Fluoropyrimidines, such as 5-fluorouracil (5-FU) and capecitabine, are widely used chemotherapeutic agents in the treatment of gastrointestinal, head and neck, and breast cancers (Masuda et al., 2017; Bang et al., 2012; Cassidy et al., 2008; Grothey et al., 2018; Geyer et al., 2006). These drugs are known to cause early-onset severe or life-threatening toxicities, including myelosuppression, mucositis, hand-foot syndrome, and diarrhea, in about 10 to 30% of patients, with reported mortality rates ranging from 0.5% to 1%] (Van Cutsem et al., 2001; Amstutz et al., 2009; Boisdron-Celle et al., 2017; Froehlich et al., 2015; Meulendijks et al., 2015; Lévy et al., 1998; Bajetta et al., 2005; Tsalic et al., 2003). The enzyme dihydropyrimidine dehydrogenase (DPD), which is coded by the DPYD gene, is the rate-limiting enzyme involved in the 5-FU metabolism (Thorn et al., 2011). Partial or complete deficiency of DPD activity is associated with life-threatening side effects among patients undergoing 5–FU–based treatment (Morel et al., 2006; Lunenburg et al., 2020). More than 450 DPYD polymorphisms have been associated with fluoropyrimidine-induced toxicity, and the incidence of DPD deficiency has been estimated at 3%–7% in the Caucasian population (Morel et al., 2006; Lunenburg et al., 2020; Amstutz et al., 2018).

Routine testing for four DPYD variants that are commonly found–DPYD2A (c.1905+1G>A), DPYD13 (c.1679T>G), c.2846A>T, and c.1129-5923C>G/hapB3 is recommended by the European Medicines Agency before the initiation of 5-FU chemotherapy (Lunenburg et al., 2020). These mutations play a role in the interindividual variability of 5-FU toxicity. The current guidelines of the Clinical Pharmacogenetics Implementation Consortium (CPIC) include 82 known DPYD variants, of which 21 are classified as poor metabolizers (no DPYD enzyme function), six as intermediate metabolizers (reduced function), and the rest as normal metabolizers or with limited evidence (Amstutz et al., 2018). The CPIC guidelines suggest avoiding 5-FU in poor metabolizers and a 25%–50 % dose reduction in intermediate metabolizers. (Lunenburg et al., 2020; Amstutz et al., 2018) While fluoropyrimidine toxicity profiles are comparable between populations, the prevalence and distribution of DPYD variants differ (White et al., 2021). Certain variants which are common in Europeans have not been reported in the African, Chinese, or Japanese populations, whilst other clinically relevant variants may be unique to non-Caucasians. Routine DPYD genotyping is not commonly practiced in India, and information on DPYD polymorphisms in the Indian population is scarce. Previously published data from India utilized PCR or Sanger 4 sequencing to examine known polymorphisms in most cases, including healthy volunteers or patients with grade 3 toxicities (Hariprakash et al., 2018; Patil et al., 2016).

To address these gaps, we conducted a single-center study using next-generation sequencing to evaluate the prevalence of DPYD variants in capecitabine-treated patients and their associated toxicity profiles.

Among the 263 patients in the D-TORCH study, DPYD genotyping was performed in 76 patients, who agreed to participate in this exploratory sub-study (Supplementary Figure S1). Baseline characteristics such as age, sex, cancer type, prior chemotherapy exposure, regimen type, and randomized arm were similar between the sequenced subgroup and the whole cohort, although a higher proportion of patients in the sequenced cohort had metastatic disease (36.4% vs. 21.4%, P = 0.007. However, with respect to toxicity, patients in the sequenced cohort experienced a higher, statistically significant proportion of grade 2–3 diarrhea, whereas the frequencies of other treatment-related toxicities were comparable between the groups. Of these, 54 (71%) harbored at least one DPYD variant, while 22 (29%) were wild-type. The median age of the participants was 47 years (range: 27–71), with a female predominance (69.7%). Most patients had an ECOG performance status of 0–1 (55.3%), and breast and gastrointestinal malignancies were similarly distributed. Most patients had metastatic breast or gastrointestinal cancer. Baseline characteristics and toxicity profiles of the sequenced subgroup versus the whole cohort are summarized in Table 1. Comparisons between DPYD variant-positive and wild-type patients are detailed in Table 2, and no statistically significant differences were observed in baseline characteristics between the variant-positive and variant-negative groups or by the metabolizer phenotype (Supplementary Table S1).

In this exploratory sub-study of the D-TORCH trial, 54 of the 76 sequenced patients (71%) harboured at least one DPYD variant, while no poor metabolizers were detected.; however, three individuals were determined to be intermediate metabolizers as per the activity values for the identified genotype, one of whom developed grade 3 toxicity. The remaining 51 individuals were classified as normal metabolizers, including one who harbored an unknown variant (no RSID) and showed no adverse effects. Among the four DPYD variants recommended by the European Medical Agency (EMA) for routine testing, rs3918290 has been the most frequently reported variant in previous Indian studies in association with grade 3 toxicity (Pavithran et al., 2021; Chan et al., 2024). However, in our cohort, only one patient carried this variant. This discrepancy is presumably due to prior studies focusing solely on patients with grade 3 toxicities, whereas we carried out NGS regardless of clinical toxicity.

The most prevalent variants found among the normal metabolizers in our study (n = 50) were rs1801265 (n = 25), rs1801159 (n = 12), rs1801160 (n = 13), and rs2297595 (n = 7). Based on CPIC guidelines, these variants do not require dose adjustment. Nevertheless, their clinical relevance is uncertain, with conflicting evidence across various ethnic populations. Interestingly, 35 out of 50 patients in our cohort, classified as normal metabolizer phenotype as per the CPIC guidelines, still developed grade 2 or 3 toxicity, emphasizing the limitation of CPIC-based phenotype prediction and dose adjustment in the Indian population. Our findings align with previous Indian studies. Patil et al. selectively tested 12 Indian patients with grade 3 toxicities during TPF (Docetaxel,5-fluorouracil (5-FU), and cisplatin) neoadjuvant therapy and reported normal metabolizers in rs1801160, rs1801265, or rs2297595 in 11 patients (Patil et al., 2016). Similarly, Sahu et al. conducted PCR-based testing in 22 gastrointestinal cancer patients with grade 3 toxicities, detecting normal metabolizer phenotypes in rs1801265 (n = 8), rs2297595 (n = 4), and rs1801160 (n = 4) (Sahu et al., 2016). Furthermore, a South Indian study of 375 patients employing PCR-based testing for four variants identified rs2297595 in 32 of 47 DPYD-mutated individuals, tying it to fluoropyrimidine toxicity (Pavithran et al., 2021). Another study using Sanger sequencing of selective variants in 1064 Indian patients receiving fluoropyrimidines reported a DPYD variant frequency of 27.2 percent (289/1064), involving either homozygous or heterozygous mutations in rs1801160 and rs1801159, though data on clinical toxicity was not described (Patil et al., 2019).

In addition, two population-based studies of DPYD polymorphisms in Indians also highlighted genetic variability. Hariprakash et al. analyzed almost 3,000 South Asians, validating their findings in 110 Indian cancer patients, and observed that certain variants were comparable to those found in individuals of European ancestry, while others were more closely related to those seen in individuals of African origin (Hariprakash et al., 2018). Heterozygous variants rs1801265, rs1801159, rs1801160, rs2297595, and rs12022243 were frequent in the general population. Notably, rs2297595, despite being a standard metabolizer variant, was associated with toxicity in the cancer cohort. The second population-based study evaluated 2000 Indian individuals, mapping DPYD variant frequencies and their functional activity using bioinformatic tools. Deleterious variants were found at a frequency of 1.89%, while normal metabolizers per CPIC were at a frequency of 58.3% in rs1801265, rs2297595, rs1801160, rs75017182, and rs1801158. In silico/CUP-SAT analyses predicted an impaired DPD enzyme nature for rs1801160 and rs1801158 but not for rs2297595, in contrast to the earlier study. Population clustering analysis of Indians was in line with South Asians being distinct from Europeans and Africans, underscoring ethnic variations in DPYD polymorphisms and 5-FU toxicity risk (Naushad et al., 2021).

In the recent systematic review by Chan et al., conducted exclusively in non-European ancestry, a higher frequency of novel DPYD variants was observed, along with an association between the normal metabolizer phenotype and fluoropyrimidine toxicity, despite overall toxicity rates being similar to those in Caucasians (Chan et al., 2024). In our study, we identified a variant of uncertain significance and a previously unreported novel variant. Similarly, Lopes et al., found that around 6% of the patient population (n = 631) had a DPYD variant that was overlooked by targeted genotyping (López López-Cepero et al., 2023). These included functionally important intronic variants within haplotype B3, which evince the limitations of conventional screening methods. NGS may better capture rare or intronic variants over PCR, though targeted panels with intronic coverage remain a viable and cost-effective alternative. Among 22 patients with DPYD wild-type, 13 experienced grade 2/3 toxicity, hinting the role of additional enzyme polymorphisms such as thymidine synthase and MTHFR involved in the 5-FU metabolism (Loganayagam et al., 2013). Routine sequencing may inform toxicity prediction by mapping gene polymorphisms that contribute to toxicity and remain uncharacterized in the Indian context.

Ethnic diversity also highlights the importance of genotype-phenotype association in decoding DPD enzyme function (White et al., 2021). The DPYD activity score, validated in the Caucasian population, remains unverified in other ethnic groups, potentially explaining toxicity differences among CPIC-classified normal metabolizers (White et al., 2021). Limited genotype-phenotype studies have also identified population-specific variations, such as the 1905 + 1G>A intronic variant, which is prevalent in Iranians but does not mirror the same toxicity profile seen in Europeans (Negarandeh et al., 2020).

Overall, in the present study, we identified exonic variants in the DPYD gene in 71% of patients (54 out of 76) in our cohort. Of these, 3.9% of patients (3 out of 76) had a DPYD exonic variant with a recommended intermediate metabolizer phenotype as per the CPIC guidelines. Further, we observed that some exonic variants detected in our cohort were also present in patients with grade 3 toxicity, however, inconsistent evidence exists for such variants. Additionally, there were patients with grade 3 or higher toxicity and no DPYD exonic variant observed. Such patients can be considered for further investigation of other underlying genetic mechanisms. Traditional PCR-based genotyping covers only targeted genetic variants and the broader gene-based variants are not captured in PCR-based assay. Our study represents the first report from India on DPYD gene-based investigation using NGS assay. However, it is limited by a smaller sample size and underrepresentation of patients with higher grade toxicity. In this context, these findings should be viewed primarily as an exploratory and descriptive assessment, rather than a definitive evaluation of the relationship between DPYD variants and clinical toxicity. Only 29% of the parent D-TORCH cohort underwent optional genotyping, and the intermediate metabolizer group was very small, limiting statistical power and producing wide confidence intervals. While baseline characteristics were broadly similar, the sequenced cohort included slightly more metastatic and combination-therapy patients and exhibited a higher proportion of ≥grade 2 diarrhea; however, no association was found with other toxicities. These factors support a cautious, hypothesis-generating interpretation of the findings. Moreover, our study has genetic analysis limited to exonic and splicing variants. Yet intronic DPYD risk alleles will not be captured directly, rather these would be tagged with exonic variants, such as exonic variants rs56038477 is often used as a tag SNP for the HapB3 haplotype. Also, in certain cases with deep intronic DPYD risk alleles, whole exome sequencing may not capture such intronic variants and true prevalence might be underreported. In this exploratory work, we also acknowledge the limitation that no orthogonal confirmation of clinically relevant calls such as rs56038477 was performed. Large-scale genotype-phenotype correlation studies are required to precisely define the impact of DPYD gene polymorphisms in the Indian population and their therapeutic implication.

In summary, the present study demonstrates the primary insights into the capecitabine-related toxicity and DPYD exonic variants among Indian patients. Ethnic differences in the DPYD polymorphic variants exist in our population, underscoring the importance of phenotype correlation and routine NGS-based testing over PCR-based screening for enhanced toxicity prediction. Findings from our study lay the groundwork for further investigation on the capecitabine-related toxicity and DPYD exonic variants. Larger studies are needed to corroborate these findings and develop population-specific recommendations for safer use of fluoropyrimidines.