Anthony P. Shuber

PCR Quantitation of Fetal Cells in Maternal Blood in Normal and Aneuploid Pregnancies

Fetal cells in maternal blood are a noninvasive source of fetal genetic material for prenatal diagnosis. We determined the number of fetal-cell DNA equivalents present in maternal whole-blood samples to deduce whether this number is affected by fetal karyotype. Peripheral blood samples were obtained from 199 women carrying chromosomally normal fetuses and from 31 women with male aneuploid fetuses. Male fetal-cell DNA-equivalent quantitation was determined by PCR amplification of a Y chromosome-specific sequence and was compared with PCR product amplified from known concentrations of male DNA run simultaneously. The mean number of male fetal-cell DNA equivalents detected in 16-ml blood samples from 90 women bearing a 46,XY fetus was 19 (range 0-91). The mean number of male fetal-cell DNA equivalents detected in 109 women bearing a 46,XX fetus was 2 (range 0-24). The mean number of male fetal-cell DNA equivalents detected when the fetus was male compared with when the fetus was female was highly significant (P = .0001). More fetal cells were detected in maternal blood when the fetus was aneuploid. The mean number of male fetal-cell DNA equivalents detected when the fetal karyotype was 47,XY,+21 was 110 (range 0.1-650), which was significantly higher than the number of male fetal-cell DNA equivalents detected in 46,XY fetuses (P = .0001). Feto-maternal transfusion of nucleated cells appears to be influenced by fetal karyotype. The sixfold elevation of fetal cells observed in maternal blood when the fetus had trisomy 21 indicates that noninvasive cytogenetic diagnosis of trisomy 21 should be feasible.

Analysis of Mutations in DNA Isolated From Plasma and Stool of Colorectal Cancer Patients

BACKGROUND & AIMS Somatic mutations provide uniquely specific markers for the early detection of neoplasia that can be detected in DNA purified from plasma or stool of patients with colorectal cancer. The primary purpose of the present investigation was to determine the parameters that were critical for detecting mutations using a quantitative assay. A secondary purpose was to compare the results of plasma and stool DNA testing using the same technology. METHODS We examined DNA purified from the stool of 25 patients with colorectal cancers before surgery. In 16 of these cases, plasma samples also were available. Mutations in stool or plasma were assessed with an improved version of the BEAMing technology. RESULTS Of the 25 stool DNA samples analyzed, 23 (92%) contained mutations that were present in the corresponding tumors from the same patients. In contrast, only 8 of the 16 (50%) plasma DNA samples analyzed had detectable levels of mutated DNA. We found that the DNA fragments containing mutations in both stool and plasma DNA typically were smaller than 150 bases in size. The sensitivity of the new method was superior to a widely used technique for detecting mutations, using single base extension and sequencing, when assessed on the same samples (92% vs 60%; P = .008, exact McNemar test). CONCLUSIONS When assessed with sufficiently sensitive methods, mutant DNA fragments are detectable in the stool of more than 90% of colorectal cancer patients. DNA purified from stool provides a better template for mutation testing than plasma.

Detection of proximal colorectal cancers through analysis of faecal DNA

Detection of mutations in faecal DNA represents a promising, non-invasive approach for detecting colorectal cancers in average-risk populations. One of the first practical applications of this technology involves the examination of microsatellite markers in sporadic cancers with mismatch-repair deficiencies. Since such cancers nearly always occur in the proximal colon, this test might be useful as an adjunct to sigmoidoscopy, which detects only distal colorectal lesions. We report here the first in-depth analysis of faecal DNA from patients with proximal cancers to determine the feasibility, sensitivity, and specificity of this approach. Using a sensitive method for microsatellite mutation detection, we found that 18 of 46 cancers had microsatellite alterations and that identical mutations could be identified in the faecal DNA of 17 of these 18 cases.

Stool screening for colorectal cancer: Evolution from occult blood to molecular markers

BACKGROUND Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. Participation rates for colorectal cancer screening remain low due, in part, to perceived discomfort, potential harm, and high costs with available tools. METHODS Stool testing, unlike other conventional screening approaches, is noninvasive and requires no cathartic preparation. However, widely used fecal blood tests yield frequent false-negative and false-positive results that lower screening effectiveness and raise program costs. There is a compelling biological rationale to target DNA alterations exfoliated from neoplasms into stool, and multiple DNA markers would need to be assayed because of the genetic heterogeneity of colorectal neoplasia. Early clinical studies with this multi-target DNA-based stool assay approach suggest high sensitivity for both colorectal cancer and premalignant adenomatous polyps while maintaining high specificity. CONCLUSIONS This apparently accurate and user-friendly new approach holds promise to improve the effectiveness, efficiency, and appeal of colorectal cancer screening. Large-scale clinical studies are clearly warranted to corroborate the early results.

DNA Integrity as a Potential Marker for Stool-based Detection of Colorectal Cancer

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.

Fetal cells in maternal blood: determination of purity and yield by quantitative polymerase chain reaction.

OBJECTIVE The detection of fetal aneuploidy and gene mutations by analysis of fetal cells in maternal blood has demonstrated the feasibility of noninvasive prenatal diagnosis. Fetal cells are rare in the maternal circulation; all current methods used for their isolation also yield maternal cells. We developed a method that permits a quantitative assessment of the relative numbers of fetal and maternal cells. STUDY DESIGN Samples from 40 pregnant women were flow sorted with different monoclonal antibodies. Deoxyribonucleic acid was subsequently purified from candidate fetal cells; polymerase chain reaction was performed with synthetic primers specific for sequences on chromosomes Y and 7. RESULTS The maximum number of fetal cells detected was 52 in 1080 maternal cells. Fetal cell purity ranged from 0.001% to 4.8%. Fetal cells were detected with antibodies to CD71, CD36, and glycophorin A. CONCLUSION Quantitative polymerase chain reaction enables the determination of the purity and yield of fetal cells remaining after isolation from maternal blood, facilitating rapid comparisons between different cell separation techniques.

Enhanced Retrieval of DNA from Human Fecal Samples Results in Improved Performance of Colorectal Cancer Screening Test

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 = 0.0005 (by McNemars test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.

DNA stabilization is critical for maximizing performance of fecal DNA-based colorectal cancer tests.

We have developed a multitarget, fecal DNA screening assay that detects the presence of gene-specific mutations and long DNA fragments associated with colorectal cancer (CRC). We continue to investigate methods that may be used to optimize clinical sensitivity. The goals of this investigation are to establish how sample handling conditions affect the stability of DNA in stool, thereby potentially limiting clinical sensitivity, and to determine conditions to ameliorate DNA degradation. A study was run comparing paired sample aliquots. Quantitative PCR data for matched aliquots was used to determine first the effect of sample incubation on total recovery and integrity of DNA, then the effect of stabilization buffer addition to stool on recoverable DNA, and finally the impact of buffer addition on assay sensitivity. Comparison of quantitative PCR data for paired aliquots shows that the amount of recoverable human DNA is negatively affected by storing stool samples (N = 43) at room temperature for ≥36 hours (P = 0.0018). However, the addition of stabilization buffer leads to a significant increase in recovery of DNA (P = 0.010), compared with samples incubated without buffer. Whereas the DNA Integrity Assay (DIA) is found to be sensitive to DNA degradation (sensitivity was reduced by 82%; P = 0.0002), point mutation marker sensitivity is more refractory. Overall, DNA can be stabilized by addition of buffer to the sample, leading to increased assay sensitivity.

Development of a model system to compare cell separation methods for the isolation of fetal cells from maternal blood

Three major methods have been described for the isolation of fetal cells from maternal blood: fluorescence‐activated cell sorting (FACS), immunomagnetic beads, and magnetic‐activated cell sorting (MACS). To date, no study has directly compared fetal cell recovery using each of these methods. Here we describe our system using a ‘model’ male fetal cell mixed into female peripheral blood mononuclear cells. Fetal cell yields and purities were assayed by a quantitative polymerase chain reaction (qPCR) using chromosomes Y‐ and 7‐specific sequences. Fetal cell recovery was investigated by selection of CD71+ cells or depletion of CD45+ cells. Our data demonstrated variation in fetal cell recovery for all methods tested, although CD71+ selection by FACS gave the best and most consistent results.

DNA Integrity Assay: A Plasma-Based Screening Tool for the Detection of Prostate Cancer

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.