B155
Screening for pancreatic cancer combining k-ras mutations in plasma and the detection of circulating tumour cells
Mavis Orizu, Li Yan, Craig Williams, Seonaid Murray, John P Neoptolemos, William Greenhalf
University of Liverpool, Liverpool, Merseyside, UK
Background
DNA appears in plasma from patients with cancer, this may originate from circulating cancer cells, necrosis of cancer cells in-situ or benign cells. K-ras codon 12 mutations are common in pancreatic diseases and are considered an early event in tumourigenesis. This may be a useful target in the molecular detection of cancer. Cancer cells can be isolated using surface antigens and DNA from these cells analysed, this can be compared to detection of these mutations from cell-free plasma DNA.
Aim
To apply Amplification Refractory Mutation System (ARMS) PCR analysis to the molecular detection of K-ras mutations in plasma and in circulating cancer cells.
Method
DNA was isolated from the plasma of 53 patients with assumed pancreatic cancer and chronic pancreatitis and was subjected to real time PCR using the ARMS method. Oligonucleotide primers specific for wild-type and mutant K-ras were utilised in ARMS-PCR of all DNA samples. Threshold cycles for each of 6 mutant specific primer sets were plotted against cycles with control primers and the sample classified on this basis as mutant or wild type. Cell lines with known mutations were spiked into blood and cancer cells identified with EpCAM and CEA antibodies.
Results and Conclusion
K-ras codon 12 mutations were detected in the plasma of 17 patients (32%). Of these, 82% were substitution of Valine for Glycine (G12V). 4 patients (23%) had G12D (aspartate) mutations. In 1 patient with chronic pancreatitis, both G12D and G12V substitutions were present. None of the other codon 12 mutations were detected in our cohort.
We have shown that gene mutation analysis can be used to identify circulating DNA and to detect K-Ras mutations in patients with pancreatic cancer and in chronic pancreatitics. We anticipate that in combination with selection of cancer cells in blood higher specificity can be obtained.
