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Supplementary Text - PDF file 99K, Contains figure legends for supplemental figures 1-4, and text describing supplemental methodology

Supplementary Figures - PDF file 441K, Four figures as follows: SF1: decreased PTEN expression in HNSCC with PTEN loss. SFs 2-4: PIK3CA mutant HNSCC tumorgrafts treatment response characterization; PI3K inhibitors +/- cetuximab, tumor measurements and western blotting for PI3K signaling

Supplementary Tables 1-3 from A Sex-Specific Association between a 15q25 Variant and Upper Aerodigestive Tract Cancers

PDF file - 2.03MB

PDF file - 69K, Results from multiple variable regression - multiply adjusted risk factor associations with overall survival.

PDF file - 61K, Risk factor distributions according to ERCC2 genotype and CCND1 genotype.

PDF file - 95K, Overall survival (Kaplan-Meier plots) for stage III-IV squamous cell carcinoma of head and neck, according to ERCC2 or CCND1 genotype and treatment.

PDF file - 45K, Tumor characteristics according to ERCC2 genotype and CCND1 genotype.

Background: Germline variation in DNA damage response may explain variable treatment outcomes in squamous cell carcinoma of the head and neck (SCCHN). By grouping patients according to stage and radiation treatment, we compared SCCHN survival with regard to ERCC2 A35931C (Lys751Gln, rs13181) and CCND1 G870A (Pro241Pro, rs9344) genotypes.Methods: In a hospital-based SCCHN case series (all white, 24.7% female, mean age 58.4 years), this treatment-outcome cohort study genotyped 275 stage III–IV cases that were initially treated with radiation (with or without chemotherapy) and 80 stage III–IV and 130 stage I–II cases that were initially treated without radiation or chemotherapy and used Kaplan–Meier and Cox regression analyses to compare genotype groups on the basis of overall, disease-specific, progression-free, and recurrence-free survival rates.Results:ERCC2 35931 AA predicted worse survival in stage III–IV cases treated with radiation [multiply-adjusted HR = 1.66, 95% confidence interval (CI), 1.15–2.40; HR over the first 3 follow-up years = 1.92; 95% CI, 1.28–2.88] and better survival in stage III–IV cases not treated with radiation (HR = 0.26; 95% CI, 0.11–0.62). Although not associated with survival in stage III–IV cancers treated with radiation (HR = 1.00; 95% CI, 0.67–1.51), CCND1-870 GG predicted better survival in stage III–IV cancers not treated with radiation (HR = 0.14; 95% CI, 0.04–0.50). Survival in stage I–II did not depend on ERCC2 A35931C or CCND1 G870A genotype.Conclusions: Although promoting tumor progression in untreated patients, germline differences in DNA-repair or cell-cycle control may improve treatment outcome in patients treated with DNA-damaging agents.Impact:ERCC2 A35931C may help distinguish advanced stage SCCHN with better outcomes from radiation treatment. Cancer Epidemiol Biomarkers Prev; 20(11); 2429–37. ©2011 AACR.

Purpose: Tumor-specific biomarkers that predict resistance to DNA damaging agents may improve therapeutic outcomes by guiding the selection of effective therapies and limiting morbidity related to ineffective approaches. XPF (ERCC4) is an essential component of several DNA repair pathways and XPF-deficient cells are exquisitely sensitive to DNA damaging agents. The purpose of this study was to determine whether XPF expression levels predict clinical response to DNA damaging agents in head and neck squamous cell carcinoma (HNSCC).Experimental Design: Quantitative immunohistochemistry was used to measure XPF expression in tumors from a cohort of 80 patients with newly diagnosed HNSCC treated with radiation therapy with or without platinum-based chemotherapy; samples were collected prospectively. Genomic DNA isolated from blood samples was analyzed for nine single nucleotide polymorphisms (SNP) in the XPF gene by using a custom array. The primary endpoint was progression-free survival (PFS).Results: XPF expression was higher in tumors from the oral cavity than from the other sites (P < 0.01). High XPF expression correlated with early time to progression both by univariate (HR = 1.87, P = 0.03) and multivariate analysis (HR = 1.83, P = 0.05). The one year PFS for high expressers was 47% (95% CI = 31–62) compared with 72% (95% CI = 55–83) for low expressers. In addition, we identified four XPF SNPs that showed marginal association with treatment failure.Conclusions: Expression level of XPF in HNSCC tumors correlates with clinical response to DNA damaging agents. XPF has potential to guide next generation personalized cancer therapy. Clin Cancer Res; 17(16); 5513–22. ©2011 AACR.

PDF file - 57K, A comparison of head and neck cancer cases included and excluded from analysis. Parentheses show numbers missing attribute data.

Background: Sequence variants located at 15q25 have been associated with lung cancer and propensity to smoke. We recently reported an association between rs16969968 and risk of upper aerodigestive tract (UADT) cancers (oral cavity, oropharynx, hypopharynx, larynx, and esophagus) in women (OR = 1.24, P = 0.003) with little effect in men (OR = 1.04, P = 0.35).Methods: In a coordinated genotyping study within the International Head and Neck Cancer Epidemiology (INHANCE) consortium, we have sought to replicate these findings in an additional 4,604 cases and 6,239 controls from 10 independent UADT cancer case–control studies.Results: rs16969968 was again associated with UADT cancers in women (OR = 1.21, 95% CI = 1.08–1.36, P = 0.001) and a similar lack of observed effect in men [OR = 1.02, 95% CI = 0.95–1.09, P = 0.66; P-heterogeneity (Phet) = 0.01]. In a pooled analysis of the original and current studies, totaling 8,572 UADT cancer cases and 11,558 controls, the association was observed among females (OR = 1.22, 95% CI = 1.12–1.34, P = 7 × 10−6) but not males (OR = 1.02, 95% CI = 0.97–1.08, P = 0.35; Phet = 6 × 10−4). There was little evidence for a sexdifference in the association between this variant and cigarettes smoked per day, with male and female rs16969968 variant carriers smoking approximately the same amount more in the 11,991 ever smokers in the pooled analysis of the 14 studies (Phet = 0.86).Conclusions: This study has confirmed a sex difference in the association between the 15q25 variant rs16969968 and UADT cancers.Impact: Further research is warranted to elucidate the mechanisms underlying these observations. Cancer Epidemiol Biomarkers Prev; 20(4); 658–64. ©2011 AACR.

Personalized cancer therapy relies on identifying patient subsets that benefit from a therapeutic intervention and suggest alternative regimens for those who don’t. A new data integrative approach, based on graphical models, was applied on our multi-modal –omics, and clinical data cohort of metastatic melanoma patients. We found that response to chemotherapy is directly linked to ten gene expression, four methylation variables and PARP1 SNP rs1805407. PARP1 is a DNA repair gene critical for chemotherapy response and for which FDA-approved inhibitors are clinically available (olaparib). We demonstrated that two PARP inhibitors (ABT-888 and olaparib) make SNP carrier cancer cells of various histologic subtypes more sensitive to alkylating agents, but they have no effect in wild-type cells. Furthermore, PARP1 inhibitors act synergistically with chemotherapy in SNP carrier cells (especially in ovarian cancer for which olaparib is FDA-approved), but they are additive at best in wild-type cancer cells. Taken together, our results suggest that the combination of chemotherapy and PARP1 inhibition may benefit the carriers of rs1805407 in the future and may be used in personalized therapy strategies to select patients that are more likely to respond to PARP inhibitors.

Non–small cell lung cancers (NSCLCs) frequently express estrogen receptor (ER) β, and estrogen signaling is active in many lung tumors. We investigated the ability of genes contained in the prediction analysis of microarray 50 (PAM50) breast cancer risk predictor gene signature to provide prognostic information in NSCLC. Supervised principal component analysis of mRNA expression data was used to evaluate the ability of the PAM50 panel to provide prognostic information in a stage I NSCLC cohort, in an all-stage NSCLC cohort, and in The Cancer Genome Atlas data. Immunohistochemistry was used to determine status of ERβ and other proteins in lung tumor tissue. Associations with prognosis were observed in the stage I cohort. Cross-validation identified seven genes that, when analyzed together, consistently showed survival associations. In pathway analysis, the seven-gene panel described one network containing the ER and progesterone receptor, as well as human epidermal growth factor receptor (HER)2/HER3 and neuregulin-1. NSCLC cases also showed a significant association between ERβ and HER2 protein expression. Cases positive for HER2 expression were more likely to express HER3, and ERβ-positive cases were less likely to be both HER2 and HER3 negative. Prognostic ability of genes in the PAM50 panel was verified in an ERβ-positive cohort representing all NSCLC stages. In The Cancer Genome Atlas data sets, the PAM50 gene set was prognostic in both adenocarcinoma and squamous cell carcinoma, whereas the seven-gene panel was prognostic only in squamous cell carcinoma. Genes in the PAM50 panel, including those linking ER and HER2, identify lung cancer patients at risk for poor outcome, especially among ERβ-positive cases and squamous cell carcinoma.

The Cancer Genome Atlas profiled 279 head and neck squamous cell carcinomas (HNSCCs) to provide a comprehensive landscape of somatic genomic alterations. Here we show that human-papillomavirus-associated tumours are dominated by helical domain mutations of the oncogene PIK3CA, novel alterations involving loss of TRAF3, and amplification of the cell cycle gene E2F1. Smoking-related HNSCCs demonstrate near universal loss-of-function TP53 mutations and CDKN2A inactivation with frequent copy number alterations including amplification of 3q26/28 and 11q13/22. A subgroup of oral cavity tumours with favourable clinical outcomes displayed infrequent copy number alterations in conjunction with activating mutations of HRAS or PIK3CA, coupled with inactivating mutations of CASP8, NOTCH1 and TP53. Other distinct subgroups contained loss-of-function alterations of the chromatin modifier NSD1, WNT pathway genes AJUBA and FAT1, and activation of oxidative stress factor NFE2L2, mainly in laryngeal tumours. Therapeutic candidate alterations were identified in most HNSCCs.

Rationale: Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects. Objectives: To test the hypothesis that MMP19 plays a significant role in the development and progression of non–small cell lung cancer (NSCLC). Methods: We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells. Measurements and Main Results: We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial–mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial–mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression. Conclusions: Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.

Introduction : Functional polymorphisms in drug metabolizing enzymes (DMEs) may be determinants of survival in oral and oropharyngeal squamous cell carcinoma (OOSCC). Methods : OOSCC cases ( N =159) with a history of either tobacco or alcohol use were genotyped for polymorphisms in eight DMEs. Overall and disease-specific survival were analyzed using Kaplan–Meier plots and the log-rank test. Cox proportional hazards regression was used to calculate hazard ratios (HR) and 95% confidence intervals (CI) in exploratory analyses of patient subgroups. Results : Kaplan–Meier analyses showed N-acteyltransferase-2 ( NAT2 ) fast acetylators experienced a 19.7% higher 5-year survival rate than slow acetylators ( P =0.03) and this association was similar in oropharyngeal and oral cancer. After multiple adjustment, including tumor site and stage, the NAT2 fast acetylator phenotype was associated with improved overall survival (vs. slow acetylators) provided chemotherapy or radiation were not used (HR, 0.26; 95% CI, 0.10–0.66). However, NAT2 phenotype was unrelated to survival in patients treated with chemoradiotherapy (HR, 1.21; 95% CI, 0.54–2.73) or radiotherapy (HR, 0.67; 95% CI, 0.31–1.59) ( P -for- NAT2 /treatment-interaction=0.04). Normal activity GSTP1 was associated with a 19.2% reduction in 5-year disease-specific survival relative to reduced activity GSTP1 ( P =0.04) but this association was not modified by treatment. Conclusions : Our results suggest that functional polymorphisms in NAT2 and GSTP1 are associated with OOSCC survival. Confirmation of these results in larger studies is required.

To investigate the association between inherited variation in the estrogen receptor beta (ERβ) gene (ESR2) and ERβ lung tumor expression, a phenotype that possibly affects survival differently in men and women.We genotyped 135 lung cancer patients for 22 ESR2 single nucleotide polymorphisms (SNPs) and measured nuclear and cytoplasmic ERβ expression by immunohistochemistry (IHC) in their primary lung tumor. Distributing Allred ERβ IHC scores according to ESR2 genotype classified under a dominant genetic model, we used rank sum tests to identify ESR2 SNPs significantly associated (p0.05) with ERβ expression.35%, 35%, and 29% of lung tumors showed no/low (Allred6), intermediate (Allred 6-7), and maximal (Allred 8) cytoplasmic ERβ expression, whereas 13%, 27%, and 60% showed no/low, intermediate, and maximal nuclear ERβ expression. For SNPs rs8021944, rs1256061 and rs10146204, ERβ expression was higher according to the rank sum test in lung tumors from patients with at least one minor allele. For each of these three SNPs, the odds of maximal (Allred 8) relative to no/low (Allred6) ERβ expression was 3-fold higher in tumors from patients with at least one minor allele than in tumors from patients homozygous for the common allele.Inherited variability in ESR2 may determine ERβ lung tumor expression.

Functional CYP2A6 genetic variation partially determines nicotine metabolism. In 2005, we examined functional CYP2A6 variants associated with reduced metabolism (CYP2A6*2, CYP2A6*9, CYP2A6*4), smoking history, and change in smoking in 878 adult smokers undergoing lung cancer screening in an urban setting. At one year, 216 quit smoking for more than 30 days while 662 continued smoking. Compared to subjects who smoked 30 cigarettes per day at baseline, the odds of a reduced metabolism genotype was 52% higher in subjects smoking 20-29 cigarettes per day and 86% higher in subjects smoking less than 20 cigarettes per day (p-trend = 0.016). Reduced metabolism genotypes appeared unrelated to quitting. Though related to smoking dose, CYP2A6 may not influence cessation.

IntroductionGenome-wide association studies (GWAS) have consistently identified specific lung cancer susceptibility regions. We evaluated the lung cancer–predictive performance of single-nucleotide polymorphisms (SNPs) in these regions.MethodsLung cancer cases (N = 778) and controls (N = 1166) were genotyped for 77 SNPs located in GWAS-identified lung cancer susceptibility regions. Variable selection and model development used stepwise logistic regression and decision-tree analyses. In a subset nested in the Pittsburgh Lung Screening Study, change in area under the receiver operator characteristic curve and net reclassification improvement were used to compare predictions made by risk factor models with and without genetic variables.ResultsVariable selection and model development kept two SNPs in each of three GWAS regions, rs2736100 and rs7727912 in 5p15.33, rs805297 and rs1802127 in 6p21.33, and rs8034191 and rs12440014 in 15q25.1. The ratio of cases to controls was three times higher among subjects with a high-risk genotype in every one as opposed to none of the three GWAS regions (odds ratio, 3.14; 95% confidence interval, 2.02–4.88; adjusted for sex, age, and pack-years). Adding a three-level classified count of GWAS regions with high-risk genotypes to an age and smoking risk factor-only model improved lung cancer prediction by a small amount: area under the receiver operator characteristic curve, 0.725 versus 0.717 (p = 0.056); overall net reclassification improvement was 0.052 across low-, intermediate-, and high- 6-year lung cancer risk categories (

Background: Germline variation in DNA damage response may explain variable treatment outcomes in squamous cell carcinoma of the head and neck (SCCHN). By grouping patients according to stage and radiation treatment, we compared SCCHN survival with regard to ERCC2 A35931C (Lys751Gln, rs13181) and CCND1 G870A (Pro241Pro, rs9344) genotypes. Methods: In a hospital-based SCCHN case series (all white, 24.7% female, mean age 58.4 years), this treatment-outcome cohort study genotyped 275 stage III–IV cases that were initially treated with radiation (with or without chemotherapy) and 80 stage III–IV and 130 stage I–II cases that were initially treated without radiation or chemotherapy and used Kaplan–Meier and Cox regression analyses to compare genotype groups on the basis of overall, disease-specific, progression-free, and recurrence-free survival rates. Results: ERCC2 35931 AA predicted worse survival in stage III–IV cases treated with radiation [multiply-adjusted HR = 1.66, 95% confidence interval (CI), 1.15–2.40; HR over the first 3 follow-up years = 1.92; 95% CI, 1.28–2.88] and better survival in stage III–IV cases not treated with radiation (HR = 0.26; 95% CI, 0.11–0.62). Although not associated with survival in stage III–IV cancers treated with radiation (HR = 1.00; 95% CI, 0.67–1.51), CCND1-870 GG predicted better survival in stage III–IV cancers not treated with radiation (HR = 0.14; 95% CI, 0.04–0.50). Survival in stage I–II did not depend on ERCC2 A35931C or CCND1 G870A genotype. Conclusions: Although promoting tumor progression in untreated patients, germline differences in DNA-repair or cell-cycle control may improve treatment outcome in patients treated with DNA-damaging agents. Impact: ERCC2 A35931C may help distinguish advanced stage SCCHN with better outcomes from radiation treatment. Cancer Epidemiol Biomarkers Prev; 20(11); 2429–37. ©2011 AACR.

Interindividual variation in genetic background may influence the response to chemotherapy and overall survival for patients with advanced-stage non-small cell lung cancer (NSCLC).To identify genetic variants associated with poor overall survival in these patients, we conducted a genome-wide scan of 307,260 single-nucleotide polymorphisms (SNPs) in 327 advanced-stage NSCLC patients who received platinum-based chemotherapy with or without radiation at the University of Texas MD Anderson Cancer Center (the discovery population). A fast-track replication was performed for 315 patients from the Mayo Clinic followed by a second validation at the University of Pittsburgh in 420 patients enrolled in the Spanish Lung Cancer Group PLATAX clinical trial. A pooled analysis combining the Mayo Clinic and PLATAX populations or all three populations was also used to validate the results. We assessed the association of each SNP with overall survival by multivariable Cox proportional hazard regression analysis. All statistical tests were two-sided.SNP rs1878022 in the chemokine-like receptor 1 (CMKLR1) was statistically significantly associated with poor overall survival in the MD Anderson discovery population (hazard ratio [HR] of death = 1.59, 95% confidence interval [CI] = 1.32 to 1.92, P = 1.42 × 10(-6)), in the PLATAX clinical trial (HR of death = 1.23, 95% CI = 1.00 to 1.51, P = .05), in the pooled Mayo Clinic and PLATAX validation (HR of death = 1.22, 95% CI = 1.06 to 1.40, P = .005), and in pooled analysis of all three populations (HR of death = 1.33, 95% CI = 1.19 to 1.48, P = 5.13 × 10(-7)). Carrying a variant genotype of rs10937823 was associated with decreased overall survival (HR of death = 1.82, 95% CI = 1.42 to 2.33, P = 1.73 × 10(-6)) in the pooled MD Anderson and Mayo Clinic populations but not in the PLATAX trial patient population (HR of death = 0.96, 95% CI = 0.69 to 1.35).These results have the potential to contribute to the future development of personalized chemotherapy treatments for individual NSCLC patients.

Purpose We hypothesized that bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), will potentiate the activity of pemetrexed, a multitargeted antifolate, in squamous cell carcinoma of the head and neck (SCCHN). Patients and Methods Patients with previously untreated, recurrent, or metastatic SCCHN were treated with pemetrexed 500 mg/m2 and bevacizumab 15 mg/kg given intravenously every 21 days with folic acid and B12 supplementation until disease progression. Primary end point was time-to-progression (TTP). DNA was isolated from whole blood samples for the detection of polymorphisms in thymidylate synthase, methylenetetrahydrofolate reductase (MTHFR), and VEGF. Results Forty patients were enrolled. The median TTP was 5 months, and the median overall survival (OS) was 11.3 months. In 37 evaluable patients, the overall response rate was 30%, including a complete response rate of 5%, and the disease control rate was 86%. Grade 3 to 5 bleeding events occurred in six patients (15%): four were grade 3, and two were fatal. Other serious toxicities in 10% or more of patients included neutropenia (10%) and infection (12.5%). One patient died of sepsis after receiving eight cycles of therapy. For the MTHFR A1298C (rs1801131) single nucleotide polymorphisms, homozygote patients with AA had worse OS (P = .034). Conclusion The addition of bevacizumab to pemetrexed resulted in promising efficacy outcomes in SCCHN. Bleeding events were frequent but some may have been due to natural history of disease. Polymorphisms in MTHFR may offer potential for treatment individualization.

Background: Computed tomography (CT) lung cancer screening offers a unique clinical setting in which to promote smoking cessation. Focusing on outcomes related to the reporting of CT abnormality, we examined the natural history of smoking in the Pittsburgh Lung Screening Study. Methods: Pittsburgh Lung Screening Study recruited 50- to 79-year-old current and former cigarette smokers living in the Pittsburgh area. We examined self-reported smoking outcomes 1 year after study entry in a subgroup that contained 2,094 active cigarette smokers without interval lung cancer diagnosis (50.7% women; median age, 57 years; 40-year median duration of cigarette smoking; and 65.2% ≥20 cigarettes/d). Analyses compared efforts to quit in relation to physician referral for abnormal CT. Results: Since study entry, 58.5% [95% confidence interval (95% CI), 56.3-60.6%] reported any quit attempt and 27.2% (95% CI, 25.3-29.1%) reported any quit interval >30 days. One year after study entry, 15.5% (95% CI, 14.0-17.1%) reported not smoking for >30 days. Comparing persons referred because of CT abnormalities creating moderate or high lung cancer suspicion (n = 156; 7.4%) to persons not referred for any reason (n = 1145; 54.7%), propensity score-adjusted fractions with any quit attempt and with any quit interval >30 days increased 18.8% (95% CI, 11.1-26.5%) and 17.7% (95% CI, 9.4-26.0%), respectively. The fraction quit >30 days at 1 year increased 12.2% (95% CI, 4.9-19.5%). Conclusions: Persons who experienced referral because of abnormal CT reported more smoking cessation. (Cancer Epidemiol Biomarkers Prev 2009;18(12):3484–9)

About 45% of head and neck squamous cell carcinomas (HNSCC) are characterized by amplification of chromosomal band 11q13. This amplification occurs by a breakage-fusion-bridge (BFB) cycle mechanism. The first step in the BFB cycle involves breakage and loss of distal 11q, from FRA11F (11q14.2) to 11qter. Consequently, numerous genes, including three critical genes involved in the DNA damage response pathway, MRE11A, ATM, and H2AFX are lost in the step preceding 11q13 amplification. We hypothesized that this partial loss of genes on distal 11q may lead to a diminished DNA damage response in HNSCC. Characterization of HNSCC using fluorescence in situ hybridization (FISH) revealed concurrent partial loss of MRE11A, ATM, and H2AFX in all four cell lines with 11q13 amplification and in four of seven cell lines without 11q13 amplification. Quantitative microsatellite analysis and loss of heterozygosity studies confirmed the distal 11q loss. FISH evaluation of a small series of HNSCC, ovarian, and breast cancers confirmed the presence of 11q loss in at least 60% of these tumors. All cell lines with distal 11q loss exhibited a diminished DNA damage response, as measured by a decrease in the size and number of γ-H2AX foci and increased chromosomal instability following treatment with ionizing radiation. In conclusion, loss of distal 11q results in a defective DNA damage response in HNSCC. Distal 11q loss was also unexpectedly associated with reduced sensitivity to ionizing radiation. Although the literature attributes the poor prognosis in HNSCC to 11q13 gene amplification, our results suggest that distal 11q deletions may be an equally significant factor. © 2007 Wiley-Liss, Inc.

Aims We investigated flurbiprofen pharmacokinetics in 12 volunteers to develop a phenotypic trait measure that correlates with the fractional clearance to 4′-hydroxyflurbiprofen. The effect of the CYP2C9 inhibitor fluconazole on flurbiprofen metabolism was also evaluated. Methods Flurbiprofen pharmacokinetics were evaluated before and after the first and seventh doses of fluconazole. The urinary recovery ratio was calculated as FLRR = 4′-OHF/[4′-OHF + Ftot] and the urinary metabolic ratio was calculated as FLMR = 4′-OHF/Ftot, where 4′-OHF and Ftot represent total (conjugated and unconjugated) amounts recovered in urine. Results There was a statistically significant relationship between the 4′-OHF formation clearance (4OHCLf) and both the 8-h FLRR and the 8-h FLMR with and without administration of fluconazole. The flurbiprofen apparent oral clearance (CL/F) was decreased by 53% [90% confidence interval (CI) −58, −48] and 64% (90% CI −69, −59), respectively, after administration of one and seven doses of fluconazole when compared with administration of flurbiprofen alone; similarly, the 4OHCLf decreased by 69% (90% CI −74, −64) and 78% (90% CI −83, −73), the 8-h FLRR decreased by 35% (90% CI −41, −29) and 40% (90% CI −46, −35) and the 8-h FLMR decreased by 61% (90% CI −65, −58) and 67% (90% CI −70, −63). The magnitude of decrease in CL/F and 4OHCLf was greater after seven doses compared with after one dose of fluconazole (P

Background Carboplatin/paclitaxel (CP), with or without sorafenib, result in objective response rates of 18–20 % in unselected chemotherapy-naïve patients. Molecular predictors of survival and response to CP-based chemotherapy in metastatic melanoma (MM) are critical to improving the therapeutic index. Intergroup trial E2603 randomized MM patients to CP with or without sorafenib. Expression data were collected from pre-treatment formalin-fixed paraffin-embedded (FFPE) tumor tissues from 115 of 823 patients enrolled on E2603. The selected patients were balanced across treatment arms, BRAF status, and clinical outcome. We generated data using Nanostring array (microRNA (miRNA) expression) and DNA-mediated annealing, selection, extension and ligation (DASL)/Illumina microarrays (HT12 v4) (mRNA expression) with protocols optimized for FFPE samples. Integrative computational analysis was performed using a novel Tree-guided Recursive Cluster Selection (T-ReCS) [1] algorithm to select the most informative features/genes, followed by TargetScan miRNA target prediction (Human v6.2) and mirConnX [2] for network inference. Results T-ReCS identified PLXNB1 as negatively associated with progression-free survival (PFS) and miR-659-3p as the primary miRNA associated positively with PFS. miR-659-3p was differentially expressed based on PFS but not based on treatment arm, BRAF or NRAS status. Dichotomized by median PFS (less vs greater than 4 months), miR-659-3p expression was significantly different. High miR-659-3p expression distinguished patients with responsive disease (complete or partial response) from patients with stable disease. miR-659-3p predicted gene targets include NFIX, which is a transcription factor known to interact with c-Jun and AP-1 in the context of developmental processes and disease. Conclusions This novel integrative analysis implicates miR-659-3p as a candidate predictive biomarker for MM patients treated with platinum-based chemotherapy and may serve to improve patient selection.

Lung cancer is the leading cause of cancer-related mortality in both sexes worldwide, mainly because of lack of established early screening strategies (1). The development of this disease over 30 to 40 years in smokers involves field cancerization, characterized as the acquisition of genetic and epigenetic changes in oncogenes and tumor suppressor genes throughout the lung epithelium (2). The epigenetic silencing of tumor suppressor genes by promoter hypermethylation has been recognized as a major and causal event for lung cancer initiation and progression (2). Moreover, the detection of gene methylation in exfoliated cells from the lungs of smokers provides an assessment of the extent of field cancerization and is a validated biomarker for predicting lung cancer risk (2–4). The precise mechanism by which cigarette carcinogens disrupt the capacity of lung cells to maintain the epigenetic code during DNA replication and repair is largely unknown. Thus, the identification of genetic determinants contributing to the propensity for acquiring gene methylation in lung epithelium of smokers should provide new insights into the mechanisms underlying epigenetic reprogramming during lung carcinogenesis. Importantly, these genetic loci may also contribute to the genetic component affecting the risk for lung cancer that includes risk loci identified in several lung cancer genome-wide association studies (GWAS [5]). Emerging evidence suggested that genome-wide landscaping of the sequence-dependent allele-specific methylation for nonimprinted genes may help pinpoint the functional regulatory polymorphisms that may influence the disease susceptibility (6). Most recently, Shi et al. (7) conducted a genome-wide assessment for methylation quantitative trait loci (meQTL) and found 34 304 cis-meQTLs, mostly localized to CpG sites outside of genes, promoters, and CpG islands, and 585 trans-meQTLs largely overrepresented in promoter CpG islands. A strong enrichment of these meQTL single nucleotide polymorphisms (SNPs) for DNase hypersensitive sites and sequences bound by CCCTC-binding factor (CTCF) or modified histones was also identified in cell lines, although the effect of methylation of these CpG sites on gene transcription regulation warrants future investigation (7). The etiology of tumor development likely requires the acquisition of the silencing of hundreds of critical genes by promoter methylation, with most gene silencing occurring independent of allele-specific methylation and/or meQTL (Leng et al., unpublished data). Previously, we conducted a candidate gene–based study that implicated genetic variation in DNA replication and apoptosis pathways in modifying the propensity for gene methylation in the aerodigestive tract of smokers (8). The current study extends this work by conducting a two-stage GWAS (9) using smokers from two geographically independent cohorts to identify low-penetrance alleles affecting the propensity for acquiring gene methylation in the lungs.

© 2014 Elsevier Ireland Ltd. EGFR polymorphisms have not been thoroughly evaluated for association with head and neck squamous cell carcinoma (HNSCC) risk. We genotyped 578 HNSCC patients and 588 cancer-free controls for 60 EGFR single nucleotide polymorphisms (SNPs) and tested associations with HNSCC risk.EGFR intronic SNPs rs12535536, rs2075110, rs1253871, rs845561 and rs6970262 and synonymous SNP rs2072454 were associated with HNSCC risk among all subjects (p < 0.05). SNPs rs12538371, rs845561, and rs6970262 were significantly associated with HNSCC risk (p < 0.05) among never tobacco users. We identified EGFR variants that likely modify risk for HNSCC including three variants that contribute to tobacco-independent risk.

Background The epidermal growth factor receptor (EGFR) is involved in the development and progression of lung cancer. Somatic EGFR mutations are predictors of response to treatment with EGFR tyrosine kinase (TK) inhibitors (TKIs) for lung cancer, especially among never smokers. EGFR mutations may occur independently of other genetic alterations. Methods The authors sequenced the EGFR -TK domain and the K-ras and p53 genes from lung tumor tissues from 44 never smokers and 46 smokers. A case-control study also was conducted to examine the relationship between an EGFR single nucleotide polymorphism in the TK domain and the lung cancer through a multivariate logistic regression analysis. In addition, the authors compared cell growth kinetics, EGFR-TKI sensitivity by MTT, and activation of signaling molecules by immunoblot in lung cancer cell lines with and without EGFR -TK mutations. Results EGFR -TK mutations were more frequently observed in never smokers (25%) than in smokers (2.2%) ( p = 0.001). Excluding cases with a K-ras mutation, the frequency of EGFR -TK domain mutation was still significantly higher in never smokers than in smokers, 26.2% versus 4.5% ( p = 0.046). EGFR -TK mutations and K-ras mutations ( p = 0.015), and p53 and K-ras mutations ( p = 0.015) were mutually exclusive, but p53 and EGFR -TK mutations were not ( p = 1.00). During sequencing of the EGFR -TK domain in tumors, an EGFR polymorphism (G2607A) was identified. The genotype AA and AA + AG occurred at a significantly higher frequency in lung cancer cases ( n = 122) when compared with controls ( n = 147) (odds ratio, 3.39 and 2.67; 95% confidence interval, 1.41–8.17 and 1.17–6.08, p = 0.006 and p = 0.02, respectively). This polymorphism was found independently of EGFR -TK mutations in lung cancer cases, indicating that it does not predispose to mutations. In vitro, lung cancer cell lines with EGFR -TK mutations also did not contain K-ras mutations and displayed a lower growth rate (50%, p = 0.013) than EGFR -TK wild-type cell lines. EGFR -TK mutant cell lines were more sensitive to both gefitinib and erlotinib, although relative sensitivity to erlotinib compared with wild-type was less pronounced than for gefitinib. Cell lines with a lower growth rate also expressed higher levels of E-cadherin than faster growing cell lines. Conclusions EGFR -TK mutation frequency is high in never-smoking lung cancer patients and is exclusive of mutation in K-ras but not p53 . In addition to somatic EGFR -TK mutations that arise in lung tumors, germline variation in the EGFR -TK domain might also be associated with an increased risk of lung cancer. Somatic EGFR -TK mutations alter cell biology and response to EGFR -TKIs and may be mutation specific.

Background We have previously shown that flurbiprofen metabolism to 4′-hydroxyflurbiprofen provides an in vivo measure of cytochrome P450 (CYP) 2C9 activity. This study evaluated the possibility of incorporating flurbiprofen into the current 5-drug Pittsburgh cocktail. Methods In a randomized, 3-way, Latin-square, crossover-design study, 24 healthy subjects (mean age [±SD], 47.8 ± 15.1 years) received flurbiprofen (50 mg) and the Pittsburgh 5-drug cocktail (100 mg caffeine, 100 mg mephenytoin, 10 mg debrisoquin [INN, debrisoquine], 250 mg chlorzoxazone, and 100 mg dapsone) separately and in combination on 3 occasions over a period of 5 weeks. Urine was collected from 0 to 8 hours, and plasma was obtained at 4 and 8 hours after drug administration. Parent drug and metabolite concentrations were measured to determine phenotypic indices for each of the metabolizing enzymes. Results The geometric mean ratio and 90% confidence interval of the phenotypic indices were included within the 80% to 125% bioequivalence range for each of the probe drugs. There were no statistically significant differences between the phenotypic indices determined after administration of the 5-drug and 6-drug cocktails. However, there was a small but statistically significant increase (7.5%, P = .03) in the 8-hour urinary flurbiprofen recovery ratio after administration of the 6-drug cocktail compared with that after administration of flurbiprofen alone. The 6-drug cocktail was well tolerated. Conclusion The results of this study show that caffeine (CYP1A2), chlorzoxazone (CYP2E1), dapsone (N-acetyltransferase 2), debrisoquin (CYP2D6), flurbiprofen (CYP2C9), and mephenytoin (CYP2C19) can be simultaneously administered in low doses without metabolic interaction. Clinical Pharmacology & Therapeutics (2006) 80, 257–263; doi: 10.1016/j.clpt.2006.06.005

Biomarkers of exposure & early effects: field studiesBiomarker: mutation frequency of glycophorin AExposure/effect represented: cigarette smoke/ DNA damageStudy design: cross-sectionalStudy size: 2405 pregnant womenAnalytical technique: The GPA somatic cell mutation assayTissue/biological material/sample size: peripheral bloodImpact on outcome (including dose-response): active smoker/non-smoker a positive significant overall association of smoking with a newborn Ψ/N Vf was observed by generalized linear model analysis (P=0.023)Quality control: reliability of maternal self-reported cigarette smoking was verified by measuring cotinine levels in maternal blood samples in a separate analysisBiomarkers of individual susceptibility: field studiesBiomarker (including alleles if genetic): CYP1A1*2A, CYP1A1*3, CYP2E1*5B, NAT2*5, *6A, *7A and *14A, GSTM1 and GSTT1Effect studied (phenotype/pathology): DNA damageMethod of analysis: RFLP-PCRImpact on outcome (including dose-response): CYP1A1* 2A or CYP1A1*3, 33.6%; CYP2E1*5B, 6.9%; GSTM1 null, 46.8%; GSTT1 null, 24.1%; and NAT2 slow, 43.1%. KEYWORDS CLASSIFICATION: Adolescent;Adult;Alleles;biomarkers of exposure & effect: field studies;biomarkers of individual susceptibility: field studies;blood;Female;Fetal Blood;genetics;Genotype;Glycophorin;Humans;Infant,Newborn;metabolism;methods;Maternal-Fetal Exchange;Mothers;Mutation;Pharmacogenetics;Polymorphism,Genetic;Pregnancy;Research;Smoking. Prenatal exposure to carcinogens results in newborn DNA damage which in turn is associated with impaired health conditions in both childhood and adulthood. The present study aimed to evaluate whether phase I and II biotransformation enzyme genetic polymorphisms in combination with environmental exposures during pregnancy result in elevated levels of newborn DNA damage. Maternal peripheral and umbilical cord blood samples from 406 mother/newborn pairs were genotyped for a panel of phase I/II metabolic enzymes (CYP1A1, CYP2E1, GSTM1, GSTT1 and NAT2) responsible for the metabolism of tobacco and lifestyle-related mutagens and carcinogens. DNA damage was measured by somatic cell mutation frequency at the glycophorin A (GPA) locus in newborns. No association with elevated somatic cell mutation frequency was observed between the combination of maternal/newborn genotypes and cigarette smoke or lifestyle exposures. The observed variation in newborn GPA frequency might be due to either environmental factors not assessed in this study or inter-individual differences in alternative metabolic or DNA repair pathways.

Previous studies have established that a subset of head and neck tumors contains human papillomavirus (HPV) sequences and that HPV-driven head and neck cancers display distinct biological and clinical features. HPV is known to drive cancer by the actions of the E6 and E7 oncoproteins, but the molecular architecture of HPV infection and its interaction with the host genome in head and neck cancers have not been comprehensively described. We profiled a cohort of 279 head and neck cancers with next generation RNA and DNA sequencing and show that 35 (12.5%) tumors displayed evidence of high-risk HPV types 16, 33, or 35. Twenty-five cases had integration of the viral genome into one or more locations in the human genome with statistical enrichment for genic regions. Integrations had a marked impact on the human genome and were associated with alterations in DNA copy number, mRNA transcript abundance and splicing, and both inter- and intrachromosomal rearrangements. Many of these events involved genes with documented roles in cancer. Cancers with integrated vs. nonintegrated HPV displayed different patterns of DNA methylation and both human and viral gene expressions. Together, these data provide insight into the mechanisms by which HPV interacts with the human genome beyond expression of viral oncoproteins and suggest that specific integration events are an integral component of viral oncogenesis.

Background Since genetic factors may play an important role in lung cancer development at low dose carcinogen exposure, non-smokers are a good model to study genetic susceptibility and its interaction with environmental factors. Materials and methods We evaluated the role of the metabolic gene polymorphisms CYP1A1MspI, CYP1A1Ile462Val, GSTM1, and GSTT1 in non-smoker lung cancer patients from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). Non-smokers (defined as subjects who never smoked on a regular basis) were selected from the GSEC database. We pooled the raw data from 21 case-control studies for a total of 2764 Caucasians (555 cases and 2209 controls) and 383 Asians (113 cases and 270 controls). Tests of heterogeneity and of inclusion bias were performed. Results A significant association between lung cancer and CYP1A1Ile462Val polymorphism was observed in Caucasians (adjusted OR = 2.04, 95% CI 1.17–3.54). GSTT1 deletion seems to be a risk factor for lung cancer in Caucasian non smokers only when the analysis was restricted to studies including healthy controls (adjusted OR = 1.66, 95% CI 1.12–2.46). A protective effect on lung cancer was observed with the combination of CYP1A1 wild type, GSTM1 null, and GSTT1 non-null genotypes. None of the analysed polymorphisms were associated with lung cancer in Asian non-smokers. Discussion Our analysis confirms previous findings that CYP1A1Ile462Val polymorphism may play a role in lung carcinogenesis in Caucasian non-smokers.

DNA repair enzyme genetic polymorphisms have been postulated to increase the risk of certain cancers in the presence of tobacco carcinogen exposures. The XPD protein is an important component of the TFIIH transcription factor complex. XPD genetic polymorphisms resulting in amino acids substitutions may lead to alterations in TFIIH helicase activity, resulting in repair and transcription defects. Cyclin D1 is a key regulatory protein for the transition of cells from the G(1)-S cell cycle phase. The CCND1 G870A polymorphism has been reported to enhance alternate splicing of a stable mRNA variant, which may result in the bypass of the G(1)/S cell cycle checkpoint. In this study, XPD G23591A (Asp312Asn) and A35931C (Lys751Gln) polymorphisms and the CCND1 G870A splice variant frequencies were determined in 273 upper aero-digestive tract cancer cases and 269 controls. The XPD Asp312Asn variant frequency was significantly different among cases and controls and conferred an odds ratio (OR) of 1.3 (95% CI 1.0-1.8). However, individuals with the CCND1 G870A and XPD Lys751Gln variants had higher age adjusted ORs of 3.2 (95% CI 2.2-4.6) and 2.2 (95% CI 1.5-3.2), respectively. Furthermore, a significant gene-gene interaction was observed among cases with at least two variant alleles for both CCND1 and XPD genes [OR 7.09 (95% CI 4.03-12.5)]. Smokers with a combination of at least one variant allele of both CCND1 and XPD genes also had an elevated risk as compared to nonsmokers. This is the first study to suggest an associative interaction between XPD and CCND1 genetic polymorphisms, tobacco exposure, and cancer risk.

Contains fulltext : 58544.pdf (Publisher’s version ) (Closed access) The objectives were to study the association between metabolic genes involved in alcohol metabolism (CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1) and alcohol consumption in a large sample of healthy controls. Healthy subjects were selected from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). Subjects with information on both alcohol consumption and at least one of the studied polymorphisms were included in the analysis (n=2224). Information on the amount of alcohol consumption was available for a subset of subjects (n=844). None of the studied genes was significantly associated with drinking habits. A significant heterogeneity with age was observed when studying the association between CYP2E1 RsaI and alcohol drinking. CYP2E1 RsaI polymorphism was significantly associated with being a never drinker at older ages (odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2-4.8; at ages above 68 years), while the association was reversed at ages below 47 years (OR 0.5, 95% CI 0.2-1.4). For subjects with detailed information on alcohol intake, no association between alcohol quantity and polymorphisms in metabolic genes was observed; subjects carrying the NQO1 polymorphism tended to drink more than subjects carrying the wild-type alleles. Therefore, no significant association between CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1 polymorphisms and alcohol consumption was observed in healthy controls.

Gene-environment interactions have been extensively studied in lung cancer. It is likely that several genetic polymorphisms cooperate in increasing the individual risk. Therefore, the study of gene-gene interactions might be important to identify high-susceptibility subgroups. GSEC is an initiative aimed at collecting available data sets on metabolic polymorphisms and the risks of cancer at several sites and performing pooled analyses of the original data. Authors of published papers have provided original data sets. The present paper refers to gene-gene interactions in lung cancer and considers three polymorphisms in three metabolic genes: CYP1A1, GSTM1 and GSTT1. The present analyses compare the gene gene interactions of the CYP1A1*2A, GSTM1 and GSTT1 polymorphisms from studies on lung cancer conducted in Europe and the USA between 1991 and 2000. Only Caucasians have been included. The data set includes 1466 cases and 1488 controls. The only clear-cut association was found with CYP1A1*2A. This association remained unchanged after stratification by polymorphisms in other genes (with an odds ratio [OR] of approximately 2.5), except when interaction with GSTM1 was considered. When the OR for CYP1A1*2A was stratified according to the GSTM1 genotype, the OR was increased only among the subjects who had the null (homozygous deletion) GSTM1 genotype (OR = 2.8, 95% CI = 0.9-8.4). The odds ratio for the interactive term (CYP1A1*2A by GSTM1) in logistic regression was 2.7 (95% CI = 0.5-15.3). An association between lung cancer and the homozygous CYP1A1*2A genotype is confirmed. An apparent and biologically plausible interaction is suggested between this genotype and GSTM1.

CYP1A1 is involved in the metabolism of benzopyrene, a suspected lung carcinogen; it is therefore conceivable that genetically determined variations in its activity modify individual susceptibility to lung cancer. The role of the CYP1A1 MspI polymorphism in lung cancer has been widely studied but has not been fully clarified. We have included 2,451 cases and 3,358 controls in a pooled analysis of 22 case-control studies on CYP1A1 and lung cancer risk. We found a clear association between the CYP1A1 homozygous MspI restriction fragment length polymorphism (RFLP) and lung cancer risk in Caucasians (age- and gender-adjusted odds ratio = 2.36; 95% confidence interval 1.16-4.81); other associations were weaker or not statistically significant. The association with the homozygous variant was equally strong for squamous cell carcinomas and adenocarcinomas among Caucasians. We analyzed the risk by duration of smoking: for Caucasian subjects with the MspI RFLP combined variants (homozygotes plus heterozygotes), the increase in the risk of lung cancer was steeper than among the individuals with the homozygous reference allele. Our analysis suggests that Caucasians with homozygous variant CYP1A1 polymorphism have a higher risk of lung cancer. The data were more consistent among Caucasians, with a strong association between the homozygous variant in both squamous cell carcinomas and adenocarcinomas, and a stronger association in men than in women. The analyses were more inconsistent and failed to reach statistical significance in Asians. This observation might be due to design specificities or unknown effect modifiers in the Asian studies.

Smoking is a known risk factor for bladder cancer. The product of the GSTM1 gene, glutathione S-transferase M1 (GSTM1), is involved in the detoxification of polycyclic aromatic hydrocarbons found in tobacco smoke; a homozygous deletion of this gene in approximately 50% of Caucasians and Asians results in a lack of GSTM1 enzyme activity. Most studies examining the relation between bladder cancer and GSTM1 have reported an increased risk associated with a lack of GSTM1 activity. The authors performed meta- and pooled analyses of published and unpublished, case-control, genotype-based studies that examined this association (17 studies, 2,149 cases, 3,646 controls) and excluded studies conducted in populations with a high prevalence of exposure to known bladder cancer risk factors other than tobacco smoke. Using random effects models in the meta-analysis, the authors obtained a summary odds ratio of 1.44 (95% confidence interval (CI): 1.23, 1.68) for GSTM1 null status with all studies included. Results from studies with at least 100 cases and 100 controls produced a summary odds ratio of 1.42 (95% CI: 1.26, 1.60). Pooled analyses using original data sets from 10 studies (1,496 cases and 1,444 controls) and adjusting for age, sex, and race produced similar results. There was no evidence of multiplicative interaction between the GSTM1 null genotype and ever smoking in relation to bladder cancer, although there was a suggestion of additive interaction (additive interaction = 0.45, 95% CI: -0.03, 0.93). These results indicate that, among populations studied to date, GSTM1 null status is associated with a modest increase in the risk of bladder cancer.

The analysis of gene expression is an integral part of any gene function research. A wide variety of techniques have been developed for this purpose, each with its own advantages and limitations. The following chapter seeks to provide an overview of some of the most recent as well as conventional methods to study gene expression. These approaches include Northern blot analysis, ribonuclease protection assay, reverse transcription polymerase chain reaction, expressed tag sequencing, differential display, cDNA arrays, serial analysis of gene expression, and transcriptome sequencing. The current applications of the information derived from gene expression studies require most of the assays to be adaptable for the quantitative analysis of a large number of samples and endpoints within a short period of time coupled with cost-effectiveness. A comparison of some of these features of each analytical approach as well as their advantages and disadvantages has also been provided.

Pharmacogenomics encompasses several major areas: the study of polymorphic variations in drug response and disease susceptibility, identification of the effects of drugs/xenobiotics at the genomic level, and genotype/phenotype associations. The most common type of human genetic variations is single-nucleotide polymorphisms (SNPs). Several novel approaches to detection of SNPs are currently available. The range of new methods includes modifications of several conventional techniques, such as PCR, mass spectrometry (ms), and sequencing, as well as more innovative technologies such as fluorescence resonance energy transfer (FRET) and microarrays. The application of each of these techniques is largely dependent on the number of SNPs to be screened and sample size. The current chapter presents an overview of the general concepts of a variety of genotyping technologies, with an emphasis on the recently developed methodologies, including a comparison of the advantages, applicability, cost efficiency, and limitations of these methods.

Purpose: All-trans-retinoic acid (ATRA) is a retinoid analogue that has been shown to be effective in acute promyelocytic leukemia. It is currently being investigated for efficacy in the treatment and prevention of various types of cancer. One of the factors limiting its use is the observed increase in ATRA clearance and elimination which occurs shortly after treatment is started, leading to reduced levels of drug in the body and loss of effectiveness. ATRA efficacy may be enhanced if this autoinduction of metabolism can be overcome, for example through the inhibition of the activity of the induced specific metabolizing enzyme(s). This requires the identification of this induced enzyme(s) and development of approaches to selectively inhibit its activity. Methods: In the course of a phase II evaluation of ATRA in prostate cancer, we investigated the activities of five specific cytochrome P450 (CYP) (CYPs 1A2, 2C19, 2D6, 2E1 and 3A4) and N-acetyltransferase enzymes using a newly developed five-drug cocktail involving caffeine, mephenytoin, debrisoquine, chlorzoxazone and dapsone respectively. Enzyme activities were assessed in 17 patients with hormone-refractory prostate cancer before the initiation of ATRA therapy, after 14 days of continuous ATRA administration and 7 days after cessation of drug therapy. Results: After 14 days of ATRA therapy, the activities of CYP2E1 (chlorzoxazone hydroxylase) and N-acetyltransferase (in fast acetylators only) were increased by 83% and 29% (P < 0.05), respectively. Both activities returned to baseline by 7 days after cessation of therapy and the profiles were similar to the changes seen in the clearance of ATRA itself. There were no changes in the activities of any of the other enzymes investigated. Conclusion: This study shows that ATRA selectively modulates the activities of specific metabolizing enzymes and that this approach may be useful in identifying enzymes that can be explored in an attempt to mitigate ATRA autoinduction through selective modulation of enzyme activities. Further investigations are required to determine whether the elevated enzymes are also responsible for the increased clearance and elimination of ATRA.

The dapsone recovery ratio (DPRR), which is determined after single oral dose administration of dapsone by measuring the parent drug and hydroxylated metabolite, provides an in vivo measure of the efficiency of the drug metabolizing enzymes responsible for this metabolic route, putatively CYP3A4. This affords the potential to evaluate the hypothesis that this drug metabolizing enzyme system is involved in the pathogenesis of human bladder cancer. The present study is a matched case-control comparison of DPRR in patients with nonaggressive bladder cancer (grades I and II or Ta, T1 and T2, n = 43), patients with aggressive bladder cancer without invasion (grade III or Ta, T1 and T2, n = 32), patients with aggressive bladder cancer and invasion (grade III or T3 and T4, n = 32), and age- and gender-matched subjects with no urologic tumor on cystoscopy from an urban U.K. community (n = 85). Demographic variables associated with aggressive bladder cancer (Gill or T3, T4, Tis) included pack-years of smoking, alcohol intake, and occupational exposure; for nonaggressive bladder cancer variables included smoking and occupational exposure. DPRR exhibited an unimodal distribution in all subjects: activity was significantly reduced in both noninvasive and invasive aggressive bladder cancer, and was a significant risk factor for cancer after adjustment for other significant risk factors. Combining the two aggressive groups, the lowest tertile of DPRR activity was associated with a sixfold increase in risk (p < 0.02) compared with the upper tertile. We conclude that a low dapsone recovery ratio is an independent risk factor for aggressive bladder cancer irrespective of its stage of invasion and suggest that the enzymes involved in its metabolism are detoxifying enzymes for unknown environmental factors to which an urban community is exposed.