Potential role of Lipocalin 2 as a biomarker of radio resistance in head and neck squamous cell carcinoma (HNSCC)

Background: HNSCC has significant burden with high morbidity and mortality. Majority of the HNSCC patients receive Radiotherapy (RT), which is associated with development of radiation resistance and disease-recurrence, which needs in-depth evaluation. Lipocalin2(LCN2), found to be dysregulated in the saliva of HNSCC patients in our previous study using LC/MS shotgun proteomics, is also found associated with radio resistance (RR). To establish the potential role as a predictor of RR, we evaluated LCN2 in HNSCC patients. Methodology: 356 biopsy-proven treatment-naïve HNSCC, 26 Oral Pre-Malignant Diseases (OPMDs) and 118 healthy subjects were recruited for the study. The patients were treated with curative intent with 66Gy of RT. 5ml of unstimulated saliva and 3ml of whole blood were collected at baseline and during follow-ups in No evidence of disease (NED) and Residual Disease (RD)patients post therapy completion. To evaluate the role LCN2 as a marker of RR, radioresistant cells were generated by repeated sub-lethal exposure of Cal27 cells to gamma-rays from radioactive Co 60 . The RR phenotype was confirmed by phosphorylated H2AX(gammaH2AX) expression using Western Blot (WB) and Immunofluorescence (IF). Various RR assays such as clonogenic survival assay, apoptosis assay and cell cycle analysis, were performed on the successfully generated RR cells, which were later analysed for the expression of LCN2. Results: Significantly higher( p≤0.0001 ) levels of LCN2 in saliva and serum (median- 609.15ng/ml & 186.18ng/ml) of patients compared to OPMD (median-358.47ng/ml & 124.05ng/ml) and healthy controls (median-95.70ng/ml & 85.53ng/ml) respectively. The increased level of these proteins also correlated with patients' response post therapy completion with significantly higher( p≤0.0001 ) LCN2 in RD (median-1919.61ng/ml & 133.48ng/ml) group compared to NED (median-537.27ng/ml & 90.68ng/ml), indicating that the increased LCN2 level estimated after therapy completion is associated with therapy resistance. Poorly differentiated patients had significantly higher( p≤0.0001 ) LCN2(H-score148.12) as compared to moderately(H-score34.04) and well differentiated(H-score17.12) on IHC analysis. In the generated RR cell line, on IF analysis gammaH2AX foci was significantly higher validated by western blot. Further using clonogenic survival assay, apoptosis and cell cycle analysis assay it was henceforth proven that the cell lines generated had RR characteristics. The proven RR cell lines were further checked for LCN2 expression, which had significantly higher (3.6 fold) expression as compared to wild type cell lines. The results were validated with western blot. Conclusion: LCN2 is proposed as a predictive biomarker to prognosticate resistance to radiation in patients with HNSCC.