Somatic Mutation Screening: Identification of Individuals Harboring K-ras Mutations With the Use of Plasma DNA

Michael S. Kopreski, Floyd A. Benko, David J. Borys, Amin Khan, Thomas J. McGarrity, Christopher D. Gocke

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 Geisinger–Hershey Medical Center, PA; C. D. Gocke, Department of Pathology, Penn State Geisinger–Hershey Medical Center and OncoMEDx, Inc.

Correspondence to: Christopher D. Gocke, M.D., Department of Pathology, Penn State Geisinger–Hershey Medical Center, P.O. Box 850, Hershey, PA 17033 (e-mail: cgocke{at}psghs.edu).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Background: Many cancers are attributed to somatic mutation of DNA. We investigated whether it is feasible to detect cancer-associated somatic mutations in patients with neoplasms by using plasma DNA. Methods: Plasma samples were prospectively collected from 240 patients undergoing colonoscopy. Colorectal biopsies were performed as clinically indicated in 135 patients, and risk factor information was available from 232 patients. DNA was extracted from plasma and colorectal tissue and was amplified by use of a polymerase chain reaction method that enriches for mutations in codon 12 of the K-ras oncogene. Molecular, histologic, and clinical data were compared by use of two-sided Fisher's exact test. Results: Mutations in the K-ras gene detected in the plasma of 64 (28%) of 232 patients were statistically significantly associated with colorectal cancer risk factors (P = .0002). Of those patients having tissue available for comparison (n = 135), mutations in the K-ras gene were found in the tissues of 35 patients, and 29 (83%) of these 35 showed mutations in plasma samples. In contrast, the plasma assay was negative in 93 of the 100 patients whose tissue K-ras was wild-type. Among patients without biopsies (n = 105), 28 had mutated K-ras in their plasma DNA, despite the absence of remarkable colonoscopy findings; 24 of these 28 patients had risk factors for colorectal cancer. Overall, 25 (39%) of 64 patients showing mutations in plasma DNA had colorectal neoplasms with K-ras mutations compared with five (3%) of 176 patients without K-ras mutations in plasma DNA. Conclusion: Plasma DNA assays for the detection of mutations in K-ras codon 12 may provide a feasible method to screen populations for somatic mutations frequently found in neoplasms. The clinical utility of using this test in screening populations requires further study.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Recent approaches to cancer-risk assessment include genetic screening of high-risk individuals for specific germline mutations (1). However, many cancers are associated with acquired somatic mutations rather than with inherited germline mutations. In colorectal cancer, while the germline mutations associated with hereditary nonpolyposis colorectal cancer occur in 2%–10% of patients (2), somatic mutations of the K-ras (also known as KRAS or Kirsten-ras) oncogene, p53 (also known as TP53) tumor suppressor gene, APC (adenomatosis polyposis coli) tumor suppressor gene, or DCC (deleted in colorectal cancer) gene individually are noted in 35%–75% of the cancers (3,4). Development of effective genetic risk-evaluation strategies for most cancers thus needs to incorporate testing for somatic and germline mutations. Unfortunately, the technical challenge of obtaining appropriate genetic material from individuals without clinically evident neoplastic tissue has limited the development of screening strategies based on detection of specific somatic mutations. Since genomic evaluation will play an increasingly important role in cancer prevention, early diagnostics, and pharmacogenomic-based therapeutics, new approaches for assessment of somatic mutations are needed.

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.


    PATIENTS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Patients and Specimens

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 Tris–HCl (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 1–2 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.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
The sensitivity of the enriched PCR assay was greater than one mutant gene of 105 normal genes as determined by serial dilution of the mutated cell line DNA into placental DNA (not shown). Patient ages ranged from 19 to 93 years (mean age, 59.6 years). There were 124 males and 116 females. For eight patients, complete risk assessment could not be made, usually reflecting an unknown family history of colorectal cancer. Of the remaining patients, 152 (66%) of 232 had one or more risk factors for colorectal cancer and 80 (34%) had no risk factors.

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. 1Go). 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. 2Go). 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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Fig. 1. K-ras mutations in plasma. Gel electrophoresis demonstrating representative samples (lanes 1–6) of extracellular DNA analyzed with the combined amplification and restriction digestion technique for K-ras codon 12 mutations. Patients 2 and 4 have mutations, while patients 1, 3, 5, and 6 do not. The uncut amplification product is 157 base pair (bp); digestion with BstNI yields a 142-bp band if mutant and a 113-bp band if wild-type. The positive control is the FET cell line DNA diluted 1:10000 and 1:100000. The negative control is placental DNA. Blank is a control run without DNA.

 


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Fig. 2. Identification of K-ras mutations in patient plasma by dot blot hybridization. Polymerase chain reaction products from plasma DNA were blotted and then probed with radiolabeled oligonucleotides specific for each possible base at the second position of codon 12 of the K-ras oncogene (relevant base listed to the left of each panel). Patient samples are arrayed in identical positions in each replicate; note that some wild-type (wt) DNA was detected in each plasma (G blot). Some patients exhibit more than one type of mutation (e.g., second row, far left: C + A). Positive and negative control DNA was applied to the bottom right corner of each blot; plasmids carrying each variant are indicated, and the wt control was placental DNA. A control blank without DNA is to the right of the G control dot on each blot.

 
Comparisons were made between histopathologic findings and the presence of mutated K-ras DNA in plasma (Table 1Go). Of the eight patients found to have colorectal cancer, five had ras mutations in their neoplasms. These five patients similarly had mutated K-ras DNA in their plasma, including the patient with carcinoma in situ. Of the 62 additional patients with adenoma only, 25 had ras mutation in the neoplasms. Mutated K-ras DNA was demonstrated in the plasma of 20 of these patients, including seven of eight patients having small (<=5 mm) ras mutation-positive adenomas only. Two additional adenoma patients also had mutated K-ras DNA in their plasma, despite the tissue samples demonstrating only the wild-type K-ras gene. Sixty-five patients had non-neoplastic tissue only. Of these, five patients were shown to have K-ras-mutations in their non-neoplastic tissue. Four of these five patients had mutated DNA in their plasma. Five additional patients with non-neoplastic tissue also had mutated K-ras DNA in their plasma, despite their tissue specimens demonstrating only wild-type K-ras. The overall sensitivity for detection of mutated K-ras DNA in the plasma of patients with ras mutations in tissue was 83% (29 of 35). In contrast, the plasma assay was negative in 93 of 100 patients having tissue demonstrating only wild-type K-ras, including 38 of 40 patients having only wild-type K-ras in their neoplasms (i.e., when the tissue is defined, the specificity is 93%). The association of K-ras mutation in DNA in the plasma of patients with ras mutation in DNA in a neoplasm was highly statistically significant (P<.001). Overall, 25 (39%) of 64 patients with K-ras-mutated DNA in their plasma had neoplasia with mutated DNA compared with five (3%) of 176 patients showing no mutations in their plasma samples.


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Table 1. K-ras mutations in tissue and plasma and pathologic findings in patients undergoing colonoscopy procedure
 
While the plasma assay appeared to be both sensitive and specific, 28 of 105 patients without evidence of neoplasia on colonoscopy who had no comparative tissue available also demonstrated mutant K-ras DNA in their plasma. Positive results seen in the plasma specimen were confirmed by blinded repeat testing, making it unlikely that they were artifactual or secondary to contamination. Twenty-four of these patients had significant risk factors for colorectal cancer: 13 with a family history of colorectal cancer and 16 with a history of polyps and/or colorectal cancer. Thus, the findings suggest that the assay is further able to stratify patients with foci of tissue that are not clinically abnormal but have mutated genes.

Overall, the presence of mutant K-ras DNA in plasma shows statistically significant association with risk factors for colorectal cancer (Table 2Go). 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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Table 2. Comparison of patient groups stratified by colorectal cancer risk factors for mutant K-ras sequences in plasma DNA samples (risk-assessable patients, n = 232)*
 
The predominant plasma mutation was GGT > GAT in 42 patients, GGT > GCT in 13, GGT > GTT in seven, GGT > CGT in one, and GGT > AGT in one. However, 51 of 64 patients with mutated DNA in their plasma demonstrated more than one K-ras mutation in their plasma. Among patients with both mutated DNA in their plasma and a colorectal cancer risk factor, 48 (89%) of 54 demonstrated multiple K-ras mutations in their plasma. This included patients in whom the neoplasm obtained from the biopsy demonstrated only a single mutation, further suggesting the presence of multiple mutated tissue foci in patients at risk. In contrast, only three (30%) of 10 patients with mutant K-ras DNA in their plasma but without risk factors had more than one mutational sequence demonstrable in their plasma. Regardless of risk status, mutations present in a neoplasm were demonstrable in the plasma, with the predominant K-ras mutation of the neoplasm identified within plasma in 24 of 25 patients.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Somatic mutation screening determines the presence of specific nongermline mutations that are possibly markers for neoplasia, with the intent of stratifying those individuals likely to benefit from chemoprevention or diagnostic strategies. Effective screening thus requires evaluation of genetic material obtained early in the premalignant–malignant process. While mutated oncogene DNA has been known to be present in the plasma and serum of patients with cancer, the ability to detect circulating mutated oncogene DNA in patients having solid tumor premalignancy has been uncertain, reflecting a paucity of knowledge regarding the pathophysiology of extracellular DNA and questions regarding applicability to premalignant cells. Our results indicate that extracellular mutated DNA circulates in the plasma of patients with premalignant colorectal tissue, being produced in sufficient amount and having sufficiently delayed clearance as to enable systemic detection. The ability of the assay to identify patients having even small adenomas with a mutated K-ras gene further suggests that DNA shed from a single neoplasm is supplemented by DNA from the total burden of colonic epithelium carrying mutations in individuals at risk. Such mechanistic processes are likely to contribute to detectable levels of circulating DNA, despite a small volume of neoplasia and a lack of invasive cellular processes or circulating tumor cells. Overall, our findings suggest that extracellular DNA not only can serve as a marker for neoplasia but also enables molecular characterization and stratification of patients at risk for malignancy. Plasma-based somatic mutation screening thus appears feasible, particularly in patient populations at risk of cancer.

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.


    NOTES
 
Supported by Clinical Research Center grant 96-49 from the M. S. Hershey Medical Center and by funding through the Ben Franklin Partnership Program of Pennsylvania (project No. 97C.1057R-1).

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 Geisinger–Hershey Medical Center, for their clinical contributions to the conduct of this study.


    REFERENCES
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 Notes
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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Manuscript received October 15, 1999; revised March 14, 2000; accepted March 30, 2000.


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