This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sturm, P. D. J. Articles by Offerhaus, G. J. A. Search for Related Content PubMed PubMed Citation Articles by Sturm, P. D. J. Articles by Offerhaus, G. J. A.

Clinical Value of K-ras Codon 12 Analysis and Endobiliary Brush Cytology for the Diagnosis of Malignant Extrahepatic BileDuct Stenosis¹

Departments of Pathology [P. D. J. S., E. C., T. B. R., L. A. N., G. J. A. O.] and Gastroenterology [E. A. J. R., K. H.] Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, the Netherlands; and Departments of Pathology [R. H. H.] and Oncology [R. H. H.], The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205

	ABSTRACT

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Extrahepatic biliary stenosis can be caused by benign and malignant disorders. In most cases, a tissue diagnosis is needed for optimal management of patients, but the sensitivity of biliary cytology for the diagnosis of a malignancy is relatively low. The additional diagnostic value of K-ras mutational analysis of endobiliary brush cytology was assessed. Endobiliary brush cytology specimens obtained during endoscopic retrograde cholangiopancreaticography were prospectively collected from 312 consecutive patients with extrahepatic biliary stenosis. The results of conventional light microscopic cytology and K-ras codon 12 mutational analysis were compared and evaluated in view of the final diagnosis made by histological examination of the stenotic lesion and/or patient follow-up. The sensitivities of cytology and mutational analysis to detect malignancy were 36 and 42%, respectively. When both tests were combined, the sensitivity increased to 62%. The specificity of cytology was 98%, and the specificity of the mutational analysis and of both tests combined was 89%. Positive predictive values for cytology, mutational analysis, and both tests combined were 98, 92, and 94%, whereas the corresponding negative predictive values were 34, 34, and 44%, respectively. The sensitivity of K-ras mutational analysis was 63% for pancreatic carcinomas compared to 27% for bile duct, gallbladder, and ampullary carcinomas. K-ras mutational analysis can be considered supplementary to conventional light microscopy of endobiliary brush cytology to diagnose patients with malignant extrahepatic biliary stenosis, particularly in the case of pancreatic cancer. The presence of a K-ras codon 12 mutation in endobiliary brush cytology per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or equivocal.

	INTRODUCTION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Stenosis of the extrahepatic bile ducts is caused by a variety of malignant and benign disorders. To optimally manage such patients, it is often important to determine the etiology of the stenosis. However, it can be difficult to differentiate malignant from benign causes of biliary stenosis, based on clinical presentation and radiological findings alone, and a definitive diagnosis of malignancy can only be established histo(cyto)-pathologically. Endobiliary brush cytology can be performed during ERCP³ to collect material for cytopathology. Despite the high specificity of brush cytology, the sensitivity is low (1 , 2) . An analysis of tumor-specific genetic alterations in these cytology specimens may add to the diagnostic value of brush cytology.

Mutations in the K-ras oncogene are attractive for such analyses for a number of reasons. (a) K-ras mutations are one of the most common genetic alterations in human cancers and are frequent in the two main malignant neoplasms that cause biliary stenosis, pancreatic carcinoma, and bile duct carcinoma (3, 4, 5, 6, 7, 8, 9) . (b) More than 90% of the K-ras mutations in these neoplasms occur in codon 12, which makes their detection relatively easy. (c) The PCR-based method used for the detection of the K-ras mutations is very sensitive and can identify rare mutant DNA copies among an abundance of wild-type DNA (3) . (d) Results from K-ras mutational analyses, as were performed here, can be obtained within 48 h, making the test suitable for routine clinical purposes.

A number of studies have emphasized the diagnostic utility of K-ras mutations in material obtained from the head of the pancreas for the diagnosis of pancreatico-biliary malignancies, but most studies were performed on small groups of selected patients (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) . Furthermore, the specificity of K-ras mutational analysis in the clinical diagnosis of neoplastic disease is unclear, because these mutations are also present in intraductal pancreatic proliferations (called "duct hyperplasia"; Refs. 23, 24, 25 ). This study has prospectively assessed the value of K-ras mutational analysis of endobiliary brush cytology as compared to conventional cytopathology for the diagnosis of a malignancy in patients with bile duct stenosis in a large series of consecutive patients with a complete follow-up.

	MATERIALS AND METHODS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients
The study population consisted of a series of consecutive patients who underwent ERCP with endobiliary brush cytology for the evaluation of an extrahepatic biliary stenosis at the Academic Medical Center in Amsterdam in the period from January 1, 1993, to February 1, 1996. The Medical Ethical Review Committee of the Academic Medical Center approved the study. If a patient underwent ERCP with brush cytology repeatedly during this period, only the first examination was included. This resulted in 312 patients with a mean age of 63 years; 172 patients were male.

A final diagnosis of the nature of biliary stenosis was based on histological and/or clinical findings (Table 1) . In the absence of a tissue diagnosis, a clinical diagnosis was established based on clinical symptomatology, the results of imaging studies prior to the ERCP procedure, and, particularly, the course of the disease. Information concerning the clinical follow-up was obtained from the patient’s physician. All patients were followed for at least 12 months. The 104 patients with a clinical diagnosis of malignant extrahepatic bile duct stenosis had rapidly progressive disease with symptoms such as jaundice, pain, cachexia, and metastases. Importantly, all these patients died of disease within a mean survival of 5.7 months (range, 0–42 months) after the ERCP procedure, which corresponds to survival rates of patients with cancer of the pancreas and extrahepatic biliary tract in general. The mean survival for all of the 220 patients with a malignant etiology of their stenosis was 9 months (range, 0–50 months). Eight of these 220 patients were still alive at the end of follow-up, and their survival ranged from 22 to 50 months: of these, 6 had a surgical resection of the carcinoma and 2 were biopsied only; thus, they were all tissue proven. In contrast, 71 of the 74 patients with benign disease (including the 10 patients with a tissue diagnosis of benign disease) were all alive after a mean follow-up period of 32 months (range, 15–54 months) and had stable disease or regression of their symptoms. Three patients with benign disease died due to unrelated causes: 2 died from heart disease 10 months and 27 months after ERCP, and 1 died following a hip fracture 18 months after ERCP, all without symptoms of obstructive biliary disease.

Tissue from the area of the bile duct stenosis was available from 71 patients with a malignant cause for their stenosis and from 10 patients with a benign stenosis. These tissues were obtained at resection of the stenotic lesion, from biopsies of the stenotic lesion with malignant findings, and at autopsy (Table 1) . In these cases, the available archival tissue blocks were analyzed for K-ras mutations, allowing us to compare directly the mutational status of the patient’s primary pathology with the analysis of the corresponding brush cytology specimens.

Methods
DNA Isolation.
One ml of each brush cytology suspension was used for DNA isolation. In case of the tissue blocks, careful microdissection from 5-µm H&E-stained sections was performed to ascertain a sample of which at least 50% of the cells comprised the tissue of interest. DNA was extracted as described previously (26) .

K-ras Mutational Analysis.
The protocol of the K-ras codon 12 mutational analysis has been described previously (26) . With this assay, DNA is subjected to PCR amplification using primers around codon 12. One of the primers generates a restriction enzyme recognition site with the wild-type codon 12 sequence but not with the mutant codon 12 sequence. Digestion of the PCR products with the restriction enzyme is followed by a second round of amplification, which then yields a PCR product enriched for K-ras codon 12 mutations. The resulting DNA fragments are denatured and dot-blotted onto nylon membranes and subjected to allele-specific oligonucleotide hybridization with radioactive labeled probes, specific for each possible K-ras codon 12 mutation, followed by autoradiography. Cell suspensions with mutant:wild-type ratios of 1:100 and 1:1000 were used as positive controls in every PCR procedure. The suspensions were made of the human colon cancer cell line SW 480 with a homozygous GGT to GTT mutation at codon 12 of K-ras and the human colon cancer cell line HT 29 with wild-type K-ras. Water was used as a control for contamination, placental DNA was used as a control for nonspecific hybridization, and cloned DNA fragments with the six different K-ras codon 12 mutations and the wild-type codon 12 were used as controls for specific hybridization. All PCR products were hybridized with oligonucleotides specific for the wild-type sequence to control for amplification of the patient samples. Both enriched and nonenriched PCR products were dot-blotted next to each other to check the digestion and mutant enrichment. Fig. 1 is an example of an autoradiogram of the K-ras analysis. The above mutational analysis has been validated through comparison with sequence analysis in a previous study (27) .

View larger version (68K): [in this window] [in a new window]   Fig. 1. Example of an autoradiogram of the K-ras mutational analysis. Seven nylon membranes each hybridized with a different radioactive labeled oligonucleotide specific for the sequence of the wild-type codon 12 (left) and the six possible mutations. For each membrane, the left lane contains the nonenriched PCR products, and the right lane contains the mutant-enriched PCR products. Lanes WT, wild-type (= glycine); Lane Cys, cysteine: Lane Ser, serine; Lane Arg, arginine; Lane Val, valine; Lane Asp, aspartic acid; Lane Ala, alanine. co, hybridization controls, on each membrane cloned DNA fragments with a known codon 12 sequence complementary to the labeled oligonucleotides used for the hybridization of that membrane; 1–8, brush cytology specimens with K-ras codon 12 sequences coding for the following amino acids: aspartic acid, arginine, glycine, glycine, valine, glycine, aspartic acid, and aspartic acid, respectively; pla, placental DNA; H2O, water; 1:100, one cell with mutant codon 12, coding for the amino acid valine, mixed in 100 cells with wild-type codon 120; 1:1000, one cell with mutant codon 12, coding for the amino acid valine, mixed in 1000 cells with wild-type codon 12.

Light Microscopy.
All of the cytology smears were independently evaluated by an experienced cytopathologist (L. A. N.). The following diagnostic categories were used: positive for carcinoma, negative for carcinoma, suspect for carcinoma, and material insufficient or not suitable for diagnosis.

Sensitivity, Specificity, and Positive and Negative Predictive Values.
The following definitions were used for evaluation. Sensitivity was defined as the percentage of patients with disease who had positive test results. Specificity was defined as the percentage of patients without disease who had negative test results. Positive predictive value was defined as the percentage of patients with positive test results who had disease. Negative predictive value was defined as the percentage of patients with negative test results who had no disease.

	RESULTS

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Of the 220 patients with a malignant etiology for their bile duct stenosis, 79 (36%) were diagnosed cytologically, and 92 (42%) had K-ras mutations detected in their cytology specimens (Table 3) . Of the 92 patients with mutant K-ras in their cytology specimens, 57 patients were not diagnosed with cytology, and thus the two tests combined were able to identify 136 (79+57) patients with malignant disease (62%). Of these 57 patients with mutant K-ras and nondiagnostic cytology, 39 had negative cytology results, 14 had suspect for carcinoma cytology, and 4 had material that was insufficient for diagnosis. Positive predictive values and negative predictive values for the cytology, K-ras mutational analysis, and both tests combined were 98 and 34%, 92 and 34%, and 94 and 44%, respectively.

Two of the eight patients with "false-positive K-ras results," both with a postsurgical stenosis, also had positive cytology (Fig. 2) .

View larger version (127K): [in this window] [in a new window]   Fig. 2. A and B, cytology positive for carcinoma from the two patients with a diagnosis of postsurgical stenosis (Giemsa stained, x132).

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The clinical value of analyzing endobiliary brush cytology specimens for K-ras codon 12 mutations in establishing the diagnosis of a malignancy in patients with extrahepatic bile duct stenosis was examined. The study materials were prospectively collected from a large series of consecutive patients who underwent ERCP with endobiliary brush cytology to rule out or confirm a neoplastic cause of their bile duct stenosis. Brush cytology accurately diagnosed malignancy in 36% of the patients with a malignant etiology for their biliary stenosis, a sensitivity comparable to two previous studies in which a large consecutive series of patients was analyzed (1 , 2) . These authors reported similar frequencies of biliary stenosis caused by malignant disease as in our study (57 and 66% versus 70%), and in these previous studies, pancreatic and bile duct carcinoma were also the most frequent carcinomas; the demographics in these two studies are comparable with this series. Thus, the study population in our series can be considered representative for patients undergoing ERCP with brush cytology for the evaluation of a potentially malignant biliary stenosis. Other studies that reported higher sensitivities of biliary cytology dealt with smaller groups of selected patients (28) .

The K-ras mutational analysis was especially valuable in the diagnosis of patients with pancreatic carcinoma (63% sensitivity versus 27% in patients with malignancy other than pancreatic carcinoma). One would expect that the K-ras mutational analysis is particularly sensitive for a stenosis caused by pancreatic carcinoma, whereas light microscopic brush cytology is the more sensitive method for carcinomas arising from the bile duct epithelium. Endobiliary brush cytology samples the bile duct epithelium most efficiently, whereas the frequency of K-ras mutations is highest in pancreatic carcinoma (3, 4, 5, 6, 7 , 9 , 26 , 28 ), and the PCR-based technique for detecting K-ras mutations is highly sensitive and thus, in contrast to cytology, less depen-dent on obtaining a large amount of tumor cells. Because cytology and K-ras mutational analysis have opposite sensitivities for the two most frequent causes of malignant biliary stenosis, the methods nicely supplement each other. Indeed, the sensitivities of cytology (36%) and K-ras mutational analysis (42%) for the diagnosis of malignant stenosis were similar but, when their sensitivities were combined, increased to 62%.

K-ras mutations were detected in the brush cytology specimens of 8 of the 74 patients (11%) with benign disease. Performing all PCR analyses in duplicate independently minimized the chance of technical errors as a cause for false-positive results. Positive results of the K-ras mutational analysis in the absence of malignancy may be caused by the presence of noninvasive "hyperplastic duct lesions" containing K-ras mutations (23 , 24 , 29 , 30 ). Hyperplastic duct lesions are frequently found together with cancer in the pancreas, and indeed, there is evidence that these duct hyperplasias can progress to infiltrating carcinoma with K-ras mutation as an early event (30, 31, 32) . K-ras mutations are also found in hyperplasias in patients with chronic pancreatitis, a condition thought to be a risk factor for developing pancreatic cancer (23 , 33) . However, it is clear that not all duct hyperplasias progress to invasive carcinoma during the life span of an average patient (24) . A longer follow-up would, therefore, be needed to better understand the meaning of the observations in the eight patients in our study without obvious neoplastic disease who harbored K-ras mutations in their brush cytology. A recent study found no cancer in 20 patients with pancreatitis and K-ras mutation in their pancreatic juice after a mean follow-up of 78 months (34) . On the other hand, Brat et al. (35) reported three patients with hyperplastic duct lesions who developed pancreatic cancer after 17 months to 10 years, and Berthelemy et al. (13) reported two patients without evidence of cancer at the time of ERCP but with mutated K-ras in their pancreatic juice who developed clinically detectable pancreatic cancers after 18 and 40 months. Nonetheless, it seems best, at present, to consider the eight patients in our study false-positives until it is proven otherwise. Only two of these patients had chronic pancreatitis. Tissue for K-ras analysis was available from one of these patients with primary sclerosing cholangitis in which the resected biliary stenosis did not harbor a K-ras mutation.

As in our study, the specificity of cytology reported in the literature is often 100% or approaching 100% (1 , 2) . Interestingly, the two patients with false-positive cytologies also had K-ras mutations detected in their cytology specimens. One patient was a 62-year-old white male. During ERCP, a regular smooth stenosis of the distal common bile duct was seen. He had undergone a cholecystectomy for cholelithiasis in the past; hence, the stenosis was diagnosed as postsurgical. The stenosis was stented, and since then, he has not been jaundiced and had any other complaints. There was no evidence of bile duct obstruction 18 months after brush cytology. The other patient was a 71-year-old white female, also with a postsurgical stenosis. The mid-common bile duct stenosis was treated with a stent. Eighteen months after brush cytology, she had no complaints of extrahepatic bile duct obstruction. These two patients clearly did not meet our criteria for a clinical diagnosis of a malignant bile duct stenosis. Nonetheless, even in retrospect, the cytologies of these two patients were considered positive for carcinoma (Fig. 2, A and B , respectively). One could speculate that these cells came from pancreatic duct lesions with high-grade dysplasia or from an in situ carcinoma, which would also explain the K-ras mutations detected in these patients. Long-term follow-up may then provide a clue to their final diagnosis.

In 60 cases, we were able to directly compare the K-ras mutations identified in brush cytology specimens to those present in the corresponding surgical specimens. We found that the results were identical in 88% (53 of 60) of the cases with malignancy. The main cause for false-negative results was the absence of K-ras mutations in the tumor (26 of 33), mostly cancers other than pancreatic cancer. The discrepant results from the seven patients in which wild-type K-ras was found in the brush cytology but mutant K-ras was detected in the patients’ carcinoma could be due to sampling error because the conventional cytology in these cases was also negative for carcinoma.

More direct sampling of the stenotic lesion could potentially improve the sensitivity of cytology but would diminish specificity of the K-ras mutational analysis. Van Laethem et al. (22) examined the diagnostic value of K-ras in pancreatic duct brushings and bile duct brushings. Sensitivity of conventional light microscopy of endobiliary brush cytology was similar in their study. They also showed the additional diagnostic value of K-ras mutational analysis in these cytology specimens, especially in the diagnosis of patients with pancreatic cancer, and the high specificity. In contrast, they found that the sensitivity of conventional cytology of pancreatic duct brushings is higher (51%), but the diagnostic value of K-ras was impaired by a high percentage (25%) of patients with chronic pancreatitis who harbored K-ras mutations in their cytology. This lower specificity of K-ras mutational analysis of pancreatic duct brushings may well be attributed to the more direct sampling of the hyperplastic duct lesions that are frequent in the pancreas with chronic inflammation. It is likely that, in brush cytology specimens from the bile duct, the yield of cells from these hyperplastic duct lesions is lower compared to cells derived from carcinomas because the cells in carcinomas grow less coherently and are easily shed. Following this reasoning, colorectal neoplasms can be diagnosed specifically with the detection of K-ras mutations in the stool despite the frequent occurrence of K-ras mutations present in aberrant crypt foci and hyperplastic polyps, two nonneoplastic lesions that are prevalent in the colorectum without neoplastic disease (36 , 37) .

In conclusion, PCR-based tests for the detection of K-ras codon 12 mutations can be a valuable diagnostic adjunct to conventional light microscopy of endobiliary brush cytology specimens obtained from patients who have a suspicious stenosis of the extrahepatic bile duct, especially in patients with pancreatic carcinoma. The presence of a mutation favors malignancy, even when the cytology reading is negative or equivocal.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by The Netherlands Foundation for Scientific Research Grant 950-10-625.

² To whom requests for reprints should be addressed, at Academic Medical Center, University of Amsterdam, Department of Pathology, Meibergdreef 9, 1105 AZ Amsterdam, the Netherlands. Phone: 3120-5665635; Fax: 3120-6960389.

³ The abbreviation used is: ERCP, endoscopic retrograde cholangiopancreaticography.

Received 10/ 2/98; revised 12/ 9/98; accepted 12/ 9/98.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Ponchon T., Gagnon P., Berger F., Labadie M., Liaras A., Chavaillon A., Bory R. Value of endobiliary brush cytology and biopsies for the diagnosis of malignant bile duct stenosis: results of a prospective study. Gastrointest. Endosc., 42: 565-572, 1995.[Medline]
2. Lee J. G., Leung J. W., Baillie J., Layfield L. J., Cotton P. B. Benign, dysplastic, or malignant—making sense of endoscopic bile duct brush cytology: results in 149 consecutive patients. Am. J. Gastroenterol., 90: 722-726, 1995.[Medline]
3. Levi S., Urbano-Ispizua A., Gill R., Thomas D. M., Gilbertson J., Foster C., Marshall C. J. Multiple K-ras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res., 51: 3497-3502, 1991.[Abstract/Free Full Text]
4. Tada M., Omata M., Ohto M. High incidence of ras gene mutation in intrahepatic cholangiocarcinoma. Cancer (Phila.), 69: 1115-1118, 1992.[Medline]
5. Tada M., Yokosuka O., Omata M., Ohto M., Isono K. Analysis of ras gene mutations in biliary and pancreatic tumors by polymerase chain reaction and direct sequencing. Cancer (Phila.), 66: 930-935, 1990.[Medline]
6. Motojima K., Tsunoda T., Kanematsu T., Nagata Y., Urano T., Shiku H. Distinguishing pancreatic carcinoma from other periampullary carcinomas by analysis of mutations in the Kirsten-ras oncogene. Ann. Surg., 214: 657-662, 1991.[Medline]
7. Watanabe M., Asaka M., Tanaka J., Kurosawa M., Kasai M., Miyazaki T. Point mutation of K-ras gene codon 12 in biliary tract tumors. Gastroenterology, 107: 1147-1153, 1994.[Medline]
8. Almoguera C., Shibata D., Forrester K., Martin J., Arnheim N., Perucho M. Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 53: 549-554, 1988.[Medline]
9. Hruban R. H., van Mansfeld A. D. M., Offerhaus G. J. A., Van Weering D. H. J., Allison D. C., Goodman S. N., Kensler T. W., Bose K. K., Cameron J. L., Bos J. L. K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am. J. Pathol., 143: 545-554, 1993.[Abstract]
10. Van Laethem J. L., Vertongen P., Deviere J., Van Rampelbergh J., Rickaert F., Cremer M., Robberecht P. Detection of c-Ki-ras gene codon 12 mutations from pancreatic duct brushings in the diagnosis of pancreatic tumours. Gut, 36: 781-787, 1994.[Abstract/Free Full Text]
11. Apple S. A., Hecht J. R., Novak J. M., Nieberg R. K., Rosenthal D. L., Grody W. W. Polymerase chain reaction-based K-ras mutation detection of pancreatic adenocarcinoma in routine cytology smears. Am. J. Clin. Pathol., 105: 321-326, 1996.[Medline]
12. Villanueva A., Reyes G., Cuatrecasas M., Martinez A., Erill N., Lerma E., Farre A., Lluis F., Capella G. Diagnostic utility of K-ras mutations in fine-needle aspirates of pancreatic masses. Gastroenterology, 110: 1587-1594, 1996.[Medline]
13. Berthelemy P., Bouisson M., Escourrou J., Vaysse N., Rumeau J. L., Pradayrol L. Identification of K-ras mutations in pancreatic juice in the early diagnosis of pancreatic cancer. Ann. Intern. Med., 123: 188-191, 1995.[Abstract/Free Full Text]
14. Iguchi H., Sugano K., Fukayama N., Ohkura H., Sadamoto K., Seo Y., Tomoda H., Funakoshi A., Wakasugi H. Analysis of Ki-ras codon 12 mutations in the duodenal juice of patients with pancreatic cancer. Gastroenterology, 110: 221-226, 1996.[Medline]
15. Caldas C., Hahn S. A., Hruban R. H., Redston M. S., Yeo C. J., Kern S. E. Detection of K-ras mutations in the stool of patients with pancreatic adenocarcinoma and pancreatic ductal hyperplasia. Cancer Res., 54: 3568-3573, 1994.[Abstract/Free Full Text]
16. van Es J. M., Polak M. M., van den Berg F. M., Ramsoekh T. B., Craanen M. E., Hruban R. H., Offerhaus G. J. A. Molecular markers for diagnostic cytology of neoplasms in the head of the pancreas: mutation of K-ras and overexpression of the p53 gene product. J. Clin. Pathol., 48: 218-222, 1995.[Abstract/Free Full Text]
17. Trümper L. H., Bürger B., Von Bonin F., Hintze A., Von Blohn G., Pfreunschuh M., Daus H. Diagnosis of pancreatic adenocarcinoma by polymerase chain reaction from pancreatic secretions. Br. J. Cancer, 70: 278-284, 1994.[Medline]
18. Tada M., Omata M., Ohto M. Clinical application of ras gene mutation for diagnosis of pancreatic adenocarcinoma. Gastroenterology, 100: 233-238, 1991.[Medline]
19. Tada M., Omata M., Kawai S., Saisho H., Ohto M., Saiki R. K., Sninsky J. J. Detection of ras gene mutations in pancreatic juice and peripheral blood of patients with pancreatic adenocarcinoma. Cancer Res., 53: 2472-2474, 1993.[Abstract/Free Full Text]
20. Watanabe H., Sawabu N., Songür Y., Yamaguchi Y., Yamakawa O., Satomura Y., Ohta H., Okai T., Wakabayashi T. Detection of K-ras point mutations at codon 12 in pure pancreatic juice for the diagnosis of pancreatic cancer by PCR-RFLP analysis. Pancreas, 12: 18-24, 1996.[Medline]
21. Ihalainen J., Taavitsainen M., Salmivaara T., Palotie A. Diagnosis of pancreatic lesions using fine needle aspiration cytology : detection of K-ras point mutations using solid phase minisequencing. J. Clin. Pathol., 47: 1082-1084, 1994.[Abstract/Free Full Text]
22. Van Laethem J-L., Bourgeois V., Parma J., Delhaye M., Cochaux P., Velu T., Deviere J., Cremer M. Relative contribution of Ki-ras gene analysis and brush cytology during ERCP for the diagnosis of biliary and pancreatic diseases. Gastrointest. Endosc., 47: 479-485, 1998.[Medline]
23. Yanagisawa A., Ohtake K., Ohashi K., Hori M., Kitagawa T., Sugano H., Kato Y. Frequent c-Ki-ras oncogene activation in mucous cell hyperplasias of pancreas suffering from chronic inflammation. Cancer Res., 53: 953-956, 1993.[Abstract/Free Full Text]
24. Tada M., Ohashi M., Shiratori Y., Okudaira T., Komatsu Y., Kawabe T., Yoshida H., Machnami R., Kishi K., Omata M. Analysis of K-ras gene mutation in hyperplastic duct cells of the pancreas without pancreatic disease. Gastroenterology, 110: 227-231, 1996.[Medline]
25. DiGiuseppe J. A., Hruban R. H., Offerhaus G. J. A., Clement M. J., Van den Berg F., Cameron J. L., Van Mansfeld A. D. M. Detection of K-ras mutations in mucinous pancreatic duct hyperplasia from a patient with a family history of pancreatic carcinoma. Am. J. Pathol., 144: 889-895, 1994.[Abstract]
26. Chung C. H., Wilentz R. E., Polak M. M., Ramsoekh T., Noorduyn L. A., Gouma D. J., Huibregtse K., Offerhaus G. J. A., Slebos R. J. C. Clinical significance of K-ras oncogene activation in ampullary neoplasms. J. Clin. Pathol., 49: 460-464, 1996.[Abstract/Free Full Text]
27. Hruban R. H., Sturm P. D. J., Slebos R. J. C., Wilentz R. E., Musler A. R., Yeo C. J., Sohn T. A., Van Velthuysen M-L. F., Offerhaus G. J. A. Can K-ras codon 12 mutations be used to distinguish benign bile duct proliferations from metastases in the liver?. Am. J. Pathol., 151: 943-949, 1997.[Abstract]
28. Kurzawinsky T., Deery A., Davidson B. R. Diagnostic value of cytology for biliary stricture. Br. J. Surg., 80: 414-421, 1993.[Medline]
29. Lemoine N. R., Jain S., Hughes C. M., Staddon S. L., Maillet B., Hall P. A., Kloppel G. K-ras oncogene activation in preinvasive pancreatic cancer. Gastroenterology, 102: 230-236, 1992.[Medline]
30. Tabata T., Fujimori T., Maeda S., Yamamoto M., Saitoh Y. The role of ras mutation in pancreatic cancer, precancerous lesions, and chronic pancreatitis. Int. J. Pancreatol., 14: 237-244, 1993.[Medline]
31. Cerny W. L., Mangold K. A., Scarpelli D. G. K-ras mutation is an early event in pancreatic duct carcinogenesis in the Syrian golden hamster. Cancer Res., 52: 4507-4513, 1992.[Abstract/Free Full Text]
32. Kozuka S., Sassa R., Taki T., Masamoto K., Nagasawa S., Saga S., Hasegawa K., Takeuchi M. Relation of pancreatic duct hyperplasia to carcinoma. Cancer (Phila.), 43: 1418-1428, 1979.[Medline]
33. Lowenfels A. B., Maisonneuve P., Cavallini G., Ammann R. W., Lankisch P. G., Andersen J. R., Dimagno E. P., Andren-Sandberg A., Domellof L. Pancreatitis and the risk of pancreatic cancer. N. Engl. J. Med., 328: 1433-1437, 1993.[Abstract/Free Full Text]
34. Furuya N., Kawa S., Akamatsu T., Furihata K. Long term follow up of patients with chronic pancreatitis and K-ras gene mutation detected in pancreatic juice. Gastroenterology, 113: 593-598, 1997.[Medline]
35. Brat D. J., Lillemoe K. D., Yeo C. J., Warfield P. B., Hruban R. H. Progression of pancreatic intraductal neoplasias to infiltrating adenocarcinoma of the pancreas. Am. J. Surg. Pathol., 22: 163-169, 1998.[Medline]
36. Villa E., Dugani A., Rebecchi A. M., Vignoli A., Grottola A., Buttafoco P., Perini M., Trande P., Merighi A., Lerose R., Manenti F. Identification of subjects at risk for colorectal carcinoma through a test based on K-ras determination in the stool. Gastroenterology, 110: 1346-1353, 1996.[Medline]
37. Yamashita N., Minamoto T., Ochiai A., Onda M., Esumi H. Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon. Gastroenterology, 108: 434-440, 1995.[Medline]

This article has been cited by other articles:

				N T van Heek, S J Clayton, P D J Sturm, J Walker, D J Gouma, L A Noorduyn, G J A Offerhaus, and J C Fox Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses J. Clin. Pathol., December 1, 2005; 58(12): 1315 - 1320. [Abstract] [Full Text] [PDF]

				T. Z. Kristiansen, J. Bunkenborg, M. Gronborg, H. Molina, P. J. Thuluvath, P. Argani, M. G. Goggins, A. Maitra, and A. Pandey A Proteomic Analysis of Human Bile Mol. Cell. Proteomics, July 1, 2004; 3(7): 715 - 728. [Abstract] [Full Text] [PDF]

				C.-Y. Chen, S.-C. Shiesh, and S.-J. Wu Rapid Detection of K-ras Mutations in Bile by Peptide Nucleic Acid-mediated PCR Clamping and Melting Curve Analysis: Comparison with Restriction Fragment Length Polymorphism Analysis Clin. Chem., March 1, 2004; 50(3): 481 - 489. [Abstract] [Full Text] [PDF]

				L. Trumper, M. Menges, H. Daus, D. Kohler, J.-O. Reinhard, M. Sackmann, C. Moser, A. Sek, G. Jacobs, M. Zeitz, et al. Low Sensitivity of the ki-ras Polymerase Chain Reaction for Diagnosing Pancreatic Cancer From Pancreatic Juice and Bile: A Multicenter Prospective Trial J. Clin. Oncol., November 1, 2002; 20(21): 4331 - 4337. [Abstract] [Full Text] [PDF]

				M. Suzuoki, Y. Hida, M. Miyamoto, T. Oshikiri, K. Hiraoka, Y. Nakakubo, T. Shinohara, T. Itoh, S. Okushiba, S. Kondo, et al. RCAS1 Expression as a Prognostic Factor After Curative Surgery for Extrahepatic Bile Duct Carcinoma Ann. Surg. Oncol., May 1, 2002; 9(4): 388 - 393. [Abstract] [Full Text] [PDF]

				K. Seki, T. Suda, Y. Aoyagi, S. Sugawara, M. Natsui, H. Motoyama, Y. Shirai, T. Sekine, H. Kawai, Y. Mita, et al. Diagnosis of Pancreatic Adenocarcinoma by Detection of Human Telomerase Reverse Transcriptase Messenger RNA in Pancreatic Juice with Sample Qualification Clin. Cancer Res., July 1, 2001; 7(7): 1976 - 1981. [Abstract] [Full Text] [PDF]

				S. Kern, R. Hruban, M. A. Hollingsworth, R. Brand, T. E. Adrian, E. Jaffee, and M. A. Tempero A White Paper: The Product of a Pancreas Cancer Think Tank Cancer Res., June 1, 2001; 61(12): 4923 - 4932. [Full Text] [PDF]

				C J R Stewart, P R Mills, R Carter, J O'Donohue, G Fullarton, C W Imrie, and W R Murray Brush cytology in the assessment of pancreatico-biliary strictures: a review of 406 cases J. Clin. Pathol., June 1, 2001; 54(6): 449 - 455. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sturm, P. D. J. Articles by Offerhaus, G. J. A. Search for Related Content PubMed PubMed Citation Articles by Sturm, P. D. J. Articles by Offerhaus, G. J. A.