Deregulation of extracellular matrix modeling with molecular prognostic markers revealed by transcriptome sequencing and validations in Oral Tongue squamous cell carcinoma

Abstract

Oral Tongue Squamous Cell Carcinoma (OTSCC), a distinct sub-group of head and neck cancers, is characteristically aggressive in nature with a higher incidence of recurrence and metastasis. Recent advances in therapeutics have not improved patient survival. The phenomenon of occult node metastasis, even among the purportedly good prognosis group of early-stage and node-negative tongue tumors, leads to a high incidence of locoregional failure in OTSCC which needs to be addressed. In the current study, transcriptome analysis of OTSCC patients identified the key genes and deregulated pathways. A panel of 26 marker genes was shortlisted and validated using real-time PCR in a prospective cohort of 100 patients. The gene expression was correlated with clinicopathological features including occult node metastasis, survival, and therapeutic outcome. The up-regulation of a panel of 6 genes namely, matrix metalloproteinase 9 (MMP9), Laminin subunit Gamma 2 (LAMC2), Desmoglein 2 (DSG2), Plasminogen Activator Urokinase (PLAU), Forkhead Box M1 (FOXM1), and Myosin 1B (MYO1B) was associated with failure of treatment in the early stage (T1, T2). Up-regulation of Tenacin C (TNC) and Podoplanin (PDPN) was significantly correlated with occult node positivity. Immunohistochemical analysis of LAMC2, MMP9, and E-Cadherin (ECAD) confirmed these markers to be indicators of poor prognosis. We propose this panel of valuable prognostic markers can be clinically useful to identify poor prognosis and occult node metastasis in OTSCC patients.

Figure 1

Box plot comparing the expression of genes in absolute normal tongue and OTSCC samples. Average log₂ fold change from tumor and adjacent normal samples have been compared and the P value from an unpaired t test is indicated in the figure. (a) MMP9 (P = 0.001) (b) LAMC2 (P = 2.10e−04) (c) DSG2 (P = 0.017) (d) FOXM1 (P = 0.002) (e) GLUT1 (P = 7.64e−06) (f) CA9 (P = 0.002) (g) HIF1A (P = 0.004) (h) S100 (P = 0.003) (i) SPP1 (P = 0.001) (j) POSTN (P = 0.009) (k) CDKN2A (P = 6.75e−06) (l) BIRC5 (P = 0.001) (m) MYO1B (P = 0.02) (n) OCT4 (P = 0.006) (o) RBP1 (P = 0.001) (p) PLAU (P = 3.20 e−05) (q) UPAR (P = 0.003) (r) TNC (P = 0.001) (s) PDPN (P = 0.01).

Figure 2

Heatmap representing the expression levels of the significant differentially expressed marker genes among the normal and OTSCC samples.

Figure 3

Immunoexpression of LAMC2: (a) WDSCC showing intense cytoplasmic positivity for LAMC2 10X. (b) WDSCC showing intense cytoplasmic positivity for LAMC2 20X. (c) MDSCC showing intense positivity for basement membrane, but cytoplasm in negative for LAMC2, 20X.

Figure 4

Immunoexpression of ECAD (a). Normal epithelium with intense E-cadherin positivity (b). Reduction of ECAD expression at ITF 10X (c). Reduction of ECAD expression at ITF 20X.

Figure 5

Immunoexpression of MMP9 (a). Normal mucosa showing minimum stain for MMP9 (b). WDSCC showing intense MMP9 stain 10X (c). WDSCC showing intense MMP9 stain 20X.

Figure 6

Kaplan–Meier survival analysis of clinicopathological variables in OTSCC patients (a). Disease free survival by clinical stage (b). Overall survival by clinical stage (c). Disease free survival by neck node status (d). Overall survival by neck node status (e). Disease free survival by occult node status (f). Overall survival by occult node status (g). Overall survival by habits status (h). Disease free survival by Perineural invasion in early stage OTSCC treated by surgery. P values correspond to the log-rank test comparing the survival curves.

Figure 7

Kaplan–Meier survival analysis of molecular markers in OTSCC patients (a). Disease free survival by LAMC2 immunoexpression (b). Overall survival by LAMC2 immunoexpression (c). Disease free survival by MMP9 immunoexpression (d). Overall survival by MMP9 immunoexpression (e). Disease free survival by ECAD immunoexpression (f). Overall survival ECAD immunoexpression. P values correspond to the log-rank test comparing the survival curves.