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Low Sensitivity of the ki-ras Polymerase Chain Reaction for Diagnosing Pancreatic Cancer From Pancreatic Juice and Bile: A Multicenter Prospective Trial

From the Department of Internal Medicine I and Department of Internal Medicine II, University of the Saarland, Homburg; Diakonissen Hospital, Ludwigslust; and Department of Medicine II, Ludwig-Maximilian University, Munich, Germany.

Address reprint requests to Lorenz Trümper, MD, PhD, Department of Hematology and Oncology, Georg-August-University, Robert-Koch-Str 40, 37075 Goettingen, Germany; email: lorenz.truemper{at}med.uni-goettingen.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: Early detection of pancreatic cancer using molecular markers may improve outcome. Mutations of the ki-ras oncogene are detected in 70% to 90% of pancreatic adenocarcinomas. A prospective, partially blinded, multicenter diagnostic trial was performed to test the sensitivity and specificity of the ki-ras polymerase chain reaction (PCR) analysis of pancreatic juice and bile specimens.

PATIENTS AND METHODS: Specimens of pancreatic juice and bile were collected from 532 consecutive patients. Mutations in codon 12 of the ki-ras gene were identified by two independent enrichment PCRs and confirmed by direct sequencing.

RESULTS: One hundred seventy-four of 532 patients were excluded from the final analysis (reasons: no amplifiable DNA, no specimen or only duodenal juice sent, lost to follow-up). Sixty-three of 358 patients had ductal pancreatic cancer. In 24 (38.1%) of 63 patients, a mutated ki-ras gene was identified in pancreatic juice and/or bile. Ki-ras mutations were found in four (8%) of 50 cases of chronic pancreatitis, in 10 (18.7%) of 53 cases of other malignancies of the pancreaticobiliary tree, and in 14 (7.3%) of 192 cases of benign diseases or normal findings. Sensitivity and specificity of the ki-ras PCR analysis for the detection of pancreatic cancer was 38.1% and 90.5%, respectively.

CONCLUSION: In this prospective trial performed in nonselected patients, mutations of the ki-ras gene were detected in 38.1% of cases with pancreatic cancer. This test in its present form is not appropriate to confirm or screen for pancreatic cancer. More sensitive and/or quantitative PCR tests may improve the molecular diagnosis of pancreatic cancer.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE INCIDENCE OF pancreatic cancer is rising in the Western world; at present, it represents the fourth most common cause of cancer-related deaths, responsible for more than 25,000 deaths per year in the United States. Only radical resection of the tumor with surrounding lymph nodes provides a curative chance for the patient; unfortunately, this option is limited to approximately 20% of the patients with pancreatic cancer, because the majority of cases are diagnosed at a late stage of disease.^1,2 Because chemotherapy and radiation do not bear a curative potential, earlier detection of the disease is the only way to improve the outcome of patients with pancreatic cancer.

In 1988, the first report on a high frequency of Kirsten-ras (ki-ras) codon 12 mutations in pancreatic cancer was published.³ The large majority of mutations have been shown to be situated in codon 12.^4-8 In several small series of nine to 33 patients, the mutations could also be detected in pure pancreatic^9-11 or in duodenal juice¹² using different polymerase chain reaction (PCR) techniques. The potential use of this molecular technique for the early detection and diagnosis of pancreatic cancer generated much interest¹³ and enthusiasm. The frequency of mutations in pancreatic cancer was estimated at 75% to 95%, when investigating histopathologic specimens.^3,4,7,14 In the largest series so far, mutated ki-ras genes were detected in 63 (75%) of 84 patients.¹⁴ Because of the high frequency of these mutations in pancreatic carcinoma, ki-ras mutations are generally believed to occur at an early stage of tumor development.¹⁵

All published data on the identification of ki-ras mutations in pancreatic or duodenal juice are based on small, probably selected, and often retrospectively analyzed series of patients. Therefore, we designed a prospective, multicenter clinical trial to study the frequency of ki-ras mutations in pancreatic juice, bile, and duodenal juice, aspirated during the process of diagnostic evaluation of unselected, consecutive patients undergoing endoscopic retrograde cholangiopancreatography (ERCP), and further, to correlate the molecular findings (obtained without prior knowledge about the clinical diagnosis on the part of the molecular biologists) with the definitive diagnoses of the patients during follow-up. The primary aim was to test the sensitivity and specificity of the ki-ras enrichment PCR in pancreatic juice or bile for pancreatic cancer and other gastrointestinal diseases including chronic pancreatitis and different benign or malignant diseases of the pancreaticobiliary tree. The secondary aim was to test the prognostic significance of the identification of a ki-ras mutation in chronic pancreatitis.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The study protocol was approved by the local ethics committee of the University of Saarland on March 9, 1995, and the respective local boards in each participating center.

Centers
Six gastroenterology departments in Germany participated in this trial (Department of Internal Medicine at the University of Bonn; Department of Internal Medicine IV, University Hospital, Cologne; Department of Internal Medicine, Salem Hospital, Heidelberg; Department of Internal Medicine II, University of the Saarland; Department of Medicine II, Ludwig-Maximilian-University, Großhadern Hospital, Munich; and Department of Internal Medicine I, University Hospital, Regensburg). All laboratory investigations were performed in the Oncology Laboratory of the Department of Internal Medicine I, University of the Saarland, Homburg, by L.T., H.D., and A.S. The evaluation and analysis of the clinical primary and follow-up data was performed by M.M., D.K., J.-O. R., and M.S.

Patients
All patients who underwent ERCP for evaluation of a suspected pathologic process in the pancreaticobiliary tree were eligible for inclusion in this study. The investigators were not involved in the definition of the indications for ERCP and there was no selection of indication criteria, so that the ERCP indications reflected the usual spectrum: the majority of patients suffered from obstructive jaundice or right upper abdominal pain or had a pancreatic mass. Patients had to give their written consent in extending the investigation to the aspiration of some pancreatic juice or bile during selective cannulation of the concerned ducts. Secretin stimulation was not used. To test the specificity of the ki-ras–enrichment PCR, patients with different benign processes (eg, suspected cholangiolithiasis or chronic pancreatitis) were also recruited. Between April 1995 and February 1999, 642 consecutive patients were enrolled in the six participating centers and assigned a patient number. Data on the ERCP findings, the suspected diagnosis, and personal data of the patients including name, age, address, the referring hospital, and the primary care physician to allow for follow-up were recorded on a separate sheet labeled with the patient number. These sheets were sent by the endoscopists in closed envelopes to the study center and were stored and analyzed separately from the samples.

Extraction of Specimens/ERCP
The ERCP was performed under conscious sedation exclusively by experienced investigators with standard duodenoscopes. Having reached the ampulla of Vater, several milliliters of duodenal juice in its surrounding was aspirated through a probe introduced through the working channel of the duodenoscope into a sterile syringe. Filling of both the pancreatic duct and the bile duct system with contrast media was attempted. After selective cannulation of the major pancreatic duct, contrast media was injected until entire filling was reached for radiographic documentation. Then, 1 to 2 mL of pancreatic juice mixed with contrast media was aspirated into a sterile syringe. The same was done with bile after selective cannulation of the common bile duct. The results of ERCP of each patient were documented on a standardized documentation sheet, labeled with the patient number. The indication for ERCP, the mode of investigation (emergency or routine case, first or follow-up investigation), the aspirated material, and the findings were noted. The specimens were labeled with the patient number corresponding to the patient sheet and sent to the laboratory (Homburg center) either immediately by courier or surface mail at room temperature or stored at -20°C after centrifugation and resuspension in 50 µL of TEN buffer until shipment on ice at a later date. Thus, samples were kept at ambient temperature for a maximum of 24 hours. In 66 cases, specimens from the same patient were sent more than once on the occasion of repeated ERCP.

Preparation of Samples
Unprocessed secretions (approximately 1 mL) were transferred into Eppendorf tubes, centrifuged at high speed, resuspended in buffer, and digested with proteinase K as described before.^11,16 Frozen samples in TEN buffer were thawed and digested as above.

PCR
Special precautions, including separate handling of PCR reagents and products in different rooms and pipetting PCR reactions under a laminar air flow hood with filtered tips, were taken to avoid contamination according to the standard operation procedures of the laboratory (available on request). Appropriate controls (PaTu cell line [mutation GGT to GTT at codon 12] and wild-type control at codon 12 [Jurkat cell line; H₂O for PCR contamination]) were included in all reactions. Ten microliters of the proteinase-digested DNA from pancreatic secretions served as a template for PCR. The PCR was performed as described previously.^11,16-18 In brief, PCR was performed in a reaction volume of 50 µL containing 200 µmol/L of dNTP (Pharmacia Biotech, Freiburg, Germany) and 20 pmol each of the primers K-ras-12-13-3' and 12-13-5' for 40 cycles and analyzed by agarose gel electrophoreses and single-strand conformation polymorphism analysis on PHAST-polyacrylamide gel (Pharmacia). Subsequently, enrichment PCR was performed as described before by Kahn et al¹⁹ with minor modifications.¹⁷ DNA contained in 10 µL (20%) of the resuspended digested cell extract was amplified by PCR for 15 cycles with 100 ng each of primers K-ras-12-13-5'-BstN1 and 12-13-3'WT in a volume of 100 µL. After digestion with the BstN1 enzyme (New England Biolabs, Germany) the digest was used as template for a second round of PCR followed by a second digestion step and analysis on native polyacrylamide gel. Selected PCR products were excised and sequenced using the SequiTherm cycle sequencing kit (Biozym Diagnostik, Germany) using oligonucleotide K-ras-12-13-3'-BstN1 as a sequencing primer. Oligonucleotide primer sequences used included the following:

k-ras-12-13-3': 5'TGT TGG ATC ATA TTC GTC CA 3'

k-ras-12-13-5': 5'CCT GCT GAA AAT GAC TGA AT 3'

k-ras-12-13-3'-WT: 5'TCA AAG AAT GGT CCT GCA CC 3'

k-ras-12-13-3'-BstN1: 5'TCA AAG AAT GGT CCT GGA CC 3'

k-ras-12-13-5'-BstN1: 5'ACT GAA TAT AAA CTT GTG GTA GTT GGA CCT 3'

Evaluation of the Results
We have previously shown¹⁷ that the rate of false-positive results of the restriction fragment length polymorphism (RFLP) PCR can be quite high and have developed the following strategy for the analysis of pancreatic and duodenal secretion samples:

1. All samples were amplified by standard ki-ras PCR to check for the presence of amplifiable DNA.
   
2. Negative samples were checked with a beta-actin PCR to confirm that no amplifiable DNA was present.
   
3. Positive samples were amplified by RFLP-PCR and analyzed by polyacrylamide gel electrophoresis analysis. Samples with clearly distinguishable mutant band (143 bp) were again amplified starting with the digested cell pellet.
   
4. Only samples showing mutation bands in two independent PCRs were scored as positive, thereby reducing the number of false-positives.¹⁷
   
5. Approximately 10% of the positive samples were randomly selected for a second (ie, in addition to the RFLP-PCR) confirmation by direct sequencing.
   

Results were reported as mutated or wild type and submitted to the study database. Bile and pancreatic juice were analyzed separately. If in a patient only one of those two contained mutated ki-ras genes, the patient was classified as positive. All secretions that we received of an individual patient at the time of entering the study were taken into account. Duodenal juice specimens were not considered in this analysis—if we received only duodenal juice from a patient, this patient was not assessable, because the study protocol did not permit duodenal juice examinations for the primary study aims.

Definitive Diagnoses
Clinicians were blinded to the PCR results and the laboratory personnel were blinded to the clinical diagnoses of the patients until the final evaluation, which was performed after the recruitment period. A definitive clinical diagnosis was established either by surgical resection or by imaging methods such as computed tomographic (CT) scanning or magnetic resonance imaging (MRI) in combination with the clinical disease course. In all cases where there was doubt, patients were followed up by contacting the referring hospital and the family physician of the individual patient to obtain the necessary information from medical records or postmortem reports. In case of a ki-ras mutation in chronic pancreatitis, this follow-up was performed repeatedly throughout the whole period of the study. Eventually, in all but 12 cases a definitive diagnosis could be established (lost to follow-up).

Statistical Calculation
On the basis of former studies,¹⁶ we presumed a sensitivity and specificity of 93% for the ki-ras enrichment PCR of pancreatic juice or bile (identification of mutations on codon 12) for the diagnosis of pancreatic cancer in a two-tailed test. The test is designed to disclose a sensitivity and specificity of more than 85% with a probability of error of 5% (alpha_nom = 0.025) and a statistical power of 80% (beta_nom = 0.10, two-tailed test). One hundred sixty assessable patients with or without pancreatic cancer were necessary to achieve this statistical power. Because ERCP is intended to clarify a pathologic pancreas process in about one third of cases, a total number of approximately 480 patients had to be recruited. Statistical evaluation was not performed until 12 months after enrollment of the last patient onto the study, so that each patient could be entered onto one of the following groups: I, pancreatic cancer without correction of diagnosis; II, initial ERCP diagnosis of pancreatic cancer was false-positive, patient suffers from another disease; III, initial ERCP diagnosis was false-negative, patient suffers from pancreatic cancer; or IV, initial diagnosis was correctly negative, patient suffers from a different disease.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eligible Patients
Between April 1995 and February 1999, 642 patient numbers were allocated to patients at the six participating centers. Because of incorrect repeated allocations of patient numbers to the same patient (n = 66), missing sheets with personal data referring to specimens sent (n = 27), or cases in which erroneously only biopsy samples were sent (n = 17), 532 patients could be enrolled on the primary analysis presented here. In a second step, a total of 174 patients were classified as not assessable when their specimens of bile or pancreatic juice did not contain DNA as judged by a negative ki-ras and beta-actin PCR (65 cases); no specimens reached the laboratory even though patient number sheets were sent (47 cases); only duodenal juice was sent to the laboratory (40 cases); the amount of secretions was not enough to perform a (second) confirmation PCR when the first PCR was interpreted as mutant ki-ras (10 cases); or no definitive diagnosis was established during follow-up (lost to follow-up) (12 cases). Eventually, 358 patients could be enrolled on the final analysis (Fig 1).

View larger version (32K): [in this window] [in a new window]   Fig 1. Study synopsis. Of 532 patients registered, 358 were enrolled on the final analysis and grouped into four clinical categories. Mutations denote the proportion of patients with mutated ki-ras genes; wild type denotes those with unmutated ras genes.

Identification of ki-ras Mutations: Per-Patient Analysis
Within the group of pancreatic cancer, there were 24 (38.1%) of 63 cases with confirmed mutations in codon 12 of the ki-ras gene, 10 of which were identified in 39 pancreatic juice specimens and 15 of which were identified in 49 bile specimens. In the majority of cases, we received either bile or pancreatic juice; and in 25 cases, bile and pancreatic juice were sent simultaneously. In a single case of those 25, both pancreatic juice and bile revealed a ki-ras mutation (Table 1).

Follow-Up of Patients With a Mutated ki-ras Gene Suffering From Benign Disease
There were four cases of mutated ki-ras oncogenes in 50 patients with chronic pancreatitis and another 14 cases with identification of a mutation in 192 cases of other benign diseases or normal findings. These 18 patients were closely followed up by phone interview of the primary care physicians or hospital chart review for 33 months (median, 13 to 54 months). None of the patients developed signs of pancreatic malignancy.

Patients With Inconsistent Results of the ki-ras PCR in Repeatedly Sent Specimens
We received specimens of 66 patients repeatedly. Of these 66, in four patients the results of single ki-ras PCR investigation differed during the course of the study. This applied exclusively to bile specimens. According to the definition, each patient with a single confirmed positive result was classified as positive.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Despite substantial progress in improving imaging techniques such as ultrasound, computed tomography, and magnetic resonance imaging, the preoperative and noninvasive diagnosis of pancreatic cancer remains difficult. With respect to the so-called tumor markers, only CA 19-9 is of substantial clinical benefit in diagnosing pancreatic malignancy. Applying the usual cutoff value of 37 U/mL, the sensitivity and specificity reach 80% and 40% to 75%, respectively.²¹ However, CA 19-9 values are strongly influenced by cholestasis, and thus are helpful only in the minority of cases without cholestasis.

Noninvasive imaging methods, especially the recent applications of helical CT scanning and MRI, are able to detect a pancreatic tumor of approximately 1 cm in diameter, but rarely discriminate between an inflammatory and a malignant pancreatic head tumor. ERCP is currently the best invasive diagnostic method, and yields a sensitivity and specificity in diagnosing pancreatic cancer of 78% to 95%,^22-24 respectively, in the hands of experienced investigators. The so-called double-duct sign, a characteristic radiographic feature of pancreatic malignancy, predicts the presence of pancreatic cancer with a specificity of about 85%,²⁵ but even the combination of the double-duct sign with a pancreatic mass seen on CT scan or MRI does not prove pancreatic cancer, because a number of cases with chronic pancreatitis meet these criteria also.

If technically possible, diagnosis may be confirmed by additional brush cytology or forceps biopsy of the stenosis of the major pancreatic or the common bile duct. However, even in the hands of experienced investigators, this procedure reaches a diagnostic yield of 60% to 70% at maximum.^26-29 In a large series of 312 consecutive patients with extrahepatic bile duct stenosis, the sensitivity of brush cytology for malignancy was not higher than 36%.³⁰

Ultrasound-guided transcutaneous fine-needle aspiration yields a specificity up to 100%,³¹ but the sensitivity is not higher than 72% to 75%^32-34 and bears the risk of spreading tumor cells along the biopsy channel. Therefore, this procedure should be avoided as long as surgical resection of the process is under discussion.

Finally, endoscopic ultrasonography (EUS) offers a precise visualization of pancreatic tumors larger than 5 to 10 mm. The sensitivity of the method in various series of more than 20 patients was consistently above 95%.^18,35,36 In a prospective and comparative study with a total of 76 patients with pancreatic carcinoma, the sensitivity of EUS was 99% compared with 77% for CT scanning.³⁷ Yet, similar to other imaging methods, EUS cannot differentiate between a benign or malignant mass of the pancreas because the malignancy of a given lesion cannot be determined on the basis of its endosonographic features.

Recently, EUS-guided fine-needle aspiration has gained interest because it allows for cytologic investigation of a pancreatic mass that is not detectable by transcutaneous ultrasound. Sensitivity and specificity in the hands of experienced investigators reach 80% and higher.^38,39 However, this method is highly sophisticated, is burdened with a considerable rate of false-negative results (especially when examining only small numbers of patients), and is available only in a few centers so far.

It was hoped that the combination of standard diagnostic procedures and novel molecular tests designed to detect presumably tumor-specific markers with a high sensitivity would greatly improve the diagnostic accuracy in pancreatic cancer. The combination of ki-ras analysis with ERCP could theoretically raise the sensitivity and specificity of the investigation to over 98%. It was also proposed that the early detection of these molecular markers in patients at risk for the development of cancer (eg, patients with chronic pancreatitis) might aid in the early diagnosis of cancer. However, only stringent diagnostic trials in analogy to clinical phase II and III trials can prove these assumptions and determine the clinical value of the respective tests.⁴⁰

Our study therefore aimed at testing the hypothesis that the majority of pancreatic cancers can be detected by ki-ras analysis in bile or pancreatic fluid under real-life conditions (multicenter analysis, necessity of sending specimens by mail with consecutive delay of processing in the laboratory, different endoscopists, blinded analysis in the laboratory with regard to the suspected clinical diagnosis) and in a prospective manner. The test was not designed to be a screening test because only patients with clinical symptoms would be tested. However, the hope was that this test would aid in the differential diagnosis in situations where imaging methods do not discriminate between inflammatory and malignant lesions. However, even after optimization of our method for the detection of ki-ras mutations, compared with our previous studies,^11,16,18,41 the sensitivity of the ki-ras PCR analysis of bile and/or pancreatic fluid did not exceed 40%. The specificity of the test is in fact (with approximately 90%) much better, but this represents no clinically relevant progress, because 10 of 100 cases of cancer would remain undetected and the specificity of ERCP alone is not much lower.

In our opinion, the major obstacles to an increased sensitivity of the ki-ras PCR analysis used here are as follows: it is difficult or impossible to obtain sufficient amounts of pancreatic juice in cases of a filiform stenosis, especially when located near the ampulla of Vater; aspirated bile will not contain tumor cells, if there is only encasement and no infiltration of the common bile duct by the tumor; not all malignant adenocarcinomas of the pancreas contain ki-ras mutations; and the sensitivity of the PCR test is limited, because of technical limitations of the assay and because application of contrast media for ERCP may further dilute the pancreatic juice containing scarce tumor cells.

The specificity of the ki-ras PCR analysis is limited by the fact that a couple of specimens of patients with different malignant and benign diseases tested positive. This may be because of false-positive PCR results that may occur when using the enrichment PCR procedure,¹⁸ or because of mutations that may be present in nonmalignant diseases of the pancreatic duct (see below).

Because chronic pancreatitis carries an increased risk for the later development of pancreatic cancer, the finding of Furuya et al,⁴² who compared patients with pancreatic cancer and chronic pancreatitis and found that up to 37% of the latter showed mutations of the ki-ras gene, generated much interest. In addition, different mutations at codon 12 in the same tumor⁴³ and mutations in adjacent inflammatory tissues¹¹ were described by others, suggesting that the detection of mutations of the ki-ras oncogene may not be sufficient to diagnose a malignancy.

In our study, four of 50 (8%) patients with chronic pancreatitis revealed a mutated ki-ras oncogene. We did not observe the development of pancreatic cancer in this group during a relatively short mean follow-up period of 33 months. Furuya et al⁴² also did not observe the development of cancer among 20 pancreatitis patients during a mean follow-up period of 78 months. Eight of 33 (24.2%) cholangiocellular, gallbladder, or periampullary carcinomas tested positive. Mutations of the ki-ras gene have been described in cases of cholangiocellular carcinomas^44,45 and periampullary carcinomas,⁴⁶ and are a frequent phenomenon in gallbladder carcinoma, especially in cases with an anomalous junction of the pancreaticobiliary tract.⁴⁷ Motojima et al,⁷ in contrast, postulated that the consideration of the ki-ras status allows for distinction of periampullary from true pancreatic carcinomas. Clinically, there should be no problem in distinguishing gallbladder from pancreatic cancer, but a distal cholangiocellular carcinoma and especially periampullary tumors are likely to be confused with pancreatic cancer in several cases.

The problem of varying results in repeatedly aspirated secretions has not been described yet. Provided that the technique of ERCP was the same (the repeatedly sent bile specimens originated from the same institutions in each case), a negative result that switched to positive in the course of the study is most likely because of an intercurrent invasion of the common bile duct wall by the tumor.

The results of this study highlight some of the major obstacles to the development of molecular tests in the diagnosis and follow-up of human cancers. Molecular markers may consist of individual molecular aberrations (such as specific immunoglobulin rearrangements in malignant lymphomas) or acquired genetic aberrations that commonly occur during the multistep development of cancer. Whereas the former are relatively specific and have been successfully applied in the clinical monitoring of lymphatic leukemias,⁴⁸ less progress has been made in transferring analyses of the latter to the clinic. Mutations or deletions of oncogenes such as the ki-ras gene or tumor suppressor genes are frequently found in human epithelial cancer. They have been successfully used in the molecular staging (eg, for head and neck cancers⁴⁹) and follow-up. However, single-oncogene mutations may neither be necessary for a given cancer (as illustrated by the rate of wild-type ras cancers in pancreatic or colorectal cancer) nor specific, as demonstrated by the presence of ki-ras mutations in hyperplastic duct cells of the pancreas without pancreatic disease.⁴³ The American Society of Clinical Oncology guidelines⁴⁰ rightly state that "new markers are frequently introduced into clinical practice without rigorous analysis, with the assumption that any information available to the clinician may help the patient." The trial reported here supports the notion that the latter assumption is indeed wrong.

Finally, the analysis of bile or pancreatic juice specimens for ki-ras mutations in detail is a costly procedure. Performance of enrichment PCR and the confirmation PCR takes about 2 days for the laboratory personnel. The currently refunded amount of approximately 210 euros per PCR in Germany only covers the costs for material and equipment used. However, the demonstration that this method has a fairly high positive predictive value may guide in the development of molecular assays that carry a higher specificity. The combination of several markers such as ki-ras with microsatellite instability⁵⁰ and other oncogene alterations such as DPC4 deletions⁵¹ may hold promise for the future. In addition, the number of mutated cells may be much lower in inflammatory lesions than in malignant lesions; therefore, quantitative analyses⁵² may also be used to define threshold values for the detection of cancer. Trials such as the one presented here are labor intensive, but are nonetheless essential in establishing or refuting presumably better diagnostic procedures. Molecular markers should not be introduced into clinical practice without such rigorous testing.

	ACKNOWLEDGMENTS

 
Supported by the Wilhelm-Sander-Stiftung, Neuburg a.d.Donau, through grant nos. 93.043.1-3 (L.T., H.D). This article contains data that are part of the MD thesis at the Medical Faculty of the University of Saarland (D.K., J.-O. R).

	NOTES

 
L.T. and M.M. contributed equally to this work.

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Submitted June 13, 2001; accepted July 10, 2002.

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