CTC together with Shh and Nrf2 are prospective diagnostic markers for HNSCC.

BACKGROUND: The lack of appropriate prognostic biomarkers remains a significant obstacle in the early detection of Head and Neck Squamous Cell Carcinoma (HNSCC), a cancer type with a high mortality rate. Despite considerable advancements in treatment, the success in diagnosing HNSCC at an early stage still needs to be improved. Nuclear factor erythroid 2-related factor 2 (Nrf2) and Sonic Hedgehog (Shh) are overexpressed in various cancers, including HNSCC, and have recently been proposed as possible therapeutic targets for HNSCC. Circulating Tumor Cell (CTC) is a novel concept used for the early detection of cancers, and studies have suggested that a higher CTC count is associated with the aggressiveness of HNSCC and poor survival rates. Therefore, we aimed to establish molecular markers for the early diagnosis of HNSCC considering Shh/Nrf2 overexpression in the background. In addition, the relation between Shh/Nrf2 and CTCs is still unexplored in HNSCC patients. METHODS: In the present study, we selected a cohort of 151 HNSCC patients and categorized them as CTC positive or negative based on the presence or absence of CTCs in their peripheral blood. Data on demographic and clinicopathological features with the survival of the patients were analyzed to select the patient cohort to study Shh/Nrf2 expression. Shh and Nrf2 expression was measured by qRT-PCR. RESULTS: Considering significant demographic [smoking, betel leaf (p-value < 0.0001)] and clinicopathological risk factors [RBC count (p < 0.05), Platelet count (p < 0.05), Neutrophil count (p < 0.005), MCV (p < 0.0001), NLR (p < 0.05), MLR (p < 0.05)], patients who tested positive for CTC also exhibited significant overexpression of Shh/Nrf2 in both blood and tissue compared to CTC-negative patients. A strong association exists between CTCs and tumor grade. Following chemotherapy (a combination of Cisplatin, 5FU, and Paclitaxel), the frequency of CTCs was significantly decreased in patients with HNSCC who had tested positive for CTCs. The Kaplan-Meier plot illustrated that a higher number of CTCs is associated with poorer overall survival (OS) in patients with HNSCC. CONCLUSIONS: Detecting CTCs, and higher expression of Shh and Nrf2 in HNSCC patients' blood, can be a promising tool for diagnosing and prognosticating HNSCC.

Role of circulating tumour cells (CTCs) in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC).

Background: Liquid biopsy is emerging as a non-invasive tool, providing a personalized snapshot of a primary and metastatic tumour. It aids in detecting early metastasis, recurrence or resistance to the disease. We aimed to assess the role of circulating tumour cells (CTCs) as a predictive biomarker in recurrent/metastatic head and neck cancer (head and neck squamous cell carcinoma (HNSCC)). Methodology: Thirty-five patients receiving palliative chemotherapy underwent blood sampling [2 mL in Ethylenediaminetetraacetic acid (EDTA) vial] at baseline and at 3 months intervals. The CTCs were isolated and evaluated using anti-epithelial cell adhesion molecule antibody-based enrichment using the OncoDiscover platform. Results: CTCs isolated from 80% of patients (n = 28) showed the sensitivity of cell detection at the baseline and 3 months intervals. The median CTC count was 1/1.5 mL of blood and the concordance with clinic-radiological outcomes was 51.4%. The median CTC count (1 (range:0-4) to 0 (range:0-1)) declined at 3 months in responders, while the non-responders had an increase in levels (0 (range :0-2) to 1 (range :0-3)). Although CTCs positively correlated with progression-free survival (PFS) and overall survival (OS), the association of CTCs did not show a significant difference with these parameters (PFS: 6 months versus 4 months; hazard ratio: 0.68; 95% confidence interval (CI): 0.29-1.58, p = 0.323; OS: 10 months versus 8 months; hazard ratio: 0.54; 95% (CI):0.18-1.57 p = 0.216) between CTC positive and CTC negative patients at 3 months. Conclusion: This study highlights the utility of CTC as a disease progression-monitoring tool in recurrent HNSCC patients. Our findings suggest the potential clinical utility of CTC and the need for exploration in upfront settings of the disease as well (NCT: CTRL/2020/02/023378).

Circulating tumor cells as a predictor for poor prognostic factors and overall survival in treatment naïve oral squamous cell carcinoma patients.

OBJECTIVE: The aim of this study was to investigate the presence of circulating tumor cells (CTCs) and their correlation with prognostic factors and clinical outcomes in treatment-naive patients with oral squamous cell carcinoma. STUDY DESIGN: CTCs were isolated using OncoDiscover technique from presurgically obtained peripheral blood of 152 patients with treatment naïve oral squamous cell carcinoma. Sensitivity analysis was performed by including 40 healthy controls. CTCs cutoff values for clinicopathologic factors were obtained from receiver operating characteristic curves. Multivariate models determined the significance of CTC as independent variables. Kaplan-Meier analysis differentiated in overall survival between CTC values corresponding to the stage. RESULTS: Sensitivity, specificity, and accuracy of CTC detection were 94.32%, 98%, and 95.17%, respectively. Platform differentiated true positives at >3.5 CTCs (P < .00001). CTCs above 20.5 were suggestive of nodal metastasis (P < .0001) with a linear trend for detecting occult metastasis (P = .061). Early and advanced stages could be differentiated by >13.5 CTCs (P < .0001). Elevated CTCs were significantly associated with extranodal extension (>21.45 CTCs, P = .025), perineural invasion (>19.35 CTCs, P = .049), and depth of invasion (>12.5 CTCs, P = .0038). Median survival was reduced by 19 months when CTCs were >13. CONCLUSIONS: Preoperative CTC levels demonstrated a strong correlation with adverse clinicopathology factors and suggested its role as a sensitive prognostic marker to predict survival outcome and disease progress.

Targeted sialic acid-doxorubicin prodrugs for intracellular delivery and cancer treatment.

PURPOSE: To evaluate a novel targeted anticancer prodrug consisting of several copies of sialic acid (SA, targeting moiety), doxorubicin (DOX), citric acid (multifunctional spacer) and poly(ethylene glycol) (PEG, carrier). METHODS: alpha, omega bis carboxyl PEG was covalently conjugated with multiple copies of SA and DOX through a citric acid spacer and characterized by proton nuclear magnetic resonance ((1)HNMR), matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF), and high-performance liquid chromatography (HPLC). The molecular models of conjugates were established using ChemDraw software. Stability, spontaneous and esterase-stimulated drug release was analyzed by HPLC. Cellular internalization (fluorescence microscopy) and cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay] of free DOX and prodrugs were evaluated. RESULTS: (1)HNMR, MALDI/TOF, and HPLC showed the formation of the PEG prodrug conjugates. More than 40% of the drug was released from its conjugate in the presence of esterase enzyme, whereas the conjugate was stable at pH 7.4 in the absence of enzyme. Molecular modeling studies showed stable conformations of conjugates. The targeted prodrug conjugates with two copies of SA and DOX showed enhanced cytotoxicity when compared with non-targeted prodrugs and free DOX. CONCLUSIONS: Targeting of the conjugate to cancer cells by SA with increased copies of targeting moiety and anticancer drug enhanced prodrug uptake by cancer cells and cytotoxicity of the prodrug.

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel.

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.