This project will investigate the oncogenic and metastatic potential of Laryngeal Squamous Cell Carcinoma (LSCC) through the analysis of p75 Neurotrophin Receptor (p75NTR) expression in Cancer Stem Cells (CSCs) and Circulating Tumour Cells (CTCs). Preliminary evidence of CSCs in LSCC has been demonstrated, while the presence and significance of CTCs in this type of cancer is still uncertain. We have recently isolated CTCs from blood of LSCC patients, and shown that they correlate well with patient post-operative outcome and disease-free survival. However, given CTCs heterogeneity in surface adhesion molecules expression, the standard CTCs isolation suffers from false negatives, with serious limitations to their study in LSCC. The p75NTR is expressed in mitotically quiescent CSCs population of the laryngeal epithelia, and also in CTCs in other cancers, thus we hypothesise that p75NTR might represent a further and efficient marker for CSCs/CTCs in LSCC. More, since p75NTR is a reliable index of tumorigenicity, invasiveness and chemotherapy resistance, it is likely that p75NTR expression correlates with the acquisition and maintenance of CSCs properties in LSCC. To test this hypothesis, the expression of full length and cleaved forms of p75NTR associated with stemness, survival, cell migration, and chemoresistance markers will be analysed in laryngeal primary tumour and locoregional lymph nodes, and in preoperative and post-operative blood-derived CTCs from LSCC patients. The molecular data will be correlated with clinico-pathological parameters, such as tumour aggressiveness and disease-free survival. This experimental approach is believed to make a better pre-operative patient stratification through prediction of tumour aggressiveness, achieve a timely diagnosis prior to metastasis through liquid biopsy, and improve post-treatment surveillance. The data obtained might also help to identify and target the p75 pathway as a promising novel approach in LSCC therapy.
The investigation of the mechanism of origin, invasion, and metastasis of the cancer is one of the emergent and most promising scientific fields in squamous cell carcinoma, especially in Laryngeal Squamous Cell Carcinoma (LSCC). In line with this, the identification of molecular pathways regulating Cancer Stem cells (CSCs) survival and/or extravasation may help the design of therapies aimed at depotentiate tumorigenicity and metastaticity. Of note, recent findings reported the expression of the same surface markers, like CD133, in CSCs within the solid tumour and in Circulating Tumor cells (CTCs) migrating from the primary tumour mass toward the general circulation, thus pinpointing the stemness nature of CTCs [Wu et al., 2009; Ricci-Vitiani et al., 2007; Brescia et al., 2012; Hsu et al., 2011; Fan et al., 2011]. Moreover, these studies opened the way to patient stratification based on CTCs-related tumorigenicity, recurrence or metastasis, and extend the research of minimal residual disease from routinely treatment of haematological malignancies to the management of squamous cell carcinomas, and in particular LSCC. The combination of conventional therapies (e.g. surgery, chemotherapy, radiotherapy) and novel CTCs-based approaches may hopefully positively impact on the long-term outcome of LSCC patients.
The Principal Investigator of this project and his collaborators have large surgical, clinical and research experience in head and neck oncology and in particular in LSCC. They have previously investigated the presence of CTCs in LSCC and found that CTCs can be isolated from blood of LSCC patients, and that their number correlates well with patients' post-operative outcome and/or disease-free survival. However, given CTCs heterogeneity in surface adhesion molecules expression, the standard isolation of CTCs by EpCAM, cytokeratine, and beta-catenin suffers from false negatives, with serious limitations to the study of CTCs in LSCC. Conversely, a novel isolation method based on the expression of the p75 Neurotrophin Receptor (p75NTR), a malignant marker in LSCC that is overexpressed by CTCs, may possibly overcome these technical limitations and allow efficient enrichment of CTCs from blood of LSCC patients.
Also, the research team led by Prof. Greco hypothesizes that the cleavage of p75NTR might differ in healthy versus tumour cells so that the expression levels of p75NTR and its cleaved products (p75CTF and p75ICD) may be a good correlate of tumour growth and metastatic potential in LSCC. The working plan is to investigate the expression and metabolism of p75NTR in solid tumour and blood of patient with LSCC before surgery and at different postoperative time points in correlation with stemness, survival, migration, and chemoresistence markers. Immunohistochemical and confocal analysis will be applied to analyse the distribution of p75NTR positive cells in the tumour mass to identify CSCs and subpopulations of cells potentially contributing to the environmental changes associated with the Epithelial-Mesenchymal Transformation process and cell extravasation. Also, the expression of p75NTR will be correlated with the expression of HMGB1 to further confirm clinical significance of aberrant p75NTR expression in LSCC patients at different disease stages.
The innovation of this research project is to address whether p75 can be considered an independent prognostic factor in LSCC, with the aim to make a better pre-operative patient stratification, achieve a timely diagnosis prior to metastasis through liquid biopsy, and improve post-treatment surveillance. Furthermore, the better understanding of the role of p75 pathway in LSCC may represent a promising novel approach in future LSCC therapy.