An 81 year-old woman was admitted to our oncology unit (Military Training Hospital Begin, Unit of Oncology, Paris, France) for the management of grade 3 diarrhea due to fluoropyrimidine-related toxicity. She suffered from an invasive ductal carcinoma of the left breast (Elston-Ellis grade 3, RH+ HER2-) diagnosed in 2007. Medical management was initially based on surgery, radiation therapy and chemotherapy (adriamycine and cyclophosphamide). Hormonal therapy (anastrozole then exemestane) was performed during the following 5 years (2008-2012), and then she entered a regular clinical and radiological follow-up program. As bone and hepatic metastases were diagnosed in 2016 and 2017, respectively, hormonal therapy (exemestane) and chemotherapy (paclitaxel) were reintroduced. In May 2018, given evidence of CNS progression with multiple cerebral metastases, capecitabine (1,500 mg twice a day) was administered. The patient presented signs of major toxicity requiring urgent hospitalization in our department 20 days after capecitabine treatment, and the main symptoms included diarrhea (grade 3) and asthenia (grade 3). She was admitted to our department for close monitoring, with intensive fluid and nutritional support.
Biological investigations performed at admission revealed hematological toxicity with grade 4 neutropenia (absolute neutrophil count: 0.31 x 109/L; reference interval, 1.5-4 x 109/L) and grade 4 thrombocytopenia (platelet count: 35 x 109/L; reference interval, 150-300 x 109/L). The consequences of the severe diarrhea resulted in low blood levels of potassium (2.8 mmol/L; reference interval, 3.5-4.5 mmol/L), phosphate (0.4 mmol/L: reference interval, 0.81-1.45 mmol/L) and magnesium (0.63 mmol/L; reference interval, 0.7-1.05 mmol/L). Moreover, prothrombin time was increased (prothrombin ratio: 41 %; reference interval, >70 %) with a mild elevation of liver transaminase levels (ALT: 60 UI/L; reference interval, <33 UI/L and AST: 106; reference interval, < 32 UI/L) and a marked hypoalbuminemia (albumin level: 22.3 g/L, reference interval, 35-52 g/L). Stool cultures failed to detect bacterial pathogens, including C. difficile strains.
Capecitabine treatment was suspended upon the patient's admission to our service. The clinical evolution was slowly favorable: no diarrhea was observed after 72 h, and cell blood counts were normalized in 1 month.
DPD deficiency was suspected as a potential explanation for the severe toxicity following the first cycle of treatment with capecitabine. DPD phenotype assessment was performed by measurements of plasma uracil (U) and dihydrouracil (UH2) using an LC-MS/MS method. Analysis revealed a partial DPD deficiency according to the established criteria (U: 40.4 ng/mL, deficiency cut-off > 16 ng/mL and/or UH2/U ratio: 5.0, deficiency cut-off < 6). The patient signed written informed consent for genotyping and related data for scientific research. The four DPYD SNPs most commonly observed in the Indo-European population were genotyped according to the current recommendations of the Clinical Pharmacogenetics Implementation Consortium. The SNPs (c.1905+1G>A, rs3918290; c.2846A>T, rs67376798; c.1679T>G; rs55886062 and c.1236G>A, rs56038477) were genotyped using LAMP Human DPD deficiency kit (LaCAR MDx Technologies, Lieges, Belgium). None of the four variant were detected. However, an atypical profile of the melting curve relative to rs3918290 genotyping was observed (Figure 1). As the presence of an uncommon SNP was suspected, sequencing of DPYD exon 14 was performed which revealed that the patient harbored a SNP in a heterozygous state: c.1903A>G (p.Asn635Asp) (chromosome position 1:97915617, A/G). To the best of our knowledge, this is a novel variant and is not identified in any of the specific Ensembl, dbSNP, ExAC, Alfred, HGMD or LOVD databases.
In order to evaluate the impact of the proximity of this variant to the intron 14 mRNA splice donor site, we used the most popular in silico tools to predict potential functional alterations involving splicing sites. SIFT (Scale-Invariant Feature Transform, http://sift.bii.a-star.edu.sg/, accessed 28 September 2018) and MutationTaster (http://www.mutationtaster.org/, accessed 28 September 2018) predictions suggested a deleterious effect for this variant, with scores of 0.002 and 23, respectively, while PolyPhen-2 prediction (http://genetics.bwh.harvard.edu/pph2/index.shtml, accessed 28 September 2018) indicated a benign effect, with a confidence score of 0.371. The PROVEAN (Protein Variation Effect Analyzer:http://provean.jcvi.org/index.php, accessed 28 September 2018) tool indicated a neutral prediction with a score of -1.770, while the Human Splicing Finder (htt://www.umd.be/HSF3/index.html, accessed 28 September 2018) tool referred to an exonic splicing enhancer (ESE) mutation "Alteration of an exonic ESE site. Potential alteration of splicing." Furthermore, the SwissModel web tools (http://swissmodel.expasy.org/, accessed 28 September 2018) indicated that the missense variant did not affect the final structure of the protein.
Therefore, in order to characterize the potential pathogenic features of the identified variant, a peripheral blood sample stored in PAXgene Blood RNA Tube (Qiagen, Hilden, Germany) was sent to the Oncogenomic Research Center (University of Bari, Italy) for further molecular analysis. Total RNA was extracted from whole blood using the PAXgene Blood RNA Kit (PreAnalytiX GmbH, Hombrechtikon, Switzerland) and reverse transcribed into cDNA with an iScript cDNA Synthesis Kit (BioRad, Hercules, CA, USA). Using a pair of primers spanning exons 13 and 15 of DPYD, the cDNA was amplified and sequenced using a 3500 Genetic Analyzer (Applied Biosystems, 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequence analysis performed on the cDNA tract including exons 13-15 did not indicate exon 14 skipping (Figure 2).
The cDNA was then used to perform real-time quantitative PCR assays using the iTaq Universal SYBR Green Supermix (BioRad) in the Step One Plus instrument (Applied Biosystems). The mRNA levels were measured using 3 replicates per sample, with the comparative threshold cycle (Ct) method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) as endogenous controls, and with related values calculated by DeltaCt. As controls, equal amounts of RNA extracted from whole blood of 3 subjects, previously genotyped and identified as wild-type for variants in the DPYD gene, were used. DPYD mRNA levels in the patient carrying the variant were reduced by an average of 45% compared to those observed in healthy subjects expressing the wild-type DPYD gene. A potential limitation in this analysis is that it is difficult to normalize across patients in samples containing multiple cell types that have different expression levels of house keeping genes, but normalizing with either a structural gene and a metabolic gene gave similar results, suggesting the effective reduction of gene expression in the patient.
As a subsequent analytical step, 5 pairs of primers were designed to perform sequencing of a 3296 bp cDNA segment, including the 3078 bp coding sequence of the DPYD gene (Table 1).
Analysis of the results allowed the identification of the variants c.85T>C (Cys29Arg) and c.496A>G (Met166Val) recognized as the reference SNP (refSNP) Cluster Report rs1801265 and rs2297595, described with clinical significance of "Pathogenic" and "With drug-response allele" respectively (http://www.ncbi.nlm.nih.gov/snp/ accessed 28 September 2018).
The re-introduction of capecitabine at lower doses (1,000 mg twice a day) was associated with recurrence of adverse effects (grade 3 diarrhea and thrombocytopenia:platelet count: 88 x 109/L) and treatment was stopped. Following this, new therapies were adopted (Palbociclib and Fulvestrant) until the patient died 3 weeks later.