Analysis of gastrinomas by immunohistochemistry and in situ hybridization histochemistry.

Gastrinomas from 25 patients were examined by immunohistochemistry (IHC) and in situ hybridization histochemistry (ISH). Most patients (84%) presented with the Zollinger-Ellison syndrome. Six had multiple endocrine neoplasia type I (MEN-I). Twelve patients (48%) had duodenal primaries and 11 of 12 of these had metastases to regional lymph nodes and/or liver in spite of the small sizes of the primary tumors (mean size of 0.9 cm). Five patients had pancreatic gastrinomas and eight patients had metastatic tumor in regional lymph nodes or liver at surgery but a primary was not found. IHC and ISH analyses showed that all cases were positive for gastrin protein and 24 of 25 (96%) expressed gastrin mRNA that was easily detected in formalin-fixed, paraffin-embedded tissue sections. Both benign and malignant tumors expressed alpha subunit of human chorionic gonadotropin protein (alpha-HCG). However, only malignant gastrinomas (29%) expressed adrenocorticotropic hormone protein or proopiomelanocortin (POMC) mRNA. ISH and Northern hybridization analysis revealed that chromogranin A mRNA was the most common member of the chromogranin/secretogranin (Cg/Sg) family which was expressed in both benign and malignant gastrinomas. These results indicate that duodenal gastrinomas are common in both sporadic and MEN-1-associated cases, and small duodenal primaries may be associated with extensive regional lymph node and liver metastases. Expression of ACTH/POMC protein and mRNA was consistently associated only with malignant gastrinomas while gastrin protein, gastrin mRNA and Cgs/Sgs mRNAs were readily detected in both benign and malignant gastrinomas.     

Multiple HPV infection: microanatomy by in situ hybridization and immunohistochemistry.

Specific human papillomavirus (HPV) types have been shown to be associated with proliferative epithelial lesions with variable biological consequences in infected patients. Simultaneous infection by more than one HPV type has been infrequently reported, and its clinical significance is unknown. We have examined four biopsies of cervical and vulvar tissue, each with evidence of infection by two different HPVs. Using both in situ hybridization and immunohistochemical techniques, we determined the cellular distribution of the viral infections. Using biotinylated type-specific probes and stringent conditions we were able to demonstrate that in each case the two HPVs occupied distinct, non-overlapping foci within the lesions. The condylomatous tissues contained DNA from HPV types that are associated with high-grade neoplasia and invasive cancer (16 and 18), as well as types commonly associated with benign proliferative lesions. Immunohistochemical analysis of the lesions with antibody to bovine papillomavirus capsid antigen failed to detect HPV in regions shown by in situ hybridization to contain HPV 16 and 18 DNA, whereas type 6 and 11 infected areas were readily identified. These results provide indirect evidence of viral interference between HPV types and indicate that interference may limit the number of HPV types that produce active infections within a single cell.     

Detection of inflammatory cytokine messenger RNA (mRNA)-expressing cells in human inflamed gingiva by combined in situ hybridization and immunohistochemistry.

Cells expressing messenger RNA (mRNA) for inflammatory cytokines [interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8] were demonstrated by in situ hybridization in human inflamed gingiva. When this technique was used in conjunction with immunohistochemistry, IL-1 alpha and/or beta and IL-8 messages were observed predominantly on macrophages infiltrating the gingiva. TNF-alpha messages were abundant on macrophages and T cells. In contrast, the IL-6 mRNA were more widely distributed on many types of cells such as macrophages, T cells, fibroblasts, endothelial cells and B cells. This study clearly identified the cells which express mRNA for inflammatory cytokines in inflamed gingiva and suggested an involvement of cytokine network in the generation of human periodontitis.     

Expression of very low density lipoprotein receptor in the vascular wall. Analysis of human tissues by in situ hybridization and immunohistochemistry.

The recently cloned very low density lipoprotein (VLDL) receptor binds triglyceride-rich, apolipoprotein-E-containing lipoproteins with high affinity. The observation that VLDL receptor mRNA is abundantly expressed in extracts of tissues such as skeletal muscle and heart, but not liver, has led to the hypothesis that this receptor may facilitate the peripheral uptake of triglyceride-rich lipoproteins. However, little information is available concerning the types of cells that express this receptor in vivo. As expression of the VLDL receptor in the vascular wall might have important implications for the uptake and transport of triglyceride-rich lipoproteins, and perhaps facilitate the development of atherosclerosis in hypertriglyceridemic individuals, we used in situ hybridization and immunohistochemistry to determine whether VLDL receptor mRNA and protein was expressed in human vascular tissue. We observed expression of the receptor by both endothelial and smooth muscle cells within normal arteries and veins, as well as within atherosclerotic plaques. In the latter, the VLDL receptor was also expressed by macrophage-derived foam cells. The widespread distribution of the VLDL receptor in vascular tissue suggests a potentially important role for this receptor in normal and pathophysiological vascular processes.     

Her2 amplification: correlation of chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

Detecting Her2 gene amplification has become routine in predicting therapeutic responsiveness in patients with breast carcinoma. Fluorescence in situ hybridization (FISH) is a common technique for detecting Her2 amplification, yet dark field fluorescence microscopy remains problematic for many pathologists. Thus, a technique such as chromogenic in situ hybridization (CISH), in which the more familiar light microscopy can be used, is appealing. Paraffin-embedded sections from 61 breast carcinomas were tested for Her2 amplification by immunohistochemistry (IHC) and CISH. FISH was used to confirm CISH results. Excellent correlation was found between IHC and CISH except in cases considered negative (1+ on the DAKO scale) by IHC. CISH detected low-level Her2 amplification in 4 of 9 of these cases. Amplification was subsequently confirmed by FISH in all but 1 case. When compared with FISH, CISH was more sensitive than IHC for detecting low levels of Her2 gene amplification. Moreover, excellent concordance was found between FISH and CISH, supporting the conclusion that the CISH assay for Her2 gene amplification provides an accurate, effective, and practical alternative to FISH.     

In situ hybridization and immunohistochemistry of p53 tumor suppressor gene in human esophageal carcinoma.

We have studied expression of p53, a tumor suppressor gene, by using both immunohistochemistry and in situ hybridization in 20 cases of squamous cell carcinoma of the esophagus. Immunohistochemical analysis was performed by using monoclonal antibody pAb1801. Immunoreactive p53 was observed in the nuclei of the tumor cells in 17 cases. We used 35S-labeled anti-sense single-stranded synthetic oligonucleotide probe ON102, which hybridized with DNA sequence near the 5' end of p53, for in situ hybridization. In all the cases of invasive squamous cell carcinoma studied, no significant accumulation of p53 hybridization signals was observed in carcinoma cells. This result indicates that overexpression of p53 observed by immunohistochemical staining is not due to an increase in the steady-state level of p53 mRNA in frank carcinoma cells. In six cases of morphologically normal esophageal mucosa distant from carcinoma, accumulation of hybridization signals was observed in basal and parabasal cells of the mucosa. The mucosa of these cases was negative for p53 immunoreactivity except for one case showing sporadic positivity. Accumulation of hybridization signals was observed in foci of squamous dysplasia not associated with invasion in three cases.     

Analysis of human pituitary tumors by in situ hybridization

A procedure for performing in situ hybridization histochemistry (ISH) on frozen and paraffin sections of human pituitary tissues is described. The use of oligonucleotide probes for hPRL and hGH labeled with 35S allowed detection of a specific messenger RNA in frozen and paraffin sections. This technique can be combined with immunochemistry to localize both the gene product and the hormone(s) produced by specific cells and should be very helpful in the characterization of normal and neoplastic human pituitary cells.     

Apolipoprotein E localization in human coronary atherosclerotic plaques by in situ hybridization and immunohistochemistry and comparison with lipoprotein lipase.

Apolipoprotein E (apo E) mediates both lipid accumulation by and removal from cells and may be secreted by both macrophages and smooth muscle cells in vitro, but its cellular source in atherosclerotic plaques is not known. Lipoprotein lipase (LPL) also enhances cell lipid accumulation and is synthesized by macrophage foam cells in atherosclerotic plaques. To determine the cellular source of apo E in human coronary atherosclerotic lesions and its relationship to LPL synthesis, in situ hybridization and immunohistochemistry were performed on 12 atherosclerotic plaques and six nondiseased coronary artery segments from 10 cardiac transplant recipients. Apo E messenger RNA was localized to both non-foam cell and foam cell macrophages in plaques, but not to other cell types, and was not detected in nonatherosclerotic arteries. Half of the regions with non-foam cell macrophages expressed neither apo E nor LPL messenger RNA, whereas 86% of macrophage foam cell-containing regions contained both messenger RNAs. Polyclonal antisera raised against human apo E localized apo E protein to the surface of macrophages and surrounding matrix in plaques but not in control coronary segments. An LPL-specific monoclonal antibody demonstrated that, similar to apo E, LPL protein on foam cell and non-foam cell macrophages was detected in atherosclerotic lesions, but LPL was also localized to intimal muscle smooth muscle cells and was not distributed as widely in association with matrix as was apo E. The expression of both apo E and LPL in atherosclerotic lesions but not in normal intima suggest that these molecules play a role in lipid metabolism in atherosclerosis.     

Detection of papilloma virus of the human uterine cervix by in situ hybridization method--comparison with immunohistochemistry

The usual methods for pathological diagnosis of HPV infection of the uterine cervix include screening in cytodiagnosis and histodiagnosis and confirmation by immunohistochemistry (IHC) method. However, some institutes have recently begun to use in situ hybridization (ISH) method for definitive diagnosis using a DNA probe. We compared IHC with ISH with regards to the localization and rate of detection of HPV in lesions of the uterine cervix such as dysplasia and squamous cell carcinoma in the present study. The cases found positive by IHC showed brownish nuclei of the epithelium and those positive in ISH showed purple to purplish-black nuclei. The comparison of cases positive by both methods revealed that the number of cells positive by IHC was smaller than that by ISH, and the cells positive by IHC were localized in the superficial layer. HPV was detected by the IHC various lesions of the uterine cervix in 13 (12.3%) of 106 patients, while it was detected by the ISH in 39 (36.8%) of 106 patients. The results of both methods were in accordance in 66.0% (77 patients; positively in 8 and negatively in 62). The detection sensitivity of IHC is lower than that of ISH. IHC cannot be used to identify the type of HPV, and it is impossible to confirm the presence or absence of virus by this method in cases of malignant changes. ISH is therefore necessary for identification of HPV and investigation of a histopathological relationship between HPV type and malignant change.     

Neural cell adhesion molecule (NCAM) in normal and neoplastic human pituitary tissues: Analysis by immunohistochemistry and in situ hybridization

The expression of the neural cell adhesion molecule (NCAM) in 6 normal human pituitaries and 25 pituitary adenomas was investigated by immunohistochemistry and in situ hybridization. NCAM protein and mRNA were present in all normal and neoplastic human pituitary tissues. There were tumor type-specific differences in the distribution of NCAM in various pituitary adenomas. Growth hormone adenomas and prolactin-producing adenomas usually expressed lower levels of NCAM, compared to null cell and gonadotroph adenomas. Adreno-corticotropic hormone adenomas expressed the highest levels of NCAM mRNA. Six freshly dissociated pituitary adenomas were cultured in serum-free medium for 7 days to analyze the regulation of NCAM mRNA by in situ hybridization. The lower levels of NCAM expression in growth hormone and prolactin adenomas were not present in cells cultured for 7 days in serum-free medium on extracellular matrix. Phorbol 12-myristate 13-acetate (PMA) stimulated NCAM mRNA expression in 5 of 6 tumors. Gonadotropin-releasing hormone and growth hormone-releasing hormone increased NCAM expression in some adenomas. This study demonstrates that there is a variable expression of NCAM in pituitary adenomas and that hypotha-lamic hormones and PMA can regulate NCAM mRNA levels in neoplastic pituitary cells.     

The utility of immunohistochemistry and in situ hybridization in placental pathology

It has long been recognized that routine histologic examination of the placenta has limitations, especially with regard to the diagnosis of infectious diseases and the concomitant cytokine response that may cause severe in utero fetal damage. Immunohistochemical testing of the placenta in such situations can be very useful in terms of identifying the infectious agent as well as in demonstrating a marked increase in cytokines such as tumor necrosis factor alpha and interleukin 8, produced primarily by cells native to the villi and fetal membranes. One hundred placentas (20 normal childbirths, 20 with severe neonatal morbidity of known cause, 25 idiopathic stillbirths where autopsy material was available, 35 with severe idiopathic neonatal morbidity) were examined for a wide variety of infectious diseases and cytokine production. An infectious agent was evident in 19 (76%) of 25 placentas from stillbirths and 28 (80%) of 35 placentas associated with idiopathic severe neonatal morbidity. No infectious agent was noted in the placentas from normal childbirths or cases of known neonatal morbidity. The most common infectious agent was coxsackie virus (51% of infections) followed by bacterial infections (24% of infections). The same infectious agent found in the placenta was found in the corresponding autopsy material from the stillbirths, with the spleen containing the greatest number of infected cells. There was a strong correlation between the number of cells demonstrating cytokine expression (tumor necrosis factor alpha and interleukin 8) and the presence of an infectious disease in the placenta and stillborn. No histologic feature was associated with an in utero infection. Immunohistochemical testing of placentas gives much insight into their structure and function, including, besides infectious disease detection, the marked diversity of function of trophoblasts, the rarity of committed B cell response, and the strong potential of contractility of villi.     

Molecular forms of tear IgA and distribution of IgA subclasses in human lacrimal glands

Tears and sera from 14 subjects were analyzed for IgA levels by radioimmunoassay by use of secretory IgA standards for tear samples and monomeric IgA standards for serum samples. [corrected] Tears from subjects without conjunctival inflammation contained 93% polymeric IgA (pIgA) and 7% monomeric IgA; tears from subjects with conjunctival inflammation had less pIgA (79%). Biopsy specimens of 11 lacrimal glands and one accessory lacrimal gland were obtained from 12 additional subjects. Tissues were stained with polyclonal antisera for immunoglobulins, J chain, and secretory component. In addition, tissues were stained for IgA subclasses with monoclonal reagents specific for IgA1 and IgA2. IgA plasma cells predominated over other types of plasma cells, and most were J chain positive. Secretory component, although absent from the interstitium of the gland, was found in most acinar and ductal cells. The average proportion of IgA1 to IgA2 cells was 56% to 44%. We concluded that the lacrimal system of the human resembles other exocrine systems in the production of predominantly pIgA and the nearly equal occurrence of IgA1 and IgA2 plasma cells.     

Occurrence of IgA subclasses (IgA1 and IgA2) in the human nervous system. Correlation with disease

The occurrence of IgA subclasses in pathological conditions of the nervous system was studied by means of monoclonal antibodies and an indirect immunofluorescence technique. IgA1- and/or IgA2-positive lymphoid (plasma) cells were found in demyelinating diseases comprising multiple sclerosis, Guillain-Barré syndrome, and adrenoleukodystrophy, in various inflammatory diseases, and in tumors, some of which exhibited labeling of tumor cells. Demyelinating and inflammatory diseases with chronic course displayed some prevalence of IgA2-, and tumors some prevalence of IgA1-positive cells. This is the first demonstration of IgA1 and IgA2 in the nervous system.     

Analysis of HER2 by chromogenic in situ hybridization and immunohistochemistry in lymph node-negative breast carcinoma: Prognostic relevance

In patients with lymph node-negative breast carcinoma (LNNBC), the prevalence of HER2 overexpression and gene amplification and their prognostic value have not been extensively evaluated. We examined 162 patients with LNNBC with complete follow-up. Immunohistochemistry (IHC) for HER2, Ki67, and p53 was performed. HER2 gene status was analyzed by chromogenic in situ hybridization (CISH) and discordant cases by fluorescence in situ hybridization. HER2 overexpression was seen in 24.7% of cases (40/162) and amplification by CISH in 17.6% (28/159). Agreement between IHC and CISH was achieved in 147 (92.5%) cases. Amplification was seen in 21 (100%) of 21 (3+), 6 (35.3%) of 17 (2+), and 1 (0.6%) of 121 (0-1+) tumors. Fluorescence in situ hybridization detected 3 (1.8%) additional cases. HER2 overexpression and amplification were present in tumors of high grade, with necrosis and lymph-vascular invasion (LVI) (all P < .027). In addition, amplified tumors showed Ki67 of more than 20% and p53 overexpression (P < .05). By univariate analysis, shorter disease-free survival (DFS) and overall survival (OS) were seen for patients with tumors showing HER2 amplification, LVI, and Ki67 of more than 20% (P < .05) (Kaplan-Meier). However, the multivariate analysis (Cox regression) demonstrated only Ki67 as an independent prognostic factor for both DFS (P = .017) and OS (P = .010), and as a trend for HER2 gene status (OS, P = .087) and LVI (DFS, P = .11; OS, P = .063). We conclude that IHC is a reliable method for detecting HER2 expression that can be complemented by CISH in nondefinitive cases (2+). Moreover, CISH is a valuable tool for the assessment of HER2 gene status with potential prognostic value and, therefore, in clinical decision making for treatment of high-risk LNNBC.     

Detection of human herpes virus 6 in AIDS-associated retinitis by means of in situ hybridization,polymerase chain reaction and immunohistochemistry

The ubiquitous nature of HHV-6 and its genomic relationship with cytomegalovirus led us to evaluate an etiological link between HHV-6 and AIDS-associated retinitis in a prospective study. HHV-6 infection was studied in patients with AIDS-associated retinitis and in two control populations. Eye pairs were obtained at necropsy from nine patients with AIDS-associated retinitis, four human immunodeficiency virus (HIV)-seropositive patients with normal fundus examination and three HIV-seronegative patients. HHV-6 infection was detected by polymerase chain reaction (PCR), in situ hybridization and immunohistochemistry. Human cytomegalovirus (CMV) and HIV-1 infections were detected in parallel by the same methods. HHV-6 infection was detected in three cases of AIDS-associated retinitis. In two of these patients, HHV-6 infection was detected both by immunohistochemistry and PCR while in the third case it was detected by in situ hybridization and PCR. In the three patients, fundus examination showed bilateral retinitis in two of them and unilateral retinitis in one of them. HHV-6 infection was not detected in the retina of the two control groups. CMV was also detected in the three cases positive for HHV-6 by all three methods. HIV DNA was detected by PCR in two of three cases and was confirmed in one of these cases by in situ hybridization. These results confirm that HHV-6 infects the retina but suggests that HHV-6 does not have an exclusive causative role in AIDS-associated retinitis, since CMV coinfection of the retina was detected in all three of the patients positive for HHV-6. © 1996 Wiley-Liss, Inc.