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.

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.

BackgroundColorectal 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 FindingsUsing 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/SignificanceAnalysis 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.

Leishmania are evolutionarily ancient protozoans (Kinetoplastidae) and important human pathogens that cause a spectrum of diseases ranging from the asymptomatic to the lethal. The Leishmania genome is relatively small [Ϸ34 megabases (Mb)], lacks substantial repetitive DNA, and is distributed among 36 chromosomes pairs ranging in size from 0.3 Mb to 2.5 Mb, making it a useful candidate for complete genome sequence determination. We report here the nucleotide sequence of the smallest chromosome, chr1. The sequence of chr1 has a 257-kilobase region that is densely packed with 79 protein-coding genes. This region is f lanked by telomeric and subtelomeric repetitive elements that vary in number and content among the chr1 homologs, resulting in an Ϸ27.5-kilobase size difference. Strikingly, the first 29 genes are all encoded on one DNA strand, whereas the remaining 50 genes are encoded on the opposite strand. Based on the gene density of chr1, we predict a total of Ϸ9,800 genes in Leishmania, of which 40% may encode unknown proteins.The Kinetoplastidae are flagellated protozoans found in terrestrial and aquatic environments that cause diseases in organisms ranging from plants to vertebrates. These diseases result in widespread human suffering and death, as well as considerable economic loss from infection of livestock, wildlife, and crops. In addition, kinetoplastids have been particularly valuable for the study of fundamental molecular and cellular phenomena, such as RNA editing (1), mRNA transsplicing (2), glycosylphosphatidylinositol-anchoring of proteins (3), antigenic variation (4), and telomere organization (5). The early evolutionary divergence of these organisms makes comparison of their sequences with those of other eukaryotes, as well as prokaryotes, useful for the identification of ancient conserved motifs, and their protein sequences may be a useful source of diversity for protein engineering.The numerous human-infective Leishmania spp. cause a spectrum of diseases with pathologies ranging from the asymptomatic to the lethal, and there are correlations between species and disease type and severity (6). The Leishmania haploid genome content is Ϸ34 megabases (Mb; ref. 7), consisting of 36 chromosomes ranging in size from 0.3 Mb to 2.5 Mb (8). It contains Ϸ30% repeated sequence (9), half of which is a series of telomeric hexamer repeats, whereas the remainder comprises other simple sequence repeats, transposons, as well as tandem and dispersed gene families such as rRNA, spliced-leader, tubulin, and gp63. The Leishmania molecular karyotype is conserved between Leishmania strains and species (10) with most genes syntenic among species (8). There are modest chromosome size polymorphisms between strains and larger size polymorphisms between species. Thus, this organism is an ideal candidate for a genome-sequencing project to elucidate its full genetic complement. The Leishmania Genome Network, established with the support of the World Health Organization, initiated a coordinated effort to map and sequenc...

BackgroundDespite 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.MethodsParticipants 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.Results63 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.Conclusions97% 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-230X-14-183) contains supplementary material, which is available to authorized users.

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