Breast Tumor-associated Antigen Research Articles

Immunomodulatory Role of Diospyros peregrina Fruit Preparation in Breast Cancer by Utilizing Macrophage Mediated Antigen Presentation and T Helper Cell (Th) Differentiation

Diospyros peregrina is dioecious plant native to India and belonging to the family of Ebenaceae, is largely utilized in treatment of various ailments. Little has been known about the antitumor activity of Diospyros peregrina with only 1 previous study on Ehrlich Ascites Carcinoma in mice. Therefore, it prompted us to extensively explore the immunomodulatory effect in various cancer forms. The focal point of this study revolves around breast cancer, which is the second most common cancer in the world. In view of the increasing demands for noninvasive treatments, natural plant-based agents open up promising applications in cancer immunotherapy METHODS: CD4+ lymphocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients and normal donor blood samples using magnetic-activated cell sorting (MACS) and cultured separately. Utilizing the plastic surface adherence property, the macrophages were isolated from CD4 negative lymphocytes of both breast cancer patients and normal donors. For the presentation of tumor antigens invitro, macrophages were pulsed with breast tumor associated antigen (BTAA) in presence or absence of Diospyros peregrina fruit preparation (DFP). Differentially pulsed and irradiated macrophages were co-cultured with autologous and allogenic lymphocytes. Supernatants hence collected from CD4+ lymphocytes were utilized for cytokine profiling using ELISA and proliferation was assessed by MTT assay. Cytotoxic T lymphocytes (CTLs) generated from CD4 negative lymphocytes culture (2×105) was incubated with MCF-7 (2×104) to check cytotoxicity using LDH release assay. CD4+ lymphocytes were treated in presence or absence of DFP, were analyzed using immunoblotting and RT-qPCR, to check DFP mediated T helper (Th) cell differentiation through investigation of signatory cytokines and transcription factors. It was found that DFP elevated the proliferation of CD4+ T lymphocytes (Th) in response to BTAA. DFP also helped in presenting BTAA pulsed macrophages directing in the cytotoxic T-lymphocyte mediated immune response. Results indicated that DFP preferentially highlighted Th1 commitment with type-1 specific cytokines IFN-g and IL-12 and was indifferent in Th2 manifestation. DFP was not only involved in the upregulation of Tbet mounted type-1 mediated immune response and activation of STAT1 but also it downregulated STAT6 and GATA3, the functional activators and regulators of type-2 immune response. Moreover, it was observed that DFP inhibited the tumor-promoting environment modulated through Tregs by downregulating Foxp3 and STAT5. Further, it was detected that DFP directs Th1 bias and results in attainment of better suppression of breast tumor CONCLUSION: The results collectively pointed out that DFP favored cell-mediated immune response from BTAA antigen presentation on macrophages and also helping in the robust proliferation of an entire spectrum of T helper lymphocytes which furthermore strengthen the underlying immune responses, hence, fencing the body, of the progression of breast cancer.

Breast Implants and Immune Modulation: Does Foreign Body-Induced Inflammation Promote Immunosurveillance of Breast Tumor Antigen?

BACKGROUND: Women with cosmetic breast implants have significantly lower rates of subsequent breast cancer than the general population.1 We hypothesize breast implant-induced local inflammation stimulates immunosurveillance recognition of breast tumor antigen. METHODS: Women with breast-related complaints were recruited from the plastic surgery clinic. Sera was collected and tested via ELISA assay for antibody responses to common breast tumor antigens: BRCA2, CEA, HER-2, mammaglobin-A and MUC-1, as well as tetanus. A cohort of patients had sera collected prospectively to allow comparison of pre- and postimplant placement. Antibody response levels between long-term breast implant (LTBI) and implant-naive (IN) groups were compared with unpaired t test. Antibody responses pre- and postimplant placement were compared with paired t test. Statistical analysis was performed with Graphpad Prism v8.0.2. RESULTS: One hundred four women were recruited. Thirty-six (34.6%) had LTBI, whereas 68 (65.4%) were IN. Women with LTBI had higher antibody responses than those with IN to mammaglobin-A (OD450, 0.33 versus 0.22; P = 0.003) and MUC-1 (OD450, 0.42 versus 0.34; P = 0.02). There was no difference in antibody responses to BRCA2, CEA, HER-2, or tetanus. In the sample of patients with longitudinal sera samples pre- and postoperative, antibody responses postimplant placement were significantly increased to mammaglobin-A (mean difference 0.13, P = 0.0002) and MUC-1 (mean difference 0.08, P = 0.02). There was no difference in postimplant responses to BRCA2, CEA, HER-2, or tetanus. There was no difference in antibody responses between women with or without capsular contracture, with or without rupture, with or without surface texture, or with subglandular versus submuscular placement. CONCLUSION: Women with LTBI have higher antibody recognition of breast tumor-associated antigens mammaglobin-A and MUC1. This study provides the first evidence of implant-related immune responses to breast cancer antigens. REFERENCE: 1. Noels EC, Lapid O, Lindeman JHN, et al. Breast implants and the risk of breast cancer: a meta-analysis of cohort studies. Aesthetic Surg J. 2015;35:55–62.

Selenocompounds mediated upregulation of HLA class I expression enhanced mammaglobin-A peptide-specific cytotoxic T lymphocyte responses against breast cancer cells

Abstract Recent phase I DNA vaccine clinical trials with Mammaglobin-A (Mam-A), a human breast tumor-associated antigen (TAA), have shown to be safe and efficient. However, the success of cancer vaccines is limited by the diminished expression of HLA class I molecules on cancer cells. In our current communication, we studied the impact of various selenocompounds towards the expression of HLA class I molecules on breast cancer cells and their impact on the cytotoxicity of MamA2.1 (HLA-A2 immunodominant epitope of Mam-A) activated human CD8+T lymphocytes (hCTLs). We noted an enhanced HLA-A2 expression along with upregulation of components involved in antigen presentation machinery in all four breast cancer cell lines, namely AU565, UACC-812, MCF-7, and MDA-MB-231, following treatment with methyselenol producing methylseninic acid (MSA) and dimethylselenide (DMDSe). Furthermore, we have demonstrated enhanced cytotoxicity of MamA2.1 activated CTLs on HLA-A2+/Mam-A+ AU565 and UACC-812 cell lines following pre-treatment with MSA and DMDSe, while no significant toxicity was noted under similar conditions on HLA-A2+/Mam-A− MCF-7 and MDA-MB-231 breast cancer cell lines. Taken together, our data demonstrated that MSA and DMDSe potentiate effector cytotoxic responses following TAA vaccine specific activation of CD8+T lymphocytes, and thus suggesting their futuristic role as vaccine adjuvants in cancer immunotherapy.

Ultrasensitive photoelectrochemical immunoassay of antibody against tumor-associated carbohydrate antigen amplified by functionalized graphene derivates and enzymatic biocatalytic precipitation

Tumor-associated carbohydrate antigens (TACAs) are often found on the surface of cancer cells. The determination of the carbohydrate components of glycoconjugates is challenging because of the chemical complexity of glycan chains. Through monitoring corresponding antibody, we can get a good solution for clinical diagnosis. Here breast tumor-associated carbohydrate antigens Tn were used as a model and a new photoelectrochemical biosensor for ultrasensitive detection of antibody against Tn was developed. To enhance the sensitivity, both graphene oxide and graphene were used during the construction of biosensor. Through the formation of immunocomplex and the insoluble biocatalytic precipitation (BCP) product, photocurrent intensity was decreased greatly and the antibody could be detected from 0.5 to 500pg/mL with a detection limit of 1.0×10−13g/mL. At the same time, the developed biosensor showed acceptable selectivity and could be used in the complex matrix. Compared with the traditional glycoarray method, this PEC method is more sensitive (5 orders of magnitude), and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.

Immunization with two recombinant Bacillus Calmette-Guérin vaccines that combine the expression of multiple tandem repeats of mucin-1 and colony stimulating-factor suppress breast tumor growth in mice

Mucin-1 (MUC1) is a breast tumor-associated antigen. However, clinical trials with MUC1 showed that, with respect to its expression levels, MUC1 is a relatively poor immunogen in human beings. Evidence showed that MUC1-specific immunodominant B and T cell epitopes are derived from the variable-number tandem repeat (VNTR) region. Therefore, immunotherapy that targets multiple VNTRs may induce anti-MUC1 immune responses. GM-CSF has been shown to increase the percentage and activity of antigen-presenting cells. In this study, we constructed two recombinant Bacillus Calmette-Guérin (BCG) vaccines that combine the expression of multiple tandem repeats of MUC1 and CSF. The effect of two novel breast cancer vaccines (rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF) on the growth of breast tumor on hu-PBL-SCID mice was evaluated. We coupled VNTRs (4 and 8) of MUC1 with GM-CSF (MVNTR4-CSF and MVNTR8-CSF). The MVNTR4-CSF and MVNTR8-CSF were inserted into the pDE22 plasmid and transformed into competent BCG by electroporation. The effect of both BCG vaccines on the growth of breast tumor on hu-PBL-SCID mice was evaluated. The growth of MUC1-positive breast tumors from hu-PBL-SCID mice immunized with two vaccines was significantly inhibited. rBCG-MVNTR4-CSF and rBCG-MVNTR8-CSF vaccines may be good candidates for breast tumor immunotherapy.

The Shaping of a Polyvalent and Highly Individual T-Cell Repertoire in the Bone Marrow of Breast Cancer Patients

We analyzed the T-cell repertoires from the bone marrow of 39 primary operated breast cancer patients and 11 healthy female donors for the presence and frequencies of spontaneously induced effector/memory T lymphocytes with peptide-HLA-A2-restricted reactivity against 10 breast tumor-associated antigens (TAA) and 3 normal breast tissue-associated antigens by short-term IFN-gamma enzyme-linked immunospot (ELISpot) analysis. Sixty-seven percent of the patients recognized TAAs with a mean frequency of 144 TAA reactive cells per 10(6) T cells. These patients recognized simultaneously an average of 47% of the tested TAAs. The T-cell repertoire was highly polyvalent and exhibited pronounced interindividual differences in the pattern of TAAs recognized by each patient. Strong differences of reactivity were noticed between TAAs, ranging from 100% recognition of prostate-specific antigen(p141-149) to only 25% recognition of MUC1(p12-20) or Her-2/neu(p369-377). In comparison with TAAs, reactivity to normal breast tissue-associated antigens was lower with respect to the proportions of responding patients (30%) and recognized antigens (27%), with a mean frequency of only 85/10(6) T cells. Healthy individuals also contained TAA-reactive T cells but this repertoire was more restricted and the frequencies were in the same range as T cells reacting to normal breast tissue-associated antigens. Our data show a highly individual T-cell repertoire for recognition of TAAs in breast cancer patients. This has potential relevance for T-cell immune diagnostics, for tumor vaccine design, and for predicting immune responsiveness.

Recognition of Prostate and Breast Tumor Cells by Helper T Lymphocytes Specific for a Prostate and Breast Tumor-Associated Antigen, TARP

T cell-based immunotherapy via the in vitro or in vivo expansion of prostate tumor-associated antigen (TAA)-specific T lymphocytes is one of the most promising therapeutic approaches to treat prostate cancer. T-cell alternate reading frame protein (TARP) is a mitochondrial protein that is specifically expressed in prostate epithelial cells. We have done experiments aimed at identifying helper T lymphocyte (HTL) epitopes for TARP for the design of T cell-based immunotherapy for prostate cancer. Dendritic cells from normal donors were pulsed with synthetic peptides derived from TARP, which were predicted to serve as HTL epitopes. These dendritic cells were used to stimulate CD4(+) T cells in vitro to trigger HTL responses against TARP. T-cell responses to these peptides were also studied with lymphocytes from prostate cancer patients. The two peptides, TARP(1-14) and TARP(14-27), were shown to elicit effective in vitro HTL responses using lymphocytes from both normal volunteers and prostate cancer patients. Peptide TARP(1-14)-reactive HTLs were found restricted by HLA-DR53 and could recognize naturally processed protein antigen derived from tumor cells, which was presented by autologous dendritic cells. Most significantly, stimulation with peptide TARP(14-27) generated four HTL lines restricted by HLA-DR1, HLA-DR9, HLA-DR13, and HLA-DR15, some of which capable of recognizing naturally processed antigens presented by dendritic cell or directly by TARP-positive tumor cells. Our results show that TARP constitutes a TAA that can be recognized by tumor-reactive HTL. The newly described TARP epitopes could be used to optimize and improve T-cell epitope-based immunotherapy against prostate and other tumors expressing TARP.

Naturally occurring B-cell responses to breast cancer.

As demonstrated by the effectiveness of trastuzumab, antibodies against breast cancer antigens are a potentially potent mechanism of tumor control. While trastuzumab is administered exogenously, its efficacy suggests that induction of very high titer antibody responses in vivo might also be therapeutic. Both naturally occurring and vaccine-induced antibody responses to some breast cancer antigens are associated with improved survival in some cases. However, the improvement in survival associated with antibody responses to breast cancer is modest, and tumor regression is not known to be associated with the natural antitumor antibody response, indicating a need for improved understanding of the natural antitumor antibody response. Naturally occurring B-cell responses in the form of serum antibody, tumor reactive lymph node B cells, and tumor-infiltrating B cells have been described, and a variety of breast tumor-associated antigens have been identified based on reactivity of patient antibodies. This review discusses current knowledge of humoral immunity to breast cancer with regard to specific antigens and the basis for their immunogenicity, and the contexts (tumor, lymph node, serum) in which responses are observed. With few exceptions, "tumor-associated antigens" identified with naturally occurring antibodies may be overexpressed on tumor but are in fact nonspecific autoantigens. This suggests that while overexpression or aberrant processing can increase immunogenicity in some cases, the immunogenicity of many or even most tumor-associated antigens is a function of expression in tumor or the result of ancillary tumor factors.

Molecular cloning of glycoprotein antigens MGP57/53 recognized by monoclonal antibodies raised against bovine milk fat globule membrane

A cDNA encoding 57 kDa and 53 kDa antigens (MGP57/53) recognized by monoclonal antibodies raised against bovine milk fat globule membrane (MFGM) (Biochim. Biophys. Acta 1199 (1994) 87–95) was cloned from lactating bovine mammary gland by a combination of reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and 3′-rapid amplification of cDNA ends (3′-RACE). The deduced amino-acid sequence showed that mature MGP57/53 consists of 409 amino-acid residues and the calculated molecular weight and isoelectric point are 45 544 and 6.42, respectively. Computer analysis reveals that it has a significant similarity to mouse mammary epithelial cell surface protein, MFG-E8 and a human breast tumor-associated glycoprotein antigen, BA46-1. An N-terminal cysteine-rich domain and a C-terminal tandemly repeated sequence were highly conserved among them, but bovine MGP57/53 lacks 36 amino-acid residues containing a cluster of 5 prolines found in mouse MFG-E8. Northern blot analysis showed that the cDNA hybridized to about 2.0 kb mRNA of lactating bovine mammary gland. These results strongly support our previous report that the two MFGM antigens originate from a single gene and are isoforms with different N-linked sugar chains.

Purification and characterization of a new 85-kDa glycoprotein antigen from human breast tumor.

A new breast-tumor-associated antigen (BTAA) was purified and partially characterized from human breast tumor. By DEAE-cellulose discontinuous NaCl-gradient chromatography of a crude extract of human malignant breast tumor, 3 major protein peaks were obtained. Circulating antibodies against one of the protein peaks, HF1, was observed in breast-cancer patients. The antibodies were absent in patients with carcinoma of the uterine cervix, lung, stomach and liver or with benign breast diseases and in healthy women. Absorption of the sera of breast-cancer patients with normal human breast tissue pellet did not remove the HF1-reactive circulating antibodies. The BTAA was partially purified from HF1 by subjecting the fraction to SDS-PAGE and eluting the band 3 (HF1-3). Western-blot analysis confirmed the presence of the BTAA in HF1-3. Using an affinity column of protein-A-Sepharose-bound IgG, purified from breast-cancer patients' sera, the BTAA was also recovered from HF1. Purification of the BTAA was achieved by subjecting HF1 to size-exclusion high-performance liquid chromatography (SE-HPLC). The antigen was characterized as a glycoprotein with MW of approximately 85 kDa and appeared not to be related either to murine mammary-tumor virus (MuMTV) structural antigens or to human fetal antigens. The BTAA-reactive circulating antibodies in the breast-cancer patients were of IgG, sub-type, and the level of these antibodies significantly decreased in patients following surgical removal of the breast tumors.

Cellular Characterization and Retroviral Transduction of Short-Term Breast Cancer Cells

The effort to develop cell lines from surgical specimens has been a difficult goal for years. We derived short-term primary human mammary carcinoma cell lines from breast tumors in 20 of 23 patients. Morphologically, cultured cells showed small cells with high nuclear cytoplasmic ratio and larger cells with abundant dense cytoplasm. The nuclei were round to oval with one to four nucleoli. Immunocytochemically, the cells stained positive for keratin. Tumor cells showed phenotypic overexpression of the breast tumor-associated antigen DF3 compared with normal mammary epithelial cells. The doubling time of tumor cells in vitro ranged from 2.6 to 3.6 days. The cultured cells were characterized as mammary carcinoma cells by their tumorigenicity in nude mice. Of 14 of the 23 short-term cell lines tested, 5 grew in nude mice and eventually regressed, 3 grew progressively in nude mice, and the remaining 6 did not grow within 3 months. To examine the feasibility of cytokine gene transfer into human mammary carcinoma cells, we introduced the cDNA for human tumor necrosis factor-alpha (TNF-alpha) into short-term cell lines with a retroviral vector. In our short-term primary breast cancer cell lines derived from breast tumors, TNF-alpha secretion ranged between 89 ng/10(6) cells/48 h and 479 ng/10(6) cells/24 h. These findings indicate that short-term primary human mammary carcinoma cell lines can be grown consistently from breast tumors, and that retroviral mediated-cytokine gene transfer into short-term human mammary carcinoma cells is feasible and may be of potential use in immunotherapy trials.

Monoclonal antibodies against human breast tumor-associated antigens: characterization of B21 antigen.

Monoclonal antibodies (MAbs) show promise in the early detection and monitoring of cancer and may have therapeutic applications as well. The purpose of this study was to characterize MAb B21, a novel murine-derived antibody that has strong reactivity with MCF-7 and T47D cell lines from human breast cancer. A number of MAbs that react with breast cancer cell lines were obtained from cultured mouse spleen cells, and one, MAb B21, was selected for detailed analysis. MAb B21 was characterized by immunocytochemical, immunofluorescence, immunoprecipitation, and Western blotting procedures. We found a strong reactivity of MAb B21 with cultured breast cancer cells and cells from human breast tumors, although some reactivity was seen sporadically in non-breast or normal tissue. Negligible reactivity was detected in a series of non-breast cell lines and with normal breast epithelial cell line MCF-10A. However, when MCF-10A cells were permeabilized, allowing the antibody to penetrate within the cells, reaction became apparent. MCF-10A cells, when transfected with the oncogene c-Ha-ras (MCF-10T), gave a positive immunostaining similar to that observed with MCF-7 and T47D cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of L-[35S]methionine-labeled MCF-7 and T47D cell extracts showed distinct immunoprecipitated components, with molecular weight values ranging from 150,000 to 20,000 with the addition of MAb B21. Western blot assays using MAb B21 of SDS-PAGE fractionated/electroblotted proteins from breast cancer cell lines and MCF-10A cells showed specific reaction with a 95,000 component. Our results indicate that B21 antigen is expressed in neoplastic cells of epithelial origin, mainly breast cancer, and to a minor extent in other cell lines. In addition, MAb B21 recognizes an antigen that is differentially localized during cell transformation. Our future studies will address the full characterization of MAb B21 and explore its capacity as a tool for therapeutic manipulation.

Potential Use of Monoclonal Antibodies in the Diagnostic Distinction of Gynecomastia from Breast Carcinoma in Men

Immunohistochemical (IHC) assays using the monoclonal antibodies (MoAbs) B72.3 and B6.2, recognizing two distinct and independently expressed breast tumor-associated antigens (BTAAs), recently have been shown to significantly improve the accuracy of cytodiagnosis of breast nodules by fine-needle aspiration (FNA). To evaluate whether the same method may be useful diagnostically in distinguishing gynecomastia from breast cancer in men, a retrospective avidin-biotin immunoperoxidase assay study was performed on 50 cases of gynecomastia and 30 cases of breast carcinoma in men, using a panel of five MoAbs known to recognize different BTAAs. The results of this study demonstrated that MoAbs B1.1, HMFG2, and MBr1 displayed a strong reactivity with gynecomastia and carcinoma, but MoAbs B72.3 and B6.2 separated benign and malignant lesions in a high percentage of cases. When used in combination, the latter two reagents reacted with 96% of the carcinomas that were analyzed but labeled only 67% of gynecomastia cases. Thus, the conjoint use of these two reagents may enhance the use of FNA biopsy as a valuable tool in the presurgical diagnosis of breast nodules in men.

Expression of a gene coding for breast tumor-associated antigen in thyroid papillary carcinoma

The expression of the H23 gene, previously shown to be overexpressed in breast cancer tissue, was examined in various thyroid pathologies. Thyroid papillary carcinomas demonstrate significant H23 mRNA levels, whereas benign thyroid pathologies have very low levels of expression. H23 gene expression in thyroid cancer inversely correlated with that of thyroperoxidase and thyroglobulin genes. Immunoblot assays of thyroid cancer tissues revealed overexpression of H23 gene at the protein level as well The data presented indicate that dedifferentiation of thyroid tissue to the malignant state is associated with increased H23 gene expression and suppression of some thyroidal differentiation marker genes.

Expression of genes coding for pS2, c-erbB2, estrogen receptor and the H23 breast tumor-associated antigen: A comparative analysis in breast cancer

Expression of the gene coding for a new breast tumor-associated antigen, H23, was compared to expression of genes coding for pS2, c-erbB2 and estrogen receptor (ER). Comparison involved mRNA expression in normal and malignant breast tissues as well as in non-breast tumors. Results obtained by RNA dot blot and Northern hybridizations showed that expression of the H23 antigen coding gene is a discriminatory marker in human breast cancer. It is expressed in 92% of breast tumors whereas 69%, 62% and 56% of breast tumors demonstrate significant mRNA levels of c-erbB2, ER and pS2, respectively. Non-malignant or normal breast tissue expresses much lower levels of the H23 antigen mRNA. From the comparative analysis presented here it is concluded that the gene coding for H23 antigen furnishes a most useful marker for human breast cancer.

Use of monoclonal antibodies to human breast‐tumor‐associated antigens in the diagnosis of fine‐needle aspirates of breast nodules: Results of a multicenter study

Fine-needle aspiration (FNA) cytology is being increasingly employed in conjunction with physical examination and mammography in the pre-surgical diagnosis of breast nodules. In the present study, we have submitted to multicenter validation an immunocytochemical test which employs monoclonal antibodies (MAbs) to breast-tumor-associated antigens (BTAA) for the diagnosis of breast cancer. The results of this analysis, which has evaluated 846 FNAs, show that the immunological test has a sensitivity of 88.62%, a specificity of 97.9% and an accuracy of 92.4%. The predictive value of a positive and a negative finding were 98.4% and 85.57% respectively. Comparison between the cytological and immunocytochemical diagnosis displayed a higher sensitivity, accuracy and predictive value of a negative result with the latter method (p less than 0.01). Our findings clearly indicate that immunocytochemical methods can complement and improve the diagnostic accuracy of FNA cytology of breast nodules.

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.

Ultrastructural localization of a breast tumor-associated antigen.

We used a monoclonal antibody to a high molecular weight glycoprotein of the milk fat globule membrane to study at the ultrastructural level the sites of accumulation of immunoreactive material in breast tumors. By light microscopy, the antigen was found only on the luminal membrane of normal resting or lactating breast cells. In tumors, antigen could be found in the cytoplasm. We used the avidin-biotin-peroxidase complex immunoperoxidase technique to localize the site of cytoplasmic accumulation of immunoreactive material in breast tumors. This technique showed that antigen is found on the membrane of cytoplasmic vesicles. Some of these vesicles were collapsed and gave rise to what appeared to be clumps of free cytoplasmic immunoreactive material. We have also documented that in some tumors the entire cytoplasmic membrane bears antigen, whereas in other tumors only areas of cytoplasmic membrane that form microscopic lumina express antigen.

The role of a new monoclonal antibody assay in the detection of recurrent breast cancer.

The immunoradiometric assay "CA 15-3", recently developed to measure a breast tumor-associated antigen, gave a mean serum value of 13.8 U/ml (S.D. 6.2) for this antigen in 156 non-cancer controls (36 biopsies for a benign breast lesion and 120 healthy controls). Setting a cut-off value of 30 U/ml (specificity 99.3%), only 3 out of 58 primary breast cancer cases were positive. In metastatic breast cancer, 11 out of 33 cases with limited recurrence (33.3%) and 36 out of 56 cases with extensive recurrence (64.3%) gave abnormal values in this assay, above the cut-off point, with an overall sensitivity of 52.8%; the difference between the sensitivity values in the two groups of recurrent cases was statistically significant (P less than 0.01). According to the findings of the present study, CA 15-3 has no role in the detection of primary breast cancer, but its usefulness in disease monitoring can be hypothesized, as circulating levels of the antigen seem to be dependent on the tumor mass.

Use of monoclonal antibodies to human breast-tumor-associated antigens in fine-needle aspirate cytology.

Fine-needle aspiration cytology (FNAC) is currently used in evaluating the nature of breast nodules. In the present study we have examined whether monoclonal antibodies (MAbs) to breast-tumor-associated antigens (BTAA) can be employed in FNAC for the diagnosis of breast cancer. For this purpose we have used 2 murine MAbs recognizing 2 distinct BTAA expressed by breast tumors, irrespective of their histotype, in an indirect avidin-biotin immunohistochemical technique on aspirate smears. The results of this study show that, while benign lesions are consistently negative, tumor cells containing aspirates are reactive with at least one MAb in 95% of the cases. Thus, selected MAbs to BTAA may be a powerful diagnostic tool in conjunction with conventional cytology, and because of the objective interpretation of the immunohistochemical findings, FNAC of the breast can eventually be extended outside specialized institutions.