Fluorouracil

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5-Fluorouracil (5-FU) is still one of the most widely used antineoplastic agents; its effect is mainly dependent on the inhibition of TS, an enzyme involved in the de novo biosynthesis of pyrimidines, by the active metabolite 2'-deoxy-5-fluorouridine monophosphate (2'-dFUMP), whereas the triphosphate metabolites interfere with nucleic acid synthesis (Fig. 7). It was evident from the time it went into use that some tumor responses to 5-FU were lower than would have been expected, although some patients suffered from severe toxicity, suggesting that genetic factors may have been responsible for the differences. High expression of TS in tumor cells is associated with an unsatisfactory response to chemotherapy (8), and the analysis of the regulatory region of the TS gene led to the discovery that variability in gene expression depends, at least in part, on the presence of a polymorphism in the 5'-untranslated region of the promoter (TSER). This consists of a sequence of 28 bp, which is repeated from two to nine times, with the number of repetitions being related to the level of gene expression (9) (Fig. 8). Clinical studies have demonstrated that the TSER *2/2 homozygous genotype is associated with lower levels of TS protein expression compared with individuals homozygous for the allele with three repeats (TSER*3¡3). Higher translation efficiency is thought to be responsible for the genotype-phenotype relationship (10). Therefore, genotyping the tumor for TSER status, in combination with the other factors that follow, is potentially useful to predict cancer sensitivity to fluoropyrimidines.

Figure 7 Overview of selected genetic determinants of activity and tolerability of fluoropyrimi-dines, including CE, CDA, DPD, hCNT1 /hENT1, TP, and TS. Abbreviations: CE, carboxylesterase; CDA, cytidine deaminase; DPD, dihydropyrimidine dehydrogenase; hCNT1/hENT1, nucleotide concentrative and equilibrative transport systems; TP, thymidine phosphorylase; TS, thymidylate synthase; 5'-dFC, 5'-deoxy-5-fluorocytidine; 5'-dFU, 5'-deoxy-5-fluorouridine; 5-FU, 5-fluorouracil; 5-FDHU, 5-fluoro-5,6-dihydrouracil; 2-dFUMP, 2'-deoxy-5-fluorouridine monophosphate.

Figure 7 Overview of selected genetic determinants of activity and tolerability of fluoropyrimi-dines, including CE, CDA, DPD, hCNT1 /hENT1, TP, and TS. Abbreviations: CE, carboxylesterase; CDA, cytidine deaminase; DPD, dihydropyrimidine dehydrogenase; hCNT1/hENT1, nucleotide concentrative and equilibrative transport systems; TP, thymidine phosphorylase; TS, thymidylate synthase; 5'-dFC, 5'-deoxy-5-fluorocytidine; 5'-dFU, 5'-deoxy-5-fluorouridine; 5-FU, 5-fluorouracil; 5-FDHU, 5-fluoro-5,6-dihydrouracil; 2-dFUMP, 2'-deoxy-5-fluorouridine monophosphate.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting step in 5-FU catabolism with 85% of the dose of 5-FU being inactivated by the enzyme; therefore, a genetically determined deficiency of the enzyme is associated with a profound alteration in metabolism and severe toxicity (Fig. 9) (11). The most common alteration associated with severe toxicity is the A —> G transition at position 1986 (DPYD*2A allele) leading

TSER*3

TSER*2

TSER*3/3 TSER*2/3 TSER*2/2 Std

248 bp 248 bp

Figure 8 Characterization of SLP variants of the TSER. In this example, PCR amplification with specific primers (top) yields fragments of different lengths; separation on an agarose gel (bottom) shows three genotypes corresponding to heterozygotes for 2/3 repeats (TSER*2/3) and homozygotes for two (TSER*2/2) and three repeats (TSER*3/3). Abbreviations: SLP, sequence length polymorphic; TSER, enhancer region of the promoter of thymidylate synthase; 5'-UTR, 5'-untranslated region of the gene; CdS, coding sequence; bp, base pair.

TSER*3/3 TSER*2/3 TSER*2/2 Std

Figure 8 Characterization of SLP variants of the TSER. In this example, PCR amplification with specific primers (top) yields fragments of different lengths; separation on an agarose gel (bottom) shows three genotypes corresponding to heterozygotes for 2/3 repeats (TSER*2/3) and homozygotes for two (TSER*2/2) and three repeats (TSER*3/3). Abbreviations: SLP, sequence length polymorphic; TSER, enhancer region of the promoter of thymidylate synthase; 5'-UTR, 5'-untranslated region of the gene; CdS, coding sequence; bp, base pair.

Deficiency

5-FU

2'-dFUMP

Tolerable toxicity

Figure 9 Mechanism of 5-FU toxicity depending on the deficiency of DPD. In the normal pheno-type (left), a substantial proportion of the 5-FU dose is metabolized to the inactive metabolite 5-FDHU, while a minor proportion is anabolized to the active metabolite 2'-dFUMP, which inhibits TS. If metabolism through DPD is impaired, an excessive amount of 5-FU is converted into 2'-dFUMP, thus resulting in marked TS inhibition and severe damage to normal tissues (right). Abbreviations: DPD, dihydropyrimidine dehydrogenase; 5-FDHU, 5-dihydro-FU; 2'-dFUMP, 2'-deoxy-5-fluorouridine monophosphate; TS, thymidylate synthase.

Normal 5-FU

Figure 9 Mechanism of 5-FU toxicity depending on the deficiency of DPD. In the normal pheno-type (left), a substantial proportion of the 5-FU dose is metabolized to the inactive metabolite 5-FDHU, while a minor proportion is anabolized to the active metabolite 2'-dFUMP, which inhibits TS. If metabolism through DPD is impaired, an excessive amount of 5-FU is converted into 2'-dFUMP, thus resulting in marked TS inhibition and severe damage to normal tissues (right). Abbreviations: DPD, dihydropyrimidine dehydrogenase; 5-FDHU, 5-dihydro-FU; 2'-dFUMP, 2'-deoxy-5-fluorouridine monophosphate; TS, thymidylate synthase.

to the skipping of exon 14 and production of an inactive enzyme (12). This variant is present in up to 3% of the individuals (13). Other mutations have also been discovered (14,15), and DPD genotyping may be a useful pharmacogenetic test for identifying patients at risk of life-threatening toxicities. Moreover, low levels of DPD expression in tumors are associated with poor 5-FU inactivation and higher efficacy rates in patients with colorectal cancer (16).

Oral bioavailability of 5-FU is poor because of high DPD activity in the gut and liver; therefore, administration of 5-FU and a DPD inhibitor (i.e., ethynyluracil) or a 5-FU prodrug, such as capecitabine, has proved to be an effective strategy. Capecitabine is a promising tumor-specific agent because it releases 5-FU in the cancer cells expressing high levels of thymidine phosphorylase (TP), an enzyme of drug anabolism (Fig. 7). TP, also known as platelet-derived-endothelial cell growth factor, is associated with high proliferation rate, angiogenesis, and inhibition of apoptosis (17). It has been demonstrated that higher expression of TP in tumors, with respect to healthy tissues, is associated with extensive metabolism of 5'-deoxy-5-fluorouridine (5'-dFU) to 5-FU (18). Therefore, genetic stratification of patients to be given capecitabine may include the analysis of TP gene expression, together with TS and DPD, to assess their likelihood of response to the treatment (Fig. 7) (16). Indeed, the probability of survival is higher in patients with metastatic colorectal cancers that have a low expression of TS, DPD, and TP genes (16). Furthermore, the TP/DPD gene expression ratio was significantly different between sensitive and resistant tumors (19).

Retrospective studies have correlated microsatellite instability (MSI) and survival with the benefit of adjuvant 5-FU chemotherapy in patients with stages II and III colon cancer. Patients not given adjuvant chemotherapy, whose tumors displayed high-frequency MSI (H-MSI), had a better five-year survival with respect to patients with low-frequency MSI (L-MSI) or microsatellite stability (MSS). On the contrary, adjuvant chemotherapy with 5-FU improved overall survival among patients with MSS or L-MSI tumors, whereas no benefit was obtained with adjuvant chemotherapy in the group with H-MSI (20). However, the predictive value of these genetic markers, including TP53, has not been fully validated, and cDNA microarray-generated gene expression profiles of tumors may allow a much more accurate analysis of 5-FU sensitivity (21).

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