The prognostic value of preoperative serum CA724 for CEA-normal colorectal cancer patients

Study population

The medical records of the CRC patients who underwent surgery at the Department of Colorectal Anal Surgery, the First Affiliated Hospital of Guangxi Medical University of between September 2012 and September 2014 were collected. The cases were chosen according to the following inclusion criteria: (1) histopathological diagnosis of colon or rectal cancer; (2) primary tumor that can be radically removed; (3) complete clinical pathology report and postoperative follow-up data; (4) complete data of preoperative serum oncologic markers. The cases were excluded according to the following criteria: (1) lack of histopathological confirmation of colon or rectal cancer; (2) preoperative neoadjuvant treatment; (3) death cause other than CRC-related; (4) other simultaneous or heterogenic malignant tumors (such as lymphoma, leiomyosarcoma, stromal tumor, melanoma, etc.); (5) no signature on the informed consent form for disposal of biological specimens.

Collection of serum tumor marker and clinicopathological characteristics

Preoperative fasting venous blood (four mL) was collected on the second day after the admission of the patients and centrifuged within 1 h after collection. Serum that had not be analyzed within 6 h was stored in a refrigerator at −20 °C. The serum levels of CEA, CA724, carbohydrate antigen 199 (CA199), carcinoembryonic antigen 125 (CA125) and carcinoembryonic antigen 242 (CA242) were measured using a chemiluminescence immunoassay and the Elecsys 2010 Immunoassay Analyzer (Roche Diagnostics, Risch-Rotkreuz, Switzerland). The following concentrations were considered to indicate high expression: CEA > 5 ng/mL, CA199 > 37 U/mL, CA724 > 5.7 U/mL, CA242 > 20.0 U/mL and CA125 > 35.0 U/mL. These clinicopathological characteristics were collected in this study including age, gender, pathological tumor stage (pT stage), pathological node stage (pN stage), metastasis, tumor location, venous invasion, perineural invasion, pathologic type, differentiation, postoperative chemoradiotherapy. The patients were staged according to the American Joint Committee on Cancer staging manual (seventh edition). Radical operation was colorectal resection plus regional lymph node dissection (Cohen, 1991; Nascimbeni et al., 2002; West et al., 2009).

Survival follow-up

Patients were followed-up by telephone or outpatient once every 3 months for the first 2 years after surgery and then every 6 months thereafter. Follow-up examinations included measurements of serum tumor marker levels, chest, abdomen, pelvic imaging by computed tomography and electronic colonoscopy. The follow-up period was from the time of the discharge to the time of death or 1st July 2019. The censored data of this study was defined as the data of the patients who were survival at the final follow-up deadline. Overall survival (OS) was defined as the time from resection of colorectal cancer to death from colorectal cancer or the censored time. Disease-free survival (DFS) was defined as the time from diagnosis to the first recurrence.

Statistical analyses

All statistical analyses were conducted using IBM SPSS Statistics for Windows, version 24.0 (IBM Corp, Armonk, NY, USA). The Chi-square test was used to identify associations between serum CA724 levels and clinicopathological features. We applied Kaplan–Meier curve to estimate the survival curve of OS and DFS, which was compared with the log-rank test. Univariate, multivariate and subgroup survival analysis were performed by using Cox proportional hazards model. All factors that were identified as significant associated with OS and DFS by univariate analysis were subjected to multivariate Cox proportional hazards analysis. Nomograms for predicting OS and DFS were developed using R v3.2.5. A probability (P) value of <0.05 was considered statistically significant. The confidence level of confidence intervals at the 95% (95% CI) was used in this study

Ethics statement

The study protocol was approved by the Hospital Ethics Committee of the First Affiliated Hospital of Guangxi Medical University, Guangxi, China-Approval number: 2018 (KY-E-086). All patients signed informed consent for collection and analysis of biological specimens. This study was conducted in accordance with the Declaration of Helsinki.

Clinical characteristics

We collected medical records of 670 CRC patients. The process of case inclusion and exclusion in this study was shown in Fig. 1. There were 394 patients whose preoperative serum CEA was normal. We excluded patients who had received preoperative adjuvant therapy, incomplete data, non-colorectal cancer deaths and not radical resection. There were 65 patients who were lost to follow-up. Finally, the medical records of 295 CRC patients with normal CEA level were retrospectively reviewed. The median follow-up time of OS was 65 months and DFS was 64 months.

The characteristics of these patients were displayed in Table 1. Among those 295 patients, there were 223 having tumors in the left side and 72 in the right side. The pathological stages of the tumors were as follows: 98 as T1-2, 197 as T3-4 and 186 as N0, 71 as N1, and 38 as N2. The tumors of 261 patients were well/moderately differentiated and those of 34 were poorly differentiated. Venous invasion or perineural invasion was detected in 48 patients and metastasis was detected in 11 patients.

The correlation between preoperative serum CA724 levels and clinical features

Chi-square test was used to test the correlation between preoperative serum CA724 levels and clinical features (Table 1), which included age, gender, tumor location, pT stage, pN stage, differentiation, venous invasion, perineural invasion, metastasis and pathologic type. Among these clinicopathological features, only pathological node stage (pN stage) (X² = 14.514, P = 0.001) and differentiation (X² = 10.712, P = 0.001) were associated with serum CA724 levels.

Prognostic values of serum tumor markers

In the Kaplan–Meier analysis, the results revealed that the OS and DFS of the patients with high CA724 was poorer than those with normal (Figs. 2B and 2F), but there was no significant difference between CA199, CA242, CA125 high patients and CA199, CA242, CA125 normal patients (Figs. 2A, 2C–2E, 2G and 2H). And there was significant difference between CA724 high patients and those with normal in the Kaplan–Meier analysis of TNM stage III (Fig. 3). As shown in Tables 2 and 3, pT stage, pN stage, metastasis, vascular or neurological invasion, differentiation and serum levels of CA724 were significantly correlated with OS and DFS by univariate analysis. In the multivariate Cox regression analysis of OS, the result indicated that only pT stage (P = 0.039), pN stage (P < 0.001), metastasis (P = 0.001) and serum CA724 (P = 0.001) were independent prognostic factors for CRC patients with normal serum CEA levels. In the multivariate Cox regression analysis of DFS, the result showed the same independent prognostic factors namely pT stage (P = 0.036), pN stage (P < 0.001), metastasis (P = 0.003) and serum CA724 (P = 0.005) for CRC patients with normal serum CEA levels.

Subgroup analysis of clinical features

The results of multivariate Cox proportional hazards analysis suggested that CA724 was an independent prognostic risk factor for CRC patients with normal CEA. In order to further assess the prognostic value of CA724 in CEA normal patients, CA724 and other factors that identified significant association with DFS and OS by univariate analysis were subjected to multivariate Cox proportional hazards analysis for subgroup analysis of each clinical feature. The statistical results of CA724 in each subgroup were demonstrated in Fig. 4. In the analysis of 11 subgroups, there were statistical significances with DFS in the subgroups of female, age group (>65), T3–T4 stage, no metastasis, right side, no venous invasion and perineural invasion, well/moderate differentiation, no postoperative chemoradiotherapy, while there were statistical significances with OS in the subgroups of gender, age group (>65), T3–T4 stage, N2 stage, tumor location, venous invasion and perineural invasion, protrude type, ulcerative typ, well/moderate differentiation, postoperative chemoradiotherapy.

Nomogram for predicting survival outcomes

Two nomograms were established to evaluate the relationship between CA724 and medical rank in CRC patients with normal CEA (Fig. 5). Based on the results of multivariate Cox proportional hazards analysis, only CA724, pT stage, pN stage and metastasis were entered the risk model. The survival rate of 1–5 years could be predicted and CA724 played an important role in the nomogram.