Identification of metastatic papillary thyroid carcinoma in FNA specimens using thyroid peroxidase immunohistochemistry.

INTRODUCTION: We evaluated immunohistochemical staining for thyroid peroxidase (TPO), a glycoprotein found in the apical plasma membrane of thyroid follicular cells, as a marker for metastatic PTC in FNA samples and compared results with thyroglobulin (Tg) and thyroid transcription factor 1 (TTF1) staining. METHODS: Cell block sections prepared from 100 FNA specimens were stained with a rabbit monoclonal antibody to TPO (EP159). The FNAs included 64 metastatic malignancies from non-thyroid primary sites, including 18 lung, and 36 cases of thyroid tumours (29 PTC, six cases of medullary thyroid carcinoma and one thyroid anaplastic carcinoma). Thyroid tumours were stained with TTF1 and Tg in addition to TPO. All cases of metastatic lung carcinoma also had TTF-1 staining results. RESULTS: TPO staining was negative in all non-thyroid malignancies. Ninety percent (26/29) of PTC were positive. All positive cases showed strong cytoplasmic staining, although 54% (14/26) showed positivity in less than half of the cells. By comparison, Tg staining of TPC cases was present in 62% and TTF-1 in 100%. In addition to showing higher sensitivity, interpretation of staining results with TPO was generally easier with than Tg. All metastatic lung adenocarcinomas were positive for TTF-1 and TPO negative. The six medullary cancers showed positivity in 17%, 0% and 83% with TPO, Tg and TTF-1, respectively. CONCLUSIONS: TPO (mAb EP159) may be a useful addition to immunohistochemical panels for FNA specimens where metastatic PTC is a consideration, particularly in cases where metastatic lung carcinoma features in the differential diagnosis.

A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome.

BACKGROUND: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (<2% of all identified mutations), yet the immunohistochemical analysis of tumour samples indicates that approximately 5% of Lynch syndrome cases are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. METHODS: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based on immunohistochemical analysis. RESULTS: We have identified a frequently occurring frame-shift mutation (c.736_741del6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n = 61). These individuals all display the rare allele (population frequency <0.05) at a single nucleotide polymorphism (SNP) in exon 11, and have been shown to possess a short common haplotype, allowing us to calculate that the mutation arose around 1625 years ago (65 generations; 95% confidence interval 22 to 120). CONCLUSION: Ancestral analysis indicates that this mutation is enriched in individuals with British and Swedish ancestry. We estimate that there are >10 000 carriers of this mutation in the USA alone. The identification of both the mutation and the common haplotype in one Swedish control sample (n = 225), along with evidence that Lynch syndrome associated cancers are rarer than expected in the probands' families, would suggest that this is a prevalent mutation with reduced penetrance.

Reduced alpha-defensin expression is associated with inflammation and not NOD2 mutation status in ileal Crohn's disease.

BACKGROUND AND AIMS: Reduced ileal Paneth cell alpha-defensin expression has been reported to be associated with Crohn's disease, especially in patients carrying NOD2 mutations. The aim of this study was to independently assess whether NOD2, alpha-defensins and Crohn's disease are linked. METHODS: Using quantitative real-time polymerase chain reaction (RT-PCR), we measured the mRNA expression levels of key Paneth cell antimicrobial peptides (DEFA5, DEFA6, LYZ, PLA2G2A), inflammatory cytokines [interkelukin 6 (IL6) and IL8], and a marker of epithelial cell content, villin (VIL1) in 106 samples from both affected ileum (inflamed Crohn's disease cases, n = 44) and unaffected ileum (non-inflamed; Crohn's disease cases, n = 51 and controls, n = 11). Anti-human defensin 5 (HD-5) and haematoxylin/eosin immunohistochemical staining was performed on parallel sections from NOD2 wild-type and NOD2 mutant ileal Crohn's disease tissue. RESULTS: In Crohn's disease patients, DEFA5 and DEFA6 mRNA expression levels were 1.9- and 2.2-fold lower, respectively, in histologically confirmed inflamed ileal mucosa after adjustment for confounders (DEFA5, p<0.001; DEFA6, p = 0.001). In contrast to previous studies, we found no significant association between alpha-defensin expression and NOD2 genotype. HD-5 protein data supports these RNA findings. The reduction in HD-5 protein expression appears due to surface epithelial cell loss and reduced Paneth cell numbers as a consequence of tissue damage. CONCLUSIONS: Reduction in alpha-defensin expression is independent of NOD2 status and is due to loss of surface epithelium as a consequence of inflammatory changes rather than being the inciting event prior to inflammation in ileal Crohn's disease.

Relationship between number of ovulatory cycles and accumulation of mutant p53 in epithelial ovarian cancer.

BACKGROUND: It has been suggested that increased numbers of ovulations might increase the risk of p53 gene (also known as TP53) mutation in the ovarian epithelium, thereby leading to the development of cancer. The data supporting this hypothesis have come from an observation that accumulation of p53 protein in epithelial ovarian cancer was strongly associated with increasing numbers of ovulatory cycles. We have further investigated the association between ovulatory history and p53 gene mutation by use of data from a large case-control study of ovarian cancer in Australia. METHODS: Tissue blocks were available for immunohistochemical analysis of p53 protein from 234 case subjects, aged 18-79 years, who had invasive epithelial ovarian cancer. Epidemiologic data were also available for these women and for 855 control subjects. Case-case comparisons were made by use of prevalence ratios and 95% confidence intervals (CIs), and case-control comparisons were made by use of odds ratios (ORs) and 95% CIs. All statistical tests were two-sided. RESULTS: There was no association between p53 accumulation and years of ovulation. Women with p53-positive cancers had undergone an average of 29.3 years of ovulation compared with 29.0 years of ovulation for women with p53-negative cancers (P=.8). Although the overall risk of ovarian cancer development was significantly increased in women who had undergone more years of ovulation (OR=2.17; 95% CI =1.54-3.05-for > or =35 years versus <23 years of ovulation), there was no difference in the risk associated with p53-positive and p53-negative cancers. CONCLUSIONS: These results confirm the association between increased ovulation and ovarian cancer risk but do not support the hypothesis that this association is due to an increased risk of p53 mutation with a greater number of ovulatory cycles.

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.

Expression of antisense CD44 variant 6 inhibits colorectal tumor metastasis and tumor growth in a wound environment.

Up-regulation of CD44 variant isoforms has been linked to the progression of epithelial tumors and the metastatic phenotype. Here we report a functional role for CD44 variant isoforms in colorectal cancer metastasis. An antisense mRNA approach was used to down-regulate CD44 variant isoforms containing CD44 variant 6 (v6) in the metastatic colorectal tumor cell line HT29. Cell lines stably expressing antisense CD44 exon 10 (v6) showed reduced expression of alternatively spliced CD44 variant isoforms but no significant change in expression of CD44 core protein, as judged by immunohistochemical analysis using CD44 domain-specific monoclonal antibodies. Expression of antisense exon 10 (v6) had no effect on HT29 tumor cell proliferation in vitro or the ability of the cells to bind immobilized hyaluronan, but it resulted in a reduced capacity to form liver metastases in nude mice following intrasplenic injection. Metastases were not detected in nude mice inoculated with antisense CD44 exon 10 (v6)-expressing cell lines after 4 months, against a background of a 30% metastasis rate in the control HT29 parental and vector alone transfected lines. Furthermore, whereas 82% of mice intrasplenically injected with control HT29 parental and vector alone cell lines developed tumors in incisional wound sites, none of the mice injected with antisense exon 10 expressing HT29 cells developed similar tumors. This is the first demonstration that antisense RNA can be used to selectively inhibit expression of specific domains of a molecule generated through alternative mRNA splicing while allowing expression of core domains to remain unaffected. Furthermore, these results provide direct evidence for a functional role of CD44 variant isoforms in the metastasis of human colorectal tumor cells and may suggest a critical role for CD44 variants in promoting cell growth specifically in the cytokine/growth factor-enriched environment of a wound site.

The deleted in colon cancer (DCC) gene is consistently expressed in colorectal cancers and metastases.

The DCC (deleted in colorectal cancer) gene was originally identified as a candidate tumour suppressor gene in colon carcinogenesis on the basis of allelic losses in chromosome 18q.21 in 70% of colon cancers. Reverse transcriptase polymerase chain reaction (RT-PCR) of DCC mRNA suggests that DCC expression may also be reduced in colon cancers. We have used monoclonal antibodies generated against the DCC immunoglobulin-like domain to investigate DCC isoforms and DCC protein expression during colon cancer progression. Normal mucosa and colonic tumour specimens representative of the range of colonic tumour progression from benign adenomatous polyps to metastases were compared by Western blot analyses. We show that while M(r) 194 000 DCC is present in normal colonic mucosa and adenomatous polyps, it is also similarly expressed in colorectal carcinomas and colonic metastases in the liver. The presence of DCC protein is consistent with the presence of DCC mRNA transcripts in the same tissue specimens. Notably DCC was not completely lost in any colonic tumour specimens examined, even those that had progressed to metastatic cancers. Quantitation of DCC protein expression in tissue specimens by densitometry demonstrated that both normal and malignant specimens exhibit a wide range of DCC protein levels and there was no significant correlation between diminished DCC protein expression and colon cancer progression. These results demonstrate the pattern of expression of the DCC gene product in colonic tumour progression and show that absence of DCC expression is not associated with colonic tumour progression.

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.

Down-regulation of the down-regulated in adenoma (DRA) gene correlates with colon tumor progression.

The down-regulated in adenoma (DRA) gene was originally identified as a gene that was down-regulated in colon tumors. It encodes a protein with anion transporter function that is expressed predominantly in the mucosa of the lower gastrointestinal tract. In this study, expression of DRA and its cellular distribution have been investigated in a series of benign adenomatous polyps and malignant colorectal tumors and in corresponding normal colonic mucosa. We show that DRA mRNA and protein are expressed in all normal colonic tissue specimens with the protein restricted primarily to the terminally differentiated columnar epithelium and some goblet cells. Apical membrane localization was especially apparent in the columnar epithelium. The levels of DRA mRNA transcripts were down-regulated in all colon tumors examined relative to matched normal mucosa, with most specimens showing undetectable levels of DRA mRNA (77 of 104 tumors). DRA down-regulation was positively associated with colonic tumor progression according to Dukes' stage and was particularly significant in the early transition from normal mucosa to polyp to adenocarcinoma. DRA expression does not appear to be strictly associated with colonic cell differentiation; rather, its absence and down-regulation were associated with the proliferating component of the crypt epithelium and with neoplastic transformation, respectively.

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.

Major mesh-related complications following hernia repair: events reported to the Food and Drug Administration.

Mesh material affects complications following hernia repair. Medical device reports on the use of surgical mesh for hernia repair were reviewed from the Food and Drug Administration's (FDA) Manufacturer User Facility Device Experience Database from January 1996 to September 2004. We analyzed 252 adverse event reports related to the use of surgical mesh for hernia repair. Adverse events included infection (42%, 107 reports), mechanical failure (18%, 46), pain (9%, 23), reaction (8%, 20), intestinal complications (7%, 18), adhesions (6%, 14), seroma (4%, 9), erosion (2%, 6), and other (4%, 9). Compared to all other mesh types, Sepra/polypropylene mesh had more mechanical failures (80 vs 14%, p < 0.05), biomaterial mesh had more reactions (57 vs 7%, p < 0.05), polytetrafluoroethylene (PTFE)/polypropylene mesh had more intestinal complications (14 vs 7%, p < 0.05), and PTFE mesh tended to have more infections (75 vs 41% all other, p = 0.07). Death occurred in 2% (5). We conclude that specific mesh materials are related to specific complications.

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.

Occult axillary lymph node metastases in breast cancer do matter: results of 10-year survival analysis.

Axillary lymph node status is one of the most powerful prognostic factors for patients with breast cancer and is often critical in stratifying patients into adjuvant treatment regimens. In 203 apparently node-negative cases of breast cancer, a combination of immunohistochemical staining and step-sectioning identified occult metastases in 25% of cases. Ten-year follow-up information is available for these patients. Histologic features of the primary tumor and immunohistochemical staining for estrogen receptor, progesterone receptor, Her-2, and p53 were also evaluated. With multivariate analysis, both occult metastases and higher histologic grade of the primary tumor were independent predictors of disease-free survival. Histologic grade was the only significant independent predictor of overall survival. Estrogen receptor, progesterone receptor, Her-2, and p53 status did not predict the presence of metastases or survival when all tumor types were considered together. Metastases >0.5 mm significantly predicted a poorer disease-free survival when invasive ductal carcinomas were considered alone. Histologic grade was significantly associated with disease-free survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients. The presence of occult metastases approached significance for overall survival in the premenopausal and perimenopausal patients but not in the postmenopausal patients.

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.

Preparation and characterization of polyclonal antibodies against human chaperonin 10.

Early pregnancy factor (EPF) has been identified as an extracellular homologue of chaperonin 10 (Cpn10), a heat shock protein that functions within the cell as a molecular chaperone. Here, we report the production of polyclonal antibodies directed against several different regions of the human Cpn10 molecule and their application to specific protein quantitation and localization techniques. These antibodies will be valuable tools in further studies to elucidate the mechanisms underlying the differential spatial and temporal localization of EPF and Cpn10 and in studies to elucidate structure and function.

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.

Prognostic significance of MUC1 epithelial mucin expression in breast cancer.

The epithelial mucin produced by the MUC1 gene is present in the apical cell membrane of normal breast epithelial cells and is highly expressed in many breast cancers. Several studies have provided conflicting evidence regarding the relationship between MUC1 expression and survival in breast cancer patients. In this study a detailed immunohistological analysis of MUC1 expression was performed using monoclonal antibody BC2 and was related to other tumor characteristics and patient survival. Patients whose tumors showed MUC1 expression in greater than 75% of tumor cells had significantly poorer disease-free and overall survival (P < .05). The proportion of cells showing cytoplasmic MUC1 expression was prognostically significant, but the proportion of cells that lined gland spaces showing apical membrane staining was of no prognostic significance. A high level of MUC1 expression was significantly associated with the presence of axillary node metastases and estrogen receptors but not with other tumor characteristics.