Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis.

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.

The potential diagnostic use of K-ras codon 12 and p53 alterations in brush cytology from the pancreatic head region.

It can be difficult to distinguish between malignant and benign disease of the region of the head of the pancreas using conventional methods. K-ras and p53 alterations occur frequently in malignancies in this region and are therefore candidate tumour markers. To define the utility of these alterations in interpreting pancreatic head cytology, the present study investigated to what extent alterations in the carcinomas were detectable on cytology and whether the alterations found in the cytology came from the carcinomas. Fifty-seven consecutive pancreaticoduodenectomy resection specimens (52 with a malignancy and five without) and the ductal brush cytology specimens collected post-operatively from these resection specimens were compared for the presence of K-ras and p53 alterations. K-ras mutations were detected using the polymerase chain reaction (PCR) and allele-specific oligonucleotide hybridization, and p53 alterations using immunochemical staining for the p53 gene product. After discrepancy analysis, the results from the resection specimens and corresponding brush cytology specimens were identical in 88 per cent for the K-ras analysis and in 84 per cent for the p53 analysis. In two cases, K-ras mutations found in the brush cytology specimens were not derived from the carcinoma but from pancreatic ductal hyperplasias. Intratumour heterogeneity and sampling error were also identified as causes for discrepant results. The five resection specimens without a malignancy and the corresponding brush cytology specimens were negative for both genetic alterations. In conclusion, the detection of K-ras and p53 alterations in cells obtained from the pancreatic head region might be a valuable adjunct to conventional cytology for the diagnosis of malignancies in the pancreatic head region. However, intratumour heterogeneity, mucinous pancreatic duct hyperplasia harbouring K-ras mutations, and sampling error will hinder their diagnostic accuracy in routine clinical use.

Clinical significance of K-ras oncogene activation in ampullary neoplasms.

AIMS: To investigate the prevalence of K-ras codon 12 point mutations in ampullary neoplasms, to explore their clinical usefulness, and to test whether the detection of these mutations could be used to identify ampullary malignancies at an early stage. METHODS: Forty one tumour specimens from 28 patients with ampullary neoplasms were analysed for activating point mutations in K-ras codon 12 using a sensitive polymerase chain reaction (PCR) based assay. RESULTS: Eleven (39%) of the 28 primary tumours harboured point mutations in K-ras. Mutations were identified in seven (41%) of the 17 carcinomas and four (36%) of the 11 adenomas. Four of the possible six permutations in codon 12 were found in these 11 samples. This spectrum of mutations is different from pancreatic carcinoma but resembles that of colorectal neoplasms. Cytological brush specimens were available in 11 cases, and in all of these specimens, the K-ras status in the primary tumour and brush specimens was identical. CONCLUSIONS: K-ras codon 12 point mutations occur in about 40% of ampullary neoplasms at a relatively early stage in tumorigenesis. The pattern of mutations in these tumours resembles that of the adenoma-carcinoma sequence in the colorectum. These results indicate that ampullary neoplasms can be detected at an early stage by searching for genetic alterations in the K-ras oncogene in cytological brush specimens.

Molecular markers for diagnostic cytology of neoplasms in the head region of the pancreas: mutation of K-ras and overexpression of the p53 protein product.

AIMS: To determine the potential efficiency of molecular markers specific for neoplastic change--mutations of the K-ras oncogene and the p53 tumour suppressor gene--in diagnosing pancreatic carcinoma. METHODS: Archival cytology samples obtained from 17 patients with established pancreatic carcinoma were assayed for alterations in K-ras and p53. To detect changes in p53 expression, immunocytochemistry with polyclonal antibody CM1 was performed on the archival cytology slides after destaining. Mutations in K-ras codon 12 were then analysed on the scrapings of the same slides using mutant enriched polymerase chain reaction (PCR) amplification and restriction fragment length polymorphism analysis with allele specific oligonucleotide hybridisation for confirmation and characterisation. RESULTS: False negative results were recorded for five of the cytology slides when compared with p53 immunostaining of the surgical resection specimen. These five cases had been stained previously with Giemsa which interacts adversely with the immunostaining in contrast to the Papanicolaou procedure. The K-ras codon 12 mutations followed the well established distribution frequency and spectrum for pancreatic cancer and corresponded with the findings in the resection specimens in all cases. Two scrapings yielded insufficient DNA for PCR. Importantly, for two cases with an inconclusive cytology diagnosis on routine light microscopy, the diagnosis was confirmed by one of the molecular markers. The application of the molecular markers increased the diagnostic accuracy of cytology in this small study from 76 to 89%. CONCLUSIONS: The study indicates that assessment of alterations in the K-ras and p53 genes may be a valuable adjunct to diagnostic cytopathology of the head region of the pancreas, although there are some difficulties which will have to be overcome.