Development of mass spectrometry-based assays to measure the ERCC1/XPF proteins as potential biomarkers in lung cancer. (Doctoral thesis)
- Proteomics of Cellular Signaling
In the last two decades, the integration of personalized medicine approaches in the management of oncology patients has led to important progress. Indeed, the use of predictive biomarkers in clinical practice to improve treatment strategies has represented an important achievement for both patient clinical outcomes and quality of medical care.
In the context of lung cancer, several studies have highlighted a potential role of the excision repair cross-complementation group 1 (ERCC1) protein as a predictive biomarker of platinum-based chemotherapy efficacy in the treatment of non-small cell lung cancer (NSCLC) patients. ERCC1 protein and its binding partner, the DNA repair endonuclease XPF, are key players in the nucleotide excision repair (NER) pathway where they repair DNA lesions caused by platinum-based drugs. However, the presence of four ERCC1 isoforms, of which only one, ERCC1-202, is functional in the NER pathway, complicates the clinical scenario. Indeed, the lack of an antibody that specifically recognizes ERCC1-202 hampers the development of a valid clinical assay to assist clinicians in therapeutic decision making. Moreover, the essential nature of the ERCC1/XPF interaction to repair DNA lesions, suggested the pivotal role of the ERCC1/XPF complex in the prediction of therapy response. Because of the need to discriminate among the four ERCC1 isoforms, to selectively quantify ERCC1-202 in an antibody-independent fashion, and because of the required contribution of the XPF protein in the DNA lesion processing, the present project aimed to develop robust mass spectrometry (MS)-based assays for the ERCC1 and XPF isoforms to provide accurate quantification of the ERCC1-202/XPF proteins as binding partners. Combining ERCC1 or XPF immunoenrichment from biological samples with targeted mass spectrometry, the selected ERCC1 and XPF proteotypic peptides (PTPs) were measured by parallel reaction monitoring (PRM) using a quadrupole-obitrap mass spectrometer. The quantification of the ERCC1-202/XPF proteins was performed using stable isotope labeled (SIL) peptides. The associated results showed that the simultaneous detection of three ERCC1 PTPs, named isoform discriminating peptides, represents a signature of ERCC1-202 and that the inclusion of control peptides allows to avoide ERCC1 isoform misclassification. The immuno-affinity studies and the evaluation of XPF stability highlighted that ERCC1-202 interacts and stabilizes XPF. Finally, a correlation between ERCC1-202/XPF protein levels was observed.
In the light of the poor analytic specificity of the current immunohistochemistry (IHC) assay for the ERCC1 protein, the use of targeted MS-based assays to detect and quantify both ERCC1-202 and XPF proteins represents a more selective approach. These findings strongly suggest to assess the role of the ERCC1-202/XPF proteins as predictive biomarkers in clinical samples with the final goal to guide the clinicians’ therapeutic decisions.