Identification of monosomy 3 in choroidal melanoma by chromosome in situ hybridisation.

BACKGROUND/AIMS: In uveal melanoma monosomy 3 is emerging as a significant indicator of a poor prognosis. To date most cytogenetic studies of uveal melanoma have utilised fresh tissue or DNA extracted from tissue sections. In this study chromosome in situ hybridisation (CISH) was used to study monosomy 3 in tissue sections. The copy number of chromosome 3 was determined and related to patient survival. METHODS: Archival glutaraldehyde or formalin fixed, paraffin embedded material was obtained from 30 metastasising and 26 non-metastasising choroidal melanomas. Hybridisations were performed using centromere specific probes to chromosomes 3 and 18. Chromosome 18 was included as a control as previous abnormalities in uveal melanoma have not been described. Chromosomal imbalance was defined on the basis of changes in both chromosome index and signal distribution. RESULTS: CISH was successfully performed on both glutaraldehyde and formalin fixed tissue. Four cases were unsuccessful because of extensive tumour necrosis. All cases were balanced for chromosome 18. Monosomy 3 was detected in 15 of the 26 cases of metastasising melanoma; the 26 non-metastasising tumours were all balanced for chromosome 3. Monosomy 3 was significantly associated with metastases related death. CONCLUSION: CISH can successfully identify monosomy 3 in archival glutaraldehyde or formalin fixed, paraffin embedded tissue sections. Similar to previous studies monosomy 3 is a significant predictor of metastases related death.

Endometrial apoptosis in patients with dysfunctional uterine bleeding.

AIMS: To assess glandular apoptosis in the zona functionalis of proliferative phase endometrium in normal individuals and in patients with dysfunctional uterine bleeding (DUB). METHODS AND RESULTS: Routinely processed, haematoxylin and eosin-stained endometrial biopsies were assessed in 26 patients with symptomatic menstrual abnormality, mainly menorrhagia, and in 24 controls. All biopsies were in the proliferative phase and had been reported as within normal limits and consistent with the menstrual cycle dates provided. Apoptotic and mitotic figures were counted in a minimum of 100 transversely sectioned endometrial glands in all cases. In 16 biopsies (12 DUB and four controls) apoptosis was further assessed using the in situ terminal deoxynucleotidyl-transferase-mediated 2'-deoxyuridine-5'-triphosphate (dUTP) nick-end labelling (TUNEL) method. Apoptotic figures were identified in most control biopsies averaging 5.6/100 glands, and were significantly increased in biopsies from patients with DUB averaging 13.9/100 glands. There was no difference in mitotic figure counts. Apoptoses tended to be clustered within adjacent glands in both groups and individual glands exhibited both mitotic and apoptotic activity. Application of the TUNEL method gave broad agreement with morphological assessment although approximately 20-25% of typical apoptotic figures were not labelled. CONCLUSIONS: Endometrial glandular apoptosis is present in most normal proliferative phase biopsies and appears increased in some cases of DUB. The significance of this finding is not known but increased apoptosis may serve as a morphological marker of abnormal endometrial development in otherwise normal biopsy specimens.

Comparison of in situ hybridisation and polymerase chain reaction in the diagnosis of B cell lymphoma.

AIM: To compare the sensitivity of the detection of immunoglobulin light chain messenger RNA (mRNA) restriction by in situ hybridisation (ISH) and clonal immunoglobulin heavy chain gene rearrangements by polymerase chain reaction (PCR) in the diagnosis of B cell lymphoma. METHODS: Analyses were applied to formalin fixed, paraffin wax embedded, routine diagnostic specimens from cases with a provisional diagnosis of reactive lymph node (n = 23), B cell lymphoma (n = 21), and T cell lymphoma (n = 4). Nonisotopic ISH for kappa and lambda immunoglobulin light chain mRNA was performed using both fluorescein and digoxigenin labelled oligodeoxynucleotide probe cocktails. PCR was carried out on DNA extracted from sections using primers to framework 3 (Fr3) of the V segments and to conserved sequences from the J regions of the immunoglobulin heavy chain genes. RESULTS: All reactive lymph nodes showed a polyclonal pattern of light chain mRNA by ISH, although one showed an excess of kappa positive cells. Nineteen of 21 (90%) cases of B cell lymphoma showed light chain restriction, and a further case showed a vast excess of kappa positive cells. By PCR, 20 of 23 reactive nodes (87%) showed a polyclonal pattern. In 13 of 21 B cell lymphomas (62%) a clonal band was detected. CONCLUSION: In the diagnosis of B cell lymphoma in routinely processed diagnostic material ISH for light chain mRNA was more sensitive (90%) than PCR for heavy chain gene rearrangement using Fr3 and J region primers (62%).

Sequential malt lymphomas of the stomach, small intestine, and gall bladder.

Low grade lymphomas of mucosa associated lymphoid tissue (MALT) are indolent neoplasms that, although tending to remain localised for many years, may spread to other mucosal sites. A 53 year old woman treated by total gastrectomy for low grade MALT lymphoma of the stomach developed a recurrence in the small bowel 18 years later, and a further recurrence involving the gall bladder after three years in complete clinical remission after chemotherapy. In situ hybridisation showed that the small intestine and gall bladder recurrences had the same pattern of light chain restriction. Tumour from all three sites was shown to be derived from a single clone by the demonstration of an identical immunoglobulin heavy chain gene rearrangement by the polymerase chain reaction. The case illustrates the propensity of MALT lymphomas to "home" to mucosal sites and gives an insight into their behavior over an extended follow up.

Detection of cytomegalovirus in upper gastrointestinal biopsies from heart transplant recipients: comparison of light microscopy, immunocytochemistry, in situ hybridisation, and nested PCR.

AIM: To establish the diagnostic value of in situ hybridisation and the nested polymerase chain reaction (PCR) in detecting clinically relevant cytomegalovirus (CMV) infection in upper gastrointestinal biopsies from heart transplant patients. METHODS: Test sensitivity and specificity for detection of CMV early gene RNA by in situ hybridisation and CMV intermediate early gene by PCR were established and then compared with haematoxylin and eosin (H&E) and immunocytochemical detection of CMV in order to establish the best pathological diagnostic approach. All investigations were carried out on formalin fixed, paraffin embedded tissue. RESULTS: Nested PCR had the highest test sensitivity, followed by in situ hybridisation and immunocytochemistry with the same sensitivity; H&E had the lowest. H&E and immunocytochemistry were the most specific but both had a significant false negative rate which was less of a problem with PCR. However, PCR gave no other diagnostic information, and in situ hybridisation was no better than immunocytochemistry. Both in situ hybridisation and PCR were technically complex and more expensive. CONCLUSIONS: H&E and immunocytochemistry represent the best initial screen for CMV and other diseases in upper gastrointestinal biopsies from heart transplant patients. If H&E and immunocytochemistry were negative, nested PCR could significantly increase the diagnostic yield of clinically relevant CMV infection. In situ hybridisation appeared to have no advantages and some drawbacks compared with immunocytochemistry and PCR.

In situ hybridization and its diagnostic applications in pathology.

In situ hybridization (ISH) is a technique by which specific nucleotide sequences are identified in cells or tissue sections. These may be endogenous, bacterial or viral, DNA or RNA. On the basis of research applications, the technique is now being translated into diagnostic practice, mainly in the areas of gene expression, infection and interphase cytogenetics. Diagnostic applications are most often based on short nucleotide sequences (oligomers) labelled with non-isotopic reporter molecules, and sites of binding may be localized by histochemical or immunohistochemical methods. The technique can be applied to routinely fixed and processed tissues; with some targets, it is even possible to obtain hybridization in autopsy material. ISH has been used to detect messenger RNA (mRNA) as a marker of gene expression, where levels of protein storage are low; for example, to confirm an endocrine tumour as the source of excess hormone production. Its application in infectious diseases has to date been mainly in viral infections, such as the typing of human papillomavirus (HPV) or the detection of Epstein-Barr virus by the presence of small nuclear RNAs (EBERs). The expression of mRNAs for histone proteins has been used to detect cells in S phase, and related methods may be applied to detect apoptotic cells. Using probes to chromosome-specific sequences, it is possible to detect aneuploidy, and to document changes in specific chromosomes, which may have prognostic significance in some tumours, such as B-cell chronic lymphatic leukaemia. Using sequence-specific probes, translocations can be identified, such as the t(11;12) of Ewing's sarcoma. This review presents an outline of the technique of in situ hybridization and discusses areas of current and potential diagnostic application.

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.

Expression of p53 in adrenocortical tumours: clinicopathological correlations.

There are limited data to suggest that abnormalities of p53 expression may be a late event in the development of adrenocortical tumours. This has been investigated further by examining a series of adrenocortical adenomas and carcinomas by immunohistochemistry for p53 expression and a subset for evidence of mutation in exons 5-8 of the p53 gene using polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP). In carcinomas, the findings have been correlated with survival data and with tumour ploidy. Immunopositivity for p53 was seen in 4 of 34 adenomas and 22 of 42 carcinomas. Mobility shifts were identified in 1 of 4 adenomas and 10 of 21 carcinomas. There was no correlation between immunostaining pattern or PCR/SSCP evidence of mutation and either survival or disease-free survival in carcinoma. There was also no correlation between p53 status and tumour ploidy. While these findings support a role for p53 in tumour progression, abnormal p53 expression does not appear to have any significant prognostic effect in carcinoma.

Demonstration of insulin production and storage in insulinomas by in situ hybridization and immunocytochemistry.

Twelve cases of insulinoma were studied to assess the amount of hormone synthesis and hormone storage by the tumour and to see what effect a hormone-producing tumour has on the adjacent normal islets. This was investigated by performing in situ hybridization, which detects hormone messenger RNA, thus giving an indication of the degree of hormone synthesis by the tumour, and immunocytochemistry, which detects the hormone itself, thus giving an indication of the amount of hormone stored in the tumour cells. It was found that in most cases there was less hormone stored within the tumour cells than in adjacent islet cells. In a minority of cases, this decrease in stored hormone was associated with reduced hormone synthesis, but the majority of cases showed either equivalent or increased levels of hormone mRNA in the tumour cells compared with adjacent islets. In addition, it was noted that, unlike some other endocrine organs, the presence of a hormone-producing tumour within the pancreas did not appear to inhibit hormone synthesis in the adjacent normal tissue.

A search for evidence of viral infection in pancreases of newly diagnosed patients with IDDM.

Techniques were developed to look for evidence of viral infection in formalin-fixed paraffin-embedded autopsy pancreatic tissues from patients who had died of recent-onset insulin-dependent diabetes mellitus. DNA extracted from 47 pancreases in which good DNA preservation was confirmed was analysed by a polymerase chain reaction for Epstein-Barr virus and by a nested polymerase chain reaction for cytomegalovirus. Histological sections from 29 pancreases in which there was good RNA preservation were tested for the presence of enterovirus and Epstein-Barr virus using in situ hybridization techniques. Seventy-five pancreases were analysed immunohistochemically for the presence of mumps virus. None of these viruses could be detected in any of the diabetic pancreases studied. Control studies suggested that the techniques employed were as sensitive as culture done at the time of autopsy. Pancreas was available for study in 9 infants who had died of myocarditis; enterovirus was demonstrable in islets in 5 of these cases. An acute or persisting infection in the pancreas at the time of clinical onset of insulin-dependent diabetes by any of the 4 virus included in this study seems unlikely.

Immunoglobulin light chain mRNA detected by in situ hybridisation in diagnostic fine needle aspiration cytology specimens.

AIMS: To demonstrate expression of immunoglobulin light chain mRNA in diagnostic fine needle aspiration (FNA) cytology specimens using an in situ hybridisation (ISH) technique; and to evaluate ISH in a series of reactive lymphoid proliferations and malignant lymphomas. METHODS: Forty diagnostic FNA specimens showing a lymphoid cell population were examined for immunoglobulin light chain mRNA expression using ISH. Aspirates were obtained from lymph node (n = 34), salivary gland (n = 3), subcutaneous tissue, thyroid and breast (n = 1 each). The cases included 20 B cell lymphomas, five cases of Hodgkin's disease and 15 reactive lymphoid proliferations. Comparison with light chain immunoreactivity was made in 36 cases and histological correlation from biopsy material was available in 24. RESULTS: Immunoglobulin light chain restriction was demonstrated in 14 of 20 B cell lymphomas using ISH and in six of 17 B cell lymphomas using immunocytochemistry. A polytypic pattern of light chain expression was observed in four of five cases of Hodgkin's disease with both techniques, and in 12 of 15 and 11 of 14 reactive lymphoid proliferations using ISH and immunocytochemistry, respectively. CONCLUSIONS: The assessment of immunoglobulin light chain expression is a useful adjunct to morphology in the diagnosis of reactive and malignant lymphoid proliferations in FNA specimens. Light chain restriction can be shown using either immunocytochemistry or ISH, but the latter is more sensitive in the diagnosis of B cell lymphoma.

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.

Coincidental occurrence of primary hyperparathyroidism and cancer-associated hypercalcaemia in a middle-aged man.

Primary hyperparathyroidism (PHPT) is found not uncommonly in patients with cancer. In this report, however, we describe a patient where both humoral hypercalcaemia of malignancy and PHPT were present coincidentally. A 47-year-old man was found to have PHPT due to parathyroid hyperplasia. Serum parathyroid hormone (PTH) levels, which were elevated before parathyroidectomy, were undetectable post-operatively; however, hypercalcaemia persisted. Nephrogenous cyclic adenosine monophosphate was elevated along with this undetectable PTH, indicative of the presence of a PTH-like factor in the serum. This was confirmed by the finding of an elevated level of PTH-related protein (PTHrP) in plasma (9.1 pmol/l, normal < 2.6 pmol/l). Secondary carcinoma was identified in a lesion in the region of the manubrium sternii. This stained positively for PTHrP by immunocytochemistry and PTHrP messenger RNA was detected by in-situ hybridization. This case illustrates the value of sensitive PTH assays in distinguishing PHPT from other causes of hypercalcaemia and also shows the importance of considering primary hyperparathyroidism in the differential diagnosis of the patient with cancer and hypercalcaemia.

Demonstration of pro-opiomelanocortin mRNA in pituitary adenomas and para-adenomatous gland in Cushing's disease and Nelson's syndrome.

Pro-opiomelanocortin (POMC) mRNA was demonstrated in pituitary adenomas from 16 patients with Cushing's disease and 10 with Nelson's syndrome. The intensity of signal was significantly greater in Nelson's syndrome than in Cushing's disease and there was a trend towards a greater proportion of positive cells. This probably reflects inhibition of POMC gene expression by the high circulating levels of cortisol in Cushing's disease. In the para-adenomatous gland, the intensity of signal was variable in cells showing Crooke's hyaline change, ranging from negative to strongly positive, in keeping with the functional heterogeneity of corticotrophs. In one case, junctional corticotrophs were present and these were more intensely stained than anterior lobe corticotrophs in the same gland. This supports the concept that these cells are subject to different regulatory influences from corticotrophs in the anterior lobe. Whether this is related to differences in embryological origins or to local factors is at present unclear.

Expression of the pro-opiomelanocortin gene in lung neuroendocrine tumours: in situ hybridization and immunohistochemical studies.

Neuroendocrine tumours of the lung may be associated with the ectopic adrenocorticotrophin (ACTH) syndrome and may synthesize and secrete ACTH-related peptides in the absence of the syndrome. However, immunocytochemical analysis may not confirm these biochemical findings, particularly in small cell carcinoma, which is poorly granulated. To investigate further the morphological evidence for expression of the pro-opiomelanocortin (POMC) gene in neuroendocrine lung tumours, we have examined a series of 46 small cell carcinomas and 13 carcinoid tumours of the lung by in situ hybridization for POMC mRNA using a digoxigenin-labelled oligoprobe. We have compared the findings with the immunocytochemical detection of ACTH and beta-endorphin. In situ hybridization was positive in 15 of 46 small cell carcinomas (33 per cent) and in 8 of 13 carcinoid tumours (62 per cent). Immunocytochemical staining was positive in only one carcinoid tumour. These in situ hybridization studies have corroborated biochemical data indicating POMC gene expression in a high proportion of lung neuroendocrine tumours. This suggests that the low levels of expression detected by immunocytochemistry may be due to low levels of hormone storage. Multivariate analysis showed a weak negative association between POMC expression and survival in small cell carcinomas, although this did not reach statistical significance.

The distribution of immunoreactive interferon-gamma-containing cells in normal human tissues.

An immunohistochemical technique has been used to study the distribution of lymphocytes expressing interferon-gamma in normal adult tissues. The greatest concentrations of these cells were seen in mucosal sites exposed to a resident microflora. It is proposed that such organisms, by eliciting immune responses, provide the stimulus for the production of 'physiological' interferon-gamma. This in turn may act to preserve the 'tone' or readiness of the immune system.

Detection of mRNA by in situ hybridisation and in northern blot analysis using oligodeoxynucleotide probes labelled with alkaline phosphatase.

AIMS: To assess whether a reduction in intensity of signal observed using an alkaline phosphatase labelled oligodeoxynucleotide probe could be explained on the basis of procedural steps rather than reduced sensitivity. METHOD: Signal intensity was assessed on in situ hybridisation for pro-opiomelanocortin (POMC) mRNA in rat pituitary and for somatostatin mRNA in human pancreas and in northern blot analysis for POMC mRNA in the presence and absence of formamide. The direct effects of formamide on the alkaline phosphatase detection step were assessed using histochemical enzyme detection in rat kidney. RESULTS: All signals were reduced in systems containing formamide. CONCLUSIONS: In the absence of formamide clear, strong signals for specific mRNAs can be obtained by in situ hybridisation and northern blot analysis using oligodeoxynucleotide probes directly labelled with alkaline phosphatase. Formamide seems to inhibit the activity of alkaline phosphatase.

Characterization of the inflammatory infiltrate in ovarian dysgerminoma: an immunocytochemical study.

The inflammatory infiltrate has been characterized in 10 cases of ovarian dysgerminoma using a panel of antisera to T-cells, B-cells and macrophages. The expression of Class II major histocompatibility complex (MHC) antigens and the distribution of interferons alpha and gamma were also examined. T-lymphocytes were present in all tumours, often closely admixed with neoplastic elements. T-cells in most areas were immunoreactive with gamma interferon. B-cells were generally scanty although germinal centres were present in three tumours. Immunocytochemistry revealed greater numbers of macrophages than had been appreciated on routinely stained sections. Macrophages were closely related to both lymphoid and tumour cells, and many macrophages were immunoreactive for alpha interferon. Class II MHC expression was mainly restricted to macrophages and B-cell areas although occasional T-cells were also stained. Dysgerminoma cells did not express Class II MHC antigens.

The distribution and possible function of gamma interferon-immunoreactive cells in normal endometrium and myometrium.

T-lymphocytes are present in normal endometrium, where they may have a role in the control of glandular maturation. T-cell activity could be related to the local secretion of cytokines such as gamma interferon, which has an anti-proliferative effect on endometrial epithelial cells in vitro. We have examined gamma interferon immunoreactivity and T-cell distribution in 24 normal pre-menopausal uteri. Endometrial appearances were representative of all stages of the menstrual cycle. Most cells in the lymphoid aggregates in the stratum basalis were stained by T-cell and gamma interferon antisera. T-lymphocytes were also scattered in glandular epithelium and throughout the stroma of basal and functional layers; immunoreactivity for gamma interferon was less consistent in these cells. There was no alteration in the intensity or distribution of gamma interferon staining in different phases of the menstrual cycle. Endometrial granulocytes (K-cells) present mainly in the late secretory endometria were not reactive with the gamma interferon antiserum. In addition to endometrial staining, T-cells were distributed in all areas of the myometrium in most uteri, and many myometrial lymphocytes were gamma interferon positive. These results support a role for gamma interferon in endometrial physiology, possibly as an inhibitor of epithelial proliferation.

Insulitis in type 1 (insulin-dependent) diabetes mellitus in man--macrophages, lymphocytes, and interferon-gamma containing cells.

This study sought to determine, firstly, the relative frequency of lymphocytes and macrophages and, secondly, the percentage of lymphocytes containing interferon-gamma in inflamed islets (insulitis) of patients with type 1 (insulin-dependent) diabetes. Autopsy pancreases of 12 patients who had died of recent-onset type 1 diabetes and one pre-diabetic patient who had died of cardiomyopathy were examined immunohistochemically. In the 87 islets that were studied, the lymphocyte macrophage ratio was 9.7:1 and approximately 40 per cent of the lymphocytes contained interferon-gamma. Interferon-gamma release in the insulitis process may be involved in the pathogenesis of type 1 diabetes.