Co-Expression of TWIST1 and ZEB2 in Oral Squamous Cell Carcinoma Is Associated with Poor Survival

<div><p>Oral squamous cell carcinoma (OSCC) is an aggressive disease accounting for more than 260,000 cancer cases diagnosed and 128,000 deaths worldwide. A large majority of cancer deaths result from cancers that have metastasized beyond the primary tumor. The relationship between genetic changes and clinical outcome can reflect the biological events that promote cancer’s aggressive behavior, and these can serve as molecular markers for improved patient management and survival. To this end, epithelial-mesenchymal transition (EMT) is a major process that promotes tumor invasion and metastasis, making EMT-related proteins attractive diagnostic biomarkers and therapeutic targets. In this study, we used immunohistochemistry to study the expression of a panel of transcription factors (TWIST1, SNAI1/2, ZEB1 and ZEB2) and other genes intimately related to EMT (CDH1 and LAMC2) at the invasive tumor front of OSCC tissues. The association between the expression of these proteins and clinico-pathological parameters were examined with Pearson Chi-square and correlation with survival was analyzed using Kaplan Meier analysis. Our results demonstrate that there was a significant differential expression of CDH1, LAMC2, SNAI1/2 and TWIST1 between OSCC and normal oral mucosa (NOM). Specifically, CDH1 loss was significantly associated with Broder’s grading, while diffused LAMC2 was similarly associated with non-cohesive pattern of invasion. Notably, co-expression of TWIST1 and ZEB2 in OSCC was significantly associated with poorer overall survival, particularly in patients without detectable lymph node metastasis. This study demonstrates that EMT-related proteins are differentially expressed in OSCC and that the co-expression of TWIST1 and ZEB2 could be of clinical value in identifying patients with poor survival for appropriate patient management.</p></div

Association of co-expression of TWIST1 and ZEB2 with survival.

<p>(a) OSCC patients with co-expression of TWIST1 and ZEB2 have poorer overall survival compared to patients with no co-expression of these two proteins. Co-expression of TWIST1 and ZEB2 is significantly associated with overall survival in node negative OSCC patients (b) but not in node positive OSCC patients (c).</p

(A) Cox regression analysis demonstrates that co-expression of TWIST1 and ZEB2 is an independent prognostic factor for poor overall survival.

<p>Abbreviations: CI, confidence intervals</p><p>*<i>p</i> < 0.05.</p><p>(B) Cox regression analysis among patients with negative cervical node demonstrates that co-expression of TWIST1 and ZEB2 is an independent prognostic factor for poor overall survival.</p

Representative immunostaining of EMT-related markers in non-malignant oral mucosa (Original magnification: 40X, 200X-Inset) and oral squamous cell carcinoma tissues (OSCC; Original magnification: 200X).

<p>Representative immunostaining of EMT-related markers in non-malignant oral mucosa (Original magnification: 40X, 200X-Inset) and oral squamous cell carcinoma tissues (OSCC; Original magnification: 200X).</p

Demographics and clinico-pathological characteristics of patients.

<p>Abbreviations: OSCC, oral squamous cell carcinoma; n, number of specimens; N/A, not available.</p><p>Demographics and clinico-pathological characteristics of patients.</p

Genome wide profiling in oral squamous cell carcinoma identifies a four genetic marker signature of prognostic significance

<div><p>Background</p><p>Cancers of the oral cavity are primarily oral squamous cell carcinomas (OSCCs). Many of the OSCCs present at late stages with an exceptionally poor prognosis. A probable limitation in management of patients with OSCC lies in the insufficient knowledge pertaining to the linkage between copy number alterations in OSCC and oral tumourigenesis thereby resulting in an inability to deliver targeted therapy.</p><p>Objectives</p><p>The current study aimed to identify copy number alterations (CNAs) in OSCC using array comparative genomic hybridization (array CGH) and to correlate the CNAs with clinico-pathologic parameters and clinical outcomes.</p><p>Materials and methods</p><p>Using array CGH, genome-wide profiling was performed on 75 OSCCs. Selected genes that were harboured in the frequently amplified and deleted regions were validated using quantitative polymerase chain reaction (qPCR). Thereafter, pathway and network functional analysis were carried out using Ingenuity Pathway Analysis (IPA) software.</p><p>Results</p><p>Multiple chromosomal regions including 3q, 5p, 7p, 8q, 9p, 10p, 11q were frequently amplified, while 3p and 8p chromosomal regions were frequently deleted. These findings were in confirmation with our previous study using ultra-dense array CGH. In addition, amplification of 8q, 11q, 7p and 9p and deletion of 8p chromosomal regions showed a significant correlation with clinico-pathologic parameters such as the size of the tumour, metastatic lymph nodes and pathological staging. Co-amplification of 7p, 8q, 9p and 11q regions that harbored amplified genes namely CCND1, EGFR, TPM2 and LRP12 respectively, when combined, continues to be an independent prognostic factor in OSCC.</p><p>Conclusion</p><p>Amplification of 3q, 5p, 7p, 8q, 9p, 10p, 11q and deletion of 3p and 8p chromosomal regions were recurrent among OSCC patients. Co-alteration of 7p, 8q, 9p and 11q was found to be associated with clinico-pathologic parameters and poor survival. These regions contain genes that play critical roles in tumourigenesis pathways.</p></div

Multivariate cox regression model analysis of four combined genetic markers consisting of EGFR, TPM2, CCND1 and LRP12 in OSCC overall survival.

<p>Multivariate cox regression model analysis of four combined genetic markers consisting of EGFR, TPM2, CCND1 and LRP12 in OSCC overall survival.</p

Top significant networks and the associated network functions linked with CNAs associated genes.

<p>Top significant networks and the associated network functions linked with CNAs associated genes.</p

Concordance percentage for amplification of LRP12 (chr 8q), CCND1 (chr 11q), TPM2 (chr 9p), FSCN1 (chr 7p), EGFR (chr 7p), CLPTM1L (chr 5p) and deletion of CHL1 (chr 3p) and CSMD1 (chr 8p) identified using array CGH and validated using qPCR copy number analysis in OSCC samples.

<p>Concordance percentage for amplification of LRP12 (chr 8q), CCND1 (chr 11q), TPM2 (chr 9p), FSCN1 (chr 7p), EGFR (chr 7p), CLPTM1L (chr 5p) and deletion of CHL1 (chr 3p) and CSMD1 (chr 8p) identified using array CGH and validated using qPCR copy number analysis in OSCC samples.</p

Top significant pathways associated with CNAs associated genes.

<p>Top significant pathways associated with CNAs associated genes.</p