Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the early onset of colorectal cancer and linked to germline defects in at least four mismatch repair genes. Although much has been learned about the molecular pathogenesis of this disease, questions related to effective presymptomatic diagnosis are largely unanswered because of its genetic complexity. In this study, we evaluated tumors from 74 HNPCC kindreds for genomic instability characteristic of a mismatch repair deficiency and found such instability in 92% of the kindreds. The entire coding regions of the five known human mismatch repair genes were evaluated in 48 kindreds with instability, and mutations were identified in 70%. This study demonstrates that a combination of techniques can be used to genetically diagnose tumor susceptibility in the majority of HNPCC kindreds and lays the foundation for genetic testing of this relatively common disease.

Genetic mapping of a locus predisposing to human colorectal cancer.

Genetic linkage analysis was used to determine whether a specific chromosomal locus could be implicated in families with a history of early onset cancer but with no other unique features. Close linkage of disease to anonymous microsatellite markers on chromosome 2 was demonstrated in two large kindreds. The pairwise lod scores for linkage to marker D2S123 in these kindreds were 6.39 and 1.45 at zero recombination, and multipoint linkage with flanking markers resulted in lod scores of 6.47 and 6.01. These results prove the existence of a genetically determined predisposition to colorectal cancer that has important ramifications for understanding and preventing this disease.

Role of RHAMM within the hierarchy of well-established prognostic factors in colorectal cancer.

OBJECTIVE: To compare the independent prognostic effect of a panel of immunohistochemical protein markers in colorectal cancer (CRC) and determine their ranking among the established prognostic factors T stage, N stage, vascular invasion, tumour budding and tumour grade. DESIGN: A tissue microarray of 1420 CRCs was immunostained for 23 markers and mismatch repair (MMR) proteins. Immunoreactivity was assessed semi-quantitatively. Receiver operating characteristic (ROC) curves were used to determine cut-off scores for tumour marker positivity. Survival time was investigated for each marker in multivariable analysis with T stage, N stage, vascular invasion, tumour budding and tumour grade. The hazard ratio (HR) was used to compare the prognostic effect of each marker on 5 year survival. RESULTS: To the standard prognostic features, only six markers added independent prognostic information including receptor for hyaluronic acid mediated motility (RHAMM) (HR = 2.39 (1.88 to 3.05)), epidermal growth factor receptor (HR = 1.65 (1.31 to 2.09)), tumour infiltrating lymphocytes (HR = 0.7 (0.54 to 0.92)), urokinase plasminogen activator (HR = 1.38 (1.09 to 1.75)), Raf-1 kinase inhibitor protein (HR = 0.75 (0.58 to 0.96)) and mammalian sterile 20-like kinase 1 (MST1) (HR = 0.75 (0.58 to 0.95). Diffuse (>90% staining) expression of RHAMM ranked above T stage, vascular invasion, tumour budding and tumour grade in terms of adverse prognostic significance and was associated with distant metastasis (p = 0.012) and with worse outcome in patients with metastatic disease (p = 0.031). CONCLUSIONS: The strong adverse effect of RHAMM on outcome in addition to its position within the hierarchy of well-established prognostic factors suggest that RHAMM should be considered a more important prognosticator than tumour grade, tumour budding and vascular invasion in patients with CRC.

Promoter methylation of the mutated in colorectal cancer gene is a frequent early event in colorectal cancer.

The mutated in colorectal cancer (MCC) gene is in close linkage with the adenomatous polyposis coli (APC) gene on chromosome 5, in a region of frequent loss of heterozygosity in colorectal cancer. The role of MCC in carcinogenesis, however, has not been extensively analysed, and functional studies are emerging, which implicate it as a candidate tumor suppressor gene. The aim of this study was to examine loss of MCC expression due to promoter hypermethylation and its clinicopathologic significance in colorectal cancer. Correspondence of MCC methylation with gene silencing was demonstrated using bisulfite sequencing, reverse transcription-polymerase chain reaction and Western blotting. MCC methylation was detected in 45-52% of 187 primary colorectal cancers. There was a striking association with CDKN2A methylation (P<0.0001), the CpG island methylator phenotype (P<0.0001) and the BRAF V600E mutation (P<0.0001). MCC methylation was also more common (P=0.0084) in serrated polyps than in adenomas. In contrast, there was no association with APC methylation or KRAS mutations. This study demonstrates for the first time that MCC methylation is a frequent change during colorectal carcinogenesis. Furthermore, MCC methylation is significantly associated with a distinct spectrum of precursor lesions, which are suggested to give rise to cancers via the serrated neoplasia pathway.

Prognostic significance of mucins in colorectal cancer with different DNA mismatch-repair status.

BACKGROUND: Expression of mucin antigen MUC1 and down regulation of MUC2 are associated with adverse prognosis in colorectal cancer (CRC), but their prognostic significance with respect to differing DNA mis- match repair (MMR) status is poorly understood. OBJECTIVE: To determine the prognostic significance of MUC1 and MUC2 in CRC with different MMR statuses. METHODS: Using the tissue microarray (TMA) technique, a series of 1420 unselected, non-consecutive CRC resections was subdivided into three groups: (1) MMR-proficient; (2) MLH1-negative; and (3) presumed hereditary non-polyposis colon cancer (HNPCC). Immunohistochemical analysis of MUC1 and MUC2 expression (>0%) and loss (0%) was performed, and the results were correlated with clinicopathological parameters. RESULTS: In MMR-proficient CRC, MUC1 expression was more frequently found in tumours with higher tumour stage (p=0.004) and higher tumour grade (p=0.041) and loss of MUC2 was associated with higher tumour stage (p=0.028), node stage (p=0.001), presence of vascular invasion (p=0.028) and worse survival (p=0.034). In MLH1-negative CRC, MUC2 loss was associated with the presence of lymph node metastasis (p=0.028) and worse survival (p=0.015), but there was no association between MUC1 expression and clinicopathological features. In presumed HNPCC, MUC1 expression and MUC2 loss were not associated with clinicopathological parameters. CONCLUSIONS: Mucins have a prognostic significance in sporadic CRC, but not in hereditary CRC. Loss of MUC2 is an adverse prognostic factor in MMR-proficient and MLH1-negative CRC, whereas MUC1 expression is associated with tumour progression in MMR-proficient CRC only.

Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis.

BACKGROUND: Hyperplastic polyposis of the colorectum is a precancerous condition that has been linked with DNA methylation. The polyps in this condition have been distinguished from typical small hyperplastic polyps and renamed sessile serrated adenomas. Sessile serrated adenomas also occur sporadically and appear to be indistinguishable from their counterparts in hyperplastic polyposis. AIMS AND METHODS: The existence of distinguishing molecular features was explored in a series of serrated polyps and matched normal mucosa from patients with and without hyperplastic polyposis by assessing mutation of BRAF, DNA methylation in 14 markers (MINTs 1, 2 and 31, p16, MGMT, MLH1, RASSF1, RASSF2, NORE1 (RASSF5), RKIP, MST1, DAPK, FAS, and CHFR), and immunoexpression of MLH1. RESULTS: There was more extensive methylation in sessile serrated adenomas from subjects with hyperplastic polyposis (p<0.0001). A more clearcut difference in patients with hyperplastic polyposis was the finding of extensive DNA methylation in normal mucosa from the proximal colon. CONCLUSIONS: A genetic predisposition may underlie at least some forms of hyperplastic polyposis in which the earliest manifestation may be hypermethylation of multiple gene promoters in normal colorectal mucosa. Additionally, some of the heterogeneity within hyperplastic polyposis may be explained by different propensities for MLH1 inactivation within polyps.

Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA microsatellite instability.

The significance of low-level DNA microsatellite instability (MSI-L) is not well understood. K-ras mutation is associated with MSI-L colorectal cancer and with the silencing of the DNA repair gene O-6-methylguanine DNA methyltransferase (MGMT) by methylation of its promoter region. MGMT methylation was studied in sporadic colorectal cancers stratified as DNA microsatellite instability-high (n = 23), MSI-L (n = 44), and microsatellite-stable (n = 23). Methylation-specific PCR was used to detect MGMT-promoter hypermethylation in 3 of 23 (13%) microsatellite instability-high, in 28 of 44 (64%) MSI-L, and in 6 of 23 (26%) microsatellite-stable cancers (P = 0.0001). K-ras was mutated in 20 of 29 (69%) methylated MSI-L cancers and in 2 of 15 (13%) unmethylated MSI-L cancers (P = 0.001), indicating a relationship between MGMT-methylation and mutation of K-ras. Loss of nuclear expression of MGMT was demonstrated immunohistochemically in 23 of 31 (74%) cancers with methylated MGMT and in 10 of 49 (20%) cancers with nonmethylated MGMT (P < 0.0001). Loss of expression of MGMT was also demonstrated in 9 of 31 serrated polyps. Silencing of MGMT may predispose to mutation by overwhelming the DNA mismatch repair system and occurs with greatest frequency in MSI-L colorectal cancers.

Replication errors in benign and malignant tumors from hereditary nonpolyposis colorectal cancer patients.

A replication error (RER) phenotype has been documented both in sporadic colorectal tumors and in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC). In the current study 8 of 49 (16%) sporadic colorectal cancers (CRCs) and 25 of 29 (86%) CRCs from HNPCC patients were found to be RER+. All 9 (100%) CRCs from HNPCC patients with germline mutations of the mismatch repair gene MSH2 were found to be RER+, while 16 of 20 CRCs from HNPCC kindreds unlinked or not studied for linkage to MSH2 were RER+. Corresponding analysis in colorectal adenomas revealed that only 1 of 33 (3%) sporadic tumors but 8 of 14 (57%) HNPCC tumors were RER+. Moreover, RER was found in all 6 extracolonic cancers (endometrium, 2; kidney, 1; stomach, 1; duodenum, 1; and ovary, 1) derived from members of HNPCC families. These data suggest the involvement of mismatch repair deficiency in the premalignant stage of tumorigenesis in HNPCC cases, and suggest that mismatch repair genes (MSH2 or others) are defective in the germline of nearly all these patients.

Expression of Bcl-2 protein is decreased in colorectal adenocarcinomas with microsatellite instability.

Bcl-2 is known to inhibit apoptosis and is thought to play a role in colorectal tumour development. Studies of the promoter region of bcl-2 have indicated the presence of a p53 responsive element which downregulates bcl-2 expression. Since p53 is commonly mutated in colorectal cancers, but rarely in those tumours showing microsatellite instability (MSI), the aim of this study was to examine the relationship of bcl-2 protein expression to MSI, as well as to other clinicopathological and molecular variables, in colorectal adenocarcinomas. Expression of bcl-2 was analysed by immunohistochemistry in 71 colorectal cancers which had been previously assigned to three classes depending upon their levels of MSI. MSI-high tumours demonstrated instability in three or more of six microsatellite markers tested, MSI-low tumours in one or two of six, and MSI-null in none of six. Bcl-2 expression in tumours was quantified independently by two pathologists and assigned to one of five categories, with respect to the number of cells which showed positive staining: 0, up to 5%; 1, 6-25%; 2, 26-50%; 3, 51-75%; and 4, > or =76%. Bcl-2 negative tumours were defined as those with a score of 0. Bcl-2 protein expression was tested for association with clinicopathological stage, differentiation level, tumour site, age, sex, survival, evidence of p53 inactivation and MSI level. A significant association was found between bcl-2 expression and patient survival (P = 0.012, Gehan Wilcoxon test). Further, a significant reciprocal relationship was found between bcl-2 expression and the presence of MSI (P = 0.012, Wilcoxon rank sum test). We conclude that bcl-2 expressing colorectal cancers are more likely to be MSI-null, and to be associated with improved patient survival.

Reciprocal relationship between the tumor suppressors p53 and BAX in primary colorectal cancers.

Though most colorectal cancers show allelic losses, a subset of colorectal cancers (microsatellite instability or MSI-positive cancers) develop numerous small insertion and deletion mutations in repetitive DNA. Some of these sequences occur in coding regions of cancer related genes which, when targeted by frameshift mutations, produce truncations in their protein product. Such a gene is the proapoptotic tumor suppressor, BAX, mutated by frameshifts within a polyG sequence in approximately 50% of MSI-positive colorectal cancers. BAX is directly transactivated by p53, a gene commonly mutated in colorectal cancers but not often in MSI-positive lesions. Here we sought to characterize the relationship between BAX and p53 by simultaneously analysing a selected series of 65 colorectal tumors for mutations in the entire coding regions of both genes. The tumors comprised 19 MSI-high, 12 MSI-low and 34 MSI-null cancers. Eight of 19 MSI-high sporadic colorectal cancers (42%) contained insertions and deletions at the polyG tract in the BAX gene. In addition, three somatic BAX missense mutations were identified in two tumors. A single missense mutation was detected in an MSI-high tumor that also contained a frameshift microdeletion, and two missense mutations were identified in an MSI-null tumor wild-type for p53. p53 mutations were detected in 5/12 MSI-low tumors (42%) and 12/34 MSI-null tumors (35%). Of significance, no p53 mutations were detected in MSI-high tumors. This study demonstrates that a reciprocal relationship exists between p53 and BAX in sporadic colorectal cancers, and further supports the hypothesis that MSI-low tumors are biologically similar to MSI-null tumors.

Mucin core protein expression in colorectal cancers with high levels of microsatellite instability indicates a novel pathway of morphogenesis.

Particular mucinous phenotypes have been associated with serrated epithelial polyps of the colon. These polyps also show a high frequency of DNA instability. The aim of this study was to examine the expression of mucins in colorectal cancers that arise through the suppressor and mutator pathways. The immunohistochemical distribution of the human apomucins MUC1, MUC2, MUC4, and MUC5AC was determined in 93 sporadic colorectal cancers classified previously (J. R. Jass et al., J. Clin. Pathol., 52: 455-460, 1999) according to levels of DNA microsatellite instability (MSI) as 22 MSI-high (MSI-H), 24 MSI-low (MSI-L), and 47 MS stable (MSS). MUC2 expression was observed in 19 (86%) MSI-H, 10 (42%) MSI-L, and 15 (32%) MSS cancers (P = 0.0001); and MUC5AC expression was observed in 17 (77%) MSI-H, 8 (33%) MSI-L, and 13 (28%) MSS cancers (P = 0.0003). There was no association between MUC1 or MUC4 expression and MSI status. The mucinous phenotype described in serrated polyps (MUC2+/MUC5AC+) was seen in 15 (68%) of 22 MSI-H and only 10 (14%) of 71 MSI-L/MSS cancers (P < 0.0001). Increased expression of the secretory mucins MUC2 and MUC5AC was observed in sporadic MSI-H cancers. Identical mucin changes and DNA MSI occurred in serrated polyps of the colorectum, which suggests that these lesions may represent precursors of MSI-H cancers.

Proliferation, apoptosis, and survival in high-level microsatellite instability sporadic colorectal cancer.

Sporadic colorectal cancer (CRC) characterized by high-level DNA microsatellite instability (MSI-H) has a favorable prognosis. The reason for this MSI-H survival advantage is not known. The aim of this study was to correlate proliferation, apoptosis, and prognosis in CRC stratified by MSI status. The proliferative index (PI) was measured by immunohistochemical staining with the Ki-67 antibody in a selected series of 100 sporadic colorectal cancers classified according to the level of MSI as 31 MSI-H, 29 MSI-Low (MSI-L), and 40 microsatellite stable (MSS). The Ki-67 index was significantly higher in MSI-H cancers (P < 0.0001) in which the PI was 90.1 +/- 1.2% (mean +/- SE) compared with 69.5 +/- 3.1% and 69.5 +/- 2.3% in MSI-L and MSS subgroups, respectively. There was a positive linear correlation between the apoptotic index (AI) and PI (r = 0.51; P < 0.001), with MSI-H cancers demonstrating an increased AI:PI ratio indicative of a lower index of cell production. A high PI showed a trend toward predicting improved survival within MSI-H cancers (P = 0.09) but did not predict survival in MSI-L or MSS cancers. The AI was not associated with survival in any MSI subgroup. In conclusion, this is the first study to show that sporadic MSI-H cancers are characterized by a higher AI:PI ratio and increased proliferative activity compared with MSI-L and MSS cancers, and that an elevated PI may confer a survival advantage within the MSI-H subset.

Colorectal neoplasms detected colonoscopically in at-risk members of colorectal cancer families stratified by the demonstration of DNA microsatellite instability.

This study compared colonoscopic findings in families meeting the Amsterdam criteria (A) for hereditary non-polyposis colorectal cancer (HNPCC) but stratified according to whether the familial cancers showed DNA microsatellite instability. DNA was extracted from paired samples of normal and cancer, and microsatellite instability was analysed at up to six loci. Families were termed replication error positive (RER+) when at least 50% of tumours tested per family were positive. Of 26 families studied 17 were RER+ and 9 were RER-. Cancers in the A/RER- families showed no right-sided predilection (P < 0.001). Colonoscopies have been performed on 182 at-risk members of A/RER+ families and 60 members of A/RER- families. More of the at-risk members of A/RER-families were found to have adenomas at colonoscopy (P = 0.095), but these were smaller than those of A/RER+ families (P = 0.19). The adenoma:carcinoma ratio was twice as high in A/RER- families (13:1) as in A/RER+ families (7:1). One of the A/RER- families had hyperplastic polyposis. The others do not appear to have attenuated familial adenomatous polyposis and are similar to the adenoma families or late-onset colorectal cancer families described by others. This study illustrates the importance of molecular technology in separating HNPCC from syndromes with overlapping phenotypes.

Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability.

Attempts to classify colorectal cancer into subtypes based upon molecular characterisation are overshadowed by the classical stepwise model in which the adenoma-carcinoma sequence serves as the morphological counterpart. Clarity is achieved when cancers showing DNA microsatellite instability (MSI) are distinguished as sporadic MSI-low (MSI-L), sporadic MSI-high (MSI-H) and hereditary non-polyposis colorectal cancer (HNPCC). Divergence of the 'methylator' pathway into MSI-L and MSI-H is at least partly determined by the respective silencing of MGMT and hMLH1. Multiple differences can be demonstrated between sporadic and familial (HNPCC) MSI-H colorectal cancer with respect to early mechanisms, evolution, molecular characterisation, demographics and morphology. By acknowledging the existence of multiple pathways, rapid advances in the fields of basic and translational research will occur and this will lead to improved strategies for the prevention, early detection and treatment of colorectal cancer.

Epithelial misplacement in Peutz-Jeghers polyps. A diagnostic pitfall.

Early difficulties with the interpretation of the histopathology caused overdiagnosis of cancer in the Peutz-Jeghers syndrome; and there is still controversy about the magnitude of risk of gastrointestinal carcinoma. Most workers now believe that there is a small but definite increase in the incidence of gastrointestinal carcinoma in Peutz-Jeghers polyps and most of these cancers occur in the upper gastrointestinal tract. In a review of 491 Peutz-Jeghers polyps in the records of St. Mark's Hospital Pathology department, misplacement of epithelium was found in approximately 10% of small intestinal polyps and closely mimicked adenocarcinoma. This "pseudoinvasion" was not observed in polyps of the stomach or colon. The epithelial misplacement may involve all layers of the bowel wall; and the most helpful histological discriminators include a lack of cytological atypia, the presence of the normal epithelial cell subtypes and a brush border, hemosiderin deposition, and intramural mucinous cysts. Epithelial misplacement may account for the overdiagnosis of carcinoma arising in Peutz-Jeghers polyps as reported in the literature.

Ultrastructural localization of epithelial mucin core proteins in colorectal tissues.

Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon.

Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum.

Promoter hypermethylation frequency and BRAF mutations distinguish hereditary non-polyposis colon cancer from sporadic MSI-H colon cancer.

BACKGROUND: Colorectal cancers resulting from defective DNA mismatch repair can occur in both hereditary non-polyposis colon cancer (HNPCC) and in the sporadic setting. They are characterised by a high level of microsatellite instability (MSI-H) and superficially resemble each other in that they are frequently located in the proximal colon and share features such as circumscribed tumour margins and tumour-infiltrating lymphocytes. However, significant differences can be demonstrated at the molecular level including widespread promoter hypermethylation and BRAF -activating mutations which occur significantly less often in HNPCC. AIMS: In this study, we sought to determine whether the presence of widespread promoter hypermethylation and BRAF mutations would exclude HNPCC. MATERIALS AND METHODS: We investigated the methylation status of four methylated in tumour markers (MINTs 1,2,12 and 31), and the promoter regions of 5 genes hMLH1, HPP1, MGMT, p16INK4A and p14ARF, in 21 sporadic MSI-H colorectal cancers and compared these with 18 cancers from HNPCC patients. The methylation status of CpG islands were determined by either methylation specific PCR (MSP) or combined bisulfite restricton analysis (COBRA). In addition we considered the BRAF mutation status of 18 HNPCC tumours and 19 sporadic MSI-H cancers which had been previously determined by RFLP analysis and confirmatory sequencing. RESULTS: Methylation of the promoter regions in target genes occurred less frequently within the HNPCC tumours (27% of analyses), compared with the sporadic MSI-H tumours (59% of analyses) (P < 0.001). Methylation of MINTs 1, 2, 12 and 31 occurred in 4% of analyses for HNPCC tumours contrasted with 73% for sporadic MSI-H tumours (P < 0.001). BRAF mutations were detected in 74% of sporadic tumours but none of the HNPCC cancers tested. CONCLUSIONS: The total number of genes and MINTs methylated in HNPCC was lower than in MSI-H colorectal tumours. No HNPCC tumour showed evidence of widespread promoter hypermethylation or BRAF mutation suggesting this feature could be used as a discriminator between familial and sporadic cases.

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC)--results of an international collaborative study.

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2-5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

Observer study of the grading of dysplasia in ulcerative colitis: comparison with clinical outcome.

Patients with extensive ulcerative colitis are entered into surveillance programs that aim to detect premalignant changes. Biopsy specimens have been collected in the St Mark's Hospital (London) surveillance program over a 22-year-period. Specimens from patients reported as having dysplasia were reexamined. A total of 207 biopsy specimens from 86 patients were graded by five experienced pathologists according to the severity of the dysplasia. The overall agreement between the pathologists grading the specimens was poor; each pair agreed on between 42% and 65% of the slides. The best agreement was for slides that were said to show no dysplasia. Comparison with clinical outcome indicated that the pathologists most likely to diagnose dysplasia in patients with carcinoma were also likely to diagnose dysplasia in patients who did not go on to develop carcinoma. Calculating an average grade of dysplasia did not significantly improve diagnostic accuracy. Despite the findings of this interobserver study, dysplasia has been a successful marker in clinical practice. Pathologists should ensure that they have access to previous slides from the same patient and adequate clinical information before reporting biopsies as positive for dysplasia. An additional biopsy should usually be undertaken before surgery is considered.