Patient and tumor characteristics
In our study we included 613 patients with CRC with a median age of 70 years (range 36–96). 53.2% of the patients were female. In 69.8% of the patients CRC was located in the left hemicolon or rectum and in the remaining 29.9% in the right hemicolon. According to the TNM-classification, most of the tumors were of pT3/pT4 stage (n = 485, 79.1%), with the minority of cancers being pT1/pT2 (n = 114, 18.6%). Among the malignancies under evaluation, 317 (51.7%) were pN0, 160 (26.1%) pN1, and 119 (19.4%) pN2 cases. Tumor-grade was G1 in 14 (2.3%) cases, G2 in 552 (90%) cases, and G3 in 33 (5.4%) cases. Furthermore 417 (68%) showed infiltrating tumor border configuration, while in the majority of cases (n = 425, 69.3%) vascular invasion was absent. Most of the tumors belonged to the microsatellite stable subgroup (n = 539, 87.9%). Within the follow-up period, median disease-specific survival was 58.4 months (range 0–152), while mean 5-year-survival was 47% (95% confidence interval: 43–51%). In Table 1, we summarized the clinico-pathological characteristics of the patients. We have to mention, that if the percentages do not add to 100% is due to missing values of same variables.
Association of clinicopathological features with SDF-1 expression and CD8 + T cell density
In Fig. 1, we illustrate representative pictures of low and high expression of SDF-1 in biopsies with high CD8 + T-cell infiltration in CRC. Furthermore, we created a table, that demonstrates the clinicopathological features under investigation and their relation to the four subgroups identified by SDF-1 expression and CD8 + T-cells density (SDF-1high/CD8 + high, SDF-1high/CD8 + low, SDF-1low/CD8 + high, SDF-1low/CD8 + low) as absolute numbers and percentages. Here we have to mention that during the procedure of TMA preparation, some of the punches may be lost.
The nature of tumor border (pushing vs. infiltrating) has been reported to impact on CRC prognosis [32]. Indeed, infiltrating tumor border, associated with poor survival, was detected with significantly (p = 0.014) higher frequency in CRC with SDF-1high/CD8 + low than in tumors with SDF-1high/CD8 + high infiltrate, thus suggesting an effect related to CD8 + T-cell infiltration. Furthermore, we observed that cases with SDF-1high/CD8 + high were characterized by a significantly lower N-stage (p < 0.001) in comparison to the cases with SDF-1high/CD8 + low. In contrast, we demonstrated that T stage, tumor grade and vascular invasion did not significantly differ in CRC with different immune environment profiles.
Interestingly, we detected a strong tendency towards statistical significance (p = 0.051) regarding the presence of PTL inflammation, which was higher in the group of SDF-1high/CD8 + high in contrast to SDF-1high/CD8 + low. This fact highlights the importance of the microenvironment and supports the hypothesis of an effective antigen-specific immune response. However, this can only be considered as a trend with a p > 0.05.
Spearman`s correlation analysis of SDF-1 and markers of the microenvironment
In order to better understand the microenvironment in the context of SDF-1 expression, we performed a Spearman`s correlation analysis with a panel of immune as well as cell signaling markers or growth factors on protein (TMA data; Table 3) and considered correlations above 0.35 to be relatively strong; correlations between 0.15 and 0.35 to be moderate, and those below 0.15 to be weak. Finally, SDF-1 tumor expression moderately correlated with CXCR4 tumor expression. SDF-1 expression by tumor-infiltrating immune cells (TIC) showed a strong correlation with CXCR4 positive TIC and a moderate correlation with CD8 T-cell density as well as pCXCR4 positive TIC on a protein level (Table 3).
Synergistic prognostic significance of SDF-1 tumor expression and CD8 + cell infiltration in the CRC microenvironment
The 5-years survival rates were significantly different depending on the nature of immune infiltrate [Table 2]. Most importantly, Kaplan–Meier plots clearly indicated that 5-year survival rate was significantly better in cases of CRC with high expression on SDF-1 and CD8 + T-cell infiltration compared to tumors showing a high infiltration of CD8 + T-cells only (66% (95%CI = 48–79%) vs 55% (95%CI = 45–64%); p = 0.0004; Fig. 2). Interestingly, there was a difference in long-term survival (> 5 years) between the test group (Fig. 2A) and the validation group (Fig. 2B). The long-term survival rate in the test group seems to be better in patients bearing tumors characterized by SDF-1low/CD8 + high in comparison to the SDF-1high/CD8 + high. However, the high expression of SDF-1 and CD8 + T-cell infiltration was significantly associated with a favorable prognosis in the validation group (p = 0.016).
Table 2
Association of SDF-1 + tumor expression and CD8 + low and high immune cell density with clinicopathological features in CRC (n = 613)*
| SDF-1high/ CD8 + high | SDF-1high/ CD8 + low | SDF-1low/ CD8 + high | SDF-1low/ CD8 + low | p-value |
N = 35 | (100%) | N = 121 | (100%) | N = 98 | (100%) | N = 359 | (100%) | |
Age | years, mean ± SD | 68.5 | ± 10.6 | 69.4 | ± 10.9 | 67.6 | ± 12.0 | 69.1 | ± 11.0 | 0.751 |
Tumor diameter | mm, mean ± SD | 53.2 | ± 14.2 | 48.2 | ± 16.1 | 52.5 | 24.4 | 49.9 | ± 20.2 | 0.253 |
Gender | Female Male | 25 10 | 71.4 28.6 | 60 61 | 49.6 50.4 | 52 46 | 53.1 46.9 | 189 170 | 52.6 47.4 | 0.146 |
Tumor location | Left-sided Right-sided | 23 12 | 65.7 34.3 | 97 24 | 80.2 19.8 | 64 33 | 65.3 33.7 | 244 114 | 68 31.8 | 0.046 |
Histologic subtype | Mucinous Non-mucinous | 2 33 | 5.7 94.3 | 3 118 | 2.5 97.5 | 3 95 | 3.1 96.9 | 22 337 | 6.1 93.9 | 0.292 |
pT stage | pT1-2 pT3-4 | 9 24 | 25.7 68.6 | 23 96 | 19.0 79.3 | 23 70 | 23.5 71.4 | 59 295 | 16.4 82.2 | 0.177 |
pN stage | pN0 pN1-2 | 23 12 | 65.7 34.3 | 61 57 | 50.4 42.1 | 67 27 | 68.4 27.6 | 166 183 | 46.2 51.0 | < 0.001 |
Tumor grade | G1 G2 G3 | 0 32 1 | 0 91.4 2.9 | 2 112 5 | 1.0 92.6 4.1 | 2 79 12 | 2.0 80.6 12.2 | 10 329 15 | 2.8 91.6 4.2 | 0.097 |
Vascular invasion | Absent Present | 26 7 | 74.3 20.0 | 88 31 | 72.7 25.6 | 67 26 | 68.4 26.5 | 244 110 | 68.0 30.6 | 0.559 |
Tumor border | Pushing Infiltrating | 13 20 | 37.1 57.1 | 37 82 | 30.6 67.8 | 39 54 | 39.8 55.1 | 91 261 | 25.3 72.7 | 0.014 |
PTL inflammation | Absent Present | 20 13 | 57.1 37.1 | 94 25 | 77.7 20.7 | 67 26 | 68.4 26.5 | 282 72 | 78.6 20.1 | 0.051 |
Microsatellite stability | Deficient Proficient | 3 32 | 8.6 91.4 | 5 116 | 4.1 95.9 | 18 80 | 18.4 81.6 | 48 311 | 13.4 86.6 | 0.004 |
5-year survival rate | (95%CI) | 0.66 | (0.48–0.79) | 0.40 | (0.31–0.48) | 0.55 | (0.45–0.64) | 0.45 | (0.40–0.50) | 0.0004 |
CXCR4 histoscore | mean ± SD | 203.5 | ± 100.2 | 177.3 | ± 95.5 | 152.9 | ± 94.4 | 143.6 | ± 99.7 | 0.001 |
CXCR4 TIC | mean ± SD | 50.7 | ± 62.3 | 53.5 | ± 130.7 | 121.9 | ± 208.8 | 70.7 | ± 145.8 | 0.016 |
pCXCR4 histoscore | mean ± SD | 76.3 | ± 97.3 | 32.4 | ± 64.2 | 54.1 | ± 81.8 | 20.9 | ± 49.2 | 0.0002 |
pCXCR4 TIC | mean ± SD | 6.2 | ± 9.1 | 4.3 | ± 10.4 | 7.6 | ± 14.0 | 4.0 | ± 6.8 | 0.001 |
* Due to missing values of the same variables, percentages may not add to 100%. Variables are indicated as absolute numbers, %, median or range; age and tumor size were evaluated using the Kruskal-Wallis test. Gender, anatomical site, T stage, N stage, grade, vascular invasion, and tumor border configuration were analyzed using the χ2 test. Survival analysis was performed using the Kaplan-Meier method. |
Table 3
Spearman’s correlation analysis of SDF-1 protein expression with CXCR4, pCXCR4 and CD8
|
SDF-1+ tumor expression | SDF-1+ TIC | CD8 | CXCR4 tumor expression | pCXCR4 tumor expression | CXCR4+ TIC | pCXCR4 TIC |
SDF-1+ tumor expression | 1.000 | | | | | | |
SDF-1+ TIC | -0.1143 0.0149 | 1.0000 | | | | | |
CD8 | -0.0359 0.4455 | 0.1648 0.0004 | 1.0000 | | | | |
CXCR4 tumor expression | 0.1864 0.0001 | 0.0571 0.2252 | 0.1244 0.0080 | 1.0000 | | | |
pCXCR4 tumor expression | 0.0296 0.5291 | -0.0245 0.6035 | 0.2601 0.0000 | 0.2764 0.0000 | 1.0000 | | |
CXCR4 + TIC | -0.0957 0.0418 | 0.3752 0.0000 | 0.2128 0.0000 | 0.1174 0.0124 | 0.1020 0.0300 | 1.0000 | |
pCXCR4 + TIC | -0.0806 0.0865 | 0.1572 0.0008 | 0.2034 0.0000 | 0.1131 0.0160 | 0.2725 0.0000 | 0.3706 0.0000 | 1.0000 |
Univariate and multivariate analysis of SDF-1 expression and CD8 + T-cell infiltration by tumor cells and tumor-infiltrating immune cells
Univariate Cox regression analysis revealed that the combination of high expression of SDF-1 and high CD8 + T cell infiltration is significantly associated with an increased overall survival (HR 0.34; 95%CI = 0.17–0.66; p = 0.002). Age, male gender, tumor grade, T-stage, N-stage, invasive margin and vascular invasion were all significantly associated with a poor prognosis in univariate analyses [Table 4].
Table 4
Uni- and multivariate Hazard Cox regression survival analysis considering the combination of both markers (n = 613 and n = 576, respectively)
| Univariate | Multivariate |
HR | 95%CI | p-value | HR | 95%CI | p-value |
Age | 1.03 | 1.02 | 1.04 | < 0.001 | 1.04 | 1.03 | 1.05 | < 0.001 |
Gender (male vs female) | 1.53 | 1.23 | 1.90 | < 0.001 | 1.61 | 1.28 | 2.02 | < 0.001 |
pT (high vs low) | 3.42 | 2.32 | 5.04 | < 0.001 | 2.41 | 1.57 | 3.71 | < 0.001 |
pN (high vs low) | 3.28 | 2.61 | 4.14 | < 0.001 | 2.36 | 1.84 | 3.02 | < 0.001 |
Grade (high vs low) | 5.31 | 1.32 | 21.33 | 0.019 | 2.88 | 0.69 | 11.95 | 0.146 |
Vascular invasion | 2.49 | 1.99 | 3.12 | < 0.001 | 1.99 | 1.56 | 2.53 | < 0.001 |
Invasive margin | 1.92 | 1.48 | 2.50 | < 0.001 | 1.41 | 1.06 | 1.88 | 0.017 |
MMR status | 1.53 | 1.07 | 2.19 | 0.021 | 1.32 | 0.91 | 1.92 | 0.149 |
SDF-1high/CD8 + low | 1.09 | 0.84 | 1.42 | 0.526 | 1.18 | 0.89 | 1.55 | 0.247 |
SDF-1low/CD8 + high | 0.66 | 0.47 | 0.92 | 0.015 | 0.89 | 0.62 | 1.29 | 0.549 |
SDF-1high/CD8 + high | 0.34 | 0.17 | 0.66 | 0.002 | 0.45 | 0.23 | 0.89 | 0.021 |
Multivariate analyses showing Hazard Ratios and p-value for all CRCs (n = 576 less than 613 due to missing values) conferred by SDF-1 expression and CD8 + cell density, age, sex, tumor size, lymph node involvement, tumor grade, vascular invasion, tumor border configuration and microsatellite stability [33]. |
Figure 1: Examples of high SDF-1 expression (Picture A) and low SDF-1 expression in CRC biopsies with high CD8 + T-cell infiltration (x200 magnification) |
In a multivariate Hazard Cox regression survival analysis the combined high expression of SDF-1 and high CD8 + T-cell infiltration in CRC succeeded to retain its role as an independent prognostic factor for overall survival (HR = 0.45, 95%CI: 0.23–0.89; p = 0.021). Moreover, we found that an increased age (HR = 1.04; 95%CI: 1.03–1.05; p < 0.001), male gender (HR = 1.61; 95%CI: 1.28–2.02; p < 0.001), a higher T-stage (HR = 2.41; 95%CI: 1.57–3.71; p = 0.001), N-stage (HR = 2.36; 95%CI = 1.84–3.02; p < 0.001), vascular invasion (HR = 1.99; 95%CI: 1.56–2.53; p < 0.001) and invasive margin (HR = 1.41; 95%CI: 1.06–1.88; p: 0.017) were independently associated with a poor prognosis [Table 4].