3.1. Serum mSEPT9-positive was closely related to the advanced-stage of CRC
Patients' complete baseline features and mSEPT9 groups of negative and positive were shown in Table 1. In the study, vascular invasion (P = 0.020), microsatellites (P < 0.001), histologic grade (P = 0.006), TNM stage (P < 0.001), tumor infiltration depth (P < 0.001) and distant metastasis (P < 0.001) were significantly associated with mSEPT9. No significant connection could be seen in gender, age, tumor location, and lymphatic metastasis with mSEPT9. Notably, we further explored the correlation between the status of mSEPT9 and TNM stage. The rate of mSEPT9+ in IV was significantly higher than that in stage I-III, especially in I (92.6% vs 17.4%, P < 0.001, Figure 2A). We also analyzed the association of the rate of positive mSEPT9 with the T stage (T1–T4), N stage (N0–N2), M stage (M0–M1), alonely. The rate of mSEPT9+ revealed a significant increase from T1 to T4 (Figure 2B), and mSEPT9+ rate in T4 was higher than that in T1 (72.2% vs 12.5%, P < 0.001). N stage with a slight change in the rate of mSEPT9+ and did not show connection (Figure 2C). As shown in Figure 2D, mSEPT9 had an excellent ability to make a distinction between local and metastatic CRC (P < 0.001).
Table 1
Correlations between serum methylated septin 9 gene and clinico-pathological features of colorectal cancer patients (n=144) (Chi square and Fisher’s tests).
Parameter
|
Negative group (%)
|
Positive group (%)
|
P
|
Gender
|
|
|
|
Male
|
31(36.0)
|
55(64.0)
|
0.121
|
Female
|
29(50.0)
|
29(50.0)
|
|
Age
|
|
|
|
≦60 yr
|
25(39.7)
|
38(60.3)
|
0.955
|
﹥60 yr
|
34(38.2)
|
55(61.8)
|
|
T stage
|
|
|
|
T1
|
7(87.5)
|
1(12.5)
|
<0.001
|
T2
|
13(76.5)
|
4(23.5)
|
|
T3
|
18(45.0)
|
22(55.0)
|
|
T4
|
22(27.8)
|
57(72.2)
|
|
N stage
|
|
|
|
N0
|
38(46.3)
|
44(53.7)
|
0.116
|
N1
|
13(46.4)
|
15(53.6)
|
|
N2
|
9(26.5)
|
25(73.5)
|
|
Distant metastasis (M)
|
|
|
|
M0
|
58(49.6)
|
59(50.4)
|
<0.001
|
M1
|
2(7.4)
|
25(92.6)
|
|
TNM stage
|
|
|
|
I
|
19(82.6)
|
4(17.4)
|
<0.001
|
II
|
19(38.8)
|
30(61.2)
|
|
III
|
20(44.4)
|
25(55.6)
|
|
IV
|
2(7.4)
|
25(92.6)
|
|
Vascular invasion
|
|
|
|
Absent
|
51(47.7)
|
56(52.3)
|
0.020
|
Present
|
9(24.3)
|
28(75.7)
|
|
Histologic grade
|
|
|
|
Low level
|
1(7.1)
|
13(92.9)
|
0.006
|
Medium level
|
57(44.9)
|
70(55.1)
|
|
High level
|
2(66.7)
|
1(33.3)
|
|
Location
|
|
|
|
Colon
|
26(31.7)
|
56(68.3)
|
0.066
|
Rectum
|
33(47.1)
|
37(52.9)
|
|
Microsatellite
|
|
|
|
pMMR
|
59(43.4)
|
77(56.6)
|
<0.001
|
dMMR
|
0(0.0)
|
16(100.0)
|
|
pMMR: proficient mismatch repair; dMMR: different mismatch repair. |
3.2. mSEPT9 was an independent risk factor for local recurrence or distant metastasis in stage II and stage III CRC patients after surgery.
At the follow-up period, 38.3% (36/94) of patients underwent local recurrence or distant progression, with 12 of stage II and 24 of stage III. The DFS rates were 73.4% and 61.4% at 12 and 24 months, respectively. The univariate analysis was revealed in Table 2. mSEPT9, TNM stage, tumor infiltration depth and lymphatic metastasis were significant with DFS (P < 0.05).
mSEPT9 showed a strong relation with DFS. Stage II and stage III patients with mSEPT9+ had a lower DFS rate than those with mSEPT9- (2-year DFS rates, 52.1% vs 73.9%, P =0.014, Figure 4A). The multivariate analysis informed that mSEPT9 had independent prognostic significance for CRC patients (HR = 2.741, P = 0.009), as well as TNM stage (HR = 3.010, P =0.006)(Table 2).
Table 2
Univariate and multivariate analysis of disease free survival in CRC patients with stage II-III.
Parameter
|
Univariate Analysis, HR (95% CI)
|
P
|
Mutivariate Analysis, HR (95% CI)
|
P
|
Gender
|
|
|
|
|
Male
|
1 (Referent)
|
0.384
|
-
|
-
|
Femal
|
0.728 (0.357-1.488)
|
-
|
-
|
-
|
Age
|
|
|
|
|
≦60 yr
|
1 (Referent)
|
0.929
|
-
|
-
|
﹥60 yr
|
0.970 (0.499-1.887)
|
-
|
-
|
-
|
T stage
|
|
|
|
|
T2-3
|
1 (Referent)
|
0.007
|
-
|
-
|
T4
|
3.346 (1.388-8.065)
|
-
|
-
|
-
|
N stage
|
|
|
|
|
N0
|
1 (Referent)
|
<0.001
|
-
|
-
|
N1
|
1.312 (0.508-3.384)
|
-
|
-
|
-
|
N2
|
5.290 (2.464-11.355)
|
-
|
-
|
-
|
TNM stage
|
|
|
|
|
II
|
1 (Referent)
|
0.005
|
1 (Referent)
|
0.003
|
III
|
2.789 (1.365-5.700)
|
-
|
3.010 (1.470-6.163)
|
|
Vascular invasion
|
|
|
|
|
Absent
|
1 (Referent)
|
0.085
|
-
|
-
|
Present
|
2.006 (1.015-3.964)
|
-
|
-
|
-
|
Location
|
|
|
|
|
Colon
|
1 (Referent)
|
0.289
|
-
|
-
|
Rectum
|
1.432 (0.738-2.780)
|
-
|
-
|
-
|
Microsatellite
|
|
|
|
|
pMMR
|
1 (Referent)
|
0.118
|
-
|
-
|
dMMR
|
2.129 (0.825-5.494)
|
-
|
-
|
-
|
mSEPT9
|
|
|
|
|
Negative
|
1 (Referent)
|
0.020
|
1 (Referent)
|
0.009
|
Positive
|
2.509 (1.175-5.359)
|
-
|
2.741 (1.281-5.865)
|
-
|
pMMR: proficient mismatch repair; dMMR: different mismatch repair;
mSEPT9: methylated septin 9 gene; CRC: colorectal cancer.
|
According to above analysis, mSEPT9 and TNM stage were selected to found the nomogram to appraise the risk of relapse or progression of CRC patients (Figure 3). Each factor corresponds to a number on the scale axis. By adding each score, clinicians and patients could easily calculate the 1-year and 2-year probabilities of DFS.
3.3 Combining mSEPT9 with TNM stage could better judge the prognosis of CRC patients.
Based on mSEPT9, the survival was further analyzed. In terms of 2-year DFS, patients with stage II mSEPT9+ performed significantly worse than those with stage II mSEPT9- (P = 0.023, Figure 4B), but no statistical difference could be seen between mSEPT9+ and mSEPT9- patients in stage III (P = 0.078, Figure 4C). Besides, although patients with stage II mSEPT9- had better survival than patients in stage III (P = 0.001, Figure 4B), patients with mSEPT9+ II had similar survival as patients in stage III (P = 0.132, Figure 4B). In the meantime, the survival rate between mSEPT9- in stage III and patients of stage II was close (P = 0.183, Figure 4C). Survival was significantly lower in stage III mSEPT9+ patients than in stage II patients (P < 0.001, Figure 4C).
DFS was further stratified by TNM staging and mSEPT9, all results were shown in Figure 4D. mSEPT9- stage II patients had better DFS than mSEPT9- patients of stage III (P = 0.012). On the survival curve, the stage II mSEPT9+ patients and stage III mSEPT9- patients almost overlapped (P = 0.761). As for mSEPT9+ patients in stage II and III, a significant difference could also be seen (P = 0.032). Overall, patients had the risk of recurrence or metastasis in the sequence of stage III mSEPT9+, stage III mSEPT9-/stage II mSEPT9+, and stage II mSEPT9- (P = 0.001), from high to low.
3.4. Low expression of septin9 protein in tumor tissues was often related to poor prognosis.
To investigate the clinical relevance between the septin 9 expression in tumor tissue and cancer progression, septin 9 protein was detected by IHC in 40 CRC patients, including 20 cases in stage II and III, separately (Table 3). The cytoplasmic region with brownish-yellow particles was positively expressed (Figure 5C). Of these tumors, 21 showed high expression, while the remaining 19 showed no or little detectable staining. Patients with low expression of septin9 in tumor tissue showed significantly poor DFS (P < 0.001, Figure 5B ). The connection between septin 9 expression and important prognostic risk factors was verified by Spearman correlation analysis, which included recurrence/progression (R = -0.523, P = 0.002), mSEPT9 status (R = -0.451, P = 0.004), and T stage(R = -0.375, P = 0.017) (Table 4).
Table 3
Clinico-pathological features and septin 9 protein expression in colorectal cancer tissue (n=40).
Parameter
|
No. of cases (%)
|
Age
|
|
≦60 yr
|
15(37.5)
|
﹥60 yr
|
25(62.5)
|
Gender
|
|
Male
|
26(65.0)
|
Female
|
14(35.0)
|
Location
|
|
Colon
|
22(55.0)
|
Rectum
|
18(45.0)
|
T stage
|
|
T2
|
2(5.0)
|
T3
|
12(30.0)
|
T4
|
26(65.0)
|
N stage
|
|
N0
|
20(50.0)
|
N1
|
7(17.5)
|
N2
|
13(32.5)
|
TNM stage
|
|
II
|
20(50.0)
|
III
|
20(50.0)
|
Septin9 of tissue
|
|
Strong
|
21(52.5)
|
Weak
|
19(47.5)
|
mSEPT9
|
|
Positive
|
23(57.5)
|
Negative
|
17(42.5)
|
Recurrence/progression
|
|
Occurrence
|
19(47.5)
|
Nothingness
|
21(52.5)
|
mSEPT9: methylated septin 9 gene. |
Table 4
The correlation between the expression of septin 9 protein in tissues and clinico-pathological features (n=40) (Spearman analysis) .
Parameter
|
The expression of septin 9 protein
|
Spearman correlation
|
P-value
|
T stage
|
-0.375
|
P=0.017
|
N stage
|
-0.258
|
P=0.108
|
Vascular invasion
|
-0.173
|
P=0.286
|
mSEPT9 status
|
-0.451
|
P=0.004
|
Recurrence/progression
|
-0.479
|
P=0.002
|
mSEPT9: methylated septin 9 gene.
|