Aberrant dUTP metabolism plays a significant role in the underlying molecular mechanisms of cell killing mediated by inhibitors of thymidylate biosynthesis. dUTP nucleotidohydrolase (
dUTPase) is the key regulator of dUTP pools, and significant evidence exists suggesting that the expression of this
enzyme may be an important determinant of cytotoxicity mediated by inhibitors of
thymidylate synthase (TS). In this study, we have determined the expression patterns of
dUTPase in normal and neoplastic tissues and examined the association between
dUTPase expression and response to
5-fluorouracil (5-FU)-based
chemotherapy and overall survival in
colorectal cancer. Immunohistochemistry was performed on
formalin-fixed,
paraffin-embedded tissue sections using a
monoclonal antibody (MAb), DUT415, that cross-reacts with both nuclear and mitochondrial
isoforms of human
dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear
dUTPase staining was observed exclusively in replicating cell types. This observation is in agreement with cell culture studies where expression of the nuclear
isoform (DUT-N) is proliferation dependent In contrast, cytoplasmic expression of
dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria. Consistent with this observation, cell culture studies reveal that the mitochondrial
isoform (DUT-M) is expressed constitutively, independent of cell cycle status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N
isoform, whereas cytoplasmic staining represents the DUT-M
isoform. In
colon cancer tumor specimens, expression of
dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns of
dUTPase protein expression observed included exclusive nuclear, exclusive cytoplasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistochemical detection of
dUTPase in
colon cancers provides distinct intracellular phenotypes of expression that may be of significant prognostic value. To examine the association between
dUTPase expression and response to 5-FU-based
chemotherapy and overall survival, we initiated a retrospective study including
tumor specimens from 20 patients who had received protracted infusion of
5-FU and
leucovorin for treatment of metastatic
colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear
dUTPase expression, 6 responded to 5-FU-based
chemotherapy, 4 had stable disease, and 2 had progressive disease. Of the patients presenting positive nuclear
dUTPase expression, 0 responded to
chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005). The median survival for patients with
tumors lacking nuclear staining was 8.5 months and 6.9 months for patients with
tumors demonstrating positive nuclear
dUTPase expression (P = 0.09). Time to progression was significantly longer for patients with
tumors lacking nuclear staining (P = 0.017). Variable cytoplasmic
dUTPase expression was observed in these
tumors; however, there was no apparent association with clinical response or survival in this limited study. Nuclear
dUTPase staining within these
tumors was also associated with TS gene expression (P = 0.06). This study demonstrates that low intratumoral levels of nuclear
dUTPase protein expression is associated with response to 5-FU-based
chemotherapy, greater time to progression, and greater overall survival in
colorectal cancer. Conversely, high levels of nuclear
dUTPase protein expression predict for
tumor resistance to
chemotherapy, shorter time to progression, and shorter overall survival. This report represents the first clinical study implicating
dUTPase overexpression as a mechanism of resistance to TS inhibitor-based
chemotherapy.