The high rate of locoregional recurrence and the development of resistance to treatment are major deterrents in the effective treatment of HNSCC [4, 6]. There is a need to identify and validate protein biomarkers which can aid in the early identification of patients with higher proclivity to developing recurrence and resistance. We have identified twelve proteins which show more than 1.5 upregulation in HNSCC patients who respond poorly to CTRT, indicating their potential to act a predictive biomarkers. In addition, some of these proteins also showed a significant pre-treatment upregulation of more than 1.5 fold in poor responders, thus, indicating their potential as prognostic markers. To date, multiple diagnostic protein biomarkers have been reported for HNSCC such as CYFRA 21 − 1, SCCA, CEA, but none has been successfully implemented in clinical practice [9]. The consistent and significant upregulation of clusterin and gelsolin at PreRT and within 48 hours to 1 week of starting RT indicates their ability to act as early prognostic and predictive markers, respectively. The upregulation of these proteins with RT is an early indication of local failure of treatment, thus, allowing the possibility of modifying the treatment appropriately. Additionally, the pre-treatment upregulation of these proteins offers the possibility of identifying patients with the highest chance of developing recurrence before the treatment is begun.
Clusterin (CLU) is a heterodimeric, secreted glycoprotein that is expressed in a wide variety of tissues [34]. It acts as an anti-apoptotic protein by enhancing tumorigenesis which leads to the cytosolic retention of Bax (by stabilizing the Ku70-Bax complex) and interfering with Bax’s proapoptotic activities [15, 35]. CLU expression is upregulated in multiple solid tumors, like bladder, breast, prostate, renal, and non-small-cell lung cancer (NSCLC) [10–12, 36, 37]. Previous studies in esophageal squamous cell cancer (ESCC) patients indicated that the serum levels of CLU were significantly elevated and the protein may function as a tumor marker and a prognostic factor for such patients [38]. Abnormal serum CLU expression was closely associated with TNM classification of malignant tumors and lymph node metastasis, with higher CLU expression correlating with poor survival of hepatocellular carcinoma patients [39]. The protective role of CLU was observed under CTRT-induced stress which led to the overexpression of CLU, leading to the emergence of a treatment-resistant phenotype [16]. In the present study, a 3.0 fold pre-treatment upregulation of CLU in the poor responders indicated that these patients may later develop resistance to treatment or a more aggressive tumor phenotype, thus indicating a poor prognosis even before treatment is begun. Once treatment is begun, poor responders exhibit a 1.7–3.9 fold upregulation of CLU within 48 hours, with a further upregulation of 5.6 fold within 1 week of RT, providing an early indication of possible treatment failure.
In addition to CLU, a consistent upregulation of gelsolin (GSN) was also observed in the poor responders. GSN is a cytoskeleton-associated and actin-binding protein that plays a role in regulating cell morphology, motility, and apoptosis [40–42]. Significantly enhanced GSN expression was observed in oral squamous cell carcinoma (OSCC) and was associated with advanced staging [43]. Enzyme-linked immunosorbent assays (ELISA) of plasma samples indicated the potential of circulating plasma GSN as a diagnostic biomarker for head and neck cancer [14]. Immunohistochemical staining of laryngeal squamous cell carcinoma (LSCC) tissue samples indicated the association of GSN expression with poor prognosis and its potential as a novel prognostic biomarker for disease-specific and overall survival [13]. In vitro studies in human oral carcinoma cells further elucidated the role of GSN in promoting apoptosis, cell proliferation, and enhancing tumor invasion and metastasis [44]. The ability of GSN to confer chemo- and radio-resistance has been reported in NSCLC, ovarian cancer, and HNSCC, all indicating that higher levels of GSN are associated with a higher risk of developing treatment resistance [17, 18, 45, 46]. We report here, for the first time, that the levels of GSN increase by 2.4 to 4.0 fold in the worst responding patients within 48 hours, which is maintained till 1 week of RT. This upregulation may be indicative of a local failure of treatment due to acquiring a more aggressive phenotype. A pre-treatment 2.0 fold upregulation of GSN further indicated its potential as an early prognostic and predictive marker for HNSCC.
Lumican, ECM1, cystatin C, and SPARC are involved in the organization of the extracellular matrix and an increase in their expression level has been reported to increased cell proliferation, migration, invasion, induction of EMT, and metastasis [47–50]. A 1.5-5.0 fold upregulation of these proteins was observed in the poor responders while downregulation of at least 2.0 fold was observed in the good responders. The insulin-like growth factor (IGF) system is involved in cell growth, differentiation, and transformation [51]. Higher levels of IGF1 and IGF2 contributed to increased differentiation and decreased apoptosis, while a high IGF1:IGFBP3 ratio in serum was shown to be indicative of a malignant HNSCC phenotype [52, 53]. Contrary to these reports, our patient cohort exhibited an overall 2.0 to 4.0 fold upregulation of IGF1, IGF2, and IGFBP3 with the progression of treatment, indicating their potential as predictive markers. The concomitant upregulation of IGFBP3 may be due to its involvement in IGF-independent pathways to regulate cancer progression. The pre-treatment levels of IGF1, IGF2, and IGFBP3 do not provide significant evidence to consider their potential as prognostic biomarkers. Proteins S100A7, S100A8, and S100A9 belong to the S100 family of calcium-binding proteins that regulate cellular processes such as differentiation, cell motility, migration, and maintain the tumor microenvironment [54]. A 2.0–5.0 fold upregulation in protein expression levels was observed in the poor responders within 48 hours of treatment.
The current prospective study is the first report of treatment dependent protein changes in HNSCC patients. As the study was carried out in HPV-negative patients, the pre-treatment and time dependent downregulation of CLU and GSN in the good responders indicated their potential to act as HPV-independent prognostic markers. Further validation of these proteins in a larger cohort, with healthy controls, would help to determine the sensitivity and specificity of the proteins as potential predictive and prognostic biomarkers.