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1.  Development of molecular methods for the identification of aspergillus and emerging moulds in paraffin wax embedded tissue sections 
Molecular Pathology  2003;56(6):368-370.
Background/Aims: Invasive infection with emerging moulds is increasing in incidence and reliable methods for speciating these organisms in tissue sections need to be developed.
Methods: Two methods for extracting fungal DNA from paraffin wax embedded tissue sections, based on the TaKaRa DEXPAT™ kit and QIAamp® DNA mini kit, were optimised and compared. DNA was amplified by PCR using pan-fungal probes, and detected by Southern blot hybridisation using a high stringency method with a probe specific for Aspergillus fumigatus and A flavus.
Results: The method based on the TaKaRa DEXPAT kit, with additional steps using lyticase and ethanol precipitation, was superior. Less than 10 conidia were detectable using spiked samples and a positive result was obtained with 100% of clinical samples known to be culture positive for A fumigatus. Other moulds could be identified by using species specific probes or by sequencing PCR products.
Conclusions: The method based on the TaKaRa DEXPAT kit could detect less than 10 conidia/sample. The method allowed accurate identification of A fumigatus and A flavus and other species could be identified using species specific probes or by DNA sequencing. These methods will provide a valuable diagnostic tool for both patient management and future antifungal and epidemiological studies.
PMCID: PMC1187358  PMID: 14645701
aspergillus; tissue; DNA extraction; polymerase chain reaction
2.  Tempero–spatial dissociation between the expression of Fas and apoptosis after coronary occlusion 
Molecular Pathology  2003;56(6):362-367.
Aims: To explore the role of Fas in cardiomyocytic apoptosis induced by ischaemia through determining the histological relation between Fas expression and apoptosis in rat myocardium during ischaemia/infarction.
Methods: The myocardial ischaemia model was produced by ligating the left coronary artery in Sprague-Dawley rats. The rats were killed from 10 minutes to seven days after surgery. Apoptotic myocardial cells were detected by the in situ terminal deoxynucleotidyl transferase mediated nick end labelling method, and the expression of Fas by immunohistochemistry and western blotting.
Results: Cardiomyocytic apoptosis appeared from three to 36 hours after ischaemia. The expression of Fas could be detected by western blot from before surgery to seven days of ischaemia. Apoptosis and the expression of Fas in the cardiomyocytes appeared in different regions of the myocardium: apoptosis in the ischaemic region, Fas in the regions surrounding ischaemic myocardium.
Conclusion: These results suggest that there is a tempero–spatial dissociation between the expression of Fas and apoptosis after coronary occlusion. Fas might not directly regulate the apoptosis of cardiomyocytes induced by ischaemia.
PMCID: PMC1187357  PMID: 14645700
apoptosis; Fas; myocytes; heart; ischaemia; infarction
3.  Inverse correlation between high level expression of cyclin E and proliferation index in transitional cell carcinoma of the bladder 
Molecular Pathology  2003;56(6):353-361.
Background/Aims: Overexpression of the G1 cyclins, D1 and E, and/or downregulation of p27Kip1 allow uncontrolled tumour cell proliferation. This study investigated the relation between these three cell cycle proteins and tumour proliferation in bladder cancer.
Method: Nuclear expression of cyclin D1, cyclin E, and p27Kip1 was determined immunohistochemically in 52 primary transitional cell carcinomas, and the Ki-67 proliferation marker was also assessed. For each protein, the percentage of positive tumour cell nuclei was determined and analysed as a continuous variable.
Results: Advancing tumour grade and pathological stage were accompanied by increasing proliferation indices, but decreasing p27Kip1 and cyclin D1 expression, with no significant change in cyclin E expression. Overall, cyclin D1 and E expression did not correlate with proliferation. However, in cyclin D1 overexpressing tumours (⩾ 5% nuclei positive), the level of cyclin D1 expression positively correlated with proliferation. The correlation between cyclin E expression and proliferation changed from positive to negative with increasing levels of cyclin E expression, accompanied by a coordinate increase in p27Kip1 expression. Overall, there was an inverse association between p27Kip1 expression and proliferation. However, a subset of tumours displayed high proliferation indices despite high p27Kip1 expression. The G1 cyclin index (sum of the level of expression of cyclins D1 and E) correlated positively with proliferation in superficial but not muscle invasive tumours. This correlation was stronger when the G1 cyclin index was adjusted for p27Kip1 expression.
Conclusion: These findings support a role for these proteins in the proliferation, differentiation, and progression of bladder transitional cell carcinomas.
PMCID: PMC1187355  PMID: 14645699
cell cycle; bladder cancer; cyclin; proliferation
4.  Nuclear β catenin as a potential prognostic and diagnostic marker in patients with colorectal cancer from Hong Kong 
Molecular Pathology  2003;56(6):347-352.
Aims: To study the expression of nuclear β 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 β 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 β 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 β 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 β 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 β catenin expression than those with colorectal adenomas alone (p < 0.0001). Moreover, those patients with colorectal cancer and high nuclear β catenin expression had a higher incidence of lymph node metastasis (χ2  =  16.99; p < 0.005) and shorter overall survival (p < 0.0001). Finally, nuclear β catenin expression in colorectal adenocarcinomas was significantly higher than in other CK20 positive adenocarcinomas.
Conclusions: Nuclear β 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.
PMCID: PMC1187354  PMID: 14645698
colorectal cancer; β catenin; nuclear translocation; cytokeratin 20; adenocarcinomas
5.  Laminin 5 γ2 chain expression: a marker of early invasiveness in colorectal adenomas 
Molecular Pathology  2003;56(6):342-346.
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 γ2 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 γ2 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 γ2 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 γ2 chain expression. Furthermore, laminin 5 γ2 chain expression correlated with lesion size. Polyps smaller than 10 mm expressed the γ2 chain less frequently than did those equal to or larger than 10 mm.
Conclusion: Laminin 5 γ2 chain expression was found to increase progressively towards a more atypical phenotype of adenoma. The results suggest that, in the future, laminin 5 γ2 chain expression may be used as an indicator of incipient malignant transformation of a benign colorectal adenoma.
PMCID: PMC1187353  PMID: 14645697
colorectal polyps; laminin-5; serrated adenoma
6.  Increased cyclin D1 expression in cancer of the ampulla of Vater: relevance to nuclear β catenin accumulation and k-ras gene mutation 
Molecular Pathology  2003;56(6):336-341.
Aims: Several studies have reported that dysregulation of β 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 β 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 β 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 β 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 β 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 β catenin expression and k-ras mutation. These results are consistent with the in vitro data that cyclin D1 can be transactivated by activated β catenin–T cell factor/LEF and k-ras pathways.
PMCID: PMC1187352  PMID: 14645696
carcinoma of ampulla of Vater; cyclin D1; β catenin; k-ras; prognosis
7.  Expression of the cell cycle regulatory proteins p34cdc2, p21waf1, and p53 in node negative invasive ductal breast carcinoma 
Molecular Pathology  2003;56(6):328-335.
Aims: To look for correlations between expression of cell cycle regulatory proteins p34cdc2, p21WAF1, and p53 in node negative invasive ductal breast carcinoma, or between these proteins and clinicopathological parameters, and to assess their prognostic value.
Methods: Immunohistochemistry using formalin fixed, paraffin wax embedded sections from 94 breast carcinomas. Adjacent benign epithelial breast tissue was available in 74 cases. Median follow up was 72 months.
Results: Nuclear and cytoplasmic p34cdc2 expression was seen in 80 and 62 tumours, respectively; nuclear expression was seen in adjacent benign epithelium in 12 cases. p21WAF1 and p53 were positive in 48 and 21 tumours, respectively. High expression of p34cdc2 in neoplastic nuclei was associated with higher histological grade and p53 expression, but not with tumour size, steroid receptor status, patient age, menopausal status, recurrence, metastasis, disease free survival (DFS), or overall survival (OS). p34cdc2 in tumour cytoplasm was associated with p34cdc2 nuclear positivity, high tumour grade, and DFS in univariate but not multivariate analysis. In contrast, p34cdc2 expression in benign tissue independently predicted DFS and OS in univariate and multivariate analysis. Expression of p53 was associated with high tumour grade and negative steroid receptors, but not with recurrence, metastasis, DFS, or OS. p21WAF1 expression was not associated with the examined parameters.
Conclusions: p34cdc2, p21WAF1, and p53 expression does not predict outcome in node negative breast carcinoma, although p34cdc2 expression in benign tissue is related to prognosis. The association between p34cdc2 and p53 implicates p53 in G2–M cell cycle checkpoint control, possibly via mediators unrelated to p21WAF1.
PMCID: PMC1187351  PMID: 14645695
breast carcinoma; p34cdc2; p21WAF1; p53; prognosis
8.  Immunohistochemical prognostic index for breast cancer in young women 
Molecular Pathology  2003;56(6):323-327.
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.
PMCID: PMC1187350  PMID: 14645694
young women; breast cancer; immunohistochemical prognostic index; c-erbB-2; p53; oestrogen receptor; progesterone receptor; Nottingham prognostic index
9.  Expression of P-cadherin, but not E-cadherin or N-cadherin, relates to pathological and functional differentiation of breast carcinomas 
Molecular Pathology  2003;56(6):318-322.
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.
PMCID: PMC1187349  PMID: 14645693
breast cancer; P-cadherin; immunohistochemistry
10.  Cell cycle regulation in patients with intestinal metaplasia at the gastro–oesophageal junction 
Molecular Pathology  2003;56(6):313-317.
Background/Aims: The incidence of oesophageal adenocarcinoma is increasing rapidly and this may be related to the presence of intestinal metaplasia (IM) at the gastro–oesophageal junction (GOJ). Recent studies have distinguished two subtypes of IM at the GOJ: short segment Barrett’s oesophagus (SSBO) and IM at a normal squamo–columnar junction (IMNSCJ). Because abnormal expression of cell cycle regulators is common in cancer and precancerous states, cell cycle regulation was studied in patients with IM at the GOJ.
Methods: Biopsy samples and resected materials were identified from patients with SSBO (10), IMNSCJ (14), a normal SCJ with (14) and without (12) inflammation, conventional Barrett’s oesophagus (BO) (12), and oesophageal adenocarcinoma (12). Sections were stained with antibodies to p21, p27, p53, Ki67, cyclin D1, and c-erbB2 and were assessed independently by two observers, using predetermined criteria.
Results: Patients with oesophageal adenocarcinoma showed high expression of c-erbB2, p53, p27, and Ki67. Patients with BO showed expression of c-erbB2 but little expression of other markers. Greatly increased expression of cyclin D1 was seen in patients with IMNSCJ. The expression of all other markers was similar in patients with IMNSCJ and those with SSBO. Cyclin D1 and c-erbB-2 were coexpressed in patients with SSBO and IMNSCJ, and their expression was associated with the presence of p53 and p21.
Conclusions: Although the proposed aetiologies of SSBO (gastro–oesophageal reflux) and IMNSCJ (Helicobacter pylori infection) differ, the cell cycle response is similar and both may have malignant potential.
PMCID: PMC1187347  PMID: 14645692
intestinal metaplasia; Barrett’s oesophagus; cell cycle regulation; p53; cyclin D1; c-erbB-2
13.  Validation of cDNA microarray gene expression data obtained from linearly amplified RNA 
Molecular Pathology  2003;56(6):307-312.
Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail.
Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR).
Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template.
Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.
PMCID: PMC1187346  PMID: 14645691
amplified RNA; in vitro transcription; microarray; expression profiling; real time PCR
14.  The type and quality of paraffin wax is important when constructing tissue microarrays 
Molecular Pathology  2003;56(5):306.
PMCID: PMC1187345  PMID: 14514928
tissue microarrays; paraffin wax; quality of wax
15.  Tumour necrosis factor microsatellite association with human papillomavirus cervical infection 
Molecular Pathology  2003;56(5):305-306.
PMCID: PMC1187344  PMID: 14514927
cervical scrapings; cytokines; human papillomavirus; microsatellites; tumour necrosis factor; polymerase chain reaction
16.  Molecular heterogeneity of meningioma with INI1 mutation 
Molecular Pathology  2003;56(5):299-301.
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.
PMCID: PMC1187342  PMID: 14514925
loss of heterozygosity; meningioma; molecular heterogeneity; INI1; hSNF5
17.  Genome wide array comparative genomic hybridisation analysis of premalignant lesions of the stomach 
Molecular Pathology  2003;56(5):293-298.
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.
PMCID: PMC1187341  PMID: 14514924
gastric polyps; adenomas; hyperplastic polyps; chromosomal changes; gastric cancer
18.  Alterations in p53 predict response to preoperative high dose chemotherapy in patients with gastric cancer 
Molecular Pathology  2003;56(5):286-292.
Aims: To evaluate the usefulness of molecular markers in predicting histopathological and clinical response to preoperative high dose chemotherapy (HDCT) and survival of patients with advanced gastric cancer.
Methods: In a phase II trial, 25 patients with metastatic gastric cancer received preoperative tandem HDCT consisting of etoposide, cisplatin, and mitomycin, followed by autologous bone marrow transplantation to achieve surgical resectability. Samples before and after treatment, from normal and tumour tissue, were characterised histopathologically, and both p53 and BAX expression was analysed by immunohistochemistry. Pretreatment formalin fixed, paraffin wax embedded samples from normal and tumour tissue were microdissected, and the extracted DNA was preamplified using improved primer extension preamplification polymerase chain reaction. Detection of microsatellite instability (MSI) or loss of heterozygosity (LOH) was performed using markers for p53, BAX, BAT25, BAT26, D2S123, D17S250, and APC. Exons 5–9 of the p53 gene were sequenced directly on ABI 373.
Results: Four parameters were significantly associated with response to chemotherapy and prolonged overall survival: positive p53 immunostaining, positive p53 mutation status before chemotherapy, strong histological regression induced by preoperative HDCT, and surgical treatment. Patients’s sex or age, tumour location or stage, lymph node status, Lauren classification, MSI, or LOH did not influence duration of survival significantly in this high risk population.
Conclusion: Positive p53 immunostaining and p53 mutation status in pretreatment tumour biopsies might be useful molecular predictors of response and prognosis in patients with advanced gastric cancer treated by preoperative HDCT.
PMCID: PMC1187340  PMID: 14514923
gastric cancer; preoperative high dose chemotherapy; molecular parameters; histological regression; p53
19.  A simple and reliable pretreatment protocol facilitates fluorescent in situ hybridisation on tissue microarrays of paraffin wax embedded tumour samples 
Molecular Pathology  2003;56(5):275-279.
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°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°C for 10 minutes, followed by hybridisation at 37°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.
PMCID: PMC1187338  PMID: 14514921
fluorescent in situ hybridisation; paraffin wax embedded tissue; tissue microarray; pretreatment; citric acid buffer
20.  Expression of cysteine dioxygenase (EC 1.13.11.20) and sulfite oxidase in the human lung: a potential role for sulfate production in the protection from airborne xenobiotica 
Molecular Pathology  2003;56(5):270-274.
Aims: The lung is one of the major sites of phase I cytochrome P450 enzyme and phase II sulfotransferase expression, which together are thought to act as an enzymic barrier against the unimpeded transfer of airborne xenobiotics into the lung parenchyma and systemic circulation. Sulfate for conjugation is produced primarily from the oxidation of cysteine, begun by cysteine dioxygenase (CDO), and completed with the conversion of sulfite to sulfate via sulfite oxidase (SO). Little is known about the site of expression of these two enzymes in the alveoli of the human lung.
Methods: Antibodies and oligonucleotide probes raised against both CDO and SO were used for immunohistochemistry and in situ hybridisation, respectively, to investigate the expression of CDO and SO in human lung alveoli.
Results: CDO and SO were expressed in alveolar epithelial cells, which is also the site of expression of cytochrome P450 1B1.
Conclusions: These results demonstrate that the two key enzymes in sulfate production are expressed in the same locale as phase I and phase II enzymes, and that these two enzymes may be involved in the production of sulfate for the maintenance of a metabolic barrier against the entry of airborne xenobiotics and the synthesis of important structural proteins and proteoglycans.
PMCID: PMC1187337  PMID: 14514920
cysteine dioxygenase; cytochrome P450; lung; alveoli; sulfation; sulfite oxidase
21.  Differential deletions of chromosome 3p are associated with the development of uterine cervical carcinoma in Indian patients 
Molecular Pathology  2003;56(5):263-269.
Background: Deletions in chromosome 3 occur frequently in uterine cervical carcinoma (CA-CX). The common consensus regions deleted during CA-CX development are not well defined, and have not been correlated with tumour progression.
Aims: To define specific regions of chromosome 3 deleted during development of CA-CX and to correlate these with clinicopathological data.
Methods: Deletion mapping of chromosome 3 was done in seven cervical intraepithelial neoplasia (CIN) and 43 primary CA-CX samples using 20 highly polymorphic microsatellite markers.
Results: Deletions of chromosome 3 were significantly associated with tumour progression. High frequencies (33–53%) of loss of heterozygosity (LOH) were found in 3p26.1, 3p22.3, 3p21.2, and 3p13, suggesting the location of putative tumour suppressor genes (TSGs) in these regions. Among these four regions, deletions in 3p21.2 were suggested to occur early during CA-CX development. A significant correlation was found between LOH at 3p26.1 and 3p22.3 with tumour progression from stage I/IIB to stage III/IV. No association was found with the highly deleted regions and human papillomavirus positivity, parity, or menopausal status. Microsatellite size alteration was seen in only seven of the samples. However, rare biallelic alterations were seen in and around the highly deleted regions. Loss of normal copy of chromosome 3 and interstitial alterations in chromosome 3p were seen in some samples.
Conclusion: These four regions on chromosome 3p may be differentially deleted during specific stages of CA-CX development. The putative TSGs located in these regions may have a cumulative effect on tumour progression.
PMCID: PMC1187336  PMID: 14514919
squamous cell carcinoma of the uterine cervix; chromosome 3 alterations; tumour suppressor gene(s); human papilloma virus infection
22.  Immunological and genetic analysis of 65 patients with a clinical suspicion of X linked hyper-IgM 
Molecular Pathology  2003;56(5):256-262.
Background: X linked hyper-IgM (XHIM) is a primary immunodeficiency caused by mutations in the tumour necrosis factor superfamily 5 gene, TNFSF5, also known as the CD40 ligand (CD40L) gene. Patients often present with recurrent infections, and confirmation of a diagnosis of XHIM enables appropriate therapeutic interventions, including replacement immunoglobulin, antibiotics, and bone marrow transplantation.
Aim: To review and optimise the institution’s diagnostic strategy for XHIM.
Method: Samples from 65 boys were referred to this centre for further investigation of suspected XHIM. The results, which included a flow cytometric whole blood assay for CD40L expression followed by mutation analysis in selected patients, were reviewed.
Results: Twenty one patients failed to express CD40L and TNFSF5 mutations were found in 20 of these patients. In contrast, no TNFSF5 mutations were found in 16 patients with weak expression of CD40L. Interestingly, one quarter of patients with confirmed XHIM who had TNFSF5 mutations had low concentrations of IgG, IgA, and IgM. Most of the remaining patients with XHIM had the classic pattern of normal or raised IgM with low concentrations of IgA and IgG.
Conclusions: This study demonstrates the usefulness of the whole blood staining method as a rapid screen to select patients for subsequent TNFSF5 mutation analysis, and shows the benefits of a unified protein/genetic diagnostic strategy.
PMCID: PMC1187335  PMID: 14514918
CD40 ligand; X linked hyper-IgM; immunodeficiency; diagnostic strategy; TNFSF5
23.  Receptor revision of immunoglobulin heavy chain genes in human MALT lymphomas 
Molecular Pathology  2003;56(5):249-255.
Background/Aims: Rearrangement of immunoglobulin gene segments, leading to B cells with functional receptors, is thought to be largely restricted to developing immature B cells in bone marrow. However, accumulating evidence suggests that mature B cells occasionally modify their antigen specificity by VH segment replacements during the germinal centre reaction to enhance antigen affinity, or to overcome self reactive antigen receptors. Although malignant B cells maintain the features of their normal counterparts in most instances, to date, such replacements have not been described for human B cell lymphomas.
Methods: Rearranged immunoglobulin heavy chain genes from two extranodal marginal zone B cell lymphomas were amplified, cloned, and sequenced. Sequences with identical CDR3 regions were selected and aligned to each other and public databases.
Results: VH replacements were seen in two extranodal marginal zone B cell lymphomas. In line with the hypothesis that in mature B cells these replacements are associated with active somatic hypermutation, in addition to VH replacement, different mutation patterns were seen in the revised VH portions. In the remaining common 3′-VH regions, these mutations could be used to establish a phylogenetic relation between the sequences, rendering the possibility of artefactual chimaeric polymerase chain reaction products very unlikely.
Conclusions: These results support the view that VH replacements are a further mechanism for reshaping antigen affinity and specificity, and indicate that these receptor modifications are not restricted to normal and reactive germinal centre B cells, but may also occur in close association with the development of malignant B cell lymphomas.
PMCID: PMC1187334  PMID: 14514917
B lymphocytes; VH replacement; immunoglobulin gene rearrangement; antibodies
24.  Wnt ligand expression in malignant melanoma: pilot study indicating correlation with histopathological features 
Molecular Pathology  2003;56(5):280-285.
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.
PMCID: PMC1187339  PMID: 14514922
Wnt; Frizzled; melanoma; naevi; signal transduction; in situ hybridisation
25.  4193delC, a common mutation causing Wilson’s disease in Saudi Arabia: rapid molecular screening of patients and carriers 
Molecular Pathology  2003;56(5):302-304.
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.
PMCID: PMC1187343  PMID: 14514926
Wilson’s disease; ATP7B gene; copper transporting ATPase; amplification refractory mutation system; mutation; Saudi tribes

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