Optimizing immunohistochemistry: antigen retrieval and signal amplification.
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Biomedical subjects
Publications and source records attributed to J A Richmond.
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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.
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.
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.
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.
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.
Increased expression of epidermal growth factor (EGF) receptor has been recorded in many types of human tumors and has been associated with reduced survival in ovarian carcinoma. The purpose of this study was to examine the immunocytochemical distribution of the EGF receptor in normal ovaries (n = 30) and in ovarian tumors (n = 126). Staining was observed in two normal ovaries, in the granulosa cells of a developing follicle, and in surface epithelium. Forty-seven of 103 malignant common epithelial tumors were immunopositive. Staining was usually focal, always confined to the neoplastic epithelium, and showed a cytoplasmic distribution. There was a slight trend for increased EGF receptor expression in more advanced common epithelial malignancies, but this was not statistically significant. No correlation between immunoreactivity and histological subtype or grade of tumor was seen. A few other tumors were also examined: one each of Brenner tumor, mature teratoma, mature teratoma with squamous carcinoma, borderline serous tumor and fibroma; all were immunopositive.
A tuberculous tuboappendiceal fistula was demonstrated on a hysterosalpingogram in an asymptomatic patient being evaluated for infertility. Antituberculous medical treatment resulted in closure of the fistula.
A 25-year computerised survey of deaths in the United Kingdom among diabetic patients of 19 years of age and under was performed. Suitable pancreatic material was available in 119 out of the 498 identified patients. The duration of diabetes was known in 95 of the 119 patients. In 60 patients it had been present for less than 1 year. Insulitis was present in 47 of the 60 patients (78%) with recent onset disease, and was also found in 3 patients who had been treated for diabetes for between 1 and 6 years. In cases in which it was identified, insulitis affected 23% of islets containing insulin, but affected only 1% of islets which were insulin deficient, thus supporting the concept that insulitis represents an immunologically mediated destruction of insulin secreting B cells. Four patients appeared to have a different disease from classical Type 1 (insulin-dependent) diabetes in that there was no evidence of insulitis and all islets contained insulin. The age of onset of diabetes was eighteen months or less in these patients.
Asphyxiating thoracic dystrophy is an autosomal recessive disorder characterised by an abnormally small thorax, variable shortening of the extremities, and pelvic anomalies. Renal and pancreatic symptoms are found in longer survivors, although most cases die in infancy of respiratory failure. Seven neonatal cases were studied at necropsy. These cases ranged in gestational age from 32 to 40 weeks. One was stillborn and the other six survived from 1 hour to 10 days. Two were sibs born to consanguineous parents. Dwarfing was not pronounced and the extremities were shortened in only one infant who also had polydactyly. All seven showed visceral changes in addition to abnormalities of bone. Endochondral ossification was irregular in sections of femur, vertebra, and rib. Pulmonary hypoplasia was associated with the small thorax typical of this disorder. Periportal fibrosis and bile duct proliferation were seen in sections of liver, and in one case cirrhosis was found. Pancreatic fibrosis was variable. These necropsy findings correlate with later clinical manifestations of the disease and emphasise the multisystem nature of this disorder.
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Pelvic tuberculosis (TBC) was diagnosed in 20 patients studied during the years 1971 to 1975. Fourteen patients were born outside the United States. The most frequent presenting complaints were infertility (14 patients), pelvic pain (6), and amenorrhea (4). Only 5 patients gave a history of previous treatment for TBC. Results of pelvic examination were normal in 11 patients; results of chest X-rays were normal in 15. Sixteen patients had endometrial biopsies, 10 of which showed granulomatous endometritis. Fifteen patients had hysterosalpingograms, all of which yielded abnormal results, and 14 were indicative of TBC. Cultures were positive for Mycobacterium tuberculosis in 6 of 16 patients. Genital TBC should be considered as a possible cause of infertility, especially in foreign-born patients. Although a conclusive diagnosis can be made only from a positive culture or histologic specimen, hysterosalpingography is a very useful aid in establishing the diagnosis.
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