Tumor-infiltrating CD8+ T cells combined with tumor-associated CD68+ macrophages predict postoperative prognosis and adjuvant chemotherapy benefit in resected gastric cancer

Gastric cancer (GC) is one of the most common malignant tumors in both sexes worldwide [1]. Regarding advanced GC, it has been suggested that a PAC regimen based on 5-fluorouracil (5-Fu) should be used as a first-line postoperative therapy [2‐6]. However, the response to PAC varies among patients. Of greater concern is the lack of a reliable criterion for identifying patients at high risk of recurrence, which in turn makes it difficult to distinguish between patients who will benefit from PAC when considering the survival rate after surgery [6‐8]. Accordingly, there is considerable interest in exploring the potential benefits of PAC for GC patients.

In recent decades, convincing evidence has emerged that the tumor microenvironment (TME) and inflammation play a key role in the development of many malignant tumors, including GC [1, 9, 10]. Tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) are two main components of the TME that have shown prognostic value in previous studies [11, 12]. Therefore, incorporating these immunological parameters into the established TNM staging system may increase the prognostic value for further stratification and better management of patients with different prognoses.

Regardless, little is known about the prognostic significance of TILs combined with TAMs in GC, and the impact of these cells on PAC benefit in GC remains unclear. In this study, we evaluated TILs and TAMs in more than 400 GC patients by immunohistochemical staining of CD8 and CD68. In addition, we used subgroup analysis to explore the relationship between TILs/TAMs and survival rates as well as the benefit of PAC based on 5-FU for survival. The results of this study will clarify the important prognostic role of lymphocyte and macrophage infiltration in GC and help to redefine subgroups of patients who are likely to benefit from PAC.

GC is an inflammation-related cancer characterized by a large degree of multinuclear and monocyte infiltration, including lymphocytes and macrophages [16, 27]. Tumor-associated immune cells affect complex microenvironments, with prognostic value in previous studies [11, 16, 28]. To date, there are several markers for TILs, such as CD3, CD4, CD8, Foxp3, and Granzyme B [29]. In the present study, we stained for CD8 on T cells, as reported [24, 30]. TAMs can be classified into two phenotypes: M1 (classically activated macrophages) and M2 (alternatively activated macrophages) [31, 32], and using immunochemistry, TAMs can be assessed by anti-CD68, M1-type TAMs by anti-HLA-DR and M2-type TAMs by anti-CD163 [32, 33]. However, it is largely unclear which macrophages have the greatest impact on the efficacy of chemotherapy for GC [34]. CD68 has been widely accepted as a specific marker of TAMs in human cancer [35, 36]. A recent report found high expression of CD68+ TAMs to be associated with recurrence of melanoma [36], and other studies have reported the clinical and functional significance of CD68+ TAMs in GC, though without addressing the M1 and M2 subsets [37, 38] . Thus, we also used CD68 as a marker of TAMs in this study.

Previous studies have explored the relationship between TIL infiltration and GC outcomes, but these studies have produced different results [39, 40]. For example, Fukuda et al. [39] found no significant difference in survival rates among patients with significant or slight TIL infiltration. In contrast, Lee et al. reported that the OS of GC patients with a high CD8+ TIL density tended to be longer than that of patients with a low TIL density at the same TNM stage, which was consistent with our findings [40]. In our study, the RFS and OS of TIL-positive cases were significantly better than those of TIL-negative cases. These conflicting results may be caused by different methods used to determine TIL strength. In addition, the function of intratumoral CD8+ lymphocytes in tumors is interlinked with other immune cells, and accumulating evidence suggests that TAMs plays an important role in cancer progression [41‐43]. However, conflicting prognostic data have been reported [33]. Ishigami et al. [44] observed a negative correlation between TAMs and the prognosis of GC patients, whereas Ohno et al. [45] concluded that the aggregation of TAMs within the tumor nest had a beneficial effect. Zhang et al. [46] also evaluated TAMs in 180 GC patients and found no significant correlation between these cells and OS. In the present study, CD68+ TAMs were found to be an independent risk factor for a worse GC prognosis.

Indeed, it is difficult to characterize complex tumor microenvironment using a single immune marker [43]. Nakanishi et al. reported that the ratio of CD68+ macrophages/CD57+ cells was closely related to prognosis of renal cell carcinoma [47]. Therefore, the main purpose of this study was to explore the effect of a combination of TILs and TAMs on the prognosis and efficacy of chemotherapy for GC, rather than the effect of different types of macrophages on prognosis. In fact, the use of multiparametric analysis to characterize immune cell types, such as CD8+ TILs and CD68+ TAMs, in the present study may provide a more comprehensive picture of the immune phenotypes within the TME, which may help in stratifying patients with the best chance of responding to immunotherapy. Using the CD8+ TIL/CD68+ TAM status, we were able to classify samples into 3 types (Type 1: CD8+ TIL positive and CD68+ TAM negative; Type 2: CD8+ TIL positive and CD68+ TAM positive or CD8+ TIL negative and CD68+ TAM negative; Type 3: CD8+ TIL negative and CD68+ TAM positive).

Traditional prognostic models for GC patients depend on the TNM staging system, which is derived from biological phenotypes centered on cancer cells. In fact, even patients with the same stage of cancer may have very different prognoses. In this study, we first found that incorporating CD8+ TILs/CD68+ TAMs into the current TNM staging system may increase prognostic value, thereby better identifying patients with different prognoses and providing better risk-oriented treatment. In general, incorporating molecular biomarkers or biologically driven classification into staging systems may better predict prognosis and treatment options and promote the development of personalized or precise medicine [48, 49]. TILs and TAMs are key in tumor-related inflammation and binding with other immune cells, which are processes considered to be evidence of host-growth interaction with tumors [31, 33, 50]. Previously, Zhang et al. generated a nomogram integrating TAMs, tumor T stage, N stage, and distant metastasis to predict the OS rate of GC [46]. In this study, we generated a novel and powerful staging system for GC patients based on TIL/TAM-based immune status and the tumor cell-centered TNM system. Our results suggest that incorporating the TIL/TAM status into the established TNM system can more accurately quantify prognostic risk.

Patients with stage II or III GC are considered to be candidates for PAC. However, it is uncertain whether all patients need PAC, as a large proportion of them do not appear to benefit from this approach. Therefore, the development of an improved PAC benefit prediction model has gained much interest [6]. Indeed, there is much evidence that the success of anticancer therapies, including traditional cytotoxic compounds, radiotherapy and targeted drugs, depends, at least in part, on activation of the anticancer immune response [51]. As previously described, CD68+ TAMs increase the response of many malignant tumors (such as stage III colorectal cancer) to 5-FU adjuvant therapy [52]. In this study, we further assessed the relationship between TILs/TAMs and survival in a subgroup of stage II/III patients receiving PAC. The results indicated that among patients receiving PAC, those with simultaneously high TIL and low TAM infiltration were more likely to have increased RFS and OS rates compared with the other 3 groups, indicating that CD8+ TILs/CD68+ TAMs may be an important factor in predicting the effect of chemotherapy. Immunotherapy, including immune checkpoint blockade, has been considered an important component of anti-cancer therapy [53, 54], and the TIL/TAM status might be an interesting target of investigation for the immunotherapy options that are emerging in this setting. A recent study found that PD-L1 is not only expressed in cancer cells but also in TILs at a high rate [55]. Furthermore, Harada et al. reported that TAMs are highly associated with PD-L1 expression in GC cells, suggesting that macrophage infiltration is a potential therapeutic target [56]. The authors also indicated that CSF1/CSF1R blockade might reduce PD-L1 expression in tumor cells [56]. Clinically, the status of CD8+ TILs/ CD68+ TAMs is helpful for stratifying patients with stage II/III GC receiving PAC, and prognostic stratification based on the TIL/TAM status can guide doctors in adopting tailor-made treatment plans according to patient performance. For example, strengthening a 5-FU-based treatment regimen or adding macrophage-targeted therapies (such as CSF1/CSF1R and/or PD-L1 inhibitors) is a potential strategy for type II or III GC with good performance. Currently, a phase 1, open-label, global study is underway to assess the combination of CSF1R antagonists and checkpoint blockades (NCT0323191). These results may help clinicians better select patients who need more aggressive adjuvant therapy or more in-depth follow-up, even though these patients might not be considered to be at high risk according to traditional clinicopathological features.

This study has some limitations. First, the study was retrospective. Although all parameters of prospective clinical trials were included, the number of patients receiving PAC was relatively small, and the prognostic significance of the PAC regimen and PAC cycle was not compared. Second, as a currently unresolved issue [24, 57], the specimens used and semi-quantitative immunohistochemical evaluation may not fully reflect the status of the tumor immune microenvironment. TAMs are more diverse and heterogeneous than cells with single markers or M1/M2 phenotypes. Further studies are needed to determine the interaction between different TAM and cancer outcomes. Third, the follow-up time was relatively short. Overall, in-depth in vitro and in vivo experiments are urgently needed to provide mechanical insight.

The TIL/TAM status is a promising biomarker for predicting the prognosis of GC. Our model incorporates immune parameters into the established TNM staging system to help clinicians and patients quantify the benefits of adjuvant chemotherapy after GC resection and formulate personalized treatment recommendations and treatment decisions. However, these results should be validated by a large, prospective, multiagency study.