Kinjal R. Patel

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.

Sialylation: an Avenue to Target Cancer Cells

Tumorigenesis and metastasis are frequently associated with altered structure and expression of oligosaccharides on cell surface glycoproteins and glycolipids. The expression of sialylated glycoconjugates has been shown to change during development, differentiation, disease and oncogenic transformation. Abnormal sialylation in cancer cell is a distinctive feature associated with malignant properties including invasiveness and metastatic potential. The alterations in sialylation is accompanied by changes in sialic acid, sialidase activity, sialyltransferase (ST) activity or sialoproteins. The present review summarizes the reports on alterations of sialic acid, linkage specific STs and sialoproteins, sialidase activity together with different subtypes of ST and sialidases mRNA expressions in various cancers like lung, breast, oral, cervical, ovarian, pancreatic etc. Sialic acids are widely distributed in nature as terminal sugars of oligosaccharides attached to proteins or lipids. The increase shedding of sialic acid observed in malignant tumors may be due to different types of sialidases. The amount of sialic acid is governed by levels of sialidases and STs. Various types of STs are also involved in formation of different types sialylated tumor associated carbohydrate antigens which plays important role in metastasis. The alterations associated with sialylation aids in early diagnosis, prognosis and post treatment monitoring in various cancers. Recently newer drugs targeting different interplays of sialylation have been developed, which might have profound effect in inhibiting sialylation and thus cancer metastasis and infiltration.

Evaluation of serum and salivary total sialic acid and α-l-fucosidase in patients with oral precancerous conditions and oral cancer

OBJECTIVES We compared serum and salivary total sialic acid/total protein (TSA/TP) ratios and α-l-fucosidase activity in patients with oral precancerous conditions (OPCs) and oral cancer to better understand the utility of saliva, in monitoring early changes occurring during oral cancer progression. STUDY DESIGN A cross-sectional study of 100 oral cancer patients, 50 patients with OPC, and 100 controls was performed. RESULTS Serum and salivary TSA/TP ratios and α-l-fucosidase activity were significantly higher in OPC and oral cancer patients compared to the controls. Also, levels were higher in controls and oral cancer patients with tobacco habits as compared to those without tobacco habits. CONCLUSION Salivary TSA/TP ratio and α-l-fucosidase activity were elevated with higher magnitude than serum levels. These results suggest that a larger study may prove the use of these saliva biomarkers as a noninvasive method for detecting early changes occurring during oral carcinogenesis.

Association between p53 Gene Variants and Oral Cancer Susceptibility in Population from Gujarat, West India

BACKGROUND p53 gene variants i.e. 16 bp duplication in intron 3, Arg72Pro in exon 4 and G>A in intron 6 have been reported to modulate susceptibility to various malignancies. Therefore, the present study evaluated the role of these p53 polymorphisms in oral cancer susceptibility in a population from Gujarat, West India. METHOD Genotype frequencies at the three p53 loci in 110 controls and 79 oral cancer cases were determined by the PCR-RFLP method. RESULTS Heterozygous individuals at exon 4 showed protection from developing oral cancer. Homozygous wild and heterozygous individuals at intron 3 and those heterozygous at exon 4 in combination appeared to be at lowered risk. Furthermore, carriers of the 16 bp duplication allele at intron 3, proline allele at exon 4 and G allele at intron 6 were protected from oral cancer development. CONCLUSION p53 polymorphisms, especially Arg72Pro in exon 4 could significantly modify the risk of oral cancer development in Gujarat, West Indian population.

Prevalence of high-risk human papillomavirus type 16 and 18 in oral and cervical cancers in population from Gujarat, West India.

BACKGROUND Oral and cervical cancers are major malignancies in men and women, respectively, in India. This study evaluated occurrence of human papillomavirus (HPV) 16 and 18 infections in oral and cervical cancers to estimate HPV-associated burden of these cancers in the population from Gujarat, West India. METHODS A total of 97 malignant oral carcinoma tissues and 52 cervical carcinoma tissues were analyzed by type-specific PCR for the presence of HPV type 16 and 18 infections. RESULTS None of the oral cancer patients revealed the presence of HPV type 16 and 18 infection. In cervical cancer, 31 (59.6%) patients were infected with HPV 16 and 18. Of these 31 HPV-positive cervical cancer patients, 28 (90.3%) were infected with HPV 16 and 3 (9.7%) were infected with HPV 18. CONCLUSION The results suggested that HPV 16 and 18 do not play an important role in oral carcinogenesis in the population from Gujarat, West India. However, HPV 16 is highly prevalent in the cervical cancer patients, which may be considered for planning of prevention programs such as screening and vaccination in women from this region.

VEGFA isoforms play a vital role in oral cancer progression

Angiogenesis plays an important role in tumor growth and prognostication. A key angiogenesis stimulator is vascular endothelial growth factor (VEGF). The present investigation aimed to study contribution of VEGFA isoforms in oral cancer progression. Reverse transcription polymerase chain reaction and ELISA were employed to analyze tissue VEGFA isoforms and serum VEGF levels, respectively, in 109 oral cancer cases and 50 controls. VEGF183 and VEGF165 were significantly downregulated in malignant tissues as compared to adjacent normal tissues. VEGF183 and VEGF189 were significantly associated with tumor differentiation and tumor size. VEGF165 was significantly higher in recurrent early stage tumors. Serum VEGF levels were significantly higher in cases as compared to the controls and were associated with tumor differentiation. Serum VEGF levels were significantly higher in patients with recurrent advanced stage tumors. Further, patients with high levels of VEGF165 and serum VEGF levels had the worst prognosis. VEGFA isoform status and serum VEGF levels play a significant role in the progression as well as prognosis of oral cancer.

“p53 mutation spectrum and its role in prognosis of oral cancer patients: A study from Gujarat, West India”

BACKGROUND AND AIM p53 mutations are critical players in etiopathogenesis of oral cancer. Interestingly, they show differences in terms of type and codon specificity. These differences might be attributed to geographical variations in tobacco use. We aimed to analyze the frequency of p53 mutations in oral cancer patients from Gujarat, India and their effect on clinico-pathological features, local recurrence and survival. MATERIAL AND METHODS p53 mutation analysis was performed on 46 paired tissue samples (adjacent normal and primary malignant) using PCR-SSCP and sequencing. RESULTS Sequencing confirmed 51 p53 mutations in 46 paired tissues. Three novel mutations (frameshift deletion in exon 4; G>T transversion at codon 117 in exon 4 and G>A transition at codon 319 in exon 9) were identified. Distinct pattern of p53 mutations was observed: more common C>T transitions and recurring mutation sites at codon 90 and 116 in exon 4. Interestingly, the probability of developing recurrence was higher in small tumors (<4 cm) with p53 mutations and in cases with p53 mutations in both adjacent normal and malignant tissues. A significant low disease free survival and overall survival was observed in cases harboring truncating and transcriptionally non-active mutations. CONCLUSION We report a very high frequency and a diverse pattern of p53 mutations in cases from this region. Interestingly, three distinct novel mutations in exons 4 and 9 were also observed. Analyzing p53 mutation status in tumor tissues at an early stage could serve as an important prognostic factor.

Salivary glyco-sialylation changes monitors oral carcinogenesis.

Alterations in cell membrane glycosylation play important role in oral carcinogenesis. The present study evaluated salivary sialylation changes i.e. total sialic acid (TSA), sialidase activity, linkage specific (α2-3 and α2-6) sialoproteins and sialyl transferase (ST) activity in controls, patients with oral precancerous conditions (OPC) and oral cancer. Subjects enrolled included 100 controls, 50 patients with OPC, 100 oral cancer patients, and 30 post treatment follow-ups. TSA was estimated by spectrophotometric method, sialidase activity by spectrofluorometric assay and linkage specific biotinylated lectins (α2-3: sambucus nigra agglutinin and α2-6: maackia amurensis agglutinin) were used to detect α-2,3 and α-2,6 STs and sialoproteins by ELISA and dot blot respectively. An increasing trend of salivary TSA/TP ratio, sialidase activity, α2-3 sialoproteins, α-2,3 and α-2,6 ST activities was observed from controls to patients with OPC to oral cancer patients and levels were significantly elevated in oral cancer patients as compared to the controls. Sialidase activity exhibited significant association with metastasis and infiltration. Sialidase activity, TSA/TP ratio, α-2,3 and α-2,6 ST activities were found to be higher in patients with metastasis as compared to patients without metastasis. A progressive increase in TSA/TP ratio, sialidase activity, α2-3 and α2-6 sialoproteins was observed from controls to early to advanced stage of the disease. Sialidase activity, α2-3 and α2-6 sialoproteins and ST activities were found to be decreased in complete responders; while levels were elevated in non-responders. The results documented utility of salivary sialylation endpoints, a non invasive tool in monitoring of oral carcinogenesis.

Glycoprotein electrophoretic patterns have potential to monitor changes associated with neoplastic transformation in oral cancer

Alterations in glycoproteins, important cell surface constituents, have long been associated with various malignancies. The present investigation therefore explored the clinical significance of a glycoproteomics approach in patients with oral precancerous conditions (OPC) and patients with oral cancer. The study included 80 oral cancer patients, 50 patients with OPC, and 84 controls. Native polyacrylamide gel electrophoresis followed by Schiffs staining was carried out to study the alterations in glycoproteins. The results showed significant elevation (p<0.0001) of 192 kDa, 170 kDa, 116 kDa and 44 kDa glycoproteins in oral cancer patients and patients with OPC compared with controls. The odds ratio indicated a significantly higher risk for oral cancer among users and especially chewers of tobacco. The levels of all the glycoprotein bands (192 kDa, 170 kDa, 116 kDa and 44 kDa) were higher in patients with a habit of tobacco use (WHT) than in patients with no habit of tobacco (NHT) and were also higher in WHT controls than in NHT controls. Moreover, a 230 kDa glycoprotein consistently appeared only in individuals with tobacco habits and an increasing trend was observed from WHT controls to patients with OPC to WHT oral cancer patients. In conclusion, the results indicated the potential utility of glycoprotein alterations in monitoring sequential changes occurring due to tobacco consumption during neoplastic transformation.

Significance of phosphorylated epidermal growth factor receptor, matrix metalloproteinases, and E-cadherin in oral cancer

Objective: The most challenging problem in oral cancer is late monitoring and disease spread (metastasis). The study aimed to simultaneously evaluate phosphorylated epidermal growth factor receptor (pEGFR), truncated E-cadherin protein, and matrix metalloproteinases (MMPs) in patients with oral cancer. Methodology: pEGFR and truncated E-cadherin protein were measured from 25 paired tissues by ELISA and Western blot, respectively. Plasma MMPs levels were studied by gelatin zymography from 100 controls and 100 patients with oral cancer. The results revealed significant higher expression of pEGFR and truncated E-cadherin protein in malignant oral cancer tissues as compared to adjacent normal. Plasma MMPs were significantly elevated in patients with oral cancer as compared to the controls. An increase in the levels of pEGFR and truncated E-cadherin protein was observed in advanced and metastatic disease as compared to early and nonmetastatic disease. The levels of MMPs were increased in advanced disease as compared to early disease. Kaplan–Meiers survival analysis indicated that elevated expression of pEGFR, truncated E-cadherin protein, active MMP-2, pro MMP-9, total MMP-2, and total MMP-9 has reduced the overall survival. Conclusion: Simultaneous elevation of pEGFR, truncated E-cadherin protein, and MMPs indicated its role in oral carcinogenesis. Further combination therapies targeting these markers might help in combating oral cancer.