Microarray analysis of the expression profile of immune-related gene in rapid recurrence early-stage lung adenocarcinoma

Lung cancer is one of the most commonly diagnosed cancer in the world, accounting for 11.6% of the total number of cancer cases, and is also the leading cause of cancer death, accounting for 18.4% of the total number of cancer deaths (Bray et al. 2018). In China, the 5-year survival rate of lung cancer is only 16.1%, and 70% of patients are initially diagnosed with advanced-stage lung cancer with a 5-year survival rate of less than 5% (Zeng et al. 2015). Lung adenocarcinoma (LUAD) is the most common histological type of lung cancer. According to the definition of American Joint Committee on Cancer (AJCC), ES-LUAD refers to tumor size ≦ 5 cm, no lymph node and distant metastasis, also indicated as T1-2N0M0 (IA-IIA).

Currently, the main treatment for ES-LUAD is surgery, and the 5-year overall survival rate is close to 67% (Yanagawa et al. 2013). However, in our clinical practice, some patients with ES-LUAD were relapsed within one year after surgery (16.2%), and the 5-year survival rate was extremely poor, well below the reported 67%, suggesting that ES-LUAD may not be a homogenous but complex disease (unpublished observation). Therefore, it is important to provide more aggressive adjuvant therapy for LUAD patients with rapid recurrence to improve their prognosis. In the past decades, various proteins (Gold et al. 2014), mRNAs (Wistuba et al. 2013), miRNAs (Lu et al. 2012), and DNA methylation (Brock et al. 2008) have been reported to predict postoperative recurrence of early-stage lung cancer, but none of these have been applied for clinical practice. Therefore, it is necessary to understand the molecular mechanism of rapid recurrence of ES-LUAD from a new perspective, and to predict the high-risk ES-LUAD patients who may develop rapid recurrence.

Microarray is a widely used technology to screen differentially expressed genes (DEGs) between cancer and normal tissues and was recently used to study hotspot molecules such as lncRNA (Bach and Lee 2018). However, to the best of our knowledge, there is no study using microarray to investigate the rapid recurrence of ES-LUAD. After hierarchical clustering analysis, we noticed that ES-LUAD patients can be divided into two different categories in the cluster analysis. GO enrichment and KEGG pathway analysis further indicated that these DEGs were mostly functionally related to immunity. Moreover, protein–protein interaction (PPI) network analysis identified many crucial nodal proteins related to immunity function, such as IL-1β, PTGS2, etc. Furthermore, ES-LUAD patients with rapid recurrence had a lower density of CD3⁺, CD4⁺, CD8⁺ and CD20⁺ tumor-infiltrating lymphocytes (TILs) subsets. These results suggested that immune-related genes and TILs participated in the rapid recurrence of ES-LUAD.

The histological subtypes of LUAD are closely related to the prognosis. Warth et al. reported that among the lepidic, acinar, papillary, micropapillary and solid predominant subtypes, the lepidic predominant subtype had the longest overall survival (OS) (Warth et al. 2012). Ujiie et al. reported that the solid predominant subtype is an independent predictor of recurrence in patients with stage I LUAD (Ujiie et al. 2015). In addition to histological subtypes, some biomarkers were also used to predict the postoperative recurrence of early-stage lung cancer (Gold et al. 2014; Wistuba et al. 2013; Lu et al. 2012; Brock et al. 2008), but they do not really distinguish patients with rapid recurrence. In our clinical practice, ES-LUAD patients had extremely poor prognosis after rapid recurrence. Therefore, it is necessary to identify these patients who will have rapid recurrence and provide them with more aggressive treatment. Thus, in this study, 20 ES-LUAD patients were classified into rapid recurrence group (RFS ≦ 1 year) and non-rapid recurrence group (RFS ≧ 3 years), and DEGs between the two groups were analyzed by microarray. 72 DEGs were clustered by the Transcriptome Analysis Console (TAC) Software 3.0.0. We found that 20 patients could be basically divided into two categories: all 8 patients with rapid recurrence were classified into one category, and 11 of 12 patients with non-rapid recurrence were classified into another category. This supported the hypothesis that from a genetic perspective, ES-LUAD patients with rapid recurrence or non-rapid recurrence may be two different disease subtypes. Thus, screening ES-LUAD patients for rapid recurrence is theoretically feasible. Next, we used the Bioconductor package limma to identify DEGs because it has been a popular choice for gene discovery through differential expression analyses of microarray over the past decade (Ritchie et al. 2015). A total of 416 DEGs were obtained by this method. It should be pointed out that the DEGs identified through the Transcriptome Analysis Console (TAC) Software 3.0.0. and the Bioconductor package limma are not completely consistent, which may be caused by different algorithms and statistical methods.

To further understand the functions of these DEGs involved in cells, GO enrichment and KEGG pathway analysis were performed on these 416 DEGs. GO and KEGG analyses are commonly used to demonstrate function enrichment of the DEGs between tumor and non-tumor tissues (Yuan et al. 2018), and can also be used to predict oncogenes (Xing et al. 2016). Thus, a functional enrichment analysis was first conducted to analyze 416 DEGs identified from rapid recurrence group and non-rapid recurrence group. In GO analysis, whether it is the cellular component aspect, the molecular function aspect or the biological process aspect, the functions of these DEGs were obviously related to immunity. In detail, in the top 10 categories of the three GO aspects, 2 of top 10 in cell components category, 2 of top 10 in the molecular function category, and 9 of 10 in the biological process category were all functionally related to immunity. Interestingly, all the three GO aspects contained humoral immunity-related terms (GO:0019814, GO:0034987, GO:0006952, etc.), suggesting that humoral immunity played a special role in the rapid recurrence of ES-LUAD. In the KEGG analysis, these DEGs were also found to be functionally involved in immune-related or inflammation-related pathways, that is, there were 6 of the top 8 pathways. Of interest, systemic lupus erythematosus (SLE) ranked first. SLE is due to the inability of individual’s immune system to distinguish between self and non-self-antigens, resulting in the production of antibodies against self-antigens and triggering an over-active inflammatory response (Hui-Yuen et al. 2016). Since impaired B cells are typical characteristics of SLE and B cells are the main cells involved in humoral immunity (Bakshi et al. 2018), this implies that in ES-LUAD patients, the occurrence of rapid recurrence may be closely related to humoral immunity. In summary, the GO enrichment results indicated that there is a large difference in the expression of immune-related genes between ES-LUAD patients with rapid recurrence and non-rapid recurrence.

We also found that among 416 DEGs, 156 genes were up-regulated and 260 genes were down-regulated. Xu et al. suggested that key genes play important biological functions, rather than those with the highest expression difference (Xu et al. 2018). Thus, the PPI network analysis was conducted using the STRING database to identify the key genes and finally obtained 216 key nodal proteins. Subsequently, 10 key genes (EGFR, MMP9, IL1B, PTGS2, MMP1, HIST1H4E, HIST2H4A, HIST1H4A, HIST1H4D, and HIST1H4J) were obtained by Cytoscape software. Interestingly, similar to the results of GO functional enrichment analysis, we found that many of these 10 key genes were functionally related to immunity or inflammation, such as IL1B and PTGS2.

IL-1β is a potent proinflammatory cytokine and was originally discovered as the main endogenous pyrogen that induces the synthesis of prostaglandin. It was later confirmed that IL-1β has multiple functions, including T cell activation and cytokine production, B cell activation and antibody production, promoting Th17 differentiation of T cells, etc. (Schett et al. 2016). Studies have also shown that sustained induction of IL-1β enhanced the intensity of the inflammatory response and generated an inflammatory microenvironment to promote the initiation and development of tumors (Bhat et al. 2014; Dinarello 2006). Furthermore, high levels of IL-1β in tumors and serum were associated with higher tumor grades and increased invasion of breast, pancreatic cancer, and myelogenous leukemia, and were also associated with poor prognosis (Setrerrahmane and Xu 2017). PTGS2, also known as COX-2, encodes the rate-limiting enzyme cyclooxygenase, which converts arachidonic acid to prostaglandins. Unlike the constitutive expression of COX-1, PTGS2 is an inducible enzyme that is activated in response to extracellular stimuli, such as growth factors and proinflammatory cytokines. Some investigators demonstrated that PTGS2 is overexpressed in a variety of epithelial malignancies, such as lung, breast, pancreas, colon, and esophagus, and is usually associated with poor prognosis (Hida et al. 1998; Hwang et al. 1998; Okami et al. 1999; Ogino et al. 2008; Takatori et al. 2008). Although high expression of either IL-1β or PTGS2 is believed to be associated with poor prognosis in many types of tumors, our microarray data showed different results indicating that IL-1β and PTGS2 expression were significantly lower in patients with rapid recurrence than in patients with non-rapid recurrence. In other words, IL-1β and PTGS2 may act as protective factors to reduce the occurrence of rapid recurrence. Because our results of IL-1β and PTGS2 were inconsistent with some literature reports, we further performed IHC analysis to examine the expression of IL-1β and PTGS2 in another group of 136 ES-LUAD patients. Consistent with our microarray data, IHC results also indicated that the expression of IL-1β and PTGS2 in the rapid recurrence group were significantly lower than in the non-rapid recurrence group. Our survival analyses of 136 patients further supported the results that patients with high expression of IL-1β or PTGS2 have a better RFS.

IL-1 promotes the expansion of natural killer (NK) cells and CD4⁺ CD8⁺ T cells by combining with IL2 (Ben Aribia et al. 1950). IL-1β down-regulated TGF-β-induced Foxp3 expression, thereby inhibiting the differentiation of regulatory T cells (Ikeda et al. 1950). These reports illustrate the potential role of IL-1β in antitumor immunity. In Allen’s in vivo studies, IL-1β also showed protective effects in mouse models of chemical colitis and colon cancer (Allen et al. 2010). Therefore, it can be reasonably assuming that IL-1β acts as a protective factor for preventing rapid recurrence in ES-LUAD patients. From the perspective of inflammation and tumors, our reasonable theory is that early inflammation can exert anti-tumor effects, but long-term persistent chronic inflammation induces tumorigenesis. Supporting this theory, high levels of IL-1 in chronic inflammation was found to promote tumor development by driving sustained NF-κB activation and MAPK activity (Bent et al. 2018). In another aspect, the LUAD patients in this study were in early tumor stage rather than late/advanced tumor stage. Thus, IL-1β may not promote tumor development through sustained long-term inflammatory stimulation, but may inhibit tumors through an anti-tumor immunity. As to PTGS2, PTGS2 encoded prostaglandin-endoperoxide synthase 2 is responsible for the production of prostaglandins, which plays a key role in the inflammatory response (Ricciotti and FitzGerald 2011). As mentioned above, inflammation is a double-edged sword that promotes or inhibits tumor development. Therefore, similar to IL-1β, PTGS2 will not promote tumor progression through sustained inflammatory stimuli in early-stage LUAD. Moreover, studies have shown that high level of PTGS2 can inhibit tumor growth and migration by regulating 8‐HOA, another derivative of PTGS2 (Hashemi Goradel et al. 2019). In summary, 2 immune-related genes of the 10 key genes, IL-1β and PTGS2, were identified in this study. They were functionally closely related to immunity or inflammatory response, suggesting a significant immune difference between ES-LUAD patients with rapid recurrence and non-rapid recurrence.

It is worth noting that EGFR is the central linker to IL1B and PTGS2, but it has not been studied in this paper because our research focuses on immune-related genes. Overexpression of EGFR in NSCLC tumors has been reported in many series, and the reported results of EGFR (over)expression range from 43 to 89% (Hirsch et al. 2003). The prognostic significance of EGFR overexpression in NSCLC has also been studied. Some studies showed that EGFR overexpression was associated with shortened survival, while others found no prognostic implication of EGFR overexpression (Hirsch et al. 2003). In our microarray data, we found that EGFR was lower in the rapid recurrence group (foldchange = − 2.11) on the level of mRNA, but the relationship between EGFR protein level and recurrence needs further research.

In addition to IL-1β and PTGS2, TILs may also be closely related to rapid recurrence in ES-LUAD patients. NSCLCs are frequently associated with prominent TILs and other inflammatory cells (Rekhtman et al. 2013). Several studies suggested that there was a positive correlation between patient survival, treatment response, and the number of TILs (Schalper et al. 2015; Donnem et al. 2015; Brambilla et al. 2016). In stage 1A NSCLC patients, the levels of intratumoral TILs are positively associated with improved RFS (Horne et al. 2011). In addition, the spatial distribution of TILs in tumors was related to the recurrence of early-stage NSCLC (Corredor et al. 2019). However, there is no report on whether TILs are involved in the rapid recurrence of ES-LUAD patients. Therefore, IHC was performed to determine the density of TILs subsets in the rapid recurrence group and the non-rapid recurrence group, and the results showed that the number of infiltrating total T cells (CD3⁺), CD4⁺ helper T cells (CD4⁺), CD8⁺ cytotoxic T cells (CD8⁺), and B cells (CD20⁺) were all significantly reduced in the rapid recurrence group. Since IL-1β can promote the proliferation and differentiation of activated B cells (Lipsky et al. 1950) and stimulate T cell replication (North et al. 1988), it is likely that IL-1β play a crucial role in the regulation the number of TILs in the recurrence of ES-LUAD patients. In this study, IL-1β expression was significantly lower in the rapid recurrence group than in the non-rapid recurrence group. It is possible that in non-rapid recurrence of ES-LUAD patients, higher level of IL-1β may promote the proliferation or replication of TILs within tumors to exert anti-tumor immunity. This warrants further investigation.

The results of the functional enrichment analysis and the identified key DEGs and TILs clearly revealed that there are significant differences in the immune level between two groups of ES-LUAD patients. Therefore, in ES-LUAD patients, rapid recurrence and non-rapid recurrence may be two different disease subtypes and have their unique phenotypes in immunity. Furthermore, our research may help broaden the application of lung cancer immunotherapy. In the past, immune checkpoint inhibitors have achieved amazing results in non-small cell lung cancer (NSCLC), whether in mono (Peters et al. 2018) or combination immunotherapy (Borghaei et al. 2019), but only for advanced NSCLC. Even recently, the combination of Neoadjuvant and consolidation immuno-oncology therapy has been propose as an effective treatment for stage III NSCLC (Yeh et al. 2018). Currently, an important predictive biomarker for lung cancer immunotherapy is PD-L1. It is reported that patients with PD-L1 overexpressing have a 67–100% response rate; whereas for PD-L1 negative, the response rate is about 0–15% (Patel and Kurzrock 2015). However, for early-stage LUAD (IA-IIA), the current treatment is still surgery-based, and immunotherapy is not recommended. Because the prognosis of patients with rapid recurrence is extremely poor, and their immune status are unique, these DEGs are very likely to become the first potential targets or biomarkers other than PD-L1 for immunotherapy in early-stage lung cancer in the future.

In conclusion, our findings provided a possible mechanism for the rapid recurrence of ES-LUAD patients and a theoretical basis for distinguishing ES-LUAD patients, who may develop rapid recurrence from an immunological perspective. Moreover, these DEGs will be the most likely potential targeting gene in future immunotherapy for ES-LUAD patients with rapid recurrence.