CONTEXT: Increased thymidylate synthase expression is a marker for decreased survival in colorectal cancer. OBJECTIVE: Thymidylate synthase localizes to both the nucleus and cytoplasm, but how the relationship of these expression levels affects colon cancer outcome has yet to be determined. DESIGN: Using AQUA, we assessed prognosis of thymidylate synthase expression as a function of subcellular localization in 2 retrospective cohorts of colorectal carcinoma. We used the first cohort (n = 599) as a training set, subsequently validating optimal expression cut points in the second cohort (n = 447). RESULTS: A significant association between decreased 5-year disease-specific survival and increased nuclear expression (16% decreased survival [72% to 56%] for the top 60% of nuclear-expressing tumors [P .001]) and cytoplasmic expression (12% decreased survival [70% to 58%] for the top 54% of cytoplasmic-expressing tumors [P = .02]) was observed for the training set. A higher nuclear to cytoplasmic ratio also correlated significantly with decreased survival (15% decreased survival [66% to 51%] for the top 25% of tumors [P .001]). Applying these findings to the validation set, as a function of time to recurrence, only the ratio (P = .03 [expression ratio]; P = .18 [nuclear]; P = .71 [cytoplasmic]) showed a significant association with decreased time to recurrence. Additionally, the expression ratio significantly added to the prognostic value given by the primary tumor pathologic classification and nodal status. CONCLUSIONS: These data suggest the relationship of nuclear to cytoplasmic thymidylate synthase expression, given as a ratio of continuous AQUA scores, to be a strong predictor of colon cancer survival.
AQUA Analysis of Thymidylate Synthase Reveals Localization to be a Key Prognostic Biomarker in 2 Large Cohorts of Colorectal Carcinoma Mark D. Gustavson, PhD; Annette M. Molinaro, PhD; Greg Tedeschi, BS; Robert L. Camp, MD, PhD; David L. Rimm, MD, PhD ● Context.—Increased thymidylate synthase expression is 58%] for the top 54% of cytoplasmic-expressing tumors a marker for decreased survival in colorectal cancer. [P .02]) was observed for the training set. A higher nu- Objective.—Thymidylate synthase localizes to both the clear to cytoplasmic ratio also correlated signiﬁcantly with nucleus and cytoplasm, but how the relationship of these decreased survival (15% decreased survival [66% to 51%] expression levels affects colon cancer outcome has yet to for the top 25% of tumors [P .001]). Applying these be determined. ﬁndings to the validation set, as a function of time to re- Design.—Using AQUA, we assessed prognosis of thymi- currence, only the ratio (P .03 [expression ratio]; P dylate synthase expression as a function of subcellular lo- .18 [nuclear]; P .71 [cytoplasmic]) showed a signiﬁcant calization in 2 retrospective cohorts of colorectal carci- association with decreased time to recurrence. Addition- noma. We used the ﬁrst cohort (n 599) as a training set, ally, the expression ratio signiﬁcantly added to the prog- subsequently validating optimal expression cut points in nostic value given by the primary tumor pathologic clas- the second cohort (n 447). siﬁcation and nodal status. Results.—A signiﬁcant association between decreased Conclusions.—These data suggest the relationship of nu- 5-year disease-speciﬁc survival and increased nuclear ex- clear to cytoplasmic thymidylate synthase expression, giv- pression (16% decreased survival [72% to 56%] for the en as a ratio of continuous AQUA scores, to be a strong top 60% of nuclear-expressing tumors [P .001]) and cy- predictor of colon cancer survival. toplasmic expression (12% decreased survival [70% to (Arch Pathol Lab Med. 2008;132:1746–1752) T hymidylate synthase (TS) catalyzes the reductive meth- ylation of deoxyuridylate in the pathway for the pro- duction of deoxythymidine triphosphate, which is critical However, considerable heterogeneity exists in the percent- age of positivity within the population as well as vari- ability in the literature about the overall prognostic value for DNA synthesis.1 Thymidylate synthase expression, as of TS expression.9 This variability is most likely due to a determinant of sensitivity to ﬂuoropyrimidines, has differences in methodologies, including differential deﬁ- been demonstrated in vitro,2,3 and TS expression in vivo nitions of TS positivity determined by subjective assess- may play an important role in determining tumor re- ments of expression levels by traditional immunohisto- sponse to 5-ﬂuorouracil (5-FU).4,5 Thymidylate synthase chemical techniques. Studies measuring mRNA have re- has been suggested to be both prognostic6,7 and predictive moved considerable subjectivity but have still failed to be- of response to therapy (see Aschele et al8 for review). come part of the routine practice for management of colon cancer.5,10,11 Accepted for publication May 6, 2008. Recently, it has been demonstrated that TS may have From the Departments of Pathology (Drs Gustavson, Camp, and other cellular functions, including translational regulation Rimm, and Mr Tedeschi) and Epidemiology and Public Health (Dr Mol- (see Liu et al12 for review). Thus the subcellular localiza- inaro), Yale University, New Haven, Conn. Dr Gustavson and Mr Te- tion of expression may be an important determinant of deschi are now with HistoRx, Inc, New Haven, Conn. Dr Gustavson and Mr Tedeschi, although not at the time of the re- the functional role of TS. Because of the potential impor- search, are currently employed by HistoRx, Inc, a private company to tance and functional consequence of TS subcellular local- which Yale University has given exclusive rights to the AQUA tech- ization, we wished to examine the role of TS localization nology. Drs Camp and Rimm are stockholders, scientiﬁc founders, and as a function of survival. We recently developed a method consultants to HistoRx, Inc. Dr Molinaro has no relevant ﬁnancial in- of automated quantitative analysis (AQUA) that allows terest in the products or companies described in this article. Sections of this article previously appeared in ‘‘Methods for Making rapid analysis of immunoﬂuorescence on tissue samples.13 Cancer Prognoses Based on the Subcellular Localization of Biomark- This method yields quantitative results comparable to en- ers,’’ p
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