Thiopurine Methyltransferase Genotype Research Articles

Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. Materials and Methods. Three major TPMT single-nucleotide polymorphisms (TPMT∗2, ∗3B, and ∗3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT∗3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT∗2, ∗3A, or ∗3B alleles were observed in this Korean population. Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

Cisplatin-induced ototoxicity and the role of pharmacogenetic testing.

A 10-year-old white male presented with a chief complaint of swelling and pain in the left distal femur that worsened when he participated in sports at school. After several weeks of continued pain and swelling an x-ray was performed and revealed the presence of a large mass. Magnetic resonance imaging confirmed a large primary bone tumor located on the left femur, and open biopsy subsequently confirmed the diagnosis of osteosarcoma. The tumor was 9 cm long in the axis and 7 cm in diameter. Further tests revealed that the disease was localized to that area. The patient weighed 36 kg and was 145 cm tall at the time of diagnosis, placing him in the 75th percentile for age and sex. The patient was started on chemotherapy based on a standard Children’s Oncology Group (COG) treatment protocol of high-dose methotrexate, doxorubicin, and cisplatin. He received cisplatin at a dose of 120 mg/m2 approximately every 5 weeks for a total of 4 treatments. For each treatment, cisplatin 60 mg/m2/day was administered via intravenous infusion on 2 consecutive days. Based on the patient’s body surface area of 1.2 m2, he received a cumulative dose of 576 mg of cisplatin (480 mg/m2). He was also placed on an appropriate antiemetic regimen per COG protocol. He was not on any other medications known to cause ototoxicity or nephrotoxicity. His renal function was normal at baseline and all subsequent lab tests thereafter. Genotyping for thiopurine S-methyltransferase (TPMT) mutations was not performed prior to initiation of cisplatin therapy. Standard audiometric monitoring was performed prior to administration of the first cisplatin dose, and the results were all within the normal range (Brock Grade 0 [Table]).1 The patient completed the full cisplatin treatment course, and audiometric monitoring was performed prior to administration of the second and third cisplatin treatments with no significant hearing impairment noted. On follow-up 2 months after completing the third treatment course, the patient reported having trouble hearing. The loss manifested initially as bilateral tinnitus and ultimately progressed to bilateral hearing loss. Audiometric monitoring performed at this visit revealed moderate to severe hearing loss, defined as a Brock score ≥2 (Table). This translates to educationally significant hearing loss presumed to be caused by the ototoxic effects of cisplatin. Hearing tests performed 6 months and 1 year later revealed that the patient’s hearing had declined slightly but remained in Brock Grade 2, suggesting that the initially observed hearing deficit was likely progressive and irreversible.

Should Thiopurine Methyltransferase Genotypes and Phenotypes be Measured Before Thiopurine Therapy in Patients With Inflammatory Bowel Disease?

Not all of the adverse effects to thiopurine therapy can be explained by thiopurine methyltransferase (TPMT) polymorphisms. This study was intended to evaluate the value of TPMT genotype and phenotype measurement during the first year of thiopurine therapy. Consecutive patients with inflammatory bowel disease (IBD) who were receiving azathioprine or 6-mercaptopurine were followed up for 12 months. TPMT genotypes and phenotypes were examined in patients with IBD before thiopurine therapy and in unrelated healthy volunteers by polymerase chain reaction and high-performance liquid chromatography. A total of 199 patients and 300 healthy volunteers were included at 2 centers. Forty-seven of the 199 patients (23.62%) exhibited adverse effects during the entire course of thiopurine therapy. Two (1%) patients carrying TPMT*3C developed leucopenia at week 4 of azathioprine treatment. The TPMT*3C had a specificity of 100% (163/163) but a sensitivity of 5.56% (2/36) for predicting leucopenia. The calculated optimal cutoff activity for high TPMT activity and decreased TPMT activity was 4.75 U/mL red blood cells. The risk of leucopenia increased in the decreased TPMT group (odds ratio: 20.25; 95% confidence interval: 2.19-187.17; P = 0.004) and increased more during the initial 3 months of thiopurine therapy (odds ratio: 34.80; 95% confidence interval: 3.71-326.77; P = 0.001). Leucopenia occurred more frequently in the patients cotreated with 5-aminosalicylates than in those not cotreated (32.81% versus 11.11%, respectively, P < 0.001). The results of this study suggest that the value of TPMT genotyping before thiopurine therapy is limited in Chinese patients with IBD, considering the low sensitivity of predicting leucopenia, and that phenotyping is a more cost-effective tool that can be successfully used in patients. The coadministration of 5-aminosalicylates results in a high frequency of leucopenia in patients receiving azathioprine or 6-mercaptopurine.

P228 Thiopurine methyltransferase genotype and thiopurine S-methyltransferase activity in Greek children with inflammatory bowel disease

Background Azathioprine (AZA) and 6-mercaptopurine (6MP) are used in the treatment of pediatric inflammatory bowel disease (IBD). Genetic variations in thiopurine S-methyltranfarase (TPMT) gene have been correlated with enzyme activity and with the occurrence of adverse events to AZA and 6MP. The aim of the present study was to examine the sensitivity and specificity of TPMT genotyping for TPMT enzymatic activity, reducing harm from thiopurine by pretesting, and the association of thiopurine toxicity with TPMT status in children with IBD. Methods TPMT red blood cell (RBC) activity was measured by using a radiochemical method and genotype was determined for the TPMT alleles *2, *3A, *3B and *3C in 108 thiopurinetreated pediatric IBD patients with a mean age of 11.3 years (range 3-16). Results Significant TPMT activity differences between wild-type and heterozygous and homozygous mutated subjects were observed. We divided TPMT activity into three categories according to frequency distribution: low (16.67%), intermediate (25.92%) and high (57.41%). The whole population included a total of 77.78% of homozygous wild-type subjects, 15.74% heterozygous variants, 1.85% homozygous variants and five (4.63%) compound heterozygous variant TPMT*3B/*3C. The overall concordance rate between TPMT genotypes and phenotypes was 88.2%. Seven carriers of at least one variant allele and low or intermediate TPMT activity developed adverse effects.

High prevalence of polymorphism and low activity of thiopurine methyltransferase in patients with inflammatory bowel disease

BackgroundGene polymorphism of thiopurine methyltransferase (TPMT) correlates with decreased enzyme activity which determines a significant risk of adverse effect reactions (ADR) in patients treated with thiopurines. The aim of this study was to investigate TPMT genotype and phenotype status in patients with inflammatory bowel diseases (IBD). MethodsFifty-one consecutive out-patients with IBD were genotyped for the following allelic variants: rs1800462 (referred as TPMT*2 allele), rs1800460 (referred as TPMT *3B allele), and 1142345 (referred as TPMT *3C allele). Red blood cell TPMT activity was measured using a competitive micro-well immunoassay for the semi-quantitative determination of TPMT activity in red blood cells (RBC) by means of a 6-MP substrate. ResultsPolymorphism of TPMT was found in 5 out of 51 patients (10%; 95% CI 2%–18%), three heterozygous and two homozygous carriers. Six patients (11.8%; 95% CI 2.4%–19.5%) displayed very low, 12 (23.5%; 95% CI 11.4%–34.5%) intermediate, and 33 (64.7%; 95% CI 52%–78%) normal/high TPMT activity. There were no differences between TPMT genotype and phenotype groups according to age, type of disease, smoking, and chronic medications. A 71% (95% CI 61%–81%; κ=0.45) concordance rate was found between genotype and phenotype status. Six out of 27 (22%) current or past users of azathioprine developed ADR, with three (50%) displaying TPMT genotype and/or phenotype alterations. ConclusionCompared to the general population, IBD patients may have significantly higher prevalence of TPMT polymorphism and, even more, low activity. Phenotypic more than genotypic TPMT analysis could be useful to better manage IBD therapy with thiopurines.

Allele frequency of inosine triphosphate pyrophosphatase (ITPA) and thiopurine-S-methyl transferase (TPMT) genes in the Tunisian population

The aim of this study was to determine the frequencies of TPMT and ITPA polymorphisms in Crohn's disease patients of Tunisian origin and to compare them with allele frequencies previously reported in other populations of various ethnic origins. ITPA (c.94C>A and IVS2+21A>C) and TPMT (c.238G>C, c.460G>A and c.719A>G) mutations and genotypes were assessed in 208 Tunisian subjects (78 males/130 females) by polymerase chain reaction-restriction fragment length polymorphism and allele-specific-PCR methods. Genotyping of ITPA revealed frequencies of 6% and 7.9% for c.94C>A and IVS2+21A>C, respectively. Accordingly, deficient or diminished ITPA phenotype can be predicted to concern 2.4% of Tunisians. The observed frequencies of the c. 238G>C, c.460G>A and c.719A>G TPMT polymorphisms were 0, 0.24 and 1.44%, respectively. This study provides the first analysis of TPMT and ITPA mutant allele frequency in individuals of Tunisian origin. Unlike in Caucasians, TPMT*3C which harbours the c.719A>G polymorphism appears to be the most common mutant allele in Tunisians. In contrast, ITPA mutant allele frequency distribution appears to be similar to that observed in Caucasians.

Genotyping should be considered the primary choice for pre-treatment evaluation of thiopurine methyltransferase function

Background and aimsA pre-treatment determination of the thiopurine S-methyltransferase (TPMT) genotype or phenotype can identify patients at risk of developing severe adverse reactions from thiopurine treatment. The risk of misclassifying a patient might be dependent on the method used. The aim of this study was to investigate the concordance between TPMT genotyping and phenotyping. MethodsThe data consist of 7195 unselected and consecutive TPMT genotype and phenotype determinations sent to the division of Clinical Pharmacology, Linköping, Sweden. TPMT activity was measured in red blood cells (RBC) and the genotype determined by pyrosequencing for the three most common TPMT variants (TPMT *2, *3A, *3C). ResultsTPMT genotyping identified 89% as TPMT wild type (*1/*1), 10% as TPMT heterozygous and 0.5% as TMPT defective. The overall concordance between genotyping and phenotyping was 95%, while it was 96% among IBD patients (n=4024). Genotyping would have misclassified 8% of the TPMT defectives as heterozygous as compared to 11% if only TPMT activity had been measured. 11% of the heterozygous patients had a normal TPMT activity (>8.9U/ml RBC) and 3% of the TPMT wild-type patients had an intermediate TPMT activity (2.5–8.9U/ml RBC). ConclusionsThere is a risk for TPMT misclassification when only genotyping or phenotyping is used, but it is not reasonable to check both in all patients. Since TPMT genotyping is the more reliable test, especially in TPMT heterozygotes, we suggest that genotyping should be considered the primary choice for the pre-treatment evaluation of TPMT function before initiation of thiopurine therapy.

The frequency and distribution of thiopurine S-methyltransferase alleles in south Iranian population

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Variability in TPMT activity is mainly due to genetic polymorphism. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in an Iranian population from south of Iran (n=500), using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. Four hundred seventy four persons (94.8%) were homozygous for the wild type allele (TPMT*1/*1) and twenty five people were TPMT*1/*3C (5%). One patient was found to be heterozygous in terms TPMT*1 and *2 alleles with genotype of TPMT*1/*2 (0.2%). None of the participants had both defective alleles. The TPMT*3C and *2 were the only variant alleles observed in this population. The total frequency of variant alleles was 2.6% and the wild type allele frequency was 97.4%. The TPMT*3B and *3A alleles were not detected. Distributions of TPMT genotype and allele frequency in Iranian populations are different from the genetic profile found among Caucasian or Asian populations. Our findings also revealed inter-ethnic differences in TPMT frequencies between different parts of Iran. This view may help clinicians to choose an appropriate strategy for thiopurine drugs and reduce adverse drug reactions such as bone marrow suppression.

Evaluating the use of metabolite measurement in children receiving treatment with a thiopurine.

Clinical response to thiopurine medication is related to the concentration of its metabolites. Proxy measures are traditionally used to assess dose adequacy. We present our experience of using tioguanine (previously known/formerly referred to as thioguanine) metabolite measurements in paediatric patients and evaluate their effect on clinical practice. To report our experience of using tioguanine metabolite measurements in paediatric patients and to evaluate their effects on clinical practice. The 6-tioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) were measured in children prescribed thiopurine medication for at least 3 months. Data were collected on thiopurine methyl transferase (TPMT) genotype, drug dose, laboratory indices and management changes. Therapeutic 6-TGN levels were defined as 235-400 pmol/8 × 10(8) RBCs. Seventy individuals (30 males) with a median age of 15 years. Underlying diagnoses were 'IBD' (68/70) and two cases of eosinophilic colitis. Sixty-three were treated with azathioprine and seven with mercaptopurine. A total of 103 separate measurements were made. On initial measurement, 68% of patients had 6-TGN levels outside therapeutic levels despite standard thiopurine dosing. Initial 6-TGN levels were significantly higher in patients with TPMT mutations. Toxicity occurred in seven cases. The 6-TGN levels were significantly higher in those with signs of marrow toxicity. The 6-TGN level correlated with WBC, leukocyte count, mean corpuscular volume (MCV) and ΔMCV; however, the ability of each of these to predict therapeutic 6-TGN levels was poor. After initial measurement, management was changed in 25/70 cases (36%). 6-TGN levels were therapeutic in a minority of those patients who were tested. Proxy measures perform poorly in predicting therapeutic 6-TGN levels. Measuring thiopurine metabolites is useful for dosage adjustment in children, and for the detection of potential toxicity.

Index of Suspicion * Case 1: Leg Cramps, Hand Spasms, Diarrhea, and Substantial Weight Loss in a 12 Year Old * Case 2: Hypothermia, Hypoglycemia, and Hyperbilirubinemia in a Neonate * Case 3: Recurrent Fevers, Abdominal Pain, and Cervical Lymphadenopathy in a 7 Year Old

A 12-year-old previously healthy boy presents with leg cramps for 2 weeks and spasms of his hands since yesterday. He also complains of nausea, bloating, epigastric pain, and recurrent watery and loose stools for 1 month. Currently, he is taking lansoprazole for epigastric pain without any relief. He also has a history of involuntary weight loss of 10 lb over 2 months. The family history contains no findings of note. On physical examination, the boy's weight is 36 kg (25th percentile) and height is 140 cm (10th percentile) and he exhibits a positive Trousseau sign and negative Chvostek sign. Remaining physical signs, including on abdominal examination, are normal. Laboratory evaluation shows hypocalcemia (total calcium of 5.3 mg/dL [1.3 mmol/L], ionized calcium of 2 mg/dL [0.5 mmol/L], and corrected calcium of 6.1 mg/dL [1.5 mmol/L]) as well as hypoalbuminemia (3 g/dL [30 g/L]). Serum magnesium measures 1.6 g/dL. CBC shows Hgb of 9.3 g/dL (93 g/L), Hct of 29% (0.29), WBC count of 9.9×103/μL (9.9×109/L), platelet count of 471×103/μL (471×109/L), mean corpuscular volume of 67 fL, and reticulocyte count of 1% (0.01). Results of the iron studies are consistent with iron deficiency anemia, with a serum iron concentration of 6 μg/dL (1.07 μmol/L) (31 to 144 μg/dL [5.5 to 25.8 μmol/L]), transferrin saturation of 3% (0.3), and serum ferritin less than 5 ng/mL (11.23 pmol/L) (21 to 275 ng/mL [47.2 to 617.9 pmol/L]). ESR is 46 mm/h and CRP is 9.5 mg/dL (normal <0.05 mg/dL). His serum 25-hydroxyvitamin D concentration is low at 5.7 ng/mL (14.2 nmol/L) (normal, 32 to 100 ng/mL [79.9 to 249.6 nmol/L]) and serum parathyroid hormone, urine calcium, and creatinine values are within normal limits. His tetany and hypocalcemia resolve with calcium replacement. Additional evaluation leads to the diagnosis. …

A Pragmatic Randomized Controlled Trial of Thiopurine Methyltransferase Genotyping Prior to Azathioprine Treatment: the TARGET Study

To conduct a pragmatic, randomized controlled trial to assess whether thiopurine methyltransferase (TPMT) genotyping prior to azathioprine reduces adverse drug reactions (ADRs). A total of 333 participants were randomized 1:1 to undergo TPMT genotyping prior to azathioprine or to commence treatment without genotyping. There was no difference in the primary outcome of stopping azathioprine due to an adverse reaction (ADR, p = 0.59) between the two study arms. ADRs were more common in older patients (p = 0.01). There was no increase in stopping azathioprine due to ADRs in TPMT heterozygotes compared with wild-type individuals. The single individual with TPMT variant homozygosity experienced severe neutropenia. Our work supports the strong evidence that individuals with TPMT variant homozygosity are at high risk of severe neutropenia, whereas TPMT heterozygotes are not at increased risk of ADRs at standard doses of azathioprine.

Thiopurine methyltransferase (TPMT) genotyping to predict myelosuppression risk.

Azathioprine (AZA), 6-mercaptopurine (6-MP), and thioguanine (TG) are thiopurine drugs. These agents are indicated for the treatment of various diseases including hematologic malignancies, inflammatory bowel disease (IBD), rheumatoid arthritis, and as immunosuppressants in solid organ transplants. Thiopurine drugs are metabolized, in part, by thiopurine methyltransferase (TPMT). TPMT displays genetic polymorphism resulting in null or decreased enzyme activity. At least 20 polymorphisms have been identified, of which, TPMT *2, *3A, *3B, *3C, and *4 are the most commonly studied. These polymorphisms have been associated with increased myelosuppression risk. TPMT genotyping may be useful to predict this risk.

Polymorphism of ITPA 94C&gt;A and risk of adverse effects among patients with acute lymphoblastic leukaemia treated with 6-mercaptopurine

Genetic polymorphisms of thiopurine S-methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase (ITPA 94C>A) contribute to variable responses, including fatal adverse effects, among subjects treated with 6-mercaptopurine (6-MP). Our objectives were to investigate the distribution of specific TPMT and ITPA genotypes in healthy subjects and patients with acute lymphoblastic leukaemia (ALL) from the three main ethnic groups (Malays, Chinese and Indians) in Malaysia and the association of the polymorphisms with adverse effects of 6-MP. Patients with ALL and healthy controls were recruited and genotyped for genetic variants of TPMT and ITPA 94C>A. The relationship between genotypes and clinical outcomes was investigated. Acute lymphoblastic leukaemia patients with allele ITPA 94A were more likely to develop fever and liver toxicity with 6-MP. The prevalence of TPMT variants was low and this makes it unlikely that testing for them would be useful in our populations. Only patients heterozygous for TPMT*3C were detected. Our results suggest that ITPA 94C>A testing, but not TPMT testing, may help in minimizing the adverse effects of 6-MP in Malaysian patients. However, whether this is true in clinical practice requires a larger study and formal randomized controlled evaluation.

Thiopurine Methyltransferase and Inosine Triphosphate Pyrophosphohydrolase Genotypes Among Patients With Inflammatory Bowel Disease Treated With Azathioprine

Thiopurine Methyltransferase and Inosine Triphosphate Pyrophosphohydrolase Genotypes Among Patients With Inflammatory Bowel Disease Treated With Azathioprine

Systematic Review of Thiopurine Methyltransferase Genotype and Enzymatic Testing Strategies

An increased understanding of the genetic basis of disease creates a demand for personalized medicine and more accurate testing for diagnosis and treatment. Thiopurine methyltransferase (TPMT) plays an important role in the metabolism of thiopurine drugs used in pediatric leukemia, rheumatoid arthritis, and inflammatory bowel disease. The objective was to review the literature systematically to ascertain the performance characteristics of current genotype and enzymatic testing technologies for TPMT. A systematic review of the literature was conducted to describe TPMT testing technologies. Eligible studies evaluated either a TPMT genotype or TPMT phenotype technology in comparison to a reference standard and expressed results in terms of sensitivity and specificity or positive/negative predictive value. The laboratory technique was recorded, and the quality of the identified studies was assessed using a modified Critical Appraisal Skills Program tool. Seventeen studies were reviewed. The sensitivity and specificity of the genotype test ranged from 55% to 100% and from 94% to 100%, respectively. The sensitivity and specificity of the phenotype test ranged from 92% to 100% and from 86% to 98%, respectively. A variety of laboratory techniques were employed. Reviewed studies were of low methodological quality. The systematic review of TPMT test strategies found that available technologies demonstrated high values for sensitivity and specificity, however, there was a lack of a single gold standard and most studies were of poor quality. Disregard for study sample size and confounding factors such as concurrent medications and blood transfusions were the main contributors to low quality. There were also inconsistencies in the selection of a reference standard which complicated the interpretation of the findings.

Severe immunosuppression in inflammatory bowel disease: opening the floodgates to opportunists?

Dear editor: Ulcerative colitis is a primarily non-infectious, chronic inflammatory bowel disease that typically affects the rectum and may involve the whole colon in an uninterrupted pattern. The disease has a high impact on the patients' health-related quality of life and is prone to repetitive hospitalisation. Therapeutic options primarily include lifestyle alterations, medical management and surgical interventions. Medical as well as surgical therapy may be associated with lifethreatening complications, which may require intensive care unit (ICU) admission. Here, we report on a female patient suffering from ulcerative colitis, whose medical therapy was complicated by toxic megacolon, and later on pulmonary aspergillosis and disseminated herpes zoster. A 55-year old woman with ulcerative colitis suffered from purulent haemorrhagic diarrhoea and weight loss of 9 kg within 2 weeks and was treated in a community general hospital for 6 weeks. Azathioprine (2.5 mg kg d p.o.) was administered for a total of 15 days and had been discontinued due to severe leukopenia. At that time, the patient had a high erythrocyte thiopurine methyl thiopurine methyltransferase (TPMT) activity (38 nmol h− gHb−; normal >23, intermediate 10–23, low <10 nmol h− gHb−), which represents the detoxifyfing enzyme for azathioprine. In addition, TPMT genotyping was performed and confirmed the absence of clinically relevant enzyme polymorphisms. Furthermore, toxic 6-thioguanine nucleotides (6-TGN) were undetectable in the patient's blood. Persistent bowel inflammation was treated with high doses of topical and systemic corticosteroids (prednisolone up to 2 mg kg− per day) and intravenous cyclosporine (3 mg kg− as continuous infusion over 24 h) in combination with antimicrobial therapy. Cyclosporine was discontinued after 6 days as leukopenia deteriorated, and subsequent immunomodulatory therapy consisted of mesalazine and regressive doses of prednisolone. At transferal to our university hospital, the patient presented the full clinical picture of toxic megacolon, which required emergency subtotal colectomy, blind closure of the rectum and percutaneous ileostomy. Following surgery, the patient was transferred to the intermediate care unit and treated with antimicrobial therapy (cefuroxime and metronidazole) and moderate doses of corticosteroids (prednisolone 1 mg kg per day). On postoperative day 3, she developed acute cholecystitis, which required urgent cholecystectomy and escalation of antimicrobial therapy (meropenem and vancomycin). Throughout the early clinical course, the patient remained leukopenic with white blood cell counts between 950 and 2,840 cells per microliter. Fractional lymphocyte count persisted below 10%. Filgrastim treatment (recombinant-methionyl human granulocyte colony-stimulating factor; r-metHuG-CSF; Christian Ertmer and Wolf A. Mardin equally contributed to this work.

Thiopurine methyltransferase polymorphisms and mercaptopurine tolerance in Turkish children with acute lymphoblastic leukemia

Thiopurine methyltransferase (TPMT) enzyme is involved in the metabolism of 6-mercaptopurine (6-MP), a key component of acute lymphoblastic leukemia (ALL) treatment protocols in children. The aims of this study were to investigate the frequency of common genetic polymorphisms associated with low TPMT activity and correlations of polymorphic variants with 6-MP tolerance in a group of Turkish children with ALL. Genotyping for G238C, A719G, and G460A mutations were performed by using NanoChip Technology. Adverse reactions during the first 6months of maintenance therapy with oral 6-MP and methotrexate were retrospectively analyzed from patient's files. Five (8.6%) of 58 children with ALL had a polymorphic TPMT allele: 4 (3.4%) were heterozygous for TPMT*3A (G460A and A719G), and one (0.9%) was heterozygous for TPMT*3C (A719G). No cases with TPMT*3B (G460A) or TPMT*2 (G238C) variants were identified. Children with TPMT*3A and *3C had significantly lower leukocyte and neutrophil counts and percentage of target 6-MP dosage, and longer periods with ≥grade 2 infections, ≥grade 2 liver toxicity, and chemotherapy interruptions than the children with wild-type TPMT during the first 24weeks of maintenance therapy. The frequency and distribution of common TPMT polymorphisms in Turkish children with ALL is similar to other Caucasian populations. Polymorphic variants were associated with excessive 6-MP toxicity supporting the suggestion that TPMT genotyping should be performed before institution of 6-MP therapy.

Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy

6-mercaptopurine (6-MP) is used in the treatment of childhood acute lymphoblastic leukaemia (ALL). Its red blood cell (RBC) metabolite concentrations (6-thioguanine [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) are related to drug response. We investigated the impact of non-genetic covariates and pharmacogenetic polymorphisms affecting thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) on 6-MP metabolism and response. Sixty-six children with ALL treated according to EORTC 58951 protocol were included in this study. Six patients had a heterozygous genotype for the most common TPMT polymorphisms, nine for ITPA 94 C > A and 17 for ITPA IVS2+21 A > C. 6-MP metabolites concentrations were analyzed by mixed model analysis. During maintenance, steady-state RBC 6-TGN concentrations were lower in patients aged 6 years or younger (493 pmol/8 × 10(8) RBC) than in older children (600 pmol/8 × 10(8) RBC). 6-MMPN concentrations were low in patients with TPMT variant/wild-type ITPA (1862 pmol/8 × 10(8) RBC), intermediate in wild-type patients and high (16468 pmol/8 × 10(8) RBC) in patients wild-type TPMT/variant ITPA. A 6-MMPN threshold of 5000 pmol/8 × 10(8) RBC was associated with an increased risk of hepatotoxicity. In this study, age and both TPMT and ITPA genotypes influenced 6-MP metabolism. High 6-MMPN was associated with hepatotoxicity. These pharmacological tools should be used to monitor ALL treatment in children.

Use of thiopurine testing in the management of inflammatory bowel diseases in clinical practice: A worldwide survey of experts

We performed a worldwide survey to evaluate the extent to which gastroenterologists who are experts in the field of inflammatory bowel diseases (IBDs) are utilizing thiopurine metabolism in practice. This was a Web-based cross-sectional survey consisting of 12 multiple-choice and open-ended questions. Between December 2009 and April 2010, 175 questionnaires were received. The proportion of practitioners with access and reimbursement for thiopurine S-methyltransferase (TPMT) genotype, TPMT phenotype, 6-thioguanine nucleotides (6-TGN) levels, and 6-methylmercaptopurine ribonucleotides (6-MMP) levels was 48%, 54%, 44%, and 35%, respectively. Before azathioprine initiation, TPMT genotype and phenotype were performed by only 30% and 43% of responders, respectively. In patients on thiopurine therapy, 6-TGN and 6-MMP levels were determined by 54% and 44% of responders, respectively. Only 27% of physicians always wait for TMPT activity/genotype results before initiating azathioprine and 81% do not routinely recheck metabolite levels after dose escalation or reduction. In cases of very high or low TPMT activity, 75% and 74% of practitioners take into account TMPT activity result, respectively. If access to all azathioprine metabolite measurements was available and if all these tests were reimbursed by public health insurance, 47% of responders would use these tests more often in their practice. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influenced experts' attitudes. Thiopurine testing is relatively underutilized by IBD gastroenterologists. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influence the management of IBD patients treated with thiopurines.

A cost effectiveness analysis of thiopurine methyltransferase testing for guiding 6‐mercaptopurine dosing in children with acute lymphoblastic leukemia

An increased understanding of the genetic basis of disease creates a demand for personalized medicine and more genetic testing for diagnosis and treatment. The objective was to assess the incremental cost-effectiveness per life-month gained of thiopurine methyltransferase (TPMT) genotyping to guide doses of 6-mercaptopurine (6-MP) in children with acute lymphoblastic leukemia (ALL) compared to enzymatic testing and standard weight-based dosing. A cost-effectiveness analysis was conducted from a health care system perspective comparing costs and consequences over 3 months. Decision analysis was used to evaluate the impact of TPMT tests on preventing myelosuppression and improving survival in ALL patients receiving 6-MP. Direct medical costs included laboratory tests, medications, physician services, pharmacy and inpatient care. Probabilities were derived from published evidence. Survival was measured in life-months. The robustness of the results to variable uncertainty was tested in one-way sensitivity analyses. Probabilistic sensitivity analysis examined the impact of parameter uncertainty and generated confidence intervals around point estimates. Neither of the testing interventions showed a benefit in survival compared to weight-based dosing. Both test strategies were more costly compared to weight-based dosing. Incremental costs per child (95% confidence interval) were $277 ($112, $442) and $298 ($392, $421) for the genotyping and phenotyping strategies, respectively, compared to weight-based dosing. The present analysis suggests that screening for TPMT mutations using either genotype or enzymatic laboratory tests prior to the administration of 6-MP in pediatric ALL patients is not cost-effective.