Immunohistochemical and genetic evaluation of deoxycytidine kinase in pancreatic cancer: relationship to molecular mechanisms of gemcitabine resistance and survival

Abstract

Gemcitabine is considered the standard first-line therapy for patients with advanced pancreatic cancer. More recent strategies have focused on improving the efficacy of gemcitabine by either improving the method of delivery or by combining gemcitabine with other non-cross-resistant chemotherapy agents or with small-molecule drugs. However, the clinical benefits, response rates, and duration of responses have been modest. Deoxycytidine kinase (dCK) is the rate-limiting enzyme involved in the metabolism of gemcitabine. The expression of dCK has been postulated to be correlative of gemcitabine resistance. We determined the relationship of dCK immunohistochemical protein expression and/or genetic status of dCK in a panel of human pancreatic cancer tissues and pancreatic cancer cell lines and determined the relationship of these variables to the clinical outcome of patients treated with gemcitabine. We report that dCK protein expression is expressed in the majority of pancreatic cancers analyzed (40 of 44 cases, 91%) and showed a range of labeling intensities ranging from 1+ (labeling weaker in intensity than normal lymphocytes present in same section) to 3+ (labeling greater in intensity than normal lymphocytes present in same section). When labeling intensity was compared with survival, low dCK expression (1+ labeling) was correlated with both overall survival (P < 0.009) and progression-free survival following gemcitabine treatment (P < 0.04). Low dCK labeling intensity was also significantly correlated with patient age (70.3 +/- 8.1 versus 59.8 +/- 7.4 years; P < 0.0006), suggesting that age-related methylation of the dCK gene may account in part for the observed differences. Sequencing of the entire dCK coding sequence in 17 cell lines and 9 patients' cancer tissues with disease progression while on gemcitabine did not identify any mutations, suggesting that genetic alterations of dCK are not a common mechanism of resistance to gemcitabine for this tumor type. Moreover, dCK labeling showed similar patterns and intensities of labeling among matched pretreatment and post-treatment tissues. In summary, pretreatment levels of dCK protein are most correlated with overall survival following gemcitabine treatment and are stable even after resistance to gemcitabine is clinically documented.

Figures

Fig. 1

Immunohistochemical labeling patterns of dCK protein in resected pancreatic cancer tissues. Examples of 1+ staining (A), 2+ staining (B), and 3+ staining (C). In all cases, note the presence of positive labeling lymphocytes within the desmoplastic stroma that serve as a useful internal control for evaluating the intensity of dCK immunolabeling.

Fig. 2

Kaplan-Meier survival curves in relation to dCK immunolabeling status. Low levels of dCK immunolabeling show a significant relationship toward shorter overall survival (A) and survival following gemcitabine (B).

Fig. 3

dCK immunolabeling patterns in an untreated primary infiltrating pancreatic cancer (A) and a matched lung metastasis that developed two years later following gemcitabine therapy (B). Strong positive (3+) immunolabeling of the nucleus and cytoplasm is seen within the neoplastic cells of the untreated primary tumor. In contrast, a significant reduction in dCK immunolabeling (1+) was found in the metastatic disease from this patient (original magnification, ×200).