An efficient strategy for evaluating new non-invasive screening tests for colorectal cancer: the guiding principles.

OBJECTIVE: New screening tests for colorectal cancer (CRC) are rapidly emerging. Conducting trials with mortality reduction as the end point supporting their adoption is challenging. We re-examined the principles underlying evaluation of new non-invasive tests in view of technological developments and identification of new biomarkers. DESIGN: A formal consensus approach involving a multidisciplinary expert panel revised eight previously established principles. RESULTS: Twelve newly stated principles emerged. Effectiveness of a new test can be evaluated by comparison with a proven comparator non-invasive test. The faecal immunochemical test is now considered the appropriate comparator, while colonoscopy remains the diagnostic standard. For a new test to be able to meet differing screening goals and regulatory requirements, flexibility to adjust its positivity threshold is desirable. A rigorous and efficient four-phased approach is proposed, commencing with small studies assessing the test's ability to discriminate between CRC and non-cancer states (phase I), followed by prospective estimation of accuracy across the continuum of neoplastic lesions in neoplasia-enriched populations (phase II). If these show promise, a provisional test positivity threshold is set before evaluation in typical screening populations. Phase III prospective studies determine single round intention-to-screen programme outcomes and confirm the test positivity threshold. Phase IV studies involve evaluation over repeated screening rounds with monitoring for missed lesions. Phases III and IV findings will provide the real-world data required to model test impact on CRC mortality and incidence. CONCLUSION: New non-invasive tests can be efficiently evaluated by a rigorous phased comparative approach, generating data from unbiased populations that inform predictions of their health impact.

Performance analysis of a machine learning flagging system used to identify a group of individuals at a high risk for colorectal cancer.

Individuals with colorectal cancer (CRC) have a tendency to intestinal bleeding which may result in mild to severe iron deficiency anemia, but for many colon cancer patients hematological abnormalities are subtle. The fecal occult blood test (FOBT) is used as a pre-screening test whereby those with a positive FOBT are referred to colonscopy. We sought to determine if information contained in the complete blood count (CBC) report coud be processed automatically and used to predict the presence of occult colorectal cancer (CRC) in the setting of a large health services plan. Using the health records of the Maccabi Health Services (MHS) we reviewed CBC reports for 112,584 study subjects of whom 133 were diagnosed with CRC in 2008 and analysed these with the MeScore tool. The odds ratio for being diagnosed with CRC in 2008 was calculated with regards to the MeScore, using cutoff levels of 97% and 99% percentiles. For individuals in the highest one percentile, the odds ratio for CRC was 21.8 (95% CI 13.8 to 34.2). For the majority of the individuals with cancer, CRC was not suspected at the time of the blood draw. Frequent use of anticoagulants, the presence of other gastrointestinal pathologies and non-GI malignancies were assocaitged with false positive MeScores. The MeScore can help identify individuals in the population who would benefit most from CRC screening, including those with no clinical signs or symptoms of CRC.

Multitarget stool DNA for colorectal cancer screening: A review and commentary on the United States Preventive Services Draft Guidelines.

Multitarget stool DNA (mt-sDNA) testing was approved for average risk colorectal cancer (CRC) screening by the United States Food and Drug Administration and thereafter reimbursed for use by the Medicare program (2014). The United States Preventive Services Task Force (USPSTF) October 2015 draft recommendation for CRC screening included mt-sDNA as an "alternative" screening test that "may be useful in select clinical circumstances", despite its very high sensitivity for early stage CRC. The evidence supporting mt-sDNA for routine screening use is robust. The clinical efficacy of mt-sDNA as measured by sensitivity, specificity, life-years gained (LYG), and CRC deaths averted is similar to or exceeds that of the other more specifically recommended screening options included in the draft document, especially those requiring annual testing adherence. In a population with primarily irregular screening participation, tests with the highest point sensitivity and reasonable specificity are more likely to favorably impact CRC related morbidity and mortality than those depending on annual adherence. This paper reviews the evidence supporting mt-sDNA for routine screening and demonstrates, using USPSTF's modeling data, that mt-sDNA at three-year intervals provides significant clinical net benefits and fewer complications per LYG than annual fecal immunochemical testing, high sensitivity guaiac based fecal occult blood testing and 10-year colonoscopy screening.

Development and validation of a predictive model for detection of colorectal cancer in primary care by analysis of complete blood counts: a binational retrospective study.

OBJECTIVE: The use of risk prediction models grows as electronic medical records become widely available. Here, we develop and validate a model to identify individuals at increased risk for colorectal cancer (CRC) by analyzing blood counts, age, and sex, then determine the model's value when used to supplement conventional screening. MATERIALS AND METHODS: Primary care data were collected from a cohort of 606 403 Israelis (of whom 3135 were diagnosed with CRC) and a case control UK dataset of 5061 CRC cases and 25 613 controls. The model was developed on 80% of the Israeli dataset and validated using the remaining Israeli and UK datasets. Performance was evaluated according to the area under the curve, specificity, and odds ratio at several working points. RESULTS: Using blood counts obtained 3-6 months before diagnosis, the area under the curve for detecting CRC was 0.82 ± 0.01 for the Israeli validation set. The specificity was 88 ± 2% in the Israeli validation set and 94 ± 1% in the UK dataset. Detecting 50% of CRC cases, the odds ratio was 26 ± 5 and 40 ± 6, respectively, for a false-positive rate of 0.5%. Specificity for 50% detection was 87 ± 2% a year before diagnosis and 85 ± 2% for localized cancers. When used in addition to the fecal occult blood test, our model enabled more than a 2-fold increase in CRC detection. DISCUSSION: Comparable results in 2 unrelated populations suggest that the model should generally apply to the detection of CRC in other groups. The model's performance is superior to current iron deficiency anemia management guidelines, and may help physicians to identify individuals requiring additional clinical evaluation. CONCLUSIONS: Our model may help to detect CRC earlier in clinical practice.

Recommendations for a step-wise comparative approach to the evaluation of new screening tests for colorectal cancer.

BACKGROUND: New screening tests for colorectal cancer continue to emerge, but the evidence needed to justify their adoption in screening programs remains uncertain. METHODS: A review of the literature and a consensus approach by experts was undertaken to provide practical guidance on how to compare new screening tests with proven screening tests. RESULTS: Findings and recommendations from the review included the following: Adoption of a new screening test requires evidence of effectiveness relative to a proven comparator test. Clinical accuracy supported by programmatic population evaluation in the screening context on an intention-to-screen basis, including acceptability, is essential. Cancer-specific mortality is not essential as an endpoint provided that the mortality benefit of the comparator has been demonstrated and that the biologic basis of detection is similar. Effectiveness of the guaiac-based fecal occult blood test provides the minimum standard to be achieved by a new test. A 4-phase evaluation is recommended. An initial retrospective evaluation in cancer cases and controls (Phase 1) is followed by a prospective evaluation of performance across the continuum of neoplastic lesions (Phase 2). Phase 3 follows the demonstration of adequate accuracy in these 2 prescreening phases and addresses programmatic outcomes at 1 screening round on an intention-to-screen basis. Phase 4 involves more comprehensive evaluation of ongoing screening over multiple rounds. Key information is provided from the following parameters: the test positivity rate in a screening population, the true-positive and false-positive rates, and the number needed to colonoscope to detect a target lesion. CONCLUSIONS: New screening tests can be evaluated efficiently by this stepwise comparative approach.

USPSTF colorectal cancer screening guidelines: an extended look at multi-year interval testing.

OBJECTIVES: The US Preventive Services Task Force (USPSTF) released draft recommendations regarding colorectal cancer (CRC) screening in October 2015. Despite evidence that annual fecal blood testing test use is uncommon in screen eligible adults, with only 10.4% reporting the use of such a test in 2012, and features poor adherence over time, the USPSTF recommended only 3 noninvasive screening strategy options, all including annual fecal occult blood testing: 1) annual fecal immunochemical test (FIT) alone; 2) annual FIT in combination with flexible sigmoidoscopy every 10 years; and 3) annual high-sensitivity fecal occult blood test (hsFOBT). Mt-sDNA is the only FDA-approved CRC screening test, is covered by Medicare every 3 years, and is included as an every-3-year (3y) option in the American Cancer Society guidelines. We demonstrate that USPSTF modeling includes an embedded sensitivity analysis of less frequent than annual test adherence, which provides support for the inclusion of mt-sDNA 3y as a recommended test. STUDY DESIGN: A descriptive analysis of USPSTF modeling of the clinical impact of various stool based CRC screening strategies. METHODS: We analyzed the data generated by the USPSTF CRC screening models describing the impact of noninvasive CRC screening strategies on CRC incidence, CRC related mortality, life years gained (LYG), colonoscopy volume and associated complication, test efficiency (a measure of benefits (LYG) and harms (colonoscopies generated), and identified strategies that provide 90% or more of the LYG by screening with colonoscopy every 10 years. We compared mt-sDNA at 3y intervals and FIT and hsFOBT at 2-year (2y) and 3y intervals and did not consider annual testing. RESULTS: We found that only mt-sDNA 3y, FIT 2y, and FIT 3y were within 98% of the efficiency frontier. However, only mt-sDNA 3y generates more than 90% of the life-years gained with screening colonoscopy. These results meet the USPSTF criteria for a recommendation for mt-sDNA 3y for routine screening. CONCLUSIONS: Given poor adherence to annual testing, any screening opportunity with a test, such as mt-sDNA, that has high sensitivity for CRC and for the most significant precancerous lesions would be an important screening option for patients for maximizing screening effectiveness in reducing CRC incidence and mortality.

HIV-specific Immunity Derived From Chimeric Antigen Receptor-engineered Stem Cells.

The human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) response is critical in controlling HIV infection. Since the immune response does not eliminate HIV, it would be beneficial to develop ways to enhance the HIV-specific CTL response to allow long-term viral suppression or clearance. Here, we report the use of a protective chimeric antigen receptor (CAR) in a hematopoietic stem/progenitor cell (HSPC)-based approach to engineer HIV immunity. We determined that CAR-modified HSPCs differentiate into functional T cells as well as natural killer (NK) cells in vivo in humanized mice and these cells are resistant to HIV infection and suppress HIV replication. These results strongly suggest that stem cell-based gene therapy with a CAR may be feasible and effective in treating chronic HIV infection and other morbidities.

Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector.

We described earlier a dual-combination anti-HIV type 1 (HIV-1) lentiviral vector (LVsh5/C46) that downregulates CCR5 expression of transduced cells via RNAi and inhibits HIV-1 fusion via cell surface expression of cell membrane-anchored C46 antiviral peptide. This combinatorial approach has two points of inhibition for R5-tropic HIV-1 and is also active against X4-tropic HIV-1. Here, we utilize the humanized bone marrow, liver, thymus (BLT) mouse model to characterize the in vivo efficacy of LVsh5/C46 (Cal-1) vector to engineer cellular resistance to HIV-1 pathogenesis. Human CD34+ hematopoietic stem/progenitor cells (HSPC) either nonmodified or transduced with LVsh5/C46 vector were transplanted to generate control and treatment groups, respectively. Control and experimental groups displayed similar engraftment and multilineage hematopoietic differentiation that included robust CD4+ T-cell development. Splenocytes isolated from the treatment group were resistant to both R5- and X4-tropic HIV-1 during ex vivo challenge experiments. Treatment group animals challenged with R5-tropic HIV-1 displayed significant protection of CD4+ T-cells and reduced viral load within peripheral blood and lymphoid tissues up to 14 weeks postinfection. Gene-marking and transgene expression were confirmed stable at 26 weeks post-transplantation. These data strongly support the use of LVsh5/C46 lentiviral vector in gene and cell therapeutic applications for inhibition of HIV-1 infection.

CD4 ligation on human blood monocytes triggers macrophage differentiation and enhances HIV infection.

UNLABELLED: A unique aspect of human monocytes, compared to monocytes from many other species, is that they express the CD4 molecule. However, the role of the CD4 molecule in human monocyte development and function is not known. We determined that the activation of CD4 via interaction with major histocompatibility complex class II (MHC-II) triggers cytokine expression and the differentiation of human monocytes into functional mature macrophages. Importantly, we determined that CD4 activation induces intracellular signaling in monocytes and that inhibition of the MAPK and Src family kinase pathways blocked the ability of CD4 ligation to trigger macrophage differentiation. We observed that ligation of CD4 by MHC-II on activated endothelial cells induced CD4-mediated macrophage differentiation of blood monocytes. Finally, CD4 ligation by MHC-II increases the susceptibility of blood-derived monocytes to HIV binding and subsequent infection. Altogether, our studies have identified a novel function for the CD4 molecule on peripheral monocytes and suggest that a unique set of events that lead to innate immune activation differ between humans and mice. Further, these events can have effects on HIV infection and persistence in the macrophage compartment. IMPORTANCE: The CD4 molecule, as the primary receptor for HIV, plays an important role in HIV pathogenesis. There are many cell types that express CD4 other than the primary HIV target, the CD4(+) T cell. Other than allowing HIV infection, the role of the CD4 molecule on human monocytes or macrophages is not known. We were interested in determining the role of CD4 in human monocyte/macrophage development and function and the potential effects of this on HIV infection. We identified a role for the CD4 molecule in triggering the activation and development of a monocyte into a macrophage following its ligation. Activation of the monocyte through the CD4 molecule in this manner increases the ability of monocytes to bind to and become infected with HIV. Our studies have identified a novel function for the CD4 molecule on peripheral monocytes in triggering macrophage development that has direct consequences for HIV infection.

Meta-analyses of colorectal cancer risk factors.

PURPOSE: Demographic, behavioral, and environmental factors have been associated with increased risk of colorectal cancer (CRC). We reviewed the published evidence and explored associations between risk factors and CRC incidence. METHODS: We identified 12 established non-screening CRC risk factors and performed a comprehensive review and meta-analyses to quantify each factor's impact on CRC risk. We used random-effects models of the logarithms of risks across studies: inverse-variance weighted averages for dichotomous factors and generalized least squares for dose-response for multi-level factors. RESULTS: Significant risk factors include inflammatory bowel disease (RR = 2.93, 95 % CI 1.79-4.81); CRC history in first-degree relative (RR = 1.80, 95 % CI 1.61-2.02); body mass index (BMI) to overall population (RR = 1.10 per 8 kg/m(2) increase, 95 % CI 1.08-1.12); physical activity (RR = 0.88, 95 % CI 0.86-0.91 for 2 standard deviations increased physical activity score); cigarette smoking (RR = 1.06, 95 % CI 1.03-1.08 for 5 pack-years); and consumption of red meat (RR = 1.13, 95 % CI 1.09-1.16 for 5 servings/week), fruit (RR = 0.85, 95 % CI 0.75-0.96 for 3 servings/day), and vegetables (RR = 0.86, 95 % CI 0.78-0.94 for 5 servings/day). CONCLUSIONS: We developed a comprehensive risk modeling strategy that incorporates multiple effects to predict an individual's risk of developing CRC. Inflammatory bowel disease and history of CRC in first-degree relatives are associated with much higher risk of CRC. Increased BMI, red meat intake, cigarette smoking, low physical activity, low vegetable consumption, and low fruit consumption were associated with moderately increased risk of CRC.

Global quantitative assessment of the colorectal polyp burden in familial adenomatous polyposis by using a web-based tool.

BACKGROUND: Accurate measures of the total polyp burden in familial adenomatous polyposis (FAP) are lacking. Current assessment tools include polyp quantitation in limited-field photographs and qualitative total colorectal polyp burden by video. OBJECTIVE: To develop global quantitative tools of the FAP colorectal adenoma burden. DESIGN: A single-arm, phase II trial. PATIENTS: Twenty-seven patients with FAP. INTERVENTION: Treatment with celecoxib for 6 months, with before-treatment and after-treatment videos posted to an intranet with an interactive site for scoring. MAIN OUTCOME MEASUREMENTS: Global adenoma counts and sizes (grouped into categories: <2 mm, 2-4 mm, and >4 mm) were scored from videos by using a novel Web-based tool. Baseline and end-of-study adenoma burden results were summarized by using 5 models. Correlations between pairs of reviewers were analyzed for each model. RESULTS: Interobserver agreement was high for all 5 measures of polyp burden. Measures that used both polyp count and polyp size had better interobserver agreement than measures based only on polyp count. The measure in which polyp counts were weighted according to diameter, calculated as (1) × (no. of polyps <2 mm) + (3) × (no. of polyps 2-4 mm) + (5) × (no. of polyps >4 mm) had the highest interobserver agreement (Pearson r = 0.978 for two gastroenterologists, 0.786 and 0.846 for the surgeon vs each gastroenterologist). Treatment reduced the polyp burden by these measurements in 70% to 89% of patients (P < .001). LIMITATIONS: Phase II study. CONCLUSION: This novel, Web-based polyp scoring method provides a convenient and reproducible way to quantify the global colorectal adenoma burden in FAP patients and a framework for developing a clinical staging system for FAP.

Using the BLT humanized mouse as a stem cell based gene therapy tumor model.

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) (1, 2) or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) (3). Both models were mainly utilized for the study of HIV infection. One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant (4). The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency (5-8). We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC (7, 9). This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9). The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (http://jaxmice.jax.org/research/immunology/005557-housing.html).

In vivo suppression of HIV by antigen specific T cells derived from engineered hematopoietic stem cells.

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.

The APC and PreSAP trials: a post hoc noninferiority analysis using a comprehensive new measure for gastrointestinal tract injury in 2 randomized, double-blind studies comparing celecoxib and placebo.

BACKGROUND: Previous gastrointestinal (GI) outcomes of nonsteroidal anti-inflammatory drug (NSAID) trials have focused on upper GI events, although recent evidence suggests NSAID-related lower GI effects are important and clinically relevant. OBJECTIVE: We assessed the long-term GI adverse event (AE) profile of celecoxib in a nonarthritis population. The aim of this post hoc analysis was to determine the incidence of serious GI AEs, using a new Clinically Significant Upper and/or Lower GI Events end point. METHODS: Patients from 2 colorectal adenoma recurrence studies were included. Patients received celecoxib 200 mg/400 mg BID, 400 mg once daily, or placebo over 3 years. The analysis measured noninferiority, using a prespecified definition of noninferiority. Celecoxib was predefined to be noninferior to placebo if the upper limit of the 95% CI for the hazard ratio (HR) with celecoxib was <1.25, at any dose, compared with the placebo (calculated using the Cox proportional hazards model). RESULTS: A total of 3588 patients were included; in the primary analysis, the HR for celecoxib (any dose) compared with placebo was 1.22 (95% CI: 0.69-2.18; P = 0.4948). In the secondary dose analyses, the HR associated with a 400-mg daily dose, compared with placebo, was 1.04 (95% CI: 0.55-1.96; P = 0.9149); for 800 mg/d, the HR was 1.79 (95% CI: 0.82-3.89; P = 0.1427). In a third covariate analysis, low-dose aspirin use (HR = 2.33; 95% CI: 1.33-4.08) and age ≥65 years (HR = 1.82; 95% CI, 1.05-3.15) was suggested to have a statistically significant association with increased risk of GI AEs. Study limitations include retrospective evaluation and small sample size of patients with GI AEs. CONCLUSIONS: The noninferiority of celecoxib to placebo was not established because the HR for the time to the first Clinically Significant Upper and/or Lower GI Event was greater than the prespecified upper limit of 95% CI for noninferiority. In addition, HRs associated with daily doses of 400 or 800 mg celecoxib compared with placebo were not significant. However, a significantly increased risk of clinically significant upper and/or lower GI events was observed in low-dose aspirin users (≤162.5 mg average daily use) and in patients ≥65 years of age.

Five-year analysis of the prevention of colorectal sporadic adenomatous polyps trial.

OBJECTIVES: Subjects in the Prevention of Colorectal Sporadic Adenomatous Polyps (PreSAP) trial (PRESAP/NCT00141193/www.clinicaltrials.gov) were studied to determine efficacy and safety at a year 5 assessment. METHODS: In this randomized, placebo-controlled, double-blind trial, 1,561 subjects with diagnosed colorectal adenomas removed within 3 months of the study's initiation were assessed after ~ 3 years on celecoxib followed by 2 years off. Studied in 107 primary and secondary care settings, subjects were stratified by cardioprotective aspirin use and randomized to receive orally 400 ng celecoxib (933 subjects) or placebo (628 subjects) once daily. Efficacy was measured by colonoscopy at years 1, 3, and 5, and safety was measured by investigators for the on-treatment period and collected by subject self-report over 2 years post-treatment. RESULTS: At year 5, the primary outcome measure was the rate of new adenomas measured cumulatively from baseline. This rate was statistically significantly lower in the celecoxib group (51.4%) than in the placebo group (57.5%; P<0.001). Similarly, the cumulative rate of new advanced adenomas was significantly lower in the celecoxib group (10.0%) than in the placebo group (13.8%; P=0.007). However, the year 5 interval measure, which was not cumulative and did not take the rates of previous years into account, showed that after 2 years off treatment, the celecoxib group (27.0%) was 1.66 times more likely to have new adenomas than the placebo group (16.3%; P<0.0001). Similarly, the percentage of patients with new advanced adenomas was significantly higher in the celecoxib group (5.0%) than in the placebo group (3.8%) (P=0.0072). The evaluation of safety from baseline through year 5 indicated that the risks of serious cardiac disorders (relative risk (RR) 1.66; 95% confidence interval (CI) 1.01-2.73), selected renal/hypertension events (RR 1.35; 95% CI 1.09-1.68), and general vascular (RR 1.34; 95% CI 1.08-1.68) and cardiac disorders (RR 1.59; 95% CI 1.12-2.26) were higher in those taking celecoxib than in those on placebo. CONCLUSIONS: The year 5 cumulative measures of the incidence of new and advanced adenomas were significantly lower in the celecoxib group than in the placebo group, but the year 5 interval rates of these measures were significantly lower in the placebo group than the celecoxib group, perhaps suggesting a release of cyclooxygenase-2 inhibition. Consistent with what has been previously reported, increased risk of renal/hypertension events and cardiac disorders associated with celecoxib therapy mandates caution in patient selection.

Sensitive digital quantification of DNA methylation in clinical samples.

Analysis of abnormally methylated genes is increasingly important in basic research and in the development of cancer biomarkers. We have developed methyl-BEAMing technology to enable absolute quantification of the number of methylated molecules in a sample. Individual DNA fragments are amplified and analyzed either by flow cytometry or next-generation sequencing. We demonstrate enumeration of as few as one methylated molecule in approximately 5,000 unmethylated molecules in DNA from plasma or fecal samples. Using methylated vimentin as a biomarker in plasma samples, methyl-BEAMing detected 59% of cancer cases. In early-stage colorectal cancers, this sensitivity was four times more than that obtained by assaying serum-carcinoembryonic antigen (CEA). With stool samples, methyl-BEAMing detected 41% of cancers and 45% of advanced adenomas. In addition to diagnostic and prognostic applications, this digital quantification of rare methylation events should be applicable to preclinical assessment of new epigenetic biomarkers and quantitative analyses in epigenetic research.