Thiopurine Methyltransferase Enzyme Research Articles

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17%). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02U/mL, P < 0.001). Twenty-seven cases (21.42%) exhibited adverse effects. All of the heterozygotes (4/4, 100%) developed severe leukopenia, and three cases (3/4, 75%) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17%, respectively, and the sensitivity was 28.57 and 60.00%, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

P-176 Thiopurine S-methyltransferase Analysis Does Not Correctly Predict the Risk of Toxicity in Hispanic Children with Inflammatory Bowel Disease

Thiopurine S-methyltransferase (TPMT) catalyzes the methlyation of thiopurine compounds, and is measured prior to using medications such as 6-mercaptopurine or Azathioprine. TPMT can be measured as blood enzyme levels or allelic variations (phenotype or genotype assay), both of which are important in distinguishing the possibility of desired clinical effects versus toxicity. The aim of our study was to evaluate the prevalence of variations in TPMT activity in pediatric Hispanic Inflammatory Bowel Disease (IBD) patients in our South Florida based Pediatric Gastroenterology practice, and to evaluate the reliability of TPMT levels in predicting toxicity risk in our studied population. Sixty-four pediatric Hispanic patients treated with either 6-mercaptopurine or Azathioprine for IBD were retrospectively studied. Either a radiochemical method to measure TPMT phenotype or TPMT genotype was analyzed to detect allelic variations and predisposition to myelotoxicity, hepatotoxicity, and pancreatotoxicity. Of the 64 Hispanic patients included (mean age: 16 years): 54.6% with Crohn's disease, 37.5% with Ulcerative Colitis, 4.6% with nonspecific colitis, and 3.1% with other unspecified abdominal complaints. TPMT phenotypes were documented in 50 patients and Genotype analysis was documented in 14 patients. TPMT phenotype was distributed as: Normal level 80% (40/50), Intermediate 18% (9/50), Low 2% (1/50). TPMT genotypic variations were distributed as: Normal 92.8% (13/14), Intermediate 7.1% (1/14), Low 0% (0/14). Toxicity was characterized by leukopenia with or without neutropenia, anemia, pancytopenia, elevated transaminases, or pancreatitis. The percentage of patients with toxicity regardless of normal or abnormal TPMT levels or alleles were 37.5%. This was further evaluated to find that 12.5% had liver toxicity, 15% had bone marrow toxicity, and 10% had both. The percentage of patients with toxicity despite normal TPMT phenotype was 22.5%. The percentage of patients with toxicity despite normal genotype was 23.07%. Our study demonstrates that 17% (11/64) of pediatric Hispanic patients in our practice have either an abnormal phenotype or genotype TPMT enzyme analysis, which is greater than previously documented in other ethnic groups. Additionally, the risk of toxicity despite normal TPMT enzyme levels and alleles was higher in our studied group compared to other previously studied populations, indicating that pediatric Hispanic patients must be monitored carefully at regular intervals to monitor for adverse reactions to thiopurine based medications.

TPMT genetic variants are associated with increased rejection with azathioprine use in heart transplantation

Azathioprine (AZA) is an important immunosuppressant drug used in heart transplantation (HTX). Consensus guidelines recommend that patients with thiopurine S-methyltransferase (TPMT) genetic variants be started on lower AZA dose because of higher active metabolite levels and risk of adverse events. However, in-vitro lymphocyte proliferation assays performed in participants with inactive TPMT alleles have suggested that AZA use may result in decreased immunosuppressant efficacy as compared with wild-type (WT) individuals. The objective of this study was therefore to determine the effect of TPMT genetic variation on AZA efficacy or prevention of rejection in HTX recipients treated with AZA. We genotyped 93 HTX recipients treated with AZA and measured erythrocyte TPMT enzyme activity. Acute rejection was monitored by routine endomyocardial biopsies. There were 83 WT and 10 heterozygote (HZ) HTX recipients. TPMT activity level was lower in HZ compared with WT (13.1±2.8 vs. 21±4.5 U/ml red blood cell, P<0.001). Despite similar AZA dose, HZ developed severe rejection earlier (P<0.001), and the total rejection score was higher (P=0.02) than WT. AZA was discontinued more frequently in HZ (P=0.01) because of rejection. The incidence of leukopenia was similar between the groups (40 vs. 43%, P=1.0). HTX recipients with TPMT genetic variant alleles who are treated with AZA develop acute rejection earlier, more frequently, and of greater severity. These patients, despite having lower TPMT enzymatic activity, should be monitored carefully for possible increased risk of acute rejection.

Comparison of three methods for measuring thiopurine methyltransferase activity in red blood cells and human leukemia cells

Thiopurine efficacy is partly reflected by the genetic polymorphism of the thiopurine methyltransferase (TPMT) enzyme, which is responsible for variation in the metabolism, toxicity and therapeutic efficacy of the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Determination of TPMT activity before administration of thiopurines is thus crucial for individualized dosing in order to prevent toxicity in TPMT deficient individuals. These individuals must be treated with markedly lower (eg, 5-10% of the standard) doses of the prescribed medications. This paper describes a comparison of three different methods for the quantification of TPMT activity in red blood cells (RBC) and cultured human cell lines. We succeeded to perform the measurement of TPMT activity in a minimum amount of 1×10(6) cultured cells with an HPLC-UV system modified and optimized in our laboratory. The TPMT activity was linearly correlated with the cell concentration of the cultured cell line in a range of 1-10×10(6) cells. A significant correlation of determination of TPMT activity in RBC between radiometric detection by HPLC, classic radiochemical detection and UV detection by HPLC, was observed, correlation coefficient (r) were 0.72 and 0.73, respectively. The within-day and day-to-day coefficients of variation of the HPLC-UV-based method were 8% and 16%, respectively. The evaluation of the methods was demonstrated by studying the TPMT activity in RBC isolated from 198 patients, as well as in MOLT4 leukemic cell line and its sub-cell lines with acquired resistance to 6-MP and 6-TG.

Aplastic anemia secondary to azathioprine in systemic lupus erythematosus: report of a case with normal thiopurine S-methyltransferase enzyme activity and review of the literature

Azathioprine-induced aplastic anemia and fatal myelosuppression is a rare occurrence in patients with systemic lupus erythematosus (SLE). We report a case of a 53-year-old female with a normal thiopurine S-methyltransferase (TPMT) level who developed aplastic anemia within 4 weeks of azathioprine initiation, resulting in death. Physicians should be vigilant in monitoring routine blood work when administering azathioprine, a relatively common drug, in patients with SLE.

Prevalence of TPMT polymorphism in Indian patients requiring immunomodulator therapy and its clinical significance

Thiopurine methyltransferase (TPMT) enzyme plays a key role in the metabolism of azathioprine/6-mercaptopurine (6-MP). Mutations in the enzyme lead to generation of excess thioguanine, which causes suppression of various cell lineages, especially neutrophils. Data on the prevalence of TPMT polymorphism are available from Western and some Asian countries; such data from India are sparse. The aim of this research is to study the prevalence of TPMT mutation in Indian patients requiring immunomodulator therapy and its relation to the development of neutropenia on azathioprine therapy. In this retrospective study, data of all patients who underwent TPMT genotyping by PCR-RFLP and allele-specific PCR prior to immunomodulator therapy were analyzed. The frequency of on-treatment development of neutropenia (total neutrophil count <1,500 per cubic millimeters) was noted. Data were available on 126 patients (mean age, 42 [SD 13.6] years; 73 men and 53 women). The disease indications included ulcerative colitis (61), Crohn's disease (43), indeterminate colitis (1), autoimmune hepatitis (16), and others (5). TPMT genotype was wild in 120 patients (95.23 %) and heterozygous in 6 patients (4.77 %); no patient had homozygous mutation. Seven of 87 patients (6.8 %) who received azathioprine developed neutropenia; blood counts normalized on cessation of the drug in all. The incidence of neutropenia in patients with wild type was 6/84 (7.14 %) and with heterozygous type 1/3 (33 %) (p = 0.5764). Nearly 5 % of this population of patients requiring immunomodulator therapy was heterozygous carriers of the TPMT gene. Neutropenia was equally common in patients without and with the mutation.

The pharmacokinetic effect of adalimumab on thiopurine metabolism in Crohn's disease patients

Background and aimsA drug interaction between infliximab and azathioprine has previously been reported in Crohn's disease patients: the concentration of the main active thiopurine metabolites, the 6-thioguanine nucleotides (6-TGN), increased 1–3weeks after the first infliximab infusion by 50% compared to baseline.The aim of this prospective study was to determine the effect of adalimumab on thiopurine metabolism in Crohn's disease patients, evaluated by 6-TGN and 6-methylmercaptopurine ribonucleotides (6-MMPR) concentration measurement. MethodsCrohn's disease patients on azathioprine or mercaptopurine maintenance therapy starting with concomitant adalimumab treatment were included. 6-TGN and 6-MMPR concentrations were determined before initiation of adalimumab and after 2, 4, 6 and 12weeks of combination therapy. The activity of three essential enzymes involving thiopurine metabolism, thiopurine S-methyltransferase (TPMT), hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and inosine-triphosphate pyrophosphatase (ITPase), was evaluated at baseline and week 4. Clinical outcome was evaluated by the Crohn's disease activity index and C-reactive protein concentrations at baseline, week 4 and week 12. ResultsTwelve Crohn's disease patients were analyzed. During the follow-up period of 12weeks the median 6-TGN and 6-MMPR concentrations did not significantly change compared to baseline. TPMT, ITPase and HGPRT enzyme activity did not change either after 4weeks. In two patients (17%) myelotoxicity was observed within 2–4weeks, in whom both low therapeutic 6-TGN and 6-MMPR concentrations were found. ConclusionsIn this study in Crohn's disease patients no pharmacokinetic interaction was shown between adalimumab and the conventional thiopurines, azathioprine and mercaptopurine.

Preanalytical stringency: what factors may confound interpretation of thiopurine S-methyl transferase enzyme activity?

Measurement of red blood cell thiopurine S-methyl transferase (TPMT) enzyme activity before commencing thiopurine therapy is recommended to avoid severe bone marrow suppression in TPMT-deficient patients. Patient's samples go through preanalytical transportation and storage steps before measurement. We studied patient's TPMT activity sample data to assess the effect of preanalytical variables including transportation time. A total of 8524 TPMT enzyme activity analyses were conducted from 2002 to 2010 in a single laboratory, with samples sent from seven centres throughout New Zealand. TPMT activity was correlated with time of arrival at the reference laboratory, patient gender and age and centre of sample collection. The 6348 (74%) selected TPMT measurements that fulfilled selection criteria ranged from 0 to 25.8 IU/mL. Median delay to sample analysis was 42 h. Median TPMT activity was significantly lower for all centres compared with the reference centre (P < 0.001). Delay in sample arrival was significantly and independently correlated with TPMT enzyme activity (ANCOVA; P < 0.001), which showed a 0.011 (95% CI, 0.008-0.014) IU/mL decrease per extra hour of delay. After correcting for these data, one centre still had a significantly lower TPMT enzyme activity compared with the reference centre. There was a significant negative correlation between TPMT enzyme activity and delay from sample collection to analysis. Transportation time is therefore an important preanalytical variable influencing TPMT activity. Samples from one centre had a lower TPMT activity after correcting for transportation delay, suggesting that other factors related to sample processing may also be relevant.

Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.

Biological variation of thiopurine methyltransferase enzyme activity: when has a significant change taken place?

Thiopurine methyltransferase (TPMT) enzyme activity is measured before initiating thiopurine therapy to reduce the risk of severe drug-associated myelotoxicity in patients with low enzyme activity. TPMT activity may vary over time in relation to drug treatment and patient clinical condition. What constitutes a significant change in TPMT activity can be derived from biological variation and analytical imprecision. A large national laboratory database was used to identify patients with three or more TPMT activity measurements. Variance of TPMT activity was analysed by determining the total coefficient of variation (CVTOT) of repeated measurements and by correlation with parameters including gender and follow-up time. Between-run analytical imprecision (CVa) was determined by replicate analysis (n = 314). Of 7383 patients with TPMT measurements, 136 were identified as having three or more measurements over time (range 3-14). Median CVTOT for individual patient results was 14.5% (range 2.5-36.7%). Analytical imprecision (CVa) was 10.3%. A reference change value (or critical difference) with 95% probability was calculated as 42%. Therefore, a change in measured TPMT activity above 42% should lead to considering sources of variation other than biological variation and analytical imprecision. TPMT enzyme activity needs to change by at least 42% to determine that a true change has taken place beyond biological variation and analytical imprecision. A single measurement of TPMT activity is sufficient for most clinical purposes.

PWE-088 Abnormal Liver Function Tests Following use of Thiopurines in a Large Cohort of Inflammatory Bowel Disease Patients-Do TPMT Levels Matter?

IntroductionThiopurine (azathioprine and 6 –Mercaptopurine (6MP)) use is one of the aetiologies for abnormal liver function tests in patients with inflammatory bowel disease. Some studies report hepatotoxicity is associated with...

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients. Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line. Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (-9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %). Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Nomenclature for alleles of the thiopurine methyltransferase gene

The drug-metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine, and azathioprine, drugs that are widely used for treatment of acute leukemias, inflammatory bowel diseases, and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants that cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pretreatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however, this has caused some problems as exemplified by two instances in which authors' articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define the nomenclature and numbering of novel variants for the TPMT gene. A website (http://www.imh.liu.se/tpmtalleles) serves as a platform for this work. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (G>A) from TPMT*24 to TPMT*30 and position 611 (T>C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged.

The Utility of Thiopurine Methyltransferase Enzyme Testing in Inflammatory Bowel Disease

To assess the levels of red blood cell thiopurine methyltransferase (TPMT) in subjects with inflammatory bowel disease (IBD) and to determine how these levels impacted thiopurine dosing and leukopenia over the first six months of therapy. A retrospective chart review was performed on all adult IBD patients (n=423, 88.2% Caucasian) who had TPMT levels measured by 11 participating gastroenterologists in Manitoba between 2008 and 2010. In addition to descriptive data, white blood cell count, dose and reason for discontinuation were analyzed for the first six months of therapy. Patients receiving ≥2.0 mg/kg of azathioprine (AZA) or ≥1.0 mg/kg of 6-mercapatopurine were considered to be 'substantially' dosed. Of the 423 patients, 8.3% had intermediate levels and 93.4% had normal levels of TPMT. Only one subject had a low level. A total of 216 patients had sufficient data to be included for full analysis. Patients with intermediate TPMT levels were generally started at lower doses of thiopurine than patients with normal TPMT levels (mean [± SD] 1.0±0.6 mg/kg versus 1.8±0.5 mg/kg). Of the subjects with normal TPMT levels, only 37.8% were dosed with ≥2.0 mg/kg of AZA. Each month, approximately 5% of subjects were leukopenic. These subjects received a mean overall AZA dose of 1.9±0.3 mg/kg and had a mean white blood cell count of 3.8±0.4×10(9)/L. Normal TPMT levels did not prevent the development of leukopenia, although life-threatening leukopenia was rare. Physicians are not using TPMT levels to substantially dose thiopurines at the outset, which may limit the speed at which adequate doses are reached to facilitate remission.

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.

Host Thiopurine Methyltransferase Status Affects Mercaptopurine Antileukemic Effectiveness

AbstractAbstract 3560Thiopurines are routinely used to treat many hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), as well as inflammatory and autoimmune diseases. Common polymorphic null alleles among humans in thiopurine methyltransferase (TPMT) render 1 in 400 patients equivalent to homozygous knock-outs (−/−), 10% of patients equivalent to hemizygous (+/−), and 90% of patients comparable to wild-type (+/+) status for TPMT enzyme activity. TPMT activity is inversely related to the accumulation of active thioguanine nucleotide metabolites among patients, resulting in a higher risk for myelosuppressive adverse effects if routine doses are given to −/− compared to +/− compared to +/+ patients. However, the impact of the host TPMT polymorphism on antileukemic efficacy in the clinic is less clear.We generated a murine model of ALL by transducing bone marrow cells of Arf-null mice on the 129X1SvJ background (Tpmt +/+) with a retroviral vector encoding p185 BCR-ABL and luciferase, and culturing them for 7–10 days. We injected 2000 BCR-ABL+ pro/pre-B cells into Tpmt-deficient (−/−), heterozygous (+/−), or wild type (+/+) unconditioned syngenic recipients and compared the antileukemic efficacy when treating with a relatively low dose of mercaptopurine (equivalent to 7–9 mg/m2—such as would be tolerated by patients homozygous for null TPMT alleles). Treatment was started one day after injection of leukemia cells and continued for 70 days or until mice were sacrificed due to moribund state (usually due to illness from leukemia). To mimic clinically relevant treatment regimens, methotrexate was injected intraperitoneally (IP) at 1 mg/kg, once a week. Mercaptopurine was administered in the drinking water at 10 mg/L, for an estimated delivered dose of 2 – 2.5 mg/kg/day of mercaptopurine.Erythrocyte TPMT activity segregated by Tpmt genotype as expected and was comparable to the differences observed in patients: 0.6 U/ml in Tpmt −/−, 6.7 U/ml in Tpmt +/−, and 11.6 U/ml in Tpmt +/+ mice (p =0.008, n=2 for each genotype). As expected, the median survival (± standard error) was similar among Tpmt genotypes in untreated mice: 16 (± 0.4) days for Tpmt +/+, 17 (± 0.7) days for Tpmt +/−, and 18 (± 0.4) days for Tpmt −/−. The difference in leukemia-free survival by Tpmt genotype was profound: at 30 days after the start of treatment, 5% (± 9%) of Tpmt +/+ mice, 47% (± 26%) of Tpmt +/− mice, and 85% (± 14%) of Tpmt −/− mice were surviving (p = 5×10−8, Figure), indicating a substantial impact of host Tpmt status on thiopurine antileukemic effectiveness. In vivo imaging of luciferase-marked ALL cells confirmed an intermediate response phenotype for the Tpmt heterozygous mice (p = 0.00001). With higher doses (closer to the doses tolerated by patients homozygous for wild-type alleles, equivalent to 50–70 mg/m2/day), leukemia-free survival improved in the Tpmt +/+ and Tpmt +/− mice, comparable to the survival of Tpmt −/− mice treated with the low dose of mercaptopurine. These findings support the notion that germline polymorphisms in Tpmt, even in the heterozygous state, affect not only host tissue toxicity, but antitumor effectiveness as well. [Display omitted] Disclosures:Evans:St. Jude Children’s Research Hospital: Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms., Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms. Patents & Royalties. Relling:St. Jude Children’s Research Hospital: Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms., Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms. Patents & Royalties; Sigma-Tau Pharmaceuticals: Research Funding.

Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5

The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

Letter: TPMT – not all that glitters is gold; authors’ reply

We appreciate the interest shown in our paper1 by Dr Gonzalez-Lama and Professor Gisbert.2 We agree that there are several aspects of the thiopurine metabolism pathway that are unexplained despite the large volume of research in this area. In particular, the cause or causes of preferential 6-MMP production is/are not yet clear, and we have shown that thiopurine methyltransferase (TPMT) enzyme activity is not the main determinant of this. However, knowledge of TPMT activity in patients is still important to predict some of the cases of severe bone marrow suppression and to help predict final metabolite concentrations, and therefore final doses.3 We agree that if more clinical data had been available to us, then more conclusions and recommendations could have been drawn from the study. Indeed, the limitations raised were already described in our discussion section. However, in response to the suggestion of possible bias due to selective measurements taken in patients with certain characteristics, TPMT determination prior to thiopurine therapy is routine in New Zealand, and so these results should not be subjected to selection bias. Moreover, the TPMT enzyme activity distribution and mean TPMT activity did not differ from that found in our previous studies among inflammatory bowel disease (IBD) patients.4, 5 In parts of New Zealand, thiopurine metabolite monitoring is now routine for all IBD patients, and in other centres, metabolites are measured only when there is nonresponse to treatment or suspected toxicity. This may have resulted in an over-representation of preferential 6-MMP production patients in our study, as we discussed in the paper. However, if anything this should have enhanced our ability to show a clinically relevant difference in TPMT enzyme activity between those with a high vs. low 6-TGN/6-MMP ratio. We demonstrated, with large patient numbers, that there was no such difference. Declaration of personal and funding interests: None.

Putting pharmacogenomics into practice

Putting pharmacogenomics into practice

High TPMT enzyme activity does not explain drug resistance due to preferential 6‐methylmercaptopurine production in patients on thiopurine treatment

Up to 20% of patients on thiopurine therapy fail to achieve adequate drug response. Many of these patients preferentially produce the toxic 6-methylmercaptopurine metabolites (6-MMP) rather than the active 6-thioguanine nucleotides (6-TGN) resulting in a high 6-MMP/6-TGN ratio (>20) and increased risk of hepatotoxicity. To determine the prevalence of preferential 6-MMP producers and define the relationships between 6-TGN, 6-MMP and thiopurine methyltransferase (TPMT). The database of 6-TGN, 6-MMP and TPMT measurements from patients throughout New Zealand was used to calculate patients' 6-MMP/6-TGN ratios and identify those with high (>20) or normal ratio (≤20).The TPMT enzyme activity was compared amongst the groups. Of 1879 patients with TPMT, 6-TGN and 6-MMP results, 349 (19%) had a 6-MMP/6-TGN ratio >20. The mean TPMT enzyme activity was slightly lower for those with a 6-MMP/6-TGN ratio ≤20 vs. >20, which achieved statistical significance (12.2 vs. 13.2; P < 0.001). However, the distributions of TPMT enzyme activity were similar, with 97% of TPMT results falling between 5.0 and 17.6 IU/mL for both groups. In all, 17% of those with 6-MMP/6-TGN ratio ≤20 were intermediate TPMT metabolisers (TPMT 5.0-9.2 IU/mL) vs. 7% in those with a ratio >20. In this patient population with measured 6-MMP/6-TGN ratios, 19% of patients were preferential 6-MMP producers. The results show that high TPMT enzyme activity is not the major reason for preferential 6-MMP production in most patients with a high metabolite ratio. This suggests that there are one or more important alternative mechanisms for preferentially producing 6-MMP.