1. Introduction

Fluoropyrimidines (FP), including 5-fluorouracil (5-FU) and its oral prodrug capecitabine, are cytotoxic antineoplastic agents belonging to the class of antimetabolites. They are commonly used to treat solid cancer types such as gastrointestinal, head-neck and breast cancers associated or not to other chemotherapeutics and both cytotoxic and biologic drugs [1,2]^[1][2]. The administration of the FP may cause severe, even life-threatening, adverse drug reactions (ADR), including myelosuppression, mucositis/stomatitis, diarrhoea and hand–foot syndrome (HFS). Indeed, it has been estimated that an increased risk of severe ADR (grade > 2) involves 10–30% of treated patients, although these data greatly depend on the therapeutic regimen used [1,3,4]^[1][3][4].

The rate-limiting step of FP catabolism is the conversion of fluorouracil to dihydrofluorouracil, which is catalysed by an enzyme called dihydropyrimidine dehydrogenase (DPD), encoded by a highly polymorphic gene (i.e., DPYD). Several single-nucleotide polymorphisms (SNPs) have been associated to an alteration of the DPYD sequence, and some of them may determine a partial or complete DPD deficiency, leading to FP severe toxicity ^[1].

The Clinical Pharmacogenetics Implementation Consortium (CPIC) and the Dutch Pharmacogenetics Working Group (DPWG) [5,6]^[5][6] have published guidelines for FP dosing based on the pharmacogenetic testing of four DPYD polymorphisms that are DPYD*2A (rs3918290), DPYD*13 (rs55886062), DPYD c.2846A>T (rs67376798) and c.1129-5923C>G (rs75017182 and HapB3). The latter is the most common variant, with ~4% allelic frequency; the DPYD*2A, c.2846A>T and DPYD*13 are present in ~2.0%, 1.4% and 0.1%, respectively, of Caucasian patients ^[5]. Recently, a new polymorphism, DPYD*6 (rs1801160), has been associated with both gastrointestinal and haematological FP-ADR ^[7].

Notably, other DPYD genetic variants may lead to dangerous clinical consequences, although their frequency is very low [5,6,8]^[5][6][8].

With the main aim of reducing the risk of severe FP-induced toxicity, the CPIC and DPWG have implemented a gene activity score (DPYD-AS), which ranges from 0 (complete DPD deficiency) to 2 (normal DPD activity). In patients who are homozygous for one or more of the aforementioned SNPs, the recommendation is to avoid the use of FP. However, if alternative drugs are not considered a suitable option, the FP dosage should be markedly reduced while establishing a therapeutic drug-monitoring (TDM) approach. Patients who are heterozygous should receive a 50% dose reduction at the first cycle of chemotherapy, followed by a titration dose, while monitoring the patient’s clinical conditions and possibly performing TDM [5,6]^[5][6].

However, it has been estimated that 30–50% of the patients experience severe ADR, despite not having a DPD deficit associated with such DPYD polymorphisms. In fact, there are several factors, including comorbidities, polytherapy, other variants in the DPYD and other genes, that can play an important role ^[9]. Besides DPYD polymorphisms, two SNPs in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene ^[10] and a tandem repeat in the thymidylate synthase enhancer region (TYMS-TSER) could concur in predicting FP-related toxicity ^[11]. Moreover, a SNP in glutathione S-transferase-p1 (GSTP1) has been suggested as a genetic factor able to influence the response to oxaliplatin, a drug frequently administered with FP ^[12].

Several strategies complementing the DPYD pharmacogenetics (DPYD-PGx) have been proposed to prevent FP-related severe ADR associated with DPD deficit. Among others, the measurement of the plasmatic dihydrouracil/uracil ratio (UH₂/U) and the monitoring of 5-FU clearance are considered valid approaches [13,14]^[13][14].

2. Cases Presentation

Case 1 was a Caucasian 55-year-old male former smoker with a history of hypertension. The patient had stage IV colorectal adenocarcinoma with metastases in the lymph nodes, lungs, liver and kidneys. The tumour mutational profile identified no mutations in the KRAS, NRAS or BRAF genes. The patient was treated with the combination of 5-FU, leucovorin and oxaliplatin (FOLFOX6) regimen, plus cetuximab. After three cycles of chemotherapy, the patient reported grade 1 thrombocytopenia and paraesthesia; grade 2 stomatitis, rash and leukopenia and grade 3 neutropenia and mucositis.

A post-therapeutic DPYD-PGx was performed, revealing that the patient was heterozygous for DPYD*2A. Moreover, the patient was homozygous (TT) for MTHFR-C677T, homozygous TYMS-TSER-2R/2R and homozygous (AA) for GSTP1-A313G. The plasmatic UH2/U ratio was 4.52. Based on these results and the reported toxicity, both the 5-FU and cetuximab doses were reduced. Specifically, the total dosage of 5-FU was reduced to 50%, according to the CPIC and DPWG guidelines.

At the fourth cycle of therapy, the pharmacokinetic analysis revealed a trough 5-FU plasma concentration of 950 ng/mL. The CT scan demonstrated an overall stable disease, according to the RECIST criteria v1.1. The patient was still treated with the same doses of 5-FU. At the sixth cycle of therapy, the 5-FU plasma concentration was 400 ng/mL. The following cycles (fifth to eighth) of chemotherapy were administered at the same drug doses. A new CT scan demonstrated no evidence of disease progression. The ADR were grade 1 leukopenia, neutropenia, thrombocytopenia and mucositis and grade 2 HFS. Following a further two cycles of therapy, the reported ADR were grade 1 paraesthesia, erythematous maculopapular rash and grade 2 cutaneous and mucous fissures. Lastly, following a further two treatment cycles, a new CT scan showed disease progression. The treatment was stopped, and the administration of a new chemotherapeutic regimen was planned. Sadly, the patient died before starting a second line of treatment.

Case 2 was a Caucasian 48-year-old male with no comorbidity. He had stage IV colorectal adenocarcinoma with metastases in the lymph nodes and liver. The tumour mutational profile highlighted the presence of a KRAS mutation; thus, a treatment with the FOLFOX6 regimen plus bevacizumab was planned. A pretherapeutic DPYD-PGx was requested, and the patient was identified as DPYD*2A heterozygous. In addition, he was wild type for MTHFR-C677T and MTHFR-A1298C, heterozygous TYMS TSER-2R/3R and homozygous (GG) for GSTP1-A313G. The plasmatic UH2/U ratio was 3.22. Based on these results, a 50% dose reduction of 5-FU was planned for the first cycle of FOLFOX administration, according to the CPIC and DPWG guidelines. After the first cycle of treatment, the plasmatic 5-FU clearance was 474 ng/mL. Following three cycles of therapy, a stable disease was found, according to the RECIST criteria v1.1, and no adverse events were reported. The patient was still treated with the same doses of chemotherapeutic agents for an additional seven cycles of therapy. Grade 1 paraesthesia and mucositis and grade 2 HFS but no severe ADR were reported, and the CT scan demonstrated a stable disease. Afterward, the patient was treated up to the twelfth cycle with a FOLFOX regimen plus bevacizumab, still obtaining, at revaluation, a stable disease. Then, he was a candidate for a maintenance therapy with capecitabine plus bevacizumab. Following 16 cycles of this therapy, the patient reported grade 1 paraesthesia and mucositis, and no severe ADR were recorded.

Case 3 was a Caucasian 60-year-old male former smoker with no comorbidities. He had stage IV rectal adenocarcinoma with liver metastases. The tumour mutational profile did not identify mutations in either KRAS, NRAS or BRAF. Based on the tumour profile and stage, the patient was a candidate for a FOLFOX regimen plus cetuximab. A pretherapeutic DPYD-PGx was performed, and the patient was found heterozygous for DPYD c2846A>T SNP. Therefore, according to the CPIC and DPWG guidelines, he started chemotherapy with a 50% dose reduction of 5-FU. Moreover, he was homozygous (TT) for MTHFR-C677T, heterozygous TYMS TSER-2R/3R and heterozygous for  GSTP1-A313G. The plasmatic UH2/U ratio was 1.77.

A grade 2 diffuse maculopapular rash was reported, and, based on such an ADR, the dose of cetuximab was also reduced to 50% for the second cycle of therapy. The plasmatic 5-FU clearance was 811 ng/mL—still high, notwithstanding the 5-FU dose reduction. The patient reported no improvement of the skin rash and grade 2 diarrhoea. At the third cycle of therapy with the same drugs doses, the 5-FU plasma level was 1093 ng/mL. Grade 1 nausea and grade 3 diarrhoea were reported. Based on these results, the 5-FU dose was further reduced by an additional 10% at the fourth cycle of therapy. However, the 5-FU plasma concentration was still high (1048 ng/mL), and grade 4 diarrhoea was reported. Hence, it was decided not to administer 5-FU in a continuous infusion, leaving the administration of 5-FU in bolus. Nevertheless, the 5-FU plasma concentration was still high (i.e., 934 ng/mL), and grade 3 diarrhoea was reported.

A CT scan showed a partial response according to the RECIST criteria with a reduction of hepatic lesions. It was decided to carry out a further cycle with oxaliplatin plus cetuximab.

After this cycle, the hepatic lesions were resected. After one month from surgery, a CT scan demonstrated the development of a new hepatic lesion. The patient was a candidate to start a new treatment with 5-FU plus irinotecan as a modified 5-FU, leucovorin and irinotecan (FOLFIRI) regimen, since 5-FU was administered with a 50% dose reduction and without continuous infusion. The patient performed six cycles of FOLFIRI plus bevacizumab. Grade 3 diarrhoea was reported. As a consequence, the 5-FU administration was stopped, and only irinotecan and bevacizumab were further administered. After four cycles of this treatment, the CT scan demonstrated a progression of the disease. The patient died after 11.2 months from starting treatment with irinotecan plus bevacizumab.

A pretherapeutic DPYD-PGx was performed in two out of three cases, while one patient (case 1) had already started chemotherapy before requesting DPYD-PGx. Importantly, the patients were monitored during all treatment cycles.

Besides these three clinical cases, the history of other eight patients is briefly reported below, and their main characteristics are listed in Table 2.

All subjects were monitored for at least four treatment cycles. In two out of eight subjects, the DPYD-PGx was required after the occurrence of severe toxicity (post-therapeutic DPYD-PGx), while in six out of eight, pharmacogenetic testing was performed before the treatment started (pretherapeutic DPYD-PGx). All patients were identified as carriers of DPYD variants—precisely, four out of eight were DPYD*2A heterozygous, and four out of eight were DPYD c.2846 heterozygous.

The two patients for whom the DPYD-PGx was performed after 5-FU administration experienced grade 3 ADR with a different timing, and both were then revealed as DPYD-variant carriers. More in detail, one patient with stage III gastric cancer, treated with FOLFOX, suffered from grade 3 vomit after the second cycle; he was then identified as DPYD*2A heterozygous and continued to be treated only with oxaliplatin. Moreover, the patient was homozygous (TT) for MTHFR-C677T, homozygous TYMS TSER-3R/3R and homozygous (AA) for GSTP1-A313G.

The other one with stage IV colon cancer, treated with FOLFIRI plus bevacizumab, showed grade 3 vomit after the eighth cycle of chemotherapy. The patient was identified as DPYD c.2846 heterozygous, and the 5-FU dosage was halved. With regards to the other SNPs, the patient was homozygous (TT) for MTHFR-C677T, heterozygous TYMS TSER-2R/3R and wild type for UGT1A1*28 SNP. The latter polymorphism is routinely analysed in patients treated with irinotecan.

Conversely, in the other patients, a pretherapeutic DPYD-PGx was performed; thus they were treated with a starting halved dose of 5-FU, and no severe ADR were reported.

3. Systematic Review

A systematic review was performed to analyse the studies investigating the variability of responses to FP-based chemotherapy by DPYD genotyping combined with phenotyping methods and/or clinical monitoring.

Of the potential 112 articles assessed for eligibility, after considering the inclusion and exclusion criteria, 22 studies were included in the analysis [15-36]. Among the studies with a DPYD-PGx/clinical monitoring combination, five out of eleven studies confirmed the importance of DPYD variants in predicting FP-related toxicity, although a too-short clinical monitoring was performed (only two treatment cycles) [16,18,24,26,34].

Lee et al., by testing 2886 patients, reported a significant association between DPYD*2A and DPYD c.2846A>T and grade 3 FP ADR [21]. Similarly, Cremolini et al., in a large cohort of colon cancer patients who were treated with FOLFIRI or FOLFOXIRI plus bevacizumab, demonstrated that DPYD*2A and DPYD c.2846A>T predicted FP-associated clinically relevant ADR [31].

A DPYD-PGx/phenotyping combined approach was performed in three studies that underlined the importance of complementing the DPYD-PGx with a phenotyping analysis. Gentile et al., by measuring the 5-FUDR (degradation rate) in peripheral blood mononuclear cells (PBMCs) utilising HPLC with MS/MS, found a significant correlation between several polymorphisms, including DPYD*2A and DPYD c.2846A>T, and this phenotyping marker [22].

Pallet et al. evaluated an approach based on the combination of the plasmatic UH2/U ratio and uracil concentration with genotyping of the four recommended DPYD SNPs. The main finding of this study was that complementing the DPYD-PGx with the plasmatic UH2/U ratio increased the possibility to identify patients at higher risks of severe FP-related toxicity [36]. Among the studies that performed a DPYD-PGx/phenotyping approach with clinical monitoring, four out of eight studies reported information about FP-related ADR until a maximum of three treatment cycles.

Galarza et al. found that salivary and plasmatic UH2 concentrations were inversely correlated with the ADR grade. However, given the low number of patients enrolled in the study, no DPYD variant allele carriers were identified [25].

Kuilenburg et al. measured the UH2/U ratio in PBMCs as an indirect assessment of DPYD activity. They demonstrated that patients with a low DPD activity experienced a more rapid onset of toxicity as compared to those with normal enzymatic activity. Moreover, grade 4 neutropenia occurred in a substantial percentage (55%) of the patients with a decreased DPD activity as compared to that (13%) of subjects with a normal DPD activity. Notably, eleven out of fourteen patients suffering from severe ADR with a decreased enzymatic activity were identified as carriers of DPYD polymorphisms. In particular, six, four and one out of eleven patients carried DPYD*2A, DPYD*9A and DPYD*6 in homozygosis, respectively [20]. Kristensen et al. also showed a significant correlation between the plasmatic UH2/U ratio and the presence of DPYD*2A [17].

Finally, in a prospective study, Henricks et al. analysed all the four recommended DPYD SNPs and performed two phenotyping tests by measuring the UH2/U ratio in PBMCs and plasmatic 5-FU pharmacokinetics (PK) by UHPLC-MS/MS. The patients carrying DPYD c.1236G>A and DPYD c.2846A>T were more likely to manifest FP-related severe toxicity as compared to wild-type subjects.

In addition, the mean DPD enzyme activity was significantly lower in patients bearing these two genetic variants, as well as DPYD*2A, as compared to other patients. Only one patient carrying DPYD*13 showed a 60% DPD activity reduction. This patient was treated with a reduced 5-FU dosage for three treatment cycles, and no severe ADR occurred. Based on these results, the authors confirmed that a dose reduction of 50% in DPYD*2A and DPYD*13 carriers is appropriate, as issued in the PGx guidelines [30].

4. Conclusions

Nowadays, the regulatory agencies recommend carrying out the DPYD-PGx, including four DPYD polymorphisms (i.e., rs3918290, rs55886062, rs67376798 and rs75017182, HapB3) in patients who need to be treated with FP.

Despite that these DPYD variants are strongly associated with treatment toxicity, other genetic and nongenetic factors concur to determine the variable response to FP-based chemotherapy.

A pretherapeutic DPYD-PGx offers the possibility to avoid early ADR. Nonetheless, severe and even fatal FP-related toxicity may happen anytime during the therapy also in subjects having no DPD deficit attributable to the four recommended DPYD SNPs.

On the other hand, measuring the plasmatic 5-FU clearance—currently, the best method to perform TDM—does not permit to diagnose a possible DPD deficit prior to starting the treatment.

Therefore, because both genetic and phenotypic tests show advantages and disadvantages, a combined genotyping/phenotyping approach, together with careful and continuous clinical monitoring, is the best diagnostic method to optimise the therapy with FP.

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