Thiopurine methyltransferase and treatment outcome in the UK acute lymphoblastic leukaemia trial ALL2003

The influence of thiopurine methyltransferase (TPMT) genotype on treatment outcome was investigated in the United Kingdom childhood acute lymphoblastic leukaemia trial ALL2003, a trial in which treatment intensity was adjusted based on minimal residual disease (MRD). TPMT genotype was measured in 2387 patients (76% of trial entrants): 2190 were homozygous wild‐type, 189 were heterozygous for low activity TPMT alleles (166 TPMT*1/*3A, 19 TPMT*1/*3C, 3 TPMT*1/*2 and 1 TPMT*1/*9) and 8 were TPMT deficient. In contrast to the preceding trial ALL97, there was no difference in event‐free survival (EFS) between the TPMT genotypes. The 5‐year EFS for heterozygous TPMT*1/*3A patients was the same in both trials (88%), but for the homozygous wild‐type TPMT*1/*1 patients, EFS improved from 80% in ALL97% to 88% in ALL2003. Importantly, the unexplained worse outcome for heterozygous TPMT*1/*3C patients observed in ALL97 (5‐year EFS 53%) was not seen in ALL2003 (5‐year EFS 94%). In a multivariate Cox regression analysis the only significant factor affecting EFS was MRD status (hazard ratio for high‐risk MRD patients 4·22, 95% confidence interval 2·97–5·99, P < 0·0001). In conclusion, refinements in risk stratification and treatment have reduced the influence of TPMT genotype on treatment outcome in a contemporary protocol.     

Thiopurine dose intensity and treatment outcome in childhood lymphoblastic leukaemia: the influence of thiopurine methyltransferase pharmacogenetics

The impact of thiopurine methyltransferase (TPMT) genotype on thiopurine dose intensity, myelosuppression and treatment outcome was investigated in the United Kingdom childhood acute lymphoblastic leukaemia (ALL) trial ALL97. TPMT heterozygotes had significantly more frequent cytopenias and therefore required dose adjustments below target levels significantly more often than TPMT wild‐type patients although the average dose range was similar for both genotypes. Event‐free survival (EFS) for patients heterozygous for the more common TPMT*1/*3A variant allele (n = 99, 5‐year EFS 88%) was better than for both wild‐type TPMT*1/*1 (n = 1206, EFS 80%, P = 0·05) and TPMT*1/*3C patients (n = 17, EFS 53%, P = 0·002); outcomes supported by a multivariate Cox regression analysis. Poor compliance without subsequent clinician intervention was associated with a worse EFS (P = 0·02) and such non‐compliance may have contributed to the poorer outcome for TPMT*1/*3C patients. Patients prescribed escalated doses had a worse EFS (P = 0·04), but there was no difference in EFS by dose intensity or duration of cytopenias. In contrast to reports from some USA and Nordic trials, TPMT heterozygosity was not associated with a higher rate of second cancers. In conclusion, TPMT*1/*3A heterozygotes had a better EFS than TPMT wild‐type patients. Thiopurine induced cytopenias were not detrimental to treatment outcome.     

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) and sulfotransferase 1A1 (SULT1A1) polymorphisms and urothelial cancer risk in Taiwan

Purpose To investigate whether the NAD(P)H:quinone oxidoreductase 1 (NQO1) and sulfotransferase 1A1 (SULT1A1) polymorphisms are associated with urothelial cancer (UC) risk in Taiwan. Methods In this study, 600 study subjects (including 300 UC patients and 300 cancer-free controls) were recruited from September 1998 to December 2005. We analyzed the NQO1 and SULT1A1 polymorphisms by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A comprehensive interview was conducted to collect information, including baseline characteristics and cigarette smoking status. We used an unconditional multivariate logistic regression to calculate the odds ratio (OR) and 95% confidence interval (CI). Results We found a significantly increased UC risk in study subjects with the NQO1 C/T and T/T genotypes (OR = 1.5; 95% CI: 1.03–2.1). A significantly increased UC risk was found in those with the SULT1A1 G/G genotype (OR = 2.0; 95% CI: 1.3–3.2). Subjects who had ever smoked with either the NQO1 C/T and T/T genotypes or the SULT1A1 G/G genotype had significantly increased UC risks, showing ORs of 3.0 and 5.3, respectively. Subjects carrying both the NQO1 C/T and T/T genotypes and the SULT1A1 G/G genotype had a significantly increased UC risk (OR = 3.7; 95% CI, 1.4–9.7). Moreover, those who had ever smoked with both the NQO1 C/T and T/T genotypes and the SULT1A1 G/G genotype had the highest UC risk (OR = 8.6; 95% CI: 2.5–29.7). Conclusions These findings suggest that NQO1 and SULT1A1 polymorphisms are associated with the risk of UC, particularly among those who have ever smoked.     

The Highly Expressed and Inducible Endogenous NAD(P)H:quinone Oxidoreductase 1 in Cardiovascular Cells Acts as a Potential Superoxide Scavenger

It has recently been demonstrated that purified NAD(P)H:quinone oxidoreductase 1 (NQO1) is able to scavenge superoxide (O₂^•−) though the rate of reaction of O₂^•− with NQO1 is much lower than the rate of enzymatic dismutation catalyzed by superoxide dismutase (SOD). This study was undertaken to determine if the endogenously expressed NQO1 in cardiovascular cells could scavenge O₂^•−. We observed that NQO1 was highly expressed in cardiovascular cells, including rat aortic smooth muscle A10 and cardiac H9c2 cells, as well as normal human aortic smooth muscle and endothelial cells. NQO1, but not SOD in the cardiovascular cells was highly inducible by 3H-1,2-dithiole-3-thione (D3T). Cytosols from H9c2 and human aortic smooth muscle cells (HASMCs) were isolated to determine the O₂^•− scavenging ability of the endogenously expressed NQO1 by using pyrogallol autooxidation assay. We showed that cytosols from the above cells inhibited pyrogallol autooxidation in an NADPH or NADH-dependent manner. The NADH/NADPH-dependent inhibition of pyrogallol autooxidation by the cytosols was completely abolished by the NQO1-specific inhibitor, ES936, suggesting that the endogenously expressed NQO1 could scavenge O₂^•−. In the presence of NADH/NADPH, cytosols from D3T-treated cells showed increased ability to scavenge O₂^•− as compared to cytosols from untreated cells. This increased ability to scavenge O₂^•− was also completely reversed by ES936. 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide spin-trapping experiments using potassium superoxide as a O₂^•− generator further confirmed the ability of NQO1 from HASMCs to scavenge O₂^•−. The spin-trapping experiments also showed that induction of NQO1 by D3T in HASMCs augmented the O₂^•− scavenging ability. Taken together, these results demonstrate that the highly expressed and inducible endogenous NQO1 in cardiovascular cells may act as a potential O₂^•− scavenger.     

Polymorphism of glutathione S-transferase Omega gene: association with risk of childhood acute lymphoblastic leukemia

Purpose  To evaluate the association between glutathione S-transferase Omega (GSTO) genes polymorphism and the susceptibility of acute lymphoblast leukemia (ALL). Methods  The polymorphism of GSTO1 and GSTO2 genes were analyzed in 99 ALL patients compared with 100 healthy children by PCR-based restriction fragment length polymorphism (RFLP) analysis. Results   GSTO1*A140D polymorphism was significantly associated with susceptibility to ALL (OR = 2.24, 95% CI = 1.16–4.35, P = 0.009) whereas, GSTO2*N142D genotype was significantly interacted with high risk group of childhood ALL (OR = 5.52, 95% CI = 1.72–17.71, P = 0.004). Conclusion  This study revealed gene polymorphism in glutathione S-transferase Omega class may be a risk factor to the development of acute childhood lymphoblastic leukemia.     

Extremely potent triterpenoid inducers of the phase 2 response: correlations of protection against oxidant and inflammatory stress

A series of synthetic triterpenoid (TP) analogues of oleanolic acid are powerful inhibitors of cellular inflammatory processes such as the induction by IFN-gamma of inducible nitric oxide synthase (iNOS) and of cyclooxygenase 2 in mouse macrophages. Here, we show that these analogues are also extremely potent inducers of the phase 2 response [e.g., elevation of NAD(P)H-quinone oxidoreductase and heme oxygenase 1], which is a major protector of cells against oxidative and electrophile stress. Moreover, like previously identified phase 2 inducers, the TP analogues use the antioxidant response element-Nrf2-Keap1 signaling pathway. Thus, induction of the phase 2 response and suppression of the iNOS induction was abrogated in nrf2(-/-) and keap1(-/-) mouse embryonic fibroblasts. The high potency of TP analogues in inducing the phase 2 response and blocking inflammation depends on the presence of activated Michael reaction (enone) functions at critical positions in rings A and C. The most potent TP doubles NAD(P)H-quinone oxidoreductase in murine hepatoma cells at 0.28 nM and has an IC(50) for suppression of iNOS induction in primary mouse macrophages of 0.0035 nM. The direct interaction of this TP with thiol groups of the Keap1 sensor for inducers is demonstrated spectroscopically. The antiinflammatory and phase 2 inducer potencies of 18 TP are closely linearly correlated (r(2) = 0.91) over 6 orders of magnitude of concentration. Thus, in addition to blocking inflammation and promoting differentiation, these TP exhibit another very important protective property: the induction of the phase 2 response.     

The Profile of Antioxidant Systems and Lipid Peroxidation Across the Crypt-Villus Axis in Rat Intestine

The distribution of lipid peroxiation and profile of antioxidant-pro-oxidant enzyme systems have been studied in rat intestinal enterocyte across the length of villi. The MDA levels estimated as a measure of lipid peroxidation, under induced or uninduced in vitro conditions, indicated markedly high levels at villus tip cells compared to that in the crypt base. The activities of glutathione-S-transferase and glutathione reductase were three- to sixfold higher in villus tip cells compared to that in the crypt base. However the levels of catalase and superoxide dismutase showed a reverse pattern, being high in the crypt base and lowest in the villus tip region. Feeding coconut oil, sunflower oil, or groundnut oil did not modify the distribution pattern of these systems across crypt-villus unit in rat intestine. These findings suggest that the large amount of free radicals generated in villus tip cells may be responsible for the release of enterocytes from the villus tip as a consequence of apoptosis.     

Microfluidic glucose stimulation reveals limited coordination of intracellular Ca2+ activity oscillations in pancreatic islets

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet beta cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over approximately 7 mM generate synchronous oscillations in beta cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that beta cell [Ca2+]i oscillations occur only within regions stimulated with more than approximately 6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems.     

Increased expression of NAD(P)H oxidase subunits,NOX4 and p22phox,in the kidney of streptozotocin-induced diabetic rats and its reversibity by interventive insulin treatment

Aim/hypothesis An increased production of reactive oxygen species (ROS) could contribute to the development of diabetic nephropathy. NAD(P)H oxidase might be an important source of ROS production in kidney as reported in blood vessels. In this study, we show the increased expression of essential subunits of NAD(P)H oxidase, NOX4 and p22phox, in the kidney of diabetic rats.Methods The levels of mRNA of both NOX4 and p22phox were evaluated in kidney from streptozotocin-induced diabetic rats and age-matched control rats at 4 and 8 weeks after onset of diabetes by Northern blot analysis. The localization and expression levels of these components and 8-hydroxy-deoxyguanosine (8-OHdG), which is a marker of ROS-induced DNA damage, were also evaluated by immunostaining.Results The levels of both NOX4 and p22phox mRNA were increased in the kidney of diabetic rats as compared with control rats. Immunostaining analysis showed that the expression levels of NOX4 and p22phox were clearly increased in both distal tubular cells and glomeruli from diabetic rats. Both the localization and the expression levels of these components were in parallel with those of 8-OHdG. Interventive insulin treatment for 2 weeks completely restored the increased levels of these components in the diabetic kidney to control levels in parallel with those of 8-OHdG.Conclusions/interpretation This study provides evidence that NAD(P)H oxidase subunits, NOX4 and p22phox, were increased in the kidney of diabetic rats. Thus, NAD(P)H-dependent overproduction of ROS could cause renal tissue damage in diabetes. This might contribute to the development of diabetic nephropathy.Abbreviations ROS reactive oxygen species - NAD(P)H oxidase nicotinamide adenine dinucleotide phosphate oxidase - 8-OHdG 8-hydroxy-deoxyguanosine - STZ streptozotocin - mtDNA mitochondrial DNA - PKC protein kinase C     

The presence of CD56/CD16 in T-cell acute lymphoblastic leukaemia correlates with the expression of cytotoxic molecules and is associated with worse response to treatment

Some cases of T-cell acute lymphoblastic leukaemia (ALL) express markers found in natural-killer (NK) cells, such as CD56 and CD16. Out of 84 T-cell ALL cases diagnosed at our Institution, CD56 and/or CD16 was detected in 24 (28·5%), which we designated T/NK-ALL group. Clinical features, laboratory characteristics, survival and expression of cytotoxic molecules were compared in T/NK-ALL and T-ALL patients. Significant differences were observed regarding age (24·9 vs. 16·4 years in T/NK-ALL and T-ALL, respectively, P  =   0·006) and platelet counts (177 × 10⁹/l vs. 75 × 10⁹/l in T/NK-ALL and T-ALL, respectively, P  =   0·03). Immunophenotypic analysis demonstrated that CD34, CD45RA and CD33 were more expressed in T/NK-ALL patients, whereas CD8 and terminal deoxynucleotidyl transferase were more expressed in T-ALL patients ( P  <   0·05 ) . The mean overall survival (863 vs. 1869 d, P  =   0·02) and disease-free survival (855 vs. 2095 d, P  =   0·002) were shorter in patients expressing CD56/CD16. However, multivariate analysis identified CD56/CD16 as an independent prognostic factor only for DFS. Cytotoxic molecules were highly expressed in T/NK-ALL compared to T-ALL. Perforin, granzyme B and TIA-1 were detected in 12/17, 4/17 and 7/24 T/NK-ALL patients and in 1/20, 0/20 and 1/20 T-ALL respectively ( P  <   0·001, P  =   0·036 and P  =   0·054). Therefore, the presence of CD56/CD16 was associated with distinct clinical features and expression of cytotoxic molecules in the blasts.     

Interactions of chemical carcinogens and genetic variation in hepatocellular carcinoma

In the etiology of hepatocellular carcinoma (HCC), in addition to hepatitis B virus and hepatitis C virus infections, chemical carcinogens also play important roles. For example, aflatoxin B(1) (AFB(1)) epoxide reacts with guanine in DNA and can lead to genetic changes. In HCC, the tumor suppressor gene p53 codon 249 mutation is associated with AFB(1) exposure and mutations in the K-ras oncogene are related to vinyl chloride exposure. Numerous genetic alterations accumulate during the process of hepatocarcinogenesis. Chemical carcinogen DNA-adduct formation is the basis for these genetic changes and also a molecular marker which reflects exposure level and biological effects. Metabolism of chemical carcinogens, including their activation and detoxification, also plays a key role in chemical hepatocarcinogenesis. Cytochrome p450 enzymes, N-acetyltransferases and glutathione S-transferases are involved in activating and detoxifying chemical carcinogens. These enzymes are polymorphic and genetic variation influences biological response to chemical carcinogens. This genetic variation has been postulated to influence the variability in risk for HCC observed both within and across populations. Ongoing studies seek to fully understand the mechanisms by which genetic variation in response to chemical carcinogens impacts on HCC risk.     

Low frequency of clonotypic Ig and T-cell receptor gene rearrangements in t(4;11) infant acute lymphoblastic leukaemia and its implication for the detection of minimal residual disease

Infant t(4;11) acute lymphoblastic leukaemia (ALL) is a rare but cytogenetically well defined subgroup of immature B-cell precursor (BCP) ALL. To date, the configuration of their antigen receptor genes has not been studied in a large group of patients so far. In this study on 27 t(4;11) infant ALL, we have used standardized primer sets for the detection of all incomplete and complete immunoglobulin (Ig) heavy chain (IGH) rearrangements, as well as for the Ig light chain kappa (IGK), T-cell receptor delta (TCRD) and gamma (TCRG) rearrangements that are most common in childhood BCP ALL. Only 52% of cases displayed clonotypic antigen receptor gene rearrangements (IGH in 48%, IGK, TCRD and TCRG in 12%, 41% and 6% respectively). This low frequency suggests, together with the findings of predominantly incomplete DJh joins and monoallelic IGH rearrangements, that they are derived from an immature progenitor cell. As 48% of the t(4;11) infant ALL cases had no detectable antigen receptor gene rearrangements that could be used for minimal residual disease (MRD) analysis, we established an expression-independent, leukaemia-specific polymerase chain reaction (PCR) using the genomic sequence of the MLL-AF4 fusion genes. This method had high sensitivity and specificity and resulted in identical estimations of tumour loads when compared with IGH targets. Thus, genomic MLL-AF4 fusion genes are a good alternative target for the analysis of MRD in patients with t(4;11) leukaemias.     

Acid-suppressive effects of rabeprazole: Comparing 10 mg and 20 mg twice daily in Japanese Helicobacter pylori-negative and -positive CYP2C19 extensive metabolisers

BACKGROUND: Rabeprazole 10mg b.i.d. is often administered as therapy for eradication of Helicobacter pylori (H. pylori) and is also proposed as therapy for refractory gastro-oesophageal reflux disease. However, there has not been a comprehensive assessment of its acid-suppressive effects. AIMS: To compare the acid-suppressive effects of rabeprazole 10mg b.i.d. with 20mg b.i.d. considering H. pylori status. SUBJECTS: Thirteen H. pylori-negative and eleven H. pylori-positive Japanese CYP2C19 extensive metabolisers (<35 years). METHODS: Intragastric pH was measured for 24h three times in a randomised manner; on day 7 of the repeated administration of rabeprazole 10mg b.i.d. or 20mg b.i.d., or a placebo. RESULTS: In median intragastric pH value and percent time of pH>3.0, >4.0, >5.0, >6.0, and >7.0 for 24h, no significant differences were observed between the two doses in either H. pylori-negative or H. pylori-positive subjects. At either dose, these parameters were significantly higher in H. pylori-positive subjects than in H. pylori-negative subjects. Nocturnal acid breakthrough occurred in seven and two of the thirteen H. pylori-negative subjects and one and two of the eleven H. pylori-positive subjects at each dose, respectively. CONCLUSIONS: The effects of rabeprazole 10mg b.i.d. were equal to those of 20mg b.i.d. in H. pylori-positive subjects; whereas in H. pylori-negative subjects, 20mg b.i.d. was superior for prevention of nocturnal acid breakthrough.     

[1,2-Bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a new compound for the therapy of ovarian cancer

Summary The enantiomeric [1,2-bis(2-hydroxyphenyl)-ethylenediamine]dichloroplatinum(II) complexes were synthesized and their configuration assessed. A preliminary test in the cisplatin-resistant human NIH:OVCAR-3 ovarian cancer cell line, which was previously characterized by its sensitivity against several therapeutically used drugs, showed that both enantiomers produce cytocidal effects in a concentration of 2.5 M. A difference between the enantiomers became evident from the faster onset of cytocidal activity of theS,S-configurated compound.Dedicated to Professor J. Knabe on the occasion of his 70th birthdayPart I: Müller et al. (1990)     

Diffuse venous thromboemboli associated with IVIg therapy in the treatment of streptococcal toxic shock syndrome: case report and review

Streptococcal toxic shock syndrome (STSS) is a severe invasive disease with a 40–80% mortality rate. Inflammatory cytokines induced by streptococcal pyrogenic exotoxins (SPEs) produce the clinical manifestations of a flu-like syndrome, followed by high fevers and multiorgan failure. Previously published reports have described the use of intravenous immunoglobulin (IVIg) as adjunctive treatment for STSS. However, concerns have been raised about the thromboembolic complications of this therapy. We report a severe case of STSS treated with two adjunctive courses of IVIg complicated by severe bilateral pulmonary thromboemboli. To our knowledge, this is the only reported case of thromboemboli associated with IVIg for STSS. The results of this case support the cautious use of IVIg for STSS and demonstrate the need for controlled trials to determine the appropriate timing, dosage, and course of treatment.