# Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency

Marie-Christine Etienne-Grimaldi, Nicolas Pallet, Valérie Boige, Joseph Ciccolini, Laurent Chouchana, Chantal Barin-Le Guellec, Aziz Zaanan, Céline Narjoz, Julien Taieb, Fabienne Thomas, et al.

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Marie-Christine Etienne-Grimaldi, Nicolas Pallet, Valérie Boige, Joseph Ciccolini, Laurent Chouchana, et al.. Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency. European Journal of Cancer, 2023, 181, pp.3-17. 10.1016/j.ejca.2022.11.028. hal-03934173

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# Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency

Marie-Christine Etienne-Grimaldi¹*, Nicolas Pallet²,³*, Valérie Boige³,⁴, Joseph Ciccolini⁵,⁶,⁷, Laurent Chouchana⁸,⁹, Chantal Barin-Le Guellec¹⁰,¹¹, Aziz Zaanan¹², Céline Narjoz²,³, Julien Taieb¹³, Fabienne Thomas ¹⁴,¶, Marie-Anne Loriot²,³,¶, on behalf of the Francophone Network of Pharmacogenetics (RNPGx) and the French Clinical Oncopharmacology Group (GPCO)-UNICANCER

1. Laboratory of Oncopharmacology, Centre Antoine Lacassagne, Nice, France.

2. Department of Clinical Chemistry, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.

3. Université de Paris, INSERM UMRS1138, Centre de Recherche des Cordeliers, F-75006 Paris, France

4. Department of Cancer Medicine, Institut Gustave Roussy, Villejuif, France

5. SMARTc, CRCM INSERM U1068, Université Aix-Marseille, Marseille, France

6. Laboratory of Pharmacokinetics and Toxicology, Hôpital Universitaire la Timone, F-13385 Marseille, France.

7. COMPO, CRCM INSERM U1068-Inria, Université Aix-Marseille, Marseille, France

8. Regional Center of Pharmacovigilance, Department of Pharmacology, Hôpital Cochin, Assistance Publique-Hopitaux de Paris, Université de Paris, Paris, France

9. French pharmacovigilance network

10. Laboratory of Biochemistry and Molecular biology, Centre Hospitalo-uinversitaire de Tours, Tours,

11. INSERM U1248, IPPRITT, University of Limoges, Limoges, France

12. Department of Gastroenterology and Digestive Oncology, Hôpital Européen Georges Pompidou, Paris University; Assistance Publique-Hôpitaux de Paris, Paris, France.

13. SIRIC CARPEM, Université de Paris; Fédération Francophone de Cancérologie Digestive (FFCD), Assistance Publique-Hôpitaux de Paris, Department of Gastroenterology and Digestive Oncology, Hôpital Européen Georges Pompidou, Paris, France.

14. Laboratory of Pharmacology, Institut Claudius Regaud, IUCT-Oncopole and CRCT, INSERM UMR1037, Université Paul Sabatier, Toulouse, France

## Correspondence

Pr. Marie-Anne Loriot
Department of clinical chemistry, Hopital Européen Georges Pompidou
20, rue Leblanc
75015 Paris, France

Phone: +33156092435; Fax: +33156093393

E-mail: marie-anne.loriot@aphp.fr

<footnote>* Co-first authors</footnote>

<footnote>¶ Co-seniors authors</footnote>
# Current diagnostic and clinical issues of screening for dihydropyrimidine dehydrogenase deficiency

Marie-Christine Etienne-Grimaldi¹*, Nicolas Pallet²,³*, Valérie Boige³,⁴, Joseph Ciccolini⁵,⁶,⁷, Laurent Chouchana⁸,⁹, Chantal Barin-Le Guellec¹⁰,¹¹, Aziz Zaanan¹², Céline Narjoz²,³, Julien Taieb¹³, Fabienne Thomas ¹⁴,¶, Marie-Anne Loriot²,³,¶, on behalf of the Francophone Network of Pharmacogenetics (RNPGx) and the French Clinical Oncopharmacology Group (GPCO)-UNICANCER

1. Laboratory of Oncopharmacology, Centre Antoine Lacassagne, Nice, France.

2. Department of Clinical Chemistry, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France.

3. Université de Paris, INSERM UMRS1138, Centre de Recherche des Cordeliers, F-75006 Paris, France

4. Department of Cancer Medicine, Institut Gustave Roussy, Villejuif, France

5. SMARTc, CRCM INSERM U1068, Université Aix-Marseille, Marseille, France

6. Laboratory of Pharmacokinetics and Toxicology, Hôpital Universitaire la Timone, F-13385 Marseille, France.

7. COMPO, CRCM INSERM U1068-Inria, Université Aix-Marseille, Marseille, France

8. Regional Center of Pharmacovigilance, Department of Pharmacology, Hôpital Cochin, Assistance Publique-Hopitaux de Paris, Université de Paris, Paris, France

9. French pharmacovigilance network

10. Laboratory of Biochemistry and Molecular biology, Centre Hospitalo-uinversitaire de Tours, Tours,

11. INSERM U1248, IPPRITT, University of Limoges, Limoges, France

12. Department of Gastroenterology and Digestive Oncology, Hôpital Européen Georges Pompidou, Paris University; Assistance Publique-Hôpitaux de Paris, Paris, France.

13. SIRIC CARPEM, Université de Paris; Fédération Francophone de Cancérologie Digestive (FFCD), Assistance Publique-Hôpitaux de Paris, Department of Gastroenterology and Digestive Oncology, Hôpital Européen Georges Pompidou, Paris, France.

14. Laboratory of Pharmacology, Institut Claudius Regaud, IUCT-Oncopole and CRCT, INSERM UMR1037, Université Paul Sabatier, Toulouse, France

## Correspondence

Pr. Marie-Anne Loriot
Department of clinical chemistry, Hopital Européen Georges Pompidou
20, rue Leblanc
75015 Paris, France

Phone: +33156092435; Fax: +33156093393

E-mail: marie-anne.loriot@aphp.fr

<footnote>* Co-first authors</footnote>

<footnote>¶ Co-seniors authors</footnote>
**Abstract**

Fluoropyrimidine drugs (FP) are the backbone of many chemotherapy protocols for treating solid tumors. The rate-limiting step of fluoropyrimidine catabolism is dihydropyrimidine dehydrogenase (DPD), and deficiency in DPD activity can result in severe and even fatal toxicity. In this review, we survey the evidence-based pharmacogenetics and therapeutic recommendations regarding *DPYD* (the gene encoding DPD) genotyping and DPD phenotyping to prevent toxicity and optimize dosing adaptation before FP administration. The French experience of mandatory DPD-deficiency screening prior to initiating FP is discussed.

**Keywords**

*DPYD*, DPD, dihydropyrimidine dehydrogenase, genotyping, phenotyping, Fluoropyrimidine, 5-fluorouracile, capecitabine, toxicity, pharmacogenetics, adverse drug reaction
**Abstract**

Fluoropyrimidine drugs (FP) are the backbone of many chemotherapy protocols for treating solid tumors. The rate-limiting step of fluoropyrimidine catabolism is dihydropyrimidine dehydrogenase (DPD), and deficiency in DPD activity can result in severe and even fatal toxicity. In this review, we survey the evidence-based pharmacogenetics and therapeutic recommendations regarding *DPYD* (the gene encoding DPD) genotyping and DPD phenotyping to prevent toxicity and optimize dosing adaptation before FP administration. The French experience of mandatory DPD-deficiency screening prior to initiating FP is discussed.

**Keywords**

*DPYD*, DPD, dihydropyrimidine dehydrogenase, genotyping, phenotyping, Fluoropyrimidine, 5-fluorouracile, capecitabine, toxicity, pharmacogenetics, adverse drug reaction
## Introduction

Fluoropyrimidine drugs (FP), 5-fluorouracil (5FU), capecitabine, and tegafur-based treatment (UFT, S1) are administered in 50% of patients receiving a chemotherapy for a solid tumors [1] mostly for treatments of digestive, breast, and head and neck cancers [2,3]. While the majority of patients can be treated safely, 10% to 40% of patients will experience severe FP-associated toxicities (grade ≥3 according to the Common Terminology Criteria for Adverse Events (CTCAE)), mainly hematological and digestive, being lethal in 0.1% to 1% of patients [1,4–11]. When not directly life-threatening, these toxicities may affect patient quality of life and treatment efficacy related due to discontinuation or postponement of chemotherapy cycles, and generate additional costs such as prolonged hospital stay [12]. The most prevalent biochemical cause of FP toxicities is a deficiency of the catabolic enzyme dihydropyrimidine dehydrogenase (DPD). Patients with partial or complete DPD deficiency have a reduced capacity to catabolize 5FU and are at risk of experiencing severe reactions, such as hematological and/or digestive toxicities [9,13]. These toxicities usually occur during the first treatment cycles, supporting the importance of screening for DPD deficiency and optimizing FP starting dose before initiating therapy. The purpose of this review is to provide an update on the scientific and clinical rationales for the implementation and interpretation of dihydropyrimidine dehydrogenase (DPYD) genotyping and phenotyping in order to secure FP-based chemotherapies.

## FP metabolism and mechanism of action

The anabolic pathways of 5FU are similar to those of uracil, and a small fraction of 5FU (1-5%) is transformed into the cytotoxic metabolites fluorodeoxyuridine-monophosphate (FdUMP), fluorodeoxyuridine-triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). Two anabolic pathways have been described. The first consists to convert 5FU by orotate phosphoribosyltransferase (OPRT) and uridine phosphorylase into fluorouridine-monophosphate (FUMP). FUMP is phosphorylated to fluorouridine-diphosphate (FUDP), which is further phosphorylated to FUTP or converted to fluorodeoxyuridine-diphosphate (FdUDP) by ribonucleotide reductase. In the second pathway, 5FU is converted by thymidine phosphorylase into FUDR, which is converted by thymidine kinase to FdUMP, which is finally phosphorylated to FdUDP and FdUTP (Figure 1).

5FU cytotoxicity is thus related to different mechanisms. FdUMP, which has a greater affinity for thymidylate synthase (TS) than the natural substrate dUMP, inhibits TS, resulting in the inhibition of DNA synthesis and promotion of thymineless stress, triggering Fas-mediated apoptosis. The ternary complex formed between FdUMP, TS, and 5-10 methylene-tetrahydrofolate is stabilized by co-administration of folinic acid, favoring TS inhibition. In
## Introduction

Fluoropyrimidine drugs (FP), 5-fluorouracil (5FU), capecitabine, and tegafur-based treatment (UFT, S1) are administered in 50% of patients receiving a chemotherapy for a solid tumors [1] mostly for treatments of digestive, breast, and head and neck cancers [2,3]. While the majority of patients can be treated safely, 10% to 40% of patients will experience severe FP-associated toxicities (grade ≥3 according to the Common Terminology Criteria for Adverse Events (CTCAE)), mainly hematological and digestive, being lethal in 0.1% to 1% of patients [1,4–11]. When not directly life-threatening, these toxicities may affect patient quality of life and treatment efficacy related due to discontinuation or postponement of chemotherapy cycles, and generate additional costs such as prolonged hospital stay [12]. The most prevalent biochemical cause of FP toxicities is a deficiency of the catabolic enzyme dihydropyrimidine dehydrogenase (DPD). Patients with partial or complete DPD deficiency have a reduced capacity to catabolize 5FU and are at risk of experiencing severe reactions, such as hematological and/or digestive toxicities [9,13]. These toxicities usually occur during the first treatment cycles, supporting the importance of screening for DPD deficiency and optimizing FP starting dose before initiating therapy. The purpose of this review is to provide an update on the scientific and clinical rationales for the implementation and interpretation of dihydropyrimidine dehydrogenase (DPYD) genotyping and phenotyping in order to secure FP-based chemotherapies.

## FP metabolism and mechanism of action

The anabolic pathways of 5FU are similar to those of uracil, and a small fraction of 5FU (1-5%) is transformed into the cytotoxic metabolites fluorodeoxyuridine-monophosphate (FdUMP), fluorodeoxyuridine-triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). Two anabolic pathways have been described. The first consists to convert 5FU by orotate phosphoribosyltransferase (OPRT) and uridine phosphorylase into fluorouridine-monophosphate (FUMP). FUMP is phosphorylated to fluorouridine-diphosphate (FUDP), which is further phosphorylated to FUTP or converted to fluorodeoxyuridine-diphosphate (FdUDP) by ribonucleotide reductase. In the second pathway, 5FU is converted by thymidine phosphorylase into FUDR, which is converted by thymidine kinase to FdUMP, which is finally phosphorylated to FdUDP and FdUTP (Figure 1).

5FU cytotoxicity is thus related to different mechanisms. FdUMP, which has a greater affinity for thymidylate synthase (TS) than the natural substrate dUMP, inhibits TS, resulting in the inhibition of DNA synthesis and promotion of thymineless stress, triggering Fas-mediated apoptosis. The ternary complex formed between FdUMP, TS, and 5-10 methylene-tetrahydrofolate is stabilized by co-administration of folinic acid, favoring TS inhibition. In
addition, the incorporation of FUTP in RNA, and FdUTP in DNA (facilitated by the depletion of
the dTTP pool as a consequence of TS inhibition), impacts RNA synthesis and damages newly
synthetized DNA [10,14].

The large majority (>85%) of 5FU is catabolized by DPD to 5-fluorodihydroouracil (5FUH2), a non-cytotoxic metabolite. DPD is ubiquitously expressed, including in tumor tissues and peripheral blood mononuclear cells (PBMC) [15–17]. Its physiological role is to convert uracil and thymine into dihydrouracil (UH2) and dihydrothymine. 5FUH2 is further converted by dihydropyrimidinase into 5-fluoroureidopropionic acid (5FUPA) in the liver. Finally, β-ureidopropionase converts 5FUPA to α-fluoro-β-alanine (FBAL), the major urinary catabolite in humans. In addition to the intensive catabolism of 5FU, ~5% of administered 5FU is excreted in the urine.

FP oral prodrugs (capecitabine, tegafur) are converted into 5FU and catabolized by DPD.
Tegafur, initially combined with uracil (UFT) in order to inhibit DPD, is now combined with the
DPD inhibitor gimeracil and the OPRT inhibitor oteracil (S-1 is the code name of the
combination of the 3 compounds). Oteracil inhibits the phosphorylation of 5FU to FUMP in the
gastrointestinal tract, thereby diminishing the toxic effects of 5FU in normal gastrointestinal
mucosa. Gimeracil is a reversible DPD inhibitor that maintains high 5FU concentrations in
blood and tumor tissues [18]. The impact of DPD deficiency on toxicities induced by tegafur
co-administered with a DPD inhibitor, is thus theoretically limited.

DPD deficiency and FP toxicity

DPD enzyme activity measured in peripheral blood mononuclear cell (PBMC) exhibits a wide
inter-patient variability [19] and a significant correlation was found between DPD enzyme
activity measured in liver and in PBMC on a limited set of 27 patients [20]. The wide inter-
subject variablity in PBMC-DPD activity leads to a large variability in 5FU systemic exposure
after administration of a standard dose [4, 19, 21, 22]. Since the publication in 1985 of the first
case of a life-threatening toxicity related to 5FU in a DPD-deficient patient [23], numerous
publications have demonstrated a causal link between DPD deficiency and the occurrence of
severe or fatal FP-related toxicities [24–28] and **Supplementary tables**. It is estimated that
approximately 3% to 5% of Caucasians are carriers of partial DPD deficiency and that 0.1 to
0.3% are completely deficient [29]. Although other genes may play a role in FP toxicity (e.g.
*TYMS* and *MTHFR*), and that complete DPD deficiency does not explain all FP toxicity-related
deaths [1], *DPYD* non-functional variants are the main identified contributors of 5FU-related
severe toxicities [30,31].

DPYD defective allelic variants
addition, the incorporation of FUTP in RNA, and FdUTP in DNA (facilitated by the depletion of
the dTTP pool as a consequence of TS inhibition), impacts RNA synthesis and damages newly
synthetized DNA [10,14].

The large majority (>85%) of 5FU is catabolized by DPD to 5-fluorodihydroouracil (5FUH2), a non-cytotoxic metabolite. DPD is ubiquitously expressed, including in tumor tissues and peripheral blood mononuclear cells (PBMC) [15–17]. Its physiological role is to convert uracil and thymine into dihydrouracil (UH2) and dihydrothymine. 5FUH2 is further converted by dihydropyrimidinase into 5-fluoroureidopropionic acid (5FUPA) in the liver. Finally, β-ureidopropionase converts 5FUPA to α-fluoro-β-alanine (FBAL), the major urinary catabolite in humans. In addition to the intensive catabolism of 5FU, ~5% of administered 5FU is excreted in the urine.

FP oral prodrugs (capecitabine, tegafur) are converted into 5FU and catabolized by DPD.
Tegafur, initially combined with uracil (UFT) in order to inhibit DPD, is now combined with the
DPD inhibitor gimeracil and the OPRT inhibitor oteracil (S-1 is the code name of the
combination of the 3 compounds). Oteracil inhibits the phosphorylation of 5FU to FUMP in the
gastrointestinal tract, thereby diminishing the toxic effects of 5FU in normal gastrointestinal
mucosa. Gimeracil is a reversible DPD inhibitor that maintains high 5FU concentrations in
blood and tumor tissues [18]. The impact of DPD deficiency on toxicities induced by tegafur
co-administered with a DPD inhibitor, is thus theoretically limited.

DPD deficiency and FP toxicity

DPD enzyme activity measured in peripheral blood mononuclear cell (PBMC) exhibits a wide
inter-patient variability [19] and a significant correlation was found between DPD enzyme
activity measured in liver and in PBMC on a limited set of 27 patients [20]. The wide inter-
subject variablity in PBMC-DPD activity leads to a large variability in 5FU systemic exposure
after administration of a standard dose [4, 19, 21, 22]. Since the publication in 1985 of the first
case of a life-threatening toxicity related to 5FU in a DPD-deficient patient [23], numerous
publications have demonstrated a causal link between DPD deficiency and the occurrence of
severe or fatal FP-related toxicities [24–28] and **Supplementary tables**. It is estimated that
approximately 3% to 5% of Caucasians are carriers of partial DPD deficiency and that 0.1 to
0.3% are completely deficient [29]. Although other genes may play a role in FP toxicity (e.g.
*TYMS* and *MTHFR*), and that complete DPD deficiency does not explain all FP toxicity-related
deaths [1], *DPYD* non-functional variants are the main identified contributors of 5FU-related
severe toxicities [30,31].

DPYD defective allelic variants
The *DPYD* gene encoding DPD, spans ~900 kb on chromosome 1 and contains 23 exons [32]. *DPYD* is highly polymorphic, with more than 200 variants described in coding regions, but their effects on DPD enzyme activity are unknown for the majority of them [33–35]. Complete DPD deficiency is a cause of familial pyrimidinemia, a rare disorder of pyrimidine metabolism characterized by a variable phenotype ranging from absence of symptoms to severe neurological involvement with developmental delay, intellectual disability, and seizures. Analysis of urine typically shows high levels of uracil and thymine, and these patients are predisposed to FP-related toxicity [23].

The first described and most studied deleterious *DPYD* variant is the splice-site *DPYD*\*2A (c.1905+1G>A; rs3918290) found in 1.5% of Caucasians, associated with reduced 5FU clearance and severe or fatal toxicity[25,36,37]. Three additional deleterious variants associated with decreased DPD activity and toxicity risk have been described: *DPYD*\*13 (c.1679T>G; p. Ile560Ser; rs55886062), c.2846A>T (p.Asp949Val, rs67376798), and the more common variant c.1129-5923C>G (rs75017182) linked to haplotype B3 (HapB3) [10](Table 1). Overall, 6-7% of Caucasians carry one of these four variants at the heterozygous state. Importantly, these four deleterious variants are not present in populations of African or Asian populations [38–40]. Other *DPYD* variants associated with reduced DPD activity (*in vitro* and/or *ex vivo*) have been reported in non-Caucasian populations, including the c.557A>G (p.Tyr186Cys, rs115232898) variant identified in African American/Afro-Caribbean with an minor allelic frequency (MAF) of 1.2% and virtually absent in Asian and European populations. *DPYD*\*5 (c.1627A>G, p.Ile543Val; rs1801159) and *DPYD*\*6 (c.2194G>A, p.Val732Ile, rs18011160) variants are detected in East-Asian populations with MAF of 25% and 18% respectively [41,42].

The four *2A, *13, c.2846A>T and HapB3 *DPYD* variants are considered clinically actionable:

* The *DPYD*\*2A variant is located in the intron boundary of exon 14 coding for a part of the pyrimidine binding site. This genetic alteration is responsible for the skipping of exon 14 (165 base pairs deletion), resulting in a truncated catalytically inactive enzyme [34,43–45]. Clinical studies have reported an association between *DPYD*\*2A variant and DPD enzyme activity, supporting the decreased function of *DPYD*\*2A. In heterozygous *DPYD*\*2A carriers, DPD activity is reduced by approximately 50%, and in homozygous carriers DPD activity is completely or nearly abolished [46,47]. Pharmacokinetic studies have demonstrated that patients carrying the *DPYD*\*2A variant are subject to significant increases in systemic 5FU exposure, more than twice averaged level [48,49].

* The *DPYD*\*13 variant (c.1679T>G; p. Ile560Ser) is a serine-for-isoleucine substitution in exon 13 corresponding to a highly conserved region of the protein. The precise functional consequence of this missense mutation is not known, but this substitution may reduce the
The *DPYD* gene encoding DPD, spans ~900 kb on chromosome 1 and contains 23 exons [32]. *DPYD* is highly polymorphic, with more than 200 variants described in coding regions, but their effects on DPD enzyme activity are unknown for the majority of them [33–35]. Complete DPD deficiency is a cause of familial pyrimidinemia, a rare disorder of pyrimidine metabolism characterized by a variable phenotype ranging from absence of symptoms to severe neurological involvement with developmental delay, intellectual disability, and seizures. Analysis of urine typically shows high levels of uracil and thymine, and these patients are predisposed to FP-related toxicity [23].

The first described and most studied deleterious *DPYD* variant is the splice-site *DPYD*\*2A (c.1905+1G>A; rs3918290) found in 1.5% of Caucasians, associated with reduced 5FU clearance and severe or fatal toxicity[25,36,37]. Three additional deleterious variants associated with decreased DPD activity and toxicity risk have been described: *DPYD*\*13 (c.1679T>G; p. Ile560Ser; rs55886062), c.2846A>T (p.Asp949Val, rs67376798), and the more common variant c.1129-5923C>G (rs75017182) linked to haplotype B3 (HapB3) [10](Table 1). Overall, 6-7% of Caucasians carry one of these four variants at the heterozygous state. Importantly, these four deleterious variants are not present in populations of African or Asian populations [38–40]. Other *DPYD* variants associated with reduced DPD activity (*in vitro* and/or *ex vivo*) have been reported in non-Caucasian populations, including the c.557A>G (p.Tyr186Cys, rs115232898) variant identified in African American/Afro-Caribbean with an minor allelic frequency (MAF) of 1.2% and virtually absent in Asian and European populations. *DPYD*\*5 (c.1627A>G, p. Ile543Val; rs1801159) and *DPYD*\*6 (c.2194G>A, p.Val732Ile, rs18011160) variants are detected in East-Asian populations with MAF of 25% and 18% respectively [41,42].

The four *\*2A*, *\*13*, c.2846A>T and HapB3 *DPYD* variants are considered clinically actionable:

* The *DPYD*\*2A variant is located in the intron boundary of exon 14 coding for a part of the pyrimidine binding site. This genetic alteration is responsible for the skipping of exon 14 (165 base pairs deletion), resulting in a truncated catalytically inactive enzyme [34,43–45]. Clinical studies have reported an association between *DPYD*\*2A variant and DPD enzyme activity, supporting the decreased function of *DPYD*\*2A. In heterozygous *DPYD*\*2A carriers, DPD activity is reduced by approximately 50%, and in homozygous carriers DPD activity is completely or nearly abolished [46,47]. Pharmacokinetic studies have demonstrated that patients carrying the *DPYD*\*2A variant are subject to significant increases in systemic 5FU exposure, more than twice averaged level [48,49].

* The *DPYD*\*13 variant (c.1679T>G; p. Ile560Ser) is a serine-for-isoleucine substitution in exon 13 corresponding to a highly conserved region of the protein. The precise functional consequence of this missense mutation is not known, but this substitution may reduce the
stability of a sensitive region of the protein [50,51]. Experimental data showed that homozygous expression of the *DPYD*\*13 variant resulted in a 75% reduction in DPD enzyme activity compared with wild-type [44]. Clinical studies have confirmed that patients carrying the *DPYD*\*13 allele (heterozygous) have a markedly reduced DPD activity [51,52].

* The missense mutation c.2846A>T (p. Asp949Val), located on exon 22, affects DPD activity through a structural change that interferes with cofactor binding and electron transport [51]. *In vitro* and *ex vivo* data generally indicate a reduction in enzyme activity in the presence of this variant. An *in vitro* assessment showed that homozygous expression of the *DPYD* c.2846A>T variant resulted in a 40 to 60% reduction in DPD activity compared to wild type [34]. This finding suggests that homozygous carriers would have a ~50% reduction in DPD activity and a heterozygous carriers may have a ~25% reduction [44].

* Haplotype B3 (Hap B3) spans from intron 5 to exon 11 [53,54]. *DPYD* c.1129-5923C>G (rs75017182) is a deep intronic mutation (intron 10) that introduces a cryptic splice site, resulting in a nonfunctional transcript. The synonymous variant c.1236G>A (p. E412E, rs56038477) is in complete linkage disequilibrium with c.1129-5923C>G and can be used as a proxy for HapB3 in Caucasians. HapB3 is also in linkage disequilibrium with 3 other intronic variants: c.483+18G>A (rs56276561), c.680+139G>A (rs6668296), and c.959-51T>G (rs115349832). HapB3 is reported to reduce mRNA splicing efficiency by 30%, and *ex vivo* data suggest a moderately reduced DPD activity of 20-35% on average in heterozygous carriers and 20-60% in homozygous carriers [54,55].

## *DPYD* gene activity score

In 2015, Henricks et al. proposed a "gene activity score" for translating the *DPYD* genotype into the DPD phenotype, based on the four most relevant *DPYD*-deficient alleles (*DPYD*\*2A, c.2846A>T, *DPYD*\*13, c.1236G>A/HapB3) (Table 1) [56]. Based on *in vitro* and clinical functionality of each deleterious allele, this approach assigns for each allele a "variant activity score" of zero for completely deficient alleles (*DPYD*\*2A and \*13), 0.5 for partially deficient alleles (c.2846A>T and HapB3) and to 1 for the wild-type \*1 allele. The gene activity score corresponds to the sum of the two lowest variant activity scores. The resulting gene activity score ranges from 0 (two completely deficient alleles) to 2 (two wild-type alleles). This gene activity score was further recommended by the Clinical Pharmacogenetics Implementation Consortium (CPIC) [41] to estimate the risk of developing severe FP-related toxicities. One limitation of this score is its restriction to four variants, since it is anticipated that other rare *DPYD* variants in Caucasians (potentially more frequent in other ethnic groups) may also influence DPD activity and severe FP-related toxicities [46]. An additional caveat is that a single variant not deleterious by itself, can nevertheless be significantly associated with DPD deficiency if it forms a haplotype with additional functional variants. Finally, another limitation
stability of a sensitive region of the protein [50,51]. Experimental data showed that homozygous expression of the *DPYD*\*13 variant resulted in a 75% reduction in DPD enzyme activity compared with wild-type [44]. Clinical studies have confirmed that patients carrying the *DPYD*\*13 allele (heterozygous) have a markedly reduced DPD activity [51,52].

* The missense mutation c.2846A>T (p. Asp949Val), located on exon 22, affects DPD activity through a structural change that interferes with cofactor binding and electron transport [51]. *In vitro* and *ex vivo* data generally indicate a reduction in enzyme activity in the presence of this variant. An *in vitro* assessment showed that homozygous expression of the *DPYD* c.2846A>T variant resulted in a 40 to 60% reduction in DPD activity compared to wild type [34]. This finding suggests that homozygous carriers would have a ~50% reduction in DPD activity and a heterozygous carriers may have a ~25% reduction [44].

* Haplotype B3 (Hap B3) spans from intron 5 to exon 11 [53,54]. *DPYD* c.1129-5923C>G (rs75017182) is a deep intronic mutation (intron 10) that introduces a cryptic splice site, resulting in a nonfunctional transcript. The synonymous variant c.1236G>A (p. E412E, rs56038477) is in complete linkage disequilibrium with c.1129-5923C>G and can be used as a proxy for HapB3 in Caucasians. HapB3 is also in linkage disequilibrium with 3 other intronic variants: c.483+18G>A (rs56276561), c.680+139G>A (rs6668296), and c.959-51T>G (rs115349832). HapB3 is reported to reduce mRNA splicing efficiency by 30%, and *ex vivo* data suggest a moderately reduced DPD activity of 20-35% on average in heterozygous carriers and 20-60% in homozygous carriers [54,55].

## *DPYD* gene activity score

In 2015, Henricks et al. proposed a "gene activity score" for translating the *DPYD* genotype into the DPD phenotype, based on the four most relevant *DPYD*-deficient alleles (*DPYD*\*2A, c.2846A>T, *DPYD*\*13, c.1236G>A/HapB3) (Table 1) [56]. Based on *in vitro* and clinical functionality of each deleterious allele, this approach assigns for each allele a "variant activity score" of zero for completely deficient alleles (*DPYD*\*2A and \*13), 0.5 for partially deficient alleles (c.2846A>T and HapB3) and to 1 for the wild-type \*1 allele. The gene activity score corresponds to the sum of the two lowest variant activity scores. The resulting gene activity score ranges from 0 (two completely deficient alleles) to 2 (two wild-type alleles). This gene activity score was further recommended by the Clinical Pharmacogenetics Implementation Consortium (CPIC) [41] to estimate the risk of developing severe FP-related toxicities. One limitation of this score is its restriction to four variants, since it is anticipated that other rare *DPYD* variants in Caucasians (potentially more frequent in other ethnic groups) may also influence DPD activity and severe FP-related toxicities [46]. An additional caveat is that a single variant not deleterious by itself, can nevertheless be significantly associated with DPD deficiency if it forms a haplotype with additional functional variants. Finally, another limitation
is the case of patients carrying at least two deleterious *DPYD* alleles because conventional genetic tests do not indicate whether they are located on the same DNA strand; for such patients, the Dutch Pharmacogenetics Working Group (DPWG) recommended an additional phenotyping test to determine DPD enzyme activity [57].

## Genotyping approaches for DPD deficiency screening

The CPIC and the Dutch Pharmacogenetics Workgroup (DPWG) proposed *DPYD* testing of *DPYD*\*2A, *DPYD*\*13, c.2846A>T and HapB3 [10,57]. Based on combined genotyping of *DPYD*\*13, c.2846A>T and HapB3 (*DPYD*\*2A not considered given that carriers received reduced FP dosing), Meulendijks et al. reported a sensitivity of 6% and a positive predictive value (PPV) of 13% for predicting global severe toxicity (grades 3-4) in a prospective study including 550 patients [58]. Toffoli et al. [33] and Etienne-Grimaldi et al. [59] showed that genotyping of *DPYD*\*2A, \*13 et c.2846A>T predicted grade 3-4 toxicities with a sensitivity of 12% and 17% and a PPV of 61% and 71%, respectively. The performance of consensual deleterious *DPYD* variants for predicting the most relevant grade 4-5 hematological and digestive toxicities was reported in the FUSAFE meta-analysis on individual patient data conducted on 6403 patients [60]: the adjusted OR associated with the presence of at least one *DPYD* variant among \*2A, \*13 and c.2846A>T was 10.6 (95%CI 7.1-15.6) and the sensitivity and PPV of the multivariate model including these 3 *DPYD* variants and clinical co-variates (age, sex, body mass index, modality of FP administration and associated anticancer drugs) were 75.5% and 15.2%, respectively. Importantly, addition of HapB3 did not significantly improve the logistic model of grade 4-5 toxicity prediction, even though HapB3 was associated with an OR of 2.1 (95%CI 1.4-3.0). Literature data consistently reported that the toxicity risk associated with HapB3 was much smaller than those associated with \*2A, \*13 and c.2846A>T [61], in line with the relative weak association between HapB3 and DPD phenotype [56]. Of note, a recent meta-analysis on resumed data focused on lethal toxicities reported a RR of death of 25.6 in patients carrying any of the 4 consensual *DPYD* variants with a mortality risk of 2.3%, while excluding HapB3 increased mortality up to 3.7% [5]. The clinical impact of HapB3 is thus conflicting and above results may question its clinical relevance for predicting grade≥3 FP-related toxicities. Of note, exhaustive exome *DPYD* sequencing led to the identification of additional rare coding variants with potent deleterious activity [59,62], indicating that extending *DPYD* targeted genotyping to whole *DPYD* gene sequencing, which is made possible by next-generation sequencing technologies, may be a promising clinically relevant approach.

## Phenotyping approaches for DPD deficiency screening
is the case of patients carrying at least two deleterious *DPYD* alleles because conventional genetic tests do not indicate whether they are located on the same DNA strand; for such patients, the Dutch Pharmacogenetics Working Group (DPWG) recommended an additional phenotyping test to determine DPD enzyme activity [57].

## Genotyping approaches for DPD deficiency screening

The CPIC and the Dutch Pharmacogenetics Workgroup (DPWG) proposed *DPYD* testing of *DPYD*\*2A, *DPYD*\*13, c.2846A>T and HapB3 [10,57]. Based on combined genotyping of *DPYD*\*13, c.2846A>T and HapB3 (*DPYD*\*2A not considered given that carriers received reduced FP dosing), Meulendijks et al. reported a sensitivity of 6% and a positive predictive value (PPV) of 13% for predicting global severe toxicity (grades 3-4) in a prospective study including 550 patients [58]. Toffoli et al. [33] and Etienne-Grimaldi et al. [59] showed that genotyping of *DPYD*\*2A, \*13 et c.2846A>T predicted grade 3-4 toxicities with a sensitivity of 12% and 17% and a PPV of 61% and 71%, respectively. The performance of consensual deleterious *DPYD* variants for predicting the most relevant grade 4-5 hematological and digestive toxicities was reported in the FUSAFE meta-analysis on individual patient data conducted on 6403 patients [60]: the adjusted OR associated with the presence of at least one *DPYD* variant among \*2A, \*13 and c.2846A>T was 10.6 (95%CI 7.1-15.6) and the sensitivity and PPV of the multivariate model including these 3 *DPYD* variants and clinical co-variates (age, sex, body mass index, modality of FP administration and associated anticancer drugs) were 75.5% and 15.2%, respectively. Importantly, addition of HapB3 did not significantly improve the logistic model of grade 4-5 toxicity prediction, even though HapB3 was associated with an OR of 2.1 (95%CI 1.4-3.0). Literature data consistently reported that the toxicity risk associated with HapB3 was much smaller than those associated with \*2A, \*13 and c.2846A>T [61], in line with the relative weak association between HapB3 and DPD phenotype [56]. Of note, a recent meta-analysis on resumed data focused on lethal toxicities reported a RR of death of 25.6 in patients carrying any of the 4 consensual *DPYD* variants with a mortality risk of 2.3%, while excluding HapB3 increased mortality up to 3.7% [5]. The clinical impact of HapB3 is thus conflicting and above results may question its clinical relevance for predicting grade≥3 FP-related toxicities. Of note, exhaustive exome *DPYD* sequencing led to the identification of additional rare coding variants with potent deleterious activity [59,62], indicating that extending *DPYD* targeted genotyping to whole *DPYD* gene sequencing, which is made possible by next-generation sequencing technologies, may be a promising clinically relevant approach.

## Phenotyping approaches for DPD deficiency screening
PBMC DPD enzyme activity is considered the reference method for investigating DPD deficiency but it is highly cost- and time-intensive. Alternate methods such as uracil breath test and uracil loading dose [63] are also cumbersome and expensive, and fail to meet current standards for large-scale DPD screening implementation. Measuring plasma uracil [U] and dihydrouracil [UH2] concentrations was proposed 25 years ago as a simple inexpensive way to evaluate DPD activity [64]. Indeed, DPD deficiency will lead to uracil accumulation in plasma, thus making [U] or [UH2]/[U] ratio convenient surrogates for detecting impaired DPD activity in patients. Many improved HPLC-UV, UPLC-UV, GC-MS and LC-MS/MS methods have been developed [65–68]. Beyond monitoring plasma levels, other groups have proposed measuring [U] and [UH2] in saliva samples to simplify the sampling process and pre-analytical steps [69–71].

Measuring [U], regardless of the analytical technique used, is the most convenient method to detect DPD deficiency, and the one retained by French Health Authorities for DPD deficiency screening. This decision was mainly based on consistent results from 3 independent prospective studies: a French study conducted on 252 colorectal cancer patients receiving i.v. 5FU [26], a more recent French study conducted on 286 breast cancer patients treated with capecitabine [59,72], and a Dutch study on 550 patients with various cancers receiving capecitabine or 5FU [58]. The last 2 studies [58,59,72] showed that [U] was a better predictor of FP-related toxicities that [UH2]/[U], while the former [26] reported similar performance. The two French studies identified a [U] threshold at risk of severe grade 3-4 toxicity at 15 ng/ml [26] and 16 ng/mL [59,72], respectively, the later yielding a RR at 3.1 (95%CI 1.7-5.9) [72]. The Dutch study showed that [U] higher than 16 ng/mL was strongly associated with severe grade 3-4 FP toxicity (OR 5.3, 95%CI 1.5-18.7) and fatal toxicities (OR 44.8, 95%CI 4.6-441), the higher the [U] , the greater the toxicity risk, with a 2.75-fold higher risk on early global toxicity grade 3-4 and a 5.1-fold higher risk on lethal toxicity, for every 10 ng/mL increase [58]. The major limitation of [U] analysis is the observance of strict pre-analytical rules requiring that blood sample must be centrifuged as quickly as possible (within 1h at ambient temperature, or 4h at +4°C), and plasma frozen at -20°C until analysis [73]. Indeed, plasma [U] increases with time likely due to conversion of blood uridine into uracil by uridine phosphorylase [74] potentially leading to falsely conclude to DPD deficiency. Recent [U] data from de With et al. [75] clearly illustrate this limitation: despite centralization of [U] measurement (performed in a single “reference hospital”), in 6 hospitals out of 16, [U] were significantly higher than those measured in the reference hospital, thus undermining the reliability of their results reporting a lack of correlation between [U] and toxicity. Also, exogenous and endogenous factors such as food intake [76], circadian variations [77], renal function [78], sampling conditions [79], and a prior FP treatment [80] may influence [U] and [UH2].
PBMC DPD enzyme activity is considered the reference method for investigating DPD deficiency but it is highly cost- and time-intensive. Alternate methods such as uracil breath test and uracil loading dose [63] are also cumbersome and expensive, and fail to meet current standards for large-scale DPD screening implementation. Measuring plasma uracil [U] and dihydrouracil [UH2] concentrations was proposed 25 years ago as a simple inexpensive way to evaluate DPD activity [64]. Indeed, DPD deficiency will lead to uracil accumulation in plasma, thus making [U] or [UH2]/[U] ratio convenient surrogates for detecting impaired DPD activity in patients. Many improved HPLC-UV, UPLC-UV, GC-MS and LC-MS/MS methods have been developed [65–68]. Beyond monitoring plasma levels, other groups have proposed measuring [U] and [UH2] in saliva samples to simplify the sampling process and pre-analytical steps [69–71].

Measuring [U], regardless of the analytical technique used, is the most convenient method to detect DPD deficiency, and the one retained by French Health Authorities for DPD deficiency screening. This decision was mainly based on consistent results from 3 independent prospective studies: a French study conducted on 252 colorectal cancer patients receiving i.v. 5FU [26], a more recent French study conducted on 286 breast cancer patients treated with capecitabine [59,72], and a Dutch study on 550 patients with various cancers receiving capecitabine or 5FU [58]. The last 2 studies [58,59,72] showed that [U] was a better predictor of FP-related toxicities that [UH2]/[U], while the former [26] reported similar performance. The two French studies identified a [U] threshold at risk of severe grade 3-4 toxicity at 15 ng/ml [26] and 16 ng/mL [59,72], respectively, the later yielding a RR at 3.1 (95%CI 1.7-5.9) [72]. The Dutch study showed that [U] higher than 16 ng/mL was strongly associated with severe grade 3-4 FP toxicity (OR 5.3, 95%CI 1.5-18.7) and fatal toxicities (OR 44.8, 95%CI 4.6-441), the higher the [U] , the greater the toxicity risk, with a 2.75-fold higher risk on early global toxicity grade 3-4 and a 5.1-fold higher risk on lethal toxicity, for every 10 ng/mL increase [58]. The major limitation of [U] analysis is the observance of strict pre-analytical rules requiring that blood sample must be centrifuged as quickly as possible (within 1h at ambient temperature, or 4h at +4°C), and plasma frozen at -20°C until analysis [73]. Indeed, plasma [U] increases with time likely due to conversion of blood uridine into uracil by uridine phosphorylase [74] potentially leading to falsely conclude to DPD deficiency. Recent [U] data from de With et al. [75] clearly illustrate this limitation: despite centralization of [U] measurement (performed in a single “reference hospital”), in 6 hospitals out of 16, [U] were significantly higher than those measured in the reference hospital, thus undermining the reliability of their results reporting a lack of correlation between [U] and toxicity. Also, exogenous and endogenous factors such as food intake [76], circadian variations [77], renal function [78], sampling conditions [79], and a prior FP treatment [80] may influence [U] and [UH2].
Thanks to the wide experience of [U] testing in France, along with the setting up of an external quality assessment scheme (EQAS) for [U] since 2019, nationwide [U] testing has been successfully implemented in France. A survey by the French Medicines Agency (ANSM) (https://ansm.sante.fr) showed that roughly 70,000 tests are carried out every year in France, which corresponds to the annual number of new patients receiving FP-based chemotherapy [1]. Currently, 47 laboratories from European countries (France, Belgium, Luxemburg, Denmark) participate to this [U] EQAS.

## Comparison and combination of genotyping and phenotyping approaches

Strengths and limitations of genotyping and phenotyping approaches are listed in **Table 2**. Due to the scarcity of the 4 deleterious *2A/*13/c.2846A>T/HapB3 *DPYD* variants (~ 6-7% of carriers in Caucasians, ~ 2-3% excluding HapB3), the sensitivity of *DPYD* genotyping, i.e. the proportion of patients with deleterious variants among patients experiencing toxicity, is inevitably low, and depends on the toxicity prevalence (severity of toxicity). In contrast, DPD phenotype is by nature a continuous variable, and as such, its performance for predicting FP toxicities depends on the applied threshold. From a large French cohort study including 3680 patients with both genotyping and phenotyping, only 10.7% of patients with [U]≥16 ng/ml carried one of the 4 deleterious variants; conversely 16% of variants carriers exhibited [U]≥16 ng/ml (26% in excluding HapB3 carriers) [81] Interestingly, the same study reported that 13.3% of patients with [UH2]/[U]<10 carried one of the 4 deleterious variants while 33% of variants carriers exhibited [UH2]/[U]<10, suggesting that [UH2]/[U] may better reflect the impact of deleterious *DPYD* variants than [U]. Regardless the phenotyping marker, some overlap is observed between deficient populations based on genotyping and phenotyping, one could thus expect to improve screening performance by combining both approaches. Results from the 3 published prospective studies testing a combined *DPYD* genotyping (2 to 3 consensual variants) plus phenotyping strategy failed to provide appropriate level of evidence for the superiority of such a combined strategy as compared to each approach alone [26,58,59]. Nevertheless, it must be underlined that complementary genetic testing might help to confirm the genetic origin of enzymatic deficiency, particularly in patients with complete DPD deficiency, and thus justify family counseling.
Thanks to the wide experience of [U] testing in France, along with the setting up of an external quality assessment scheme (EQAS) for [U] since 2019, nationwide [U] testing has been successfully implemented in France. A survey by the French Medicines Agency (ANSM) (https://ansm.sante.fr) showed that roughly 70,000 tests are carried out every year in France, which corresponds to the annual number of new patients receiving FP-based chemotherapy [1]. Currently, 47 laboratories from European countries (France, Belgium, Luxemburg, Denmark) participate to this [U] EQAS.

## Comparison and combination of genotyping and phenotyping approaches

Strengths and limitations of genotyping and phenotyping approaches are listed in **Table 2**. Due to the scarcity of the 4 deleterious *2A/*13/c.2846A>T/HapB3 *DPYD* variants (~ 6-7% of carriers in Caucasians, ~ 2-3% excluding HapB3), the sensitivity of *DPYD* genotyping, i.e. the proportion of patients with deleterious variants among patients experiencing toxicity, is inevitably low, and depends on the toxicity prevalence (severity of toxicity). In contrast, DPD phenotype is by nature a continuous variable, and as such, its performance for predicting FP toxicities depends on the applied threshold. From a large French cohort study including 3680 patients with both genotyping and phenotyping, only 10.7% of patients with [U]≥16 ng/ml carried one of the 4 deleterious variants; conversely 16% of variants carriers exhibited [U]≥16 ng/ml (26% in excluding HapB3 carriers) [81] Interestingly, the same study reported that 13.3% of patients with [UH2]/[U]<10 carried one of the 4 deleterious variants while 33% of variants carriers exhibited [UH2]/[U]<10, suggesting that [UH2]/[U] may better reflect the impact of deleterious *DPYD* variants than [U]. Regardless the phenotyping marker, some overlap is observed between deficient populations based on genotyping and phenotyping, one could thus expect to improve screening performance by combining both approaches. Results from the 3 published prospective studies testing a combined *DPYD* genotyping (2 to 3 consensual variants) plus phenotyping strategy failed to provide appropriate level of evidence for the superiority of such a combined strategy as compared to each approach alone [26,58,59]. Nevertheless, it must be underlined that complementary genetic testing might help to confirm the genetic origin of enzymatic deficiency, particularly in patients with complete DPD deficiency, and thus justify family counseling.
## Cost-effectiveness of upfront DPD deficiency testing to guide FP dose adjustment

The cost benefit of an upfront DPD screening strategy has been evaluated in several studies [82-84] that focused on *DPYD* genotyping only. Although toxicity-related costs may depend on national health systems, these Irish, English and Dutch studies concluded that targeted *DPYD* genotyping is cost-effective. For instance, the cost-efficiency study by Henricks et al. conducted on 1103 patients prospectively enrolled in the ALPE trial, showed that upfront genotyping of the 4 *DPYD* variants followed by FP dose reduction according to CPIC guidelines improves patient safety and quality of life, without additional costs (author's conclusion is “cost-saving or cost-neutral”) [82]. Cost-effectiveness of phenotyping-guided dosing remains to be evaluated so as to provide a comprehensive comparison of both genotyping, phenotyping, and combined approaches.

## Pharmacokinetic-guided dosing of 5FU

5FU clearance has a very large inter-patient variability, up to a 10 to 100-fold range, and drug dosing based on body surface area (BSA) does not reduce the resulting inter-patient variability in 5FU systemic exposure [21]. Importantly, many studies (reviewed in Beumer et al. [21]) have demonstrated relationships between 5FU systemic exposure (mainly area under the curve (AUC) of concentration x time) and hematologic and/or digestive severe FP-related toxicities, as well as treatment efficacy (tumor response and survival). Irrespective to the 5FU-based containing regimen, an optimal target 5FU AUC of 20 to 30 ng.h/mL has been defined for digestive cancers [21]. Of note, Saam et al. [85] reported that BSA-based dosing results in 5FU AUC below 20 ng.h/ml in 53% of patients receiving FOLFIRI (29/55) and in 51% of patients treated with FOLFOX6 regimen (153/302), highlighting potential relevance of 5FU therapeutic drug monitoring (TDM) not only for reducing, but also increasing 5FU dosing. Two randomized studies [86,87] have demonstrated that individual 5FU dosing based on significantly improved 5FU therapeutic index, allowing validation of TDM for continuous i.v. 5FU. In the trial of Gamelin et al. [87] in CRC patients treated with a weekly 8-hour 5-FU 1500 mg/m² single agent dosing regimen, the TDM performed weekly allowed to reach the target AUC in 94% of patients in a mean time of four cycles. 5FU TDM improved overall response rates from 18% with BSA-based dosing of 5FU to 33% with PK-guided dosing of 5-FU while incidence of toxicity was lower. In the Fety et al. [86] study in HNSCC, TDM was performed at cycle 1 to adjust the doses for cycles 2 and 3. Hematological toxicity and mucositis were significantly reduced in the PK adjusted arm with a comparable objective response rate to the standard treatment arm. A large number of nonrandomized or single-arm clinical studies (reviewed in Beumer et al [21]) have consistently described a reduction of toxicity when applying 5FU TDM. A 5FU dose adjustment algorithm for FOLFOX6 regimen has been
## Cost-effectiveness of upfront DPD deficiency testing to guide FP dose adjustment

The cost benefit of an upfront DPD screening strategy has been evaluated in several studies [82-84] that focused on *DPYD* genotyping only. Although toxicity-related costs may depend on national health systems, these Irish, English and Dutch studies concluded that targeted *DPYD* genotyping is cost-effective. For instance, the cost-efficiency study by Henricks et al. conducted on 1103 patients prospectively enrolled in the ALPE trial, showed that upfront genotyping of the 4 *DPYD* variants followed by FP dose reduction according to CPIC guidelines improves patient safety and quality of life, without additional costs (author's conclusion is “cost-saving or cost-neutral”) [82]. Cost-effectiveness of phenotyping-guided dosing remains to be evaluated so as to provide a comprehensive comparison of both genotyping, phenotyping, and combined approaches.

## Pharmacokinetic-guided dosing of 5FU

5FU clearance has a very large inter-patient variability, up to a 10 to 100-fold range, and drug dosing based on body surface area (BSA) does not reduce the resulting inter-patient variability in 5FU systemic exposure [21]. Importantly, many studies (reviewed in Beumer et al. [21]) have demonstrated relationships between 5FU systemic exposure (mainly area under the curve (AUC) of concentration x time) and hematologic and/or digestive severe FP-related toxicities, as well as treatment efficacy (tumor response and survival). Irrespective to the 5FU-based containing regimen, an optimal target 5FU AUC of 20 to 30 ng.h/mL has been defined for digestive cancers [21]. Of note, Saam et al. [85] reported that BSA-based dosing results in 5FU AUC below 20 ng.h/ml in 53% of patients receiving FOLFIRI (29/55) and in 51% of patients treated with FOLFOX6 regimen (153/302), highlighting potential relevance of 5FU therapeutic drug monitoring (TDM) not only for reducing, but also increasing 5FU dosing. Two randomized studies [86,87] have demonstrated that individual 5FU dosing based on significantly improved 5FU therapeutic index, allowing validation of TDM for continuous i.v. 5FU. In the trial of Gamelin et al. [87] in CRC patients treated with a weekly 8-hour 5-FU 1500 mg/m² single agent dosing regimen, the TDM performed weekly allowed to reach the target AUC in 94% of patients in a mean time of four cycles. 5FU TDM improved overall response rates from 18% with BSA-based dosing of 5FU to 33% with PK-guided dosing of 5-FU while incidence of toxicity was lower. In the Fety et al. [86] study in HNSCC, TDM was performed at cycle 1 to adjust the doses for cycles 2 and 3. Hematological toxicity and mucositis were significantly reduced in the PK adjusted arm with a comparable objective response rate to the standard treatment arm. A large number of nonrandomized or single-arm clinical studies (reviewed in Beumer et al [21]) have consistently described a reduction of toxicity when applying 5FU TDM. A 5FU dose adjustment algorithm for FOLFOX6 regimen has been
proposed [88] and validated [89], based on the 5FU AUC measured at the previous cycle. Of note, the benefit of TDM performed at cycle 1 has been demonstrated in elderly patients [90]: the percentage of elderly patients within the therapeutic range significantly increased from 29% at C1 to 64% at C2 (p = 0.011). Since TDM is not a pre-emptive strategy, it does not allow to prevent life-threatening toxicities occurring at first cycle [91]. Alternative is the administration of a 5FU test dose, as described by Bocci et al. [92] although a low 5FU dose may induce severe toxicities in case of profound DPD deficiency. Despite being time-consuming and requiring specific resources and expertise, pharmacokinetic-guided dosing of 5FU has proven its clinical relevance and is cost-effective [93]. 5FU TDM is thus a relevant strategy for adjusting 5FU dose as soon as second cycle in patients with partial DPD deficiency

## Recommendations

Despite extensive research on FP pharmacogenetics since 1985, controversy remains among the medical community about the usefulness of systematic DPD deficiency screening prior to the initiation of FP-based treatments.

### Recommendations from scientific consortia

Current CPIC guidelines provide FP dosing recommendations at first cycle for *DPYD* normal, intermediate, and poor metabolizers [10], based on the DPYD activity score proposed by Henricks [56]. Poor metabolizers with a gene activity score at zero should avoid 5FU and prodrugs (Figure 2). Poor metabolizers with a gene activity score at 0.5 and no alternate therapeutic option should receive <25% of the standard dose with early TDM if feasible, and additional DPD phenotype testing to better assess DPD activity. Intermediate metabolizers with a gene activity score at 1 or 1.5 should receive 50% of the standard dose, with a special attention in patients homozygous for c.2846A>T (gene score 1) who may require a reduction of more than 50% (2018 update). CPIC recommendations only applied for i.v. 5FU and capecitabine.

In 2019 the DPWG published guidelines [57] also based on the DPYD activity score by Henricks [56]. DPWG recommendations differ from CPIC for patients carrying two decreased function alleles (gene activity score 1) or one decreased and one non-functional allele (gene activity score 0.5): DPGW considers that enzyme activity cannot be accurately predicted and that additional phenotyping (ideally DPD enzyme activity measured in PBMCs) is required to adjust FP dosing. For other patients with gene activity score at 1 (i.e. patient with one non-functional allele) or 1.5, dosing recommendations for 5FU (i.v. and cutaneous) and capecitabine are similar to those of CPIC (50% of standard dose). Second difference is that DPWG contra-indicates tegafur in patients with a gene activity score at 0, 1 or 1.5.

In 2018, the French National Pharmacogenetic Network (Réseau Francophone de Pharmacogénétique (RNPGx)) and the Clinical Oncopharmacology Group (Groupe de
proposed [88] and validated [89], based on the 5FU AUC measured at the previous cycle. Of note, the benefit of TDM performed at cycle 1 has been demonstrated in elderly patients [90]: the percentage of elderly patients within the therapeutic range significantly increased from 29% at C1 to 64% at C2 (p = 0.011). Since TDM is not a pre-emptive strategy, it does not allow to prevent life-threatening toxicities occurring at first cycle [91]. Alternative is the administration of a 5FU test dose, as described by Bocci et al. [92] although a low 5FU dose may induce severe toxicities in case of profound DPD deficiency. Despite being time-consuming and requiring specific resources and expertise, pharmacokinetic-guided dosing of 5FU has proven its clinical relevance and is cost-effective [93]. 5FU TDM is thus a relevant strategy for adjusting 5FU dose as soon as second cycle in patients with partial DPD deficiency

## Recommendations

Despite extensive research on FP pharmacogenetics since 1985, controversy remains among the medical community about the usefulness of systematic DPD deficiency screening prior to the initiation of FP-based treatments.

### Recommendations from scientific consortia

Current CPIC guidelines provide FP dosing recommendations at first cycle for *DPYD* normal, intermediate, and poor metabolizers [10], based on the DPYD activity score proposed by Henricks [56]. Poor metabolizers with a gene activity score at zero should avoid 5FU and prodrugs (Figure 2). Poor metabolizers with a gene activity score at 0.5 and no alternate therapeutic option should receive <25% of the standard dose with early TDM if feasible, and additional DPD phenotype testing to better assess DPD activity. Intermediate metabolizers with a gene activity score at 1 or 1.5 should receive 50% of the standard dose, with a special attention in patients homozygous for c.2846A>T (gene score 1) who may require a reduction of more than 50% (2018 update). CPIC recommendations only applied for i.v. 5FU and capecitabine.

In 2019 the DPWG published guidelines [57] also based on the DPYD activity score by Henricks [56]. DPWG recommendations differ from CPIC for patients carrying two decreased function alleles (gene activity score 1) or one decreased and one non-functional allele (gene activity score 0.5): DPGW considers that enzyme activity cannot be accurately predicted and that additional phenotyping (ideally DPD enzyme activity measured in PBMCs) is required to adjust FP dosing. For other patients with gene activity score at 1 (i.e. patient with one non-functional allele) or 1.5, dosing recommendations for 5FU (i.v. and cutaneous) and capecitabine are similar to those of CPIC (50% of standard dose). Second difference is that DPWG contra-indicates tegafur in patients with a gene activity score at 0, 1 or 1.5.

In 2018, the French National Pharmacogenetic Network (Réseau Francophone de Pharmacogénétique (RNPGx)) and the Clinical Oncopharmacology Group (Groupe de
Pharmacologie Clinique Oncologique (GPCO-UNICANCER)) published guidelines for systematic upfront DPD-deficiency screening for 5FU and capecitabine [94]. The RNPGx/GPCO recommended [U] as the preferred approach, with FP contra-indication for [U] >100 ng/mL [94] and FP dose reduction from 25% to 50% for [U] ≥16 ng/mL depending on the [U] and patient profile. Targeted genotyping of the 4 consensual deleterious *DPYD* alleles was recommended in addition to phenotyping, along with dose reductions based on CPIC recommendations.

## Recommendations from medical societies

The National Comprehensive Cancer Network (NCCN) guidelines point the risk of severe FP toxicities induced by deleterious *DPYD* variants but does not support pre-emptive *DPYD* genotyping due to levels of evidence deemed controversial. The American Society of Clinical Oncology (ASCO) does not provide any guidelines for DPD deficiency testing, arguing a lack of standardized approaches. European Society of Molecular Oncology (ESMO) guidelines in localized colon cancer strongly recommend DPD genotyping or phenotyping before initiating FP-based adjuvant therapy, and ESMO guidelines in metastatic colorectal cancer patients states that DPD-deficiency testing should be conducted before initiating 5FU or capecitabine [95,96]. Asian medical societies do not recommend DPD testing since DPD deficiency is rare in Asian patients.

## Recommendations from health authorities

The conflicting positions taken by scientific consortia and medical societies have led health authorities to remain cautious in making clear decisions. Under pressure of 5FU victim patient's associations, particularly in France and the United Kingdom, European health authority's position regarding DPD testing has recently moved.

In 2018, French Health Authorities set up a working group to deeply analyze DPD phenotyping and genotyping performances, with the aim to identify complete DPD-deficient patients and prevent life-threatening toxicities [97]. On April 2019, France was the first country to mandate DPD-deficiency testing in all patients before initiating FP-based chemotherapy, based on [U] along with strict pre-analytical requirements. Since 2021, prescription and dispensation assistance softwares must include a specific pop-up, warning the physician and pharmacist on the need to be aware of [U]. Due to scarcity of published data on complete deficient patients, the [U] threshold for complete deficiency was set at 150 ng/ml, based on data from 38,862 patients tested in French laboratories between 2000 and 2018, corresponding to 0.08% of patients. The threshold for partial deficiency was set at 16 ng/ml. French HAS-INCa recommendations are the following : i- FP are contra-indicated in patients with [U] ≥150 ng/mL; ii- FP dose reduction should be considered in patients with [U] between 16 and 150 ng/ mL on the basis of a clinical-biological reflection depending on the depth of DPD deficiency, on the chemotherapy protocol and the pathophysiological criteria (Figure 2). In the absence of
Pharmacologie Clinique Oncologique (GPCO-UNICANCER)) published guidelines for systematic upfront DPD-deficiency screening for 5FU and capecitabine [94]. The RNPGx/GPCO recommended [U] as the preferred approach, with FP contra-indication for [U] >100 ng/mL [94] and FP dose reduction from 25% to 50% for [U] ≥16 ng/mL depending on the [U] and patient profile. Targeted genotyping of the 4 consensual deleterious *DPYD* alleles was recommended in addition to phenotyping, along with dose reductions based on CPIC recommendations.

### Recommendations from medical societies

The National Comprehensive Cancer Network (NCCN) guidelines point the risk of severe FP toxicities induced by deleterious *DPYD* variants but does not support pre-emptive *DPYD* genotyping due to levels of evidence deemed controversial. The American Society of Clinical Oncology (ASCO) does not provide any guidelines for DPD deficiency testing, arguing a lack of standardized approaches. European Society of Molecular Oncology (ESMO) guidelines in localized colon cancer strongly recommend DPD genotyping or phenotyping before initiating FP-based adjuvant therapy, and ESMO guidelines in metastatic colorectal cancer patients states that DPD-deficiency testing should be conducted before initiating 5FU or capecitabine [95,96]. Asian medical societies do not recommend DPD testing since DPD deficiency is rare in Asian patients.

### Recommendations from health authorities

The conflicting positions taken by scientific consortia and medical societies have led health authorities to remain cautious in making clear decisions. Under pressure of 5FU victim patient's associations, particularly in France and the United Kingdom, European health authority's position regarding DPD testing has recently moved.

In 2018, French Health Authorities set up a working group to deeply analyze DPD phenotyping and genotyping performances, with the aim to identify complete DPD-deficient patients and prevent life-threatening toxicities [97]. On April 2019, France was the first country to mandate DPD-deficiency testing in all patients before initiating FP-based chemotherapy, based on [U] along with strict pre-analytical requirements. Since 2021, prescription and dispensation assistance softwares must include a specific pop-up, warning the physician and pharmacist on the need to be aware of [U]. Due to scarcity of published data on complete deficient patients, the [U] threshold for complete deficiency was set at 150 ng/ml, based on data from 38,862 patients tested in French laboratories between 2000 and 2018, corresponding to 0.08% of patients. The threshold for partial deficiency was set at 16 ng/ml. French HAS-INCa recommendations are the following : i- FP are contra-indicated in patients with [U] ≥150 ng/mL; ii- FP dose reduction should be considered in patients with [U] between 16 and 150 ng/ mL on the basis of a clinical-biological reflection depending on the depth of DPD deficiency, on the chemotherapy protocol and the pathophysiological criteria (Figure 2). In the absence of
validated dose-guided reduction according to [U], HAS-INCa recommendations emphasize that FP dose must be re-evaluated as soon as second cycle, depending on tolerance at first cycle, for ensuring an efficient dose-intensity. In addition, for i.v. 5FU, TDM is recommended for adjusting 5FU dose in partial deficient patients. Importantly, since 2020, no preventable toxicity-related death has been declared to the French Pharmacovigilance Network in patients with complete or partial DPD deficiency (data available on https://ansm.sante.fr).

On April 2020, the European Medical Agency (EMA) updated its recommendations on upfront DPD testing for 5FU, capecitabine and tegafur chemotherapy, notifying that DPD deficiency can be tested not only by checking for the presence of certain mutations in the *DPYD* gene, but also by measuring [U] [98]. The EMA recommends considering a reduced starting FP dose in partially-deficient patients, and contra-indicates FP in patients with complete DPD deficiency. The European summaries of product characteristics (SmPCs) of 5FU and capecitabine mention a contra-indication in patients with known complete DPD deficiency, and a special warning and reduced dose recommendation in patients with partial DPD deficiency. In line with EMA, SmPCs of 5FU and capecitabine recommend upfront DPD phenotyping and/or genotyping but does not make it mandatory.

In contrast, in the US package inserts of 5FU and capecitabine, the Food and Drug Administration does not recommend DPD-deficiency testing, and does not contra-indicate these drugs in patients with complete DPD deficiency; the “Warning and Precautions” section indicates an increased risk of serious or fatal reactions in patients with low or absent DPD activity and states that “There is insufficient data to recommend a specific dose in patients with partial DPD activity as measured by any specific test” without mention on the tests [99].

## Benefits and risks for patients

There are no randomized clinical studies comparing up-front DPD deficiency screening (whatever the approach) followed by FP dose adjustment *versus* standard FP dose. Nowadays such a study would not be ethical. A few non-randomized interventional clinical studies have shown that pre-emptive DPD testing, regardless of the approach, contributed to a significantly reduced incidence of drug-related severe toxicity or death compared with historical groups, or through matched pair study [100–103]. A large prospective study conducted in 17 Dutch hospitals (1103 patients) assessed the effect of pre-emptive screening for the four consensual deleterious *DPYD* variants on patient safety [103]. Heterozygous *DPYD* variant carriers received an initial dose reduction of 25% (c.2846A>T and HapB3) or 50% (*DPYD*\*2A and \*13), and *DPYD* wild-type patients were treated according to current standard of care. Despite dose reduction, overall grade 3-4 toxicity was significantly higher in variant carriers as compared to wild-type patients (39% *versus* 23%, respectively). The authors further compared, for each variant, the relative risk (RR) of severe toxicities between this *DPYD*-guided dose study and a
validated dose-guided reduction according to [U], HAS-INCa recommendations emphasize that FP dose must be re-evaluated as soon as second cycle, depending on tolerance at first cycle, for ensuring an efficient dose-intensity. In addition, for i.v. 5FU, TDM is recommended for adjusting 5FU dose in partial deficient patients. Importantly, since 2020, no preventable toxicity-related death has been declared to the French Pharmacovigilance Network in patients with complete or partial DPD deficiency (data available on https://ansm.sante.fr).

On April 2020, the European Medical Agency (EMA) updated its recommendations on upfront DPD testing for 5FU, capecitabine and tegafur chemotherapy, notifying that DPD deficiency can be tested not only by checking for the presence of certain mutations in the *DPYD* gene, but also by measuring [U] [98]. The EMA recommends considering a reduced starting FP dose in partially-deficient patients, and contra-indicates FP in patients with complete DPD deficiency. The European summaries of product characteristics (SmPCs) of 5FU and capecitabine mention a contra-indication in patients with known complete DPD deficiency, and a special warning and reduced dose recommendation in patients with partial DPD deficiency. In line with EMA, SmPCs of 5FU and capecitabine recommend upfront DPD phenotyping and/or genotyping but does not make it mandatory.

In contrast, in the US package inserts of 5FU and capecitabine, the Food and Drug Administration does not recommend DPD-deficiency testing, and does not contra-indicate these drugs in patients with complete DPD deficiency; the “Warning and Precautions” section indicates an increased risk of serious or fatal reactions in patients with low or absent DPD activity and states that “There is insufficient data to recommend a specific dose in patients with partial DPD activity as measured by any specific test” without mention on the tests [99].

## Benefits and risks for patients

There are no randomized clinical studies comparing up-front DPD deficiency screening (whatever the approach) followed by FP dose adjustment *versus* standard FP dose. Nowadays such a study would not be ethical. A few non-randomized interventional clinical studies have shown that pre-emptive DPD testing, regardless of the approach, contributed to a significantly reduced incidence of drug-related severe toxicity or death compared with historical groups, or through matched pair study [100–103]. A large prospective study conducted in 17 Dutch hospitals (1103 patients) assessed the effect of pre-emptive screening for the four consensual deleterious *DPYD* variants on patient safety [103]. Heterozygous *DPYD* variant carriers received an initial dose reduction of 25% (c.2846A>T and HapB3) or 50% (*DPYD*\*2A and \*13), and *DPYD* wild-type patients were treated according to current standard of care. Despite dose reduction, overall grade 3-4 toxicity was significantly higher in variant carriers as compared to wild-type patients (39% *versus* 23%, respectively). The authors further compared, for each variant, the relative risk (RR) of severe toxicities between this *DPYD*-guided dose study and a
historical patient cohort receiving standard FP doses: for *DPYD*2A, RR was 1.3 (95%CI 0.6-2.7) for genotype-guided dosing compared with 2.87 (95%CI 2.1-3.9) in the historical cohort; for *DPYD*13 no toxicity occurred with genotype-guided dosing compared with 4.3 (95%CI 2.1-8.8); for HapB3 RR was 1.69 (95%CI 1.2-2.4) compared with 1.72 (95%CI 1.2-2.4); and for c.2846A>T RR was 2.0 (95%CI 1.2-3.3) compared with 3.1 (95%CI 2.3-4.3), suggesting that a dose reduction of 25% is too low for c.2846A>T carriers, in line with sparse pharmacokinetics data collected in five c.2846A>T carriers [103].

A major issue of FP dose reduction in partial deficient patients is its potential deleterious impact of treatment efficacy, this being particularly relevant for adjuvant colorectal cancer [104,105]. Only one retrospective study specifically examined efficacy in DPD-deficient patients receiving reduced FP dose [101]. Efficacy was assessed in 40 prospectively identified heterozygous *DPYD*2A carriers treated with an ~50% reduced FP dose (5FU, capecitabine) who were matched (according to hospital, tumor type, treatment, disease stage, age) with wild-type *DPYD*2A patients receiving standard dose. Overall and progression-free survivals were not significantly different between *2A carriers (medians 27 and 14 months, respectively) and wild-type patients (medians 24 and 10 months, respectively).

The risk of undertreating partial deficient patients with DPD-guided dose is of great concern since pharmacokinetics studies have demonstrated significant relationships between 5FU systemic exposure and treatment efficacy. Two French studies recently investigated 5FU systemic exposure in cancer patients with pre-emptive uracilemia testing and subsequent FP dose reduction [106,107]. In contrast to a previous historical study [26], both reported the lack of significant correlation between 5FU clearance and [U]. The study of Dolat et al. involving 169 patients showed that patients with [U] ≥16 ng/mL had significantly lower 5FU AUC than patients with [U] <16 ng/mL (p=.0016), in line with significantly lower 5FU doses administered in deficient patients (mean 3540 mg) relative to non-deficient patients (mean 4050 mg, p<0.001) [106]. Such results show the difficulty to assess the optimal FP starting dose based on [U].

Another aspect of DPD phenotyping is to help identifying DPD proficient patients. A recent retrospective study conducted on 136 cancer patients treated with various FP-based regimens showed that the 18% of patients with the highest DPD enzyme activity measured in PBMCs had a significantly lower overall survival, progression-free survival and response rate as compared to remaining patients with normal or deficient DPD activity [108].

## Conclusions

Cancer patients treated with FP might experience severe, life-threatening toxicities due to impaired DPD activity. France remains currently the only country that makes upfront DPD-deficiency testing mandatory. EMA recommends DPD deficiency testing for all patients before FP-based chemotherapy by checking the four consensual deleterious *DPYD* variant or by
historical patient cohort receiving standard FP doses: for *DPYD*2A, RR was 1.3 (95%CI 0.6-2.7) for genotype-guided dosing compared with 2.87 (95%CI 2.1-3.9) in the historical cohort; for *DPYD*13 no toxicity occurred with genotype-guided dosing compared with 4.3 (95%CI 2.1-8.8); for HapB3 RR was 1.69 (95%CI 1.2-2.4) compared with 1.72 (95%CI 1.2-2.4); and for c.2846A>T RR was 2.0 (95%CI 1.2-3.3) compared with 3.1 (95%CI 2.3-4.3), suggesting that a dose reduction of 25% is too low for c.2846A>T carriers, in line with sparse pharmacokinetics data collected in five c.2846A>T carriers [103].

A major issue of FP dose reduction in partial deficient patients is its potential deleterious impact of treatment efficacy, this being particularly relevant for adjuvant colorectal cancer [104,105]. Only one retrospective study specifically examined efficacy in DPD-deficient patients receiving reduced FP dose [101]. Efficacy was assessed in 40 prospectively identified heterozygous *DPYD*2A carriers treated with an ~50% reduced FP dose (5FU, capecitabine) who were matched (according to hospital, tumor type, treatment, disease stage, age) with wild-type *DPYD*2A patients receiving standard dose. Overall and progression-free survivals were not significantly different between *2A carriers (medians 27 and 14 months, respectively) and wild-type patients (medians 24 and 10 months, respectively).

The risk of undertreating partial deficient patients with DPD-guided dose is of great concern since pharmacokinetics studies have demonstrated significant relationships between 5FU systemic exposure and treatment efficacy. Two French studies recently investigated 5FU systemic exposure in cancer patients with pre-emptive uracilemia testing and subsequent FP dose reduction [106,107]. In contrast to a previous historical study [26], both reported the lack of significant correlation between 5FU clearance and [U]. The study of Dolat et al. involving 169 patients showed that patients with [U] ≥16 ng/mL had significantly lower 5FU AUC than patients with [U] <16 ng/mL (p=.0016), in line with significantly lower 5FU doses administered in deficient patients (mean 3540 mg) relative to non-deficient patients (mean 4050 mg, p<0.001) [106]. Such results show the difficulty to assess the optimal FP starting dose based on [U]. Another aspect of DPD phenotyping is to help identifying DPD proficient patients. A recent retrospective study conducted on 136 cancer patients treated with various FP-based regimens showed that the 18% of patients with the highest DPD enzyme activity measured in PBMCs had a significantly lower overall survival, progression-free survival and response rate as compared to remaining patients with normal or deficient DPD activity [108].

## Conclusions

Cancer patients treated with FP might experience severe, life-threatening toxicities due to impaired DPD activity. France remains currently the only country that makes upfront DPD-deficiency testing mandatory. EMA recommends DPD deficiency testing for all patients before FP-based chemotherapy by checking the four consensual deleterious *DPYD* variant or by
measuring [U] and contraindicates FP in case of complete deficiency or recommends reducing the FP starting dose in case of partial DPD deficiency. In patients with partial deficiency, the FP dose reduction applied at first cycle should be followed by a dose increase according to tolerance, as soon as cycle 2 in order to ensure treatment efficacy. The impact of DPD-guided FP dose reduction on efficacy is poorly documented. FP-dose recommendations should also consider baseline characteristics such as age, sex, performance status, body mass index, FP-based regimen and associated anticancer drugs, since they are significantly related to the toxicity risk. Importantly for i.v. 5FU, FP dose adaptation based on pharmacokinetics should be implemented in partially deficient patients since it is currently the dose-individualization strategy with the highest level of evidence.

Both phenotyping and genotyping approaches have demonstrated their feasibility for large scale screening. Compared to phenotyping, genotyping has no pre-analytical constraints, is easier to perform, and gives unequivocal results. However, due to scarcity of deleterious DPYD variants, consensual genotyping has a lower sensitivity than phenotyping for identifying patients at-risk of severe FP-related toxicities. Moreover, current genotyping recommendations is only relevant in Caucasian population. DPYD genotype-guided FP dose adaptation has been validated in prospective studies. There is currently no validated dosing-guidelines based on [U]. A clinical trial in the Netherlands is ongoing to investigate whether upfront DPD genotyping and phenotyping, and subsequent dose reductions based on CPIC and French guidelines, reduce severe toxicities in patients treated with 5FU or capecitabine (ClinicalTrials.gov NCT04269369). Additional prospective studies are needed to define a dose reduction algorithm based on [U] and associated with the best clinical benefit for the patient. Hopefully, such future studies will lead to the harmonization of clinical guidelines for DPD testing across oncology organizations.
measuring [U] and contraindicates FP in case of complete deficiency or recommends reducing the FP starting dose in case of partial DPD deficiency. In patients with partial deficiency, the FP dose reduction applied at first cycle should be followed by a dose increase according to tolerance, as soon as cycle 2 in order to ensure treatment efficacy. The impact of DPD-guided FP dose reduction on efficacy is poorly documented. FP-dose recommendations should also consider baseline characteristics such as age, sex, performance status, body mass index, FP-based regimen and associated anticancer drugs, since they are significantly related to the toxicity risk. Importantly for i.v. 5FU, FP dose adaptation based on pharmacokinetics should be implemented in partially deficient patients since it is currently the dose-individualization strategy with the highest level of evidence.

Both phenotyping and genotyping approaches have demonstrated their feasibility for large scale screening. Compared to phenotyping, genotyping has no pre-analytical constraints, is easier to perform, and gives unequivocal results. However, due to scarcity of deleterious DPYD variants, consensual genotyping has a lower sensitivity than phenotyping for identifying patients at-risk of severe FP-related toxicities. Moreover, current genotyping recommendations is only relevant in Caucasian population. DPYD genotype-guided FP dose adaptation has been validated in prospective studies. There is currently no validated dosing-guidelines based on [U]. A clinical trial in the Netherlands is ongoing to investigate whether upfront DPD genotyping and phenotyping, and subsequent dose reductions based on CPIC and French guidelines, reduce severe toxicities in patients treated with 5FU or capecitabine (ClinicalTrials.gov NCT04269369). Additional prospective studies are needed to define a dose reduction algorithm based on [U] and associated with the best clinical benefit for the patient. Hopefully, such future studies will lead to the harmonization of clinical guidelines for DPD testing across oncology organizations.
References

[1] Barin-Le Guellec C, Lafay-Chebassier C, Ingrand I, Tournamille J-F, Boudet A, Lanoue M-C, et al. Toxicities associated with chemotherapy regimens containing a fluoropyrimidine: A real-life evaluation in France. Eur J Cancer 2020;124:37-46.
https://doi.org/10.1016/j.ejca.2019.09.028.

[2] Diasio RB, Harris BE. Clinical pharmacology of 5-fluorouracil. Clin Pharmacokinet 1989;16:215-37. https://doi.org/10.2165/00003088-198916040-00002.

[3] Marshall JL, Hwang JJ. Capecitabine: fulfilling the promise of oral chemotherapy. Expert Opinion on Pharmacotherapy 2002;3:733-43.
https://doi.org/10.1517/14656566.3.6.733.

[4] Ciccolini J, Mercier C, Evrard A, Dahan L, Boyer J-C, Duffaud F, et al. A rapid and inexpensive method for anticipating severe toxicity to fluorouracil and fluorouracil-based chemotherapy. Ther Drug Monit 2006;28:678-85.
https://doi.org/10.1097/01.ftd.0000245771.82720.c7.

[5] Sharma BB, Rai K, Blunt H, Zhao W, Tosteson TD, Brooks GA. Pathogenic DPYD Variants and Treatment-Related Mortality in Patients Receiving Fluoropyrimidine Chemotherapy: A Systematic Review and Meta-Analysis. Oncologist 2021;26:1008-16.
https://doi.org/10.1002/onco.13967.

[6] Taieb J, Tabernero J, Mini E, Subtil F, Folprecht G, Van Laethem J-L, et al. Oxaliplatin, fluorouracil, and leucovorin with or without cetuximab in patients with resected stage III colon cancer (PETACC-8): an open-label, randomised phase 3 trial. Lancet Oncol 2014;15:862-73. https://doi.org/10.1016/S1470-2045(14)70227-X.

[7] Van Cutsem E, Twelves C, Cassidy J, Allman D, Bajetta E, Boyer M, et al. Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 2001;19:4097-106. https://doi.org/10.1200/JCO.2001.19.21.4097.

[8] Schmoll H-J, Cartwright T, Tabernero J, Nowacki MP, Figer A, Maroun J, et al. Phase III trial of capecitabine plus oxaliplatin as adjuvant therapy for stage III colon cancer: a planned safety analysis in 1,864 patients. J Clin Oncol 2007;25:102-9.
https://doi.org/10.1200/JCO.2006.08.1075.

[9] Froehlich TK, Amstutz U, Aebi S, Joerger M, Largiadèr CR. Clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early-onset fluoropyrimidine toxicity. Int J Cancer 2015;136:730-9. https://doi.org/10.1002/ijc.29025.

[10] Amstutz U, Henricks LM, Offer SM, Barbarino J, Schellens JHM, Swen JJ, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing: 2017 Update. Clin Pharmacol Ther 2018;103:210-6. https://doi.org/10.1002/cpt.911.

[11] Meta-Analysis Group In Cancer, Lévy E, Piedbois P, Buyse M, Pignon JP, Rougier P, et al. Toxicity of fluorouracil in patients with advanced colorectal cancer: effect of administration schedule and prognostic factors. J Clin Oncol 1998;16:3537-41.
https://doi.org/10.1200/JCO.1998.16.11.3537.

[12] Delea TE, Vera-Llonch M, Edelsberg JS, McGarry L, Anton S, Ulcickas-Yood M, et al. The incidence and cost of hospitalization for 5-FU toxicity among Medicare beneficiaries with metastatic colorectal cancer. Value Health 2002;5:35-43. https://doi.org/10.1046/j.1524-4733.2002.51083.x.

[13] van Kuilenburg ABP. Dihydropyrimidine dehydrogenase and the efficacy and toxicity of 5-fluorouracil. Eur J Cancer 2004;40:939-50. https://doi.org/10.1016/j.ejca.2003.12.004.

[14] Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3:330-8. https://doi.org/10.1038/nrc1074.

[15] Guimbaud R, Guichard S, Dusseau C, Bertrand V, Aparicio T, Lochon I, et al. Dihydropyrimidine dehydrogenase activity in normal, inflammatory and tumour tissues of colon and liver in humans. Cancer Chemother Pharmacol 2000;45:477-82.
https://doi.org/10.1007/s002800051022.

[16] Hirota T, Date Y, Nishibatake Y, Takane H, Fukuoka Y, Taniguchi Y, et al.
References

[1] Barin-Le Guellec C, Lafay-Chebassier C, Ingrand I, Tournamille J-F, Boudet A, Lanoue M-C, et al. Toxicities associated with chemotherapy regimens containing a fluoropyrimidine: A real-life evaluation in France. Eur J Cancer 2020;124:37-46.
https://doi.org/10.1016/j.ejca.2019.09.028.

[2] Diasio RB, Harris BE. Clinical pharmacology of 5-fluorouracil. Clin Pharmacokinet 1989;16:215-37. https://doi.org/10.2165/00003088-198916040-00002.

[3] Marshall JL, Hwang JJ. Capecitabine: fulfilling the promise of oral chemotherapy. Expert Opinion on Pharmacotherapy 2002;3:733-43.
https://doi.org/10.1517/14656566.3.6.733.

[4] Ciccolini J, Mercier C, Evrard A, Dahan L, Boyer J-C, Duffaud F, et al. A rapid and inexpensive method for anticipating severe toxicity to fluorouracil and fluorouracil-based chemotherapy. Ther Drug Monit 2006;28:678-85.
https://doi.org/10.1097/01.ftd.0000245771.82720.c7.

[5] Sharma BB, Rai K, Blunt H, Zhao W, Tosteson TD, Brooks GA. Pathogenic DPYD Variants and Treatment-Related Mortality in Patients Receiving Fluoropyrimidine Chemotherapy: A Systematic Review and Meta-Analysis. Oncologist 2021;26:1008-16.
https://doi.org/10.1002/onco.13967.

[6] Taieb J, Tabernero J, Mini E, Subtil F, Folprecht G, Van Laethem J-L, et al. Oxaliplatin, fluorouracil, and leucovorin with or without cetuximab in patients with resected stage III colon cancer (PETACC-8): an open-label, randomised phase 3 trial. Lancet Oncol 2014;15:862-73. https://doi.org/10.1016/S1470-2045(14)70227-X.

[7] Van Cutsem E, Twelves C, Cassidy J, Allman D, Bajetta E, Boyer M, et al. Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 2001;19:4097-106. https://doi.org/10.1200/JCO.2001.19.21.4097.

[8] Schmoll H-J, Cartwright T, Tabernero J, Nowacki MP, Figer A, Maroun J, et al. Phase III trial of capecitabine plus oxaliplatin as adjuvant therapy for stage III colon cancer: a planned safety analysis in 1,864 patients. J Clin Oncol 2007;25:102-9.
https://doi.org/10.1200/JCO.2006.08.1075.

[9] Froehlich TK, Amstutz U, Aebi S, Joerger M, Largiadèr CR. Clinical importance of risk variants in the dihydropyrimidine dehydrogenase gene for the prediction of early-onset fluoropyrimidine toxicity. Int J Cancer 2015;136:730-9. https://doi.org/10.1002/ijc.29025.

[10] Amstutz U, Henricks LM, Offer SM, Barbarino J, Schellens JHM, Swen JJ, et al. Clinical Pharmacogenetics Implementation Consortium (CPIC) Guideline for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing: 2017 Update. Clin Pharmacol Ther 2018;103:210-6. https://doi.org/10.1002/cpt.911.

[11] Meta-Analysis Group In Cancer, Lévy E, Piedbois P, Buyse M, Pignon JP, Rougier P, et al. Toxicity of fluorouracil in patients with advanced colorectal cancer: effect of administration schedule and prognostic factors. J Clin Oncol 1998;16:3537-41.
https://doi.org/10.1200/JCO.1998.16.11.3537.

[12] Delea TE, Vera-Llonch M, Edelsberg JS, McGarry L, Anton S, Ulcickas-Yood M, et al. The incidence and cost of hospitalization for 5-FU toxicity among Medicare beneficiaries with metastatic colorectal cancer. Value Health 2002;5:35-43. https://doi.org/10.1046/j.1524-4733.2002.51083.x.

[13] van Kuilenburg ABP. Dihydropyrimidine dehydrogenase and the efficacy and toxicity of 5-fluorouracil. Eur J Cancer 2004;40:939-50. https://doi.org/10.1016/j.ejca.2003.12.004.

[14] Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3:330-8. https://doi.org/10.1038/nrc1074.

[15] Guimbaud R, Guichard S, Dusseau C, Bertrand V, Aparicio T, Lochon I, et al. Dihydropyrimidine dehydrogenase activity in normal, inflammatory and tumour tissues of colon and liver in humans. Cancer Chemother Pharmacol 2000;45:477-82.
https://doi.org/10.1007/s002800051022.

[16] Hirota T, Date Y, Nishibatake Y, Takane H, Fukuoka Y, Taniguchi Y, et al.
Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues. Lung Cancer 2012;77:16-23.
https://doi.org/10.1016/j.lungcan.2011.12.018.

[17] Mori K, Hasegawa M, Nishida M, Toma H, Fukuda M, Kubota T, et al. Expression levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in various human tumor tissues. Int J Oncol 2000;17:33-8. https://doi.org/10.3892/ijo.17.1.33.

[18] Uesaka K, Boku N, Fukutomi A, Okamura Y, Konishi M, Matsumoto I, et al. Adjuvant chemotherapy of S-1 versus gemcitabine for resected pancreatic cancer: a phase 3, open-label, randomised, non-inferiority trial (JASPAC 01). Lancet 2016;388:248-57.
https://doi.org/10.1016/S0140-6736(16)30583-9.

[19] Etienne MC, Lagrange JL, Dassonville O, Fleming R, Thyss A, Renée N, et al. Population study of dihydropyrimidine dehydrogenase in cancer patients. J Clin Oncol 1994;12:2248-53. https://doi.org/10.1200/JCO.1994.12.11.2248.

[20] Chazal M, Etienne MC, Renée N, Bourgeon A, Richelme H, Milano G. Link between dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells and liver. Clin Cancer Res 1996;2:507-10.

[21] Beumer JH, Chu E, Allegra C, Tanigawara Y, Milano G, Diasio R, et al. Therapeutic Drug Monitoring in Oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology Recommendations for 5-Fluorouracil Therapy. Clinical Pharmacology & Therapeutics 2019;105:598-613. https://doi.org/10.1002/cpt.1124.

[22] Lunar N, Etienne-Grimaldi M-C, Macaire P, Thomas F, Dalenc F, Ferrero J-M, et al. Population pharmacokinetic and pharmacodynamic modeling of capecitabine and its metabolites in breast cancer patients. Cancer Chemother Pharmacol 2021;87:229-39.
https://doi.org/10.1007/s00280-020-04208-8.

[23] Tuchman M, Stoeckeler JS, Kiang DT, O'Dea RF, Ramnaraine ML, Mirkin BL. Familial pyrimidinemia and pyrimidinuria associated with severe fluorouracil toxicity. N Engl J Med 1985;313:245-9. https://doi.org/10.1056/NEJM198507253130407.

[24] van Kuilenburg ABP, Meinsma R, Zonnenberg BA, Zoetekouw L, Baas F, Matsuda K, et al. Dihydropyrimidinase deficiency and severe 5-fluorouracil toxicity. Clin Cancer Res 2003;9:4363-7.

[25] Morel A, Boisdron-Celle M, Fey L, Soulie P, Craipeau MC, Traore S, et al. Clinical relevance of different dihydropyrimidine dehydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther 2006;5:2895-904.
https://doi.org/10.1158/1535-7163.MCT-06-0327.

[26] Boisdron-Celle M, Remaud G, Traore S, Poirier AL, Gamelin L, Morel A, et al. 5-Fluorouracil-related severe toxicity: a comparison of different methods for the pretherapeutic detection of dihydropyrimidine dehydrogenase deficiency. Cancer Lett 2007;249:271-82.
https://doi.org/10.1016/j.canlet.2006.09.006.

[27] Mercier C, Ciccolini J. Severe or lethal toxicities upon capecitabine intake: is DPYD genetic polymorphism the ideal culprit? Trends Pharmacol Sci 2007;28:597-8.
https://doi.org/10.1016/j.tips.2007.09.009.

[28] Deenen MJ, Tol J, Burylo AM, Doodeman VD, de Boer A, Vincent A, et al. Relationship between single nucleotide polymorphisms and haplotypes in DPYD and toxicity and efficacy of capecitabine in advanced colorectal cancer. Clin Cancer Res 2011;17:3455-68. https://doi.org/10.1158/1078-0432.CCR-10-2209.

[29] Ciccolini J, Gross E, Dahan L, Lacarelle B, Mercier C. Routine dihydropyrimidine dehydrogenase testing for anticipating 5-fluorouracil-related severe toxicities: hype or hope? Clin Colorectal Cancer 2010;9:224-8. https://doi.org/10.3816/CCC.2010.n.033.

[30] Rosmarin D, Palles C, Church D, Domingo E, Jones A, Johnstone E, et al. Genetic markers of toxicity from capecitabine and other fluorouracil-based regimens: investigation in the QUASAR2 study, systematic review, and meta-analysis. J Clin Oncol 2014;32:1031-9.
https://doi.org/10.1200/JCO.2013.51.1857.

[31] Loganayagam A, Arenas Hernandez M, Corrigan A, Fairbanks L, Lewis CM, Harper P, et al. Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity. Br J Cancer 2013;108:2505-15.
Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues. Lung Cancer 2012;77:16-23.
https://doi.org/10.1016/j.lungcan.2011.12.018.

[17] Mori K, Hasegawa M, Nishida M, Toma H, Fukuda M, Kubota T, et al. Expression levels of thymidine phosphorylase and dihydropyrimidine dehydrogenase in various human tumor tissues. Int J Oncol 2000;17:33-8. https://doi.org/10.3892/ijo.17.1.33.

[18] Uesaka K, Boku N, Fukutomi A, Okamura Y, Konishi M, Matsumoto I, et al. Adjuvant chemotherapy of S-1 versus gemcitabine for resected pancreatic cancer: a phase 3, open-label, randomised, non-inferiority trial (JASPAC 01). Lancet 2016;388:248-57.
https://doi.org/10.1016/S0140-6736(16)30583-9.

[19] Etienne MC, Lagrange JL, Dassonville O, Fleming R, Thyss A, Renée N, et al. Population study of dihydropyrimidine dehydrogenase in cancer patients. J Clin Oncol 1994;12:2248-53. https://doi.org/10.1200/JCO.1994.12.11.2248.

[20] Chazal M, Etienne MC, Renée N, Bourgeon A, Richelme H, Milano G. Link between dihydropyrimidine dehydrogenase activity in peripheral blood mononuclear cells and liver. Clin Cancer Res 1996;2:507-10.

[21] Beumer JH, Chu E, Allegra C, Tanigawara Y, Milano G, Diasio R, et al. Therapeutic Drug Monitoring in Oncology: International Association of Therapeutic Drug Monitoring and Clinical Toxicology Recommendations for 5-Fluorouracil Therapy. Clinical Pharmacology & Therapeutics 2019;105:598-613. https://doi.org/10.1002/cpt.1124.

[22] Lunar N, Etienne-Grimaldi M-C, Macaire P, Thomas F, Dalenc F, Ferrero J-M, et al. Population pharmacokinetic and pharmacodynamic modeling of capecitabine and its metabolites in breast cancer patients. Cancer Chemother Pharmacol 2021;87:229-39.
https://doi.org/10.1007/s00280-020-04208-8.

[23] Tuchman M, Stoeckeler JS, Kiang DT, O'Dea RF, Ramnaraine ML, Mirkin BL. Familial pyrimidinemia and pyrimidinuria associated with severe fluorouracil toxicity. N Engl J Med 1985;313:245-9. https://doi.org/10.1056/NEJM198507253130407.

[24] van Kuilenburg ABP, Meinsma R, Zonnenberg BA, Zoetekouw L, Baas F, Matsuda K, et al. Dihydropyrimidinase deficiency and severe 5-fluorouracil toxicity. Clin Cancer Res 2003;9:4363-7.

[25] Morel A, Boisdron-Celle M, Fey L, Soulie P, Craipeau MC, Traore S, et al. Clinical relevance of different dihydropyrimidine dehydrogenase gene single nucleotide polymorphisms on 5-fluorouracil tolerance. Mol Cancer Ther 2006;5:2895-904.
https://doi.org/10.1158/1535-7163.MCT-06-0327.

[26] Boisdron-Celle M, Remaud G, Traore S, Poirier AL, Gamelin L, Morel A, et al. 5-Fluorouracil-related severe toxicity: a comparison of different methods for the pretherapeutic detection of dihydropyrimidine dehydrogenase deficiency. Cancer Lett 2007;249:271-82.
https://doi.org/10.1016/j.canlet.2006.09.006.

[27] Mercier C, Ciccolini J. Severe or lethal toxicities upon capecitabine intake: is DPYD genetic polymorphism the ideal culprit? Trends Pharmacol Sci 2007;28:597-8.
https://doi.org/10.1016/j.tips.2007.09.009.

[28] Deenen MJ, Tol J, Burylo AM, Doodeman VD, de Boer A, Vincent A, et al. Relationship between single nucleotide polymorphisms and haplotypes in DPYD and toxicity and efficacy of capecitabine in advanced colorectal cancer. Clin Cancer Res 2011;17:3455-68. https://doi.org/10.1158/1078-0432.CCR-10-2209.

[29] Ciccolini J, Gross E, Dahan L, Lacarelle B, Mercier C. Routine dihydropyrimidine dehydrogenase testing for anticipating 5-fluorouracil-related severe toxicities: hype or hope? Clin Colorectal Cancer 2010;9:224-8. https://doi.org/10.3816/CCC.2010.n.033.

[30] Rosmarin D, Palles C, Church D, Domingo E, Jones A, Johnstone E, et al. Genetic markers of toxicity from capecitabine and other fluorouracil-based regimens: investigation in the QUASAR2 study, systematic review, and meta-analysis. J Clin Oncol 2014;32:1031-9.
https://doi.org/10.1200/JCO.2013.51.1857.

[31] Loganayagam A, Arenas Hernandez M, Corrigan A, Fairbanks L, Lewis CM, Harper P, et al. Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity. Br J Cancer 2013;108:2505-15.
https://doi.org/10.1038/bjc.2013.262.

[32] Wei X, Elizondo G, Sapone A, McLeod HL, Raunio H, Fernandez-Salguero P, et al. Characterization of the human dihydropyrimidine dehydrogenase gene. Genomics 1998;51:391-400. https://doi.org/10.1006/geno.1998.5379.

[33] Toffoli G, Giodini L, Buonadonna A, Berretta M, De Paoli A, Scalone S, et al. Clinical validity of a DPYD-based pharmacogenetic test to predict severe toxicity to fluoropyrimidines. Int J Cancer 2015;137:2971-80. https://doi.org/10.1002/ijc.29654.

[34] Offer SM, Fossum CC, Wegner NJ, Stuflesser AJ, Butterfield GL, Diasio RB. Comparative Functional Analysis of DPYD Variants of Potential Clinical Relevance to Dihydropyrimidine Dehydrogenase Activity. Cancer Res 2014;74:2545-54. https://doi.org/10.1158/0008-5472.CAN-13-2482.

[35] Van Kuilenburg AB, Vreken P, Abeling NG, Bakker HD, Meinsma R, Van Lenthe H, et al. Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency. Hum Genet 1999;104:1-9. https://doi.org/10.1007/pl00008711.

[36] Saif MW, Ezzeldin H, Vance K, Sellers S, Diasio RB. DPYD*2A mutation: the most common mutation associated with DPD deficiency. Cancer Chemother Pharmacol 2007;60:503-7. https://doi.org/10.1007/s00280-006-0392-5.

[37] McLeod HL, Sargent DJ, Marsh S, Green EM, King CR, Fuchs CS, et al. Pharmacogenetic predictors of adverse events and response to chemotherapy in metastatic colorectal cancer: results from North American Gastrointestinal Intergroup Trial N9741. J Clin Oncol 2010;28:3227-33. https://doi.org/10.1200/JCO.2009.21.7943.

[38] Hishinuma E, Narita Y, Saito S, Maekawa M, Akai F, Nakanishi Y, et al. Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals. Drug Metab Dispos 2018;46:1083-90. https://doi.org/10.1124/dmd.118.081737.

[39] Offer SM, Lee AM, Mattison LK, Fossum C, Wegner NJ, Diasio RB. A DPYD variant (Y186C) in individuals of african ancestry is associated with reduced DPD enzyme activity. Clin Pharmacol Ther 2013;94:158-66. https://doi.org/10.1038/clpt.2013.69.

[40] da Rocha JEB, Lombard Z, Ramsay M. Potential Impact of DPYD Variation on Fluoropyrimidine Drug Response in sub-Saharan African Populations. Front Genet 2021;12:626954. https://doi.org/10.3389/fgene.2021.626954.

[41] Caudle KE, Thorn CF, Klein TE, Swen JJ, McLeod HL, Diasio RB, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing. Clin Pharmacol Ther 2013;94:640-5. https://doi.org/10.1038/clpt.2013.172.

[42] Elraiyah T, Jerde CR, Shrestha S, Wu R, Nie Q, Giama NH, et al. Novel Deleterious Dihydropyrimidine Dehydrogenase Variants May Contribute to 5-Fluorouracil Sensitivity in an East African Population. Clin Pharmacol Ther 2017;101:382-90. https://doi.org/10.1002/cpt.531.

[43] Vreken P, Van Kuilenburg AB, Meinsma R, Smit GP, Bakker HD, De Abreu RA, et al. A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency. J Inherit Metab Dis 1996;19:645-54. https://doi.org/10.1007/BF01799841.

[44] Offer SM, Wegner NJ, Fossum C, Wang K, Diasio RB. Phenotypic profiling of DPYD variations relevant to 5-fluorouracil sensitivity using real-time cellular analysis and in vitro measurement of enzyme activity. Cancer Res 2013;73:1958-68. https://doi.org/10.1158/0008-5472.CAN-12-3858.

[45] McLeod HL, Collie-Duguid ES, Vreken P, Johnson MR, Wei X, Sapone A, et al. Nomenclature for human DPYD alleles. Pharmacogenetics 1998;8:455-9. https://doi.org/10.1097/00008571-199812000-00001.

[46] van Kuilenburg AB, Haasjes J, Richel DJ, Zoetekouw L, Van Lenthe H, De Abreu RA, et al. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clin Cancer Res 2000;6:4705-12.

[47] Van Kuilenburg AB, Vreken P, Beex LV, Meinsma R, Van Lenthe H, De Abreu RA, et
https://doi.org/10.1038/bjc.2013.262.

[32] Wei X, Elizondo G, Sapone A, McLeod HL, Raunio H, Fernandez-Salguero P, et al. Characterization of the human dihydropyrimidine dehydrogenase gene. Genomics 1998;51:391-400. https://doi.org/10.1006/geno.1998.5379.

[33] Toffoli G, Giodini L, Buonadonna A, Berretta M, De Paoli A, Scalone S, et al. Clinical validity of a DPYD-based pharmacogenetic test to predict severe toxicity to fluoropyrimidines. Int J Cancer 2015;137:2971-80. https://doi.org/10.1002/ijc.29654.

[34] Offer SM, Fossum CC, Wegner NJ, Stuflesser AJ, Butterfield GL, Diasio RB. Comparative Functional Analysis of DPYD Variants of Potential Clinical Relevance to Dihydropyrimidine Dehydrogenase Activity. Cancer Res 2014;74:2545-54. https://doi.org/10.1158/0008-5472.CAN-13-2482.

[35] Van Kuilenburg AB, Vreken P, Abeling NG, Bakker HD, Meinsma R, Van Lenthe H, et al. Genotype and phenotype in patients with dihydropyrimidine dehydrogenase deficiency. Hum Genet 1999;104:1-9. https://doi.org/10.1007/pl00008711.

[36] Saif MW, Ezzeldin H, Vance K, Sellers S, Diasio RB. DPYD*2A mutation: the most common mutation associated with DPD deficiency. Cancer Chemother Pharmacol 2007;60:503-7. https://doi.org/10.1007/s00280-006-0392-5.

[37] McLeod HL, Sargent DJ, Marsh S, Green EM, King CR, Fuchs CS, et al. Pharmacogenetic predictors of adverse events and response to chemotherapy in metastatic colorectal cancer: results from North American Gastrointestinal Intergroup Trial N9741. J Clin Oncol 2010;28:3227-33. https://doi.org/10.1200/JCO.2009.21.7943.

[38] Hishinuma E, Narita Y, Saito S, Maekawa M, Akai F, Nakanishi Y, et al. Functional Characterization of 21 Allelic Variants of Dihydropyrimidine Dehydrogenase Identified in 1070 Japanese Individuals. Drug Metab Dispos 2018;46:1083-90. https://doi.org/10.1124/dmd.118.081737.

[39] Offer SM, Lee AM, Mattison LK, Fossum C, Wegner NJ, Diasio RB. A DPYD variant (Y186C) in individuals of african ancestry is associated with reduced DPD enzyme activity. Clin Pharmacol Ther 2013;94:158-66. https://doi.org/10.1038/clpt.2013.69.

[40] da Rocha JEB, Lombard Z, Ramsay M. Potential Impact of DPYD Variation on Fluoropyrimidine Drug Response in sub-Saharan African Populations. Front Genet 2021;12:626954. https://doi.org/10.3389/fgene.2021.626954.

[41] Caudle KE, Thorn CF, Klein TE, Swen JJ, McLeod HL, Diasio RB, et al. Clinical Pharmacogenetics Implementation Consortium Guidelines for Dihydropyrimidine Dehydrogenase Genotype and Fluoropyrimidine Dosing. Clin Pharmacol Ther 2013;94:640-5. https://doi.org/10.1038/clpt.2013.172.

[42] Elraiyah T, Jerde CR, Shrestha S, Wu R, Nie Q, Giama NH, et al. Novel Deleterious Dihydropyrimidine Dehydrogenase Variants May Contribute to 5-Fluorouracil Sensitivity in an East African Population. Clin Pharmacol Ther 2017;101:382-90. https://doi.org/10.1002/cpt.531.

[43] Vreken P, Van Kuilenburg AB, Meinsma R, Smit GP, Bakker HD, De Abreu RA, et al. A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency. J Inherit Metab Dis 1996;19:645-54. https://doi.org/10.1007/BF01799841.

[44] Offer SM, Wegner NJ, Fossum C, Wang K, Diasio RB. Phenotypic profiling of DPYD variations relevant to 5-fluorouracil sensitivity using real-time cellular analysis and in vitro measurement of enzyme activity. Cancer Res 2013;73:1958-68. https://doi.org/10.1158/0008-5472.CAN-12-3858.

[45] McLeod HL, Collie-Duguid ES, Vreken P, Johnson MR, Wei X, Sapone A, et al. Nomenclature for human DPYD alleles. Pharmacogenetics 1998;8:455-9. https://doi.org/10.1097/00008571-199812000-00001.

[46] van Kuilenburg AB, Haasjes J, Richel DJ, Zoetekouw L, Van Lenthe H, De Abreu RA, et al. Clinical implications of dihydropyrimidine dehydrogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPD gene. Clin Cancer Res 2000;6:4705-12.

[47] Van Kuilenburg AB, Vreken P, Beex LV, Meinsma R, Van Lenthe H, De Abreu RA, et
al. Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity. Eur J Cancer 1997;33:2258-64.
https://doi.org/10.1016/s0959-8049(97)00261-x.

[48] van Kuilenburg ABP, Maring JG, Schalhorn A, Terborg C, Schmalenberg H, Behnke D, et al. Pharmacokinetics of 5-fluorouracil in patients heterozygous for the IVS14+1G > A mutation in the dihydropyrimidine dehydrogenase gene. Nucleosides Nucleotides Nucleic Acids 2008;27:692-8. https://doi.org/10.1080/15257770802145009.

[49] van Kuilenburg ABP, Häusler P, Schalhorn A, Tanck MWT, Proost JH, Terborg C, et al. Evaluation of 5-fluorouracil pharmacokinetics in cancer patients with a c.1905+1G>A mutation in DPYD by means of a Bayesian limited sampling strategy. Clin Pharmacokinet 2012;51:163-74. https://doi.org/10.1007/BF03257473.

[50] Collie-Duguid ES, Etienne MC, Milano G, McLeod HL. Known variant DPYD alleles do not explain DPD deficiency in cancer patients. Pharmacogenetics 2000;10:217-23.
https://doi.org/10.1097/00008571-200004000-00002.

[51] van Kuilenburg ABP, Dobritzsch D, Meinsma R, Haasjes J, Waterham HR, Nowaczyk MJM, et al. Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure. Biochem J 2002;364:157-63.

[52] Johnson MR, Wang K, Diasio RB. Profound dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype. Clin Cancer Res 2002;8:768-74.

[53] Seck K, Riemer S, Kates R, Ullrich T, Lutz V, Harbeck N, et al. Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals. Clin Cancer Res 2005;11:5886-92. https://doi.org/10.1158/1078-0432.CCR-04-1784.

[54] van Kuilenburg ABP, Meijer J, Mul ANPM, Meinsma R, Schmid V, Dobritzsch D, et al. Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity. Hum Genet 2010;128:529-38. https://doi.org/10.1007/s00439-010-0879-3.

[55] Nie Q, Shrestha S, Tapper EE, Trogstad-Isaacson CS, Bouchonville KJ, Lee AM, et al. Quantitative Contribution of rs75017182 to Dihydropyrimidine Dehydrogenase mRNA Splicing and Enzyme Activity. Clin Pharmacol Ther 2017;102:662-70.
https://doi.org/10.1002/cpt.685.

[56] Henricks LM, Lunenburg CATC, Meulendijks D, Gelderblom H, Cats A, Swen JJ, et al. Translating DPYD genotype into DPD phenotype: using the DPYD gene activity score. Pharmacogenomics 2015;16:1277-86. https://doi.org/10.2217/pgs.15.70.

[57] Lunenburg CATC, van der Wouden CH, Nijenhuis M, Crommentuijn-van Rhenen MH, de Boer-Veger NJ, Buunk AM, et al. Dutch Pharmacogenetics Working Group (DPWG) guideline for the gene-drug interaction of DPYD and fluoropyrimidines. Eur J Hum Genet 2020;28:508-17. https://doi.org/10.1038/s41431-019-0540-0.

[58] Meulendijks D, Henricks LM, Jacobs BAW, Aliev A, Deenen MJ, de Vries N, et al. Pretreatment serum uracil concentration as a predictor of severe and fatal fluoropyrimidine-associated toxicity. Br J Cancer 2017;116:1415-24. https://doi.org/10.1038/bjc.2017.94.

[59] Etienne-Grimaldi M-C, Boyer J-C, Beroud C, Mbatchi L, Kuilenburg A van, Bobin-Dubigeon C, et al. New advances in DPYD genotype and risk of severe toxicity under capecitabine. Plos One 2017;12:e0175998. https://doi.org/10.1371/journal.pone.0175998.

[60] https://www.annalsofoncology.org/issue/S0923-7534(19)X9100-0 (accessed September 30, 2021).

[61] Meulendijks D, Henricks LM, Sonke GS, Deenen MJ, Froehlich TK, Amstutz U, et al. Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data. Lancet Oncol 2015;16:1639-50.
https://doi.org/10.1016/S1470-2045(15)00286-7.

[62] Boige V, Vincent M, Alexandre P, Tejpar S, Landolfi S, Le Malicot K, et al. DPYD Genotyping to Predict Adverse Events Following Treatment With Fluorouracil-Based Adjuvant Chemotherapy in Patients With Stage III Colon Cancer: A Secondary Analysis of
al. Heterozygosity for a point mutation in an invariant splice donor site of dihydropyrimidine dehydrogenase and severe 5-fluorouracil related toxicity. Eur J Cancer 1997;33:2258-64.
https://doi.org/10.1016/s0959-8049(97)00261-x.

[48] van Kuilenburg ABP, Maring JG, Schalhorn A, Terborg C, Schmalenberg H, Behnke D, et al. Pharmacokinetics of 5-fluorouracil in patients heterozygous for the IVS14+1G > A mutation in the dihydropyrimidine dehydrogenase gene. Nucleosides Nucleotides Nucleic Acids 2008;27:692-8. https://doi.org/10.1080/15257770802145009.

[49] van Kuilenburg ABP, Häusler P, Schalhorn A, Tanck MWT, Proost JH, Terborg C, et al. Evaluation of 5-fluorouracil pharmacokinetics in cancer patients with a c.1905+1G>A mutation in DPYD by means of a Bayesian limited sampling strategy. Clin Pharmacokinet 2012;51:163-74. https://doi.org/10.1007/BF03257473.

[50] Collie-Duguid ES, Etienne MC, Milano G, McLeod HL. Known variant DPYD alleles do not explain DPD deficiency in cancer patients. Pharmacogenetics 2000;10:217-23.
https://doi.org/10.1097/00008571-200004000-00002.

[51] van Kuilenburg ABP, Dobritzsch D, Meinsma R, Haasjes J, Waterham HR, Nowaczyk MJM, et al. Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure. Biochem J 2002;364:157-63.

[52] Johnson MR, Wang K, Diasio RB. Profound dihydropyrimidine dehydrogenase deficiency resulting from a novel compound heterozygote genotype. Clin Cancer Res 2002;8:768-74.

[53] Seck K, Riemer S, Kates R, Ullrich T, Lutz V, Harbeck N, et al. Analysis of the DPYD gene implicated in 5-fluorouracil catabolism in a cohort of Caucasian individuals. Clin Cancer Res 2005;11:5886-92. https://doi.org/10.1158/1078-0432.CCR-04-1784.

[54] van Kuilenburg ABP, Meijer J, Mul ANPM, Meinsma R, Schmid V, Dobritzsch D, et al. Intragenic deletions and a deep intronic mutation affecting pre-mRNA splicing in the dihydropyrimidine dehydrogenase gene as novel mechanisms causing 5-fluorouracil toxicity. Hum Genet 2010;128:529-38. https://doi.org/10.1007/s00439-010-0879-3.

[55] Nie Q, Shrestha S, Tapper EE, Trogstad-Isaacson CS, Bouchonville KJ, Lee AM, et al. Quantitative Contribution of rs75017182 to Dihydropyrimidine Dehydrogenase mRNA Splicing and Enzyme Activity. Clin Pharmacol Ther 2017;102:662-70.
https://doi.org/10.1002/cpt.685.

[56] Henricks LM, Lunenburg CATC, Meulendijks D, Gelderblom H, Cats A, Swen JJ, et al. Translating DPYD genotype into DPD phenotype: using the DPYD gene activity score. Pharmacogenomics 2015;16:1277-86. https://doi.org/10.2217/pgs.15.70.

[57] Lunenburg CATC, van der Wouden CH, Nijenhuis M, Crommentuijn-van Rhenen MH, de Boer-Veger NJ, Buunk AM, et al. Dutch Pharmacogenetics Working Group (DPWG) guideline for the gene-drug interaction of DPYD and fluoropyrimidines. Eur J Hum Genet 2020;28:508-17. https://doi.org/10.1038/s41431-019-0540-0.

[58] Meulendijks D, Henricks LM, Jacobs BAW, Aliev A, Deenen MJ, de Vries N, et al. Pretreatment serum uracil concentration as a predictor of severe and fatal fluoropyrimidine-associated toxicity. Br J Cancer 2017;116:1415-24. https://doi.org/10.1038/bjc.2017.94.

[59] Etienne-Grimaldi M-C, Boyer J-C, Beroud C, Mbatchi L, Kuilenburg A van, Bobin-Dubigeon C, et al. New advances in DPYD genotype and risk of severe toxicity under capecitabine. Plos One 2017;12:e0175998. https://doi.org/10.1371/journal.pone.0175998.

[60] https://www.annalsofoncology.org/issue/S0923-7534(19)X9100-0 (accessed September 30, 2021).

[61] Meulendijks D, Henricks LM, Sonke GS, Deenen MJ, Froehlich TK, Amstutz U, et al. Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data. Lancet Oncol 2015;16:1639-50.
https://doi.org/10.1016/S1470-2045(15)00286-7.

[62] Boige V, Vincent M, Alexandre P, Tejpar S, Landolfi S, Le Malicot K, et al. DPYD Genotyping to Predict Adverse Events Following Treatment With Fluorouracil-Based Adjuvant Chemotherapy in Patients With Stage III Colon Cancer: A Secondary Analysis of
the PETACC-8 Randomized Clinical Trial. JAMA Oncol 2016;2:655-62.
https://doi.org/10.1001/jamaoncol.2015.5392.

[63] Mattison LK, Fourie J, Hirao Y, Koga T, Desmond RA, King JR, et al. The uracil breath test in the assessment of dihydropyrimidine dehydrogenase activity: pharmacokinetic relationship between expired 13CO2 and plasma [2-13C]dihydroouracil. Clin Cancer Res 2006;12:549-55. https://doi.org/10.1158/1078-0432.CCR-05-2020.

[64] Sumi S, Kidouchi K, Ohba S, Wada Y. Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography. J Chromatogr B Biomed Appl 1995;672:233-9. https://doi.org/10.1016/0378-4347(95)00228-b.

[65] Jiang H, Jiang J, Hu P, Hu Y. Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002;769:169-76.
https://doi.org/10.1016/s1570-0232(02)00009-0.

[66] Remaud G, Boisdron-Celle M, Hameline C, Morel A, Gamelin E. An accurate dihydrouracil/uracil determination using improved high performance liquid chromatography method for preventing fluoropyrimidines-related toxicity in clinical practice. J Chromatogr B Analyt Technol Biomed Life Sci 2005;823:98-107.
https://doi.org/10.1016/j.jchromb.2005.05.044.

[67] Marin C, Krache A, Palmaro C, Lucas M, Hilaire V, Ugdonne R, et al. A Simple and Rapid UPLC-UV Method for Detecting DPD Deficiency in Patients With Cancer. Clin Transl Sci 2020;13:761-8. https://doi.org/10.1111/cts.12762.

[68] Kuhara T, Ohdoi C, Ohse M, van Kuilenburg ABP, van Gennip AH, Sumi S, et al. Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci 2003;792:107-15. https://doi.org/10.1016/s1570-0232(03)00044-8.

[69] Carlsson G, Odin E, Gustavsson B, Wettergren Y. Pretherapeutic uracil and dihydrouracil levels in saliva of colorectal cancer patients are associated with toxicity during adjuvant 5-fluorouracil-based chemotherapy. Cancer Chemother Pharmacol 2014;74:757-63. https://doi.org/10.1007/s00280-014-2553-2.

[70] Galarza AFA, Linden R, Antunes MV, Hahn RZ, Raymundo S, da Silva ACC, et al. Endogenous plasma and salivary uracil to dihydrouracil ratios and DPYD genotyping as predictors of severe fluoropyrimidine toxicity in patients with gastrointestinal malignancies. Clin Biochem 2016;49:1221-6. https://doi.org/10.1016/j.clinbiochem.2016.07.004.

[71] Antunes MV, Raymundo S, Cezimbra da Silva AC, Muller VV, Vicente Neto OJ, Schwartsmann G, et al. Determination of Endogenous Concentrations of Uracil and Dihydouracil in Dried Saliva Spots by LC-MS/MS: Method Development, Validation, and Clinical Application. Ther Drug Monit 2019;41:383-90.
https://doi.org/10.1097/FTD.0000000000000615.

[72] Milano G, Ferrero J-M, Thomas F, Bobin-Dubigeon C, Merlin J-L, Pinguet F, et al. Abstract P3-15-04: A French prospective pilot study to identify dihydropyrimidine dehydrogenase (DPD) deficiency in breast cancer patients receiving capecitabine. Cancer Res 2013;73:P3-15. https://doi.org/10.1158/0008-5472.SABCS13-P3-15-04.

[73] Maillard M, Launay M, Royer B, Guitton J, Gautier-Veyret E, Broutin S, et al. Quantitative impact of pre-analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency. British Journal of Clinical Pharmacology n.d.;n/a. https://doi.org/10.1111/bcp.15536.

[74] Pizzorno G, Cao D, Leffert JJ, Russell RL, Zhang D, Handschumacher RE. Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update. Biochim Biophys Acta 2002;1587:133-44. https://doi.org/10.1016/s0925-4439(02)00076-5.

[75] de With M, Knikman J, de Man FM, Lunenburg CATC, Henricks LM, van Kuilenburg ABP, et al. Dihydropyrimidine Dehydrogenase Phenotyping Using Pretreatment Uracil: A Note of Caution Based on a Large Prospective Clinical Study. Clinical Pharmacology & Therapeutics n.d.;n/a. https://doi.org/10.1002/cpt.2608.

[76] Henricks LM, Jacobs BAW, Meulendijks D, Pluim D, van den Broek D, de Vries N, et
the PETACC-8 Randomized Clinical Trial. JAMA Oncol 2016;2:655-62.
https://doi.org/10.1001/jamaoncol.2015.5392.

[63] Mattison LK, Fourie J, Hirao Y, Koga T, Desmond RA, King JR, et al. The uracil breath test in the assessment of dihydropyrimidine dehydrogenase activity: pharmacokinetic relationship between expired 13CO2 and plasma [2-13C]dihydroouracil. Clin Cancer Res 2006;12:549-55. https://doi.org/10.1158/1078-0432.CCR-05-2020.

[64] Sumi S, Kidouchi K, Ohba S, Wada Y. Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography. J Chromatogr B Biomed Appl 1995;672:233-9. https://doi.org/10.1016/0378-4347(95)00228-b.

[65] Jiang H, Jiang J, Hu P, Hu Y. Measurement of endogenous uracil and dihydrouracil in plasma and urine of normal subjects by liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2002;769:169-76.
https://doi.org/10.1016/s1570-0232(02)00009-0.

[66] Remaud G, Boisdron-Celle M, Hameline C, Morel A, Gamelin E. An accurate dihydrouracil/uracil determination using improved high performance liquid chromatography method for preventing fluoropyrimidines-related toxicity in clinical practice. J Chromatogr B Analyt Technol Biomed Life Sci 2005;823:98-107.
https://doi.org/10.1016/j.jchromb.2005.05.044.

[67] Marin C, Krache A, Palmaro C, Lucas M, Hilaire V, Ugdonne R, et al. A Simple and Rapid UPLC-UV Method for Detecting DPD Deficiency in Patients With Cancer. Clin Transl Sci 2020;13:761-8. https://doi.org/10.1111/cts.12762.

[68] Kuhara T, Ohdoi C, Ohse M, van Kuilenburg ABP, van Gennip AH, Sumi S, et al. Rapid gas chromatographic-mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency. J Chromatogr B Analyt Technol Biomed Life Sci 2003;792:107-15. https://doi.org/10.1016/s1570-0232(03)00044-8.

[69] Carlsson G, Odin E, Gustavsson B, Wettergren Y. Pretherapeutic uracil and dihydrouracil levels in saliva of colorectal cancer patients are associated with toxicity during adjuvant 5-fluorouracil-based chemotherapy. Cancer Chemother Pharmacol 2014;74:757-63. https://doi.org/10.1007/s00280-014-2553-2.

[70] Galarza AFA, Linden R, Antunes MV, Hahn RZ, Raymundo S, da Silva ACC, et al. Endogenous plasma and salivary uracil to dihydrouracil ratios and DPYD genotyping as predictors of severe fluoropyrimidine toxicity in patients with gastrointestinal malignancies. Clin Biochem 2016;49:1221-6. https://doi.org/10.1016/j.clinbiochem.2016.07.004.

[71] Antunes MV, Raymundo S, Cezimbra da Silva AC, Muller VV, Vicente Neto OJ, Schwartsmann G, et al. Determination of Endogenous Concentrations of Uracil and Dihydroouracil in Dried Saliva Spots by LC-MS/MS: Method Development, Validation, and Clinical Application. Ther Drug Monit 2019;41:383-90.
https://doi.org/10.1097/FTD.0000000000000615.

[72] Milano G, Ferrero J-M, Thomas F, Bobin-Dubigeon C, Merlin J-L, Pinguet F, et al. Abstract P3-15-04: A French prospective pilot study to identify dihydropyrimidine dehydrogenase (DPD) deficiency in breast cancer patients receiving capecitabine. Cancer Res 2013;73:P3-15. https://doi.org/10.1158/0008-5472.SABCS13-P3-15-04.

[73] Maillard M, Launay M, Royer B, Guitton J, Gautier-Veyret E, Broutin S, et al. Quantitative impact of pre-analytical process on plasma uracil when testing for dihydropyrimidine dehydrogenase deficiency. British Journal of Clinical Pharmacology n.d.;n/a. https://doi.org/10.1111/bcp.15536.

[74] Pizzorno G, Cao D, Leffert JJ, Russell RL, Zhang D, Handschumacher RE. Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update. Biochim Biophys Acta 2002;1587:133-44. https://doi.org/10.1016/s0925-4439(02)00076-5.

[75] de With M, Knikman J, de Man FM, Lunenburg CATC, Henricks LM, van Kuilenburg ABP, et al. Dihydropyrimidine Dehydrogenase Phenotyping Using Pretreatment Uracil: A Note of Caution Based on a Large Prospective Clinical Study. Clinical Pharmacology & Therapeutics n.d.;n/a. https://doi.org/10.1002/cpt.2608.

[76] Henricks LM, Jacobs BAW, Meulendijks D, Pluim D, van den Broek D, de Vries N, et
al. Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers. Br J Clin Pharmacol 2018;84:2761-9. https://doi.org/10.1111/bcp.13719.

[77] Jiang H, Lu J, Ji J. Circadian rhythm of dihydrouracil/uracil ratios in biological fluids: a potential biomarker for dihydropyrimidine dehydrogenase levels. Br J Pharmacol 2004;141:616-23. https://doi.org/10.1038/sj.bjp.0705651.

[78] Gaible C, Narjoz C, Loriot M-A, Roueff S, Pallet N. Pretherapeutic screening for Dihydropyrimidine deshydrogenase deficiency in measuring uracilemia in dialysis patients leads to a high rate of falsely positive results. Cancer Chemother Pharmacol 2021;88:1049-53. https://doi.org/10.1007/s00280-021-04354-7.

[79] Coudoré F, Roche D, Lefeuvre S, Faussot D, Billaud EM, Loriot M-A, et al. Validation of an ultra-high performance liquid chromatography tandem mass spectrometric method for quantifying uracil and 5,6-dihydrouracil in human plasma. J Chromatogr Sci 2012;50:877-84. https://doi.org/10.1093/chromsci/bms085.

[80] Thomas F, Maillard M, Launay M, Tron C, Etienne-Grimaldi M-C, Gautier-Veyret E, et al. Artificial increase of uracilemia during fluoropyrimidine treatment can lead to DPD deficiency misinterpretation. Ann Oncol 2021;32:810-1. https://doi.org/10.1016/j.annonc.2021.02.020.

[81] Pallet N, Hamdane S, Garinet S, Blons H, Zaanan A, Paillaud E, et al. A comprehensive population-based study comparing the phenotype and genotype in a pretherapeutic screen of dihydropyrimidine dehydrogenase deficiency. Br J Cancer 2020;123:811-8. https://doi.org/10.1038/s41416-020-0962-z.

[82] Henricks LM, Lunenburg CATC, de Man FM, Meulendijks D, Frederix GWJ, Kienhuis E, et al. A cost analysis of upfront DPYD genotype-guided dose individualisation in fluoropyrimidine-based anticancer therapy. Eur J Cancer 2019;107:60-7. https://doi.org/10.1016/j.ejca.2018.11.010.

[83] Deenen MJ, Meulendijks D, Cats A, Sechterberger MK, Severens JL, Boot H, et al. Upfront Genotyping of DPYD*2A to Individualize Fluoropyrimidine Therapy: A Safety and Cost Analysis. J Clin Oncol 2016;34:227-34. https://doi.org/10.1200/JCO.2015.63.1325.

[84] Tsiachristas A, Vallance G, Koleva-Kolarova R, Taylor H, Solomons L, Rizzo G, et al. Can upfront DPYD extended variant testing reduce toxicity and associated hospital costs of fluoropyrimidine chemotherapy? A propensity score matched analysis of 2022 UK patients. BMC Cancer 2022;22:458. https://doi.org/10.1186/s12885-022-09576-3.

[85] Saam J, Critchfield GC, Hamilton SA, Roa BB, Wenstrup RJ, Kaldate RR. Body surface area-based dosing of 5-fluorouracil results in extensive interindividual variability in 5-fluorouracil exposure in colorectal cancer patients on FOLFOX regimens. Clin Colorectal Cancer 2011;10:203-6. https://doi.org/10.1016/j.clcc.2011.03.015.

[86] Fety R, Rolland F, Barberi-Heyob M, Hardouin A, Campion L, Conroy T, et al. Clinical impact of pharmacokinetically-guided dose adaptation of 5-fluorouracil: results from a multicentric randomized trial in patients with locally advanced head and neck carcinomas. Clin Cancer Res 1998;4:2039-45.

[87] Gamelin E, Delva R, Jacob J, Merrouche Y, Raoul JL, Pezet D, et al. Individual fluorouracil dose adjustment based on pharmacokinetic follow-up compared with conventional dosage: results of a multicenter randomized trial of patients with metastatic colorectal cancer. J Clin Oncol 2008;26:2099-105. https://doi.org/10.1200/JCO.2007.13.3934.

[88] Kaldate RR, Haregewoin A, Grier CE, Hamilton SA, McLeod HL. Modeling the 5-fluorouracil area under the curve versus dose relationship to develop a pharmacokinetic dosing algorithm for colorectal cancer patients receiving FOLFOX6. Oncologist 2012;17:296-302. https://doi.org/10.1634/theoncologist.2011-0357.

[89] Wilhelm M, Mueller L, Miller MC, Link K, Holdenrieder S, Bertsch T, et al. Prospective, Multicenter Study of 5-Fluorouracil Therapeutic Drug Monitoring in Metastatic Colorectal Cancer Treated in Routine Clinical Practice. Clin Colorectal Cancer 2016;15:381-8. https://doi.org/10.1016/j.clcc.2016.04.001.

[90] Macaire P, Morawska K, Vincent J, Quipourt V, Marilier S, Ghiringhelli F, et al.
al. Food-effect study on uracil and dihydrouracil plasma levels as marker for dihydropyrimidine dehydrogenase activity in human volunteers. Br J Clin Pharmacol 2018;84:2761-9. https://doi.org/10.1111/bcp.13719.

[77] Jiang H, Lu J, Ji J. Circadian rhythm of dihydrouracil/uracil ratios in biological fluids: a potential biomarker for dihydropyrimidine dehydrogenase levels. Br J Pharmacol 2004;141:616-23. https://doi.org/10.1038/sj.bjp.0705651.

[78] Gaible C, Narjoz C, Loriot M-A, Roueff S, Pallet N. Pretherapeutic screening for Dihydropyrimidine deshydrogenase deficiency in measuring uracilemia in dialysis patients leads to a high rate of falsely positive results. Cancer Chemother Pharmacol 2021;88:1049-53. https://doi.org/10.1007/s00280-021-04354-7.

[79] Coudoré F, Roche D, Lefeuvre S, Faussot D, Billaud EM, Loriot M-A, et al. Validation of an ultra-high performance liquid chromatography tandem mass spectrometric method for quantifying uracil and 5,6-dihydroouracil in human plasma. J Chromatogr Sci 2012;50:877-84. https://doi.org/10.1093/chromsci/bms085.

[80] Thomas F, Maillard M, Launay M, Tron C, Etienne-Grimaldi M-C, Gautier-Veyret E, et al. Artificial increase of uracilemia during fluoropyrimidine treatment can lead to DPD deficiency misinterpretation. Ann Oncol 2021;32:810-1. https://doi.org/10.1016/j.annonc.2021.02.020.

[81] Pallet N, Hamdane S, Garinet S, Blons H, Zaanan A, Paillaud E, et al. A comprehensive population-based study comparing the phenotype and genotype in a pretherapeutic screen of dihydropyrimidine dehydrogenase deficiency. Br J Cancer 2020;123:811-8. https://doi.org/10.1038/s41416-020-0962-z.

[82] Henricks LM, Lunenburg CATC, de Man FM, Meulendijks D, Frederix GWJ, Kienhuis E, et al. A cost analysis of upfront DPYD genotype-guided dose individualisation in fluoropyrimidine-based anticancer therapy. Eur J Cancer 2019;107:60-7. https://doi.org/10.1016/j.ejca.2018.11.010.

[83] Deenen MJ, Meulendijks D, Cats A, Sechterberger MK, Severens JL, Boot H, et al. Upfront Genotyping of DPYD*2A to Individualize Fluoropyrimidine Therapy: A Safety and Cost Analysis. J Clin Oncol 2016;34:227-34. https://doi.org/10.1200/JCO.2015.63.1325.

[84] Tsiachristas A, Vallance G, Koleva-Kolarova R, Taylor H, Solomons L, Rizzo G, et al. Can upfront DPYD extended variant testing reduce toxicity and associated hospital costs of fluoropyrimidine chemotherapy? A propensity score matched analysis of 2022 UK patients. BMC Cancer 2022;22:458. https://doi.org/10.1186/s12885-022-09576-3.

[85] Saam J, Critchfield GC, Hamilton SA, Roa BB, Wenstrup RJ, Kaldate RR. Body surface area-based dosing of 5-fluorouracil results in extensive interindividual variability in 5-fluorouracil exposure in colorectal cancer patients on FOLFOX regimens. Clin Colorectal Cancer 2011;10:203-6. https://doi.org/10.1016/j.clcc.2011.03.015.

[86] Fety R, Rolland F, Barberi-Heyob M, Hardouin A, Campion L, Conroy T, et al. Clinical impact of pharmacokinetically-guided dose adaptation of 5-fluorouracil: results from a multicentric randomized trial in patients with locally advanced head and neck carcinomas. Clin Cancer Res 1998;4:2039-45.

[87] Gamelin E, Delva R, Jacob J, Merrouche Y, Raoul JL, Pezet D, et al. Individual fluorouracil dose adjustment based on pharmacokinetic follow-up compared with conventional dosage: results of a multicenter randomized trial of patients with metastatic colorectal cancer. J Clin Oncol 2008;26:2099-105. https://doi.org/10.1200/JCO.2007.13.3934.

[88] Kaldate RR, Haregewoin A, Grier CE, Hamilton SA, McLeod HL. Modeling the 5-fluorouracil area under the curve versus dose relationship to develop a pharmacokinetic dosing algorithm for colorectal cancer patients receiving FOLFOX6. Oncologist 2012;17:296-302. https://doi.org/10.1634/theoncologist.2011-0357.

[89] Wilhelm M, Mueller L, Miller MC, Link K, Holdenrieder S, Bertsch T, et al. Prospective, Multicenter Study of 5-Fluorouracil Therapeutic Drug Monitoring in Metastatic Colorectal Cancer Treated in Routine Clinical Practice. Clin Colorectal Cancer 2016;15:381-8. https://doi.org/10.1016/j.clcc.2016.04.001.

[90] Macaire P, Morawska K, Vincent J, Quipourt V, Marilier S, Ghiringhelli F, et al.
Therapeutic drug monitoring as a tool to optimize 5-FU-based chemotherapy in gastrointestinal cancer patients older than 75 years. Eur J Cancer 2019;111:116-25.
https://doi.org/10.1016/j.ejca.2019.01.102.

[91] Bocci G, Danesi R, Paolo AD, Innocenti F, Allegrini G, Falcone A, et al. Comparative Pharmacokinetic Analysis of 5-Fluorouracil and Its Major Metabolite 5-Fluoro-5,6-dihydroouracil after Conventional and Reduced Test Dose in Cancer Patients. Clin Cancer Res 2000;6:3032-7.

[92] Bocci G, Barbara C, Vannozzi F, Di Paolo A, Melosi A, Barsanti G, et al. A pharmacokinetic-based test to prevent severe 5-fluorouracil toxicity. Clin Pharmacol Ther 2006;80:384-95. https://doi.org/10.1016/j.clpt.2006.06.007.

[93] Goldstein DA, Chen Q, Ayer T, Howard DH, Lipscomb J, Harvey RD, et al. Cost effectiveness analysis of pharmacokinetically-guided 5-fluorouracil in FOLFOX chemotherapy for metastatic colorectal cancer. Clin Colorectal Cancer 2014;13:219-25.
https://doi.org/10.1016/j.clcc.2014.09.007.

[94] Loriot M-A, Ciccolini J, Thomas F, Barin-Le-Guellec C, Royer B, Milano G, et al. [Dihydropyrimidine déhydrogenase (DPD) deficiency screening and securing of fluoropyrimidine-based chemotherapies: Update and recommendations of the French GPCO-Unicancer and RNPGx networks]. Bull Cancer 2018;105:397-407.
https://doi.org/10.1016/j.bulcan.2018.02.001.

[95] Cervantes A, Adam R, Roselló S, Arnold D, Normanno N, Taïeb J, et al. Metastatic colorectal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up†. Annals of Oncology 2022;0. https://doi.org/10.1016/j.annonc.2022.10.003.

[96] Argilés G, Tabernero J, Labianca R, Hochhauser D, Salazar R, Iveson T, et al. Localised colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Annals of Oncology 2020;31:1291-305.
https://doi.org/10.1016/j.annonc.2020.06.022.

[97] Des recommandations pour prévenir certaines toxicités sévères des chimiothérapies par fluoropyrimidines. Haute Autorité de Santé n.d. https://www.has-sante.fr/jcms/c_2892234/fr/des-recommandations-pour-prevenir-certaines-toxicites-severes-des-chimiotherapies-par-fluoropyrimidines (accessed August 31, 2021).

[98] EMA. EMA recommendations on DPD testing prior to treatment with fluorouracil, capecitabine, tegafur and flucytosine. European Medicines Agency 2020.
https://www.ema.europa.eu/en/news/ema-recommendations-dpd-testing-prior-treatment-fluorouracil-capecitabine-tegafur-flucytosine (accessed November 28, 2022).

[99] Reference ID: 4394864 - Accessdata.fda.gov

[100] Launay M, Ciccolini J, Fournel C, Blanquicett C, Dupuis C, Fakhry N, et al. Upfront DPD deficiency detection to secure 5-fu administration: part 2- application to head-and-neck cancer patients. Clin Cancer Drugs 2017;4:122-8.
https://doi.org/10.2174/2212697x04666170817123425.

[101] Henricks LM, van Merendonk LN, Meulendijks D, Deenen MJ, Beijnen JH, de Boer A, et al. Effectiveness and safety of reduced-dose fluoropyrimidine therapy in patients carrying the DPYD*2A variant: A matched pair analysis. Int J Cancer 2019;144:2347-54.
https://doi.org/10.1002/ijc.32022.

[102] Yang CG, Ciccolini J, Blesius A, Dahan L, Bagarry-Liegey D, Brunet C, et al. DPD-based adaptive dosing of 5-FU in patients with head and neck cancer: impact on treatment efficacy and toxicity. Cancer Chemother Pharmacol 2011;67:49-56.
https://doi.org/10.1007/s00280-010-1282-4.

[103] Henricks LM, Lunenburg CATC, de Man FM, Meulendijks D, Frederix GWJ, Kienhuis E, et al. DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis. Lancet Oncol 2018;19:1459-67.
https://doi.org/10.1016/S1470-2045(18)30686-7.

[104] Biagi JJ, Raphael MJ, Mackillop WJ, Kong W, King WD, Booth CM. Association between time to initiation of adjuvant chemotherapy and survival in colorectal cancer: a systematic review and meta-analysis. JAMA 2011;305:2335-42.
https://doi.org/10.1001/jama.2011.749.
Therapeutic drug monitoring as a tool to optimize 5-FU-based chemotherapy in gastrointestinal cancer patients older than 75 years. Eur J Cancer 2019;111:116-25.
https://doi.org/10.1016/j.ejca.2019.01.102.

[91] Bocci G, Danesi R, Paolo AD, Innocenti F, Allegrini G, Falcone A, et al. Comparative Pharmacokinetic Analysis of 5-Fluorouracil and Its Major Metabolite 5-Fluoro-5,6-dihydroouracil after Conventional and Reduced Test Dose in Cancer Patients. Clin Cancer Res 2000;6:3032-7.

[92] Bocci G, Barbara C, Vannozzi F, Di Paolo A, Melosi A, Barsanti G, et al. A pharmacokinetic-based test to prevent severe 5-fluorouracil toxicity. Clin Pharmacol Ther 2006;80:384-95. https://doi.org/10.1016/j.clpt.2006.06.007.

[93] Goldstein DA, Chen Q, Ayer T, Howard DH, Lipscomb J, Harvey RD, et al. Cost effectiveness analysis of pharmacokinetically-guided 5-fluorouracil in FOLFOX chemotherapy for metastatic colorectal cancer. Clin Colorectal Cancer 2014;13:219-25.
https://doi.org/10.1016/j.clcc.2014.09.007.

[94] Loriot M-A, Ciccolini J, Thomas F, Barin-Le-Guellec C, Royer B, Milano G, et al. [Dihydropyrimidine déhydrogenase (DPD) deficiency screening and securing of fluoropyrimidine-based chemotherapies: Update and recommendations of the French GPCO-Unicancer and RNPGx networks]. Bull Cancer 2018;105:397-407.
https://doi.org/10.1016/j.bulcan.2018.02.001.

[95] Cervantes A, Adam R, Roselló S, Arnold D, Normanno N, Taïeb J, et al. Metastatic colorectal cancer: ESMO Clinical Practice Guideline for diagnosis, treatment and follow-up†. Annals of Oncology 2022;0. https://doi.org/10.1016/j.annonc.2022.10.003.

[96] Argilés G, Tabernero J, Labianca R, Hochhauser D, Salazar R, Iveson T, et al. Localised colon cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up†. Annals of Oncology 2020;31:1291-305.
https://doi.org/10.1016/j.annonc.2020.06.022.

[97] Des recommandations pour prévenir certaines toxicités sévères des chimiothérapies par fluoropyrimidines. Haute Autorité de Santé n.d. https://www.has-sante.fr/jcms/c_2892234/fr/des-recommandations-pour-prevenir-certaines-toxicites-severes-des-chimiotherapies-par-fluoropyrimidines (accessed August 31, 2021).

[98] EMA. EMA recommendations on DPD testing prior to treatment with fluorouracil, capecitabine, tegafur and flucytosine. European Medicines Agency 2020.
https://www.ema.europa.eu/en/news/ema-recommendations-dpd-testing-prior-treatment-fluorouracil-capecitabine-tegafur-flucytosine (accessed November 28, 2022).

[99] Reference ID: 4394864 - Accessdata.fda.gov

[100] Launay M, Ciccolini J, Fournel C, Blanquicett C, Dupuis C, Fakhry N, et al. Upfront DPD deficiency detection to secure 5-fu administration: part 2- application to head-and-neck cancer patients. Clin Cancer Drugs 2017;4:122-8.
https://doi.org/10.2174/2212697x04666170817123425.

[101] Henricks LM, van Merendonk LN, Meulendijks D, Deenen MJ, Beijnen JH, de Boer A, et al. Effectiveness and safety of reduced-dose fluoropyrimidine therapy in patients carrying the DPYD*2A variant: A matched pair analysis. Int J Cancer 2019;144:2347-54.
https://doi.org/10.1002/ijc.32022.

[102] Yang CG, Ciccolini J, Blesius A, Dahan L, Bagarry-Liegey D, Brunet C, et al. DPD-based adaptive dosing of 5-FU in patients with head and neck cancer: impact on treatment efficacy and toxicity. Cancer Chemother Pharmacol 2011;67:49-56.
https://doi.org/10.1007/s00280-010-1282-4.

[103] Henricks LM, Lunenburg CATC, de Man FM, Meulendijks D, Frederix GWJ, Kienhuis E, et al. DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis. Lancet Oncol 2018;19:1459-67.
https://doi.org/10.1016/S1470-2045(18)30686-7.

[104] Biagi JJ, Raphael MJ, Mackillop WJ, Kong W, King WD, Booth CM. Association between time to initiation of adjuvant chemotherapy and survival in colorectal cancer: a systematic review and meta-analysis. JAMA 2011;305:2335-42.
https://doi.org/10.1001/jama.2011.749.
[105] Des Guetz G, Nicolas P, Perret G-Y, Morere J-F, Uzzan B. Does delaying adjuvant chemotherapy after curative surgery for colorectal cancer impair survival? A meta-analysis. Eur J Cancer 2010;46:1049–55. https://doi.org/10.1016/j.ejca.2010.01.020.

[106] Dolat M, Macaire P, Goirand F, Vincent J, Hennequin A, Palmier R, et al. Association of 5-FU Therapeutic Drug Monitoring to DPD Phenotype Assessment May Reduce 5-FU Under-Exposure. Pharmaceuticals (Basel) 2020;13:416. https://doi.org/10.3390/ph13110416.

[107] Tron C, Lemaitre F, Boisteau E, Sourd SL, Lièvre A. When helping the minority of patients may hurt the majority: The case for DPD phenotyping and 5-fluorouracil therapeutic drug monitoring. Digestive and Liver Disease 2021;53:258–60. https://doi.org/10.1016/j.dld.2020.11.004.

[108] Chamorey E, Francois E, Etienne M-C, Ferrero J-M, Peyrade F, Barranger E, et al. DPD status and fluoropyrimidines-based treatment: high activity matters too. BMC Cancer 2020;20:436. https://doi.org/10.1186/s12885-020-06907-0.

[109] Boisdron-Celle M, Capitain O, Faroux R, Borg C, Metges JP, Galais MP, et al. Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach. Semin Oncol 2017;44:13–23. https://doi.org/10.1053/j.seminoncol.2017.02.008.

[110] Meulendijks D, Cats A, Beijnen JH, Schellens JHM. Improving safety of fluoropyrimidine chemotherapy by individualizing treatment based on dihydropyrimidine dehydrogenase activity - Ready for clinical practice? Cancer Treat Rev 2016;50:23–34. https://doi.org/10.1016/j.ctrv.2016.08.002.
[105] Des Guetz G, Nicolas P, Perret G-Y, Morere J-F, Uzzan B. Does delaying adjuvant chemotherapy after curative surgery for colorectal cancer impair survival? A meta-analysis. Eur J Cancer 2010;46:1049–55. https://doi.org/10.1016/j.ejca.2010.01.020.

[106] Dolat M, Macaire P, Goirand F, Vincent J, Hennequin A, Palmier R, et al. Association of 5-FU Therapeutic Drug Monitoring to DPD Phenotype Assessment May Reduce 5-FU Under-Exposure. Pharmaceuticals (Basel) 2020;13:416. https://doi.org/10.3390/ph13110416.

[107] Tron C, Lemaitre F, Boisteau E, Sourd SL, Lièvre A. When helping the minority of patients may hurt the majority: The case for DPD phenotyping and 5-fluorouracil therapeutic drug monitoring. Digestive and Liver Disease 2021;53:258–60. https://doi.org/10.1016/j.dld.2020.11.004.

[108] Chamorey E, Francois E, Etienne M-C, Ferrero J-M, Peyrade F, Barranger E, et al. DPD status and fluoropyrimidines-based treatment: high activity matters too. BMC Cancer 2020;20:436. https://doi.org/10.1186/s12885-020-06907-0.

[109] Boisdron-Celle M, Capitain O, Faroux R, Borg C, Metges JP, Galais MP, et al. Prevention of 5-fluorouracil-induced early severe toxicity by pre-therapeutic dihydropyrimidine dehydrogenase deficiency screening: Assessment of a multiparametric approach. Semin Oncol 2017;44:13–23. https://doi.org/10.1053/j.seminoncol.2017.02.008.

[110] Meulendijks D, Cats A, Beijnen JH, Schellens JHM. Improving safety of fluoropyrimidine chemotherapy by individualizing treatment based on dihydropyrimidine dehydrogenase activity - Ready for clinical practice? Cancer Treat Rev 2016;50:23–34. https://doi.org/10.1016/j.ctrv.2016.08.002.
**Conflict of interest statement:**

The authors declare no conflict of interest

**Funding sources**

None

**Figure legends**

**Figure 1. 5FU metabolism and mechanism of action**

Only a small fraction of 5FU (1–5%) is metabolized to the cytotoxic metabolites fluorodeoxyuridine-monophosphate (FdUMP), fluorodeoxyuridine-triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). The main mechanism of action of FP is thymidylate synthase (TS) inhibition, preventing dTMP formation. Alternative pharmacodynamic pathways involve incorporation of 5FU metabolites into DNA and RNA, resulting in cytotoxicity as a consequence of RNA and DNA damage. UP, uridine phosphorylase; OPRT: Orotate phosphoribosyl-transferase; TP, thymidylate phosphorylase; UCK, uridine-cytidine kinase; RR, ribonucleotide reductase; TK, thymidylate kinase; FUrd: fluorouridine; FdUrd: fluorodesoxyuridine; TS: thymidylate synthase; DPD: dihydropyrimidine dehydrogenase.

**Figure 2. Current decision-making algorithms for DPD deficiency screening and subsequent FP dose adaptation at first cycle**

Phenotyping approach corresponds to HAS/INCa French recommendations. Measurement of plasma [UH2]/[U] ratio is not mandatory but may help to classify patients as deficient/not deficient. Genotyping approach corresponds to the most recent CPIC and DPWG recommendations. The gene activity score is obtained by adding the two lowest activity score variants.

* For patients with no alternative therapeutic option and a gene score at 0.5, additional DPD phenotyping is recommended, along with TDM to refine FP dose adjustment.

* For patients with a gene activity score at 1 resulting from homozygosity for HapB3 or c.2846A>T, or the presence of these 2 variants, it is advised to perform DPD phenotyping to refine FP dose adjustment. Literature data suggest that patients homozygous for c.2846A>T should receive a starting dose considerably lower than 50%.

* Based on conflicting data about HapB3 impact on DPD activity, the starting dose to apply for HapB3 heterozygous patients is unclear.

§ HapB3: c.1236G>A in complete linkage disequilibrium with c.1129-5923C>G.
**Conflict of interest statement:**

The authors declare no conflict of interest

**Funding sources**

None

**Figure legends**

**Figure 1. 5FU metabolism and mechanism of action**

Only a small fraction of 5FU (1–5%) is metabolized to the cytotoxic metabolites fluorodeoxyuridine-monophosphate (FdUMP), fluorodeoxyuridine-triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). The main mechanism of action of FP is thymidylate synthase (TS) inhibition, preventing dTMP formation. Alternative pharmacodynamic pathways involve incorporation of 5FU metabolites into DNA and RNA, resulting in cytotoxicity as a consequence of RNA and DNA damage. UP, uridine phosphorylase; OPRT: Orotate phosphoribosyl-transferase; TP, thymidylate phosphorylase; UCK, uridine-cytidine kinase; RR, ribonucleotide reductase; TK, thymidylate kinase; FUrd: fluorouridine; FdUrd: fluorodesoxyuridine; TS: thymidylate synthase; DPD: dihydropyrimidine dehydrogenase.

**Figure 2. Current decision-making algorithms for DPD deficiency screening and subsequent FP dose adaptation at first cycle**

Phenotyping approach corresponds to HAS/INCa French recommendations. Measurement of plasma [UH2]/[U] ratio is not mandatory but may help to classify patients as deficient/not deficient. Genotyping approach corresponds to the most recent CPIC and DPWG recommendations. The gene activity score is obtained by adding the two lowest activity score variants.

* For patients with no alternative therapeutic option and a gene score at 0.5, additional DPD phenotyping is recommended, along with TDM to refine FP dose adjustment.

* For patients with a gene activity score at 1 resulting from homozygosity for HapB3 or c.2846A>T, or the presence of these 2 variants, it is advised to perform DPD phenotyping to refine FP dose adjustment. Literature data suggest that patients homozygous for c.2846A>T should receive a starting dose considerably lower than 50%.

* Based on conflicting data about HapB3 impact on DPD activity, the starting dose to apply for HapB3 heterozygous patients is unclear.

§ HapB3: c.1236G>A in complete linkage disequilibrium with c.1129-5923C>G.
<table><caption>Table 1. Current consensual deleterious *DPYD* variants and prevalence in populations of Caucasian origin</caption><thead><tr><th rowspan="2">DPYD variants</th><th rowspan="2">Minor allelic frequency</th><th colspan="2">Proportion of carriers</th><th rowspan="2">Allele activity score*</th></tr><tr><th>heterozygous</th><th>homozygous</th></tr></thead><tbody><tr><td>DPYD*2A<br>(IVS14+1G>A,<br>c.1905G>A)</td><td>0.8%<br>[10,83,109,110]</td><td>1.5%</td><td>0.01%</td><td>0</td></tr><tr><td>DPYD*13<br>(c.1679T>G; p.I560S)</td><td>0.1%<br>[10,56,61,109,110]</td><td>0.2%</td><td>0.0001%</td><td>0</td></tr><tr><td>c.2846A>T<br>(p.D949V)</td><td>0.6%<br>[9,10,56,109,110]</td><td>1%</td><td>0.004%</td><td>0.5</td></tr><tr><td>HapB3<br>(c.1129-5923C>G,<br>c.1236G>A)</td><td>2.4%<br>[10]</td><td>4.6%</td><td>0.06%</td><td>0.5</td></tr></tbody></table>

<footnote>*According to Henricks et al [Pharmacogenomics 2015]</footnote>
<table><caption>Table 1. Current consensual deleterious *DPYD* variants and prevalence in populations of Caucasian origin</caption><thead><tr><th rowspan="2">DPYD variants</th><th rowspan="2">Minor allelic frequency</th><th colspan="2">Proportion of carriers</th><th rowspan="2">Allele activity score*</th></tr><tr><th>heterozygous</th><th>homozygous</th></tr></thead><tbody><tr><td>DPYD*2A<br>(IVS14+1G>A,<br>c.1905G>A)</td><td>0.8%<br>[10,83,109,110]</td><td>1.5%</td><td>0.01%</td><td>0</td></tr><tr><td>DPYD*13<br>(c.1679T>G; p.I560S)</td><td>0.1%<br>[10,56,61,109,110]</td><td>0.2%</td><td>0.0001%</td><td>0</td></tr><tr><td>c.2846A>T<br>(p.D949V)</td><td>0.6%<br>[9,10,56,109,110]</td><td>1%</td><td>0.004%</td><td>0.5</td></tr><tr><td>HapB3<br>(c.1129-5923C>G,<br>c.1236G>A)</td><td>2.4%<br>[10]</td><td>4.6%</td><td>0.06%</td><td>0.5</td></tr></tbody></table>

<footnote>*According to Henricks et al [Pharmacogenomics 2015]</footnote>
<table><caption>Table 2. Strengths and limitations of phenotyping and genotyping for DPD-deficiency screening</caption><thead><tr><th></th><th>Strengths</th><th>Limitations</th></tr></thead><tbody><tr><td>Genotyping of DPYD *2A, p.D949V, *13 and Haplotype B3</td><td>- Low cost<br>- Easy to perform and implement<br>- Unequivocal result<br>- No pre-analytical requirement<br>- Validated genotype-guided FP dosing</td><td>- No relevant for Asian and African populations<br>- Low sensibility for identifying patients at risk of toxicity<br>- Account for genetics only<br>- Uncertain enzyme activity reliability</td></tr><tr><td>Phenotyping of [U] ± [UH<sub>2</sub>]/[U]</td><td>- Apply to all populations<br>- Takes into account all parameters influencing DPD enzyme activity<br>- Higher sensitivity as compared to genotyping</td><td>- Time-consuming and highly specialized non-standardized analytical method<br>- Strict pre-analytical requirements<br>- Circadian variability<br>- Possible influence of food intake<br>- Uncertain cutoff value<br>- No validated phenotype-guided FP dosing<br>- Lack of specificity</td></tr></tbody></table>
<table><caption>Table 2. Strengths and limitations of phenotyping and genotyping for DPD-deficiency screening</caption><thead><tr><th></th><th>Strengths</th><th>Limitations</th></tr></thead><tbody><tr><td>Genotyping of DPYD *2A, p.D949V, *13 and Haplotype B3</td><td>- Low cost<br>- Easy to perform and implement<br>- Unequivocal result<br>- No pre-analytical requirement<br>- Validated genotype-guided FP dosing</td><td>- No relevant for Asian and African populations<br>- Low sensibility for identifying patients at risk of toxicity<br>- Account for genetics only<br>- Uncertain enzyme activity reliability</td></tr><tr><td>Phenotyping of [U] ± [UH<sub>2</sub>]/[U]</td><td>- Apply to all populations<br>- Takes into account all parameters influencing DPD enzyme activity<br>- Higher sensitivity as compared to genotyping</td><td>- Time-consuming and highly specialized non-standardized analytical method<br>- Strict pre-analytical requirements<br>- Circadian variability<br>- Possible influence of food intake<br>- Uncertain cutoff value<br>- No validated phenotype-guided FP dosing<br>- Lack of specificity</td></tr></tbody></table>
<figure><img src="image_1.png" /><figcaption>FIGURE 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIGURE 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>FIGURE 2</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>FIGURE 2</figcaption></figure>