Oral cancer screening: serum Raman spectroscopic approach.

Serum Raman spectroscopy (RS) has previously shown potential in oral cancer diagnosis and recurrence prediction. To evaluate the potential of serum RS in oral cancer screening, premalignant and cancer-specific detection was explored in the present study using 328 subjects belonging to healthy controls, premalignant, disease controls, and oral cancer groups. Spectra were acquired using a Raman microprobe. Spectral findings suggest changes in amino acids, lipids, protein, DNA, and ß-carotene across the groups. A patient-wise approach was employed for data analysis using principal component linear discriminant analysis. In the first step, the classification among premalignant, disease control (nonoral cancer), oral cancer, and normal samples was evaluated in binary classification models. Thereafter, two screening-friendly classification approaches were explored to further evaluate the clinical utility of serum RS: a single four-group model and normal versus abnormal followed by determining the type of abnormality model. Results demonstrate the feasibility of premalignant and specific cancer detection. The normal versus abnormal model yields better sensitivity and specificity rates of 64 and 80%; these rates are comparable to standard screening approaches. Prospectively, as the current screening procedure of visual inspection is useful mainly for high-risk populations, serum RS may serve as a useful adjunct for early and specific detection of oral precancers and cancer.

Association of differential ß-catenin expression with Oct-4 and Nanog in oral squamous cell carcinoma and their correlation with clinicopathological factors and prognosis.

BACKGROUND: The re-expression of pluripotent markers (Oct-4 and Nanog) and the reactivation of stem cell-related pathways in oral carcinoma have been well researched. However, the relationship between the stem cell signaling molecule ß-catenin and pluripotent markers Oct-4 and Nanog in oral cancer is yet to be studied in detail. Therefore, we have investigated the correlation among Oct-4, Nanog, and ß-catenin in oral squamous cell carcinoma, which, in turn, could provide valuable insight into its prognostic significance. METHODS: The immunohistochemical analysis was performed for 60 cases of oral cancer to study the expression pattern of Oct-4, Nanog, and ß-catenin. Whereas immunofluorescence analysis was used to investigate the co-localization of ß-catenin with Oct-4 and Nanog in oral carcinoma tissues and H314 cell line. Finally, co-immunoprecipitation analysis was used to study the possible interaction between ß-catenin and Oct-4 in oral carcinoma cells. RESULTS: ß-catenin, Oct-4, and Nanog showed significant correlation with lymph node metastasis, stage, grade, and prognosis in oral squamous cell carcinoma. Interestingly, a significant positive correlation was found among the expression of Oct-4, Nanog, and ß-catenin. Moreover, the interaction between ß-catenin and Oct-4 was observed in oral cancer. CONCLUSION: The positive correlation among Oct-4, Nanog, and ß-catenin suggests their coordinated role in maintaining proliferation in oral carcinoma cells. The interaction between ß-catenin and Oct-4 may be a crucial event in oral carcinogenesis. On the other hand, ß-catenin, Oct-4, and Nanog could be used as independent prognostic markers of oral squamous cell carcinoma.

Understanding the role of keratins 8 and 18 in neoplastic potential of breast cancer derived cell lines.

BACKGROUND: Breast cancer is a complex disease which cannot be defined merely by clinical parameters like lymph node involvement and histological grade, or by routinely used biomarkers like estrogen receptor (ER), progesterone receptor (PGR) and epidermal growth factor receptor 2 (HER2) in diagnosis and prognosis. Breast cancer originates from the epithelial cells. Keratins (K) are cytoplasmic intermediate filament proteins of epithelial cells and changes in the expression pattern of keratins have been seen during malignant transformation in the breast. Expression of the K8/18 pair is seen in the luminal cells of the breast epithelium, and its role in prognostication of breast cancer is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have modulated K8 expression to understand the role of the K8/18 pair in three different breast epithelium derived cell lines: non-transformed MCF10A, transformed but poorly invasive MDA MB 468 and highly invasive MDA MB 435. The up-regulation of K8 in the invasive MDA MB 435 cell line resulted in a significant decrease in proliferation, motility, in-vitro invasion, tumor volume and lung metastasis. The down-regulation of K8 in MDA MB 468 resulted in a significant increase in transformation potential, motility and invasion in-vitro, while MCF10A did not show any changes in cell transformation assays. CONCLUSIONS/SIGNIFICANCE: These results indicate the role of K8/18 in modulating invasion in breast cancer -its presence correlating with less invasive phenotype and absence correlating with highly invasive, dedifferentiated phenotype. These data may have important implications for prognostication of breast cancer.

Alterations in keratins and associated proteins during 4- Nitroquinoline-1-oxide induced rat oral carcinogenesis.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is the sixth largest group of malignancies globally and the single largest group of malignancies in the Indian subcontinent. Despite the advances in treatment and therapeutic modalities the five year survival rate of OSCC has not changed in the last few decades, and remains less than 40%. Several studies have focused on defining molecular markers that can either detect cancer at an early stage or can predict patient's outcome. However, such markers are still undefined. Keratins (K) are epithelia predominant intermediate filament proteins which are expressed in a differentiation dependent and site specific manner. Keratins are being used as biomarkers in different epithelial disorders including cancer. They are associated with desmoplakin and α6ß4 integrin which are components of desmosomes and hemidesmosomes respectively. MATERIALS AND METHODS: 4-Nitroquinoline 1-Oxide (4NQO) was used as a carcinogen for the development of various stages of oral carcinogenesis in rat lingual mucosa. Two-Dimentional gel electrophoresis was performed for the separation of Keratins followed by western blotting for their specific identification. Western blotting and RT PCR was carried out for desmoplakin and α6ß4 integrin respectively to understand their levels. Immunohistochemical analysis was carried out to further study the localization of desmoplakin and α6 integrin. RESULTS: In this study we have analysed the alterations in Keratins and associated proteins during sequential stages of 4NQO induced rat oral carcinogenesis. Our results showed that the alterations primarily begin after the dysplastic changes in the lingual epithelium like the elevation of Keratins 5/6a, ectopic expression of Keratin 8, increase in suprabasal expression of α6 integrin and increase in desmoplakin levels. Most of these alterations persisted till the development of SCC except desmoplakin, the levels of which were downregulated in papillomatous lesions and SCC. Many of these alterations have also been documented in human oral carcinogensis. CONCLUSION: Thus, 4NQO model of rat lingual carcinogenesis reproduces majority of the changes that are seen in human oral carcinogenesis and it can be exploited for the development of biomarkers.

Loss of keratin 8 phosphorylation leads to increased tumor progression and correlates with clinico-pathological parameters of OSCC patients.

BACKGROUND: Keratins are cytoplasmic intermediate filament proteins expressed in tissue specific and differentiation dependent manner. Keratins 8 and 18 (K8 and K18) are predominantly expressed in simple epithelial tissues and perform both mechanical and regulatory functions. Aberrant expression of K8 and K18 is associated with neoplastic progression, invasion and poor prognosis in human oral squamous cell carcinomas (OSCCs). K8 and K18 undergo several post-translational modifications including phosphorylation, which are known to regulate their functions in various cellular processes. Although, K8 and K18 phosphorylation is known to regulate cell cycle, cell growth and apoptosis, its significance in cell migration and/or neoplastic progression is largely unknown. In the present study we have investigated the role of K8 phosphorylation in cell migration and/or neoplastic progression in OSCC. METHODOLOGY AND PRINCIPAL FINDINGS: To understand the role of K8 phosphorylation in neoplastic progression of OSCC, shRNA-resistant K8 phospho-mutants of Ser73 and Ser431 were overexpressed in K8-knockdown human AW13516 cells (derived from SCC of tongue; generated previously). Wound healing assays and tumor growth in NOD-SCID mice were performed to analyze the cell motility and tumorigenicity respectively in overexpressed clones. The overexpressed K8 phospho-mutants clones showed significant increase in cell migration and tumorigenicity as compared with K8 wild type clones. Furthermore, loss of K8 Ser73 and Ser431 phosphorylation was also observed in human OSCC tissues analyzed by immunohistochemistry, where their dephosphorylation significantly correlated with size, lymph node metastasis and stage of the tumor. CONCLUSION AND SIGNIFICANCE: Our results provide first evidence of a potential role of K8 phosphorylation in cell migration and/or tumorigenicity in OSCC. Moreover, correlation studies of K8 dephosphorylation with clinico-pathological parameters of OSCC patients also suggest its possible use in prognostication of human OSCC.

Prognostic value of tissue polypeptide antigen in oral squamous cell carcinoma.

Human oral cancer has a high risk of locoregional relapse which is difficult to diagnose early due to lack of prognostic markers. We and others have shown aberrant expression of cytokeratin (CK) 8 and 18 in human oral cancer. Aberrant supra-basal expression of CK19 has been shown earlier. In this study, our aim was to develop a non-invasive test for prognostication of human oral cancer. Immunoradiometric assay (IRMA) was used to measure the circulating levels of TPA in the sera of 80 oral cancer patients before surgery and seven days after surgery. Elevated serum TPA levels were noted in the post surgery samples of 28 out of the 50 patients for whom clinical follow-up was available, as compared to their pre-surgery samples. TPA levels in the pre-surgery serum samples of patients were significantly higher than levels in the sera of healthy controls (p=0.001). Elevated levels in patients correlated significantly with stage (p=0.02), development of recurrence (p<0.006), and impacted survival (p<0.033). IHC analysis showed statistically significant correlation between expression of CK8, 18 and 19 in surrounding uninvolved tissues of the tumour and post surgery elevated serum TPA levels (p<0.001). This suggests the possibility that CK fragments are released from the surrounding uninvolved tissues into the sera of patients after surgical removal of the tumour. Thus, our study indicates that TPA can be a useful tumour marker for the prediction of recurrence and poor prognosis in human oral cancer.

Cytokeratin fragments in the serum: their utility for the management of oral cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality worldwide. Oral cancer is the most predominant malignancy in the Indian subcontinent due to the widespread habits of chewing tobacco and related products. Patients with oral tumours have a high risk of early locoregional relapse. Early detection of disease progression remains a challenging task mainly due to the lack of adequate early prognostic markers. CEA, SCC Ag, CA-125, serum cytokeratin (CK) fragments, Cyfra 21-1 (CK 19), TPS (CK 18), TPA (CK 8, 18, and 19) etc. are being used as serum markers for the prediction of prognosis of various malignancies. This review presents the available literature on serum CK markers in different malignancies evaluates their utility in the management of oral cancer, and identifies the lacunae which need to be addressed to develop sensitive and specific assays for early detection of recurrence, prognosis, and treatment monitoring.