Abstract
Background
The Immunoscore was initially established to evaluate the prognosis of stage I/II/III colorectal cancer patients. However, the feasibility of the Immunoscore for the prognosis of colorectal cancer liver metastases (CRCLM) has not been reported.
Methods
Liver metastases in 249 CRCLM patients were retrospectively analyzed. The Immunoscore was assessed according to the counts and densities of CD3+ and CD8+ T cells in the central- and peritumoral areas by immunohistochemistry. The prognostic role of the Immunoscore for relapse–free survival (RFS) and overall survival (OS) was analyzed with Kaplan–Meier curves and Cox multivariate models, and confirmed via an internal validation. Receiver operating characteristic (ROC) curves were plotted to compare the prognostic values of the Immunoscore and the clinical risk score (CRS) system.
Results
CRCLM patients with high Immunoscores (> 2) had significantly longer RFS [median RFS (95% confidence interval; 95% CI) 21.4 (7.8–35.1) vs. 8.7 (6.8–10.5) months, P < 0.001] and OS [median OS (95% CI): not reached vs. 28.7 (23.2–34.2) months, P < 0.001] than those with low Immunoscores (≤ 2). After stratification by CRS, the Immunoscore retained a statistically significant prognostic value for OS. The areas under the ROC curves (AUROCs) of the Immunoscore and the CRS system for RFS were 0.711 [95% CI 0.642–0.781] and 0.675[95% CI 0.601–0.749] (P = 0.492), whereas the AUROC of the Immunoscore system for OS was larger than that of the CRS system [0.759 (95% CI 0.699–0.818) vs. 0.660 (95% CI 0.592–0.727); P = 0.029].
Conclusions
The Immunoscore of liver metastases can be applied to predict the prognosis of CRCLM patients following liver resection.
Electronic supplementary material
The online version of this article (10.1007/s00262-017-2094-8) contains supplementary material, which is available to authorized users.
Keywords: Colorectal cancer liver metastases, Tumor-infiltrating lymphocyte, Immunoscore, Prognosis
Introduction
Colorectal cancer (CRC) is the third most common malignancy and the fourth leading cause of cancer death worldwide [1]. Approximately 35–55% of CRC patients develop liver metastases synchronously or metachronously [2], and liver resection has been proven to be the only curative treatment for colorectal cancer liver metastases (CRCLM) [3, 4]. However, more than 50% of patients experience recurrence after initial liver resection, with a 5-year survival rate of only 25–50% [5–9]. Postoperative survival is highly related to clinical variables, such as the number of liver lesions, the diameter of the largest metastasis, preoperative serum carcinoembryonic antigen (CEA) levels, the lymph node status of the primary tumor, the disease-free interval from the resection of the primary tumor to the occurrence of liver metastases, and the presence of extra-hepatic metastases. Various scoring systems have utilized these variables to predict patient survival. The most widely used system is the clinical risk score (CRS) system proposed by Fong et al., which includes the size of the largest metastasis, the number of metastases, the disease-free interval from the resection of the primary tumor to the occurrence of liver metastases, preoperative CEA levels, and the lymph node status of the primary tumor [10]. However, survival varies even among patients with the same CRS, implying that these clinical risks are not the only factors in survival.
The immune microenvironment plays an important role in the occurrence and progression of CRC. Studies that aimed to establish prognostic models utilizing peritumoral immune biomarkers for early stage CRC and liver metastasis have been reported. Tumor-infiltrating lymphocyte (TIL) counts and T-cell subsets, such as helper T cells (CD4+), cytotoxic T cells (CD8+) and T regulatory cells (Tregs, FOXp3+), in liver lesions have recently been reported to be independent prognostic indicators of survival after CRC liver resection [11–14]. Although these reports above indicated the important role of TILs, no convenient but effective scoring system has yet to be proposed for CRCLM patients who underwent liver metastasectomy. An Immunoscore system established by Galon J et al., which takes into account the densities of CD3+ and CD8+ immune cells in the center tumor (CT) and invasive margin (IM) of the primary tumors, showed encouraging performance for predicting the postoperative outcome of stage I–III CRC patients after surgery [15–19]. The system was proposed as a necessary complement to the TNM staging for CRC, which is widely accepted by clinical physicians due to its simplicity, repeatability, accuracy and feasibility.
Considering these previous findings, we questioned whether the Immunoscore, which was initially defined to quantify in situ immune infiltrates, can be extended to liver lesions and applied to predict the recurrence of CRC after liver metastasectomy. The present study was performed (1) to evaluate the prognostic performance of the Immunoscore on liver metastatic lesions of CRCLM patients following liver metastasectomy and (2) to compare the prognostic sensitivity and specificity between the Immunoscore system and Fong’s CRS system.
Methods and materials
Patient selection
Consecutive patients with CRC liver metastases who underwent liver metastasectomy were recruited between June 2002 and December 2015 at Sun Yat-sen University Cancer Center in China. The eligibility criteria were as follows: (1) pathologically and radiologically diagnosed with CRC liver metastases; (2) underwent liver metastasectomy with curative intent; and (3) presence of adequate pathological information and metastasis specimens for analysis. The exclusion criteria included those with (1) the presence of metastases in sites other than the liver and (2) a history of prior liver resections.
The current study was approved by the institutional ethical review board of Sun Yat-sen University Cancer Center and was conducted in accordance with the Helsinki declaration of the World Medical Association. Informed consent was obtained from all patients for the use of medical information and specimens for clinical research.
Treatment protocols
The administration of perioperative chemotherapy was determined according to the tumor burden and European Society for Medical Oncology (ESMO) guidelines by the physicians of Sun Yat-Sen University Cancer Center [20]. The perioperative chemotherapy regimens administered in this study included XELIRI (irinotecan and capecitabine), CAPEOX (oxaliplatin and capecitabine), FOLFOX (oxaliplatin, 5-Fu and leucovorin [LV]), FOLFIRI (irinotecan, 5-Fu and LV), FOLFOXIRI (oxaliplatin, irinotecan, 5-Fu and LV), 5-Fu/LV, single-agent capecitabine and a combination of above regimens and hepatic arterial infusion (HAI) or bevacizumab or cetuximab. No patients with Ras mutation were treated with cetuximab. In addition, bevacizumab was suspended within 6 weeks of surgery.
Data collection and follow-up
The patients were followed-up until February 2017 by hospital records or phone contact with the patients or relatives who were aware of their illness. Clinical information, including the factors involved in the Fong CRS system, was collected and recorded. The CRS was calculated according to five risk factors: positive lymph nodes at the primary site, disease-free interval shorter than 12 months, number of metastases > 1, diameter of maximum metastases > 5 cm, and serum CEA levels > 200 ng/mL. Each risk factor was counted as one point, corresponding to a possible score of 0–5 [10].
Immunoscore evaluation
The Immunoscores of liver metastases were evaluated in accordance with the method reported by Galon et al. [19, 21–23]. Paraffin-embedded slides of liver metastatic lesion specimens were stained using an immunohistochemical technique that labeled the CD3+ (ZSGS-BIO, Beijing, China; Catalog No. ZA0503) and CD8+ (ZSGS-BIO, Beijing, China; Catalog No. ZA0508) T cells with specific antibodies. Computer-assisted calculations of the density of CD3+ and CD8+ T cells in both the center tumor (CT) and the invasive margin (IM) of the liver metastases were conducted using Image J software (National Institute of Health, Bethesda, MD, USA), as described by Gabrielson et al. (example provided in Supplemental Figure 1) [24]. Two independent pathologists, who were blinded to the patients’ clinical information, participated in the analysis to avoid the interference of necrotic areas and to verify the location of the CT/IM. The Immunoscore evaluations were performed based on the densities of CD3+ and CD8+ T cells in both the CT and IM regions and by the cut-off of the median of each index (CD3+ cells in the CT, CD3+ cells in the IM, CD8+ cells in the CT and CD8+ cells in the IM) [24–26]. High value of each index was scored 1, whereas low value was scored 0. All scores were calculated as the final Immunoscore. A final score of 0 corresponded to low densities of CD3+ and CD8+ T cells found in both the CT and IM areas. A score of 1 indicated a high density of either type of lymphocytes found in either the CT or IM region. And a maximum score of four indicated high densities of both types of lymphocytes observed in both regions. Furthermore, patients with an Immunoscore > 2 were defined as having high Immunoscores, while those with an Immunoscore ≤ 2 were defined as having low Immunoscores.
Statistical analysis
Kaplan–Meier curves with a log-rank test were used to analyze the prognostic role of the Immunoscore in predicting relapse-free survival (RFS) and overall survival (OS). RFS was defined as the duration from the date of liver metastasectomy to the date of the first relapse at any site or death due to any cause other than relapse. The OS was calculated from the liver metastasectomy to death due to any cause. Univariate and multivariate analyses were performed to determine the influence of the prognostic factors for survival. A re-sampling procedure was conducted to evaluate the robustness of the multivariate model and to validate the results. Briefly, a less-powered dataset (the internal validation set) was generated by randomly selecting 70% of the original dataset using SPSS software. Cox multivariate models for RFS and OS were used on the internal validation set to validate the consistency of the results. The Mann–Whitney test was used to further analyze the associations between the Immunoscore and clinicopathological factors. Receiver operating characteristic (ROC) curves were also used to compare the predictive ability of the Immunoscore system with the CRS system for RFS and OS. All statistical examinations were performed with IBM SPSS software (version 22) using two-tailed tests. P values < 0.05 were considered statistically significant.
Results
Patient characteristics
Two-hundred forty-nine CRCLM patients with radical resection of the liver metastases were retrospectively analyzed in this study. One-hundred forty-four (57.8%) patients received preoperative chemotherapy, and 166 (66.7%) underwent postoperative chemotherapy. Only 33 (13.3%) patients did not receive perioperative chemotherapy treatment. The baseline information of the patients is presented in Table 1.
Table 1.
Association of the Immunoscore (high/low) with clinicopathological factors in CRCLM patients
| Low Immunoscore | High Immunoscore | P value | |
|---|---|---|---|
| Patients | 159 (63.9) | 90 (36.1) | |
| Age | |||
| ≤ 65 years | 132 (83.0) | 71 (78.9) | 0.421 |
| > 65 years | 27 (17.0) | 19 (21.1) | |
| Gender | |||
| Male | 96 (60.4) | 62 (68.9) | 0.181 |
| Female | 63 (39.6) | 28 (31.1) | |
| Primary tumor site | |||
| Colon | 97 (61.0) | 62 (68.9) | 0.214 |
| Rectum | 62 (39.0) | 28 (31.1) | |
| Primary tumor grade | |||
| G1–2 | 117 (73.6) | 75 (83.3) | 0.079 |
| G3 | 42 (26.4) | 15 (16.7) | |
| Histological subtype | |||
| Non-mucinous | 146 (91.8) | 84 (93.3) | 0.667 |
| Mucinous | 13 (8.2) | 6 (6.7) | |
| Primary tumor T-stagea | |||
| Tis-2 | 29 (18.2) | 11 (12.2) | 0.215 |
| T3-4 | 130 (81.8) | 79 (87.8) | |
| Primary tumor N-stagea | |||
| N0 | 59 (37.1) | 37 (41.1) | 0.534 |
| N1–2 | 100 (62.9) | 53 (58.9) | |
| Preoperative CEA | |||
| ≤ 200 ng/ml | 149 (93.7) | 86 (95.6) | 0.545 |
| > 200 ng/ml | 10 (6.3) | 4 (4.4) | |
| Interval from primary tumor resection to liver metastases | |||
| ≤ 12 months | 34 (21.4) | 21 (23.3) | 0.722 |
| > 12 months | 125 (78.6) | 69 (76.7) | |
| Number of metastases per patient | |||
| ≤ 1 | 49 (30.8) | 41 (45.6) | 0.020 |
| > 1 | 110 (69.2) | 49 (54.4) | |
| Size of the max metastases | |||
| ≤ 5 cm | 119 (74.8) | 78 (86.7) | 0.028 |
| > 5 cm | 40 (25.2) | 12 (13.3) | |
| Preoperative chemotherapy | |||
| No | 69 (43.4) | 36 (40.0) | 0.603 |
| Yes | 90 (56.6) | 54 (60.0) | |
| Postoperative chemotherapy | |||
| No | 55 (34.6) | 28 (31.1) | 0.576 |
| Yes | 104 (65.4) | 62 (68.9) | |
| CRS | |||
| 0–2 | 82 (51.6) | 59 (65.6) | 0.033 |
| 3–5 | 77 (48.4) | 31 (34.4) | |
Data are presented as the number of cases with percentage in parentheses
Bold value indicates P < 0.05
CEA carcinoembryonic antigen, CRS clinical risk score
aAccording to the Union International Control Cancer (UICC) staging system (7th version)
The median densities of CD3+ cells were 56.5/mm2 (IQR 19.9–144.5/mm2) in the CT and 1051.9/mm2 (IQR 500.0–1778.7/mm2) in the IM. The median densities of CD8+ cells were 43.2/mm2 (IQR 15.2–134.6/mm2) in the CT and 787.9/mm2 (IQR 530.3–1125.4/mm2) in the IM (Fig. 1). Of the 249 patients, 159 (63.9%) had a low Immunoscore, whereas 90 (36.1%) had a high Immunoscore. High Immunoscores were associated with fewer liver metastatic lesions (P = 0.020), smaller lesion sizes (P = 0.028) and lower CRS (P = 0.033; Table 1).
Fig. 1.
Representative immunohistochemical images of CD3+ and CD8+ T cells in the core center tumor (CT) and in the invasive margin (IM) of the liver metastases. a–b Representative images of high-density and low-density CD3+ cells in the center of the liver metastases; c–d Representative images of high-density and low-density CD3+ cells in the invasive margin of the liver metastases; e–f Representative images of high-density and low-density CD8+ cells in the center of the liver metastases; g–h Representative images of high-density and low-density CD8+ cells in the invasive margin of the liver metastases
Prognostic role of the Immunoscore in CRCLM after liver surgery
Up to February 2017, 180 of the 249 patients (72.3%) exhibited disease recurrence, and 126 (50.6%) died. The median follow-up time was 46.4 months (interquartile range, IQR 27.3–83.0 months). The five Immunoscore levels corresponded to marked survival differences, with an increasing risk of relapse (3-year RFS rates: 50.6% I 4, 32.6% I 3, 25.8% I 2, 17.5% I 1, 3.0% I 0; P < 0.001 for all comparisons) and death (5-year OS rates: 73.7% I 4, 45.9% I 3, 41.1% I 2, 21.3% I 1, 15.9% I 0; P < 0.001 for all comparisons) with decreasing Immunoscores 4–0 (Supplemental Table 1 and Supplemental Figure 2). We further analyzed outcomes according to the definitions of high Immunoscore (> 2) and low Immunoscore (≤ 2). Patients with a high Immunoscore had a significantly longer RFS [median RFS (95% confidence interval; 95% CI): 21.4 (7.8–35.1) vs. 8.7 (6.8–10.5) months, P < 0.001] and a higher 3-year RFS rate (42.4 vs. 17.0%, respectively; P < 0.001) than those with a low Immunoscore (Fig. 2a). Similarly, patients with a high Immunoscore also had a significantly longer OS [median OS (95% CI): not reached vs. 28.7 (23.2–34.2) months; P < 0.001] and a higher 5-year OS rate (59.7 vs. 25.9%, P < 0.001) than patients with a low Immunoscore (Fig. 2b). After stratifying by preoperative chemotherapy (with/without), the Immunoscore remained a clinically and statistically significant predictor of prognosis (Supplemental Table 2 and Supplemental Figure 3).
Fig. 2.
Kaplan–Meier survival curves of CRCLM patients after liver metastasectomy stratified by the Immunoscore (high/low). Both relapse-free survival rate (a) and overall survival rate (b) are higher in patients with a high Immunoscore
Prognostic factors in the univariate and multivariate analyses
After adjusting for other clinicopathological factors in the Cox multivariate models, an increase in the Immunoscore by one point (analyzed as a ranked variable with 5 as the maximum score) was estimated to correspond to an average 27.0% RFS advantage [hazard ratio (HR) 0.73, 95% CI 0.64–0.83] and 31.0% OS advantage (HR 0.69, 95% CI 0.59–0.80) compared to the next lower Immunoscore (P < 0.001; Table 2). The internal re-sampling validation analysis revealed that a higher Immunoscore level conferred a 38.0% increased RFS advantage (HR 0.62, 95% CI 0.52–0.73) and a 32.0% increased OS advantage (HR 0.68, 95% CI 0.56–0.82) compared to a lower level (P < 0.001; Supplemental Table 3).
Table 2.
Prognostic factors for survival of CRCLM patients after liver metastasectomy as determined by univariate and multivariate analyses
| Relapse-free survival | Overall survival | |||||||
|---|---|---|---|---|---|---|---|---|
| Univariate analysis | Multivariate analysis | Univariate analysis | Multivariate analysis | |||||
| HR (95% CI) | P value | HR (95% CI) | P value | HR (95% CI) | P value | HR (95% CI) | P value | |
| Age | ||||||||
| > 65 vs. ≤ 65 | 0.73 (0.49–1.08) | 0.115 | 0.86 (0.57–1.29) | 0.461 | 1.01 (0.65–1.57) | 0.968 | 1.19 (0.74–1.93) | 0.470 |
| Gender | ||||||||
| Male vs. female | 1.08 (0.80–1.47) | 0.604 | 1.18 (0.86–1.64) | 0.303 | 0.94 (0.65–1.35) | 0.727 | 0.95 (0.65–1.39) | 0.781 |
| Primary tumor | ||||||||
| Rectal vs. colon | 1.01 (0.74–1.36) | 0.974 | 0.82 (0.60–1.13) | 0.232 | 0.90 (0.63–1.29) | 0.564 | 1.03 (0.70–1.53) | 0.866 |
| Tumor grade | ||||||||
| G3 vs. G1–2 | 1.50 (1.06–2.11) | 0.021 | 1.17 (0.80–1.69) | 0.418 | 1.76 (1.19–2.61) | 0.005 | 1.83 (1.18–2.82) | 0.007 |
| T-stage | ||||||||
| T3–4 vs. Tis-2 | 1.15 (0.78–1.71) | 0.476 | 1.46 (0.95–2.26) | 0.085 | 1.12 (0.70–1.77) | 0.645 | 1.40 (0.83–2.36) | 0.211 |
| N-stage | ||||||||
| N1–2 vs. N0 | 1.53 (1.12–2.09) | 0.007 | 1.47 (1.06–2.04) | 0.021 | 1.79 (1.21–2.61) | 0.002 | 1.68 (1.13–2.48) | 0.010 |
| Preoperative CEA | ||||||||
| > 200 vs. ≤ 200 ng/ml | 1.75 (0.97–3.15) | 0.062 | 2.28 (1.22–4.23) | 0.009 | 1.96 (0.99–3.87) | 0.053 | 3.53 (1.67–7.46) | 0.001 |
| Interval from primary tumor resection to liver metastases | ||||||||
| > 12 vs. ≤ 12 months | 0.90 (0.64–1.27) | 0.538 | 0.73 (0.51–1.06) | 0.099 | 1.11 (00.73–1.69) | 0.634 | 1.42 (0.90–2.24) | 0.128 |
| Number of metastases | ||||||||
| > 1 vs. ≤ 1 | 2.28 (1.64–3.16) | < 0.001 | 1.55 (1.06–2.25) | 0.023 | 1.83 (1.23–2.71) | 0.003 | 1.18 (0.74–1.89) | 0.485 |
| Size of the largest metastasis (cm) | ||||||||
| > 5 vs. ≤ 5 | 1.91 (1.36–2.67) | < 0.001 | 1.56 (1.07–2.26) | 0.021 | 2.75 (1.89–4.01) | < 0.001 | 2.14 (1.41–3.24) | < 0.001 |
| Preoperative chemotherapy | ||||||||
| Yes vs. no | 2.22 (1.62–3.04) | < 0.001 | 2.22 (1.55–3.17) | < 0.001 | 1.41 (0.98–2.03) | 0.064 | 1.44 (0.95–2.21) | 0.090 |
| Postoperative chemotherapy | ||||||||
| Yes vs. no | 0.65 (0.48–0.89) | 0.007 | 0.69 (0.50–0.97) | 0.032 | 0.50 (0.35-0.72) | < 0.001 | 0.46 (0.30–0.69) | < 0.001 |
| Immunoscorea | 0.69 (0.61–0.77) | < 0.001 | 0.73 (0.64–0.83) | < 0.001 | 0.65 (0.56–0.75) | < 0.001 | 0.69 (0.59–0.80) | < 0.001 |
Bold value indicates P < 0.05
HR hazard ratio, CI confidence interval, CEA carcinoembryonic antigen
aAnalyzed as a ranked variable
Other clinicopathological variables that were associated with both inferior RFS and OS in the multivariate Cox analysis included a primary tumor stage of N1–2, preoperative serum CEA levels > 200 ng/mL, and diameter of the largest metastasis > 5 cm and postoperative chemotherapy (Table 2).
Prognostic value of the Immunoscore among patients with the same CRS
Among the 249 CRCLM patients, 141 (56.6%) had a low CRS (0–2), whereas 108 (43.4%) had a high CRS (3–5). Among patients with a low CRS, 82 (58.2%) and 59 (41.8%) were classified into the low and high Immunoscore subgroups, whereas among patients with a high CRS, 77 (71.3%) and 31 (28.7%) were classified into the low and high Immunoscore subgroups.
In the low CRS group, patients with high Immunoscores obtained both a higher 3-year RFS rate (54.9 vs. 24.2%; P < 0.001; Fig. 3a) and a higher 5-year OS rate (66.0 vs. 39.9%; P = 0.002; Fig. 3b) than those with low Immunoscores. However, among patients with a high CRS, the Immunoscore only conferred a prognostic value for OS (5-year OS rate: 10.3 vs. 44.2% for the low vs. high Immunoscore groups, P = 0.002; Fig. 3d). The difference in RFS between low and high Immunoscore groups did not reach statistical significance (3-year RFS rate: 8.5 vs. 19.5% for the low vs. high Immunoscore groups, P = 0.160; Fig. 3c).
Fig. 3.
Kaplan–Meier curves of RFS and OS of the high and low Immunoscore subgroups corresponding to CRS 0–2 and CRS 3–5. RFS (a) and OS (b) of the high/low Immunoscore groups of CRCLM patients with CRS 0–2. CRCLM patients with high Immunoscores obtained both superior RFS and OS than those with low Immunoscores; RFS (c) and OS (d) of the high/low Immunoscore groups of CRCLM patients with CRS 3–5. The Immunoscore only conferred a prognostic value for OS. The difference in RFS between low and high Immunoscore groups did not reach statistical significance
Comparison of the Immunoscore and CRS
ROC curves were used to compare the sensitivity and specificity of survival prediction between the CRS and the Immunoscore (maximum score of 5) (Fig. 4). In the ROC analysis for RFS prediction, both the CRS, with an area under the ROC curve (AUROC) of 0.675(95% CI 0.601–0.749, P < 0.001), and the Immunoscore, with an AUROC of 0.711 (95% CI 0.642–0.781, P < 0.001), had significant predictive values (Fig. 4a). Although the Immunoscore corresponded to a relatively larger AUROC for RFS prediction compared with the CRS system, the difference was not statistically significant (P = 0.492; Fig. 4a). In comparing the sensitivity and specificity for OS prediction, both the CRS system (AUROC: 0.660, 95% CI 0.592–0.727, P < 0.001) and the Immunoscore system (AUROC: 0.759, 95% CI: 0.699-0.818, P < 0.001) exhibited significant predictive values (Fig. 4b). Furthermore, the Immunoscore showed a superior prognostic value for predicting OS compared with the CRS (P = 0.029, Fig. 4b).
Fig. 4.
Comparison of the sensitivity and specificity for predicting RFS and OS of CRCLM patients after liver metastasectomy with the Immunoscore system and the CRS system. a ROC curves indicated the significant predictive values of both systems for RFS prediction, but the difference between the two systems was insignificant; b ROC curves indicated the significant predictive values of both systems for OS prediction, and the Immunoscore system was superior to the CRS system in OS prediction. The predictive ability of the Immunoscore was represented by the inverse of the Immunoscore (-Immunoscore) in the ROC curves
Discussion
In the present study, the Immunoscore system was first applied to evaluate liver metastatic lesions in CRC patients and showed both a convenient and effective prognostic value in patients who underwent liver metastasectomy. Among patients with the same CRS, the Immunoscore further played a predictive role in OS prediction. When compared with the traditional CRS scoring system, the Immunoscore exhibited a better predictive value for OS.
For CRCLM patients who underwent liver resection, several studies have reported that individual T-cell counts and subset ratios in liver metastatic lesions were significant predictors of survival and recurrence [12, 27–30]. Katz SC et al. compared T-cell counts between patients who survived ≤ 2 and ≥ 10 years following resection of CRCLM and found that high CD8+ T cell counts and low CD4+ T-cell counts were associated with 10-year survival rate following liver resection [27]. Furthermore, low CD45RO(+) cell infiltration at the peritumoral margin in the distant liver stroma and fibrotic capsule formation were both demonstrated to independently predict prolonged survival [28]. The absolute number of peritumoral Tregs was shown to predict cancer-specific survival and disease-free survival in CRCLM patients undergoing liver resection [31]. Subsequently, a predictive scoring system using TIL densities based on CD3+, CD8+, granzyme B (GZMB)-positive, and FOXp3-positive immune cells at the invasive margin of liver metastases was developed to predict the response to chemotherapy (with a sensitivity of 79% and specificity of 100%) [32]. Although there is a strong association between local immune cells and the chemotherapy response in patients with CRCLM, the clinical performance of this scoring system was still complex, thereby limiting its value. In addition, only some CRCLM patients underwent liver resection in that study.
In the present study, we used a simple method, the Immunoscore system, to predict the outcomes of patients with CRCLM after liver resection. The Immunoscore is based on the density of CD3+ and CD8+ immune cells in two regions (CT and IM). It is derived from the immune contexture, which is defined as the type, functional orientation, density and location of adaptive immune cells within distinct tumor regions. It was demonstrated that the Immunoscore system was superior to the American Joint Committee on Cancer/Union for International Cancer Control TNM classification system in patients with stage I/II/III CRC. In addition, the Immunoscore system is a convenient, fast, robust and economical method, which can be widely applied in routine clinical practice. An international consortium has also been initiated to validate this score system and promote the Immunoscore as a new factor in the CRC TNM classification. The results of the present study may extend the applications of the Immunoscore from early stage CRC patients to CRCLM patients who have undergone liver resection.
The results of the present study also indicated that high Immunoscores may be closely associated with several superior clinicopathological factors, such as a smaller size of the largest metastasis, fewer metastatic lesions, and a lower CRS, compared with low Immunoscores. This can be explained by the hypothesis that a superior host immune response may limit the progression and invasion of malignancies, and thus, patients with a higher Immunoscore may be commonly characterized by better prognostic factors. This has been supported by the previous study in which a strong association between immune cytotoxicity and metastasis was reported [33]. Patients with more distant metastases presented a significantly lower density of lymphocytes (CD3, GZMB, CD8, T-Bet, CD57, and CD45RO) in tumors [33]. Therefore, considering the relations among several factors of the CRS and the Immunoscore, it can be easily understood that the CRS was significantly and negatively associated with the Immunoscore in our study. Although the Immunoscore still retained significant prognostic value both in patients that did and did not receive preoperative chemotherapy, the use of preoperative chemotherapy may impair the prognostic performance of the Immunoscore. This was consistent with the findings of the previous study that chemotherapy and radiotherapy may affect the immune cell population and clinical implications of TIL accumulation [34].
Furthermore, when CRCLM patients were stratified by low and high CRSs, the Immunoscore showed a significant prognostic value for both RFS and OS in the low CRS (0–2) group and a correlation with OS in the high CRS (3–5) group, indicating that although an association with CRS existed, the Immunoscore still had an additional significant prognostic value in patients with the same clinical risk conditions, especially in patients with a low CRS. The Immunoscore and CRS may be considered as complements for predicting the prognosis of CRCLM. However, the equivocal prognostic ability of the Immunoscore for RFS prediction in patients with a high CRS suggested that the host’s immune system may have a relatively greater influence on OS than on RFS.
The current study also compared the sensitivity and specificity of the Immunoscore and CRS systems for survival prediction. Both the CRS and Immunoscore systems exhibited significant effects in predicting RFS, while the difference between the two scoring systems did not reach statistical significance, implying that the Immunoscore and CRS systems may not have a marked difference in predicting short-term survival. Both the CRS and the Immunoscore presented significant prognostic values in predicting OS, and the AUROC of the Immunoscore system was significantly larger than that of the CRS system, indicating that the Immunoscore system may be superior to the CRS system for predicting long-term survival. This finding was in line with previous studies that analyzed the association between the immune indexes and survival. The magnitude of T-cell infiltration in the IM of metastases was positively associated with long-term survival following resection of CRC liver metastases but had a relatively weak association with short-term survival [27].
This study represented the first use of the Immunoscore in patients with CRC liver metastases, and the internal validation enhanced the confidence of the results. Nonetheless, the study was limited by the small sample size, participant of only one institution, and its retrospective nature. Although the Immunoscore proposed by Galon et al. [15] was developed with the use of automated tissue microarray and the image software SpotBrowser, the present study used the free software image J, instead of the specified software. Inconsistencies across the enrolled patients, which included patients who underwent either surgery alone or in combination with perioperative chemotherapy, also contributed to unavoidable confounding factors. Therefore, the results should be interpreted with caution. Moreover, comparisons between the Immunoscore and other score systems that contain other TILs were not performed in the present study.
In conclusion, the present study confirmed that the Immunoscore system can be adapted to predict the prognosis of patients with CRCLM after liver resection. Furthermore, the Immunoscore was associated with the size and number of metastatic lesions and the CRS of patients. Compared with the traditional CRS system, the Immunoscore showed better prognostic value for long-term survival prediction. The Immunoscore may be used as a complement to the CRS system for predicting the prognosis of patients with CRCLM after liver resection.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
We express our gratitude to all the patients for their participation in this study. We also appreciate all our colleagues at the Sun Yat-sen University Cancer Center who participated in administering the treatment in the current study. Medbanks (Beijing) Network Technology Co., Ltd. is thanked for data collection.
Abbreviations
- AUROC
Area under the ROC curve
- CAPEOX
Oxaliplatin and capecitabine
- CEA
Carcinoembryonic antigen
- CI
Confidence interval
- CRC
Colorectal cancer
- CRCLM
Colorectal cancer liver metastases
- CRS
Clinical risk score
- CT
Center tumor
- ESMO
European society for medical oncology
- FOLFIRI
Irinotecan, 5-Fu and leucovorin
- FOLFOX
Oxaliplatin, 5-Fu and leucovorin
- FOLFOXIRI
Oxaliplatin, irinotecan, 5-Fu and leucovorin
- GZMB
Granzyme B
- HAI
Hepatic arterial infusion
- HR
Hazard ratio
- IM
Invasive margin
- IQR
Interquartile range
- LV
Leucovorin
- OS
Overall survival
- RFS
Relapse-free survival
- ROC
Receiver operating characteristic
- TIL
Tumor-infiltrating lymphocyte
- Tregs
T-regulatory cells
- XELIRI
Irinotecan and capecitabine
Author contribution
YL and RX were involved in the study design, protocol development, and data analysis and interpretation. MH and QS performed the immunohistochemical staining. SY and PS contributed to the Immunoscore evaluation. YW and HL performed the literature search for the study and were involved in data interpretation and writing the report. ZW, FW, YY, BL, DW, PD, GC, XW, ZL, LL, ZP and DW contributed to data collection. All authors reviewed and approved the manuscript for submission.
Funding
The work was supported by the Science and Technology Planning Project of Guangdong Province, China (201508020247).
Compliance with ethical standards
Conflicts of interest
The authors declare no conflicts of interest.
Ethical approval
All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional research committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
Yun Wang and Hao-cheng Lin contributed equally to this work.
Contributor Information
Rui-hua Xu, Email: xurh@sysucc.org.cn.
Yu-hong Li, Email: liyh@sysucc.org.cn.
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