dUTP nucleotidohydrolase isoform expression in normal and neoplastic tissues: Association with survival and response to 5-fluorouracil in colorectal cancer

Aberrant dUTP metabolism plays a significant role in the underlying molecul ar mechanisms of cell killing mediated by inhibitors of thymidylate biosynt hesis, dUTP nucleotidohydrolase (dUTPase) is the key regulator of dUTP pool s, and significant evidence exists suggesting that the expression of this e nzyme may be an important determinant of cytotoxicity mediated by inhibitor s of thymidylate synthase (TS), In this study, we have determined the expre ssion patterns of dUTPase in normal and neoplastic tissues and examined the association between dUTPase expression and response to 5-fluorouracil (5-F U)-based chemotherapy and overall survival in colorectal cancer. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tis sue sections using a monoclonal antibody (MAb), DUT415, that cross-reacts w ith both nuclear and mitochondrial isoforms of human dUTPase, Nuclear and c ytoplasmic staining was observed in both normal and neoplastic: tissues. In normal tissues, nuclear dUTPase staining was observed exclusively in repli cating cell types, This observation is in agreement with cell culture studi es where expression of the nuclear isoform (DUT-N) is proliferation depende nt. In contrast, cytoplasmic expression of dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria, Cons istent with this observation, cell culture studies reveal that the mitochon drial isoform (DUT-M) is expressed constitutively, independent of cell cycl e status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N isoform, whereas cytoplasmic stain ing represents the DUT-M isoform, In colon cancer tumor specimens, expression of dUTPase was shown to be high ly variable in both amount and intracellular localization. Patterns of dUTP ase protein expression observed included exclusive nuclear, exclusive cytop lasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistoche mical 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 inf usion of 5-FU and leucovorin for treatment of metastatic colon cancer. Posi tive nuclear staining was found in 8 patients, whereas 12 lacked nuclear ex pression. Of the patients lacking nuclear dUTPase expression, 6 responded t o 5-FU-based chemotherapy, 4 had stable disease, and 2 had progressive dise ase. Of the patients presenting positive nuclear dUTPase expression, 0 resp onded to chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005), The median survival for patients with tumors lacking nuclear s taining was 8.5 months and 6.9 months for patients with tumors demonstratin g positive nuclear dUTPase expression (P = 0.09), Time to progression was s ignificantly longer for patients with tumors lacking nuclear staining (P = 0.017), Variable cytoplasmic dUTPase expression was observed in these tumor s; however, there was no apparent association with clinical response or sur vival in this limited study. Nuclear dUTPase staining within these tumors w as also associated with TS gene expression (P = 0.116), This study demonstrates that low intratumoral levels of nuclear dUTPase pro tein expression is associated with response to 5-FU-based chemotherapy, gre ater 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 implicat ing dUTPase overexpression as a mechanism of resistance to TS inhibitor-bas ed chemotherapy.