Affiliations of authors: M. S. Kopreski, OncoMEDx, Inc., Columbia, MD; F. A. Benko, D. J. Borys (Department of Pathology), A. Khan, T. J. McGarrity (Department of Medicine), Penn State GeisingerHershey Medical Center, PA; C. D. Gocke, Department of Pathology, Penn State GeisingerHershey Medical Center and OncoMEDx, Inc.
Correspondence to: Christopher D. Gocke, M.D., Department of Pathology, Penn State GeisingerHershey Medical Center, P.O. Box 850, Hershey, PA 17033 (e-mail: cgocke{at}psghs.edu).
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ABSTRACT |
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INTRODUCTION |
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It has been demonstrated that mutated oncogene DNA sequences can be detected in the plasma and serum of patients with cancer (511). Extracellular DNA appears to be present regardless of mutation or tumor type; mutations in K-ras, N-ras, or p53 genes are demonstrable in the DNA extracted from plasma or serum of patients with advanced pancreatic cancer (5,6), colorectal cancer (710), and leukemia (11). Similarly, circulating immunoglobulin heavy chain DNA has been shown in patients with lymphoma (12), and microsatellite DNA alterations have been detected in plasma or serum from patients with lung cancer (13), head and neck cancer (14), and renal cancer (15). These studies demonstrate that plasma and serum provide readily accessible genetic material in patient populations having clinically obvious disease. However, it remains be clarified whether mutated oncogene DNA sequences are detectable in the plasma or serum of patients with solid tumors at a premalignant stage or with clinically nonevident altered tissue, a likely requirement for screening strategies based on detection of somatic mutations. We define somatic mutation screening as the identification of individuals or populations who carry specific somatic mutations in their blood or other body fluids.
In this study, we evaluate the use of plasma DNA analysis as a method for somatic mutation screening. Mutations in the K-ras oncogene are seen in a variety of malignancies, including those of the lung (16), the pancreas (17), and the colon (18). K-ras mutations are common in both premalignant and malignant colorectal tissues and are present in 13%73% of aberrant crypt foci (19,20), in 15%75% of adenomatous polyps (21), and in approximately 40% of colorectal cancers (18). We thus anticipated that, by assaying for K-ras gene mutations in DNA obtained from the plasma of individuals undergoing colorectal evaluation, the feasibility of plasma-based somatic mutation screening could be assessed.
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PATIENTS AND METHODS |
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Five to 10 milliliters of peripheral blood was collected in EDTA-coated siliconized glass Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) from 240 patients referred to our colonoscopy clinic either for symptomatic evaluation (143 patients), for evaluation of asymptomatic high-risk individuals (92 patients), or for evaluation of asymptomatic individuals without risk factors (five patients). Blood was obtained immediately prior to the colonoscopy procedure. All protocols were approved by the Institutional Review Board (Penn State College of Medicine) and all patients signed a written informed consent. Plasma was fractionated from blood on the day of collection by centrifugation at 400g at room temperature for 10 minutes, divided into aliquots, and then stored frozen at -20°C until assayed.
One hundred thirty-five patients had tissue specimens obtained after polypectomy, colorectal biopsy, or colorectal surgery. Tissue specimens were obtained only from sites of suspected pathologic abnormalities, including those of suspected neoplasia, colitis, inflammation, or abnormal mucosal appearance. Diagnosis of neoplasia was based solely on histopathologic findings. If multiple adenomas were diagnosed in a given patient, effort was made to assay all available tissue specimens.
DNA Extraction
Extracellular DNA was extracted from plasma by use of a gelatin precipitation method as described previously (7). Tissue DNA was obtained from available paraffin blocks. Two paraffin sections were cut at a thickness of 15 µm with the use of ethanol-cleaned microtome blades. The tissue was placed in microfuge tubes, the paraffin was removed, and the DNA was extracted as described previously (7).
K-ras Gene Amplification
DNA extracted from plasma and tissue was amplified with a polymerase chain reaction (PCR)-based assay that enriches for mutations in K-ras codon 12. The assay employed simultaneous restriction enzyme digestion and PCR amplification (combined amplification and restriction digestion), thereby shortening the overall time required for analysis, and is similar to previously reported assays (7,22).
A reaction mixture was prepared containing 35 µL of cold-extracted DNA solution from patient plasma or from patient tissue; 50 mM KCl; 10 mM TrisHCl (pH 9.0); 0.1% Triton X-100; 1.5 mM MgCl2; 200 µmol/L each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate; 22.8 pmol of K-ras primer 1 (5'-ACTGAATATAAACTTGTGGTAGTTGGACCT-3'); 11.4 pmol of K-ras primer 2 (5'-TCAAAGAATGGTCCTGGACC-3'); 4 U BstNI restriction endonuclease (Stratagene Cloning Systems, La Jolla, CA); and 1 U Taq polymerase (Promega Corp., Madison, WI). This reaction mixture was overlaid with mineral oil and cycled in a PHC-2 thermocycler (Techne, Princeton, NJ) where the reaction was incubated at 94°C for 7 seconds, then at 60°C for 3 minutes, then at 94°C for 6 seconds, then again annealed, extended, and digested at 60°C for 3 minutes, then incubated at 94°C for 5 seconds, and so on, decreasing the duration of the 94°C denaturation by 1 second each cycle until after six cycles the denaturation lasts only 1 second. Thereafter, cycles with 1-second denaturation steps and 3-minute extension and digestion steps were performed, for a total of 40 cycles in the amplification reaction. After the initial eight cycles, the reaction was paused at 60°C, and an additional 10 U of BstNI restriction enzyme was added. At the completion of the 40-cycle reaction, 10 U of BstNI restriction enzyme was again added directly to the cycling reaction tube, and the mixture was incubated at 60°C for 12 hours.
All amplification assays included a K-ras mutation-positive control consisting of the colon carcinoma cell line FET (K-ras codon 12 mutation; GGT > GCT), a wild-type K-ras (mutation-negative) control consisting of DNA from normal placenta tissue, and a negative control lacking DNA. Particular attention was paid to prevent contamination (23). The risk of contamination yielding false-positive results was further minimized by repeating the assay on all patient plasma samples for a minimum of two separate times on different days by use of different aliquots of the frozen plasma specimen. Results from both the gel electrophoresis and the dot blot hybridization were always interpreted with the investigator blinded to the information regarding clinical findings, corresponding tissue analysis, and previously collected data on patient plasma.
Detection of K-ras Mutations
Mutations in the final digested amplified product were identified by agarose gel electrophoresis as described previously (7). In all plasma specimens, the identification of altered bases was performed by use of dot blot hybridization. Following amplification and before any final enzyme digestion, 5 µL was applied to a nylon membrane (MSI, Westboro, MA). Replicate blots were hybridized to 32P-radiolabeled oligonucleotides designed to identify point mutations in positions 1 and 2 of codon 12 of the K-ras gene (Mutaprobe Human K-ras 12 set; Oncogene Science, Inc., Uniondale, NY). Hybridization and wash conditions were as specified by the membrane's manufacturer. Blots were exposed to x-ray film for 10 hours at -80°C. All blots included positive controls for each point mutation and negative controls consisting of placental DNA and aliquots from the amplification-processed water blanks. Tissue specimens that were detected as mutation positive by gel electrophoresis were also characterized by dot blot hybridization.
Clinical Comparisons
Specific clinical data were obtained by direct questioning of the patient and by confirmatory or supplemental retrospective review of patient records. These data included patient age, sex, colonoscopy indication, and risk factors for colorectal cancer (defined as history of colorectal cancer, history of colorectal polyps, history of inflammatory bowel disease, or family history of colorectal cancer in either a first-degree relative or in a second-degree relative under age 35 years). Colonoscopy findings were recorded at the time of evaluation by the endoscopist. Pathology reports on all tissue biopsy specimens were reviewed.
Statistical Analysis
Groups were compared by use of Fisher's exact test unless otherwise specified. All tests of statistical analysis were two-sided.
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RESULTS |
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Colonoscopy
Neoplasms were demonstrated by colonoscopy in 70 (29%) of 240 patients. Of these, eight patients had colorectal cancer (seven invasive and one carcinoma in situ), and 62 patients had adenomatous polyps but no cancer. The number of adenomas varied from one to six, with approximately one half of these patients having a single adenoma. Seven of eight patients with cancer had concurrent adenoma. In 65 additional patients, pathologic review indicated non-neoplastic tissue only, including hyperplastic tissue, colitis, or nondiagnostic histopathology. Overall, 170 patients had either normal colonoscopy not requiring biopsy or biopsy but no neoplastic findings in pathology. Since repeat tandem colonoscopies were not performed, the rate of missed neoplasia (false-negative colonoscopy) in this group could not be determined. However, one patient showing mutated K-ras DNA in the plasma who was being evaluated for metastatic adenocarcinoma of the liver had an initial negative colonoscopy. A repeat colonoscopy within a brief interval revealed a primary colorectal carcinoma with a mutation in the K-ras gene. This patient is reported among the eight cancer patients.
Tissue Mutations
A tissue was defined as ras mutation positive if amplified mutated gene sequences were detectable by gel electrophoresis and the mutation was confirmed by dot blot hybridization. In patients having multiple neoplasms, demonstration of a K-ras mutation in a single neoplasm was sufficient to characterize the patient as having K-ras mutation-positive tissue. Overall, K-ras mutations in tissue were demonstrated in 35 patients, including 30 (43%) of 70 patients having neoplasia. Mutations were demonstrated in tissue from five of eight patients with colorectal cancer, including four patients with carcinomas with K-ras mutations and a fifth patient with a concurrent mutation-positive adenoma but mutation-negative carcinoma. Of 62 patients with adenoma alone, 25 (40%) had a K-ras mutation.
Tissue specimens were available from 65 additional patients without evidence of neoplasia. K-ras mutations were demonstrated in non-neoplastic tissue from five of these patients. Two patients were found to harbor a K-ras mutation in their hyperplastic polyps. A K-ras mutation was demonstrated in the tissue of one patient with colitis. In two additional patients, a histologically normal-appearing mucosal specimen had tissue with mutated K-ras.
Sequence analysis by hybridization confirmed mutations to be present in all of the tissues deemed mutant K-ras positive. While a single mutation was found in some neoplastic tissue, multiple mutations consistent with more than one clone were demonstrated in 20 (67%) of 30 neoplasms with K-ras mutation. Other investigators (24,25) have reported observing multiple K-ras mutations in the same neoplastic tissue. The predominant mutation detected in neoplastic tissue in this study was GGT > GAT in 20 patients, GGT > GCT in eight, and GGT > GTT in two.
Plasma K-ras DNA
Mutant K-ras oncogene DNA was demonstrated in the plasma of 64 patients (27%) (Fig. 1). In 59 patients with mutated K-ras DNA in their plasma, results were consistently reproducible on retesting of paired specimen aliquots. In an additional five patients, only one aliquot tested positive. The placenta and non-DNA-containing negative controls always tested negative, while the positive control always tested positive. Overall assay sensitivity varied with the method utilized in detecting the amplified PCR product. A mutated K-ras product from plasma was detectable by gel electrophoresis in 52 patients, with all results reproducible. Dot blot hybridization reproducibly detected a mutated product in the same 52 patients (Fig. 2
). However, dot blot hybridization permitted detection of mutated K-ras DNA in the plasma of an additional 12 patients not otherwise detectable by gel electrophoresis, including plasma from five patients having neoplasia containing a mutant ras gene. In five of these 12 patients, the dot blot was positive in only one of two aliquots, although three of these five had ras-mutated adenoma. These results suggest that, in some cases, circulating levels of mutant ras DNA were near the threshold of assay detection. In all patients, some wild-type K-ras DNA was identifiable by dot blot hybridization.
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Overall, the presence of mutant K-ras DNA in plasma shows statistically significant association with risk factors for colorectal cancer (Table 2). K-ras mutations were detectable in the plasma of 20 (61%) of 33 patients with a history of colorectal cancer, in 38 (38%) of 101 with a history of colorectal polyps, in 25 (42%) of 59 with a family history of colorectal cancer, in three (20%) of 15 with inflammatory bowel disease, and in 26 (57%) of 46 having two or more risk factors. Only seven patients with mutated DNA in the plasma and normal colonoscopies were without risk factors. Of interest, in five of these patients, the mutated plasma DNA could only be detected with the use of dot blot hybridization. It is possible that quantitative differences in circulating levels of mutated DNA sequences might exist between patients with and without risk factors, perhaps reflecting the overall burden of mutation-containing tissue. The finding of mutated K-ras DNA in plasma was not associated either with age (the mean age of patients with mutated DNA was 62.4 years [standard deviation, 14.8] and without mutated DNA was 58.5 years [standard deviation, 15.1] [P = .08, two-sided t test]) or with sex (mutated DNA in plasma in 33 males and 31 females).
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DISCUSSION |
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As shown in this study, individuals with mutant oncogene DNA in their plasma often harbor neoplasms having the same mutation. The ability of plasma DNA to serve as a marker for neoplasia enables somatic mutation screening to delineate a patient population likely to benefit from additional diagnostic evaluation and may further provide a prognostic marker. While K-ras mutations are common to multiple tumor types, in our patient population, the comparison between mutated DNA in plasma and colorectal neoplasia or colorectal cancer risk factors supports a colorectal origin for the mutated DNA. However, since oncogene mutations may occur in cancers arising at any of multiple sites, acceptable and cost-effective paradigms will be needed to evaluate the asymptomatic patient who tests positive for circulating mutant oncogenes during screening. In this study, only five patients were asymptomatic and without risk factors, suggesting caution be used in extrapolating our study results to the more general population. Furthermore, should plasma-based DNA assays be employed as a primary screen for specific cancers, optimization of diagnostic sensitivity would likely require a multiplexed approach employing multiple oncogene markers. In this regard, we have similarly been able to demonstrate P53 and APC mutations in the plasma and serum of patients with colorectal neoplasia (26).
While plasma DNA appears to offer a sensitive means of detecting neoplastic disease, this study also suggests the ability to detect mutant gene DNA in plasma derived from patients with non-neoplastic or clinically nonevident mutated tissue. Among patients having mutated K-ras DNA in the plasma, 58% failed to demonstrate neoplasia on colonoscopy. While it is recognized that colonoscopy may miss frank neoplasia (27,28), with reported overall miss rates for adenoma of 24% (28), it is likely that, in the majority of cases, our findings reflect the presence of clinically nonevident abnormal tissue with mutated DNA. It is unlikely that these plasma findings are artifactual, since results were reproducible, controls always tested appropriately, and mutated gene sequences could always be confirmed. The significant association between plasma with mutated DNA and cancer risk factors in this group with normal findings on colonoscopy is noteworthy and implies that the assay is stratifying a population likely to carry foci of tissue with mutated genes. One might anticipate that patients having risk factors for cancer would be more likely to develop such foci. These results are consistent with results obtained in other studies (25,29,30) that demonstrate K-ras mutations in biopsy specimens from otherwise normal-appearing colonic mucosa. K-ras mutations have been demonstrated in human aberrant crypt foci (19,20), which might explain reports of K-ras mutations in endoscopically normal-appearing mucosa. Recently, Takayama et al. (31) confirmed that aberrant crypt foci are common, even in a population having normal colonoscopies, being demonstrated in 56% of otherwise normal subjects, in 88% of patients having adenoma, and in 100% of patients having colorectal cancer. Ras mutations were common in these aberrant crypt foci. Other investigators have similarly detected the presence of mutated K-ras DNA in colonic effluent samples (32) and stool (33) from patients with colorectal cancer risk factors who have normal colonoscopies. The ability of our assay to detect circulating mutated oncogene DNA in patients having clinically occult tissue with mutations may hinder identification of the true assay specificity and predictive values because insufficient tissue sampling or inadequate tissue localization could incorrectly suggest false positivity.
At the present time, the prognostic significance of finding mutated oncogene DNA in patients without demonstrable neoplasia is unknown. Furthermore, whether detecting multiple mutated oncogenes within plasma carries additional prognostic implication is not known. Long-term follow-up is not presently available in our patient population. Whether such patients are at increased risk for the development of cancer remains to be clarified by future studies. Plasma-based assays thus offer a minimally invasive method for somatic mutation screening.
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NOTES |
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We thank T. Rinehart for assistance in manuscript preparation. We also thank the attending physicians, fellows, and nurses of the Division of Gastroenterology, Penn State GeisingerHershey Medical Center, for their clinical contributions to the conduct of this study.
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Manuscript received October 15, 1999; revised March 14, 2000; accepted March 30, 2000.
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