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Melphalan is a bifunctional alkylating agent that elicits its cytotoxic activity by rapidly forming an initial DNA monoadduct, which then produces an inter-strand crosslink. Most studies exploring the role of inherited differences in DNA repair and melphalan outcomes focus on inter-strand crosslink repair, however, monoadduct repair likely plays a key role since it minimises the ultimate production of these crosslinks. The purpose of this systematic review was to assess evidence of an association between variation in monoadduct repair pathways and melphalan response. A literature search was undertaken using Medline, Embase, Scopus and PubMed databases. Duplicates were removed and only full-text articles were included. To be included for critique in this systematic review, articles were assessed for relevance using strict inclusion/exclusion criteria. Fourteen studies were identified that involved patients treated with melphalan, however, in 3, only a minority of the cohort received melphalan. Across the remaining 11 studies, 61 genes/proteins in DNA monoadduct repair pathways were assessed. Both germline SNP (CDKN1A, ERCC1, ERCC2, ERCC4, ERCC6, EXO1, MLH1, MNAT1, MUTYH, PARP4, PCNA, POLE, POLR1G, RAD23B, RFC1, RFC3, RPA1, RPA3, TREX1, UNG, XPC, XRCC1) and somatic expression (CDKN1A, PARP1, PCNA, MGMT, RECQL, RFC5) were associated with melphalan outcomes in ≥ 1 study. It appears that inherited germline differences in monoadduct repair genes may be a risk factor for poor outcomes. However, the diversity of study design, patient cohorts, genes assessed and lack of replication, preclude any meta-analysis. Further prospective studies are required to validate these findings.

Genetic variation in the activation of the prodrug cyclophosphamide (CP) by cytochrome P450 (CYP) enzymes has been shown to influence outcomes. However, CYP are also subject to phenoconversion due to either the effects of comedications or cancer associated down-regulation of expression. The aim of this study was to assess the relationship between CP bioactivation with CYP2B6 and CYP2C19 genotype, as well as CYP2C19 phenotype, in breast cancer patients. CP and the active metabolite levels were assessed in breast cancer patients (n = 34) at cycle 1 and cycle 3 of treatment. Patients were genotyped for a series of SNP known to affect CYP2B6 and CYP2C19 function. The activity of CYP2C19 was also assessed using a probe drug. We found a significant linear gene-dose relationship with CYP2B6 coding SNP and formation of 4-hydroxycyclophosphamide. A possible association with CYP2C19 null genotype at cycle 1 was obscured at cycle 3 due to the substantial intra-individual change in CP bioactivation on subsequent dosing. Comedications may be the cause for this inter-occasion variation in bioactivation of cyclophosphamide and the ensuing phenoconversion may account for the conflicting reports in the literature about the relationship between CYP2C19 genotype and CP bioactivation pharmacokinetics. Trial registration ANZCTR363222 (6/11/2012, retrospectively registered).

Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC–MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.

AIMS: A previous study suggested that a thymine (THY) challenge dose could detect aberrant pharmacokinetics in known cases of fluoropyrimidine toxicity compared with healthy volunteers. The preliminary data suggested that urine sampling also could detect this aberrant disposition. The aim of this case–control study was to assess the ability of the urinary THY challenge test to discriminate cases of severe gastrointestinal toxicity in a cohort of patients treated with 5‐fluorouracil or capecitabine. METHODS: Patients (n = 37) received a 250 mg (per os) dose of THY and a cumulative urine sample was collected for 0–4 h. The urinary amounts of THY and metabolite dihydrothymine (DHT) were determined by liquid chromatography/mass spectrometry. Genomic DNA was analysed for DPYD gene variants. Renal function was estimated from blood creatinine levels. Cases (n = 9) and noncases (n = 23) of severe (grade ≥ 3) gastrointestinal toxicity were defined based on Common Terminology Criteria for Adverse Events. RESULTS: The median THY/DHT ratios were 6.2 (interquartile range 2.9–6.4) in cases, including the 2 patients who were DPYD heterozygous carriers. However, this was not significantly different (P = .07) from the THY/DHT in noncases (median 2.6, interquartile range 2.8–4.2). Although creatinine clearance was lower (P = .001) in cases, renal function could not discriminate cases from noncases. However, logistic regression analysis using both of these explanatory variables could discriminate most cases (receiver operating characteristic area 0.8792, 95% confidence interval 0.72–1.00). CONCLUSIONS: The THY challenge test combined with a patient's renal function may be useful as a phenotypic diagnostic test to detect risk of life‐threatening fluoropyrimidine gastrointestinal toxicity.

Cyclophosphamide is an alkylating agent used in the treatment of solid and haematological malignancies and as an immunosuppressive agent. As a prodrug, it is dependent on bioactivation to the active phosphoramide mustard metabolite to elicit its therapeutic effect. This focused review will highlight the evidence for the role of germline pharmacogenetic variation in both plasma pharmacokinetics and clinical outcomes. There is a substantial indication from 13 pharmacokinetic and 17 therapeutic outcome studies, in contexts as diverse as haematological malignancy, breast cancer, systemic lupus erythematosus and myeloablation, that pharmacogenetic variation in both CYP2C19 and CYP2B6 influence the bioactivation of cyclophosphamide. An additional role for pharmacogenetic variation in ALDH1A1 has also been reported. Future studies should comprehensively assess these 3 pharmacogenes and undertake appropriate statistical analysis of gene-gene interactions to confirm these findings and may allow personalised treatment regimens.

Fluorouracil (5-FU), a chemotherapeutic agent widely used in the treatment of numerous common malignancies, causes oral mucositis in a proportion of patients. The contribution of drug transport processes to the development of this toxicity is currently unknown. This work aimed to establish and optimise a simple phenotyping assay for 5-FU uptake into primary buccal mucosal cells (BMC). The uptake kinetics of radiolabelled 5-FU were determined in pooled BMC freshly collected from healthy volunteers. The inter- and intra-individual variability in 5-FU uptake was then assessed across a cohort that included both healthy volunteers and cancer patients. 5-FU uptake into pooled primary BMC was both time and concentration dependent. An Eadie–Hofstee analysis suggested two components; a high-affinity (KM = 3.3 µM) low-capacity ( $$V_{\text{MAX}}$$ = 57.8 pmol min−1 105 viable cells−1) transporter, and a high-capacity ( $$V_{\text{MAX}}$$ = 1230 pmol min−1 105 viable cells−1) low-affinity (KM = 3932 µM) transporter. There was 180-fold variation in the rate of 5-FU uptake into BMC (0.10–17.86 pmol min−1 105 viable cells−1) across the 34 subjects (healthy participants N = 24, cancer patients N = 10). Notably, retesting of a subset of these participants (N = 16) multiple times over a period of up to 140 days demonstrated poor stability of the uptake phenotype within individuals. The uptake of 5-FU into healthy oral mucosal cells is a highly variable process facilitated by membrane transporters at pharmacologically relevant concentrations. This bioassay is simple, minimally invasive, and suitable for phenotypic analysis of drug transport in healthy primary cells.

Purpose: This work investigated the utility of circulating microRNA (miRNA) as biomarkers of clozapine (CLZ)-induced cardiotoxicities: serious adverse events with an unusually high incidenc...

CYP2C19 is a hepatic drug metabolizing enzyme that contributes to the metabolism of chemotherapeutic agents such as cyclophosphamide, thalidomide, bortezomib, icotinib, tivantinib and indisulam. CYP2C19 null function genotype strongly predicts phenotype in healthy populations. However we have previously shown an additional acquired loss of CYP2C19 metabolic function in up to one third of patients with advanced solid-tumor cancers. It is not known whether this effect also occurs in earlier stage disease. The aim of this study was to investigate whether this acquired loss of CYP2C19 function was detectable in patients with stage III-IV and resected disease. Patients undergoing treatment for gastro-intestinal tumors (n=50) with either no evaluable disease (resected) or stage III-IV disease were recruited following informed consent and probed for CYP2C19 activity (200 mg proguanil p.o.) on three test occasions. CYP2C19 metabolic activity was assessed using HPLC analysis of the 3h post dose plasma drug and metabolite concentrations. The log metabolic ratio (drug/metabolite ≤ 1 ≥) was used to categorize individuals as either extensive or poor metabolizers. CYP2C19 genotype was determined by PCR-RFLP to identify individuals homozygous for null function mutations (CYP2C19*2 and CYP2C19*3). One patient had a poor metabolizer genotype (CYP2C19*2*2) and was excluded. An acquired loss of CYP2C19 activity was observed in 23% (n=6) stage III-IV and 13% (n=3) of resected patients at the first test. This genotype-phenotype discordance was a significant in both groups (p This study demonstrated that an acquired loss of CYP2C19 activity was present in patients with stage III-IV disease as well as in those subjects who had undergone resection. This work confirms our previous finding of a significant level of CYP2C19 genotype-phenotype discordance in patients with advanced end-stage cancer. This discordance is unlikely to be due to rare null mutations in this gene as dynamic changes in metabolic activity were observed in some individuals. While the use of genotyping is often suggested as a method to personalize treatment, this data indicates that this approach could significantly underestimate the number of phenotypic CYP2C19 poor metabolizers. An acquired loss of function may have implications for the safety and efficacy of chemotherapeutic agents metabolized by CYP2C19. Further work is required to elucidate the mechanisms behind this down-regulation of drug metabolism in cancer and to determine whether this phenomenon adversely affects therapeutic outcomes. Citation Format: Kathryn E. Burns, Wing-Yee Lo, Michael P. Findlay, George Laking, Nuala A. Helsby. Genotype-phenotype discordance of the hepatic drug metabolism enzyme CYP2C19 in gastrointestinal cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5548. doi:10.1158/1538-7445.AM2014-5548