Goal: Identify a serum biomarker(s) of hepatocellular carcinoma that originates from cancer tissue.
Methods: Formalin-fixed human liver tissue from patients classified as healthy, cirrhotic, or hepatocellular carcinoma (HCC) were evaluated using N-glycan MALDI imaging mass spectrometry on a Solarix dual source 7T MALDI-FTICR (Bruker-Daltonics). A coating of PNGase F was applied to the tissue using a TMSprayer (HTX Technologies LLC). Data was analyzed using FlexImaging and SCiLS Lab software (Bruker-Daltonics). In total, 188 HCC tissue samples and 145 control tissue samples were analyzed. Subsequently, serum glycoproteomics was performed using a recombinant lectin with greater affinity toward branched and fucosylated glycan. Proteins identified as containing the same glycans observed in HCC tissue were further examined in four independent sample sets consisting of 225 patients with liver cirrhosis and 326 with HCC including 240 with early stage cancer.
Results: Through tissue-based glycan imaging, increased fucosylation was observed in 96% of HCC tissue. The glycan most often observed was a tetra-antennary glycan with one to three fucose residues. Using a recombinant AAL lectin that has increased affinity towards fucosylated and branched glycan, we identified low molecular weight kininogen (LMWK) as a serum protein that contained this glycan. Subsequently a plate based assay using high affinity antibodies, modified to lack glycosylation, and recombinant lectins with high affinity towards the specific fucose change observed was used to test the performance of fucosylated LMWK as a biomarker of HCC. In four studies, a fucosylated LMWK based biomarker algorithm had a median AUC of 0.9575 in the detection of all HCC and 0.945 for early stage HCC.
Conclusions: Using a novel tissue-based glycan imaging platform, we were able to identify glycan changes that occur directly in cancer tissue and used this information to identify serum based biomarkers of HCC that are superior to those currently used.