Molecular pathology : MP最新文献

Background: A novel single nucleotide polymorphism (SNP), G(-248)A, in the 5' untranslated region of the BAX promoter and its association with reduced protein expression, progression beyond Rai stage 0, and treatment resistance in chronic lymphocytic leukaemia (CLL) has been reported previously.

Aim: To develop a restriction enzyme analysis (REA) based method for routine detection of BAX promoter SNP in a clinical laboratory.

Methods: The BAX promoter was analysed in duplicate by REA and sequencing in 90 samples (from 45 patients with CLL, 43 controls, and two cell lines). The promoter region was amplified, digested with restriction endonucleases (Aci I and Tau I), and separated by gel electrophoresis.

Results: After digestion, the normal GG genotype samples produced three distinct bands. The homozygous AA replacement abolished the cleavage site, resulting in a single band. Although the heterozygous samples produced three bands, the two smaller visible bands were reduced in intensity (> 50%). The test characteristics of Aci I REA were better than those of Tau I REA, in terms of sensitivity (100% v 77.8%), specificity (98.6% v 92.3%), positive predictive value (95.03% v 87.4%), and negative predictive value (100% v 85.83%).

Conclusions: REA using Aci I is a highly sensitive and specific method for detecting the BAX G(-248)A SNP in CLL.

Background: Laser microbeam microdissection has greatly facilitated the procurement of specific cell populations from tissue sections. However, the fact that a coverslip is not used means that the morphology of the tissue sections is often poor.

Aims: To develop a mounting method that greatly improves the morphological quality of tissue sections for laser microbeam microdissection purposes so that the identification of target cells can be facilitated.

Methods: Fresh frozen tissue and formalin fixed, paraffin wax embedded tissue specimens were used to test the morphological quality of mounted and unmounted tissue. The mounting solution consisted of an adhesive gum and blue ink diluted in water. Interference of the mounting solution with DNA quality was analysed by the polymerase chain reaction using 10-2000 cells isolated by microdissection from mounted and unmounted tissue.

Results: The mounting solution greatly improved the morphology of tissue sections for laser microdissection purposes and had no detrimental effects on the isolation and efficiency of amplification of DNA. One disadvantage was that the mounting solution reduced the cutting efficiency of the ultraviolet laser. To minimise this effect, the mounting solution should be diluted as much as possible. Furthermore, the addition of blue ink to the mounting medium restores the cutting efficiency of the laser.

Conclusions: The mounting solution is easy to prepare and apply and can be combined with various staining methods without compromising the quality of the DNA extracted.

Background: The E-cadherin-catenin adhesion complex is crucial for intercellular adhesiveness and maintenance of tissue architecture. Its impairment is associated with poorly differentiated phenotype and increased invasiveness of carcinomas.

Aims: To evaluate E-cadherin, beta catenin, gamma catenin, and ezrin expression and its relation to histopathological features of primary and metastatic Wilms's tumours.

Methods: Immunohistochemistry was used to determine the expression and cellular distribution of E-cadherin, beta catenin, gamma catenin, and ezrin in primary and metastatic Wilms's tumours. Western blotting was used to determine polypeptide size and expression of E-cadherin and beta catenin in Wilms's tumours compared with normal kidney.

Results: Moderate expression of E-cadherin was found mainly in cytoplasm and occasionally cell membranes of dysplastic tubules, whereas low expression was seen in cytoplasm of blastemal cells. Primary and metastatic tumours showed moderate to high beta catenin expression in blastemal and epithelial cells, with predominantly membranous and cytoplasmic staining. Occasional nuclear staining was noted in metastatic tumours. Low to high gamma catenin and ezrin expression was seen in cytoplasm of blastemal and epithelial cells of primary and metastatic tumours. Higher amounts of 92 kDa beta catenin were detected in tumours than in normal kidney. Low expression of 120 kDa E-cadherin was seen in moderately differentiated tumours, whereas expression was lacking in poorly differentiated tumours.

Conclusions: Compared with primary tumours, metastatic tumours showed lower expression of E-cadherin and gamma catenin, with nuclear staining for beta catenin. Low E-cadherin was associated with poorly differentiated tumours. These results suggest that abnormal expression of adhesion proteins correlates with the invasive and metastatic phenotype in Wilms's tumours.

Aims: To compare circulating matrix metalloproteinase (MMP) concentrations with antibodies to single and double stranded DNA (ssDNA and dsDNA) to determine their relation in inflammatory arthritic diseases, such as systemic lupus erythematosus (SLE).

Methods: Fibroblast MMP-2 and neutrophil MMP-9 were resolved by gelatin zymography and measured by densitometry. Anti-ssDNA and anti-dsDNA were determined by enzyme immunoassay and samples grouped on antibody content as follows: low anti-ssDNA/low anti-dsDNA antibodies (group 1); high anti-ssDNA/low anti-dsDNA antibodies (group 2); and high anti-ssDNA/high anti-dsDNA antibodies (group 3).

Results: Group 3 samples contained significantly lower amounts of MMP-9 when compared with group 1 samples. Higher molecular weight MMP-9 forms (130 and 225 kDa) were virtually absent. Group 2 samples contained intermediate MMP-9 concentrations. Fibroblast MMP-2 was unchanged in all groups. Mean complement C3 and C4 concentrations showed a consistent, but variably significant, decrease with increasing anti-ssDNA and anti-dsDNA antibodies. The mean erythrocyte sedimentation rate was raised in all patient groups.

Conclusions: Neutrophil MMP-9, an inflammatory marker, inversely correlates with anti-dsDNA antibodies, which are a specific marker for SLE, and may be important in monitoring disease activity during antibody deposition in tissues.

Aims: To define regions of loss on the distal portion of chromosome 12q in gastric adenocarcinoma.

Methods: Microsatellite analysis on chromosome 12 was performed on 19 human gastric cancer cell lines using 77 markers, 71 of which were within or distal to 12q21; some portions of this region showed extended regions of homozygosity (ERHs) in 10 of 19 gastric cancer cell lines. In addition, microdissected tumour cells from 76 primary gastric adenocarcinomas were examined using 13 markers of interest implicated by the cell line data; 70% of these showed allelic imbalance (AI) at one or more markers in or distal to 12q21.

Results: Mapping ERHs in the cell lines and sites of AI in the tumours identified three regions that contain putative tumour suppressor genes: region A is located within 2.8 Mb between markers D12S1667 and D12S88; region B, within 1.9 Mb between markers D12S1607 and D12S78; and region C, in 0.74 Mb between markers D12S342 and D12S324. Fluorescence in situ hybridisation (FISH) analysis in two cell lines confirmed that two of the ERHs reflected deletions, not amplifications, of D12S81 in region A and D12S340 in region C. FISH analysis of marker D12S1075 within an ERH containing region B in one cell line showed neither amplification nor deletion. AI on 12q was not associated with prognosis, but was associated with ethnicity of the patient.

Conclusions: These results identify regions on chromosome 12 that appear to contain tumour suppressor genes important in the development of gastric cancer.