Cell-free circulating tumour-derived DNA (ctDNA) can be detected by testing for methylated BCAT1 and IKZF1 DNA, which has proven sensitivity for colorectal cancer (CRC). A prospective correlative biomarker study between presence of methylated BCAT1 and IKZF1 in tissue and blood was conducted in cases with CRC to explore how detection of such ctDNA biomarkers relates to cancer characteristics, methylation in tissue and surgical resection of the primary cancer.

BACKGROUNDColvera™ is a test that detects circulating tumor-derived DNA in patients with colorectal cancer by assaying for the presence of methylated BCAT1 and IKZF1 in blood. This study describes the analytical and clinical performance characteristics of the test.METHODSValidation was performed in accordance with ISO15189 and National Pathology Accreditation Advisory Council requirements. Spiked samples including 264 plasma and 120 buffer samples were randomized, divided into 8 batches of 48 samples, and processed over 8 days using 2 equipment lines (each line consisting of a QIAsymphony SP/AS, QIACube HT, and LC480); 2 reagent batches; and 2 operators to determine limit of detection, selectivity/specificity, precision, reproducibility, ruggedness, and susceptibility to commonly known interfering substances. Clinical performance was validated by assaying 222 archived plasma samples from subjects (n = 26 with cancer) enrolled in a previous prospective trial.RESULTSThe limit of detection for Colvera was 12.6 pg/mL (95% CI, 8.6-23.9 pg/mL), which equates to 2 diploid genome copies per milliliter plasma. No statistically significant difference was determined between testing days (n = 8), instrumentation, operators, or reagent batches in precision studies for the methylation-specific assays. The assay performance was unaffected by 9 commonly known interference substances, variations in bisulfite conversion, or quantitative PCR settings (cycling temperatures, incubation times, and oligonucleotide concentrations). For this clinical cohort, sensitivity and specificity estimates for Colvera were 73.1% (19 of 26; 95% CI, 52.2-88.4) and 89.3% (175 of 196; 95% CI, 84.1-93.2), respectively.CONCLUSIONColvera is a robust test and suitable for detection of circulating tumor-derived DNA by measuring levels of methylated BCAT1 and IKZF1 in human blood plasma.

Recurrence will develop in 30–50% of colorectal cancer (CRC) cases despite apparent clearance following treatment. Carcinoembryonic antigen (CEA) is the only guideline‐recommended blood test for monitoring cases for recurrence, but its sensitivity and specificity are suboptimal. This observational study compared a novel 2‐gene (methylated BCAT1 and IKZF1 DNA) blood test with CEA for detection of recurrent CRC. We conducted a paired comparison of the BCAT1/IKZF1 test with CEA (cut‐off 5 ng/mL) in blood from patients in remission after treatment for primary CRC and undergoing surveillance. Blood collected in the 12 months prior to or 3 months after complete investigational assessment of recurrence status were assayed and the results compared by McNemar's test. Of 397 patients enrolled, 220 underwent satisfactory assessment for recurrence and 122 had blood testing performed within the prescribed period. In 28 cases with recurrent CRC, CEA was positive in 9 (32%; 95% CI 16–52%) compared to 19 (68%; 95% CI 48–84%) positive for methylated BCAT1/IKZF1 (P = 0.002). All samples that were CEA positive were also BCAT1/IKZF1 positive. In 94 patients without clinically detectable recurrence, CEA was positive in 6 (6%, 95% CI 2–13%) and BCAT1/IKZF1 in 12 (13%, 95% CI 7–21%), P = 0.210. The odds ratio of a positive CEA test for recurrence was 6.9 (95% CI 2–22) compared to 14.4 (5–39) for BCAT1/IKZF1. The BCAT1/IKZF1 test was more sensitive for recurrence than CEA and the odds of recurrence given a positive test was twice that of CEA. The BCAT1/IKZF1 test should be further considered for monitoring cases for recurrence.

OBJECTIVES: To compare the performance of a new blood test for colorectal cancer (CRC) to an established fecal immunochemical test (FIT) in a study population with the full range of neoplastic and non-neoplastic pathologies encountered in the colon and rectum.METHODS: Volunteers were asked to complete a FIT prior to colonoscopy. Blood was collected after bowel preparation but prior to colonoscopy, and plasma was assayed for the presence of methylated BCAT1 and IKZF1 DNA using a multiplex real-time PCR assay. Sensitivity and specificity estimates for the blood test were calculated from true-and false-positive rates for neoplasia and compared with FIT at a range of fecal hemoglobin (Hb) concentration positivity thresholds.RESULTS: In total, 1,381 volunteers (median age 64 years; 49% male) completed both tests prior to colonoscopy. Estimated sensitivity of the BCAT1IKZF1 blood test for CRC was 62% (4166; 95% confidence interval 49-74%) with a specificity of 92% (12071315; 90-93%). FIT returned the same specificity at a cutoff of 60 mu g Hbg, at which its corresponding sensitivity for cancer was 64% (4266; 51-75%). In the range of commonly used FIT cutoffs, respective cancer sensitivity and specificity estimates with FIT were: 59% (46-71%) and 93% (92-95%) at 80 mu g Hbg, and 79% (67-88%) and 81% (78-83%) at 10 mu g Hbg. Although estimated sensitivities were not significantly different between the two tests for any stage of cancer, FIT showed a significantly higher sensitivity for advanced adenoma at the lower cutoffs. Specificity of FIT, but not of the BCAT1IKZF1 blood test, deteriorated substantially in people with overt blood in the feces. When combining FIT (cutoff 10 mu g Hbg) with the BCAT1IKZF1 blood test, sensitivity for cancer was 89% (79-96%) at 74% (72-77%) specificity.CONCLUSIONS: A test based on detection of methylated BCAT1IKZF1 DNA in blood has comparable sensitivity but better specificity for CRC than FITat the commonly used positivity threshold of 10 mu g Hbg. Further evaluation of the new test relative to FIT in the population screening context is now required to fully understand the potential advantages and disadvantages of these biomarkers in screening.

Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA) has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA) isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC), it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes) was analyzed in WBC DNA and eight differentially-methylated regions (DMRs) were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

Background Specific genes are methylated with high frequency in colorectal neoplasia, and may leak into blood. Detection of multiple methylated DNA biomarkers in blood may improve assay sensitivity for colorectal cancer (CRC) relative to a single marker. We undertook a case-control study evaluating the presence of two methylation DNA markers, BCAT1 and IKZF1, in circulation to determine if they were complementary for detection of CRC. Methods Methylation-specific PCR assays were developed to measure the level of methylated BCAT1 and IKZF1 in DNA extracted from plasma obtained from colonoscopy-confirmed 144 healthy controls and 74 CRC cases. Results DNA yields ranged from 2 to 730 ng/mL plasma (mean 18.6ng/mL; 95% CI 11-26 ng/mL) and did not correlate with gender, age or CRC status. Methylated BCAT1 and IKZF1 DNA were detected in respectively 48 (65%) and 50 (68%) of the 74 cancers. In contrast, only 5 (4%) and 7 (5%) controls were positive for BCAT1 and IKZF1 DNA methylation, respectively. A two-gene classifier model (“either or” rule) improved segregation of CRC from controls, with 57 of 74 cancers (77%) compared to only 11 of 144 (7.6%) controls being positive for BCAT1 and/or IKZF1 DNA methylation. Increasing levels of methylated DNA were observed as CRC stage progressed. Conclusions Detection of methylated BCAT1 and/or IKZF1 DNA in plasma may have clinical application as a novel blood test for CRC. Combining the results from the two methylation-specific PCR assays improved CRC detection with minimal change in specificity. Further validation of this two-gene blood test with a view to application in screening is now indicated.

clinical trial, we found that the sensitivity of sDNA testing for detection of advanced lesions or all adenomas in African Americans was similar to or exceeded that of other racial groups, with comparable specificity.Given the known racial disparities in receipt of colonoscopy, this noninvasive technology may offer a promising screening alternative.

It is recently reported that microRNAs (miRNAs) are stably present in serum and potentially useful in the diagnosis of cancer. Using highly sensitive microarray, 3D-Gene®, we examined comprehensive miRNA expression profiles in serum obtained from 66 pancreatic cancer patients, 66 bile duct cancer patients and 100 matched healthy volunteer as the study set, and found that 122 miRNA markers for pancreatic cancer and 125 miRNA markers for bile duct cancer differentiated pancreatic / bile duct cancer patients from healthy control with a statistical significance. We then validated the diagnostic performance of these markers using serum obtained from 33 pancreatic cancer patients, 33 bile duct cancer patients and 50 healthy controls. One of the single miRNA markers for pancreatic cancer yielded more than 90% diagnostic sensitivity and specificity, and combined use of two markers perfectly differentiated cancer and healthy samples. We also found that over 100 miRNA markers of pancreatic cancer overlapped with miRNA markers of bile duct cancer. Our results suggest that these miRNA markers have clinical values to screen out pancreatic and bile duct cancer patients.

Background: A recently developed multiplex serology method has been used to identify new virulence factors of Helicobacter pylori.The results of associations with gastric cancer have not been consistent across studies, possibly due to differences in the studied populations and/or the genetic diversity of H. pylori.We aimed to investigate the association between seropositivity to fifteen different H. pylori antigens using the multiplex serology method and the risk of gastric cardia (GCA) and gastric non-cardia (GNCA) adenocarcinomas in northeastern Iran, where the population has both a high prevalence of H. pylori infection and a high incidence of gastric adenocarcinoma.Methods: We included 272 cases of gastric adenocarcinoma (142 GCA, 103 GNCA, and 22 unspecified tumors) and 524 controls who were individually matched to cases for age, sex, and place of residence in this populationbased case-control study.Seropositivity to H. pylori was assessed using both multiplex serology and H. pylori IgG ELISA (BioHit, Finland).Results: H. pylori positivity based on the whole-cell ELISA test and also multiplex serology assay; defined as recognizing antibodies to ≥ 4 antigens; were not significantly associated with any of study groups; and its positivity rate was generally lower in ELISA assay than multiplex serology; GCA cases (79.6% vs. 95.1%),GNCA cases (74.8% vs. 95.2%),and controls (82.4% vs. 94.7%).Of the 15 antibodies in the multiplex assay, 10 showed no significant association with all gastric adenocarcinoma, GCA, or GNCA; while CagA and VacA were associated with a significantly increased risk of these cancers in multivariate adjusted models.Conversely, GroEL and NapA were significantly associated with a reduced risk of all gastric adenocarcinoma and GNCA.Conclusion: Our study showed no association between ELISA test positivity and gastric cancer risk, which implies that this method should not be used for risk stratification and referral for H. pylori eradication.Rather, effective risk stratification would require further characterization of H. pylori strain exposure with virulence factors such as CagA and VacA antigens.Our finding regarding lower risk of gastric adenocarcinoma among GroEL and NapA antigen positive subjects deserves additional investigation.

Specific genes, such as BCAT1 and IKZF1, are methylated with high frequency in colorectal cancer (CRC) tissue compared to normal colon tissue specimens. Such DNA may leak into blood and be present as cell-free circulating DNA. We have evaluated the accuracy of a novel blood test for these two markers across the spectrum of benign and neoplastic conditions encountered in the colon and rectum.

The CAHM gene (Colorectal Adenocarcinoma HyperMethylated), previously LOC100526820, is located on chromosome 6, hg19 chr6:163 834 097-163 834 982. It lacks introns, encodes a long non-coding RNA (lncRNA) and is located adjacent to the gene QKI, which encodes an RNA binding protein. Deep bisulphite sequencing of ten colorectal cancer (CRC) and matched normal tissues demonstrated frequent hypermethylation within the CAHM gene in cancer. A quantitative methylation-specific PCR (qMSP) was used to characterize additional tissue samples. With a threshold of 5% methylation, the CAHM assay was positive in 2/26 normal colorectal tissues (8%), 17/21 adenomas (81%), and 56/79 CRC samples (71%). A reverse transcriptase-qPCR assay showed that CAHM RNA levels correlated negatively with CAHM % methylation, and therefore CAHM gene expression is typically decreased in CRC. The CAHM qMSP assay was applied to DNA isolated from plasma specimens from 220 colonoscopy-examined patients. Using a threshold of 3 pg methylated genomic DNA per mL plasma, methylated CAHM sequences were detected in the plasma DNA of 40/73 (55%) of CRC patients compared with 3/73 (4%) from subjects with adenomas and 5/74 (7%) from subjects without neoplasia. Both the frequency of detection and the amount of methylated CAHM DNA released into plasma increased with increasing cancer stage. Methylated CAHM DNA shows promise as a plasma biomarker for use in screening for CRC.

Background The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9 , VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. Methods Combined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). Results Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. Conclusions This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes ( SOX21 , SLC6A15 , NPY , GRASP , ST8SIA1 and ZSCAN18) show very low methylation in non-neoplastic colorectal tissue and are candidate biomarkers for stool-based assays, while 11 genes ( BCAT1 , COL4A2 , DLX5 , FGF5 , FOXF1 , FOXI2 , GRASP , IKZF1 , IRF4 , SDC2 and SOX21 ) have very low methylation in peripheral blood DNA and are suitable for further evaluation as blood-based diagnostic markers.

Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80-90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development. PUBLICATION HISTORY : Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).