Molecular determination of carriage of the mecA locus in coagulase negative staphylococci in screening swabs from patients in an intensive care unit
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:To describe mutations in the PAX6 gene in five patients with aniridia from three unrelated families.METHODS:The PAX6 gene was analysed using single stranded conformational polymorphism analysis and direct sequencing.RESULTS:In one family, three individuals from two generations had aniridia, whereas in each of the other families only one member was affected. The first patient had the heterozygous Q221X (1023C --> T) nonsense mutation in exon 8. The same mutation was found in his mother and sister. Another patient had a heterozygous Q297X (1252C --> T) mutation in exon 10. The third patient carried a heterozygous IVS5+2T --> C mutation leading to aberrant splicing of mRNA.CONCLUSIONS:These findings provide further examples of haploinsufficiency of PAX6 in aniridia.
Aims: To compare the molecular genetic changes in the Ki-ras and adenomatous polyposis coli (APC) genes between colorectal carcinomas and synchronous metastases, and then to compare and contrast those changes with previously reported changes in the two genes between these carcinomas and accompanying adenomas. This expanded comparison would provide greater understanding of the progression of molecular changes in neoplastic tissue during the development of malignancy from a benign adenoma to carcinoma and then to metastatic spread of the malignancy.Methods: DNA was extracted from paraffin wax embedded tissue. This was followed by polymerase chain reaction and gel electrophoresis for mutations in the Ki-ras gene using single stranded conformational polymorphism analysis. Amplification of a CA repeat marker was used to assess loss of heterozygosity (LOH) at the APC gene.Results: The findings for the Ki-ras gene in 42 paired carcinomas and synchronous metastases were identical, regardless of whether or not the carcinoma and its companion adenoma had identical Ki-ras findings. The results of APC LOH for 39 paired carcinomas and synchronous metastases were also identical, whether or not the carcinoma and its companion adenoma had identical APC LOH findings. Results were uninformative for three pairs.Conclusions: With respect to these two genes, a carcinoma may be discordant from its companion adenoma, but the metastasis remains consistent with the colonic carcinoma.
Aims: Chondroblastoma is a rare, locally aggressive bone tumour that causes osteolytic destruction at the epiphyseal end of the affected bone. It is possible that tumour cells may stimulate osteoclastogenesis and osteolytic destruction through the production of receptor activator of NF-κB ligand (RANKL), which is a key molecule essential for regulating osteoclast formation and activity. Therefore, the expression of RANKL at both the mRNA and the protein level was investigated in chondroblastoma tumour tissue obtained from patients. Methods: The expression of RANKL gene transcripts was analysed by the reverse transcription-polymerase chain reaction (RT-PCR), and the cellular localisation of RANKL mRNA and protein was demonstrated by means of in situ hybridisation and immunohistochemistry. Results: RT-PCR analysis indicated that RANKL mRNA was present in all chondroblastoma specimens and normal cancellous bone samples, but not in normal articular cartilage and chondrosarcoma tissues. In contrast, gene transcripts of osteoprotegerin (OPG), the decoy receptor of RANKL, were detected in all types of tissues. The chondroid origin of neoplastic mononuclear cells in chondroblastoma was confirmed by positive S-100 immunohistochemical staining. Both RANKL mRNA and protein were exclusively expressed in these neoplastic mononuclear cells. Conclusions: These findings suggest that RANKL may be involved in the tumour cell induced recruitment of osteoclast-like cells and consequent osteolytic bone destruction in chondroblastoma.
AIM:Polyps of the colon and rectum are considered to be premalignant lesions in the development of colorectal cancer. However, knowledge of how normal epithelial cells gain invasive properties is limited. Laminin 5 gamma 2 chain expression was investigated to determine the role of laminin 5 as a marker of potential invasiveness in colorectal polyps.MATERIAL/METHODS:Sixty seven polyps of different types (15 hyperplastic polyps, 12 serrated adenomas, 16 tubular adenomas, and 24 adenomas with a villous component) were assessed for gamma 2 chain expression of laminin 5 by immunohistochemistry on archival, paraffin wax embedded sections.RESULTS:Ten polyps stained positive and the number of polyps expressing the laminin 5 gamma 2 chain increased significantly as the phenotype of the adenomas became more atypical: none of the 15 hyperplastic polyps, two of the 16 tubular adenomas (12.5%), and six of the 24 adenomas with a villous component (25%) were positive. Two of 12 (17%) serrated adenomas, regarded as a distinct form of colorectal neoplasia, showed gamma 2 chain expression. Furthermore, laminin 5 gamma 2 chain expression correlated with lesion size. Polyps smaller than 10 mm expressed the gamma 2 chain less frequently than did those equal to or larger than 10 mm.CONCLUSION:Laminin 5 gamma 2 chain expression was found to increase progressively towards a more atypical phenotype of adenoma. The results suggest that, in the future, laminin 5 gamma 2 chain expression may be used as an indicator of incipient malignant transformation of a benign colorectal adenoma.
AIMS:To study the expression of nuclear beta catenin in patients with colorectal cancer, colorectal adenoma, and colorectal polyps to elucidate its role in carcinogenesis, and its potential for prognosis and diagnosis. METHODS:The expression of nuclear beta catenin was studied by immunohistochemistry using paraffin wax embedded specimens. Sixty specimens each of colorectal carcinoma, colorectal adenoma, colorectal polyp, and normal colorectal specimens were analysed. The potential for prognosis was assessed by correlating nuclear beta catenin expression in 60 and 75 patients with colorectal cancer with lymph node metastasis and survival, respectively. The diagnostic capacity was explored by comparing nuclear beta catenin expression in 60 patients with colorectal cancer with other cytokeratin 20 (CK20) positive adenocarcinomas, namely: 30 colonic mucinous adenocarcinomas, 30 gastric adenocarcinomas, 27 pancreatic adenocarcinomas, and 12 ovarian mucinous adenocarcinomas. RESULTS:Nuclear beta catenin expression was highly associated with progression of colorectal tissue from normal epithelial tissue, polyps, adenomas, to carcinomas (r = 0.875; p < 0.0001). Nineteen patients with colorectal adenoma who subsequently developed colorectal carcinoma had higher nuclear beta catenin expression than those with colorectal adenomas alone (p < 0.0001). Moreover, those patients with colorectal cancer and high nuclear beta catenin expression had a higher incidence of lymph node metastasis (chi(2) = 16.99; p < 0.005) and shorter overall survival (p < 0.0001). Finally, nuclear beta catenin expression in colorectal adenocarcinomas was significantly higher than in other CK20 positive adenocarcinomas. CONCLUSIONS:Nuclear beta catenin expression is a potential prognostic factor in patients with colorectal cancer, and together with CK20, it could be used to identify colorectal carcinoma in the Hong Kong population.
Aims: To compare the expression of the cell adhesion molecules P-cadherin, N-cadherin, and E-cadherin in invasive and in situ breast carcinomas relative to clinicopathological features (size, node status, type, grade, and receptors) to determine whether expression patterns relate to specific tumour characteristics. Methods: Using immunohistochemistry, 110 invasive and in situ breast carcinomas were examined for the presence, extent, and localisation of all three cadherins. Findings were related to tumour size, type, grade, node status, oestrogen (ER), progesterone, and epidermal growth factor receptor (EGFR) expression for invasive carcinomas and to grade and receptors for in situ carcinomas. Results: P-cadherin was detected in 40% of invasive carcinomas, N-cadherin in 30%, and E-cadherin in 81%. For invasive carcinomas, the presence of P-cadherin significantly correlated with high grade, lack of ER and presence of EGFR, but not tumour size or node status. Carcinomas containing P-cadherin could be put into three categories dependent upon receptor and E-cadherin profile. There were no correlations between E/N-cadherin and size, grade, node status, or receptors. Three of 16 infiltrating lobular carcinomas expressed cytoplasmic but none membranous E-cadherin, and P-cadherin and N-cadherin were present in four carcinomas of this type. E-cadherin was found in all ductal carcinomas in situ, P-cadherin in a proportion of high grade tumours, and N-cadherin in a mixture of grades. Conclusion: P-cadherin but not E/N-cadherin expression in breast carcinomas shows a strong correlation with higher grade (poorer differentiation), lack of ERs, and presence of EGFR, and its expression may aid in the further subdivision of high grade carcinomas.
Aims: Several studies have reported that dysregulation of beta catenin or k-ras mutation promotes cyclin D1 expression. This study investigated the relation between cyclin D1 expression and clinicopathological parameters in carcinoma of the ampulla of Vater (CAV), and also assessed the relation between increased cyclin D1 expression and beta catenin/k-ras status in this series.Methods: Thirty CAVs were evaluated for cyclin D1 expression by immunohistochemistry in relation to patient clinicopathological features. Aberrant beta catenin expression and k-ras mutation were also investigated by immunostaining and direct sequencing, and related to cyclin D1 expression.Results: Increased cyclin D1 expression was seen in 17 of 30 CAVs and was significantly correlated with tumour cell proliferation and disease free survival time (p = 0.018, p = 0.018, respectively). Nuclear accumulation of beta catenin was found in nine of 30 cases, including four cases with missense mutations in exon 3 of CTNNB-1, and was significantly correlated with increased cyclin D1 expression (p = 0.003). k-ras gene mutation was detected in 12 of 30 cases, and was also significantly correlated with increased cyclin D1 expression (p = 0.026). Overall, 14 of 17 CAVs with increased cyclin D1 expression showed nuclear accumulation of beta catenin and/or k-ras mutation.Conclusions: Increased cyclin D1 expression appears to be associated with tumour proliferation and poorer clinical outcome in CAV. It is also associated with both aberrant beta catenin expression and k-ras mutation. These results are consistent with the in vitro data that cyclin D1 can be transactivated by activated beta catenin-T cell factor/LEF and k-ras pathways.
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.
AIMS:To design and validate a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene of Mycoplasma genitalium.METHODS:Primers were designed that were complementary to the 16S rRNA gene sequence of M genitalium. After optimisation of the reaction conditions, the PCR was tested against nine M genitalium strains, a dilution series of M genitalium DNA, and a panel of common microorganisms. The PCR was also challenged in parallel with a published assay against 54 urine specimens from men with urethritis.RESULTS:The expected 341 bp product was produced on amplification of material from all M genitalium strains and from none of the other microorganisms tested. The lower limit of detection was 50 genome copies. The new assay detected M genitalium DNA in nine of 54 men with urethritis, in comparison with eight positive specimens detected with the alternative PCR.CONCLUSIONS:This novel PCR targeting the M genitalium 16S rRNA gene has been optimised and now provides a sensitive and specific alternative or addition to the available MgPa gene targeting assays.
Background: INI1 (hSNF5) mutations are linked to rhabdoid tumours, but mutations in meningiomas with hot spot mutations in position 377 have also been reported. Aims: To analyse the INI1 gene in meningioma. Methods: Exons 1, 4, 5, and 9 of the INI1 gene were analysed by the polymerase chain reaction and direct sequencing in 80 meningiomas. For all cases, western blotting of the INI1 protein was performed. Results: Only one of the 80 samples showed a cytosine insertion in codon 376. This mutation changed the open reading frame in almost the whole exon 9 and resulted in a longer hSNF5 protein. Complex analysis of the above described tumour sample by western blotting, DNA sequencing, and loss of heterozygosity (LOH) analysis showed that this particular meningioma consisted of heterogeneic cellular components. One of these components had a mutated INI1 gene, whereas in the other component INI1 was intact. Conclusions: INI1 mutation is a rare event in the molecular pathology of meningiomas. It is possible for the INI1 gene to be mutated in only a proportion of meningioma cells.
Aims: Women under 35 years of age comprise a small proportion of patients with breast cancer, but determining their prognosis can be difficult. This prospective, multivariate study looked at several factors with the aim of obtaining a useful index to evaluate the prognosis of these women. Methods: In total, 108 patients below 35 years of age affected by invasive ductal carcinoma without distant metastasis were studied. The mean duration of the follow up period was six years. Histopathological (tumour size, histological grade, and lymph node stage) and immunohistochemical (c-erbB-2, p53, oestrogen receptor, and progesterone receptor) factors were measured in all patients, and the Nottingham prognostic index (NPI) was then calculated. An immunohistochemical prognostic index (IHPI) was created using the arithmetic sum of the four individual immunohistochemical factors. Results: In univariate assessment of survival, all the studied factors yielded a significant association with either overall survival or disease free survival, except for c-erbB-2 and p53 with disease free survival. In univariate calculation of risk, all the factors gave significant results; however, in multivariate analysis only tumour size, histological grade, and progesterone receptor were significant. Both NPI and IHPI correlated significantly with prognosis. In multivariate regression analysis, IHPI correlated with tumour size and there was a significant interaction between both variables. Conclusion: IHPI is very useful in determining the prognosis of tumours ⩽ 2 cm and of moderate use for tumours > 2, although it has no use in tumours > 5 cm.
Cervical cancer is the second most common cancer in the female population worldwide, and human papillomavirus (HPV) DNA has been isolated from more than 90% of these carcinomas. Immunoregulatory/antitumour mechanisms include cytokines that interfere directly with HPV harbouring cells. Among these cytokines, tumour necrosis factor α (TNFα) is released by HPV infected cells and inhibits the growth of transformed cell lines.1 TNFα is a proinflammatory/antitumour cytokine that is indispensable to the inflammatory response. The TNF locus contains several polymorphic areas, including five microsatellite markers—a, b, c, d, and e—which contain 14, 7, 2, 7, and 3 alleles, respectively. These microsatellites are associated with different degrees of TNFα secretion, and are related to a greater susceptibility to …
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.
Apolipoprotein E (APOE) is thought to be responsible for the transportation of lipids within the brain, maintaining structural integrity of the microtubule within the neurone, and assisting with neural transmission. Possession of the APOE epsilon4 allele has also been shown to influence neuropathological findings in patients who die from traumatic brain injury, including the accumulation of amyloid beta protein. Previous clinical studies reporting varying outcome severities of traumatic brain injury, including cognitive and functional recovery, all support the notion that APOE epsilon4 allele possession is associated with an unfavourable outcome. Evidence from experimental and clinical brain injury studies confirms that APOE plays an important role in the response of the brain to injury.
BACKGROUND/AIM:It has been reported previously in cases of adenosquamous carcinoma of the lung in Okinawa, a subtropical island 2000 km south of mainland Japan, that the squamous cell carcinoma components were positive for human papillomavirus (HPV) by non-isotopic in situ hybridisation (NISH). The adenocarcinoma cells adjacent to the squamous cell carcinoma components were enlarged and also positive for HPV. This is thought to indicate that after adenocarcinoma cells are infected with HPV, they undergo morphological changes, and that "squamous metaplasia" follows. In this present study, the effects of HPV transfection into adenocarcinoma cells were examined. The relation between the region expressing the HPV gene and squamous metaplasia was also studied.METHODS:Plasmid pBR322 containing HPV type 16 (HPV-16) was transfected into cultured colonic adenocarcinoma (DLD-1) and lung adenocarcinoma (PC-14) cells using the calcium phosphate method. Neomycin was used as a selection marker. The presence of HPV E1, E2, E4, E5, E6, E7, L1, and L2 mRNAs and also transglutaminase 1, involucrin, cyclin dependent kinases (CDKs), cyclins, caspases, apoptosis inducing factor, DNase gamma, Fas, and Fas ligand mRNAs in HPV transfected cells was investigated by means of reverse transcription polymerase chain reaction (RT-PCR). The G0-G1 cell population was analysed by flow cytometry. Morphological examination under light and electron microscopes was also carried out.RESULTS:The virus transfected cells showed squamous metaplasia when they were injected into severe combined immunodeficient mice, expressing the high molecular weight keratin (Moll's number 1 keratin) and involucrin molecules immunohistochemically, and involucrin and transglutaminase I mRNAs by RT-PCR. The squamous metaplasia was most conspicuous in the HPV transfected DLD-1 cell when compared with HPV transfected PC-14 cells. Squamous metaplasia was most clearly demonstrated in one HPV transfected DLD-1 cell clone, which expressed not only E2 but also E6-E7 fusion gene mRNA. Viral L1 mRNA expression was absent in HPV transfected cell clones, and was not related to squamous metaplasia. The growth rate of HPV transfected cells was reduced. Transfection of the virus into the cultured adenocarcinoma cells increased the G0-G1 cell population greatly, as assessed by flow cytometer analysis. Furthermore, in the virus transfected cells, apoptosis was also observed by means of the terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling method.CONCLUSION:HPV transfection into adenocarcinoma cells induced clear squamous metaplasia. One of the HPV transfected cell clones that expressed E2 and E6-E7 fusion gene mRNA showed the squamous metaplasia particularly clearly, and apoptosis was also demonstrated.
AIMS:Monitoring treated patients with thyroid cancer for recurrent or metastatic disease is currently based upon the serial measurement of circulating plasma thyroglobulin (Tg) concentrations. However, the clinical usefulness of Tg immunoassays is limited by poor sensitivity and interference from anti-Tg antibodies. This study investigated whether the detection of Tg mRNA in peripheral blood, using reverse transcriptase polymerase chain reaction (RT-PCR), is of value in the biochemical surveillance of patients with thyroid cancer.METHODS:RNA was extracted from peripheral blood of five normal controls, six patients with abnormal thyroid function tests, and 28 patients who had undergone thyroidectomy for well differentiated thyroid cancer. From each, an 87 bp product from base pair 262 to 348 in the cDNA sequence of the thyroglobulin gene was amplified by RT-PCR.RESULTS:Tg mRNA was detected in normal individuals and patients with thyroid cancer. In the group of patients studied, identification of metastatic thyroid tissue by radioiodine scanning correlated better with Tg mRNA assay results than with serum Tg concentrations (accuracy 84% v 75%). No interference from circulating Tg antibodies was apparent. In patients studied prospectively over a 12 month period, there was a significant correlation between detectable Tg mRNA in peripheral blood and the presence or absence of metastatic disease, as demonstrated by radioiodine scanning.CONCLUSIONS:These results suggest that detection of Tg mRNA in blood is a more sensitive marker for metastatic thyroid disease than Tg immunoassay, and appears to be unaffected by the presence of circulating anti-Tg antibodies.
segment polarity genes that regulate segmental and imaginal disc patterning in the fruit fly, Drosophila melanogaster. Unlike drosophila and other invertebrates, which only have a single hh gene, vertebrates have a family of genes that are homologous to the hh gene. Mammals have three genes with homology to the hh gene. These comprise Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). All hedgehog genes encode signalling molecules that are involved in short and long range patterning processes during embryogenesis. Like all hedgehog proteins Shh protein also undergoes molecular processing in the endoplasmic reticulum. This involves cleavage of its signal peptide, followed by autocatalytic cleavage of the hedgehog protein precursor into a 19 kDa N-terminal domain (Shh-N) and a 25 kDa C-terminal domain (Shh-C). The signalling activity of hedgehog proteins resides in Shh-N. Shh-C has intramolecular cholesterol transferase activity and is responsible for covalently attaching a cholesterol molecule to the C-terminal end of Shh-N. The addition of cholesterol plays an important role in spatially restricting the zone of activity of Shh-N by anchoring it to the cell membrane and restricting its diffusion from the site of secretion. It is believed that inborn errors of cholesterol synthesis such as Smith–Lemli–Opitz syndrome (microcephaly, growth and mental retardation, facial dysmorphism, syndactyly of the second and third toes, congenital heart disease, hypotonia, and genital abnormalities in males) can interfere with SHH signalling by interfering with its molecular processing, in particular with the cholesterol modification of Shh-N.
Background/Aims: The p73 gene encodes a protein that shares structural and functional homology with the p53 gene product. The highest degree of homology is in the DNA binding domain, which is the region of p53 that is most frequently mutated in cancer. In contrast to p53 there is little evidence that p73 acts as a classic tumour suppressor gene. Because of the similarities between the p53 and p73 genes and the high frequency of mutation of p53, this study was designed to investigate the p73 gene in patients with gastric adenocarcinoma.Methods: The mutational status of the p73 gene was investigated in a series of 13 cases of gastric adenocarcinoma from the antro-pyloric region and the gastro-oesophageal junction, using the polymerase chain reaction, single strand conformational polymorphism, and direct DNA sequencing.Results: A glutamine to arginine mutation was detected in exon 5 of the p73 gene in a case of adenocarcinoma at the gastro-oesophageal junction.Conclusion: Although limited to a small series of cases, these results suggest that p73 may have a potential pathogenetic role in this tumour.
