To Tumor-associated Antigens Research Articles

Different Forms of Helper Tolerance to Carcinoembryonic Antigen: Ignorance and Regulation

Understanding the mechanisms of immune tolerance to tumor-associated antigens (TAA) is an important step in the design of cancer immunotherapy. The aim was to determine how T helper (Th) cell tolerance is mediated for a prototypic TAA, carcinoembryonic antigen (CEA). Peripheral blood mononuclear cells from 50 healthy volunteers were stimulated with CEA, and the type and fine specificity of any Th cell responses were identified. The inhibitory effects of T regulatory (Tr) populations were determined by depleting "natural" CD25(+) Tr cells or neutralizing cytokine produced by the "induced" Tr form. Proliferative Th cell responses were consistently induced by CEA in 22 of 50 individuals. Responding cells were drawn from the CD45RA(+) "naive" or quiescent population. Depleting the CD25(+) fraction did not enhance CEA responsiveness. However, CEA elicited secretion of the Tr cytokine interleukin-10 (IL-10) in 23 of 50 donors, including 20 of 22 where no proliferation was induced. Neutralizing IL-10 revealed previously unseen proliferation to CEA by CD45RO(+) "memory" Th cells. Epitope maps revealed differences in the fine specificities of Th cells capable of proliferating or secreting IL-10. There are at least two major forms of CEA tolerance in different individuals. One is "ignorance," a failure of specific Th cells to respond to antigen presented in vivo. The other, seen when ignorance is lost, is mediated by IL-10-secreting Tr cells that recognize CEA. TAA tolerance, for example to colorectal carcinoma cells expressing CEA, may be overcome by peptide vaccines that exploit the differences in epitopes recognized by effector and Tr responses.

GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy.

Method of treating tumor growth and metastasis by using trifunctional antibodies to reduce the risk for graft-versus-host disease in allogeneic antitumor cell therapy

The patent claims that trifunctional (TrFAb) and trispecific antibodies (TrSAb) will redirect immune effectors to tumor associated antigens (TAAs) while downregulating allo-activation of donor T cells, leading to reduced graft- versus-host disease (GvHD). A TrFAb is a bispecific antibody (BiAb) that binds to immune effector cells and targets a TAA, whilst binding of the Fc-portion of the TrFAb to Fcγ receptors (FcγR) on immune effector cells leads to downregulation of allo-activation of donor T cells responsible for mediating GvHD. Furthermore, a TrSAb consists of a proposed construct that binds to immune effectors and to two separate TAAs while the Fc-portion of the TrSAb constructs interacts to downregulate allo-activation as proposed in the TrFAb construct. The patent claims that infusion of TrFAb or TrSAb would markedly improve antitumor activity whilst maintaining potent anti-GvHD activity when used after allogeneic stem cell transplantation for both hematologic malignancies and solid tumors.

Xenovaccinotherapy for colorectal cancer

The objectives of this phase I–II trial were to assess the toxicity, immunological and clinical responses induced in 37 patients with stage IV colorectal cancer by the subcutaneous administration of a xenogenic polyantigenic vaccine (XPV) prepared from disrupted murine melanoma (B16) and carcinoma (LLC) cells. An inducing course of vaccinotherapy consisted of 10 immunizations (5 at weekly and 5 at fortnight intervals). Twenty-four hours later each of first 5 vaccinations the patient was subcutaneously given a low dose of the recombinant interleukin-2 (IL-2). A consolidating course of vaccinotherapy consisted of monthly vaccinations. No grade III or IV toxicities, but also laboratory and clinical signs of developing systemic autoimmune disorders were noted in any XPV-treated patient. A significant increase in cell-mediated immunoreactivity to both LLC and B16 antigens (Ags) occurred in the patients after inducing vaccinations, as determined by delayed-type hypersensitivity (DTH) skin reactions, as well as by blood lymphocyte proliferation responses. Vaccinations also led to increased cell-mediated reactivity to murine non-tumor, spleen cell (SC)-associated Ags. This reactivity, however, was not as significant as that to tumor-associated antigens (TAAs). XPV was also found to capable of generating IgG antibody-mediated responses. With immunotherapy concentrations of both interferon-gamma (IFN-γ) and interleukin-4 (IL-4) detectably elevated in patients' sera, suggesting intensification of T helper 1-/T helper 2-mediated responses in the XPV-treated patients. The average survival of the XPV-treated patients was noticeably superior than was that of the clinically comparable control patients (17 vs 7 months). Collectively the results suggest that xenogenic TAAs are safe to use, able to induce measurable cellular and humoral immune responses, and may be clinically effective in certain colorectal cancer patients.

Serum antibodies specific for tumor antigens in colorectal cancer may be useful diagnostic biomarkers

10041 Background: Colorectal cancer is immunogenic as evidenced by the presence of antibodies specific to tumor antigens in patient serum and T cell infiltration at the tumor site predicting favorable disease outcome. We questioned whether antibody immunity to tumor associated antigens (TAA) in patients with colorectal cancer could be used as a biomarker to predict disease. Methods: We evaluated serum samples for the presence of antibody immunity to six tumor antigens, cyclin D1, HER-2/neu, insulin-like growth factor binding protein 2 (IGFBP2), NY-ESO-1, p53, and topoisomerase-IIα (TOPO2α), in colorectal cancer patients (n=40) and normal donors (n=200) using ELISA. We then evaluated the diagnostic value of these potential biomarkers by measuring antibody levels to these TAA in an independent sample set which included 30 colorectal cancer patients and 135 normal donors. The samples were judged as positive or negative for colorectal cancer using measurements derived either from a single marker or a combination of the markers. Receiver operating characteristic (ROC) plots were generated to determine the diagnostic accuracy of each test. Results: Colorectal cancer patients had increased levels of antibodies to p53 (p=0.002), TOPO2α (p<0.001), and IGFBP2 (p<0.001) compared to controls. By using a combination of just two antibody measurements, TOPO2α and IGFBP2, we could discriminate colorectal cancer patients from controls with a diagnostic power of 0.775 as estimated by the area under ROC curve. At a cutoff point of 0.03 mcg/ml TAA specific IgG, the sensitivity was 100% and the specificity was 58%. Conclusions: Serum antibodies to colorectal cancer related antigens may serve as useful biomarkers for colorectal cancer diagnosis. No significant financial relationships to disclose.

Application of Bayesian Modeling of Autologous Antibody Responses against Ovarian Tumor-Associated Antigens to Cancer Detection

Biomarkers for early detection of epithelial ovarian cancer (EOC) are urgently needed. Patients can generate antibodies to tumor-associated antigens (TAAs). We tested multiplex detection of antibodies to candidate ovarian TAAs and statistical modeling for discrimination of sera of EOC patients and controls. Binding of serum antibody of women with EOC or healthy controls to candidate TAA-coated microspheres was assayed in parallel. A Bayesian model/variable selection approach using Markov Chain Monte Carlo computations was applied to these data, and serum CA125 values, to determine the best predictive model. The selected model was subjected to area under the receiver-operator curve (AUC) analysis. The best model generated an AUC of 0.86 [95% confidence interval (95% CI), 0.78-0.90] for discrimination between sera of EOC patients and healthy patients using antibody specific to p53, NY-CO-8, and HOXB7. Inclusion of CA125 in the model provided an AUC of 0.89 (95% CI, 0.84-0.92) compared with an AUC of 0.83 (95% CI, 0.81-0.85) using CA125 alone. However, using TAA responses alone, the model discriminated between independent sera of women with nonmalignant gynecologic conditions and those with advanced-stage or early-stage EOC with AUCs of 0.71 (95% CI, 0.67-0.76) and 0.70 (95% CI, 0.48-0.75), respectively. Serum antibody to p53 and HOXB7 is positively associated with EOC, whereas NY-CO-8-specific antibody shows negative association. Bayesian modeling of these TAA-specific serum antibody responses exhibits similar discrimination of patients with early-stage and advanced-stage EOC from women with nonmalignant gynecologic conditions and may be complementary to CA125.

Spotlight

Zeng et al., pp.268–273 The human immune system can recognize and react to tumor-associated antigens (TAA). Identifying TAA based on their recognition by antibodies provides molecular details of the immune response and suggests that detection, diagnosis and prognosis of cancer by serological markers may yet be possible. Most antigens react with only a few, if any, allogeneic sera, making it difficult and expensive to investigate antibody responses against a large panel of TAA that covers a wide spectrum of patients with a particular cancer. The authors propose that purifying individual proteins might not be necessary if dominant B-cell epitopes could be identified. Using the antigen NY-ESO-1 as a prototype, they tested a synthetic peptide of the epitope ESO:1–40 and found that the peptide was as sensitive as the recombinant protein for detecting antibodies against the tumor antigen. Previous studies indicate that such antibodies appear mainly in patients with advanced disease, and this particular antigen is expressed in a variety of human cancers, including lung, breast, ovarian, prostate, melanoma and others. The authors suggest that the epitope-based approach could dramatically simplify antibody detection in cancer patients. de Jong et al., pp.274–282 Human papillomavirus (HPV) type 16 is the high risk type found most frequently in cervical cancer; HPV oncoproteins E6 and E7 are constitutively expressed in tumor cells and are required to maintain malignant phenotype, making them attractive targets for immunotherapy. Tumor-related immunosuppression often accompanies advanced cervical cancer, leading researchers to suspect that adoptive transfer of tumor-specific T cells might have a better chance of working than a therapeutic vaccine. Previous work in mice has shown that adoptive T cell therapy can eradicate solid tumors. The technique has also been applied to patients with metastatic melanoma, with mixed success. The authors investigated the feasibility of enriching for T cells that recognize the E6 antigen from the peripheral blood of healthy donors on the basis of interferon-gamma secretion. The obtained T cell populations successfully recognized distinct epitopes within the E6 antigen. There are several advantages to this approach. Enrichment of antigen-specific T cells secreting IFN-gamma ensures selection of T cells with an optimal cytokine profile for anti-tumor immunity, and enriching for antigen-specific T cells helps minimize alloreactivity and risk of graft-vs.-host disease. These results represent a strong first step toward developing a T cell-based therapy for advanced cervical cancer by showing that HPV16E6-specific T cells, which may be useful for eradicating tumor cells, can be collected from the peripheral blood cells of healthy donors. Increased purity of HPV16E6-specific CD4+ T-cells by extended culture period before isolation of antigen-specific cells Hanaoka et al., pp.317–322 The role of tobacco smoking in breast cancer has been of great interest to the public health community, particularly since tobacco smoke contains known carcinogens, and most established risk factors for breast cancer cannot be modified. However, the relationship between smoking and breast cancer has been foggy at best; some studies have shown a link, while others found no relationship and still others have suggested a protective effect from smoking. Previous analyses have demonstrated that tobacco-related carcinogens do reach human breast tissue. However, the antiestrogenic effects of tobacco smoke could decrease breast cancer risk, particularly among postmenopausal women. The authors studied a large cohort of middle-aged Japanese women and attempted to determine the risk of breast cancer as it relates to smoking status: e.g., active smokers, nonsmokers who have not been exposed to tobacco smoke regularly, and nonsmokers who are regularly exposed to smoke in their homes or workplaces (“passive smokers”). Active smoking increased breast cancer risk among premenopausal women; premenopausal, passive smokers showed a smaller increase in risk. No increase in risk associated with smoking was found for postmenopausal women. This is the first prospective study to link active smoking with breast cancer risk in Asian women.

Multistep process through which adenoviral vector vaccine overcomes anergy to tumor-associated antigens

AbstractOur goal in the present work was to characterize the multiple steps involved in overcoming the anergy that exists in tumor hosts to tumor-associated antigen (TAA). Our studies showed that the subcutaneous injection of the Ad-sig-TAA/ecdCD40L vector resulted in secretion of the TAA/ecdCD40L protein for at least 10 days from infected cells. Binding of the TAA/ecdCD40L protein to dendritic cells (DCs) resulted in the induction of CCR-7 chemokine receptor expression and cytokine release. This was followed by migration of the DCs to regional lymph nodes. Tetramer staining, enzyme-linked immunospot (ELISPOT) assay, and cytotoxicity assay all showed that the Ad-sig-TAA/ecdCD40L vector increased the levels of splenic CD8+ T cells specific for the 2 TAAs (human MUC1 [hMUC1] and HPV E7) tested. Vaccination with the Ad-sighMUC1/ecdCD40L vector suppressed the growth of hMUC1 antigen-positive tumor cells in 100% of the test mice that were previously anergic to the hMUC1 antigen. These data suggest that Ad-sig-TAA-ecd/ecdCD40L vector injections may be of value in treating the many epithelial malignancies in which TAA-like hMUC1 is overexpressed. (Blood. 2004;104:2704-2713)

Restored immune response to an MHC-II-Restricted antigen in tumor-bearing hosts after elimination of regulatory T cells

Background/purpose Pediatric sarcomas have a poor prognosis, recur frequently, and are not effectively treated by currently available therapy. Immunotherapy is a promising treatment modality; however, to be successful, immune tolerance must be overcome. CD4 +CD25 + T cells are immunosuppressive. The authors hypothesize that immune tolerance may be overcome by eliminating the regulatory CD4 +CD25 + T cells, which are induced by tumor. Methods A murine fibrosarcoma (MF), which expresses an MHC-II-restricted tumor antigen (mL26), was injected subcutaneously. CD4 + T cells were isolated and CD25 + population examined. Monoclonal antibody was used to deplete CD25 + T cells. Proliferation to mL26 was used to determine CD4 + T cell response to tumor-associated antigen (TAA). Results Depletion of CD25 + cells prevented tumor establishment. Tumor-infiltrating CD25 + T cells, which madeup 48% of CD4 + T cells in tumors, suppressed proliferation in allogeneic mixed lymphocyte reactions. Draining lymph node cells from tumor-bearing (TB) mice did not proliferate in response to mL26, whereas those from naive mice responded vigorously. Depletion of CD25 + cells before immunization restored response to mL26 in TB mice. Conclusions CD4 +CD25 + T cells induced by MF facilitate tumor establishment and maintain immune tolerance. Depletion of CD4 +CD25 + T cells may be an effective means for reversing tumor tolerance and enhancing cancer vaccines.

Monoclonal antibody drug immunoconjugates for targeted treatment of cancer.

Monoclonal antibodies (mAb) directed to tumor-associated antigens (TAA) or antigens differentially expressed on the tumor vasculature have been covalently linked to drugs that have different mechanisms of action and various levels of potency. The use of these mAb immunoconjugates to selectively deliver drugs to tumors has the potential to both improve antitumor efficacy and reduce the systemic toxicity of therapy. Several immunoconjugates, particularly those that incorporate internalizing antibodies and tumor-selective linkers, have demonstrated impressive activity in preclinical models. Immunoconjugates that deliver doxorubicin, maytansine and calicheamicin are currently being evaluated in clinical trials. The feasibility of using immunoconjugates as cancer therapeutics has been clearly demonstrated. Gemtuzumab ozogamicin, a calicheamicin conjugate that targets CD33, has recently been approved by the Food and Drug Administration (FDA) for treatment of acute myelogenous leukemia (AML). This review concentrates on the properties of the tumor and the characteristics of the mAb, linker, and drugs that influence the efficacy, potency, and selectivity of immunconjugates selected for cancer treatment.

Probing Dendritic Cell Function by Guiding the Differentiation of Embryonic Stem Cells

Publisher Summary Dendritic cells (DC) are unique among the populations of antigen presenting cells by virtue of their capacity to direct the outcome of antigen recognition by naive T cells. By serving as messengers direct from the site of infection, DC inspect the T cell repertoire, identifying those cells specific for the cargo of antigens they display on their surface as peptide-MHC complexes. Having encountered T cells with a complementary receptor for antigen, DC deliver instructions directing the expansion of relevant clones and their deployment to the front line of the immune response. Should these T cells pose a threat to the integrity of the host, DC may decommission them from active service, thereby minimizing any damage from friendly fire. The role played by DC in fine-tuning these opposing forces of self-tolerance and immunity makes them attractive candidates for immune intervention in a variety of disease states. Alternatively, by specifically enhancing their immunogenicity, DC might be coerced into breaking the natural state of self-tolerance to tumor-associated antigens (TAA) and directing the full force of the immune response towards transformed cells and established tumors. Despite their clinical potential, very little is known of the molecular basis of DC function, which permits them to fulfill these conflicting roles. An approach has been developed to the study of DC, which draws on the unique features of embryonic stem (ES) cells: their self-renewal, pluripotency, and tractability for genetic modification. This chapter describes in detail the protocols involved and discusses the advantages such an experimental system provides over the current art.

Multiparameter cytokine-specific affinity matrix assay for the determination of frequencies and phenotype of antigen-reactive T cells

A major challenge in Phase I clinical trials of cancer vaccines is how to identify the optimal regimen and/or dosing for Phase II and III trials. Immunologic responses to tumor-associated antigens (TAA) could potentially serve as surrogate markers. Assays which detect cytokine production and/or secretion by antigen (Ag)-reactive T cells are increasingly being used to monitor such responses in clinical trials. A potential problem of these assays is high non-specific background and/or inability to easily identify the T-cell phenotype. We designed an assay, termed cytokine-specific affinity matrix (Multi-CSAM™) assay, 1 1 Provisional U.S. Patent and Trademark's Application Number 60/263,929. which can determine the frequencies of phenotypically identifiable antigen (Ag)-reactive T cells. The assay relies on the tightly regulated production and secretion of cytokine by Ag-activated cells, capture of secreted cytokine by an artificial cytokine-specific cell-surface affinity matrix and flow-cytometric detection of bound cytokine with a fluorochrome-labeled anti-cytokine mAb. In an attempt to minimize non-specific background, the assay excludes non-viable cells by using the cell-impermeant DNA-binding dye TO-PRO-3, and selects activated cells on the basis of CD69 expression. Furthermore, the phenotype of the Ag-reactive T cells is identified using the CD4 and CD8 T-cell markers. Thus, using an appropriately modified two-laser benchtop instrument, a single sample can be evaluated by five-color multiparametric flow-cytometry that employs a combination of appropriately labeled mAbs. The five colors selected are anti-IFNγ-PE, the dye TO-PRO-3, anti-CD69-PerCP, anti-CD4-FITC and anti-CD8-PE/Texas Red. Although our ultimate goal is to identify T cells reactive to TAA, as a first step this report demonstrates that Multi-CSAM can be used to detect the reactivity to TT and Candida.

Cell-mediated immunity to tumor-associated antigens is a better predictor of survival in early stage breast cancer than stage, grade or lymph node status.

Cell-mediated immune (CMI) responses to tumor-associated antigens (TAA) in the early postoperative period were examined for correlations with disease recurrence and survival in a 13-year-prospective study of 77 stage 1 and 2 breast cancer patients treated with modified radical or radical mastectomy alone. Among the 21 patients who had positive lymphoproliferative tests using patients' peripheral blood mononuclear cells and autologous TAA of breast cancer cells, only one died from metastatic disease (5%). Among the 56 patients who had a negative test, 23 died from metastatic disease (41%). This difference is statistically significant (p = 0.002) Three other risk factors including tumor size, nodal status and cell differentiation patterns were also analyzed. When these three clinical-pathologic criteria were analyzed individually, none reliably predicted disease recurrence and survival. Nodal status was the most predictive clinical-pathologic risk factor, but was not significant (p = 0.089). The results of this study demonstrate the detection of CMI responses against autologous TAA by lymphoproliferative assays identifies a sub-set of stage 1 and 2 breast cancer patients who are at minimal risk of developing metastatic disease. This testing also identifies immunologically unreactive patients who are at risk for disease recurrence.

Internal image anti‐idiotype antibodies related to renal‐cell carcinoma‐associated antigen G250

The potential usefulness of internal-image anti-idiotype antibodies (Ab2s) in modulating hosts' immune responses to tumor-associated antigen (TAA) have stimulated considerable interest in the development and characterization of Ab2s. Six different mouse monoclonal Ab2s (NUH31, 44, 51, 71, 82 and 91) were generated against murine monoclonal antibody G250 (MAbG250) which recognizes a human renal-cell carcinoma-associated antigen. All 6 Ab2s showed specificity for the MAbG250 paratope in Western-blot analysis. In inhibition assays, all Ab2s were able to compete with the nominal antigen, albeit with differing efficiency. Based on cross-blocking studies for idiotope mapping, the Ab2s could be divided into 2 groups (group I; NUH31, 51, 71, group 2; NUH44, 82, 91). However, cross-reactivity between these 2 groups was observed, indicating that they recognized partly overlapping epitopes on the paratope of MAbG250. Sera from rabbits immunized with Ab2s showed reactivity with G250 antigen-positive cell lysates, but not with antigen-negative cell lysates. Additional studies revealed that all Ab2s were able to induce anti-anti-idiotype antibodies resembling MAbG250 (Ab1'). These findings suggest that the Ab2s functionally mimic the original G250 antigen and may be of use in the immunotherapy of human renal-cell carcinoma.

Immunotherapy in a Human Ovarian Cancer Xenograft Model with Two Bispecific Monoclonal Antibodies: OV-TL 3/CD3 and OC/TR

The bispecific antibodies (bs-mAbs) OV-TL 3/CD3 and OC/TR (MOv18/CD3) efficiently mediate ovarian tumor cell lysis by cytotoxic T cells and activated peripheral blood lymphocytes (PBL) in vitro. OV-TL 3/CD3 and OC/TR are reactive with tumor-associated antigens on ovarian carcinoma cells (OA3 and CA-MOv18, respectively), and CD3 on activated PBL, bridging both cells and simultaneously inducing activation of the effector cells. In a comparative study we investigated the therapeutic efficacy of OV-TL 3/CD3 and OC/TR by targeting activated PBL with the bs-mAbs against intraperitoneally growing NIH:OVCAR-3 human ovarian carcinoma cells. As they have good tumor localization characteristics, HPLC-purified bispecific F(ab')2 fragments were used to target highly active PHA and IL-2-stimulated PBL effector cells. The efficacy of OV-TL 3/CD3 was compared to OC/TR with respect to tumor-associated antigen (TAA) binding on NIH:OVCAR-3 ascites cells and NIH:OVCAR-3 tumor cell lysis in vitro. In this report we show that ip ovarian cancer-bearing nude mice treated with IL-2 and activated PBL coated with bispecific F(ab')2 had a significantly longer survival than the untreated mice. No significant difference in survival was found between the OC/TR or OV-TL 3/CD3 bispecific antibody, although MOv18 expression was higher on NIH:OVCAR-3 ascites cells and PBL targeted with OC/TR induced slightly higher tumor cell lysis in vitro. Thus, the therapeutic efficacy of these bs-mAbs in vivo could not be predicted by TAA expression or bs-mAb-mediated tumor cell lysis in vitro.

Selected monoclonal antibodies can increase the accuracy of cytodiagnosis of neoplastic effusions of cryptic origin expanded in a short term culture.

The use of a selected panel of monoclonal antibodies (MoAb) to tumor associated antigens (TAA) in immunocytochemical (IIC) tests has been shown, in a preliminary study, to be a powerful diagnostic tool for the identification of the primary solid tumor causing metastatic effusion. Despite this improvement in a minority of neoplastic fluids a number of different causes may still determine false negative (FN) immunocytochemical diagnoses. The aim of the present study was to confirm the diagnostic accuracy of this panel of MoAb. This was done by analyzing in IIC tests a larger number of effusions and by evaluating whether the expansion in short term culture of those fluids with an uncertain malignant morphology could provide an adequate cellular substrate for immunocytodiagnosis. The analysis of 314 effusions confirmed the results of the pilot study and demonstrated that the combination of short term culture and immunocytochemical assays can further increase the sensitivity of this novel diagnostic procedure from 84.3% to 95.3%.

Cell kinetics and tumor-associated antigen expression in human mammary carcinomas.

Twenty-six primary breast carcinomas were studied to evaluate cell proliferation as assessed by thymidine labeling index (TLI), and antigenic phenotype, as defined by immunohistochemistry using eight monoclonal antibodies (MAbs) to tumor-associated antigens (TAAs). The majority of tumors had low TLI values. Reactivity to MAbs B72.3, CC49, CC83 (anti TAG 72), COL-12 (anti CEA) and MOv2 (against a tumor-associated mucoprotein) was restricted to less than 50% of the tumors studied, while MAbs B1.1 (anti CEA), MBrl and MBr8 (to tumor-associated carbohydrates) reacted with greater than 50% of the cases. Correlations between expression of TAAs and proliferative activity showed that the tumors could be divided into three groups, two characterized by either high proliferative activity and absence of antigenic expression or low proliferative activity and strong antigenic expression, and the third showing no relation between these two biological features. We defined two antigenic phenotypes associated with specific cellular kinetics: one characterized by negative immunoreaction with MAbs, CC49, CC83 and COL-12 and high proliferative activity; the other characterized by intense immunoreactivity with these antibodies and low proliferative activity. The data suggest that cell proliferation and antigenic phenotype may define biologic subsets of breast carcinomas.

Combinations of monoclonal antibodies can distinguish primary lung tumors from metastatic lung tumors sampled by fine needle aspiration biopsy

Transthoracic fine needle aspiration (FNA) biopsies performed under computed tomography (CT) scan (CT-FNA) have greatly improved the cytodiagnosis of lung tumors. However, the distinction between a primary lesion and a metastatic lesion still may be difficult on the basis of morphologic criteria. To evaluate whether a selected panel of monoclonal antibodies (MoAb) to tumor-associated antigens (TAA) can improve the diagnostic potential of FNA, we have immunocytochemically analyzed 122 pulmonary CT-FNA. Whereas conventional cytology was capable of recognizing only the neoplastic nature of the lesions, the immunocytochemical diagnosis could identify the primary or metastatic nature of the pulmonary masses in 92.5% of the cases. The immunocytochemical findings were confirmed by clinical-histopathologic data. The current results demonstrate that the use of immunocytochemical methods can significantly improve the diagnostic accuracy of conventional cytology of lung masses.

Use of selected combinations of monoclonal antibodies to tumor associated antigens in the diagnosis of neoplastic effusions of unknown origin

While conventional cytodiagnosis can, in most instances, recognize cancer cells in metastatic effusions from solid tumors, the cellular type or the organ of origin of the primary neoplasia can rarely be determined only on the basis of their morphology. In the present study we have evaluated whether immunocytochemical techniques can be used to overcome this limitation by employing a panel of monoclonal antibodies (MoAbs) to tumor associated antigens (TAA) which lack detectable reactivity with mesothelial cells. To this end we have analyzed, by indirect immunofluorescence, cytospins of 60 malignant effusions of unknown origin. The results of this study have shown that the definition of the origin of the primary tumor, which was subsequently confirmed histologically and/or clinically, could be reached in 87% of the cases. These findings demonstrate that selected combinations of MoAbs, when used in immunocytochemical tests, can provide a powerful diagnostic tool in defining the site of cryptic primary neoplasias causing metastatic effusions.

A Human Monoclonal Antibody to Cytokeratin Intermediate Filament Antigens Derived from a Tumor Draining Lymph Node

Human lymphocytes derived from a lymph node draining a primary breast adenocarcinoma were fused with the mouse myeloma P3X63Ag8.653 to generate human-mouse hybridomas secreting human monoclonal antibodies (MAbs) to tumor associated antigens (TAAs). One of the resulting human MAbs, YBB 190 (IgM) is described. Enzyme-linked immunosorbent assays (ELISA) employing membrane and cytosol fractions of human tissues demonstrated YBB 190 reactivity against cytosol but not membrane components of malignant and normal epithelial tissues. When tested by an indirect immunoperoxidase staining method against fresh frozen human tissue sections, YBB 190 reacted with malignant cells in 26 of 28 epithelial cancers and with normal epithelia in 11 different benign tissues. Preliminary western blot antigen characterization indicated that YBB 190 recognizes cytokeratin intermediate filaments, or a protein that is closely associated with cytokeratins. These data indicate that B cells with specificity for intermediate filaments are present in tumor draining lymph nodes. Our findings provide insights into the nature of potential autoimmune responses in cancer patients and suggest that improved tumor directed sensitization procedures may be required to more effectively utilize lymphocytes from tumor draining lymph nodes to generate therapeutically useful human MAbs to TAAs.