ArticleLiterature Review

Routine Dihydropyrimidine Dehydrogenase Testing for Anticipating 5-Fluorouracil-Related Severe Toxicities: Hype or Hope?

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

5-Fluorouracil (5-FU) is a mainstay for treating colorectal cancer, alone or more frequently as part of combination therapies. However, its efficacy/toxicity balance is often limited by the occurrence of severe toxicities, showing in about 15%-20% of patients. Several clinical reports have shown the deleterious effect of dihydropyrimidine dehydrogenase (DPD) genetic polymorphism, a condition that reduces the liver detoxification step of standard dosages of 5-FU, in patients undergoing fluoropyrimidine-based therapy. Admittedly, DPD deficiency accounts for 50%-75% of the severe and sometimes life-threatening toxicities associated with 5-FU (or oral 5-FU). However, technical consensus on the best way to identify patients with DPD deficiency before administrating 5-FU is far from being achieved. Consequently, no regulatory step has been undertaken yet to recommend DPD testing as part of routine clinical practice for securing the administration of 5-FU. This review covers the limits and achievements of the various strategies proposed so far for determining DPD status in patients scheduled for 5-FU therapy.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Folfox), and can be further combined with the latest available targeted therapies such as anti-VEGF or anti-EGFR1 monoclonal antibodies, making this drug the backbone of most treatments in digestive oncology [1]. Owing to the number of patients treated worldwide, limiting the occurrence of severe 5-FU-related side effects is a major issue, and it is known that patients with dihydropyrimidine dehydrogenase (DPD) deficiency, a pharmacogenetic syndrome resulting in partial or total loss of ability to detoxify 5-FU in the liver (Figure 1), will experience severe/lethal toxicities [2]. Depending on the regimen, administration of standard 5-FU usually leads to 15-30% of severe toxicities, and about 1% of toxic deaths have been regularly reported in the literature [3]. ...
... DPD deficiency is a pharmacogenetic syndrome associated with increased risk of developing life-threatening toxicities in patients receiving a 5-FU-containing regimen. Admittedly, 30 to 80% of the severe toxicities recorded after 5-FU intake could be attributable to impaired DPD activity in the liver [2,11] and DPD deficiency is an issue in patients undergoing oral capecitabine as well [9,12]. In this respect, developing strategies to anticipate treatment-related toxicities by the pre-identification of DPD deficient patients and subsequent dose tailoring should improve the efficacytoxicity balance of this widely prescribed anticancer drug [7]. ...
... In this respect, developing strategies to anticipate treatment-related toxicities by the pre-identification of DPD deficient patients and subsequent dose tailoring should improve the efficacytoxicity balance of this widely prescribed anticancer drug [7]. Defining the best strategy to evaluate the actual DPD status is a controversial issue, and the utility of screening patients on the DPYD genotype remains widely debated today [2,13]. Several variants (i.e. ...
Article
Full-text available
Aims: 5-FU is the backbone of most regimens in digestive oncology. Administration of standard 5-FU leads to 15-30% of severe side-effects, and lethal toxicities are regularly reported with fluoropyrimidine drugs. DPD deficiency is a pharmacogenetic syndrome responsible for most cases of life-threatening toxicities upon 5-FU intake, and pre-treatment checking for DPD status should help to reduce both incidence and severity of side-effects through adaptive dosing strategies. Methods: We have used a simple method for rapidly establishing the DPD phenotype of patients with cancer and used it prospectively in 59 routine patients treated with 5-FU-based therapy for digestive cancers. No patient with total DPD deficiency was found but 23 % of patients exhibited poor metabolizer phenotype, and one patient was phenotyped as profoundly deficient. Consequently, 5-FU doses in poor metabolizer patients were cut by an average 35% (2390 ± 1225 mg VS. 3653 ± 1371 mg, p < 0.003, t test). Results: Despite this marked reduction in 5-FU dosing, similar efficacy was achieved in the two subsets (clinical benefit: 40 VS. 43 %, stable disease: 40 VS. 37%, progressive disease: 20 % in both subsets, p = 0.893, Pearson's Chi-Square). No difference in toxicities was observed (p = 0.104, Fischer Exact Test). Overall, only 3% of early severe toxicities were recorded, a value markedly lower than the ones usually reported with 5-FU. Conclusions: This feasibility study shows how simplified DPD-based adaptive dosing of 5-FU can reduce sharply the incidence of treatment-related severe toxicities while maintaining efficacy as part of routine clinical practice in digestive oncology.
... Over the last two decades, the association between DPD-enzyme deficiency and the occurrence of severe fluorouracil-related toxicity has been extensively studied. Patients receiving 5-FU-based chemotherapy may develop severe to life-threatening adverse events, including neutropenia, neutropenic infections, stomatitis, diarrhea, and alopecia, and it is estimated that DPD deficiency accounts for 50-75% of the cases of severe side effects [9]. ...
... Over 50 genetic variants have been identified in the DPYD gene coding regionhowever, the majority without functional consequences on enzymatic activity [9,10]. The most prominent and most studied DPYD variant is a point mutation in the splice site of intron 14 (c.1905 + 1G > A, synonyms IVS14 + 1G > A or DPYD*2A), responsible for up to 29% of reported grade III-V toxicities following fluorouracil administration [13]. ...
... Conflicting results were seen in a more recent prospective trial, which concluded that severe toxicities could only be marginally attributed to DPYD gene polymorphism [14]. Furthermore, it is suggested that additional enzymes and polymorphisms in various downstream acting genes may also play a role in 5-FU degradation and toxicity [9]. The pronounced variability in the DPYD coding sequence, together with contradictory results from genetic studies, causes marked difficulties in genotype-phenotype correlations and presents a major limitation to the application of a genotype-based strategy to predict severe fluorouracil toxicity in daily practice [9]. ...
Chapter
Full-text available
The chemotherapeutic options have increased dramatically in patients with gastrointestinal cancer and have led to an improved outcome. With this, an in-depth understanding of the side effects of chemotherapy is becoming increasingly important in order to minimize the negative impact of the use of these agents. Chemotherapeutic agents have a long list of potential side effects. In this chapter, we focus specifically on some of the more common and/or more relevant and challenging side effects related to frequently used agents in gastrointestinal cancer. The fluoropyrimidines may cause cardiac toxicity, most frequently angina-like chest pain. The knowledge of the catabolism of fluorouracil has led to the possibility of testing for dihydropyrimidine dehydrogenase (DPD) in order to avoid serious fluorouracil-related toxicity in patients with DPD deficiency. Oxaliplatin-induced neurotoxicity is probably the most important clinical problem associated with the administration of oxaliplatin. With the increasing use of oxaliplatin, hypersensitivity reactions are more frequently reported and become challenging in clinical practice. The introduction of the targeted agents in colorectal cancer led also to specific problems: the anti-VEGF-related side effects, of which arterial thrombosis and gastrointestinal perforation, although relatively rare, are very relevant for the patient, and the anti-EGFR-related side effects, including skin rash, hypomagnesemia, and allergic reactions, are common. Understanding the underlying causes, mechanisms, risk factors, and developing treatment guidelines has made these side effects often more acceptable for many patients. However, the side-effect profile always has to be balanced against the activity and benefit of the anticancer agents.
... A major mechanism of resistance to 5-FU is TS overexpression [8]. Higher activity of the rate-limiting enzyme for 5-FU catabolism dihydropyrimidine dehydrogenase (DPD) was also associated with reduced response to 5-FU [9,10]. However, a previous prospective trial using the expression of TS and DPD to tailor the choice of first-line chemotherapy with 5-FU/LV in patients with advanced CRC resulted in a response rate (RR) of 35%, which is double than the expected RR in unselected patients, but did not confirm the high RR reported in retrospective studies. ...
... Less DNA aberrations were observed in the non-responders compared with the responders, especially for losses (P < 0.03). The median number of chromosomal alterations of the responders was 7.0 (range [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], with a median number of 3.0 gains (range 1-8) and 3.5 losses (range 0-12). For the non-responders, the median chromosomal alterations was 4.0 (range 0-14), with a median number of 2.5 gains (range 0-9) and 0 losses (range Fig. (4). ...
Article
PURPOSE: High-throughput arrays are opening new opportunities for our understanding of the molecular aberrations underlying the outcome of colorectal cancer (CRC) in response to specific therapies. The aim of the present study was to evaluate whether genome-wide DNA copy number profiles of CRC specimens can predict the clinical outcome to irinotecan and 5-fluorouracil (5-FU) or pemetrexed treatments. EXPERIMENTAL DESIGN: High-resolution DNA copy number profiles were obtained by 44K oligonucleotide-based array-comparative-genomic-hybridization (aCGH). DNA was isolated from formaldehyde-fixed paraffin-embedded primary tumors of 34 patients with advanced CRC, treated within a randomized phase II trial of pemetrexed+irinotecan (ALIRI) vs. leucovorin-modulated-5-FU + irinotecan (FOLFIRI). RESULTS: Unsupervised hierarchical cluster analysis of the aCGH data revealed two clusters significantly correlated with response status (P=0.03). Most frequently observed chromosomal aberrations (>50%) in the 9 responders were losses of 18q (78%), losses of 18p (72%), 8p22-p23.1 (56%), and 17p11.2 (67%), and gains of 20p13-q13.3 (73%), the whole chromosome 13 (78%), 7p11.2-22.3 (56%), and 8q24.21 (56%). After multiple correction gains in 8q24.21and in 20p13-q13 as well as losses of 18p and 17p11 were significantly associated with response. The analysis of patients categorized for different treatments showed that tumors of patients with better response after FOLFIRI had specific profiles of chromosomal aberrations, including significantly more losses at chromosome 18, fewer losses at chromosome 17, and more gains at chromosomes 20 and 8. Similar results were observed in the ALIRI arm. However, in contrast to the results observed in the patients treated in the ALIRI arm, response to FOLFIRI was correlated with a higher number of losses at chromosome 1, including the 1p36 region (P<0.001). The aberrations in this region were also significantly different in the non-responders in the ALIRI (N=15) vs FOLFIRI (N=10) arm (P=0.02). However, due to the small sample size of the ALIRI and FOLFIRI groups, the relevance of these findings should be interpreted with caution CONCLUSIONS: Genome-wide DNA profiling of CRC revealed several genomic loci, of which the copy number status may serve as predictive marker for clinical outcome after the commonly used FOLFIRI regimen, and large prospective studies are ongoing. Some of these aberrations, but also other specific aberrations, affected the activity of the pemetrexed-irinotecan combination, and should be further studied to explain the biological basis of response to these drugs and optimize their use. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2146.
... In a retrospective study, Ciccolini et al. demonstrated that 71% of severe toxicities and 80% of toxic deaths Clinical Biochemistry xxx (2016) xxx-xxx could be related to functional DPD deficiency [8]. Thus, the assessment of individual DPD activity has been proposed as a means to individualize 5-FU administration before the first dose [4,[8][9][10][11][12]. ...
... Alongside DPYD genotyping, several phenotypic methods have been proposed for establishing, indirectly, the presence or absence of a DPD deficiency status. These approaches have manly focused on the measurement of endogenous plasma uracil (U) to dihydrouracil (UH 2 ) ratio [8][9][10]18,19] or, alternatively, the ratio at defined time points after administration of a loading dose of U [20,21]. Monitoring the [UH 2 ]/[U] ratio and comparing it with a toxicity cut-off value, determined from a reference population, should allow the detection of patients at risk and subsequently lead to a possible dose adjustment [4]. ...
Article
Objective: The aim of this study was to evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratio and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. Methods: Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer, before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *7, *2A, *13 and Y186C. Data on toxicity included grade 1 to 4 neutropenia, mucositis, diarrhea, nausea/vomiting and cutaneous rash. Results: 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P<0.01). A cut-off of 1.16 for salivary ratio was set (AUC 0.842), with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 (AUC 0.746), revealed a 71% sensitivity and 76% specificity. Conclusions: DPYD genotyping for alleles 7, *2A, *13 and Y186C was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.
... DPYD gene alterations are present in approximately 3-5% of the Caucasian population, with some debate over the effect of sex and ethnicity [7]. The most common mutation (1% of the general population), DPYD*2A (c.1905+1G>A), results in a guanine base being replaced by adenine at the intron boundary of exon 14, resulting in a splicing defect [8,9]. This leads to skipping of an entire exon (165 bp mRNA deletion) and consequently formation of a non-functioning enzyme [6]. ...
... Broad gene analysis by next-generation sequencing has led to the possibility of evaluating entire genome sequencing with an affordable cost and a short turnaround time. However, the high number of genetic polymorphisms in DPYD makes it difficult to make genotype-phenotype correlations, because not all these SNPs will lead to the same reduction in enzymatic activity [8,16]. Recommendations by groups such as the CPIC have based at least some of their guidelines on in vitro work, with the resulting conclusions that the variants DPYD*4, DPYD*5, DPYD*6 and DPYD*9A confer normal enzyme activity [19]. ...
Article
Background: 5-Fluorouracil (5-FU) is an agent frequently used in the treatment of solid cancers. A deficiency in the enzyme that catabolizes 5-FU leads to severe toxicity. The gene responsible for this enzyme is DPYD, located on chromosome 1q22. The most prevalent alteration described is DPYD*2A, which leads to a splicing defect and thus skipping of the translation of an entire exon. The objectives of this retrospective study were to describe the frequencies of DPYD gene mutations in a Belgian population and to correlate them with the grade of toxicity. Methods: This was a retrospective, single-center study conducted at the University Hospitals Leuven, by reviewing a database of patients screened for DPYD gene mutations between May 2009 and June 2015 after prolonged grade 3-4 toxicity. Polymerase chain reaction sequencing of exons 2, 6, 10, 11, 13, 18, 19 and 22, and pyrosequencing of exon 14 were performed by an in-house laboratory. Results: Of the 80 patients screened, 65 were heterozygous or compound heterozygous for DPYD and 3 had a homozygous mutation. The most prevalent mutation in our population was DPYD*9A. Conclusions: Despite previous reports, in our small retrospective study the most prevalent variation in patients with severe adverse events was DPYD*9A. As this variant has previously been reported to be benign, we suggest that screening for dihydropyrimidine dehydrogenase deficiency should be extended across multiple exons of the DPYD gene.
... It is the rate limiting enzyme degrading over 80% of the drug to its inactive metabolite 5-fluoro-5,6-dihydrouracil [9,14,15]. Because of this, DPD is an important factor for efficacy [16,17], as well as the development of toxicity [10]. DPD is encoded by the gene DPYD, which consists of 23 exons on chromosome 1p22 [18]. ...
... With the increasing incidence of cancer the number of patients who are likely to be treated with a fluoropyrimidine drug will increase, as well as the number of patients that would be saved from 5-FU or CAP induced severe toxicity when using pre-treatment genetic screening. In 2010 Ciccolini et al. already pointed out that it was time to mandate the integration of systematic prospective testing for DPYD as part of routine clinical practice in oncology [10]. Based on the arguments given above we truly believe it is time to add upfront DPYD genotyping to the current guidelines and to start implementation of DPYD screening without further delay. ...
Article
5-Fluorouracil (5-FU) and capecitabine (CAP) are among the most frequently prescribed anticancer drugs. They are inactivated in the liver by the enzyme dihydropyrimidine dehydrogenase (DPD). Up to 5% of the population is DPD deficient and these patients have a significantly increased risk of severe and potentially lethal toxicity when treated with regular doses of 5-FU or CAP. DPD is encoded by the gene DPYD and variants in DPYD can lead to a decreased DPD activity. Although prospective DPYD genotyping is a valuable tool to identify patients with DPD deficiency, and thus those at risk for severe and potential life-threatening toxicity, prospective genotyping has not yet been implemented in daily clinical care. Our goal was to present the available evidence in favour of prospective genotyping, including discussion of unjustified worries on cost-effectiveness, and potential underdosing. We conclude that there is convincing evidence to implement prospective DPYD genotyping with an upfront dose adjustment in DPD deficient patients. Immediate benefit in patient care can be expected through decreasing toxicity, while maintaining efficacy.
... Most reports associate 5-FU toxicity with DPYD deficiency [4]. However, DPYD deficiency occurs in about 3 % of all cancer patients [5], whereas 5-FU-related side effects take place in 15-20 % of all patients [6]. Thus, DPYD deficiency only partially explains 5-FU-related toxicity. ...
... Particularly cardiac toxicity does not correlate with DPYD deficiency [10]. Clinical decisions based solely on the screening of DPYD deficiency might therefore be misleading [5], [11]. ...
Conference Paper
Identification of patients with increased risk of 5-fluorouracil (5-FU)-related toxicity is an important challenge for cancer treatment. Research often focus on dihydropyrimidine dehydrogenase (DPYD) deficiency in this context. However, patients with normal DPYD activity may also develop life-threatening 5-FU adverse effects. DPYD initiates the catabolic route of 5-FU generating metabolites such as fluoroacetate (FAC). The catabolite FAC is known to inhibit the TCA cycle enzyme aconitase, which is supposed to impair mitochondrial energy metabolism. Therefore, we aim for a systems understanding of the association of 5-FU-related cardiac side effects with aconitase inhibition caused by FAC. Using a mitochondrial model of cardiomyocytes we found strong depletion of ATP production and citrate accumulation as main effects of aconitase inhibition. Shadow price analysis revealed that the uptakes of valine, arginine, proline and glutamate are most effective in compensating the impairment of energy metabolism. Our findings suggest that 5-FU catabolism contributes to the occurrence of cardiac adverse effects and are the basis for further biomarker identifications and development of side effect treatment.
... Reported interpatient variability in BSA is usually in the range of 10-15%, whereas the observed variation in drug-clearance rate is frequently >30%, suggesting that adjusting drug dosing according to BSA is unlikely to significantly narrow the variability in efficacy and toxicity 40 . For instance, the widely prescribed chemotherapeutic agent 5-fluorouracil (5-FU) is typically proscribed on a mg/m² basis, despite several clinical reports demonstrating that 5-FU clearance is not related to patient BSA 41 , and that other covariates (sex or dihydropyrimidine dehydrogenase status) should instead be used to determine dosage 41,42 . ...
Article
Full-text available
Computational oncology is a generic term that encompasses any form of computer-based modelling relating to tumour biology and cancer therapy. Mathematical modelling can be used to probe the pharmacokinetics and pharmacodynamics relationships of the available anticancer agents in order to improve treatment. As a result of the ever-growing numbers of druggable molecular targets and possible drug combinations, obtaining an optimal toxicity-efficacy balance is an increasingly complex task. Consequently, standard empirical approaches to optimizing drug dosing and scheduling in patients are now of limited utility; mathematical modelling can substantially advance this practice through improved rationalization of therapeutic strategies. The implementation of mathematical modelling tools is an emerging trend, but remains largely insufficient to meet clinical needs; at the bedside, anticancer drugs continue to be prescribed and administered according to standard schedules. To shift the therapeutic paradigm towards personalized care, precision medicine in oncology requires powerful new resources for both researchers and clinicians. Mathematical modelling is an attractive approach that could help to refine treatment modalities at all phases of research and development, and in routine patient care. Reviewing preclinical and clinical examples, we highlight the current achievements and limitations with regard to computational modelling of drug regimens, and discuss the potential future implementation of this strategy to achieve precision medicine in oncology.
... Rappelons que, hélas, un anticalcique (Pexid) et un antidiabétique (Phenformine) ont par méconnaissance des mutations liées au CYP2D6, entraînésp ar accumulation dans le foie et le cerveau un grand nombre de décès. [20] AIrinotecan (UDPGT-1A1) [17] AInterferon (IL28B) [24] Ces examens sont actuellement essentiellement demandéspar les services de cardiologie, de psychiatrie, de cancérologie, de médecine générale et de transplantation d'organes. ...
... The DPD gene is well-characterized and a number of singlenucleotide polymorphisms (SNPs) have been associated with reduced DPD activity. Genetic screening of DPD before administering 5-FU to patients may be of value in avoiding toxicity and enabling more appropriate chemotherapeutic choices, although no regulatory steps dictate testing is yet mandatory (60,61). A number of screening strategies for DPD mutations are available (62). ...
Article
Personalized Medicine evolved from the genomics era and allows disease prediction based on genetic mutation, and development of individualized health care, both preventive and responsive, as a consequence of a patient's own genetic features. Personalized medicine for cancer is similarly proactive and individualized, based on genetic information and used to manage cancer risk and disease for solid and lymphoproliferative cancers, those with a hereditary basis and those arising spontaneously. In the developing world, cancers now kill more people than infectious disease and while many resource-limited countries still lack basic facilities to care for cancer patients, middleincome countries, such as Panama, are beginning to make simple applications of personalized medicine for cancer diagnosis and treatment. These applications focus on cancers which affect the greatest number of people and those for which proven tests and therapies already exist. Three such cancers are lung, breast and colorectal cancers, which have similarities in the biochemical dysfunctions at their foundations. A limited and affordable portfolio of genetic tests could be established by developing world diagnostic laboratories to aid oncologists in risk assessment, diagnosis, prognosis and pharmaceutical choice in the personalized management of cancer. The establishment and clinical use of these tests in developing world nations will require innovative models of financing and strengthening of human resources and technical and legislative infrastructure.
... Por otro lado, se han desarrollado técnicas cromatográficas para detectar diferencias en la actividad de la DPD que han sido causadas por polimorfismos genéticos. 15 Estas técnicas ofrecen herramientas tanto para el médico como para el paciente antes de comenzar el tratamiento con 5-FU, ya que el grupo de riesgo se determina a priori, a partir de la detección de alelos de riesgo o anormalidades en la actividad enzimática. De esta forma se pueden modificar las dosis o incluso considerar otras vías terapéuticas para la enfermedad. ...
Article
Recent progress in medical knowledge has indicated that both clinical and biological markers will determine the response to different medical treatments: age, gender and genetics will determine the success of treatment. Genetic variability in this respect is fundamental and determines efficiency and safety of drugs, as well as susceptibility and illness' development. Fortunately, personalized medicine now offers individually tailored treatment strategies for each patient's needs. This is of outmost importance in oncology, since treatment is per se toxic and the commonly found low serum drug concentrations result in low treatment efficacy. Personalized medicine will allow a better approach to this, until now, a poorly managed disease. In this review we intent to raise awareness of personalized medicine and of clinical pharmacologic monitoring, with the aim to achieve adequate levels of efficacy and safety in the use of the cytotoxic drug 5-Fluorouracil (5-FU). Additionally, the importance of pharmacogenomics for the use of 5-FU is discussed. We designed this discussion towards medical practitioners challenged with treatment decisions every day, together with their patients.
... (The project focuses on gene-, cell-and pharmaceutical therapy for rare diseases and is planned for five years (2015–2019) [22]. In general, as for now, despite high expectation in the U.S., Germany and other countries, Personalized Medicine has not become a medical standard for many conditions [23,24] and there is an ongoing discussion as to whether Personalized Medicine is a hope or hype [25][26][27][28]. In fact, Personalized Medicine is now widely used only in oncology [2,29,30], particularly for treatment of melanoma, metastatic lung, breast, or brain cancer and leukemia. ...
Article
Full-text available
The aim of our research was to collect comprehensive data about the public and physician awareness, acceptance and use of Personalized Medicine (PM), as well as their opinions on PM reimbursement and genetic privacy protection in the U.S. and Germany. In order to give a better overview, we compared our survey results with the results from other studies and discussed Personalized Medicine preconditions for its wide implementation into the medical standard. For the data collection, using the same methodology, we performed several surveys in Pennsylvania (U.S.) and Bavaria (Germany). Physicians were contacted via letter, while public representatives in person. Survey results, analyzed by means of descriptive and non-parametric statistic methods, have shown that awareness, acceptance, use and opinions on PM aspects in Pennsylvania and Bavaria were not significantly different. In both states there were strong concerns about genetic privacy protection and no support of one genetic database. The costs for Personalized Medicine were expected to be covered by health insurances and governmental funds. Summarizing, we came to the conclusion that for PM wide implementation there will be need to adjust the healthcare reimbursement system, as well as adopt new laws which protect against genetic misuse and simultaneously enable voluntary data provision.
... Consequently, no regulatory step has been undertaken yet to recommend DPD testing as part of routine clinical practice for securing the administration of 5-FU. Owing to the wide range of phenotype-based techniques presently available in addition to specific genotype screening, the amount of clinical evidence warranting preliminary screening for DPD status as a way to ensure better safety in administering 5-FU is increasing recently [30,31]. Our life-threatening capecitabine-related toxicity case provides another stark reminder that integration of systematic testing for DPD as part of routine clinical practice may be ideal. ...
Article
Full-text available
Background Capecitabine is a prodrug that is enzymatically converted to its active form, fluorouracil (also called 5-fluorouracil), which is commonly used as adjuvant chemotherapy in colorectal cancer patients. Severe gastrointestinal bleeding induced by capecitabine is rare. Here, we are presenting the first case report of surgery specimen assisted diagnosis of this uncommon condition. Case presentation A 63-year-old Chinese male with a history of colon adenocarcinoma and right hemicolectomy presented with severe lower gastrointestinal bleeding 2 days after finishing capecitabine administration during the first cycle of XELOX adjuvant chemotherapy. Because of the negative findings of active bleeding points by digital subtraction angiography (DSA) or colonoscopy, emergency laparotomy and partial enterectomy were performed. The bloody diarrhea had resolved after surgery and a terminal ileitis was diagnosed after pathological examination of the surgical specimen. Conclusions Terminal ileitis induced by capecitabine is likely to be underreported. It should be considered more often as a cause of severe gastrointestinal bleeding during or after treatment with capecitabine agents. Emergency surgery may achieve satisfactory outcomes if endoscopic hemostasis is ineffective. Highlights of this case 1. Gastrointestinal bleeding following capecitabine treatment in colorectal cancer patients might be life-threatening. 2. Terminal ileitis induced by capecitabine should always be considered in the differential diagnosis of severe gastrointestinal bleeding. 3. Awareness of the risk factors such as deficiency of dihydropyrimidine dehydrogenase, advanced age, or right colectomy may aid in reducing capecitabine-related morbidity. 4. When severe bleeding occurs, emergency surgery may achieve satisfactory outcomes if medical and endoscopic interventions are ineffective.
... Our group has developed a simplified method to establish, on a phenotyping basis, the DPD status prior to administrate fluoropyrimidine drugs. Upfront detection allows preventive cut in dosing, so as to prevent severe toxicities to show [6]. Determining the best strategy to sort patients on their DPD status is a long and still ongoing story (refer to "Upfront DPD Deficiency Detection to secure 5-Fu Administration: Part 1-Where Do We Stand?" elsewhere in this issue). ...
Article
Background: Upfront screening for dihydropyrimidine dehydrogenase (DPD) deficiency in patients scheduled for 5-FU should help reduce the risk of toxicities by preventive adaptive dosing. Our group has developed a simple functional testing categorizing patients upon their DPD status, i.e. extensive metabolizer (EM) or poor metabolizer (PM) patients, using UH2/U ratio measurement in plasma as a surrogate for DPD activity. 5-FU dosing can then be tailored according to DPD deficiency status. Objectives: We present here an observational study of this strategy implemented in routine clinical practice when treating head-and-neck cancer patients. Results: A total of 218 evaluable adult patients were treated with a 5-FU-regimen, with DPD-based adaptive dosing. Among them, 20 (9%) were identified as PM and received subsequently a 20-50% reduced dosing of 5-FU as compared with EM patients (2102 ±254 mg VS. 2577 ±353mg, p<0.001 ttest). Gender (Female) was associated with higher risk for being PM (p=0.01, Pearson's Chi squared test). Overall, early severe toxicities were seen only in 5% of patients, all being EM with standard dosing. Similarly, overall severe toxicities were observed in 12.8% of patients only, both figures being markedly lower than usually reported with standard 5-FU. Despite the average -20% reduction in 5-FU dosing between PM and EM patients, clinical efficacy was not statistically different between the two groups (p = 0.2774, chi-square test). Conclusion: This study shows that 5-FU-related toxicities can be greatly reduced in routine clinical practice by the upfront detection of DPD deficient patients with simple adaptive dosing strategy.
... However, drug overexposure in patients with impaired liver detoxification could be a critical condition triggering severe toxicities, as observed with major cytotoxics such as fluorouracil, capecitabine, gemcitabine, mercaptopurine, and irinotecan. [13][14][15] With these drugs, drug elimination is also dependent on a single liver enzyme (ie, DPD, TPMT, and UGT1A1, respectively) coded by a polymorphic gene affecting enzymatic activity and liver clearance. ...
Article
Full-text available
Key Points Ara-C is the mainstay of treatment for patients with AML, and life-threatening toxicities are common. We demonstrated that cytidine deaminase downregulation predicts severe/lethal toxicities with cytarabine.
... DPD is also responsible for the conversion of the pyrimidine base uracil (U) to 5,6-dihydrouracil (UH 2 ). Thus, the assessment of the ratio between their concentrations has been proposed as a pre-chemotherapy indicative of DPD activity, as a means to improve 5FU therapy outcomes and reduce toxicity rates [12,13].The [UH 2 ]/[U] metabolic ratios have been typically measured in plasma samples, with significant association with 5FU concentrations and the occurrence of severe toxicity [11,[14][15][16][17][18][19]. Recently, a relation between salivary [UH 2 ]/[U] ratio and severe toxicity after different fluoropyrimidine-based regimens was reported [20,21], with diagnostic performance comparable to plasma. ...
Article
Objective: to evaluate plasma and salivary uracil (U) to dihydrouracil (UH2) ratios as tools for predicting 5-fluorouracil systemic exposure and drug-related severe toxicity, and clinically validate the use of dried saliva spots (DSS) as an alternative sampling strategy for dihydropyrimidine dehydrogenase (DPD) deficiency assessment. Methods: Pre-chemotherapy plasma, fresh saliva and DSS samples were obtained from gastrointestinal patients (N = 40) for measurement of endogenous U and UH2 concentrations by LC-MS/MS. A second plasma sample collected during 5FU infusion was used for 5FU area under the curve (AUC) determination by HPLC-DAD. Data on toxicity was reported according to CTCAE. Results: 15% of the patients developed severe 5FU-related toxicity, with neutropenia accounting for 67% of the cases. U, UH2 and [UH2,]/[U] were highly correlated between fresh and dried saliva samples (rs = 0.960; rs = 0.828; rs = 0.910, respectively). 5FU AUC ranged from 11.3 to 37.31 mg h L-1, with 46.2% of under-dosed and 10.3% over-dosed patients. The [UH2]/[U] ratios in plasma, fresh saliva and dried saliva samples were moderately correlated with 5FU AUC and adverse events grade, indicating a partial contribution of the variables to drug exposure (r = -0.412, rs = -0.373, rs = 0.377) and toxicity (r = -0.363, rs = -0.523, rs = 0.542). Metabolic ratios were lower in patients with severe toxicity (P < .01 salivary ratios, and P < .5 plasma ratios), and 5FU AUC were in average 47% higher in this group than in moderate toxicity. The diagnostic performance of [UH2]/[U] ratios in fresh saliva and DSS for the identification of patients with severe toxicity were comparable. Conclusions: The [UH2]/[U] metabolic ratios in plasma, fresh saliva and DSS were significantly associated with 5FU systemic exposure and toxicity degree. This study also demonstrated the applicability of DSS as alternative sampling for evaluating DPD activity.
... DPD is also responsible for the conversion of the pyrimidine base uracil (U) to 5,6-dihydrouracil (UH 2 ). Thus, the assessment of the ratio between their concentrations has been proposed as a pre-chemotherapy indicative of DPD activity, as a means to improve 5FU therapy outcomes and reduce toxicity rates [12,13].The [UH 2 ]/[U] metabolic ratios have been typically measured in plasma samples, with significant association with 5FU concentrations and the occurrence of severe toxicity [11,[14][15][16][17][18][19]. Recently, a relation between salivary [UH 2 ]/[U] ratio and severe toxicity after different fluoropyrimidine-based regimens was reported [20,21], with diagnostic performance comparable to plasma. ...
Article
In this study, a LC-MS/MS method for the measurement of docetaxel in Dried Blood Spots (DBS) samples was developed and validated. Docetaxel was extracted from 8 mm DBS punch with a mixture of methanol and acetonitrile (9:1, v/v). The chromatographic separation occurred in an Acquity® C18 column (150 × 2.1 mm, 1.7 μm) eluted with a mixture of water and acetonitrile plus 0.1% formic acid (45:55, v/v). Total analytical run time was 7 min. The method was linear from 50 to 3000 ng ml-1. Precision assays showed CV% < 9.8% and accuracy between 99 and 103%, mean recovery was 81%. The method was applied in the determination of the docetaxel in 31 patients, after collection of two paired venous blood and DBS samples, following a limited sampling strategy protocol. The analyte was stable in DBS for 18 days at 25 °C and 9 days at 45 °C. The interval of docetaxel concentrations measured in DBS collected before the end of the infusion was 756-3047 ng ml-1 and 60 ± 10 min after the end of the infusion was 57-331 ng ml-1. AUC values calculated from DBS-derived estimated plasma concentrations (EPC) represented on average 100% of those obtained in plasma samples of 3.1 mgh/l (2.4-4.9 mg h/l). There was a 93% agreement between the classification of patients as within or without the therapeutic range by plasma and EPC AUC. These findings support the clinical use of DBS sampling for routine therapeutic drug monitoring of docetaxel.
... In general, the testing approach for DPD deficiency can be genotyping based or functional measurement/phenotype of DPD. Supplementary Table 2 summarizes the advantages and limitations of tests with these two approaches (10,11). ...
Article
Full-text available
Background: The fluoropyrimidine anticancer drug, especially 5- fluorouracil (5-FU) and its prodrug capecitabine are still being the backbone of chemotherapeutic regimens for colorectal cancer. Dihydropyrimidine dehydrogenase (DPD) is the crucial enzyme in the catabolism of 5-FU. Over the past 30 years, there is substantial clinical evidence showing that DPD deficiency is strongly associated with severe and fatal fluoropyrimidine-induced toxicity. Patients and methods: A 49-year-old lady with resected stage III carcinoma of sigmoid colon was scheduled to have a course of 5-FU based adjuvant chemotherapy. She developed unexpected acute severe (grade 4) toxicity after the first cycle of chemotherapy. Genomic DNA was isolated from 3 ml peripheral blood cells for full sequencing of DPYD (the gene encoding DPD). Results: Exome sequencing confirmed that she is heterozygous for NM_000110.3: c.321+2T>C of the DPYD gene. To the best of our knowledge, this variant is a novel pathogenic splicing variant of the DPYD gene resulting in a non-functional allele. As she has a heterozygous genotype and considered having decreased DPD activity, we followed the international recommendation and restart chemotherapy with at least 50% reduction for 5-FU dose. We then titrated the 5-FU dose, and she tolerated the subsequent cycles of chemotherapy and completed the whole course of adjuvant chemotherapy. Conclusions: With a pre-emptive test on DPD deficiency before the administration of the fluoropyrimidine drugs, the aforementioned patient's life-threatening event could be avoided. This clinical utility has been confirmed by two recent large-scale studies and called for a drug label update.
... 5-fluorouracil is metabolized mainly in the liver and has a half-life of about 10 minutes (5). Dihydropyrimidine dehydrogenase (DPD) found in the liver and it is the initial and rate-limiting enzyme in 5-FU catabolism, therefore 5-FU should be used with a great care in patients who are known or suspected to have a DPD deficiency as they are at a greater risk of 5-FU induced toxicity (6). The common clinical adverse effects of 5-FU include myelosuppression, diarrhea, vomiting and mucositis (7). ...
Article
Full-text available
The main histological and histochemical changes in the rat's liver tissue induced by intraperitoneal injection of 5-FU were investigated in this study. Twelve adult female rats were divided into two groups of 6 rats each, group I was given 2 ml/kg body weight of normal saline for 7 consecutive days and served as the control group, while group II was given 20 mg/kg of 5- fluorouracil intraperitoneally for 7 consecutive days. Specimens of the liver tissue from the two groups were taken and prepared for light microscopic examination. Results showed the appearance of some histological changes in the5-FU recipient group (group II) compared to the control group (group I) including congestion of central vein, dilatation and congestion of the hepatic sinusoids, vacuolar degeneration of the hepatocytes, loss of normal histological architecture of the liver, in addition to sever fatty changes and apoptosis of the hepatocytes. Histochemical examination of the liver of the treated group revealed a marked decrease in the carbohydrates including glycogen manifested as weak positive reaction to PAS and Best's carmine stains compared to the control group in addition to marked increase in the activity of alkaline phosphatase manifested as a strong reaction to Gomori's alkaline phosphatase stain as compared to the control group. We conclude that 5-fluorouracil has toxic effects on the liver tissue causing vacuolar degeneration of the hepatocytes, sever fatty changes and apoptosis. © 2014 University of Mosul - College of Veterinary Medicine. All rights reserved.
... Upfront detection of DPD deficiency is thus critical to customize dosing and ensure optimal treatment without triggering potentially lethal toxicities 4 . To this end, establishing DPD phenotype is appealing because genotyping DPYD and search for allelic variants usually associated with PM phenotype is highly specific but hindered by poor sensitivity 5,6,7 . Several methods have thus been proposed over the last 20 years to establish DPD status on a functional, rather than genetic, basis. ...
Article
Full-text available
Detecting patients with DPD deficiency is becoming a major concern in clinical oncology. Monitoring physiological plasma uracil and/or plasma uracil‐to‐dihydrouracil metabolic ratio is a common surrogate frequently used to determine DPD phenotype without direct measurement of the enzymatic activity. With respect to the rising number of patients to be analyzed, developing simple, rapid and affordable methods suitable to routine screening is critical. We have developed and validated a simple and robust UPLC‐UV method with shorten (i.e., 12 minutes) analytical run times, compatible with these requirements of large‐scale upfront screening. The method enabled detection of U over a 5‐500 ng.mL‐1 range (265 nm) and of UH2 over a 40‐500 ng.mL‐1 range (210 nm) in plasma with no chromatographic interference. When used as part of routine screening for DPD deficiency, this method was fully able to discriminate non‐deficient patients (i.e., with U levels below 16 ng.mL‐1) from deficient patients at risk of severe toxicities (i.e., U > 16 ng.mL‐1). Results from one month of routine testing are presented and although no complete deficit were detected, 10,7% of the screened patients presented DPD deficiency and would thus require cut in dosing. Overall, this new method, using a simple pre‐analytical solid‐phase extraction procedure and based upon the use of standard UPLC apparatus is both cost‐ and time‐effective and can be easily implemented at low cost in any laboratory about to start DPD testing in routine.
... Several approaches to prediction of FP toxicity and dose individualization have been developed, including DPYD genotyping to predict DPD activity. [7][8][9][10][11] The most well-known practically relevant DPYD variant is DPYD*2A, a splice-site variant (c.1905+1G>A; IVS14 +1G>A; rs3918290). 12 Upfront genotyping for DPYD*2A and consequent FP dose-adjustment improves patient safety and is costeffective. ...
Article
Aim: Cancer patients with reduced dihydropyrimidine dehydrogenase (DPD) activity are at increased risk of severe fluoropyrimidine (FP)-related adverse events (AE). Guidelines recommend FP dosing adjusted to genotype-predicted DPD activity based on four DPYD variants (rs3918290, rs55886062, rs67376798, rs56038477). We evaluated relationship between three further DPYD polymorphisms [c.496A>G (rs2297595), *6 (c.2194G>A, rs1801160) and *9A (c.85T>C, rs1801265)] and the risk of severe AEs. Methods: Consecutive FP-treated adult patients were genotyped for "standard" and tested DPYD variants, and for UGT1A1*28 if irinotecan was included, and were monitored for the occurrence of grade ≥3 (National Cancer Institute Common Terminology Criteria) vs. grade 0-2 AEs. For each of the tested polymorphisms, variant allele carriers were matched to respective wild type controls (optimal full matching combined with exact matching, in respect to: age, sex, type of cancer, type of FP, DPYD activity score, use of irinotecan/UGT1A1, adjuvant therapy, radiotherapy, biological therapy and genotype on the remaining two tested polymorphisms). Results: Of the 503 included patients (82.3% colorectal cancer), 283 (56.3%) developed grade ≥3 AEs, mostly diarrhea and neutropenia. Odds of grade ≥3 AEs were higher in c.496A>G variant carriers (n=127) than in controls (n=376) [OR=5.20 (95%CI 1.88-14.3), Bayesian OR=5.24 (95% CrI 3.06-9.12)]. Odds tended to be higher in c.2194G>A variant carries (n=58) than in controls (n=432) [OR=1.88 (0.95-3.73), Bayesian OR=1.90 (1.03-3.56)]. c.85T>G did not appear associated with grade ≥3 AEs (206 variant carriers vs. 284 controls). Conclusion: DPYD c.496A>G and possibly c.2194G>A variants might need to be considered for inclusion in the DPYD genotyping panel.
... Several approaches to prediction of FP toxicity and dose individualization have been developed, including DPYD genotyping to predict DPD activity. [7][8][9][10][11] The most well-known practically relevant DPYD variant is DPYD*2A, a splice-site variant (c.1905+1G>A; IVS14 +1G>A; rs3918290). 12 Upfront genotyping for DPYD*2A and consequent FP dose-adjustment improves patient safety and is costeffective. ...
Preprint
Aim. Cancer patients with reduced dihydropyrimidine dehydrogenase (DPD) activity are at increased risk of severe fluoropyrimidine (FP)-related adverse events (AE). Guidelines recommend FP dosing adjusted to genotype-predicted DPD activity based on four DPYD variants (rs3918290, rs55886062, rs67376798, rs56038477). We evaluated relationship between three further DPYD polymorphisms [ c.496A>G (rs2297595), *6 c.2194G>A (rs1801160) and *9A c.85T>C (rs1801265)] and the risk of severe AEs. Methods. Consecutive FP-treated adult patients were genotyped for “standard” and tested DPYD variants, and for UGT1A1*28 if irinotecan was included, and were monitored for the occurrence of grade ≥3 (National Cancer Institute Common Terminology Criteria) vs. grade 0-2 AEs. For each of the tested polymorphisms, variant allele carriers were matched to respective wild type controls (optimal full matching combined with exact matching, in respect to: age, sex, type of cancer, type of FP, DPYD activity score, use of irinotecan/ UGT1A1 , adjuvant therapy, radiotherapy, biological therapy and genotype on the remaining three tested polymorphisms). Results. Of the 503 included patients (82.3% colorectal cancer), 283 (56.3%) developed grade ≥3 AEs, mostly diarrhea and neutropenia. Odds of grade ≥3 AEs were higher in c.496A>G variant carriers (n=127) than in controls (n=376) [OR=5.20 (95%CI 1.88-14.3), Bayesian OR=5.24 (95% CrI 3.06-9.12)]. Odds tended to be higher in *6 c.2194G>A variant carries (n=58) than in controls (n=432) [OR=1.88 (0.95-3.73), Bayesian OR=1.90 (1.03-3.56)]. *9A c.85T>G did not appear associated with grade ≥3 AEs (206 variant carriers vs. 284 controls). Conclusion. DPYD c.496A>G variant might need to be considered for inclusion in the DPYD genotyping panel.
... Le cas le plus emblématique est celui du 5-FU, dont la détoxica- tion hépatique est gouvernée par une enzyme unique, la DPD, affectée par un polymorphisme génétique. Les patients touchés par ce polymorphisme et présen- tant un phénotype poor-metabolizer (PM) sont à risque de développer des toxicités chimio-induites potentiel- lement létales, un fait connu depuis le milieu des années 1980 [8]. Il faudra attendre toutefois plus de 20 ans pour que le premier essai prospectif rando- misé démontre l'intérêt clinique du typage de la DPD et d'individualisation posologique du 5-FU, avec une réduction marquée de l'incidence des toxicités sévères et une amélioration de l'efficacité thérapeutique [8,9]. Outre le bénéfice clinique pour le patient, le déve- loppement de la pharmacogénétique s'accompagne également d'un bénéfice pharmacoéconomique. ...
... Ther. Patents (2015) 25 (10) relationship between 5-FU-related toxicities, such as diarrhea, mucositis and stomatitis, and DPD [35]. Low intratumor 5-FU levels and its high degradation depend on dihydropyrimidine dehydrogenase (DPYD) activity, an enzyme responsible of drug catabolism [36]. ...
Article
Full-text available
Introduction: 5-Fluorouracil (5-FU)-based chemotherapy is the most widely prescribed treatment for gastrointestinal solid tumors, but there are several drawbacks such as toxicities, lack of selectivity and effectiveness as well as the development of resistance that need to be overcome. Areas covered:In this review, the authors present the latest innovations in 5-FU derivatives or combinations with: i) other chemotherapeutic drugs; ii) novel targeted compounds; iii) radiotherapy; iv) mAbs; v) siRNA strategies; and vi) traditional Chinese medicine extracts. Moreover, advances to overcome or determine 5-FU adverse effects and effectiveness are described. Finally, the authors introduce the ongoing clinical trials and highlight the main challenges to be addressed in the future. Expert opinion: Although in the past few years there has been a great advancement in the antitumor effectiveness and selectivity of 5-FU-based therapies, it is envisaged that future approaches using 'omics' technologies that could determine the tumor heterogeneity may help in identifying additional candidate genes, microRNAs or cytokines involved in both the path mechanisms of 5-FU-related toxicity and its therapeutic efficacy. Moreover, the development of novel targeted 5-FU derivatives or 5-FU-based therapies tailored to individual patients opens up new possibilities in the improvement of the quality of life and survival for those suffering from this devastating disease.
... DPD is also responsible for the conversion of the pyrimidine base uracil (U) to 5,6-dihydrouracil (UH 2 ). Thus, the assessment of the ratio between their concentrations has been proposed as a pre-chemotherapy indicative of DPD activity, as a means to improve 5FU therapy outcomes and reduce toxicity rates [12,13].The [UH 2 ]/[U] metabolic ratios have been typically measured in plasma samples, with significant association with 5FU concentrations and the occurrence of severe toxicity [11,[14][15][16][17][18][19]. Recently, a relation between salivary [UH 2 ]/[U] ratio and severe toxicity after different fluoropyrimidine-based regimens was reported [20,21], with diagnostic performance comparable to plasma. ...
... As a result, this toxicity limits or delays the administration of optimal or successive courses, which impacts the clinical outcome of patients with cancer. The mechanisms of fluoropyrimidines-based chemotherapies toxicity and the different strategies to prevent this toxicity are of great importance and numerous works have been published [5][6][7][8][9][10]. The dihydropyrimidine dehydrogenase (DPD), a rate-limited enzyme, is responsible of 80% of the catabolism of fluoropyrimidines. ...
Article
Fluoropyrimidines-based chemotherapies are the backbone in the treatment of many cancers. However, the use of 5-fluorouracil and its oral pre-prodrug, capecitabine, is associated with an important risk of toxicity. This toxicity is mainly due to a deficiency of dihydropyrimidine dehydrogenase (DPD). This deficiency may be detected by using a phenotypic approach that consists in the measurement of uracilemia or the calculation of dihydrouracil (UH2)/uracil (U) ratio. For uracilemia, a threshold value of 16 ng/ml has been proposed for partial deficiency, while a value of 150 ng/ml has been proposed for complete deficiency. We have developed a rapid, accurate and fully-automated procedure for the quantification of U and UH2 in plasma. Sample extraction was carried out by a programmable liquid handler directly coupled to a liquid chromatography – tandem mass spectrometry (LC-MS/MS) system. The method was validated according to the EMA guidelines and ISO 15189 requirements and was applied to real patient samples (n = 64). The limit of quantification was 5 and 10 ng/ml for U and UH2 respectively. Imprecision and inaccuracy were less than 15% for inter and intra-assay tests. Comparison with dedicated routine method showed excellent correlation. An automated procedure perfectly fulfills the need of low inaccuracy and CVs at the threshold values (less than 5% at 16 ng/ml) and is highly suitable for the characterization of DPD deficiency. Automatization should guaranty reliable and robust performances by minimizing the sources of variation such as volume inaccuracies, filtration or manual extraction related errors.
... Hence, the DPD phenotype status in patients was necessary for the efficacy and toxicity of 5-FU in clinical treatment [85]. A previous study showed that patients with DPD deficiency caused severe side effects due to the lack of capacity to detoxify 5-FU in the liver [86]. However, it was found that low expression of tumoral DPD contributed to 5-FU sensitivity in gastric cancer patients (Table 2) [87]. ...
Article
Full-text available
Background Cancer is one of the most serious diseases that threatening human health with high morbidity and mortality in the world. For the treatment of cancer, chemotherapy is one of the most widely used strategies, which is widely used for almost all kinds of tumors and diverse stages of tumor development. The efficacy of chemotherapy not only depends on the activity of the drug administrated, but also on whether the compound could reach the effective therapeutic concentration in tumor cells. Therefore, expression and activity of drug-metabolizing enzymes (DMEs) in tumor tissues and metabolic organs of cancer patients is important for dispositional behavior of anticancer drugs as well as the clinical response of chemotherapy. Method This review summarizes the recent advancement on the DMEs expression and activity in various cancers, as well as the potential regulatory mechanisms of major DMEs in cancer and cancer therapy. Results Compared to normal tissues, expression and activity of major DMEs are significantly dysregulated in patients by various factors including epigenetic modification, ligand-activated transcriptional regulation, signaling pathways. Additionally, DMEs play an important role in anticancer drug efficacy, chemoresistance as well as the activation of prodrugs. Conclusion This review reinforces a more comprehensive understanding of DMEs in cancer and cancer therapy, and provides more opportunities for cancer therapy.
... 7,32 The question of the best screening method for DPD deficiency has been assessed in many reviews. 33,34 The genotyping approach based on three selected variants presents some limitations. For instance, in our study focusing on the three variants would have led us to misclassify 6 out of 22 individuals: mother I.1 and patients #3, #4, and #12 would have been classified as "normal," although they have a partial deficiency (linked with an heterozygous genotype for deleterious mutation) and individuals II.1 and II.4 would have been considered partially deficient and then treated with a half-dose of 5-FU, 6 whereas they are completely DPD-deficient. ...
Article
Despite the growing evidence that dihydropyrimidine dehydrogenase deficiency (DPD encoded by DPYD gene) confers a higher risk of developing severe toxicity, most patients are not screened for DPD deficiency before fluoropyrimidine treatment. We report here the genetic and phenotypic analyses of DPD in a family related to a patient who died after a first cycle of 5-fluorouracil and in 15 additional retrospective patients having a partial DPD deficiency (as measured by plasma dihydrouracil/uracil ratio). The patient with lethal toxicity was found to be a compound heterozygote for two DPYD mutations: a novel 8-bp duplication (c.168_175dupGAATAATT, p.Phe59Ter) and c.1679T>G (Ile560Ser). The patient's dihydrouracil/uracil ratio indicates complete DPD deficiency. The novel mutation was found in 2 members of the patient's family. Deleterious DPYD mutations were identified in 9 out of the 15 patients. The relationship between genotype and dihydrouracil/uracil values in the 22 patients of the present study was significant (p=0.01). This article is protected by copyright. All rights reserved. © 2015 American Society for Clinical Pharmacology and Therapeutics.
... The clearance of 5FU depends on DPD activity. Hence, reduced DPD activity leads to both increased toxicity and efficacy of the drug 70 . Till date at atleast 30 polymorphism in DPD gene have been described 71 . ...
Article
Full-text available
Capecitabine an oral prodrug of 5- fluorouracil is widely used for the treatment of variety of solid tumors, particularly colorectal cancer. However it is not devoid of toxicities and may limit therapy. A little is known about predictors of toxicity, response and survival in patients treated with capecitabine. The pharmacogenetic testing methods can identify such variants and thus indicate those patients who are at risk for adverse effects with capecitabine. Various studies have been carried out to assess the various genetic predictive and prognostic markers with treatment. The purpose of this review is to describe the comprehensive reports and draw conclusion with the available information on capecitabine pharmacogenetics and future directions on ongoing research. An extensive literature search was carried out on the genes encoding the enzymes involved in the metabolism of capecitabine. Overall, evidence indicates multiple genes associated with the response/toxicity with capecitabine therapy, however majority of reports indicate DPD deficiency as a source of life-threatening toxic effects. Hence, prospective studies correlating the enzymes and the concentration of the drug and its metabolites in the body are needed before validated SNP tests can enter routine clinical practice. � 2013 are reserved by International Journal of Pharmaceutical Sciences and Research.
... This mechanism further explains the observation that EOC cells manifest significantly decreased apoptosis and increased survival [32,33,35,36]. Interestingly, the evaluation of mutations in the various redox enzymes in the form of SNPs is an active area of scientific research [37][38][39][40][41][42][43][44][45]. Genetic polymorphisms are known to be associated with cancer susceptibility and can be determined by studying functional polymorphisms in genes that control the levels of cellular reactive oxygen species and oxidative damage, including SNPs for genes involved in carcinogen metabolism (detoxification and/or activation), antioxidants, and DNA repair pathways [46]. ...
Article
Full-text available
Ovarian cancer is the deadliest of all gynecologic cancers. Recent evidence demonstratesan association between enzymatic activity altering single nucleotide polymorphisms (SNP)with human cancer susceptibility. We sought to evaluate the association of SNPs in key oxi-dant and antioxidant enzymes with increased risk and survival in epithelial ovarian cancer.Individuals (n = 143) recruited were divided into controls, (n = 94): healthy volunteers, (n =18), high-risk BRCA1/2 negative (n = 53), high-risk BRCA1/2 positive (n = 23) and ovariancancer cases (n = 49). DNA was subjected to TaqMan SNP genotype analysis for selectedoxidant and antioxidant enzymes. Of the seven selected SNP studied, no association withovarian cancer risk (Pearson Chi-square) was found. However, a catalase SNP was identi-fied as a predictor of ovarian cancer survival by the Cox regression model. The presence ofthis SNP was associated with a higher likelihood of death (hazard ratio (HR) of 3.68 (95%confidence interval (CI): 1.149–11.836)) for ovarian cancer patients. Kaplan-Meier survivalanalysis demonstrated a significant median overall survival difference (108 versus 60months, p<0.05) for those without the catalase SNP as compared to those with the SNP.Additionally, age at diagnosis greater than the median was found to be a significant predic-tor of death (HR of 2.78 (95% CI: 1.022–7.578)). This study indicates a strong associationwith the catalase SNNP and survival of ovarian cancer patients, and thus may serve as aprognosticator.
... Over 80% of 5-FU is metabolized by DPD in the liver. The conversion of 5-FU to the inactive dihydrofluorouracil (DHFU) through DPD is the rate-limiting step of the 5-FU catabolism [2,3]. TS catalyzes the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP), which is critical for DNA replication. ...
Article
Full-text available
Abstract: Fluoropyrimidine-based chemotherapy is extensively used for the treatment of solid cancers, including colorectal cancer. However, fluoropyrimidine-driven toxicities are a major problem in the management of the disease. The grade and type of the toxicities depend on demographic factors, but substantial inter-individual variation in fluoropyrimidine-related toxicity is partly explained by genetic factors. The aim of this study was to investigate the effect of dihydropyrimidine dehydrogenase (DPYD), thymidylate synthase (TYMS), and methylenetetrahydrofolate reductase (MTHFR) polymorphisms in colorectal cancer patients. Eighty-five patients who were administered fluoropyrimidine-based treatment were included in the study. The DPYD, TYMS and MTHFR polymorphisms were scanned by a next generation Sequenom MassARRAY. Fluoropyrimidine toxicities were observed in 92% of all patients. The following polymorphisms were detected: DPYD 85T>C (29.4% heterozygote mutants, 7.1% homozygote mutants), DPYD IVS 14+1G>A (1.2% heterozygote mutants), TYMS 1494del TTAAAG (38.4% heterozygote mutants, 24.7% homozygote mutants), MTHFR 677C>T (43.5% heterozygote mutants, 9.4%homozygote mutants) and MTHFR 1298A>C (8.2% heterozygote mutants, 2.4% homozygote mutants). A statistically significant association was demonstrated between MTHFR 677C>T and fluoropyrimidine-related toxicity (p value = 0.007). Furthermore, MTHFR 1298A>C was associated with hematopoietic toxicity (p value = 0.008). MTHFR polymorphisms may be considered as related factors of fluoropyrimidine toxicity and may be useful as predictive biomarkers for the determination of the colorectal cancer patients who can receive the greatest benefit from fluoropyrimidine-based treatments. Keywords: DPYD; TYMS and MTHFR genes; polymorphisms; pharmacogenetics; colorectal cancer
Article
"Our ultimate aim is to achieve the perfect balance, allowing maximal effectiveness and minimal toxicity."
Chapter
The chemotherapeutic options have increased dramatically in patients with gastrointestinal cancer and have led to an improved outcome. With this, an in-depth understanding of the side effects of chemotherapy is becoming increasingly important in order to minimize the negative impact of the use of these agents. Chemotherapeutic agents have a long list of potential side effects. In this chapter, we focus specifically on some of the more common and/or more relevant and challenging side effects related to frequently used agents in gastrointestinal cancer. The fluoropyrimidines may cause cardiac toxicity, most frequently angina-like chest pain. The knowledge of the catabolism of fluorouracil has led to the possibility of testing for dihydropyrimidine dehydrogenase (DPD) in order to avoid serious fluorouracil-related toxicity in patients with DPD deficiency. Oxaliplatin-induced neurotoxicity is probably the most important clinical problem associated with the administration of oxaliplatin. With the increasing use of oxaliplatin, hypersensitivity reactions are more frequently reported and become challenging in clinical practice. The introduction of the targeted agents in colorectal cancer led also to specific problems: the anti-VEGF-related side effects, of which gastrointestinal perforation, although relatively rare, is very relevant for the patient, and the anti-EGFR-related side effects, including skin rash, hypomagnesemia, and allergic reactions, are common. Understanding the underlying causes, mechanisms, risk factors, and developing treatment guidelines has made these side effects often more acceptable for many patients. However, the side-effect profile always has to be balanced against the activity and benefit of the anticancer agents.
Article
Dihydropyrimidine dehydrogenase (DPD) activity determination in peripheral blood mononuclear cells of DPD deficient patients was hitherto inaccurate due to hemoglobin (Hb) contamination. We developed an improved method for accurate measurement of DPD activity in patients. DPD activity was determined by HPLC with online radioisotope detection using liquid scintillation counting. Hb was determined spectrophotometrically. Method accuracy and precision were significantly improved by using cumulative area of all peaks as IS. Peripheral blood mononuclear cell lysates from DPD deficient patients were highly contaminated with on average 23.3% (range 2.7-51%) of Hb resulting in up to twofold underestimated DPD activity. DPD activities were corrected for Hb contamination. The method was validated and showed good long-term sample stability. This method has increased specificity allowing accurate identification of DPD deficient patients.
Article
The systemic treatment of patients with colorectal cancer (CRC) has traditionally been based on clinical and tumor histological criteria. Recently however, several prognostic and predictive biomarkers have been proposed for patients with newly diagnosed CRC, including the subgroup with stage II disease. Among the best validated prognostic biomarkers for CRC are CEA levels, MS instability status and certain gene signatures. Although no biomarker currently exists for identifying patients likely to benefit from chemotherapy, the mutational status of KRAS and NRAS is used to predict response to cetuximab and panitumumab. For upfront identification of patients at high risk of suffering from severe therapy-related toxicity, specific variants of dihydropyrimidine dehydrogenase may be measured for predicting toxicity from fluoropyrimidines and uridine diphosphate glucuronosyltransferase*28 (UGT1A1*28) for predicting toxicity from irinotecan.
Article
5-Fluorouracil (5FU) and capecitabine are the cornerstones of all currently applied regimens for the treatment of patients with cancers of the gastrointestinal tract, breast, head and neck. Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the metabolism of 5FU and as such, a deficiency of DPD has been recognised as an important risk factor, predisposing patients to develop severe 5FU-associated toxicity. In this manuscript, we discuss a wide range of methods that have been established to assess the genetic and functional status of DPD. Genotyping of DPYD is used to identify DPD deficient patients. However, its suitability for pre-treatment testing is under debate, not least due to conflicting genotype-phenotype relations in mutation carriers and relatively low positive predictive values. In addition to genetic screening, a number of phenotype-based methods have been introduced which appear to be well suited for clinical laboratories and which are an attractive option for monitoring of the DPD status. These phenotype-based screening approaches to detect DPD-deficient patients warrant further clinical validation.
Chapter
Full-text available
Personalized medicine is a rapidly expanding field offering patient-specific therapies to treat disease. Occasionally, the emergence of new technology or knowledge propels medicine across a threshold that is so monumental that it mandates changes in the structure of health-care delivery. Today, medical science is at another such threshold with the advent of individualized medicine. The Mayo Clinic Center for Individualized Medicine (CIM), for instance, is designed to discover and integrate the latest in genomic, molecular, and clinical science into personalized care for patients across a multiple-site academic medical center. Major investments in basic science have created an opportunity for significant progress in clinical medicine. Individualized medicine has advanced because researchers have discovered hundreds of genes that harbor variations contributing to human illness and identified genetic variability in patients’ responses to dozens of treatments and have begun to target the molecular causes of some diseases. In addition, scientists are developing and using diagnostic tests based on genetics or other molecular mechanisms to better predict patients’ responses to targeted therapy. Genome-wide association studies involve hundreds of thousands of single-nucleotide polymorphisms (SNPs) which are tested for association with a disease in hundreds or thousands of persons. The genome-wide association study has therefore revolutionized the search for genetic influences on complex traits. It has been discovered that many diseases are caused by many genetic and environmental factors working together, each having a relatively small effect.
Article
5-fluorouracil (5-Fu) as the most common adjuvant chemotherapy has been widely used for patients with colorectal cancer. As reported dihydropyrimidine dehydrogenase (DPD) has been known as an initial rate-limiting enzyme to catalyze approximately 80% of administered dose of 5-Fu. However, patients with a partial or complete DPD deficiency because of the mutation of DPYD gene may suffer from diarrhea, stomatitis, hand-foot syndrome or neurotoxicity and even death. In this article, the application of DPYD gene screening in patients with colorectal cancer of current status and recent clinical application are reviewed.
Article
Dihydropyrimidine dehydrogenase (DPD, E.C. 1.3.1.2) was purified from sheep liver with a yield of 16.7%, purification fold of 407.5 and specific activity of 0.705 EU/mg proteins. The purification procedure consisted of ammonium sulphate fractionation, DEAE ion exchange chromatography and 2',5'-ADP Sepharose-4B affinity chromatography. The molecular weight determined by SDS-PAGE and was found 111 kDa. Optimum pH, ionic strength temperature and stable pH were determined as 8.0, 0.9 mM, 50 °C and 6.0, respectively. The kinetic parameters (Km and Vmax) of the enzyme were determined with NADPH as 22.97 μM and 0.17 EU/mL, respectively. The same parameters were determined with uracil as 17.46 μM and 0.14 EU/mL, respectively. Additionally, in vitro inhibitory effects of some antidepressant drugs including escitalopram, fluoxetine, mirtazapine, haloperidol and some anaesthetic drugs including propofol and lidocaine were investigated against DPD. In addition, IC50 values for each active drug obtained for escitalopram, fluoxetine, mirtazapine, haloperidol, propofol and lidocaine were determined as 1736.11, 13.24, 86.65, 99.03, 0.21 and 15.07 μM, respectively.
Article
We aimed to investigate the association between dihydropyrimidine dehydrogenase (DPYD) gene polymorphisms and the risk of pediatric acute lymphoblastic leukemia (ALL) and its prognosis after chemotherapy. A total of 147 pediatric ALL patients diagnosed by our hospital between January 2011 and December 2014 were included in the case group, and 102 healthy people who received a physical examination during the same time frame in our hospital were included in the control group. DNA sequencing was applied for site determination and genotyping of the DPYD 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms. The genotype and allele frequencies of the two groups were compared. A significant difference was found in the comparison of the mutant gene and allele frequencies of the 85T > C polymorphism between the case and control groups (P < 0.05). The CT and CC genotypes in the 85T > C polymorphism were associated with the risk of the disease (OR = 1.592, 95 % CI = 1.010-2.509), suggesting that the recessive gene (85C) was more likely to lead to the occurrence of ALL compared with the dominant gene (85T) (P < 0.05). Patients carrying the C allele of the 85T > C polymorphism presented higher damage of their liver functions and higher infection rates compared with patients carrying the non-C allele (P < 0.05). A higher proportion of liver function damage and a higher infection rate were found in patients with the GA genotype in the IVS14 + 1G > A polymorphism compared with the GG genotype (P < 0.05). The complete remission (CR) rate in patients with the GG genotype in the IVS14 + 1G > A polymorphism was higher than in patients with the GA genotype (P = 0.020). After 5-fluorouracil/calcium folinate (5-FU/CF)-based chemotherapy, the event-free survival (EFS) rate of patients with the TT genotype was higher than patients with the CT and CC genotypes (P < 0.05). Our results revealed that the C allele of the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Patients carrying the C allele may have an increased risk of ALL. Thus, the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients.
Chapter
The modern treatment of locoregionally advanced disease often requires a multimodality combination approach. A number of chemotherapeutic agents can be combined with radiation, but the platinum agent cisplatin, a potent radiation sensitizer, is best studied in head and neck cancer. Newer agents such as cetuximab can be used in combination with radiation therapy for those patients who cannot tolerate cisplatin. For chemotherapy-naïve patients with metastatic head and neck cancer who demonstrate a good performance status, platinum doublet regimens are commonly used. Doublet regimens generally improve response rates compared to single-agent chemotherapies, although they have not demonstrated a survival benefit over single agents and they have added toxicity. Immunotherapies, alternative cytotoxic chemotherapies, and targeted therapies are second-line options for patients with disease that has progressed on platinum-based therapy. Immunotherapy, in particular, has gained focus by enhancing the ability of the immune system to recognize and destroy malignant cells. When multimodal approaches are used, as in combined chemotherapy and radiation therapy, toxicities are increased. It is imperative that patients are followed closely in order to maximize treatment benefit while minimizing complications.
Article
Aims Most clinical trials exclude elderly people, leading to a limited understanding of the benefit-to-risk ratio in this population. Despite existing data regarding the oncological management of elderly receiving fluorouracil (5-FU)-based regimen, our objective was to investigate 5-FU exposure/toxicity relationship in patients ≥75 years and compare the effectiveness of 5-FU therapeutic drug monitoring between elderly and younger patients. Methods Hundred fifty-four patients (31 of whom are older than 75 years) with gastrointestinal cancers, who were to receive 5-FU–based regimens, were included in our study. At cycle 1 (C1), the 5-FU dose was calculated using patient's body surface area, then a blood sample was drawn to measure 5-FU concentration and 5-FU dose was adjusted at the subsequent cycles based on C1 concentration. Assessments of toxicity were performed at the beginning of every cycle. Results Seventy-one percent of elderly patients required dose adjustments after C1, compared with 50% for younger patients. Percentages of patients within 5-FU area under the curve range at cycle 2 were 64% and 68%, respectively, for elderly and younger patients. The proportion of elderly patients experiencing severe toxicities fell from 15% at C1 to only 5% at cycle 3. Conclusion Pharmacokinetic-guided 5-FU–dosing algorithm, leading to an improved tolerability while remaining within therapeutic concentration range, is even more valuable for patients older than 75 years than in younger patients.
Article
Aims: Despite fluoropyrimidines (FPs) constituting the main component of the chemotherapy combination protocols in 50% of chemotherapies for solid tumour treatments, incidence data for FP-related toxicity are poorly documented in real life. This study evaluated the number of patients receiving FP-based chemotherapies in France, along with the true incidence of FP-related serious adverse effects (SAEs) before the recent mandatory dihydropyrimidine dehydrogenase (DPD)-screening was introduced by French health authorities, DPD being the rate-limiting enzyme of 5-fluorouracil (5-FU) catabolism. Methods: Exhaustive data on the number of patients treated with FP-based chemotherapy in 2013-2014 were collected in the Centre-Val de Loire region of France. True incidence of SAEs was extracted from a cohort of 513 patients with incident solid tumours receiving first-line FP-based chemotherapy. Results: After extrapolation at national level, we estimated that 76,200 patients are currently treated annually with 5FU (53,100 patients, 62% digestive system-related versus 26% breast cancers versus 12% head and neck cancers) or capecitabine (23,100 patients, 45% digestive system-related versus 37% breast cancers versus 18% non-documented). Earlier (in the first two cycles) the SAE incidence rate was 19.3% (95% confidence interval (CI) 16-23%) including one toxic death (0.2%, 95%CI 0-1%). SAE incidence rate was 32.2% (95%CI 28-36%) over the first 6 months of treatment. Incidence of death, life-threatening prognosis or incapacity/disability was 1.4% (95%CI 0.4-2.4%) and 1.6% (95%CI 0.5-2.6%) during first two cycles and first 6 months, respectively. Conclusion: These data highlight the significant public health issue related to FP toxicity, with around 1200 patients developing FP-related life-threatening prognosis or incapacity/disability annually in France, including 150 toxic deaths. It is hoped that DPD-deficiency screening will reduce such iatrogenic events and eradicate toxic deaths.
Chapter
The initial conventional chemotherapy of colorectal cancer (CRC) always consists of a fluoropyrimidine, either the parent drug 5-fluorouracil (5FU), the prodrug capecitabine, or the triple combination S-1, in which Ftorafur serves as a prodrug for 5FU. Initially, 5FU was combined with leucovorin, which potentiates the inhibition of the target thymidylate synthase (TS). Since TS inhibition leads to depletion of TTP, essential for DNA synthesis, this results in an increased incorporation of dUTP into DNA, together leading to a so-called thymine-less death. In advanced CRC both low TS expression and a high inhibition are related to a longer survival of patients. Other resistance parameters include decreased intratumoral activation to its nucleotide, and increased systemic degradation by dihydropyrimidine dehydrogenase. In the commonly used combinations (FOLFIRI and FOLFOX) with the topoisomerase I inhibitor, irinotecan, and the DACH-modified platinum analog, oxaliplatin, the relation with TS became less clear. Resistance to irinotecan is mainly associated with its systemic degradation by cytochrome P450 enzymes or conjugation by the UDP-glucuronyl transferase, while resistance to oxaliplatin is associated with increased repair of oxaliplatin-DNA adducts by the repair enzyme ERCC1. With the introduction of capecitabine, the limiting activation catalyzed by carboxyl-esterase and cytidine deaminase was associated with resistance, although the last step in the activation to 5FU catalyzed by both uridine phosphorylase and thymidine phosphorylase (TP) has more often been associated with resistance. Recently, another fluoropyrimidine, TAS-102, containing trifluorothymidine, was registered as third-line treatment. For this drug, decreased cellular uptake by the equilibrative nucleoside transporter, activation by thymidine kinase 1, and decreased incorporation into DNA have been associated with resistance. Analysis of these parameters should enable to guide treatment.
Article
Purpose: Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. Methods: Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC-MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. Results: There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). Conclusions: The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.
Chapter
5-Fluorouracil (5-FU) and its oral prodrug capecitabine are mainstays of oncologic therapy. They are fluoropyrimidine derivatives and function as antimetabolites. Fluoropyrimidines may be administered as monotherapy or in combination with other chemotherapeutic agents. They are predominantly used for the treatment of gastrointestinal, breast, and head and neck cancers. The therapeutic and toxicologic effects of 5-FU and capecitabine are attributed to their deleterious incorporation into RNA and DNA, and to their inhibition of thymidylate synthetase. Studies differ, but it is estimated that somewhere between 10% and 40% of patients on 5-FU and capecitabine develop some form of severe or life-threatening toxicity. Life-threatening toxicity may result from myelosuppression, diarrhea, mucositis, neurotoxicity, and cardiotoxicity. Hand-and-foot syndrome is also a characteristic adverse effect of 5-FU and capecitabine. Toxicity may result from intentional overdose, iatrogenic overdose, or therapeutic administration. A significant proportion severe toxicity occurs in the setting of therapeutic administration rather than acute overdose and is generally attributed to genetic polymorphisms. Treatment of toxicity is largely supportive. The antidote for fluoropyrimidine toxicity is uridine triacetate, but its indications are limited, and ideally the treating oncologist or a toxicologist should be consulted prior to its administration.
Article
Urological malignancies, represented mainly by prostate, bladder, and renal cancers, are some of the leading causes of cancer-related mortalities worldwide. Despite various efforts over decades to develop early detection tests and effective therapeutic paradigms, the response rate to the existing treatments remains low for both primary and late stage/recurrent phases of these cancers. The evolving landscape of molecular diagnostics, aiming to make the diagnosis and treatment more patient-driven, underpins precision oncology and particularly intends to rationally profile individual tumors and highlight the mechanistic insight and complexity of tumor microenvironment in order to develop biomarkers of toxicity risks and response prediction in a clinically oriented dynamical setting. The present review is an effort to capture some of the recent developments in the area of molecular diagnostics and functional testing platforms and their potential application in clinical decision making in the premises of precision oncology of urological malignancies.
Article
Full-text available
Background and Objectives: Dihydropyrimidine dehydrogenase (DPD) plays a key role in the catabolism of 5-fluorouracil (5-FU) to 5-fluoro-5,6-dihydrouracil (5-FDHU), and as such, an impairment of DPD has been recognized as an important factor for altered 5-FU and 5-FDHU pharmacokinetics, predisposing patients to the development of severe 5-FU-associated toxicity. Our objectives were to avoid severe 5-FU toxicities in patients with greatly impaired 5-FU and 5-FDHU pharmacokinetics after the administration of a reduced test dose of 5-FU and to investigate possible 5-FU or 5-FDHU pharmacokinetic parameters of the test dose related to the most common drug toxicities that affect patients after the first cycle of 5-FU chemotherapy. Methods: Pharmacokinetics of 5-FU/5-FDHU and DPD activity in peripheral blood mononuclear cells (PBMCs) were examined in 188 gastrointestinal cancer patients given a test dose of 5-FU, 250 mg/m2 ,2 weeks before starting the planned 5-FU treatment of 370 mg/m2 plus L-folinic acid, 100 mg/m2, for 5 days every 4 weeks. Drug levels were examined by HPLC, and toxicities were graded according to World Health Organization criteria. Results: The 5-FU test dose was well tolerated in all patients. Of 188 patients, 3 (1.6%) had marked alterations of 5-FU/5-FDHU pharmacokinetics (ie, 5-FU half-life (t½) >5 hours, 5-FU total body clear- ance (CLTB) < 1 L ·h 1 ·m 2, and 5-FDHU time to reach maximum plasma concentration (tmax) >45 minutes); they were excluded from 5-FU treatments and treated with irinotecan, which was well tolerated. The plasma disposition of 5-FU in the remaining 185 patients revealed an area under the curve (AUC) of 3.73 2.18 h · g/mL (mean SD), maximum plasma concentration (Cmax) of 16.78 8.61 g/mL, and t½ of 0.16 0.15 hour, whereas the CLTB was 65.67 31.86 L · h 1 ·m 2. The 5-FDHU plasma profile showed a Cmax value of 3.64 1.94 g/mL, whereas the tmax value was 26.63 10.06 minutes, with an AUC value of 3.71 1.90 h · g/mL. The PBMC DPD activity was 202.15 141.14 pmol 5-FDHU · min1 ·m g1 protein (95% confidence interval, 165-239.3 pmol 5-FDHU · min1 ·m g1 pro- tein). A significant correlation between 5-FU AUC and 5-FDHU AUC was found (r 0.5492, P < .0001),
Article
Full-text available
Fluoropyrimidine drugs are widely used in head and neck cancer (HNC). DPD deficiency is a pharmacogenetics syndrome associated with severe/lethal toxicities upon 5-FU or capecitabine intake. We have developed a simple, rapid, and inexpensive functional testing for DPD activity, as a means to identify deficient patients and to anticipate subsequent 5-FU-related toxicities. We present here the impact of fluoropyrimidine dose tailoring based on DPD functional screening in a prospective, open, non-controlled study, both in term of reduction in severe toxicities and of treatment efficacy. About 65 patients with HNC (59 ± 9 years, 52M/13F, Prospective Group) were entered into the study. Screening for DPD deficiency was performed prior to the beginning of the chemotherapy or radiochemotherapy. DPD status was evaluated by monitoring U/UH2 ratio levels in plasma as a surrogate marker for enzymatic functionality. 5-FU doses were reduced according to the extent of the detected DPD impairment, and adjusted on the basis of age, general condition, and other clinical/paraclinical covariates, if required. Treatment-related toxicities and subsequent impact on treatment delay were carefully monitored next for comparison with a retrospective, Reference subset of 74 other patients with HNC (mean age: 59 ± 10, 58M/16F, Reference Group), previously treated in the same institute with similar schedule but using standard 5-FU dosage. Thirty-one out of 65 patients (48%) were identified as mildly (28%) to markedly (20%) DPD deficient. Subsequently, dose reductions ranging from 10 to 100% with 5-FU were applied in those patients. In this group, six patients (9%) experienced severe toxicities, none of them being life threatening, and no toxic death was encountered. In comparison, 16 out of 74 patients (22%) of the Reference Group displayed severe side effects after standard 5-FU administration, 13% being life-threatening toxicities (e.g., G4 neutropenia + sepsis). Moreover, one toxic death was observed in this Reference Group. No postponement or cancelation of forthcoming chemoradiotherapy courses occurred in the Prospective Group, whereas treatment had to be disrupted in six patients (8%) from the Reference Group. No difference in first-line therapy efficacy was evidenced between the two subsets (78 vs. 79% response, P = 0.790). Although non-randomized, this study strongly suggests that prospective determination of DPD status has an immediate clinical benefit by reducing the drug-induced toxicities incidence in patients treated with 5-FU, allowing an optimal administration of several courses in a row, while maintaining efficacy. Our preliminary results thus advocate for systematic DPD screening in patients eligible for treatment with fluoropyrimidine drugs in HNC.
Article
Full-text available
5-Fluorouracil (5-FU) is the most widely used chemotherapy drug, primarily against gastrointestinal, head and neck, and breast cancers. 5-FU has large pharmacokinetic variability resulting in unexpected toxicity or ineffective treatment. Therapeutic drug management of 5-FU minimizes toxicity and improves outcome. A nanoparticle-based immunoassay was developed to provide oncologists with a rapid, cost-effective tool for determining 5-FU plasma concentrations. Monoclonal antibodies, bound to nanoparticles, were used to develop an immunoassay for the Olympus AU400. Assay precision, linearity, calibration stability, and limit of detection were run at multiple centers; interference, cross-reactivity, lower limit of quantitation and recovery at 1 center. Clinical samples collected from 4 cancer centers were analyzed for 5-FU concentrations by liquid chromatography-tandem mass spectrometry and compared with the immunoassay results. With calibrators from 0 to 1800 ng/mL 5-FU and autodilution, concentrations up to 9000 ng/mL could be determined. Time to first result was 10 minutes, and 400 samples per hour could be quantitated from a standard curve stored for >30 days. Imprecision across all laboratories was <5%, and the assay was linear upon dilution over the entire range. Cross-reactivities for dihydro-5-FU, uracil, capecitabine, and tegafur were <1%, 9.9%, 0.05%, and 0.23%, respectively. The limit of detection was 52 ng/mL with a lower limit of quantitation of 86 ng/mL. Assay results of clinical samples (93-1774 ng/mL) correlated with liquid chromatography-tandem mass spectrometry results: (R = 0.9860, slope 1.035, intercept 10.87 ng/mL). This novel immunoassay is suitable for quantitating 5-FU plasma concentrations with advantages of speed, small sample size, minimal sample pretreatment, and application on automated instrumentation. These advantages enable efficient therapeutic drug management of 5-FU in clinical practice.
Article
Full-text available
Chemotherapy dosing of the fluoropyrimidine 5-fluorouracil (5-FU) is currently based on body surface area. However, body surface area-based dosing has been associated with clinically significant pharmacokinetic variability, and as such, dosing based on body surface area may be of limited use. The clinical activity of 5-FU is modest at standard doses, and in general, dosing is limited by the safety profile, with myelosuppression and gastrointestinal toxicity being the most commonly observed side effects. Various strategies have been developed to enhance the clinical activity of 5-FU, such as biochemical modulation, alterations in scheduling of administration, and the use of oral chemotherapy. Studies that have shown an association between plasma concentration with toxicity and clinical efficacy have shown that pharmacokinetically guided dose adjustments can substantially improve the therapeutic index of 5-FU treatment. These studies have shown that only 20%-30% of patients treated with a 5-FU-based regimen have 5-FU levels that are in the appropriate therapeutic range--approximately 40%-60% of patients are underdosed and 10%-20% of patients are overdosed. To date, 5-FU drug testing has not been widely used because of the lack of a simple, fast, and inexpensive method. Recent advances in testing based on liquid chromatography-mass spectroscopy and a nanoparticle antibody-based immunoassay for 5-FU may now allow for routine monitoring of 5-FU in clinical practice. We review the data on pharmacokinetically guided dose adjustment of 5-FU and discuss the potential of this approach to advance therapeutic outcomes.
Article
Full-text available
Decreased 5-fluorouracil catabolism has been considered a major factor contributing to fluoropyrimidine (FP)-related toxicity. Alterations in the dihydropyrimidine dehydrogenase gene coding for the first and rate-limiting enzyme of FP catabolic pathway could explain toxicity in only a limited proportion of FP-treated patients. The importance of gene variants in dihydropyrimidinase (DPYS) coding for subsequent catabolic enzyme of FP degradation is not fully understood. We performed genotyping of DPYS based on denaturing high-performance liquid chromatography in 113 cancer patients including 67 with severe FP-related toxicity and 46 without toxicity excellently tolerating FPs treatment. We detected nine DPYS variants including four located in non-coding sequence (c.-1T>C, IVS1+34C>G, IVS1-58T>C, and novel IVS4+11G>T), four silent (c.15G>A, c.216C>T, and novel c.105C>T and c.324C>A), and one novel missense variant c.1441C>T (p.R481W). All novel alterations were detected once only in patients without toxicity. The c.-1T>C and IVS1-58T>C variants were found to modify the risk of toxicity. The CC carriers of the c.-1C alleles were at higher risk of mucositis (OR = 4.13; 95% CI = 1.51-11.31; P = 0.006) and gastrointestinal toxicity (OR = 3.54; 95% CI = 1.59-7.88; P = 0.002), whereas the presence of the IVS1-58C allele decreased the risk of gastrointestinal toxicity (OR = 0.4; 95% CI = 0.17-0.93; P = 0.03) and leucopenia (OR = 0.29; 95% CI = 0.08-1.01; P = 0.05). Our results indicate that missense and nonsense variants in DPYS are infrequent, however, the development of serious primarily gastrointestinal toxicity could be influenced by non-coding DPYS sequence variants c.-1T>C and IVS1-58T>C.
Article
Full-text available
The importance of polymorphisms in the dihydropyrimidine dehydrogenase gene (DPYD) for the prediction of severe toxicity in 5-fluorouracil (5-FU)-based chemotherapy is still unclear. This study aims to assess the predictive value of DPYD variation with respect to previously described DPYD variants for 5-FU toxicity. It represents the first analysis of the gene at the haplotype level, also capturing potentially important genetic variation located outside the coding regions of DPYD. The entire coding sequence and exon-flanking intronic regions of DPYD were sequenced in 111 cancer patients receiving fluoropyrimidine-based chemotherapy. DPYD haplotypes were inferred and their associations with severe 5-FU toxicity were assessed. None of the previously described deleterious variants (IVS14+1G>A, c.2846A>T and c.1679T>G) were detected in 24 patients who experienced severe 5-FU toxicity. A potential association was observed between a haplotype containing three novel intronic polymorphisms (IVS5+18G>A, IVS6+139G>A and IVS9-51T>G) and a synonymous mutation (c.1236G>A), which was observed five- out of eight-times in patients with severe adverse effects. The association of a haplotype containing no nonsynonymous or splice-site polymorphisms indicates that additional important genetic variation may be located in noncoding gene regions. Furthermore, a comparison with other studies suggests that the relative importance of particular DPYD mutations (IVS14+1G>A and c.2846A>T) for predicting severe 5-FU toxicity differs geographically across Europe.
Article
Full-text available
Alterations in dihydropyrimidine dehydrogenase gene (DPYD) coding for the key enzyme (DPD) of fluoropyrimidines (FPs) catabolism contribute to the development of serious FPs-related toxicity. We performed mutation analysis of DPYD based on cDNA sequencing in 76 predominantly colorectal cancer patients treated by FPs with early development of high (grade 3-4) hematological and/or gastrointestinal toxicity. Six previously described [85T>C (C29R), 496A>G (M166V), 775A>G (K259E), 1601G>A (S534N), 1627A>G (I543V), IVS14+1G>A, 2194G>A (V732I)] and two novel [187A>G (K63E) and 1050 G>A (R357H)] non-synonymous DPYD variants were found in 56/76 (73.7%) high-toxicity patients. Subsequently, these alterations were analyzed in 48 patients with excellent long-term tolerance of FPs and in 243 controls and were detected in 37/48 (77.1%) and 166/243 (68.3%) cases, respectively. Analysis of these alterations as risk factors for development of toxicity in pooled FPs-treated population demonstrated that C29R negatively correlated with overall gastrointestinal toxicity (OR = 0.48; 95%CI 0.23-1.0) and M166V in women protected against overall hematological toxicity and neutropenia (both OR = 0.26; 95%CI 0.07-0.89), whereas IVS14+1G>A (found in five high-toxicity patients only) increased risk of mucositis in overall population (OR = 7.0; 95%CI 1.1-44.53), and thrombocytopenia in women (OR = 10.8; 95%CI 1.24-93.98). Moreover, we identified a strong association of V732I with leucopenia (OR = 8.17; 95%CI 2.44 - 27.31) and neutropenia (OR=2.78; 95% CI 1.03-7.51). Our data enabled characterization of "high risk" haplotypes (carriers of IVS14+1G>A or V732 lacking M166V) representing small (22% female and 11% male patients), population in high risk of serious hematological toxicity development, and in patients with "lower risk" that unlikely develop serious hematological toxicity [carriers of M166V without IVS14+1G>A and V732I in females (32% women), and non-carriers of C29R, M166V, IVS14+1G>A, and V732I in males (46% men)]. Our results indicate that genotyping of several DPYD variants may lead to stratification of patients with respect to the risk of serious hematological toxicity development during FPs treatment.
Article
Full-text available
Cancer patients carrying mutations in the dihydropyrimidine dehydrogenase gene (DPYD) have a high risk to experience severe drug-adverse effects following chemotherapy with fluoropyrimidine drugs such as 5-fluorouracil (5-FU) or capecitabine. The pretreatment detection of this impairment of pyrimidine catabolism could prevent serious, potentially lethal side effects. As known deleterious mutations explain only a limited proportion of the drug-adverse events, we systematically searched for additional DPYD variations associated with enhanced drug toxicity. We performed a whole gene approach covering the entire coding region and compared DPYD genotype frequencies between cancer patients with good (n = 89) and with poor (n = 39) tolerance of a fluoropyrimidine-based chemotherapy regimen. Applying logistic regression analysis and sliding window approaches we identified the strongest association with fluoropyrimidine-related grade III and IV toxicity for the non-synonymous polymorphism c.496A>G (p.Met166Val). We then confirmed our initial results using an independent sample of 53 individuals suffering from drug-adverse-effects. The combined odds ratio calculated for 92 toxicity cases was 4.42 [95% CI 2.12-9.23]; p (trend)<0.001; p (corrected) = 0.001; the attributable risk was 56.9%. Comparing tumor-type matched sets of samples, correlation of c.496A>G with toxicity was particularly present in patients with gastroesophageal and breast cancer, but did not reach significance in patients with colorectal malignancies. Our results show compelling evidence that, at least in distinct tumor types, a common DPYD polymorphism strongly contributes to the occurrence of fluoropyrimidine-related drug adverse effects. Carriers of this variant could benefit from individual dose adjustment of the fluoropyrimidine drug or alternate therapies.
Article
Full-text available
The activity of dihydropyrimidine dehydrogenase (DPD), the key enzyme of pyrimidine catabolism, is thought to be an important determinant for the occurrence of severe toxic reactions to 5-fluorouracil (5-FU), which is one of the most commonly prescribed chemotherapeutic agents for the treatment of solid cancers. Genetic variation in the DPD gene (DPYD) has been proposed as a main factor for variation in DPD activity in the population. However, only a small proportion of severe toxicities in 5-FU based chemotherapy can be explained with such rare deleterious DPYD mutations resulting in severe enzyme deficiencies. Recently, hypermethylation of the DPYD promoter region has been proposed as an alternative mechanism for DPD deficiency and thus as a major cause of severe 5-FU toxicity. Here, the prognostic significance of this epigenetic marker with respect to severe 5-FU toxicity was assessed in 27 cancer patients receiving 5-FU based chemotherapy, including 17 patients experiencing severe toxic side effects following drug administration, none of which were carriers of a known deleterious DPYD mutation, and ten control patients. The methylation status of the DPYD promoter region in peripheral blood mononuclear cells was evaluated by analysing for each patient between 19 and 30 different clones of a PCR-amplified 209 base pair fragment of the bisulfite-modified DPYD promoter region. The fragments were sequenced to detect bisulfite-induced, methylation-dependent sequence differences. No evidence of DPYD promoter methylation was observed in any of the investigated patient samples, whereas in a control experiment, as little as 10% methylated genomic DNA could be detected. Our results indicate that DYPD promoter hypermethylation is not of major importance as a prognostic factor for severe toxicity in 5-FU based chemotherapy.
Article
Full-text available
The pig and human dihydropyrimidine dehydrogenase (DPD) cDNAs were cloned and sequenced. The pig enzyme, expressed in Escherichia coli, catalyzed the reduction of uracil, thymine, and 5-fluorouracil with kinetics approximating those published for the enzyme purified from mammalian liver. DPD could be expressed in significant quantities only when uracil was added to the bacterial growth medium. The pig and human enzymes contained 1025 amino acids and calculated M(r) = 111,416 and 111,398, respectively. Conserved domains corresponding to a possible NADPH binding site and FAD binding site were found in the NH2-terminal half of the proteins and two motifs of putative [4Fe-4S] binding sites were found near to the carboxyl terminus of the enzyme. The latter corresponds to the labile COOH-terminal fragment previously shown to contain the iron sulfur centers. A sequence encompassing a peptide corresponding to the uracil binding site was found between the NADPH/FAD-containing NH2-terminal portion of the protein and the iron-sulfur binding sites near to the COOH terminus. Thus, the DPD appears to be derived from at least three distinct domains. The DPYD gene was localized to the centromeric region of human chromosome 1 between 1p22 and q21.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU), and it is suggested that patients with a partial deficiency of this enzyme are at risk from developing a severe 5FU-associated toxicity. In this study, we demonstrated that a lethal toxicity after a treatment with 5FU was attributable to a complete deficiency of DPD. Analysis of the DPD gene for the presence of mutations showed that the patient was homozygous for a G-->A mutation in the invariant GT splice donor site flanking exon 14 (IVS14+1G>A). As a consequence, no significant residual activity of DPD was detected in peripheral blood mononuclear cells. To determine the frequency of the IVS14+1G>A mutation in the Dutch population, we developed a novel PCR-based method allowing the rapid analysis of the IVS14+1G>A mutation by RFLP. Screening for the presence of this mutation in 1357 Caucasians showed an allele frequency of 0.91%. In our view, the apparently high prevalence of the IVS14+1G>A mutation in the normal population, with 1.8% heterozygotes, warrants genetic screening for the presence of this mutation in cancer patients before the administration of 5FU.
Article
Full-text available
Previous work demonstrated that 5-fluorouracil (5-FU) metabolism is a critical factor for treatment tolerability. In order to study the predictivity of pharmacokinetics with respect to the occurrence of 5-FU toxicity, this study investigates the relationship between the pharmacokinetics of 5-FU and its metabolite 5-fluoro-5,6-dihydrouracil (5-FDHU), dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells (PBMNC) and treatment tolerability. Pharmacokinetics and metabolism of 5-FU and activity of DPD in PBMNC were examined in 110 colorectal cancer patients given adjuvant 5-FU 370 mg/m2 plus L-folinic acid 100 mg/m2 for five days every four weeks. Drug levels were examined by HPLC. while toxicities were graded according to WHO criteria. DPD activity in patients with mild toxicities (WHO grade < or = 1) was 197.22 < or = 11.34 pmol of 5-FDHU/min/ mg of protein, while in five patients with grade 3-4 gastrointestinal toxicity, DPD ranged from low to normal values (range 31.12-182.37 pmol/min/mg of protein). In these patients. 5-FU clearance (CL) was lower (range 14.12-25.17 l/h/m2), and the area under the curve (AUC) was higher (range 14.70-26.20 h x microg/ml) than those observed in 84 patients with mild toxicities (CL, 56.30 +/- 3.60 l/h/M2; AUC, 7.91 +/- 0.44 h x microg/ml). The severity of adverse events was associated with increased 5-FU/5-FDHU AUC ratio and reduced 5-FU CL, while 5-FU and 5-FDHU pharmacokinetics were not related to DPD activity. This study shows that DPD activity in PBMNC is unrelated to 5-FU/5-FDHU disposition and patients with severe toxicity display marked pharmacokinetic alterations while a reduction of DPD activity may not occur.
Article
Full-text available
A familial approach was used to elucidate the genetic determinants of profound and partial dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) deficiency in an Alabama family. In 1988, our laboratory diagnosed profound DPD deficiency in a breast cancer patient with grade IV toxicity after cyclophosphamide/methotrexate/5-fluorouracil chemotherapy (R. B. Diasio et al., J. Clin. Investig., 81: 47-51, 1988). We now report the genetic analysis of archived genomic DNA that reveals that the proband was a compound heterozygote for two different mutations, one in each allele: (a) a G to A mutation in the GT 5' splicing recognition sequence of intron 14, which results in a 165-bp deletion (corresponding to exon 14) in the DPD mRNA (DPYD*2A); and (b) a T1679G mutation (now designated DPYD*13), which results in a I560S substitution. Sequence analysis revealed segregation of both mutations with the son and the daughter each inheriting one mutation. Phenotype analysis (DPD enzyme activity) confirmed that both children were partially DPD deficient. Plasma uracil and DPD mRNA levels were found to be within normal limits in both children. We conclude that profound DPD deficiency in the proband resulted from a combination of two mutations (one mutation in each allele) and that heterozygosity for either mutation results in partial DPD deficiency. Lastly, we identified two variant alleles reported previously as being associated with DPD enzyme deficiency [T85C resulting in a C29R substitution (DPYD*9A) and A496G (M166V) in a family member with normal DPD enzyme activity]. These data suggest that both variant alleles are unrelated to DPD deficiency and emphasize the need to perform detailed familial genotypic and phenotypic analysis while characterizing this pharmacogenetic syndrome.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) enzyme deficiency is a pharmacogenetic syndrome with possible fatal outcome following 5-fluorouracil (5-FU) treatment. Several studies examining the molecular basis for DPD deficiency have identified over 30 sequence variations in the DPYD gene (which codes for the DPD enzyme). Our laboratory has recently developed and validated a denaturing high performance liquid chromatography method capable of identifying both known and unknown sequence variations in the DPYD gene. In the present study, we used this denaturing high performance liquid chromatography approach to examine the DPYD genotype of three patients who experienced lethal toxicity after administration of 5-FU. DPD enzyme activity could only be measured in one patient before death and demonstrated that lethal toxicity can occur in a partially DPD-deficient individual. Multiple heterozygous sequence variations (both known and unknown) were detected in all three patients including the novel variants 545T>A, M182K and 2329G>T, A777S. We conclude that (a) lethal toxicity can occur in partially DPD-deficient individuals after administration of 5-FU and is not exclusive to profoundly DPD-deficient individuals as suggested previously, (b) the complicated heterozygote genotype seen in these patients, combined with DPD deficiency being an autosomal codominant inherited syndrome, precludes the use of simple genotyping assays that identify only one or two mutations as a method for identifying DPD-deficient individuals; and (c) these multiple heterozygote genotypes (which are more difficult to accurately characterize) may be responsible for some of the conflicting reports which suggests a lack of correlation between phenotype and genotype.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11 + 1G-->T and the missense mutations 731A-->C (E244V) and 1651G-->A (A551T). The G-->T mutation in the invariant GT splice donor site flanking exon 11 (IVS11 + 1G-->T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400-446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.
Article
Full-text available
Complete or partial loss of dihydropyrimidine dehydrogenase (DPD) function has been described in cancer patients with intolerance to fluoropyrimidine drugs like 5-fluorouracil (5-FU) or Xeloda. The intention of this population study is to assess and to evaluate gene variations in the entire coding region of the dihydropyrimidine dehydrogenase gene (DPYD), which could be implicated in DPD malfunction. A cohort of 157 individuals was genotyped by denaturing high-performance liquid chromatography; 100 of these genotypes were compared with functional studies on DPD activity and mRNA expression. Twenty-three variants in coding and noncoding regions of the DPYD gene were detected, giving rise to 15 common haplotypes with a frequency of >1%. Rare sequence alterations included a frameshift mutation (295-298delTCAT) and three novel point mutations, 1218G>A (Met406Ile), 1236G>A (Glu412Glu), and 3067C>T (Pro1023Ser). DPD enzyme activity showed high variation in the analyzed population and correlated with DPD mRNA expression. In particular, the novel variants were not accompanied with decreased enzyme activity. However, a statistically significant deviation from the median DPD activity of the population was associated with the mutations 1601G>A (Ser534Asn) and 2846A>T (Asp949Val). This work presents an analysis of DPYD gene variations in a large cohort of Caucasians. The results reflect the genetic and enzymatic variability of DPD in the population and may contribute to further insight into the pharmacogenetic disorder of DPD deficiency.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) deficiency, a known pharmacogenetic syndrome associated with 5-fluorouracil (5-FU) toxicity, has been detected in 3% to 5% of the population. Genotypic studies have identified >32 sequence variants in the DPYD gene; however, in a number of cases, sequence variants could not explain the molecular basis of DPD deficiency. Recent studies in cell lines indicate that hypermethylation of the DPYD promoter might down-regulate DPD expression. The current study investigates the role of methylation in cancer patients with an unexplained molecular basis of DPD deficiency. DPD deficiency was identified phenotypically by both enzyme assay and uracil breath test, and genotypically by denaturing high-performance liquid chromatography. The methylation status was evaluated in PCR products (209 bp) of bisulfite-modified DPYD promoter, using a novel denaturing high-performance liquid chromatography method that distinguishes between methylated and unmethylated alleles. Clinical samples included five volunteers with normal DPD enzyme activity, five DPD-deficient volunteers, and five DPD-deficient cancer patients with a history of 5-FU toxicity. No evidence of methylation was detected in samples from volunteers with normal DPD. Methylation was detected in five of five DPD-deficient volunteers and in three of five of the DPD-deficient cancer patient samples. Of note, one of the two samples from patients with DPD-deficient cancer with no evidence of methylation had the mutation DPYD*2A, whereas the other had DPYD*13. Methylation of the DPYD promoter region is associated with down-regulation of DPD activity in clinical samples and should be considered as a potentially important regulatory mechanism of DPD activity and basis for 5-FU toxicity in cancer patients.
Article
Full-text available
The purpose of this study was to determine simple genetic factors helpful to tailor 5-FU administration and determine strategy in first-line chemotherapy of advanced colorectal cancer. In 76 patients initially treated by 5-FU, thymidylate synthase, dihydropyrimidine dehydrogenase and methylene tetrahydrofolate reductase germinal polymorphisms, dihydrouracil/uracil plasma ratio and 5-FU plasma clearance were investigated and correlated for tolerance (10.5% grade 3 and 4 toxicity) and efficacy (32.9% objective response rate and 20 months median overall survival time). Toxicity was linked to performance status >2 (P=0.004), low UH2/U ratio, 2846 A>T, IVS 14+1G>A for DPD (P=0.031), and homozygoty C/C for MTHFR 1298 A>C (P=0.0018). The overall survival of the patients with a 3R/3R TS genotype associated with C/C for 677 C>T or A/A for 1298 A>C was statistically shorter (log-rank test P=0.0065). Genetic factors permit the tailoring of 5-FU treatment. They should occupy center stage in future clinical trials for specifically designing treatment for patients with a given biologic feature.
Article
Full-text available
Dihydropyrimidine dehydrogenase (DPD) is an inactivating and rate-limiting enzyme for 5-fluorouracil (5-FU), and its deficiency is associated with a risk for developing a severe or fatal toxicity to 5-FU. In this study, to search for genetic variations of DPYD encoding DPD in Japanese, the putative promoter region, all exons, and flanking introns of DPYD were sequenced from 341 subjects including cancer patients treated with 5-FU. Fifty-five genetic variations, including 38 novel ones, were found and consisted of 4 in the 5'-flanking region, 21 (5 synonymous and 16 nonsynonymous) in the coding exons, and 30 in the introns. Nine novel nonsynonymous SNPs, 29C>A (Ala10Glu), 325T>A (Tyr109Asn), 451A>G (Asn151Asp), 733A>T (Ile245Phe), 793G>A (Glu265Lys), 1543G>A (Val515Ile), 1572T>G (Phe524Leu), 1666A>C (Ser556Arg), and 2678A>G (Asn893Ser), were found at allele frequencies between 0.15 and 0.88%. Two known nonsynonymous variations reported only in Japanese, 1003G>T (*11, Val335Leu) and 2303C>A (Thr768Lys), were found at allele frequencies of 0.15 and 2.8%, respectively. SNP and haplotype distributions in Japanese were quite different from those reported previously in Caucasians. This study provides fundamental information for pharmacogenetic studies for evaluating the efficacy and toxicity of 5-FU in Japanese and probably East Asians.
Article
2541 Background: DPD deficiency is a pharmacogenetic syndrome associated with severe toxicities upon 5-FU/capecitabine intake. To detect patients at risk upon fluoropyrimidines administration we developed a simple, rapid and inexpensive phenotypic method for DPD status evaluation. Here, clinical relevance of DPD deficiency a priori determination was studied. Methods: This method was used prospectively in 50 head and neck cancers patients (60±7 years, 37M/13F). Screening for DPD deficiency was performed prior to the beginning of the platinum + 5-FU (± radiation therapy) administration. DPD status was evaluated by HPLC monitoring U/UH2 ratio levels in plasma as a surrogate marker. Only 5-FU doses were adjusted according to the level of detected impairment (mild deficiency: -20%, severe: - 50%, total: no 5-FU). Drug-induced toxicities and subsequent impact on treatment schedule were carefully monitored next for comparison with a retrospective subset of 50 patients (mean age: 58±9 years, 40M/10F), treated with standard dosage of the same chemotherapy or radio- chemotherapy schedule. Results: DPD determination permitted the detection of 11 patients with DPD impairment out of 50 (22%). These 11 patients had their 5-FU doses customized and, consequently, neither severe toxicity nor delays in the administration radiation therapy or chemotherapy were reported in this group. Conversely, 7 out of 50 (14%) patients administered with standard dosage of 5-FU displayed severe toxicities (>grade-3, WHO grading), with subsequent postponement of the following courses. One patient (2%) did not recover from his toxicities. Of these 8 patients, retrospective determination of DPD status suggested impaired function in 7 of them (87%), including the toxic- death one. Conclusions: Although non randomized, this study suggests that prospective use of DPD status has an immediate clinical benefit by, reducing the drug-induced toxicities incidence in patients treated with 5-FU, and allowing an optimal administration of several courses in a row. Our preliminary results advocate systematic DPD status screening in patients eligible for treatment with fluoropyrimidine drugs. Survival and response study still on going. No significant financial relationships to disclose.
Article
6515 Background: Dihydropyrimidine dehydrogenase (DPD) plays a pivotal role in the detoxification of 5-FU. We studied the impact of screening DPD impairment in head and neck cancer (HNC) patients, both on reduction of drug-related toxicities and as a pharmacoeconomic endpoint. Methods: A total of 148 consecutive patients with HNC treated with 5-FU+platinum were monitored. Seventy-four patients (Arm A - before 2006) were treated with standard dosage, whereas 74 other patients (Arm B - after 2006) had their DPD status phenotypically evaluated prior to receiving 5-FU, with subsequent dose reduction if DPD deficiency were suspected. Severe toxicities and response were compared. Additionally, direct and indirect costs required to manage the treatment-related toxicities and to establish DPD status were calculated. Results: Sepsis was observed in 16.2% of patients treated with standard dosage. In Arm B, DPD deficiency was suspected in 35% of the patients and 5-FU dosage was subsequently reduced. Consequently, only 1.8% of them experienced sepsis. Of note, response rates were comparable between Arm A and B (62 vs 61%, p>0.05), thus demonstrating that 5-FU dose tailoring did not negatively impact on treatment efficacy, while reducing the occurrence of severe toxicities. Managing toxicities required an average 23-days of extra-hospitalization (4–96 days), including an average 1.6-day stay in ICU. No patients from Arm B had to stay in ICU. Drugs required for managing toxicities cost an average of $339 per patient (Arm A) and was reduced down to $38 per patient (Arm B). Similarly, mean extra-hospitalization cost was $5,940/patient in Arm A and $245/patient in Arm B. Testing DPD cost $49/patient in Arm B. Conclusions: Developing an adaptative dosing strategy based upon DPD status evaluation led to a dramatic reduction of the incidence of 5-FU-related severe toxicities, while maintaining optimal efficacy. Subsequently, extra-cost (medication + hospitalization costs) required to manage the toxicities fell down from $6,279 to $294/patient. Overall, this study advocates that systematic screening for DPD deficiency could be cost-efficient in the setting of 5-FU-based chemotherapies, with a reduction of 95% of the extra-costs. No significant financial relationships to disclose.
Article
5-Fluorouracil (5FU) is most commonly used in chemotherapy for human malignancy. Over 80% of administered 5FU is metabolically degraded by dihydropyrimidine dehydrogenase (DPD), a primary and rate-limiting enzyme in the 5FU metabolic pathway. A DPD-deficient phenotype among cancer patients, which has posed a serious problem in 5FU-based chemotherapy, was reported to be in part ascribed to germline mutations in dihydropyrimidine dehydrogenase (DPYD) gene. Therefore, we for the first tune examined the frequencies and types of germline mutations in the DPYD gene among a total of 107 Japanese cancer patients and healthy volunteers. Of 214 alleles examined among them, 181 alleles were of the same type, which was assigned as wild type; 21 alleles revealed a nucleotide substitution resulting in silent mutation; and the remaining 12 alleles showed five types of nucleotide deletion or substitutions resulting in one frameshift and four missense mutations. Three of them, A74G, 812delT and L572V, were novel mutations. None of the study subjects showed homozygous frameshift or missense mutated alleles. We also studied the association between toxic response to 5FU and heterozygous frame shift or missense mutation of the DPYD gene among eight cancer patients who had received 5FU-based chemotherapy. These patients did not show any adverse effects higher than grade 3, suggesting that heterozygotes are not associated with increased toxicity to 5FU. Our results indicate that a very small percentage, about 0.2%, of the Japanese population seems to carry homozygous mutations in DPYD gene, mutations which possibly indicate genetically increased toxicity of 5FU-based chemotherapy.
Article
Background and objective: 5-fluorouracil (5-FU) is still a widely used anticancer drug. More than 85% of the 5-FU administered is catabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The purpose of this study was to determine the mutation frequency of four exons in Chinese cancer patients and the relationship between genotype and DPD activity. Methods: Samples from 142 cancer patients were investigated in this study. The DPD activity was determined by reversed-phase HPLC. Exons 2, 13, 14 and 18 were amplified by polymerase chain reaction (PCR), sequenced and analysed from both sense and antisense directions. Nonparametric one-sample Kolmogorov–Smirnov test was used for distribution analysis; two independent samples t-test and one-way anova was performed for two groups and three groups analyses, respectively. Results and discussion: Plasma-DPD activities in the 142 cancer patients followed a Gaussian distribution. The mean plasma-DPD activity in women was lower than that in men (P = 0·006). Four mutations, 85T>C(DPYD*9A), 1627A>G(DPYD*5), 1896T>C and 2194G>A(DPYD*6), were found in the 142 cancer patients. The following mutations reported by others were not detected: 61C>T, 62G>A, 74A>G, 1601G>A(DPYD*4), 1679T>G(DPYD*13), 1714C>G, 1897delC(DPYD*3) and IVS 14 + 1G>A. No significant correlation was found between three mutations [85T>C(DPYD*9A), 1627A>G (DPYD*5) and 1896T>C], and DPD activity was found. Conclusion: No clear correlation between the mutations studied and DPD activity could be established in this study. However, larger-scale prospective studies are needed to better assess the reported genotype–phenotype correlations.
Article
Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterised by thymine-uraciluria in homozygous deficient patients and has been associated with a variable clinical phenotype. In order to understand the genetic and phenotypic basis for DPD deficiency, we have reviewed 17 families presenting 22 patients with complete deficiency of DPD. In this group of patients, 7 different mutations have been identified, including 2 deletions [295–298delTCAT, 1897delC], 1 splice-site mutation [IVS14+1G>A)] and 4 missense mutations (85T>C, 703C>T, 2658G>A, 2983G>T). Analysis of the prevalence of the various mutations among DPD patients has shown that the G→A point mutation in the invariant splice donor site is by far the most common (52%), whereas the other six mutations are less frequently observed. A large phenotypic variability has been observed, with convulsive disorders, motor retardation and mental retardation being the most abundant manifestations. A clear correlation between the genotype and phenotype has not been established. An altered β-alanine, uracil and thymine homeostasis might underlie the various clinical abnormalities encountered in patients with DPD deficiency.
Article
Dihydropyrimidine dehydrogenase (DPD) catabolizes endogenous pyrimidines and pyrimidine-based antimetabolite drugs. A deficiency in human DPD is associated with congenital thymine-uraciluria in pediatric patients and severe 5-fluorouracil toxicity in cancer patients. The dihydropyrimidine dehydrogenase gene (DPYD) was isolated, and its physical map and exon–intron organization were determined by analysis of P1, PAC, BAC, and YAC clones. TheDPYDgene was found to contain 23 exons ranging in size from 69 bp (exon 15) to 961 bp (exon 23). A physical map derived from a YAC clone indicated thatDPYDis at least 950 kb in length with 3 kb of coding sequence and an average intron size of about 43 kb.The previously reported 5′ donor splice site mutation present in pediatric thymine-uraciluria and cancer patients can now be assigned to exon 14. All 23 exons were sequenced from a series of human DNA samples, and three point mutations were identified in three racial groups as G1601A (exon 13, Ser534Asn), A1627G (exon 13, Ile543Val), and G2194A (exon 18, Val732Ile). These studies, which have established that theDPYDgene is unusually large, lay a framework for uncovering new mutations that are responsible for thymine-uraciluria and toxicity to fluoropyrimidine drugs.