Fifty primary CRC tissues and 50 matched adjacent normal mucosa tissues were collected from the patients who underwent surgical resection at the Sichuan Provincial People’s Hospital (Chengdu, China). The original tumours were staged on the basis of the tumour-node-metastasis (TNM) classification system of the International Union Against Cancer[17]. Tumour differentiation was scored according to Edmondson Steiner scoring by senior pathologists. Detailed clinicopathological information was excerpted from the clinical data and a summary of the specific CRC demographics is displayed in Table 1. Informed prior to analysis, all patients consented to the tissue procurement, and the study was approved by the Institutional Ethics Committee of Sichuan Provincial People’s Hospital.

Human cancer tissue sections were subjected to immunohistochemistry analysis using a Dako Envision System (Dako Cytomation GmbH, Hamburg, Germany Denmark) according to the manufacturer’s instructions. In brief, tumour blocks were formalin-fixed, paraffin-embedded, and cut into 4-μm-thick sections. The sections were deparaffinized in xylene and rehydrated through diminishing concentrations of ethanol (100%, 95%, 85%, and 75%). This was followed by subsequent incubation in 3% H₂O₂ for 10 min in the dark at room temperature to eliminate endogenous peroxidase activity. Antigen-retrieval was performed by heating the sections for 5 min in citrate buffer (pH 6.0) using the autoclave sterilizer method. The sections were then allowed to cool at room temperature for 60 min, and rinsed twice for 5 min with fresh PBS. Thereafter, the slides were preincubated with healthy bovine or goat serum albumin diluted in PBS (pH 7.4) for 15 min at 37 °C, and then incubated overnight at 4 °C with primary antibody specific for IL-6 (mouse anti-IL-6, dilution 1:100, Proteintech). After three rinses in fresh PBS, the slides were incubated for 40 min at 37 °C with horseradish peroxidase-coupled secondary antibody. Following three additional washes, all specimens were stained with 3,3’-diaminobenzidine (DAB) substrate chromogen system (Dako Cytomation GmbH). Finally, the sections were rinsed in distilled water, and counterstained with Mayer’s haematoxylin according to the manufacturer’s instructions. Non-immune rabbit IgG at the same dilution as the primary antibody was used as a negative control.

Cells with observable brown particles in the cytoplasm were taken as positive. All sections were assessed by two professional pathologists who were blinded to patient outcomes and all clinicopathologic data. The immunohistochemical staining was evaluated according to the intensity (weak = 1, intense = 2) of IL-6 immunostaining and the density (0% = 0, 1%-50% = 1, 51%-75% = 2, > 76% = 3) of positive carcinoma cells[5]. The eventual score of each specimen was calculated by multiplying intensity and density, and the tumours were finally determined as negative expression: score = 0; low expression: score ≤ 3; or high expression: score > 3. If the two assessments did not agree for a sample, the sample was re-evaluated and classified based on the evaluations given most frequently by the experts.

The data were analysed with the SPSS 16.0 for Windows (SPSS Inc). Pearson χ² test and Fisher’s exact test were used to compare qualitative variables, and quantitative variables were analysed by the t-test. The correlation between clinicopathological factors and IL-6 expression was evaluated by the Spearman test for non-parametric variables. P-values less than 0.05 were considered statistically significant.

To explore the underlying clinical role of IL-6 in CRC, the expression of IL-6 was examined by immunohistochemical staining in 50 CRC tissue samples and 50 matched adjacent normal mucosa tissue samples. Strong IL-6 staining was mostly located in the cytoplasm of CRC cells (Figure 1A), while partial IL-6 staining was observed in the normal mucosa. Strong IL-6 expression was observed in 8% (4/50) of the normal colorectal mucosa samples and in 46% (23/50) of primary CRC samples. IL-6 expression was significantly increased in the CRC tissues compared with the normal mucosa tissues (P < 0.001; Figure 1B).

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Figure 1 Interleukin-6 expression is elevated in human colorectal cancer tissue samples. A: Individual FFPE sections demonstrated that colorectal cancer cells had invaded underneath the colorectal mucosa. The IL-6 expression level was elevated in colorectal tumour tissues compared to adjacent non-tumour tissues. “T” refers to tumour tissue, and “N” indicates adjacent non-tumour tissue from the same patient; B: IL-6 expression scores are shown as box plots, with the horizontal lines representing the median, the bottom and top of the boxes representing the 25^th and 75^th percentiles, respectively, and the vertical bars representing the range of data.

When analysing the levels of IL-6 expression in colorectal tumour cells by immunohistochemistry, we observed that IL-6 in tumour cells showed stronger immunoreactivity as tumour cells invaded more deeply (Figure 2A). This means that the tumour regions that were closer to the invasion front showed higher IL-6 expression levels. In addition, the majority of tumor cells in lymph node metastases were also IL-6-immunopositive (Figure 2B).

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Figure 2 Interleukin-6 expression is associated with invasion depth and lymph node metastasis in colorectal cancer. A: Increased expression of IL-6 at the invasive front of human CRC samples compared with tumour centre; B: The tumor cells in lymph node metastases were also IL-6-immunopositive. IL: Interleukin.

We next analysed the association between the levels of IL-6 expression and clinicopathologic parameters in CRC, including TNM stage (stages I, II, III, and IV) and histological differentiation (well, moderately, and poorly differentiated). The results showed that the levels of IL-6 expression were inversely associated with histological differentiation (P < 0.05, Figure 3A and B), but positively correlated with TNM stage (P < 0.001, Figure 3C and D). Of the 50 IL-6-positive CRC cases, 14 were well-differentiated, 27 moderately differentiated and 9 poorly differentiated. In addition, 29 cases were classified as stage I-II, while 21 cases were classified as stage III-IV. Taken together, these analyses indicated that the upregulation of IL-6 in CRC cells correlates with tumour progression.

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Figure 3 Interleukin-6 expression is significantly correlated with histological differentiation and TNM stage. A: Poorly differentiated colorectal tumours showed higher expression of IL-6 than moderately and well differentiated colorectal tumours; B: Comparison of immunohistochemical scores of IL-6 according to histological differentiation (P < 0.05); C: Strong immunoreactivity was more likely to present with advanced-stage (stage III/IV) CRC compared to early-stage (stage I/II); D: Comparison of immunohistochemical scores of IL-6 according to TNM stage (P < 0.001).

Colorectal cancer (CRC) is the fourth leading cause of cancer-related death worldwide. Previous studies have demonstrated that IL-6 is a critical tumour promoter during early CRC tumourigenesis. However, there have been few studies regarding the expression of IL-6 and its prognostic role in CRC. The correlation between the expression levels of IL-6 and the clinicopathological features of CRC specimens has not yet been investigated.

Previous studies have demonstrated that IL-6 is a critical tumour promoter during early CRC tumourigenesis and that the IL-6/STAT3 signaling pathway plays an important role in the progression of CRC.

To the best of our knowledge, this is the first analysis of the expression of IL-6 in resected CRC samples and its prognostic role using immunohistochemistry combined with biostatistics.

The results showed that the expression levels of IL-6 were correlated with TNM stage and histological differentiation. Furthermore, IL-6 in tumour cells showed stronger immunoreactivity as tumour cells invaded more deeply. These data indicate that IL-6 might be used as a potential target for postoperative adjuvant therapy in patients with CRC.

Invasion depth, also called the depth of tumour invasion, is the most significant histological predictor of lymph node metastasis in colorectal cancer. Increasing evidence indicates that colorectal cancer with submucosal deep invasion correlates with a poor prognosis.

This is an interesting paper. The method they used to quantify IL-6 in CRC specimens is only a qualitative method.