LncRNA profile study reveals a three-lncRNA signature associated with the survival of patients with oesophageal squamous cell carcinoma

Abstract

Background: Oesophageal cancer is one of the most deadly forms of cancer worldwide. Long non-coding RNAs (lncRNAs) are often found to have important regulatory roles.

Objective: To assess the lncRNA expression profile of oesophageal squamous cell carcinoma (OSCC) and identify prognosis-related lncRNAs.

Method: LncRNA expression profiles were studied by microarray in paired tumour and normal tissues from 119 patients with OSCC and validated by qRT-PCR. The 119 patients were divided randomly into training (n=60) and test (n=59) groups. A prognostic signature was developed from the training group using a random Forest supervised classification algorithm and a nearest shrunken centroid algorithm, then validated in a test group and further, in an independent cohort (n=60). The independence of the signature in survival prediction was evaluated by multivariable Cox regression analysis.

Results: LncRNAs showed significantly altered expression in OSCC tissues. From the training group, we identified a three-lncRNA signature (including the lncRNAs ENST00000435885.1, XLOC_013014 and ENST00000547963.1) which classified the patients into two groups with significantly different overall survival (median survival 19.2 months vs >60 months, p<0.0001). The signature was applied to the test group (median survival 21.5 months vs >60 months, p=0.0030) and independent cohort (median survival 25.8 months vs >48 months, p=0.0187) and showed similar prognostic values in both. Multivariable Cox regression analysis showed that the signature was an independent prognostic factor for patients with OSCC. Stratified analysis suggested that the signature was prognostic within clinical stages.

Conclusions: Our results suggest that the three-lncRNA signature is a new biomarker for the prognosis of patients with OSCC, enabling more accurate prediction of survival.

Keywords: Oesophageal Cancer.

Figure 1

Identification of the long non-coding RNA (lncRNA) signature in the training set. (A) After microarray processing, the microarray data was described by an 60×8900 matrix with a ‘good’ or ‘poor’ label column. (B) After two filtering procedures, 909 lncRNAs remained for further analysis. (C) Selection process for the nine lncRNAs with highest classification power for patient survival. A random Forest supervised classification algorithm was used to narrow down the number of lncRNAs by several iterative steps, in which one-third of the least important lncRNAs were discarded at each step according to their importance score. (D) Development of prognostic classifier for all combinations (N=2⁹−1=511) of the nine lncRNAs using the nearest shrunken centroid algorithm. V_g and V_p are the mean expression profiles of the lncRNA combination (g₁ g₃ g₄ g₆) for good-prognostic samples and poor-prognostic samples, respectively. V_i is the expression profile of sample i. The Euclid distances d(V_i,V_g) and d(V_i,V_p) are used to classify sample i into a low- or high-risk group. (E) The procedure for identifying the final signature. The accuracies of all 511 signatures were calculated and the nine highest accuracies for k=1, 2, …, 9 are shown in the plot. The signature containing three lncRNAs was selected as the final signature.

Figure 2

Unsupervised hierarchical clustering of the 119 pairs of tissues. The normalised expression data of the 6389 lncRNAs with coefficient of variance >0.10 was used for clustering analysis. Hierarchical clustering clearly separated tumour (blue bar) and normal (yellow bar) samples. Only six tumour samples and six normal samples were misclassified.

Figure 3

The three-lncRNA signature predicts overall survival of patients with OSCC. Heat maps (A–C) of the relative expression level (tumour minus normal) after z-score transformation for each lncRNA, and Kaplan–Meier survival curves (D–F) of patients classified into high- and low-risk groups using the three-lncRNA signature. p Values were calculated by log-rank test. (A, D) Training set, 60 patients. (B, E) Test set, 59 patients. (C, F) Independent cohort, 60 patients. OSCC, oesophageal squamous cell carcinoma.

Figure 4

Survival prediction in stage II and III patients. Kaplan–Meier survival curves of stage II and III patients with OSCC classified into high- and low-risk groups based on the three-lncRNA signature. (A) Stage II patients, training set (n=22). (B) Stage II patients, combined test set and independent cohort (n=55). (C) Stage III patients, training set (n=36). (D) Stage III patients, combined test set and independent cohort (n=56). OSCC, oesophageal squamous cell carcinoma.

Figure 5

Comparison of sensitivity and specificity for survival prediction by the three-lncRNA signature, TNM stage and combination of the two factors. The three receiver operating characteristics (ROC) curves in the training set (A) and test set (B). p Values show the area under the ROC (AUROC) of TNM stage versus the AUROC of the three-lncRNA signature, or the combination of signature and TNM. TNM, tumour node metastasis.