Prospective evaluation of methylated SEPT9 in plasma for detection of asymptomatic colorectal cancer.

BACKGROUND: As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase both, but none has yet been tested in a screening setting. OBJECTIVE: We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population. DESIGN: Asymptomatic individuals ≥50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates. RESULTS: 7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude rate 50.9%); for CRC stages I-IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%). CONCLUSIONS: Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas. CLINICAL TRIAL REGISTRATION NUMBER: NCT00855348.

Endoscopic detection of proximal serrated lesions and pathologic identification of sessile serrated adenomas/polyps vary on the basis of center.

BACKGROUND & AIMS: We investigated rates of detection of proximal serrated lesions in a cohort of average-risk patients undergoing screening colonoscopies. METHODS: We reviewed results from screening colonoscopies performed by attending gastroenterologists at 32 endoscopy centers from 2008-2010. Pathology slides were interpreted at the individual centers. For this analysis, serrated lesions included hyperplastic polyps larger than 10 mm, those interpreted as sessile serrated adenomas (or sessile serrated polyp), and traditional serrated adenomas. Rates of detection for conventional adenomas and serrated lesions were compared among centers. RESULTS: A total of 5778 lesions were detected in 7215 screening colonoscopies. Of the 5548 lesions with pathology results, 3008 (54.2%) were conventional adenomas, 350 (6.3%) were serrated, and 232 (4.2%) were proximal serrated. The proportion of colonoscopies with at least 1 proximal serrated lesion was 2.8% (range among centers, 0%-9.8%). The number of serrated lesions per colonoscopy ranged from 0.00-0.11 (average, 0.05 ± 0.25). Overall lesion detection rates correlated with proximal serrated lesion detection rates (R = 0.91, P < .0001); conventional adenoma and proximal serrated lesion detection rates also correlated (R = .43, P = .025). The detection rate of proximal serrated lesions differed significantly among centers (P < .0001); odds ratios for detection ranged from 0-0.79. Some centers' pathologists never identified proximal serrated lesions as sessile serrated adenomas/polyps. CONCLUSIONS: In an average-risk screening cohort, detection of proximal serrated lesions varied greatly among endoscopy centers. There was also substantial variation among pathologists in identification of sessile serrated adenomas/polyps. Nationally, a significant proportion of proximal serrated lesions may be missed during colonoscopy examination or incorrectly identified during pathology assessment. ClinicalTrials.gov Number: NCT00855348.

Pathway-specific tumor suppression. Reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice.

Expression of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) is frequently reduced in human colorectal cancer, and this correlates with poor patient prognosis. To clarify the role of p27 in gastrointestinal (GI) cancer, we measured p27 expression, as well as the effect of germline deletion of p27, in 3 different mouse models of GI neoplasia. p27 expression was frequently reduced in GI tumors arising in 1,2-dimethylhydrazine (DMH) treated mice, and in Apc mutant Min/+ mice, but not in GI tumors arising in Smad3 mutant mice. Germline deletion of p27 resulted in accelerated tumor development and increased tumor cell proliferation in both DMH treated and Min/+ mice, but not in Smad3 mutant mice. p27 deficiency also led to increased adenoma to adenocarcinoma progression. These results indicate that reduction of p27 cooperates with mutations in Apc but not in Smad3 during GI tumorigenesis. Thus, tumor suppression by p27 is contingent on the specific oncogenic pathway that drives tumor development.

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.

From discovery to the clinic: the novel DNA methylation biomarker (m)SEPT9 for the detection of colorectal cancer in blood.

Detection of colorectal cancer at an early stage has been shown to significantly decrease mortality from the disease, while the advent of effective therapies for late-stage colorectal cancer make the detection of colorectal cancer at any stage a critical step in further reducing colorectal cancer mortality. Availability of a blood-based test for colorectal cancer is expected to improve screening compliance in the general population. Through DNA methylation-sensitive, restriction enzyme-based biomarker discovery, we identified a region of the Septin 9 gene that is methylated in over 90% of colorectal cancer tissues with little or no methylation seen in normal colon tissue and other controls. Specific detection of colorectal cancer DNA using the Septin 9 methylation biomarker ((m)SEPT9) was demonstrated in multiple studies of plasma from colorectal cancer patients and colonoscopy-verified negative controls. A prospective, population-based trial to determine the clinical performance of (m)SEPT9 in colorectal cancer screening guideline-eligible individuals has recently been completed, with the results to be published in the near future. The potential pitfalls and lessons learned in the multiyear process of developing the (m)SEPT9 biomarker from initial discovery to commercialization are described in this article.

p27kip1 deficiency impairs G2/M arrest in response to DNA damage, leading to an increase in genetic instability.

p27(kip1) is a cyclin-dependent kinase inhibitor and a tumor suppressor. In some tumors, p27 suppresses tumor growth by inhibition of cell proliferation. However, this is not universally observed, implying additional mechanisms of tumor suppression by p27. p27-deficient mice are particularly susceptibility to genotoxin-induced tumors, suggesting a role for p27 in the DNA damage response. To test this hypothesis, we measured genotoxin-induced mutations and chromosome damage in p27-deficient mice. Both p27(+/-) and p27(-/-) mice displayed a higher N-ethyl-N-nitrosourea-induced mutation frequency in the colon than p27(+/+) littermates. Furthermore, cells from irradiated p27-deficient mice exhibited a higher number of chromatid breaks and showed modestly increased micronucleus formation compared to cells from wild-type littermates. To determine if this mutator phenotype was related to the cell cycle-inhibitory function of p27, we measured cell cycle arrest in response to DNA damage. Both normal and tumor cells from p27-deficient mice showed impaired G(2)/M arrest following low doses of ionizing radiation. Thus, p27 may inhibit tumor development through two mechanisms. The first is by reducing the proliferation of cells that have already sustained an oncogenic lesion. The second is by transient inhibition of cell cycle progression following genotoxic insult, thereby minimizing chromosome damage and fixation of mutations.

Tumor suppressor genetics.

The observation that mutations in tumor suppressor genes can have haploinsufficient, as well as gain of function and dominant negative, phenotypes has caused a reevaluation of the 'two-hit' model of tumor suppressor inactivation. Here we examine the history of haploinsufficiency and tumor suppressors in order to understand the origin of the 'two-hit' dogma. The two-hit model of tumor suppressor gene inactivation was derived from mathematical modeling of cancer incidence. Subsequent interpretations implied that tumor suppressors were recessive, requiring mutations in both alleles. This model has provided a useful conceptual framework for three decades of research on the genetics and biology of tumor suppressor genes. Recently it has become clear that mutations in tumor suppressor genes are not always completely recessive. Haploinsufficiency occurs when one allele is insufficient to confer the full functionality produced from two wild-type alleles. Haploinsufficiency, however, is not an absolute property. It can be partial or complete and can vary depending on tissue type, other epistatic interactions, and environmental factors. In addition to simple quantitative differences (one allele versus two alleles), gene mutations can have qualitative differences, creating gain of function or dominant negative effects that can be difficult to distinguish from dosage-dependence. Like mutations in many other genes, tumor suppressor gene mutations can be haploinsufficient, dominant negative or gain of function in addition to recessive. Thus, under certain circumstances, one hit may be sufficient for inactivation. In addition, the phenotypic penetrance of these mutations can vary depending on the nature of the mutation itself, the genetic background, the tissue type, environmental factors and other variables. Incorporating these new findings into existing models of the clonal evolution will be a challenge for the future.

p27(Kip1) (Cdkn1b)-deficient mice are susceptible to chemical carcinogenesis and may be a useful model for carcinogen screening.

The two-year rodent bioassay is one of several tests that are widely used by governmental regulatory agencies as well as pharmaceutical and chemical companies to determine the carcinogenic potential of chemicals or environmental agents where human exposure is anticipated. That this assay has remained relatively unchanged for the last 25 years is a testament to the power of this approach to identify carcinogens and thus to minimize human exposure. However, there has long been controversy over the specificity and relevance of the rodent bioassay as well as its high cost in terms of time, expense, and numbers of mice. Much discussion has been generated in recent years over how to improve the 2-year rodent bioassay for more accurate and faster detection of potential human carcinogens. Here, we argue that the use of p27(Kip1) (Cdkn1b) knockout mouse for carcinogen screening may solve several shortcomings inherent in the conventional bioassay while preserving its best quality, that is, protecting public health by providing reliable in vivo information on the potential of chemicals to cause cancer.