Introduction

Colorectal cancer is one of the most common malignant diseases worldwide. Although a variety of anticancer drugs have been developed, the death rates of CRC have not been obviously decreased [1, 2]. Expression of PD-L1 in intratumoral compartment has been suggested to influence immune response [3] and serve as a prognostic marker in CRC [4]. PD-L1 is not solely considered as a result of an increased immune inhibiting PD/PD-L1 interplay but rather is viewed as a reflection of adaptive antitumor immunity, where tumor-infiltrating lymphocytes are activated in response to tumor antigens [4]. It has been reported that PD-L1 on either tumor cells or host immune cells contributes to tumor escape, and the relative contributions of PD-L1 on these cells seem to be context-dependent [5]. Recent study showed that tumoral PD-L1 is a favorable prognostic factor in early stage of non-small cell carcinoma [6]. It was also reported that there were differences in outcome in triple-negative breast cancer depending on the expression of PD-L1 in the tumoral cell membrane, cytoplasm, and stromal cellular compartments [7]. Yaqi Li et al. reported that tumoral PD-L1 correlated with better prognosis of CRC patients [8]. Whereas some studies found that PD-L1 was associated with deleterious effect on survival [9, 10], these studied did not distinguish PD-L1 expression in tumoral or stromal cells. Therefore, PD-L1 expression used as a predictor factor is also controversial.

Studies reported that CD8+ TIL induces PD-L1 expression in tumor cells by producing IFNγ [11,12,13]. CD8+ T cells are thought to have antitumor functions during tumor development in a tumor microenvironment. Evidence has shown that activated CD8+ cytotoxic T lymphocytes were correlated with favorable survival of CRC patients and gastric cancer patients [14,15,16,17]. Therefore, further detailed analysis is needed to confirm the prognostic significance of PD-L1 and CD8+ TILs in CRC and to investigate the relationship between PD-L1 and CD8+ T cells.

The Qingpu District of Shanghai in China was one of the endemic areas. Schistosomiasis, which is an infectious disease [18], is considered as a risk factor for CRC [19]. Schistosomiasis is correlated with inflammation [20,21,22]. CD8+ TILs are the main force involved in inflammatory response. In addition, PD-L1 was involved in immune microenvironment and upregulated by CD8+ TILs. With these considerations, we wonder to investigate the relationship between schistosomiasis and CD8+ TILs and PD-L1.

In short, this study aimed primarily to investigate the effect of schistosoma infection on CD8+ TILs and PD-L1 expression and the relationship between schistosomiasis and CD8+ TILs and PD-L1 expression. Besides, we proposed to further to compare the prognostic role of PD-L1 and CD8+ TILs in SCRC and NSCRC.

Methods and materials

Patients

This retrospective analysis includes 338 patients with resected primary CRC at Qingpu Branch of Zhongshan Hospital affiliated to Fudan University, from January 2008 to August 2016. All of the operations followed the principle described previously [23]. The inpatient medical records and pathological reports were reviewed from the pathological system and Qingpu District Center for Disease Control and Prevention, and the patients were followed up by telephone. OS is defined as the interval from the surgical operation date to the last follow-up or death caused by CRC. Inclusion criteria are as previously described [23]. Two expert pathologists reviewed HE-stained slides to determine the diagnosis and to restage the tumors according to the eighth edition of American Joint Committee on Cancer (AJCC). This study is approved by the medical ethics committee of Fudan University, in accordance with the Helsinki Declaration of 1975. Prior written informed consent was obtained from all patients.

Tissue microarrays (TMA)

The TMA blocks were manufactured from the most representative areas of individual paraffin blocks, as previously described [24]. Briefly, reviewed HE-stained slides and marked the represented areas in tumor tissues, and the single core (2 mm wide and 6 mm long) for each case was precisely arrayed into a new recipient paraffin block. The cores containing more than 20% tumor cells were considered as valid cores.

Immunohistochemical (IHC)

All the tissue slides were stained by the fully automated Bond-III system (Leica Microsystems, Newcastle-upon-Tyne, UK) according to the manufacturer’s instructions. The following primary antibodies were used: PD-L1 (MXR003; 1:750; MXB Biotechnologies, Fuzhou, China) and CD8 (clone NCL-L-CD8-4B11; 1: 100; DAKO, Minneapolis, MN, USA).

Pathological assessment of PD-L1 expression and CD8+ T cell density

PD-L1 IHC was analyzed independently by two experienced pathologists, who were unaware of the clinical data. The results were evaluated according to the percentage of the stained cells. Scoring was assessed in both tumoral membranous and stromal immune cell membranous compartments. Tumors were classified as PD-L1 positive if there was ≥ 1% tumoral membranous PD-L1 expression (tPD-L1+) or ≥ 1% stromal PD-L1 expression (sPD-L1+).

The TMA slides were scanned using a scanner system (PRECICE 500B) at × 40 magnification. For CD8, the densities of positively stained cells were evaluated on whole section slides using an image analysis system (Image J software, USA) (cells per square millimeter) (Fig. 1C). At least half of the core area was selected randomly, and the results of the calculated densities were extracted and put into an Excel file. Measurements were recorded as the mean number of positive cells per tissue unit in square millimeters as well as the number of positive cells among each 1-mm2 tissue units.

Fig. 1
figure 1

Immunohistochemical staining of representative programmed death-ligand 1 (PD-L1) expression (× 200) and CD8 (× 200) positivity. A PD-L1 expression positivity on tumor cells. B PD-L1 expression positivity on tumor-infiltrating mononuclear cells. C PD-L1 expression positivity both on tumor cells and within the immune stroma. D Immunohistochemical staining of representative CD8 positivity (× 200)

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Statistical analysis

Data were analyzed using SPSS (version 20.0; IBM Corp.) and Graphpad 5.0. Every variable was analyzed using univariate analysis to identify all potentially important predictors and then variables with p ≤ 0.05 in the univariate analysis were included in a multivariate analysis. Finally, multivariate Cox regression analysis was performed to identify predictive factors for OS.

Results

Patient characteristics

The clinical characteristics of the 338 patients are shown in Table 1. The median age of the patients at diagnosis was 67 years (range, 33–91 years). According to AJCC Staging Manual (seventh edition), there were very few highly differentiated cases in the follow-up data. Seventy-six percent cases were well/moderate differentiated, and 24% were poorly differentiated. Intriguingly, schistosoma infection was observed in 38% (128 out of 338) CRC patients (Supplementary Fig. 1). And the diagnosis of schistosomiasis was done by finding schistosome eggs in HE-stained slides.

Table 1 Clinicopathological characteristics of the CRC cohort
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Staining results of each marker

Figure 1 shows representative PD-L1-stained images on both tumor cells and tumor-infiltrating mononuclear cells. Among 338 cases analyzed, 41% of cases showed tumoral PD-L1 expression (tPD-L1+: defined as ≥ 1%), and 64% showed PD-L1 expression within the immune stroma (sPD-L1+: defined as ≥ 1%) (Table 1 and Fig. 1A, B). There were 38% (129 out of 338) of cases expressing PD-L1 both in tumoral and immune stroma (Table 1 and Fig. 1C). The median value of CD8+ density was 405 cell/mm2 (range, 0–2466 cell/mm2) (Table 1 and Fig. 1D).

Relationship between schistosomiasis and CD8+ TIL density and PD-L1 expression

Patients were divided into two groups: schistosomal-associated colorectal cancer (SCRC) patients and non-schistosomal-associated colorectal cancer (NSCRC) patients. As shown in Fig. 2A, there were no significant correlation between CD8+ TILs density and schistosomiasis (p > 0.05).

Fig. 2
figure 2

A The relationship between schistosomal infection and CD8+ TILs density (p > 0.05). B PD-L1 expression by immune stroma (p = 0.0001) increased with increasing CD8 density. C PD-L1 expression by tumor cells (p = 0.0102) increased with increasing CD8 density. Correlation between CD8 density and PD-L1 expression by location were examine using the Mann-Whitney test

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We next compared the correlation of CD8+ T cell density with PD-L1 on the tumor cells or in the immune stroma, respectively. As shown in Fig. 2B, C and Table 2, CD8+ T cell density was significantly higher within sPD-L1+ group than that within sPD-L1- group (Fig. 2B, p < 0.0001) (sPD-L1- group versus sPD-L1+ group, median 347 versus 460 cell/mm2), and it was also obviously higher within the tPD-L1+ group than within the tPD-L1- group (Fig. 2C, p = 0.0102) (tPD-L1+ group versus tPD-L1- group, median 371 versus 454 cell/mm2).

Table 2 The association between clinicopathological characteristics and PD-L1
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Correlation between PD-L1 expression and patient characteristics

The relationships of tPD-L1 and sPD-L1 expression with clinicopathologic features are detailed in Table 2. One hundred thirty-eight patients (41%) and 142 (42%) were placed in the tPD-L1high (expression level ≥ 2%) and sPD-L1high group (expression level ≥ 2%) based on the optimum cutoff point, respectively. Stromal PD-L1 positivity were significantly associated with less aggressive tumor features, including early pathological T stage (p < 0.001), absence of lymph node metastasis (p = 0.031), absence of tumor deposit (p = 0.012), early TNM Stage (p = 0.034), less tumor budding (p = 0.039), and less bowel perforation (p < 0.001). Meanwhile, tumoral PD-L1 positivity were significantly associated with early TNM Stage (p = 0.020) (Table 2).

Prognostic significance of PD-L1 expression and CD8+ T cells density

Mean and median time to OS was 62.54 and 62.85(1.25–134.4) months, respectively. During the follow-up, there were 42% (141 out of 338) patients died. Higher PD-L1 expression on both tumor cells (expression level ≥ 2%, tPD-L1high) and within the immune stroma (expression level ≥ 2%, sPD-L1high) was associated with better OS in CRC patients, but only the sPD-L1 reached statistical significance (p = 0.0023, Fig. 3A for sPD-L1; p = 0.3693, Fig. 3B for tPD-L1).

Fig. 3
figure 3

Kaplan-Meier curves for overall survival (OS) of CRC patients, OS was calculated using the Kaplan–Meier method and analyzed using the log-rank test. A OS of CRC patients with expression of PD-L1 on the immune stroma cells (sPD-L1 positive expressing ≥ 2%) (p = 0.0023). B OS of CRC patients with expression of PD-L1 on tumoral cells (tPD-L1 positive expressing ≥ 2%) (p = 0.3693). C OS of CRC patients with CD8+ cells density (p < 0.0001). The optimum cutoff value of CD8+T cell density were determined by X-tile program, which were 279 cell/mm2. CD8low group was defined as CD8+ T cells density < 279, and CD8high group was defined as CD8+ T cells density ≥ 279 cell/mm2

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With regard to CD8+T cell, the optimum cutoff value, which was determined by X-tile program, was 279 cell/mm2 (Supplementary Fig. 2). Patients were divided into two groups for further analysis (CD8low < 279 and CD8high ≥ 279 cell/mm2). Tumors with higher CD8+ T cell density had better OS compared with that of with lower CD8+ T cell density (p < 0.0001, respectively, Fig. 3C).

The univariate Cox regression model indicated that age, gender, pathological T stage, lymph node metastasis, TNM stages, tumor differentiation, vessel invasion, tumor deposit, tumor budding, Schistosomiasis, CD8+ T cells, and sPD-L1 were significantly associated with OS (p < 0.05, Table 3). Multivariate analysis after adjustment indicated that gender, TNM stage, tumor deposit, Schistosomiasis, and CD8+ T cells were independent prognostic factors for OS of CRC patients (p < 0.05, Table 3).

Table 3 Univariate and multivariate Cox regression of clinicopathological for overall survival
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Survival analysis based on subgroups

Kaplan-Meier analysis demonstrated that merely sPD-L1 expression level was associated with favorable OS in the NSCRC group (p = 0.0040) (Fig. 4A), sPD-L1 expression level in the SCRC group and tPD-L1 in the both groups were not correlated with OS (p > 0.05) (Fig. 4B–D). In the NSCRC set, the univariate Cox regression model revealed that gender, TNM stage, pathological T stage, lymph node metastasis, tumor differentiation, tumor budding, vessel invasion, tumor deposit, sPD-L1 expression level, and CD8+ T cells density were associated with OS (p < 0.05) (Table 4), and the multivariate Cox regression analysis showed that gender, pathological T stage, TNM stage, tumor deposit, and CD8+ T cells density were independent prognosis factors (p < 0.05) (Table 4). In the SCRC set, the univariate analysis demonstrated that lymph node metastasis, TNM stage, tumor differentiation, tumor deposit, and CD8+ T cell density were associated with OS (p < 0.05), and multivariate analysis results showed that only TNM stage, tumor deposit, and CD8+ T cell density were independent factors for OS (p < 0.05).

Fig. 4
figure 4

Kaplan-Meier curves for overall survival OS of CRC patients, OS was calculated using the Kaplan–Meier method and analyzed using the log-rank test. A PD-L1 expressing on the immune stroma cells (sPD-L1 positive expressing ≥ 2%) for OS of schistosomal-associated-colorectal cancer patients (SCRC) (p = 0.0040) or for OS of non-schistosomal-associated colorectal cancer patients (NSCRC) (B) (p = 0.1949). C PD-L1 expressing on the tumoral cells (tPD-L1 positive expressing ≥ 2%) for OS of SCRC patients (p = 0.8926) or for OS of NSCRC patients (D) (p = 0.8228)

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Table 4 Univariate and multivariate analysis for overall survival in SCRC set and NSCRC set
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Discussion

Various tumor entities with elevated immune response have dense CD8 pos T cell infiltrates in common, which are responsible for a local production of interferon gamma (IFNγ) [25, 26]. IFNγ, in turn, provokes the adaptive upregulation of PD-L1 on nearby tumor cells via NFκB [27]. Our results showed that PD-L1 expression in tumoral cells and stromal cells were positively correlated with CD8+ TILs density.

In this study, the expression of PD-L1 in tumor cells and immune stroma were associated with less aggressive tumor features and translated into favorable OS in patients with CRC cancer. These were consistent with J Wyss et al.’s findings [4]. The association of PD-L1 expression with beneficial clinical outcome has been reported in a diverse set of tumor types, such as NSCLC [28], melanoma [29], breast cancer [7, 30], and including CRC [8]. This might seem inconsistent with the immunosuppressive function of PD-L1. However, this might be explained that PD-L1 expression within tumor microenvironment is not only as an immunosuppression factor, but rather acts as a reflection of adaptive antitumor immunity, where tumor-infiltrating lymphocytes are activated in response to tumor antigens. Contrary to our findings, Thompson et al. [13] showed that in patients with locally advanced gastric cancer, tumoral, and stromal PD-L1 expression and CD8+ TILs were associated with unfavorable outcome. These opposite results might explained by the interaction between tumor and tumor-associated stroma and TILs might be different among different tumor types.

Our results showed that CD8 density was also an independent predictor for CRC patients. CD8, which is predominantly expressed on cytotoxic T cells, is a crucial component of the cellular immune system and is pivotal for cell-mediated anti-tumor immune response [31, 32]. Previous studies demonstrated that patients whose tumors contained infiltrating CD8+ TIL showed better survival in non-small cell lung cancer (NSCLC) [33,34,35,36,37,38,39,40]. These results further suggest that PD-L1 expression may reflects an association with a TIL-mediated antitumor inflammatory response, rather than always being associated with tumor immune evasion [7]. Unexpectedly, there were no correlation between CD8+ TILs and PD-L1 and schistosomiasis. It was possible that the patients in the cohort with schistosomiasis are obviously older than patients without schistosomiasis (median age of patients with schistosomiasis was 74 years old and that of patients without schistosomiasis was 64.5 years old, p < 0.0001). And the vigor of immunity of older people is weak [41]. In order to confirm this speculation, we excluded patients younger than 60 years old, then to analyze the relationship between schistosomiasis and CD8+ TILs. However, the small percentage of SCRC patients did not allow us to perform further analysis stratified by age. Thus, further works in larger cohort are still needed to investigate the impact of S. japonicum on CD8+ TILs density and PD-L1 expression.

Our retrospective study had several limitations. First, we do recognized the limitation of utilizing a TMA approach to assess expression of a biomarker that may only be locally present in samples, raising the possibility of false negatives, which could possibly change the significance of PD-L1 expression in CRC. Second, we speculated that IFNγ which secreted by CD8+ T cells upregulated the expression of PD-L1. However, further studies needed to clarify the association between PD-L1 expression and CD8+ TILs and to determine whether this combination has predictive relevance as a biomarker for selecting individual patients for treatment involving PD-1/PD-L1 blockade or for selection of certain tumor types for development. Third, determination of PD-L1 expression in tumor samples was generally performed by immunohistochemistry using various antibodies. Fourth, the threshold for positivity was not formally assessed.

In conclusion, the results in the present study demonstrated that stomal PD-L1 expression but not tumoral PD-L1 expression in the whole cohort and in the NSCRC set were associated with less aggressive tumor feature and translated into better OS. And the expression of PD-L1 was positively associated with CD8+ TILs density.