Nowadays, mass-spectrometry-based methods are continuing to make major contributions to the discovery of fundamental biological processes due to requirements of high sensitivity, selectivity and capability to identify and measure hundreds of compounds simultaneously. Nevertheless, problems related to small amount/volume of biological samples as well as interfering compounds co-extracted with target analytes make it difficult to sort out controversial matter whose resolution is crucial for understanding specific questions. Some of the technical approaches that were at an experimental stage back then are considered the golden standard today, but many questions have still remained unsolved due to the need of further analytical advancements. Ion mobility mass spectrometry is an appealing solution which offers exclusive advantages such as an extra separation dimension, indispensable for the distinction of isomers and isobars compounds, and chemical noise reduction, essential to improve sensitivity. On a hybrid quadrupole linear ion trap mass spectrometer ¿ the top of technology regarding reliability of qualitative screening and quantitative analysis, Sciex has developed the SelexION Technology, consisting in a compact device integrated in the ion source region, named ¿differential mobility separation¿ (DMS) cell. This instrumental platform permitting an important advancement of the analytical potentials in the life sciences sector allowing to open new ways to more and more complex analysis. The main research program concerns the polifunctional application of the required instrumentation to the study of ultra micro concentration of specific markers in the fields of neonatology, oncology and ophthalmology.
Clinicians currently have access to a limited number of molecules for early diagnosis or intervention in preterm neonates with free radical diseases. In consequence, there is a critical need to identify other reliable biomarkers among prostanoids and furanoids in biological samples that can be obtained from preterm new-borns in a non-invasive way.
Although several national and international research groups have actively been engaged in the discovery and quantification of such biomarkers, critical and compulsory endeavours remains:
(i) Selection of biological matrices alternative to plasma for non-invasiveness of the sampling method and availability from preterm neonates.
ii) Screening of BOSs and/or their metabolites in meconium/faeces and saliva, matrices that are suitable in clinical studies involving infants and that have not been considered till the moment.
(iii) Development of a multi-analyte method including a panel of BOSs useful to monitor type and severity of the oxidative damage.
All these points will be considered and faced in this research project, based on the collaboration with the Dpt.s of Pediatria e Neuropsichiatria Infantile. Recently, hematologists and neonatologists demonstrated that allogeneic Umbilical Cord Blood Red Cell Concentrates (CB RBC) is an innovative blood product which might be safer than adult blood for the transfusion therapy of preterm infants (Bianchi M et al. Neonatology 107 (2015) 81). The analytical methodologies and results obtained from this research project will be fundamental to investigate if this transfusion practice might also favorably impact on oxidative injury and O2 radical disease morbidity.
The future scientific impact of this project is very high and able to expand current knowledge on mechanisms responsible for the detrimental effect of blood product transfusion; in particular, the oxidative stress caused by adult blood transfusions in preterm neonates has been scarcely investigated. Regarding the immediate technological impact, the development of simple, fast, reproducible, and standardized multi-analyte methods to investigate BOSs in biological samples alternative to blood could be applicable to the routine monitoring of these patients and allow an earlier identification of the oxidative damage. Finally, the project might have also an important socio-economic impact. Complications of preterm birth are the single largest direct cause of neonatal deaths, responsible for 35% of the world¿s 3.1 million deaths a year. The effect of pre-term birth amongst survivors may continue throughout life, impairing neurodevelopmental functioning, increasing the risk of learning impairment and visual disorders and affecting long-term physical health with a higher risk of non-communicable disease. These effects exert a heavy burden on families, society and the health system.
The combination of therapies aimed at debulking the tumour and novel stem cells-based approaches might improve long-term outcome of LSCC patients.
Our working hypothesis is to investigate the expression and metabolism of p75NTR fragments in solid tumour and blood of patient with LSCC before surgery and at different postoperative time points, in association with stemness, survival, migration, and chemoresistence markers. In preliminary experiments, we were able to isolate CTCs from LSCC patients, and correlate them with the post-surgery follow-up parameters.
The hypothesis of p75NTR cleavage in the regulation of CSC survival and behaviour in LSCC open a new perspective in the management of disease allowing liquid biopsy based prediction of tumour progression, and amelioration of patient life quality also in advantage tumour stage.
Overall, we hypothesize that the p75 pathway must be a finely regulated process in normal and malignant laryngeal epithelia and that targeting of p75 pathway by pharmacological inhibition or specific enzymes is a promising novel approach in squamous carcinoma therapy.
The quantitative proteomic profiling of p75 fragments in rare cells, like CTCs, and in limited volumes of physiological fluids by the automated TripleQuad/SelexION integrated platform (SCIEX) will allow the analysis of hundreds samples with higher sensitivity and specificity, as compared to traditional technology.