TP53 gene deletion in esophageal cancer tissues of patients and its clinical significance
- Authors:
- Published online on: November 1, 2012 https://doi.org/10.3892/mmr.2012.1162
- Pages: 122-126
Abstract
Introduction
Esophageal cancer (EC) is one of the most common gastrointestinal cancers in the world and there is a clear regional and ethnic difference in terms of its incidence rate and etiology factors (1). The incidence of EC has been as high as 68.88/100,000 among the Kazakhs living in the Xinjiang Uyghur Autonomous Region (Northwest region of China). In China, the histopathological type of EC is different from that found in Europe and America and majority of cases are squamous cell carcinomas. EC is related to the activation of multiple genes and is a multi-step process. Oncogene activation and tumor suppressor gene inactivation are the basis for the development of molecular genetics. Currently, the p53 gene is the most important tumor suppressor gene and is located on human chromosome 17p13.1, which regulates the cell cycle and induces cell apoptosis (2,3).
Fluorescence in situ hybridization (FISH) is a cytogenetic technique that has gradually developed in recent years, involving the use of fluorescent labeled DNA probes to detect cell changes within the chromosome (4). Several studies have demonstrated that p53 gene deletion is important in the development and progression of EC (5–7). However, reports on the p53 gene deletion associated with Xinjiang Kazakh EC patients are absent.
In this study, we used FISH to detect TP53 gene deletion in 40 esophageal carcinoma cases in Kazakh patients, in order to analyze its clinical significance in evaluating patients’ prognosis based on molecular pathology.
Patients and methods
From October 2010 to December 2011, 40 Kazakh EC patients were admitted to the Department of Thoracic Surgery, First Affiliated Hospital of Xinjiang Medical University, and underwent surgical resection. There were 32 male and 8 female patients, with an average age of 56 years (ranging from 31 to 82). None of the patients had received any preoperative radiotherapy, chemotherapy or other special treatment.
Pathological diagnosis confirmed esophageal squamous cell carcinoma in 40 cases post-operatively, including well-differentiated tumors in 14 cases, moderately differentiated tumors in 12 cases and poorly differentiated tumors in 14 cases (Table I). Ten samples of normal esophageal tissues (>5 cm from the tumor) were collected as normal controls. Informed consent was obtained from all patients.
Touch preparations of cells were made on glass slides from fresh specimens and air-dried overnight at room temperature and then stored at −80°C ready for FISH. The same specimens were stained with hematoxylin and eosin (H&E) for pathological evaluation. FISH detection was followed by Nakamura’s improved Vysis protocol (8). Direct fluorochrome-labeled centromeric probes were used for enumeration of different chromosomes. Spectrum orange-labeled and spectrum green-labeled probes for the TP53 and centromere of chromosome 17 (CEP17) were purchased from the manufacturer (Vysis, Inc., Downers Grove, IL, USA).
Cells were denatured with 70% formamide and then washed twice in standard saline citrate (SSC) at 74°C and at room temperature, respectively, for 2 min in a water bath. The slides were then dehydrated through a graded ethanol series (70, 85 and 100%, each for 2 min). We then applied 10 μl of hybridization solution containing 1 μl of each of the DNA probes, 7 μl of hybridization buffer and 1 μl of double distilled water. This was covered with a cover slip and sealed with rubber cement. Following incubation for 16 h at 42°C in a humidity-controlled chamber, the slides were washed with an SSC solution for 5 min at 74°C and at room temperature for 2 min. Then 5 μl diamidinophenylindole (DAPI, II) was applied to each spot and covered with a cover slip. The slides were observed under a fluorescence microscope that was connected to a cooled charge-coupled device camera. According to the kit instructions, under a fluorescence microscope, a special image acquisition and analysis system (Leica Microsystems, Ltd., Germany) was used for the signal count. In total, 100 nuclei were observed to obtain the number of p53 gene signals, such as 1 or 0; when nucleus fluorescence was >30%, we determined p53 gene deletion.
Microsoft Social Sciences 15.0 software (SPSS software, Chicago, IL, USA) was used for statistical analysis. The corrected χ2 test or the Scheffe method were used for univariate analysis of data from each group. P<0.05 was considered to indicate a statistically significant difference.
Results
Orange (TP53) and green (CEP17) fluorescence signals were detected in all EC specimens. CEP17 hybridization signals were larger and brighter than TP53 gene signals. The representative FISH models included: normal, 2 intranuclear orange signals and 2 intranuclear green signals; partial deletion, ≥2 intranuclear green signals and 1 intranuclear orange signal; complete deletion, ≥2 intranuclear green signals and 0 orange signals (Fig. 1). Table I shows the TP53 gene deletion in the 40 esophageal carcinoma cases.
Table II shows the correlation between clinicopathological factors and TP53 deletion. Statistical analysis indicated that there were no significant correlations among TP53 gene deletion rate, age and gender (P>0.05). TP53 gene deletion rates were 28% (4/14) and 78% (11/14) in well-differentiated and poorly-differentiated EC, respectively (P=0.028). TP53 gene deletion rates were 38% (7/18) and 72% (16/22) in the lymph node metastasis and no lymph node metastasis groups, respectively (P=0.0313). A high frequency of CEP17 hyperdisomy was detected.
Discussion
The causes of EC are complicated. At present, EC is considered as a multi-factor, polygenic variant, multi-staged disease. The variation of correlated proto-oncogenes and tumor suppressor genes is the key to tumorigenesis and development. Deletion of the tumor suppressor gene is the major mode of variation (9).
The p53 gene is currently thought to be the most important tumor suppressor gene and more than 50% of tumors are associated with its abnormality. p53 genetic mutation is important in the development of EC. Studies have indicated that patients with p53 genetic mutation had poorer prognosis and greater malignancy (10,11). Multiple studies have proven that p53 genetic mutation is an independent and reliable biological parameter for evaluating the disease prognosis (12,13). Due to p53 gene deletion or abnormality, cells with injured DNA enter into S stage, resulting in changes to hereditary characteristics, chromosomal aberration and, ultimately, carcinomatous change.
FISH is a technique using fluorescently labeled single-chain nucleotide DNA probes, based on the base complementarity principle, to form double-stranded nucleic acid with complementary single chain nucleotides by specific binding, and to detect cell chromosome changes and definite gene copy number changes in order to diagnose malignant cells. FISH is capable of detecting all types of cytogenetic changes, including aneusomy, amplification and deletion. Recently, FISH has been used in the diagnosis of hematologic malignancy, lung, breast and renal cancer, and it is a technique with high sensitivity and specificity (4,14–17).
In the past, there were limitations in the detection of mutant p53 protein by immunohistochemical methods since viral infection and stress may also produce p53 protein aggregation, and therefore samples may have contained both mutant and wild-type variants. Several studies found that the p53 protein positive rate was lower than that observed using PCR-SSCP detection, which indicates that p53 mutation or abnormality in the malignant tumor is much higher than that shown by immunohistochemisty detection in real situations (18). The sensitivity of FISH is similar to isotope hybridization in situ, but the space resolution and gene mapping accuracy is higher; therefore, FISH plays a significant role in tumor studies (8,18,19).
In this study, FISH was applied using double-color DNA probes (fluorescent orange-labeled LSI TP53 probe and fluorescent green-labeled CEP17 probe) to detect TP53 gene deletion in Kazakh esophageal squamous cell cancer patients (7,20).
In the normal controls, in over 90% of nuclei, CEP17 and TP53 genes displayed 2 signals, indicating that FISH was successful in our study (data not shown). Among 40 EC cases, the mean TP53 gene deletion rate was 55% (22/40), and 28% (4/14), 58% (7/12) and 78% (11/14) in well-differentiated, moderately differentiated and poorly differentiated cases, respectively. This indicates that the TP53 gene deletion rate is correlated with the level of differentiation; the higher the differentiation, the higher the TP53 deletion rate. Moreover, the TP53 gene deletion rate also correlated with lymph node metastasis (P=0.0313). Similar studies support our results (21,22).
Our study used FISH to detect the TP53 gene in Kazakh patients with EC, and the results show for the first time that the TP53 gene deletion rate is correlated with the level of differentiation and lymph node metastasis in ECs, and may be an important molecular biological marker for evaluating the condition of ECs in Kazakh patients. Further study on the post-operative life span of these patients is required to confirm the correlation of TP53 gene deletion and EC prognosis.
Acknowledgements
This study was funded by the Xinjiang Uyghur Autonomous Region Key Discipline and Specialties Foundation (2010-09), and the First Affiliated Hospital of Xinjiang Medical Univeristy Research Award Foundation (2012-YFY-27). Special thanks to the Department of Hematology of the First Affiliated Hospital of Xinjiang Medical University for technical support.
Abbreviations:
|
FISH |
fluorescence in situ hybridization |
|
EC |
esophageal cancer |
|
CEP17 |
centromere of chromosome 17 |
|
SSC |
standard saline citrate |
References
|
Lu XM, Zhang YM, Lin RY, Arzi G, Wang X, Zhang YL, Zhang Y, Wang Y and Wen H: Relationship between genetic polymorphisms of metabolizing enzymes CYP2E1, GSTM1 and Kazakh’s esophageal squamous cell cancer in Xinjiang. World J Gastroenterol. 11:3651–3654. 2005.PubMed/NCBI | |
|
Schmale H and Bamberger C: A novel protein with strong homology to the tumor suppressor p53. Oncogene. 15:1363–1367. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Graesslin O, Chantot-Bastaraud S, Lorenzato M, Birembaut P, Quéreux C and Daraï E: Fluorescence in situ hybridization and immunohistochemical analysis of p53 expression in endometrial cancer: prognostic value and relation to ploidy. Ann Surg Oncol. 15:484–492. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Halling KC and Kipp BR: Fluorescence in situ hybridization in diagnostic cytology. Hum Pathol. 38:1137–1144. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Li WJ and Duan JX: Detecting of p53 gene heterozygosity loss in esophageal carcinoma tissues. JZU Med Sci. 39:499–501. 2004. | |
|
Hollstein MC, Metcalf RA, Welsh JA, Montesano R and Harris CC: Frequent mutation of the p53 gene in human esophageal cancer. Proc Natl Acad Sci USA. 87:9958–9961. 1990. View Article : Google Scholar : PubMed/NCBI | |
|
Audrézet MP, Robaszkiewicz M, Mercier B, et al: TP53 gene mutation profile in esophageal squamous cell carcinomas. Cancer Res. 53:5745–5749. 1993.PubMed/NCBI | |
|
Nakamura H, Saji H, Idiris A, et al: Chromosomal instability detected by fluorescence in situ hybridizationin surgical specimens of non-small cell lung cancer is associated with poor survival. Clin Cancer Res. 9:2294–2299. 2003. | |
|
Cheng ZQ, Wang XM, Shan J, et al: Detection of p53 gene deletion in primary lung cancer by dual-FISH technique and its clinical significance. Clin Med Chin. 26:368–371. 2010. | |
|
Uchino S, Saito T, Inomata M, Osawa N, Chikuba K, Etoh K and Kobayashi M: Prognostic significance of the p53 mutation in esophageal cancer. Jpn J Clin Oncol. 26:287–292. 1996. View Article : Google Scholar : PubMed/NCBI | |
|
da Manoel-Caetano FS, Borim AA, Caetano A, Cury PM and Silva AE: Cytogenetic alterations in chagasic achalasia compared to esophageal carcinoma. Cancer Genet Cytogenet. 149:17–22. 2004.PubMed/NCBI | |
|
Curtis C, Harris MD and Hollstein MC: Clinical implications of the p53 tumor suppessor gene. N Eng J Med. 329:1318–1325. 1993. View Article : Google Scholar : PubMed/NCBI | |
|
Smith DR, Ji CY and Coh HS: Prognostic significance of p53 over expression and mutation in colorectal adrecarcinomas. Br J Cancer. 74:216–223. 1996. View Article : Google Scholar : PubMed/NCBI | |
|
Fritcher EG, Brankley SM, Kipp BR, et al: A comparison of conventional cytology, DNA ploidy analysis, and fluorescence in situ hybridization for the detection of dysplasia and adenocarcinoma in patients with Barrett’s esophagus. Hum Pathol. 39:1128–1135. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Kawai T, Hiroi S, Nakanishi K, Sakurai Y and Torikata C: Abnormalities in chromosome 17 and p53 in lung carcinoma cells detected by fluorescence in situ hybridization. Pathol Int. 54:413–419. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Varshney D, Zhou YY, Geller SA and Alsabeh R: Determination of HER-2 status and chromosome 17 polysomy in breast carcinomas comparing Hercep Test and PathVysion FISH assay. Am J Clin Pathol. 121:70–77. 2004. View Article : Google Scholar : PubMed/NCBI | |
|
Fujiwara T, Cai DW, Georges R, et al: Therapeutic effect of a retroviral wild-type p53 expression vector in an orthotopic lung cancer model. J Natl Cancer Inst. 86:1458–1462. 1994. View Article : Google Scholar : PubMed/NCBI | |
|
Lu SM, Cheng CL and Sheng BY: Immunohistochemical detection of p53 protein altered expression in 1364 patients with maligant tumors and its clinicopathological significance. Chin J Cancer. 20:620–623. 2001. | |
|
Li JD, Li H and Shen ZY: Esophageal spontaneous apoptosis and nuclear value-added antigen, relationship with p53. Chin J Oncol. 20:415–417. 1998. | |
|
Idiris A, Madiniyet N, Hadeti B, Zhang Z, Ilyar S and Wen H: Molecular pathological diagnosis for early esophageal cancer in Kazakh patients. Oncol Lett. 13:549–553. 2012. | |
|
Abedi-Ardekani B, Kamangar F, Sotoudeh M, et al: Extremely high Tp53 mutation load in esophageal squamous cell carcinoma in Golestan Province, Iran. PLoS One. 6:e294882011. View Article : Google Scholar : PubMed/NCBI | |
|
King SI, Purdie CA, Bray SE, Quinlan PR, Jordan LB, Thompson AM and Meek DW: Immunohistochemical detection of Polo-like Kinase-1 (PLK1) in primary breast cancer is associated with TP53 mutation and poor clinical outcome. Breast Cancer Res. 14:402012. View Article : Google Scholar : PubMed/NCBI |
