Molecular pathology : MP最新文献

Background: Gastric cancer is one of the most frequent malignancies in the world, ranking fifth in the Netherlands as a cause of cancer death. Surgery is the only curative treatment for advanced cases, but results of gastrectomy largely depend on the stage of the disease. A better understanding of the mechanisms of progression from a preneoplastic condition through intraepithelial neoplasia to invasive cancer may provide information relevant to designing focused prevention strategies.

Methods: Because the pattern of chromosomal aberrations in precursors of gastric cancer is unclear, 11 gastric polyps with intraepithelial neoplasia (three hyperplastic polyps and eight adenomas) were analysed by microarray comparative genomic hybridisation to study chromosomal instability in precursors of gastric cancer.

Results: Chromosomal aberrations were detected in all specimens. Adenomas showed no more chromosomal aberrations than did the hyperplastic polyps. The most frequent aberrations were gain of 7q36 and 20q12, and loss of 5q14-q21 in the adenomas, and loss of 15q11-14, 1p21-31, and 21q11-21.2 in the hyperplastic polyps. The most frequent chromosomal aberration in common to both types was loss of 9p21.3.

Conclusion: Hyperplastic polyps showed many chromosomal aberrations, confirming that neoplastic transformation can occur in these lesions. These observations are consistent with the existence of two morphologically and genetically distinct pathways to gastric cancer-the hyperplastic polyp pathway and the (intestinal type) adenoma pathway. The relative contribution of each to gastric carcinogenesis in general, and how they compare to patterns of chromosomal aberrations in the more prevalent flat foci of intraepithelial neoplasia remain to be determined.

Aims: Secreted Wnt ligands are key proteins regulating cell-cell interactions and cell growth and differentiation. These proteins, along with other components of the Wnt signalling pathway, are involved in the malignant transformation of various human cancers, including malignant melanoma. This study defines the expression of several members of the Wnt ligand family and correlates their expression with histological characteristics.

Methods: The expression of Wnt2, Wnt5a, Wnt5b, Wnt7b, and Wnt10b was defined by in situ, antisense RNA hybridisation of paraffin wax embedded sections of benign naevi and malignant melanoma. Immunoperoxidase based antibody staining was used to define the expression of frizzled (Fz) receptors.

Results: All naevi tested strongly expressed Wnt2, Wnt5a, Wnt7b, and Wnt10b. Melanomas characterised by small, uniform cells expressed each of the Wnts in a pattern similar to that seen for benign naevi. In contrast, melanomas characterised by large, pleomorphic cells expressed Wnt10b but did not express Wnt2 and had low levels of expression of Wnt5a. Expression of Wnt7b was variable in these melanomas. Fz receptor expression was present at a low level in normal epithelium and all naevi and melanomas.

Conclusions: The expression pattern of Wnt ligands in malignant melanoma correlates with histopathological features and may provide a basis for the molecular classification of this disease.

Background: In patients with Wilson's disease (WD), an autosomal recessive disorder, toxic accumulation of copper results in fatal liver disease and irreversible neuronal degeneration. ATP7B, the gene mutated in WD, contains 21 exons and encodes a copper transporting ATPase. A novel disease causing mutation (4193delC) in exon 21 of the ATP7B gene has previously been detected by heteroduplex analysis and DNA sequencing.

Aims: To screen for the above mutation in patients with WD and carriers using an amplification refractory mutation system (ARMS).

Methods: ARMS was used to screen for the 4193delC mutation in 30 patients with WD and their relatives.

Results: A homozygous mutation was detected in 16 of 30 patients with WD.

Conclusions: This polymerase chain reaction based method, which has been known for years, is a simple, inexpensive, and rapid method for screening common and specific mutations in patients with WD and carriers.

Aims: To describe a robust pretreatment protocol for preparing paraffin wax embedded tissues on tissue microarrays for fluorescence in situ hybridisation (FISH). The newly developed pretreatment protocol described here was compared with the commonly used sodium thiocyanate based protocol and two different heating methods used in standard antigen unmasking protocols for immunohistochemistry (pressure cooking and microwaving in citrate acid buffer).

Methods: Dewaxed tissue sections were incubated in 10mM citric acid buffer at 80 degrees C for 30 minutes to two hours, followed by a short pepsin digestion (1-5 mg/ml). Pretreated tissues were co-denatured with DNA probes at 80 degrees C for 10 minutes, followed by hybridisation at 37 degrees C for 48-72 hours.

Results: The three protocols using citrate acid buffer produced FISH signals with superior signal to noise ratios compared with sodium thiocyanate pretreatment. Most importantly, the best tissue attachment was achieved using the newly developed pretreatment protocol: on tissue microarrays less than 1% of cores were lost. To date, a total of 30 probes have been successfully hybridised on to breast tissue and multi-tissue microarrays.

Conclusion: This pretreatment protocol is easy, reproducible, and facilitates FISH on tissue microarrays, with potential for widespread application in cancer research.

Several "high throughput methods" have been introduced into research and routine laboratories during the past decade. Providing a new approach to the analysis of genomic alterations and RNA or protein expression patterns, these new techniques generate a plethora of new data in a relatively short time, and promise to deliver clues to the diagnosis and treatment of human cancer. Along with these revolutionary developments, new tools for the interpretation of these large sets of data became necessary and are now widely available. Tissue microarray (TMA) technology is one of these new tools. It is based on the idea of applying miniaturisation and a high throughput approach to the analysis of intact tissues. The potential and the scientific value of TMAs in modern research have been demonstrated in a logarithmically increasing number of studies. The spectrum for additional applications is widening rapidly, and comprises quality control in histotechnology, longterm tissue banking, and the continuing education of pathologists. This review covers the basic technical aspects of TMA production and discusses the current and potential future applications of TMA technology.

Aim: To purify and partially characterise a fraction from human leucocytes containing a substance cytotoxic to Candida albicans.

Methods: Leucocytes were isolated from the buffy coats of healthy blood donors. The cytotoxic factor (CF) was isolated from the soluble fraction of the cells. A cell lysate was passed through a filter with a cut off value of 3 kDa, and the filtrate was processed by anionic exchange chromatography and gel filtration. The purified CF was analysed for its chemical and biological properties. The cytotoxicity of CF was tested on C albicans grown on agar plates.

Results: Mass spectrometry showed a molecular mass of 2.148 kDa. CF was found in polymorphonuclear neutrophilic cells only. No amino acids were detected, and a low ultraviolet absorbance at 260 nm and resistance to nuclease indicate the absence of nucleic acids. An anthrone test was positive for carbohydrate. The substance was soluble in water. CF showed a dose related cytotoxicity in the range of 0.1-1 mg/ml. The cytotoxic effect was abrogated by zinc ions. Preliminary testing indicated that CF also had cytotoxic effects against some bacteria.

Conclusions: This report describes a factor from isolated human leucocytes that is cytotoxic to C albicans. The substance contains a carbohydrate moiety, whereas no amino acids were detected. The cytotoxicity can be abrogated by zinc ions in vitro. This substance is probably part of the repertoire by which leucocytes prevent infections.

Germline mutations of the BRCA1 gene predispose individuals mainly to the development of breast and/or ovarian cancer. However, the exact function of the gene is still unclear, although the encoded proteins are involved in various cellular processes, including transcriptional regulation and DNA repair pathways. Several BRCA1 splice variants are found in different tissues, but in spite of intense investigations, their regulation and possible functions are poorly understood at the moment. This review summarises current knowledge on the roles of these splice variants and the mechanisms responsible for their formation. Because alternative splicing is now widely accepted as an important source of genetic diversity, elucidating the functions of the BRCA1 splice variants would help in the understanding of the exact role(s) of this tumour suppressor. This should help to resolve the current paradox that, despite its seemingly vital cellular functions, mutations of this gene are associated with tissue specific tumour formation predominantly in the breast and the ovary.

Aims: To describe a cytogenetic technique suitable for the rapid assessment of global gene expression that is based on comparative genomic hybridisation (CGH), and to use it to understand the relation between genetic amplifications and gene expression.

Methods: Whereas traditional CGH uses DNA as test and reference in hybridisations, expressive genomic hybridisation (EGH) uses globally amplified mRNA as test and normal DNA as reference. EGH is a rapid and powerful tool for localising and studying global gene expression profiles and correlating them with loci of genetic amplifications using traditional CGH.

Results: EGH was used to correlate genetic amplifications detected by CGH with the expression profile of two independent cell lines-Colo320 and T47D. Although many amplifications resulted in overexpression, other amplifications were partially or completely silenced at the cytogenetic level.

Conclusion: This technique will assist in the analysis of overexpressed genes within amplicons and could resolve a controversial issue in cancer cytogenetics; namely, the relation between genetic amplifications and overexpression.

Aims: Molecular genetic changes involved in tumorigenesis and malignant transformation of human tumours are novel targets of cancer diagnosis and treatment. This study aimed to analyse the expression of putative tumour suppressor genes, FHIT and WT-1, and tumour rejection genes, BAGE, GAGE-1/2, MAGE-1, MAGE-3, and HAGE (which are reported to be important in human cancers), in salivary gland neoplasms.

Methods: Gene expression was analysed by reverse transcription polymerase chain reaction (RT-PCR) in normal salivary gland tissue and 44 benign and malignant salivary gland tumours.

Results: Aberrant FHIT transcripts were found in one of 38 normal salivary glands, three of 28 adenomas, and two of 16 carcinomas. WT-1 mRNA was detectable in two adenomas and five carcinomas. Immunoblotting showed that WT-1 mRNA expression was associated with raised WT-1 protein concentrations. RT-PCR for detection of BAGE, GAGE, and MAGE gene expression was positive in two adenomas and nine carcinomas, but negative in normal salivary gland tissue. HAGE mRNA was found in two normal salivary glands, 11 benign, and eight malignant tumours.

Conclusions: FHIT mRNA splicing does not appear to be involved in the genesis of salivary gland neoplasms. The upregulation of WT-1 mRNA in tumours of epithelial/myoepithelial phenotype may imply a potential role of WT-1 in the genesis and/or cellular differentiation of these salivary gland tumours. The tumour rejection genes were more frequently, but not exclusively, expressed in malignant salivary gland tumours than in benign neoplasms, although none was suitable as a diagnostic marker of malignancy in salivary gland neoplasms.