The prognostic value of p53 positive in colorectal cancer: A retrospective cohort study

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1 703651TUB / Tumor BiologyWang et al. research-article2017 Original Article The prognostic value of p53 positive in colorectal cancer: A retrospective cohort study Tumor Biology May 2017: 1 7 The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalspermissions.nav DOI: journals.sagepub.com/home/tub Peng Wang 1, Jianwei Liang 1, Zheng Wang 1, Huirong Hou 2, Lei Shi 1 and Zhixiang Zhou 1 Abstract This retrospective cohort study aimed to discuss the prognostic value of p53 positive in colorectal cancer. A total of 124 consecutive patients diagnosed with colorectal cancer were evaluated at the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College from 1 January 2009 to 31 December The expression of p53 in colorectal cancer was examined by immunohistochemistry. Based on the expression levels of p53, the 124 patients were divided into a p53 positive group and a p53 negative group. In this study, 72 patients were in the p53 positive group and 52 in the p53 negative group. The two groups were well balanced in gender, age, body mass index, American Society of Anesthesiologists scores, and number of lymph nodes harvested. p53 positive was associated with carcinoembryonic antigen 5 ng/ml (p = 0.036), gross type (p = 0.037), degree of tumor differentiation (p = 0.026), pathological tumor stage (p = 0.019), pathological node stage (p = 0.004), pathological tumor node metastasis stage (p = 0.017), nerve invasion (p = 0.008), and vessel invasion (p = 0.018). Tumor site, tumor size, and pathological pattern were not significantly different between these two groups. Disease-free survival and overall survival in the p53 positive group were significantly shorter than the p53 negative group (p = and 0.025, respectively). Colorectal cancer patients with p53 positive tended to be related to a higher degree of malignancy, advanced tumor node metastasis stage, and shorter disease-free survival and overall survival. p53 positive was independently an unfavorable prognostic marker for colorectal cancer patients. Keywords p53 positive, colorectal cancer, prognostic value, survival Date received: 24 October 2016; accepted: 17 January 2017 Introduction Colorectal cancer (CRC) is one of the most common cancers in China. According to a recent survey, 1 the estimated number of newly diagnosed cases in China in 2015 was 376,300. With a more Westernized diet and improved life expectancy for Chinese, there will be a higher incidence of CRC. 2 Although improvements in surgical procedures and advances in chemotherapy and radiotherapy methods have been made, the 5-year survival rate for CRC patients is still unsatisfactory. 3 With the advent of the era of precision medicine, it is urgent that we identify new prognostic factors and choose individualized treatments to improve this bad situation. The occurrence of CRC is a multiphase procession involving many biomarkers, of which p53 abnormal is one of most important and familiar events. The latest studies 1 Department of Colorectal Surgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China 2 The Overall Planning Office, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Corresponding author: Zhixiang Zhou, Department of Colorectal Surgery, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , China. Zhouzx001@163.com Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License ( which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (

2 2 Tumor Biology have demonstrated that p53 abnormal was detected among more than 50% of the CRC patients and was closely connected to the occurrence, development, infiltration, and metastasis of CRC. 4 6 The p53 protein coded by abnormal p53 gene had a longer half-life period so that it could be detected by immunohistochemistry (IHC). Several clinical studies have proved the prognostic value of p53 positive (overexpression or positivity) in patients with esophageal squamous cell carcinoma, 7 gastric cancer, 8,9 gallbladder cancer, 10 pancreatic cancer, 11 and non small cell lung cancer. 12 But the prognostic value of p53 positive in CRC remains unclear. Thus, a retrospective cohort study involving 124 patients with CRC was conducted to study the prognostic value of p53 positive by IHC and to analyze related pathological factors. Patients and methods Patients From 1 January 2009 to 31 December 2010, a total of 137 consecutive patients with CRC underwent surgery at National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. All patients underwent the same preoperative examinations, including routine blood test, carcinoembryonic antigen (CEA) test, chest X-ray, electrocardiogram examination, computerized tomography of abdominal cavity, and magnetic resonance imaging of pelvic cavity (for patients with rectal cancer). Every patient was given a preoperative grade by anesthetists according to the American Society of Anesthesiologists (ASA). The American Joint Committee on Cancer (AJCC, the 7th ed.) 13 staging system was used for tumor staging. The reasonable advice of postoperative therapy was based on National Comprehensive Cancer Network (NCCN) guides corresponding time points. Postoperative therapies, including chemotherapy and chemoradiotherapy, were all performed by oncology clinicians and radiation therapists at our institution. Patients were asked to visit doctors every 3 months within the first 2 years, every 6 months for the next 3 years, and every year following those 5 years. The end of follow-up time was set at 30 June A total of 13 patients were excluded from this study for the following reasons: 6 patients underwent neoadjuvant therapy, 2 patients with number of lymph nodes harvested less than 12, 3 patients with no formal postoperative treatments according to NCCN guidelines, 2 patients with palliative resection. So, 124 patients with CRC were included in our study. The study was approved by the ethics committee at the National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. The demographic and pathological results were recorded in our database: gender, age, body mass index (BMI), ASA scores, CEA level, tumor site, gross type, tumor size, number of lymph nodes harvested, tumor differentiation degree, pathological pattern, pathological tumor node metastasis (ptnm), nerve invasion, and vessel invasion. Disease-free survival (DFS) was defined as the period from surgery to death or disease recurrence. Overall survival (OS) was defined as the period from surgery to death. Immunohistochemical staining Surgical tissues of CRC were fixed in paraformaldehyde for 24 h. Tissue sections of 4 µm thickness were made after paraffin embedding and then were placed at room temperature for 12 h. After being dewaxed in xylene and rehydrated in alcohol, endogenous peroxidase activity was eliminated by 3% hydrogen peroxide for 15 min at room temperature. Then, the tissue sections were immersed in citrate buffer (ph 6.0) and incubated in a microwave oven at 100 C for 10 min. The sections were then washed with double-distilled water (ddh 2 O) followed by phosphate-buffered saline (PBS) washing for three times. A volume of 5% bovine serum albumin (BSA) was used for blocking tissue sections for about 1 h; the p53 primary antibody was diluted at the rate of 1:100 and incubated with the sections at 4 C overnight. The secondary antibody was incubated for 1 h. After being dyed with 3,3 -diaminobenzidine (DAB), the sections were counterstained with hematoxylin, washed with PBS, dehydrated with alcohol (sweetening alcohol by xylene), and sealed with neutral resin. The results were observed by three different pathologists. Based on the Rommel score, 14 p53 expression levels were graded semiquantitatively, and five grades were divided according to positive percentage (0: 0%, 1: 10%, 2: 50%, 3: 80%, 4: >80%). p53 positive was defined as a staining of more than 10% of tumor cells. Statistical analysis All data were analyzed using Statistical Package for the Social Sciences (SPSS) 21.0 for Windows (SPSS Inc., Chicago, IL, USA). Quantitative data following normal distribution were given as a form of mean ± standard deviation, and they were analyzed by t test. Qualitative data were given as a form of number, and they were analyzed using the chi-square test or Fisher s exact test. The Kaplan Meier method was used for analyzing the survival distribution, and the survival curve was analyzed by logrank test. Cox regression was used to analyze multivariate factors in evaluating prognostic factors, and hazard ratio (HR) and 95% confidence interval (CI) were used to assess the relationships between these factors and DFS and OS. A two sided p value less than 0.05 was considered as statistically significant.

3 Wang et al. 3 Figure 1. Representative images of p53 staining in (a) 0%, (b) 10%, (c) 50%, (d) 80%, and (e) >80% (bar: 100 µm). Results Patient characteristics and pathological results Representative images of p53 staining were shown in Figure 1. A total of 124 patients with CRC were finally included in our study: 72 p53 positive patients (age (years): ± 12.33) and 52 p53 negative patients (age (years): ± 11.54). Differences in gender, age, BMI, and ASA scores between the two groups were not statistically significant. But patients with CEA 5 ng/ml in the p53 positive group were more than in the p53 negative group (p = 0.036; Table 1). The comparisons of pathological factors were also shown in Table 1. There were no statistically significant differences in tumor site (p = 0.948) and number of lymph nodes harvested (p = 0.216) between the two groups. CRC patients with p53 positive tended to have poor differentiation degree (p = 0.026); bigger tumor size (p < 0.001); advanced pt, pn, and ptnm stage (p = 0.019, 0.004, and 0.017, respectively); nerve invasion (p = 0.008); and vessel invasion (p = 0.018). Patients with infiltrative type and mucinous adenocarcinoma occupied the majority in the p53 positive group, while protruded type and canalicular adenocarcinoma occupied the majority in the p53 negative group and the differences were statistically significant (p = and 0.021, respectively). Recurrence analysis Among the 124 CRC patients, 26 patients in the p53 positive group and 9 patients in the p53 negative group underwent recurrence after surgery, of which the p value was Time interval from surgery to recurrence of p53 positive patients was shorter than for the p53 negative group (36.44 ± vs ± months), but the differences were not statistically significant (p = 0.062). Survival time from recurrence to death or the end of follow-up of p53 positive patients was also shorter than patients in the p53 negative group (3.92 ± 1.29 vs 7.50 ± 2.07 months, p < 0.001; Table 2). Prognosis analysis The follow-up time was between 66 and 90 months. The survival time was between 8 and 90 months. Univariate analysis by the Kaplan Meier method have shown that gross type, differentiation degree, pathological pattern, pt stage, pn stage, nerve invasion, vessel invasion, and p53 positive were all related to the prognosis of CRC patients (p < 0.05; Table 3). CRC patients with p53 positive had significantly shorter OS and DFS than those with p53 negative (p = and 0.021, respectively; Figure 2). Multivariable analysis by Cox regression showed that p53 positive, together with higher malignant pathological pattern, poor differentiation degree, advanced pt stage and pn stage, and nerve invasion were all independently unfavorable prognosis predictors of patients with CRC (Table 4). Discussion With improvements in lifestyle, there has been a dramatic increase in patients with CRC all over the world, especially

4 4 Tumor Biology Table 1. Demographics and pathological results. Parameters p53(+) (n = 72) p53( ) (n = 52) p value Gender Male Female Age (mean ± standard deviation, years) ± ± BMI (mean ± standard deviation, kg/m 2 ) ± ± ASA score CEA level (ng/ml) < Site Ascending colon 14 8 Transverse colon 3 2 Descending colon 4 3 Sigmoid colon Rectum Gross type Protruded type Ulcerative type 7 11 Infiltrative type Colloid type 9 3 Tumor size (mean ± standard deviation, cm) 4.81 ± ± 1.24 <0.001 Number of lymph nodes harvested (mean ± standard deviation) ± ± Differentiation degree Good Moderate Poor 25 8 Pathological pattern Canalicular adenocarcinoma Mucinous adenocarcinoma 29 9 Papillary adenocarcinoma 14 9 Signet-ring cell carcinoma 3 2 pt stage pn stage ptnm stage I 5 6 II III IV 8 3 Nerve invasion Vessel invasion BMI: body mass index; ASA: American Society of Anesthesiologists; CEA: carcinoembryonic antigen; ptnm: pathological tumor node metastasis; pt: pathological tumor; pn: pathological node.

5 Wang et al. 5 Table 2. Comparisons of recurrence after surgery. Variable p53(+) group p53( ) group p value Number Time interval (months) ± ± Survival time after recurrence (months) 3.92 ± ± 2.07 <0.001 Table 3. Univariate analysis of the prognostic roles in CRC patients. Parameters DFS (p value) OS (p value) Gender Age ( 60 years) CEA ( 5 ng/ml) Site Gross type <0.001 <0.001 Tumor size ( 5 cm) Differentiation degree <0.001 <0.001 Pathological pattern <0.001 <0.001 pt stage pn stage <0.001 <0.001 Nerve invasion Vessel invasion p53 positive CRC: colorectal cancer; DFS: disease-free survival; OS: overall survival; CEA: carcinoembryonic antigen; pt: pathological tumor; pn: pathological node. in China. In spite of improvements in comprehensive treatment of CRC, including surgery, chemotherapy, and radiotherapy, the 5-year OS and DFS remain about 45% and 40%, respectively, 15 so it remains a crucial health problem. As to tumor heterogeneity, it is necessary to detect new prognostic factors in order to achieve precise treatment to improve the poor prognosis at a molecular level. The occurrence of CRC is multiphased, with many factors involved, of which p53 mutation is an important incident from advanced adenoma to cancer (Figure 3). p53, as one of most widely studied cancer suppressor genes, plays an important role in regulating the cell cycle, repairing DNA damage, eliminating free radicals, regulating immune responses, and so on. 4,16 18 However, p53 abnormal has close biological connections with the occurrence, development, infiltration, and metastasis of over 50% of CRC patients. 4 6 After eliminating several factors mentioned above that might affect the outcome, 124 patients diagnosed with CRC were included into our study, and 72 and 52 patients were divided into a p53 positive group and a p53 negative group, respectively, based on their p53 expression levels. The two groups were well balanced in gender, age, BMI, ASA scores, and number of lymph nodes harvested. Compared with the p53 negative group, patients in the p53 positive group showed a greater tendency toward CEA 5 ng/ml, poor differentiation, bigger tumor size, advanced pt stage and pn stage, nerve invasion, and vessel invasion. In the p53 positive group, the mucinous adenocarcinoma type and signetring cell carcinoma type, which meant a higher malignant degree compared with other pathological patterns, took up a larger proportion than in the p53 negative group (44.44% vs 21.54%, p = 0.021). Meanwhile, multivariable analysis showed that p53 positive together with higher malignant pathological pattern, poor differentiation degree, advanced pt stage and pn stage, and nerve invasion were all unfavorable prognostic predictors of CRC patients. Figure 2. DFS (left) and OS (right) survival curve of CRC patients in relation to p53 expression.

6 6 Tumor Biology Table 4. Multivariable analysis of the prognostic roles in CRC patients. Parameters DFS OS HR 95% CI p value HR 95% CI p value Gross type Differentiation degree < <0.001 Pathological pattern pt stage pn stage < <0.001 Nerve invasion < <0.001 Vessel invasion p53 positive CRC: colorectal cancer; DFS: disease-free survival; OS: overall survival; HR: hazard ratio; CI: confidence interval; pt: pathological tumor; pn: pathological node. Figure 3. Multiphase ideograph of the occurrence of CRC. Cai et al. 19 showed that p53 expression was detected in 61.3% of their 117 patients with CRC with a signet-ring cell (SRC) component. The positive expression of p53 in CRC cases with an SRC component showed a significant association with decreased OS (p = 0.017). Perraud et al. 20 also found that p53 was an important marker of advanced tumor stages, and it appeared useful as a prognostic factor to predict recurrence-free survival. Similarly, Liu et al. 21 contained 62 (40.5%) patients with p53 positive protein expression. The log-rank test showed that there was an obvious difference in the OS between the p53 positive group and the p53 negative group (p < 0.001). Multivariable analysis by the Cox regression model further showed that p53 protein expression was an independent predictor of shorter OS in patients with completely resected CRC (HR = 1.77, 95% CI = , p = 0.009). Although several clinical studies revealed results corresponding with our study, there were also inconsistent conclusions. McGregor et al. 22 showed that aberrant p53 expression was found in 65% (482/740) of patients, and it was associated with distal location (p < 0.001) and stage III disease (p < 0.001) which was similar to our study results. However, no difference was observed in DFS and OS, which was inconsistent with our discovery. Furthermore, overexpression of p53 also predicted colorectal neoplasia risk in patients with inflammatory bowel disease (IBD) and mucosa changes indefinite for dysplasia (IND). Horvath et al. 23 conducted a study including 44 cases with IBD and IND diagnosed by colon biopsy. After a median follow-up of 101 months (range: 6 247), 11 (25%), progressed to neoplasia (low-grade dysplasia = 6, high-grade dysplasia = 2, cancer = 3) at a median follow-up of 66 months (range: ). Univariate analysis demonstrated that p53 overexpression was associated with progression to neoplasia. Cooks et al. 24 found that subjects with p53 mutation developed severe chronic inflammation and persistent tissue damage and were highly prone to inflammation-associated colon cancer for the reason that mutant p53 further prolonged activation of tumor necrosis factor alpha (TNF-α)-induced nuclear factor kappa B (NF-κB). These findings might explain the early appearance of p53 mutations in human colitis associated CRC. Detecting new prognostic markers is of great importance toward achieving precise treatment in order to improve the DFS and OS of patients with CRC. The tumor heterogeneity is partially determined by the differences in biomarkers. In this study, we only studied p53 as one possible microscopical prognostic marker which could be considered as a limitation of this study, but the incidence of other biological marker abnormalities, including K-ras, 25 BRAF, 26 DNA mismatch repair (MMR), 27 changed much less than p53. Furthermore, we excluded several macroscopical factors that would have influenced results, and we had a longer median follow-up. In addition, the two groups were well balanced in gender, age, BMI, ASA scores, and number of lymph nodes harvested. The final results were reliable, while bias might exist. So, further randomized controlled trials are needed to confirm this result. Conclusion In conclusion, CRC patients with p53 positive tended to be related to a higher degree of malignancy, advanced TNM stage, and shorter DFS and OS. p53 positive was independently an unfavorable prognostic marker for CRC patients.

7 Wang et al. 7 Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by The Capital Health Research and Development of Special (Grant No ). References 1. Chen W, Zheng R, Baade PD, et al. Cancer statistics in China, CA Cancer J Clin 2016; 66(2): Zeng WG, Zhou ZX, Hou HR, et al. Outcome of laparoscopic versus open resection for rectal cancer in elderly patients. J Surg Res 2015; 193(2): Tejpar S, Shen L, Wang X, et al. Integrating biomarkers in colorectal cancer trials in the West and China. Nat Rev Clin Oncol 2015; 12(9): Li T, Kon N, Jiang L, et al. Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and Senescence. Cell 2012; 149(6): Muller PA and Vousden KH. p53 mutations in cancer. Nat Cell Biol 2013; 15(1): Muller PA and Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell 2014; 25(3): Yao W, Qin X, Qi B, et al. Association of p53 expression with prognosis in patients with esophageal squamous cell carcinoma. Int J Clin Exp Pathol 2014; 7(10): Babacan NA, Egilmez HR, Yücel B, et al. The prognostic value of UHRF-1 and p5w3 in gastric cancer. Saudi J Gastroenterol 2016; 22(1): Çalik M, Demirci E, Altun E, et al. Clinicopathological importance of Ki-67, p27, and p53 expression in gastric Cancer. Turk J Med Sci 2015; 45(1): Cai W, Li Q, Yang Z, et al. Expression of p53 upregulated modulator of apoptosis (PUMA) and C-myb in gallbladder adenocarcinoma and their pathological significance. Clin Transl Oncol 2013; 15(10): Xiang JF, Wang WQ, Liu L, et al. Mutant p53 determines pancreatic cancer poor prognosis to pancreatectomy through upregulation of cavin-1 in patients with preoperative serum CA19-9 1,000 U/mL. Sci Rep 2016; 6: Xie D, Lan L, Huang K, et al. Association of p53/p21 expression and cigarette smoking with tumor progression and poor prognosis in non-small cell lung cancer patients. Oncol Rep 2014; 32(6): The American Joint Committee on Cancer (AJCC). 7th ed. New York, NY: Springer, Vieth M, Kushima R, Mukaisho K, et al. Immunohistochemical analysis of pyloric gland adenomas using a series of Mucin 2, Mucin 5AC, Mucin 6, CD10, Ki67 and p53. Virchows Arch 2010; 457(5): Binefa G, Rodríguez-Moranta F, Teule À, et al. Colorectal cancer: from prevention to personalized medicine. World J Gastroenterol 2014; 20(22): Vaseva AV, Marchenko ND, Ji K, et al. p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell 2012; 149(7): Riley T, Sontag E, Chen P, et al. Transcriptional control of human p53-regulated genes. Nat Rev Mol Cell Biol 2008; 9(5): Suzuki S, Tanaka T, Poyurovsky MV, et al. Phosphateactivated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactiveoxygen species. Proc Natl Acad Sci U S A 2010; 107(16): Cai X, Qi WX, Wang L, et al. Correlation of multiple proteins with clinic-pathological features and its prognostic significance in colorectal cancer with signet-ring cell component. Eur Rev Med Pharmacol Sci 2016; 20(16): Perraud A, Akil H, Nouaille M, et al. Expression of p53 and DR5 in normal and malignant tissues of colorectal cancer: correlation with advanced stages. Oncol Rep 2011; 26(5): Liu BW, Liu Y, Liu JR, et al. Prognostic effect of p53 expression in patients with completely resected colorectal cancer. Tumour Biol 2014; 35(10): McGregor MJ, Fadhil W, Wharton R, et al. Aberrant P53 expression lacks prognostic or predictive significance in colorectal cancer: results from the VICTOR trial. Anticancer Res 2015; 35(3): Horvath B, Liu GL, Wu XR, et al. Overexpression of p53 predicts colorectal neoplasia risk in patients with inflammatory bowel disease and mucosa changes indefinite for dysplasia. Gastroenterol Rep 2015; 3(4): Cooks T, Pateras IS, Tarcic O, et al. Mutant p53 prolongs NF-κB activation and promotes chronic inflammation and inflammation- associated colorectal cancer. Cancer Cell 2013; 23(5): Nagaoka T, Kitaura K, Miyata Y, et al. Downregulation of epidermal growth factor receptor family receptors and ligands in a mutant K-ras group of patients with colorectal cancer. Mol Med Rep 2016; 13(4): Pikoulis E, Margonis GA, Andreatos N, et al. Prognostic role of BRAF mutations in colorectal cancer liver metastases. Anticancer Res 2016; 36(9): Li SK and Martin A. Mismatch repair and colon cancer: mechanisms and therapies explored. Trends Mol Med 2016; 22(4):