From the present study, it appears that total DPD deficiency is a rare event. Although pretreatment DPD activity cannot be a useful indicator for improving FU dose adaptation strategy, the identification of severe DPD deficiency (< 0.100 nmol/min/mg protein) could lead to starting the treatment with a markedly reduced FU dose or even to using an alternative chemotherapy regimen.

Although resistance to FU is multifactorial, the present clinical study suggests that FU catabolism in target cells is probably a determinant factor for FU responsiveness in cancer patients and justifies the clinical use of specific DPD inhibitors as FU biomodulators.

No abstract

More than 80% of an administered dose of fluorouracil (FU) is eliminated by catabolism through dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of pyrimidines (Diasio and Harris, 1989). Tuchman and colleagues (1985) were the first to describe a patient with severe FU toxicity associated with a pyrimidine metabolism disorder. On the basis of another case report, Diasio et al (1988) conducted a familial study, the conclusions of which suggested an autosomal recessive pattern of inheritance for DPD deficiency. Since then, similar pharmacogenetic syndromes have been reported by the same group (Harris et al, 1991;Lu et al, 1993), others (Houyau et al, 1993) and ourselves (Fleming et al, 1993). We recently performed a review analysis on previously published cases of DPD deficiency associated with FU toxicity. It appeared that a complete absence of DPD activity is extremely rare and even partial enzyme activity might result in more or less severe FU toxicity . In addition, the fact that among 15 cumulated cases (most with digestive cancer) 13 (87%) were women was very striking and suggestive of a sex-linked deficiency in DPD activity. We report on the clinical and pharmacological results from 19 cancer patients with more or less intense FU-related toxicity, who were phenotyped in our centre as carrying a DPD deficiency diagnosed in peripheral blood mononuclear cells. Different features were analysed including sex ratio, the toxicity profile, and a possible link between the intensity of DPD deficiency and the severity of FU toxicity. MATERIAL AND METHODS PatientsThis study represents a 3-year collection of blood lymphocytes taken from 53 consecutive patients treated by FU-based chemotherapy in different French institutions (general hospitals, cancer centres, private hospitals). These patients had experienced unpredicted more or less severe FU-related toxicity. Blood lymphocytes were collected in the respective hospitals within a period of 1 month after the FU toxicity episode. All lymphocyte samples were shipped in dry-ice to our laboratory. Among this group of 53 patients (23 men, 30 women, mean age 58, range 36Ð73), 19 exhibited a moderate or marked DPD deficiency (less than 70% of the mean population value, i.e. less than 150 fmol min Ð1 mg Ð1 protein; Etienne et al, 1994). A complete description of these 19 case reports is given in Table 1. We defined a toxicity score which was the sum of the toxicity grades (WHO classification) for mucositis, neutropenia, thrombocytopenia and digestive toxicity. The presence of neurotoxicity was graded 4. The maximal toxicity score was 20. Determination of DPD activityLymphocyte preparation was carried out in the hospital where the patient received the FU-based treatment. Because of a known circadian pattern of DPD activity (Harris et al, 1990), blood samples were drawn between 8 a.m. and 10 a.m. to minimize the influence of circadian variability. When patients developed severe haematological toxicity, a normalization of blood cell count (white blood cells) was awai...

Dihydropyrimidine dehydrogenase (DPD) degrades over 80% of administered 5-fluorouracil (5FU), thereby regulating the efficacy of this commonly used anticancer agent. DPD activity is highly variable (8-21-fold) and individuals with reduced activity have a high risk of 5FU toxicity. DPYD encodes DPD protein and 13 different mutations have been reported in DPD-deficient subjects. However, the contribution of these variant genotypes to polymorphic DPD activity in vivo is not clear. The previously described DPYD mutations are contained in 10 exons. These 10 exons were sequenced in a cohort of cancer patients with reduced (n = 23) or normal (n = 14) DPD activity to determine the contribution of each variant allele to low DPD activity in vivo. Eight of the 13 previously defined DPYD mutations (G62A, delta TCAT295-298, C703T, G1003T, G1156T, delta C1897, G2657A, and G2983T) were not detected. A previously defined exon 13 mutation (G1601A) was detected in three individuals with reduced DPD activity. An exon 14 splice donor site mutation (intron14 G1A) was detected in a normal DPD activity individual. It was demonstrated that T85C, A1627G and G2194A are common polymorphisms. A novel exonic mutation (T1679G) was detected in a patient with reduced DPD activity and 5FU toxicity. In addition, three novel common polymorphisms were detected in introns 10 and 13. Only three patients did not have any mutations and 30 had multiple DPYD mutations in the regions examined. However, only 17% (4/23) of the patients with a low DPD phenotype have a molecular basis for reduced activity. Although novel DPYD variants have been identified in this study, the 17 DPYD mutations now described do not entirely explain polymorphic DPD activity and toxic response to 5FU. These data emphasize the complex nature of the molecular mechanisms controlling polymorphic DPD activity in vivo.

These data indicate that the capacities to clear 5-FU are lower in women compared with men and are not influenced by age. It would be of interest to know whether this sex-related difference in 5-FU Cl may be clinically relevant by considering both toxicity and tumor response to 5-FU treatment.

Methylenetetrahydrofolate reductase (MTHFR) controls intracellular CH2FH4 concentrations (required for optimal fluoropyrimidine efficacy) by irreversibly converting CH2FH4 into 5-methyltetrahydrofolate. MTHFR 677C>T and 1298A>C polymorphisms are linked to altered enzyme activity. Thus, mutated MTHFR tumours should, in theory, be more sensitive to 5-fluorouracil (5FU) than wild-type tumours. MTHFR polymorphisms in position 677 and 1298 were analysed in 98 colorectal cancer patients with unresectable liver metastases (57 men, 41 women, mean age 64 years) receiving 5FU-folinic acid. 677C>T and 1298A>C genotypes were determined simultaneously by melting curve analyses on liver metastases. 677C>T genotype distribution was 46.9% wt/wt, 34.7% wt/mut and 18.4% mut/mut; that of 1298A>C was 52.0% wt/wt, 35.7% wt/mut and 12.3% mut/mut. The response rate was not related to 1298A>C genotype but was significantly linked to 677C>T genotype (response rate: 40%, 21% and 56% in wt/wt, wt/mut and mut/mut, respectively; P = 0.040), with an increased response rate in mut/mut tumours relative to wt/wt (odds ratio = 1.88). Thymidylate synthase activity measured in metastases was a significant predictor of 5FU responsiveness and the addition of the 677C>T genotype improved model prediction. MTHFR 1298A>C polymorphism was significantly linked to specific survival, with homozygous mutated patients having the worst prognosis (P = 0.009, relative risk = 2.48 in mut/mut versus wt/wt). MTHFR 1298A>C genotype remained a significant predictor in a multivariate analysis including metastasis characteristics. The results suggest that MTHFR genotypes are relevant and independent factors of patient outcome in 5FU-based treatment of advanced colorectal cancer.

P r o g n o s t i c V a l u e o f T u m o r a l T h y m i d y l a t e S y n t h a s e a n d p 5 3 i n M e t a s t a t i c C o l o r e c t a l C a n c e r P a t i e n t s R e c e i v i n g F l u o r o u r a c i l -B a s e d C h e m o t h e r a p y : P h e n o t y p i c a n d G e n o t y p i c A n a l y s e sBy Marie-Christine Etienne, Maurice Chazal, Pierre Laurent-Puig, Nicolas Magné, Christophe Rosty, Jean-Louis Formento, Mireille Francoual, Patricia Formento, Nicole Renée, Emmanuel Chamorey, André Bourgeon, Jean-François Seitz, Jean-Robert Delpero, Christian Letoublon, Denis Pezet, and Gérard MilanoPurpose: The aim of this multicenter prospective study was to evaluate the role of intratumoral parameters related to fluorouracil (FU) sensitivity in 103 metastatic colorectal cancer patients receiving FU-folinic acid.Patients and Methods: Liver metastatic biopsy specimens were obtained for all patients and primary tumor biopsy specimens for 54 patients. Thymidylate synthase (TS), folylpolyglutamate synthetase, and dihydropyrimidine dehydrogenase were measured by radioenzymatic assays; TS promoter polymorphism (2R/2R v 2R/3R v 3R/3R) was determined by polymerase chain reaction; and p53 protein and mutations were analyzed by immunoluminometric assay and denaturing gradient gel electrophoresis, respectively.Results: p53 mutations were observed in 56.7% of metastases. TS activity was significantly higher in 2R/3R tumors as compared with 2R/2R or 3R/3R. TS activity in metastasis was the only parameter linked to clinical responsiveness (responders exhibited the lower TS, P ‫؍‬ .047). Univariate Cox analyses demonstrated that TS activity in primary tumor (the greater the TS, the poorer the survival; P ‫؍‬ .040), TS promoter polymorphism in primary tumor (risk of death of 2R/3R v 2R/2R, 2.68; P ‫؍‬ .035), and p53 stop mutation in metastasis (risk of death of stop mutations v wild type, 3.14; P ‫؍‬ .018) were the only significant biologic predictors of specific survival. Stepwise analysis did not discriminate between TS activity and TS polymorphism.Conclusion: Present results confirm the value of tumoral TS activity for predicting FU responsiveness, point out the importance of detailed p53 mutation analysis for predicting survival, and suggest that TS genotype in primary tumor carries a prognostic value similar to that of TS activity. T HE BASIS OF THE chemotherapeutic strategy for the treatment of advanced colorectal cancer is currently broadening with the clinical emergence, among a plethora of potential candidates, of new active drugs such as irinotecan 1 and oxaliplatin. 2 It follows that fluorouracil (FU) is no longer the sole agent with significant antitumor activity in colorectal cancer. This new therapeutic context heightens the need for objective arguments when choosing the most active drug for a given colorectal tumor. Concerning FU itself 3 or new promising FU prodrugs such as capecitabine, 4 several tumoral candidates able to predict FU efficacy have been identified.Thymidylate synthase (TS), one of the key enz...