Spatially regulated differentiation of endometrial vascular smooth muscle cells.

Angiogenesis within the human endometrium involves the development of arterioles and elaboration of a capillary network. It was postulated that maturation of these arterioles involves a spatially regulated process of vascular smooth muscle cell (VSMC) differentiation. The endometrial vascular tree was therefore examined immunohistochemically for evidence of longitudinal and radial gradients of VSMC phenotype. Twenty-three hysterectomy specimens and 15 first trimester decidual tissues were studied. Five cytoskeletal markers (alpha and gamma-smooth muscle (sm) actin, sm myosin, desmin, vimentin), three endothelial markers (CD31, CD34, factor VIII related antigen) and two steroid receptors (oestrogen and progesterone) were detected immunohistochemically. alpha-sm actin was present throughout the wall of basal arterial segments and extended longitudinally towards the endometrial surface. Sm myosin expression was more restricted longitudinally and radially within in the vascular tree. The expression of gamma-sm actin was even more restricted than myosin. In first trimester decidua, however, gamma-sm actin was widely distributed within the wall of spiral arteries that were not invaded by trophoblast. Oestrogen and progesterone receptors were present in peri-vascular stromal cells but absent from vascular smooth muscle and endothelium. Endometrial VSMC differentiation involves a progressive increase in cytoskeletal complexity and occurs in a spatially regulated fashion.

Fine needle aspiration cytology in the investigation on non-Hodgkin's lymphoma.

AIMS: To assess the value of flow cytometry (FCM) in the diagnosis and classification of reactive lymphoid hyperplasia and malignant lymphoma by fine needle aspiration (FNA) cytology. METHODS: Forty six fine needle aspirates of lymphoproliferative disorders were examined by FCM as well as routine cytological assessment. An immunoglobulin light chain ratio (LCR) was calculated for clonality analysis. Additional immunophenotyping was performed in 15 cases. RESULTS: All 25 cases of reactive lymphoid hyperplasia were polyclonal by FCM (LCR < 2/1); 17 of 20 cases of B cell non-Hodgkin's lymphoma were monoclonal (LCR > 3/1). Analysis of cells based on size facilitated detection of small populations of clonal neoplastic cells. Analysis of CD5, CD10, and CD23 expression by FCM facilitated subclassification of mantle cell lymphoma, small lymphocytic lymphoma, and some lymphomas of follicle centre cell origin. One case of T cell non-Hodgkin's lymphoma was correctly classified by FCM. CONCLUSIONS: FNA cytology is a reliable method for investigation of lymphoproliferative disorders. Although excision biopsy and histopathological examination remain the gold standard for primary diagnosis and classification of non-Hodgkin's lymphoma, FNA cytology with clonality analysis and immunophenotyping by FCM is useful for distinguishing reactive from neoplastic lymphoid populations, and can facilitate lymphoma classification.

Diagnostic value of inhibin immunoreactivity in ovarian gonadal stromal tumours and their histological mimics.

AIMS: Inhibin is a heterodimeric protein hormone which appears to be a sensitive serological and immunohistochemical marker of ovarian granulosa cell tumours. The purposes of this study were to examine inhibin immunoreactivity in a wide range of gonadal stromal neoplasms and to assess its value in the differential diagnosis of problematic ovarian tumours. METHODS AND RESULTS: Inhibin immunostaining was identified in eight cases of stromal hyperthecosis, 24 adult-type granulosa cell tumours, two juvenile granulosa cell tumours, nine Sertoli cell or Sertoli-Leydig cell tumours, one gynandroblastoma, three gonadoblastomas, two cases of sex cord tumour with annular tubules, two steroid cell tumours and two sclerosing stromal tumours. Inhibin was also present in 4/6 fibrothecomas and 2/3 unclassified gonadal stromal tumours. There was no staining of neoplastic cells in any of the problematic ovarian tumours, in many of which a diagnosis of gonadal stromal tumour had been initially considered. However, inhibin was detected in reactive stromal cells in many cases. CONCLUSIONS: Inhibin is a sensitive immunohistochemical marker of a wide range of gonadal stromal tumours and is of value in the differential diagnosis of ovarian neoplasia.

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR).

Immunoglobulin heavy chain (IgH) gene rearrangement analysis was performed on 27 fine needle aspiration (FNA) specimens (13 reactive hyperplasia, 11 B cell non-Hodgkin's lymphoma (B-NHL), one Hodgkin's disease and two suspicious of non-Hodgkin's lymphoma). Satisfactory amplification was achieved in 23/27 cases. A polyclonal pattern was seen in 14 cases (11 reactive hyperplasia, one B-NHL, one suspicious of lymphoma, one Hodgkin's disease). A monoclonal band was seen in nine cases (eight B-NHL, one reactive hyperplasia). Amplification was unsuccessful in four cases. Clonal analysis by PCR-based IgH gene rearrangement analysis can be successfully applied to FNA material and can be useful in diagnosis, but the results must be interpreted in conjunction with morphology and other ancillary information.

E-cadherin and integrin cell adhesion molecule expression in invasive and in situ carcinoma of the cervix.

Integrin and E-cadherin cell adhesion molecules are important in the maintenance of normal epithelial structures, and altered expression of these molecules may be important in epithelial tumors, particularly in the processes of invasion and metastasis. This study examines the immunohistochemical expression of the alpha2beta1, alpha3beta1, alpha6, and beta4 integrins and of E-cadherin in high-grade cervical intraepithelial neoplasia (CIN) and in invasive carcinoma of the cervix. Abnormal expression of E-cadherin and of all of the integrin subunits examined was detected in the dysplastic epithelium in CIN in the majority of cases, but in a minority of cases this expression was lost in the superficial layers of the dysplastic epithelium. Diffuse expression of E-cadherin and of all of the integrins was seen in the majority of cases of invasive cervical carcinoma. Abnormal expression of integrin cell adhesion molecules occurs in the majority of cases of high-grade CIN, and E-cadherin expression is retained in the dysplastic epithelium. Diffuse expression of integrins and of E-cadherin also occurs in invasive carcinoma, but as the expression pattern was similar in all cases of carcinoma, no correlation with outcome is possible and the significance of this expression is unclear.

Proliferation, ploidy and prognosis in uterine smooth muscle tumours.

DNA ploidy, mitotic rate (per 10 high power fields), mitotic index (per 1000 tumour nuclei), Ki-67 labelling index and S phase fraction were measured in 23 uterine leiomyosarcomas and 10 tumours of uncertain malignant potential. Correlations were calculated by Spearmann rank correlation. Univariate survival analysis was performed by log rank analysis and multivariate analysis performed by the Cox linear regression method. Ki-67 index and S phase fraction were significantly higher in leiomyosarcomas than in tumours of uncertain malignant potential. There was significant correlation between mitotic rate, mitotic index, Ki-67 index and S phase fraction in cases of leiomyosarcoma. Fifteen of 22 leiomyosarcomas and one of 10 tumours of uncertain malignant potential were DNA aneuploid. On univariate analysis of all the smooth muscle tumours, DNA ploidy, presence of significant nuclear atypia and presence of coagulative tumour cell necrosis were associated with outcome. Only DNA ploidy was associated with outcome in the group of leiomyosarcomas. On multivariate analysis of all of the smooth muscle tumours, DNA ploidy, age and grade of atypia were independently associated with outcome. No single factor was independently predictive of outcome in the group of leiomyosarcomas. Alternative indices of cell proliferation correlate with mitotic rate in uterine leiomyosarcoma and do not provide additional useful prognostic information. DNA ploidy, age and grade of atypia are independently associated with outcome in uterine smooth muscle tumours and measurement of DNA ploidy may be useful in identification of cases with an adverse prognosis.

Localization of endothelin receptors in human uterus throughout the menstrual cycle.

Quantitative autoradiography employing the ETA selective ligand [125I]PD 151242 and the ETB selective ligand [125I]BQ3020 was used to assess the localization of ETA and ETB receptors in human uterus throughout the menstrual cycle. ETA and ETB receptors were present in endometrium and myometrium across the menstrual cycle. In myometrium, neither ETA nor ETB receptor density showed any detectable change across the menstrual cycle. ETA receptors were expressed in stroma throughout the endometrium and showed an increase in density in proliferative endometrium compared with secretory and menstrual endometrium. Endometrial ETB receptors were expressed at low density in the proliferative phase. In the early secretory phase there was an increase in ETB receptor density in the glandular epithelium of the basal region of the endometrium but not in functional endometrium. In the late secretory phase ETB receptor expression was increased in glandular epithelium throughout the endometrium. The highest density of ETB receptors was seen in menstrual endometrium, where they were present in stromal as well as epithelial cells. These results suggest that ovarian steroid hormones may play a role in the control of expression of ETA and ETB receptors in endometrial stromal and epithelial cells respectively.

Trophoblast proliferation rate does not predict progression to persistent gestational trophoblastic disease in complete hydatidiform mole.

Persistent gestational trophoblastic disease (PGTD) is a significant complication of complete hydatidiform mole. There are, however, no reliable clinical or pathological parameters to predict the development of PGTD following the evacuation of a complete mole. In this study we examined the value of Ki67 expression in the prediction of PGTD following complete mole. Immunohistochemistry for Ki67 was performed on 21 cases of complete mole complicated by PGTD and 17 cases that resolved spontaneously after evacuation. Strong Ki67 expression was seen in both villous cytotrophoblast and extravillous trophoblast of the complete moles. The Ki67 index of extravillous trophoblast was significantly higher than that of villous cytotrophoblast. There was no significant difference in Ki67 index of either villous cytotrophoblast or extravillous trophoblast between cases of complete mole complicated by PGTD and those that resolved spontaneously. Trophoblast proliferation rate does not predict outcome following complete hydatidiform mole. Other factors involved in myometrial invasion may be of importance in the development of this complication, but serum chorionic gonadotrophin level is the only reliable predictor of the development of persistent gestational trophoblastic disease.

Comparison of ploidy analysis by flow cytometry and image analysis in hydatidiform mole and non-molar abortion.

Determination of DNA ploidy is useful in the diagnosis and classification of hydatidiform mole. Most reports of ploidy analysis in molar tissue have used DNA flow cytometry. Although image analysis cytometry offers theoretical advantages over flow cytometry, there have been few reports of ploidy analysis by image analysis in hydatidiform mole. We selected 47 cases and measured DNA ploidy by flow cytometry and image analysis cytometry in complete hydatidiform mole, partial hydatidiform mole and non-molar abortion. The two cytometry modalities were compared using kappa statistics. There was reasonable overall agreement between the two modalities (kappa = 0.69) and when ploidy was stratified into diploid/polyploid and triploid categories there was near perfect agreement (kappa = 0.93). Aneuploid cell populations, which were not evident on flow cytometry, were identified by image analysis in a significant proportion of complete and partial hydatidiform moles and in a small number of non-molar abortions. Flow cytometry and image analysis cytometry yield comparable ploidy information, useful in the diagnosis and classification of hydatidiform mole. Image analysis cytometry offers greater sensitivity in the detection of small non-diploid cell populations but the significance of this latter finding is uncertain.

Clear cell sarcoma of the kidney in an adult.

An adult case of clear cell sarcoma of the kidney is described. The clinical, histological, immunohistochemical and electron microscopical features of the tumour are described.

Expression of cell adhesion molecules in placentae from pregnancies complicated by pre-eclampsia and intrauterine growth retardation.

Platelets and neutrophils are involved in maternal placental vascular damage in pre-eclampsia. Recruitment of these cells is probably mediated by cell adhesion molecules expressed at the uteroplacental bed. It remains controversial as to whether platelets and neutrophils mediate damage to trophoblast or villous vasculature. The purpose of this study was to determine the expression of cell adhesion molecules in placentae from normal pregnancies and pregnancies complicated by pre-eclampsia and intrauterine growth retardation (IUGR). Immunostaining for platelet endothelial cell adhesion molecule (PECAM) and intercellular adhesion molecule-1 (ICAM-1) was localized mainly to the endothelium of stem villi, intermediate villi, terminal villi and decidual vessels. Scattered staining for ICAM-1 was also evident in the stroma and fetal membranes. The endothelium of stem villi, intermediate villi and terminal villi were all negative for vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin. PECAM, ICAM-1 and ICAM-2 mRNA were all detectable in normal placentae using northern blotting analysis whereas mRNA for E-Selectin and VCAM-1 were both undetectable. There were no differences in cell adhesion molecule immunostaining or mRNA expression in placentae from pregnancies complicated by pre-eclampsia and IUGR inconclusion, expression of cell adhesion molecules in placentae from pre-eclampsia and IUGR are consistent with a normal physiological role in vascular function.

Overexpression of the c-myc proto-oncogene occurs frequently in uterine sarcomas.

Overexpression of the c-myc proto-oncogene occurs in carcinoma of the ovary, endometrium, and cervix, and is associated with an adverse prognosis, but little is known about the pattern of c-myc expression in uterine sarcomas. This study investigates the expression of c-myc in uterine smooth muscle tumors and malignant mixed müllerian tumors. Twenty-three leiomyosarcomas, 10 leiomyomas, and 9 malignant mixed müllerian tumors were examined for c-myc overexpression by immunohistochemistry. Differences in mitotic rate and in survival were compared in c-myc positive and negative cases of leiomyosarcoma. Overexpression of c-myc was seen in 6/12 leiomyomas, 11/23 leiomyosarcomas, and 9/9 malignant mixed müllerian tumors. Positive staining was restricted to a perinuclear location in all of the leiomyomas and one leiomyosarcoma. Diffuse cytoplasmic staining was seen in the remaining 10 positive leiomyosarcomas. Positive staining was seen in both epithelial and stromal elements of malignant mixed müllerian tumors, including homologous and heterologous areas of stromal differentiation. There was no significant difference in mitotic rate or in survival between c-myc positive and negative cases of leiomyosarcoma. Overexpression of c-myc occurs in many uterine leiomyosarcomas and the majority of malignant mixed müllerian tumors. Overexpression of c-myc also occurs in benign uterine smooth muscle tumors but with a different pattern than that seen in malignant tumors. This overexpression does not correlate with survival and the significance of overexpression of c-myc in these tumors is unclear.

p53 immunoreactivity and mutation of the p53 gene in smooth muscle tumours of the uterine corpus.

Mutation and overexpression of p53 have been described in uterine malignant mixed Müllerian tumours and in endometrial adenocarcinoma, where it has been associated with a poor prognosis. This study examines p53 expression and mutation of the p53 gene in benign and malignant smooth muscle tumours of the uterine corpus. p53 expression was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded tissue from 23 leiomyosarcomas, 10 tumours of uncertain malignant potential (TUMPs), and 18 leiomyomas. Single-stranded conformational polymorphism, (SSCP) analysis of exons 5-8 of the p53 gene was performed on 13 leiomyosarcomas, nine TUMPs, and eight leiomyomas. With microwave antigen retrieval, p53 immunoreactivity was seen in 13/23 microwave treatment, staining was abolished in three leiomyosarcomas, all immunoreactive TUMPs, and the single positive leiomyoma. SSCP analysis revealed mutation in three leiomyosarcomas. There was one mutation in exon 5 in a case with positive immunohistochemistry. Two cases with negative staining showed mutation, one in exon 7 and one in exon 8. Mutation was present in exon 7 in 4/9 and in exon 6 in 1/9 TUMPs. All of these cases showed positive immunohistochemistry. There was no significant difference in outcome between cases with and without positive immunohistochemistry. p53 expression is seen in a significant proportion of uterine leiomyosarcomas. Microwave antigen retrieval increases the proportion of positive cases and also results in positive staining in TUMPs. Mutation of the p53 gene occurs in only a minority of leiomyosarcomas and in a significant proportion of TUMPs. Positive immunohistochemistry does not, however, correlate with the presence of mutation and other factors may be responsible for p53 detection in many cases.

p53 immunoreactivity in cervical intraepithelial neoplasia and non-neoplastic cervical squamous epithelium.

AIMS: To determine the pattern of p53 immunoreactivity in cervical squamous epithelium and to investigate the relation between p53 immunostaining and human papillomavirus (HPV) infection. METHODS: Immunocytochemistry for p53 was performed in 65 specimens of formalin fixed, paraffin wax embedded cervical tissue using a polyclonal antibody against recombinant p53. Microwave oven heating was used for antigen retrieval. Eight normal biopsy specimens, eight cases with histological features of HPV infection, and 49 cases of cervical intraepithelial neoplasia (CIN) were examined. Thirty one cases of CIN were examined. Thirty one cases of CIN were examined for evidence of HPV infection using in situ hybridisation with probes directed against wide spectrum HPV, HPV 16 and HPV 18. RESULTS: p53 immunoreactivity was seen in seven of eight (87%) of specimens with histological features of HPV infection, five of eight (62%) normal specimens, 13 of 22 (59%) CIN III, three of 14 (21%) CIN II and five of 13 (38%) CIN I specimens. The numbers of positive nuclei were small in cases of CIN and the location of positive nuclei within the epithelium paralleled the degree of dysplasia. Eleven of 15 (73%) CIN specimens which were immunoreactive for p53 yielded a positive signal for HPV by in situ hybridisation. A positive signal for HPV was also seen in 10 of 16 (63%) of CIN specimens in which p53 staining was absent. CONCLUSIONS: p53 immunoreactivity can be demonstrated in a small proportion of cells in the cervical squamous epithelium in a significant proportion of cases of CIN. This immunoreactivity seems to be independent of the presence of HPV, as assessed by in situ hybridisation. p53 immunoreactivity also occurs in non-neoplastic cervical squamous epithelium with a pattern of distribution within the epithelium which differs from that seen in CIN. Antigen retrieval by microwave oven heating enhances p53 immunostaining and may result in visualisation of cellular p53 in the absence of mutation.

Comparison of villous trophoblast proliferation rate in hydatidiform mole and non-molar abortion by assessment of proliferating cell nuclear antigen expression.

This study examines the proliferative activity of trophoblast in hydatidiform mole, non-molar hydropic abortion and non-molar spontaneous abortion. Nine cases of complete mole, 10 cases of partial mole, eight cases of non-molar hydropic abortion and six cases of non-hydropic second trimester abortion were examined by routine histopathology and the rate of cell proliferation was assessed by immunoreactivity for proliferating cell nuclear antigen (PCNA). Hydropic abortion showed a significantly lower PCNA index than complete mole and partial mole. There was no significant difference in PCNA index between partial mole and non-hydropic abortion. The trophoblast of partial hydatidiform mole demonstrates significant cell proliferation but this, although higher than that of hydropic abortion, is no higher than that of non-hydropic abortion of a similar gestational age. The role of partial mole as a precursor of persistent gestational trophoblastis disease remains unclear.

The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma.

Micrometastases have been detected by immunocytochemical means in the lymph nodes of patients with otherwise node-negative cancer of the colon and rectum. This study examines the incidence and prognostic significance of nodal micrometastases in Dukes' B carcinoma. Five hundred and fifty-nine lymph nodes from 77 cases of Dukes' B carcinoma were examined for lymph node micrometastases by immunocytochemical staining for cytokeratin AE1:AE3. Micrometastases were detected in 19 cases (25 per cent). Cell clusters were present in ten cases, the remaining nine cases displaying only single cells. The presence of micrometastases was unrelated to age (P = 0.06), sex (P = 0.32), tumour site (P = 0.37), tumour size (P = 0.67), or tumour differentiation (P = 0.66). Ten-year survival estimates by the Kaplan-Meier lifetable method was 47 per cent in patients with and without micrometastases (chi 2 = 0.35 and 1 df, P = ns). The presence of nodal micrometastases detectable only by immunocytochemistry in patients with Dukes' B colorectal cancer does not justify reassignment to a more advanced disease stage.

Partial hydatidiform mole: a common but underdiagnosed condition. A 3-year retrospective clinicopathological and DNA flow cytometric analysis.

This retrospective study reports the incidence of hydatidiform mole in a population of 19,457 pregnancies over a 3-year period. During the study period all "products of conception" from first and second trimester abortions were referred for pathological examination. Coded histologic sections were reviewed using the published histologic criteria for hydatidiform mole. Ploidy was estimated by DNA flow cytometry. Clinical data were retrieved from maternal case notes. Thirty-eight cases were confirmed as hydatidiform mole, 10 (26%) as complete mole, and 28 (74%) as partial mole. Twenty-three cases of partial mole (88%) were triploid, and nine of 10 complete moles were diploid. The incidence of hydatidiform mole was 1:512 pregnancies, (complete mole, 1:1,945; partial mole, 1:695). Only one case (3.5%) of partial mole was suspected clinically. One case of persistent gestational trophoblastic disease occurred following a complete mole. No sequelae were encountered following partial mole. We conclude that hydatidiform mole is a common condition and the majority of cases are partial moles. Quantitatively imprecise morphologic criteria contribute to the inaccuracy in reporting of partial mole; analysis of ploidy is useful in the evaluation of problem cases. Follow-up of partial mole is warranted because its true biological potential is as yet unclear.

Coeliac disease with histological features of peptic duodenitis: value of assessment of intraepithelial lymphocytes.

AIMS: To determine if a clinically important polymorphonuclear leucocyte infiltrate and surface gastric epithelial metaplasia occur in the second part of the duodenum in coeliac disease; to evaluate the utility of these morphological criteria in the differential diagnosis of coeliac disease and peptic duodenitis. METHODS: 49 mucosal biopsy specimens of the second part of the duodenum reported as showing inflammation were reviewed. Sections were prepared with haematoxylin and eosin, periodic acid Schiff, and Warthin-Starry stains. Clinical presentation, outcome, and immunological investigations were assessed. RESULTS: Four cases confirmed as coeliac disease on clinical and immunological grounds showed acute inflammation and surface epithelial gastric metaplasia. Increased intraepithelial lymphocytes (IELs) were found in each of the four. CONCLUSIONS: Clinically important polymorphonuclear leucocyte infiltration and surface epithelial gastric metaplasia may occur in the duodenal mucosa in coeliac disease and should not be used as diagnostic features to exclude the diagnosis of coeliac disease in the absence of confirmatory clinical and immunological information.