An insight into the genetic pathway of adenocarcinoma of the small intestine.

BACKGROUND: Although the adenoma to carcinoma pathway in colorectal cancer is well described, the mechanisms of carcinogenesis in the small intestine remain unclear. AIMS: The aim of this study was to investigate candidate genes in the genetic pathway of adenocarcinoma of the small intestine. SUBJECTS AND METHODS: A total of 21 non-familial, non-ampullary adenocarcinomas of the small intestine were analysed. DNA was extracted from formalin fixed paraffin wax embedded tissue using standard techniques. The replication error (RER) status was determined by amplification of BAT26. The mutation cluster region (MCR) of the adenomatous polyposis coli (APC) gene was screened using polymerase chain reaction single strand conformational polymorphism and direct sequencing. Immunohistochemistry was performed on formalin fixed paraffin wax embedded tissue using monoclonal antibodies for hMLH1, hMSH2, beta-catenin, E-cadherin, and p53. RESULTS: Fourteen male and seven female patients with a median age of 64 years (range 21-85) presented with adenocarcinoma of the duodenum (10), jejunum (7), and ileum (4). One cancer (5%) was found to be RER+, and all tumours stained positive for hMLH1 and hMSH2. No mutations were detected in the MCR of the APC gene. beta-Catenin showed increased nuclear expression with loss of membranous staining in 10 cancers (48%). Absent or decreased membrane expression of E-cadherin was found in eight cancers (38%). Strong staining of p53 was found in the nucleus of five cancers (24%). CONCLUSION: We did not detect mutations in the MCR of the APC gene, and this suggests that adenocarcinoma of the small intestine may follow a different genetic pathway to colorectal cancer. Abnormal expression of E-cadherin and beta-catenin was common and reflects an early alternative to APC in this pathway in which mutations may be found in adenocarcinoma of the small intestine.

Inhibin expression in normal and pre-eclamptic placental tissue.

Serum inhibin levels increase during normal pregnancy, but are significantly higher in patients with pre-eclampsia. The aim of this study was to demonstrate possible increased expression of inhibin within the placentas of women with pre-eclampsia compared with non-pre-eclamptic controls. Cellular expression of inhibin alpha and beta A subunits was studied using immunohistochemistry on formalin-fixed, paraffin-embedded placental sections from cases of pre-eclampsia (n = 23) and gestational age-matched non-pre-eclamptic controls (n = 16). Immunohistochemistry was performed using monoclonal antibodies against inhibin alpha and beta A subunits by the indirect immunoperoxidase technique. Intensity of staining was graded by a semiquantitative scoring method. Differences in distribution and intensity of staining between control and pre-eclamptic placentas were analyzed using a nonparametric Mann-Whitney U test. Staining for both inhibin alpha and beta A was predominantly confined to the cytoplasm of syncytiotrophoblast, with weak expression within intermediate trophoblast. The intensity of staining for inhibin alpha was significantly greater in the syncytiotrophoblast of pre-eclamptic patients (mean staining intensity controls = 0.97, disease = 1.87; p < 0.001). Inhibin beta A staining was generally stronger than for the alpha subunit, and was also significantly increased in pre-eclamptic patients compared with controls (mean controls = 1.72, disease 2.19; p < 0.05). This is the first evidence for increased placental inhibin presence in pre-eclampsia, suggesting increased inhibin production within the placenta, a finding that could account for increased serum inhibin levels in pre-eclampsia.

Proliferating cell nuclear antigen expression grossly over-estimates cellular proliferation in cardiac myxomas.

AIMS: Myxomas are thought to be slowly growing benign neoplasms. Presentation is often due to embolic phenomena, though rapid increase in size is sometimes seen. Such an increase may be due to proliferation of cellular components, an increase in matrix due to synthesis or oedema, haemorrhage into the lesion, or the addition of surface thrombus. Routine microscopy suggests a low proliferation rate. The aim of this study was to investigate cellular proliferation, and to assess its contribution to tumour growth. METHODS: The antibodies JC1, Ki67, PC10, and MIB1 were used to make an immunohistochemical assessment of proliferation in five cases of cardiac myxoma. RESULTS: A significant difference was seen between number and type of cells stained with PC10 and the other markers. Whilst PC10 stained the nuclei of most (60 - 95%) endothelial and stromal cells in all cases, the other markers stained far fewer cells (up to 5%). All markers stained varying numbers of lymphoid cells. CONCLUSIONS: Proliferation in cardiac myxomas is unlikely to be rapid. The widespread positivity for PC10 suggests that PCNA is not a reliable marker in such tissues. Clinical cases in which myxomas have grown rapidly are probably due to changes in intercellular matrix rather than cellular proliferation.

Prevalence of Alzheimer plaques in AIDS.

OBJECTIVES: Both genetic and environmental risk factors for Alzheimer's disease have been identified. The best established environmental risk factor, head trauma, is thought to act through the triggering of an inflammatory response. Another stimulus to an inflammatory response in the brain is AIDS. Whether there is an increased prevalence of beta/A4 amyloid deposits in the form of argyrophilic plaques in the brains of patients with AIDS has therefore been investigated. METHODS: The prevalence of argyrophilic amyloid plaques in the cerebral cortex of frontal and temporal lobes was compared in 97 cases of AIDS dying at ages 30-69 years with that in 125 age matched, non-HIV infected controls. RESULTS: In the control group, and in AIDS, the prevalence of plaques increased with age (p=0.005 and 0.048 respectively). There was a significantly greater prevalence of argyrophilic plaques in the AIDS group as a whole (29%) (p < 0.004) and in those in the fourth decade (18%) (p < 0.014) than in control subjects (13% and 0% respectively). CONCLUSION: There is a predisposition to argyrophilic plaque formation in the brain in AIDS. The findings support the view that a stimulus to an inflammatory response in the brain favours argyrophilic plaque formation. The clinical relevance of our findings is, as yet, unclear.

Apparent deficiency of mucosal vascular collagen type IV associated with angiodysplasia of the colon.

AIMS: To investigate the presence and distribution of vascular collagen type IV in colonic tissue in cases of angiodysplasia and age and sex matched controls. METHODS: Sections of colon from seven cases of colonic angiodysplasia and eight age and sex matched controls were examined for the presence of collagen type IV in vessels of the mucosa and submucosa. Immunohistochemical staining was performed on paraffin wax embedded sections, and the degree of vascular staining for each marker compared between mucosa and submucosa and between cases and controls. Staining for endothelial markers P-selection and factor VIII was used to control for non-specific differences in immunostaining. RESULTS: In both the angiodysplastic tissues and approximately half the control tissues, staining for collagen type IV was considerably weaker in vessels in the mucosa than in the submucosa. In angiodysplasia, ectatic vessels in the mucosa appeared to contain less collagen type IV than similarly sized vessels in the submucosa, and perforating vessels appeared in many cases to lose staining at the level of the muscularis mucosae. No differences were found in staining intensity for the control endothelial markers between cases and controls. CONCLUSIONS: The apparent relative deficiency of collagen type IV in the mucosal vessels in angiodysplasia may be related to their susceptibility to ectasia and haemorrhage. The finding of a similar deficiency in half of the control cases may reflect a population at risk of this relatively common condition.

Induction of myocardial heat shock protein 70 during cardiac surgery.

Animal experiments have shown that members of the heat shock protein (HSP) family have cytoprotective properties against ischaemia. In experimentally induced cardiac ischaemia, the induction of HSP70s correlates with reduced infarct size and enhanced myocardial function and endothelial recovery. Direct evidence that increased myocardial HSP70 expression result in cytoprotection during ischaemia has also been obtained using transgenic mice overexpressing either rat or human HSP72. This study examined the induction and expression of myocardial HSP70s after an obligatory period of ischaemia in patients during cardiac surgery. The level of HSP72/HSC73 protein in Tru-cut biopsies of the myocardium, taken before and after an acute ischaemic insult, was examined using a polyclonal antibody. The amount of HSP72 mRNA in the biopsies was also determined by reverse transcriptase polymerase chain reaction (RT-PCR) and correlated HSP72/HSC73 protein expression. In four patients subjected to brief alternating periods of normothermic ischaemia and reperfusion, the amount of myocardial HSP72/HSC73 protein was increased several fold after ischaemic insult. This was accompanied by increased expression of HSP72 mRNA. In contrast, the amounts of myocardial HSP72/HSC73 protein and HSP72 mRNA were unchanged in a patient subjected to a single prolonged period of hypothermic ischaemia. Given the proven myocardial protective properties of HSP72 in experimental models, it is postulated that the observed induction of HSP72 may have a similar function in man.

TAL-1 protein expression in vascular lesions.

The distribution of TAL-1 protein, an important vascular promoter in mice, has been examined immunohistochemically in a range of human vascular lesions and normal tissues. Formalin-fixed, paraffin-embedded vascular lesions including granulation tissue, haemangiomas, Kaposi's sarcomas, spindle cell haemangioendotheliomas, and angiosarcomas, were examined using a monoclonal antibody to recombinant TAL-1. Endothelial cells in all lesions gave positive immunostaining of variable intensity. Granulation tissue and spindle cell areas of the vascular tumours gave the strongest staining (nuclear and cytoplasmic). The better-differentiated endothelial cells within the tumours and resident well-formed vessels were less positive and some cells were in fact negative. The malignant endothelial cells in angiosarcomas showed less intense positive staining than KS cells. This study has shown TAL-1 protein expression in a range of reactive, benign, and malignant vascular lesions. Protein expression appears to be stronger in the spindle cell areas, perhaps reflecting greater expression in less-differentiated endothelial cells.

The immunoexpression of bcl-2 and p53 in Kaposi's sarcoma.

The aim of this study was to examine the immunohistochemical expression of p53 and bcl-2 in Kaposi's sarcoma and relate this with proliferation index (as measured by MIB-1 staining) and clinicopathological subtypes. Twenty formalin-fixed, paraffin-embedded cases of Kaposi's sarcoma were stained with commercially available antibodies to p53, bcl-2 and MIB-1, after pressure cooking antigen retrieval. All cases were strongly positive for bcl-2 with the majority containing more than 75% positive cells. In comparison, p53 expression was less striking. Eleven cases contained less than 24% (+1) of cells staining positively. Only two cases showed greater than 75% of positive cells, and both of these latter two lesions had metastasized. The MIB-1 staining in all cases of Kaposi's sarcoma was strongly positive, irrespective of clinicopathological type, in keeping with the highly proliferative nature of this lesion. Thus, we have demonstrated uniformly increased expression of bcl-2 protein in Kaposi's sarcoma irrespective of clinicopathological subtype and MIB-1 staining, while p53 expression is relatively less common, except in those cases which have metastasized. This may help identify those cases that will behave in a more aggressive manner. However, more cases need to be evaluated to verify this.

EBV latent membrane protein (LMP-1) and bcl-2 protein expression in Reed-Sternberg-like cells in post-transplant lymphoproliferative disorders.

An inconsistent association exists between EBV-LMP-1 and bcl-2 protein expression in Reed-Sternberg cells seen in Hodgkin's disease. In fact, many studies have concluded that there is no correlation between EBV-LMP and bcl-2 expression in Hodgkin's disease. We undertook an analysis of post-transplant lymphoproliferative disorders to explore the relationship between EBV-LMP and bcl-2 in Reed-Sternberg-like cells found in this condition, given the strong association between this disorder and EBV. Reed-Sternberg-like cells were found histologically in 11 of 28 cases of renal, heart and heart-lung post-transplant lymphoproliferative disorders. Formalin-fixed, paraffinembedded sections were stained with monoclonal antibodies to EBV-LMP-1 and bcl-2 proteins. Reed-Sternberg-like cells in all 11 cases co-expressed EBV-LMP and bcl-2. A similar relationship was noted with large, mononuclear cells and occasional small lymphoid cells. The staining pattern seen with both antibodies was of similar intensity and both displayed cytoplasmic Golgi accentuation. In the setting of post-transplant lymphoproliferative disorders. Reed-Sternberg-like cells exhibit strong co-expression of EBV-LMP-1 and bcl-2 proteins, supporting a positive correlation between them. This is in contrast to the findings in Hodgkin's disease. The reason for this discrepancy may be due to the iatrogenic immunosuppression and resultant severe EBV infection, together with other cellular events.

Immunohistochemical detection of p53 and Bcl-2 proteins in Hashimoto's thyroiditis and primary thyroid lymphomas.

AIMS: To investigate whether immunohistochemical staining using p53 and/or bcl-2 distinguishes between florid Hashimoto's thyroiditis and low grade mucosa associated lymphoid tissue (MALT) lymphoma of the thyroid. METHODS: Ten cases of Hashimoto's thyroiditis and eight of primary thyroid lymphoma were stained with monoclonal antibodies directed against p53 and bcl-2. RESULTS: In Hashimoto's thyroiditis most small lymphoid cells in mantle zones, within the thyroid parenchyma and in lymphoepithelial lesions expressed bcl-2 protein. Very occasional centroblasts in reactive germinal centres were positive for p53, but all other lymphoid cells from cases of Hashimoto's disease were negative for p53. In diffuse, low grade lymphomas bcl-2 protein was uniformly expressed by most tumour cells. However, low grade lymphomas with a follicular pattern did not express bcl-2. The diffuse, low grade lymphomas were negative for p53, while occasional larger cells in the follicular subtype were positive. Both high grade lymphomas were bcl-2 negative but strongly p53 positive. CONCLUSIONS: This study indicates that there is an inverse correlation between p53 and bcl-2 immunostaining in thyroid lymphomas (low grade lymphomas: bcl-2 positive, p53 negative; high grade lymphomas: bcl-2 negative, p53 positive). Furthermore, immunohistochemical staining for bcl-2 and p53 proteins does not distinguish florid Hashimoto's thyroiditis from diffuse, low grade thyroid lymphoma.

Heterogeneity of bcl-2 expression in MALT lymphoma.

Bcl-2 protein expression was studied in a series of 58 MALT lymphomas using a monoclonal antibody which recognises this protein in routinely processed paraffin embedded tissue. Thirty-three of 58 cases showed heterogeneity for bcl-2 expression, 18 of 58 cases were bcl-2 positive and 7 of 58 were bcl-2 negative. High grade and low grade MALT lymphomas showed different patterns of staining. All 21 low grade tumours were positive for bcl-2, though in seven cases only a proportion of the neoplastic cells expressed this protein. In the 37 high grade tumours the majority of the neoplastic cells were negative with seven cases showing no reactivity at all. These findings give further support to the theory that MALT lymphomas differ in pathogenesis to nodal lymphomas and suggest that the good prognosis of MALT lymphomas may partly be explained by the fact that they maintain a normal pattern of bcl-2 expression.