We investigated whether polymorphisms in reduced folate carrier (SLC19A1 G80A) and gamma-glutamyl-hydrolase (GGH-401C/T) are predictive of methotrexate polyglutamate (MTXPG) levels in patients with rheumatoid arthritis treated with weekly low-dose methotrexate (MTX). Adult patients treated with MTX were enrolled in a multicentred study. Blood was drawn at the time of the visit, DNA was extracted and red blood cell (RBC) MTXPG levels (up to the penta-order of glutamation) were measured by high-performance liquid chromatography-fluorometry. A G80A polymorphism in SLC19A1 and a -401C/T promoter polymorphism in GGH were measured by polymerase chain reaction-restriction fragment length polymorphism. Multivariate linear and logistic regressions were used to predict long-chain RBC MTXPG3-5. In 226 adult patients receiving MTX (median 15 mg range: 5-25 mg) median RBC long-chain MTXPG3-5 was 56 nmol/l (range < 5-224 nmol/l). A total of 35 patients carried the SLC19A1 80AA genotype whereas 36 patients carried the GGH-401TT genotype. Weekly MTX dose, age, presence of the SLC19A1 80AA and GGH-401TT genotypes predicted independently and significantly MTXPG3-5 levels (global r = 0.38; P < 0.0001). Patients with the GGH-401TT genotype were 4.8-fold [odds ratio (OR) 95% confidence interval (CI) 1.8-13.0; P = 0.002] more likely to have MTXPG3-5 below the group median compared to patient carriers of the GGH-401CC or CT genotype. Conversely, those with the SLC19A1 80AA genotype were 3.4-fold more likely to have MTXPG3-5 levels above the group median compared to those with the SLC19A1 80GG or 80GA genotype (OR CI 95% 1.4-8.4; P = 0.007). These data demonstrate that polymorphisms in SLC19A1 and GGH affect polyglutamation of MTX.

Background: Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity.Methods: We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C 18 column with an ammonium acetate-formic acid-acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy. Results: Intra-and interday imprecision were <10% at low and high analyte concentrations. The conversion of

BackgroundThe clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment.MethodsWe have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity.ResultsCEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development.ConclusionCEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.

Background: Women treated with early stage breast cancer often gain weight in the two years following diagnosis, with adjuvant chemotherapy, younger age, lower body mass index (BMI) and pretreatment menopausal status being associated factors (Breast J 2007; 13:258). The effect of insulin resistance on treatment-related weight gain, and of treatment on measures of insulin resistance, are unclear.Purpose: To prospectively study weight gain in women receiving adjuvant therapy for early stage breast cancer and assess changes in body mass index, body composition, and explore the relationship between markers of insulin resistance, cancer therapy, exercise and weight gain.Methods: Prospective, observational study of non-diabetic women with early-stage breast cancer receiving adjuvant chemotherapy and/or hormonal therapy. Fasting insulin, glucose, and triglycerides, weight, BMI, waist and hip circumference were obtained at baseline before adjuvant therapy, and at 6, and 12 months. Patients completed nutrition and exercise logs using the Nutritionist-Pro program. Insulin resistance was calculated using the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR). Waist/hip ratio (WHR) was used as a surrogate for percent body fat.Results: Complete data available for 95 patients are reported regarding weight change at 6 months (mean 0.4 kg, 95%CI -0.3, 1.0; p=.23 compared to baseline) and 12 months (0.9 kg, 95%CI 0.4, 1.8, p=.04 compared with baseline). Mean baseline HOMA-IR was 2.3 (95%CI 1.9, 2.7); mean baseline WHR was 0.82 (95%CI 0.81, 0.84). Six-month HOMA-IR tended to increase (by mean 0.34, p=.06) as did glucose/insulin ratio (p=.05). Waist measurement (p=.96) and WHR (p=.52) were unchanged. Chemotherapy-treated patients exhibited adverse changes compared with others (mean change for chemotherapy-treated and no chemotherapy for HOMA-IR: 0.53 vs. -0.64, p=.005; glucose/insulin: -2.1 vs. 2.7, p=.003; waist: 0.37 vs. -1.94 cm, p=.08; WHR: 0.001 vs. -0.024, p=.06). Age, BMI, hormonal therapy, or types of breast surgery were not associated with significant changes. Exercising patients demonstrated lower HOMA-IR and WHR, and greater glucose/insulin ratios at baseline, but exercise (aerobic, strength, or any exercise) was not associated with significant change at 6 and 12 months for any of these variables. While differences disappeared at 12 months despite further weight gain repeated measures analysis of variance confirmed the effect of chemotherapy over time for HOMA-IR (p=.048), glucose/insulin (p=.038) and WHR (p=.050).Conclusions: These preliminary data suggest that patients completing chemotherapy within 6 months of baseline appear to experience at least short-term changes in metabolism suggestive of insulin resistance. These adverse changes may help explain weight gain associated with adjuvant chemotherapy, and merit further prospective study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1054.

In this triple of papers, we examine when two cycle-free partial orders can share an abstract automorphism group. This question was posed by M. Rubin in his memoir concerning the reconstruction of trees.In this first paper, we give a variety of conditions that guarantee when a CFPO shares an automorphism group with a tree. Some of these conditions are conditions on the abstract automorphism group, while some are one the CFPO itself. Some of the lemmas used have corollaries concerning the model theoretic properties of a CFPO.