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Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA

Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection... British Journal of Cancer (2002) 87, 551 – 554 ã 2002 Cancer Research UK All rights reserved 0007 – 0920/02 $25.00 www.bjcancer.com Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA ,1 2 1 3 1 4 1 2,4 F Maire* , S Micard , P Hammel , H Voitot ,P Le ´vy , P-H Cugnenc , P Ruszniewski and P Laurent Puig 1 2 Fe ´de ´ration Me ´dico-Chirurgicale d’He ´pato-Gastroente ´rologie, Ho ˆpital Beaujon, AP-HP, 92110 Clichy, France; U490 INSERM Laboratoire de Toxicologie 3 4 Mole ´culaire, 45 rue des Saints-Pe `res 75006 Paris, France; Laboratoire de Biochimie, Ho ˆpital Beaujon, 92110 Clichy, France; Service de Chirurgie Digestive et Oncologie, Ho ˆpital Europe ´en Georges Pompidou, 75015 Paris, France KRAS2 mutations in codon 12 have been detected in about 80% of pancreatic cancers. The aim of this study was to evaluate the value of KRAS2 mutations detection in circulating deoxyribo nucleic acid to differentiate pancreatic cancer from chronic pancreatitis. Circulating deoxyribo nucleic acid was isolated from serum in 47 patients with histologically proven pancreatic adenocarcinomas (26 males, median age 65 years) and 31 controls with chronic pancreatitis (26 males, median age 48 years). Mutations at codon 12 of KRAS2 gene were searched for using polymerase chain reaction and allele specific amplification. Serum carbohydrate antigen 19.9 levels were also determined. KRAS2 mutations were found in 22 patients (47%) with pancreatic cancer and in four controls with chronic pancreatitis (13%) (P50.002). None of the latter developed a pancreatic cancer within the 36 months of median follow-up. The sensitivity, specificity, positive and negative predictive values of serum serum KRAS2 mutations for the diagnosis of pancreatic cancer were 47, 87, 85 and 52%, respectively. KRAS2 mutations were not related to age, gender, smoking habit, tumour stage, or survival. Among the 26 patients with normal or non-contributive (due to cholestasis) serum carbohydrate antigen 19.9 levels, 14 (54%) had KRAS2 mutations. The combination of KRAS2 and carbohydrate antigen 19.9 gave a sensitivity, specificity, positive and negative predictive values for the diagnosis of pancreatic cancer of 98, 77, 87 and 96%, respectively. Detection of KRAS2 mutations in circulating deoxyribo nucleic acid has a low sensitivity but a specificity about 90% for the diagnosis of pancreatic cancer. It seems particularly useful when serum carbohydrate antigen 19.9 levels are normal or inconclusive. A combined normal serum carbohydrate antigen 19.9 and absence of circulating KRAS2 mutations makes the diagnosis of pancreatic cancer extremely unlikely. British Journal of Cancer (2002) 87, 551 – 554. doi:10.1038/sj.bjc.6600475 www.bjcancer.com ã 2002 Cancer Research UK Keywords: KRAS2 mutations; circulating DNA; pancreatic adenocarcinoma; chronic pancreatitis Five-year survival in patients with pancreatic adenocarcinomas is tions were first reported in surgically removed pancreatic tumoural less than 5%, partly due to advanced disease at diagnosis. The tissue or at autopsy (Almoguera et al, 1988; Tada et al, 1991). differentiation between pancreatic cancer and chronic pancreatitis Thereafter mutations were discovered in 63 to 83% of samples of can be particularly difficult leading to inappropriate treatment. pure pancreatic juice or main pancreatic duct brushing obtained Serum carbohydrate antigen 19.9 (Ca 19.9) levels are elevated in during endoscopic retrograde pancreatography (Iguchi et al, 80% of pancreatic cancer patients, but can also be increased in 1996; Kondo et al, 1997; Tada et al, 1998; Van Laethem et al, 20% of patients with chronic pancreatitis (Satake and Takeuchi, 1998; Okai et al, 1999; Watanabe et al, 1999; Ha et al, 2001; Pugli- 1994; Nouts et al, 1998). Moreover, pancreatic inflammation, as ese et al, 2001; Seki et al, 2001) or at fine-needle tumour aspiration (Pabst et al, 1999; Puig et al, 2000), and in 20 to 54% of stools observed in chronic pancreatitis, can be mistaken on imaging as cancer and inversely. An accurate and non-invasive test to differ- (Caldas et al, 1994; Wenger et al, 1999) from patients with entiate pancreatic cancer from chronic pancreatitis would be pancreatic cancer. Circulating deoxyribo nucleic acid (DNA) was extremely helpful. first detected in serum or plasma of normal subjects in 1975 (Stein- Previous studies have reported KRAS2 gene mutations (almost man, 1975). Since then, mutations in the KRAS2 gene have been always confined to codon 12) in 75 to 95% of exocrine pancreatic detected in the plasma of patients with colorectal, lung and haema- cancer (Caldas and Kern, 1995). KRAS2 mutations provoke activa- tological cancers (Anker et al, 1997). To date, few studies have tion of nuclear transcriptor factors, resulting in cellular reported KRAS2 mutations in circulating DNA in patients with proliferation and also in tumour angiogenesis as reported recently pancreatic cancer with a wide spectrum of sensitivity (27 to (Banerjee et al, 2000; Ikeda et al, 2001). Detection of KRAS2 muta- 81%) (Sorenson et al, 1994; Mulcahy et al, 1998; Yamada et al, 1998; Castells et al, 1999; Porta et al, 1999; Theodor et al, 2000). The aim of our study was to evaluate the value of KRAS2 mutation *Correspondence: F Maire; E-mail: [email protected] detection in circulating DNA in a large series of patients to differ- Revised 16 May 2002; accepted 23 May 2002 entiate pancreatic adenocarcinoma from chronic pancreatitis. Molecular and Cellular Pathology Molecular and Cellular Pathology KRAS2 mutations in circulating DNA in pancreatic adenocarcinoma F Maire et al positive controls were performed for each set of PCR reactions. PATIENTS AND METHODS PCR products were separated by electrophoresis in a 6% acryla- mide gel and stained with ethidium bromide. Selection and outcome of patients Between January 1995 and 1999, 47 patients (26 males and 21 Serum Ca 19.9 dosage females, median age 65 years (range 39 – 84)) with pancreatic ductal adenocarcinoma were included in the study. In all of them, The serum value of Ca 19.9 was measured with a commercial solid- diagnosis was confirmed by pathological examination of pancreatic phase double-antibody sandwich immunoassay (Roche Labora- tumour obtained by fine needle aspiration during endoscopic ultra- tories, Basel, Switzerland). The upper limit of normal value was sonography (n=42) or operative procedure (n=5). Tumour staging 37 UI ml . All patients and controls also underwent biochemical was established by abdominal computed tomography, endoscopic liver tests. Cholestasis was defined by alkaline phosphatase levels ultrasonography or operative findings: stage I (n=5, 11%), stage above twice the normal value. II or III (n=19, 40%) and stage IV (n=23, 49%) according to the TNM classification (UICC, 1997). Five patients underwent surgical Statistical analysis resection, 32 patients received systemic chemotherapy and/or radiotherapy and 10 symptomatic treatment. Median follow-up For each marker (serum KRAS2 mutations and Ca 19.9), sensitiv- was 6 months (range 1 – 24). At the end of the study, all but four ity, specificity, positive and negative predictive values were patients with pancreatic cancer were dead. calculated. Thereafter, the combination of both markers (i.e., one and/or the other positive) was studied. The chi-squared test was used to compare the occurrence of KRAS2 mutations. Differences Control group were considered significant when P50.05. A control group was recruited during the same time in the same centre and included 31 patients with chronic pancreatitis (26 RESULTS men and five women, median age 48 years (range 20 – 64)). Diag- nosis of chronic pancreatitis relied upon the presence of pancreatic Circulating DNA quantification calcifications and/or irregularity of pancreatic ducts, according to Cambridge Classification (Axon et al, 1984) on computed tomo- Adequate DNA was extracted from the serum in sufficient quanti- graphy scan and endoscopic retrograde pancreatography, ties for analysis in all patients and controls. All patients but one respectively. Etiology of chronic pancreatitis was alcoholic in 30 had a serum DNA concentration higher than the threshold of patients and idiopathic in one patient. No case of pancreatic cancer detection of 100 ng ml . The mean concentrations of DNA occurred during the 36-month follow-up in these 31 patients. extracted from serum of patients with pancreatic cancer and 71 71 chronic pancreatitis were 730+90 ng ml and 560+93 ng ml , respectively (P=0.19). DNA extraction and quantification Peripheral venous blood samples were collected after informed KRAS2 mutations in circulating DNA consent in patients and controls. Blood samples were centrifuged, serum was removed and stored at 7208C until use. DNA was KRAS2 mutations were identified in the serum of 22 patients extracted from serum by using the QIAmp Blood Kit (Qiagen, (47%) with pancreatic adenocarcinoma and in four patients Courtaboeuf, France) according to the blood and body fluid proto- (13%) with chronic pancreatitis (P50.002). The sensitivity, speci- col recommended by the manufacturer. Two millilitres of serum ficity, positive and negative predictive values of serum KRAS2 were used, and a DNA elution volume of 50 ml was obtained by mutations for the diagnosis of pancreatic cancer were 47, 87, 85 extraction. The DNA elution was then concentrated to a final and 52%, respectively. There were no statistically significant differ- volume of 15 ml. ences in age, gender, smoking, tumour stage and survival, Quantification of serum DNA was performed for all samples according to presence or absence of plasma KRAS2 mutations. using a volume of 3 ml of DNA elution obtained after Qiagen Among patients with chronic pancreatitis, no cancer occurred extraction by fluorescence emission after intercalation of Hœschst after a mean follow-up of 36 months (range 13 – 64), even in those dye. The fluorescence was read by DyNA Quanto 200 fluorimeter, with positive KRAS2 mutations (with follow-up of 40 months, using the cuve and capillary DyDNA Capillary Cuvette Adaptor Kit range 20 – 61), assessed by clinical observation and abdominal (Pharmacia Biotech, Orsay, France). The threshold of DNA detec- computed tomography scan. tion established by the manufacturer is 2 ng ml , which corresponds to 100 ng ml serum in our extraction protocol Serum Ca 19.9 levels (Coulet et al, 2000). The sensitivity, specificity, positive and negative predictive values of abnormal Ca 19.9 levels for the diagnosis of pancreatic cancer were Detection of KRAS2 gene mutations 91, 87, 91 and 87%, respectively (Table 1). Serum Ca 19.9 levels Only G12D mutations in codon 12 of the KRAS2 gene were were normal or non interpretable due to cholestasis in 26 patients. searched for using allele-specific amplification, with the following Among them, 14 (54%) patients had serum KRAS2 mutations. primers: 5'-CTTGTGGTAGTTGGAGCTAA-3',5'-AATGGTCCT- Combination of both tests increased sensitivity to 98% with a GCACCAGTAATATG-3'. Amplifications were performed with negative predictive value of 96% for the diagnosis of pancreatic 0.3 mM of each primers, 200 mM of each deoxynucleotide tripho- cancer (Table 1). sphate (dNTP), 1.5 mM of MgCl , 0.025 units per mlof AmpliTaq Gold polymerase Cetus (Perkin Elmer), 2.5 mlor5 ml DISCUSSION of 106 buffer, 5 ml of the concentrated DNA elution was used as template in a 50 ml volume reaction. Polymerase chain reaction In the present study, only the most frequent KRAS2 gene muta- (PCR) with serum DNA was performed 10 min at 948C, followed tion G12D (aspartic acid) observed in pancreatic cancer (Iguchi by 60 cycles of 948C for 30 s, 618C for 30 s, 728C for 1 min and a et al, 1996; Tada et al, 1998; Castells et al, 1999; Watanabe et final extension of 10 min at 728C. Controls without DNA and al, 1999) was analysed in the serum of patients and controls, in British Journal of Cancer (2002) 87(5), 551 – 554 ã 2002 Cancer Research UK KRAS2 mutations in circulating DNA in pancreatic adenocarcinoma F Maire et al Table 1 Accuracy of serum KRAS2 mutation detection, serum Ca19.9 is not informative in 5% of population who cannot express serum levels, and both for the diagnosis of pancreatic cancer Ca 19.9 due to Lewis a negative status (Narimatsu et al, 1996). However, serum Ca 19.9 lacks specificity (70 to 80%): it can be Serum KRAS2 Serum Both tests increased in cholestasis, diabetes mellitus or chronic pancreatitis mutations Ca19.9 combined (Nouts et al, 1998). In the present study, serum Ca 19.9 had very good specificity (87%), similar to that of serum KRAS2 mutations. Sensitivity (%) 47 91 98 Combination of both tests could be useful to assess cancer diagno- Specificity (%) 87 87 77 Positive predictive value (%) 85 91 87 sis in patients with normal or non contributive Ca 19.9 due to Negative predictive value (%) 52 87 96 cholestasis or negative Lewis a antigen status, and to exclude cancer diagnosis when both tests are negative (predictive negative value of 96%). In the present study, the presence of KRAS2 mutations in order to limit the cost of such test and to validate it in clinical serum was not correlated to age, gender and smoking habit. It practice. was neither correlated to tumour stage since mutations were Our study underlines the high feasibility of KRAS2 mutations detected in plasma of patients with non metastatic tumours, analysis, as circulating DNA was obtained in sufficient quantities which supports the hypothesis that KRAS2 mutations are early in all patients. Mean serum DNA concentration were 730 ng ml , events in pancreatic carcinogenesis (Jimenez et al, 1999). Two which is comparable to that observed in the series of Mulcahy et al studies in the literature are in agreement with this result (Yamada (1998). et al, 1998; Theodor et al, 2000), but another one found a statis- In the present study, detection of KRAS2 mutations in circulat- tically significant relation between circulating DNA KRAS2 ing DNA had a low sensitivity but a high specificity for the mutations and poor prognosis (Castells et al, 1999). Yamada et diagnosis of pancreatic cancer. The sensitivity (47%) was in agree- al (1998) have reported disappearance of detectable mutation in ment with previous studies (27 to 81%) (Mulcahy et al, 1998; plasma after tumoural resection or radio-chemotherapy in six of Yamada et al, 1998; Castells et al, 1999; Porta et al, 1999; Theodor nine patients, suggesting KRAS2 mutations may be used as a et al, 2000; Zambon et al, 2000), although the search for five other tumour relapse marker. possible KRAS2 mutations in codon 12 was not performed. Higher Screening for malignancy in patients with chronic pancreatitis is KRAS2 mutation prevalences have been reported in pancreatic or a difficult challenge. Patients with chronic pancreatitis have an duodenal juice (63 to 87%), due probably to higher DNA tumour increased risk of pancreatic cancer, estimated at 1.8% at 10 years content in pancreatic juice as compared to plasma (Wilentz et al, and 4% at 20 years (Lowenfels et al, 1993). Three studies have eval- 1998; Van Laethem et al, 1998; Watanabe et al, 1999). Use of uated occurrence of pancreatic cancer in patients with chronic samples of pancreatic juice however requires invasive procedures pancreatitis with respect to KRAS2 mutations in pancreatic juice: (fine-needle aspiration during endoscopic ultrasonography or only one found an increase in pancreatic cancer in patients with endoscopic retrograde pancreatography). Three studies have KRAS2 mutations with methodological limitations (few cases, early reported a higher specificity of serum KRAS2 mutations compared diagnosis of cancer after inclusion) (Furuya et al, 1997; Lohr et al, to the current study (100 vs 87%), but their control groups 2001; Queneau et al, 2001). To our knowledge, no study focused included few patients and essentially healthy subjects (Mulcahy et on the incidence of pancreatic cancer in patients with chronic al, 1998; Porta et al, 1999; Theodor et al, 2000). Since KRAS2 pancreatitis according to serum KRAS2 mutations. In the present mutations have been reported in pancreatic tissue or juice from study, follow-up was too short to demonstrate an increased risk 6 – 42% of patients with chronic pancreatitis (Furuya et al, 1997; of cancer in patients with chronic pancreatitis and serum KRAS2 Mulligan et al, 1999; Lu ¨ ttges et al, 2000; Ha et al, 2001), and know- mutations. ing that a part of this mutated DNA can be released into In conclusion, although detection of plasma KRAS2 mutations circulation (Yamada et al, 1998), the control group should include in circulating DNA is not a definitive argument for malignancy, patients with chronic pancreatitis. In the series published by it could contribute to cancer diagnosis. This test seems particularly Castells et al (1999) with the largest control group (including interesting in patients with normal or inconclusive Ca 19.9 levels patients with chronic pancreatitis), specificity was comparable due to cholestasis or Lewis a negative status. In patients with (94%) to the present study, and increased in the presence of a normal serum Ca 19.9 levels and no KRAS2 mutation, the diagno- pancreatic mass. The accuracy of serum KRAS2 mutation detection sis of pancreatic cancer can be excluded with almost certainty. in the differential diagnosis between pancreatic cancer and chronic pancreatitis may be improved by performing quantitative PCR measurement of mutated DNA, in order to discriminate patients with unspecific low levels of mutated DNA (as supposed in chronic pancreatitis) from patients with high levels (pancreatic cancer), as suggested in pancreatic juice analysis by Tada et al (1998). 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Gastrointest Endosc 50: 797 – 803 tisfactory technique for clinical applications. Clin Chim Acta 302: 35 – 48 British Journal of Cancer (2002) 87(5), 551 – 554 ã 2002 Cancer Research UK http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Cancer Springer Journals

Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA

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Springer Journals
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Copyright © 2002 by The Author(s)
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Biomedicine; Biomedicine, general; Cancer Research; Epidemiology; Molecular Medicine; Oncology; Drug Resistance
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0007-0920
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10.1038/sj.bjc.6600475
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British Journal of Cancer (2002) 87, 551 – 554 ã 2002 Cancer Research UK All rights reserved 0007 – 0920/02 $25.00 www.bjcancer.com Differential diagnosis between chronic pancreatitis and pancreatic cancer: value of the detection of KRAS2 mutations in circulating DNA ,1 2 1 3 1 4 1 2,4 F Maire* , S Micard , P Hammel , H Voitot ,P Le ´vy , P-H Cugnenc , P Ruszniewski and P Laurent Puig 1 2 Fe ´de ´ration Me ´dico-Chirurgicale d’He ´pato-Gastroente ´rologie, Ho ˆpital Beaujon, AP-HP, 92110 Clichy, France; U490 INSERM Laboratoire de Toxicologie 3 4 Mole ´culaire, 45 rue des Saints-Pe `res 75006 Paris, France; Laboratoire de Biochimie, Ho ˆpital Beaujon, 92110 Clichy, France; Service de Chirurgie Digestive et Oncologie, Ho ˆpital Europe ´en Georges Pompidou, 75015 Paris, France KRAS2 mutations in codon 12 have been detected in about 80% of pancreatic cancers. The aim of this study was to evaluate the value of KRAS2 mutations detection in circulating deoxyribo nucleic acid to differentiate pancreatic cancer from chronic pancreatitis. Circulating deoxyribo nucleic acid was isolated from serum in 47 patients with histologically proven pancreatic adenocarcinomas (26 males, median age 65 years) and 31 controls with chronic pancreatitis (26 males, median age 48 years). Mutations at codon 12 of KRAS2 gene were searched for using polymerase chain reaction and allele specific amplification. Serum carbohydrate antigen 19.9 levels were also determined. KRAS2 mutations were found in 22 patients (47%) with pancreatic cancer and in four controls with chronic pancreatitis (13%) (P50.002). None of the latter developed a pancreatic cancer within the 36 months of median follow-up. The sensitivity, specificity, positive and negative predictive values of serum serum KRAS2 mutations for the diagnosis of pancreatic cancer were 47, 87, 85 and 52%, respectively. KRAS2 mutations were not related to age, gender, smoking habit, tumour stage, or survival. Among the 26 patients with normal or non-contributive (due to cholestasis) serum carbohydrate antigen 19.9 levels, 14 (54%) had KRAS2 mutations. The combination of KRAS2 and carbohydrate antigen 19.9 gave a sensitivity, specificity, positive and negative predictive values for the diagnosis of pancreatic cancer of 98, 77, 87 and 96%, respectively. Detection of KRAS2 mutations in circulating deoxyribo nucleic acid has a low sensitivity but a specificity about 90% for the diagnosis of pancreatic cancer. It seems particularly useful when serum carbohydrate antigen 19.9 levels are normal or inconclusive. A combined normal serum carbohydrate antigen 19.9 and absence of circulating KRAS2 mutations makes the diagnosis of pancreatic cancer extremely unlikely. British Journal of Cancer (2002) 87, 551 – 554. doi:10.1038/sj.bjc.6600475 www.bjcancer.com ã 2002 Cancer Research UK Keywords: KRAS2 mutations; circulating DNA; pancreatic adenocarcinoma; chronic pancreatitis Five-year survival in patients with pancreatic adenocarcinomas is tions were first reported in surgically removed pancreatic tumoural less than 5%, partly due to advanced disease at diagnosis. The tissue or at autopsy (Almoguera et al, 1988; Tada et al, 1991). differentiation between pancreatic cancer and chronic pancreatitis Thereafter mutations were discovered in 63 to 83% of samples of can be particularly difficult leading to inappropriate treatment. pure pancreatic juice or main pancreatic duct brushing obtained Serum carbohydrate antigen 19.9 (Ca 19.9) levels are elevated in during endoscopic retrograde pancreatography (Iguchi et al, 80% of pancreatic cancer patients, but can also be increased in 1996; Kondo et al, 1997; Tada et al, 1998; Van Laethem et al, 20% of patients with chronic pancreatitis (Satake and Takeuchi, 1998; Okai et al, 1999; Watanabe et al, 1999; Ha et al, 2001; Pugli- 1994; Nouts et al, 1998). Moreover, pancreatic inflammation, as ese et al, 2001; Seki et al, 2001) or at fine-needle tumour aspiration (Pabst et al, 1999; Puig et al, 2000), and in 20 to 54% of stools observed in chronic pancreatitis, can be mistaken on imaging as cancer and inversely. An accurate and non-invasive test to differ- (Caldas et al, 1994; Wenger et al, 1999) from patients with entiate pancreatic cancer from chronic pancreatitis would be pancreatic cancer. Circulating deoxyribo nucleic acid (DNA) was extremely helpful. first detected in serum or plasma of normal subjects in 1975 (Stein- Previous studies have reported KRAS2 gene mutations (almost man, 1975). Since then, mutations in the KRAS2 gene have been always confined to codon 12) in 75 to 95% of exocrine pancreatic detected in the plasma of patients with colorectal, lung and haema- cancer (Caldas and Kern, 1995). KRAS2 mutations provoke activa- tological cancers (Anker et al, 1997). To date, few studies have tion of nuclear transcriptor factors, resulting in cellular reported KRAS2 mutations in circulating DNA in patients with proliferation and also in tumour angiogenesis as reported recently pancreatic cancer with a wide spectrum of sensitivity (27 to (Banerjee et al, 2000; Ikeda et al, 2001). Detection of KRAS2 muta- 81%) (Sorenson et al, 1994; Mulcahy et al, 1998; Yamada et al, 1998; Castells et al, 1999; Porta et al, 1999; Theodor et al, 2000). The aim of our study was to evaluate the value of KRAS2 mutation *Correspondence: F Maire; E-mail: [email protected] detection in circulating DNA in a large series of patients to differ- Revised 16 May 2002; accepted 23 May 2002 entiate pancreatic adenocarcinoma from chronic pancreatitis. Molecular and Cellular Pathology Molecular and Cellular Pathology KRAS2 mutations in circulating DNA in pancreatic adenocarcinoma F Maire et al positive controls were performed for each set of PCR reactions. PATIENTS AND METHODS PCR products were separated by electrophoresis in a 6% acryla- mide gel and stained with ethidium bromide. Selection and outcome of patients Between January 1995 and 1999, 47 patients (26 males and 21 Serum Ca 19.9 dosage females, median age 65 years (range 39 – 84)) with pancreatic ductal adenocarcinoma were included in the study. In all of them, The serum value of Ca 19.9 was measured with a commercial solid- diagnosis was confirmed by pathological examination of pancreatic phase double-antibody sandwich immunoassay (Roche Labora- tumour obtained by fine needle aspiration during endoscopic ultra- tories, Basel, Switzerland). The upper limit of normal value was sonography (n=42) or operative procedure (n=5). Tumour staging 37 UI ml . All patients and controls also underwent biochemical was established by abdominal computed tomography, endoscopic liver tests. Cholestasis was defined by alkaline phosphatase levels ultrasonography or operative findings: stage I (n=5, 11%), stage above twice the normal value. II or III (n=19, 40%) and stage IV (n=23, 49%) according to the TNM classification (UICC, 1997). Five patients underwent surgical Statistical analysis resection, 32 patients received systemic chemotherapy and/or radiotherapy and 10 symptomatic treatment. Median follow-up For each marker (serum KRAS2 mutations and Ca 19.9), sensitiv- was 6 months (range 1 – 24). At the end of the study, all but four ity, specificity, positive and negative predictive values were patients with pancreatic cancer were dead. calculated. Thereafter, the combination of both markers (i.e., one and/or the other positive) was studied. The chi-squared test was used to compare the occurrence of KRAS2 mutations. Differences Control group were considered significant when P50.05. A control group was recruited during the same time in the same centre and included 31 patients with chronic pancreatitis (26 RESULTS men and five women, median age 48 years (range 20 – 64)). Diag- nosis of chronic pancreatitis relied upon the presence of pancreatic Circulating DNA quantification calcifications and/or irregularity of pancreatic ducts, according to Cambridge Classification (Axon et al, 1984) on computed tomo- Adequate DNA was extracted from the serum in sufficient quanti- graphy scan and endoscopic retrograde pancreatography, ties for analysis in all patients and controls. All patients but one respectively. Etiology of chronic pancreatitis was alcoholic in 30 had a serum DNA concentration higher than the threshold of patients and idiopathic in one patient. No case of pancreatic cancer detection of 100 ng ml . The mean concentrations of DNA occurred during the 36-month follow-up in these 31 patients. extracted from serum of patients with pancreatic cancer and 71 71 chronic pancreatitis were 730+90 ng ml and 560+93 ng ml , respectively (P=0.19). DNA extraction and quantification Peripheral venous blood samples were collected after informed KRAS2 mutations in circulating DNA consent in patients and controls. Blood samples were centrifuged, serum was removed and stored at 7208C until use. DNA was KRAS2 mutations were identified in the serum of 22 patients extracted from serum by using the QIAmp Blood Kit (Qiagen, (47%) with pancreatic adenocarcinoma and in four patients Courtaboeuf, France) according to the blood and body fluid proto- (13%) with chronic pancreatitis (P50.002). The sensitivity, speci- col recommended by the manufacturer. Two millilitres of serum ficity, positive and negative predictive values of serum KRAS2 were used, and a DNA elution volume of 50 ml was obtained by mutations for the diagnosis of pancreatic cancer were 47, 87, 85 extraction. The DNA elution was then concentrated to a final and 52%, respectively. There were no statistically significant differ- volume of 15 ml. ences in age, gender, smoking, tumour stage and survival, Quantification of serum DNA was performed for all samples according to presence or absence of plasma KRAS2 mutations. using a volume of 3 ml of DNA elution obtained after Qiagen Among patients with chronic pancreatitis, no cancer occurred extraction by fluorescence emission after intercalation of Hœschst after a mean follow-up of 36 months (range 13 – 64), even in those dye. The fluorescence was read by DyNA Quanto 200 fluorimeter, with positive KRAS2 mutations (with follow-up of 40 months, using the cuve and capillary DyDNA Capillary Cuvette Adaptor Kit range 20 – 61), assessed by clinical observation and abdominal (Pharmacia Biotech, Orsay, France). The threshold of DNA detec- computed tomography scan. tion established by the manufacturer is 2 ng ml , which corresponds to 100 ng ml serum in our extraction protocol Serum Ca 19.9 levels (Coulet et al, 2000). The sensitivity, specificity, positive and negative predictive values of abnormal Ca 19.9 levels for the diagnosis of pancreatic cancer were Detection of KRAS2 gene mutations 91, 87, 91 and 87%, respectively (Table 1). Serum Ca 19.9 levels Only G12D mutations in codon 12 of the KRAS2 gene were were normal or non interpretable due to cholestasis in 26 patients. searched for using allele-specific amplification, with the following Among them, 14 (54%) patients had serum KRAS2 mutations. primers: 5'-CTTGTGGTAGTTGGAGCTAA-3',5'-AATGGTCCT- Combination of both tests increased sensitivity to 98% with a GCACCAGTAATATG-3'. Amplifications were performed with negative predictive value of 96% for the diagnosis of pancreatic 0.3 mM of each primers, 200 mM of each deoxynucleotide tripho- cancer (Table 1). sphate (dNTP), 1.5 mM of MgCl , 0.025 units per mlof AmpliTaq Gold polymerase Cetus (Perkin Elmer), 2.5 mlor5 ml DISCUSSION of 106 buffer, 5 ml of the concentrated DNA elution was used as template in a 50 ml volume reaction. Polymerase chain reaction In the present study, only the most frequent KRAS2 gene muta- (PCR) with serum DNA was performed 10 min at 948C, followed tion G12D (aspartic acid) observed in pancreatic cancer (Iguchi by 60 cycles of 948C for 30 s, 618C for 30 s, 728C for 1 min and a et al, 1996; Tada et al, 1998; Castells et al, 1999; Watanabe et final extension of 10 min at 728C. Controls without DNA and al, 1999) was analysed in the serum of patients and controls, in British Journal of Cancer (2002) 87(5), 551 – 554 ã 2002 Cancer Research UK KRAS2 mutations in circulating DNA in pancreatic adenocarcinoma F Maire et al Table 1 Accuracy of serum KRAS2 mutation detection, serum Ca19.9 is not informative in 5% of population who cannot express serum levels, and both for the diagnosis of pancreatic cancer Ca 19.9 due to Lewis a negative status (Narimatsu et al, 1996). However, serum Ca 19.9 lacks specificity (70 to 80%): it can be Serum KRAS2 Serum Both tests increased in cholestasis, diabetes mellitus or chronic pancreatitis mutations Ca19.9 combined (Nouts et al, 1998). In the present study, serum Ca 19.9 had very good specificity (87%), similar to that of serum KRAS2 mutations. Sensitivity (%) 47 91 98 Combination of both tests could be useful to assess cancer diagno- Specificity (%) 87 87 77 Positive predictive value (%) 85 91 87 sis in patients with normal or non contributive Ca 19.9 due to Negative predictive value (%) 52 87 96 cholestasis or negative Lewis a antigen status, and to exclude cancer diagnosis when both tests are negative (predictive negative value of 96%). In the present study, the presence of KRAS2 mutations in order to limit the cost of such test and to validate it in clinical serum was not correlated to age, gender and smoking habit. It practice. was neither correlated to tumour stage since mutations were Our study underlines the high feasibility of KRAS2 mutations detected in plasma of patients with non metastatic tumours, analysis, as circulating DNA was obtained in sufficient quantities which supports the hypothesis that KRAS2 mutations are early in all patients. Mean serum DNA concentration were 730 ng ml , events in pancreatic carcinogenesis (Jimenez et al, 1999). Two which is comparable to that observed in the series of Mulcahy et al studies in the literature are in agreement with this result (Yamada (1998). et al, 1998; Theodor et al, 2000), but another one found a statis- In the present study, detection of KRAS2 mutations in circulat- tically significant relation between circulating DNA KRAS2 ing DNA had a low sensitivity but a high specificity for the mutations and poor prognosis (Castells et al, 1999). Yamada et diagnosis of pancreatic cancer. The sensitivity (47%) was in agree- al (1998) have reported disappearance of detectable mutation in ment with previous studies (27 to 81%) (Mulcahy et al, 1998; plasma after tumoural resection or radio-chemotherapy in six of Yamada et al, 1998; Castells et al, 1999; Porta et al, 1999; Theodor nine patients, suggesting KRAS2 mutations may be used as a et al, 2000; Zambon et al, 2000), although the search for five other tumour relapse marker. possible KRAS2 mutations in codon 12 was not performed. Higher Screening for malignancy in patients with chronic pancreatitis is KRAS2 mutation prevalences have been reported in pancreatic or a difficult challenge. Patients with chronic pancreatitis have an duodenal juice (63 to 87%), due probably to higher DNA tumour increased risk of pancreatic cancer, estimated at 1.8% at 10 years content in pancreatic juice as compared to plasma (Wilentz et al, and 4% at 20 years (Lowenfels et al, 1993). Three studies have eval- 1998; Van Laethem et al, 1998; Watanabe et al, 1999). Use of uated occurrence of pancreatic cancer in patients with chronic samples of pancreatic juice however requires invasive procedures pancreatitis with respect to KRAS2 mutations in pancreatic juice: (fine-needle aspiration during endoscopic ultrasonography or only one found an increase in pancreatic cancer in patients with endoscopic retrograde pancreatography). Three studies have KRAS2 mutations with methodological limitations (few cases, early reported a higher specificity of serum KRAS2 mutations compared diagnosis of cancer after inclusion) (Furuya et al, 1997; Lohr et al, to the current study (100 vs 87%), but their control groups 2001; Queneau et al, 2001). To our knowledge, no study focused included few patients and essentially healthy subjects (Mulcahy et on the incidence of pancreatic cancer in patients with chronic al, 1998; Porta et al, 1999; Theodor et al, 2000). Since KRAS2 pancreatitis according to serum KRAS2 mutations. In the present mutations have been reported in pancreatic tissue or juice from study, follow-up was too short to demonstrate an increased risk 6 – 42% of patients with chronic pancreatitis (Furuya et al, 1997; of cancer in patients with chronic pancreatitis and serum KRAS2 Mulligan et al, 1999; Lu ¨ ttges et al, 2000; Ha et al, 2001), and know- mutations. ing that a part of this mutated DNA can be released into In conclusion, although detection of plasma KRAS2 mutations circulation (Yamada et al, 1998), the control group should include in circulating DNA is not a definitive argument for malignancy, patients with chronic pancreatitis. In the series published by it could contribute to cancer diagnosis. This test seems particularly Castells et al (1999) with the largest control group (including interesting in patients with normal or inconclusive Ca 19.9 levels patients with chronic pancreatitis), specificity was comparable due to cholestasis or Lewis a negative status. In patients with (94%) to the present study, and increased in the presence of a normal serum Ca 19.9 levels and no KRAS2 mutation, the diagno- pancreatic mass. The accuracy of serum KRAS2 mutation detection sis of pancreatic cancer can be excluded with almost certainty. in the differential diagnosis between pancreatic cancer and chronic pancreatitis may be improved by performing quantitative PCR measurement of mutated DNA, in order to discriminate patients with unspecific low levels of mutated DNA (as supposed in chronic pancreatitis) from patients with high levels (pancreatic cancer), as suggested in pancreatic juice analysis by Tada et al (1998). 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Gastrointest Endosc 50: 797 – 803 tisfactory technique for clinical applications. Clin Chim Acta 302: 35 – 48 British Journal of Cancer (2002) 87(5), 551 – 554 ã 2002 Cancer Research UK

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British Journal of Cancer – Springer Journals

Published: Aug 27, 2002

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