Estimating drug concentration-response relationships by applying causal inference methods for continuous point exposures and time-to-event outcomes.

In clinical development, it is useful to characterize the causal relationship between individual drug concentrations and clinical outcomes in large phase III trials of new therapeutic agents because it can provide insights on whether increasing the currently administered drug dose may lead to better outcomes. However, estimating causal effects of drug concentration is complicated by the fact that drug concentration is a continuous measure and it is usually influenced by patient-level prognostic characteristics such as body weight and sex. In this article, we compare two approaches to estimate causal effects of continuous point exposures on time-to-event outcomes: (a) outcome regression (OR) and (b) weighting. In particular, we make the first direct comparison of the balancing weights, inverse probability weighting and OR methods for estimating the effects of continuous exposures on time-to-event outcomes in simulations and demonstrate that these methods can exhibit markedly different behaviours that subsequently lead to a change in the conclusions. To improve weighted exposure effect estimators, we also propose a new simple-to-apply diagnostic to detect when such estimators might be subject to severe bias, and demonstrate its effectiveness in simulations. Finally, we apply these methods to an example of multiple sclerosis drug development by providing causal effect estimates of average ocrelizumab concentrations on time-to-event disability progression outcomes.

Blood neurofilament light levels predict non-relapsing progression following anti-CD20 therapy in relapsing and primary progressive multiple sclerosis: findings from the ocrelizumab randomised, double-blind phase 3 clinical trials.

BACKGROUND: Neurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear. METHODS: We examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models. FINDINGS: In persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS. INTERPRETATION: Highly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression. FUNDING: F. Hoffmann-La Roche Ltd.

Association of Higher Ocrelizumab Exposure With Reduced Disability Progression in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Ocrelizumab improved clinical and MRI measures of disease activity and progression in three phase 3 multiple sclerosis (MS) studies. Post hoc analyses demonstrated a correlation between the ocrelizumab serum concentration and the degree of blood B-cell depletion, and body weight was identified as the most influential covariate on ocrelizumab pharmacokinetics. The magnitude of ocrelizumab treatment benefit on disability progression was greater in lighter vs heavier patients. These observations suggest that higher ocrelizumab serum levels provide more complete B-cell depletion and a greater delay in disability progression. The current post hoc analyses assessed population exposure-efficacy/safety relationships of ocrelizumab in patients with relapsing and primary progressive MS. METHODS: Patients in OPERA I/II and ORATORIO were grouped in exposure quartiles based on their observed individual serum ocrelizumab level over the treatment period. Exposure-response relationships were analyzed for clinical efficacy (24-week confirmed disability progression (CDP), annualized relapse rate [ARR], and MRI outcomes) and adverse events. RESULTS: Ocrelizumab reduced new MRI lesion counts to nearly undetectable levels in patients with relapsing or primary progressive MS across all exposure subgroups, and reduced ARR in patients with relapsing MS to very low levels (0.13-0.18). A consistent trend of higher ocrelizumab exposure leading to lower rates of CDP was seen (0%-25% [lowest] to 75%-100% [highest] quartile hazard ratios and 95% confidence intervals; relapsing MS: 0.70 [0.41-1.19], 0.85 [0.52-1.39], 0.47 [0.25-0.87], and 0.34 [0.17-0.70] vs interferon ß-1a; primary progressive MS: 0.88 [0.59-1.30], 0.86 [0.60-1.25], 0.77 [0.52-1.14], and 0.55 [0.36-0.83] vs placebo). Infusion-related reactions, serious adverse events, and serious infections were similar across exposure subgroups. DISCUSSION: The almost complete reduction of ARR and MRI activity already evident in the lowest quartile, and across all ocrelizumab-exposure groups, suggests a ceiling effect. A consistent trend of higher ocrelizumab exposure leading to greater reduction in risk of CDP was observed, particularly in the relapsing MS trials, and was not associated with a higher rate of adverse events. Higher ocrelizumab exposure may provide improved control of disability progression by reducing disease activity below that detectable by ARR and MRI, and/or by attenuating other B-cell-related pathologies responsible for tissue damage. CLASSIFICATION OF EVIDENCE: This analysis provides Class III evidence that higher ocrelizumab serum levels are related to greater reduction in risk of disability progression in patients with multiple sclerosis. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in the open-label extension. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT01247324 (OPERA I), NCT01412333 (OPERA II), and NCT01194570 (ORATORIO).

Recurrent disability progression endpoints in multiple sclerosis clinical trials.

BACKGROUND: The current standard endpoint to assess disability accumulation in multiple sclerosis (MS) clinical trials is the time to the first confirmed disability progression, which excludes subsequent progression events. Including recurrent progression events may permit a more comprehensive assessment of treatment effects on disability progression. OBJECTIVE: To propose a definition of recurrent disability progression events and to compare time-to-first and recurrent event analysis. METHODS: Recurrent disability progression events were defined by expanding the recommended first event definition. Marginal recurrent event methods (negative binomial model, Lin-Wei-Yang-Ying model) were compared with Cox regression in data from three randomized controlled trials in relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS), and in simulated randomized controlled trial data. RESULTS: The recurrent event analyses included a substantially larger number of progression events compared with the time-to-first-event analyses (+7.5% and +9.9% in the RMS trials and +22.7% in the PPMS trial). The increase in the number of events resulted in more precise treatment effect estimates and a corresponding gain in statistical power. CONCLUSION: Our results support the use of recurrent event data analysis, especially in progressive MS trials, to improve estimates of treatment effects, increase statistical power, and better capture the clinically meaningful long-term disability progression experience.

Risk of requiring a wheelchair in primary progressive multiple sclerosis: Data from the ORATORIO trial and the MSBase registry.

BACKGROUND AND PURPOSE: Reaching Expanded Disability Status Scale (EDSS) ≥7.0 represents the requirement for a wheelchair. Here we (i) assess the effect of ocrelizumab on time to EDSS ≥7.0 over the ORATORIO (NCT01194570) double-blind and extended controlled periods (DBP+ECP), (ii) quantify likely long-term benefits by extrapolating results, and (iii) assess the plausibility of extrapolations using an independent real-world cohort (MSBase registry; ACTRN12605000455662). METHODS: Post hoc analyses assessing time to 24-week confirmed EDSS ≥7.0 in two cohorts of patients with primary progressive multiple sclerosis (baseline EDSS 3.0-6.5) were investigated in ORATORIO and MSBase. RESULTS: In the ORATORIO DBP+ECP, ocrelizumab reduced the risk of 24-week confirmed EDSS ≥7.0 (hazard ratio = 0.54, 95% confidence interval [CI]: 0.31-0.92; p = 0.022). Extrapolated median time to 24-week confirmed EDSS ≥7.0 was 12.1 and 19.2 years for placebo and ocrelizumab, respectively (7.1-year delay [95% CI: -4.3 to 18.4]). In MSBase, the median time to 24-week confirmed EDSS ≥7.0 was 12.4 years. CONCLUSIONS: Compared with placebo, ocrelizumab significantly delayed time to 24-week confirmed wheelchair requirement in ORATORIO. The plausibility of the extrapolated median time to reach this milestone in the placebo group was supported by observed real-world data from MSBase. Extrapolated benefits for ocrelizumab over placebo could represent a truly meaningful delay in loss of ambulation and independence.

Ocrelizumab in relapsing and primary progressive multiple sclerosis: Pharmacokinetic and pharmacodynamic analyses of OPERA I, OPERA II and ORATORIO.

AIMS: Ocrelizumab is a humanized monoclonal antibody that selectively targets CD20-positive B cells and is indicated for treatment of patients with relapsing forms of multiple sclerosis (RMS) or primary progressive multiple sclerosis (PPMS). The pharmacokinetics and pharmacodynamics of ocrelizumab in patients with RMS or PPMS were assessed. METHODS: A population pharmacokinetic model was developed based on data from the Phase II study and the Phase III studies OPERA I and OPERA II in patients with RMS. Data from the ORATORIO Phase III study in patients with PPMS became available after model finalization and was used for external model evaluation. RESULTS: The ocrelizumab serum concentration vs time course was accurately described by a 2-compartment model with time-dependent clearance. Body weight was found to be the main covariate. The area under the concentration-time curve over the dosing interval was estimated to be 26% higher for patients with RMS weighing <60 kg and 21% lower for patients weighing >90 kg when compared with the 60-90 kg group. The terminal half-life of ocrelizumab was estimated as 26 days. The extent of B-cell depletion in blood, as the pharmacodynamic marker, was greater with increasing ocrelizumab exposure. CONCLUSION: The pharmacokinetics of ocrelizumab was described with pharmacokinetic parameters typical for an immunoglobulin G1 monoclonal antibody, with body weight as the main covariate. The pharmacokinetics and B-cell depletion in blood were comparable across the RMS and PPMS trials, and the extent of blood B-cell depletion was greater with higher exposure.

Five years of ocrelizumab in relapsing multiple sclerosis: OPERA studies open-label extension.

OBJECTIVE: To assess over 3 years of follow-up the effects of maintaining or switching to ocrelizumab (OCR) therapy on clinical and MRI outcomes and safety measures in the open-label extension (OLE) phase of the pooled OPERA: I/II studies in relapsing multiple sclerosis. METHODS: After 2 years of double-blind, controlled treatment, patients continued OCR (600 mg infusions every 24 weeks) or switched from interferon (IFN)-ß-1a (44 µg 3 times weekly) to OCR when entering the OLE phase (3 years). Adjusted annualized relapse rate, time to onset of 24-week confirmed disability progression (CDP)/improvement (CDP), brain MRI activity (gadolinium-enhanced and new/enlarging T2 lesions), and percentage brain volume change were analyzed. RESULTS: Of patients entering the OLE phase, 88.6% completed year 5. The cumulative proportion with 24-week CDP was lower in patients who initiated OCR earlier vs patients initially receiving IFN-ß-1a (16.1% vs 21.3% at year 5; p = 0.014). Patients continuing OCR maintained and those switching from IFN-ß-1a to OCR attained near complete and sustained suppression of new brain MRI lesion activity from years 3-5. Over the OLE phase, patients continuing OCR exhibited less whole brain volume loss from double-blind study baseline vs those switching from IFN-ß-1a (-1.87% vs -2.15% at year 5; p < 0.01). Adverse events were consistent with past reports and no new safety signals emerged with prolonged treatment. CONCLUSION: Compared with patients switching from IFN-ß-1a, earlier and continuous OCR treatment up to 5 years provided sustained benefit on clinical and MRI measures of disease progression. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that earlier and continuous treatment with OCR provided sustained benefit on clinical and MRI outcomes of disease activity and progression compared with patients switching from IFN-ß-1a. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in OLE. CLINICAL TRIAL IDENTIFIERS: NCT01247324/NCT01412333.

Contribution of Relapse-Independent Progression vs Relapse-Associated Worsening to Overall Confirmed Disability Accumulation in Typical Relapsing Multiple Sclerosis in a Pooled Analysis of 2 Randomized Clinical Trials.

Importance: Accumulation of disability in multiple sclerosis may occur as relapse-associated worsening (RAW) or steady progression independent of relapse activity (PIRA), with PIRA regarded as a feature of primary and secondary progressive multiple sclerosis. Objective: To investigate the contributions of relapse-associated worsening vs relapse-independent progression to overall confirmed disability accumulation (CDA) and assess respective baseline prognostic factors and outcomes of 2 treatments. Design, Setting, and Participants: Analyses occurred from July 2015 to February 2020 on pooled data from the intention-to-treat population of 2 identical, phase 3, multicenter, double-blind, double-dummy, parallel-group randomized clinical trials (OPERA I and II) conducted between August 2011 and April 2015. In the trials, patients with relapsing multiple sclerosis (RMS), diagnosed using the 2010 revised McDonald criteria, were randomized from 307 trial sites in 56 countries; resulting data were analyzed in the pooled data set. Interventions: Participants were randomized 1:1 to receive 600 mg of ocrelizumab by intravenous infusion every 24 weeks or subcutaneous interferon ß-1a 3 times a week at a dose of 44 µg throughout a 96-week treatment period. Main Outcomes and Measures: Confirmed disability accumulation was defined by an increase in 1 or more of 3 measures (Expanded Disability Status Scale, timed 25-ft walk, or 9-hole peg test), confirmed after 3 or 6 months, and classified per temporal association with confirmed clinical relapses (PIRA or RAW). Results: In the pooled OPERA I and II population (1656 of 2096 eligible participants), baseline demographics and disease characteristics were similar for patients randomized to interferon ß-1a vs ocrelizumab (mean [SD] age, 37.2 [9.2] vs 37.1 [9.2] years; 552 [66.6%] vs 541 women [65.4%]). After 96 weeks, 12-week composite CDA had occurred in 223 (29.6% by Kaplan-Meier estimate) randomized to interferon ß-1a and 167 (21.1%) randomized to ocrelizumab; 24-week composite CDA had occurred in 170 (22.7%) taking interferon ß-1a and 129 (16.2%) taking ocrelizumab. The PIRA events were the main contributors to 12-week and 24-week composite CDA after 96 weeks in patients treated with interferon ß-1a (174 of 223 [78.0%] and 137 of 170 [80.6%], respectively) and ocrelizumab (147 of 167 [88.0%] and 115 of 129 [89.1%], respectively); a minority had CDA explained by RAW events (69 of 390 [17.7%] and 52 of 299 [17.4%], respectively). Very few patients with composite CDA experienced both RAW and PIRA events (17 of 390 [4.4%] for 12-week and 15 of 299 [5.0%] for 24-week composite CDA). Ocrelizumab (vs interferon ß-1a) was associated with reduced risk of composite CDA (hazard ratio [HR], 0.67) and confirmed PIRA (HR, 0.78) and RAW (HR, 0.47) events. Conclusions and Relevance: Most disability accumulation in RMS is not associated with overt relapses. This indicates an underlying progression in this typical RMS population and challenges the current clinical distinction of relapsing and progressive forms of multiple sclerosis. Ocrelizumab was superior to interferon ß-1a in preventing both RAW and PIRA. Trial Registration: ClinicalTrials.gov Identifiers: OPERA I (NCT01247324) and OPERA II (NCT01412333).

Initiation of symptomatic medication in Alzheimer's disease clinical trials: Hypothetical versus treatment policy approach.

In clinical trials in populations with mild cognitive impairment, it is common for participants to initiate concurrent symptomatic medications for Alzheimer's disease after randomization to the experimental therapy. One strategy for addressing this occurrence is to exclude any observations that occur after the concurrent medication is initiated. The rationale for this approach is that these observations might reflect a symptomatic benefit of the concurrent medication that would adversely bias efficacy estimates for an effective experimental therapy. We interrogate the assumptions underlying such an approach by estimating the effect of newly prescribed concurrent medications in an observational study, the Alzheimer's Disease Neuroimaging Initiative.

Chronic white matter lesion activity predicts clinical progression in primary progressive multiple sclerosis.

Chronic active and slowly expanding lesions with smouldering inflammation are neuropathological correlates of progressive multiple sclerosis pathology. T1 hypointense volume and signal intensity on T1-weighted MRI reflect brain tissue damage that may develop within newly formed acute focal inflammatory lesions or in chronic pre-existing lesions without signs of acute inflammation. Using a recently developed method to identify slowly expanding/evolving lesions in vivo from longitudinal conventional T2- and T1-weighted brain MRI scans, we measured the relative amount of chronic lesion activity as measured by change in T1 volume and intensity within slowly expanding/evolving lesions and non-slowly expanding/evolving lesion areas of baseline pre-existing T2 lesions, and assessed the effect of ocrelizumab on this outcome in patients with primary progressive multiple sclerosis participating in the phase III, randomized, placebo-controlled, double-blind ORATORIO study (n = 732, NCT01194570). We also assessed the predictive value of T1-weighted measures of chronic lesion activity for clinical multiple sclerosis progression as reflected by a composite disability measure including the Expanded Disability Status Scale, Timed 25-Foot Walk and 9-Hole Peg Test. We observed in this clinical trial population that most of total brain non-enhancing T1 hypointense lesion volume accumulation was derived from chronic lesion activity within pre-existing T2 lesions rather than new T2 lesion formation. There was a larger decrease in mean normalized T1 signal intensity and greater relative accumulation of T1 hypointense volume in slowly expanding/evolving lesions compared with non-slowly expanding/evolving lesions. Chronic white matter lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in slowly expanding/evolving lesions and in non-slowly expanding/evolving lesion areas of pre-existing lesions predicted subsequent composite disability progression with consistent trends on all components of the composite. In contrast, whole brain volume loss and acute lesion activity measured by longitudinal T1 hypointense lesion volume accumulation in new focal T2 lesions did not predict subsequent composite disability progression in this trial at the population level. Ocrelizumab reduced longitudinal measures of chronic lesion activity such as T1 hypointense lesion volume accumulation and mean normalized T1 signal intensity decrease both within regions of pre-existing T2 lesions identified as slowly expanding/evolving and in non-slowly expanding/evolving lesions. Using conventional brain MRI, T1-weighted intensity-based measures of chronic white matter lesion activity predict clinical progression in primary progressive multiple sclerosis and may qualify as a longitudinal in vivo neuroimaging correlate of smouldering demyelination and axonal loss in chronic active lesions due to CNS-resident inflammation and/or secondary neurodegeneration across the multiple sclerosis disease continuum.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

BACKGROUND: A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome-wide search. METHODS: Real-time PCR was used to measure four DNA methylation biomarkers: GSTP1 and three previously unreported candidates associated with the genes RASSF2, HIST1H4K, and TFAP2E in sodium bisulfite-modified DNA. Matched plasma and urine collected prospectively from 142 patients referred for prostate biopsy and 50 young asymptomatic males were analyzed. RESULTS: Analysis of all biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers discriminated PCa from biopsy negative patients with AUCs ranging from 0.64 for HIST1H4K (95% CI 0.55-0.72, P < 0.00001) to 0.69 for GSTP1 (95% CI 0.60-0.77, P < 0.00001). All biomarkers showed minimal correlation with PSA. Multivariate analysis did not yield a panel that significantly improved performance over that of single biomarkers. All biomarkers showed greater sensitivity for PCa in urine than in plasma DNA. CONCLUSIONS: Analysis of the biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers provided information independent of PSA and may warrant inclusion in nomograms for predicting prostate biopsy outcome. The biomarkers' PCa sensitivity was greater for urine than plasma DNA. The biomarker performances in urine DNA should next be validated in formal training and test studies.

Circulating methylated SEPT9 DNA in plasma is a biomarker for colorectal cancer.

BACKGROUND: The presence of aberrantly methylated SEPT9 DNA in plasma is highly correlated with the occurrence of colorectal cancer. We report the development of a new SEPT9 biomarker assay and its validation in case-control studies. The development of such a minimally invasive blood-based test may help to reduce the current gap in screening coverage. METHODS: A new SEPT9 DNA methylation assay was developed for plasma. The assay comprised plasma DNA extraction, bisulfite conversion of DNA, purification of bisulfite-converted DNA, quantification of converted DNA by real-time PCR, and measurement of SEPT9 methylation by real-time PCR. Performance of the SEPT9 assay was established in a study of 97 cases with verified colorectal cancer and 172 healthy controls as verified by colonoscopy. Performance based on predetermined algorithms was validated in an independent blinded study with 90 cases and 155 controls. RESULTS: The SEPT9 assay workflow yielded 1.9 microg/L (CI 1.3-3.0) circulating plasma DNA following bisulfite conversion, a recovery of 45%-50% of genomic DNA, similar to yields in previous studies. The SEPT9 assay successfully identified 72% of cancers at a specificity of 93% in the training study and 68% of cancers at a specificity of 89% in the testing study. CONCLUSIONS: Circulating methylated SEPT9 DNA, as measured in the new (m)SEPT9 assay, is a valuable biomarker for minimally invasive detection of colorectal cancer. The new assay is amenable to automation and standardized use in the clinical laboratory.

Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

DNA methylation biomarkers for blood-based colorectal cancer screening.

BACKGROUND: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. METHODS: We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. RESULTS: Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). CONCLUSIONS: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

DNA methylation analysis as novel tool for quality control in regenerative medicine.

Cell-based regenerative medicine, including tissue engineering, is a novel approach to reconstituting tissues that do not spontaneously heal, such as damaged cartilage, and to curing diseases caused by malfunctioning cells. Typically, manufacturing processes to generate cartilage for replacement therapies involve isolation and expansion of cells from cartilage biopsies. A challenge in the field is potential contamination by other cell types (e.g., fibroblast-like cells), which can overgrow the desired cells during culturing and may ultimately compromise clinical efficacy. No standard analytical system has been absolutely effective in ensuring the identity of these cell-based products. Therefore, we tested deoxyribonucleic acid methylation analysis as a quality assessment tool, applying it to Genzyme's Carticel product, a chondrocyte implant that the Food and Drug Administration has approved. We identified 7 potent discriminators by assaying candidate genomic regions derived from methylation discovery approaches and literature searches regarding a functional role of genes in chondrocyte biology. Using a support vector machine, we trained an optimal cell type classifier that was absolutely effective in discriminating chondrocytes from synovial membrane derived cells, the major potential contaminant of chondrocyte cultures. The abundant marker availability and high quality of this assay format also suggest it as a potential quality control test for other cell types grown or manipulated in vitro.

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease.

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.

Aristaless-like homeobox-4 gene methylation is a potential marker for colorectal adenocarcinomas.

BACKGROUND & AIMS: The identification of novel genetic and epigenetic markers indicative of changes in the pathogenesis of colon cancer, along with easier-to-use, more sensitive assay methods, may improve the detection, treatment, and overall prognosis of this malignancy. METHODS: Using methylation-specific arbitrarily primed polymerase chain reaction, a fragment of the Aristaless-like homeobox-4 (ALX4) gene that was highly methylated in colon adenomas and cancer was identified. Methylation of ALX4 was analyzed in colorectal adenomas and cancers, in the liver metastases of patients with colorectal cancer, and in 61 other neoplasias, including gastric, esophageal, and hepatocellular cancer and cholangiocarcinoma. ALX4 methylation was also analyzed in the serum of 30 patients with colon cancer. RESULTS: ALX4 gene methylation was confirmed in colon adenomas (11/13) and more frequently present in primary colorectal cancers (30/47) compared with the normal colon mucosa (0/21) (P < .0001). In addition, ALX4 methylation was frequently observed in adenocarcinomas of the esophagus (12/14), stomach (11/15), and bile ducts (4/5) compared with all other cancers (P < .001). ALX4 gene methylation was also more frequently found in sera of patients with colon cancer compared with noncancer controls (P < .0001). Using a cutoff of 41.4 pg/mL, sensitivity and specificity were 83.3% and 70%, respectively. CONCLUSIONS: Apart from colon adenomas and primary and metastatic colorectal cancers, ALX4 is frequently methylated in adenocarcinomas of the gastrointestinal tract. ALX4 gene methylation in sera of patients with cancer may thus serve as a methylation-specific test for colon and other gastrointestinal cancers.

Association of DNA methylation of phosphoserine aminotransferase with response to endocrine therapy in patients with recurrent breast cancer.

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor-positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor-positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor-positive patients with recurrent breast cancer.