Background & Aims: Assay of altered DNA exfoliated into stool represents an intriguing approach to screen for colorectal neoplasia, but multiple markers must be targeted because of genetic ...heterogeneity. We explored the feasibility of a stool assay panel of selected DNA alterations in discriminating subjects with colorectal neoplasia from those without. Methods: Freezer-archived stools were analyzed in blinded fashion from 22 patients with colorectal cancer, 11 with adenomas ≥1 cm, and 28 with endoscopically normal colons. After isolation of human DNA from stool by sequence-specific hybrid capture, assay targets included point mutations at any of 15 sites on K-ras, p53, and APC genes; Bat-26, a microsatellite instability marker; and highly amplifiable DNA. Results: Analyzable human DNA was recovered from all stools. Sensitivity was 91% (95% confidence interval, 71%–99%) for cancer and 82% (48%–98%) for adenomas ≥1 cm with a specificity of 93% (76%–99%). Excluding K-ras from the panel, sensitivities for cancer were unchanged but decreased slightly for adenomas to 73% (39%–94%), while specificity increased to 100% (88%–100%). Conclusions: Assay of altered DNA holds promise as a stool screening approach for colorectal neoplasia. Larger clinical investigations are indicated.
GASTROENTEROLOGY 2000;119:1219-1227
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.
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.
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.
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.
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 ≥7. 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.
ABSTRACT Trinucleotide CAG repeats in the X-linked human an drogen 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 Ina 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 inter molecular 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.
Progressive microsatellite changes in replication error positive (RER+) endometrium were used to reconstruct evolutionary stages of nonfamilial adenocarcinoma. RER+ putative endometrial precancers ...(atypical endometrial hyperplasias) progress to RER+ carcinomas, which retain some of the altered microsatellites acquired in earlier precursor stages. The RER+ phenotype may provide a specific marker for early-stage endometrial neoplasms that cannot be resolved by routine histopathology and may be a useful tool to stratify stages in the evolution of RER+ tumors.
We have tested the hypothesis that endometrial precancers persist in uteri of patients with endometrial carcinoma and are monoclonal. Twenty-two hysterectomies with both well-differentiated ...endometrial adenocarcinoma and adjacent (normal or abnormal) noncancerous endometrium underwent successful clonal analysis using a PCR assay for nonrandom X chromosome inactivation. Monoclonal lesions included endometrial carcinoma, endometrial polyps, and atypical endometrial hyperplasias, whereas normal and anovulatory endometrium were polyclonal. Comparison of the specific X chromosome copy preferentially inactivated by the matched monoclonal cancers and associated monoclonal lesions allowed us to exclude polyps, but not endometrial hyperplasias, as potential precancers. The repetitive genetic marker (HUMARA) for X inactivation was altered in some cancers, permitting identification of microsatellite instability (RER+). Two patients with RER+ cancers also had adjacent RER+ hyperplasias. The seven monoclonal and two RER+ hyperplasias had focal or diffuse cytological atypia, a feature previously associated with risk for endometrial cancer. We conclude that: (a) putative endometrial precancers and cancers share a monoclonal growth pattern; (b) cancers with microsatellite instability may acquire this feature as precancers; and (c) monoclonal endometrial precancers have the morphology of hyperplasias, which vary in the extent of cytological atypia and degree of architectural complexity.
Squamous neoplasms of the female genital tract, including vulvar intraepithelial neoplasia, presumably are derived from a single cell. This study addressed this hypothesis and determined the clonal ...status of other squamous epithelial alterations associated with vulvar carcinoma, including hyperplasia and lichen sclerosis. X chromosome inactivation patterns of 22 epithelial lesions and matched normal epithelium were determined using a polymerase chain reaction (PCR)-based assay targeting the X-linked human androgen receptor gene (HUMARA). Clonality was inferred by comparing matched lesional and control tissues as follows: 1) monoclonal, if intensity of either PCR product was skewed relative to normal reference epithelium (control), 2) polyclonal, if both lesional and control were unskewed, and 3) unknown, if both lesion and control tissues were skewed toward the same allele. Two cases were excluded because of noninformative homozygous HUMARA alleles. Of 8 vulvar intraepithelial neoplasias analyzed, 7 were scored monoclonal and 1 polyclonal. Of 12 hyperplasias, 6 were monoclonal, including one with lichen sclerosis, 2 were polyclonal, and in 4, the clonal status could not be determined. The PCR-based clonal assay supports a monoclonal derivation for vulvar intraepithelial neoplasia and, in some cases, vulvar hyperplasia, and lichen sclerosis. The finding of monoclonal hyperplasia and lichen sclerosis suggests that clonal expansion may evolve before the development of morphological atypia in these epithelia.
The pathogenesis of carcinoma of the vulva is diverse and includes both human papilloma virus (HPV)-positive and HPV-negative pathways. The objective of this study was to correlate the morphology ...with patterns of loss of heterozygosity (LOH) within four vulvar carcinomas and in adjacent vulvar epithelia. Tumors were categorized as HPV positive or negative by polymerase chain reaction (PCR) analysis. Forty-one different sites of normal squamous mucosa, hyperplasia, vulvar intraepithelial neoplasia (VIN), and carcinoma were microdissected in duplicate, and each extracted DNA was analyzed in duplicate for LOH at 10 chromosomal loci by PCR and polyacrylamide gel electrophoresis. Patterns of LOH were compared within different sites of tumors and between the tumor and the noninvasive epithelia. Of three tumors with multiple invasive foci analyzed, divergent patterns of LOH were identified in two, correlating in one with differences in tumor grade. In one HPV-16-positive case, multiple sites of VIN displayed heterogeneity for LOH consistent with divergent clonal or subclonal populations, some of which were not shared by the tumor. In one HPV-negative case, LOH was found in foci of hyperplasia and differentiated VIN (atypical hyperplasia), the latter sharing LOH with the invasive carcinoma at some but not all chromosomal loci. This study suggests that a genetic relationship exists between VIN and carcinoma, irrespective of HPV involvement. It also suggests that in HPV-negative tumors, allelic loss may predate the onset of invasive carcinoma and, in some cases, cellular atypia (VIN). However, the divergent patterns of LOH observed imply that many genetic alterations in the adjacent vulvar epithelium are not directly related to the invasive carcinoma.
An early genetic change in the pathway to colorectal cancer is a mutation in the adenomatous polyposis coli (
APC
) gene. On the basis of the premise that cells with mutant
APC
genes are shed into ...the feces, these investigators devised a powerful molecular method to find such genes in feces from patients with colorectal cancer. Whereas feces from normal subjects had no detectable mutant
APC
genes, stools from over half the patients with colorectal cancer or colonic polyps contained such genes.
Stools from over half the patients with colorectal cancer or colonic polyps contained mutant
APC
genes.
Several strategies for the early detection of colorectal tumors have been devised. Colonoscopy, sigmoidoscopy, and barium enemas are highly specific and sensitive tests for neoplasia,
1
–
4
but they are invasive and limited by the availability of experts in the procedures and patient compliance.
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,
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Testing for occult blood in the stool has been shown in some studies to reduce the incidence of and morbidity and mortality from colorectal cancer.
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11
These fecal occult-blood tests are noninvasive and extremely useful but not sufficiently sensitive or specific for neoplasia.
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15
Furthermore, some fecal occult-blood tests require patients to change their diet before . . .
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