Overexpression of
aspartyl (asparaginyl) beta-hydroxylase (AAH) has been demonstrated in
hepatocellular carcinoma,
cholangiocarcinoma, and
pancreatic carcinoma. AAH has an important role in regulating cell motility and invasiveness. Humbug is a truncated homolog of AAH, with a role in
calcium regulation. The present study examines the prognostic use of AAH and humbug gene expression in stage II
colon cancer. One hundred thirty cases of TNM stage II colon
carcinoma were retrieved from the Rhode Island Hospital pathology archives. Tissue microarrays were immunostained with the FB50 and 15C7
monoclonal antibodies generated to recombinant AAH. However, FB50 also recognizes humbug. In addition, AAH and humbug expression was analyzed in samples of
colon cancer and adjacent normal mucosa by real-time quantitative
reverse transcriptase-polymerase chain reaction. Humbug (FB50) expression was localized to the
tumor cytoplasm, whereas normal colonic epithelium did not exhibit significant immunoreactivity. Humbug staining was detected in 85% of the
neoplasms, 23% of which stained strongly. Strong humbug immunoreactivity positively correlated with nuclear grade (P = .006) and inversely with survival (P = .027). In contrast to humbug, AAH (15C7) immunoreactivity was seen in normal and neoplastic epithelium. There was no correlation between AAH immunoreactivity and
tumor grade, or survival. Correspondingly,
reverse transcriptase-polymerase chain reaction studies demonstrated up-regulation of humbug but not AAH in 95% of colon
carcinomas relative to adjacent
colon cancer-free mucosa (P < .0001). This study demonstrates that high levels of humbug immunoreactivity in colon
carcinomas correlate with histologic grade and
tumor behavior, suggesting that humbug can serve as a prognostic
biomarker of TNM stage II
colon cancers. In addition, molecular studies demonstrated that the increased levels of FB50 detected were due to humbug, as opposed to AAH overexpression.