Vol. 4, 271-2 75, February 1998 Clinical Cancer Research 271 Advances in Brief A Prospective Study of K-ras Mutations in the Plasma of Pancreatic Cancer Patients1 Hugh E. Mulcahy, Jacqueline Lyautey, Christine Lederrey, Xu qi Chen, Philippe Anker, Elspeth M. Alstead, Anne Ballinger, Michael J. G. Farthing, and Maurice Stroun2 Digestive Diseases Research Centre, St. Bartholomew’s and The Royal London School of Medicine and Dentistry, London El 2AD, United Kingdom [H. E. M., E. M. A., A. B., M. J. G. F.], and D#{233}partementde Biochimie et de Physiologic V#{233}g#{233}tale, Universit#{233}de Gen#{232}ve, 121 1 Geneva, Switzerland [J. L., C. L., X. q. C., P. A.. M. S.] Abstract K-ras mutations are frequently found in primary pan- creatic adenocarcinomas. In this prospective study, we looked for K-ras mutations in the plasma of patients with pancreatic cancer. We isolated plasma DNA from 21 pan- creatic cancer patients using a simple and rapid extraction technique and detected K-ras alterations with a PCR assay and subsequent product sequencing. Patients were followed up to determine their clinical outcome We found K-ras mutations in the plasma of 17 patients (81 %). In cases in which both plasma and pancreatic tissue were available, DNA mutations were similar in corresponding plasma and tissue samples. Plasma DNA alterations were found 5-14 months before clinical diagnosis in four patients. Mutant DNA was not found in the plasma of two patients with chronic pancreatitis or in five healthy controls. Our results indicate that K-ras mutations are often found in DNA iso- lated from the plasma of pancreatic cancer patients and that a noninvasive plasma-based assay may provide qualitative diagnostic information to clinicians in the future Larger studies are required to further assess the relevance of our findings to clinical practice. Introduction Pancreatic adenocarcinoma is the fourth most common cause of cancer death in the Western world. Median survival after diagnosis is less than 6 months (1), and long-term survival rates are less than 5% (2), partly because symptoms usually occur only at a late pathological stage. Even when symptoms are Received 6/23/97; revised I 1/12/97; accepted I 1/12/97. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I Supported by the Joint Research Board of St. Bartholomew’s Hospital, the Ligue Suisse Contre Ic Cancer (Grant SKL 294-2-1996), and the 0. J. Isvet Fund (Grant 747). 2 To whom requests for reprints should be addressed, at Laboratoire de Biochimie et de Physiologic V#{233}g#{233}tale, Universit#{233}de Gen#{232}ve, PavilIon des Isotopes, 20 Boulevard d’Yvoy, 121 1 Geneva, Switzerland. present, pancreatic adenocarcinoma can be difficult to differen- tiate from other conditions on the basis of clinical features and imaging investigations. Thus, a false positive diagnosis is corn- mon (3-5), whereas fine-needle aspiration or biopsy yields a false negative result in 10-40% of cases (6). Serum markers have been developed to complement conventional diagnostic tests (2), but even the most promising quantitative assays rely on a cutoff point above or below which a diagnosis of pancreatic cancer is more or less likely. The K-ras gene is mutated in over 90% of pancreatic cancers (7). These mutations are well defined, reliably detected by DNA amplification assays, and occur early in the genesis of pancreatic cancer (8), indicating that mutant K-ras might serve as a qualitative marker for pancreatic cancer (9-12). K-ras alterations have been found in pancreatic cancer tissue obtained by fine-needle aspirate (9, 13) and in pancreatic duct secretions obtained endoscopically (10, 1 1). Mutant DNA has also been extracted from pancreatic cancer cells found circulating in pe- ripheral blood (1 2). However, this phenomenon seems to be restricted to intraoperative and early postoperative periods (14), making it unsuitable for diagnostic purposes. In addition to its presence within circulating cells, DNA is also found circulating in blood plasma. Nanogram quantities are present in the plasma of normal subjects (15, 16), and increased quantities circulate in patients with chronic autoimmune disor- ders such as systemic lupus erythematosus ( I 6). Cancer patients have even higher quantities of serum or plasma DNA (17-20); the highest levels are found in those with pancreatic cancer (21). DNA extracted from the plasma of cancer patients also displays neoplastic characteristics such as decreased strand stability (22). Studies using PCR have identified microsatellite alterations in the plasma and serum DNA of patients with head and neck cancer (23) and lung cancer (24). In addition, ras gene muta- tions have been detected in the plasma of patients with colorec- tal cancer (25) and hematological malignancies (26). Finally, Sorenson et a!. (27) found mutant plasma DNA in three patients with pancreatic cancer. In our prospective study, we isolated DNA from the plasma of patients with a suspected diagnosis of pancreatic cancer and analyzed this DNA for K-ras mutations. Materials and Methods Patients. This study included 21 patients with pancreatic cancer (17 with a firm diagnosis at the time of plasma sampling and 4 who were undergoing investigations for possible pancre- atic cancer at the time of sampling). The median age ofthe study population was 60 years (range, 41-88 years; 9 males and 12 females). Diagnosis was established by a combination of pan- creatic biopsy or aspirate, abdominal ultrasound, computed to- mography, and endoscopic retrograde cholangiopancreatogra- phy. All patients had unresectable disease at diagnosis and were treated by endoscopic biliary stenting and/or chemotherapy and radiotherapy. Patients were followed for a median of 5.5 months (range, 1.2-16.7 months) and follow-up ended in March 1997. Research. on November 28, 2021. © 1998 American Association for Cancer clincancerres.aacrjournals.org Downloaded from