Patients
A total of 310 patients with CRC were included in this study. The detailed patient characteristics are listed in Table 1. CTCs (≥ 1 per 5 mL blood) were detected in 272 patients (87.74%) with a median count of 6.00 CTCs (range 0–88 per 5 mL blood). Gli1-positive CTCs were detected in 221 patients (71.29%).
Table 1
Patient characteristics and CTCs
Characteristics | Total CTCs (tCTCs) | P | mCTCs | P | Gli1expression in tCTCs | P | Gli1expression in mCTCs | P |
≥ 1 | < 1 | ≥ 1 | < 1 | ≥ 1 | < 1 | ≥ 1 | < 1 |
Age (years) |
≥ 60 | 143 | 19 | 0.766 | 106 | 56 | 0.148 | 122 | 40 | 0.102 | 62 | 100 | 0.146 |
< 60 | 129 | 19 | 85 | 63 | 99 | 49 | 45 | 103 |
Sex |
Female | 173 | 24 | 0.957 | 125 | 72 | 0.379 | 138 | 59 | 0.524 | 66 | 131 | 0.620 |
Male | 99 | 14 | 66 | 47 | 83 | 30 | 41 | 72 |
Differentiation grade |
poor | 13 | 1 | 0.491 | 8 | 6 | 0.133 | 11 | 3 | 0.752 | 6 | 8 | 0.050* |
medium | 241 | 28 | 156 | 86 | 173 | 69 | 90 | 152 |
high | 45 | 9 | 27 | 27 | 37 | 17 | 11 | 43 |
Tumor size (cm) |
≥ 5 | 68 | 12 | 0.385 | 49 | 31 | 0.938 | 55 | 25 | 0.560 | 27 | 53 | 0.867 |
< 5 | 204 | 26 | 142 | 88 | 166 | 64 | 80 | 150 |
CEA (µg/ mL) |
≥ 5 | 78 | 10 | 0.762 | 57 | 31 | 0.471 | 67 | 21 | 0.235 | 38 | 50 | 0.043* |
< 5 | 194 | 28 | 134 | 88 | 154 | 68 | 69 | 153 |
TNM stage |
1–2 | 140 | 25 | 0.098 | 105 | 60 | 0.435 | 113 | 52 | 0.244 | 53 | 112 | 0.344 |
3–4 | 132 | 13 | 86 | 59 | 108 | 37 | 54 | 91 |
Lymph node metastasis |
None | 145 | 25 | 0.004* | 109 | 61 | 0.105 | 117 | 53 | 0.018* | 55 | 115 | 0.132 |
Single | 38 | 10 | 23 | 25 | 29 | 19 | 13 | 35 |
Multiple | 89 | 3 | 59 | 33 | 75 | 17 | 39 | 53 |
Distance metastasis |
Yes | 9 | 0 | 0.255 | 6 | 3 | 0.752 | 7 | 2 | 0.662 | 3 | 6 | 0.940 |
No | 263 | 38 | 185 | 166 | 214 | 87 | 104 | 197 |
Associations between CTC counts and patient characteristics
We found that the total CTCs (tCTCs) was correlated with lymph node metastasis (P = 0.004); however, no significant difference was found between CTCs EMT subtypes and any of the patient characteristics (P > 0.05; Table 1). Nonetheless, we found that the count of tCTCs was associated with advanced stage disease and lymph node metastasis (P < 0.05; Fig. 2A, 2B). The median CTC count in patients with advanced stage CRC (stage III or IV) was 10.87 ± 14.165, which was remarkably higher than that in patients with stage I or II disease (8.59 ± 10.833, P = 0.031; Fig. 2A). These data suggested a positive correlation between the CTC counts and the progression of cancer, as reflected in the increasing stages of CRC. We observed a positive trend in the correlation between the count of tCTCs and metastasis, with 8.57 ± 10.689 in patients with non-metastatic CRC, 11.73 ± 18.266 in patients with single lymph node metastasis, and 10.58 ± 12.014 in multiple metastasis; however, the difference was not significant (P > 0.05).
Associations between Gli1 expression in CTCs and patient characteristics
Among the 272 patients with ≥ 1 CTCs per 5 mL blood eligible for Gli1 expression analysis, there were a total of 221 (81.25%) patients positive for Gli1. Gli1 was expressed in all three subtypes of CTCs (epithelial CTCs, hybrid CTCs, and mesenchymal CTCs; Table 2). The Gli1 expression was 80.77% (84/104) positive among ECTCs, 84.43% (206/244) among HCTCs, and 89.86% (248/276) among combined bi-phenotypic and mesenchymal CTCs (mCTCs).
Table 2
Gli1 expression status in tCTCs and different EMT subtypes of CTCs
CTCs characteristics | Gli1 expression negative | Gli1 expression positive |
Total CTCs (tCTCs) | 18.75% (51/272) | 81.25% (221/272) |
Epithelial CTCs (ECTCs) | 19.23% (20/104) | 80.77% (84/104) |
Hybrid CTCs (HCTCs) | 15.57% (38/244) | 84.43% (206/244) |
Mesenchymal CTCs (mCTCs) | 16.75% (32/191) | 83.25% (159/191) |
The associations between Gli1 expression status and patient characteristics are listed in Table 1. We found that tCTCs with positive expression of Gli1 were significantly associated with lymph node metastasis (P = 0.018), while no significant correlation was observed regarding age, sex, TNM stage, or tumor differentiation. We did find, however, that the count of tCTCs with positive expression of Gli1 correlated with the advanced disease (Fig. 2C, 2D). The median count of Gli1+tCTCs was found to be 5.74 ± 10.462 in stage III and IV patients, which was significantly higher than that in stage I or II patients (3.57 ± 6.250, P = 0.002). Likewise, the median count of Gli1+tCTCs was 3.61 ± 6.217 in patients with non-metastatic CRC, 6.21 ± 12.647 in patients with single lymph node metastasis, and 5.53 ± 9.441 in patients with multiple lymph node metastasis. These data suggested that the count of Gli1+tCTCs increases along with increasing disease stages and the tumor progression, although there was no significant association. No significant difference was observed in the count of Gli1+mCTCs regarding differentiation grade and CEA level; however, patients in the medium differentiation grade group presented an increased level of Gli1+mCTCs compared with those in the group of poor differentiation grade (Fig. 2E, 2F).
CTC counts and Gli1 expression were valuable indicators for the prognosis of CRC patients
Among the 310 patients with a disease follow-up assessments, the follow-up period ranged from 7 to 62 months (median, 21 months). We analyzed the PFS of all CRC patients, taking into consideration tCTCs, mCTCs, Gli1+tCTCs, and Gli1+mCTCs (Fig. 3). As expected, we found that patients with ≥ 1 Gli1+mCTCs per 5 mL blood exhibited a poorer PFS than those with < 1 Gli1+mCTCs per 5 mL blood (P = 0.049, HR = 2.065, 95%CI = 0.969–4.399). Multivariate Cox regression analysis revealed that the count of Gli1+mCTCs was not an independent prognostic biomarker for CRC survival (P = 0.272; Table 3); however, age (P = 0.040, HR = 0.440, 95%CI = 0.220–1.532; Table 3) and CEA level (P = 0.024, HR = 2.387, 95%CI = 1.122–5.080; Table 3) were identified as significant factors impacting the PFS of CRC patients.
Table 3
Multivariate Cox regression analysis of PFS
Variables | HR | 95%CI | P |
Age, years (≥ 60 vs. <60) | 0.440 | 0.220–1.532 | 0.040 |
Tumor size, cm (≥ 5 vs. <5) | 0.450 | 0.169–1.197 | 0.110 |
CEA, µg /mL (≥ 5 vs. <5) | 2.387 | 1.122–5.080 | 0.024 |
TNM stage (III + IV vs. I + II) | 3.932 | 0.988–15.641 | 0.052 |
Lymph node metastasis (multiple vs. single vs. none) | 0.785 | 0.391–1.577 | 0.497 |
Distance metastasis (Yes vs. No) | 3.294 | 0.881–12.313 | 0.076 |
tCTCs count, /5 mL (≥ 6 vs. <6) | 1.100 | 0.508–2.382 | 0.809 |
mCTCs count, /5 mL (≥ 1 vs. <1) | 0.593 | 0.216–1.631 | 0.311 |
Gli1+tCTCs count, /5 mL (≥ 1 vs. <1) | 2.131 | 0.728–6.237 | 0.167 |
Gli1+mCTCs count, /5 mL (≥ 6 vs. <6) | 0.580 | 0.220–1.532 | 0.272 |