Isabelle Laverdière

Genetic variants in microRNAs and microRNA target sites predict biochemical recurrence after radical prostatectomy in localized prostate cancer

Recent evidence indicates that microRNAs might participate in prostate cancer initiation, progression and treatment response. Germline variations in microRNAs might alter target gene expression and modify the efficacy of prostate cancer therapy. To determine whether genetic variants in microRNAs and microRNA target sites are associated with the risk of biochemical recurrence (BCR) after radical prostatectomy (RP). We retrospectively studied two independent cohorts composed of 320 Asian and 526 Caucasian men with pathologically organ‐confined prostate cancer who had a median follow‐up of 54.7 and 88.8 months after RP, respectively. Patients were systematically genotyped for 64 single‐nucleotide polymorphisms (SNPs) in microRNAs and microRNA target sites, and their prognostic significance on BCR was assessed by Kaplan–Meier analysis and Cox regression model. After adjusting for known clinicopathologic risk factors, two SNPs (MIR605 rs2043556 and CDON rs3737336) remained associated with BCR. The numbers of risk alleles showed a cumulative effect on BCR [perallele hazard ratio (HR) 1.60, 95% confidence interval (CI) 1.16–2.21, p for trend = 0.005] in Asian cohort, and the risk was replicated in Caucasian cohort (HR 1.55, 95% CI 1.15–2.08, p for trend = 0.004) and in combined analysis (HR 1.57, 95% CI 1.26–1.96, p for trend <0.001). Results warrant replication in larger cohorts. This is the first study demonstrating that SNPs in microRNAs and microRNA target sites can be predictive biomarkers for BCR after RP.

Molecular Markers in Key Steroidogenic Pathways, Circulating Steroid Levels, and Prostate Cancer Progression

Purpose: Prostate cancer is a heterogeneous genetic disease, and molecular methods for predicting prognosis in patients with aggressive form of the disease are urgently needed to better personalize treatment approaches. The objective was to identify host genetic variations in candidate steroidogenic genes affecting hormone levels and prostate cancer progression. Experimental Design: The study examined two independent cohorts composed of 526 Caucasian men with organ-confined prostate cancer and 601 Taiwanese men on androgen–deprivation therapy. Caucasians were genotyped for 109 haplotype-tagging single-nucleotide polymorphisms (SNP) in CYP17A1, ESR1, CYP19A1, and HSD3B1, and their prognostic significance on disease progression was assessed using Kaplan–Meier survival curves and Cox regression models. Positive findings, including previously identified SRD5A1, SRD5A2, HSD17B2, HSD17B3, and HSD17B12 polymorphisms, were then explored in Taiwanese men (n = 32 SNPs). The influence of positive markers on the circulating hormonal levels was then appraised in Caucasians using specific and sensitive mass spectrometry–based methods. Results: After adjusting for known risk factors, variants of CYP17A1 (rs6162), HSD17B2 (rs4243229 and rs7201637), and ESR1 (rs1062577) were associated with progressive disease in both cohorts. Indeed, the presence of these variations was significantly associated with progression in Caucasians (HR, 2.29–4.10; P = 0.0014–2 × 10−7) and survival in Taiwanese patients [HR = 3.74; 95% confidence interval (CI): 1.71–8.19, P = 0.009]. Remarkably, the CYP17A1 rs6162 polymorphism was linked to plasma dehydroepiandrosterone-sulfate (DHEA-S) levels (P = 0.03), HSD17B2 rs7201637 with levels of dihydrotestosterone (P = 0.03), and ESR1 rs1062577 with levels of estrone-S and androsterone-glucuronide (P ≤ 0.05). Conclusion: This study identifies, in different ethnic groups and at different disease stages, CYP17A1, HSD17B2, and ESR1 as attractive prognostic molecular markers of prostate cancer progression. Clin Cancer Res; 19(3); 699–709. ©2012 AACR.

In vitro investigation of human UDP-glucuronosyltransferase isoforms responsible for tacrolimus glucuronidation: predominant contribution of UGT1A4.

Tacrolimus (Tacro) is a potent immunosuppressant and a central agent in the prevention of posttransplantation rejection. Tacro is characterized by a narrow therapeutic index and wide interindividual pharmacokinetic fluctuation. The contribution of human UDP-glucuronosyltransferase (UGT) in its metabolism has not been extensively studied. In vitro metabolism studies support that the liver produced Tacro-glucuronide (Tacro-G) while its formation was minimal or undetectable in the presence of intestine and kidney microsomes. Among 16 human UGTs tested, UGT1A4 was the sole enzyme involved in Tacro-G formation. This conclusion is supported by the finding of inhibition with a specific substrate of UGT1A4 lamotrigine with Ki values similar for both human liver and UGT1A4 microsomes and the correlation with trifluoperazine-glucuronide formation by liver microsomes (rs = 0.551; p = 0.02). Formation of Tacro-G by liver samples varied among individuals (6.4-fold variation; n = 16), and common nonsynonymous variants may contribute to this variability. In the human embryonic kidney 293 cellular model, no significant differences in enzyme kinetics could be revealed for UGT1A4*2 (P24T) and *3 (L48V), whereas the allozyme *4 (R11W) displayed a 2-fold higher velocity (p < 0.01) compared with the UGT1A4*1 enzyme preparation. In human liver samples, carriers of the UGT1A4 variants did not display statistically different efficiency in Tacro-G formation compared with homozygote for the reference genotype UGT1A4*1/*1. We conclude that UGT1A4 is the major isoform involved in Tacro glucuronidation, whereas additional studies are required to assess the contribution of UGT1A4 genetic factors in tacrolimus glucuronidation variability.

Steroidogenic Germline Polymorphism Predictors of Prostate Cancer Progression in the Estradiol Pathway

Purpose: Reliable biomarkers that predict prostate cancer outcomes are urgently needed to improve and personalize treatment approaches. With this goal in mind, we individually and collectively appraised common genetic polymorphisms related to estradiol metabolic pathways to find prostate cancer prognostic markers. Methods: The genetic profiles of 526 men with organ-confined prostate cancer were examined to find common genetic polymorphisms related to estradiol metabolic pathways and these findings were replicated in a cohort of 213 men with more advanced disease (follow-up time for both cohorts, >7.4 years). Specifically, we examined 71 single-nucleotide polymorphisms (SNP) in SULT2A1, SULT2B1, CYP1B1, COMT, CYP3A4, CYP3A5, CYP3A43, NQO1, and NQO2 and assessed the impact of the SNPs alone and in combination on prostate cancer progression and on circulating hormone levels. Results: According to a multivariate analysis, CYP1B1 (rs1800440), COMT (rs16982844), and SULT2B1 (rs12460535, rs2665582, rs10426628) were significantly associated with prostate cancer progression and hormone levels. Remarkably, by combining the SNP information with previously identified HSD17B2 markers, the patients could be stratified into four distinct prognostic subgroups. The most prominent association was observed for the eight-marker combination [CYP1B1 (rs1800440), SULT2B1 (rs12460535, rs2665582, and rs10426628), and HSD17B2 (rs4243229, rs1364287, rs2955162, and rs1119933)]. Conclusion: This study identified specific germline variations in estradiol metabolism–related pathways, namely CYP1B1, SULT2B1, and HSD17B2, as novel prognostic markers that are cumulatively associated with increased risk of prostate cancer progression. This panel of markers warrants additional investigation and validation to help stratify patients according to their risk of progression. Clin Cancer Res; 20(11); 2971–83. ©2014 AACR.

Importance of 5α-reductase gene polymorphisms on circulating and intraprostatic androgens in prostate cancer.

Purpose: Polymorphisms in the genes SRD5A1 and SRD5A2 encoding androgen biosynthetic 5α-reductase enzymes have been associated with an altered risk of biochemical recurrence after radical prostatectomy in localized prostate cancer. Experimental Design: To gain potential insights into SRD5A biologic effects, we examined the relationship between SRD5A prognostic markers and endogenous sex-steroid levels measured by mass spectrometry in plasma samples and corresponding prostatic tissues of patients with prostate cancer. Results: We report that five of the seven SRD5A markers differentially affect sex-steroid profiles of dihydrotestosterone and its metabolites in both the circulation and prostatic tissues of patients with prostate cancer. Remarkably, a 32% increase in intraprostatic testosterone levels was observed in the presence of the high-risk SRD5A rs2208532 polymorphism. Moreover, SRD5A2 markers were associated predominantly with circulating levels of inactive glucuronides. Indeed, the rs12470143 SRD5A2 protective allele was associated with high circulating androstane-3α, 17β-diol-17-glucuronide (3α-diol-17G) levels as opposed to lower levels of both 3α-diol-17G and androsterone-glucuronide observed with the rs2208532 SRD5A2 risk allele. Moreover, SRD5A2 rs676033 and rs523349 (V89L) risk variants, in strong linkage disequilibrium, were associated with higher circulating levels of 3α-diol-3G. The SRD5A2 rs676033 variant further correlated with enhanced intraprostatic exposure to 5α-reduced steroids (dihydrotestosterone and its metabolite 3β-diol). Similarly, the SRD5A1 rs166050C risk variant was associated with greater prostatic exposure to androsterone, whereas no association was noted with circulating steroids. Conclusions: Our data support the association of 5α-reductase germline polymorphisms with the hormonal milieu in patients with prostate cancer. Further studies are needed to evaluate if these variants influence 5α-reductase inhibitor efficacy. Clin Cancer Res; 20(3); 576–84. ©2013 AACR.

Extensive splicing of transcripts encoding the bile acid-conjugating enzyme UGT2B4 modulates glucuronidation

Background and aims UGT2B4 is a member of the UDP-glucuronosyltransferase (UGT) superfamily, a major detoxifying system in humans. UGT2B4 is involved in bile acids metabolism and highly expressed in liver and extrahepatic tissues. The aim of this study was to uncover new molecular mechanisms underlying interindividual variability in the UGT2B4 pathway. Methods We carried out a comprehensive scan for additional exons at this locus and discovered multiple alternative splicing events. We then assessed the expression profile of alternatively spliced transcripts in human tissues and the activity of the corresponding overexpressed proteins toward bile acids. Results We discovered three previously unidentified UGT2B4 exons, increasing the total known gene length to 46 kb. Molecular analyses revealed at least eight distinct mRNAs produced by (i) alternative promoter usage, (ii) complete and partial exon skipping, and (iii) use of alternative 3′ splice sites. These splice variants were predominantly expressed in liver, gastrointestinal tract, and other extrahepatic tissues. Quantitative analyses of splicing events further sustain their prevalence in the liver. UGT2B4 proteins produced from these mRNA variants had undetectable transferase activity in human cells. However, when stably co-expressed with the active UGT2B4 isoform 1, three newly identified UGT2B4 isoforms (i2, i3, and i5) were found to negatively regulate glucuronidation. Conclusion In addition to heritable genetic mutations and control of gene expression, the newly discovered diversity of UGT2B4 mRNAs may introduce variability in this glucuronidation pathway.

ABCC5 and ABCG1 polymorphisms predict irinotecan-induced severe toxicity in metastatic colorectal cancer patients

Objective Irinotecan is a cytotoxic agent used widely for the treatment of solid tumors, particularly for metastatic colorectal cancers. Treatment with this drug frequently results in severe neutropenia and diarrhea that can markedly impact the course of treatment and patients’ quality of life. Pharmacogenomic tailoring of irinotecan-based chemotherapy has been the subject of several investigations, but with limited data on ATP-binding cassette (ABC) and solute carrier (SLC) transporter genes. Materials and methods In this study, we aimed to discover toxicity-associated markers in seven transporter genes participating in irinotecan pharmacokinetics involving the ABC transporter genes ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, and ABCG2 and the solute carrier organic anion transporter gene SLCO1B1 and using a haplotype-tagging single-nucleotide polymorphisms (n=210 htSNPs) strategy. The profiles of 167 metastatic colorectal cancer Canadian patients treated with FOLFIRI-based regimens were examined and the findings were replicated in an independent cohort of 250 Italian patients. Results In combined cohorts, a two-marker ABCC5 rs3749438 and rs10937158 haplotype (T–C) predicted lower risk of severe diarrhea [odds ratio (OR) of 0.43; P=0.001]. The co-occurrence of ABCG1 rs225440T and ABCC5 rs2292997A predicted the risk of severe neutropenia (OR=5.93; P=0.0002), which was further improved when incorporating the well-known risk marker UGT1A1*28 rs8175347 (OR=7.68; P<0.0001). In contrast, carriers of one protective marker (UGT1 rs11563250G) but none of these risk alleles experienced significantly less severe neutropenia (8.2 vs. 34.0%; P<0.0001). Conclusion This combination of predictive genetic markers could potentially lead to better risk assessment and may thus improve personalized treatment.

A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for monitoring drug exposure in hematopoietic stem cell transplant recipients

A liquid chromatography-tandem mass spectrometry method was developed for the quantification of circulating levels of multiple immunosuppressant drugs including cyclosporine (CsA), tacrolimus, methotrexate (Mtx), prednisone, prednisolone, methylprednisone, total and free mycophenolic acid (MPA), as well as MPA phenolic (MPAG) and acyl (AcMPAG) glucuronide metabolites. Linearity, precision and accuracy were validated within the typical therapeutic range of concentrations for each compound. The assay was linear over 0.125-25ng/mL for tacrolimus, 1-500ng/mL for prednisone/methylprednisone, 2-400ng/mL for Mtx, 2-1000ng/mL for prednisolone and from 7.5 to 1500ng/mL for CsA with the lowest limit of quantification (LLOQ) being 0.125, 1.00, 2.00, 2.00 and 7.5ng/mL, respectively. The calibration curve concentrations for MPA and MPAG ranged from 50 to 50,000ng/mL (LLOQ: 50ng/mL) and 10 to 10,000ng/mL (LLOQ: 10ng/mL) for AcMPAG. Mean recoveries in blood and plasma were 84%±5.7%. The method could measure individual drugs with high sensitivity, accuracy (bias≤14%), and reproducibility (CV≤12.8%). Its clinical application was validated by measuring levels of these drugs in samples obtained from hematopoietic stem cell transplant recipients treated with combined immunosuppressive drug therapy. Our results indicate that this approach is suitable for simultaneous determination of in vivo levels of immunosuppressive drugs commonly used in combined therapies.

A novel UGT1 marker associated with better tolerance against irinotecan-induced severe neutropenia in metastatic colorectal cancer patients

The risk of severe irinotecan-induced neutropenia has been shown to be related to the UGT1 variant UGT1A1*28, which increases exposure to the potent metabolite SN-38. Our goal was to identify a novel UGT1 marker(s) using 28 haplotype-tagged single nucleotide polymorphisms genotyped by mass spectrometry. By characterizing the UGT1 sequence from a cohort of 167 Canadian metastatic colorectal cancer (mCRC) patients and a validation cohort of 250 Italian mCRC patients, we found rs11563250G, located in the intergenic region downstream of UGT1, to be significantly associated with reduced risk of severe neutropenia (odds ratio (OR)=0.21; P=0.043 and OR=0.27; P=0.036, respectively, and OR=0.31 when combined; P=0.001), which remained significant upon correction for multiple testing in the combined cohort (P=0.041). For the two-marker haplotype rs11563250G and UGT1A1*1 (rs8175347 TA6), the OR was of 0.17 (P=0.0004). Genetic testing of this marker may identify patients who might benefit from increased irinotecan dosing.

The UGT1 locus is a determinant of prostate cancer recurrence after prostatectomy.

The prognostic significance of common deletions in uridine diphospho-glucuronosyltransferase 2B (UGT2B) genes encoding sex steroid metabolic enzymes has been recently recognized in localized prostate cancer (PCa) after radical prostatectomy (RP). However, the role of germline variations at the UGT1 locus, encoding half of all human UGTs and primarily involved in estrogen metabolism, remains unexplored. We investigated whether variants of UGT1 are potential prognostic markers. We studied 526 Caucasian men who underwent RP for clinically localized PCa. Genotypes of patients for 34 haplotype-tagged single-nucleotide polymorphisms (htSNPs) and 11 additional SNPs across the UGT1 locus previously reported to mark common variants including functional polymorphisms were determined. The risk of biochemical recurrence (BCR) was estimated using adjusted Cox proportional hazards regression and Kaplan-Meier analysis. We further investigated whether variants are associated with plasma hormone levels by mass spectrometry. In multivariable models, seven htSNPs were found to be significantly associated with BCR. A greater risk was revealed for four UGT1 intronic variants with hazard ratios (HRs) of 1.59-1.88 (P<0.002) for htSNPs in UGT1A10, UGT1A9, and UGT1A6. Conversely, decreased BCR was associated with three htSNPs in introns of UGT1A10 and UGT1A9 (HR=0.56-058; P≤0.01). An unfavorable UGT1 haplotype comprising all risk alleles, with a frequency of 14%, had a HR of 1.68 (95% CI=1.13-2.50; P=0.011). Significant alteration in circulating androsterone levels was associated with this haplotype, consistent with changes in hormonal exposure. This study provides the first evidence, to our knowledge, that germline polymorphisms of UGT1 are potential predictors of recurrence of PCa after prostatectomy.