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 2010. 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 = 0.021 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.
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 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 2016.
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 (ddH2O) 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 log-rank 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.
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): 56.86 ± 12.33) and 52 p53 negative patients (age (years): 57.40 ± 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).
Representative images of p53 staining in (a) 0%, (b) ≤10%, (c) ≤50%, (d) ≤80%, and (e) >80% (bar: 100 µm).
Demographics and pathological results.
BMI: body mass index; ASA: American Society of Anesthesiologists; CEA: carcinoembryonic antigen; pTNM: pathological tumor–node–metastasis; pT: pathological tumor; pN: pathological node.
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 = 0.037 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 0.022. Time interval from surgery to recurrence of p53 positive patients was shorter than for the p53 negative group (36.44 ± 22.44 vs 52.60 ± 21.37 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).
Comparisons of recurrence after surgery.
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 = 0.025 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).
Univariate analysis of the prognostic roles in CRC patients.
CRC: colorectal cancer; DFS: disease-free survival; OS: overall survival; CEA: carcinoembryonic antigen; pT: pathological tumor; pN: pathological node.
DFS (left) and OS (right) survival curve of CRC patients in relation to p53 expression.
Multivariable analysis of the prognostic roles in CRC patients.
CRC: colorectal cancer; DFS: disease-free survival; OS: overall survival; HR: hazard ratio; CI: confidence interval; pT: pathological tumor; pN: pathological node.
Discussion
With improvements in lifestyle, there has been a dramatic increase in patients with CRC all over the world, especially 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
Multiphase ideograph of the occurrence of CRC.
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 signet-ring 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.
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 = 1.15–2.71, p = 0.009). Although several clinical studies19–21 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: 19–145). 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.
Footnotes
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. 2016-2-4022).