Significance of plasma MACC1 levels on the prognostic stratification in patients with colorectal cancer

Abstract The clinical significance of metastasis‐associated in colon cancer‐1 (MACC1) has been investigated but the relevance of peripheral MACC1 levels was rather limited. Herein, our data revealed that plasma MACC1 levels in 117 colorectal cancer patients (CRC) were dramatically higher than that in normal controls (P < 0.001), and with a strong discrimination power between the two groups (AUC = 0.960, P < 0.001). Moreover, MACC1 is an independent prognostic factor for CRC patients. When clinical parameters stratified by MACC1low and MACC1high, MACC1 levels exhibited further significant predictive value. Summary, plasma MACC1 levels could be a useful prognostic and diagnostic biomarker, and could improve the prognostic value of traditional prognosticators for colorectal cancer patients.


| CRC patients
In total,117 consecutive CRC pre-operative plasma were included from April 2007 to May 2013 at Taizhou Hospital of Zhejiang province (National human genetic resources platform of China YCZYPT [2017]02). Clinical stage was according to the AJCC 7th TNM staging system. 6 Patient's overall survival was defined from the data of surgical operation to the last follow-up.
Written informed consent was obtained from each participant prior to the surgery, and this study was approved by the Institu-

| Statistical analysis
Group MACC1 comparison was analysed with Mann-Whitney U-test.
Receiver operating characteristics (ROC) analysis was performed and the cut-off value was determined by Youden's index. Survival probabilities were evaluated with Kaplan-Meier method, and differences in survival were analysed by the log-rank test. Statistical analysis was performed with SPSS v.13.0 (SPSS, Inc., Chicago, IL, USA). All statistical tests were two-sided and P < 0.05 was considered statistically significant.

| Relationship between plasma MACC1 levels and clinical variables in CRC patients
Plasma MACC1 levels in CRC patients and normal controls and comparison between the groups were detailed in Table S1. The median levels of MACC1 in CRC patients were notably increased than normal controls (16.91 ng/mL vs 1.51 ng/mL; P < 0.001), which could effectively distinguish the CRC patients from normal controls with the ROC curve (AUC = 0.960; P < 0.001). An optimal cut-off value was determined with the Youden's index for MACC1 was 3.43 ng/ ml, with the sensitivity (0.897) and specificity (0.948; Figure 1A).
In CRC patients, no significant association was found for MACC1 levels to gender, age, and primary tumour status (T). However, MACC1 levels were significantly associated with regional lymph node status (N). MACC1 in patients with N 0 , N 1 and N 2 was 15.56 ng/mL, 20.02 ng/mL and 23.43 ng/mL respectively (P = 0.010).
Much higher MACC1 levels were also observed in patients with M 1 than those with M 0 (45.21 ng/mL vs 16.77 ng/mL; P = 0.034), and in patients with AJCC III+IV than those with AJCC I+II (22.13 ng/mL vs 14.30 ng/mL; P = 0.004). Moreover, MACC1 levels in died CRC patients were dramatically higher than that in live CRC patients (25.99 ng/mL vs 10.84 ng/mL; P < 0.001; Table S1).   Figure 1B). Moreover, a worse survival was observed between patients with age above median (>67 years) vs younger (P = 0.037), N 1+2 vs N 0 (P < 0.001) and AJCC III+IV vs AJCC I+II (P < 0.001). However, no statistical difference was observed between male vs female (P = 0.509), T 3+4 vs T 2 (P = 0.508). Although the survival in patients with M 1 (n = 3) was much less than that with M 0 (n = 113), no significance was observed (P = 0.158), which may be caused by only three M 1 patients were included in the study (Table 1).
Among these variables, Cox's proportional hazards model analysis showed that plasma MACC1 levels is an independent prognostic marker for CRC patients (HR = 2.121, P = 0.004; Table S2).

| DISCUSSION
A wealth of studies have been carried out and strengthen the potential application of both MACC1 transcripts and protein expression as a novel diagnostic and prognostic indicator in various malignancies. 9,10 However, "liquid biopsies" such as peripheral circulating cellfree nucleic acids and soluble proteins, being minimal invasive, economical and routinely applicable, their special interest has emerged.

In this context, Stein et al firstly reported that circulating MACC1
transcripts is associated with metastasis and prognosis in CRC patients, where high MACC1 transcript levels are correlated with worse survival. 11 In this study, our data showed plasma MACC1 levels were markedly elevated in CRC patients, which could powerfully discriminate the CRC patients from normal controls. With the cut-off value of 3.43 ng/mL determined by Youden's index, the sensitivity and specificity was 0.897 and 0.948 respectively. In breast cancer, Tan et al 12 reported that, with the cut-off value 59.05 pg/mL (AUC = 0.757), the sensitivity and specificity were 0.714 and 0.891 respectively.
Though has a similar diagnostic power, the cut-off value between our study and that in Tan et al 12  Our results further revealed that patients with MACC1 high had an obviously worse survival and a 5-year survival rate compared with those with MACC1 low , and plasma MACC1 was an significant independent prognostic biomarker for CRC patients, strengthening the issue that MACC1 was a novel and reliable molecule in prognostic prediction as earlier studies emphasized. 1,10 Finally, we evaluate whether MACC1 levels in stratified clinical parameters could improve their prognostic power in CRC patients.
Data showed that patients with MACC1 high has much worse survival than those with MACC1 low in the male or female, younger or elder patients. This trend was also observed in subgroup patients with M 0 , N 0 , N 1 , T 2 , T 3+4 and stage AJCC III+IV .
In conclusion, our study revealed that plasma MACC1 is a clinical relevant biomarker in diagnosis and prognosis in CRC, and the incorporation of MACC1 levels with other stratified clinical parameters could improve their prognostic values for CRC subpopulations.