Analyzing LSCC RNA sequencing (RNA-seq) data set and relevant clinical parameter of 109 LSCC patients from TCGA, we identified thirteen lncRNAs related to OS. On the basis of these lncRNAs, we developed a lncRNAs signature, which could accurately categorized patients into high-risk status and low-risk status. Additionally, we built a visually inclusive nomogram, integrating lncRNAs signature and clinicopathologic variables to predict survival in LSCC patients underwent surgery resection. The nomogram effectively predicted survival rate, with a bootstrapped corrected C-index of 0.73 and AUC of 0.938, which possessed better predictive ability and clinical usability than TNM stage alone.
Increasing number of studies have found that lncRNAs may be exploited as potential effective biomarkers in diagnosis, progression and prognosis of LSCC [20-22]. Basing a comprehensive lncRNAs profile for LSCC, Shen et al  identified AC026166.2-001 and RP11-169D4.1-001 as new lncRNAs with accurate diagnosis ability for LSCC, were independent factors for prognosis and may be potential therapeutic targets. A study of lncRNAs microarray by Chen et al.  uncoverd that lncRNA AC 008440.10 was significantly related to LSCC stage, lymph node metastasis (LNM) and survival time. Recently, Zhao et al.  confirmed that LINC00668, was up-regulated in LSCC, probably in vitro promote the malignant phenotypes of cells and the author deduced that LINC00668 may enhance the stability of RAB3B mRNA by binding its 3’UTR. Notably, He et al.  collected data from the open Gene Expression Omnibus(GEO), reported that 18-mRNA and one-lncRNA module were correlated with disease-free survival (DFS) of LSCC patients and it effectively divided patients into high-risk group and low-risk group with different DFS outcomes, independent of patient age and tumor grade. Similarly, Wu et al. , using data from GEO, constructed a potential panel of two-lncRNAs signature, including RP11-169K16.4 and RP11-107E5.3, to predict recurrence of patients with laryngeal carcinoma and confirmed that it was independent predictors of laryngeal cancer patients. These studies suggested the potential clinical implications of lncRNA in improving the prognosis prediction of LSCC. However, it should be noted that the lncRNAs signature predicting the overall survival (OS) outcome of LSCC has not been reported yet. Hence, in the current study, using TCGA database containing large-scale lncRNAs expression data, we aimed to identify OS-related lncRNAs and establish a lncRNAs signature, which may be more valuable for LSCC patients to optimize tailored treatment in the era of precision medicine.
To our knowledge, this is the first study constructed an inclusive nomogram, combining lncRNAs signature and clinicopathologic factors, for predicting survival probability in patients with LSCC. We built a lncRNAs signature, consist of AC007907.1, AC025419.1, AC078993.1, AC090241.2, AL158166.1, AL355974.2, AL596330.1, HOXB-AS4, KLHL6-AS1, LHX1-DT, LINC00528, LINC01436 and TTTY14, could effectively classified patients into high-risk group with shorter OS and low-risk group with longer OS. Using stratified analysis, lncRNAs signature shown perfect discrimination ability, regardless of tumor size, node status, and TNM stage. Additionally, we identified three independent predictors that margin status, tumor status and lncRNAs signature, which were all embedded into the nomogram. In this study, in consideration of homogeneity, and ability of discrimination and risk stratification of the model, the performance of the nomogram in predicting survival probability is superior to the TNM staging system. The advantage of the current nomogram is that it integrated genomic and clinicopathological variables, which are important for predicting survival risk, but cannot be obtained by TNM stage system. Remarkably, DCA results showed that LSCC survival-related treatment decision based on the nomogram led to more net benefit than treatment decision based on TNM stage or lncRNAs signature, or treating either all patients or none. Taken together, the present nomogram would be clinically useful for the clinicians in tailoring survival-associated treatment decision.
It is also worth to mention that an important feature of our comprehensive nomogram may be the ability to stratify clinical subgroups, including early LSCC and advanced LSCC. Patients diagnosed with early LSCC are generally considered to have a low survival risk, and therefore do not receive adjuvant treatment after radical resection. Nevertheless, some patients in the clinically low-risk subgroup (early LSCC) are at high risk of survival, and they are likely to benefit from adjuvant treatment or intensive follow-up plan. Likewise, Patients diagnosed with advanced LSCC are usually identified as high risk survival status and need to receive adjuvant therapy underwent laryngectomy. Nevertheless, several patients in the clinically high-risk subgroup (advanced LSCC) are at low risk of survival, and they may not benefit from adjuvant therapy or intensive follow-up plan. It is an arduous challenge to accurately predict survival risk of patients. Encouragingly, our nomogram presented perfect discrimination capacity in early LSCC and advanced LSCC subgroups. Hence, our nomogram probably benefit a large proportion of the patients who might be considered at high risk of survival in early LSCC subgroup or might be considered at low risk of survival in advanced LSCC subgroup.
Among the thirteen OS-related LINC01436, and TTTY14 have been previously reported to be related with cancers, such as in non-small cell lung cancer (NSCLC), oral squamous cell carcinoma (OSCC) and gastric cancer [25-27]. LINC01436 has been revealed to be potential prognostic biomarkers for NSCLC . In terms of mechanism, LINC01436, acted as a microRNA (miR)-30a-3p sponge, regulated the expression level of its target gene EPAS1. TTTY14 (testis-specific transcript, Y-linked 14) was thought to be associated with human papillomavirus (HPV) induced tumorigenesis, the expression level of TTTY14 was obsearvedly differed between HPV inactive/negative group and HPV active group , in accordance with our functional enrichment analysis. In additional, TTTY14 was identified as significantly correlated with overall survival in OSCC and gastric cancer [26, 27]. Hence, further characterization of molecules such as AC007907.1, AC025419.1, AC078993.1, AC090241.2, AL158166.1, AL355974.2, AL596330.1, HOXB-AS4, KLHL6-AS1, LHX1-DT, LINC00528, LINC01436 and TTTY14 will provide new perspective for the development and progress of LSCC, and aided to find potential therapeutic targets for LSCC patients.
Consistent with previous studies, margin status was found to be significant association with survival among patients with LSCC in the present study [29, 30]. Published trials about LSCC did not reported that tumor status which was an independent risk factor for OS in our study was related to prognosis of LSCC. However, tumor status has been confirmed that it was independent prognostic factors for survival in hepatocellular carcinoma (HCC) . Additionally, we identified male was positively associated with OS probability in the univariate Cox analysis, inconsistent with previous trials that male was poor prognosis for LSCC [32, 33]. Nevertheless, the effect of gender on the prognosis of OS was not statistically significant in multivariable Cox analysis. In addition to these clinical factors, as expected, the lncRNAs signature was an effective independent prognostic factor for the prediction of patients with LSCC.
Although our nomogram demonstrated impressive performance in LSCC survival prediction, there are specific limitations associated with our trial. First, the presented nomogram based only on TCGA database with limited simple sizes for LSCC, are not yet suitable for general application prior to validation of the predictive models with independent testing cohort. So external and multicenter prospective cohorts with large sample sizes are still needed to validate the clinical application of our model.
Second, Missing variables were a source of defect in this evaluation. Such as extracapsular spread [34, 35], lymphovascular invasion status , perineural invasionas  and human papillomavirus (HPV) [36, 37] as important prognostic parameters for LSCC patients, weren’t well recorded in TCGA database. Notably, our functional enrichment analysis found that the prognostic lncRNAs was significantly associated with human papillomavirus. Published researches and meta-analysis indicated HPV positive laryngeal cancer patients is sensitive to radiotherapy and chemotherapy and showed inferior survival [36, 37]. Hence, we recommend that future studies should added value of those factors in a multivariable prediction model to improve the accuracy of prediction in LSCC patients
Third, our selection of factors was limited to those available in our database. On account of the anonymous database, we cannot extend our database with characteristics such as race, insurance status, comorbidity, hemoglobin level, albumin, tumor hypoxia, and TP53 mutation, which were frequently reported prognostic factors of patients with LSCC [38-40]. Further efforts to incorporate more patient-specific, tumor-specific and molecular factors will potentially help to improve the performance of the present model.
Fourth, we do not explore the underlying biological function and pathways of the prognostic lncRNAs, so further studies are needed to uncover the related mechanisms.