No abstract

We were able to detect the majority of colorectal cancers by analyzing stool DNA for just three genetic markers. Additional work is needed to determine the specificity of these genetic tests for detecting colorectal neoplasia in asymptomatic patients and to more precisely estimate the prevalence of the mutations and sensitivity of the assay.

APC mutations can be detected in fecal DNA from patients with relatively early colorectal tumors. This feasibility study suggests a new approach for the early detection of colorectal neoplasms.

Trinucleotide CAG repeats in the X-linked human androgen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles in a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under optimal conditions amplification efficiency of two HUMARA alleles is near-equivalent, generating PCR products in a ratio proportional to that of the genomic template. In contrast, reduction of template quantity, damage of template by ultraviolet irradiation or addition of monovalent salts (sodium chloride, sodium acetate or ammonium acetate) produces highly variable imbalances of allelic PCR products, with a strong tendency to preferentially amplify lower molecular weight alleles. Variability and biasing was diminished by substitution of 7-deaza-2'-dGTP for dGTP during amplification, an intervention which reduces stability of intramolecular and intermolecular GC base pairing. We conclude that DNA which is scanty, damaged or salt contaminated may display amplification bias of GC-rich PCR targets, potentially confounding accurate interpretation or reproducibility of assays which require co-amplification of alleles.

Background: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa. Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity. Results: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group. Conclusions: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.

No abstract

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P ‫؍‬ Colorectal cancer (CRC) is curable in more than 90% of cases when caught in the earliest stages. Current colorectal cancer screening guidelines include a variety of options. Colonoscopy may be the most sensitive screening test, 1 however its invasiveness (including bowel preparation and the procedure itself) present major barriers to its implementation for large-scale, nationwide screening.2 An improved non-invasive screening option could address many of the issues associated with colonoscopy. Non-invasive screening is available today through assessment of occult blood in fecal samples, but this test has relatively low sensitivity, especially for early stage cancer, limiting its impact on cancer mortality. However, analysis of DNA from stool provides an attractive, alternative, non-invasive means for CRC screening if scalable, sensitive, and specific tests can be developed.We have previously described 3 a stool-based screening test for early detection of colorectal cancers. The multi-target nucleic acid assay consists of a panel of 21 specific mutations in adenomatous polyposis coli (APC), 4 p53 5,6 , and K-ras 7 genes, a microsatellite instability marker (BAT-26), 8 and a marker for genomic integrity (DNA Integrity assay; DIA). 9 As reported in separate studies, 3,10,11,12 the multi-target assay has an aggregate sensitivity of 67% (95% CI: 60.3 to 73.9%) and specificity of 97% (95% CI: 92.9 to 99.2%), a major improvement to the current screening methods of the fecal occult blood test (25 to 40% sensitivity). 13,14 In the multi-target assay studies human DNA was recovered and purified using streptavidin-bound magnetic beads. 3,15 We have reported on the use of separate components of this multi-target test elsewhere. 9,11,16,17,18 The mutation panel portion of the multi-target assay relies on detecting mutations in several well-documented colorectal cancer-associated genes. 19,20 The DNA integrity portion of the test consists of a set of markers that serve as surrogate markers for the presence of long DNA fragments. The principles and...

Purpose: The aim of this study was to evaluate the utility of the DNA integrity assay (DIA) as a plasma-based screening tool for the detection of prostate cancer. Experimental Design: Blood samples were collected from patients with biopsy-proven prostate cancer prior to prostatectomy (n = 123) and processed as two-spin plasma preparations. The three control groups included: males <40 years old with no history of cancer (group 1, n = 20); cancer-free postprostatectomy patients (group 2, n = 25), and patients with a negative prostate biopsy (group 3, n = 22). DNA in plasma preparations were isolated, hybrid-captured, and DNA fragments (200 bp, 1.3, 1.8, and 2.4 kb) were multiplexed in real-time PCR. A baseline cutoff was determined for individual fragment lengths to establish a DIA score for each patient sample. Results: Patients with prostate cancer (86 of 123; 69.9%) were determined to have a positive DIA score of z7. The DIA results from control groups 1, 2, and 3 showed specificities of 90%, 92%, and 68.2%, respectively. Of the patients with negative age-adjusted prostate-specific antigen (PSA) and prostate cancer, 19 of 30 (63%) had a positive DIA score. The area under the receiver operating characteristic curve for DIA was 0.788. Conclusion: While detecting 69.9 % of those with prostate cancer, DIA maintained an overall specificity of 68.2% to 92%, a range favorably comparable to that currently accepted for PSA (60-70%). The variability in specificity between control groups is likely explained by the established 19% to 30% detection of prostate cancer on subsequent biopsies associated with control group 3. DIA detected 63% of the prostate cancers undetected by currently accepted PSA ranges.