factors influencing the detection of mutant k-ras in the serum of patients with colorectal cancer
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Factors Influencing the Detection of MutantK-ras in the Serum of Patients with Colorectal Cancer
ROBYN WARD,a,b CATHERINE SHEEHAN,a MARK NORRIE,a TANYA APPLEGATE,c CAROLINE FUERY,c HELEN IMPEY,c
NICHOLAS HAWKINS,a AND ALISON TODDc
aDepartment of Medical Oncology, St. Vincent’s Hospital, Darlinghurst 2010, AustraliacJohnson and Johnson Research, Rushcutters Bay 2010, Australia
DNA from neoplastic cells can be found in the serum of individuals with cancer. TheK-ras oncogene has been used as a marker for tumor DNA circulating in the serum.The aim of this study was to identify those pathological characteristics of colorectalcancer that may affect the ability to detect the presence of this marker in the serum.In addition, we describe a novel technology, known as DzyNA-PCR, which allowsaccurate quantification of the levels of DNA circulating in serum.
Tumors were collected from 100 individuals with colorectal cancer, and serumwas collected preoperative from these same patients. DNA extracted from tumor andserum samples from these patients was analyzed for the presence of mutations of K-ras using REMS-PCR.1 Tumors from 28 patients were shown to harbor a point mu-tation at codon 12 of K-ras. Analysis of the serum DNA from patients with ras-positive tumors demonstrated the presence of circulating mutant K-ras in 8 of the 28samples, including 5 of the 12 individuals with Astler Coller B2 cancer (42%) andall of the 3 patients with metastatic disease (Astler Coller D). Interestingly, only 1of the 8 seropositive individuals had nodal metastases. There was a significant cor-relation between the presence of detectable circulating mutant K-ras DNA and in-creasing Astler Coller stage (p = 0.003). Overall survival in the patients withcirculating mutant DNA was worse than that in the group in which this marker wasnot detectable in the serum (p = 0.005). There was no significant correlation with Tstage, tumor volume or grade, or patient age. Circulating mutant K-ras was more fre-quent in those tumors where p53 function was normal (p = 0.03).
The overall level of DNA circulating in the serum was also assessed usingDzyNA-PCR. In this assay, PCR reactions are performed with a primer that containsthe complementary sequence of a DNAzyme. Amplicons generated in this reactioncontain active copies of DNAzymes that cleave fluorescent reporter substratespresent in the reaction mix. Real-time fluorometric measurements were performedon the ABI Prism 7700 Sequence Detection System. Experiments where a K-rasplasmid was amplified by DzyNA-PCR indicated that the technology allows quanti-fication of DNA over at least seven orders of magnitude. When the log of the copy
bAddress for correspondence: Department of Medical Oncology, St. Vincent’s Hospital, Victo-ria Street, Darlinghurst 2010, Australia. Voice: 61-2-92958412; fax: 61-2-92958451.
18 ANNALS NEW YORK ACADEMY OF SCIENCES
number was plotted against the Ct value (i.e., the cycle number at which a thresholdvalue is reached), a standard curve (r = 0.990) was generated. This standard curvewas used to estimate the number of copies of K-ras (wild type and mutant) in serumsamples from seropositive individuals in order to ascertain the overall levels of DNAin the circulation of these patients. In preliminary experiments, the assay detectedbetween 103 and 104 copies of K-ras in DNA from an equivalent of 5 µL of serum.The REMS assay is able to detect 1 mutant K-ras allele in a background of 1000wild-type alleles. Therefore, it is possible that, in at least some serum samples, theabsolute number of mutant alleles in the equivalent of 5 µL of serum may be belowthe level of detection of the REMS-PCR assay.
We conclude that detection of mutant K-ras in the serum of patients with colorec-tal cancer may indicate an adverse prognosis. It is interesting that patients withoutnodal metastases exhibit circulating mutant DNA and a worse prognosis, and thismay suggest a predilection for hematogenous metastases in this group. The impor-tance of quantification has been illustrated through the use of a novel DzyNA-PCRmethodology. The development of accurate methods for the measurement of tumorDNA in serum will be necessary before the true significance of this phenomenon canbe determined.
REFERENCE
1. WARD, R.L., N.J. HAWKINS, R. O’GRADY, C. SHEEHAN, T. O’CONNOR, H. IMPEY, C.FUREY & A. TODD. 1998. REMS-PCR—a novel assay for the detection of K-rasmutations in clinical samples. Am. J. Pathol. 153: 373–379.