Cowden disease, also known as multiple hamartoma syndrome, is an autosomal dominant cancer syndrome with a high risk of breast and thyroid cancer. The gene involved has been localized to chromosome 10q22-23. Recently, the tumour suppressor gene PTEN/MMAC1, encoding a putative protein tyrosine or dual-specificity phosphatase, was cloned from that region and three mutations were detected in patients with Cowden disease. We confirmed that the PTEN/MMAC1 gene is indeed the gene for Cowden disease by a refined localization of the gene to the interval between D10S1761 and D10S541, which contains the PTEN/MMAC1 gene and, by mutation analysis in eight unrelated familial and 11 sporadic patients with Cowden disease. Eight different mutations were detected in various regions of the PTEN/MMAC1 gene. One mutation was detected twice. All detected changes in the gene can be predicted to have a very deleterious effect on the putative protein. Five of the nine patients have a mutation in exon 5 coding for the putative active site and flanking amino acids. Evaluation of the clinical data of the patients in which a mutation could be detected gives no clear indications for a correlation between the genotype and phenotype. In 10 patients no mutation could be detected so far. In support of the linkage data, no evidence has emerged from the phenotype of these patients suggestive for genetic heterogeneity.

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Aims: To compare commercially available Ki-67 equivalent antibodies with regard to qualitative and quantitative immunohistochemical staining characteristics. Methods: The following antibodies were used: monoclonal MIB-1 (Immunotech), monoclonal MM1 (Novocastra), polyclonal NCL-Ki-67p (Novocastra), and polyclonal Rah Ki-67 (Dako). All immunostainings were evaluated in squamous epithelium from formalin fixed and paraffin wax embedded pharyngeal tonsils. Labelling indices (LIs) were recorded twice to test their reproducibility. Results: By application of all four antibodies the nuclear staining could be either diffuse, granular, or a combination of both (classified as granular in this study). The diffuse pattern generally showed a strong or moderate staining intensity, whereas the granular pattern displayed a continuum from strong to very weak, making it difficult to discriminate between positive and negative nuclei. The diffuse staining pattern was seen in approximately 59% of the nuclei with the MIB-1 antibody and in 35-45% when the other antibodies were used. The following mean LIs were recorded: MIB-1, 31%; NCL-Ki-67p, 21%; Rah Ki-67, 17%; and MM1, 14%. The reproducibility was excellent for all four antibodies, with the mean of differences between the two runs of counts ranging from 1.1% to 1.5%. Conclusions: The four tested Ki-67 equivalent antibodies revealed differences in qualitative and quantitative staining characteristics, which resulted in considerable variations in registered LIs. The MIB-1 antibody appears to have a higher sensitivity for detecting the Ki-67 antigen than the other three tested antibodies. These differences are important to consider when proliferative activity is determined by the Ki-67 LI.

A review of 1050 pathology reports from colorectal adenocarcinoma specimens examined at the Department of Pathology, Sørlandet sykehus HF, Kristiansand, Norway during the period 1995-2006 revealed a poor performance of most doctors concerning lymph node harvest. A mean of 8.1 nodes per specimen (range 12.3-2.1) and a mean proportion of 22.3% of specimens with ≥12 lymph nodes (range 47.1-0%) were found. A small pilot study was undertaken in 2007 to evaluate the effect of prolonged formalin fixation and the use of a special lymph node fixative [glacial acetic acid, ethanol, water and formaldehyde (GEWF) solution] with regard to the number of retrieved nodes. This showed that one extra day formalin fixation and the use of GEWF solution considerably enhanced the detection of lymph nodes, particularly those of smaller size. Based on these findings, our routines concerning handling of colorectal cancer specimens were changed during 2007. After this time all specimens have been fixed in a mixture of GEWF solution and formalin for at least 48 h and the doctors have been encouraged to find as many lymph nodes as possible. In cases revealing <12 nodes after microscopical examination, the specimens have been re-examined and searched for additional nodes. A review of lymph node retrieval in 423 cases of colorectal cancer during the period 2008-2010 showed that the mean number of nodes per specimen had increased to 16.8 (range 29.0-13.3) and the proportion of specimens with ≥12 nodes to 78.0% (range 96.8-63.6%). Thus, these changes of routines which were easy to implement without significant extra costs have considerably improved lymph node harvest at our department. The use of a special lymph node fixative (e.g. GEWF solution) is highly recommended not only for detection of nodes in colorectal specimens, but also for retrieval of lymph nodes embedded in fat tissue generally.

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