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High pretreatment serum CA19-9 level predicts a poor prognosis for patients with stage III colon cancer after curative resection and adjuvant chemotherapy

Carbohydrate antigen 19-9 (CA19-9) is one of the most widely used tumor markers in gastrointestinal cancer. However, serum CA19-9 is not a recommended routine measurement in colon cancer. In this study, we evaluated the value of the preoperative serum CA19-9 level for the prediction of postoperative prognosis in stage III colon cancer. The medical records of 367 consecutive patients with stage III colon cancer who underwent curative resection followed by adjuvant chemotherapy with oxaliplatin and capecitabine between December 2007 and April 2015 were retrospectively reviewed. We determined the optimal cutoff value of CA19-9 for 3-year recurrence using the receiver operating characteristic (ROC) method. Differences in disease-free survival (DFS) and overall survival (OS) rates stratified by CA19-9 level were compared by using Kaplan-Meier and log-rank tests. A Cox proportional hazards model was used to identify prognostic variables for DFS and OS. The statistically determined best cutoff value for CA19-9 was 24 U/ml. High CA19-9 levels (> 24 U/ml) were significantly associated with poorly differentiated tumors, abnormal carcinoembryonic antigen (CEA) levels, and a high cumulative incidence of lung metastasis. Additionally, compared with low CA19-9 levels, high preoperative CA19-9 levels were associated with inferior 3-year DFS and OS rates, especially for high-risk patients (T4Nany or TanyN2). Multivariate analyses revealed that CA19-9 was an independent factor associated with both DFS (hazard ratio [HR], 2.248; 95% confidence interval [CI], 1.393-3.628; P = 0.001) and OS (HR: 2.081; 95% CI: 1.137-3.808; P = 0.017). The results of this study showed that high levels of preoperative serum CA19-9 indicated a worse prognostic outcome for stage III colon cancer patients. An early follow-up protocol to assess lung metastasis and a full course of adjuvant chemotherapy should be used for these patients.


Introduction
Colorectal cancer (CRC) has been one of the leading malignant tumors that threaten human health in both the United States and China [1,2]. At diagnosis, approximately one-third of patients exhibit regional lymph node metastasis and stage III colon cancer [3,4]. As a standard of care, curative tumor resection combined with adjuvant chemotherapy is well established and widely accepted for the Ivyspring International Publisher treatment of patients with stage III colon cancer [5,6]. Due to improvements in adjuvant chemotherapy, the 5-year relative survival rate for patients with stage III colon cancer increased from 55% during the years 1975-1977 to 74% during the years 2006-2012 [7]. However, not all patients derived benefit from curative-intent adjuvant chemotherapy; 20% to 30% of patients ultimately developed distant metastases [8,9]. Herein, an abbreviated shortened duration of adjuvant therapy might be a feasible way to avoid or reduce toxicities in some patients without impairing oncologic outcomes. As a result, the adjuvant chemotherapy needs an optimization of the duration according to the post-operative recurrence risk of the patients. It was necessary to explore effective prognostic markers for risk stratification and the further individualization of postoperative follow-up and postoperative therapy regimens.
Peripheral blood can be obtained noninvasively through a simple procedure that is easily accepted by patients. Therefore, the detection of serum tumor biomarkers was in line with the requirements of tumor specificity and the principle of noninvasive examination. Serum carbohydrate antigen 19-9 (CA19-9) has been known as a tumor biomarker in colon carcinoma for almost 40 years [10]. In addition, CA 19-9 is secreted by normal biliary epithelium, and its level can be markedly elevated because of benign biliary disease and inflammatory processes [11,12]. To date, serum CA19-9 is one of the most frequently applied tumor biomarkers in gastrointestinal cancers and is used particularly for detecting and monitoring pancreatic cancer [13,14] and bile duct cancer [15]. However, the current guidelines of the European Society of Medical Oncology (ESMO) and the National Comprehensive Cancer Network (NCCN) recommended the routine measurement of preoperative CEA levels but not of CA19-9 levels for postoperative surveillance [5,16]. In fact, previous studies have demonstrated that preoperative CA19-9 levels can be used as an additional marker to monitor the disease process after CRC resection in patients who do not exhibit an increased CEA level [17,18]. Nevertheless, evidence for the prognostic or predictive values of the preoperative CA19-9 level in patients with stage III colon cancer receiving curative treatment remains unavailable. Accordingly, we hypothesized that the preoperative serum CA19-9 level was able to divide patients with stage III colon cancer into subgroups with different prognoses. Therefore, the current study aimed to determine whether preoperative serum CA19-9 levels presented prognostic value for stage III colon cancer patients undergoing curative resection followed by oxaliplatin-containing adjuvant chemotherapy.
Accordingly, we determined the optimal CA19-9 cutoff value and subsequently evaluated the association of CA19-9 level with postoperative metastatic patterns and long-term survival rates in order to stratify risk and optimize postoperative therapy.

Patient selection
In this study, the medical records of 450 consecutive patients were reviewed. All patients were diagnosed with stage III colon cancer and underwent tumor resection and followed by adjuvant chemotherapy between December 2007 and April 2015 at Sun Yat-sen University Cancer Center. Patients who met the following inclusion criteria were enrolled: (1) histologically confirmed adenocarcinoma; (2) American Society of Anesthesiologists (ASA) class I-II status; (3) performance of a complete curative resection for colon tumor; (4) treatment with XELOX adjuvant chemotherapy (oxaliplatin and capecitabine); (5) absence of preoperative anticancer treatment; and (6) a postoperative follow-up period of at least 3 months. The patient demographics, tumor characteristics, and adjuvant chemotherapy cycles were carefully recorded. The tumor stage was classified according to the 2010 American Joint Committee on Cancer staging system. All procedures were performed according to the ethical standards of the 2013 World Medical Association Declaration of Helsinki. The informed consent requirement was waived based on the nature of this retrospective study, in which patient data was kept confidential. This study was approved by the Institutional Review Board and Independent Ethics Committee of Sun Yat-sen University Cancer Center.

Perioperative evaluation and treatment
Preoperative blood tests, including measurement of CA19-9 and CEA levels, at the time of diagnosis were carried out within 2 weeks of tumor resection. Preoperative imaging by contrast-enhanced computed tomography (CT) of the chest, abdomen, and pelvis was performed to confirm the preoperative stage of the patients. All patients underwent standard complete mesocolic excision (CME) with regional lymphadenectomy for colon tumor. XELOX adjuvant chemotherapy with 130 mg/m 2 oxaliplatin administered intravenously on Day 1 and 1000 mg/m 2 capecitabine administered orally twice daily on Days 1-14 for a 3-week cycle was recommended to begin 3-6 weeks after surgery. The postoperative tumor pathological staging was confirmed by two independent pathologists. Patients with tumors staged as T4Nany or TanyN2 were classified as the high-risk group, while patients with tumors staged as T1-3 and N1 were classified as the low-risk group.

Follow-up
Follow-up data were recorded from the tracking system of our center. Patients were monitored through subsequent visits every 3 months for the first 2 years and then semiannually for 5 years after tumor resection. Clinical examination, assessment of CEA and CA19-9 levels, and chest radiography were conducted every 3 months. Chest/abdominal/pelvic CT and colonoscopy were performed annually. Overall survival (OS) was defined as the interval from the date of tumor resection to the date of death from any cause or to the last follow-up. Disease-free survival (DFS) was defined as the interval from tumor resection to the date of disease recurrence, death or the last follow-up. The final follow-up visit occurred in April 2018.

Statistical analysis
The optimal CA19-9 cutoff values for the prediction of 3-year DFS and OS were identified by receiver operating characteristic (ROC) curve analysis according to the highest Youden index by using the Cutoff Finder software program (http://molpath.charite.de/cutoff/). The rest of the data were analyzed using IBM SPSS statistics software, version 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 6.01 (GraphPad, Inc., La Jolla, CA, USA). Comparisons between the groups of patients with high and low levels of CA19-9 according to the clinicopathological variables were performed with chi-squared or Fisher's exact tests. The OS and RFS rates were estimated with the Kaplan-Meier method, and the differences between groups were then assessed with the log-rank test. Parameters for which P < 0.05 for OS and DFS in the univariate Cox models were further assessed in multivariate Cox models. Hazard ratios (HRs) and 95% confidence intervals (CIs) were subsequently calculated. All of the statistical tests were two-sided; P < 0.05 was considered significant.

Relationships between CA19-9 level and clinicopathological characteristics
Relationships between CA19-9 level and different clinicopathological characteristics are presented in Table 1. High CA19-9 levels had a significantly greater association with poorly differentiated and undifferentiated tumors than did low CA19-9 levels (35.1% vs. 25.0%, P = 0.047). In addition, patients with a high CA19-9 level were more likely than patients with a low CA19-9 level to exhibit an abnormal CEA level (64.0% vs. 32.8%; P < 0.001). No significant differences were observed between patients with low and high CA19-9 levels in terms of age, sex, baseline hemoglobin, tumor location, TNM stage, or number of cycles of adjuvant chemotherapy.

Discussion
Our study evaluated the prognostic value of the preoperative serum CA19-9 level in stage III colon cancer patients receiving curative treatment. In total, we presented three major findings. First, we found that a high preoperative CA19-9 level indicated a higher risk of postoperative lung metastasis. Furthermore, our final results also revealed a significant correlation between elevated pretreatment serum CA19-9 levels and poor prognosis in stage III colon cancer patients, especially high-risk patients. Finally, our multivariate analysis identified a high preoperative CA19-9 level as an independent prognostic factor for 3-year OS and DFS. To the best of our knowledge, the current study is the first to demonstrate the prognostic value of the preoperative CA19-9 level in patients with curatively resected stage III colon cancer treated with the XELOX regimen.  Previous studies identified that CA19-9 was a prognostic factor for colorectal cancer in that patients with elevated baseline CA19-9 probably showed a high risk of postoperative metastasis, which contributed to a worse survival rate [19,20]. Our results were consistent with those of these previous reports; we showed that patients with a high CA19-9 level presented a significantly increased incidence of postoperative metastasis (32.4% vs. 15.2%, P < 0.001) compared with patients with a low CA19-9 level. CA19-9 clearly belongs to a cell surface glycoprotein and is involved in cellular adhesion. Cancer cells expressing this protein may present a greater metastatic and invasive potential [21,22]. Moreover, the CA19-9 monosialoganglioside may be involved in the occurrence of tumor cell-induced platelet aggregation, which promotes the process of distant metastases in CRC [23]. Based on the involvement of CA19-9 in tumorigenesis, the malignant biological behavior of elevated CA19-9 might contribute to tumor metastasis and chemotherapeutic resistance.
With respect to the metastatic pattern, we found that high preoperative levels of CA19-9 indicated a higher incidence of lung metastasis (12.6% vs. 3.9%, P = 0.002) and abdominopelvic metastasis (10.8% vs. 3.5%, P = 0.006) than did low preoperative levels of CA19-9. As shown in our results, high CA19-9 levels were significantly associated with poorly differentiated and undifferentiated tumors. According to the results of a population-based study, poor differentiation, which is a feature of aggressive and advanced tumors, was identified as a high-risk factor for peritoneal carcinomatosis of colorectal origin [24,25]. In line with our results, a previous study reported that high levels of CA19-9 were associated with a higher risk of lung metastasis and that the detection of preoperative CA19-9 levels might help predict lung metastasis [26,27]. Furthermore, our results revealed that the high-CA19-9 group had a threefold higher cumulative incidence of lung metastasis than did the low-CA19-9 group (12.0% vs. 3.9%, P < 0.001). Although the mechanisms underlying the increase in the incidence of lung metastasis remain unclear, early postoperative screening for lung metastasis with chest CT in patients with high levels of CA19-9 should be required. Early surveillance by a CT scan within 6 months after curative resection may be helpful for the treatment of stage III disease; salvage surgery for disease recurrence could thus be performed in a timely manner [28].
Interestingly, our result indicated that CA19-9 had a different impact on survival in high-and low-risk patients. There were no differences in the 3-year survival rate among the low-risk patients with different CA19-9 levels, while among the high-risk patients, the survival rates were significantly worse in those with high CA19-9 levels than in those with low CA19-9 levels. This difference could be mainly attributed to the disparate characteristics between colon cancer patients with different risk levels. In general, patients with advanced disease were more likely to suffer from aggressive tumors, a high tumor burden, and aggravated immunosuppression [8,29].
Additionally, previous studies have demonstrated that serum CA19-9 levels could accurately reflect tumor metabolism and tumor burden and thus may be suitable for monitoring treatment response and disease progression of pancreatic cancer [30,31]. Therefore, the preoperative serum CA19-9 level was more sensitive for predicting survival outcomes in patients with high-risk colon cancer.
Our present study also reveals that the completion of oxaliplatin-containing adjuvant chemotherapy was a protective factor for 3-year OS in all patients. However, the possible efficacy of the treatment choice was associated with multiple factors, including patient age, comorbidities, compliance to therapy, and disease status [32][33][34]. Recently, the results of the International Duration Evaluation of Adjuvant Therapy (IDEA) project recommended that the division of stage III colon cancer into a low-risk (T1-3N1) and a high-risk (T4N1-2 or TanyN2) group was necessary; this division could improve adjuvant treatment selection for the patients [35]. Patients in the high-risk group should be given a full 6 months of the FOLFOX or XELOX regimen [16]. Nevertheless, TNM stage was not sufficient to warrant the division of the patients with different survival rates and chemotherapeutic benefits. Serum CA19-9 level was identified as a biological characteristic that could be incorporated into a standard TNM staging system to further refine risk stratification and predict benefits in OS from adjuvant chemotherapy in patients with resected pancreatic cancer [36]. Once patients were found as having high-risk disease with a high serum CA19-9 level, the intensified or full course of adjuvant chemotherapy should be strongly recommended. Besides, serum CA19-9 measurement is very easily performed in clinical settings and has a low cost. Therefore, we recommend that the preoperative serum CA19-9 level should be applied as a prognostic tool in clinical practice in order to provide useful information for therapeutic choices for patients with stage III colon cancer.
Some limitations of this study should be acknowledged. First, this retrospective study was conducted with an uncontrolled methodology with a limited number of patients from a single center. Second, the lack of the validation for the main findings and conclusions was another limitation of the study. Therefore, the findings need to be validated in multiple-center or larger cohort studies in the future. Third, the short duration of follow-up was insufficient to evaluate 5-year survival outcomes. This limitation may have led to an underestimation of the prognostic impact of serum CA19-9 level on long-term survival. In addition, our study assessed the CA19-9 level at a single time point before surgery, but the relative changes in the CA19-9 level during adjuvant chemotherapy and follow-up were not evaluated. Previous studies have reported that a decrease in the CA19-9 level at week 8 can serve as an early marker for predicting chemotherapeutic efficacy [37]. Moreover, molecular features of tumors, such as microsatellite instability (MSI), CpG island methylator phenotype (CIMP) level, and BRAF and KRAS mutational status, were not evaluated in this study; an analysis of the association of CA19-9 with these molecular markers would help us further understand the impact of CA19-9 on tumorigenesis in colon cancer. Despite these limitations, our findings suggested that the serum CA19-9 level should be considered a routine preoperative measurement for patients with stage III colon cancer.

Conclusion
The preoperative serum CA19-9 level is an independent prognostic factor for patients with stage III colon cancer undergoing curative resection followed by adjuvant chemotherapy. Measurement of the preoperative serum CA19-9 level has clinical implications for risk stratification and the individualization of the follow-up procedures as well as for adjuvant therapeutic strategies.