Brent H. Dorsett

Identification of prekeratin by immunofluorescence staining in the differential diagnosis of tumors

Fibrillar proteins with a role in cellular shape and motility are present in the cytoplasm of most animal cells. They vary greatly in size, organization, and reactivity according to cell type and can be separately identified by the use of recently developed monospecific antisera and indirect immunofluorescence staining. Prekeratin, a structural protein in the form of intermediate sized filaments is present exclusively in cells of epithelial origin. In the present study 41 human tumors of various organs and their normal tissue counterparts were reacted with prekeratin antiserum and examined by immunofluorescence staining in paraffin embedded sections. Prekeratin was identified in all epithelial cells of the squamous type, which gave the strongest staining reaction, and in smaller amounts in epithelial cells of other histologic types. Cells of lymphoid, melanic, neural, and connective tissue origin were not stained. Thus, combining the specificity of antiprekeratin sera with the selectivity of immunofluorescence staining resulted in a new method of identifying tissues that is applicable to the differential diagnosis of tumors.

EBV-associated anorectal lymphomas in patients with acquired immune deficiency syndrome.

Primary lymphomas of the gastrointestinal tract represent 9% of all non-Hodgkin lymphomas, and of these only 3% arise in the rectum or anus. In contrast to their rare occurrence in the general population, the incidence of anorectal lymphomas in patients with acquired immune deficiency syndrome (AIDS), particularly homosexual patients, may be as high as 26% as reported in our own series of AIDS-associated lymphomas. To determine the characteristics of this entity, we studied 15 cases of primary anorectal lymphoma in AIDS patients and compared them with four cases of anorectal lymphoma unrelated to AIDS. The cases in our study were also compared with the reports of rectal lymphoma in the medical literature over the past 30 years. In the present series, the AIDS patients were all male with a median age of 34 years, human immunodeficiency virus (HIV)-positive, with homosexuality as the main risk factor. The four non-AIDS patients included a woman and had a median age of 66.5 years. Histologically, the anorectal lymphomas in AIDS patients were all high grade, predominantly immunoblastic, and polymorphous. In the non-AIDS patients, only two of four lymphomas were high grade, including one Burkitt type. All tumors were of B-cell phenotype. In the AIDS-associated anorectal lymphomas, the presence of Epstein-Barr virus (EBV) in a latent form was demonstrated by an abundance of Epstein-Barr-encoded RNA (EBER) in 14 of 15 cases and latent membrane protein (LMP) in four cases. All anorectal lymphomas unrelated to AIDS were negative for EBV. The unusual anorectal location of AIDS-associated lymphomas is explainable by the high incidence of preceding traumatic lesions and chronic infections in the area. As a result, EBV-carrying B cells may be attracted to the field providing the cell population that, under the conditions of immune deficiency, is able to give rise to high-grade lymphomas.

Lymphoid monoclonal antibodies reactive with lung tumors : diagnostic applications

In the course of investigating 30 monoclonal antibodies (MAbs) for their potential reactivity with 25 lung tumors of different histologic types, we found that three MAbs commonly used for their specificities for lymphoid markers were highly reactive with non-small-cell carcinomas (NSCLC) and totally nonreactive with small-cell carcinomas (SCLC). Immunostaining was performed by the standard streptavidin-biotin-peroxidase method after microwave antigen retrieval on formalin-fixed, paraffin-embedded tissue sections. LN2 (CD74), LN3 (HLA-DR), and BLA-36, which are commonly used for the identification of B-lymphocytes, strongly immunostained 19 of 25 squamous and adenocarcinomas and none of 34 small-cell carcinomas and carcinoids. Moreover, in combined tumors, these MAbs selectively stained the adenocarcinoma cells but not the adjacent small-cell carcinoma cells. A cocktail mixture of LN2, LN3, and BLA-36 assayed on 24 additional lung tumors produced similar results with even stronger and sharper stainings. Other lymphoid MAbs showed some selective staining but to a lesser degree. Among nonlymphoid MAbs, the results were as expected, with MAbs for cytokeratin (B72.3) and epithelial membrane antigen staining NSCLC but also some SCLC. The MAbs for chromogranin and neuron-specific enolase were not entirely specific, whereas some nerve-cell adhesion molecule MAbs showed good specificity for SCLC. In a field with few specific MAbs, the newly discovered ability of these lymphoid MAbs to discriminate between SCLC and NSCLC may prove useful in the immunohistochemical diagnosis of lung tumors.

EBV-associated Primary Lymphomas in Salivary Glands of HIV-infected Patients

The lymph nodes within and around salivary glands are commonly involved in inflammatory processes, but rarely the site of primary lymphomas. We observed six cases of primary salivary gland lymphoma in HIV-infected patients and studied them in parallel with three cases of primary salivary gland lymphoma unrelated to HIV and three cases of HIV-related salivary gland lymphadenopathies in order to characterize this new entity. We found that all salivary gland lymphomas in HIV-infected patients were of high histologic grade while salivary gland lymphomas unrelated to HIV were predominantly of low grade MALT type. All lymphomas in both categories expressed the B-cell phenotype. Just as HIV-unrelated lymphomas frequently arise on the background of chronic inflammatory lymphoid processes, lesions characteristic of HIV-lymphadenopathy were still present in some lymphomas of HIV-infected patients. EBV RNA transcripts (EBER) were demonstrated in three, and latent membrane protein (LMP) in two of the six HIV-related and in none of the three HIV-unrelated lymphomas. The three EBER-positive lymphomas were of the histologic types known to express the virus in most cases. The presence of HIV in the form of the core protein p24 and envelope glycoprotein gp41 on the dendritic reticular cells of germinal centers was ascertained in the cases of HIV-related lymphadenopathies but also in the coexistent lymphadenopathies of lymphomas. The practical importance of diagnosing the salivary lymphadenopathies and lymphomas associated with the HIV-infection resides in avoiding their misdiagnosis and surgical removal as tumors of salivary glands.

Lymphoid proliferations and lymphomas associated with gastric metaplasia, dysplasia, and carcinoma.

Gastric carcinomas are invariably accompanied by lymphoid proliferations. We studied their features in 22 resected gastric carcinomas in which the lymphoid proliferations ranged from reactive lymphoid follicles to mucosa-associated lymphoid tissue (MALT) lymphomas. In most cases, the collections of lymphocytes were abundant, which is remarkable considering the lack of lymphoid tissue in the normal stomach. They were not haphazardly located but in direct contact with the metaplastic, dysplastic, and neoplastic epithelial cells, in positions suggestive of defense barriers. They consisted of newly formed lymphoid follicles with reactive germinal centers sometimes high up in the superficial mucosa, collections of plasma cells beneath the surface epithelium, and large aggregates of B cells above and below the muscularis mucosae as well as abundant T cells. The latter, both CD4+ and CD8+, were seen within metaplastic epithelial cells as well as within carcinomatous glands that were partially destroyed, resembling apparent neoplastic lympho-epithelial lesions (LEL). In three cases, the B cells infiltrating the gastric muscular layers represented MALT-lymphomas adjacent to gastric carcinomas, as confirmed by polymerase chain reaction (PCR) analysis in two cases. In a case of lymphoepithelioma-like carcinoma, the excessive lymphoid cells were predominantly of T-CD8+ type. In this case, EBV identified by EBV-encoded RNA and latent membrane protein was present in large amounts. Helicobacter pylori was seen in only six cases in areas of chronic gastritis that were distant from carcinoma. H. pylori was not present in the areas of metaplasia, dysplasia, or carcinoma. It appears that the lymphoid proliferations accompanying these gastric changes do not arise in response to the pathogenic agent H. pylori, which caused the persistent infection leading to them yet is no longer present, but rather in response to the existence of the abnormal epithelial cells. Thus the lymphoid proliferations consistently associated with gastric metaplasia, dysplasia, and neoplasia may be regarded as immune reactions to the long-term cellular changes triggered by the initial chronic gastritis. On rare occasions, the exaggerated lymphoid proliferations may reach the end of the spectrum, resulting in MALT lymphomas coexistent with gastric carcinomas.

Description of an endometrioid ovarian cancer cell line

Abstract A human cell line, designated L-1, has been established from the ascites of an untreated patient with stage IV (FIGO) endometrioid ovarian cancer. This cell line initially grew uninterrupted for 6 months without fibroblast contamination and contact inhibition, and has been subcultured weekly for the past 7 years. L-1 does not contain steroid hormone receptors nor does it demonstrate the presence of oncofetal antigens by immunohistochemical techniques. The doubling time of L-1 is 11.8 hr. Flow cytometric analysis reveals an aneuploid DNA peak, and an abnormal karyotype demonstrates hyperdiploidy, translocations, and deletions. Morphology, growth patterns, cytogenetic analysis, and other features of L-1 are characterized.

Immunohistochemical characterization of a monoclonal antibody detecting an endometrioid ovarian cancer-associated antigen.

Murine monoclonal antibody FEN-1 was derived by immunizing Balb/c mice with an affinity-purified endometrioid ovarian cancer-associated antigen recovered from ascites-derived immune complexes. Splenic lymphocytes from the immunized mouse were fused with the myeloma cells SP2/0-AG14 in the presence of PEG 1500. The hybrid cultures were screened for production of immunoglobulins reactive with an extract preparation of an endometrioid ovarian tumor by enzyme-linked immunosorbent assay and flow cytometry. One of the hybrids secretes a monoclonal antibody of the IgG3 subtype designated FEN-1, which reacts with 100% of endometrioid ovarian cancer containing adenoacanthoma by indirect immunoperoxidase on paraffin-embedded tissue. No detectable levels of antigen were found in squamous metaplasia associated with nonendometrioid tumors, and no reactivity occurred against endometrial adenocarcinomas, endometriosis, or normal ovary and endometrium. The antibody does not cross-react with mucinous tumors, nonepithelial tumors of the ovary, or gastrointestinal tissue. This antibody may be used as an aid in the diagnosis of nonmucinous ovarian carcinomas by immunohistology.

Ovarian cancer-associated antibodies recovered from ascites: Their use for the isolation of ovarian cancer-associated antigen to produce monoclonal antibodies

Immune complexes (ICs) were recovered from the ascites of a is similar in composition to serum, and is in immediate patient with stage IV endometrioid ovarian cancer by sequential contact with the tumor; therefore, it might be expected precipitation with 33% saturated ammonium sulfate and 2.5% to contain substantial levels of ICs. In our laboratory, polyethylene glycol 6000 (PEG 6000), followed by affinity chro- we have previously demonstrated that ovarian cancermatography on protein A-Sepharose CL4B. The IgG-containing associated antibodies (OCAAbs) recovered from such ICs were dissociated using 8 M urea, separated by ion-exchange ICs are tumor associated, and that the corresponding chromatography on Sephadex QAEJO, and subsequently ana- antigens are immunoreactive [7,8,15-171. We have delyzed for purity by immunoelectrophoresis (IEP) and radial im- veloped new methods allowing the recovery of sizable munodlffusion (RID). Recovered antibody was tested for reactivity by immunohistologlc techniques against paraffin-embedded quantities of IC-associated antibodies and antigens from tumor tissue and acetone-fixed cell suspensions of epithelial tu- malignant effusions in cancer patients. These antigens mors. The antibody which demonstrated ovarian cancer-associ- were successfully employed as immunogens to prepare ated activity was absorbed with antigen extracts of breast, colon, heterologous lung tumor-associated antisera [ 181. The and lung cancers as well as keratin to reduce cross-reactivity. production of monoclonal antibodies with tumor speciThe absorbed endometrioid ovarian cancer-associated antibody ficity holds great potential for immunodiagnosis, nuclear (CXXAb) was used to produce an immunoadsorbent column for imaging, and immunotherapy of malignant disease. The the recovery of tumor-associated antigens. A mouse monoclonal selection of suitable immunogens for preparing such anantibody designated FEN-1 was produced using this antigen- tibodies is of critical importance. Immunization with containing fraction, and preliminary screening has demonstrated crude tumor cell extracts containing unknown quantities ovarian tumor-associated reactivity. The use of autologous ICs of antigens may limit the success of these procedures. as reagents for preparing tumor antigen-rich immunogens may provide a valuable tool in the search for tumor-associated anti- The finding that autologous ICs from malignant effusions contain substantial amounts of tumor-related antibodies

Anti HTLV-III and Anti T-Cell Antibodies in AIDS and ARC Patients

The number of surface immunoglobulins (SIg)-positive lymphocytes was found to increase when normal lymphocytes were incubated with sera of AIDS or ARC patients. These circulating antilymphocyte antibodies were selectively reactive with 0KT4- but not with 0KT8-staining T-cells. In the present study, AIDS, ARC and control patients were evaluated for antilymphocyte antibodies, anti HTLV-III antibodies and helper/suppressor (h/s) T-cell ratios. In the AIDS group, 23 of 24 patients had circulating HTLV-III antibodies, 24 of 24 showed markedly decreased h/s T-cell ratios and 19 of 21 patients showed marked elevation of anti-lymphocyte antibodies. In contrast, in control patients, none of 14 had HTLV-III antibodies, 13 of 14 had normal h/s T-cell ratios and 3 of 11 showed small increases in antilymphocyte antibodies. In the ARC group, 16 of 16 had circulating anti HTLV-III antibodies. 11 of 15 had decreased h/s T-cell ratios and 6 of 11 showed elevated anti T-cell antibodies. The correlations obtained between these tests and clinical diagnoses indicate their potential usefulness in the detection and monitoring of AIDS and ARC patients.