Standard-dose tegafur-uracil (UFT) is not a safe alternative in partial dihydropyrimidine dehydrogenase-deficient patients

Dihydropyrimidine dehydrogenase (DPD) deficiency is prevalent in 3-5% of the Caucasian population; however, the frequency of this pharmacogenetic syndrome in the Indian population and other racial and ethnic groups remains to be elucidated. We describe an Indian patient who presented to clinic for the treatment of gastric adenocarcinoma with 5-flurouracil (5-FU) therapy who subsequently was diagnosed with DPD deficiency by using the peripheral blood mononuclear cell (PBMC) DPD radioassay. This observation prompted us to examine the data generated from healthy (cancer-free) Indian subjects who were enrolled in a large population study to determine the sensitivity and specificity of the uracil breath test (UraBT) in the detection of DPD deficiency. Thirteen Indian subjects performed the UraBT. UraBT results were confirmed by PBMC DPD radioassay. The Indian cancer patient demonstrated reduced DPD activity (0.11 nmol/min/mg protein) and severe 5-FU toxicities commonly associated with DPD deficiency. Of the 13 Indian subjects [ten men and three women; mean age, 26 years (range: 21-31 years)] enrolled in the UraBT, 12 Indian subjects demonstrated UraBT breath profiles and PBMC DPD activity within the normal range; one Indian subject demonstrated a reduced breath profile and partial DPD deficiency. DPD deficiency is a pharmacogenetic syndrome which is also present in the Indian population. If undiagnosed, the DPD deficiency can lead to death. Future epidemiological studies would be helpful to determine the prevalence of DPD deficiency among racial and ethnic groups, allowing for the optimization of 5-FU chemotherapy.

e13134 Background: Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the metabolism of 5-fluorouracil (5-FU). Patients with a partial or complete DPD deficiency are at risk to develop severe toxicity after 5-FU administration. Uracil (U) is degraded in dihydrouracil (DHU) in a similar way as 5-FU. An oral uracil test dose might be useful to determine the systemic DPD activity by measuring uracil and its metabolite dihydrouracil in plasma. DPD deficiency is hypothesized to result in higher uracil levels and a reduced turnover of uracil into dihydrouracil. Methods: Uracil (500 mg/m2) was administered to 11 healthy volunteers with normal DPD activity (≥ 6 nmol/mg/hour) and 1 patient with colorectal carcinoma with a novel DPYD mutation and decreased DPD activity (4.6 nmol/mg/hour). DPD activity was measured in PBMC, as determined as described earlier. Blood samples were taken at t= 15, 30, 45, 60, 80, 100, 120, 150, 180, and 220 or 240 min after oral uracil intake. U and DHU pla...

Dihydropyrimidine dehydrogenase (DPD) deficiency is critical in the predisposition to 5-fluorouracil dose-related toxicity. We recently characterized the phenotypic [2-(13)C]uracil breath test (UraBT) with 96% specificity and 100% sensitivity for identification of DPD deficiency. In the present study, we characterize the relationships among UraBT-associated breath (13)CO(2) metabolite formation, plasma [2-(13)C]dihydrouracil formation, [2-(13)C]uracil clearance, and DPD activity. An aqueous solution of [2-(13)C]uracil (6 mg/kg) was orally administered to 23 healthy volunteers and 8 cancer patients. Subsequently, breath (13)CO(2) concentrations and plasma [2-(13)C]dihydrouracil and [2-(13)C]uracil concentrations were determined over 180 minutes using IR spectroscopy and liquid chromatography-tandem mass spectrometry, respectively. Pharmacokinetic variables were determined using noncompartmental methods. Peripheral blood mononuclear cell (PBMC) DPD activity was measured using the DPD radioassay. The UraBT identified 19 subjects with normal activity, 11 subjects with partial DPD deficiency, and 1 subject with profound DPD deficiency with PBMC DPD activity within the corresponding previously established ranges. UraBT breath (13)CO(2) DOB(50) significantly correlated with PBMC DPD activity (r(p) = 0.78), plasma [2-(13)C]uracil area under the curve (r(p) = -0.73), [2-(13)C]dihydrouracil appearance rate (r(p) = 0.76), and proportion of [2-(13)C]uracil metabolized to [2-(13)C]dihydrouracil (r(p) = 0.77; all Ps < 0.05). UraBT breath (13)CO(2) pharmacokinetics parallel plasma [2-(13)C]uracil and [2-(13)C]dihydrouracil pharmacokinetics and are an accurate measure of interindividual variation in DPD activity. These pharmacokinetic data further support the future use of the UraBT as a screening test to identify DPD deficiency before 5-fluorouracil-based therapy.

Lethal 5-fluorouracil toxicity associated with a novel mutation in the dihydropyrimidine dehydrogenase gene

BackgroundPretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive.MethodsAmong a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated.ResultsWhen [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a “wild-type” genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant.ConclusionsFrequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.

Pour ou contre le phénotypage/génotypage des patients traités par le 5-fluorouracile pour prévenir les effets indésirables ?

e19560 Background: Dihydropyrimidine dehydrogenase (DPD) catalyzes degradation of 5-fluorouracil (5FU). Patients with complete or partial DPD deficiency (0.1 and 3% of the population, respectively) can experience severe or lethal toxicities to 5FU, and impaired clearance may underlie many of the 1300 US 5FU toxicity deaths each year. Uridine triacetate prevents or diminishes toxicities when orally administered after 5FU overexposure, and has been used successfully as an investigational antidote in >50 patients following accidental overdoses. Methods: A colorectal cancer patient experienced Grade 3 and 4 GI and hematologic toxicities after a 96 hour infusion of 1000 mg/m2 5FU and was found to be DPD deficient (double mutation). The patient subsequently tolerated 50 and 100 mg 5FU bolus treatments in a modified FOLFOX regimen. However, during the second cycle, this DPD-deficient patient inadvertently received 1000 mg 5FU in 1 minute. Life-threatening toxicity was expected. Treatment with uridine triacetate (10 g q6h for 20 total doses) began within 8 hours. Results: In marked contrast to his first exposure to 5FU, the patient experienced no mucositis or additional cytopenia. Preclinical and mechanistic data predicted and support this observation. In a mouse model of DPD deficiency using ethynyluracil (2 mg/kg i.p.), a standard therapeutic 5FU dose of 100 mg/kg i.p. was lethal. Treatment with oral uridine triacetate (2 g/kg t.i.d. x 5 d) starting 2, 24 or 72 hours after 5FU resulted in 100%, 80% or 70% survival, respectively. Conclusions: Timely treatment with uridine triacetate appeared to prevent severe 5FU toxicity in a DPD-deficient patient and reduced mortality in DPD-inhibited mice receiving 5FU. Uridine triacetate has been shown previously to protect patients (and mice) from 5FU toxicities following 5FU overdose. Therefore, DPD-deficient patients who have received 5FU should also benefit from uridine triacetate treatment if the overexposure is identified soon enough after 5FU dosing. Therapeutic monitoring of 5FU during or after infusions would permit rapid detection of 5FU overexposure due to DPD-deficiency or other clearance defects, enabling the use of uridine triacetate as a potential antidote.

To the editor, We appreciate the comments from Dr Deenen and colleagues regarding their concerns on the management of dihydropyrimidine dehydrogenase (DPD)-deficient patients [Deenen et al. 2013], in response to our article [Cubero et al. 2012]. In fact, we agree that standard-dose tegafur-uracil (UFT) is not safe as a starting dose for those patients experiencing severe toxicity after receiving 5-fluorouracil (5-FU). However, the aim of our article is to clarify that UFT can still be an option for selected patients if a careful dosing schedule is employed. The statement that the presence of uracil in UFT creates an artificial DPD activity deficiency is a theoretical hypothesis mentioned by other authors [Niederhuber et al. 2004], including Dr Deenen in his previous case report [Deenen et al. 2010]. Interestingly, there seems to be a difference in safety profiles between 5-FU and UFT as it was demonstrated in at least two other randomized controlled trials. In the first, Douillard and colleagues described a multicenter phase III trial comparing UFT plus leucovorin (UFT/LV) and fluorouracil plus leucovorin (5-FU/LV) in previously untreated metastatic colorectal cancer. After including 380 patients (147 patients in each arm without prior adjuvant therapy), they found that there was statistically significantly less severe leukopenia (2% versus 12%; p < 0.001), less severe mucositis (2% versus 16%; p < 0.001) and lower rates of febrile neutropenia (1% versus 8%; p < 0.001) in the UFT/LV arm as compared with the 5-FU/LV arm [Douillard et al. 2002]. Likewise, Carmichael and colleagues presented similar results in a study including 816 patients (652 patients without previous chemotherapy), also in untreated metastatic colorectal cancer. Once again, but in a much larger sample size, there was less severe leukopenia (<1% versus 19%; p < 0.001), less severe mucositis (1% versus 19%; p < 0.001) and lower rates of febrile neutropenia (0% versus 13%; p < 0.001) in UFT/LV arm in comparison with the 5-FU/LV arm [Carmichael et al. 2002]. Considering that the prevalence of partial DPD deficiency in the White population is approximately 3–5% [Lu et al. 1993, 1998; Etienne et al. 1994], reaching up to 12.3% in specific groups such as African-American women [Mattison et al. 2006], the lower percentages of severe side effects in studies of Douillard and colleagues and Carmichael and colleagues raise the possibility that some of the patients with partial DPD deficiency are not experiencing severe toxicity. Checking for mutations and polymorphisms in the coding region of the DPD gene is only a surrogate of DPD activity. Even patients heterozygote for some known mutations can have very low activity of DPD because there are other mechanisms involved in gene expression (epigenetic mechanisms). In our pilot study [Cubero et al. 2012], in order to corroborate the DPD-gene sequencing, we selected only patients presenting severe toxicity (grade 3 and 4) after the first cycle of 5-FU-based chemotherapy, another surrogate of DPD deficiency. The fact that not one patient developed any severe toxicity in any of the UFT cycles is a proof-of-principle that this drug is still feasible for some patients with partial DPD deficiency. However, after a severe toxicity due to 5-FU and a DPD-gene polymorphism is identified, if UFT is still considered, we strongly recommend an empirical reduction in UFT dose in the first cycle of chemotherapy. We do not recommend the use of UFT for those patients proven homozygote for DPD-gene mutation.

The objective of this study was to evaluate the safety of using tegafur-uracil (UFT) in colorectal cancer patients with partial dihydropyrimidine dehydrogenase (DPD) deficiency. The study included five colorectal cancer patients who presented with acute toxicity (grades 3 and 4) after being given the first cycle of chemotherapy using 5-fluorouracil. The DPD deficiency was confirmed by gene sequencing. After a full recovery from all side effects, we changed the regimen to UFT (300 mg/m(2)/day) associated with leucovorin (90 mg/day) for 21 days, with an empirical dose reduction of at least 10% in the first cycle. We prospectively analysed 22 UFT cycles in 5 patients. We did not observe any episodes of grade 3 or 4 toxicity. The predominant toxicities were of grades 1 and 2 (nausea, vomiting and diarrhoea). Here, we demonstrate a complete absence of severe toxicity in all patients and cycles analysed. We believe that UFT is a safe alternative for the treatment of patients with partial DPD deficiency.

Dihydropyrimidine dehydrogenase (DPD) catalyzes the degradation of thymine, uracil, and the chemotherapeutic drug 5-fluorouracil. To identify patients suffering from complete or partial DPD deficiency and to identify pitfalls that can preclude the proper diagnosis of patients with partial DPD deficiency, a sensitive and accurate assay is necessary. The activity of DPD was measured using [4-(14)C]thymine followed by separation of substrate and products with reversed-phase HPLC with on-line detection of the radioactivity. Complete baseline separation of radiolabeled thymine and all degradation products was achieved within 15 min. The detection limit for dihydrothymine was 0. 4 pmol. In lymphocytes, the DPD activity deviated from linearity at low protein concentrations (<0.2 g/L). Profoundly decreased activity of DPD was detected in the peripheral blood mononuclear cells (PBM cells) of two tumor patients when measured at low protein concentrations. Low DPD activity comparable to that observed in obligate heterozygotes was initially detected in PBM cells, containing substantial amounts of myeloid cells, from a patient suffering from 5-fluorouracil toxicity. However, after the patient experienced full clinical recovery, normal DPD activity was observed in the PBM cells. No significant differences in DPD activity were observed between exponentially growing fibroblasts and those at confluence. The range of DPD activities of obligate heterozygotes overlaps the range of DPD activities of controls. The low activity of DPD measured in PBM cells containing myeloid cells or that measured at a low protein concentration in the assay mixture is not indicative of heterozygosity for a mutant DPD allele. Although fibroblasts are suitable to establish a complete deficiency of DPD, unambiguous detection of heterozygotes is not possible.

We conducted a prospective study on a large set of cancer patients in an attempt to evaluate the incidence of complete or partial dihydropyrimidine dehydrogenase (DPD) deficiency as found in peripheral mononuclear cells (PMNC). One hundred eighty-five unselected consecutive cancer patients were included. The population consisted of 152 men (mean age, 62.1 years; range, 35 to 90) and 33 women (mean age, 59.2 years; range, 36 to 77). Sixty-eight were head and neck patients treated by a 5-day continuous infusion of fluorouracil (FU; starting dose, 1 g/m2/d, with dose adaptation based on pharmacokinetics) for which DPD activity was measured 2 to 3 days before FU administration (94 cycles analyzed). PMNC-DPD activity was measured by a radio-enzymatic assay using carbon-14-FU. DPD activity in the entire population showed a unimodal distribution, which globally fits a gaussian distribution. Mean and median DPD activity values were 0.222 and 0.211 nmol/min/mg protein, respectively (range, 0.065 to 0.559). No total DPD deficiency was found. Multifactor analysis of variance showed that liver function (biologic evaluation) and age did not influence DPD activity, but that DPD activity was, on average, 15% lower in women (0.194 nmol/min/mg protein) than in men (0.228 nmol/min/mg protein) (P = .03). No difference was demonstrated between premenopausal and postmenopausal women. In patients treated with FU, the risk of developing side effects was not linked to pretreatment DPD activity. FU-related toxicity was linked to FU systemic exposure. The correlation between pretreatment DPD activity and FU systemic clearance (CI) was weak (n = 90, linear regression r = .31, P = .002). Pretreatment DPD activity in patients who required a dose reduction was not significantly different from DPD activity in patients who did not require dose modification. 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.

e13505 Background: The enzyme dihydropyrimidine dehydrogenase (DPD) catalyzes the first step in degradation of 5-fluorouracil (5FU). Patients with complete or partial DPD deficiency (about 0.1% and 3% of the population, respectively) can experience severe or lethal toxicities after receiving standard doses of 5FU, and impaired clearance may underlie a large fraction of the ∼1,300 deaths per year due to 5FU toxicity in the US. Orally-administered uridine triacetate (vistonuridine) prevents or diminishes toxicities when administered after 5FU overexposure, and has been used successfully as an antidote in more than 28 patients to date who had received accidental overdoses of 5FU. Because DPD deficiency may alter 5FU clearance kinetics (versus 5FU overdoses in the setting of normal DPD activity), studies were conducted on reversal of 5FU toxicity in mice pretreated with the potent DPD inhibitor ethynyluracil (eniluracil; EU) to model DPD deficiency. Methods: Balb/C mice received 2 mg/kg EU i.p., followed by 100 mg/kg 5FU (weekly bolus MTD in mice). Groups of 10 mice each then received either vehicle or oral uridine triacetate (2,000 mg/kg t.id. × 5) beginning at a range of times after 5FU. Survival was monitored. Results: 100 mg/kg 5FU was lethal in mice pretreated with EU. Uridine triacetate administration beginning within 24 hours after EU + 5FU resulted in survival of all the mice in the treatment groups. Fewer mice survived if treated with uridine triacetate at later time points after EU plus 5FU. Conclusions: Timely treatment with uridine triacetate reduced 5FU toxicity and mortality in DPD-inhibited mice. Its benefit has previously been demonstrated in patients (and mice) overdosed with 5FU. Therefore, DPD-deficient patients who have received 5FU should also benefit from treatment with uridine triacetate if the deficiency is identified soon enough after 5FU dosing. Therapeutic monitoring of 5FU during or after infusions could permit rapid detection of 5FU overexposure due to DPD-deficiency, enabling the use of uridine triacetate as an antidote in DPD-deficient patients. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Wellstat Therapeutics Corporation

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases and it catalyzes the reduction of uracil and thymine to 5,6-dihydrouracil and 5,6-dihydrothymine, respectively. In children, a deficiency of DPD is often accompanied by a neurological disorder but a considerable variation in the clinical presentation among these patients has been reported1. In these patients, a large accumulation of uracil and thymine has been detected in urine, blood and in cerebrospinal fluid whereas no activity of DPD could be detected in fibroblasts and mononuclear cells . The detection of more than 30 patients of various nationalities with a (partial) DPD deficiency within 15 years in The Netherlands alone suggest that this type of inborn error is less rare than previously assumed 1, 2 The recent cloning of the cDNA coding for human DPD and the sequence of the entire human DPD gene3 (DPYD) has allowed the detection of the defects at the molecular level. Identification of disease-causing mutations in the DPD gene will allow rapid pre-screening of patients at risk.

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk of developing severe 5-FU-associated toxicity. We evaluated the importance of DPD deficiency, gender and the presence of the IVS14+1G>A mutation in the etiology of 5-FU toxicity. In 61% of cases, decreased DPD activity could be detected in peripheral blood mononuclear cells. Furthermore, the number of females (65%) in the total group of patients appeared to be higher than the number of males (35%) (p = 0.03). Patients with partial DPD deficiency appeared to have a 3.4-fold higher risk of developing grade IV neutropenia than patients with normal DPD activity. Analysis of the DPYD gene of patients suffering from grade IV neutropenia for the presence of the IVS14+1G>A mutation showed that 50% of the patients investigated were heterozygous or homozygous for the IVS14+1G>A mutation. Adopting a threshold level for DPD activity of 70% of that observed in the normal population, 14% of the population is prone to the development of severe 5-FU-associated toxicity. Below this threshold level, 90% of individuals heterozygous for a mutation in the DPYD gene can be identified. Considering the common use of 5-FU in the treatment of cancer, the severe 5-FU-related toxicities in patients with low DPD activity and the apparently high prevalence of the IVS14+1G>A mutation, screening of patients at risk before administration of 5-FU is warranted.

2003 Background: Although DPD deficiency is a well established cause of severe FU-related toxicities, relationships between the depth of the deficiency and the intensity of the toxicity is still poorly documented. We analyzed DPD activity in a large population of patients with FU-related toxicities. Methods: Blood lymphocytes from 144 consecutive cancer patients having developed FU toxicities were collected from different French institutions between January 1993 and July 2004 (53 men, 91 women; mean age 57, extremes 31–94). DPD activity was measured with a radioenzymatic HPLC assay. The IVS14+1G>A mutation was analyzed in 102 patients (RFLP assay). Results: Grade 3–4 toxicity (WHO classification) was 64% for mucositis, 58% for neutropenia, 42% for thrombopenia, 19% for diarrhea and 14% for neurotoxicity. Toxicity led to patient death in 9 cases (8 women, 1 man). DPD activity ranged from 8 to 504 pmol/min/mg (mean 200, median 188, N=144). Nine patients had an activity < 50 pmol/min/mg (severe deficiency) and 19 had an activity between 50 and 100 pmol/min/mg (partial deficiency), thus accounting for a total of 19% deficient patients. The relative risk of developing grade 3–4 toxicity in DPD deficient patients relative to non-deficient patients was 7.69 for neurotoxicity (Fisher’s Exact test, p< 0.001), 2.93 for diarrhea (p<0.001), 1.73 for thrombopenia (p=0.01), 1.63 for mucositis (p<0.001) and 1.59 for neutropenia (p=0.005). The lower the DPD activity, the higher the mucositis, neutropenia or diarrhea grading (Spearman rank correlations, p< 0.03). Toxic deaths were significantly related to low DPD activity (Mann-Whitney p=0.002), with 7 patients out of 9 exhibiting a DPD deficiency. The DPYD mutation (wt/mut) was detected in only 2 patients; both exhibited a low DPD activity (44 and 142 pmol/min/mg) and developed grade 4 mucositis, neutropenia, thrombopenia and grade 3 neurotoxicity without toxic death. Conclusions: 1- Patients with normal DPD activity may develop FU-related toxicities. 2- The intensity of the FU toxicity is related to the severity of the DPD deficiency. No significant financial relationships to disclose.