Serum albumin-bound proteomic signature for early detection and staging of hepatocarcinoma: sample variability and data classification.

BACKGROUND: Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) proteomic signature might be of interest for the early detection and staging of hepatocellular carcinoma (HCC). However, published procedures have been criticized for the lack of data about analytical reproducibility, and the use of inadequate data processing. METHODS: MALDI-TOF profiling of peptides bound to serum albumin ("albuminome") was performed using 90 µL of serum from 45 study subjects (HCV-related cirrhosis, small, unifocal HCCs and advanced HCCs). To overcome the large intra-sample variability, a Quality Assurance protocol was implemented, and 4-8 samples for each subject were processed and analyzed. Overall, 522 subject samples and 299 quality-control spectra were analyzed. A machine-learning approach (Random Forest) was applied to analyze the data sets. RESULTS: Mean intra-sample coefficient of variation (CV) of the analytical procedure was 17.6%-30.0%; inter-subject CV was in the range 48.8%-71.3% among the three study groups. The Random Forest procedure correctly classified 433/522 "patient samples" and 295/299 "reference samples"; 43/45 patients were correctly classified following this approach. CONCLUSIONS: Our data suggest that, notwithstanding the large analytical variability found, multiple proteomic profiles obtained from each subject can differentiate cirrhosis with and without HCC, and HCCs with and without vascular invasion, warranting further investigation in a prospective setting.

A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate.

The expression of the asialoglycoprotein receptor on the cells of the large majority of the well differentiated hepatocellular carcinomas can be exploited to improve the chemotherapy of these tumours by coupling anticancer agents to macromolecules taken up by the receptor. In line with this approach, in previous experiments we coupled doxorubicin (DOXO) to lactosaminated human albumin (L-HSA) using the (6-maleimidocaproyl)hydrazone derivative of the drug as an acid sensitive linker. Encouraging results were obtained in laboratory animals using L-HSA-DOXO. This conjugate, however, has the disadvantage of a difficult synthesis, which requires protein thiolation with iminothiolane and can hinder its preparation on a large scale. Here we describe a very simple method of coupling. The HS-groups required for the reaction with the maleimide moiety of DOXO-EMCH are made available in L-HSA by a cleavage of the protein disulphides achieved with tris(2-carboxyethyl) phosphine (TCEP). Contrary to thiolic reducing agents, the use of TCEP eliminates the need of an inert atmosphere and allows a one-step coupling reaction, without purification of the reduced protein before the addition of DOXO-EMCH. As the previous L-HSA-DOXO conjugate, the new conjugate accomplishes a very efficient liver targeting of the drug. This novel method of synthesis should facilitate the preparation of L-HSA-DOXO in the amounts required for clinical studies.