Shilin N. Shukla

Electrochemical Sensor for Multiplex Biomarkers Detection

Purpose: Multiplexing assay of biomarkers at the point-of-care is an elusive goal for molecular diagnostics. Experimental Design: Here, we report an electrochemical (EC) sensor for oral cancer detection based on the simultaneous detection of two salivary biomarkers: interleukin (IL)-8 mRNA and IL-8 protein. Results: Under the multiplexing mode, the limit of detection of salivary IL-8 mRNA reaches to 3.9 fM and 7.4 pg/mL for IL-8 protein in saliva. Multiplex assay of these 2 biomarkers directly from 28 cancer and 28 matched control saliva samples shows significant difference between the two groups. From the receiver operating characteristic analysis, the EC sensor yields around 90% sensitivity and specificity for both IL-8 mRNA and IL-8 protein, which are very close to the data measured by traditional assays (ELISA and PCR) with the same group of saliva. Combined IL-8 mRNA and protein show better AUC compared with single biomarker. Conclusions: We show, for the first time, concurrently multiplexing detection of salivary mRNA and protein biomarkers using point-of-care EC sensor.

PROGNOSTIC SIGNIFICANCE OF MOLECULAR MARKERS IN ORAL SQUAMOUS CELL CARCINOMA: A MULTIVARIATE ANALYSIS

Multiple marker accumulation impacts tumor progression and biologic phenotypes affect clinical outcome of patients with head and neck cancer. Hence, this study investigated a battery of molecular markers that may help to reflect biologic aggressiveness and predict prognosis.

CLINICAL SIGNIFICANCE OF MMP-2 AND MMP-9 IN PATIENTS WITH ORAL CANCER

Factors that represent the potential for invasion and metastasis, such as matrix metalloproteinases (MMPs), could predict prognosis of cancer. Therefore, the authors studied plasma and tissue levels of MMP‐2 and MMP‐9 in oral cancer, the leading malignancy in India.

Lipid Peroxidation, Total Antioxidant Status, and Total Thiol Levels Predict Overall Survival in Patients With Oral Squamous Cell Carcinoma

Tobacco is the major etiological factor for oral cancer development through the generation of oxidative stress. Therefore, markers of oxidative stress such as total antioxidant status, lipid peroxidation, and total thiol levels might be useful to monitor oxidative stress and predict overall survival in oral cancer patients. The study included 140 oral cancer patients and 50 healthy controls, who were classified as with the habit of tobacco and no habit of tobacco. Adjacent normal and malignant tissue samples were collected from oral cancer patients. Plasma and tissue levels of lipid peroxidation, thiol, and total antioxidant status were assayed by spectrophotometric methods. Thiol levels were significantly lower in controls with the habit of tobacco (P = .033), oral cancer patients ( P = .0001), and malignant tissues (P = .015) as compared to controls with no habit of tobacco, controls with the habit of tobacco, and adjacent normal tissues, respectively. Tobacco exposure was higher in oral cancer patients than controls with the habit of tobacco. Controls with the habit of tobacco who had lower thiol (odds ratio [OR] = 10.58, P = .008) and high tobacco exposure (OR = 0.251, P = .05) showed an elevated risk of oral cancer development. Patients showing a lipid peroxidation level above the cutoff level as compared to patients below the cutoff level showed poor overall survival, whereas those with thiol and total antioxidant status levels below the cutoff level as compared to their respective counterparts showed poor overall survival. In conclusion, lipid peroxidation and thiol could be useful for predicting the risk of oral carcinogenesis in healthy tobacco consumers and predicting overall survival of oral cancer patients.

A Review on Salivary Genomics and Proteomics Biomarkers in Oral Cancer

Oral cancer has emerged as an alarming public health problem with increasing incidence and mortality rates all over the world. Therefore, the implementation of newer screening and early detection approaches are of utmost importance which could reduce the morbidity and mortality associated with this disease. Sensitive and specific biomarkers for oral cancer are likely to be most effective for screening, diagnosis, staging and follow-up for this dreaded malignancy. Unlike other deep cancers, oral cancer is located in oral cavity. Hence, the direct contact between saliva and oral cancer lesion makes the measurement of tumor markers in saliva an attractive alternative to serum and tissue testing. The DNA, RNA and protein molecules derived from the living cancer cells can be conveniently obtained from saliva. Thus, salivary biomarkers, a non-invasive alternative to serum and tissue-based biomarkers may be an effective modality for early diagnosis, prognostication and monitoring post therapy status. In the current post-genomic era, various technologies provide opportunities for high-throughput approaches to genomics and proteomics; which have been used to evaluate altered expressions of gene and protein targets in saliva of oral cancer patients. The emerging field of salivary biomarkers has great potentials to prove its clinical significance to combat oral cancer. Hence, we have reviewed importance of several salivary genomics and proteomics biomarkers for oral cancer.

Significance of alterations in plasma lipid profile levels in breast cancer.

Hypotheses. The relationship between lipids and breast cancer is obscure. Until now, conflicting results have been reported on the association between lipids and risk of breast cancer in women. Therefore, the major aim of this study is to examine the role of alterations in lipid profile in breast cancer. Study Design. Plasma lipids (ie, total cholesterol [TC], high-density lipoprotein [HDL], low-density lipoprotein [LDL], very-low-density lipoprotein [VLDL], and triglycerides [TG]) were analyzed from 70 controls, 30 patients with benign breast disease (BBD), 125 untreated breast cancer patients, and 93 posttreatment follow-up samples. Methods. Samples were analyzed using highly sensitive and specific spectrophotometric methods. Results. Plasma TC, LDL, VLDL, and TG were significantly lower (p = .042, p = .003, p = .024, p = .014, respectively) in patients with BBD compared with controls. Plasma TC and HDL were significantly lower (p = .026, p = .0001, respectively), and VLDL and TG were significantly higher (p = .009, p = .05) in breast cancer patients as compared with controls. Plasma VLDL and TG were significantly higher in breast cancer patients as compared with patients with BBD. The receiver-operating characteristic curve showed that plasma TC, LDL, VLDL, and TG levels could significantly discriminate (p = .001, p = .005, p = .005, p = .005, respectively) between controls and patients with BBD. Plasma levels of TC, HDL, VLDL, and TG could significantly distinguish (p = .01, p = .002, p = .001, p = .002, respectively) between controls and breast cancer patients. Plasma levels of VLDL and TG could significantly discriminate (p = .000, p = .000, respectively) between patients with BBD and breast cancer patients. Odds ratio analysis revealed that higher levels of TC and HDL were significantly associated with a reduction in breast cancer risk (p = .01 and p = .0001, respectively), whereas higher levels of VLDL and TG were significantly associated with increased breast cancer risk (p = .001 and p = .002, respectively). Plasma VLDL and TG levels were significantly lower in complete responders as compared with pretreatment levels (p = .000, p = .000, respectively), and plasma TC and LDL levels were significantly lower in nonresponders as compared with pretreatment levels (p = .015, p = .009, respectively). Conclusion. The alterations in lipid profile levels showed a significant correlation with breast cancer risk, disease status, and treatment outcome.

Molecular alterations in oral carcinogenesis: significant risk predictors in malignant transformation and tumor progression.

In this study an attempt was made to establish the significance of a battery of molecular alterations and thereby identify risk predictors in oral carcinogenesis. For this purpose, EGFR, Stat3, H-ras, c-myc, p53, cyclin D1, p16, Rb, Ki-67 and Bcl-2 were localized immunohistochemically in normal mucosa (n=12), hyperplasia (n=35), dysplasia (n=25), early stage carcinoma (n=65) and advanced stage carcinoma (n=70). Deregulation occurred at an early stage and the number of alterations increased with disease progression. Using multivariate logistic regression analysis, the significant risk predictor for hyperplasia from normal mucosa was Ki-67 (OR=5.75, p=0.021); the significant risk predictors for dysplasia from hyperplasia were EGFR (OR=12.96, p=0.002), Stat3 (OR=17.16, p=0.0001), p16 (OR=5.50, p=0.039) and c-myc (OR=5.99, p=0.052); the significant risk predictors for early stage carcinoma from dysplasia were p53 (OR=6.63, p=0.0001) and Rb (OR=3.81, p=0.056); and the significant risk predictors for further progression were EGFR (OR=5.50, p=0.0001), Stat3 (OR=4.49, p=0.0001), H-ras (OR=4.05, p=0.001) and c-myc (OR=2.99, p=0.015). Cyclin D1 holds a key position linking upstream signaling pathways to cell cycle regulation. Gene products of the mitogenic signaling pathway play an equally significant role as cell cycle regulatory proteins in the hyperplasia-dysplasia-early-advanced-carcinoma sequence and together may provide a reference panel of markers for use in defining premalignant lesions and predicting the risk of malignant transformation and tumor progression.

Cytokeratin and vimentin expression in breast cancer

BACKGROUND The transition from epithelial keratin to mesenchymal vimentin expression marks an important step in the malignant progression of breast cancer. This study analyzed the clinical significance of cytokeratin and vimentin in patients with breast cancer. MATERIALS AND METHODS Expression of cytokeratin and vimentin was evaluated by immunohistochemistry on paraffin-embedded tissue sections of patients with breast cancer. RESULTS Loss of cytokeratin was seen in 11% of the patients. A clearer trend towards loss of cytokeratin was observed in patients with stage IV disease and PR negativity. Weak cytokeratin expression was present in patients who developed recurrence or metastatic disease. Loss of cytokeratin was associated with reduced overall survival in univariate and multivariate analysis, gain of vimentin expression was seen in 57% of breast carcinoma patients. It was higher in patients with lymph node positivity, advanced stage, HER2 positivity, and disease recurrence or metastasis. Multivariate survival analysis indicated that gain of vimentin expression was associated with reduced relapse-free survival. CONCLUSION Loss of cytokeratin and gain of vimentin expression are indicators of biologically aggressive breast carcinoma.

Tobacco, antioxidant enzymes, oxidative stress, and genetic susceptibility in oral cancer.

Objectives:Oral cancer accounts third of all malignancies in India. Tobacco use, the major etiological factor for oral cancer is known to generate free radicals resulting in alterations in antioxidant enzymes like, glutathione-S-transferase (GST), glutathione reductase, superoxide dismutase, catalase, and glutathione peroxidase as well as lipid peroxidation and total thiol. Therefore, it is of fundamental importance to evaluate the role of tobacco and antioxidant enzymes and oxidative stress markers in oral carcinogenesis. Materials and Methods:One hundred forty oral cancer patients and 50 healthy controls, classified as “habitual controls” and “nonhabitual controls” having tobacco habits and no tobacco habits, respectively, were included in the study. Adjacent normal and malignant tissue samples were also collected. Erythrocyte, plasma, and tissue levels of antioxidant enzymes and total thiol were assayed by spectrophotometric methods. GSTM1 genotype was analyzed using polymerase chain reaction. Results:Antioxidant enzymes were significantly higher whereas glutathione peroxidase and thiol levels were lower in patients as compared with habitual controls. Habitual controls with higher tobacco exposure and lower antioxidant enzymes as well as thiol showed higher risk of oral cancer development. Antioxidant enzymes were higher, whereas catalase and thiol levels were lower in malignant as compared with adjacent normal tissues. Sixty-three percent of the patients showed GSTM1 null genotype. Conclusion:The study showed risk of oral cancer development in habitual controls with lower antioxidant enzymes, lower oxidative stress markers, and higher lifetime tobacco exposure. Individuals with GSTM1 null genotype may be at higher risk of oral cancer development.

Matrix Metalloproteinases and Their Inhibitors: Correlation with Invasion and Metastasis in Oral Cancer

Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of various malignancies. The study evaluated a comprehensive profile of MMP-2 and MMP-9 and their inhibitors, tissue inhibitor of metalloproteinases-2 (TIMP-2) and tissue inhibitor of metalloproteinases-1 (TIMP-1), respectively in 50 controls and 75 patients with oral squamous cell carcinoma (OSCC). Blood samples from controls and patients as well as malignant and adjacent normal tissues from the patients were collected. The study examined pro, active and total forms of MMP-2 and MMP-9 using zymography. Enzyme-linked immunoassay (ELISA) and reverse transcription polymerase chain reaction were carried out to evaluate protein levels and mRNA expression; respectively, for the MMPs and TIMPs. Plasma pro, active and total MMP-2, MMP-9 as well as TIMP-1 and TIMP-2 levels were significantly higher in oral cancer patients as compared to the controls. mRNA expression of the MMPs and TIMPs was significantly higher in malignant tissues as compared to adjacent normal tissues. A significant positive correlation was observed between levels of proMMP-9 and active MMP-9 with differentiation, stage and infiltration. ProMMP-2 and active MMP-2 exhibited significant positive correlation with differentiation and lymph node involvement. The multivariate analysis of ELISA results revealed a significant positive correlation between MMP-2, TIMP-1 and TIMP-2 levels with lymph node involvement, stage and differentiation. The receiver operating characteristic curve (ROC) analysis showed that the levels of MMPs and TIMPs have significant discriminatory efficacy to differentiate between controls and patients. The results indicate that MMP-2, MMP-9, TIMP-1 and TIMP-2 have significant clinical usefulness for oral cancer patients. Zymographic analysis is a simple, cost effective, rapid and sensitive alternative assay.