Laurent Chouchana

Review article: the benefits of pharmacogenetics for improving thiopurine therapy in inflammatory bowel disease.

Aliment Pharmacol Ther 2012; 35: 15–36

Phenome-Wide Association Studies on a Quantitative Trait: Application to TPMT Enzyme Activity and Thiopurine Therapy in Pharmacogenomics

Phenome-Wide Association Studies (PheWAS) investigate whether genetic polymorphisms associated with a phenotype are also associated with other diagnoses. In this study, we have developed new methods to perform a PheWAS based on ICD-10 codes and biological test results, and to use a quantitative trait as the selection criterion. We tested our approach on thiopurine S-methyltransferase (TPMT) activity in patients treated by thiopurine drugs. We developed 2 aggregation methods for the ICD-10 codes: an ICD-10 hierarchy and a mapping to existing ICD-9-CM based PheWAS codes. Eleven biological test results were also analyzed using discretization algorithms. We applied these methods in patients having a TPMT activity assessment from the clinical data warehouse of a French academic hospital between January 2000 and July 2013. Data after initiation of thiopurine treatment were analyzed and patient groups were compared according to their TPMT activity level. A total of 442 patient records were analyzed representing 10,252 ICD-10 codes and 72,711 biological test results. The results from the ICD-9-CM based PheWAS codes and ICD-10 hierarchy codes were concordant. Cross-validation with the biological test results allowed us to validate the ICD phenotypes. Iron-deficiency anemia and diabetes mellitus were associated with a very high TPMT activity (p = 0.0004 and p = 0.0015, respectively). We describe here an original method to perform PheWAS on a quantitative trait using both ICD-10 diagnosis codes and biological test results to identify associated phenotypes. In the field of pharmacogenomics, PheWAS allow for the identification of new subgroups of patients who require personalized clinical and therapeutic management.

Interindividual variability in TPMT enzyme activity: 10 years of experience with thiopurine pharmacogenetics and therapeutic drug monitoring

BACKGROUND & AIMS TPMT activity and metabolite determination (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) remain controversial during thiopurine management. This study assessed associations between patient characteristics and TPMT activity, and their impact on metabolite levels. PATIENTS & METHODS A retrospective review of the laboratory database from a French university hospital identified 7360 patients referred for TPMT phenotype/genotype determination, and/or for 6-TGN/6-MMPN monitoring. RESULTS Four TPMT phenotypes were identified according to TPMT activity distribution: low, intermediate, normal/high and very high. Based on 6775 assays, 6-TGN concentrations were 1.6-fold higher in TPMT-deficient patients compared with TPMT-normal patients. Azathioprine dose and TPMT genotype were significant predictors of metabolite levels. Furthermore, 6-MMPN and 6-MMPN: 6-TGN ratios were, respectively, 1.6- and 2.2-fold higher in females than in males, despite similar TPMT, 6-TGN and azathioprine doses. An unfavorable ratio (≥20) was associated with a slightly higher TPMT activity. CONCLUSION These results illustrate the usefulness of pharmacogenomics and metabolite measurement to improve the identification of noncompliance and patients at high risk for toxicity or therapeutic resistance. Original submitted 13 November 2013; Revision submitted 30 January 2014.

Chronic voluntary ethanol intake hypersensitizes 5-HT1A autoreceptors in C57BL/6J mice

Alcoholism is a complex disorder involving, among others, the serotoninergic (5‐HT) system, mainly regulated by 5‐HT1A autoreceptors in the dorsal raphe nucleus. 5‐HT1A autoreceptor desensitization induced by chronic 5‐HT reuptake inactivation has been associated with a decrease in ethanol intake in mice. We investigated here whether, conversely, chronic ethanol intake could induce 5‐HT1A autoreceptor supersensitivity, thereby contributing to the maintenance of high ethanol consumption. C57BL/6J mice were subjected to a progressive ethanol intake procedure in a free‐choice paradigm (3–10% ethanol versus tap water; 21 days) and 5‐HT1A autoreceptor functional state was assessed using different approaches. Acute administration of the 5‐HT1A receptor agonist ipsapirone decreased the rate of tryptophan hydroxylation in striatum, and this effect was significantly larger (+75%) in mice that drank ethanol than in those drinking water. Furthermore, ethanol intake produced both an increased potency (+45%) of ipsapirone to inhibit the firing of 5‐HT neurons, and a raise (+35%) in 5‐HT1A autoreceptor‐mediated stimulation of [35S]GTP‐γ‐S binding in the dorsal raphe nucleus. These data showed that chronic voluntary ethanol intake in C57BL/6J mice induced 5‐HT1A autoreceptor supersensitivity, at the origin of a 5‐HT neurotransmission deficit, which might be causally related to the addictive effects of ethanol intake.

Poor Response to Thiopurine in Inflammatory Bowel Disease: How to Overcome Therapeutic Resistance?

A 24-year-old woman (53 kg) with a 5-year history of steroid-dependent ulcerative colitis with mild and extensive ulcerations presented to the gastroenterology clinic for symptom recurrence. She was given 100 mg/day (1.9 mg · kg−1 · day−1) azathioprine (AZA)5 for 1 month, after which the dose was increased to 125 mg/day (2.3 mg · kg−1 · day−1). Four months later, the patient was tapered off steroid therapy. Her symptoms persisted after 7 months of AZA therapy, however, and she experienced gastrointestinal side effects. The patient was switched to another thiopurine drug, 6-mercaptopurine (6-MP), at 75 mg/day (1.4 mg · kg−1 · day−1), which was well tolerated but similarly ineffective (8 stools daily). A brief course of steroid therapy rapidly produced a substantial but short-lived clinical improvement. To understand this patients unresponsiveness to 2 thiopurine agents, we quantified thiopurine metabolites (1) 1 year after initiating AZA therapy. Intraerythrocyte concentrations of 6-thioguanine nucleotides (6-TGNs) were low (132 pmol/8 · 108 erythrocytes; therapeutic interval, 230–400 pmol/8 · 108 erythrocytes), and 6-methylmercaptopurine ribonucleotides (6-MMPRs) were very high (11 666 pmol/8 · 108 erythrocytes; therapeutic interval, <5800 pmol/8 · 108 erythrocytes). A second quantification of thiopurine metabolites 3 months later confirmed these results (6-TGNs, 127 pmol/8 · 108 erythrocytes; 6-MMPR, 26 304 pmol/8 · 108 erythrocytes). The patient had an unusual and extremely high thiopurine S -methyltransferase (TPMT) activity in erythrocytes [61.5 nmol · h−1 · (mL erythrocytes)−1; reference interval, 8.5–15 nmol · h−1 · (mL erythrocytes)−1]. The lack of clinical efficacy for 6-MP, together with the evidence of pharmacologic resistance, prompted discontinuation of 6-MP therapy. Thereafter, we administered the tumor necrosis factor-α (TNF-α) antagonist adalimumab, but we quickly replaced it with infliximab, which has a …

TPMT status determination: the simplest is the most effective?

Dear Sir, We carefully read the article by Hindorf and Appell stating that genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase (TPMT) function.1 As evidence since many years, TPMT status should be determined before a thiopurine treatment to identify patients at risk for severe adverse events, as bone marrow suppression, and to propose an individualized dosage.2 Hindorf and Appell mentioned “it is not reasonable to check both genotype and phenotype in all patients”. We agree …

Molecular insight into thiopurine resistance: transcriptomic signature in lymphoblastoid cell lines

BackgroundThere has been considerable progress in the management of acute lymphoblastic leukemia (ALL) but further improvement is needed to increase long-term survival. The thiopurine agent 6-mercaptopurine (6-MP) used for ALL maintenance therapy has a key influence on clinical outcomes and relapse prevention. Genetic inheritance in thiopurine metabolism plays a major role in interindividual clinical response variability to thiopurines; however, most cases of thiopurine resistance remain unexplained.MethodsWe used lymphoblastoid cell lines (LCLs) from healthy donors, selected for their extreme thiopurine susceptibility. Thiopurine metabolism was characterized by the determination of TPMT and HPRT activity. We performed genome-wide expression profiling in resistant and sensitive cell lines with the goal of elucidating the mechanisms of thiopurine resistance.ResultsWe determined a higher TPMT activity (+44%; P = 0.024) in resistant compared to sensitive cell lines, although there was no difference in HPRT activity. We identified a 32-gene transcriptomic signature that predicts thiopurine resistance. This signature includes the GTPBP4 gene coding for a GTP-binding protein that interacts with p53. A comprehensive pathway analysis of the genes differentially expressed between resistant and sensitive cell lines indicated a role for cell cycle and DNA mismatch repair system in thiopurine resistance. It also revealed overexpression of the ATM/p53/p21 pathway, which is activated in response to DNA damage and induces cell cycle arrest in thiopurine resistant LCLs. Furthermore, overexpression of the p53 target gene TNFRSF10D or the negative cell cycle regulator CCNG2 induces cell cycle arrest and may also contribute to thiopurine resistance. ARHGDIA under-expression in resistant cell lines may constitute a novel molecular mechanism contributing to thiopurine resistance based on Rac1 inhibition induced apoptosis and in relation with thiopurine pharmacodynamics.ConclusionOur study provides new insights into the molecular mechanisms underlying thiopurine resistance and suggests a potential research focus for developing tailored medicine.

Letter: thiopurine blood monitoring for patients with inflammatory bowel disease - authors' reply

SIRS, We read carefully the letter from Dr El-Matary regarding our review on the benefits of pharmacogenetics for improving thiopurine therapy in inflammatory bowel disease. 2 We agree that performing blood testing remain a matter of debate for the prevention of thiopurine toxicity. First, neutropenia, strongly linked to high 6-thioguanine nucleotide blood level, is a frequent and serious adverse event of thiopurine therapy, potentially lethal. It frequently occurs in the first months of therapy, starting from 2 weeks, mostly in thiopurine S-methyltransferase deficient patients. However, neutropenia can happen anytime during therapy, until 27 years in long-term follow-up. Moreover, as Connell et al. showed, blood count is often normal, 1 month before the occurrence of a moderate or severe leucopenia. Nevertheless, this suggests that performing blood counts in a 3 months basis may have limited efficacy to predict early bone marrow suppression. Actually, early neutropenia probably differs from delayed toxicity, in which a dosage alteration, a therapy change or a deficiency in vitamin B12 or folates can be involved. Second, hepatotoxicity, probably related to elevated and lasting 6-methylmercaptopurine blood level, is commonly represented as mild, transient or reversible, elevation in serum transaminase. Thiopurine-induced liver injury occurs more frequently within the first months of treatment between, 1.5 and 5 months, although it can also occur after long-term treatment. 7 Thus, despite no consensus, liver tests can be easily added to routine blood count determinations. A recent meta-analysis did not conclude for clear benefits to maintain thiopurine therapy after 18 months. However, relapse occurs between 14% and 41% for the first subsequent year after therapy cessation. 10 In this meta-analysis, as the authors acknowledged, only two studies with high heterogeneity and poor statistical power were included for the assessment of relapse until 5 years. Finally, stopping an effective and well-tolerated thiopurine therapy is still questionable.

Letter: TPMT activity and age in IBD patients – authors' reply

1. Chouchana L, Narjoz C, Beaune P, Loriot MA, Roblin X. Review article: the benefits of pharmacogenetics for improving thiopurine therapy in inflammatory bowel disease. Aliment Pharmacol Ther 2012; 35: 15–36. 2. Serpe L, Calvo PL, Muntoni E, et al. Thiopurine S-methyltransferase pharmacogenetics in a large-scale healthy Italian-Caucasian population: differences in enzyme activity. Pharmacogenomics 2009; 10: 1753–65. 3. McLeod HL, Krynetski EY, Wilimas JA, Evans WE. Higher activity of polymorphic thiopurine S-methyltransferase in erythrocytes from neonates compared to adults. Pharmacogenetics 1995; 5: 281–6. 4. Pettersson B, Almer S, Albertioni F, Soderhall S, Peterson C. Differences between children and adults in thiopurine methyltransferase activity and metabolite formation during thiopurine therapy: possible role of concomitant methotrexate. Ther Drug Monit 2002; 24: 351–8. 5. Ferroni MA, Marchi G, Sansone E, et al. Variability in the rate of 6-mercaptopurine methylation in the erythrocytes, liver and kidney in an Italian population. Eur J Clin Pharmacol 1996; 51: 23–9. 6. Grossman AB, Noble AJ, Mamula P, Baldassano RN. Increased dosing requirements for 6-mercaptopurine and azathioprine in inflammatory bowel disease patients six years and younger. Inflamm Bowel Dis 2008; 14: 750–5.