Genome-wide expression and copy number analysis identifies driver genes in gingivobuccal cancers.

The molecular mechanisms contributing to the development and progression of gingivobuccal complex (GBC) cancers-a sub-site of oral cancer, comprising the buccal mucosa, the gingivobuccal sulcus, the lower gingival region, and the retromolar trigone-remain poorly understood. Identifying the GBC cancer-related gene expression signature and the driver genes residing on the altered chromosomal regions is critical for understanding the molecular basis of its pathogenesis. Genome-wide expression profiling of 27 GBC cancers with known chromosomal alterations was performed to reveal differentially expressed genes. Putative driver genes were identified by integrating copy number and gene expression data. A total of 315 genes were found differentially expressed (P ≤ 0.05, logFC > 2.0) of which 11 genes were validated by real-time quantitative reverse transcriptase-PCR (qRT-PCR) in tumors (n = 57) and normal GBC tissues (n = 18). Overexpression of LY6K, in chromosome band 8q24.3, was validated by immunohistochemical (IHC) analysis. We found that 78.5% (2,417/3,079) of the genes located in regions of recurrent chromosomal alterations show copy number dependent expression indicating that copy number alteration has a direct effect on global gene expression. The integrative analysis revealed BIRC3 in 11q22.2 as a candidate driver gene associated with poor clinical outcome. Our study identified previously unreported differentially expressed genes in a homogeneous subtype of oral cancer and the candidate driver genes that may contribute to the development and progression of the disease. © 2011 Wiley Periodicals, Inc.

Detection of HPV E6/E7 mRNA in Clinical Samples Using RNA In Situ Hybridization.

Detection of human papilloma virus (HPV) in tissue specimens has been a clinical challenge since last 2 decades; however, screening for presence of E6/E7 transcripts is regarded as the gold standard, and it verifies the active HPV infection. Here, we describe "RNAscope® assay" a novel RNA in situ hybridization (ISH) technology; which detects E6/E7 mRNA of seven high risk HPV subtypes (HPV 16, 18, 31, 33, 35, 52, and 58) in formalin fixed paraffin embedded (FFPE) tissue samples.

Recurring Amplification at 11q22.1-q22.2 Locus Plays an Important Role in Lymph Node Metastasis and Radioresistance in OSCC.

A key feature in the pathogenesis of OSCC is genetic instability, which results in altered expression of genes located in amplified/deleted chromosomal regions. In a previous study we have shown that the amplification of the 11q22.1-q22.2 region, encoding cIAP1 and cIAP2, is associated with lymph node metastasis and poor clinical outcome in OSCC. Here, we validate the aCGH results by nuc ish and detect a weak amplification at the 11q22.1-q22.2 locus in 37% of the 182 samples tested. We find positive correlation of 11q22.1-q22.2 amplification with lymph node metastasis, reduced survival, and increased cancer recurrence, and we observe that patients with 11q22.1-q22.2 amplification fail to respond to radiotherapy. We confirm the concurrent overexpression of cIAP1 and cIAP2 and observe differential subcellular localization of the two proteins in OSCC. To ascertain the roles of cIAP1/cIAP2 in lymph node metastasis and radioresistance, we use an in vitro pre-clinical model and confirm the role of cIAP1 in invasion and the role of cIAP2 in invasion and migration. Studies of other tumor types in which cIAP1 is overexpressed suggest that multi-regimen treatments including SMAC mimetics may be effective. Thus, the evaluation of 11q22.1-q22.2 amplifications in OSCC patients may help choose the most effective treatment.

Low prevalence of transcriptionally active human papilloma virus in Indian patients with HNSCC and leukoplakia.

OBJECTIVES: In the present study, we comprehensively analyzed the prevalence of transcriptionally active human papilloma virus (HPV) in tissue samples of Indian patients with leukoplakia, predominantly hyperplastic lesions and head and neck squamous cell carcinoma (HNSCC). In addition, saliva samples from patients with HNSCC were screened for HPV detection. STUDY DESIGN: P16 overexpression was analyzed by immunohistochemistry. Tissue samples of leukoplakia (n = 121) and HNSCC (n = 427) and saliva from patients with HNSCC (n = 215) were tested for HPV using nested polymerase chain reaction. Positive samples were sequenced for subtyping. The presence of HPV E6/E7 mRNA was confirmed by RNA in situ hybridization. RESULTS: P16 expression and HPV DNA were not detected in any of the leukoplakia specimens. Of the 427 HNSCC tumors, 9 showed p16 overexpression and 7/427 cases were positive for HPV16 DNA, in saliva or tissue. E6/E7 mRNA positivity was observed in 8 HNSCC samples, primarily from patients with no habit of tobacco consumption. The prevalence of high-risk HPV was restricted to oropharynx and larynx, with very little concordance between p16 overexpression and HPV positivity. All patients with HPV-positive saliva samples had transcriptionally active HPV present in their tumors. CONCLUSION: The presence of HPV DNA does not necessarily reflect transcriptionally active virus in tumors; hence, it is important to consider this fact while categorizing HPV-associated tumors.

Downregulation of keratin 76 expression during oral carcinogenesis of human, hamster and mouse.

BACKGROUND: Keratins are structural marker proteins with tissue specific expression; however, recent reports indicate their involvement in cancer progression. Previous study from our lab revealed deregulation of many genes related to structural molecular integrity including KRT76. Here we evaluate the role of KRT76 downregulation in oral precancer and cancer development. METHODS: We evaluated KRT76 expression by qRT-PCR in normal and tumor tissues of the oral cavity. We also analyzed K76 expression by immunohistochemistry in normal, oral precancerous lesion (OPL), oral squamous cell carcinoma (OSCC) and in hamster model of oral carcinogenesis. Further, functional implication of KRT76 loss was confirmed using KRT76-knockout (KO) mice. RESULTS: We observed a strong association of reduced K76 expression with increased risk of OPL and OSCC development. The buccal epithelium of DMBA treated hamsters showed a similar trend. Oral cavity of KRT76-KO mice showed preneoplastic changes in the gingivobuccal epithelium while no pathological changes were observed in KRT76 negative tissues such as tongue. CONCLUSION: The present study demonstrates loss of KRT76 in oral carcinogenesis. The KRT76-KO mice data underlines the potential of KRT76 being an early event although this loss is not sufficient to drive the development of oral cancers. Thus, future studies to investigate the contributing role of KRT76 in light of other tumor driving events are warranted.