P5-01-06: Gene Copy Number and Expression of TYMP and TYMS Are Predictive of Outcome in Breast Cancer Patients Treated with Capecitabine.
Authors: Audet RM , Changyu S , Duchnowska R , Adamowicz K , Zok J , Rogowski W , Litwiniuk M , Debska S , Jaworska M , Foszczynska-Kloda M , Kulma-Kreft M , Zabkowska K , Jassem J , Edgerton S , Vang Nielsen K , Thor A , Chang J , Miller K , Sledge GW , Leyland-Jones B 
Published: June 2012

Abstract
Background: The efficacy of the pro-drug capecitabine (C) may be affected by the expression of thymidylate synthase (TYMS) and thymidine phosphorylase (TYMP), respectively the target and the activating enzyme of C, as well as dihydrofolate reductase (DHFR) that provides a key intermediate.
Method: In this retrospective study, expression and gene copy number (GCN) of the above enzymes were assessed and correlated with time-to progression (TTP) and progression-free survival (PFS) of adult female patients with pathologically confirmed breast cancer and locally advanced or metastatic disease treated with C 1000 mg/m2 BID days 1–14 of a 21-day cycle. TYMS, TYMP and DHFR GCN were scored using custom made FISH probes (Dako, Denmark) in at least 60 morphologically intact non-overlapping nuclei. FISH markers dichotomized as high/low by the median were correlated with TTP and PFS using Cox proportional hazard models in 65, 57 and 24 patients for TYMS, TYMP and DHFR probes respectively. Gene expression was analyzed using the whole genome cDNA-mediated annealing, selection extension and ligation (DASL) platform on total RNA extracted from FFPE tissue samples and correlated with TTP and PFS using Cox models in 75 patients. Correlations between GCN and expression were measured using both Pearson's and Spearman's correlation coefficients.
Results: Our interim results (ASCO 2011) showed that higher TYMS GCN was significantly associated with both decreased TTP (HR 1.76, 95% CI 1.07 to 2.90, p=0.026) and PFS (HR 1.86, 95% CI 1.14 to 3.04, p=0.036) in the overall patient population. We now observe 1) a statistically significant positive correlation between TYMS GCN and expression (Pearson 0.26, p=0.049; Spearman 0.25, p=0.056); 2) a trend towards worse outcome with higher TYMS expression in a) the overall population (TTP-HR 1.23, 95% CI 0.93 to 1.64, p=0.148), b) in ER+ patients (TTP-HR 1.46, 95% CI 0.97 to 2.19, p=0.07) as well as in c) HER2− patients (TTP-HR 1.17, 95% CI 0.85 to 1.61, p=0.343), 3) a statistically significant association between higher TYMP expression and longer PFS, but not TTP in ER+ and HER2− patients (table).

Discussion: Our ASCO 2011 analysis showed that high TYMS GCN is predictive of poor outcome in ER+ and HER2− patients, consistent with the fact it is the target of C. Here, we show that 1) expression of TYMS is significantly correlated with GCN 2) higher TYMS expression demonstrates the same trend towards poor outcome in ER+ and HER2− patients as in FISH, 3) higher TYMP expression is significantly associated with longer PFS in ER+ and HER2−patients, consistent with its C activating role. Differential sensitivity between FISH and DASL might be explained by the fact that DASL is performed in a pool of RNA coming from many cellular types, whereas FISH is scored selectively in tumor cells. These findings suggest that TYMS and TYMP GCN and expression can be useful predictive markers of C sensitivity in human breast cancer.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-06.