In the 21st century, cancer is expected to become the leading cause of death and the leading obstacle to increased life expectancy in countries around the world. According to the 2020 Cancer Statistics report, there were approximately 19.3 million new cancer cases and 10.0 million cancer deaths worldwide (1). The most commonly diagnosed cancer types are female breast cancer (11.7% of all cases), lung cancer (11.4%), colorectal cancer (10.0%) and prostate cancer (7.3%) (1). The leading cause of cancer-related death is lung cancer (18% of all cancer-related deaths), followed by colorectal cancer (9.4%), liver cancer (8.3%) and stomach cancer (7.7%) (1). With the increase in the screening rate and advancements in diagnosis and treatment technology, the prognosis of cancer patients has been greatly improved. However, it was reported that the overall 5-year relative survival rate of all cancers diagnosed between 2009 and 2015 was only 67%, with pancreatic cancer (9%), liver cancer (18%), lung cancer (19%) and esophageal cancer (20%) having the lowest 5-year survival rates (2).

With the in-depth understanding of the tumor microenvironment, immunotherapy has become a new standard treatment strategy in addition to traditional therapies such as surgery, radiotherapy and chemotherapy. Immune checkpoint inhibitors (ICIs) have made a breakthrough in clinical treatment. ICIs mainly include CTLA-4 inhibitors and PD1/PD-L1 inhibitors, which have antitumor effects by blocking inhibitory receptors on T cells and reactivating T cells. Currently, ICIs are approved for melanoma, lung cancer, bladder cancer, kidney cancer, lymphoma and so on and have significantly improved the survival rate of patients with many kinds of cancer (3). Unfortunately, only a small subset of patients can benefit from these drugs. For example, the objective response rates (ORRs) of pembrolizumab or nivolumab in the first-line treatment of melanoma and the second-line treatment of non-small-cell lung cancer (NSCLC) are 40-45% and 20%, respectively (4–6). Therefore, it is necessary to identify biomarkers for predicting the efficacy of ICIs to screen the patients who may benefit from ICIs.

Many molecular biological markers related to the prognosis of ICIs have been gradually established. Changes in the intrinsic factors of tumor cells, such as increased tumor mutation burden (TMB) and increased expression of PD-L1, are usually associated with increased clinical benefits in patients (3). Specific gene mutations can also affect the response to ICIs. For example, patients with LRP1B-mutant cancer showed longer progression-free survival (PFS) and overall survival (OS) than wild-type patients when treated with ICIs (7). In addition, the degree of infiltration of all kinds of immune cells and the expression of various cytokines in the tumor microenvironment also affects the results of immunotherapy (3).

In addition to tumor-related factors, host characteristics such as age and sex may also be predictive markers for the efficacy of ICIs (3). Among them, age, as one of the most important clinical features of cancer patients, has shown a positive impact on prognosis in some studies. For example, studies by Kugel et al. showed that melanoma patients aged ≥60 years responded better to PD1 inhibitors than younger patients (8). Another meta-analysis of melanoma showed that patients over 75 years old were more likely to benefit from ICIs (9). For pan-cancer patients, Wu et al. found that the prognosis of older patients (≥65 years old) using ICIs was better than that of younger patients (<65 years old) (10). However, some studies have revealed the adverse effect of age on prognosis. For example, Li et al. showed that older cancer patients tend to have better OS and PFS (11). In addition, some studies have shown that age is not an independent predictor of prognosis in cancer patients treated with ICIs. For example, Sun et al. revealed that there was no significant difference in the OS of patients with NSCLC using PD-1/PD-L1 inhibitors between the young group (< 65 years old) and the old group (≥65 years old) (12). At the cutoff point of 70 years old, the PFS and OS of patients treated with ICIs in the melanoma, NSCLC and pan-cancer datasets were similar (13–15). Another analysis of renal cell carcinoma (RCC) and urothelial carcinoma (UC) as a whole or in subgroups showed that older patients over 75 years old had disease control rates (DCRs) comparable to those of younger patients treated with ICIs (16). The controversial results mentioned above may be related to the cancer type, sample size, and age cutoff point. At present, the selection of the age cutoff point is still controversial, leading to discrepancies in the age cutoff points presented in different studies. In addition, we found that for a single cancer type, such as melanoma, or for a collection of cancer types, the selection of different age cutoff points is likely to affect the predictive impact of age on the prognosis of patients treated with ICIs. Therefore, the selection of the appropriate age cutoff point is still worthy of further exploration.

In summary, this study aims to explore the appropriate age cutoff point for ICI efficacy prediction. Considering that the effect of age on the prognosis of patients using ICIs may be impacted by various age-related negative/positive predictive markers, we intended to use the data obtained from the database and literature to try to reveal the biomarkers related to age from the perspective of gene mutations, gene expression levels, immune cell infiltration and related pathways.

ICIs are effective against various cancers (27), lack known cumulative toxicity and are safer than conventional cytotoxic chemotherapies (15). Therefore, ICIs represent an attractive choice for the treatment of elderly patients. In previous clinical studies, the ages of 65 or 70 years are often used to distinguish between the elderly and the young. However, any certain age cutoff is insufficient for characterizing elderly individuals because aging is a highly variable physiological process (28). To reduce the possible deviation caused by a specific age limit, we set a number of different age cutoff points (50, 55, 60, 65, 70, and 75) and consequently observed the differences in survival outcomes brought about by different cutoff points. Our results showed that the 50 years old is a good age cutoff point for prognosis. Therefore, using the data in the database and literature, we divided people into the elderly group and the young group according to the cutoff of 50 years old and further explored the biomarkers related to age.

In this study, we found that TMB, NAL and the number of DDR pathway mutations in the elderly group were significantly higher than those in the young group of pan-cancer patients. TMB refers to the total number of nonsynonymous mutations present in tumors (20), which produce abnormal proteins by altering amino acid sequences. If abnormal proteins are eventually recognized by immune cells, they are likely to become neoantigens that facilitate the subsequent immune response (29). High-TMB tumors tend to generate more neoantigens and are more immunogenic, thus responding better to ICIs (30). Samstein et al. included a large cohort of 1662 cancer patients receiving ICIs and demonstrated that high TMB can predict better OS across multiple cancer types, such as colorectal cancer, NSCLC, and head and neck cancer (18). Another comprehensive analysis suggested that for patients treated with ICIs in the pan-cancer dataset, high TMB was significantly related to favorable OS (HR=0.40; 95% CI: 0.30-0.53; p<0.00001) and a low risk of disease progression (HR=0.37; 95% CI: 0.26-0.53; p<0.00001) (31). Subgroup analysis showed that the prognostic effect of TMB was independent of TMB detection method and tumor type (31). A number of clinical trials, such as Keynote-001, also showed a positive correlation between TMB and ICI response (32). Other studies have shown that the number of mutations targeted by T cells, namely, the true NAL, may be more associated with the ICI response than TMB (33). In addition, DDR mutations usually lead to DNA damage and repair defects, resulting in genomic instability and TMB increase, and may enhance the antitumor immune response through neoantigen-dependent and neoantigen-independent mechanisms (34, 35). Several studies have revealed that DDR mutations are related to better prognosis of patients receiving ICIs (36, 37). In conclusion, the increase in efficacy of ICIs in the elderly group relative to that of the young group may be attributed to an increase in TMB, NAL and DDR mutations in the elderly group.

From the perspective of gene mutations, our results suggested that there were 166 significantly different genes between the elderly group and the young group, and almost all of genes had higher mutation frequencies in the elderly group. TP53 was the most common mutation in both the elderly and young groups. Studies have shown that the correlation between TP53 mutation and tumor immunity is related to the cancer type. For example, TP53-mutated lung adenocarcinoma and breast cancer exhibit enhanced PD-L1 expression and immune cell infiltration, which indicate a good response to ICIs, while in colon adenocarcinoma, head and neck squamous cell carcinoma and stomach adenocarcinoma, TP53 mutation showed the opposite effect (38). This may be explained by the fact that TP53 mutation can lead to chromosome/genomic instability, thus increasing TMB and tumor aneuploidy level (TAL) (39). Since TMB and TAL are positively and negatively correlated with the immunotherapy response, respectively, the therapeutic response of TP53 mutant cancer to ICIs may be influenced by both the TMB and TAL (40). Chen et al. analyzed the immunotherapy cohort of melanoma and NSCLC patients and found that the LRP1B-mutant group had higher TMB and NAL and better prognosis than the wild-type group (41). This may be attributed to the characteristics of LRP1B mutant tumors, that is, the abundance of genes involved in antigen processing and presentation and cell cycle checkpoints (41). In the 2020 American Society of Clinical Oncology (ASCO), a multicenter study including an ICI cohort of different cancer species showed that the ORR in patients with LRP1B mutations was significantly higher than that in wild-type patients (57% vs 18%), independent of TMB/microsatellite instability (MSI) status. Moreover, LRP1B mutations were associated with longer OS (HR= 0.58, 95% CI: 0.36-0.95) and PFS (HR=0.39, 95% CI: 0.24-0.63) (7). As a result, LRP1B may be an independent prognostic indicator for predicting the efficacy of ICIs. In addition, studies have reported that in the pan-cancer immunotherapy dataset, MUC16 and TTN mutations were associated with higher TMB and better OS (42). Using the TIDE algorithm, Li et al. found that the SYNE1-mutant group had a stronger response to ICI treatment than the wild-type group (43). In tumors with RYR2 or CSMD3 mutations, the CXCL9 expression level is elevated, which can promote T cell migration and activation and facilitate the antitumor immune response (44, 45). In summary, the specific gene mutations mentioned above may explain the better prognosis of patients aged ≥50. It is worth noting that considering the dual role of TP53 mutation, TP53 mutation may result in different outcomes in pan-cancer datasets consisting of different cancer types or frequencies.

The frequency of mutations in some genes was higher in the younger group; for example, the BRAF mutation, which mainly occurs in melanoma and is conducive to the generation of an immunosuppressive microenvironment, was more common in the younger group. It has been reported that BRAF mutation could inactivate the antitumor immune response by upregulating the MAPK signaling pathway in melanoma (46). BRAF-mutant melanoma may achieve immune escape through several mechanisms, including preventing antigen-presenting cells from presenting tumor antigens and subsequently activating T cells, resulting in the low expression of human leucocyte antigen (HLA) class I molecules and melanoma differentiation antigens, and promoting the accumulation of myeloid-derived suppressor cells and regulatory T cells (47–49). In conclusion, BRAF mutation appears to be detrimental to patients’ response to ICIs, which is consistent with the trend of our findings. Since BRAF inhibitors may induce increased PD-L1 expression in tumor cells and help restore the immune-stimulating microenvironment of BRAF-mutant melanoma (50), the combination of BRAF inhibitors and ICIs may provide a stronger antitumor effect.

Next, we analyzed the difference in the expression of 75 immune-related genes between the elderly group and the young group. The results showed that the immune genes related to antigen presentation, such as HLA-I (HLA-A, HLA-B and HLA-C) and HLA-II (HLA-DR, HLA-DQ and HLA-DP), which are responsible for presenting antigens to CD8+ T cells and CD4+ T cells, respectively, were highly expressed in the elderly group (51). The interaction between MICA/B and NKG2D activates the cytotoxicity of natural killer (NK) cells, which has been reported as an important costimulation signal of T cells (52). The expression of cell adhesion-related genes was significantly increased in the elderly group. As one of the cell adhesion molecules, ICAM-1 can bind to LFA-1 on CD8+ T cells and promote the activation and cytotoxicity of CD8+ T cells in a TCR-dependent manner (53). Consistent with this, the expression of ICAM-1 is negatively correlated with the incidence of lymph node or distant metastasis in patients with breast cancer and colorectal cancer (54, 55), suggesting a favorable prognosis. However, it has also been reported that the expression of ICAM-1 is positively correlated with the metastatic potential of some tumors (56). The mechanism by which ICAM-1 plays a dual role in the development of tumors is still unclear, and the relationship between ICAM-1 and the therapeutic response of ICIs remains to be further explored. Immune genes associated with chemokines, such as CCL5, CXCL9 and CXCL10, were highly expressed in the elderly group. Increased levels of CXCL9 and CXCL10 have been reported to be related to elevated tumor CD8+ T cell density and improved survival in patients with a variety of cancers (57, 58). CXCR3 is the coreceptor of CXCL9/10/11. In the tumor microenvironment, the CXCL9/10/11-CXCR3 signaling pathway can exert antitumor immunity through multiple mechanisms (i.e., promoting the chemotaxis movement of CXCR3-activated immune cells to tumor sites (59) and activating the STAT and PI3K-Akt signaling pathways, thus upregulating PD-L1 expression, which usually means a good response to ICIs) (60). It is well known that the sustained antitumor effect of ICIs is closely related to high expression levels of immune checkpoint molecules. In line with this, our results suggested that the expression of immune checkpoint-related genes, such as PDCD1 [also known as PD1/CD279; ligand: PDL1, PDL2 (PDCD1LG2)], CD274 (also known as PDL1; ligand: PD1), CTLA4 (also known as CD152, ligand: CD80, CD86), LAG3 (ligand: MHC-II) and TIGIT (ligand: CD155, CD112, CD113), was higher in the elderly group. In addition, LAG3 and TIGIT are usually coexpressed and upregulated along with PD1 on tumor-infiltrating lymphocytes (TILs) (61).

After analyzing the degree of immune cell infiltration, we found that most of the immune cell subtypes, such as CD8+ T lymphocytes, Tregs and M1 macrophages, were highly infiltrated in the elderly group, while M2 macrophages were highly infiltrated in the young group. TILs are a predictive biomarker of response to ICI treatment. The greater the number of TILs, the stronger the patients’ response to ICIs (62). Among TILs, CD8+ T cells are the immune cells that have the strongest positive effect on the survival of cancer patients. Bruni et al. confirmed the positive prognostic value of CD8+ T cells in 18700 patients across 17 cancer types (63). As a subtype of CD4+ T cells, Tregs suppress the antitumor immune response and enhance the immune escape of tumor cells. To date, the association between Tregs and poor prognosis in renal and cervical cancer has been well established (64, 65), while the positive impact of Tregs on survival has been demonstrated in bladder cancer, hematological malignancies and head and neck cancer (66–68). The controversial result may contribute to the fact that the definition of Tregs is inconsistent in previous studies, most of which only rely on FOXP3 as a marker for Tregs (63). The dual role of macrophages in the process of tumor development is related to their polarization state. For example, M1 macrophages release IL-1 and IL-12 under stimulation by factors such as interferon-γ and tumor necrosis factor-α to play proinflammatory and antitumoral roles (69). In contrast, under the stimulation of IL-4 and IL-13, M2 macrophages secrete mediators, such as IL-10 and transforming growth factor β, that contribute to the establishment of a tolerable microenvironment to exert anti-inflammatory and protumoral effects (69). Through experimental research, Du et al. found that exosomes derived from M1 macrophages can directly regulate T cells, promoting Th1 cell differentiation (increasing the proportion) and effector functions (increasing IFN-γ intensity) and increasing the production of IFN-γ by CD8+ T cells (70). Moniek’s data on the existence, induction, and plasticity of antigen-presenting cells in cervical cancer indicated that tumor-infiltrating Th1 cells could stimulate a tumor-rejecting environment by converting M2 macrophages to M1 macrophages (71). Thus, the better prognosis of elderly cancer patients receiving ICI therapy may be closely related to the type and degree of immune cell infiltration in the immune microenvironment.

By analyzing immune cell-related pathways, we found that the TCR signaling pathway, CD28 costimulation signaling pathway, IL-2 signaling pathway, IL-12 signaling pathway, PD1 signaling pathway and NF-κB signaling pathway were significantly positively correlated with CD8+ T lymphocytes, Tregs and M1 macrophages and significantly negatively correlated with M2 macrophages. It is well known that the effective activation of T cells depends on the recognition of MHC-binding antigenic peptides by TCRs and the role of costimulatory signals. Subsequently, activated T cells promote IL-2 signaling, which promotes the proliferation, differentiation and survival of T lymphocytes, as well as the killing activity of NK cells (72). NF-κB can be activated either by the classical pathway under a variety of inflammatory signals, such as proinflammatory cytokines and Toll-like receptors, or by the nonclassical pathway, which is induced by ligands from the tumor necrosis factor receptor family (73). It has been reported that NF-κB can induce the production of chemokines and cytokines, attract immune cells, and maintain a proinflammatory tumor immune microenvironment (74, 75). At the same time, the NF-κB signaling pathway is also involved in the regulation of tumor cell immune checkpoint expression, which can induce PD-L1 expression, indicating a good response to ICIs (76, 77).

The advantages of this study are listed as follows. We tried to explore the influence of different age cutoff points on the prognosis of cancer patients treated with ICIs. According to the results, a suitable cutoff point of 50 years old was selected, and patients were grouped accordingly. In addition, we used the database and data from related literature to try to reveal the biological factors related to age. The limitations of this paper are listed as follows. Due to limited data sources, it is not clear whether patients in the MSKCC clinical cohort received other treatments before ICIs. In addition, our exploration of genetic and other biological factors lacks direct prognostic data support.

Cancer patients aged ≥50 years can benefit more from ICIs than younger patients. This may be related to specific gene mutations, gene expression levels and the degree of immune cell infiltration related to age. In the future, prognostic data are still needed to directly verify the underlying mechanisms behind the age cutoff point of 50 years.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Mutations-mc3.v0.2.8.PUBLIC.maf. (https://gdc.cancer.gov/about-data/publications/pancanatlas), RNA (Final) -EBPlusPlusAdjustPANCAN_IlluminaHiSeq_RNASeqV2.geneExp.tsv (https://gdc.cancer.gov/about-data/publications/pancanatlas), https://www.cbioportal.org/study?id=tmb_mskcc_2018.

Study concept and design: JZ and PL. Data analysis: RG, QL, AL, and JL. Drafting of the manuscript: RG. Critical revision of the manuscript for important intellectual content: WD and MC. All authors contributed to the article and approved the submitted version.

This study was supported by grant from Natural Science Foundation of Guangdong Province (2018A030313846).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.