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© 1999 American Society for Clinical Oncology K-ras Mutations in DNA Extracted From the Plasma of Patients With Pancreatic Carcinoma: Diagnostic Utility and Prognostic SignificanceFrom the Institut Clínic de Malalties Digestives and Department of Pathology, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Hospital Clínic i Provincial, University of Barcelona; and Departments of Biochemistry, Gastroenterology, and Surgery, and Laboratori d'Investigació Gastrointestinal, Hospital de la Santa Creu i Sant Pau, Barcelona, Catalonia, Spain. Address reprint requests to Antoni Castells, MD, Molecular Neurogenetics Unit, Massachusetts General Hospital, 149 13th St, 6th Floor, Charlestown, MA 02129; email castells{at}helix.mgh.harvard.edu
PURPOSE: Previous studies have demonstrated the presence of K-ras mutations in the plasma of patients with pancreatic carcinoma. However, the diagnostic utility and the prognostic significance of this finding have never been addressed. PATIENTS AND METHODS: Forty-four consecutive patients with histologically confirmed primary pancreatic ductal adenocarcinoma were included. A control group of 37 patients with chronic pancreatitis, 10 patients with other tumors of the pancreatic area, nine patients with acute pancreatitis, and four healthy volunteers was also included. Plasma DNA was isolated and K-ras codon-12 mutations were analyzed by means of restriction fragment length polymorphism-polymerase chain reaction and single-strand conformation polymorphism techniques. Patients were followed up to establish their clinical outcome. RESULTS: The mutant-type K-ras gene was found in plasma DNA samples of 12 (27%) of 44 patients with pancreatic ductal adenocarcinoma; this finding was related to the tumor stage (P = .05), mainly in the presence of distant metastases (P = .02). In addition, K-ras mutations were detected in the plasma DNA of two (5%) of 37 patients with chronic pancreatitis. In the subset of patients with pancreatic masses, the sensitivity and specificity of plasma K-ras analysis for pancreatic adenocarcinoma were 27% and 100%, respectively. Finally, pancreatic carcinoma patients with the mutant-type K-ras gene in plasma DNA exhibited a shorter survival time than patients with the wild-type gene (P < .005), and plasma K-ras mutations were identified as the only independent prognostic factor (odds ratio, 1.51; 95% confidence interval, 1.02 to 2.23). CONCLUSION: Plasma K-ras analysis is a highly specific, low-sensitivity approach that has diagnostic and prognostic clinical implications in patients with pancreatic carcinoma.
PANCREATIC CARCINOMA represents the fourth leading cause of cancer death in Western countries.1 Patients with this neoplasm have a median survival time of 6 months after their initial diagnosis.2,3 Although there have been advances in imaging techniques, diagnosing the disease remains difficult, and as a result, most patients exhibit disseminated disease.2-4 Additionally, abdominal or endoscopic ultrasound-guided, fine-needle aspiration biopsy specimens often constitute the only sample amenable for cytologic confirmation of the disease.5 ras mutations are found in 80% to 90% of pancreatic carcinoma patients, usually at codon 12 of the K-ras gene.6-9 This high incidence of mutations suggests that the K-ras gene may constitute a useful tumor marker. In fact, in vitro amplification of DNA has been used extensively in pancreatic cancer from fine-needle aspirates,10-12 pancreatic juice,13-15 and feces,16 thus increasing the diagnostic utility of aspiration biopsy or endoscopic retrograde cholangiopancreatography. On the other hand, it is well known that small amounts of free DNA are found in the plasma or serum of neoplastic patients.17,18 With the use of polymerase chain reaction (PCR) techniques, microsatellite alterations have been detected in the plasma DNA of patients with small-cell lung cancer19 and in the serum DNA of patients with head and neck cancer,20 and K-ras mutations have been found in the plasma DNA of patients with colorectal21 and pancreatic neoplasms.13,22,23 Preliminary studies in pancreatic cancer, however, have not assessed the relevance of this finding in clinical practice. The present prospective investigation was aimed at determining the presence of K-ras mutations in the peripheral blood of pancreatic carcinoma patients. The accuracy of this approach was established by comparing a large series of patients in different stages of pancreatic adenocarcinoma with a control group that included not only healthy subjects but also patients with other pancreatic disorders. Inclusion of patients with chronic pancreatitis was important because K-ras mutations have been found in the pancreas24,25 and pancreatic juice from nonneoplastic cases.15,26 The diagnostic utility of this procedure was addressed in the setting of those patients with pancreatic masses, and its prognostic significance was established in a follow-up study by means of a multivariate analysis. Finally, K-ras mutations were looked for both in the plasma and in the buffy coat layer in order to determine whether they corresponded to naked DNA released from the primary tumor or, conversely, whether they derived from whole neoplastic circulating cells.
Patients Between July 1996 and August 1997, 44 consecutive patients with histologically confirmed primary pancreatic ductal adenocarcinoma were included in the study. The mean age was 62 ± 12 years, and there were 25 men and 19 women. Tumors were staged by means of dynamic computed tomography, magnetic resonance imaging, and angiography and/or endoscopic ultrasonography. When patients underwent surgery, definitive tumor stage was established on the basis of operative findings. On the basis of the tumor-node-metastasis (TNM) system,27 four patients were classified in stage I, 11 in stage II, five in stage III, and 23 in stage IV. Tumor stage could not be assessed in the remaining patient because of the lack of accurate information. Control group subjects were recruited during the same time period and included 37 patients with chronic pancreatitis, 10 patients with other tumors of the pancreatic area (five had cholangiocarcinoma of the distal intrapancreatic common bile duct, two had mucinous adenoma, one had pancreatic metastasis, one had a neuroendocrine tumor, and one had an estromal tumor of the duodenal wall), nine patients with acute pancreatitis, and four healthy volunteers. The diagnosis of chronic pancreatitis was based on radiologic data obtained by means of either endoscopic retrograde cholangiopancreatography or computed tomography.28 Peripheral venous blood samples (20 mL) were obtained by means of a standard venipuncture technique, using heparinized tubes. In patients with pancreatic tumors, the blood sample was collected before any therapeutic procedure. In patients who underwent endoscopic retrograde cholangiopancreatography, the sample was collected before this diagnostic technique was performed. The study protocol was approved by the ethics of research committees of both institutions (Hospital Clínic i Provincial and Hospital de la Santa Creu i Sant Pau), and informed consent was obtained from each patient. The study was performed in a blind fashion, so that patients' clinical characteristics were unknown by the investigator performing the molecular analysis on the blood samples. In addition, each sample was analyzed twice. All patients were followed up to determine their clinical outcome. Survival analysis was done in December 1997.
DNA Extraction From the Plasma
DNA Extraction From the Buffy Coat Layer
DNA Extraction From Cytohistologic Specimens The pancreatic juice collected at endoscopic retrograde cholangiopancreatography was centrifuged at 12,000 rpm for 5 minutes. Aspirates from pancreatic tissue were fixed in 50% ethanol and centrifuged, and a paraffin cell block was made. Several sections of the cell block were examined after staining with hematoxylin and eosin. One hematoxylin-and-eosin–stained, 5- to 10-µm slice was used for microscopic examination, and DNA was extracted from the adjacent slide, after paraffin was removed with 500 µL of xylene. The sample was then washed twice with 100% ethanol and dried. In all samples obtained by fine-needle aspiration biopsy, a minimum of 30% of tumor cells was present. Tissue DNA extraction was performed by using proteinase K treatment followed by phenol-chloroform extraction and isopropanol precipitation.
Detection of K-ras Codon-12 Mutations by Restriction Fragment Length Polymorphism-PCR
Characterization of K-ras Mutations by the Single-Strand Conformation Polymorphism Method
Statistical Analysis The prognostic value of K-ras mutations in peripheral blood was assessed with a multivariate approach including other parameters with a predictive influence on survival, such as age, sex, tumor size and location, presence of vascular invasion, tumor stage according to the TNM system,27 and K-ras status in tissue samples. All data were obtained at inclusion. Survival time was considered from the date of diagnosis. Univariate analysis was performed by computing survival curves according to the Kaplan-Meier method32 and comparing them through the Mantel-Cox and Breslow tests. For continuous variables, the cutoff level chosen was their median value. Variables that achieved statistical significance (P < .05) in the univariate analysis were subsequently included in a multivariate analysis using a stepwise forward Cox regression procedure.33 All calculations were performed by using the SPSS statistical software package (SPSS Inc, Chicago, IL).
K-ras Mutations in Pancreatic Adenocarcinoma Patients The mutant-type K-ras gene was found in plasma DNA samples of 12 (27%) of 44 patients with pancreatic ductal adenocarcinoma (Table 1 and Fig 1). K-ras alterations included aspartic acid (n = 5), valine (n = 3), alanine (n = 2), and arginine (n = 2) (Table 2). The wild-type K-ras gene was found in all DNA samples extracted from the buffy coat layers of those 44 patients.
The presence of the mutant-type K-ras gene in the plasma DNA of these patients was not related to age, sex, tumor site, tumor size, nor vascular involvement (Table 3). In contrast, K-ras mutations in plasma were correlated with tumor stage (P = .05), mainly in the presence of distant metastases (P = .02) (Table 3).
In the 39 pancreatic carcinoma patients in whom both plasma and tissue samples were available, the correlation of K-ras mutations was analyzed. Twenty-eight patients (72%) exhibited K-ras mutations in their primary tumor, with nine (32%) of them showing an identical alteration in the corresponding plasma DNA sample. Otherwise, mutations were not detected in plasma samples of the remaining 11 patients whose primary tumor was negative for K-ras.
K-ras Mutations in the Control Group The correlation of K-ras mutations between plasma and pancreatic juice or tissue was analyzed in those 26 chronic pancreatitis patients in whom both samples were available. No plasma K-ras mutations were observed in 21 patients with negative pancreatic juice. Of the two patients with K-ras mutations in their plasma DNA, one patient exhibited the same alteration in the pancreatic tissue specimen obtained when he underwent a partial pancreatectomy as a consequence of untreatable abdominal pain. No mutation was detected in DNA extracted from the pancreatic juice of the second patient with the mutant-type K-ras gene in plasma DNA (Table 2). Finally, three patients showed K-ras mutations in DNA obtained from the pancreatic juice, but no alteration was detected in the corresponding plasma sample. The above-mentioned five chronic pancreatitis patients with K-ras mutations in DNA extracted from either plasma, pancreatic juice, or pancreatic tissue were studied extensively by means of endoscopic retrograde cholangiopancreatography, abdominal computed tomography, and endoscopic ultrasonography. No evidence of neoplastic disease was found. Pathologic examination of a resected pancreatic specimen from the patient who underwent surgery confirmed the absence of malignant proliferation. In addition, after a median follow-up of 9 months (range, 6 to 17 months), no chronic pancreatitis patient with K-ras mutations had developed a pancreatic carcinoma. No K-ras mutations were found in the plasma (Table 1) or buffy coat layer DNA samples of any patient with acute pancreatitis or other neoplasms of the pancreatic area, nor in healthy volunteers.
Diagnostic Utility of Plasma K-ras Mutations in Pancreatic Masses
Prognostic Significance of Plasma K-ras Mutations in Pancreatic Carcinoma Patients
Finally, when both parameters selected in the univariate analysis were included in a stepwise Cox regression model, plasma K-ras mutations were identified as the only independent predictive factor of survival (odds ratio, 1.51; 95% confidence interval, 1.02 to 2.23).
The results of the present investigation confirm that it is possible to identify K-ras mutations in DNA extracted from the plasma of patients with pancreatic carcinoma. The plasma DNA of these patients, as well as the plasma DNA from patients with small-cell lung cancer, head and neck neoplasms, and colorectal carcinoma,19-23 shared identical molecular characteristics (gene mutations or microsatellite alterations) with patients' primary tumors, thus suggesting its neoplastic origin. In that sense, without considering any clinical usefulness, the analysis of plasma DNA represents a worthwhile, noninvasive way to obtain genetic material suitable for molecular investigations in patients with malignant neoplasms. This approach is especially significant in pancreatic carcinoma patients, because obtaining cytohistologic specimens from these patients is arduous. Our data show that detection of plasma K-ras mutations is a high-specificity, low-sensitivity method of diagnosing pancreatic carcinoma. Accordingly, it may become a confirmatory tool in those cases in which a more invasive approach is contraindicated. The absence of plasma K-ras mutations in early tumor stages would preclude the possibility of carrying out screening strategies on in-risk populations on the basis of the detection of mutant plasma DNA. Advances in this setting should be focused on improving the sensitivity of the technique and identifying new targets involved in early neoplastic stages. Finally, if one takes into account that plasma K-ras mutations are correlated with the presence of distant metastases, this approach may be especially useful in tumor staging and in monitoring both disease spread and tumor recurrence. On the other hand, it is important to note that five of 37 patients with chronic pancreatitis and with no evidence of neoplastic degeneration showed K-ras mutations in either their plasma or pancreatic juice. Similarly, recent studies have demonstrated the presence of K-ras mutations in hyperplastic mucous cells of the pancreas with chronic inflammation.16,25 The exact significance of this observation is unclear; some authors suggest that K-ras mutations are an early event in pancreatic carcinogenesis. However, K-ras mutations have also been found in hyperplastic duct cells of the pancreas without pancreatic disease,24 and long-term follow-up of patients with chronic pancreatitis carrying this mutated gene failed to demonstrate any relationship with the development of pancreatic neoplasm.26 To clarify such contradictory data, a prospective study of a large number of patients is mandatory. In this way, the possibility of detecting K-ras mutations in peripheral blood by means of a noninvasive technique may help to clarify the clinical significance of this finding. The origin of mutated plasma DNA in patients with cancer is uncertain. Because the mutated plasma DNA carries the same molecular alterations as the primary tumor, it is tempting to hypothesize that neoplastic cell lysis leads to the release of naked DNA into the circulation.19-21 Nevertheless, its derivation from whole circulating cancer cells needs to be considered. In fact, several authors, using PCR and targeting tissue-specific gene transcription, have identified circulating cancer cells in patients with different neoplasms,34-36 including pancreatic adenocarcinoma.13,37 The correlation observed between plasma DNA mutations and distant metastases in our study supports this possibility. By contrast, the lack of K-ras mutations in DNA extracted from the buffy coat layer makes the eventual presence of whole cancer cells in peripheral blood very unlikely. Still, it should be kept in mind that neoplastic cells are supposed to be circulating in clusters, and consequently, their recognition could be hampered by a single blood sample extraction. Finally, large amounts of nonmutated DNA coming from leukocytes held in the buffy coat layer might also mask some vestiges of the mutant-type K-ras gene. The presence of plasma K-ras mutations was identified as an independent prognostic factor in pancreatic adenocarcinoma patients. In fact, subjects with mutated plasma DNA exhibited a shorter survival time than those with the wild-type K-ras gene. However, this result needs to be qualified. First, the plasma K-ras mutation variable was included in the final equation of the Cox analysis with a weak statistical significance. This result suggests a strong collinearity between this parameter and the tumor stage. Second, the present study was composed primarily of patients with very advanced disease. As a result, a bias due to an unbalanced tumor stage could not be ruled out. Finally, the statistical power of the present analysis is undoubtedly hampered by the grim prognosis of the overall series. Accordingly, a definitive conclusion with regard to the prognostic significance of plasma DNA mutations should be postponed until the end of follow-up studies that include a larger number of patients in different tumor stages. In conclusion, the results of the present study indicate that plasma K-ras analysis is a high-specificity, low-sensitivity approach in pancreatic carcinoma patients and suggest that a plasma-based assay may have a role in the diagnostic and prognostic assessment of these patients. Further investigations are needed to confirm these results, to improve the technique's sensitivity, and to establish its usefulness in the early diagnosis and in-risk group screening of pancreatic cancer.
Supported in part by a grant from the Plan Nacional de Investigación Científica y Desarrollo Tecnológico (SAF97-0107). P.P. and L.B. received research grants (95/26 and 95/3008) from the Marató TV3-Càncer. We thank Dr Anil K. Rustgi for his critical review of the manuscript, Laura Gargallo for her efficient nursing support, and Paula Simpson-O'Brien for editorial assistance.
The first two authors contributed equally to this study.
1. Boring C, Squires TS, Taong T: Cancer statistics, 1993. CA Cancer J Clin 43:7-26, 1993[Medline] 2. Warshaw AL, Fernández-del-Castillo C: Pancreatic carcinoma. N Engl J Med 326:455-465, 1992[Medline] 3. Rosewicz S, Wiedenmann B: Pancreatic carcinoma. Lancet 349:485-489, 1997[Medline] 4. Rösch T Braig C, Gain T, et al: Staging of pancreatic and ampullary carcinoma by endoscopic ultrasonography. Gastroenterology 102:188-199, 1992[Medline]
5.
Brandt KR, Vcharboneau JW, Stephens DH, et al: CT- and US-guided biopsy of the pancreas. Radiology 187:99-104, 1993 6. Almoguera C, Shibata D, Forrester K, et al: Most human carcinomas of the exocrine pancreas contain mutant K-ras genes. Cell 53:549-553, 1988[Medline]
7.
Smit V, Boot A, Smits A, et al: K-ras codon 12 mutations occur very frequently in pancreatic adenocarcinomas. Nucleic Acids Res 16:7773-7782, 1988 8. Grünewald K, Lyons J, Fröhlich A, et al: High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas. Int J Cancer 43:1037-1041, 1989[Medline] 9. Shibata D, Capellà G, Perucho M: Mutational activation of the K-ras gene in human pancreatic carcinoma. Baillieres Clin Gastroenterol 4:151-169, 1991
10.
Shibata D, Almoguera C, Forrester K, et al: Detection of K-ras mutations in fine needle aspirates from human pancreatic cancer. Cancer Res 50:1279-1283, 1990 11. Tada M, Omata M, Ohto M: Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma. Gastroenterology 100:233-238, 1991[Medline] 12. Villanueva A, Reyes G, Cuatrecasas M, et al: Diagnostic utility of K-ras mutations in fine-needle aspirates of pancreatic masses. Gastroenterology 110:1587-1594, 1996[Medline]
13.
Tada M, Omata M, Kawai S, et al: Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res 53:2472-2474, 1993
14.
Berthélemy P, Bouisson M, Escourrou J, et al: Identification of K-ras mutations in pancreatic juice in the early diagnosis of pancreatic cancer. Ann Intern Med 123:188-191, 1995 15. Boadas J, Mora J, Roca M, et al: Analysis of the utility of K-ras mutations in pancreatic juice in the diagnosis of pancreatic cancer. Pancreas 15:464, 1997
16.
Caldas C, Hahn SA, Hruban RH, et al: Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res 54:3568-3573, 1994
17.
Leon SA, Shapiro B, Sklaroff DM, et al: Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 37:646-650, 1977 18. Shapiro B, Chakrabarty M, Cohn EM, et al: Determination of circulating DNA levels in patients with benign or malignant gastrointestinal disease. Cancer 51:2116-2120, 1983[Medline] 19. Chen XQ, Stroun M, Magnenat JL, et al: Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 2:1033-1035, 1996[Medline] 20. Nawroz H, Koch W, Anker P, et al: Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 2:1035-1037, 1996[Medline] 21. Anker P, Lefort F, Vasioukhin V, et al: K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 112:1114-1120, 1997[Medline] 22. Sorenson GD, Pribish DM, Valone FH, et al: Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 3:67-71, 1994[Abstract]
23.
Mulcahy HE, Lyautey J, Lederrey C, et al: A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res 4:271-275, 1998 24. Tada M, Ohashi M, Shiratori Y, et al: Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology 110:227-231, 1996[Medline]
25.
Yanagisawa A, Ohtake K, Ohnishi K, et al: Frequent c-Ki-ras oncogene activation in mucous cell hyperplasia of pancreas suffering from chronic inflammation. Cancer Res 53:953-956, 1993 26. Furuka N, Kawa S, Akamatsu T, et al: Long-term follow-up of patients with chronic pancreatitis and K-ras mutation detected in pancreatic juice. Gastroenterology 113:593-598, 1997[Medline] 27. Beahrs OH, Henson DE, Hutter RVP, et al: Manual for Staging of Cancer (ed 4). Philadelphia, PA, Lippincott, 1993 28. Sarner M, Cotton PB: Definitions of acute and chronic pancreatitis. Clin Gastroenterol 13:865-870, 1984[Medline] 29. Sambrook J, Frisch FF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989 30. Jiang W, Kahn SM, Guillem JG, et al: Rapid detection of ras oncogenes in human tumors: Applications to colon, esophageal, and gastric cancer. Oncogene 4:923-928, 1989[Medline] 31. Matias-Guiu X, Villanueva A, Cuatrecasas M, et al: p53 in a tyroid follicular carcinoma with foci of poorly differentiated and anaplastic carcinoma. Pathol Res Pract 192:1-11, 1996[Medline] 32. Kaplan GL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958 33. Cox DR: Regression models and life tables. J R Stat Soc (B) 34:187-220, 1972 34. Smith B, Selby P, Southgate J, et al: Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. Lancet 338:1227-1229, 1991[Medline] 35. Burchill SA, Bradbury MF, Pittman K, et al: Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br J Cancer 71:278-281, 1995[Medline] 36. Castells A, Boix L, Bessa X, et al: Detection of colonic cells in peripheral blood of colorectal cancer patients by means of reverse transcriptase and polymerase chain reaction. Br J Cancer 78:1368-1372, 1998[Medline] 37. Mohiuddin M, Ahmed MM, Venkatasubbarao K: c-Ki-ras mutations in peripheral blood of pancreatic cancer patients: A marker for early tumor metastasis. Int J Radiat Oncol Biol Phys 34:161-166, 1996[Medline] Submitted June 25, 1998; accepted October 8, 1998.
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
2 test; the Yates correction was applied when necessary. Continuous variables with nonparametric distribution were compared by means of the Mann-Whitney U test. 
