Tailored treatment and clinical management for DPYD compound heterozygous: a multidisciplinary teamwork

Abstract

Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme governing fluoropyrimidines (FPs) catabolism. Impaired or abrogated DPD enzyme activity is often caused by genetic polymorphisms in the DPYD gene that are well-validated predictors of FP-associated toxicity. Presently, four DPYD variants are included in FP genetic-based dosing guidelines. Patient safety of FP treatment has been significantly improved by pre-emptive screening for DPYD genotype variants and dose adjustments in individuals carrying heterozygous DPYD variant alleles. Nonetheless, managing carriers of multiple DPYD gene variants remains still challenging We conducted a study involving patients undergoing standard-of-care fluoropyrimidine treatment who underwent preemptive DPYD genotyping for DPYD*2A, DPYD*13, D949V, and IVS10. Additionally, patients were screened for the DPYD*6. Adverse drug reactions (ADRs) were graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Adverse events (AEs) ≥ grade 3 were considered severe. Herein, we report 4 cases of patients carrying double-site heterozygous variants of the DPYD gene (IVS10 and DPYD*6), diagnosed with either colon adenocarcinoma or breast cancer. These patients underwent pharmacogenetic-guided dose reduction of the standard by 25–50%, showing varying treatment responses. In conclusion, the management of patients carrying double-site heterozygous IVS10 and DPYD*6 variants should be performed by a multidisciplinary team due to the need for tailored treatment approaches including precision dosing, integrative deep analysis and therapeutic drug monitoring for early detection of AEs in order to maintain effectiveness and safety for each case.

Full text

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Tailored treatment and clinical management for DPYD
compound heterozygous: a multidisciplinary teamwork
Laura Simone
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Brigida Anna Maiorano
IRCCS Ospedale San Raffaele
Raffaela Barbano
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Tommaso Mazza
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Tommaso Biagini
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Gabriele Di Maggio
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Maria Grazia Rodriquenz
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Luciano Nanni
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Grazia Ciavarella
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Antonio Rinaldi
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Marzia Del Re
University of Pisa
Massimo Carella
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Giuseppe Fania
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Evaristo Maiello
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Giuseppe Miscio
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)
Tiziana Latiano
Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo (FG)

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Article
Keywords:
Posted Date: October 30th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-5310617/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full
License
Additional Declarations: No competing interests reported.

Page 3/16
Abstract
Dihydropyrimidine dehydrogenase (DPD), encoded by the DPYD gene, is the rate-limiting enzyme governing
uoropyrimidines (FPs) catabolism. Impaired or abrogated DPD enzyme activity is often caused by genetic
polymorphisms in the DPYD gene that are well-validated predictors of FP-associated toxicity. Presently, four
DPYD variants are included in FP genetic-based dosing guidelines. Patient safety of FP treatment has been
signicantly improved by pre-emptive screening for DPYD genotype variants and dose adjustments in
individuals carrying heterozygous DPYD variant alleles. Nonetheless, managing carriers of multiple DPYD gene
variants remains still challenging
We conducted a study involving patients undergoing standard-of-care uoropyrimidine treatment who
underwent preemptive DPYD genotyping for DPYD*2A, DPYD*13, D949V, and IVS10. Additionally, patients were
screened for the DPYD*6. Adverse drug reactions (ADRs) were graded according to the Common Terminology
Criteria for Adverse Events (CTCAE) version 5.0. Adverse events (AEs) ≥ grade 3 were considered severe.
Herein, we report 4 cases of patients carrying double-site heterozygous variants of the DPYD gene (IVS10 and
DPYD*6), diagnosed with either colon adenocarcinoma or breast cancer. These patients underwent
pharmacogenetic-guided dose reduction of the standard by 25–50%, showing varying treatment responses.
In conclusion, the management of patients carrying double-site heterozygous IVS10 and DPYD*6 variants
should be performed by a multidisciplinary team due to the need for tailored treatment approaches including
precision dosing, integrative deep analysis and therapeutic drug monitoring for early detection of AEs in order to
maintain effectiveness and safety for each case.
INTRODUCTION
Fluoropyrimidines (FPs) are still the backbone of chemotherapeutic two-drug or three-drug regimens for the
treatment of many solid tumours, including colorectal cancer (CRC), gastrointestinal, hepato-biliary tumours,
pancreatic and breast cancer (BCa) either in the adjuvant or in the metastatic setting 1.
The mainstays of FPs are 5-uorouracil (5-FU), commonly given intravenously (IV), and its orally active prodrug
Capecitabine, developed to mimic the continuous infusion of 5-FU while avoiding IV administration 2.
The mechanism of action of FPs entails the misincorporation of 5-FU metabolites into RNA and DNA and the
inhibition of thymidylate synthase (TYMS) by 5-uoro-2’-deoxyuridine-5’-monophosphate (FdUMP). The
catabolism and excretion are managed by dihydropyrimidine dehydrogenase enzyme (DPD) converting 5-FU into
dihydrouorouracil (DHFU) in the liver 3.
Although chemotherapeutic drugs and dosing schemes have become more tolerable, Adverse drug reactions
(ADRs) still affect 10–40% of patients and, depending on the treatment regimen, can produce hospitalization,
discontinuation of treatment, or even death in approximately 1% of cases 4,5. The most common FP-induced
adverse drug reactions (ADRs) are diarrhoea, nausea, vomiting, mucositis, myelosuppression, and hand-foot
syndrome ≥ Grade 3 according to Common Terminology Criteria for Adverse Events (CTCAE), and up to 10% of
cases also cardiological toxicity 6.

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Inter-patient variability of the toxicity prole of FPs can be partially accounted for by variable expression of DPD
which is genetically determined by its extremely polymorphic coding gene DPYD impact 7. Deciency in DPD
enzymes leads to prolonged FPs half-life and potentially increased toxicity 8,9.
Given that up to 9% of the Caucasian population exhibits a partial DPD enzyme deciency and 0.1–0.2% has a
complete DPD enzyme deciency, the European Medical Agency (EMA) recommends the genotypic or
phenotypic assessment of the DPD gene before initiating FPs treatment (note EMA/229267/2020,
www.ema.europa.eu) 10.
DPYD genotyping, therefore, include the main polymorphisms recognized as clinical markers of ADRs such as
IVS10 (max allele frequency (AF) in the general population = 2.4%; AF in patient cohort = NA), followed by D949V
(max AF = 0.4%; AF patient = 2.4%), DPYD*2A (max AF = 0.8%; AF patients = 6.2%) and DPYD*13 (max AF =
0.06%; AF patients = NA) (as detailed in 11). Moreover, scientic societies, such as the Associazione Italiana di
Oncologia Medica (AIOM) and Società Italiana di Farmacologia (SIF), recently recommended the genotyping of
DPYD*6 variant (max AF = 9.5%; AF patients = 19.7%) 12, after patients develop FP-induced ADRs (AIOM-SIF)
(https://www.aiom.it/raccomandazioni-per-analisi-farmacogenetiche/) 13.
A rare event is the occurrence of double heterozygote variant carriers of the DPYD gene, the so-called
‘compounds heterozygous’.
There is limited
in vivo
data on treatment tolerability in compound heterozygote patients with solid tumours
treated with FPs.
Here, we report a case series of 4 cases of patients with compounds heterozygous DPYD variants, who were
diagnosed and treated with pre-emptive dose reductions of FPs, analysing the treatment tolerability. Our
ndings highlight the inter-variability in the response of compound heterozygous patients to FP treatment,
underscoring the importance of implementing guidelines for diplotypes. This would enable pharmacologists
and physicians to better evaluate the DPYD prole, to reduce ADRs occurrence thereby improving the tolerability
of FPs in clinical practice.
METHODS
Study design and patients
After the acquisition of written and informed consent from each patient following the guidelines approved by
the IRCCS “Casa Sollievo della Sofferenza” Ethical Committee, 993 patients were enrolled by the Oncologic Unit
and screened for DPYD polymorphism at Clinical Laboratory Analysis and Transfusion Medicine of our institute.
All patients aged 40–70 years were diagnosed with solid tumours and genotyped and in advance of
uoropyrimidine treatment according to the standard of care. The uoropyrimidine dosage was determined at
the discretion of the treating physician according to published clinical trials and guidelines.
DNA extraction and genotyping

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Molecular analysis of the DPYD gene was performed on ethylenediaminetetraacetic acid (EDTA) whole blood
processed within 30 minutes, or within 5 days from storage at 4°C. DNA extraction from peripheral blood
nucleated cells (PBMC) was performed using a Qiagen’s QIAamp DNA Blood Kit (51104, Qiagen) according to
the manufacturer’s protocol. DNA was subsequently quantied using the Invitrogen Qubit 4 Fluorometer
(Q33238, Thermosher Scientic).
DNA samples were genotyped for 4 DPYD gene polymorphism as indicated in Table1
Table 1
DPYD genotypes details, related patient’s phenotypic traits and dosing indication As indicated in each lane: rs
ID, variant SNPs, effect of the variant on DPD enzyme, alternative names. Black circle: DPD activity score according
CPIC® Guideline for FPs and DPYD, update 2020 (ref), asterisk: recommended dose reducing of standard
according AIOM guidelines (ref), and: note for rs1801160 that allows dose reduction after ADR events during
treatment.
rsID SNPs Other
names Effect
on
protein
Position at
NC_000001.11
(GRCh38.p2)
DPD
Activity
score°
Metabolic
Phenotype/Risk° %
Dose
of
FPs*
rs75017182 c.1129-
5923C >
G
IVS10 aberrant
splicing g.97579893G
> C 1,5 Intermediate/High 75
rs3918290 c.1905
+ 1G > A DPYD*2A splicing
defect g.97450058C
> T 1 Intermediate/High 50
rs67376798 c.2846A
> T D949V D949V g.97082391T
> A 1,5 Intermediate/High 50
rs55886062 c.1679T
> G DPYD*13 I560S g.97515787A
> C 1 Intermediate/High 50
rs1801160 c.2194G
> A DPYD*6 V732I g.97305364C
> T 2 Normal/Low 85%^
using EasyPGX® ready DPYD kit (
RT026
, Diatech Pharmacogenetics) according to the manufacturer’s protocol.
Using “EasyPGX DPYD Analysis Software” version 4.0.1 according to CE IVD validated and certied procedures
to automatically carry out analysis of results. Through allelic discrimination, the test allows the detection of the
4 main polymorphisms validated as clinical markers associated with drug toxicity according to the European
Medical Agency (EMA), and the Agenzia Italiana del Farmaco (AIFA) and AIOM-SIF recommendations. Patients
were additionally analysed for the DPYD*6 variant allele (Table1).
Data Collection
Patient and disease characteristics were obtained from the patient records, that is age, sex, body surface area,
Eastern Cooperative Oncology Group-Performance Status (ECOG-PS), tumour type, treatment schedule, FPs
type and dose, concomitant anticancer treatment, and FP-related ADRs. ADRs were recorded according to the
CTCAE, version 5.0 (grade 1 to 5).
Variant annotations tools

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The putative functional effect of DPYD*6 was assessed through a pool of seventeen
in-silico
predictor tools, i.e.,
AlphaMissense, CADD, DANN, DEOGEN 2, Eigen-PC, FATHMM, LRT, MutationTaster, MutationAssessor, MetaLR,
PROVEAN, Polyphen2, SIFT, SIFT4G, VEST4, fathmm-MKL, and GERP++. Conservation of the corresponding
genomic site through 100 vertebrates was assessed by using PhyloP 100V. The effect of deep intronic variants
on the splicing machinery was predicted by SpliceAI, Pangolin, and TraP v3.0
in-silico
web applications. Allelic
frequency (AF) values of the examined variants were retrieved from the Genome Aggregation Database
(gnomAD) v4.0.0.
RESULTS
Frequency of DPYD variants in our cohort
From February 2020 to December 2023, a total of 993 patients diagnosed with colon, gastrointestinal, head and
neck, or breast cancer were enrolled in the study. As per guidelines, the patients were genotyped for DPYD
variant alleles before FP treatment according to the standard of care. Using real-time PCR technique, the
variants IVS10, DPYD*13, DPYD*2A, and D949V were analysed according to AIOM/SIF guidelines 14. Moreover,
to implement pre-treatment genotyping, the DPYD*6 allele variant was also screened 8,12,15.
The analysis showed that 809 patients were wild-type (DPYD*1/*1), while 184 patients were mutated. In detail,
136 patients were mutated for the DPYD*6 variant, 21 for IVS10, 17 for DPYD*2A, and 3 for D949V. Notably, the
DPYD*13 allele variant was not found in our cohort. Moreover, we identied 6 carriers of the double-site variant
DPYD*6/IVS10 and 1 double-site variant carrier of DPYD*6/ DPYD*2A (Fig.1a).
In our cohort, DPYD variant carriers represented 18% of the enrolled patients (Fig.1b). The most prevalent
variant was DPYD*6, with an AF of 13.7%, followed by IVS10 with 2% AF, DPYD*2A and D949V with AF of 1.7%
and 0.3% respectively. The DPYD*6/IVS10 DPYD*6/ DPYD*2A double-site variant represented 0.6% and 0.1% out
of the total, respectively (Fig.1c).
It is noteworthy that DPYD genetic variants were often denoted as using the star (*) nomenclature for alleles,
SNP identier, nucleotide base change of the DNA, or amino acid change.
In Table 1, we provided all the nomenclatures, the enzymatic activity score, and treatment-related indications for
these variants.
As detailed in Table 1, mutated patients for each variant should be subjected to a reduction of the standard
dose of FPs due to the reduced DPD enzyme activity compared to wild-type, alteration in metabolic phenotype,
and associated toxicity risks.
We focused on the double-variant DPYD*6/IVS10. IVS10 is predicted to cause a gain of an in-frame nearby
donor site (1 bp, SpliceAI score = 0.77) and a gain of a distant acceptor site (44 bp, SpliceAI score = 0.68) (Fig.
2a). This evidence was conrmed by Pangolin that predicted a nearby (1 bp) splice gain with a score of 0.78.
Changes in the splicing machinery were further conrmed by TraP v3.0, which returned a score relative to this
variant of 0.693 (~ 99.9% of non-coding score percentiles) that is compatible with a functional intronic variant
assessed with high reliability. This variant was found in 1993 alleles over a total of 152204 genotyped alleles in
gnomAD 4.0.0, equally balanced between males and females and with an AF = 1.3% in the general population.

Page 7/16
However, it was prevalently found in the European (non-Finnish) ancestry group. DPYD*6, on the other hand, is
reported in ClinVar as a benign, drug-response variant, even if eight, i.e., CADD, DANN, Eigen-PC, LRT,
MutationTaster, PolyPhen2, fathmm-MKL, and GERP++, on seventeen
in-silico
pathogenicity predictors classify
it as possibly functional. The wild-type genomic site is deeply conserved through vertebrates (PhyloP = 7.49);
however, this variant is as frequent in the general population as 75368 alleles (AF = 4.7% for 1612218 total
alleles genotyped) and it is equally balanced between sexes. It is prevalently represented in the European (non-
Finnish) ancestry group as well (Fig. 2b).
Being more than 275 kbp apart, the real-time PCR method was unable to discriminate the phase of the two
variants. As depicted in Fig.2c, DPYD*6/IVS10 could, in fact, be located on one allele (
in cis
) or on different
alleles (
in trans
) resulting in differences in enzyme function.
Case analysis
4 out of 6 compound heterozygous carriers of DPYD*6/IVS10 variants underwent a FP-based treatment and
therefore were included in our case description.
The rst patient was a 46-year-old Caucasian woman who underwent a right nipple-sparing mastectomy 6 years
before, with a diagnosis of ductal inltrating carcinoma (ER 60%, PgR 0%, ki67 40%, HER2+), followed by
radiotherapy and adjuvant treatment with epirubicin, cyclophosphamide, trastuzumab and tamoxifen. After
developing bone metastases, the patient was subsequently treated with docetaxel + trastuzumab + pertuzumab
(for two years), T-DM1 (for two years), and trastuzumab deruxtecan (for one year). In 2020, after bone and brain
progression, capecitabine (1000 mg/mq bis-in-die [BID] days 1–14) + trastuzumab (8 then 6 mg/kg) + tucatinib
(300 mg BID) q21 were planned to be administered until disease progression or unacceptable toxicity as per
guidelines. The patient was in good general conditions, asymptomatic, ECOG-PS was 0.
Due to the presence of capecitabine in the treatment schedule, a genotypic test of DPYD before treatment
started was performed. Mutational DPYD analysis showed a double-site DPYD gene mutation IVS10C > G (also
called c.1129-5923C > G, rs75017182) and DPYD*6 (also called c.2194G > A, V732I, rs1801160). Due to the
alteration, a clinical pharmacology consultation was requested to determine the most appropriate dose of
capecitabine for the patient’ individualized treatment.
Due to the largely unknown impact of double-site variations on DPD deciency status, a 25% dose reduction of
capecitabine (750 mg/mq BID d.1–14 q21) was performed to ensure the ecacy of the proposed treatment.
The treatment has been ongoing for 24 months, without any ADRs. The best response achieved with the therapy
was a stable disease. In December 2023, due to a further disease progression, eribulin was started, which is
ongoing in April 2024.
The second case is about a 58-year-old Caucasian female patient who had already undergone a total
thyroidectomy plus radio-iodine for a papillary thyroid carcinoma in 2004, and a left quadrantectomy for ductal
BCa in 2018. In 2020, the patient had a colonoscopy performed for rectorrhagia and pelvic pain which revealed
a grade 2 adenocarcinoma. The computerized tomography (CT) scan ruled out a thickness of the distal rectum,
and multiple nodal metastases in the meso-rectal, iliac-obturator and presacral areas. The multidisciplinary
team decided to start pre-operative capecitabine plus pelvic radiotherapy, and then rectal resection. Before

Page 8/16
capecitabine started, a genotypic assessment of DPD deciency was performed, and a double heterozygous
alteration of the DPYD gene was found: IVS10C > G (c.1129-5923C > G, rs75017182) and DPYD*6 (c.2194G > A,
V732I, rs1801160).
After pharmacological evaluation, and due to the best response achieved in the previous compound
heterozygous patient, capecitabine dosage was reduced by 25% of the standard dose (to 600 mg/mq BID during
radiotherapy administration). Unlike the previous patient, after the rst week of treatment, the patient developed
a G4 palmo-plantar erythrodysesthesia, and capecitabine was withdrawn. The patient was evaluated by the
dermatologist and treated with topical steroid and antimicrobial drugs. After a careful risk/benet assessment,
considering the known and unknown toxicity, and the intent of not compromising the ecacy of the combined
treatment, we decided to interrupt capecitabine denitively. In the next weeks, the toxicity completely resolved,
the patient completed the radiotherapy schedule and subsequently underwent surgery. The patient was alive in
April 2024 with no evidence of disease (NED).
The third case was a 70-year-old male Caucasian, with multiple cardiovascular comorbidities (dilatative
cardiomyopathy, carotid and inferior legs arterial atheroma, abdominal aortic aneurysm) with a diagnosis of a
right colon neoplasia with multiple bilateral liver metastases in October 2023. The histologic exam evidenced an
adenocarcinoma, with KRAS mutation of exon 2. Also, in this patient a double heterozygotes DPYD mutation
was found: IVS10C > G (c.1129-5923C > G, rs75017182) and DPYD*6 (c.2194G > A, V732I, rs1801160). ECOG-PS
was 1. Due to the histologic, molecular and instrumental disease prole, we chose to start FOLFOX-6 regimen
(5-Fluorouracil 400 mg/mq bolus then 2400 mg/mq IV over 48 hours, Leucovorin 400 mg/mq, Oxaliplatin 85
mg/mq) q14. The patient's cardiac function was assessed with electrocardiogram and echocardiogram results
in the normal range (ejection fraction 61%).
Nevertheless, considering the comorbities, after the genotyping and the update of CPIC guidelines for this
diplotype (ref), the patient started FOLFOX-6 with dose reduction of 50%. The treatment is ongoing in April 2024,
and no toxicity has been developed.
The latter case is about a 67-year-old Caucasian male patient that was being followed up at our Oncologic Unit
for a multi-treated Kaposi sarcoma for about 10 years. He had also a history of cardiovascular disease. He
developed a positive foecal occult blood test (FOBT) in May 2023, and was diagnosed with a localised right
colon adenocarcinoma. A right hemicolectomy was performed, and the histologic exam showed an
adenocarcinoma pT4a pN1b LVI/PnI negative. According to the disease stage, adjuvant treatment with
capecitabine (2000 mg/mq, days 1–14) + oxaliplatin (130 mg/mq) q3w for 6 cycles should have been
administered. ECOG-PS was 2. As per guidelines DPYD assessment deciency test before treatment started
was performed. A double-site mutation was found: IVS10C > G (c.1129-5923C > G, rs75017182) and DPYD*6
(c.2194G > A, V732I, rs1801160).
After pharmacological consulting, and considering the comorbidities and general conditions, the treatment was
started with 5FU reduced by 50% compared to the standard dose. However, after the rst cycle of therapy, the
patient developed nausea and diarrhea G3. The treatment was withdrawn. Anti-emetics, anti-diarrhoics, and
rehydration with electrolytes supplementation were administered. After treatment restarted, G2 gastrointestinal
toxicity developed again, capecitabine was interrupted and symptomatic therapy was administered. Thus, given
the unknown risk for further toxicity due to the double DPYD alteration, it was decided, in accordance with the

Page 9/16
patient, to discontinue capecitabine. Only oxaliplatin was administered in the last 4 cycles, and no ADRs
presented. The patient terminated the adjuvant treatment and entered a 6-months oncological follow up
program without evidence of disease at April 2024.
All patient factors, FPs treatment, and AEs are summarized in Table2.
Table 2
Compounds Heterozygous patient Factors, FPs Treatment, and AEs
All initial dose reductions were performed because of the IVS10 variant allele. Adverse events of grade 3 or
higher are in bold. Abbreviation: see below
Characteristic Patient #1 Patient #2 Patient #3 Patient #4
Age, years 40 58 70 67
Gender Female Female Male Male
ECOG
performance
status
0 1 2 1
BSA°, m21,84 1,77 1,99 1,85
EGFR° 95 90 88 72
Tumour type Breast, Ductal
inltrating
carcinoma
Colon
Adenocarcinoma G2
ypT0 N1c
Colon
Adenocarcinoma
Colon
adenocarcinoma
pT4a pN1b
FP
treatment/dose capecitabine capecitabine 5-FU capecitabine
Combination trastuzumab +
tucatinib RT Oxaliplatin oxaliplatin (130
mg/mq
Administered
dosage (% of
standard dose)
75% 75% 50% 50%
Treatment
durations 24 months 1 week 5 months 2 months
Modality of FP
administration Oral Oral IV Oral
Dose reduction
during treatment No No No No
Early
discontinuation of
treatment
No Yes No Yes
ADR—highest
grade (start
cycle)
No G4 palmo-plantar
erythrodysesthesia
G4 (1st cycle)
No G3 nausea and
diarrhea (1st cycle),
G2 nausea and
diarrhea (2nd cycle)

Page 10/16
DISCUSSION
In this study of 993 patients, six individuals yielding 0.6% of the cohort, carried IVS10C > G and DPYD*6 variants
of
DPYD
gene heterozygously.
This allowed us to characterize four patients with this polymorphism, who were treated with a different FP-
containing chemotherapy regimen and who showed varying responses to FPs dose reduction and different FPs-
related ADRs.
In the rst group, patients received a 25% dose reduction of FPs, to preserve treatment ecacy, following the
AIOM-SIF guidelines for the IVS10 variant.
The clinical outcome showed that patient #1 had not experienced ADRs with NED whereas patient #2 showed
G4 palmo-plantar erythrodysesthesia determining treatment withdrawal.
In the second group, patients received a 50% dose reduction of FPs due to the toxicity of the previous cases,
following updated CPIC indication and PharmGBK for IVS10 variant (detailed at
https://cpicpgx.org/guidelines/guideline-for-uoropyrimidines-and-dpyd/ and
https://www.pharmgkb.org/guidelineAnnotation/PA166109594).
In a mirror-like image of the rst group, patient #3 did not experience ADRs and achieved NED. The treatment is
ongoing, and no toxicity has been developed. On the contrary, patient #4 encountered G3 nausea and diarrhoea
imposing to withdraw the treatment.
These ndings suggest that managing heterozygous patients is a clinical challenge.
We aimed highlight the role, management and clinical implications of a double heterozygote variant of the
DPYD
gene, which was rarely described in the scientic literature. Johnson et al. using a familial approach describe a
case of DPYD deciency in a breast cancer patient with grade IV toxicity after chemotherapy with
cyclophosphamide/methotrexate/5-uorouracil; the patient was a compound heterozygote for two different
mutations, DPYD*2A and DPYD*13, one in each allele. In addition, the authors identied two allelic variants
previously considered to be associated with DPD enzyme deciency (DPYD*9A and M166V) in a family member
who nevertheless maintained normal DPYD enzyme activity 16. These data suggest that a genotypic alteration
does not always cause a phenotypic consequence in terms of deciency in enzyme activity. At the same time, it
would be important to collect the data of any DPYD mutations in the family if available.
Indeed, only one other paper has described a case with the same variants 17 as our case series. Baiardi et al.
describe the case of a patient carrying the compound heterozygous variant of the DPYD gene (IVS10C > G and
DPYD*6) diagnosed with adenocarcinoma of the left colon for whom a 25% dose reduction of the standard
adjuvant treatment with capecitabine was chosen. At the end of the 4th cycle, the patient suffered increased
serum amylase G1, hepatobiliary disorders G1, diarrhoea G1 and fatigue G2 so treatment was discontinued 16.
Many efforts have been made to study the carriers of these isolated variants; however, the impact of the two
variants together is unknown. Even less is known about DPYD variant-derived mutated proteins.

Page 11/16
Structurally, the IVS10 variant has been suggested to activate a splice donor site within intron 10 18. Here, we
predict it to also cause a gain of a distant acceptor site, thereby promoting alternative aberrant splicing and
potentially affecting DPD enzyme activity. However, the quantitative contribution of IVS10 to alternative splicing
and DPD function has yet to be demonstrated. Despite the unknown impact on the protein structure, DPD
activity was reduced by 35% compared to the non-carrier population, carriers of rs7501718 19.
Conversely, the variant DPYD*6 (c.2194G > A, V732I, rs1801160) is a single nucleotide variant (SNV) resulting in
the substitution of Valine for Isoleucine in the DPD enzyme. Although its clinical impact is still debated, it has
been linked to ADRs and toxicity 12 20 and it has been classied here as possibly functional by eight on
seventeen in-silico pathogenicity predictors.
Considering the variants individually, the carriers of the IVS10 variant are dened as intermediate metabolizers
(AS = 1.5). In contrast, carriers of DPYD*6 variant are dened as normal/low metabolizers (AS = 2) according to
DPYD gene activity score (DPYD-AS) proposed by Henricks et al. to translate genotype into phenotype 21.
Despite the large number of studies and classication of carriers of a single DPYD variant no studies have
assessed the cumulative activity of the two variants and/or the metabolizing ecacy of the compound
heterozygous.
Methodologically, current real-time methods cannot distinguish between variants located on a single allele (in
cis) or different alleles (in trans). In the former case, one functionally active allele remains, whereas in the latter
case, both alleles are affected, potentially resulting in decreased enzyme activity 22.
We can speculate that patients #1 and #3 could be cis-compounds heterozygous with a normal-functioning
allele that allows correct catabolism of FPs without toxicity, while patients #2 e #4 could be trans- compound
heterozygous with both affected alleles unable to perform catabolism of FPs leading to metabolites
accumulation and ADRs onset despite the dose reduction.
In agreement with previous works 17, we can assert that these variants should be managed by a
multidisciplinary team with a dose reduction ranging from 25 to 50% to maintain treatment ecacy, strict
clinical monitoring for early ADRs detection.
Therefore, our data suggest that compound heterozygous patients require thorough analysis, as they were only
genotyped for four DPYD variants, and the effects of additional deleterious DPYD variants and the location of
polymorphisms cannot be ruled out. Furthermore, besides genetic polymorphism, DPYD is strictly regulated by
transcription factors such as miRNA 27a, 27b, 494 23–25.
Thus, in the presence of compound heterozygosity, to minimize uoropyrimidine-based chemotherapy toxicity
without altering treatment ecacy, it may be benecial to enhance routine genotyping tests by:
Incorporating additional DPD phenotyping tests, such as the measurement of DPD activity in PBMCs;
utilizing innovative next-generation sequencing (NGS) approaches could unveil additional rare,
integrating rare genetic variants into DPYD pharmacogenetic testing using NGS 26;
implementing therapeutic drug-monitoring approach.

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This work, as well as the parallel publications, will contribute to further discussion on the implementation of
DPYD
testing and the available guidelines for physicians.
CONCLUSIONS
A multidisciplinary teamwork in the management of patients carrying compounds heterozygous DPYD variants
should be standardised. Genotypic data (status of DPYD gene and eventual family history of DPYD mutation)
should be interpreted and integrated with phenotypic (status of DPD activity) and clinical data (comorbidities
and concomitant drug intake). Application of tailored treatment approaches including integrative deep analysis,
precision dosing and early detection of ADRs is the cornerstone in order to ensure therapeutic ecacy and
safety for each case.
Abbreviations
DPYD: Dihydropyrimidine dehydrogenase, gene;DPD: Dihydropyrimidine dehydrogenase, enzyme;5-FU: 5-
Fluorouracil; IV: intravenous; FPs: Fluoropyrimidine; DHFU: dihydrouorouracil; FdUMP: 5-uoro-2’-deoxyuridine-
5’-monophosphate; TYMS: Thymidylate Synthase; CRC: colorectal cancer; BCa: breast cancer; CPIC: Clinical
Pharmaceutical Implementation Consortium; CTCAE: Common Terminology Criteria for Adverse Events; EMA:
European Medicynes Agency; AIOM: Associazione Italiana di Oncologia Medica;SIF: Società Italiana di
Farmacologia;AIFA: Agenzia Italiana del Farmaco; SNP: Single Nucleotide Polymorphism; SNV: single
nucleotide variant; ADRs Adverse drug reactions; NED: no evidence of disease; ECOG-PS Eastern Cooperative
Oncology Group Performance Status; BSA: Body Surface Area; EGFR: Estimated Glomerular Filtration Rate.
Declarations
Data availability statement
This manuscript does not report data generation or analysis
Ethics statement
The study was conducted following the Italian (D. Lgs. 30/06/2003, n. 196) regulations for research on human
subjects. The informed consent was obtained from all patients following the protocol approved by the Ethical
Committee of IRCCS Casa Sollievo della Sofferenza Hospital, Italy (Mo/CSS/C.I.TestGen).
Acknowledgements
This study was funded by Italian Ministry of Health (Current Research funds) to LS, MC, TM and 5 per 1000
voluntary contributions to TM. The funder played no role in study design, data collection, analysis and
interpretation of data, or the writing of this manuscript. 
Contributions
Conceptualization: GM, LS
Experiment design: GM, RB, TM, MC

Page 13/16
Experiment implementation: GM, RA, CG, LS, TM, TB
Clinical data: BAM, LN, TL, MGR, GDM, MDR
Result investigation: GM, RB, LS,
Funding acquisition: EM, TL
Project administration: GM, MC
Supervision: GM, RB, LS
Writing—original draft: LS
Writing—review & editing: LS, GM, RB, GF, TM, MC
Corresponding authors
LS l.simone@operapadrepio.it
Competing interests
All authors declare no nancial or non-nancial competing interests.

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Figures
Figure 1
DPYD variants frequency in enrolled patients
a. Flowchart of patients enrolled in the study. b. Pie-chart illustrating the percentage of all identied
DPYD
genetic variants. c. Histogram showing the distribution of all identied
DPYD
genetic variants (percentage of
patients and percentage are reported).

Page 16/16
Figure 2
Illustration of zygosity
a.Localization of the gained donor and acceptor sites on the NM_000110.4 MANE transcript. b. Pie chart
reporting
in-silico
predictors supporting the pathogenicity of DPYD*6. Considered pathogenicity thresholds:
CADD > 20, DANN > 0.9, Eigen-PC > 2, LRT = Categorical (D = deleterious), MutationTaster > 0.5, PolyPhen2 >
0.85, fathmm-MKL > 0.5, and GERP++ > 2. c. Boxes represent alleles: light blue circle represents IVS10 variant;
green circle represents DPYD*6 variant.