Search

The therapeutic efficacy of tamoxifen is predominantly mediated by its active metabolites 4-hydroxy-tamoxifen and endoxifen, whose formation is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6). Yet, known CYP2D6 polymorphisms only partially determine metabolite concentrations in vivo. We performed the first cross-ancestry genome-wide association study with well-characterized patients of European, Middle-Eastern, and Asian descent (N = 497) to identify genetic factors impacting active and parent metabolite formation. Genome-wide significant variants were functionally evaluated in an independent liver cohort (N = 149) and in silico. Metabolite prediction models were validated in two independent European breast cancer cohorts (N = 287, N = 189). Within a single 1-megabase (Mb) region of chromosome 22q13 encompassing the CYP2D6 gene, 589 variants were significantly associated with tamoxifen metabolite concentrations, particularly endoxifen and metabolic ratio (MR) endoxifen/N-desmethyltamoxifen (minimal P = 5.4E-35 and 2.5E-65, respectively). Previously suggested other loci were not confirmed. Functional analyses revealed 66% of associated, mostly intergenic variants to be significantly correlated with hepatic CYP2D6 activity or expression (ρ = 0.35 to -0.52), and six hotspot regions in the extended 22q13 locus impacting gene regulatory function. Machine learning models based on hotspot variants (N = 12) plus CYP2D6 activity score (AS) increased the explained variability (~ 9%) compared with AS alone, explaining up to 49% (median R2 ) and 72% of the variability in endoxifen and MR endoxifen/N-desmethyltamoxifen, respectively. Our findings suggest that the extended CYP2D6 locus at 22q13 is the principal genetic determinant of endoxifen plasma concentration. Long-distance haplotypes connecting CYP2D6 with adjacent regulatory sites and nongenetic factors may account for the unexplained portion of variability.

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is a key to tumor progression. Here, we deconvoluted bulk tumor transcriptomes to infer cross-talk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in situ hybridization and IHC data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune cross-talk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of cross-talk in the TME.Significance:This study provides deconvoluted bulk tumor transcriptomes across multiple cancer types to infer cross-talk in the tumor microenvironment.

Supplementary results and methods

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and debilitating disease with limited therapeutic options. The aim of this clinical study was to evaluate the safety, efficacy and pharmacokinetics of a novel regimen comprised of metronomic oxaliplatin (O), chronomodulated capecitabine (X) and UGT1A1 genotype-guided dosing of irinotecan (IRI) [OXIRI] as well as its immunomodulatory effects. Thirty-six patients were enrolled into either dose-escalation or expansion cohorts. In the dose escalation phase, capecitabine doses (2000, 2650, 3500 and 4500 mg/day) were administered at midnight on days 1 to 14 while oxaliplatin and irinotecan were intravenously infused at fixed doses of 50 and 75 mg/m

Endoxifen is one of the most important metabolites of the prodrug tamoxifen. High interindividual variability in endoxifen steady-state concentrations (CSS,min ENDX ) is observed under tamoxifen standard dosing and patients with breast cancer who do not reach endoxifen concentrations above a proposed therapeutic threshold of 5.97 ng/mL may be at a 26% higher recurrence risk compared with patients with endoxifen concentrations exceeding this value. In this investigation, 10 clinical tamoxifen studies were pooled (1,388 patients) to investigate influential factors on CSS,min ENDX using nonlinear mixed-effects modeling. Age and body weight were found to significantly impact CSS,min ENDX in addition to CYP2D6 phenotype. Compared with postmenopausal patients, premenopausal patients had a 30% higher risk for subtarget CSS,min ENDX at tamoxifen 20 mg per day. In treatment simulations for distinct patient subpopulations, young overweight patients had a 3.1-13.8-fold higher risk for subtarget CSS,min ENDX compared with elderly low-weight patients. Considering ever-rising obesity rates and the clinical importance of tamoxifen for premenopausal patients, this subpopulation may benefit most from individualized tamoxifen dosing. ispartof: CLINICAL PHARMACOLOGY & THERAPEUTICS vol:108 issue:3 pages:661-670 ispartof: location:United States status: published

AIM This pilot study aimed to compare the pharmacokinetic profiles of oral (PO) and intravenous (IV) ibuprofen for treatment of patent ductus arteriosus (PDA) in preterm neonates. METHODS In a single-centre, parallel, randomised open-label trial, neonates ≤35 weeks, weight

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is a key to tumor progression. Here, we deconvoluted bulk tumor transcriptomes to infer cross-talk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. This approach recovered known transcriptional hallmarks of cancer and stromal cells and was concordant with single-cell, in situ hybridization and IHC data. Inferred autocrine cancer cell interactions varied between tissues but often converged on Ephrin, BMP, and FGFR-signaling pathways. Analysis of immune checkpoints nominated interactions with high levels of cancer-to-immune cross-talk across distinct tumor types. Strikingly, PD-L1 was found to be highly expressed in stromal rather than cancer cells. Overall, our study presents a new resource for hypothesis generation and exploration of cross-talk in the TME. Significance: This study provides deconvoluted bulk tumor transcriptomes across multiple cancer types to infer cross-talk in the tumor microenvironment.

Current technologies to measure drug–target interactions require complex processing and invasive tissue biopsies, limiting their clinical utility for cancer treatment monitoring. Here we develop an analytical platform that leverages circulating extracellular vesicles (EVs) for activity-based assessment of tumour-specific drug–target interactions in patient blood samples. The technology, termed extracellular vesicle monitoring of small-molecule chemical occupancy and protein expression (ExoSCOPE), utilizes bio-orthogonal probe amplification and spatial patterning of molecular reactions within matched plasmonic nanoring resonators to achieve in situ analysis of EV drug dynamics. It measures changes in drug occupancy and protein composition in molecular subpopulations of EVs. When used to monitor various targeted therapies, the ExoSCOPE revealed EV signatures that closely reflected cellular treatment efficacy. We further applied the technology for clinical cancer diagnostics and treatment monitoring. Using a small volume of blood, the ExoSCOPE accurately classified disease status and rapidly distinguished between targeted treatment outcomes, within 24 h after treatment initiation. In this work, the authors develop a platform that leverages extracellular vesicles to measure drug–target engagement and apply it to monitor the outcomes of targeted treatments in lung cancer patients.

Background: Genetic variants of TPMT and NUDT15 have been reported to predict the inter-patient variability in response and toxicity profiles of patients receiving thiopurine therapy. However, the clinical utility of TPMT genotyping in guiding thiopurine doses has been questionable, in part due to underlying differences in the prevalence of TPMT variants in both Caucasian and Asian populations. Several NUDT15 variants have been associated with thiopurine-induced leukopenia, particularly in Asian cohorts. So far, none have been reported in a multiethnic Asian population. Aim: To investigate the associations between TPMT and NUDT15 variants with thiopurine-induced myelotoxicity in 129 Asian inflammatory bowel disease patients. Materials & methods: Pyrosequencing was performed to screen for TPMT and NUDT15 variants. Intracellular steady-state metabolite concentrations were quantified using liquid chromatography–tandem mass spectrometry. Results: Significant declines in nadir white blood cell, absolute neutrophil count and platelet counts were observed with increasing copy numbers of the risk T allele at NUDT15 c.415C>T locus (overall p < 0.05) within 4, 8 and 12 weeks and 6 months after thiopurine initiation. Patients with low and intermediate NUDT15 activity, as inferred from haplotype pairs, had significantly higher risks of leukopenia (p = 0.000253) and neutropenia (p = 0.002) compared with patients with normal NUDT15 activity. Conclusion: These findings highlight the critical relevance of NUDT15 pharmacogenetics in predicting for thiopurine-induced myelotoxicity and confirm the lack of significance of TPMT variants in Asian inflammatory bowel disease patients.

International audience; Exfoliation syndrome (XFS) is the commonest known risk factor for secondary glaucoma and a significant cause of blindness worldwide. Variants in two genes, LOXL1 and CACNA1A have been previously associated with XFS. To further elucidate the genetic basis of XFS, we collected a global sample of XFS cases to refine the association at LOXL1, which previously showed inconsistent results between populations,and to identify new variants associated with XFS. We identified a rare, protective allele at LOXL1 (p.407Phe, OR= 25, P=2.9 x 10-14) through deep resequencing of XFS cases and controls from 9 countries. This variant results in increased cellular adhesion strength compared to the wild-type (p.407Tyr) allele. A genome-wide association study (GWAS) of XFS cases and controls from 24 countries followed by replication in 18 countries identified seven genome-wide significant loci (P < 5 x 10-8). Index variants at the new loci map to chromosomes 13q12 (POMP), 11q23.3 (TMEM136), 6p21 (AGPAT1), 3p24 (RBMS3) and 5q23 (near SEMA6A). These findings provide biological insights into the pathology of XFS, and highlight a potential role for naturally occurring rare LOXL1 variants in disease biology.

The critical role of lysine demethylase 4A (KDM4A), in regulating chromatin structure and consequently in driving cellular proliferation and oncogenesis has been the focus of recent studies. Non-small-cell lung cancer (NSCLC) patients with adenocarcinoma histology who were homozygous for KDM4A single nucleotide polymorphism (SNP)-A482 (rs586339) were recently shown to have significantly worse overall survival (OS) compared with patients with the wild-type or the heterozygous genotype at this locus (hazard ratio=1.68, P=0.042). In the current study, we investigated the association between the same polymorphism with OS in our Asian NSCLC-adenocarcinoma patients comprising Chinese (N=572), Malays (N=50), and Indians (N=22). KDM4A SNP-A482 genotype status was determined by Sanger sequencing. OS was calculated from the date of diagnosis to date of death or censored at the date of last follow-up. Kaplan-Meier analysis, log-rank test, and Cox regression methods were utilized to evaluate OS outcomes. KDM4A SNP-A482 had a minor allele (C) frequency of 18.8% and a major allele (A) frequency of 81.2% in our Asian NSCLC (adenocarcinoma) patients. However, the OS in our Asian NSCLC patients homozygous for KDM4A SNP-A482 was not significantly different from those who were wild type or heterozygous at this locus [CC vs. AA/AC: median OS (95% confidence interval): 40.2 (18.7-61.6) vs. 29.6 (26.9-32.3) months; P=0.858]. The results remained statistically nonsignificant even after adjustment for epidermal growth factor receptor mutational status, suggesting that KDM4A SNP-A482 does not significantly influence OS in Asian NSCLC patients.

Background Clozapine remains the only antipsychotic with unique efficacy in treatment resistant schizophrenia (TRS). Considerable ongoing research and clinical efforts have focused on reducing barriers to clozapine use in a bid to increase rates of clozapine use to improve outcomes in TRS. However, less than half of individuals with TRS respond to clozapine, with the remainder categorised as clozapine resistant (CRS) or Ultra-resistant. It is important to deepen understanding on the development of CRS so we can identify them early, attempt to prevent/delay its onset and also explore novel treatments. In the present study, we sought to compare clozapine responders from CRS and identify factors which might be associated with CRS. Methods This study was conducted at the Institute of Mental Health, the only psychiatric facility with clozapine services in Singapore. Individuals with TRS on clozapine for at least 12 weeks and the capacity to give informed consent were enrolled into the study. Each participant underwent a clinical assessment on the Structured Clinical Interview DSM-IV-TR (SCID), Positive and Negative Syndrome Scale (PANSS) and Social Occupational Functioning Assessment Scale (SOFAS). We applied the Treatment Response and Resistance In Psychosis consensus criteria to define clozapine response and resistance. Brief Neurocognitive Assessment was done using the digit sequencing and symbol coding tasks from the Brief Assessment of Cognition in Schizophrenia (BACS). A fasting sample of blood was collected; assays for clozapine and norclozapine were performed using high performance liquid chromatography. To be classified as CRS, a participant must meet the below criteria: (i) not meeting symptom remission criteria on the PANSS using the Remission in Schizophrenia Working Group criteria, (ii) moderate and lower level of functioning on the SOFAS, and (iii) at least 12 weeks of clozapine treatment and plasma clozapine levels ≥ 350 ng/ml. Results A total of 91 participants were enrolled in this study and 67 (73.6%) met criteria for CRS. 1 in 4 clozapine responders had plasma clozapine levels not exceeding 350 ng/ml. There were no significant differences in age, sex, smoking status, age at onset of illness, clozapine adherence, antipsychotic polypharmacy rates and duration of clozapine use between clozapine responders and CRS. Compared to clozapine responders, individuals with CRS had statistically significantly lower BMI (26.4 vs. 23.4, p=0.008) lower rates of employment (79.2% vs. 35.8%, p Discussion Our study highlighted a high rate of CRS and suggests that poorer cognitive function, specifically in processing speed, might be associated with the development of clozapine resistance in schizophrenia. Additionally, it was interesting to note that a higher BMI was associated with clozapine response; this lends weight to existing metabolic and lipid hypotheses of schizophrenia.

Background Thiopurines are recommended for maintenance of steroid-free remission in inflammatory bowel disease (IBD). Thiopurine metabolite monitoring (MM) is increasingly used in the Western population. However, it remains a new strategy in Singapore with limited information on its therapeutic and economic benefits. Hence, this study aims to investigate the clinical utility of MM and its impact on healthcare resource utilisation in Singaporean IBD patients. Methods A retrospective observational study was conducted at the Singapore General Hospital outpatient IBD Centre. Patients with IBD, baseline MM during 2014–2017 and weight-based thiopurine doses for ≥4 weeks were followed up for 1 year. Actions taken to optimise therapy, metabolite levels before and after the first action were documented. Outcomes assessed included steroid-free remission (SFR), clinical remission (defined as SFR, CRP Results Ninety IBD patients (50 CD, 40 UC) were included. Among them, 40% had baseline metabolite levels within therapeutic range, 31.1% sub-therapeutic, 21.1% supra-therapeutic and 7.8% shunters. Repeated MM with subsequent dose optimisation helped 67.2% of patients achieve therapeutic levels after 1 year. In particular, dose optimisation and reinforcement of adherence in the sub-therapeutic group recaptured clinical remission in almost 50% of the patients. Overall, 86.7% of patients achieved steroid-free remission and 90% had no therapy escalation or surgery (Table 1). Despite greater outpatient visits and laboratory investigations with MM, the median total healthcare costs at 1 year only increased marginally (S$6,407.66 [shunters] vs. S$5,215.20 [supra-therapeutic] vs. S$4,970.80 [sub-therapeutic] vs. S$4,370.48 [control (within therapeutic range)], p = 0.592) (Table 2). Conclusion MM guided timely dose optimisation or therapy escalation for non-responders, identification of non-adherence and reversal of shunting. Therefore, it is a useful clinical tool to optimise thiopurines without significantly increasing healthcare resource costs.

Signaling between cancer and nonmalignant (stromal) cells in the tumor microenvironment (TME) is key to tumorigenesis yet challenging to decipher from tumor transcriptomes. Here, we report an unbiased, data-driven approach to deconvolute bulk tumor transcriptomes and predict crosstalk between ligands and receptors on cancer and stromal cells in the TME of 20 solid tumor types. Our approach recovers known transcriptional hallmarks of cancer and stromal cells and is concordant with single-cell and immunohistochemistry data, underlining its robustness. Pan-cancer analysis reveals previously unrecognized features of cancer-stromal crosstalk. We find that autocrine cancer cell cross-talk varied between tissues but often converged on known cancer signaling pathways. In contrast, many stromal cross-talk interactions were highly conserved across tumor types. Interestingly, the immune checkpoint ligand PD-L1 was overexpressed in stromal rather than cancer cells across all tumor types. Moreover, we predicted and experimentally validated aberrant ligand and receptor expression in cancer cells of basal and luminal breast cancer, respectively. Collectively, our findings validate a data-driven method for tumor transcriptome deconvolution and establishes a new resource for hypothesis generation and downstream functional interrogation of the TME in tumorigenesis and disease progression.

Thiopurines are used in the treatment of inflammatory bowel disease (IBD) but remain clinically challenging to manage due to wide interpatient variability in clinical outcomes and adverse events. Apart from genetic variants in thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) genes, polymorphisms in FTO alpha-ketoglutarate dependent dioxygenase (FTO) were found predictive of thiopurine-induced leukopenia, albeit with conflicting results. To clarify the role of FTO variants in a multiethnic Asian IBD cohort, we recruited 149 patients on thiopurine-based therapy and genotyped two FTO variants p.Ala134Thr (rs79206939) and rs16952570 T C using Sanger sequencing. FTO p.Ala134Thr (rs79206939) was non-polymorphic and absent whereas intronic rs16952570 T C was equally prevalent in Chinese (22%) and Indians (18%) and higher in Malays (28%). Higher nadir white blood cell (WBC) and absolute neutrophil count (ANC) levels were observed in patients harboring FTO rs16952570 CC genotypes compared with TT carriers at 4, 8, and 12 weeks after start of thiopurine therapy (P 0.05). A similar trend was observed in patients carrying the previously well-characterized NUDT15 rs116855232 wild-type CC genotypes. Further in silico analysis suggests that FTO variants linked to rs16952570, particularly rs74018601, may play a regulatory role in altering the FTO expression. The findings from this study indicate a novel protective association with the FTO variant rs16952570 CC genotype and hematological parameters.

Drug efflux and influx transporters play critical roles in regulating the cellular drug disposition and modulating the pharmacokinetics and pharmacodynamics of anti-cancer agents, which may potentially alter treatment outcomes. The efficiency of drug transport is often dependent on the expression and activity of these membrane-bound proteins, factors which have been shown to be regulated by genes that are known to be highly polymorphic in different ethnic populations. The role of drug transporters becomes even more critical for anti-cancer agents due to the narrow therapeutic windows that separate treatment response and toxicities for these agents. Moreover, high inter-individual variability in the disposition of anti-cancer agents often results in variable treatment outcomes among patients receiving standard doses of the same drug. Such variability has been attributed at least in part to polymorphisms in genes encoding drug-metabolizing enzymes and transporter. To date, numerous pharmacogenetic studies have investigated the associations between variants in the ABC and SLC transporters genes with drug disposition, treatment outcomes and drug-induced toxicities. However, the strengths of these associations and their clinical relevance in different ethnic populations have not been critically examined. This review aims to summarize and evaluate the implications of pharmacogenetic variants in the ABC and SLC transporters genes on the pharmacokinetics and clinical outcomes of three anti-cancer agents: irinotecan, docetaxel and doxorubicin in Caucasian and Asian patients.

Des-aspartate-angiotensin I (DAA-I) is an endogenous angiotensin peptide and a prototype angiotensin receptor agonist (ARA). It acts on the angiotensin AT1 receptor and antagonises the deleterious actions of angiotensin II. DAA-I attenuates animal models of human disease in which angiotensin II has been implicated, such as cardiac hypertrophy, neointima formation, arteriosclerosis, renal failure, post-infarction injuries, diabetes, viral infection, chemical-induced inflammation, heat stroke, cancer, and gamma radiation lethality. DAA-I crosses Caco-2 cells and is effective at sub-nanomolar concentrations. These two properties are responsible for its oral efficacy. A single dose-escalation study was conducted to evaluate the safety, tolerability and pharmacokinetics of orally administered DAA-I in 18 healthy subjects. DAA-I was safe and well tolerated by the subjects, who were administered either 0.08, 0.70 or 1.50 mg/kg of the compound. The heart rate and systolic and diastolic blood pressures determined at each post-dose measurement remained within the clinically acceptable range. Across all cohorts, DAA-I had no substantial effect on blood pressures compared with placebo. Electrocardiographs (ECGs) were normal, and none of the subjects complained of chest discomfort. All clinical laboratory tests obtained before and after DAA-I and placebo treatment were normal. Pharmacokinetic analysis over a 12-h period following DAA-I administration did not show any increase of its level beyond basal concentration. This is in line with studies showing that intravenously administered DAA-I is rapidly metabolized and has a short half-life. We postulate that, during its short systemic sojourn, DAA-I exerts its actions via biased agonism on the angiotensin AT1 receptor. The ClinicalTrial.gov assignment number for this study is NCT02666196.

This study investigated the impact of ABCB5, ABCC5 and RLIP76 polymorphisms on doxorubicin pharmacokinetics in Asian breast cancer patients (N=62). Direct sequencing was performed to screen for previously identified ABCC5 polymorphisms as well as polymorphisms in the exons and exon-intron boundaries of ABCB5 and RLIP76 genes. Genotype-phenotype correlations were analyzed using Mann-Whitney U-test. The homozygous variant allele at the ABCC5 g.+7161G>A (rs1533682) locus was significantly associated with higher doxorubicin clearance (g.+7161AA vs g.+7161GG, CL/BSA (Lh-1m-2): 30.34 (25.41-33.60) vs 22.46 (15.04-49.4), P=0.04). Homozygosity for the reference allele at the ABCC5 g.-1679T>A locus was associated with significantly higher doxorubicinol exposure (g.-1679TT vs g.-1679TA, AUC0-∞/dose/BSA (hm-5): 15.48 (6.18-67.17) vs 8.88 (3.68-21.71), P=0.0001). No significant influence of the three newly identified ABCB5 polymorphisms (c.2T>C, c.343A>G and c.1573G>A) on doxorubicin pharmacokinetics was observed. No polymorphisms were identified in the RLIP76 gene. These findings suggest that ABCC5 polymorphisms may explain partially the interpatient variability in doxorubicin disposition.

AIM The aim was to examine the influence of CYP2C19 variants and associated haplotypes on the disposition of tamoxifen and its metabolites, particularly norendoxifen (NorEND), in Asian patients with breast cancer. METHODS Sixty-six CYP2C19 polymorphisms were identified in healthy Asians (n = 240), of which 14 were found to be tightly linked with CYP2C19*2, CYP2C19*3 and CYP2C19*17. These 17 SNPs were further genotyped in Asian breast cancer patients receiving tamoxifen (n = 201). Steady-state concentrations of tamoxifen and its metabolites were quantified using liquid chromatography–mass spectrometry. Non-parametric tests and regression methods were implemented to evaluate genotypic–phenotypic associations and haplotypic effects of the SNPs. RESULTS CYP2C19 functional polymorphisms and their linked SNPs were not significantly associated with plasma concentrations of tamoxifen and its main metabolites N-desmethyltamoxifen, (Z)-4-hydroxytamoxifen and (Z)-Endoxifen. However, CYP2C19*2 and its seven linked SNPs were significantly associated with lower NorEND concentrations, MRNorEND/NDDM and MRNorEND/(Z)-END. Specifically, patients carrying the CYP2C19*2 variant allele A had significantly lower NorEND concentrations [median (range), GG vs. GA vs. AA: 1.51 (0.38–3.28) vs. 1.28 (0.30–3.36) vs. 1.15 ng ml−1 (0.26–2.45, P = 0.010)] as well as significantly lower MRNorEND/(Z)-END [GG vs. GA vs. AA: 9.40 (3.27–28.35) vs. 8.15 (2.67–18.9) vs. 6.06 (4.47–14.6), P < 0.0001] and MRNorEND/NDDM [GG vs. GA vs. AA: 2.75 (0.62–6.26) vs. 2.43 (0.96–4.18) vs. 1.75 (1.10–2.49), P < 0.00001]. CYP2C19 H2 haplotype, which included CYP2C19*2, was also significantly associated with lower NorEND concentrations (P = 0.0020), MRNorEND/NDDM (P < 0.0001) and MRNorEND/(Z)-END (P < 0.0001), indicating significantly lower formation rates of NorEND. CONCLUSION These data highlight the potential relevance of CYP2C19 pharmacogenetics in influencing NorEND concentrations in tamoxifen-treated patients, which may influence treatment outcomes.

730 Background: Tumoral KRAS mutations (KRAS mt) are detected in ~80% of APC and associated with a negative prognosis. Digital droplet PCR (ddPCR) has high sensitivity for circulating KRASmtbut narrower gene coverage. NGS using hybrid-capture methods has reported ctDNA KRASmtin ~30% of patients (pt). We previously presented clinical results of the Phase I trial of OXIRI (GI ASCO 18 #411). We now present the first prospective evaluation of ctDNA in APC by an amplicon-based NGS approach in this Phase I trial. Methods: Paired ctDNA and CA19-9 samples were taken at baseline, C2D1, C3D1 and end of trial. A targeted panel with error-correction (Lucence Diagnostics) was used to detect for mtin KRAS, TP53, SMAD4, CDKN2A, CTNNB1, GNAS, APC and MYC. CT scans were performed every 2 cycles. Survival curves by Kaplan Meier were compared by mutational status, ctDNA and CA19-9 response using the log-rank test. Spearman correlation of ctDNA and CA19-9 changes was performed. Results: ctDNA mtwas detected at baseline in 19/23 (83%) samples, comprising KRAS 17/23 (73%), TP53 (61%), SMAD4 (48%) and CDKN2A (30%). KRAS mtand SMAD4 mt conferred a negative prognosis for overall survival with a hazard ratio of 4.2 (CI: 1.6-10.4; p = 0.01) and 2.8 (CI: 0.9-8.65; p = 0.01) respectively. Drop in ctDNA and CA19-9 was associated with a trend for longer progression free survival at C2D1 (both) and C3D1 (ctDNA only). Radiological partial response (PR) was associated with lower ctDNA in 5/5 pt and CA 19-9 in 4/5 pt. Decrease in ctDNA and CA19-9 was associated with disease control (PR/SD) at C2D1 in 11/14 pt and 10/10 pt; at C3D1 in 11/12 pt and 6/7 respectively. No significant correlation between the amplitude of CA19-9 and ctDNA changes was found. Conclusions: ctDNA could be detected in 83% of pts of whom KRAS mtrates were similar to reports using tissue NGS. Determination of RAS mtand SMAD4 mtin ctDNA may aid in the prognostication of pts and decrease in ctDNA levels may predict for treatment benefit, similar in extent to CA 19-9. This may be particularly useful in non-CA19-9 secreting APC as an adjunct to imaging. Clinical trial information: NCT02368860.

Background To date, biomarkers to predict benefit from anti-angiogenic therapy are still lacking. Sorafenib and metronomic oral vinorelbine combination was studied and changes in blood and DCE-MRI parameters were investigated as biomarkers for benefit. Material and methods Patients with advanced NSCLC were recruited to 3 successive cohorts. Each cohort was given a fixed metronomic (3 times a week) dose of oral vinorelbine at 60 mg/week, 90 mg/week, or 120 mg/week respectively. Within each cohort, patients received a starting dose of sorafenib at 200 mg bid for 4 weeks. In the absence of dose-limiting toxicities, each patient's dose of sorafenib was escalated to 400 mg bid for 4 weeks, 600 mg bid for 4 weeks and finally 800 mg bid. Biomarkers measured include DCE-MRI parameters, circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPs), and plasma thrombospondin (TSP-1). Results 48 evaluable patients were analyzed. There were 4 (8.9%) patients with partial response (PR) and 7 (15.2%) with cavitary response (CaR). Two subpopulations of CECs (CEChi, CEClo) were identified that trended in opposite directions during treatment, with CEChi demonstrating an upward trend in contrast to CEClo. Higher baseline CEChi and lower baseline blood flow (F) and fractional intravascular blood volume (V1) predicted for response. Multivariate analysis revealed a lower baseline V1, and dynamic changes of CEC during treatment (CEC increase, sum of CEChi and CEClo) predicted for improved survival. Conclusions Sorafenib and metronomic oral vinorelbine is active in advanced NSCLC. Baseline levels and changes in DCE parameters and CEC may be useful predictive biomarkers.

Summary Objectives Uric acid may activate an innate immune response in osteoarthritis (OA), contributing to disease pathology and progression. We evaluated the effectiveness of colchicine on pain and function in symptomatic knee OA (KOA) and the underlying mechanism of action. Methods Colchicine effectiveness in symptoms and inflammation modification in knee osteoarthritis (COLKOA) was a double-blind, placebo-controlled, randomized trial comparing 16 weeks of treatment with 0.5 mg twice-daily oral colchicine to placebo for knee osteoarthritis (KOA). The primary endpoint was ≥30% improvement in total Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score at week 16. Secondary endpoints included improvement in pain (0–10 Likert scales); WOMAC pain; patient global assessment (0–100); physical function; the OARSI-OMERACT response; quality of life; and change in serum, urine, synovial fluid (SF) biomarkers of cartilage metabolism and inflammation, and plasma/SF colchicine concentrations. Results Of 109 randomly assigned participants, 39% (95% confidence interval (CI) 27–52%) and 49% (95% CI 36–62%) in the colchicine and placebo arms respectively met the primary endpoint at study end ( P = 0.284, odds ratio 0.66, 95% CI 0.31–1.41). No strong evidence of treatment differences was identified on clinical secondary endpoints. Treatment significantly reduced mean serum hs-CRP ( P = 0.008) and SF CTXI ( P = 0.002); treatment tended to reduce inflammatory markers (SF IL-6, IL8, TNFα, CD14 and IL-18), but these differences were not statistically significant. Conclusion Colchicine (0.5 mg twice-daily orally) reduced inflammation and high bone turnover biomarkers known to be associated with OA severity and progression risk, but did not reduce KOA symptoms over a 16-week study period. A longer-term study to evaluate for slow-acting disease modifying effects is warranted. Trial registration The trial has been registered at clinicaltrials.gov as NCT02176460. Date of registration: June 26, 2014.

Introduction: Most international clinical practice guidelines for prostate cancer (PCa) are driven by data derived in a Western setting. However, tumour biology and clinical disease progression are likely to differ in the Asian population. We compare the performance of the revised American Joint Committee on Cancer (AJCC) prognostic groups with the commonly used D’Amico Risk Classification and conventional predictors for PCa, in a large cohort of Asian patients. Materials and Methods: We retrospectively reviewed data for 404 consecutive Singaporean patients receiving definitive radiotherapy at our centre between December 1996 and October 2006. The primary outcome was biochemical relapse-free survival (BRFS), defined using the Phoenix definition. The secondary outcome was overall survival (OS). Prognostic risk groups were defined using AJCC 7th edition (AJCC7) and 6th edition (AJCC6). Univariate analysis (UVA) and multivariate analysis (MVA) were performed for the following putative risk factors: age, Gleason score, prognostic grouping, tumour classification, radiation delivery technique, radiotherapy dose, hormonal therapy and initial PSA value. Results: For the cohort, median age was 69 years. Median follow-up was 66.3 months. Five-year BRFS rate was 84.3% with 71 biochemical relapses and 5-year OS rate was 89.1% with 54 deaths. The concordance-indices for BRFS prediction were 0.588, 0.550 and 0.567 for AJCC7, AJCC6 and D’Amico respectively. Initial PSA, T-stage and AJCC7 were prognostic for BRFS on UVA. Comparison of AJCC7 vs. D’Amico showed no statistical additional value of either classification system although D’Amico was superior when compared to AJCC6 in predicting BRFS. T-stage ≥3 and D’Amico were significant prognostic factors for BRFS on MVA. Conclusion: In our local, predominantly Chinese population, neither AJCC6 nor AJCC7 demonstrated a high predictive accuracy for BRFS although AJCC7 has a slightly better predictive ability than AJCC6. Key words: D'amico, Intensity modulated radiotherapy, Prognosis, Risk stratification

The present application relates to methods of detecting a mutation in a target nucleic acid molecule. Two phosphorodiamidate morpholino oligomer probes that differ by at least one base are each covalently coupled to a nano article and hybridised to a target sequence. The melting temperature of the complexes between each of the two probes and the target nucleic acid are measured and compared to determine whether the sample contains a nucleic acid with the mutation. Further, the present invention relates to kits comprising a first and second conjugate as described herein and to the use of such kits for the detection of mutations in a target nucleic acid molecule or for assigning a genotype to a target nucleic acid molecule.

Recently, several genome-wide association studies have demonstrated a cumulative association of 5 polymorphic variants in chromosomes 8q24 and 17q with prostate cancer (CaP) risk in Caucasians, particularly those harboring aggressive clinicopathologic characteristics. The purpose of this study was to evaluate the influence of these variants on CaP susceptibility in Singaporean Asian men.We performed a case-control study in 289 Chinese CaP patients and 412 healthy subjects (144 Chinese, 134 Malays, and 134 Indians), and examined the association of the 5 single nucleotide polymorphisms (SNPs) with CaP.In the healthy subjects, rs16901979 A-allele frequency was highest amongst Chinese (0.32) compared with Malays (0.13; P0.0001) or Indians (0.09; P0.0001); rs6983267 G-allele was highest in Indians (0.51) compared with Chinese (0.42; P = 0.041) or Malays (0.43; P = 0.077); whereas rs1859962 G-allele frequency was highest amongst Indians (0.56) compared with Chinese (0.40; P = 0.0002) or Malays (0.38; P0.0001). Individuals with the rs4430796 TT genotype were at increased CaP risk in the Chinese via a recessive model (odds ratios (OR) = 1.56, 95% CI = 1.04-2.33). Significant associations were observed for rs4430796 TT with Gleason scores of ≥ 7 (OR = 1.76, 95% CI = 1.14-2.73) and prostate-specific antigen (PSA) levels of ≥ 10 ng/ml at diagnosis (OR = 1.63, 95% CI = 1.01-2.63), as well as for rs6983267 GG with stage 3-4 CaPs (OR = 1.91, 95% CI = 1.01-3.61). A cumulative gene interaction influence on disease risk, which approximately doubled for individuals with at least 2 susceptibility genotypes, was also identified (OR = 2.18, 95% CI = 1.10-4.32).This exploratory analysis suggests that the 5 genetic variants previously described may contribute to prostate cancer risk in Singaporean men.

Summary: Several nuclear receptors are being increasingly recognized for their role as master xenosensors. Among them, CAR-RXRα heterodimer, as encoded by NR1I3 and NR2B1, responds to the presence of drug compounds and regulates the transcription of a wide array of genes involved in their disposition. To investigate the frequency distribution and linkage disequilibrium patterns of NR1I3 and NR2B1 genetic variations, these genes were screened in 168 healthy local Asian subjects, namely Chinese, Malays, and Indians ( n = 56 subjects each). A total of 38 and 88 SNPs were identified in NR1I3 and NR2B1, respectively. Among them, there were 13 and 43 novel SNPs present at low allelic frequencies ( NR1I3 and NR2B1, respectively. Notably, the genetic variations in the NR1I3 and NR2B1 genes were mainly confined to the introns whilst the exons were highly conserved across the ethnic populations. Indians harboured distinct frequency distributions from Chinese and Malays in both genes. Based on the linkage disequilibrium patterns of both genes, a number of tag-SNPs were selected for each population ( n = 8-13 for NR1I3; n = 12-18 for NR2B1 ) . In-silico prediction analyses revealed a number of possible functional SNPs. Our data would be valuable for future pharmacogenetic studies on the drug substrates of CAR-RXRa target genes.

The use of imatinib in the treatment of BCR-ABL-positive chronic myeloid leukemia and gastrointestinal stromal tumors has significantly improved survival outcomes in patients afflicted by these malignancies. However, a substantial proportion of imatinib-treated patients still experience treatment failure. Suboptimal concentrations of imatinib have been postulated to contribute at least partially to the development of resistance against imatinib. Indeed, variations in the genes encoding drug transporters have been reported to markedly influence imatinib disposition and treatment outcomes in various populations. This review aims to consolidate and critically assess the studies conducted to date which have investigated the influence of pharmacogenetic variants in drug transporters on the disposition of imatinib and treatment outcomes in Asians and other populations.

Summary Background Extranodal natural killer T-cell lymphoma (NKTCL), nasal type, is a rare and aggressive malignancy that occurs predominantly in Asian and Latin American populations. Although Epstein-Barr virus infection is a known risk factor, other risk factors and the pathogenesis of NKTCL are not well understood. We aimed to identify common genetic variants affecting individual risk of NKTCL. Methods We did a genome-wide association study of 189 patients with extranodal NKTCL, nasal type (WHO classification criteria; cases) and 957 controls from Guangdong province, southern China. We validated our findings in four independent case-control series, including 75 cases from Guangdong province and 296 controls from Hong Kong, 65 cases and 983 controls from Guangdong province, 125 cases and 1110 controls from Beijing (northern China), and 60 cases and 2476 controls from Singapore. We used imputation and conditional logistic regression analyses to fine-map the associations. We also did a meta-analysis of the replication series and of the entire dataset. Findings Associations exceeding the genome-wide significance threshold (p −8 ) were seen at 51 single-nucleotide polymorphisms (SNPs) mapping to the class II MHC region on chromosome 6, with rs9277378 (located in HLA-DPB1 ) having the strongest association with NKTCL susceptibility (p=4·21 × 10 −19 , odds ratio [OR] 1·84 [95% CI 1·61–2·11] in meta-analysis of entire dataset). Imputation-based fine-mapping across the class II MHC region suggests that four aminoacid residues (Gly84-Gly85-Pro86-Met87) in near-complete linkage disequilibrium at the edge of the peptide-binding groove of HLA-DPB1 could account for most of the association between the rs9277378*A risk allele and NKTCL susceptibility (OR 2·38, p value for haplotype 2·32 × 10 −14 ). This association is distinct from MHC associations with Epstein-Barr virus infection. Interpretation To our knowledge, this is the first time that a genetic variant conferring an NKTCL risk is noted at genome-wide significance. This finding underlines the importance of HLA-DP antigen presentation in the pathogenesis of NKTCL. Funding Top-Notch Young Talents Program of China, Special Support Program of Guangdong, Specialized Research Fund for the Doctoral Program of Higher Education (20110171120099), Program for New Century Excellent Talents in University (NCET-11-0529), National Medical Research Council of Singapore (TCR12DEC005), Tanoto Foundation Professorship in Medical Oncology, New Century Foundation Limited, Ling Foundation, Singapore National Cancer Centre Research Fund, and the US National Institutes of Health (1R01AR062886, 5U01GM092691-04, and 1R01AR063759-01A1).

Introduction Activating mutations in the EGFR gene have been shown to confer sensitivity to EGFR tyrosine kinase inhibitors in patients with advanced NSCLC. However, wide interpatient variability in treatment outcomes in response to EGFR tyrosine kinase inhibitors in these patients remains unaccounted for. This study aimed to evaluate the influence of EGFR mutation types and subtypes on survival outcomes in advanced Asian patients with NSCLC receiving first-line gefitinib therapy. Methods Patients with stage IIIB or IV NSCLC who were harboring EGFR mutations, receiving first-line gefitinib treatment, and of Asian descent (N = 383) were evaluated. Overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier analysis. Log-rank tests and Cox proportional hazards models were implemented to evaluate the differences in PFS and OS. Results Significant differences in PFS were observed between patients carrying EGFR mutations in exons 18, 19, 20, and 21, with patients carrying EGFR exon 19 mutations having the longest median PFS (overall p = 8.88 × 10 -15 ). Comparison of PFS among the five different exon 19 mutation subtypes and among the two exon 19 deletion start codons did not reveal any significant differences. No significant difference was observed in OS among patients carrying EGFR mutations on different exons (overall p = 0.054); however, OS was found to be significantly different among the various subtypes of exon 19 mutations, with the 15–nucleotide deletion "non-ELREA" group having the shortest OS of 11.3 months (overall p = 0.025). Conclusions EGFR mutation types and subtypes significantly influence survival outcomes in patients with advanced NSCLC who are receiving first-line gefitinib treatment.

Targeting EGFR is a validated approach in the treatment of squamous-cell cancers (SCCs), although there are no established biomarkers for predicting response. We have identified a synonymous mutation in EGFR, c.2361G>A (encoding p.Gln787Gln), in two patients with head and neck SCC (HNSCC) who were exceptional responders to gefitinib, and we showed in patient-derived cultures that the A/A genotype was associated with greater sensitivity to tyrosine kinase inhibitors (TKIs) as compared to the G/A and G/G genotypes. Remarkably, single-copy G>A nucleotide editing in isogenic models conferred a 70-fold increase in sensitivity due to decreased stability of the EGFR-AS1 long noncoding RNA (lncRNA). In the appropriate context, sensitivity could be recapitulated through EGFR-AS1 knockdown in vitro and in vivo, whereas overexpression was sufficient to induce resistance to TKIs. Reduced EGFR-AS1 levels shifted splicing toward EGFR isoform D, leading to ligand-mediated pathway activation. In co-clinical trials involving patients and patient-derived xenograft (PDX) models, tumor shrinkage was most pronounced in the context of the A/A genotype for EGFR-Q787Q, low expression of EGFR-AS1 and high expression of EGFR isoform D. Our study reveals how a 'silent' mutation influences the levels of a lncRNA, resulting in noncanonical EGFR addiction, and delineates a new predictive biomarker suite for response to EGFR TKIs.

Tamoxifen (TAM) is an established endocrine treatment for all stages of oestrogen receptor (ER)-positive breast cancer. Its complex metabolism leads to the formation of multiple active and inactive metabolites. One of the main detoxification and elimination pathways of tamoxifen and its active metabolites, 4-hydroxytamoxifen (4-OHT) and endoxifen, is via glucuronidation catalysed by uridine 5'-diphospho-glucuronosyltransferases (UGTs). However, few studies have comprehensively examined the impact of variations in the genes encoding the major hepatic UGTs on the disposition of tamoxifen and its metabolites. In the present study, we systematically sequenced exons, exon/intron boundaries, and flanking regions of UGT1A4, UGT2B7 and UGT2B15 in 240 healthy subjects of different Asian ethnicities (Chinese, Malays and Indians) to identify haplotype tagging single nucleotide polymorphisms. Subsequently, 202 Asian breast cancer patients receiving tamoxifen were genotyped for 50 selected variants in the three UGT genes to comprehensively investigate their associations with steady-state plasma levels of tamoxifen, its active metabolites and their conjugated counterparts. The UGT1A4 haplotype (containing variant 142T>G, L48 V defining the *3 allele) was strongly associated with higher plasma levels of TAM-N-glucuronide, with a twofold higher metabolic ratio of TAM-N-glucuronide/TAM observed in carriers of this haplotype upon covariate adjustment (P

Purpose This study aimed to evaluate the safety, tolerability and pharmacokinetics of the combination of oral vinorelbine with erlotinib using the conventional (CSV) and metronomic (MSV) dosing schedules in patients with advanced non-small cell lung cancer (NSCLC). Methods This was an open-label, multiple dose-escalation phase I study. An alternating 3+3 phase I design was employed to allow each schedule to enroll three patients sequentially at each dose level. Thirty patients with Stage IIIB/IV NSCLC were treated with escalating doses of oral vinorelbine starting at 40 mg/m2 on day 1 and 8 in the CSV group (N = 16) and at 100 mg/week in the MSV group (N = 14). Erlotinib was administered orally daily. Results The maximum tolerated dose was vinorelbine 80 mg/m2 with erlotinib 100 mg in the CSV group and vinorelbine 120 mg/week with erlotinib 100 mg in the MSV group. Grade 3/4 toxicities included neutropenia (N = 2; 13%) and hyponatremia (N = 1; 6%) in the CSV group, and neutropenia (N = 5; 36%) in the MSV group. Objective response was achieved in 38% and 29% in the CSV and MSV groups respectively. Vinorelbine co-administration did not significantly affect the pharmacokinetics of erlotinib and OSI-420 after initial dose. However, at steady-state, significantly higher Cmax, higher Cmin and lower CL/F of erlotinib were observed with increasing dose levels of vinorelbine in the CSV group. Significantly higher steady-state Cmin, Cavg and AUCss of erlotinib were observed with increasing dose levels of vinorelbine in the MSV group. Conclusions Combination of oral vinorelbine with erlotinib is feasible and tolerable in both the CSV and MSV groups. Trial Registration ClinicalTrials.gov NCT00702182

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • SLCO1B3 is an influx transporter located at the hepatocyte basolateral membrane and it is involved in the uptake of a broad range of drug substrates including docetaxel. • The pharmacogenetics of SLCO1B3 is not well characterized and previous in vivo and in vitro studies reported conflicting results with regards to the functional effects of the limited number of SLCO1B3 polymorphisms that were studied. • Docetaxel displays a wide interindividual variability in its pharmacokinetics and pharmacodynamics and an understanding of SLCO1B3 pharmacogenetics might provide clinical benefits in guiding docetaxel dosing. WHAT THIS STUDY ADDS • The SLCO1B3 gene was comprehensively screened in the local healthy Asian populations (n= 168). A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15 haplotype-tag SNPs (htSNPs) were identified. These htSNPs were profiled in a cohort of Chinese nasopharyngeal cancer (NPC) patients (n= 50). • Genotypic-phenotypic analysis showed that a haplotypic construct comprising of four variants [IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA] was the critical determinant of docetaxel disposition. • This study suggests that the comprehensive screening and haplotypic linkage analysis of SLCO1B3 can better elucidate its pharmacogenetic effects on interpatient variability of docetaxel and other putative drug substrates. Further studies are warranted in cancer patients belonging to other ethnic groups. AIMS To completely screen the SLCO1B3 gene in three distinct healthy Asian populations (Chinese, Malay and Indian, n= 168) and investigate the influence of haplotype-tag SNPs (htSNPs) on docetaxel disposition in 50 nasopharyngeal carcinoma patients. METHODS Genomic DNA of individuals was screened for SLCO1B3 polymorphisms by direct sequencing. htSNPs were derived based on the sequence clustering algorithm and profiled in the patients. Population based genetic association analysis was performed using Haplostats package implemented in R and PLINK. RESULTS A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15-htSNPs were identified. The SLCO1B3 haplotypic region comprising seven htSNPs was found to be significantly associated with docetaxel clearance (P= 0.003). Conditional haplotype analyses revealed that the haplotypic constructs comprising the IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA polymorphisms were critical determinants of variability in docetaxel disposition [clearance and area under the plasma concentration−time curve (AUC(0,∞)): r2= 29% and 22%, respectively]. Patients harbouring the GAG*347insA haplotype were significantly associated with a 30% decrease in clearance and a 40% increase in AUC(0,∞) of docetaxel compared with patients harbouring the reference haplotype, GGA*347wt (P= 0.025 and 0.018, respectively). In contrast, a 50% higher clearance was observed in patients carrying the GAG*347wt haplotype compared with those with the reference haplotype (P= 0.002). The functional SLCO1B3 haplotypic constructs included the widely studied Met233Ile variant and *347_*348insA located in the putative miR-890 binding site in the 3′-untranslated region which may influence the transport characteristics of SLCO1B3. CONCLUSIONS This study highlights the importance of SLCO1B3 polymorphic variations in influencing docetaxel disposition in nasopharyngeal carcinoma patients.

A systematic review and a meta-analysis were performed to quantify the accumulated information from genetic association studies investigating the impact of the CYP4F2 rs2108622 (p.V433M) polymorphism on coumarin dose requirement. An additional aim was to explore the contribution of the CYP4F2 variant in comparison with, as well as after stratification for, the VKORC1 and CYP2C9 variants. Thirty studies involving 9,470 participants met prespecified inclusion criteria. As compared with CC-homozygotes, T-allele carriers required an 8.3% (95% confidence interval (CI): 5.6�11.1%; P < 0.0001) higher mean daily coumarin dose than CC homozygotes to reach a stable international normalized ratio (INR). There was no evidence of publication bias. Heterogeneity among studies was present (I2 = 43%). Our results show that the CYP4F2 p.V433M polymorphism is associated with interindividual variability in response to coumarin drugs, but with a low effect size that is confirmed to be lower than those contributed by VKORC1 and CYP2C9 polymorphisms. Wiley Online Library