The Role of TPMT, ITPA, and NUDT15 Variants during Mercaptopurine Treatment of Swedish Pediatric Patients with Acute Lymphoblastic Leukemia.

OBJECTIVE: To evaluate the roles of thiopurine methyltransferase (TPMT), inosine triphosphatase (ITPA), and Nudix hydrolase 15 (NUDT15) in 6-mercaptopurine (6-MP) sensitivity during treatment of pediatric patients with acute lymphoblastic leukemia (ALL). STUDY DESIGN: The study included 102 pediatric patients with ALL subject to the Nordic society Of Paediatric Haematology and Oncology (NOPHO) ALL-2000 and ALL-2008 protocols. Episodes of neutropenia and febrile neutropenia, TPMT sequence variants, as well as 6-MP end doses, were collected retrospectively from medical records. TPMT, ITPA, and NUDT15 sequence variants were analyzed using pyrosequencing. RESULTS: TPMT variants were associated with a reduced risk of neutropenia and febrile neutropenia during the maintenance II period (P = .019 and P < .0001, respectively). In addition, a NUDT15 variant was associated with a lower end dose of 6-MP (P = .0097), but not with neutropenia and febrile neutropenia. ITPA variants were not associated with an increased risk of neutropenia, febrile neutropenia, nor lower end dose of 6-MP. However, when analyzing the entire treatment period, ITPA variants were associated with a decreased risk of febrile neutropenia. CONCLUSIONS: White blood cell count-based dose adjustments are regularly performed for known TPMT- deficient patients and results in a reduced risk of neutropenia and febrile neutropenia. Also in NUDT15-deficient patients dose adjustments are performed as indicated by low end dose of 6-MP. ITPA-deficient patients had a decreased risk of febrile neutropenia when analyzing the entire treatment period. Our data suggest that NUDT15 plays an important role in 6-MP treatment and the results should be confirmed in larger cohorts. Future studies should also follow up whether white blood cell count-based dose adjustments affect the risk of relapse.

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

BACKGROUND: Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy. METHODS: We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor. RESULTS: The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h). CONCLUSIONS: This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Squamous cell dysplasia in the proximal rectum of three patients treated for ulcerative colitis on immunomodulators.

BACKGROUND: Anal canal high-grade squamous intraepithelial lesion (HSIL) is the precursor to anal cancer. Immunocompromised patients are at increased risk and disease is usually within 3 cm from the anal verge. High-resolution anoscopy (HRA) with an 8-cm anoscope is used to identify and guide cautery treatment of HSIL. PURPOSE: We report three patients with a long-term history of ulcerative colitis (UC) treated with systemic immunomodulators who developed proximally located rectal HSIL. RESULTS/OUTCOMES: Two patients were HIV-negative women, 63 and 48 years old, and the third was a 51-year-old HIV-positive man with underlying UC for 10, 16, and 3 years, respectively. They each presented with a HPV-positive HSIL visibly extending above the limits of the anoscope used for HRA. None developed cancer. All had episodes of active UC. It is unclear what causative role systemic immunomodulators play in predisposing UC patients to proximal HSIL. HSIL probably developed on a tongue of HPV-infected squamous epithelium growing proximally over the inflamed rectum. Islands developed when areas of squamous epithelium degenerated, creating skip areas. DISCUSSION: This study highlights the potential for HSIL to extend into the rectum either as a contiguous patch or isolated islands and the need for heightened surveillance in patients with extensive anal canal HSIL treated with immunodulator therapy. HSIL identified at the limit of the anoscope should be investigated further with colonoscopy, and argon plasma coagulation (APC) ablation can serve as an effective treatment option. Patients are at risk for stricture, but it is unclear what role the UC or the ablation played in stricture formation.

Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia.

Genotyping of TPMT prior to 6-mercaptopurine (6-MP) administration in acute lymphoblastic leukaemia (ALL) patients has been integrated into clinical practice in some populations of European ancestry. However, the comparable rates of 6-MP myelotoxicity, but rarity of TPMT variants, in Asians suggest that major determinants have yet to be discovered in this population. We genotyped 92 Japanese paediatric ALL patients for NUDT15 rs116855232, a 6-MP toxicity-related locus discovered in Asians. Logistic regression and survival analysis were used to evaluate its association with leucopenia, hepatotoxicity, 6-MP dose reduction, therapy interruption and event-free survival. The allele frequency of rs116855232 was 0·16, and leucopenia was more common in carriers of the T allele (odds ratio, 7·20; 95% confidence interval, 2·49-20·80; P = 2·7 × 10(-4) ). As leucopenia results in 6-MP dose reduction, we observed average doses during maintenance therapy of 40·7, 29·3 and 8·8 mg/m(2) for patients with CC, CT and TT genotypes, respectively (P < 0·001). Hepatotoxicity was observed only in CC genotype patients. Event-free survival did not significantly differ by NUDT15 genotype. rs116855232 is an important determinant of 6-MP myelotoxicity in Japanese children with ALL and may represent the most robust toxicity-related locus in Asians to date. Considerations for clinical application may be warranted.

A novel turn-on fluorescence sensor based on the Nd (III) complex for the ultrasensitive detection of 6-mercaptopurine.

6-mercaptopurine (6MP) is a chemotherapeuticdrug widely used for treating inflammatory bowel diseases and several cancers. Nevertheless, determining and monitoring its concentration in the human body is highly important because over or under-doses of 6MP can lead to critical health issues. In this paper, we have developed a turn-on fluorescent probe for the determination of the anticancer drug 6-mercaptopurine (6-MP) based on coordination complex [Nd (Anth)3 (H2O)3]. [Nd (Anth)3 (H2O)3] has been synthesized through a simple precipitation process taking the stoichiometric ratio of Nd (III) nitrate hexahydrate and 2-aminobenzoic acid (2-ABA), commonly known as anthranilic acid (Anth). The synthesis and structure have been investigated and validated by different characterizations like UV-visible spectroscopy, FT-IR, HRMS, XPS, and SEM. The synthesized complex displayed excellent fluorescence properties, and the fluorescence intensity was enhanced with the addition of 6MP in the form of a [Fe (6MP)3]2+ mixed complex (Fe-6MP), which is formed by dissolving it in FeCl3. The fabricated sensors displayed the best linear response in a wide range of concentrations from 2.55 µM to 45.51 µM of 6MP. The lower limit of detection (LOD) of the developed sensor was found to be 0.26 µM with a linear correlation coefficient (R2) of 0.99. The synthesized probe gives an acceptable response for the sensing of 6MP in the presence of several interfering agents.

Inflammation is an independent risk factor for colonic neoplasia in patients with ulcerative colitis: a case-control study.

BACKGROUND & AIMS: An association between inflammatory activity and colorectal neoplasia (CRN) has been documented in patients with ulcerative colitis (UC). However, previous studies did not address the duration of inflammation or the effects of therapy on risk for CRN. We investigated the effects of inflammation, therapies, and characteristics of patients with UC on their risk for CRN. METHODS: We collected data from 141 patients with UC without CRN (controls) and 59 matched patients with UC who developed CRN (cases), comparing disease extent and duration and patients' ages. We used a new 6-point histologic inflammatory activity (HIA) scale to score biopsy fragments (n = 4449). Information on medications, smoking status, primary sclerosing cholangitis, and family history of CRN were collected from the University of Chicago Inflammatory Bowel Disease Endoscopy Database. Relationships between HIA, clinical features, and CRN were assessed by conditional logistic regression. RESULTS: Cases and controls were similar in numbers of procedures and biopsies, exposure to steroids or mesalamine, smoking status, and family history of CRN. They differed in proportion of men vs women, exposure to immune modulators, and primary sclerosing cholangitis prevalence. In univariate analysis, HIA was positively associated with CRN (odds ratio [OR], 2.56 per unit increase; P = .001), whereas immune modulators (including azathioprine, 6-mercaptopurine, and methotrexate) reduced the risk for CRN (OR, 0.35; P < .01). HIA was also associated with CRN in multivariate analysis (OR, 3.68; P = .001). CONCLUSIONS: In a case-control study, we associated increased inflammation with CRN in patients with UC. Use of immune modulators reduced the risk for CRN, indicating that these drugs have chemoprotective effects. On the basis of these data, we propose new stratified surveillance and treatment strategies to prevent and detect CRN in patients with UC.

Efficacy and safety of treatment of hepatitis C in patients with inflammatory bowel disease.

BACKGROUND & AIMS: There is uncertainty about the efficacy and safety of treatment for hepatitis C virus (HCV) infection in patients with inflammatory bowel disease (IBD). IBD can become exacerbated during treatment with interferon (IFN), and serious adverse events, such as pancytopenia or hepatotoxicity, can be compounded by drug interactions. We investigated the risk of exacerbation of IBD during HCV therapy and the rate of adverse effects of concomitant therapy for HCV and IBD. We also evaluated the efficacy of HCV treatment in the IBD population. METHODS: We conducted a retrospective review of all patients who underwent IFN-based treatment for HCV at the Mayo Clinic in Rochester, Minnesota from 2001 to 2012. Exacerbation of IBD was evaluated by clinical, endoscopic, and histologic parameters during antiviral therapy and the ensuing 12 months. Hematologic toxicity was assessed by levels of all 3 cell lineages at baseline and during therapy. Efficacy of antiviral treatment was assessed by serum levels of HCV RNA until 24 weeks after completion of therapy. We also conducted a detailed MEDLINE database search and reviewed the literature on this topic. RESULTS: We identified 15 subjects with concomitant IBD (8 with ulcerative colitis and 7 with Crohn's disease). Only 1 patient experienced exacerbation of the disease during therapy; symptoms were controlled with mesalamine enemas. Another patient developed a flare shortly after completing antiviral therapy; symptoms returned spontaneously to baseline 2 weeks later. All subjects experienced an anticipated degree of pancytopenia while on IFN-based therapy. The rate of sustained virologic response was 67%. A concise review of available literature regarding the safety and efficacy of HCV treatment in IBD patients is also presented; although limited, the published data appear to support the safety of treatment with IFN in patients whose IBD is under control. CONCLUSIONS: In conjunction with data from the literature, our findings indicate that the efficacy and safety of HCV therapy with IFN and ribavirin for patients with IBD are comparable to those of subjects without IBD.

Histologic markers of inflammation in patients with ulcerative colitis in clinical remission.

BACKGROUND & AIMS: Mucosal healing, based on histologic analysis, is an end point of maintenance therapy for patients with ulcerative colitis (UC). There are few data on how histologic signs of inflammation correlate with endoscopic and peripheral blood measures of inflammation in these patients. We investigated patterns of histologic features of inflammation in patients with UC in clinical remission, and correlated these with endoscopic and biochemical measures of inflammation. METHODS: We performed a prospective observational study of 103 patients with UC in clinical remission undergoing surveillance colonoscopy while receiving maintenance therapy with mesalamine or thiopurines; 2674 biopsy specimens were collected from 708 colonic segments. Each colonic segment was evaluated based on the Mayo endoscopic subscore and the Geboes histology score (range, 0-5.4). Biomarkers were measured in peripheral blood samples. RESULTS: Histologic features of inflammation were found in 54% of patients receiving maintenance therapy; 37% had at least moderate inflammation based on histology scores. Of the 52 patients with endoscopic evidence only of left-sided colitis, 34% had histologic features of inflammation in their proximal colon. Histology scores correlated with endoscopic scores for per-segment inflammation (Spearman ρ = 0.65; P < .001). Patients with histology scores greater than 3.1 had a significantly higher mean level of C-reactive protein than those with scores less than 3.1. There were no differences among treatment groups in percentages of patients with histologic scores greater than 3.1. CONCLUSIONS: Patients in clinical remission from UC still frequently have histologic features of inflammation, which correlate with endoscopic appearance. Patients with at least moderate levels of inflammation, based on histologic grading (score >3.1), have higher serum levels of C-reactive protein, which could be used as a surrogate marker of histologic inflammation.

Diagnosis and treatment of gastrointestinal disorders in patients with primary immunodeficiency.

Gastrointestinal disorders such as chronic or acute diarrhea, malabsorption, abdominal pain, and inflammatory bowel diseases can indicate immune deficiency. The gastrointestinal tract is the largest lymphoid organ in the body, so it is not surprising that intestinal diseases are common among immunodeficient patients. Gastroenterologists therefore must be able to diagnose and treat patients with primary immunodeficiency. Immune-related gastrointestinal diseases can be classified as those that develop primarily via autoimmunity, infection, an inflammatory response, or malignancy. Immunodeficient and immunocompetent patients with gastrointestinal diseases present with similar symptoms. However, intestinal biopsy specimens from immunodeficient patients often have distinct histologic features, and these patients often fail to respond to conventional therapies. Therefore, early recognition of symptoms and referral to an immunologist for a basic immune evaluation is required to select appropriate treatments. Therapies for primary immunodeficiency comprise immunoglobulin replacement, antibiotics, and, in severe cases, bone marrow transplantation. Treatment of immunodeficient patients with concomitant gastrointestinal disease can be challenging, and therapy with immunomodulators often is required for severe disease. This review aims to guide gastroenterologists in the diagnosis and treatment of patients with primary immunodeficiency.

Bioequivalence of Two 6-Mercaptopurine Tablet Formulations in Healthy Fasting Chinese Volunteers.

The supply of branded 6-mercaptopurine (6-MP) is limited in China, necessitating the local production and clinical evaluation of generic alternatives. We evaluated the in vivo bioequivalence (BE) of a new generic mercaptopurine tablet (50 mg) formulation by comparing peak plasma concentration and area under the concentration-time curve (AUC) with a branded 6-MP formulation as the reference in 36 healthy fasting Chinese adults. The in vivo BE was evaluated by the average BE test. The safety parameters of the test and reference formulations were also evaluated. The geometric mean ratios for AUC over the dosing interval and AUC from time zero to infinity were 104% and 104%, respectively, of the reference values, while the point estimate of the geometric mean ratio for peak plasma concentration was 104% of the reference value. The test and reference formulations in this study were both deemed safe as only 23 Grade 1 adverse events were observed in 13 of 36 subjects. The test and reference formulations of 6-MP tablets meet the regulatory criteria for BE in healthy fasting Chinese adults.

Immunomodulators: still having a role?

Immunomodulators, particularly the thiopurines and to a lesser extent methotrexate, were standard of care for inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, for >40 years. While there has been a renaissance in available therapies with the advent of biologics and small molecules, an impetus remains for the ongoing use of thiopurines and methotrexate. This is particularly true for the maintenance of remission and when used in combination therapy with infliximab to suppress anti-biologic antibodies. This article summarizes the data behind immunomodulator use in Crohn's disease, focusing on the beneficial role these drugs still have while acknowledging their clinical limitations.

NUDT15 is a key genetic factor for prediction of hematotoxicity in pediatric patients who received a standard low dosage regimen of 6-mercaptopurine.

6-Mercaptopurine (6-MP) is commonly used for treatment of acute lymphoblastic leukemia (ALL). The incidence of hematotoxicity caused by this drug is quite high in Asians even using a standard low dosage regimen. The present study was aimed to elucidate the impact of thiopurine S-methyltransferase (TPMT), a nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15), inosine triphosphatase (ITPA) and ATP Binding Cassette Subfamily C Member 4 (ABCC4) polymorphisms on hematotoxicity in pediatric patients who received a standard low starting dose of 6-MP. One hundred and sixty-nine pediatric patients were enrolled and their genotypes were determined. Patients who carried NUDT15∗3 and NUDT15∗2 genotypes were at a 10-15 fold higher risk of severe neutropenia than those of the wild-type during the early months of the maintenance phase. Risk of neutropenia was not significantly increased in patients with other NUDT15 variants as well as in patients with TPMT, ITPA or ABCC4 variants. These results suggest that NUDT15 polymorphisms particularly, NUDT15∗3 and NUDT15∗2, play major roles in 6-MP-induced severe hematotoxicity even when using a standard low dosage of 6-MP and genotyping of these variants is necessary in order to obtain precise tolerance doses and avoid severe hematotoxicity in pediatric patients.

ITPA:c.94C>A and NUDT15:c.415C>T Polymorphisms and Their Relation to Mercaptopurine-Related Myelotoxicity in Childhood Leukemia in Thailand.

BACKGROUND: Mercaptopurine is a key agent in childhood leukemia treatment. Genetic polymorphism in the genes involving thiopurine metabolisms is related to 6-MP related toxicity. OBJECTIVE: This study aimed to determine the prevalence of ITPA:c.94C>A and NUDT15:c.415C>T polymorphisms among Thai children diagnosed with leukemia and their association with mercaptopurine-related myelotoxicity. METHODS: Patients and survivors with a diagnosis of leukemia treated with mercaptopurine-containing chemotherapy regimens were enrolled. Clinical data and laboratory parameters during treatment as well as ITPA:c.94C>A and NUDT15:c.415C>T genotypes were analyzed. RESULTS: In all, 99 patients with acute leukemia or survivors were enrolled in the study. The prevalences of ITPA:c.94C>A, NUDT15:c.415C>T, and co-occurrence of ITPA:c.94C>A and NUDT15:c.415C>T polymorphisms were 34, 17, and 4%, respectively. Numbers of absolute neutrophil count (ANC) and platelet count significantly decreased among patients carrying NUDT15:c.415C>T compared with NUDT15 wild type patients with p-values<0.001 and 0.019, respectively. The differences were not observed among patients carrying ITPA:c.94C>A compared with ITPA wild type patients. According to multivariate GEE, NUDT15:c.415C>T and co-occurrence of ITPA:c.94C>A and NUDT15:c.415C>T had a significant negative effect on ANC during treatment (coefficient: -463.81; CI: -778.53, -149.09; p-value=0.004 and coefficient: -527.56; CI: -1045.65, -9.48; p-value=0.046). No significant effect of ITPA:c.94C>A on ANC during treatment was observed. CONCLUSION: ITPA:c.94C>A and NUDT15:c.415C>T polymorphisms are common among Thai children with leukemia. A strong association with mercaptopurine-related myelotoxicity was observed among patients carrying either NUDT15:c.415C>T alone or combined with ITPA:c.94C>A.

Interactions between PAMAM-NH2 and 6-Mercaptopurine: Qualitative and Quantitative NMR studies.

Despite being used as an anti-leukemic drug, the poor solubility of 6-mercaptopurine (6-MP) limits its use in topical and parenteral applications. Dendrimers are commonly used as drug carriers to improve their solubility in aqueous solution. In this work, the interactions between 6-MP and the amine-terminated poly(amidoamine) dendrimers (PAMAM-NH2 ) were investigated by various NMR technology. The chemical shift titrations disclosed that the 6-MP interacted with the surface of PAMAM-NH2 mainly through electrostatics. The determination of diffusion coefficient and relaxation measurements further confirmed the presence of interactions in 6-MP/PAMAM-NH2 complexes. In addition, the encapsulation of 6-MP within the cavity of PAMAM-NH2 was revealed through nuclear Overhauser effect spectroscopy and Saturation Transfer Double Difference analysis. Finally, the binding strength (H-8 is 100% and H-2 is 70%) of 6-MP to PAMAM-NH2 was quantitatively expressed using epitope maps. This study provides a systematic methodology for qualitative and quantitative studies of the interactions between dendrimers and drug molecules in general.

Mercaptopurine-Loaded Sandwiched Tri-Layered Composed of Electrospun Polycaprolactone/Poly(Methyl Methacrylate) Nanofibrous Scaffolds as Anticancer Carrier with Antimicrobial and Antibiotic Features: Sandwich Configuration Nanofibers, Release Study and in vitro Bioevaluation Tests.

BACKGROUND: 6-Mercaptopurine (6-MP) is a potential anti-cancer agent which its therapeutic and limitation applicability due to its high toxicity. OBJECTIVE: Herein, 6-MP was loaded into tri-layered sandwich nanofibrous scaffold (the top layer composed of poly methyl methacrylate/polycaprolactone (PMMA/PCL), the middle layer was PCL/PMMA/6-MP, and the bottom layer was PCL/PMMA to improve its bioactivity, adjusting the release-sustainability and reduce its toxicity. METHODS: Electrospun tri-layered nanofibers composed of PCL/PMMA were utilized as nano-mats for controlling sustained drug release. Four groups of sandwich scaffold configurations were investigated with alteration of (PMMA: PCL) composition. RESULTS: The sandwich scaffold composed of 2%PCL/4%PMMA/1%6-MP showed the best miscibility, good homogeneity and produced the smoothest nanofibers and low crystallinity. All fabricated 6-MP-loaded-PCL/PMMA scaffolds exhibited antimicrobial properties on the bacterial and fungal organisms, where the cytotoxicity evaluation proved the safety of scaffolds on normal cells, even at high concentration. Scaffolds provided a sustained-drug release profile that was strongly dependent on (PCL: PMMA). As (PCL: PMMA) decreased, the sustained 6-MP release from PCL/PMMA scaffolds increased. Results established that ~18% and 20% of 6-MP were released after 23h from (4%PCL/4%PMMA/1%6-MP) and (2%PCL/4%PMMA/1%6-MP), respectively, where this release was maintained for more than 20 days. The anti-cancer activity of all fabricated scaffolds was also investigated using different cancerous cell lines (e.g., Caco-2, MDA, and HepG-2) results showed that 6-MP-loaded-nanofibrous mats have an anti-cancer effect, with a high selective index for breast cancer. We observed that viability of a cancer cell was dropped to about 10%, using nanofibers containing 2%PCL/4%PMMA/1%6-MP. CONCLUSION: Overall, the PCL: PMMA ratio and sandwich configuration imparts a tight control on long-term release profile and initial burst of 6-MP for anticancer treatment purposes.

Nur77 promotes embryo adhesion by transcriptionally regulating HOXA10 expression.

HOXA10 is an important regulator of embryo adhesion. Reduced expression may contribute to embryo implantation failure. The expression of Nur77 and HOXA10 has been shown to be reduced in the endometrium of patients with recurrent implantation failure. In this study, we found that Nur77 was directly bound to the HOXA10 promoter and promoted HOXA10 protein expression in a dose-dependent manner in Ishikawa cells. Furthermore, the promoting effect of Nur77 on embryo adhesion was significantly blocked when endogenous HOXA10 expression was knocked down in Ishikawa cells. Moreover, the Nur77 agonist 6-MP was also confirmed to promote embryo adhesion. This study is the first to show that Nur77 promotes embryo adhesion by transcriptionally regulating HOXA10 expression. Nur77 and 6-MP may provide a basis to develop a new treatment for patients who suffer from embryo adhesion dysfunction.Abbreviations: HB-EGF: heparin-binding epidermal growth factor-like growth factor; HOXA10: homeobox A10; NGFI-B: nerve growth factor-induced gene B; RIF: recurrent implantation failure; RPL: recurrent pregnancy loss; 6-MP: 6-mercaptopurine; IGFBP-1: insulin-like growth factor binding protein-1; LIF: leukemia inhibitory factor; IL-1ß: Interleukin - 1ß; CRISPR/Cas9: Clustered Regularly Interspaced Short Palindromic Repeats; AF-1: activation function-1 domain; HIF-1α: hypoxia-inducible factor 1α; CREB: cAMP response element binding.

Ancestry and TPMT-VNTR Polymorphism: Relationship with Hematological Toxicity in Uruguayan Patients with Acute Lymphoblastic Leukemia.

6-Mercaptopurine (6-MP) is a thiopurine drug widely used in childhood acute lymphoblastic leukemia (ALL) therapy. Genes such as TPMT and NUDT15 have an outstanding role in 6-MP metabolism. Mutations in both genes explain a significant portion of hematological toxicities suffered by ALL Uruguayan pediatric patients. A variable number tandem repeat in the TPMT promoter (TPMT-VNTR) has been associated with TPMT expression. This VNTR has a conservative architecture (AnBmC). To explore new causes of hematological toxicities related to ALL therapy, we genotyped the TPMT-VNTR of 130 Uruguayan pediatric patients. Additionally, individual genetic ancestry was estimated by 45 ancestry-informative markers (AIMs). Hematological toxicity was measured as the number of leukopenia events and 6-MP dose along the maintenance phase. As previously reported, we found TPMT*2 and TPMT*3C alleles were associated to TPMT-VNTR A2BC and AB2C, respectively. However, contrasting with other reports, TPMT*3A allele was found in a heterogeneous genetic background in linkage equilibrium. Patients carrying more than 5 A repeats present a significant higher number of leukopenia events among patients without TPMT and/or NUDT15 variants. Native American ancestry and the number of A repeats were significantly correlated with the number of leukopenia events. However, the correlation between Native American ancestry and the number of leukopenia events was lost when the number of A repeats was considered as covariate. This suggests that TPMT-VNTR alleles are more relevant than Native American ancestry in the hematological toxicity. Our results emphasize that TPMT-VNTR may be used as a pharmacogenetic biomarker to predict 6-MP-related hematological toxicity in ALL childhood therapy.

Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes.

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 µm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.

Variants in TPMT, ITPA, ABCC4 and ABCB1 Genes As Predictors of 6-mercaptopurine Induced Toxicity in Children with Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia is the most common childhood malignancy. Optimal use of anti leukemic drugs has led to less toxicity and adverse reactions, and a higher survival rate. Thiopurine drugs, including 6-mercaptopurine, are mostly used as antileukemic medications in the maintenance phase of treatment for children with acute lymphoblastic leukemia. For those patients, TPMT genotype- tailored 6-mercaptopurine therapy is already implemented in the treatment protocols. We investigated the role of TPMT, ITPA, ABCC4 and ABCB1 genetic variants as predictors of outcome and 6-mercaptopurine induced toxicity during the maintenance phase of treatment in pediatric acute lymphoblastic leukemia. METHODS: Sixty-eight children with acute lymphoblastic leukemia were enrolled in this study. Patients have been treated according to ALL IC-BFM 2002 or ALL IC-BFM 2009 protocols. Toxicity and adverse events have been monitored via surrogate markers (off-therapy weeks, episodes of leu - ko penia and average 6-mercaptopurine dose) and a prob- abilistic model was employed to predict overall 6-mercaptopurine related toxicity. RESULTS: We confirmed that patients with acute lymphoblastic leukemia that carry inactive TPMT allele(s) require 6- mercaptopurine dose reduction. ITPA and ABCC4 genetic variants failed to show an association with 6-mercapto - purine induced toxicity during the maintenance phase. Carriers of ABCB1 variant allele experienced greater hepatotoxicity. The probabilistic model Neural net which considered all the analysed genetic variants was assessed to be the best prediction model. It was able to discriminate ALL patients with good and poor 6-mercaptopurin tolerance in 71% of cases (AUC=0.71). CONCLUSIONS: This study contributes to the design of a panel of pharmacogenetic markers for predicting thiopurineinduced toxicity in pediatric ALL.