Objectives

To establish a rapid, highly specific, efficient, sensitive, and affordable CTC enumeration liquid biopsy technology and to validate its efficacy to isolate CTCs disseminating from epithelial tumors of the HNC subpopulation in India. Furthermore, the study aimed to investigate the correlation of CTC distribution from peripheral blood with respect to various clinicopathologic factors in these patients.

Materials and Methods

A cross-sectional study was conducted using peripheral blood from 350 enrolled HNC patients. CTCs were isolated using DCGI (India) approved technology that exploits EpCAM-based immunomagnetic separation. EpCAM+ tumor cells were isolated from only 1.5 ml of blood and critically assayed for cytokeratin 18 (CK18) expression. These cells were quantified using fluorescence imaging to obtain a threshold to further minimize nonspecific and false-positive enumeration. CTC enumeration was subsequently subjected to statistical correlation with various clinical and pathologic parameters.

Results

CTCs were detected in all HNC patients across various subsites. There was a minimum threshold of at least 12 CTCs in early oral cancer patients according to their clinicopathologic signatures. Compared to early oral cancer patients, advanced nodal patients showed a 40% escalation in CTC count, while an increase of up to 80% was observed when associated with aggressive features such as lymphovascular emboli (LVE) and extranodal extensions. Notably, laryngopharyngeal primary cases had the highest mean CTC count of 33 in 1.5 ml of blood.

Conversely, patients with advanced disease had higher CTC counts, and this was staggered in comparison with nearly—but not all—clinical features. Remarkably, a higher clinical N (nodal) stage statistically correlated with increased CTC counts. A marked increase in CTCs was also seen in tumors that showed lymphovascular emboli on histopathology and extranodal extension. The CTC counts were independent of parameters such as age, sex, T stage, perineural invasion, bone involvement, or skin involvement. There was a notable trend toward reduced CTC counts after chemotherapy; however, it was not statistically significant.

Conclusion

This rapid and efficient CTC platform has been clinically validated for use in Indian HNC phenotypes. This is the first comprehensive study to show a staggering positive correlation between CTCs and various clinicopathologic factors, encompassing the largest number of oral cancer patients across the entire spectrum of HNSCC—the most common cancer in India. High CTC counts among HNC patients could possibly be one of the reasons for dismal outcomes, and further studies correlating CTCs with patient survival in HNC are warranted. However, this study strongly implicates the prospective utility of CTCs as a tumor marker in establishing clinical staging for HNC patients.