Background Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients. Methods DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms. Results The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9). Conclusions Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples. Trial Registration: Test set study-ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

BACKGROUND:Screening for colorectal cancer (CRC) is effective at reducing mortality, but nearly 35% of eligible patients do not get screened. New noninvasive screening methods may help increase CRC screening participation. Current CRC screening methods include blood-based screening with methylated Septin 9 (SEPT9) DNA (Epi proColon), stool-based screening with fecal immunochemical testing (FIT), and the multianalyte fecal test combining FIT and stool DNA (Cologuard).OBJECTIVES:To estimate the cost and clinical implications to health plans, including the clinical and fiscal implications of the use of blood-based screening with SEPT9 DNA, FIT, and FIT/stool DNA, for patients who are unwilling or unable to undergo other recommended screening methods, and to quantify the clinical and fiscal impacts on health plans of expanding CRC screening participation from today's level of 65% up to 80%.METHODS:We designed a simulation model to estimate the 3-year clinical and economic impacts for noninvasive screening scenarios and for no screening in the screening-nonadherent population. Clinical inputs were derived from SEPT9, FIT, and FIT/stool DNA validation studies in the peer-reviewed literature, the US census, and other sources in the peer-reviewed literature. We modeled a population of 1 million covered lives (aged 0-64 years) in a hypothetical health plan to estimate CRC, advanced adenoma, and nonadvanced adenoma diagnoses for different screening scenarios. We also modeled the expenditures related to screening, diagnostic follow-up, and treatment costs for CRC for a 15% increase (34,800 members) to 80% screening over the course of 3 years.RESULTS:In the health plan population, 232,000 members aged 50 to 64 years were eligible for screening, of whom 81,200 (35%) were unscreened. The number of cases of CRC that were detected was similar for each screening scenario, including 221 for SEPT9, 216 for FIT, and 193 for FIT/stool DNA versus 49 for no screening. The 3-year per-member per-month (PMPM) cost impact for screening versus no screening and the evaluation of positive tests for the scenarios was $0.67 for SEPT9, $0.33 for FIT, and $0.69 for FIT/stool DNA. Including the treatment costs for CRC, the PMPM costs increased to $1.08, $0.71, and $0.98, respectively.CONCLUSIONS:Our simulation model suggests that similar clinical detection rates are achievable with the 3 noninvasive blood- and stool-based screening methods. These results support a role for blood- and stool-based screening to increase participation in CRC screening.

Screening for colorectal cancer is widely considered a cost effective intervention with strong evidence supporting mortality reduction in the screened population. Despite this, global screening rates remain low, even in developed countries. Blood based screening tests have the potential to overcome resistance to screening, thereby increasing overall participation, and saving lives. Here we review the development of a recently US FDA approved blood based epigenetic screening test for colorectal cancer. The Epi proColon test is based on the Septin 9 (SEPT9) gene promoter methylation status measured in cell free DNA from plasma. While the assessment of DNA methylation has been practiced in laboratories for some time, the approved test provides a standardized and kitted method for isolation of cell free DNA from plasma, reagents and methods for bisulfite conversion and purification of converted DNA (bisDNA) in preparation for DNA methylation analysis by real time PCR. This enables broad dissemination of DNA methylation based testing to laboratories approved to perform standard molecular diagnostics. In clinical trials, the approved Epi proColon test had sensitivity for colorectal cancer of 68-72%, comparable to commonly used stool tests, at a specificity of 80-82%. The test was well received, with 99.5% of patients in a participation trial agreeing to testing, and of these, when the test was positive, 67% scheduled colonoscopy. Finally, 59% of patients who had a colonoscopy in this study had a finding requiring polypectomy or biopsy. Based on these data, the test was approved as a screening test in the US, for patients who are otherwise unscreened, a key advance for molecular diagnostics applications in the field of clinical epigenetics.

Colorectal cancer screening programs seek to maintain patient adherence to repeated tests over the duration of patients’ screening eligibility. This study compares uptake of an experimental blood test with that of a FIT for colorectal cancer screening in an office setting. This small two-site randomized controlled trial (ClinicalTrials.gov, ID NCT02251782) assigned 413 average-risk adults overdue for screening in an integrated health system to one of two arms: Offer of a FIT versus offer of a blood test for colorectal cancer screening. The primary outcome was completion of the offered test within six weeks. Secondary outcomes were the proportion of participants with positive test results, and the proportion of participants with positive results who completed colonoscopy within 3 months of referral. 99.5% (CI95: 97.3%-100%) of participants in the blood test arm and 88.1% (CI95: 83.0% − 91.8%) of participants in the FIT arm completed the offered test, a difference of 11.4% (CI95: 6.9% − 15.9%, p<.001). 16.5% of participants in the blood test arm and 1.7% of participants in the FIT arm had a positive test. 20 of 30 and 2 of 3 in the blood test and FIT arms, respectively, either scheduled or completed a colonoscopy within 3 months of the positive test result. Uptake of a blood test was higher than that of a FIT among participants in a randomized controlled trial. A blood test may be a useful option for those hesitant to screen with currently available tests.

e15072 Background: Colonoscopy and fecal tests are effective at reducing colorectal cancer (CRC) related mortality, yet nearly 35% of those eligible are not screened, in part due to aversion to the test procedures. Accordingly, new blood-based methods may have a role in reducing the unscreened proportion of the population. The objective of this study was to estimate the potential clinical and fiscal impacts of adding blood-based CRC screening with methylated Septin9 DNA (hereafter ‘Septin9’) to reduce the unscreened proportion to 20%, a goal set by the American Cancer Society. Methods: We developed a simulation model to estimate 3 year health outcomes with and without availability of Septin9 in a commercial health plan population (age < 65) with 1m enrollees. The base case compares current U.S. screening distributions (53% colonoscopy, 12% fecal testing, 35% not screened) vs a scenario where Septin9 is covered and used by an additional 15% of the unscreened. Age distributions and CRC incidence were derived from the US census and SEER, respectively. Screening test performance was based on validation studies of Septin9. We included costs of the screening tests, diagnostic colonoscopy, pathologic evaluation of positive colonoscopy findings, and stage-specific treatment of CRC. We calculated the number of expected advanced adenomas (AA), non-advanced adenomas (NAA), and CRC cases in each scenario, as well as total and per-member per-year (PMPY) cost—a measure of health plan expenditure adjusted for plan size and duration of cost impacts. Results: Over a 3-year time horizon, use of Septin9 by 7600 previously unscreened plan members resulted in 4000 more diagnostic colonoscopies, 1900 more pathologic evaluations, 350 more AA findings, 1300 more NAA findings, and 65 more CRC cases. Additional health plan expenditures totaled $18m (PMPY = $6). For reference, a recent study estimated that increasing the colonoscopy screened proportion from 50% to 70% would cost $11 PMPY. Conclusions: Blood-based testing has the potential to improve CRC screening rates and improve cancer and adenoma detection. The fiscal impact of blood-based testing to reach unscreened individuals is similar to other accepted screening tests.

Background: Twenty percent of advanced gastric cancers are HER2-positive.Trastuzumab(Tmab), a monoclonal antibody that interferes with the HER2 receptor, combined with cisplatin and a fluoropyrimidine has been shown to increase survival in HER2-positive gastric cancer.Tmab induces reactive oxygen species (ROS) in lung adenocarcinoma in vitro, and may have similar effects in other cancer types as well.CD44 variant 9 (CD44v9) expression is associated with a poor prognosis in gastric adenocarcinomas.The CD44v9 isoform interacts with and stabilizes xCT, a glutamate-cystine transporter, resulting in increased intracellular levels of reduced glutathione (GSH), and increased defense against ROS.The relationship between CD44v9 expression and HER2 status in the response of gastric cancer to various treatments, including Tmab has not been reported.The present study was conducted to investigate the role of CD44v9 in the effectiveness of Tmab treatment on gastric adenocarcinomas.We hypothesize that CD44v9 causes resistance to Tmab via induction of ROS resistance, and targeting CD44v9 may increase effectiveness of Tmab therapy.Methods: NCI-N87(high HER2), a gastric cancer cell line, was used for this study.Anti-HER2 siRNA was used to knockdown HER2 expression in NCI-N87 cells.The pRc/CMV expressing plasmid encoding human CD44v9 was transfected into NCI-N87 cells (CD44v9-NCI-N87).N-acetyl cysteine (NAC) (15mM) was used as a ROS scavenger for 72h in vitro.Cells were incubated for 72h with different concentrations of Tmab (a range of doses between 0, 10, 50 μg/ml), and cell viability was determined by MTS assay.Intracellular GSH levels were measured by luminescent based assay.Intracellular ROS were measured by flow cytometry with CM-H2DCFDA.Cells were cultured for 24h with different concentrations of Tmab (a range of doses between 0, 40, 80, 120, 200 μg/ml) in this assay.Results: Tmab caused a dose-dependent decrease in cell viability in NCI-N87 cells(10μg/ml 88.2%(p=0.015),50μg/ml 73.3%(p<0.001)),but did not in anti-HER2 siRNA treated NCI-N87(10μg/ml 99.0%(p=0.879),50μg/ml 93.0%(p= 0.495) and CD44v9-NCI-N87(10μg/ml 104.4%(p=0.442),50μg/ml 97.3%(p=0.623)).Tmab increased intracellular ROS level in NCI-N87 cells (p=0.007), but did not in anti-HER2 siRNA treated NCI-N87 and CD44v9-NCI-N87.Intracellular GSH levels were significantly higher in CD44v9-NCI N87 than NCI-N87 (p=0.004).NAC decreased Tmab-induced cell death in NCI-N87.These data indicated that CD44v9 is associated with increased intracellular GSH level, and may protect against Tmab induced ROS damage and cell death.Conclusion: Knock down of CD44v9 expression in HER2 positive gastric cells restores Tmab sensitivity through the restoration of ROS mediated cell death.

BACKGROUND:Despite strong recommendations for colorectal cancer (CRC) screening, participation rates are low. Understanding factors that affect screening choices is essential to developing future screening strategies. Therefore, this study assessed patient willingness to use non-invasive stool or blood based screening tests after refusing colonoscopy.METHODS:Participants were recruited during regular consultations. Demographic, health, psychological and socioeconomic factors were recorded. All subjects were advised to undergo screening by colonoscopy. Subjects who refused colonoscopy were offered a choice of non-invasive tests. Subjects who selected stool testing received a collection kit and instructions; subjects who selected plasma testing had a blood draw during the office visit. Stool samples were tested with the Hb/Hp Complex Elisa test, and blood samples were tested with the Epi proColon® 2.0 test. Patients who were positive for either were advised to have a diagnostic colonoscopy.RESULTS:63 of 172 subjects were compliant to screening colonoscopy (37%). 106 of the 109 subjects who refused colonoscopy accepted an alternative non-invasive method (97%). 90 selected the Septin9 blood test (83%), 16 selected a stool test (15%) and 3 refused any test (3%). Reasons for blood test preference included convenience of an office draw, overall convenience and less time consuming procedure.CONCLUSIONS:97% of subjects refusing colonoscopy accepted a non-invasive screening test of which 83% chose the Septin9 blood test. The observation that participation can be increased by offering non-invasive tests, and that a blood test is the preferred option should be validated in a prospective trial in the screening setting.

Publisher Summary The human genome contains four bases—guanine, adenine, thymine, and cytosine. The cytosines can be either methylated or unmethylated at the fifth carbon position in the pyrimidine ring. In general, they can only be methylated when they are in the context of a CpG dinucleotide that involves a cytosine immediately followed by a guanine. The methylation status of a CpG island is correlated with the chromatin structure and expression levels of nearby genes. CpG islands associated with actively transcribed genes are typically unmethylated. When a CpG island is methylated, methyl-CpG-binding domain proteins recognize the methylated CpG and recruit the necessary factors for chromatin condensation and gene inactivation. This DNA methylation state is maintained during cell division by a family of enzymes called DNA methyltransferases. Cancer was viewed as an accumulation of chromosomal aberrations and, therefore, called a “genetic disease.” However, it has become clear over time that epigenetic changes play a crucial role in carcinogenesis. While attention is focused on methylation in carcinogenesis, a similar groundswell of research is emerging on methylation in other diseases, especially autoimmune and cardiovascular conditions.

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a survival rate of over 90% when detected early. Currently, less than half of guideline eligible individuals are screened regularly for CRC, highlighting a need for improved non-invasive methods of early detection. We previously identified and validated a blood-based biomarker, Septin 9, which identifies methylated DNA in plasma from all stages of CRC. We have now conducted a genome-wide screen to identify additional blood-based biomarkers that either augment the performance of Septin 9 for CRC detection or perform individually as well as Septin 9. Methods: Differential methylation hybridization (DMH) via customized high-density microarrays with >50,000 features was used to explore methylation differences between Septin 9 (-) CRC (n = 13), Septin 9 (+) CRC (n = 14), normal adjacent to tumor (NAT, n = 11) and whole blood (n = 8) DNA. Significant probesets were identified using a Benjamin-Hochberg false discovery rate of 0.25 and ranked by t statistic. The top 65 DMH and 62 literature candidates were chosen for real-time PCR assay development and validated using tissues from 51 Septin 9 (-) CRCs, 147 Septin 9 (+) CRCs, 8 NATs and 14 normal colon epithelia in addition to 94 other disease and normal tissues. Ten of these candidates were chosen for HMO assay development and the performances of the four most promising candidates were evaluated in plasma from 136 CRC patients and 170 colonoscopy-verified controls using a case-control study design. Results: 7026 statistically significant probesets for all CRC tissues versus NAT and whole blood and 7280 significant probesets for Septin 9 (+) CRC versus Septin 9 (-) CRC were identified. Comparison of the top 150 candidates for Septin 9 (-) CRC versus whole blood and versus Septin 9 (+) CRC yielded 224 unique candidates. Results of real-time PCR analysis validated 26 of 45 biomarker candidates, with significant overlap between those CRC tissues identified by Septin 9 and those identified by the new candidates (all Spearman R values > 0.48). Using HM real-time PCR analysis in DNA extracted from plasma, Septin 9 detected 85/136 CRC patients (63%) and 27/170 controls (16%). The four new biomarkers detected from 54/136 CRC patients (40%) to 74/136 CRC patients (54%) and from 18/170 controls (11%) to 42/170 controls (25%). Conclusions: By using a systematic screen, we identified 26 biomarkers that specifically discriminated CRC from normal colon tissues, although complementarity to Septin 9 was limited. Plasma-based, highly sensitive real-time PCR assays were successfully developed for four of these biomarkers. All biomarkers identified a similar subset of CRC patient plasmas, with slight variations in sensitivity and specificity. Septin 9 remains the best biomarker for maximizing sensitivity and specificity on plasma DNA. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1326.

Introduction: The low sensitivity of the fecal occult blood tests (FOBT) based on the guaiac have made to develop new immunochemical tests with an efficiency to detect advanced neoplasia (advanced adenoma and colorectal cancer) still not well established. Aim: Determine the sensitivity and specificity of a FOBT based on the guaiac (FOBTg) with a quantitative immunochemical FOBT (FOBTi) to detect advanced neoplasia. Material and methods: Patients: all individuals with a colonoscopy programmed by any cause (screening, surveillance or symptoms) were included. Individuals with personal history of inflammatory bowel diseases or colorectal cancer were excluded. During the days previous to the exploration three high sensitivity FOBTg and two FOBTi (positive test ≥100ng/ml) were completed. The sample was calculated for demonstrating the superiority of the FOBTi of 15% of sensitivity with a final sample of 700 individuals. Results We presented the results of the first 140 individuals. The colonoscopy was performed for symptoms in 66% of cases and for screening or surveillance in 34% of cases. The colonoscopy detected 16 individuals (11%) with an advanced neoplasia (2 infiltrant carcinoma and 14 advanced adenomes). Other finds were: 20 patients with non advanced adenomes, 11 with polyps not adenomatous and 4 inflammatory colitis. The colonoscopy was normal in 88 (63%) individuals. The percentage of positive test was from 3,5% (5/140) and 16,5% (23/140) for the FOBTg and FOBTi, respectively. The sensitivity, specificity, positive predictive value and negative predictive value to detect an advanced neoplasia were 12%, 97%, 40% and 89% with the FOBTg and of 62,5%, 89%, 60,8%, 81% for the FOBTi. If considered only the first immunochemical test, the sensitivity, specificity, positive predictive value and negative predictive value were 43,7%, 92,7%, 43,7%, 92,7%, respectively. Conclusion The FOBTi is very superior to the FOBTg to detect advanced neoplasia although the high number of false positives could limit its use. The utilization of one or two FOBTi should be appraised depending on the disposable endoscopic resources.

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.

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 ∼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.

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.