A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva

Head and neck cancers, including oral squamous cell carcinoma (OSCC), are the sixth most common malignancy in the world and are characterized by poor prognosis and a low survival rate. Saliva is oral fluid with intimate contact with OSCC. Besides non‐invasive, simple, and rapid to collect, saliva is a potential source of biomarkers. In this study, we build an SRM assay that targets fourteen OSCC candidate biomarker proteins, which were evaluated in a set of clinically‐derived saliva samples. Using Skyline software package, we demonstrated a statistically significant higher abundance of the C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1 candidate biomarkers in the saliva of OSCC patients. Furthermore, our study also demonstrated that CFB, C3, C4B, SERPINA1 and LRG1 are associated with the risk of developing OSCC. Overall, this study successfully used targeted proteomics to measure in saliva a panel of biomarker candidates for OSCC.     

Development of selected reaction monitoring‐MS methodology to measure peptide biomarkers in prostate cancer

Prostate cancer is a leading cause of cancer‐related death. The current modality of diagnosis, the measurement of serum PSA, not only suffers from lack of specificity, but does not distinguish clinical cases in which current treatment measures would be most successful, i.e. aggressive, life‐threatening tumors. A multiplexed MS methodology, selected reaction monitoring‐MS/MS coupled with stable isotope dilution (SID), was developed and tested in both cells lines and clinical tissue samples. Standard curves were generated for two peptides representing PSA and one peptide from each of two additional orthogonally validated biomarkers, AMACR and EZH2. The standard curves show high reproducibility, sensitivity, and good linearity. All four peptides were then measured in six clinically relevant cell lines and are in agreement with the biochemical characteristics of each individual cell line. The SID selected reaction monitoring‐MS/MS methodology was then transferred to tissue samples, in which the assay shows potential to differentiate benign disease from localized cancer and localized cancer from aggressive metastatic disease. These results establish the preliminary development of a rational targeted MS platform that strives to bridge the gap between discovery and validation of biomarkers for the detection of prostate cancer.     

Identification of novel candidate circulating biomarkers for malignant soft tissue sarcomas: Correlation with metastatic progression

Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors for which identification and validation of biological markers may improve clinical management. The fraction of low‐molecular‐weight (LMW) circulating proteins and fragments of proteins is a rich source of new potential biomarkers. To identify circulating biomarkers useful for STS early diagnosis and prognosis, we analyzed 53 high‐grade STS sera using hydrogel core‐shell nanoparticles that selectively entrap LMW proteins by size exclusion and affinity chromatography, protect them from degradation and amplify their concentration for mass spectrometry detection. Twenty‐two analytes mostly involved in inflammatory and immunological response, showed a progressive increase from benign to malignant STS with a relative difference in abundance, more than 50% when compared to healthy control. 16 of these were higher in metastatic compared to non‐metastatic tumors. Cox‘s regression analysis revealed a statistical significant association between the abundance of lactotransferrin (LTF) and complement factor H‐related 5 (CFHR5) and risk of metastasis. In particular, CFHR5 was associated with the risk of metastasis. The role of circulating proteins involved in metastatic progression will be crucial for a better understanding of STS biology and patient management.     

LC‐MS/MS‐based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label‐free proteomic analysis by LC‐MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC‐associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 ( http://proteomecentral.proteomexchange.org/dataset/PXD001171 ).     

Genetic variants associated with the progression of hepatocellular carcinoma in hepatitis C Egyptian patients

Background

Hepatocellular carcinoma (HCC) associated to infection with hepatitis C virus (HCV) has become the fastest-rising cause of cancer-related deaths. Genetic variations may play an important role in the development of HCC in HCV patients. Ghrelin exerts anti-inflammatory, antifibrotic and hepatoprotective effects on chronically injured hepatic tissues. Ghrelin gene shows several single nucleotide polymorphisms (SNPs) including − 604G/A, Arg51Gln, and Leu72Met. Hemochromatosis gene (HFE) mutations namely C282Y and H63D may cause hepatic iron overload, thus increasing the risk of HCC in HCV patients.

Aim

To investigate the association of progression of HCC with ghrelin and HFE gene polymorphisms in HCV Egyptian patients.

Methods

Seventy-nine chronic HCV patients (thirty-nine developed HCC and forty did not), and forty healthy control subjects were included in the study. The polymorphisms were evaluated by PCR/RFLP analysis, and related protein levels were measured by either ELISA or colorimetric assays.

Results

The three tested SNPs on ghrelin gene were detected in the studied groups, only one SNP (Arg51Gln) showed significantly higher GA, AA genotypes and A allele frequencies in hepatitis C patients who developed HCC than in hepatitis C patients without HCC and controls. Of the two mutations studied on HFE gene only H63D heterozygous allele was detected, and its frequency did not statistically differ among studied groups.

Conclusion

Our results suggest that A allele at position 346 of the ghrelin gene is associated with susceptibility to HCC in hepatitis C patients.