Interpretation and quantification of immunostains.

Despite the fact that immunohistochemistry is widely used in routine diagnostic work and is a very common part of scientific reports in pathology and cytology, its standardization still lags behind. Interpretation of immunostains should be based on microanatomic distribution of the staining, proportion of positively stained cells, staining intensity, if relevant, and cutoff levels. These parameters should be shown to be reasonably reproducible and should be clearly defined in publications. Uniformity in the setting of thresholds could probably benefit from interlaboratory control materials containing defined amounts of the target antigen. Reliable and precise quantitative immunohistochemistry requires the use of control materials containing defined amounts of the target antigen and processed alongside the specimen combined with automated computer-assisted microspectrophotometry. Application of these suggestions is hoped to improve standardization and to facilitate communication in the field of immunohistochemistry.

Effect of formalin tissue fixation and processing on immunohistochemistry.

Although immunohistochemistry is routinely performed by many pathology laboratories, its standardization still lags behind. A major cause of variation in the reproducibility of immunohistochemical staining is induced by tissue fixation and, to a lesser degree, tissue processing. This report, stemming from the first meeting of the International Consensus Group on Standardization and Quality Control (ICGSQC) in Nice, France, summarizes the problem and suggests solutions to begin to achieve standardization of fixation and processing. Most laboratories use neutral-buffered formalin (10%) for tissue fixation which introduces cross-links, whereas coagulative fixatives are less popular. Problems with formalin fixation comprise delay of fixation and variations in the duration of the fixation mainly. Solutions to these problems could be to start fixation soon (<30 min) after surgical removal of the tissue and to avoid overfixation (>24-48 hrs). For tissue processing, the most important problem is inadequate tissue dehydration prior to paraffin embedding. This can be prevented by preparing all solutions freshly every week, depending on the volume of tissue processed. If consistently applied, these procedures could eliminate some of the sources of variation in immunohistochemical stains.

Lymphohistiocytoid mesothelioma. An often misdiagnosed variant of sarcomatoid malignant mesothelioma.

Three cases of lympho-histiocytoid mesothelioma, a rare variant of pleural sarcomatoid malignant mesothelioma, are described. Histologically, the neoplasms were characterized by a diffuse discohesive proliferation of atypical histiocytoid cells intermixed with a marked lymphocytic and lesser plasmacytic infiltrate. One case initially was misdiagnosed as a ganglioneuroma, a second case was misinterpreted as malignant lymphoma, and a third case was sent in consultation with the differential diagnosis of inflammatory pseudotumor vs mesothelioma. Immunohistochemical studies showed strong and generalized expression of cytokeratins and vimentin by the neoplastic histiocytoid cells in all 3 cases. Two cases were positive for calretinin, one of which also was positive for HBME-1, thrombomodulin, and LeuM1. None of the cases stained with the epithelial glycoprotein markers carcinoembryonic antigen, B72.3, and Ber-EP4, or the blood group antigen, BG-8. The immunophenotype of the lymphoplasmacytic infiltrate revealed predominantly reactive, mature T cells, with fewer polytypic plasma cells, histiocytes, and B cells. In lymphohistiocytoid mesothelioma, as in the usual examples of sarcomatoid mesothelioma, the demonstration of cytokeratin expression by the neoplastic cells is the most useful diagnostic finding that allows exclusion of other neoplasms with which this entity may be confused.

Follicle center lymphoma and Warthin tumor involving the same anatomic site. Report of two cases and review of the literature.

We report 2 cases of follicle center non-Hodgkin lymphoma (NHL) and Warthin tumor involving the same site. Case 1 is a 68-year-old woman with Warthin tumor and grade 1 follicular NHL involving a periparotid lymph node. She had localized NHL and was treated with radiation therapy; dissemination developed 54 months later. Case 2 is a 55-year-old man with a 17-year history of a parotid mass with gradual enlargement during the last 5 years. Surgical excision revealed Warthin tumor and grade 1 follicular NHL involving the right parotid gland and surrounding lymph nodes. Immunohistochemical studies supported the diagnosis of NHL in both cases; the neoplasms were positive for CD20 and BCL-2 and negative for CD3. Polymerase chain reaction analysis done on paraffinembedded tissue of case 1 revealed monoclonal immunoglobulin heavy chain gene rearrangement and bcl-2/JH fusion DNA sequences diagnostic of the t(14;18)(q32;q21). The small size of the Warthin tumor in case 1, clearly arising in lymph node, supports the hypothesis that Warthin tumor arises from heterotopic salivary gland ducts within lymph nodes. The localized NHL in both patients suggests that the NHL initially arose in the lymph node involved by Warthin tumor, and, thus, the Warthin tumor may have provided a source of long-term antigenic stimulation from which a monoclonal B-cell population subsequently arose.

Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer. The Quicgel method.

Variation in tissue fixation, processing, and staining is largely responsible for poor reproducibility of estrogen receptor (ER) immunohistochemistry assays. A frozen, agar-suspended pellet of MCF-7 cells with known ER content was added to each of 55 samples of invasive breast carcinoma (IBC), serving as a control. Image analysis determined percentages of positive area (positive nuclei per total nuclei analyzed) and positive stain (sum of optical density of the positive nuclear area divided by sum of the optical density of all nuclei studied) of MCF-7 cells and IBC. MCF-7 cells had a mean value of 150 fmol/mg of ER by dextran-coated charcoal analysis. Image analysis of MCF-7 cells included with the 55 cases showed a mean positive area of 70.81. Positive staining from the IBC cases ranged from 0 to 98.5. By using the known ER content and the positive area of the MCF-7 cells, a conversion factor was used to translate the positive area of the clinical specimens to a femtomole equivalent, which for the 55 IBCs ranged from 0 to 1,790 (mean, 187). Inclusion of a control with known femtomole quantity of ER provides an internal standard for quality control and ER quantitation.

Intravascular large B-cell lymphoma: the CD5 antigen is expressed by a subset of cases.

The CD5 antigen is a T-cell associated marker that is also usually expressed by two B-cell neoplasms, chronic lymphocytic leukemia/small lymphocytic lymphoma and mantle cell lymphoma. We observed CD5 antigen expression in a subset of cases of intravascular large B-cell lymphoma (IVLBL), and we report here five cases. The patients, two men and three women, ranged in age from 59 to 81 years. Biopsy specimens were obtained from kidney, lung, bone marrow, abdominal wall, and neck, the latter involving a lymphangioma. All of the cases had histologic features typical of IVLBL, with large and atypical lymphoid cells located predominantly within blood vessels. Immunohistochemical studies performed using routinely fixed, paraffin-embedded tissue sections showed that the neoplastic cells were B cells, positive for the CD20 antigen and negative for the CD3 or CD43 antigens. All cases were also positive for the CD5 antigen. One case had an immunoglobulin heavy chain gene rearrangement shown by using a polymerase chain reaction method. The finding of CD5 antigen expression in a subset of IVLBL cases adds to other evidence in the literature suggesting that IVLBL is a heterogeneous entity. We considered the possibility that these cases were related to or represented unusual histologic forms of transformation from either chronic lymphocytic leukemia/small lymphocytic lymphoma or mantle cell lymphoma. All of the cases, however, were negative for the CD23 antigen and cyclin D1 (bcl-1) protein, which is evidence against this interpretation. The biologic significance of CD5 antigen expression in cases of IVLBL is uncertain. These neoplasms might arise from a separate lineage of CD5-positive B cells or from a specific, early stage of B-cell differentiation. Alternatively, some investigators have suggested that CD5 antigen expression by B cells is a marker of activation.

The immunohistochemical diagnostic panel for epithelial mesothelioma: a reevaluation after heat-induced epitope retrieval.

The immunohistochemical diagnosis between epithelial mesothelioma and adenocarcinoma is currently based on the use of a panel of antibodies to adenocarcinoma-associated antigens and a few antibodies to mesothelial-associated antigens. Since the introduction of epitope retrieval methods, the sensitivity of many antibodies has been enhanced. Thus, a reevaluation of the mesothelioma/adenocarcinoma diagnostic panel becomes necessary. We studied 268 paraffin-embedded formalin-fixed tumor samples that included 57 epithelial mesotheliomas and 211 adenocarcinomas of various origins, comparing an extensive antibody panel with and without heat-induced epitope retrieval (HIER). Marked increase in the sensitivity of several antibodies, with no loss of specificity, was found when HIER was used. After statistical analysis, the antibodies to the epithelial glycoproteins carcinoembryonic antigen, BerEp4, and Bg8 emerged as the best discriminators between adenocarcinoma and epithelial mesothelioma within the entire panel. The mesothelium-associated antibodies, HBME-1, calretinin, and thrombomodulin were less sensitive and less specific than the former, although they were found to be useful on certain cases. Antibodies to cytokeratins and vimentin, although of minor diagnostic value in this context, may be helpful to evaluate the quality of antigen preservation. This study confirms the value of immunohistochemistry to accurately distinguish mesothelioma from adenocarcinoma when an antibody panel approach is used. The addition of heat-induced epitope retrieval methods increases the effectiveness of the procedure and is recommended for most of the antibody panel members.

The effect of decalcification on in situ hybridization.

In situ hybridization (ISH) for mRNA polyadenylated sequences was performed on 25 non-neoplastic and neoplastic decalcified, paraffin-embedded tissues using a poly d(T) oligonucleotide probe to assess the efficacy of this molecular diagnostic tool on decalcified tissue samples. Three commercially available decalcifying agents were used, including one EDTA-based solution (Versenate) and two hydrochloric acid-based solutions (S/P Decal, RBD). Before decalcification, the tissues were fixed in formalin for 6, 24, and 72 hours, respectively. The results of ISH performed on decalcified tissues were compared with the results from the nondecalcified control samples for each tissue using a numeric scoring system (0, negative; 4, strong positivity equal to control; 5, stronger than control). There was generally excellent reactivity when using Versenate (mean, 4.15), good reactivity with S/P Decal (mean, 3.17), and fair-to-poor reactivity with RBD (mean, 1.69) (all P values < .0001). The length of time in formalin did not affect the outcome of ISH on these tissues. We conclude that ISH can be performed with success on decalcified, paraffin-embedded tissues when using Versenate, an EDTA-based agent. Although accurate results might be obtained with S/P Decal and RBD, caution should be exercised when using these two hydrochloric acid-based solutions because they might produce false-negative results.

Solitary fibrous tumor of the skin.

Solitary fibrous tumor (SFT) is a rare mesenchymal neoplasm that most commonly involves the pleura, but is increasingly more often observed in extrapleural locations. A 37-year-old woman presented with an SFT involving the skin and subcutaneous tissue of the scalp. Histologically, SFT is well circumscribed and composed of uniform spindle cells arranged in interlacing fascicles. It exhibits alternating hypercellular and hypocellular areas with abundant thick, often keloid-like, hyalinized collagen. Hemangiopericytoma-like areas are frequently prominent. Immunohistochemical markers for smooth muscle, neural, and epithelial differentiation are negative, but generalized positivity for CD-34 is characteristic. Because of the expanding spectrum of anatomic involvement of SFT, it is not surprising that on rare occasions this tumor may involve the skin.

Intravascularly disseminated angiosarcoma: true neoplastic angioendotheliomatosis? Report of two cases.

Although vascular invasion is common in many malignant tumors, disseminated intravascular anaplastic neoplasms with occult primary tumor are rare occurrences. Intravascular malignant lymphoma, also called angiotropic lymphoma, is a rare variant of large cell lymphoma predominantly involving vessels in multiple organs, and usually without significant nodal involvement. Although initially misinterpreted as an endothelial neoplasm-angioendotheliomatosis-immunohistochemical studies subsequently proved it to represent a peculiar form of malignant lymphoma. In this report, we describe two patients with extensive intravascular dissemination of angiosarcoma initially without clinically obvious primary tumor. These may be interpreted as examples of true angioendotheliomatosis. In each case the immunohistochemical studies ruled out the most common intravascular malignant neoplasms. The diagnosis of intravascular angiosarcoma was confirmed by the immunoreactivity of the tumor cells to several markers of endothelial lineage in both cases. Thus, angiosarcoma may present with intravascular dissemination and occult primary tumor and closely resemble metastatic carcinoma, melanoma, or angiotropic lymphoma. Immunohistochemical studies are crucial in ruling out these possibilities and in confirming the endothelial origin of the neoplastic cells.

HBME-1 immunostaining in thyroid fine-needle aspirations: a useful marker in the diagnosis of carcinoma.

The monoclonal antibody, HBME-1, generated against the microvillous surface of mesothelial cells, has been shown to have significant reactivity in histologic sections of follicular-derived thyroid malignancies. We examined the diagnostic utility of HBME-1 in thyroid fine-needle aspiration (FNA) specimens. Twenty-four aspirates from 23 patients were evaluated. Only cases with adequate cell blocks and tissue follow-up were studied. Immunocytochemical analysis was performed on air-dried, direct smears and on sections from Bouin's-fixed, paraffin-embedded cell blocks with a standard avidin-biotin peroxidase complex method with epitope retrieval. The same immunostaining technique was applied to the corresponding formalin-fixed tissue sections. Eight (57%) of the 14 malignant aspirates showed strong cytoplasmic and/or membrane immunoreactivity for HBME-1. The cell-block and direct-smear preparations were positive in five of seven papillary carcinomas (one follicular variant), one of one minimally invasive follicular carcinoma, and one of four hybrid tumors with mixed papillary and follicular features. An additional hybrid tumor case was focally positive only in the smear slide. The eighth positive case was an adenosquamous carcinoma of the larynx that invaded into the thyroid (smear preparation was negative for this case). The 10 benign lesions were negative. All of the malignant-tumor tissue sections were positive for HBME-1, and focal positivity was seen in 5 of 10 benign resection specimens. We conclude that a positive immunostain for HBME-1 on a thyroid FNA is supportive evidence that the lesion is a carcinoma, that a negative result for HBME-1 does not preclude the diagnosis of thyroid carcinoma, and that HBME-1 can be effectively applied to thyroid FNA specimens and can be a valuable adjunct in the cytologic diagnosis of thyroid malignancies.

Absence of Kaposi's sarcoma-associated herpesvirus-like DNA sequences in malignant vascular tumors of the serous membranes.

Reports connect Kaposi's sarcoma-associated herpesvirus (KSHV) with various types of Kaposi's sarcoma (KS) and body-cavity-based lymphomas (primary effusion lymphomas). KSHV DNA sequences have also been detected in a few non-KS malignant vascular tumors. To determine whether KSHV is associated with malignant vascular tumors involving the body cavities, 11 primary vascular tumors of the serous membranes (4 angiosarcomas, 7 hemangioendotheliomas) were tested for KSHV sequences by polymerase chain reaction and Southern blot hybridization. The tumors were from patients who were not known to be immunocompromised. KSHV sequences were detected in the penile KS control but were absent in all 11 specimens studied. Our results were consistent with previous reports that KSHV sequences were rarely detected in non-KS vascular lesions. The results also suggested that although KSHV sequences might be present in primary effusion tumors such as primary effusion lymphoma, they might not be found in other tumors related to body cavities or to serous membranes.

Malignant vascular tumors of the serous membranes mimicking mesothelioma. A report of 14 cases.

Malignant endothelial neoplasms involving the serous membranes are rare, and only a few cases have been documented. We report 14 patients with epithelioid hemangioendothelioma (EHE) or epithelioid angiosarcoma (EA) diffusely involving the pleural, peritoneal, or pericardial cavities, resulting in a picture closely resembling mesothelioma. The mean age at diagnosis was 52 (range, 34-85). The patients included two women and one man with peritoneal tumors, eight men with pleural tumors, and three men with pericardial tumors. A shared histological appearance was a diffuse sheet-like and clustered pattern of tumor growth with variable degrees of vascular differentiation. A tubulopapillary growth pattern, often seen in mesothelioma, was prominent in four cases. Nine cases showed a variable number of spindle cells, some neoplastic, others reactive, focally producing a biphasic growth pattern, further suggesting mesothelioma. Initial interpretations included mesothelioma, adenocarcinoma, and, in one case with prominent spindle-cell components, leiomyosarcoma. Immunohistochemically, strong vimentin staining and negative or weak to moderate cytokeratin staining were observed in all 14 cases. The tumor cells coexpressed at least two of the four endothelial markers used in the study (CD31, CD34, von Willebrand factor, and Ulex europaeus agglutinin-I [UEA-I)]. Detection of abortive vessel formation was facilitated by staining for collagen type IV. Markers of mesothelial, epithelial, muscular, and neuronal differentiation were all negative in the subset of cases studied. As a control group, 39 mesotheliomas and more than 60 adenocarcinomas of various origins were studied using the same antibody panel. This group revealed strong keratin staining, moderate or negative vimentin staining, and no expression of any of the endothelial-lineage markers, with the exception of positive staining for UEA-I in occasional adenocarcinomas. Clinically, these endothelial tumors were highly aggressive; 12 patients presented with disseminated disease, and most died within months of the initial presentation. These findings indicate that, although uncommon, EHE/EA should be included in the differential diagnosis of serous membrane neoplasms with histological and clinical features of malignant mesothelioma. The diagnosis of an endothelial neoplasm can be suspected by the presence of abortive vessel formation and by the strong expression of vimentin, with absent or low-level expression of cytokeratin. The demonstration of immunoreactivity for two or more endothelial-associated markers is essential in confirming the diagnosis.

Endothelial area as a prognostic indicator for invasive breast carcinoma.

BACKGROUND: Vascular enumeration using antibodies to Factor VIII has been reported to be an independent prognostic indicator of invasive breast carcinoma. METHODS: To eliminate potential subjectivity in distinguishing between individual vessels, especially in areas of tangled capillaries, total endothelial area (EA) was assessed using a Samba 4000 image analyzer. One hundred seventy-eight invasive breast carcinomas (Stage 1 and 2, mean follow-up: 71 months) were immumostained for the presence of CD34, the human hematopoietic progenitor cell antigen also present in endothelium, and EA was quantitated within 5 adjacent 20X fields (0.74 mm2). Additionally, these same vessels were manually counted from the image analyzer. Manual counts were also made from a photomicrograph representative of a single 10X field (1.06 mm2). RESULTS: High grade carcinomas contained greater endothelial area than low grade carcinomas (P = 0.0001). Endothelial area was prognostically significant (P = 0.004) in univariate analysis of disease-free survival (DFS) and overall survival (OS), as were stage of disease, tumor size, and combined histologic grade (P < or = 0.024). Manual vessel counts from the monitor were significant for OS only. Manual vessel counts from photomicrographs showed no statistically significant association with DFS or OS. In multivariate analysis, EA, but not vessel enumeration, remained as an independent predictor for OS (lymph node negative patients only, n = 87) and for DFS (lymph node positive patients only, n = 91). For the entire group of patients (lymph node negative and lymph node positive) independent predictors of DFS and OS were tumor grade and size (P < or = 0.006). CONCLUSIONS: Of the three methods used to evaluate tumor angiogenesis, total endothelial area, as objectively evaluated by image analysis, was the only independent prognostic indicator for OS for patients with lymph node negative invasive breast carcinoma.

Expression of bcl-2 by breast cancer: a possible diagnostic application.

Expression of bcl-2 is most commonly associated with the t(14;18) translocation present in most folicular lymphomas (1). More recently, bcl-2 oncoprotein has been identified in normal tissues and in nonhematologic malignancies. In this study, we investigate the use of bcl-2 as a marker to distinguish metastatic breast carcinoma from primary lung and gastric cancers, and we evaluate the role of bcl-2 as an independent prognostic factor in breast carcinoma and its relationship to other breast cancer markers. bcl-2 immunostains were done on 371 adenocarcinomas of the breast, lung, and stomach. Additionally, 231 samples of metastases from patients with breast or gastric cancer were evaluated for bcl-2 expression. All breast cancer tissue samples had immunohistochemical data on expression of estrogen and progesterone receptors, p53, neu/cerb2, and MIB-1. A large proportion (79.3%) of invasive breast carcinomas expressed bcl-2, whereas only 5.6% and 8.3% of pulmonary and gastric carcinomas did. Moreover, staining was moderate to intense in 70.2% of the breast cancers, compared with only one specimen of lung carcinoma (1.9%) and gastric carcinoma (0.9%) that showed moderate staining. There was agreement of bcl-2 expression between primary and metastatic sites in all specimens except one. Expression of bcl-2 in breast adenocarcinomas was significantly associated with hormone receptor positivity and low histologic grade. Nonetheless, 20.6% of bcl-2-positive specimens were estrogen receptor negative and 24.2% of bcl-2-positive specimens were progesterone receptor negative. Neither the presence nor the absence of bcl-2 expression significantly predicted disease-free survival or overall survival in patients with breast cancer. We conclude that adenocarcinomas with intense bcl-2 staining are more likely to be of breast than of pulmonary or gastric origin. We recommend the addition of bcl-2 to a panel of antibodies (estrogen receptor, GCDFP-15, and S100) that might contribute to the identification of a larger proportion of metastatic breast carcinomas, because almost one-half of the estrogen-receptor negative cancers were bcl-2 positive.