Distinctive Expression and Amplification of Genes at 11q13 in Relation to HPV Status with Impact on Survival in Head and Neck Cancer Patients.

Clear differences have been established between head and neck squamous cell carcinomas (HNSCC) depending on human papillomavirus (HPV) infection status. This study specifically investigated the status of the CTTN, CCND1 and ANO1 genes mapping at the 11q13 amplicon in relation to the HPV status in HNSCC patients. CTTN, CCND1 and ANO1 protein expression and gene amplification were respectively analyzed by immunohistochemistry and real-time PCR in a homogeneous cohort of 392 surgically treated HNSCC patients. The results were further confirmed using an independent cohort of 279 HNSCC patients from The Cancer Genome Atlas (TCGA). The impact on patient survival was also evaluated. CTTN, CCND1 and ANO1 gene amplification and protein expression were frequent in HPV-negative tumors, while absent or rare in HPV-positive tumors. Using an independent validation cohort of 279 HNSCC patients, we consistently found that these three genes were frequently co-amplified (28%) and overexpressed (39⁻46%) in HPV-negative tumors, whereas almost absent in HPV-positive tumors. Remarkably, these alterations (in particular CTTN and ANO1 overexpression) were associated with poor prognosis. Taken together, the distinctive expression and amplification of these genes could cooperatively contribute to the differences in prognosis and clinical outcome between HPV-positive and HPV-negative tumors. These findings could serve as the basis to design more personalized therapeutic strategies for HNSCC patients.

Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts.

The miR-196 family members have been found dysregulated in different cancers. Therefore, they have been proposed as promising biomarkers and therapeutic targets. This study is the first to investigate the role of miR-196b in the development and progression of head and neck squamous cell carcinomas (HNSCC), and also the impact on the surrounding tumor microenvironment. Increased miR-196b levels were detected in 95% of primary tumors and precancerous lesions, although no significant differences were observed between non-progressing versus progressing dysplasias. Furthermore, increased levels of both miR-196a and miR-196b were successfully detected in saliva samples from HNSCC patients. The functional consequences of altered miR-196 expression were investigated in both HNSCC cell lines and cancer-associated fibroblasts (CAFs) by transfection with specific pre-miR precursors. Results showed that both miR-196a and miR-196b elicit cell-specific responses in target genes and downstream regulatory pathways, and have a distinctive impact on cell proliferation, migration and invasion. These data reveal the early occurrence and prevalence of miR-196b dysregulation in HNSCC tumorigenesis, suggesting its utility for early diagnosis and/or disease surveillance and also as a non-invasive biomarker in saliva. The pleiotropic effects of miR-196a/b in HNSCC cell subpopulations and surrounding CAFs may complicate a possible therapeutic application.

Clinical significance of Anoctamin-1 gene at 11q13 in the development and progression of head and neck squamous cell carcinomas.

This study investigates the clinical significance of Anoctamin-1 gene mapping at 11q13 amplicon in both the development and progression of head and neck squamous cell carcinomas (HNSCC). ANO1 protein expression was evaluated by immunohistochemistry in a cohort of 372 surgically treated HNSCC patients and also in 35 laryngeal precancerous lesions. ANO1 gene amplification was determined by real-time PCR in all the laryngeal premalignancies and 60 of the HNSCCs, and molecular data correlated with clinical outcome. ANO1 gene amplification was frequently detected in both premalignant lesions (63%) and HNSCC tumours (58%), whereas concomitant ANO1 expression occurred at a much lower frequency (20 and 22%). Interestingly, laryngeal dysplasias harbouring ANO1 gene amplification showed a higher risk of malignant transformation (HR = 3.62; 95% CI 0.79-16.57; P = 0.097; Cox regression). ANO1 expression and gene amplification showed no significant associations with clinicopathological parameters in HNSCC. However, remarkably ANO1 expression differentially influenced patient survival depending on the tumour site. Collectively, this study provides original evidence demonstrating the distinctive impact of ANO1 expression on HNSCC prognosis depending on the tumour site.

Immunohistochemical markers of distant metastasis in laryngeal and hypopharyngeal squamous cell carcinomas.

Metastasis remains a major cause of mortality in head and neck squamous cell carcinoma (HNSCC). Current clinicopathological features have shown limited predictability for the risk of distant metastasis in individual patients, and therefore more accurate and reliable markers are needed. The aim of this study was to investigate the ability of various molecular markers present in primary tumors to predict the risk of developing distant metastasis. Restrictive clinical criteria were applied for patient selection in order to carry out a case-control study with comparable clinical features on a group-wide basis and a similar risk of metastasis. All patients were surgically treated (with postoperative radiotherapy when appropriate) and classified as stage IV disease. Immunohistochemical analysis was performed for a panel of proteins known to participate in cellular processes relevant to metastatic dissemination (E-cadherin, annexin A2, cortactin, FAK, EGFR, p53, and p-AKT). Results showed that the loss of E-cadherin expression was significantly correlated with the risk of distant metastasis (P = 0.002; log-rank test), while the loss of annexin A2 expression was nearly statistically significant (P = 0.06). None of the other protein markers assessed were associated with the development of distant metastasis. Therefore, according to our data the loss of epithelial adhesion seems to play a central role in the development of metastasis in HNSCC, and more importantly, immunohistochemical assessment of key proteins involved in cell adhesion regulation, such as E-cadherin could represent a useful tool to evaluate easily and routinely the metastatic potential of these carcinomas.

In oral squamous cell carcinoma, high FAK expression is correlated with low P53 expression.

Focal adhesion kinase (FAK) and p53 have been associated with metastatic activity and a poor prognosis in oral squamous cell carcinoma (OSCC). Recently, a feedback mechanism in which FAK regulates p53 has been proposed. The present study aims to determine the role of p53 in FAK regulation in these tumors. FAK and p53 expression was examined by immunohistochemistry in normal oral mucosa and in 67 oral squamous cell carcinomas. p16(INK4a) was also studied in view of its association with human papillomavirus infection. The association between FAK and p53 was subsequently analyzed. FAK expression in OSCCs was heterogeneous: 22 (33 %) cases showed weak expression, 16 (24 %) showed moderate expression, and 22 (33 %) cases showed high expression. Regarding p53, 31 of 67 (46 %) available tumor specimens showed negative staining, and 36 of 67 (54 %) showed positive nuclear staining for p53. FAK expression was inversely correlated with p53 expression (Fisher's exact test, p = 0.005). There was no association between p16(INK4a) and p53 or FAK expression. In conclusion, our results support the hypothesis that FAK activity might be involved in the down-regulation of p53 expression in OSCC.