Prognostic significance of SDF-1 in colorectal cancer depends on CD8+ T-cell density

Background: Since colorectal cancer (CRC) remains one of the most common malignancies, a tremendous amount of studies keep taking place in this field. Over the past 25 years, a notable part of the scientific community has focused on the association between the immune system and colorectal cancer. A variety of studies have shown that high densities of infiltrating CD8+ T cells are associated with improved disease-free and overall survival in colorectal cancer (CRC). Stromal cell-derived factor-1 (SDF-1) is a protein that regulates leukocyte trafficking and is variably expressed in several healthy and malignant tissues. There is strong evidence that SDF-1 has a negative prognostic impact on colorectal cancer (CRC). However, based on a significant correlation of SDF-1 and CD8+ T cells in a previous study (r=0.53, p<0.0001), we hypothesized that the prognostic significance of SDF-1 in CRC could depend on the immune microenvironment. Therefore, we explored the combined prognostic significance of SDF-1 expression and CD8+ T cell density in a large CRC collective. Methods: We analyzed a tissue microarray (TMA) of 613 patient specimens of primary CRCs by immunohistochemistry (IHC) for the expression of SDF-1 by tumor cells and tumor-infiltrating immune cells (TICs) and CD8+ T-cells. Besides, we analyzed the expression of SDF-1 at the RNA level in The Cancer Genome Atlas cohort (TCGA). Results: We found that the the combined high expression of SDF-1 and CD8+ T-cell infiltration shows a favorable 5-year overall survival rate (66%; 95%CI=48–79%) compared to tumors showing a high expression of CD8+ T-cells only (55%; 95%CI=45–64%; p=0.0004). High expression of SDF-1 and CD8+ T-cells infiltration was significantly associated with a favorable prognosis also in a validation group (p=0.016). Univariate and multivariate

Conclusions: SDF-1 high /CD8 high density represents an independent, favorable, prognostic condition in CRC, most likely due to an effective antigen-specific immune response.

Background
Colorectal cancer (CRC) is the third most common malignancy to be diagnosed.
Furthermore, CRC remains the second most common cause of death from cancer worldwide, despite the screening and new methods of treatment [1,2]. These facts explain why so many studies have taken place to identify the mechanisms of CRC development. Understanding these features could lead to new concepts in the approach of diagnosis, prognosis, and even treatment of CRC.
As with the majority of cancers, the Tumor Node Metastasis (TNM) classification serves as the gold standard tool for the staging of CRC [3]. By every newly diagnosed CRC, an interdisciplinary team of specialists in several fields (Visceral Surgery, Oncology, Gastroenterology, Radiology/Nuclear Medicine, Radiation Oncology and Pathology) takes into account the TNM classification to determine the treatment. According to this system, they decide which of the patients are suitable for surgical resection and which of them are candidates for adjuvant chemotherapy following the resection of the primary tumor [4,5].
However, we repeatedly observe that patients with identical stages and treatments have a completely different outcome in terms of survival and recurrence. This fact indicates that TNM classification alone in the vast majority of cases is not sufficient for the prognosis of colorectal cancer [6,7]. Subsequently, an enormous amount of studies have investigated and still investigate other features, which could be an excellent additional tool for this classification. The crucial role of the microenvironment in CRC was thoroughly explored and showed that the high immune cell infiltration by cytotoxic CD8 + cells and memory CD45RO + cells has a favorable prognostic significance [8][9][10][11][12].
CD8 (cluster of differentiation 8) is a well-known protein that serves as a co-receptor for the T-cell receptor (TCR) and binds to the major histocompatibility complex (MHC) molecule [13]. When the cytotoxic T cells are combined with CD8 surface protein, produce the CD8 + T cells and play a vital role in antigen recognition. Naito et al. proved that the infiltration of tumors with CD8 + T-cells has a beneficial prognostic influence in CRC [14].
Since then, a substantial number of studies have also examined the role of CD8 + T-cells, and nowadays, the scientific society has accepted its positive prognostic impact on CRC [15][16][17][18][19]. These results highlight the fact that the infiltration of the tumor with CD8 + Tcells is associated with a better prognosis not only in CRCs but also in other malignancies such as lung [20], renal [21] and endometrial [22]. Following these positive results, various immune cells were investigated with the aim of finding a better and more sufficient way to evaluate the prognosis of CRC. The stromal cell-derived factor 1 (SDF1) is a chemokine protein, which is strongly chemotactic for lymphocytes [23]. SDF-1 and one of its receptors, CXCR4, have been shown to play a crucial role in the tumor-stromal communication affecting cancer growth, angiogenesis, and metastasis formation [24]. Samarenda et al. performed a meta-analysis of 38 studies that evaluated the association between SDF-1 expression and cancer survival. The authors showed that a high SDF-1 expression was associated with significantly reduced overall survival in patients with lung, pancreatic, and esophagus-gastric cancer. Yet, there was no correlation between SDF-1 expression and overall survival in colorectal cancer [25].
In the study we conducted, we hypothesized that the prognostic significance of SDF-1 could depend on the immune microenvironment in CRC [26][27][28][29]. To explore this hypothesis, we comparatively investigated the prognostic role of SDF-1 in human CRC in the context of CD8 + T-cell density.

Tissue microarray construction
In our study, we included 613 patients with unselected, clinically annotated primary CRC specimens in a tissue microarray (TMA). Our study had the approval of the local ethics committee (EKBB). As far as it concerns the technique used for the TMA construction, we have described this in previous studies of our team [30]. Our specialist prepared the tissue cylinders of formalin-fixed, paraffin-embedded tissue blocks according to standard procedures. The tissue cylinders had a diameter of 0.6 mm, and were punched from morphologically representative areas of each donor block and finally brought into one recipient paraffin block (30 × 25 mm), using a semi-automated tissue microarray arrayed.
It is crucial to mention that each punch was made from the center of the tumor to enable each TMA spot to include more than 50% of tumor cells.

Clinico-pathological Features
Clinico-pathological data were collected retrospectively in a non-stratified and random manner. Annotation included patient age and gender, tumor diameter in mm, site of the tumor, pT-stage, pN-stage, grade, stage according to TNM-classification, tumor border configuration (infiltrative vs pushing), vascular invasion, overall survival time (months), 5years survival in % (95%CI) and the presence of peri-tumoral lymphocytic inflammation at the invasive tumor front (Table 1). Tumor border configuration and peri-tumoral lymphocytic inflammation were evaluated using the original hematoxylin-eosin slides of the resection specimens corresponding to each TMA punch.

Immunohistochemistry
We used standard indirect immunoperoxidase procedures (IHC; ABC-Elite, Vector Laboratories, Burlingame, CA) as we have already described previous studies of our team [31]. Our specialists dewaxed and rehydrated slides in distilled water. Afterward, endogenous peroxidase activity was blocked using 0.5% H2O2. Sections were incubated with 10% normal goat serum (DakoCytomation, Carpinteria, CA) for 20 minutes and incubated with primary antibody at room temperature. We used primary antibodies that were specific for CD8 (Ventana 790-4460) and SDF-1 (Abcam ab9797). Subsequently, these parts were incubated with peroxidase-labeled secondary antibody (DakoCytomation) for 30 min at room temperature. For visualization of the antigen, these parts were immersed in 3-amino-9-ethylcarbazole plus substrate-chromogen (DakoCytomation) for 30 minutes and counterstained with Gill's hematoxylin.

Evaluation Of Immunohistochemistry
Two Gene Expression levels (FPKM values) for the genes were downloaded using TCGAbiolinks R package.

Statistical analysis
All statistical analyses were made using STATA software version 13 (StataCorp, College Station, TX, USA). We explored associations with survival using the Cox proportional hazard regression model. Cut-off values used to classify CRC with low or high immune cell infiltration were available from previous publications [32] or generated by applying regression tree analysis. Threshold value for CD8 + was 10 cells/TMA-punch. Threshold value for SDF-1 histoscore was 200 (= 75th percentile). Chi-square, Fisher's exact, and Kruskal-Wallis tests were used to determine the association of SDF-1 tumor expression and CD8 + T-cell infiltration and clinical-pathological features.
For survival analysis, the study population was randomly assigned to a test and validation group. Univariate survival analysis was performed by Kaplan-Meier and log-rank tests.
The assumption of proportional hazards was verified for all markers by analyzing correlation of Schoenfeld residuals and ranks of individual failure times. Any missing clinicopathological information was assumed to be missing at random. Subsequently, SDF- Association of clinicopathological features with SDF-1 expression and CD8 + T cell density In Fig. 1 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-1 high /CD8 + high in contrast to SDF-1 high /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 (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

Discussion
A significant amount of studies showed that the infiltration of CRC by CD8 + T-cells represents a favorable prognostic factor for the clinical outcome. Since then, more and more scientists are exploring the role of the microenvironment and immune response in the development of malignancies [11][12][13][14][15][16][17][18][19][20]. A great variety of immunocompetent cells, cytokines, and chemokines are currently investigated. Our team has already tested some of these factors in previous studies [34][35][36]. Furthermore, a study of Pagès et al. from May 2018 showed that the Immunoscore could determine the risk of recurrence in patients with colon cancer [44].
Among other immune markers, several studies have investigated the expression of SDF-1 and its role in tumor immunobiology. However, these studies came to conflicting data.
Some of them showed that the high expression of SDF-1 is associated with reduced overall survival in patients with lung, pancreatic, and esophagus-gastric cancer. In contrast to these results, it was observed that in breast cancer, the high expression of SDF-1 was associated with increased overall survival [37][38][39][40]. In the case of colorectal cancer, there is a high heterogeneity across existing studies [26][27][28][29].
Some of the cells that produce the SDF-1 are the endothelial and bone marrow cells, mucosal epithelial cells, tumor cells, and T-lymphocytes [41]. SDF-1 expression is increased in tissues characterized by neo-angiogenesis and inflammation, supporting chemotactic gradients attracting immune cells. In a previous study of our team, we investigated the SDF-1-CXCR4 chemokine axis in cell trafficking as well as in tumor progression [42]. In that study, we showed that the activation of CXCR4, which is suggested by the presence of its phosphorylated form (pCXCR4), in CRC tumors and in infiltrating immune cells is associated with a significant favorable prognosis. According to our data, Stanisavljevic et al. have also shown that SDF-1 expression represents a favorable prognostic factor for disease-free survival in CRC [29].
In the presented study we explored if the prognostic role of SDF-1 expression in CRC depends on the tumor microenvironment, especially on CD8 + T-cell infiltration. Finally, we found that a better prognosis characterizes CRC showing SDF-1 high tumor expression and CD8 + high density in contrast to CRC having a SDF-1 high /CD8 + low or SDF-1 low /CD8 + high pattern, most likely due to an effective antigen-specific immune response. Our data provide novel insights into the prognostic relevance of the interaction between the innate and adaptive immune system in CRC microenvironment. Our results could be the reason to When it comes to limitations, we have to begin with the fact that our study is a retrospective one. Nevertheless, by using the data that emerge from extensive retrospective analyses, we may, in the future, be able to develop prospective studies.
Secondary, TMA technology may fail to represent tumor tissue heterogeneity. Yet, the blocks included in our TMA were derived from tumor centers and included more than 50% of cancer cells. Additionally, the large number of individual CRC specimens (> 600) may partly compensate for the heterogeneity of the immune contexture in different tumor areas. Another point to consider is that by analyzing the Kaplan Meier curves of our study, we observe a difference in the long-term survival (> 5 years) rates between the testing and validation group. As the cohort was randomly split; it seems that in the validation group, CD8 + T cells appear to be the dominant marker. However, in the dichotomized cox regression, the combination still had a significantly lower HR. Yet, to overcome this criticism, a prospective validation study is planned on an independent cohort. Finally, the group investigated in this study includes CRC patients that were operated between 1985 and 1998. At that time, the use of neoadjuvant therapy was not part of the treatment of CRC. Overall-survival according to SDF-1 expression and CD8+ cell density in a test A (n=289) and validation B group (n=317)