Abstract
Background/Aim: Identifying useful biomarkers is central to selecting optimal therapeutic strategies for esophageal squamous cell carcinoma (ESCC). Serum p53 antibody (S-p53Ab), squamous cell carcinoma antigen (SCC-Ag), and carcinoembryonic antigen (CEA) were investigated to evaluate the significance of single and combined tumor markers in determining the diagnosis and prognosis of ESCC. Materials and Methods: Serum samples were obtained preoperatively from 133 patients with histologically-confirmed ESCC, including 32 patients with stage I (24.1%). Levels of S-p53Ab were assessed by enzyme-linked immunosorbent assay, using a new version of a highly specific, quantitative kit. The cut-off value for S-p53Ab was 1.3 U/ml. Results: S-p53Ab was detected in 39.1% (52 out of 133) of patients with ESCC, including 40.0% (20 out of 50) of patients with early-stage ESCC. Positive rates for S-p53Ab, CEA, and SCC-Ag among patients with stage I ESCC (n=32) were 40.6%, 12.5%, and 31.3%, respectively. Positivity for S-p53Ab was not associated with positivity for CEA or SCC-Ag (p=0.249 and 0.747, respectively). The positive rate for diagnosis of ESCC increased from 39.1% to 65.4% when S-p53Ab was combined with SCC-Ag in this study. We found no significant correlation between the presence of S-p53Ab in ESCC and overall survival. Conversely, Cox regression analysis revealed that the International Union Against Cancer/TNM classification and systemic inflammation score were independent prognostic factors for ESCC in this series (hazard ratio(HR)=3.811, 95% confidence interval(CI)=1.548-9.378, p=0.004; and HR=2.218; 95% CI=1.087-4.523, p=0.029, respectively). Kaplan–Meier analysis revealed significant differences between patients with elevated S-p53Ab and SCC-Ag and patients with elevated levels of only one or neither of these factors (p=0.009). Conclusion: The diagnostic rate with S-p53Ab was better than that with SCC-Ag and CEA in patients with early-stage ESCC. Combined detection of S-p53Ab and SCC-Ag can markedly improve diagnostic sensitivity and may permit more accurate stratification of patients with ESCC.
Esophageal cancer is the eighth most common cancer worldwide and the sixth most common cause of cancer-related death (1). Although the standard techniques for detection and diagnosis, chemoradiotherapy, and surgery have all improved over time, the prognosis of advanced esophageal cancer remains poor. Histologically, esophageal cancer is classified into subtypes of squamous cell carcinoma (SCC) and adenocarcinoma, each with a specific cellular origin, pathogenesis and epidemiology. In the United States and other Western countries, about 50% of esophageal cancers are adenocarcinomas. On the other hand, in Asian countries, including Japan, about 90% of esophageal cancers are SCC. The predominant histological subtype of esophageal cancer is SCC, which comprises about 80% of all esophageal cancers worldwide (2). Thus, interest exists in new and useful biological markers that will permit more accurate stratification of patients with esophageal SCC (ESCC), improve clinical decision-making, and possibly contribute to more rational study designs and analysis. Circulating p53 antibodies in patients have been investigated for various types of carcinoma, including ESCC (3-9). Several studies have identified serum p53 antibody (S-p53Ab) as an early marker of malignant disease, an indicator for monitoring patients with malignant tumors during treatment, and a prognostic factor for patients with several types of tumors (10, 11). Although previous studies have attempted to evaluate the clinical value of S-p53Ab under different conditions, the role of S-p53Ab in patients with ESCC has not been clearly established. The present study therefore evaluated the clinical relevance of S-p53Ab and investigated whether its diagnostic value could be improved when combined with other biomarkers in 133 patients with ESCC, including 32 (24.1%) with stage I ESCC.
Materials and Methods
Patients. We retrospectively reviewed a database of 133 patients with primary ESCC who received treatment at the Department of Surgical Oncology at Nagasaki University Hospital (Japan) between September 2008 and May 2017. Written informed consent was obtained from each patient. This study complied with the standards of the Declaration of Helsinki and the current ethical guidelines and was approved by the Institutional Ethics Board (number 09062633) at the Nagasaki University Hospital. Patients who had received preoperative radiotherapy or who died within 30 days after treatment were excluded from this study.
Serum and tumor samples. Clinical investigation included staging in accordance with the International Union Against Cancer (UICC)/TNM classification (12). Serum samples were collected from the peripheral vein of each patient before treatment and stored at −80°C until assay.
Enzyme-linked immunosorbent assay (ELISA) for S-p53Ab. Levels of S-p53Ab were assessed by ELISA using an anti-p53 detection kit (MESACUP anti-p53 Test; Medical and Biological Laboratories, Nagoya, Japan). In brief, samples were added to microtiter wells coated with wild-type human p53 protein or control protein to detect nonspecific interactions and incubated for 1 h at 37°C. After washing, peroxidase-conjugated goat antihuman immunoglobulin G that binds to p53-Ab was added and plates were incubated for 1 h at 37°C. Substrate solution was then added and incubation continued for 30 min at 37°C. After the addition of stop solution, color development was assessed by measuring absorption at 450 nm using a spectrophotometer. Levels of S-p53Ab were determined from a calibration curve constructed from the specific signals of standards. S-p53Ab levels ≥1.3 U/ml were considered as positive, according to the instructions from the manufacturer (13).
Values for systemic inflammation score (SIS), carcinoembryonic antigen (CEA), and squamous cell cancer antigen (SCC-Ag). Routine laboratory measurements including albumin, differential white blood count, lymphocyte-to-monocyte ratio (LMR), and tumor markers such as CEA and SCC-Ag were performed on the same day to exclude any inflammatory-inducing effects of sequential preoperative examinations such as esophagogastroduodenoscopy or barium enema. According to our institutional criteria, cut-off values for serum CEA and SCC-Ag were 5.0 ng/ml and 1.5 U/ml, respectively. SIS was estimated as described previously (14). Briefly, patients with both decreased LMR (<4.44) and hypoalbuminemia (<4.0 g/dl) were assigned a score of 2. Patients with only one of these biochemical abnormalities were assigned a score of 1. Patients with neither abnormality were assigned a score of 0.
Relationships between pretreatment clinicolaboratory characteristics and serum p53 antibody (S-p53Ab) in patients with esophageal squamous cell carcinoma.
Statistical analysis. The significance of differences among groups was determined using the chi-squared test and Mann–Whitney U-test. Hazard ratios (HR) with 95% confidence intervals (CI) were calculated by uni- and multivariate analyses using Cox proportional hazards models. To assess the clinical characteristics most closely related to overall survival, multivariate analysis was performed using characteristics with values of p<0.05 in univariate analysis. Kaplan–Meier analysis and the log-rank test were used to compare survival curves among groups. Deaths before May 31, 2017 were included in this analysis. All data were statistically analyzed using JMP® version 10 software (SAS Institute, Cary, NC, USA) at a significance level of 0.05.
Results
Association between presence of S-p53Ab and clinicopathological features in patients with ESCC. Serum samples from 52 (39.1%) of 133 patients with ESCC were positive for S-p53Ab. The mean age of this group was 67.1 years (range=44-83 years), and the male-to-female ratio was 2.4:1. The remaining 81 patients (60.9%) were negative for S-p53Ab and had a mean age of 65.3 years (range=44-82 years) and a male-to-female ratio of 16:1. We analyzed clinicopathological features of these patients stratified by the presence of S-p53Ab. No significant relationships, except for sex, age, LMR, and SIS, were identified regardless of positive or negative results for S-p53-Ab (Table I).
Positive rates of serum p53 antibody (S-p53Ab), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC-Ag) in each classification stage.
Sensitivity of S-p53Ab, CEA and SCC-Ag in patients with ESCC. The sensitivity of a single serum tumor marker for detection of ESCC was highest for SCC-Ag (Figure 1). Sensitivities of S-p53Ab, CEA, and SCC-Ag according to UICC/TNM stage were as follows. Among the 32 patients with stage I ESCC, 13 (40.6%), 4 (12.5%), and 10 patients (31.3%) were positive for S-p53Ab, CEA, and SCC-Ag, respectively. Among the 18 patients with stage II ESCC, 7 (38.9%), 1 (5.6%), and 4 patients (22.2%) were positive for S-p53Ab, CEA, and SCC-Ag, respectively. We investigated the clinical relevance of S-p53Ab levels as a marker of early ESCC in patients with stages I and II ESCC. Figure 1 shows the positive rate for S-p53Ab, CEA, and SCC-Ag. Detection sensitivities of S-p53Ab plus SCC-Ag, CEA plus SCC-Ag, and S-p53Ab plus CEA combinations in the 133 patients were 65.4% (n=87), 49.6% (n=66), and 48.9% (n=65), respectively, with S-p53Ab plus SCC-Ag being the most sensitive set of biomarkers (p<0.01). Figure 2 shows the detection sensitivities of S-p53Ab plus SCC-Ag, CEA plus SCC-Ag, and S-p53Ab plus CEA according to UICC/TNM stage.
Characteristics of patients and surgical outcomes. Results from univariate analysis identified SIS, Glasgow prognostic score, CEA, SCC-Ag, C-reactive protein, and serum albumin as continuous variables that were prognostic factors for overall survival, along with sex, tumor depth, TNM stage, and treatment (Table II). Age at surgery, Charlson comorbidity index, tumor site, LMR and S-p53Ab showed no prognostic significance for overall survival. Based on our multivariate analysis, TNM stage and SIS were independent prognostic factors for overall survival (HR=3.811; 95% CI=1.548-9.378, p=0.004; HR=2.218; 95% CI=1.087-4.523; p=0.029, respectively). Median and minimum follow-up times for survivors were 1,092 days and 70 days, respectively.
Kaplan–Meier analysis demonstrated a significant difference in survival between two groups of patients: those negative for S-p53Ab or SCC-Ag, who had a mean survival of 2129 days (95% CI=1731-2527 days), and those positive for S-p53Ab and SCC-Ag, with mean survival of 1037 days 95% CI=619-1455 days; p=0.016) (Figure 3).
Discussion
Recently, a large-scale genomic analysis of ESCCs from patients in Japan has been conducted to determine the mutational landscape of this cancer (15). In that study, biallelic inactivation of TP53 was observed in most samples, suggesting its essential role in tumor initiation. A mutated TP53 gene leads to the synthesis of a mutant protein with an unusually long half-life, and massive overexpression of the protein products. Mutant p53 accumulation is immunogenic and often leads to p53Ab production in the serum of patients with various types of carcinoma (16-18). Several studies have demonstrated that S-p53Ab can serve as an early marker for detecting malignant disease, as an indicator of treatment effects in patients with malignant tumors, and as a prognostic factor for patients with several types of cancer (19-22). ESCC can be diagnosed at an early stage due to advances in medical technology, and testing potential biological markers for early diagnosis of ESCC is also important. Although previous studies have evaluated the clinical value of S-p53Ab under different conditions, the role of S-p53Ab in patients with ESCC has not been clearly established.
Positive rates of combinations of serum p53 antibody (S-p53Ab), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC-Ag) in each classification stage.
Uni- and multivariate Cox proportional hazard regression analysis for OS.
A: Overall survival among patients with esophageal squamous cell carcinoma according to positivity for serum p53 antibody (S-p53Ab), carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC-Ag). B: Disease-specific survival among patients with esophageal squamous cell carcinoma according to positivity for serum p53 antibody and squamous cell carcinoma antigen. Positivity for both markers was associated with significantly reduced overall survival.
The present study showed that ESCC was detectable in 52 (39.1%) of 133 patients before treatment using an ELISA kit for S-p53Ab, which is comparable with previous findings in patients with ESCC (2). Among the 32 patients with stage I ESCC, 13 (40.6%), four (12.5%), and 10 patients (22.2%) were positive for S-p53Ab, CEA, and SCC-Ag, respectively. We investigated the clinical relevance of S-p53Ab level as a marker of early ESCC in patients with stage I and II ESCC. Figure 1 shows that among 50 patients with these stages, 20 (40.0%), 5 (10.0%), and 14 (28.0%) were positive for S-p53Ab, CEA, and SCC, respectively (p=0.006). These data suggest that the S-p53Ab test would appear to be most sensitive for detecting ESCC at an early stage. On the other hand, the positive rate of S-p53Ab was lower than that for SCC-Ag in patients with stage III and IV ESCC, probably because the S-p53Ab test depends on the response of the immune system to mutant p53 protein (23). Notably, 90.4% of patients who were positive for S-p53Ab did not have a high CEA level, and only 40.4% of those positive for S-p53Ab had a high SCC-Ag level. The presence of S-p53Ab was not associated with serum CEA or SCC-Ag (p=0.249 and p=0.747, respectively), and S-p53Ab may thus serve as an early detection marker for ESCC independent of CEA or SCC-Ag. The positive rate for a diagnosis of ESCC increased from 39.1% to 65.4% when detection of S-p53Ab was combined with SCC-Ag. The positive rate of S-p53Ab plus SCC-Ag was significantly higher than that of CEA plus SCC-Ag and S-p53Ab plus CEA, suggesting that the combination of S-p53Ab and SCC-Ag was a very sensitive biomarker set for detecting ESCC (Figure 2). Although several studies have identified S-p53Ab as a prognostic factor for patients with various types of tumor, its prognostic value in patients with ESCC remains controversial (20-22). The combination of S-p53Ab and SCC-Ag can thus serve as a sensitive biomarker for ESCC and may permit more accurate stratification of patients with ESCC. A potential limitation of this study was the retrospective, single-center design. A large-scale prospective validation study is thus needed to confirm these results.
In conclusion, our study demonstrated that the combination of S-p53Ab and SCC-Ag can offer a useful biomarker set for ESCC and should be incorporated into traditional clinical and pathological variables to complement outcome prediction for patients with SCC after treatment. This, in turn, should improve clinical decision-making, and may contribute to more rational study design and analysis.
Footnotes
Conflicts of Interest
No financial or other potential conflicts of interest exist for any of the Authors.
- Received December 26, 2017.
- Revision received January 25, 2018.
- Accepted January 29, 2018.
- Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
