Genetic Factors Influencing Pyrimidine-Antagonist Chemotherapy

Genetic factors influencing pyrimidine-antagonist chemotherapy

Jan Gerard Maring1, Harry J.M. Groen2, Floris M. Wachters2, Donald R.A. Uges3, Elisabeth G.E. de Vries4

1Department of Pharmacy, Diaconessen Hospital Meppel and Bethesda Hospital Hoogeveen; Departments of 2Pulmonary Diseases, 3Pharmacy and 4Medical Oncology , University Hospital Groningen, The Netherlands

Pharmacogenetics J, revised resubmitted Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

Grandmother & Children. Almeria, Spain 1996.

16 17 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

Abstract

Pyrimidine antagonists, e.g. 5-fluorouracil, cytarabine and gemcitabine, are widely used in chemotherapy regimes for colorectal, breast, head and neck, non-small cell lung cancer, pancreatic cancer and leukaemias. Extensive metabolism is a prerequisite for conversion of these pyrimidine prodrugs into active compounds. Interindividual variation in the activity of metabolising enzymes can affect the extent of pro-drug activation and, as a result, act on the efficacy of chemotherapy treatment. Genetic factors at least partly explain interindividual variation in anti-tumour efficacy and toxicity of pyrimidine antagonists. In this review, proteins relevant for the efficacy and toxicity of pyrimidine antagonists will be summarised. In addition, the role of germline polymorphisms, tumour specific somatic mutations and protein expression levels in the metabolic pathways and clinical pharmacology of these drugs are described. Germ line polymorphisms of uridine monophosphate kinase (UMPK), orotate phosphoribosyl transferase (OPRT), thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and methylene tetrahydrofolate reductase (MTHFR) and gene expression levels of OPRT, UMPK, TS, DPD, uridine phosphorylase, uridine kinase, thymidine phosphorylase, thymidine kinase, dUTP nucleotide hydrolase are discussed in relation to 5-FU efficacy. Cytidine deaminase (CDA) and 5’- nucleotidase (5NT) gene polymorphisms and CDA, 5NT, deoxycytidine kinase and MRP5 gene expression levels and their potential relation to gemcitabine and cytarabine cytotoxicity are reviewed.

Introduction

Pyrimidine antagonists belong to the group of antimetabolite anti-cancer drugs and show structural resemblance with naturally occurring nucleotides (see figure 1). Their action is accomplished through incorporation as false precursor in DNA or RNA or through inhibition of proteins involved in nucleotide metabolism. The most commonly used pyrimidine antagonists are 5-fluorouracil (5-FU), gemcitabine and cytarabine (ara-C). Newer oral variants of 5-FU are capecitabine and tegafur. 5-FU and its analogues are used e.g. in the treatment of colorectal-, breast- and head and neck cancer [1-3], whereas gemcitabine is especially prescribed for non-small cell lung cancer and pancreatic cancer [4,5]. Ara-C is used in the treatment of leukaemia [6]. All pyrimidine antagonists are prodrugs and intracellular conversion into cytotoxic nucleosides and nucleotides is needed to produce cytotoxic metabolites. Proteins, involved in pyrimidine metabolism handle these synthetic drugs, as if they were naturally occurring substrates. The extensive metabolism of pyrimidine antagonists implies that the intracellular concentrations of cytotoxic metabolites, thus indirectly the potential anti-tumour effects, largely depend on intracellular metabolic enzyme activity. The aim of this review is to summarise pharmacogenomic data regarding

16 17 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

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Figure 1 Overview of the chemical structures of the naturally occurring pyrimidines cytosine and uracil and the synthetic pyrimidine analogues cytarabine, gemcitabine, capecitabine, 5-fluorouracil and tegafur.

proteins related to the efficacy and toxicity of pyrimidine antagonists and to identify potential predictive and/or prognostic genetic factors for toxicity and treatment outcome. The impact of germ line polymorphisms as well as tumour specific somatic mutations and protein expression levels on the clinical pharmacology and metabolic pathways of these drugs will be discussed.

Metabolic pathways of pyrimidine antagonists

5-Fluorouracil The initial metabolism of 5-FU into nucleotides is essential for its action. Several enzymes of the pyrimidine metabolic pathway are required for the conversion of 5-FU to nucleotides [7]. Cytotoxic nucleotides can be formed by three routes as is illustrated in figure 2: 1. Conversion of 5-FU to 5-fluoro-uridine-monophosphate (5-FUMP) by orotate phosphoribosyl transferase (OPRT); 2. Sequential conversion of 5-FU to 5-FUMP by uridine phosphorylase and uridine kinase; 3. Sequential conversion of 5-FU to 5-fluoro-deoxy- uridine-monophosphate (FdUMP) by thymidine phosphorylase and thymidine kinase [8]. The antitumour activity results from inhibition of thymidylate synthase (TS) by FdUMP, as well as from incorporation of 5-FU metabolites into RNA and DNA. Only a small part

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of the dose is activated via these routes, as in humans 80-90% of the administered dose is degraded by dihydropyrimidine dehydrogenase (DPD). 5-FU degradation occurs in all tissues, including tumour tissue, but is highest in the liver [9].

Capecitabine and tegafur Capecitabine is an oral prodrug of 5-FU. After absorption from the gut capecitabine is converted into 5’-deoxy-5-fluorocytidine (5’-dFCR) by carboxyl-esterase in the liver, and subsequently further converted into 5’-deoxy-5-fluorouridine (5’-dFUR) by cytidine deaminase (CDA). Finally, 5-FU results from bioconversion of 5’-dFUR by thymidine phosphorylase [10] (see figure 2). Tegafur is another oral 5-FU prodrug, that is converted into 5-FU by cytochrome P450 (CYP450) enzymes in the liver. CYP2A6 is the main CYP450 enzyme involved in tegafur metabolism, but CYP1A2 and CYP2C8 also play a role (see

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�� Figure 2 Metabolism of 5-FU and 5-FU analogues. For explanation of symbols and metabolic routes see text.

18 19 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

figure 2). Tegafur is combined with uracil in a molar proportion of 1:4 available in the com- mercial preparation UFT®. Uracil is a competitive substrate for DPD and its role in UFT® is to diminish 5-FU catabolism by DPD.

Gemcitabine and cytarabine The metabolic pathways of gemcitabine and ara-C are almost alike [11,12] (see figure 3). Membrane transport of both gemcitabine and ara-C is mediated by equilibrative nucleoside transporters. Subsequently gemcitabine (dFdC) is phosphorylated into gemcitabine monophosphate (dFdCMP) by deoxycytidine kinase (dCK). The same enzyme is responsible for the intracellular phosphorylation of ara-C into cytarabine monophosphate (ara- CMP). dCK is the rate limiting enzyme in the biotransformation of both gemcitabine and ara-C. Inactivation of dFdCMP and ara-CMP can occur through dephosphorylation by 5’- nucleotidase (5NT). Monophosphates, escaping from dephosphorylation are available for further phosphorylation into di- and triphospates by dCMP kinase and nucleoside diphosphate kinase, respectively. Gemcitabine diphosphate (dFdCDP) is a potent inhibitor of the enzyme ribonucleotide reductase (RNR), which will lead to depletion of dCDP and dCTP in the cell. This may favour the incorporation of dFdCTP into DNA. Moreover, gemcitabine has the unique property that after incorporation of gemcitabine monophosphate in DNA, one deoxynucleotide molecule more can be inserted. This stops DNA polymerase [13]. This pattern is distinct from that of ara-C, which halts polymerase progression at the analogue insertion site. The “masked termination” of gemcitabine makes the inserted analogue more resistant to removal from DNA [13]. Other differences with ara-C are the faster membrane transport velocity of dFdC, the greater effectiveness of dFdC phosphorylation by dCK and the longer intracellular retention of dFdCTP. These factors may, at least in part, explain the different spectra of antitumour activity of both drugs. Only a small part of the gemcitabine and ara-C dose is responsible for the cytotoxic effects, since more than 90% of the dose is inactivated by the enzyme cytidine deaminase into dFdU and ara-U respectively.

Germline polymorphisms and fluoropyrimidine efficacy

5-fluorouracil Genetic polymorphisms of enzymes involved in the metabolic activation pathway of 5-FU have been described for the enzymes uridine kinase (UMPK) and orotate phosphorylase transferase (OPRT). Three allelic variants of UMPK have been recognised in the human population: UMPK1, UMPK2 and UMPK3 [14]. The UMPK1 allele is associated with about 3 times the catalytic activity of the UMPK2 allele. Therefore UMPK2 homozygotes are relatively deficient of total UMPK enzyme. The allele frequency of UMPK1 is about 95-97% and that of UMPK2 3-5%, in both Caucasians and Asians [15-17]. UMPK3 is rarely seen. The consequences of this polymorphism for 5-FU chemotherapy have not yet been studied.

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Figure 3 Metabolism of gemcitabine and cytarabine. For explanation of symbols and metabolic routes see text.

In the OPRT gene a G213A mutation in exon 3 and a 440G mutation in exon 6 have been observed, with allele frequencies of 26% and 27% respectively [18]. Both mutations do not significantly compromise in vitro OPRT activity, and therefore it seems unlikely that they affect 5-FU anti-tumour efficacy. Contrary to 5-FU activating enzymes, much more information is available regarding

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polymorphisms of target enzyme TS and 5-FU degrading enzyme DPD. For TS, at least two genotypes have been identified, characterised by triple and double tandem repeat sequences in the DNA promoter enhancer region (TSER). The triple tandem 3R genotype is associated with higher TS mRNA and TS protein levels [19-22]. The allele frequency of the 3R genotype is subject to considerable ethnic variation, being about 67% in Asians and 38% in Caucasians [23]. The consequences of the TSER genotype for fluoropyrimidine chemosensitivity have been studied in tumour cell lines and in clinical trials. In the only study performed in tumour cell lines, the TSER polymorphism had no effect on 5- FU chemosensitivity [24]. In 65 patients with rectal cancer, tumour downstaging after preoperative 5-FU based chemoradiation was observed in only 22% of homozygotes for triple tandem repeat sequences (3R/3R genotype) compared to 60% of patients who were heterozygous (3R/2R) or homozygous for double tandem repeats (2R/2R) [25]. In another small retrospective study of 24 patients with metastatic colorectal cancer, 3 out of 4 patients with the 2R/2R genotype responded on capecitabine, whereas only 1 out of 12 of heterozygotes and 2 out of 8 patients with the 3R/3R genotype responded [26]. Furthermore, in a study of 221 patients with Dukes C colorectal cancer, patients with the 3R/3R genotype gained no survival benefit from 5-FU treatment, whereas survival was increased in patients with a 2R/2R or 2R/3R genotype [27]. However, the TSER genotype did not seem to be an efficacious marker for tumour sensitivity to 5-FU based oral adjuvant chemotherapy in 135 Japanese colorectal patients [28]. Surprisingly, in a small study of 54 colorectal cancer patients, not the 2R but the 3R/3R genotype correlated with an increased disease free survival after adjuvant 5-FU chemotherapy [29]. Several factors can possibly explain these inconclusive data. Firstly, within the second 28 bp tandem repeat sequence, a G/C polymorphism was recently discovered. Functional analysis revealed that the 3R/G genotype has three to four times greater efficiency of translation than other polymorphic sequences. [30,31]. In a study of the G/C polymorphism in 258 primary colorectal tumours was found that all low expression genotypes were associated with longer survival after adjuvant fluoropyrimidine chemotherapy, whereas no benefit was seen for high expression genotypes [30]. Secondly, a 6bp deletion genotype (TS1494del6) in the 3’ untranslated region of the TS gene has been associated with reduced TS mRNA stability [32,33]. The frequency of this genotype was found to be 41% in non-Hispanic whites, 26% in Hispanic whites, 52% in African-Americans and 76% in Singapore Chinese. The relative instability of TSmRNA may have its effect on TS protein activity and thus indirectly on fluoropyrimidine chemosensitivity. However, this polymorphism was not directly related to in vitro chemosensitivity in tumour cell lines [24] or disease free survival after adjuvant 5-FU chemotherapy in a small study of 54 colorectal cancer patients [29]. Another cause of conflicting data is the loss of heterozygosity (LOH), that is frequently observed for TS [34]. The TS gene is located on chromosome 18p11.32, a region most frequently lost in colorectal cancer. In a small study of 30 stage IV colorectal cancer patients, LOH was observed in 17 out of 22 heterozygotes

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[35]. The presence of a 3R allele (3R/3R, 3R/loss, 2R/3R) was associated with lower survival on fluoropyrimidine chemotherapy. LOH in tumour tissue is a significant factor, to be taken into account when TS genotypes are to be considered for outcome prediction. It is important to bear in mind that LOH limits the use of germ line DNA from e.g. blood for predictive genotyping. Finally, it must be stressed that many studies of the TS polymorphism were underpowered to detect relations of the different genotypes with clinical outcome. Hence, the exact consequences of the triple tandem repeat genotypes for 5-FU chemosensitivity are not yet clear. Sufficiently powered clinical trials and additional mechanistic studies may be needed to elucidate the cause of so far unexpected outcomes. Besides the TS polymorphisms, the phenomenon of inherited DPD deficiency is an important issue. DPD deficiency is caused by molecular defects in the DPD gene that result in complete or partial loss of DPD activity [36]. This can cause extreme 5-FU toxicity. So far, in patients suffering from severe 5-FU associated toxicity, 11 different mutations in the dihydropyrimidine dehydrogenase (DPYD) gene have been identified, including at least 1 polymorphism [36-38]. In patients who are deficient for DPD, 5-FU clearance is dra- matically reduced and standard doses of 5-FU cause excessive toxicity in these patients [36,39,40]. The frequency of DPD deficiency has been estimated to be as high as 2-3% in Caucasians based on measurements of DPD activity in peripheral mononuclear cells in patients and healthy volunteers [41,42]. This percentage is probably high enough to justify screening on DPD deficiency prior to 5-FU based chemotherapy. Instead of screening for specific mutations in the DPYD gene, Mattison et al. developed a simple uracil breath test 13 13 for DPD phenotyping, based on the release of CO2 from 2- C uracil in the presence of intact DPD [43]. Expired air was collected 5-90 min after oral ingestion of 6 mg/kg 2-13C uracil. Partially deficient DPD breath profiles were well differentiated from normal profiles. An oral challenge with uracil, prior to chemotherapy, with subsequent measurement of the uracil clearance in plasma, might also give a good prediction of the patient’s DPD status. Such tests are currently developed, but not yet available [44]. A polymorphism that might also influence the efficacy of 5-FU and its analogues is that of the methylene tetrahydrofolate reductase (MTHFR) gene. MTHFR catalyses the conversion of 5,10-methyltetrahydrofolate to 5-methyltetrahydrofolate. 5,10-methyltetrahydrofolate is an essential cofactor in the biosynthesis of dTMP (see figure 2). The dissociation of FdUMP from the ternairy complex with thymidylate synthase and 5,10-methyltetrahydrofolate is suppressed when levels of 5,10- methyltetrahydrofolate are increased [45]. A common C677T transition in the MTHFR gene results in a variant with lower specific activity [46]. The geographic and ethnic distribution of this genotype was studied by Wilcken et al. [47]. The homozygote T genotype was found to be particularly common in northern China (20%), southern Italy (26%) and Mexico (32%). In two studies with respectively 45 and 51 colorectal cancer patients, the MTHFR genotype has been shown to affect the folate pool [48,49]. In the latter study, no effect was seen of the MTHFR genotype on overall survival after oral 5-FU based chemotherapy [49]. Contrary to this, in another study of 43 meta-

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static colorectal cancer patients receiving fluoropyrimidine-based chemotherapy, the presence of one or two mutant alleles was associated with increased tumour response [50]. In vitro transfection of mutant 677T MTHFR cDNA in colon and breast cancer cells increased the chemosensitivity of these cells to 5FU [51], suggesting at least a modulating role of the MTHFR genotype on cellular fluoropyrimidine sensitivity. The exact role of the MTHFR genotype has to be elucidated in larger clinical trials.

Capecitabine and tegafur Polymorphisms in capacitabine metabolising enzymes have only been reported for cytidine deaminase. Since this polymorphism may also be relevant for gemcitabine and ara-C metabolism it will be discussed in the next paragraph. Furthermore, mutations in CYP2A6 have recently been identified to affect tegafur metabolism [52]. Two mutant alleles, CYP2A6*4C and CYP2A6*11 were detected in a patient with largely reduced tegafur clearance. The prevalence of these mutations and its impact on clinical outcome are not yet clear.

Gemcitabine and cytarabine There are at least two polymorphisms in the metabolising pathway, that might be relevant for gemcitabine and ara-C toxicity and efficacy. Firstly, cloning of human cytidine deaminase revealed two protein variants (CDD1 and CDD2) with different in vitro deamination rates of ara-C [53]. Theoretically, patients who deaminate gemcitabine or ara-C more efficiently, are shorter exposed to the parent drugs, while faster metabolism of capecitabine results in increased 5-FU levels. In vitro transfection of human bladder cancer cells with CDD2 cDNA did increase CDD activity, and indeed made the cells more sensitive to 5’dFUR and capecitabine, but resistant to gemcitabine [54]. This warrants further investigation of the possible role of the CDD genotype in fluoropyrimidine chemosensitivity. Secondly, 5’-nucleotidase deficiency, is an autosomal recessive condition, known to cause haemolytic anaemia. Several mutations causing 5’-nucleotidase deficiency have been identified [55]. On theoretical grounds, in patients with 5’-nucleotidase deficiency, treatment with gemcitabine or ara-C may result in increased toxicity although this has not yet been published. An overview of polymorphic genes and related enzymes with possible relevance for fluoropyrimidine chemotherapy is presented in table 1.

24 25 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy - Consequences of toxicity Increased 5-FU decreased Possibly efficacy increased Possibly efficacy decreased Possibly efficacy Unknown Unknown Decreased metabo into lism of tegafur 5-FU Unknown Unknown Patient data miscellaneous typescancer colorectal cancer colorectal cancer colorectal cancer colorectal cancer colorectal cancer not available leukaemia Drug 5-FU and analogues 5-FU and analogues 5-FU and analogues 5-FU and analogues 5-FU and analogues tegafur gemci- Capecitabine, ara-C tabine, Gemcitabine and ara-C A mutation A mutation → Allele frequency IVS14+1G most frequent 0.5-1% 45% in northern China 51% in southern Italy 56% in Mexico 26% 38% in Caucasians 67% in Asians 29% 95-97% 3-5% <1% Unknown 70% 30% unknown - Polymorphism in par found 11 mutations patients tial DPD deficient 3 C667T in exon MFHFR activitydecreased 3 G213A in exon OPRT activityincreased TSER*3 activityTS increased 1494del6 UMPK1 variant UMPK2 variant UMPK3 variant CYP2A6*4C CYP2A6*19 CDA1 variant CDA2 variant mutations Several - Overview of proteins that potentialy may affect drug treatment outcome due to genetic polymorphisms genetic to due outcome affect may treatment drug potentialy that Overview proteins of

Enzyme dehy Dihydropyrimidine (DPD) drogenase tetrahydrofolate Methylene reductase (MFHFR) phosphoribosyl Orotate (OPRT)transferase (TS) synthase Thymidylate Uridine monophosphate kinase (UMPK) P450 2A6 Cytochrome (CYP2A6) deaminase (CDA) Cytidine 5’-nucleotidase (5NT) Table 1 Table

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Impact of somatic mutations and gene expression levels: 5-fluorouracil and its metabolic enzymes

5-Fluorouracil Data on expression levels of enzymes involved in the metabolic activation of 5-FU in relation to chemosensitivity is sparse. Low UMPK levels were observed in 29 colorectal tumour samples of patients with acquired 5-FU resistance [56]. Low OPRT as well as UP and UK activity have been associated with 5-FU resistance in tumour cell lines [57-60]. The role of OPRT has also been investigated in a few clinical studies. High OPRT activity was associated with good in vitro sensitivity to 5-FU, measured by Collagen Gel Droplet Embedded Culture Drug Sensitivity test, Fluoresceine Diacetate Assay or Histoculture Drug Response Assay, in human colorectal cancer tissues [61,62]. Furthermore, in 37 patients treated with oral tegafur-uracil for disseminated colorectal cancer, responding tumours had higher expressions of OPRT than non-responding tumours [63]. In the same study, there was no difference in UP mRNA between responding and non-responding tumours. The role of UP in 5-FU sensitivity is probably marginal, since transfection of tumour cells with UP cDNA, did not affect fluoropyrimidine sensitivity [64]. More research is needed to establish the exact role of OPRT in 5-FU sensitivity. Most research of fluoropyrimidine activation pathways has been focused on the role of TP. Results regarding the role of tumoural TP expression appear to be contradictory. In patients, high as well as low TP mRNA expression levels have been correlated with response to 5-FU therapy. An increase in both relapse free survival and overall survival was suggested in TP-positive compared to TP-negative breast tumours in a small study of 109 patients treated with adjuvant cyclophosphamide, methotrexate and 5-FU (CMF) [65]. Furthermore, in 38 metastatic colorectal cancer patients treated with 5-FU and leucovorin, TP mRNA levels in non-responding tumours were higher than in responding patients [66]. In 28 patients treated for advanced gastric cancer with 5-FU plus pirarubicin and cisplatin, high tumour tissue TP expression was associated with response on chemotherapy [67]. In another 126 advanced gastric cancer patients, TP overexpression was associated with increased patient survival after fluoropyrimidine chemotherapy, but correlated with unfa- vourable prognosis in patients not treated with fluoropyrimidines [68]. Consequently, it is hard to draw conclusions from current, in general small trials. Taking a close look at 5-FU metabolism, one might expect that cells with higher TP levels would be more sensitive to 5-FU, due to higher FdUMP levels resulting from increased 5-FU activation (see figure 2). However, TP also functions as angiogenesis factor since TP and platelet derived endothelial cell growth factor (ECGF1) have been recognised as the same protein. High TP gene expression might therefore be associated with a more aggres- sive and malignant tumour phenotype [66]. Transfection of cancer cells with TP cDNA has been performed in several in vitro studies to investigate the effect of TP overexpression on chemosensitivity. A clear correlation was found between TP activity and in vitro sensi-

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tivity to the 5-FU analogue 5’-dFUR, and to a lesser extent to 5-FU itself [69-74]. Marchetti et al. also studied the effect of TP overexpression on in vivo tumour growth in a rat model [69]. The impact on tumour growth turned out to be relatively modest and only involved the initial stages of tumour growth. These results suggest that tumour growth and chemosensitivity are independently related to TP expression. A major point of interest in interpreting the clinical data in relation to in-vitro data is the role of infiltrating stromal fi- broblasts and macrophages in TP expression. In most tumour types, TP expression is much higher in infiltrating cells compared to tumour cells [75]. This may largely trouble the interpretation of data of in vivo PCR as well as IHC studies. Consequently, this aspect particulary has to be taken into account in study designs to validate TP as predictive marker. Data on the role of TK in 5-FU chemosensitivity are also contradictory. Chemosensitivity was unchanged in TK deficient colon cancer cells compared to parent cells, suggesting only a marginal role of TK in 5FU sensitivity [76]. In another study, TK overexpression has been associated with 5-FU resistance in gastric tumour cell lines [59], but other groups found a reduction of TK activity in chemoresistant colon cancer cell lines [58,60]. Intra- cellular availability of deoxyribose-1-phosphate might account for these differences, since the activation of 5-FU by TP and TK is fully dependent on the availability of this co-substrate. If cellular levels of this co-substrate are too low, hardly any 5-FU can be me- tabolised via this route. Only one patient study is available regarding tumour TK activity in relation to treatment efficacy. In this study, tumour TK activity could not be related to the efficacy of second line 5-FU (poly)chemotherapy in 121 advanced breast cancer patients who failed on first-line tamoxifen treatment [77]. Thus, TK is probably not a strong factor related to 5-FU sensitivity. A route that has been extensively evaluated in relation to 5-FU chemoresistance is the intracellular biodegradation of 5-FU by DPD. In vitro experiments in human tumour cell lines demonstrated an inverse correlation between both DPD mRNA expression and activity with 5-FU response [78]. DPD mRNA levels were also related to primary 5-FU resistance in 7 human gastrointestinal cell lines [79]. Furthermore, high DPD activity and DPD mRNA levels were correlated with low sensitivity to 5FU in 3 human gastric, 2 colon, 1 breast and 1 pancreatic carcinoma xenografts in the nude mice model [80]. However, data of presently available clinical studies are less unequivocal. An overview of clinical relevant studies is presented in table 2. Most studies have been performed in colorectal cancer [61,81-84 ], some in gastric [85-87], head and neck [88-90], and non-small cell lung cancer [91-93], and few in breast and bladder cancer [94,95]. A number of studies have indicated that there is no strong link between DPD mRNA levels and DPD protein activity levels. It has been reported that DPD mRNA, but not DPD activity, is reduced in colorectal tumours compared with normal mucosa, although considerable overlap exists in measured mRNA levels [96-103]. Reduced mRNA levels have also been reported in ovarian cancer [104]. DPD activity appears to be increased in breast cancer (see table 3) [94,105]. This suggests that DPD is not only regulated at transcriptional and translational but also at the post-

26 27 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy reference [81] [82] [83] [61] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] outcome response to DPD related Low found correlations No found correlations No found correlations No longer survival to DPD related Low poor survival to High DPD related DFUR sensitivity to DPD related Low found correlations No found correlations No found correlations No with UFT response DPD correlated Low prognosis with better DPD correlated Low with poor survival High DPD correlated found correlations No poor DFS to High DPD related 5-FU sensitivity to DPD related Low assay mRNA ELISA mRNA activity mRNA Activity ELISA IHC activity activity IHC IHC IHC activity activity activity chemotherapy regimen 5-FU/LV 5-FU 5-FU 5-FU 5-FU 5-FU DFUR 5-FU/MTX 5-FU 5-FU/LV/cisplatin UFT UFT 5-FU 5-FU 5-FU 5-FU patients 33 100 348 54 309 81 22 38 62 82 109 54 68 60 191 74 Overview of studies regarding intratumorial DPD expression levels in relation to chemosensivity to relation in levels expression DPD intratumorial regarding Overview studies of

cancer Colorectal Gastric head and neck NSCLC breast bladder Table 2 Table MTX = methotrexate;; = immunohistochemistry IHC

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transcriptional level. This hampers the use of DPD mRNA as predictive marker for 5-FU efficacy. Inhomogeneity of tumour tissue samples may, in part, also account for conflicting results. Summarising, it can be concluded that data on tumour expression levels of 5-FU metabolising enzymes in relation to chemosensitivity is sparse and that studies show conflicting results. This may at least partly be due to small numbers and therefore lack of statistical power in the studies.

Capecitabine/tegafur In the animal model, high TP/DPD ratios have been associated with antitumour efficacy of capecitabine and its metabolite 5’dFUR in human tumour xenografts [106]. In vivo, high TP/DPD ratios suggested a better clinical outcome after adjuvant treatment with 5’dFUR in a study of 88 colorectal cancer patients, and in 17 metastatic gastric cancer patients [107,108]. TP expression was examined by immunohistochemistry in 650 breast tumours of patients with early breast cancer receiving adjuvant 5’dFUR [109]. Eight-year follow-up data showed that high TP expression in the tumour was a favourable prognostic indicator. Thus, TP expression seems to be a predictive factor for 5’dFUR efficacy. Since the activation of tegafur depends on several CYP450 enzymes, interindividual variations in enzyme expression may affect the rate of tegafur metabolism. Indeed, the relative contribution of each enzyme is known to differ among patients but the clinical consequences of this phenomenon are unclear [110].

Impact of somatic mutations and gene expression levels: 5-fluorouracil and its target enzymes

Contrary to enzymes related to the 5-FU metabolic activation pathway, the target enzyme TS has been extensively studied in relation to 5-FU efficacy. A large amount of preclinical in vitro data suggest a correlation between TS activity and sensitivity to 5-FU. In a panel of 19 nonselected breast, digestive tract and head and neck cancer cell lines as well as in a panel of 13 non selected human colon cancer cell lines, the TS activity was inversely correlated with 5-FU sensitivity [78,111]. Several in vitro studies in tumour cell lines also suggest TS overexpression as a mechanism of resistance after repeated exposure to 5-FU [79,112,113]. Chemosensitivity to 5-FU was also increased after transfection of human colon cancer cells with antisense TS cDNA [114]. However, conflicting data have been observed in clinical studies evaluating the prognostic role of mRNA, protein and activity levels of TS in relation to tumour response and clinical outcome to 5FU based chemotherapy. An overview of relevant clinical studies is presented in table 4. Most studies regarding TS expression in relation to chemosensitivity have been performed in patients with disseminated colorectal cancer [61,84,115-127], some in gastric cancer

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Table 3 DPD expression and activity in different tissue types

Tumour type Samples Parameter Tumour vs. Normal reference

Gastrointestinal 51, matched mRNA lower [96] activity no difference

41, matched mRNA lower [97] activity no difference

10 tumours + 7 me- mRNA lower [98] tastases, matched

15 tumours + 10 protein lower [99] metastases, 25 non- matched controls

36, matched protein no difference [100]

43, matched mRNA lower [101] protein no difference activity no difference [102] 36, matched protein lower

40, matched mRNA lower [103] protein lower activity no difference

Ovarian 85 tumour + 27 non- mRNA lower [104] matched controls

Breast 26, matched activity higher [105] 49, matched activity higher [94]

[87,128-131] and few studies in head and neck cancer [88,89,132], non-small cell lung cancer [92,93] pancreatic and breast cancer [77,133,134]. Several factors may account for the difficult interpretation of combined clinical data. Firstly, it has been reported that TS protein can down-regulate its own translation, whereas its transcription is regulated by E2F, a cell cycle checkpoint regulator. Together, this results in low TS levels in stationary phase cells. Although cells with a low TS might theoretically be more sensitive to 5-FU, the low proliferation rate prevents induction of DNA damage and 5-FU antitumour efficacy [135]. Secondly, a specific interaction exists between oncogenes and TS, by binding of TS protein to the p53 and c-myc RNA, while wild type p53 can also inhibit TS promotor activity. TS inhibition by 5-FU can also result in a depression of TS protein mediated inhibition of TS mRNA translation leading to induction of more TS protein synthesis, and p53 protein may further deregulate this process. These complex indirect and direct interactions between oncogenes and TS may have as yet

30 31 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy [93] [120] [77] [89] [124] [123] [92] [87] [133] reference [114] et al. Peters [115] et al. Leichman [116] et al. Lenz [117] et al. Cascinu [118] et al. Wong [119] et al. Aschele et al. Paradiso [121] et al. Aschele [122] Kornmann et al. et al. Shirota et al. Findlay [125] et al. Aschele [84] Kornmann et al. [61] et al. Fujii [126] et al. Noordhuis [127] et al. Lenz [128] Metzger et al. [129] et al. Yeh [130] Boku et al. et al. Tahara [131] et al. Johnston et al. Etienne [88] et al. Etienne et al. Higashiyama Huang et al. [132] et al. Lizard-Nacol et al. Foekens Hu et al. - outcome non-response to related TS high non-response to related TS high response to related TS low response to related TS low non-response to related TS high response to related TS low response to related TS low response to related meta-TS low response to related TS low non-response to related TS high found no correlations 5FU/LV response to related TS low longer survival to related TS high 5-FU sensitivity to related TS low 5-FU response to related TS low longer survival to related TS low non-response to related TS high non-response to related TS high response to related TS low found no correlations non-response to related TS high found no correlations found no correlations 5-FU sensitivity to not related TS longer survival to related TS low found no correlations ef treatment better to related TS high ficacy on tamoxifen after failure 5-FU response to related TS High assay activity mRNA TS106 mRNA + IHC IHC polyclonal TS106 IHC IHC polyclonal TS106 IHC IHC polyclonal mRNA mRNA IHC polyclonal IHC MRNA Activity activity mRNA mRNA TS106 IHC IHC IHC TS106 IHC activityTS activityTS FdUMP binding IHC polyclonal mRNA activityTS mRNA TS chemotherapy regimen bolus 5-FU/LV protracted 5-FU/LV 5-FU/LV bolus 5-FU/LV protracted 5-FU/LV 5-FU/LV/MTX 5-FU/LV/MTX 5-FU/LV/MTX or MITOX 5-FU/LV/MITO 5-FU/LV/oxaliplatin 5-FU/IFN alfa-2b 5-FU/MTX 5-FU/LV; 5FU/LV 5-FU 5-FU/LV 5-FU/LV/cisplatin 5-FU/LV/cisplatin 5-FU/LV/cisplatin 5-FU/cisplatin 5-FU/MTX 5-FU/LV/cisplatin/MTX/IFN 5-FU/LV/cisplatin 5-FU 5-FU sensitivityin vitro 5-FU or UFT® CAF regimen CAF or CMF regimen 5-FU based patients 47 46 36 41 52 48 108 27 29 50 134 124 309 54 54 65 38 30 39 38 70 82 52 60 68 75 121 73 Overview of studies regarding intratumorial TS expression levels in relation to chemosensivity to relation in levels TS expression intratumorial regarding Overview studies of

cancer colorectal gastric head and neck NSCLC breast pancreatic Table 4 Table TS; = interferon against IFN antibody = monoclonal TS106 = immunohistochemistry; IHC = mitoxantrone; MITOX = mitomycine; MTX MITO = methotrexate;

30 31 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

unclear clinical implications [136]. Finally, the assay type may have influenced the outcome of some studies, since immunohistochemistry as well as rt-PCR were used to measure TS expression. Immunohistochemistry has been performed using different types of antibodies, including monoclonal TS106 was well as polyclonal antibodies. Differences in binding specificity and selectivity may have had a decisive impact on study outcome. Furthermore, the issue of infiltrating cells disturbing tumour tissue homogeneity and thus troubling PCR data, as mentioned earlier when interpreting TP data, may also hold for TS. Acknowledging the importance of TS expression in colorectal cancer, another determinant for 5-FU cytotoxicity may be the enzyme dUTP nucleotidohydrolase (dUTPase), which is the key regulator of dUTP pools. There are at least two isoforms of dUTPase, a nuclear and a mitochondrial form, encoded by the same gene. In tumour cell lines, increased dUTPase levels accounted for resistance to the 5-FU metabolite 5’dFUR [136,137]. In a small retrospective study in tumours of 20 metastatic colorectal cancer patients, high nuclear dUTPase protein expression was associated with poor tumour response on 5-FU therapy [138]. However, larger studies are needed to establish the role of dUTPase in 5-FU chemoresistance.

Impact of somatic mutations and gene expression levels: gemcitabine and cytarabine

Resistance to nucleoside analogues can be due to a number of factors, affecting drug metabolism, including increased deamination, loss of expression of activating kinases, and increased activity of nucleotidases. In vitro, gene transfer of CDA cDNA into murine fibroblast cells was shown to induce cellular resistance to ara-C through increased deamination [140]. In patients with acute myeloid leukaemia (AML), pre-treatment CDA activity did correlate with response on ara-C induction treatment in two studies both involving 36 patients [141,142]. Ara-C resistance has also been associated with low dCK activity both in vitro [143,144] and in 21 AML patients [145]. In turn, increased dCK activity was associated with increased activation of both compounds to cytotoxic nucleoside triphosphate derivates. However, mutational inactivation of dCK is not thought to confer resistance to ara-C in AML patients because mutations in the dCK gene are rarely found in refractory or relapsed AML patients [146,147]. Alternatively, inactivation of dCK by the formation of alternatively spliced dCK transcripts has been demonstrated in 7 out of 12 patients with resistant AML compared to 1 out of 10 patient with sensitive AML [148]. This mechanism is probably a cause of therapy failure in patients with resistant AML [149]. Finally, high 5NT expression in blast cells was shown to be an independent prognostic factor for poor outcome in 108 AML patients after ara-C containing regimens [150]. The balance between dCK and 5NT might predict drug toxicity, since strongly increased 5NT activity combined

32 33 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

with reduced dCK activity was observed in a human leukaemic cell line, resistant to ara-C and gemcitabine [151]. Apart from resistance to nucleoside analogues caused by aberrant drug metabolism, altered drug transport may cause decreased chemosensitivity. Recently, overexpression of the human multidrug resistance protein 5 (MRP5), an ABC transporter known to transport nucleotide monophosphates, has been associated with resistance to gemcitabine in vitro [152]. MRP5 mediated efflux of gemcitabine metabolites may lower the accumulation of gemcitabine in cells. So far, other chemoresistance related transport mechanisms have not been identified for pyrimidine antagonists.

Other proteins related to pyrimidine antagonist chemosensitivity

As mentioned in the previous sections, expression levels of specific proteins, directly involved in drug metabolism of pyrimidine antagonists may affect treatment efficacy. However, expression of genes related to cell growth and the apoptotic pathway may also affect chemosensitivity. This interaction of apoptosis regulator proteins with chemosensitivity is an intriguing issue, but an extensive analysis of current literature on this subject falls outside the scope of this review. The most extensively studied genes with regard to pyrimidine antagonist chemotherapy are bcl-2, bax, c-myc and p53 [153-161]. Here, we will only briefly report on the putative role of p53 with respect to 5-FU efficacy, as this relationship has been most extensively studied. Mutations in the p53 gene and p53 overexpression have been associated with 5-FU chemoresistance both in vitro [162-164] and in vivo in colorectal [117,165-168], head and neck [169,170], and breast cancer [171]. However, results in some other studies are less unequivocal [172,173]. Recently, it has been suggested that only patients whose primary colorectal tumour contain amplified c-myc and wild-type p53 might benefit from 5-FU based chemotherapy [174]. Interestingly, there is an increasing evidence of interactions between TS and p53. P53 gene mutation and protein overexpression are associated with increased TS mRNA levels and TS cyto- plasmatic protein in colorectal tumours [117,121,175]. It has been demonstrated that wild-type p53 is able to bind TS mRNA which results in a feedback regulation of TS protein synthesis [176]. In cells with mutated p53 this feedback mechanism fails and, as previously mentioned, overexpression of TS is associated with poor clinical outcome.

Impact of current data on clinical practice

Summarising, we conclude that in tumour material none of the enzymes discussed above can currently function as predictive marker by itself. So far, only small studies have been performed and drawing conclusions from combined data of these often statistically

32 33 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy - Consequences High DPD activity with in tumour tissue associated 5-FU efficacyreduced activity with reduced be associated may High dUTPase 5-FU efficacy data inconclusive Unknown, data inconclusive Unknown, 5-FU ef with reduced associated expression TS High ficacy efficacy with reduced associated High CDA expression of ara-C to with resistence associated dCK expression Low ara-C efficacy with reduced associated High 5NT expression of ara-C Patient data types cancer miscellaneous colorectal cancer cancer breast cancer Colorectal Gastric cancer cancer Breast colorectal cancer leukaemia myeloid Acute leukaemia myeloid Acute leukaemia myeloid Acute Drug 5-FU 5-FU and analogues 5-FU and analogues 5-FU 5-FU and analogues Ara-C (gemcitabine) Ara-C (gemcitabine) Ara-C (gemcitabine) Overview of proteins that potentialy may affect drug treatment outcome due to altered gene expression gene altered to due outcome affect may treatment drug potentialy that Overview proteins of

Protein (DPD) dehydrogenase Dihydropyrimidine (dUTPase) dUTP nucleotidohydrolase kinase (TK)Thymidine phosphorylaseThymine / factor ECGF1) (TP; growth Endothelial cell (TS) synthase Thymidylate deaminase (CDA) Cytidine Deoxycytidine kinase (dCK) 5’-nucleotidase (5NT) Table 5 Table

34 35 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

underpowered studies is fairly impossible due to differences in applied techniques or patient groups. Clinical practice affecting studies are not available yet. Good predictive genetic markers or marker sets are lacking. The specificity of a predictive marker needs to be high, as it will be used to discriminate between treatment options. Probably, panels of genes will be needed to obtain an adequate specificity. To find such gene panels, far more studies are needed. Looking at current data, a combination of the expression of DPD, TS, TP and OPRT, all four crucial enzymes in the metabolism of 5-FU, at least appears to be a promising approach in colorectal cancer [61-63,177-179] (table 5).

Future perspectives: the polygenetic approach

Despite the here summarised potential genes and proteins interfering with pyrimidine antagonist metabolism, only few factors indeed have been proven to affect chemotherapy efficacy and/or toxicity. Most consistent data is available regarding the role of TS, DPD and p53 in 5-FU chemotherapy, and that of TP in capecitabine chemotherapy (see table 1 and table 5). The impact of germ line DPD deficiency on 5-FU pharmacokinetics and the development of severe life threatening toxicity is obvious. Although screening on germ line DPD deficiency is currently believed to be too expensive, it should become standard clinical practice as soon as a rapid and cheap test becomes available. Regarding 5-FU efficacy, some attempts recently have been made to identify putative markers of response in tumour material for use in patient treatment guidance [89,131,176,180]. However, despite some promising studies, the overall data are difficult to interpret and not always in line with previous results in larger patient groups. This might, at least in part, be due to the complexity in transcription and translation of some of the genes discussed above (e.g. TS). Additional mechanistic studies are needed to explore this issue more in detail. Furthermore, as mentioned before, many available studies are insufficiently powered to reach statistical significance. Therefore, incorporation of pharmacogenomic issues in future phase II and III clinical trials should be encouraged. Another cause for inconsistency in current data may be found in the applied scientific methodology. Crucial factor in the search for predictive markers is the availability of a standardised, accurate, precise and robust test. Method validation, such as performed for the TS106 antibody for TS immunohistochemistry, is a prerequisite for standardised testing [181]. Subsequently, these quality controlled diagnostics should be tested not only retrospectively, but also in prospective multi-center (and multi-laboratory) clinical trials. Unfortunately, such tests are not available yet. Also causing inconsistency in the outcome of monogenetic studies may be the fact that the impact of other genes related to response and survival is ignored in the final calcula- tions. Since cancer is a genetically heterogeneous disease, the monogenetic approach is likely too simple. This implies that far more research is needed to gain more insight in the

34 35 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

exact role of genetic polymorphisms and gene expression levels on pyrimidine antagonist chemotherapy. The interaction between TS and p53, combined with the impact of several TS polymorphisms illustrates the complexity of pharmacogenomic research. The two most important approaches in current and near future research to find predictive marker sets will be the candidate gene and the microarray strategies. A broad range of candidate genes to focus on in future research has been summarised in this review. On the other hand, further developments in microarray techniques and proteomics may eventually lead to the identification of a subset of relevant genes for a certain drug in a certain tumour type. In the near future, identification of relevant germline polymorphisms, combined with relevant mRNA expression levels in tumour tissue, might permit more effective, individualised cancer chemotherapy. Ideally, a process of “chemotyping”, i.e. choosing the right chemotherapy regimen in the right dose, based on selected geno- typical and phenotypical information, should precede drug prescribing. The interdiscipli- nary Pharmacogenetics Anticancer Agents Research (PAAR) group is an example of an excellent initiative to coordinate research on these issues [182]. Eventually, well-controlled prospective clinical trials with adequate sample size and statistical power will be needed to demonstrate the surplus value of new concepts above current practice.

36 37 Chapter 2 Genetic factors influencing pyrimidine-antagonist chemotherapy

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