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. 2004 Mar 2;101(9):3089-94.
doi: 10.1073/pnas.0308716101. Epub 2004 Feb 17.

Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients

Tian-Li Wang  1 , Luis A Diaz Jr, Katharine Romans, Alberto Bardelli, Saurabh Saha, Gennaro Galizia, Michael Choti, Ross Donehower, Giovanni Parmigiani, Ie-Ming Shih, Christine Iacobuzio-Donahue, Kenneth W Kinzler, Bert Vogelstein, Christoph Lengauer, Victor E Velculescu
Affiliations

Digital karyotyping identifies thymidylate synthase amplification as a mechanism of resistance to 5-fluorouracil in metastatic colorectal cancer patients

Tian-Li Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Resistance to chemotherapy is a major cause of mortality in advanced cancer patients. In this study, digital karyotyping was used to search for genomic alterations in liver metastases that were clinically resistant to 5-fluorouracil (5-FU). In two of four patients, we identified amplification of an approximately 100-kb region on 18p11.32 that was of particular interest because it contained the gene encoding thymidylate synthase (TYMS), a molecular target of 5-FU. Analysis of TYMS by fluorescence in situ hybridization identified TYMS gene amplification in 23% of 31 5-FU-treated cancers, whereas no amplification was observed in metastases of patients that had not been treated with 5-FU. Patients with metastases containing TYMS amplification had a substantially shorter median survival (329 days) than those without amplification (1,021 days, P <0.01). These data suggest that genetic amplification of TYMS is a major mechanism of 5-FU resistance in vivo and have important implications for the management of colorectal cancer patients with recurrent disease.

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Figures

Fig. 1.
Fig. 1.
Overlapping regions of amplification on chromosome 18p identified by DK. Bitmap views comprised of 18,431 pixels representing tag density values at the chromosomal position of each virtual tag on chromosome 18. Yellow regions indicate tag densities that were not amplified, and black regions represent areas with genomic tag densities indicating amplification. Genomic tag densities were determined as described in Materials and Methods and had maximal values of 10 and 6 copies per diploid genome for the amplifications in FU-M2 and FU-M4, respectively. Genes present within overlapping amplified regions are indicated below on a high-resolution map. Only TYMS and rTS were entirely contained within the regions that were amplified in both FU-M2 and FU-M4.
Fig. 2.
Fig. 2.
Quantitative PCR analysis of genomic DNA from colorectal metastases. Quantitive PCR analysis of TYMS (right curves) and LINE element control (left curves) performed on genomic DNA from colorectal cancer metastases FU-M2 (A) and FU-M4 (B) and normal (nontumor) DNA (C). (D) Differences in threshold cycle numbers between LINE element and TYMS confirm that TYMS is present at increased gene copy numbers in colorectal metastases.
Fig. 3.
Fig. 3.
TYMS amplification assessed by interphase FISH. Analysis of interphase nuclei from a colorectal cancer metastasis to the liver after 5-FU treatment (A). Matched colorectal adenoma obtained before 5-FU treatment (B) and colorectal cancer obtained after 5-FU neoadjuvant treatment (C) from a patient with familial adenomatous polyposis. Nuclei are visualized with 4′,6′-diamidino-2-phenylindole stain (blue); TYMS probe (located on chromosome 18p, 0.8-1.0 Mb from the telomere) is visualized by using FITC-avidin (green), and chromosome 18 control probe (located on chromosome 18p, 13.0-13.2 Mb from the telomere) is visualized by using tetramethylrhodamine B isothiocyanate-conjugated antibodies (red). Increased TYMS gene copy number was observed only in patients previously treated with 5-FU (A and C). (Magnification: ×600.)
Fig. 4.
Fig. 4.
Five-year survival curve for patients with and without TYMS amplification.
See this image and copyright information in PMC

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