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.

Abnormal subcellular distribution of mature MUC2 and de novo MUC5AC mucins in adenomas of the rectum: immunohistochemical detection using non-VNTR antibodies to MUC2 and MUC5AC peptide.

Anti-mucin variable number tandem repeat (VNTR) antibodies have been used previously to demonstrate the de novo presence of MUC5AC and MUC6 mucin in colorectal adenomas and increased synthesis of MUC2, the major secreted mucin in normal colorectal mucosa. Here we examined secreted mucins in tubular, tubulovillous and villous adenomas of the rectum using non-VNTR antibodies designed to assess mature mucin. Mucin gene messenger RNAs were detected by in situ hybridization. The anti-MUC2 non-VNTR antibody in the goblet cells of adenomas revealed a staining pattern of increased cytoplasmic, Golgi and membrane staining with no change in goblet vesicle reactivity compared with normal controls. In addition, blank goblet cell vesicle immunostaining for MUC2 was found in the transitional mucosa adjacent to all types of adenoma. Although a trend to overexpression of MUC2 was observed with in situ hybridization this was not detected with immunohistology. De novo synthesis of MUC5AC, but not MUC5B or MUC6 mucin was seen in all adenomas and transitional mucosa using immunohistochemistry. There was no correlation of MUC2 or MUC5AC mucin with polyp size or the grade of dysplasia using the non-VNTR antibodies. This study demonstrates that anti-mucin non-VNTR antibodies reveal a different subcellular-localization in rectal adenomas compared with normal colorectal mucosa. Further, this pattern is in contrast to that reported for anti-mucin VNTR antibodies. Combined use of these reagents may benefit future assessment of these cancers.

Overexpression of H-Ryk in epithelial ovarian cancer: prognostic significance of receptor expression.

H-Ryk is an atypical receptor tyrosine kinase that is expressed in a differentiation-specific manner in epithelial tissues. We have previously shown by in situ hybridization and immunohistochemistry that H-Ryk is overexpressed in malignant ovarian tumors. In addition, we have demonstrated that overexpression of H-Ryk is transforming in vitro and in vivo. To evaluate whether expression of H-Ryk is a prognostic factor in epithelial ovarian cancer, we carried out a retrospective study of 88 primary malignant ovarian tumors (28 serous tumors, 11 mucinous tumors, 29 endometrioid tumors, 13 clear cell tumors, 3 malignant mixed Mullerian tumors, 1 mixed epithelial tumor, 1 primary peritoneal tumor, 1 undifferentiated tumor, and 1 transitional carcinoma) diagnosed between 1990 and 1993 using immunohistochemistry. On univariate analysis, overall survival decreased significantly with age (P = 0.01); in patients with International Federation of Gynecology and Obstetrics (FIGO) stage II (P = 0.008), FIGO stage III (P < 0.001), and FIGO stage IV (P < 0.001) disease; and in patients with residual disease (residual disease < or = 2 cm, P = 0.007; residual disease > 2 cm, P < 0.001) after surgery. In addition, overexpression of the H-Ryk receptor in malignant epithelium (P = 0.04) and blood vessel (P = 0.01) was associated with a significantly decreased overall survival. H-Ryk blood vessel overexpression (P = 0.03), residual disease > 2 cm (P = 0.006), and residual disease < or = 2 cm (P = 0.01) conferred a significantly shorter progression-free survival. No correlation was found between H-Ryk overexpression and age, histological subtype, degree of differentiation, FIGO stage, or residual disease. Overall, after adjustment for all of the prognostic factors by multivariate analysis (Cox proportional hazards model), residual disease was the most powerful prognostic indicator for overall survival (P < 0.001) and progression-free survival (P = 0.01) in this patient subset. This implies that H-Ryk acts cooperatively with other biological factors in the pathogenesis of ovarian cancer.

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.

Cellular localisation of HHV-8 in Castleman's disease: is there a link with lymph node vascularity?

AIMS: Human herpesvirus 8 (HHV-8) has been identified in multicentric Castleman's disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis. METHODS: Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as "low" or "high". RESULTS: Five multicentric Castleman's disease and two solitary Castleman's disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10-50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status. CONCLUSION: The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.

Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue.

BACKGROUND: The mutational mechanism responsible for cyst formation in polycystic kidney disease 1 gene (PKD1) remains controversial, with data indicating a two-hit mechanism, but also evidence of polycystin-1 expression in cystic tissue. METHODS: To investigate this apparent paradox, we analyzed polycystin-1 expression in cystic renal or liver tissue from 10 patients with truncating PKD1 mutations (including one early-onset case) and 2 patients with severe disease associated with contiguous deletions of TSC2 and PKD1, using monoclonal antibodies (mAbs) to both extreme N-(7e12) and C-terminal (PKS-A) regions of the protein. Truncation of the C-terminal epitope from the putative mutant proteins in each case allowed exclusive assessment of the nontruncated protein with PKS-A. RESULTS: In adult PKD1 tissue, the majority of cysts (approximately 80%) showed polycystin-1 expression, although staining was absent in a variable but significant minority (approximately 20%), in spite of the normal expression of marker proteins. Unlike adult PKD1, however, negative cysts were rarely found in infantile PKD1 or TSC2/PKD1 deletion cases. CONCLUSIONS: If a two-hit mutational mechanism is operational, these results suggest that the majority of somatic mutations in adult PKD1 are likely to be missense changes. The low level of polycystin-1-negative cysts in the three "early-onset" cases, however, suggests that a somatic PKD1 mutation may not always be required for cyst formation.

CD40 upregulation is independent of HHV-8 in the pathogenesis of Kaposi's sarcoma.

AIMS: Human herpesvirus 8 (HHV-8) is now acknowledged as the infective cofactor in the pathogenesis of Kaposi's sarcoma. The mode by which HHV-8 causes Kaposi's sarcoma is unresolved and it is probable that it acts in conjunction with other factors including cytokines, anti-apoptosis proteins, and cell surface receptors. CD40, a cell membrane receptor belonging to the tumour necrosis factor receptor super family, promotes B cell survival and is expressed constitutively on endothelial cells. It is upregulated on cytokine treatment and has been documented recently in Kaposi's sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposi's sarcoma correlated with HHV-8 status, using a unique set of HHV-8 positive and negative specimens. METHODS: Twenty one paraffin wax embedded samples of Kaposi's sarcoma were selected, of which 18 were screened for the presence of HHV-8 using both conventional solution phase and TaqMan polymerase chain reaction (PCR). CD40 immunohistochemistry was assessed using a biotinylated amplification system. Staining was scored semiquantitatively. RESULTS: The results indicated that the expression of CD40 is independent of viral status, being present in both HHV-8 positive and negative specimens. CONCLUSIONS: This suggests that HHV-8 promotes Kaposi's sarcoma cell survival following infection by mechanisms other than those involving CD40.

Immunohistochemical screening for oncogenic tyrosine kinase activation.

Tyrosine kinases causing the abnormal phosphorylation of intracellular proteins have been shown to contribute to oncogenic transformation in a number of human neoplasms. Immunohistological staining of routine biopsy sections for increased levels of phosphotyrosine may therefore provide a simple means of screening for tumours containing activated tyrosine kinases. In this study, monoclonal antibodies to phosphotyrosine were used to immunostain a cell line and tumour biopsies from lymphomas known to contain the activated anaplastic-lymphoma-kinase (ALK) tyrosine kinase. A range of normal and other neoplastic tissues were also immunostained for comparison. An anaplastic large cell lymphoma (ALCL) cell line carrying the (2;5) translocation, which creates the activated nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) tyrosine kinase, was strongly labelled. Routine tissue biopsies from five cases of ALK-positive ALCL were also strongly positive for phosphotyrosine. The characteristic granular cytoplasmic labelling pattern for phosphotyrosine observed in a B-cell lymphoma (expressing full length ALK kinase) was identical to that obtained using an ALK-specific antibody, thus confirming that labelling for phosphotyrosine in lymphoma cells reflects the presence of an activated kinase. When normal lymphoid tissues were stained, there was little or no labelling for phosphotyrosine, but stronger labelling was seen in other cells and tissues; for example, endothelial cells and some carcinoma samples. Whilst the strong labelling for phosphotyrosine observed in the lymphoma cells is due to the presence of activated ALK, the strong staining of some normal cells presumably represents physiologically active kinases and this should be taken into account when interpreting the immunostaining of non-lymphoid tumours. The simplicity of this method, however, means that it offers a new rapid approach to the screening of large numbers of tumours for the presence of aberrant tyrosine kinase activation, particularly if they arise from tissues which normally contain only background levels of phosphotyrosine.

Coordinate expression of the autosomal dominant polycystic kidney disease proteins, polycystin-2 and polycystin-1, in normal and cystic tissue.

A second gene for autosomal dominant polycystic kidney disease (ADPKD), PKD2, has been recently identified. Using antisera raised to the human PKD2 protein, polycystin-2, we describe for the first time its distribution in human fetal tissues, as well as its expression in adult kidney and polycystic PKD2 tissues. Its expression pattern is correlated with that of the PKD1 protein, polycystin-1. In normal kidney, expression of polycystin-2 strikingly parallels that of polycystin-1, with prominent expression by maturing proximal and distal tubules during development, but with a more pronounced distal pattern in adult life. In nonrenal tissues expression of both polycystin molecules is identical and especially notable in the developing epithelial structures of the pancreas, liver, lung, bowel, brain, reproductive organs, placenta, and thymus. Of interest, nonepithelial cell types such as vascular smooth muscle, skeletal muscle, myocardial cells, and neurons also express both proteins. In PKD2 cystic kidney and liver, we find polycystin-2 expression in the majority of cysts, although a significant minority are negative, a pattern mirrored by the PKD1 protein. The continued expression of polycystin-2 in PKD2 cysts is similar to that seen by polycystin-1 in PKD1 cysts, but contrasts with the reported absence of polycystin-2 expression in the renal cysts of Pkd2+/- mice. These results suggest that if a two-hit mechanism is required for cyst formation in PKD2 there is a high rate of somatic missense mutation. The coordinate presence or loss of both polycystin molecules in the same cysts supports previous experimental evidence that heterotypic interactions may stabilize these proteins.

Overexpression of H-Ryk in mouse fibroblasts confers transforming ability in vitro and in vivo: correlation with up-regulation in epithelial ovarian cancer.

Abnormalities in the function of receptor tyrosine kinases (RTKs) have been demonstrated to be important in the pathogenesis of cancer. H-Ryk, a new member of the RTK family, is an unusual RTK in that it is catalytically inactive because of amino acid substitutions of conserved residues in the catalytic domain. We show by immunohistochemistry that it is expressed in the epithelium, stroma, and blood vessels of normal tissues. Evaluation of a panel of 33 primary ovarian tumors (2 benign, 8 borderline, and 23 malignant) was performed. H-Ryk was overexpressed in borderline and malignant ovarian tumors. In serous and clear cell subtypes, there was increased expression in the epithelium, stroma, and blood vessels. Consistent with this observation, overexpression of H-Ryk in the mouse fibroblast cell line NIH3T3 induces anchorage-independent growth and tumorigenicity in nude mice. This implies that overexpression of the receptor can be transforming and may therefore be significant in the pathogenesis of ovarian cancer.