The percentage of stained nuclei was scored as per standard procedure11. Briefly, expression of antigen in stained cellular components were estimated and scored based on positive or negative reaction with respective antibody. For p53 immunoreactions, the percentage of positive tumour nuclei were evaluated based on following score level: None (0%), weak (<10%), moderate (10-30%) or strong staining intensity (>50%). Tumors exhibiting more than 15% (cutoff values) positive reactions were considered p53 positive. For evaluation of PTEN expression a histoscore (H-score) was used. The intensity of staining score used in the study were 0, 1, 2 and 3 for none, weak, moderate and strong staining intensity. The proportion of positive cells was divided as follows: 1-10% (score 1), 11-50% (score 2) and more than 50% (score 3). The final numerical H-score for each case was obtained by multiplication of scores obtained in above categories.
RESULTS
Deleterious PTEN and TP53 gene mutation in Saudi breast cancer patients: The study included 342 samples with 130 male and 212 female breast cancer samples. The age at diagnoses of breast cancer for PTEN and TP53 mutation carriers were within 40 years. The complete coding region was screened for deleterious mutations in each sample, using both heteroduplex DNA analysis (HAD) and single strand conformation analysis (SSCA). Deleterious gene mutations were found in 114 of the 342 malignant Saudi studied (33.3%). Of the mutations identified, 36.0% were located in PTEN gene and 64.0% in TP53 gene. The frequency of PTEN mutation in male and female was 22.0 and 78.0%, respectively. Similarly, the frequency of TP53, mutation among male and female was 19.2 and 80.8%, respectively. Most of the mutations identified were missense or frame shift mutations. Interestingly, most of the mutations were detected within exon 8 of PTEN gene and exon 9 of TP53 gene. Seven distinct mutations of PTEN gene were identified, out of which 68T>C and 697C>G (exon 8) and 567AC>TG (exon 5) substitution mutation occurred in maximum number of individuals under study. The frequency of mutations ranged from 4.9-29.3% with most frequent mutation (29.3%) being the 68T>C substitution in exon 8. Similarly, in TP53 gene seven mutations were identified. The frequencies of these mutations varied from 2.7-32.9% among the TP53 gene mutations (Fig.1, Table 3). In TP53 gene the most frequent mutation being the 308 AC> GG substitution (32.9%) and 311 ins T (23.4%) in exon 9 and 119 del C (23.4%) in exon 4. The SSCA shift in exon 3 was detected in 2 breast carcinoma samples for 113 del C mutation, in 15 samples in exon 4 for 119 del C mutation and in 17 samples in exon 9 for 311 ins C mutation. None of these mutations were detected from 20 randomly selected benign samples. No frameshift mutation was detected in PTEN gene. No family history of carcinoma had been reported by the individuals included under study.
| Table 3: | The PTEN and TP53 sequence mutations with clinicopathological types in breast cancer |
| Fig. 1: | Frequencies of different mutations in PTEN and TP53 genes in Saudi breast cancer patients |
| Fig. 2: | Distribution of PTEN and TP53 mutations in Saudi male and female breast cancer patients |
Figure 2 shows distribution of PTEN and TP53 mutations in Saudi male and female breast cancer patients. Of the seven PTEN mutations detected, 697 C>G substitution mutations were predominantly found in male breast cancer patients. Similarly, out of seven TP53 mutations 113 del C mutations were found to be predominant in males. The 700 T>C, 697 C>G and 68 T>C substitution mutations of PTEN gene were detected in both male and female. In TP53 gene the 314 A>G substitution and119 del C mutations were found in both the sexes.
All PTEN mutations occurring between exons 1 and 9 and TP53 mutations occurring between exons 4 and 19 from breast cancer tumors were screened for mutational analysis. Mutations identified were transitions, transversions and additions or deletions (Fig. 3). The frequencies of these mutations were compared between male and female. Transitions occurred in 55.6% and transversions in 44.6% of male PTEN mutation carriers. Conversely, in female PTEN mutation carriers, transitions and transversions accounted for 43.8 and 56.2%, respectively (Fig. 4). No, addition or deletion was noted in either male or female PTEN mutation carriers. In males with TP53 mutation carriers only transitions and deletions were recorded. The frequency of transitions in male TP53 mutation carriers was 21.4%. Complex mutation like deletion accounted for 78.6%. In females with TP53 mutation carriers, the frequency of transitions and transversions accounts for 20.3 and 40.7%, respectively. Conversely, complex mutations like insertion and deletions accounted for 28.9 and 10.1%, respectively.
Histopathology and immunohistochemistry: The PTEN mutation carriers showed poorly differentiated infiltrating ductal carcinomas, higher mitotic counts and pleomorphism, few tubule formations compared to non PTEN mutation carrier or control patient (benign). Moreover, carriers of PTEN exhibited typical or atypical medullary carcinomas and are high-grade tumours. The H and E stained sections from the breast cancer patients showed increase in mitotic activity combined with dysregulation of proliferation in surrounded epithelial cells, nuclear pleomorphism, disruption in histological patterns involving invasive marked in the ductal breast carcinoma with irregular patterns of breast tumors and cytological atypia (Fig. 5a,b). The carrier of TP53 germline mutation showed intense and widespread staining and exhibited invasive lobular and tubular carcinomas. Strong nuclear staining was observed on majority of the tumor cells.
The expression of PTEN and TP53 was assessed using immunohistochemistry (Fig. 6, 7). Overall 41% of the analyzed breast cancer tumors displayed loss of PTEN expression. The PTEN immunohistochemistry revealed strong staining intensities in 21% and moderate staining in 10% of the tumor tissues. The TP53 over expression was observed in 73% of the tumors (Table 4).
| Fig. 3(a-b): | Sanger sequencing electropherograms of (a) PTEN and (b) TP53 gene sequence mutations from genomic DNA samples taken from male and female Saudi patients with breast cancer |
Respective exons are presented in Table 3 |
|
| Fig. 4: | Characterization of PTEN and TP53 mutations identified in Saudi male and female breast cancer patients |
| Fig. 5(a-b): | Photomicrographs of H and E stained sections from breast cancer patients carrying, (a) PTEN mutation and (b) TP53 mutation, showing increased mitotic activity combined with dysregulation of proliferation in surrounded epithelial cells, nuclear pleomorphism, disruption in histological patterns involving invasive marked in the ductal breast carcinoma (arrows) with irregular patterns of breast tumors and cytological atypia is lacking |
| Fig. 6(a-c): | Photomicrographs of immunostained labeling sections with DAPI dye from the breast cancer patients of p53 antibody were taken by confocal microscope with scale bar: 100 μm, (a) Immunohistochemical staining slide with p53 mutant (green) revealed overexpression of p53 protein in breast cancer tissue, (b) Slide stained with DAPI nuclear fluorescent staining dye (blue) and (c) combined slide of a and b slides |
| Fig. 7(a-c): | Photomicrographs of immunostained labeling sections with DAPI dye from the breast cancer patients of PTEN antibody were taken by confocal microscope with scale bar: 100 μm, (a) Immunohistochemical staining slide with PTEN mutant (green) revealed overexpression of PTEN protein in breast cancer tissue, (b) Slide stained with DAPI nuclear fluorescent staining dye (blue) and (c) Combined slide of a and b slides |
| Table 4: | Proportion of cancers showing greater than 75% of cells positive by immunohistochemistry (IHC) |
DISCUSSION
In the present study, the frequency of PTEN and TP53 gene in Saudis breast cancer patient was strongly positive. In normal conditions these genes maintain balance of the cell population thereby preventing tumor formation exhibiting its tumor suppressor activities. Mutation in these genes are known to predispose early-onset breast cancer1. The PTEN functions are down regulated due to mutations leading to stoppage or reduction of apoptosis and induction of cell cycle, both of which are involved in development of carcinoma12. The association of germline mutations of TP53 with early onset of breast cancer has been well eastablished13. Women with TP53 mutations were reported to have 50-90% life time risk of developing breast cancer and that with PTEN mutations to have 25-85% risk14.
Traditional Sanger DNA sequencing was used to characterize the mutational status of the PTEN and TP53 genes and deleterious gene mutations accounts for 33.3%. Seven distinct mutations of PTEN gene were identified, out of which 68T>C and 697C>G in exon 8) and 567AC>TG in exon 5 occurred in maximum number of individuals under study. Similarly, 7 distinct mutations of TP53 gene were identified, the most frequent mutation being the 308 AC>GG and 311 ins T in exon 9 and 119 del C (23.4%) in exon 4. The results corroborate with one of the earlier findings15. The TP53 is considered to be one of the frequently mutated gene in Arab breast cancer patients16. The prevalence rate of TP53 mutations being 29% which is at par to the present result. Mutational analysis of PTEN and TP53 revealed transitions, transversions and additions or deletions. Most of the mutations were single base pair substitutions. The finding is in consistence with some of the previous reports17.
Besides molecular analyses for tumor suppressor gene (TSG) mutation, histopathological phenotypes are successfully used to determine the carrier of PTEN and TP53 germline mutation. Breast cancer patients with germline mutation in TSG often exhibit different histological phenotypes with variable proliferation rates. In the present study breast cancer samples with PTEN and TP53 mutation displayed different histological features suggesting an alternative mechanism of molecular pathogenesis. Thus, the impact of germline mutation in variability of histological phenotype cannot be ruled out18.
The histological features of malignant breast tissues were stained with hematoxylin and eosin stains (H and E). The histological types investigated in the study were infiltrative ductal carcinoma, lobular ductal carcinoma and in situ ductal carcinoma. The malignant tissues were tested with anti-PTEN monoclonal antibody 6.H2.1 and anti-p53 monoclonal antibody DO7 to assess immunohistochemical expression. Patient with TP53 mutation carrier were found to exhibit higher level of p53 expression and high proliferation rate of histological features. Thus, overexpression of p53 protein is an indicative of TP53 alterations18. Over expression of p53 has been attributed to early-onset of breast cancers compared to later-onset breast cancers. Previous studies on TP53 mutation also identified increment in p53 expressions in TP53 mutation carriers. Majority of the TP53 mutations were IHC positive. Moderate to strong p53 expression was observed in 65.7% of tumors, while weak to complete absence of expression was observed in 34.2% of tumors. The present result is in concordance to one of the earlier studies19. However, expression of p53 was absent in benign control. Contrary to this, absence or decrement of PTEN expression was an indicative of PI3K-AKT pathway activation 20. The loss of PTEN expression is often invariably linked to breast cancers21. The monoclonal antibody 6H2.1 exhibited presence of molecular alteration of PTEN as manifested by variable immunostaining reactions. In contrast variable histological features were exhibited by PTEN and TP53 mutation carriers. More lobular and intraductal carcinomas were noted in patient with TP53 mutation carriers. No such variations were observed in tumours from control patients. Thus, it has been established from the present study that breast cancer tumours in carriers of PTEN and TP53 gene mutations differ morphologically and histopathologically. The association between PTEN loss or p53 over expression with pathogenesis of breast cancer has been well attributed.
SIGNIFICANCE STATEMENT
This study discovers tumor suppressor genes like PTEN and TP53 have been identified in breast cancer samples, their contribution to breast cancer in Saudi population is not well established. This study will help the researcher to use the molecular analysis of TSG mutations to be exploited as molecular biomarkers for effective management of breast cancer patients through surgical or chemotherapeutic interventions.
CONCLUSION
Although germline mutations of tumor suppressor genes (TSG) like PTEN and TP53 have been identified in breast cancer samples, their contribution to breast cancer in Saudi population is not well established. The present study revealed association of PTEN down regulation and positive p53 expression with the pathogenesis of breast cancer in Saudi population. The findings may help in the development of sensitive diagnostic tests for identification and screening of PTEN and TP53 mutation in breast cancer patients. The specific tests may be used in the early diagnosis and efficient prognosis of breast cancer. Moreover, molecular analysis of TSG mutations can be exploited as molecular biomarkers for effective management of breast cancer patients through surgical or chemotherapeutic interventions.
ACKNOWLEDGMENT
The authors would like to thank the Deanship of Scientific Research, Princess Nourah bint Abdulrahman University for research project was supported by a grant from funding center ID: (233-ص-38) .This research project and approved from IRB institutional review board NO.(18-0278).