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Consensus guidelines exist for genotype-guided fluoropyrimidine dosing based on variation in the gene dihydropyrimidine dehydrogenase (DPYD). However, these guidelines have not been widely implemented in North America and most studies of pretreatment DPYD screening have been conducted in Europe. Given regional differences in treatment practices and rates of adverse events (AEs), we investigated the impact of pretreatment DPYD genotyping on AEs in a Canadian context. Patients referred for DPYD genotyping prior to fluoropyrimidine treatment were enrolled from December 2013 through November 2019 and followed until completion of fluoropyrimidine treatment. Patients were genotyped for DPYD c.1905+1G>A, c.2846A>T, c.1679T>G, and c.1236G>A. Genotype-guided dosing recommendations were informed by Clinical Pharmacogenetics Implementation Consortium guidelines. The primary outcome was the proportion of patients who experienced a severe fluoropyrimidine-related AE (grade ≥3, Common Terminology Criteria for Adverse Events version 5.0). Secondary outcomes included early severe AEs, severe AEs by toxicity category, discontinuation of fluoropyrimidine treatment due to AEs, and fluoropyrimidine-related death. Among 1394 patients, mean (SD) age was 64 (12) years, 764 (54.8%) were men, and 47 (3.4%) were DPYD variant carriers treated with dose reduction. Eleven variant carriers (23%) and 418 (31.0%) noncarriers experienced a severe fluoropyrimidine-related AE (p = 0.265). Six carriers (15%) and 284 noncarriers (21.1%) experienced early severe fluoropyrimidine-related AEs (p = 0.167). DPYD variant carriers treated with genotype-guided dosing did not experience an increased risk for severe AEs. Our data support a role for DPYD genotyping in the use of fluoropyrimidines in North America., (© 2021 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Purpose: Female patients with breast cancer frequently develop arthralgia when treated with aromatase inhibitors (AI). Although the mechanism of AI-induced arthralgia is unknown, potential biomarkers have been identified. The purpose of this study was to investigate the clinical and genetic predictors of AI-induced arthralgia in a prospective cohort of patients with estrogen receptor-positive breast cancer., Methods: One hundred and ninety-six patients were enrolled at initiation of AI therapy with either letrozole or anastrozole. Patients completed two validated self-report questionnaires assessing pain, stiffness, and physical function at baseline, and repeated the questionnaires at two and at six months after the initiation of treatment with an AI. Germline DNA of all patients was genotyped for seven single-nucleotide polymorphisms (SNPs) previously identified by genetic screens and genome-wide association studies as associated with AI-induced arthralgia., Results: More than 50% of the study group experienced arthralgia symptoms. Genetic analysis revealed that four SNPs, in CYP19A1 (rs4775936) and ESR1 (rs9322336, rs2234693, rs9340799), were associated with the development of arthralgia (adjusted P = 0.016, 0.018, 0.017, 0.047). High body mass index (BMI) was also associated with the development of arthralgia symptoms (adjusted P = 0.001). Patients prescribed letrozole were significantly more likely to develop arthralgia than patients on anastrozole (P = 0.018), and also more likely to discontinue AI therapy due to arthralgia. The CYP19A1 (rs4775936) SNP was significantly associated with discontinuation of therapy due to intolerable arthralgia., Conclusions: Our results suggested that BMI and AI drug (letrozole versus anastrozole) were clinical predictors of arthralgia, while genetic variants rs4775936, rs9322336, rs2234693, and rs9340799 were genetic predictors of AI-induced arthralgia. Significantly, rs4775936 was also a predictor of discontinuation of therapy.

Bile acids are endogenous ligands of nuclear receptors pregnane X (PXR) and farnesoid X (FXR). PXR and FXR regulate pathways that are impaired in inflammatory bowel disease (IBD). Decreases in PXR and FXR activity are documented in IBD; however reasons for this are unknown. We aimed to assess the effect of Crohn's disease (CD) on the plasma bile acid composition in vivo and the resultant impact on PXR and FXR activation. A cross-sectional study evaluated the plasma concentrations of 12 bile acids in addition to 4β-hydroxycholesterol (4βOHC), an in vivo probe of the PXR target-gene cytochrome 3A4 (CYP3A4) and the FXR target-gene, fibroblast growth factor (FGF) 19 in individuals with (n = 74) and without (n = 71) CD. An in vitro model was used to assess the impact of CD-specific changes in the plasma bile acid composition on PXR and FXR activation. Decreases in glycochenodeoxycholic acid, taurocholic acid and lithocholic acid were seen in CD with increases in glycodeoxycholic acid and glycocholic acid relative to the total plasma bile acid profile. In vitro, increasing concentrations of bile acids applied in the same ratio as seen in the study cohorts resulted in decreased activation of both PXR and FXR in the CD model. In vivo, plasma 4βOHC (CD = 18.68 ng/ml ± 13.02 ng/ml, non-CD = 46.38 ng/ml ± 40.70 ng/ml, p ≤ 0.0001) and FGF19 (CD = 0.276 pg/L ± 0.189 pg/L, non-CD = 0.485 pg/L ± 0.42 pg/L, p = 0.0002) concentrations were lower in CD versus controls. Ultimately, CD-specific changes in the plasma bile acid composition lead to reduced activation of FXR and PXR target genes in vitro and in vivo.

Background: Targeted next-generation sequencing (NGS) enables rapid identification of common and rare genetic variation. The detection of variants contributing to therapeutic drug response or adverse effects is essential for implementation of individualized pharmacotherapy. Successful application of short-read based NGS to pharmacogenes with high sequence homology, nearby pseudogenes and complex structure has been previously shown despite anticipated technical challenges. However, little is known regarding the utility of such panels to detect copy number variation (CNV) in the highly polymorphic cytochrome P450 (CYP) 2D6 gene, or to identify the promoter (TA) 7 TAA repeat polymorphism UDP glucuronosyltransferase (UGT) 1A1*28. Here we developed and validated PGxSeq, a targeted exome panel for pharmacogenes pertinent to drug disposition and/or response., Methods: A panel of capture probes was generated to assess 422 kb of total coding region in 100 pharmacogenes. NGS was carried out in 235 subjects, and sequencing performance and accuracy of variant discovery validated in clinically relevant pharmacogenes. CYP2D6 CNV was determined using the bioinformatics tool CNV caller (VarSeq). Identified SNVs were assessed in terms of population allele frequency and predicted functional effects through in silico algorithms., Results: Adequate performance of the PGxSeq panel was demonstrated with a depth-of-coverage (DOC) ≥ 20× for at least 94% of the target sequence. We showed accurate detection of 39 clinically relevant gene variants compared to standard genotyping techniques (99.9% concordance), including CYP2D6 CNV and UGT1A1*28. Allele frequency of rare or novel variants and predicted function in 235 subjects mirrored findings from large genomic datasets. A large proportion of patients (78%, 183 out of 235) were identified as homozygous carriers of at least one variant necessitating altered pharmacotherapy., Conclusions: PGxSeq can serve as a comprehensive, rapid, and reliable approach for the detection of common and novel SNVs in pharmacogenes benefiting the emerging field of precision medicine.

Objectives: Cisplatin-induced ototoxicity is a common permanent consequence of curative chemoradiation for locally advanced head and neck squamous cell carcinoma (HNSCC). Predictors of ototoxicity in HNSCC were examined., Materials and Methods: In this prospective, observational cohort study, 206 adult HNSCC patients underwent audiometric testing at baseline, during and after treatment with cisplatin-based chemoradiation. Ototoxicity was defined as ≥grade 2 audiometric change from baseline (CTCAE v4.02). Relationships between clinical and pharmacogenetic (TPMT, COMT, ACYP2, CTR1, OCT2, MATE1, ABCC2, ABCC3, and ABCG2) covariates and ototoxicity, progression-free (PFS) and overall survival (OS) were assessed by Cox regression., Results: Weekly cisplatin resulted in lower ototoxicity risk while PFS and OS were similar compared to high dose cisplatin (P = 0.00035; HR = 0.18; 95% CI, 0.07-0.46). COMT (rs9332377) carriers had higher ototoxicity risk (P = 0.00556; HR = 1.72; 95% CI, 1.17-2.52) while MATE1 (rs2289669) A/A carriers were protected from ototoxicity (P = 0.01062; HR = 0.46; 95% CI, 0.26-0.84). Absence of the protective MATE1 allele among those who carry the risk allele in COMT predicted increased ototoxicity risk, (P = 0.00414; HR = 3.22; 95% CI, 1.45-7.17 and P = 0.00022; HR = 4.89; 95% CI, 2.11-11.36). Survival outcomes did not differ between carriers of protective or risk alleles., Conclusions: Weekly cisplatin dosing, COMT and MATE1 are predictors of ototoxicity without affecting treatment efficacy. COMT and MATE1 genotyping and weekly dosing may be a potential strategy for mitigating cisplatin-induced ototoxicity in HNSCC., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: The aromatase inhibitor (AI) letrozole is a first-line drug in the adjuvant treatment of breast cancer in postmenopausal women. Adherence to AI therapy, including letrozole, remains problematic due to the development of debilitating AI-induced arthralgia. Letrozole is metabolized in the liver by CYP2A6. It remains unknown if plasma letrozole levels or CYP2A6 genetic variation is associated with the development of arthralgia., Methods: We enrolled 126 female breast cancer patients initiated on letrozole therapy and prospectively collected blood samples at baseline and two follow-up time points to determine letrozole plasma concentrations and CYP2A6 genotype. At each visit, participants completed two validated questionnaires to assess the severity of arthralgia symptoms., Results: More than half (55%) of patients experienced a significant increase in their arthralgia symptoms after initiation of treatment. The clinical variables of body mass index (P = 0.0003) and age (P = 0.0430) were negatively and positively associated with plasma letrozole concentrations, respectively. CYP2A6 genotype was significantly associated with letrozole levels (P < 0.0001), and increased plasma letrozole levels were observed in patients with CYP2A6 reduced-function genotypes. Plasma levels of letrozole and CYP2A6 genotype were not significantly associated with a change in pain score from baseline., Conclusions: CYP2A6 genotype was a significant predictor of letrozole plasma levels, but was not associated with the development of arthralgia.

Purpose: Tamoxifen is frequently prescribed to prevent breast cancer recurrence. Tamoxifen is a prodrug and requires bioactivation by CYP2D6. Tamoxifen use is often limited by adverse effects including severe hot flashes. There is paucity of prospectively collected data in terms of CYP2D6 genotype and measured tamoxifen, 4-hydroxytamoxifen and endoxifen concentrations in relation to hot flash severity during tamoxifen therapy., Methods: We conducted a longitudinal prospective study of breast cancer patients on tamoxifen (n = 410). At each visit, blood samples were collected, and patients completed a standardized hot flash survey (n = 1144) that reflected hot flash severity during the 7 days prior to the visit. Plasma concentrations of tamoxifen, 4-hydroxytamoxifen, and endoxifen were measured using liquid chromatography-tandem mass spectrometry and genotyping was carried out for CYP2D6. A linear mixed-effects regression analysis assessed the association of covariates in relation to the hot flash severity score (HFSS)., Results: Median age at first assessment was 50 years with 61.9% of patients considered peri-menopausal. Most patients (92.2%) experienced hot flash symptoms with 51.0% having low HFSS (0-4) and 7.32% experiencing HFSS > 25. Age was significantly associated with hot flash severity, with patients aged 45-59 more likely to have higher HFSS. Neither duration of tamoxifen therapy nor observed tamoxifen, endoxifen and 4-hydroxy tamoxifen plasma concentration predicted hot flash severity. Genetic variation in CYP2D6 or CYP3A4 was not predictive of hot flash severity., Conclusions: Hot flash severity during tamoxifen therapy can not be accounted for by CYP2D6 genotype or observed plasma concentration of tamoxifen, 4-hydroxytamoxifen, or endoxifen.

Rosuvastatin is commonly prescribed for the treatment of hypercholesterolemia and hepatic transporter-mediated accumulation in the liver enhances its efficacy. Current guidelines indicate no preference for fed or fasted rosuvastatin administration. We investigated the association between food intake and rosuvastatin disposition in healthy subjects and low-density lipoprotein cholesterol (LDL-C)-lowering effects among patients taking rosuvastatin. We demonstrate that administration with food resulted in a near 40% reduction of rosuvastatin exposure in healthy Asian (n = 12) and Caucasian (n = 11) subjects. Higher rosuvastatin concentrations in Asian subjects also correlated with higher allele frequency of ABCG2 c.421C>A. In mice, a greater rosuvastatin liver:plasma ratio was noted when administered with food. Among rosuvastatin patients (n = 156), there was no difference in dose needed to reach target LDL-C, measured LDL-C, or lathosterol concentrations, when administered in a fed or fasting state. Therefore, taking rosuvastatin with food could reduce systemic concentrations, and subsequent myopathy risk, without compromising LDL-C-lowering benefit., (© 2018 American Society for Clinical Pharmacology and Therapeutics.)

Background: Azathioprine (AZA)-induced pancreatitis is an unpredictable and dose-independent adverse event affecting 2%-7% of patients with inflammatory bowel disease (IBD) patients treated with AZA. There are no tools in clinical practice to identify at-risk individuals; however, a genome wide association study (GWAS) identified a strong association between the Class II HLA gene region polymorphism (rs2647087) and thiopurine-induced pancreatitis., Aim: To independently confirm the findings of the GWAS in an IBD cohort, to evaluate its utility in clinical practice and to offer a novel AZA treatment algorithm for IBD based on pharmacogenomic principles., Methods: A retrospective cohort study evaluated 373 AZA-exposed IBD patients from a tertiary care academic centre in London, Canada. Due to the limited number of patients taking mercaptopurine (MP), such patients were not included this cohort. All subjects underwent screening for the single nucleotide polymorphism (SNP) rs2647087 mapped to the HLA-DQA1*02:01-HLA-DRB1*07:01 haplotype and were sub-divided based on the presence (n = 13) or absence (n = 360) of an AZA-induced pancreatitis diagnosis. The risk of AZA-induced pancreatitis was assessed based on rs2647087 genotype., Results: The risk of pancreatitis during AZA-therapy was highly predictable and genotype dependent: 0.53% for wild type (A/A), 4.25% (OR = 4.19, 95% CI 1.02-36.45, P = 0.044) for heterozygous (A/C), and 14.63% (OR = 15.83, 95% CI 3.80-145.26, P = 0.0001) for homozygous variant (C/C) patients., Conclusions: The class II HLA region (at rs2647087) is an important marker of AZA-induced pancreatitis risk. We propose a simple and clinically implementable algorithm based on rs2647087 and TPMT genotypes for AZA selection and dosing for patients with IBD., (© 2017 John Wiley & Sons Ltd.)

The use of pharmacogenomics to personalize drug therapy has been a long-sought goal for warfarin and tamoxifen. However, conflicting evidence has created reason for hesitation in recommending pharmacogenomics-guided care for both drugs. This review will provide a summary of the evidence to date on the association between cytochrome P450 enzymes and the clinical end points of warfarin and tamoxifen therapy. Further, highlighting the clinical experiences that we have gained over the past ten years of running a personalized medicine program, we will offer our perspectives on the utility and the limitations of pharmacogenomics-guided care for warfarin and tamoxifen therapy., Competing Interests: The authors declare no conflict of interest.

Background: Direct-acting oral anticoagulants (DOACs) are widely prescribed for stroke prevention in patients with atrial fibrillation (AF). An important advantage of DOACs is that routine monitoring of an anticoagulation response is not necessary. Nevertheless, because of their mechanism of action, a DOAC anticoagulation effect can be inferred based on the observed plasma concentration. However, there is a paucity of data relating to observed interpatient variation in DOAC plasma concentrations in the postmarket clinical setting., Methods: We determined rivaroxaban and apixaban plasma concentrations in patients with AF during routine clinic visits., Results: Among 243 patients (rivaroxaban, n = 94; apixaban, n = 149) enrolled in this study, a 60- and 50-fold interpatient variation in plasma concentration was observed for rivaroxaban and apixaban, respectively. Approximately 12% of patients receiving rivaroxaban and 13% of patients receiving apixaban exceeded the 95th percentile for predicted maximum plasma concentration observed in clinical trials., Conclusions: In this routine-care setting, rivaroxaban and apixaban plasma concentrations tended to be more variable than those observed in clinical trials. Identification of additional clinical and molecular determinants that more fully predict patients at risk for excessively high or low DOAC concentrations may enable a more precise DOAC dosing regimen for the individual patient., (Copyright © 2017 Canadian Cardiovascular Society. Published by Elsevier Inc. All rights reserved.)

Trimethylamine-N-oxide (TMAO) is a recently identified predictor of cardiovascular and chronic kidney disease. TMAO is primarily generated through gut-microbiome mediated conversion of dietary choline and carnitine to TMA, which is converted to TMAO by hepatic flavin monooxygenase 3 (FMO3) and subsequently undergoes renal elimination. We investigated the role of uptake and efflux drug transporters in TMAO disposition in vitro and in vivo. After screening a large array of uptake transporters, we show organic cation transporter 2 (OCT2) is the key transporter for TMAO cellular uptake. In Oct1/2 knockout mice, we observed increased plasma TMAO levels with reduced renal retention, suggesting the importance of Oct2 in facilitating the uptake of TMAO into renal tubular cells in vivo. Multiple transporters of the ATP-binding cassette (ABC) family, including ABCG2 (BCRP) and ABCB1 (MDR1), were capable of TMAO efflux. In human subjects, clinical, dietary, and pharmacogenetic covariates were evaluated for contribution to TMAO levels in a cohort of dyslipidemic patients (n = 405). Interestingly, genetic variation in ABCG2, but not other transporters, appeared to play a role in modulating TMAO exposure.

Background: Tamoxifen, a common anti-estrogen breast cancer medication, is a prodrug that undergoes bioactivation via cytochrome P450 enzymes, CYP2D6 and to a lesser degree, CYP3A4 to form the active metabolite endoxifen. With an increasing use of oral anti-cancer drugs, the risk for drug-drug interactions mediated by enzyme inhibitors and inducers may also be expected to increase. Here we report the first case demonstrating a potent drug-drug interaction in a real-world clinical setting between tamoxifen and rifampin in a breast cancer patient being treated concurrently for ulcerative colitis., Case Presentation: We describe a patient on adjuvant tamoxifen therapy for breast cancer that was prescribed rifampin for TB prophylaxis prior to initiation of an anti-tumor necrosis factor (TNF)-α agent due to worsening ulcerative colitis. This 39 year old Caucasian woman had been followed by our personalized medicine clinic where CYP2D6 genotyping and therapeutic monitoring of tamoxifen and endoxifen levels had been carried out. The patient, known to be a CYP2D6 intermediate metabolizer, had a previous history of therapeutic endoxifen levels. Upon admission to hospital for a major flare of her ulcerative colitis a clinical decision was made to initiate an anti-TNFα biological agent. Due to concerns regarding latent TB, rifampin as an anti-mycobacterial agent was initiated which the patient was only able tolerate for 10 days. Interestingly, her plasma endoxifen concentration measured 2 weeks after cessation of rifampin was sub-therapeutic at 15.8 nM and well below her previous endoxifen levels which exceeded 40 nM., Conclusion: Rifampin should be avoided in patients on tamoxifen therapy for breast cancer unless continued tamoxifen efficacy can be assured through endoxifen monitoring. Drug-drug interactions can pose a significant risk of sub-therapeutic benefit in tamoxifen patients.

Background: The gastrointestinal (GI) microbiome is recognized for potential clinical relevance in inflammatory bowel disease (IBD). Data suggest that there is a disease-dependent loss of microbial diversity in IBD. Trimethylamine-N-oxide (TMAO) is generated by GI anaerobes through the digestion of dietary phosphatidylcholine and carnitine in a microbial-mammalian co-metabolic pathway. IBD-related changes in the gut microbiome may result in disease-specific changes in TMAO plasma concentrations., Aim: To determine whether TMAO plasma levels in IBD are altered compared to controls and whether they correlate with disease presence or activity., Methods: Liquid chromatography-tandem mass spectrometry was used to measure TMAO, choline, and carnitine plasma levels in 479 subjects (373 non-IBD controls, 106 IBD). Subjects were also genotyped for the flavin monooxygenase (FMO)3 variants, E158K and E308G., Results: Plasma TMAO levels were 2.27 µM lower in the IBD population compared to the control population (p = 0.0001). Lower TMAO levels were similarly seen in active ulcerative colitis (UC) (1.56 µM) versus inactive disease (3.40 µM) (p = 0.002). No difference was seen in active Crohn's disease (CD) versus inactive CD. No intergroup variation existed in plasma TMAO levels based on FMO3 genotype. Choline levels were higher in IBD, while carnitine levels were similar between the two groups, suggesting that lower TMAO levels in IBD were not due to dietary differences., Conclusions: Decreased TMAO levels are seen in IBD compared to a non-IBD population. These data suggest that TMAO may have potential as a biomarker to support IBD diagnosis as well as to assess disease activity in UC.

Background: Treatment of advanced and metastatic colorectal cancer with irinotecan is hampered by severe toxicities. The active metabolite of irinotecan, SN-38, is a known substrate of drug-metabolising enzymes, including UGT1A1, as well as OATP and ABC drug transporters., Methods: Blood samples (n=127) and tumour tissue (n=30) were obtained from advanced cancer patients treated with irinotecan-based regimens for pharmacogenetic and drug level analysis and transporter expression. Clinical variables, toxicity, and outcomes data were collected., Results: SLCO1B1 521C was significantly associated with increased SN-38 exposure (P<0.001), which was additive with UGT1A1*28. ABCC5 (rs562) carriers had significantly reduced SN-38 glucuronide and APC metabolite levels. Reduced risk of neutropenia and diarrhoea was associated with ABCC2-24C/T (odds ratio (OR)=0.22, 0.06-0.85) and CES1 (rs2244613; OR=0.29, 0.09-0.89), respectively. Progression-free survival (PFS) was significantly longer in SLCO1B1 388G/G patients and reduced in ABCC2-24T/T and UGT1A1*28 carriers. Notably, higher OATP1B3 tumour expression was associated with reduced PFS., Conclusions: Clarifying the association of host genetic variation in OATP and ABC transporters to SN-38 exposure, toxicity and PFS provides rationale for personalising irinotecan-based chemotherapy. Our findings suggest that OATP polymorphisms and expression in tumour tissue may serve as important new biomarkers.

Tamoxifen is a prodrug, requiring cytochrome P450 enzyme-mediated metabolism to form the active metabolite endoxifen. We identified a case of drug-drug interaction involving tamoxifen and phenytoin, associated with a markedly lower endoxifen level than predicted. The patient is a 49-year-old woman, genotyped as a cytochrome P450 2D6 (CYP2D6) extensive metabolizer, chronically taking phenytoin for a seizure disorder. The plasma endoxifen level 2 months after starting tamoxifen was 4.72 nmol/l, the lowest level we have seen in our clinic among patients with CYP2D6 extensive metabolizer genotypes (n=195). To our knowledge, this is the first report documenting the extent of induction in terms of both tamoxifen and endoxifen levels during concomitant phenytoin therapy, and this effect would likely result in loss of therapeutic benefit from tamoxifen. Phenytoin should therefore not be used concurrently with tamoxifen for extended periods of time unless a therapeutic drug (endoxifen) monitoring strategy is utilized.

Tamoxifen use is often limited in some patients due to adverse effects including severe hot flash symptoms. Tamoxifen undergoes hepatic bioactivation by CYP2D6 and CYP3A4 to form the active metabolite endoxifen. It remains unclear whether the extent of attained endoxifen level or genetic polymorphisms in drug metabolizing enzymes is associated with the frequency and severity of hot flashes. We conducted a prospective study using self-reported surveys to assess tamoxifen side effects experienced during the week prior to clinic visits of 132 female breast cancer patients on tamoxifen therapy, and hot flash severity scores were tabulated. At the time of clinic visit, blood samples were obtained to determine tamoxifen and its metabolite levels and to determine CYP2D6 and CYP3A4 genotypes. The majority of participants (77 %) experienced hot flashes, with 11 % experiencing severe or very severe symptoms. We observed an inverse correlation between endoxifen concentration and hot flash severity score following adjustment for age, BMI, and menopausal status in patients with non-zero scores (p < 0.001). Interestingly, CYP2D6 genotype was not significantly associated with hot flash scores in patients on no known inhibitory medications. However, CYP3A4*22 carriers were less likely to have hot flashes with an odds ratio of 8.87 (p < 0.01) even when compared to a cohort with similar endoxifen levels. Our data demonstrate that patients with higher endoxifen levels tended to predict lower hot flash severity scores. Importantly, this is the first study to show CYP3A4*22 genotype as an independent predictor of hot flash severity during tamoxifen therapy.

Developmental competence is the response of a cell(s) to information. Determination of adult labial identity in Drosophila requires Proboscipedia (PB) and Sex combs reduced (SCR); however, co-ectopic expression of PB and SCR is not sufficient for induction of ectopic adult labial identity, because the developmental information supplied by PB and SCR is suppressed. The evolutionarily conserved LASCY, DYTQL, NANGE motifs, and the C-terminal domain of SCR are sequence elements that mediate some, or all, of the suppression of ectopic proboscis determination. Therefore, the developmentally competent primordial proboscis cells provide an environment devoid of suppression, allowing PB and SCR to determine proboscis identity. SCR derivatives lacking suppression sequences weakly induce ectopic proboscis transformations independently of PB, suggesting that SCR may be the activity required for induction of adult labial identity, as is the case for larval labial identity. A possible explanation for PB independence of SCR in determination of adult and embryonic labial identity is PB operates as a competence factor that switches SCR from determining T1 identity to labial identity during metamorphosis. Lastly, labial determination is not conserved between SCR and murine HOXA5, suggesting that SCR has acquired this activity during evolution.

Tamoxifen is a widely utilized adjuvant anti-estrogen agent for hormone receptor-positive breast cancer, known to undergo CYP2D6-mediated bioactivation to endoxifen. However, little is known regarding additional genetic and non-genetic determinants of optimal endoxifen plasma concentration. Therefore, 196 breast cancer patients on tamoxifen were enrolled in this prospective study over a 24-month period. Blood samples were collected for pharmacogenetic and drug-level analysis of tamoxifen and metabolites. Regression analysis indicated that besides CYP2D6, the recently described CYP3A4*22 genotype, seasonal variation, and concomitant use of CYP2D6-inhibiting antidepressants were significant predictors of endoxifen concentration. Of note, genetic variation explained 33 % of the variability while non-genetic variables accounted for 13 %. Given the proposed notion of a sub-therapeutic endoxifen concentration for predicting breast cancer recurrence, we set the therapeutic threshold at 18 nM, the 20th percentile for endoxifen level among enrolled patients in this cohort. Nearly 70 % of CYP2D6 poor metabolizers as well as extensive metabolizers on potent CYP2D6-inhibiting antidepressants exhibited endoxifen levels below 18 nM, while carriers of CYP3A4*22 were twofold less likely to be in sub-therapeutic range. Unexpectedly, endoxifen levels were 20 % lower during winter months than mean levels across seasons, which was also associated with lower vitamin D levels. CYP3A4*22 genotype along with sunshine exposure and vitamin D status may be unappreciated contributors of tamoxifen efficacy. The identified covariates along with demographic variables were integrated to create an endoxifen concentration prediction algorithm to pre-emptively evaluate the likelihood of individual patients falling below the optimal endoxifen concentration.

Tamoxifen is a widely prescribed adjuvant anti-estrogen agent for estrogen receptor-positive breast cancer. Tamoxifen is known to undergo CYP2D6-mediated bioactivation to the active metabolite endoxifen. Endoxifen concentrations exhibit high interindividual variability, contributing to either sub-optimal tamoxifen efficacy or side effects in subsets of patients. However, the relationship between endoxifen exposure and tumor growth inhibition has not been well-characterized and little is known regarding the optimal in vivo endoxifen plasma level required for tumor inhibition. Pharmacokinetics-Pharmacodynamics (PK-PD) modeling was carried out to characterize the relationship between endoxifen concentration and tumor growth inhibition (TGI) in dose-ranging experiments in the human MCF7 xenograft bearing mouse model. Subsequently, simulations using human PK were used to determine the efficacious clinically relevant endoxifen concentration required to produce optimal tumor suppression. Based on the PK-PD model and simulations using clinical PK/concentration data of endoxifen, C stasis (100 % TGI) is observed at 53 nM, a concentration attained by many tamoxifen-treated patients. Importantly, PK-PD simulations indicate that mean steady-state levels observed in CYP2D6 extensive metabolizers are expected to result in optimal tumor suppression while mean concentrations observed in poor metabolizers are predicted to result in suboptimal TGI. Our study is the first to characterize the in vivo PK-PD relationship for endoxifen where clinically observed endoxifen concentrations are associated, in an exposure-dependent manner, with % TGI measured in a xenograft model. It is anticipated that endoxifen concentration achieved in individual patients is the limiting factor for achieving optimal tumor growth suppression.

Tamoxifen is widely prescribed to patients with estrogen receptor-positive breast cancer, and it is a prodrug that requires bioactivation by cytochrome P450 enzymes CYP2D6 and 3A4 to generate the active metabolite, endoxifen. Large interpatient variability in endoxifen plasma levels has been reported, and polymorphisms in CYP2D6 have been implicated as a major determinant of such variability. However, little is known regarding the role of drug transporters such as P-glycoprotein [multidrug resistance 1 (MDR1), ATP-binding cassette B1 (ABCB1)] to endoxifen disposition and response. Therefore, we determined the ability of P-glycoprotein to transport endoxifen in vitro, using a polarized human P-glycoprotein-overexpressing cell line. Markedly higher transport of endoxifen was observed in the basal-to-apical direction, which was abrogated in the presence of the potent and specific P-glycoprotein inhibitor (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). To validate the in vivo relevance of P-glycoprotein to endoxifen disposition, plasma and tissue concentrations were also determined in Mdr1a-deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wild-type and Mdr1a-deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a-deficient mice compared to wild-type mice. Because P-glycoprotein is highly expressed at the blood-brain barrier and in some breast cancer tumors, variation in expression and function of this transporter may alter central nervous system entry and the attained intracellular concentration in such breast cancer cells and therefore may prove to be of relevance to therapeutic outcome.

Background: CTLA-4 functions primarily as an inhibitor of T cell activation. There are several candidate explanations as to how CTLA-4 modulates T cell responses, but the exact mechanism remains undefined. The tail of CTLA-4 does not have any intrinsic enzymatic activity but is able to associate with several signaling molecules including the serine/threonine phosphatase PP2A. PP2A is a heterotrimeric molecule comprised of a regulatory B subunit associated with a core dimer of a scaffolding (A) and a catalytic (C) subunit., Results: Here, we performed an analysis of the human CTLA-4 interface interacting with PP2A. We show that PP2A interacts with the cytoplasmic tail of CTLA-4 in two different sites, one on the lysine rich motif, and the other on the tyrosine residue located at position 182 (but not the tyrosine 165 of the YVKM motif). Although the interaction between CTLA-4 and PP2A was not required for inhibition of T cell responses, it was important for T cell activation by inverse agonists of CTLA-4. Such an interaction was functionally relevant because the inverse agonists induced IL-2 production in an okadaic acid-dependent manner., Conclusion: Our studies demonstrate that PP2A interacts with the cytoplasmic tail of human CTLA-4 through two motifs, the lysine rich motif centered at lysine 155 and the tyrosine residue 182. This interaction and the phosphatase activity of PP2A are important for CTLA-4-mediated T cell activation.

Although cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) inhibits T cell activation when ligated by B7 molecules on antigen-presenting cells, it can also act as an activating receptor when binding certain soluble recombinant ligands known as inverse agonists. Following ligation with an inverse agonist, we observed CTLA-4 microclusters evenly distributed on the T cell surface over a 60-min period. We have previously shown that the inverse agonist properties of these ligands correlate with their capacity to induce the formation of large CTLA-4 oligomers that are distinctly different from those resulting by CTLA-4 engagement with membrane-bound B7. These oligomers are composed of CTLA-4 molecules expressed on the cell surface and decrease from both the soluble cell lysate and lipid rafts upon cellular fractionation. Formation of these inverse agonist-induced CTLA-4 oligomers does not require an intact actin cytoskeleton. However, modulation of these oligomers was partially blocked upon actin depolymerization. Retention of CTLA-4 oligomers on the cell surface correlated with enhanced T cell signaling. Together, our data further characterize the structural basis of inverse agonist properties for CTLA-4 ligands that may be used in the design and screening of therapeutic biologicals targeting this receptor.

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.

The onset of adaptive immune responses includes the presentation of foreign antigenic peptides to T-cells, and the formation of a T-cell-antigen-presenting cell interface termed the immunological synapse (IS). Although the generation of a mature IS is thought to be the hallmark of T-cell activation, new evidence suggests that microclusters ofat signaling molecules at the periphery of the IS are responsible for initiating and maintaining T-cell activation while the core of the IS provides a platform for signal downregulation. In this context, costimulatory molecules and self-peptides contribute to sustain the signaling required for T-lymphocyte differentiation into effector cells. This review discusses these aspects in the identification of novel candidates for therapeutic modulation of immune responses.

TCRalphabeta+CD4-CD8- double-negative (DN) T regulatory (Treg) cells have recently been shown to suppress Ag-specific immune responses mediated by CD8+ and CD4+ T cells in humans and mice. Our previous study using cDNA microarray analysis of global gene expression showed that FcRgamma was the most highly overexpressed gene in functional DN Treg cell clones compared with nonfunctional mutant clones. In this study, we demonstrate that FcRgamma-deficient DN T cells display markedly reduced suppressive activity in vitro. In addition, unlike FcRgamma-sufficient DN T cells, FcRgamma-deficient DN T cells were unable to prolong donor-specific allograft survival when adoptively transferred to recipient mice. Protein analyses indicate that in addition to FcRgamma, DN Treg cell clones also express higher levels of TCRbeta, while mutant clones expressed higher levels of Zap70 and Lck. Within DN Treg cells, we found that FcRgamma associates with the TCR complex and that both FcRgamma and Syk are phosphorylated in response to TCR cross-linking. Inhibition of Syk signaling and FcRgamma expression were both found to reduce the suppressive function of DN Treg cells in vitro. These results indicate that FcRgamma deficiency significantly impairs the ability of DN Treg cells to down-regulate allogeneic immune responses both in vitro and in vivo, and that FcRgamma plays a role in mediating TCR signaling in DN Treg cells.

Within the paradigm of the two-signal model of lymphocyte activation, the interest in costimulation has witnessed a remarkable emergence in the past few years with the discovery of a large array of molecules that can serve this role, including some with an inhibitory function. Interest has been further enhanced by the realization of these molecules' potential as targets to modulate clinical immune responses. Although the therapeutic translation of mechanistic knowledge in costimulatory molecules has been relatively straightforward, the capacity to target their inhibitory counterparts has remained limited. This limited capacity is particularly apparent in the case of the cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), a major negative regulator of T cell responses. Because there have been several previous comprehensive reviews on the function of this molecule, we focus here on the physiological implications of its structural features. Such an exercise may ultimately help us to design immunotherapeutic agents that target CTLA-4.

Arista versus tarsus determination is well investigated in Drosophila, yet it remains unresolved whether Antennapedia (ANTP) cell autonomously or noncell autonomously determines tarsus identity and whether Sex combs reduced (SCR) is the HOX protein required for normal tarsus determination. Three observations rule out a cell autonomous role for ANTP in tarsus determination. (i) Clonal ectopic overexpression of ANTP did not repress the expression of the arista determining protein Homothorax (HTH) in early 3rd stadium antennal imaginal discs. (ii) Clonal ectopic expression of ANTP did not transform the arista to a tarsus. (iii) Ectopic overexpression of ANTP, Labial (LAB), Deformed (DFD), SCR, Ultrabithorax (UBX), Abdominal-A (ABD-A), or Abdominal-B (ABD-B), using the dppGAL4 driver, resulted in arista-to-tarsus transformations, and repressed HTH/Extradenticle (EXD) activity noncell autonomously in early 3rd stadium antennal imaginal discs. SCR may not be the HOX protein required for normal tarsus determination, because co-ectopic expression of Proboscipedia (PB) inhibited the arista-to-tarsus transformations induced by ectopic expression of DFD, SCR, ANTP, UBX, ABD-A, and ABD-B. The proposal that SCR is the HOX protein required for normal tarsus determination is dependent on SCR being the sole target of PB suppression, which is not the case. Therefore, the possibility exists that normal tarsus determination is HOX independent.

Abs or their recombinant fragments against surface receptors of the Ig superfamily can induce or block the receptors' native function depending on whether they induce or prevent the assembly of signalosomes on their cytoplasmic tails. In this study, we introduce a novel paradigm based on the observation that a bispecific tandem single-chain variable region fragment ligand of CTLA-4 by itself converts this inhibitory receptor into an activating receptor for primary human T lymphocytes. This reversal of function results from increased recruitment of the serine/threonine phosphatase 2A to the cytoplasmic tail of CTLA-4, consistent with a role of this phosphatase in the regulation of CTLA-4 function, and assembly of a distinct signalosome that activates an lck-dependent signaling cascade and induces IL-2 production. Our data demonstrate that the cytoplasmic domain of CTLA-4 has an inherent plasticity for signaling that can be exploited therapeutically with recombinant ligands for this receptor.