Prevalence of HTLV-I-associated T-cell lymphoma.

In order to assess the prevalence rate of HTLV-1-associated T-cell lymphomas and human retrovirus infection in general, approximately 21,000 individuals representing various patient populations, retroviral risk groups, and blood donors were examined for HTLV-I, HTLV-II, HIV-1, or HIV-2 infection using serologic and PCR assays. The prevalence rates among volunteer blood donors were 0.02% and 0% for HTLV and HIV, respectively. Significantly increased HTLV prevalence rates were observed among paid blood donors, African American health care clinic patients, Amerindians, recipients of HTLV-positive cellular blood products, intravenous drug users, sexual contacts and family members of HTLV-positive people, and patients with primary thrombocytosis and other-than-low-grade non-Hodgkin's lymphoma (NHL). Among some of these groups there were significant differences in the prevalence of HTLV-I versus HTLV-II. The eight HTLV-positive NHL patients all had mature, high-grade, CD4+ T-cell lymphomas with clonally integrated HTLV-I, for a prevalence of 4% among other-than-low-grade NHL patients. Seven of the eight died from their disease within 2 years despite treatment. Interestingly, two groups at risk for HTLV infection, namely needle stick victims and recipients of HTLV-infected and/or pooled plasma products, showed no evidence for infection. Significantly increased HIV-1 prevalence was observed among paid blood donors, African Americans, homosexuals, female prostitutes, hemophiliacs, and other-than-low-grade NHL patients. Only one patient was infected with HIV-2. Of the nine HIV-positive, other-than-low-grade NHL patients, seven HIV-1 positives had B-cell lymphomas, one HIV-1 positive had an HTLV-I-positive CD4+ T-cell lymphoma, and one infected with HIV-2 had a CD4+ T-cell lymphoma that was HTLV negative. The data indicate that HTLV-I lymphoma, while uncommon, is not necessarily rare among other-than-low-grade NHL cases in the United States and, given its poor prognosis, should probably be studied separately in clinical trials.

Microplate-based DNA hybridization assays for detection of human retroviral gene sequences.

Nonisotopic, microwell-based DNA hybridization assays for the specific detection of human immunodeficiency virus type 1 (HIV-1) gag, human T-cell lymphotropic virus type I (HTLV-I) pol, and HTLV-II pol DNA sequences were evaluated. The performances of these detection kits (Gene Detective enzyme oligonucleotide assays; Cellular Products, Inc., Buffalo, N.Y.) were assessed by using clinical samples whose infection status were established by amplification by PCR and then liquid hybridization detection by using virus-specific probes. Peripheral blood mononuclear cell lysates from 59 HIV-1-, 35 HTLV-I-, and 19 HTLV-II-infected individuals and from 15 healthy blood donors were used as substrates for PCR amplification. The results of the study demonstrated a clinical sensitivity of 100%. In addition, the enzyme oligonucleotide assays were able to detect 1 to 10 proviral copies subsequent to PCR amplification, indicating an analytical sensitivity comparable to that of liquid hybridization.

Monoclonal antibodies and chemiluminescence immunoassay for detection of the surface protein of human T-cell lymphotropic virus.

Monoclonal antibodies (MAbs) raised against human T-cell lymphotropic virus type I (HTLV-I) recognized five distinct antigenic domains of viral env gene-encoded proteins. By using recombinant env proteins and synthetic peptides as mapping antigens, it was determined that the most immunogenic region represented a central portion of the retroviral surface protein (domain 2; amino acids 165 to 191). However, only a single MAb was able to react strongly with native viral proteins. This antibody (clone 6C2) was directed to an epitope within domain 4 (amino acids 210 to 306) of the retroviral env gene and reacted with envelope proteins in both HTLV-I and HTLV-II, as determined by immunoprecipitation, solid-phase binding, and immunoblotting. No reactivity against envelope components of other human retroviruses, including human immunodeficiency virus types 1 and 2, was present. Flow cytometry data demonstrated that MAb 6C2 reacted with cell lines chronically infected with HTLV-I or HTLV-II and also with surface antigens expressed on fresh adult T-cell leukemia cells, following up-regulation with interleukin-2. By a chemiluminescence immunoassay procedure, picogram amounts of viral surface protein could be detected in the unconcentrated supernatants of HTLV-infected cell lines and in diagnostic cultures. Levels of env and gag proteins released by cells into culture supernatants were not directly related to percent expression of cell surface viral-coat proteins. Further, the molar ratio of p19 to gp46 in conditioned media varied from strain to strain, possibly reflecting differences in viral assembly or packaging mechanisms. MAb 6C2 will be of value in characterizing the biochemical and immunological behavior of retroviral env gene proteins and in studying the interaction of HTLV-I and HTLV-II with their receptors.

HTLV-I DNA sequences in CNS tissue of a patient with tropical spastic paraparesis and HTLV-I-associated myelopathy.

Human T cell lymphotrophic virus type I (HTLV-I) is the etiologic agent of adult T cell lymphoma/leukemia (ATLL) and tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM). We studied an HTLV-I-seropositive, white man diagnosed in 1977 with ATLL and 10 years later, 6 months prior to his death, with TSP/HAM. Sections of brain, spinal cord, and visceral tissues were examined histologically, immunohistochemically, by in situ hybridization, and by the polymerase chain reaction (PCR). PCR amplification of a region of the polymerase (pol) gene of HTLV-I from visceral tissue demonstrated the presence of proviral HTLV-I DNA in paraffin-embedded sections from the liver and in DNA extracted from frozen sections of kidney and spleen, but failed to demonstrate viral sequences in paraffin sections of the lung and a lymph node. PCR analysis of CNS tissue demonstrated viral sequences in regions of the brain including frozen samples from cerebellum and cerebral cortex and paraffin sections of the thoracic spinal cord, but failed to detect proviral DNA in sections from a region in the lumbar cord. These results map the distribution of HTLV-I DNA sequences in the CNS of a patient with TSP/HAM for 3 months.

Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a Cancer and Leukemia Group B study.

Three monoclonal antibodies (MAbs) (DF3, F36/22, CU18) were used to monitor expression of distinct epitopes present within a family of mucin-like, breast carcinoma-associated molecules. Primary tumor specimens from more than 190 stage II breast cancer patients were evaluated for expression of the high molecular weight antigens. With a median follow-up of 6 years, patients whose tumors exhibited high immunoperoxidase staining scores (greater than 50% positive cells) with MAb DF3 had a superior disease-free survival ([DFS] 56% +/- 6% v 37% +/- 5% at 6 years; P = .0088) and overall survival ([OS] 72% +/- 5% v 59% +/- 5% at 6 years; P = .025). Staining scores with the other two antibodies did not correlate with improved prognosis. For MAbs DF3 and CU18, patients whose tumors exhibited predominantly apical cellular reactivity patterns had improved DFS, although differences reached conventional levels of statistical significance only with MAb CU18. In multivariate analyses, the prognostic value of MAb DF3 staining was independent of other identified prognostic factors. Furthermore, the concordance between primary and axillary lymph node metastases staining with each MAb was 73%, 80%, and 85% for MAbs DF3, F36/22, and CU18, respectively. These results suggest that staining with MAb DF3 identifies a group of node-positive women with a relatively favorable prognosis. Expression of the DF3 mucin-like glycoprotein is related to better differentiation, and staining with MAb DF3 provides an accurate and objective estimate of clinical outcome independent of histopathologic evaluation.

Human immunodeficiency virus type 1-neutralizing monoclonal antibodies which react with p17 core protein: characterization and epitope mapping.

Monoclonal antibodies (MAbs) to human immunodeficiency virus type 1 were produced. Two antibodies reacted with the 17-kilodalton core protein (p17) of the virus and with its polyprotein precursor. To various degrees, each MAb neutralized infection by the cell-free virus. With a series of sequential overlapping hexapeptides which represent the p17 gene product, the epitopes identified by the MAbs were defined. The epitopes localize to overlapping regions near the amino terminus of the protein. Soluble synthetic peptides which span the antibody-binding sites of interest were demonstrated to competitively inhibit the reactivity of p17 MAbs, thus confirming the location of virus-neutralizing sites within the core protein.

Monoclonal antibody identifies a highly conserved and immunodominant epitope of the human immunodeficiency virus transmembrane protein.

A murine monoclonal antibody (MAb), 10E9, has been generated which identifies a conserved and immunodominant epitope of the human immunodeficiency virus (HIV) transmembrane protein, gp41. The MAb reacts with the protein backbone of the mature env gene product and also with polyprotein precursor, gp160. Human sera were tested for their ability to competitively inhibit the immunoreactivity of MAb 10E9. Of 100 serum samples obtained from patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC), all showed strong inhibition to the reaction. In contrast, sera obtained from normal donors or those with other viral infections failed to perturb the binding activity of MAb 10E9. The geographic diversity of the AIDS/ARC patients studied provides evidence that the 10E9 epitope of gp41 is highly conserved.

Physicochemical purification and immunological characteristics of ductal carcinoma antigen.

Ductal carcinoma antigen (DCA), as recognized by monoclonal antibody (MAb) F36/22, was purified in high yield and to homogeneity from malignant effusions by means of physicochemical techniques. Fractionation procedures were monitored by immunoenzymometric assays. Preparations purified over 2,000-fold were obtained through acid-extraction, lectin-chromatographies and gel filtration steps. Purified antigen demonstrated a single component upon electrophoretic examination. This material had a high molecular weight and high immunoreactivity. Neuraminidase treatments failed to perturb the antigenicity of the component, indicating an absence of sialic acid residues at the antibody-combining site. Further, extensive proteolytic digestion effected the release of heterogeneous glycopeptides which retained immunologic activity, suggesting the presence of carbohydrate at the active site. Immunologically, DCA was compared with other tumor-associated mucin-like glycoproteins which have been detected in the circulation of patients. Results indicated no cross-reactivity between DCA and other mucins, including CA19-9 and CA-125. Further, no antigenic relationship was noted for purified carcinoembryonic antigen (CEA), also a heavily-glycosylated structure. These data thus suggest that immunological monitoring of disease may be approached using a selected panel of antigenically distinct reagents.

Immunohistochemical demonstration of prostate-specific antigen in metastases with the use of monoclonal antibody F5.

With the use of a murine monoclonal antibody (F5), a panel of metastatic tumors was evaluated for the expression of prostate-specific antigen (PA) under immunoperoxidase staining procedures. Specimens studied included 25 of prostatic origin and 73 originating from nonprostatic primary sites. Regardless of the site of dissemination or the malignancy grade, all metastases from the prostate were antibody-reactive. In contrast, nonprostatic metastases were negative in each case, including those originating from other genitourinary neoplasms. Thus, PA expression as detected with monoclonal antibody F5 is a stable characteristic of disseminated prostatic tumors.

Prognostic importance of prostate-specific antigen for monitoring patients with stages B2 to D1 prostate cancer.

To evaluate the prognostic value of prostate-specific antigen (PA) for detection of tumor growth after definitive therapy, 602 sera from 70 patients with stages B2 to D1 prostate cancer (26 of whom recurred) were analyzed in a blind study. Using Cox's proportional-hazards model, a highly significant association was found between serially measured PA and disease-free survival time (p = 0.0002). A positive predictive value of 100% was found for some markedly elevated PA levels and confirmed recurrence of disease. In fact, this study suggested that once a PA level of 88 ng/ml was reached, there was an average time of less than 2 months before a recurrence was clinically confirmed. Tumor growth in patients who recurred was indicated by a PA elevation before recurrence in 92% (24 of 26) as opposed to 20% (9 of 44) in disease-free patients. Additionally, in these 24 of 26 patients, levels of PA were elevated 12 months (mean lead time) before a confirmed disease recurrence. In patients who were still disease free, serial PA appeared to increase concurrently with putative tumor growth as shown by the initial surgical stage. Generally, the greater the PA level the more advanced was the stage of disease (B2 to D1). These data suggest that PA may be a useful adjuvant marker for monitoring tumor growth in patients with regionally confined prostate cancer.

Recent progress in the immunological monitoring of carcinomas using monoclonal antibodies.

In efforts to improve the specificity and sensitivity of tests to monitor patients with carcinoma, monoclonal antibody technology has been widely embraced by the scientific community. Results clearly indicate that novel antigen determinants have been identified and that significant improvements have been realized for the study of "classical" tumor antigens previously detected with polyclonal reagents. The concept of tumor specificity has also been amenable to an increased level of objectivity as based upon the probelike characteristic of monoclonal antibodies. It is apparent from this work that an antigen does not have to be tumor specific to be useful as a marker.

Immunoaffinity isolation of ductal carcinoma antigen using monoclonal antibody F36/22.

Monoclonal antibody (McAb) F36/22, raised against a human breast tumor line, identifies an antigen found in the circulation of cancer patients. Antigen was purified from malignant effusions using McAb-affinity chromatography followed by adsorption-desorption from immobilized wheat germ lectin. Electrophoretic analysis demonstrated the isolation of a single high mol. wt glycoprotein exhibiting an isoionic point near pH 4.2 and a density of approx. 1.45 g/ml. Although highly reactive with wheat germ lectin, a negligible or weak interaction was observed with concanavalin A, lentil and peanut agglutinin. The antigen was immune-precipitable, indicating the occurrence of multiple McAb-binding sites, and was resistant to heat and acid treatments. Antigenicity was not perturbed following protease or neuraminidase treatments, but was affected upon exposure to alkaline conditions. Taken together, these data suggest that McAb F36/22 recognizes a high mol. wt component occurring in circulation as a mucin-like glycoprotein.

Expression of ductal carcinoma antigen in breast cancer sera as defined using monoclonal antibody F36/22.

A quantitative immunoassay procedure has been constructed to evaluate levels of ductal carcinoma antigen recognized by murine McAb F36/22. Using this method, 3% of 64 apparently healthy individuals and 13% of 40 patients with benign breast disease expressed serum antigen levels above 70 units/ml. Greater than 50% of 116 patients with clinical evidence of breast cancer demonstrated circulating ductal carcinoma antigen levels above 70 units/ml. Patients with ductal carcinomas of other sites, including prostate and gastrointestinal tumors, also demonstrated elevated levels of antigen (11 and 27%, respectively). The incidence of elevated serum ductal carcinoma antigen levels correlated significantly with the incidence of intratumoral antigen expression. Lectin binding, molecular weight, and density measurements indicated that circulating antigen occurs as a high-molecular-weight glycoprotein with mucin-like characteristics.

Reactivity of monoclonal antibody F36/22 with human ovarian adenocarcinomas.

Monoclonal antibody F36/22 recognizes high-molecular-weight glycoprotein components associated with neoplastic development of the ovary. Indirect immunoperoxidase staining techniques were performed on a panel of nonmalignant ovarian tissues, primary ovarian tumors, exfoliated ascitic tumor cells, and metastatic lesions. Normal ovarian tissue components (n = 20) failed to exhibit detectable levels of antigen, whereas benign ovarian tissues show a low incidence of immunostaining (three of 26) restricted to some ductal elements. One hundred % (19 of 19) of the immunopositive primary malignant tumors were histologically classified as adenocarcinomas. Each of the predominant adenocarcinoma histotypes consistently showed expression of the antigen with 30 to 100% of the tumor cells scored as immunopositive. Ascitic tumor cells obtained from all of the ovarian adenocarcinoma patients examined (47 of 47) displayed immunopositive reactions, whereas normal mesothelial cells in these specimens exhibited undetectable staining. In addition, ovarian adenocarcinoma metastases (12 of 12) exhibited very intense immunoreaction products. No detectable antigen was expressed by nonadenocarcinoma ovarian tumor cells.

Relationship between antigen density and immunotherapeutic response elicited by monoclonal antibodies against solid tumors.

The relationship between the level of cell surface antigen expression and solid tumor immunotherapy with monoclonal antibody (MoAb) was evaluated. Two MoAb's that were shown effective in the passive therapy of breast carcinomas of human origin, established and growing in female Swiss nude mice, were used for these studies. Several groups of tumors were produced from cell cultures of different passages; each cell culture possessed a distinct target antigen level. Results from immunotherapy experiments demonstrated that the amount of tumor reduction response after MoAb therapy was proportional to the antigen density at the cell surface. Analysis of these data indicated a theoretical improbability of a single MoAb treatment being able to completely eradicate solid tumors and may necessitate the use of multiple MoAb's to circumvent this problem.

Experimental tumoricidal effects of monoclonal antibody against solid breast tumors.

Two distinct monoclonal antibodies (mAbs) were effective in the therapy of breast carcinomas of human origin established and growing in nude mice. Passive administration of either of the antibodies produced very rapid (less than 1 week) and significant reduction of in vivo tumor volume. Each of the mAbs showed in vivo targeting of the tumors. Histological analysis of mAb-treated tumors revealed extensive cellular necrosis. Each of the antibodies in vitro was effective in complement-mediated cytolysis at a concentration less than 1 ng/ml. The tumoricidal responses show that this is a useful model for passive human immunotherapy using mAbs.

Tissue distribution of an epithelial and tumor-associated antigen recognized by monoclonal antibody F36/22.

Murine monoclonal antibody F36/22 was derived by immunizing BALB/c mice with human breast cancer cells. This antibody reacts with an antigen located both on differentiated mammary ductal epithelia and on breast carcinomas, as examined by indirect immunoperoxidase techniques. Although the expression of this antigen correlated with estrogen receptor levels of breast tumors, antibody F36/22 did not directly react with estrophilin. In contrast to the expression of classical differentiation antigens, this antigen was found in a high percentage of poorly differentiated carcinomas of the breast. Staining intensities were similar for well- and poorly differentiated tumors; thus, antigen expression was not related to tumor grade. Intratumoral heterogeneity of antigen expression was observed in the majority of tumors. Since a subset (64 of 80) of the breast carcinomas examined have expressed the antigen, McAb 36/22 was of use for the immunological subclassification of tumors which were indistinguishable by conventional histopathological staining techniques. The antigen was also present on other adenocarcinomas (ovary, colon, stomach, pancreas, and prostate); however, these tumors usually exhibited reduced staining intensity compared with that observed in breast cancer. The normal counterpart tissues at these histotypes contained no detectable levels of the antigen, and increased expression of the antigen was associated with tumorigenesis at these sites. Tumors of mesenchymal origin and carcinomas other than adenocarcinomas exhibited undetectable levels of the antigen. Therefore, depending on the organ site, McAb F36/22 recognizes an epithelial and/or tumor-associated antigen.

Monoclonal antibodies (F36/22 and M7/105) to human breast carcinoma.

Cloned hybridoma cell lines were obtained from fusions of murine myeloma cells with lymphocytes of mice immunized against human breast cancer cells. Hybridomas F36/22 and M7/105 produced antibodies whose binding to breast cancer cells could not be inhibited by prior absorptions with fibroblasts, lymphoblastoid cells, or erythrocytes. Results from cell surface binding assays using a panel of tumor cell lines indicated that antibodies F36/22 and M7/105 recognized determinants expressed maximally on breast cancer cells. Antibody F36/22 reacted with normal mammary epithelial membranes and milk fat globule membranes, whereas antibody M7/105 produced no detectable binding to these specimens. Antigens carrying these epitopes each showed reactivity with concanavalin A lectin. The determinant corresponding to antibody F36/22 was detectable in histological sections of a subset of breast tumors obtained at surgery.