Helper-Dependent Chain Reaction (HDCR) for Selective Amplification of Methylated DNA Sequences.

Over the last few years a number of restriction enzymes that cut DNA only if cytosines within their recognition sequences are methylated have been characterized and become commercially available. Cleavage with these enzymes to release DNA fragments in a methylation-dependent manner can be combined with a novel method of amplification, Helper Dependent Chain Reaction (HDCR), to selectively amplify these fragments. HDCR uses "Helper" oligonucleotides as templates for extension of the free 3' end of target fragments to incorporate tag sequences at the ends of fragments. These tag sequences are then used for priming of amplification of target fragments. Modifications to the amplification primers (Drivers) and the Helpers ensure that there is selection for the sequences within target fragments with each cycle of amplification. The combination of methylation-dependent enzymes and HDCR allows the sensitive and selective amplification of methylated DNA sequences without the need for bisulfite treatment.

Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer.

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.

Little evidence for association between the TGFBR1*6A variant and colorectal cancer: a family-based association study on non-syndromic family members from Australia and Spain.

BACKGROUND: Genome-wide linkage studies have identified the 9q22 chromosomal region as linked with colorectal cancer (CRC) predisposition. A candidate gene in this region is transforming growth factor ß receptor 1 (TGFBR1). Investigation of TGFBR1 has focused on the common genetic variant rs11466445, a short exonic deletion of nine base pairs which results in truncation of a stretch of nine alanine residues to six alanine residues in the gene product. While the six alanine (*6A) allele has been reported to be associated with increased risk of CRC in some population based study groups this association remains the subject of robust debate. To date, reports have been limited to population-based case-control association studies, or case-control studies of CRC families selecting one affected individual per family. No study has yet taken advantage of all the genetic information provided by multiplex CRC families. METHODS: We have tested for an association between rs11466445 and risk of CRC using several family-based statistical tests in a new study group comprising members of non-syndromic high risk CRC families sourced from three familial cancer centres, two in Australia and one in Spain. RESULTS: We report a finding of a nominally significant result using the pedigree-based association test approach (PBAT; p = 0.028), while other family-based tests were non-significant, but with a p-value <; 0.10 in each instance. These other tests included the Generalised Disequilibrium Test (GDT; p = 0.085), parent of origin GDT Generalised Disequilibrium Test (GDT-PO; p = 0.081) and empirical Family-Based Association Test (FBAT; p = 0.096, additive model). Related-person case-control testing using the "More Powerful" Quasi-Likelihood Score Test did not provide any evidence for association (MQLS; p = 0.41). CONCLUSIONS: After conservatively taking into account considerations for multiple hypothesis testing, we find little evidence for an association between the TGFBR1*6A allele and CRC risk in these families. The weak support for an increase in risk in CRC predisposed families is in agreement with recent meta-analyses of case-control studies, which estimate only a modest increase in sporadic CRC risk among 6*A allele carriers.

A panel of genes methylated with high frequency in colorectal cancer.

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.

Discovery and validation of molecular biomarkers for colorectal adenomas and cancer with application to blood testing.

BACKGROUND & AIMS: Colorectal cancer incidence and deaths are reduced by the detection and removal of early-stage, treatable neoplasia but we lack proven biomarkers sensitive for both cancer and pre-invasive adenomas. The aims of this study were to determine if adenomas and cancers exhibit characteristic patterns of biomarker expression and to explore whether a tissue-discovered (and validated) biomarker is differentially expressed in the plasma of patients with colorectal adenomas or cancer. METHODS: Candidate RNA biomarkers were identified by oligonucleotide microarray analysis of colorectal specimens (222 normal, 29 adenoma, 161 adenocarcinoma and 50 colitis) and validated in a previously untested cohort of 68 colorectal specimens using a custom-designed oligonucleotide microarray. One validated biomarker, KIAA1199, was assayed using qRT-PCR on plasma extracted RNA from 20 colonoscopy-confirmed healthy controls, 20 patients with adenoma, and 20 with cancer. RESULTS: Genome-wide analysis uncovered reproducible gene expression signatures for both adenomas and cancers compared to controls. 386/489 (79%) of the adenoma and 439/529 (83%) of the adenocarcinoma biomarkers were validated in independent tissues. We also identified genes differentially expressed in adenomas compared to cancer. KIAA1199 was selected for further analysis based on consistent up-regulation in neoplasia, previous studies and its interest as an uncharacterized gene. Plasma KIAA1199 RNA levels were significantly higher in patients with either cancer or adenoma (31/40) compared to neoplasia-free controls (6/20). CONCLUSIONS: Colorectal neoplasia exhibits characteristic patterns of gene expression. KIAA1199 is differentially expressed in neoplastic tissues and KIAA1199 transcripts are more abundant in the plasma of patients with either cancer or adenoma compared to controls.

Reversing the course of forgetting.

Forgetting functions were generated for pigeons in a delayed matching-to-sample task, in which accuracy decreased with increasing retention-interval duration. In baseline training with dark retention intervals, accuracy was high overall. Illumination of the experimental chamber by a houselight during the retention interval impaired performance accuracy by increasing the rate of forgetting. In novel conditions, the houselight was lit at the beginning of a retention interval and then turned off partway through the retention interval. Accuracy was low at the beginning of the retention interval and then increased later in the interval. Thus the course of forgetting was reversed. Such a dissociation of forgetting from the passage of time is consistent with an interference account in which attention or stimulus control switches between the remembering task and extraneous events.

Reversing the signaled magnitude effect in delayed matching to sample: delay-specific remembering.

Pigeons performed a delayed matching-to-sample task in which large or small reinforcers for correct remembering were signaled during the retention interval. Accuracy was low when small reinforcers were signaled, and high when large reinforcers were signaled (the signaled magnitude effect). When the reinforcer-size cue was switched from small to large partway through the retention interval, accuracy accordingly changed from low to high. The opposite happened when the cue was switched from large to small. This dissociation of forgetting from the passage of time raises the possibility that remembering is delay-specific. The reversal of the signaled magnitude effect during the retention interval is consistent with an attentional account in which the stimulus control of remembering is influenced by extraneous events.

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas.

An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5'-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2(-ΔCt) = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers.

A computational technique for improving estimates of discriminability and bias across multiple dimensions of choice.

Brown and White (2009) proposed measures of discriminability and bias that accommodate additional dimensions of choice--and hence, bias--in conditional discriminations such as matching-to-sample and the yes-no signal detection task. Their proposed measures increase the statistical independence of discriminability and bias estimates, thus improving their accuracy. Because Brown and White's (2009) equations partition response data more than do standard equations, however, their measures have a slightly lower ceiling. Consequently, measurements can be less accurate when there are few trials and discriminability and bias are extreme. We introduce a computational estimation technique that overcomes this limitation. It estimates Brown and White's (2009) discriminability and bias measurements from an array of related measures that have a higher ceiling. Simulations show that resulting estimates of discriminability and bias are either comparable to or more accurate than measurements calculated from traditional equations or Brown and White's (2009) direct measures, even with few trials. A worked example of our technique may be downloaded from brm.psychonomic-journals.org/content/supplemental.

Reinforcer probability, reinforcer magnitude, and the reinforcement context for remembering.

Traditional theories of delayed matching-to-sample performance do not predict that accuracy will improve when absolute levels of reinforcement are increased. This prediction emerges only when reinforcement context is considered (J. A. Nevin, M. Davison, A. L. Odum, & T. A. Shahan, 2007). To provide quantitative data, the authors factorially manipulated between conditions the probability and duration of reinforcement for correct choices by pigeons. In Experiment 1, increasing the value of either variable improved initial discriminability of the forgetting functions, but did not affect the rate of forgetting. In Experiment 2, initial discriminability covaried with changes in choice immediacy and trial completion rate, suggesting a relationship with response strength consistent with Nevin et al.'s behavioral momentum model. Adding reinforcement context to K. G. White and J. T. Wixted's (1999) model also generates predictions consistent with the present experiments and with the effects of manipulating extraneous reinforcement. The inclusion of reinforcement context thus improves predictions of delayed matching-to-sample performance.

Measuring discriminability when there are multiple sources of bias.

Performance measures such as log d and d' aim to measure stimulus discriminability independently of response bias in conditional discrimination tasks, including the yes/no signal-detection procedure. However, they assume only one dimension of bias (e.g., response color) and do not account for bias on additional dimensions (e.g., response side). Such bias reduces log d, thus violating the statistical independence of discriminability and bias measurements. We modified log d to account for side bias and reanalyzed previous side-biased data. With strong side bias, the modified log d differed enough from the standard log d to potentially alter the conclusions of an experiment. Simulations showed that the modified log d produces discriminability estimates that are more accurate and bias-independent than the standard log d calculation.

Map of differential transcript expression in the normal human large intestine.

While there is considerable research related to using differential gene expression to predict disease phenotype classification, e.g., neoplastic tissue from nonneoplastic controls, there is little understanding of the range of expression in normal tissues. Understanding patterns of gene expression in nonneoplastic tissue, including regional anatomic expression changes within an organ, is vital to understanding gene expression changes in diseased tissue. To explore the gene expression change along the proximal-distal axis of the large intestine, we analyzed microarray data in 184 normal human specimens using univariate and multivariate techniques. We found 219 probe sets that were differentially expressed between the proximal and distal colorectal regions and 115 probe sets that were differentially expressed between the terminal segments, i.e., the cecum and rectum. We did not observe any probe sets that were statistically different between any two contiguous colorectal segments. The dominant expression pattern (65 probe sets) follows a dichotomous expression pattern consistent with the midgut-hindgut embryonic origins of the gut while a second pattern (50 probe sets) depicts a gradual change in transcript levels from the cecum to the rectum. While the dichotomous pattern includes roughly equal numbers of probe sets that are elevated proximally and distally, nearly all probe sets that show a gradual change demonstrate increasing expression levels moving from proximal to distal segments. These patterns describe an expression map of individual transcript variation as well as multigene expression patterns along the large intestine. This is the first gene expression map of an entire human organ.

Seasonal variation in pigeon body weight and delayed matching-to-sample performance.

The weights of 5 pigeons with free access to food, monitored over 3 calendar years in the laboratory, were found to fluctuate with season. All pigeons were at their heaviest in the winter and were lightest in the summer. Five different pigeons performed a standard delayed matching-to-sample task for 44 weeks from January to November. Their weights were held at 85% of their summer free-feeding weights, making their predicted deprivation level higher in the winter relative to predicted winter free-feeding weights. Slopes of forgetting functions fit to weekly response totals for each pigeon were shallower in winter, showing an improvement in accuracy with longer delays. Thus, delayed matching-to-sample performance may have been affected by the practice of maintaining the pigeons at a constant body weight throughout the calendar year.

On the effects of signaling reinforcer probability and magnitude in delayed matching to sample.

Two experiments examined whether postsample signals of reinforcer probability or magnitude affected the accuracy of delayed matching to sample in pigeons. On each trial, red or green choice responses that matched red or green stimuli seen shortly before a variable retention interval were reinforced with wheat access. In Experiment 1, the reinforcer probability was either 0.2 or 1.0 for both red and green responses. Reinforcer probability was signaled by line or cross symbols that appeared after the sample had been presented. In Experiment 2, all correct responses were reinforced, and the signaled reinforcer durations were 1.0 s and 4.5 s. Matching was more accurate when larger or more probable reinforcers were signaled, independently of retention interval duration. Because signals were presented postsample, the effects were not the result of differential attention to the sample.

Remembering: the role of extraneous reinforcement.

In two experiments, pigeons' responding on an extraneous task was explicitly reinforced during delayed matching-to-sample trials. In Experiment 1, red or green sample stimuli were followed by retention intervals of 0.2, 1, 4, or 12 sec, during which pecks to a white center key were reinforced with 2.5-sec access to wheat according to extinction, variable-interval 30-sec, and variable-interval 15-sec schedules in different conditions. A proportion of .2, .5, .7, or .9 of subsequent red or green choice responses that matched the sample were reinforced with 3-sec access to wheat. The result was that increasing center key reinforcement, or reducing reinforcer probability, lowered overall accuracy. Initial discriminability fell, but with no change in the rate of forgetting. In Experiment 2, initial discriminability was affected by extraneous reinforcers that were contingent on center key pecking, but not by noncontingent reinforcers. A plausible conclusion is that initial discriminability decreases when reinforcers strengthen competing behaviors.

The optimal correction for estimating extreme discriminability.

Discriminability measures such as d' and log d become infinite when performance is extremely accurate and no errors are recorded. Different arbitrary corrections can be applied to produce finite values, but how well do these values estimate true performance? To answer this question, we directly calculated the effects of a range of different corrections on the sampling distributions of d' and log d. Many arbitrary corrections produced better estimates of discriminability than did the intuitively plausible technique of rerunning problem conditions. We concluded that when it is not possible to run more trials and when other techniques are not appropriate, the best correction overall is to add a correction constant between 0.25 and 0.5 to all response counts, regardless of their value.

Local proactive interference in delayed matching to sample: the role of reinforcement.

Discriminability in delayed matching to sample was lower when the samples on consecutive trials differed compared with when samples on consecutive trials were the same. This local proactive interference occurred when correct choices on the previous trial were reinforced but not when correct choices on the previous trial were not reinforced. When the choice on the previous trial was incorrect, discriminability was higher on different consecutive trials than on same trials. These effects were amplified by varying the ratio of reinforcers for correct choices, as predicted by a model that attributes local proactive interference to an interaction between control by the sample on the current trial and the influence of reinforcers for correct choices on previous trials.