Creation of an ustekinumab external control arm for Crohn's disease using electronic health records data: A pilot study.

BACKGROUND: Randomized trials are the gold-standard for clinical evidence generation, but they can sometimes be limited by infeasibility and unclear generalizability to real-world practice. External control arm (ECA) studies may help address this evidence gaps by constructing retrospective cohorts that closely emulate prospective ones. Experience in constructing these outside the context of rare diseases or cancer is limited. We piloted an approach for developing an ECA in Crohn's disease using electronic health records (EHR) data. METHODS: We queried EHR databases and manually screened records at the University of California, San Francisco to identify patients meeting the eligibility criteria of TRIDENT, a recently completed interventional trial involving an ustekinumab reference arm. We defined timepoints to balance missing data and bias. We compared imputation models by their impacts on cohort membership and outcomes. We assessed the accuracy of algorithmic data curation against manual review. Lastly, we assessed disease activity following treatment with ustekinumab. RESULTS: Screening identified 183 patients. 30% of the cohort had missing baseline data. Nonetheless, cohort membership and outcomes were robust to the method of imputation. Algorithms for ascertaining non-symptom-based elements of disease activity using structured data were accurate against manual review. The cohort consisted of 56 patients, exceeding planned enrollment in TRIDENT. 34% of the cohort was in steroid-free remission at week 24. CONCLUSION: We piloted an approach for creating an ECA in Crohn's disease from EHR data by using a combination of informatics and manual methods. However, our study reveals significant missing data when standard-of-care clinical data are repurposed. More work will be needed to improve the alignment of trial design with typical patterns of clinical practice, and thereby enable a future of more robust ECAs in chronic diseases like Crohn's disease.

Characterization of N-acetyltransferase 1 and 2 polymorphisms and haplotype analysis for inflammatory bowel disease and sporadic colorectal carcinoma.

BACKGROUND: N-acetyltransferase 1 (NAT1) and 2 (NAT2) are polymorphic isoenzymes responsible for the metabolism of numerous drugs and carcinogens. Acetylation catalyzed by NAT1 and NAT2 are important in metabolic activation of arylamines to electrophilic intermediates that initiate carcinogenesis. Inflammatory bowel diseases (IBD) consist of Crohn's disease (CD) and ulcerative colitis (UC), both are associated with increased colorectal cancer (CRC) risk. We hypothesized that NAT1 and/or NAT2 polymorphisms contribute to the increased cancer evident in IBD. METHODS: A case control study was performed with 729 Caucasian participants, 123 CRC, 201 CD, 167 UC, 15 IBD dysplasia/cancer and 223 controls. NAT1 and NAT2 genotyping were performed using Taqman based techniques. Eight single nucleotide polymorphisms (SNPs) were characterized for NAT1 and 7 SNPs for NAT2. Haplotype frequencies were estimated using an Expectation-Maximization (EM) method. Disease groups were compared to a control group for the frequencies at each individual SNP separately. The same groups were compared for the frequencies of NAT1 and NAT2 haplotypes and deduced NAT2 phenotypes. RESULTS: No statistically significant differences were found for any comparison. Strong linkage disequilibrium was present among both the NAT1 SNPs and the NAT2 SNPs. CONCLUSION: This study did not demonstrate an association between NAT1 and NAT2 polymorphisms and IBD or sporadic CRC, although power calculations indicate this study had sufficient sample size to detect differences in frequency as small as 0.05 to 0.15 depending on SNP or haplotype.

Association between manganese superoxide dismutase promoter gene polymorphism and breast cancer survival.

BACKGROUND: Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk and survival in breast cancer. This base pair transition (-9 T > C) leads to a valine to alanine amino acid change in the mitochondrial targeting sequence. A polymorphism has also been identified in the proximal region of the promoter (-102 C>T) that alters the recognition sequence of the AP-2 transcription factor, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the -102 C>T polymorphism with overall and relapse-free breast cancer survival in a hospital-based case-only study. MATERIALS AND METHODS: The relationship between the MnSOD -102 C>T polymorphism and survival was examined in a cohort of 291 women who received chemotherapy and/or radiotherapy for incident breast cancer. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. Patient survival was evaluated according to the MnSOD genotype using Kaplan-Meier survival functions. Hazard ratios were calculated from adjusted Cox proportional hazards modeling. All statistical tests were two-sided. RESULTS: In an evaluation of all women, there was a borderline significant reduction in recurrence-free survival with either one or both variant alleles (CT + TT) when compared with patients with wild-type alleles (CC) (odds ratio, 0.65; 95% confidence interval, 0.42-1.01). When the analysis was restricted to patients receiving radiation therapy, there was a significant reduction in relapse-free survival in women who were heterozygous for the MnSOD -102 genotype (relative risk, 0.40; 95% confidence interval, 0.18-0.86). Similarly, when the homozygous and heterozygous variant genotypes were combined, there remained a significant reduction in relapse-free survival in this group (hazard ratio, 0.42; 95% confidence interval, 0.20-0.87). CONCLUSION: The MnSOD -102 variant allele appears to be associated with an improved recurrence-free survival in all patients, and more dramatically in subjects who received adjuvant radiation therapy.

Functional properties of an alternative, tissue-specific promoter for human arylamine N-acetyltransferase 1.

Variable expression of human arylamine N-acetyltransferase 1 (NAT1) due to genetic polymorphism, gene regulation or environmental influences is associated with individual susceptibility to various cancers. Recent studies of NAT1 transcription showed that most mRNAs originate at a promoter, P1, located 11.8 kb upstream of the single open reading frame (ORF) exon. We have now characterized an alternative NAT1 promoter lying 51.5 kb upstream of the NAT1 ORF. In the present study, analysis of human RNAs representing 27 tissue types by reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative RT-PCR showed the upstream 51.5 kb promoter, designated P3, to be most active in specific tissues, including kidney, liver, lung, and trachea. All NAT1 P3 mRNAs included 5'-untranslated region (5'-UTR) internal exons of 61 and 175 nucleotides in addition to the 79 nucleotide 5'-UTR exon present in P1 mRNA. CAP-dependent amplification of 5'-P3 mRNA termini defined an 84 bp transcription start region in which most start sites are centrally clustered. The hepatoma-derived HepG2 cell line expressed a high level of P3 mRNA with the same spliced structure and start site pattern as found in normal tissues. A 435-bp minimal promoter was defined by transfection of HepG2 with luciferase expression constructs containing genomic fragments from the P3 start region. These findings imply a fundamental role for P3 in NAT1 regulation and define additional regions for genetic polymorphisms associated with enhanced cancer risk.

No apparent association between genetic polymorphisms (-102 C>T) and (-9 T>C) in the human manganese superoxide dismutase gene and gastric cancer(1).

BACKGROUND: Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species, constituting a major cellular defense mechanism against agents that induce oxidative stress. A genetic polymorphism in the mitochondrial targeting sequence of this gene has been associated with increased cancer risk. This one base pair transition (-9 T>C) leads to a Val to Ala amino acid change in the mitochondrial targeting sequence. In addition, the MnSOD promoter contains an activator protein-2 (AP-2) binding site that modifies transcription of MnSOD. Mutations have been identified in the proximal region of the promoter in human tumor cell lines. One of these mutations (-102 C>T) has been shown to change the binding pattern of AP-2, leading to a reduction in transcriptional activity. The aim of our study was to investigate possible associations of the (-9 T>C) and (-102 C>T) polymorphisms with gastric cancer in a population-based case-control study conducted in Warsaw, Poland. MATERIALS AND METHODS: DNA was obtained from a population based case-control study of stomach cancer conducted in Warsaw, Poland, between 1994 and 1996. The MnSOD -9 T>C genotype was determined by PCR-RFLP assay. The MnSOD -102 C>T genotype was determined using a TaqMan allele discrimination assay. RESULTS: The frequency of the -102 C>T polymorphism was 41% (38/91) in gastric cancer cases and 38% (50/130) in the controls (odds ratio [OR] 1.1, 95% confidence interval [CI] 0.6-2.1). The frequency of the -9 T>C polymorphism was 44% (202/464) in cases and 56% (262/464) in controls (OR 1.1; 95% CI 0.9-1.37). The lack of association was observed in both non-smokers (OR 1.5; 95% CI 0.7-2.34) and smokers (OR 1.1; 95% CI 0.7-1.7). Furthermore, the association was not significant when smokers were segregated by extent of smoking history. CONCLUSION: The association of the manganese superoxide dismutase polymorphisms at -102 C>T and the -9 T>C were not found to be associated with gastric cancer in a Polish case-control study.

Method for determination of (-102C>T) single nucleotide polymorphism in the human manganese superoxide dismutase promoter.

BACKGROUND: Manganese superoxide dismutase (MnSOD) plays a critical role in the detoxification of mitochondrial reactive oxygen species constituting a major cellular defense mechanism against agents that induce oxidative stress. The MnSOD promoter contains an activator protein-2 (AP-2) binding site that modifies transcription of MnSOD. Mutations have been identified in the proximal region of the promoter in human tumor cell lines. One of these mutations (-102C>T) has been shown to change the binding pattern of AP-2 leading to a reduction in transcriptional activity. The aim of our study was to develop a method to identify and determine the frequency of this (-102C>T) polymorphism in human tissues. RESULTS: A new TaqMan allelic discrimination genotype method was successfully applied to genomic DNA samples derived from blood, buccal swabs, snap frozen tissue and paraffin blocks. The polymorphism was shown to be in Hardy-Weinberg Equilibrium in an evaluation of 130 Caucasians from Warsaw, Poland: 44 (33.8%) were heterozygous and 6 (4.6%) were homozygous for -102T. CONCLUSION: This report represents the first description of the MnSOD -102C>T polymorphism in human subjects by a novel Taqman allelic discrimination assay. This method should enable molecular epidemiological studies to evaluate possible associations of this polymorphism with malignancies and other diseases related to reactive oxygen species.