A robust six-gene prognostic signature for prediction of both disease-free and overall survival in non-small cell lung cancer

Lung cancer is the leading cause of cancer death worldwide, and non-small cell lung cancer (NSCLC) composes the majority (approximately 85%) of all lung cancers [1, 2]. Despite advances in treatment strategies, the high mortality rate for lung cancer patients has not considerably declined, due to the late diagnosis of the disease [3]. The major clinical determinants of NSCLC prognosis include tumor extension, performance status and histological type [4, 5]. However, various disease outcomes have been identified in patients with similar clinical and pathological features, suggesting that the current clinical prognostic factors used may be insufficient to consistently predict individual clinical outcomes [6]. This emphasizes the necessity of identifying robust and reliable prognostic markers with higher sensitivity and accuracy in NSCLC.

Transcriptome profiling has widely been used to characterize prognostic signatures in patients with lung cancer, and has generated a number of candidate biomarkers with potential clinical values [7‐9]. However, the suggested signatures lack consistency among studies and provide limited prognostic information, partially due to the limited sample size and technical factors. Moreover, NSCLC is a highly heterogeneous disease, thus it is critical to identify a reliable signature that can define patients who are at a high-risk to develop disease recurrence. To this end, integrating the results from multiple studies holds promise for more robust prognostic signatures. In addition, most investigations used overall survival (OS) rather than tumor recurrence as an end point [9‐11]. Disease-free survival (DFS) is defined as the interval from surgery to the first diagnosis of any type of relapse or death, and is used as a possible alternative for OS.

Therefore, we attempted to identify and validate a robust and reliable prognostic signature for DFS and OS prediction by integrating multiple datasets of NSCLC patients. In the present study, we revealed a six-gene signature with a reliable prognostic value in NSCLC, which might complement conventional clinical prognostic factors, and further provide more effective therapeutic interventions and personalized therapies for NSCLC patients.

Numerous reports have indicated that disturbed gene expression may be implicated in various aspects of tumor, including tumorigenesis, progression and prognosis [19‐21]. Some genes have been considered as prospective biomarkers to predict prognosis in NSCLC patients [14, 22, 23]. However, several concerns limit their prognostic and predicative power, such as inadequate samples, lack of DFS prediction, and lack of effective validation. In this study, we developed and validated a novel prognostic six-gene signature that was found to be significantly associated with both the DFS and OS of NSCLC patients. Our results revealed that this classifier could successfully identify high-risk and low-risk NSCLC patients with significant differences in both DFS and OS. In addition, the prognostic value of the six-gene signature was verified in three GEO datasets and an independent TCGA dataset, suggesting the reproducibility and reliability of the six-gene signature for both DFS and OS prediction in NSCLC.

The clinical prognostic factors in NSCLC include stage, age, gender and performance status [6, 24]. Our study showed that stage and age were significantly correlated with both the DFS and OS of patients in GSE31210; performance status was significantly associated with DFS, and stage and performance status were related to OS of patients in GSE37745; stage and gender were significantly involved in OS of patients in GSE50081. In the entire patient cohort, stage was identified as an independent prognostic factor for DFS, and age and stage were associated with OS of NSCLC patients. Furthermore, we performed a stratification analysis on the entire cohort, and found that the prognostic power of the six-gene signature was independent of age, gender and stage. Interestingly, Birim et al. [24] indicated that non-squamous cell histology was a risk factor for postoperative outcome in NSCLC. Our study showed that histological type had no significant association with either DFS or OS in NSCLC. While stratification analysis indicated that high-risk group had significantly shorter DFS and OS than low-risk group for patient with adenocarcinoma but not squamous carcinoma.

Currently, tumor stage has been broadly utilized as a strong indicator of survival in NSCLC [25]. However, the current staging system is far from accurate in the aspect of survival prediction at the individual level [26]. As expected in our study, univariate and multivariate analysis showed that stage II, stage III, and stage IV were significantly associated with OS and DFS in the entire GEO cohort. However, stage II, and stage III were found to be the independent prognostic factors for prediction of both DFS and OS in the TCGA database. As documented, age is a main indicator of patient survival, and younger patients are tended to survive longer than the older ones [27‐29]. Nevertheless, age alone is not a survival indicator for cancer patients because older patients are less likely to receive adjuvant therapy [30]. Multivariate analysis showed that age was significantly associated with OS in the entire GEO cohort and the TCGA cohort, but there was no correlation between DFS and age in these two cohorts. Thus, compared to age and stage, the risk score was a more reliable prognostic factor for NSCLC patients.

Several gene mutations have been revealed to be associated with the pathogenesis of NSCLC, such as EGFR and KRAS mutations [31]. Notably, EGFR or KRAS mutated lung cancer accounts for a significant subgroup of NSCLC, especially in adenocarcinoma [32, 33]. Markedly, targeting EGFR mutations has changed the therapeutic paradigm in NSCLC patients harboring EGFR mutations [34]. Over the last decade, multiple EGFR tyrosine kinase inhibitors (TKIs) have been developed to target mutated EGFR, and have achieved a better survival in patients with EGFR mutations than in those with the wild type [35]. Whereas, KRAS mutations predict a worse prognosis among NSCLC patients treated by chemotherapy and EGFR-TKIs [36, 37]. In the present study, univariate analysis indicated that EGFR mutations, but not KRAS mutations, were correlated with both DFS and OS, whereas multivariate analysis indicated that EGFR mutation status did not act as an independent prognostic factor.

In this study, we developed a prognostic six-gene signature for both DFS and OS prediction in NSCLC. Most of these genes have not been well characterized in tumor biology, except for CDCP1. CDCP1, also known as CUB domain-containing protein 1, is a transmembrane glycoprotein, whose phosphorylation is linked with the progression and metastasis of several cancers [38, 39]. In addition, blocking of CDCP1 has been shown to be a potential mode for therapeutic intervention against metastatic disease [38, 40]. Chiu et al. [41] showed that the ADAM9 metalloprotease enhanced CDCP1 expression via activating EGFR signaling pathways in advanced lung cancer disease. Ikeda et al. [42] revealed that CDCP1 expression was an independent prognostic factor for both OS and DFS, and could be used as an useful marker for survival prediction of patients with lung adenocarcinoma. Our study combined these 6 genes into a single panel, and established its prognostic value in both DFS and OS in NSCLC.

As reported, the complexity of cancer can be decreased and presented by a few cancer hallmarks that enable cancer cell proliferation and metastasis. These hallmarks can offer a framework to understand the cancer diversity. Hence, we focused on detecting the association of prognostic gene signature and cancer hallmarks [43]. Studies have revealed that DNA replication, cell cycle, DNA damage repair, apoptosis, chromosome and gene instability, energy supply play important roles in cancer progression [44‐47]. Coincidentally, the GSVA results demonstrated that the six-gene signature was remarkably connected with these biological processes. Specifically, E2F targets have been demonstrated to participate in DNA replication, cell cycle, DNA damage repair, apoptosis [48]. G2M checkpoint, mitotic spindle, and MYC targets have been reported to contribute to the instability of chromosome and gene [49‐51]. Utilization of glycolysis-related metabolic pathway has been implicated to provide ATP as a main source of energy supply for cancers [52]. Moreover, mTORC1 signaling has been suggested to be activated in human fibrolamellar liver carcinoma [53]. Demonstrated here, the results of present study demonstrated that the six-gene signature correlated with several cancer-progression associated biological processes which supported the DFS/OS predictive ability of the signature. Significantly, the correlation analysis in our study showed that patients having these activated biological processes tended to have adverse outcomes. Thus, this further confirmed that our six-gene signature used for predicting prognosis was reasonable and reliable.

In this work, some limitations need to be acknowledged. First, a few clinical characters presented an unbalanced distribution, such as an overwhelming majority of patients in stage I/II and presenting a histological adenocarcinoma type. Thus, the robustness of the six-gene signature requires further validation in large-scale prospective investments. Second, most of the genes identified here are rarely reported in the academic literature, and there are no experimental data regarding the identified signature, thus more evidence is needed to elucidate the inherent correlation between the six-gene signature and the prognosis of NSCLC patients. Despite these drawbacks, our results demonstrate valuable information on the importance and significance of the six-gene signature in both DFS and OS prediction in NSCLC.

In this study, we developed an innovative six-gene prognostic signature for both DFS and OS prediction in NSCLC patients. The six-gene signature was an independent prognostic factor, and might complement clinicopathological factors and facilitate the personalized treatment of NSCLC patients. Large-scale prospective investments should be applied for further assessment of the robustness of this signature in future studies.