Studies have shown that the tumor suppressor gene p53 may regulate thymidylate synthase (TS) activity in colorectal cancer (CRC) cells, hence attributed to chemo-resistance to 5-flurouracil in CRC. In this study, a total of 299 primary CRC patients who underwent surgery alone or received an adjuvant 5-FU-based chemotherapy were retrospectively studied. TS expression and p53 nuclear accumulation on paraffin embedded primary tumor tissue arrays were immunohistochemically assessed, and their relationship to patient overall survival (OS) and disease free survival (DFS) were analyzed. No correlation was found between TS and p53 expression. p53 nuclear accumulation was significantly correlated with tumor location. In all, multivariate analysis shows that TNM stage is a good indicator of patient survival. TS or p53 is not an independent prognostic or predictive factor in the CRCs. In chemotherapy-treated group, simultaneous analysis of TS and p53 indicates patients in the p53-/TS- or p53+/TS+ group have significant better OS and DFS than the group p53-/TS+ or p53+/TS- (P < 0.01). Thus, our study suggests that simultaneous evaluation of both TS and p53 can help to predict the therapeutic effect of CRCs with 5-FU-based adjuvant chemotherapy.
Colorectal cancer ranks the third most common cancer globally. Over 1.2 million new cases and 608,700 deaths were estimated to have occurred in 2008 [
Identifying molecular markers that can predict clinical outcomes would be of most importance to optimize individual therapy for CRC patients. The mechanism of cytotoxicity of 5-FU has been partially ascribed to the inhibition of the nucleotide synthetic enzyme TS, which consequently induces p53-dependent apoptosis [
In an effort to investigate the relationship of TS and p53, and their potential role as a prognostic or predictive factor, we analyze TS expression and p53 nuclear accumulation in colorectal cancer from 299 patients treated with surgery alone or with adjuvant 5-FU-based chemotherapy.
The patient cohort consisted of 299 patients, who underwent tumor resection between 2001 and 2006 at the First Affiliated Hospital of Sun Yat-sen University (SYSU). Among them, 75 (25.1%) patients received adjuvant 5- FU-based chemotherapy. Patients who received preoperative chemotherapy and/or radiotherapy were excluded from this study. Clinicopathologic data and follow-up data of enrolled patients were maintained by specialists. The study was approved by the Ethics Review Board of the Sixth Affiliated Hospital of SYSU. A written informed consent from each patient regarding tissue sampling had been obtained.
The paraffin-embedded tissue blocks and the corresponding histological H&E stained slides were overlaid for tissue TMA sampling. Duplicates of 1 mm diameter cylinder were punched from representative tumor areas of individual donor tissue block and re-embedded into a recipient paraffin block at a defined position using a tissue-arraying instrument (MiniCore, ALPHELYS, France).
Immunohistochemical staining was performed using the Polink-2 plus® Polymer HRP Detection System (GBI, WA, USA) according to the manufacturer’s instructions. After deparaffinization in xylene and rehydration through a graded alcohol series, slides were transferred to sodium citrate buffer (Beijing Dingguo Changsheng Biotech Co. Ltd, #AR-0511, China) for 15 min in the microware and left at room temperature for 30 min. Endogenous peroxidase was blocked with 0.3% hydrogen peroxide for 10 min at room temperature, then the slides were incubated with primary antibody to human TS (1:100, Proteintech #15047-1-AP, USA) or p53 (1:50, CST #2527, USA) overnight, respectively. The p53 (7F5) rabbit monoclonal antibody detects endogenous levels of both wild-type and mutant human p53 protein. Slides were washed three times with phosphate-buffered saline (PBS) and incubated with Polymer Helper (reagent 1, Polink-2 plus® supply) and Poly-HRP anti-Goat IgG (reagent 2, Polink-2 plus® supply) for 30 min. Then the slides were stained with DAB and counterstained with hematoxylin. For negative control, isotype-matched antibodies were applied. Staining results were reviewed and scored independently by two pathologists (Y.B. and L.F).
For p53 nuclear staining, cells were assessed according to the proportion of nuclear positive cells. The staining pattern was graded from 0 to 4, with 0 being no staining; 1 when < 25% of the cell nuclei stained positive; 2 when 25% - 50% of cell nuclei stained positive; 3 when 50% - 75% cells displayed nuclear staining; and 4 when >75% cells displayed nuclear staining.
To evaluate TS expression level, each slide was assigned a score for intensity and staining positive pattern. The percentage of positive tumor cells as follows: 1 (up to 25% of positive cells), 2 (25% - 50% of positive cells), 3 (50% - 75% of positive cells) and 4 (>75% of positive cells). Intensity scores ranged from 0 - 3: 0, no staining; 1, weak; 2, moderate, 3, strong. Multiplication of the two scores resulted in a final score ranging from 0 to 12.
Overall survival (OS) was calculated from the date of surgery to the date of death or the last follow-up time if follow-up was more than 5-years. For disease-free survival (DFS), an event was defined as the first clinical or pathologic evidence of local or distant recurrence.
Receiver Operation Characteristic (ROC) curve analysis was applied to determine the cut off point for tumor “high expression” by using the 0, 1-criterion. Under this condition, a score value of 8 was adopted as cut-off for stratification of TS expression into low (≤8, TS−) and high (>8, TS+); for p53 analysis, tumors were classified as p53-positive (p53+) if 50% or more cells demonstrated p53 nuclear accumulation (score > 2).
The relationship between TS, p53 and clinicopathologic features of CRC patients were analyzed by χ2-test. The Kaplan-Meier method was used for the univariate survival analysis, and the differences between compared groups were assessed by the log-rank test. The Cox proportional hazards regression model was used to compare OS and DFS between marker categories and to obtain risk ratios. All statistical analyses were performed using SPSS software version 16 (Chicago, IL, USA). A value of P < 0.05 was considered statistically significant (bilateral).
IHC stainings were assessed using anti-p53 and anti-TS antibodies (
The expressions of TS and nuclear staining of p53, as well as other clinicopathologic factors were further examined by Cox regression analysis. Univariate analysis in all patients indicated a significant association between TNM stage and the survival of patients (P < 0.001 for both OS and DFS). Tumor located in rectum was associated with a significantly worse OS (P = 0.004) and DFS (P = 0.004). These were confirmed in a multivariate analysis, which revealed that TNM stage and tumor site were independent prognostic factors for CRC patients (
In order to assess the relationship of TS and p53 level with treatment outcome, patients with 5-FU based chemotherapy were analyzed separately. In the chemotherapy-treated patients, 47 (72.3%) and 33 (44.0%) of 75
Parameter | n | p53 nuclear accumulation | TS expression | |||||
---|---|---|---|---|---|---|---|---|
Low | High | P | Low | High | P | |||
All patient | 299 | 161 | 138 | 124 | 175 | |||
Sex | 0.307 | 0.610 | ||||||
Male | 166 | 94 | 72 | 71 | 95 | |||
Female | 136 | 69 | 67 | 53 | 80 | |||
Age | 0.429 | 0.610 | ||||||
≥59 | 166 | 86 | 80 | 53 | 80 | |||
<59 | 133 | 75 | 58 | 71 | 95 | |||
Tumor location | 0.013 | 0.957 | ||||||
Colon | 138 | 85 | 53 | 57 | 81 | |||
Rectum | 161 | 76 | 85 | 67 | 94 | |||
TNM stage | 0.064 | 0.317 | ||||||
1 - 2 | 178 | 88 | 90 | 78 | 100 | |||
3 - 4 | 121 | 73 | 48 | 46 | 75 | |||
Chemotherapy | 0.484 | 0.401 | ||||||
No | 224 | 118 | 106 | 96 | 128 | |||
Yes | 75 | 43 | 32 | 28 | 47 | |||
Parameter | Value | Univariate | Multivariate | |||||||
---|---|---|---|---|---|---|---|---|---|---|
OS | DFS | OS | DFS | |||||||
HR (95% CI) | P | HR (95% CI) | P | HR (95% CI) | P | HR (95% CI) | P | |||
Sex | Male | 0.94 (0.61, 1.44) | 0.775 | 0.93 (0.61, 1.43) | 0.749 | |||||
Age | <59 | 1.47 (0.94, 2.32) | 0.201 | 1.26 (0.82, 1.96) | 0.292 | |||||
Tumor location | Rectum | 1.96 (1.24, 3.11) | 0.004 | 1.94 (1.23, 3.05) | 0.004 | 1.71 (1.07, 2.73) | 0.025 | 1.88 (1.20, 2.96) | 0.006 | |
TNM stage | TNM1/2 | 0.36 (0.23, 0.56) | <0.001 | 0.37 (0.24, 0.57) | <0.001 | 0.36 (0.23, 0.57) | <0.001 | 0.38 (0.25, 0.59) | <0.001 | |
Chemotherapy | Yes | 1.07 (0.65, 1.78) | 0.788 | 1.05 (0.64, 1.72) | 0.855 | |||||
p53 expression | Low | 0.96 (0.62, 1.49) | 0.853 | 0.93 (0.61, 1.43) | 0.739 | |||||
TS expression | Low | 1.11 (0.71, 1.74) | 0.638 | 1.14 (0.74, 1.76) | 0.548 | |||||
p53/TS level | p53−/TS+ | 1.72 (0.91, 3.25) | 0.098 | 1.61 (0.88, 2.95) | 0.125 | |||||
p53+/TS− | 1.63 (0.81, 3.26) | 0.170 | 1.42 (0.73, 2.77) | 0.307 | ||||||
p53+/TS+ | 1.13 (0.56, 2.26) | 0.741 | 1.10 (0.57, 2.13) | 0.774 | ||||||
tumor samples showed TS+ and p53+, respectively. No significant association was found between TS/p53 expression and patient characteristics, such as patient gender, age, TNM stage, and tumor location (
According to the p53 and TS statues, patients were divided into the following four groups: p53 positive and TS high (p53+/TS+, n = 19), p53 negative and TS low (p53−/TS−, n = 14), p53 negative and TS high (p53−/ TS+, n = 28), p53 positive and TS low (p53+/TS−, n = 14). There was no evidence of interaction between TS expression or p53 nuclear accumulation for either OS (P = 0.805 for p53, and P = 0.778 for TS) or DFS (P = 0.620 for p53, and P = 0.899 for TS). However, multivariate analysis revealed a significant association of combined phenotypes p53+/TS− with a worse OS (P = 0.022) and DFS (P = 0.025) (
Parameter | n | p53 nuclear accumulation | TS expression | |||||
---|---|---|---|---|---|---|---|---|
Low | High | P | Low | High | P | |||
All patient | 75 | 42 | 33 | 28 | 47 | |||
Sex | 0.954 | 0.301 | ||||||
Male | 48 | 27 | 21 | 20 | 28 | |||
Female | 27 | 15 | 12 | 8 | 19 | |||
Age | 0.439 | 0.446 | ||||||
≥59 | 31 | 19 | 12 | 18 | 26 | |||
<59 | 44 | 23 | 21 | 10 | 21 | |||
Tumor location | 0.424 | 0.571 | ||||||
Colon | 38 | 23 | 15 | 13 | 25 | |||
Rectum | 37 | 19 | 18 | 15 | 22 | |||
TNM stage | 0.525 | 0.244 | ||||||
1 - 2 | 39 | 21 | 18 | 17 | 22 | |||
3 - 4 | 36 | 22 | 14 | 11 | 25 | |||
Parameter | Value | Univariate | Multivariate | |||||||
---|---|---|---|---|---|---|---|---|---|---|
OS | DFS | OS | DFS | |||||||
HR (95% CI) | P | HR (95% CI) | P | HR (95% CI) | P | HR (95% CI) | P | |||
Sex | Male | 0.59 (0.25, 1.43) | 0.243 | 0.53 (0.27, 1.26) | 0.370 | |||||
Age | <59 | 1.43 (0.59, 3.43) | 0.427 | 1.58 (0.67, 3.73) | 0.124 | |||||
Tumor location | Colon | 2.02 (0.81, 5.06) | 0.135 | 2.08 (0.84, 5.18) | 0.092 | |||||
TNM stage | TNM1/2 | 0.40 (0.16, 1.01) | 0.054 | 0.43 (0.18, 1.05) | 0.045 | 0.34 (0.13, 0.88) | 0.026 | 0.36 (0.14, 0.91) | 0.031 | |
p53 nuclear accumulation | Low | 0.89 (0.37, 2.19) | 0.805 | 0.80 (0.33, 1.93) | 0.499 | |||||
TS expression | Low | 1.14 (0.45, 2.86) | 0.778 | 1.06 (0.44, 2.56) | 0.863 | |||||
p53/TS level | p53−/TS+ | 1.95 (0.75, 5.12) | 0.056 | 4.41 (0.95, 20.48) | 0.058 | 6.95 (0.90, 23.92) | 0.064 | 4.36 (0.93, 20.53) | 0.062 | |
p53+/TS− | 2.15 (0.82, 5.60) | 0.040 | 5.10 (1.00, 25.91) | 0.050 | 12.12 (1.42, 33.23) | 0.022 | 6.67 (1.27, 25.05) | 0.025 | ||
p53+/TS+ | 0.34 (0.12, 0.96) | 0.680 | 0.87 (0.12, 6.22) | 0.893 | ||||||
In the present study, we find that the immunohistochemical analysis of p53 and TS in colorectal cancer may be useful to predict the survival of patients who have received 5-FU based chemotherapy. TS or p53 alone is not an independent marker for the survival in CRC patients. However, the combination of the two markers acts as a good indicator for predicting 5-FU based chemotherapy outcome.
The tumor suppressor p53 is one of the most frequently mutated genes in human CRC [
TS is a key enzyme in catalyzing the methylation of deoxythymidine to thymidylate, an essential precursor for DNA replication [
Our results demonstrate that the analysis of TS and p53 together can be important in predicting chemotherapeutic efficacy in CRC patients to 5-FU based chemotherapy. Further studies on larger cohort are needed to verify the correlation between p53 and TS and clinical outcome in CRC patients treated with 5-FU based chemotherapy. If our observation is confirmed, TS and p53 status will need to be evaluated as a stratification criterion in future adjuvant treatment.
This study was supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars of Ministry of Education (No. 201300000237) and Medical Scientific Research Foundation of Guangdong Province (No. B2013161).