Mirjam M. Polak

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.

Expression of mutant p53 protein and CD44 variant proteins in colorectal tumorigenesis.

Colorectal tumorigenesis evolves through a series of molecular genetic changes, providing putative markers for tumour progression. This study investigated the relation between expression of the tumour suppressor gene p53 and splice variants v5 and v6 of the cell adhesion molecule CD44 by immunohistochemistry on tissue samples of early adenomas (n = 12), late adneomas (n = 12), Dukess A and B carcinomas (n = 21), and Dukess C and D carcinomas (n = 22) and compared these results with expression of these proteins in normal colonic mucosa (n = 17). A statistically significant trend of increasing expression was seen for both p53 (p < 0.005) and CD44 variant exon v6 (p < 0.0005) in subsequent stages of this tumour progression model. High expression of CD44 v5 was seen in most colorectal neoplasms (83%-96%), independent of stage. A statistically significant correlation was present between p53 expression and expression of variant v6 of CD44 (p < 0.01). Both p53 expression and CD44 v6 expression in adenomas increased with the degree of dysplasia (p < 0.05). The results of this study show that mutant p53 protein and variant v6 of the CD44 glycoprotein are markers of tumour progression in colorectal cancer.

A Serrated Colorectal Cancer Pathway Predominates over the Classic WNT Pathway in Patients with Hyperplastic Polyposis Syndrome

Hyperplastic polyposis syndrome (HPS) is characterized by the presence of multiple colorectal serrated polyps and is associated with an increased colorectal cancer (CRC) risk. The mixture of distinct precursor lesion types and malignancies in HPS provides a unique model to study the canonical pathway and a proposed serrated CRC pathway in humans. To establish which CRC pathways play a role in HPS and to obtain new support for the serrated CRC pathway, we assessed the molecular characteristics of polyps (n = 84) and CRCs (n = 19) in 17 patients with HPS versus control groups of various sporadic polyps (n = 59) and sporadic microsatellite-stable CRCs (n = 16). In HPS and sporadic polyps, APC mutations were exclusively identified in adenomas, whereas BRAF mutations were confined to serrated polyps. Six of 19 HPS CRCs (32%) were identified in a serrated polyp. Mutation analysis performed in the CRC and the serrated component of these lesions showed identical BRAF mutations. One HPS CRC was located in an adenoma, both components harboring an identical APC mutation. Overall, 10 of 19 HPS CRCs (53%) carried a BRAF mutation versus none in control group CRCs (P = 0.001). Six BRAF-mutated HPS CRCs (60%) were microsatellite unstable owing to MLH1 methylation. These findings provide novel supporting evidence for the existence of a predominant serrated CRC pathway in HPS, generating microsatellite-stable and microsatellite-instable CRCs.

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.

Genetic defects underlying Peutz–Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates

LKB1/STK11 germline inactivations are identified in the majority (66–94%) of Peutz–Jeghers syndrome (PJS) patients. Therefore, defects in other genes or so far unidentified ways of LKB1 inactivation may cause PJS. The genes encoding the MARK proteins, homologues of the Par1 polarity protein that associates with Par4/Lkb1, were analyzed in this study because of their link to LKB1 and cell polarity. The genetic defect underlying PJS was determined through analysis of both LKB1 and all four MARK genes. LKB1 point mutations and small deletions were identified in 18 of 23 PJS families using direct sequencing and multiplex ligation‐dependent probe amplification analysis identified exon deletions in 3 of 23 families. In total, 91% of the studied families showed LKB1 inactivation. Furthermore, a MARK1, MARK2, MARK3 and MARK4 mutation analysis and an MARK4 quantitative multiplex polymerase chain reaction analysis to identify exon deletions on another eight PJS families without identified LKB1 germline mutation did not identify mutations in the MARK genes. LKB1 defects are the major cause of PJS and genes of the MARK family do not represent alternative PJS genes. Other mechanisms of inactivation of LKB1 may cause PJS in the remaining families.

Intestinal and pancreatic metaplasia at the esophagogastric junction in patients without Barrett's esophagus.

OBJECTIVES:A distinctive type of columnar epithelium with intestinal metaplasia is considered diagnostic for Barretts esophagus. The neoplastic potential of pancreatic metaplasia at the esophagogastric junction is unknown. The aims of the present study were: 1) to characterize both forms of metaplasia at the esophagogastric junction, and to estimate their prevalence; 2) to investigate c-erbB-2 expression and K-ras mutations in pancreatic metaplasia; and 3) to study the relationship between metaplasia, inflammatory changes in the cardiac mucosa, and presence of H. pylori.METHODS:A total of 76 esophagogastrectomy specimens of patients with a normally located squamocolumnar junction, were investigated immunohistochemically. K-ras mutations were evaluated using PCR.RESULTS:Intestinal metaplasia in the cardia was found in 12% of patients: six complete-type, and three incomplete-type. Pancreatic metaplasia was demonstrated in 14% of patients, and neither c-erbB-2 expression nor K-ras mutations were found. Intestinal and pancreatic metaplasia were associated with mucosal inflammation. In contrast to generalized gastritis, isolated “carditis” was not associated with H. pylori infection.CONCLUSIONS:When intestinal metaplasia occurs in a biopsy from the esophagogastric junction, it is not necessarily a marker for Barretts esophagus. No indication was found that pancreatic metaplasia has neoplastic potential. Both forms of metaplasia reflect mucosal inflammation. Carditis may be a distinct inflammatory condition of the gastric mucosa that is not related to H. pylori infection.

Diagnostic and prognostic value of incidence of k-ras codon 12 mutations in resected distal bile duct carcinoma

Background and Objectives: The K‐ras gene is one of the most extensively investigated oncogenes in a wide variety of human tumors, but has rarely been studied in distal bile duct carcinoma (DBDC). We sought to investigate the diagnostic and prognostic value of K‐ras codon 12 mutations in this type of tumor.

Exclusion of RUNX3 as a tumour-suppressor gene in early-onset gastric carcinomas

Recent studies claim a critical role for RUNX3 in gastric epithelial homeostasis. However, conflicting results exist regarding RUNX3 expression in the stomach and its potential role as a tumour-suppressor gene (TSG) in gastric carcinogenesis. Our aim was to evaluate the role of RUNX3 in early-onset gastric carcinomas (EOGCs). We analysed 41 EOGCs for RUNX3 aberrations using loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) analyses. LOH of markers flanking RUNX3 was relatively common, indicating that loss of the gene may play a role in gastric carcinogenesis. However, FISH analysis of selected cases and a panel of 14 gastric carcinoma-derived cell lines showed widespread presence of multiple copies of centromere 1. While RUNX3 copy numbers were generally equal to or fewer than those of centromere 1, at least two copies were present in almost all cells analysed. Accordingly, a subpopulation of tumour cells in 12/37 cases showed RUNX3 protein expression. However, expression was not detected in the adjacent nontumorous mucosa of any case. Together, these observations indicate that chromosome 1 aberrations occur frequently in EOGCs and are reflected in the LOH and IHC patterns found. Our findings refute a role for RUNX3 as a TSG in EOGCs.

The COX-2 promoter polymorphism -765 G > C is associated with early-onset, conventional and stump gastric cancers

COX-2 overexpression is known to be an important mechanism in gastric carcinogenesis. Previously we have found that early-onset gastric cancer has a unique COX-2 low-expressing phenotype that differs significantly from that of the frequent overexpression seen in conventional gastric cancers. To investigate whether the COX-2 –765 G>C promoter polymorphism (known to lead to a reduction of COX-2 promoter activity in the colon) may explain this difference in expression, we carried out single-nucleotide polymorphism (SNP) analysis of 241 gastric cancers, including early-onset gastric cancer, conventional gastric cancers and gastric stump cancers, as well as in 100 control patients, using real-time PCR and sequence analysis, and correlated these findings with COX-2 expression using immunohistochemistry. We found that the C allele was present in 30% of early-onset gastric cancers, 24% of conventional gastric cancer, 23% of stump cancers, in contrast to 41% in the control group. There was a statistically significant difference in the presence of the C allele in patients with gastric cancer compared with the control group (P=0.007), with the C allele being associated with protection against gastric cancer. However, there was no significant difference between the early-onset, conventional and stump gastric cancer groups. Interestingly, there was no correlation between the presence of the C allele and a difference in COX-2 expression. In summary, we show that the COX-2 –765 G allele promoter polymorphism is significantly associated with gastric cancer when compared with the normal control group, but does not appear to be related directly to COX-2 expression pattern in gastric cancer. Although early-onset gastric cancers appear to have a unique COX-2 expression pattern when compared with conventional gastric cancer, the exact mechanism by which this occurs is yet to be elucidated.

Diagnostic p53 Immunostaining of Endobiliary brush cytology : Preoperative cytology compared with the surgical specimen

Endobiliary brush cytology is important in the distinction of malignant and benign causes of extrahepatic bile duct obstruction. The additional diagnostic value of p53 immunostaining on these cytology specimens was assessed.