Detection of Circulating Tumor Cells in Oral Squamous cell Carcinoma Patients Using Cytokeratin-19 Real-Time PCR

Abstract

Background: Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the oral cavity, accounting for more than 90% of all oral malignancies. Detection of disseminated circulating tumor cells (CTC) in the peripheral blood aids in early diagnosis and better prognosis. Hence, this Prospective study was conducted to evaluate the expression of CK 19 in the blood of patients with Oral Squamous Cell Carcinoma before biopsy, before surgery and 3 months after their surgical management and also co-relate the quantitative expression of CK19 with 6 months post-operative status in patients treated for OSCC.

Materials & methods: A total of 80 patients were screened by a thorough case history and cinical examination. To evaluate the presence of circulating tumour cells, blood samples were collected  from the patients before biopsy, before surgery and 3 months after surgery. Enrichment of Circulating Tumor Cells (CTCs): CTCs present in the peripheral blood samples were enriched with Lymphoprep (Cat#07801, Stemcell technologies, Canada). Melt curve or High Resolution Melt Curve analysis were performed after amplification of the samples to confirm the specificity of amplification of each of mRNA. Briefly, samples were subjected to a ramp step that ranged between 60°C to 95°C with an initial hold for 90 seconds followed by a rise of: a) 1°C at each step with a 5 second hold to enable the melting process or b) 0.1°C at each step with a 5 second hold to enable high resolution melt (HRM) curve analysis.

Results:  Recorded results unravel the expression pattern of Ck19 by using 2 different primer set. Color code of samples, cycle threshold value (ct) and calculated concentration (copies) indicates the expression frequency of cytokeratin 19. The Primer 1 & 2 were represented as “a, b, c” and “A,B,C” respectively in which aA , bB, cC refers to before biopsy, before surgery and 3 months after surgery respectively. Expression of CK 19 of all samples has been depicted graphically. The cytokeratin 19 expression before biopsy (aA), before surgery (bB) and 3 months after surgery(cC) have been compared  in which samples 5 and 9 ,showed a massive increase in rate of expression of cytokeratin 19 , three months after surgery when compared with before biopsy and surgery in both primer sets.

Conclusion: The expression of CK-19 in CTCs has a certainty towards detection of metastasis and loco regional recurrence in patients with OSCC.

Keywords: Oral squamous cell carcinoma, Circulating, Tumour cells