Translocation of an intracellular antigen to the surface of medullary breast cancer cells early in apoptosis allows for an antigen-driven antibody response elicited by tumor-infiltrating B cells

Tumor-infiltrating lymphoplasmacytic cells are a key feature of medullary carcinoma of the breast (MCB), a distinct subtype of human breast cancer that, despite cytologically anaplastic characteristics, has a more favorable prognosis than other types of breast cancer. Since it has been proposed that the improved clinical outcome is due at least in part to the presence of a prominent lymphoplasmacytic cell infiltrate in the tumor stroma, we recently examined the tumor-infiltrating B cell response in MCB and showed that it is oligoclonal and directed against an intracellular protein translocated to the cell surface upon MCB cell apoptosis. Human Abs cloned from MCB lymphoplasmacytic infiltrate-derived phage display libraries and reflecting the dominant part of the response were used to identify the target Ag as actin. Here, we have characterized in detail the cloned human IgG Abs and the translocation process of actin to the cell surface of apoptotic MCB cells. Our analysis shows that the cloned Abs bind specifically and with high affinity to actin, as determined by ELISA and surface plasmon resonance. Sequence analysis revealed that the Abs are highly somatically mutated, with high replacement to silent ratios, indicative of an Ag-driven, affinity-matured response. Interestingly, the tumor-infiltrating B cells in half the MCB patients mainly exhibited an IgG2 response, while IgG1 dominated in the others. To gain insight to the molecular events that may elicit such an Ab response, we examined the translocation of actin to the cell surface of apoptotic MCB cells using flow cytometry and laser scanning cytometry. Our results show that actin becomes exposed on the cell surface of a large proportion of apoptotic MCB cells as an early apoptotic event. We propose that the Ab response against actin produced by tumor-infiltrating B lymphoplasmacytic cells is Ag-driven, affinity-matured, and elicited due to the increased rate of apoptosis occurring within the MCB tumor that facilitates the translocation and proteolytic fragmentation of intracellular proteins.     

CD20+ B cells: the other tumor-infiltrating lymphocytes

Tumor-infiltrating CD8(+) T cells are strongly associated with patient survival in a wide variety of human cancers. Less is known about tumor-infiltrating CD20(+) B cells, which often colocalize with T cells, sometimes forming organized lymphoid structures. In autoimmunity and organ transplantation, T cells and B cells collaborate to generate potent, unrelenting immune responses that can result in extensive tissue damage and organ rejection. In these settings, B cells enhance T cell responses by producing Abs, stimulatory cytokines, and chemokines, serving as local APCs, and organizing the formation of tertiary lymphoid structures that sustain long-term immunity. Thus, B cells are an important component of immunological circuits associated with persistent, rampant tissue destruction. Engagement of tumor-reactive B cells may be an important condition for generating potent, long-term T cell responses against cancer.     

Sialosyllactotetraosylceramide, a novel ganglioside antigen detected in human carcinomas by a monoclonal antibody

A novel ganglioside was detected in a small cell lung carcinoma by TLC-immunostaining of gangliosides with a monoclonal antibody, the C-50 MAb. Structural characterization showed this ganglioside to be IV3NeuAc-LcOse4Cer, a hitherto unknown ganglioside. This ganglioside has also been detected as a minor component in many different carcinomas using the C-50 MAb. The normally dominant CA-50 ganglioside antigen is IV3NeuAc, III4Fuc-LcOse4Cer. Based upon solid-phase binding to IV3NeuAc, III4-LcOse4Cer and IV3NeuAc-LcOse4Cer it is concluded that the C-50 MAb recognizes an epitope present in sialylated type I carbohydrate chains.     

Activation of human B lymphocytes by a particulate antigen

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.     

Antigen-driven oligoclonal expansion of tumor-infiltrating B cells in infiltrating ductal carcinoma of the breast

The objective of this study was to determine whether tumor-infiltrating B cells (TIL-B) of infiltrating ductal carcinoma (IDC) of the breast represent a tumor-specific humoral immune response. Immunohistochemical analysis of three Her-2/neu-negative IDC tumors from geriatric patients showed that TIL-B cluster in structures similar to germinal centers containing CD20(+) B lymphocyte and CD3(+) T lymphocyte zones with interdigitating CD21(+) follicular dendritic cells, suggesting an in situ immune response. A total of 29, 31, and 58 IgG1 H chain clones was sequenced from the three IDC tumors, respectively. Intratumoral oligoclonal expansion of TIL-B was demonstrated by a preponderance (45-68%) of clonal B cells. In contrast, only 7% of tumor-draining lymph node and 0% of healthy donor PBL IgG H chains were clonal, consistent with the larger repertoires of node and peripheral populations. Patterns and levels of TIL-B IgG H chain somatic hypermutation suggested affinity maturation in intratumoral germinal centers. To examine the specificity of TIL-B Ig, a phage-displayed Fab library was generated from the TIL-B of one IDC tumor. Panning with an allogeneic breast cancer cell line enriched Fab binding to breast cancer cells, but not nonmalignant cell lines tested. However, panning with autologous tumor tissue lysate increased binding of Fab to both tumor tissue lysate and healthy breast tissue lysate. These data suggest an in situ Ag-driven oligoclonal B cell response to a variety of tumor- and breast-associated Ags.     

Direct activation of human B lymphocytes by Candida albicans-derived mannan antigen

We have studied the activation of human resting B cells by a carbohydrate antigen, mannan, with a polymannose branched repetitive structure. Mannan has been extracted from the cell wall of the Candida albicans yeast. For this purpose, dense G0 B lymphocytes were purified from tonsils. Mannan antigen was shown to trigger B cell activation, since an increase of cell volume and RNA synthesis occurred. B cell proliferation was observed following addition of recombinant interleukin 2, but not following addition of recombinant interleukin 4 or low-molecular-weight BCGF. The B cell activation appears to be mannan-specific since B cells obtained from mannan-sensitized subjects but not from unsensitized subjects were responsive. The observation that mannan antigen can directly activate specific dense B lymphocytes can be related to the previous observation that the in vitro anti-mannan antibody production does not require a cognate T-B cell interaction.     

Clonal analysis and in situ characterization of lymphocytes infiltrating human breast carcinomas

Summary T lymphocytes were isolated from tumor biopsies in 13 patients with breast carcinomas. Immunohistology with monoclonal antibodies confirmed the presence of mononuclear cell infiltrates composed primarily of T lymphocytes in all tumors studied. While the proportion of T lymphocytes expressing the T4 or the T8 surface marker varied from tumor to tumor as determined by morphometric analysis, T8+ cells were more numerous than T4+ cells in 8/12 breast tumors studied. Relatively few T cells (<10% in 11/12 tumors) were in an activated state as judged by the surface expression of HLA-DR antigens or the receptor for interleukin-2 (IL-2). In 1 case 20% of the infiltrating mononuclear cells were expressing the IL-2 receptor. The tumor infiltrating lymphocytes (TIL) recovered from 10 tumors were cloned in a microculture system that permits proliferation of nearly 100% of normal peripheral blood T lymphocytes (PBL-T). In contrast to normal and autologous PBL-T, frequencies of proliferating T lymphocyte precursors (PTL-P) were depressed (<0.01) in 7/10 TIL preparations indicating a decreased responsiveness of TIL to phytohemagglutinin at the single-cell level. The frequency of PTL-P was noticeably higher in 2 cases (0.03 and 0.09) and close to normal in 1 case (0.39).A total of 170 clones were expanded in vitro and analyzed for different functional capabilities. Most of these clones expressed the T4+/T8-phenotype (73%) and strikingly 53% of these T4+/T8– clones were cytolytic in a lectin-dependent assay, a functional subset which is uncommon among normal PBL-T. Some clones (10%) lysed allogeneic breast tumor cells (MCF7). Only 15% of the clones displayed natural killer activity. Among the cytolytic clones, 17 of 31 tested were also IL-2 producers irrespective of the T4 or T8 phenotype. Our results show that human mammary carcinomas contain many infiltrating T cells with cytolytic potential. Interestingly, among the proliferating cytolytic T cell clones (56% of the microcultures), many expressed the T4+/T8– phenotype. These findings may indicate that the in situ cytolytic reaction (against unknown antigens) is associated preferentially with class II antigens.Fogarty International Fellow of NIH, 1984–1985; on leave from Dept of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pa, USA     

Editing antigen presentation: antigen transfer between human B lymphocytes and macrophages mediated by class A scavenger receptors

B lymphocytes can function independently as efficient APCs. However, our previous studies demonstrate that both dendritic cells and macrophages are necessary to propagate immune responses initiated by B cell APCs. This finding led us to identify a process in mice whereby Ag-specific B cells transfer Ag to other APCs. In this study, we report the ability and mechanism by which human B lymphocytes can transfer BCR-captured Ag to macrophages. The transfer of Ag involves direct contact between the two cells followed by the capture of B cell-derived membrane and/or intracellular components by the macrophage. These events are abrogated by blocking scavenger receptor A, a receptor involved in the exchange of membrane between APCs. Macrophages acquire greater amounts of Ag in the presence of specific B cells than in their absence. This mechanism allows B cells to amplify or edit the immune response to specific Ag by transferring BCR-captured Ag to other professional APCs, thereby increasing the frequency of its presentation. Ag transfer may perpetuate chronic autoimmune responses to specific self-proteins and help explain the efficacy of B cell-directed therapies in human disease.     

Cytolytic antitumor effector cells in long-term cultures of human tumor-infiltrating lymphocytes in recombinant interleukin 2

Summary Lymphocytes infiltrating human solid tumors (TIL) and autologous peripheral blood lymphocytes (A-PBL) were cultured with 1000 units/ml of recombinant interleukin 2 (rIL2) in long-term cultures. TIL isolated from 26 primary squamous cell carcinomas of the head and neck expanded better (P<0.01) and achieved higher total lytic units of activity against fresh tumor cell targets (P<0.05) than A-PBL. TIL obtained from primary hepatocellular carcinomas (n=7) showed a higher degree of expansion than those from metastatic liver tumors (n=7). Further, TIL from metastatic tumors of the head and neck, liver, and ovary were delayed up to 50 days in their proliferative response to rIL2. Long-term mass cultures in rIL2 of TIL, A-PBL, or normal PBL were serially monitored for cytotoxicity with different cultured and fresh tumor cell targets and for phenotypic markers of the predominating cell populations. Antitumor cytotoxicity was found in cultures enriched in CD3 + Leu19 + and/or CD3-Leu19 + cells. Two-color sorting of such cultures followed by cytotoxicity assays confirmed that the human antitumor effectors expressed either the CD3 + Leu19 + or CD3-Leu19 + phenotype. CD3 + Leu19- cells had little or no antitumor cytotoxicity. The two types of Leu19 + effector cells were present in low numbers in fresh TIL, A-PBL, or normal PBL; in contrast, in some rIL2-expanded long-term cultures, they represented a majority of proliferating cells. This study identifies for the first time two types of antitumor effector cells in rIL2 cultures of human TIL, one of which may represent activated natural killer cells on the basis of the absence of the CD3 and expression of the Leu19 antigen. These antitumor effector cells mediate non-MHC-restricted cytotoxicity of fresh or cultured tumor cell targets of different histologic types.     

Signet ring cells in fine needle aspiration cytology of breast carcinomas: review of the cytological findings in ten cases identified by histology

Objective:  To establish whether the presence of signet ring cells (SRCs) in histology sections of breast carcinoma cases was reflected by their presence in fine needle aspiration cytology (FNAC) smears, correlating to the histological type of breast carcinoma.
Methods:  We reviewed the FNAC findings of ten cases that had been diagnosed as primary breast carcinoma with SRCs on histological sections between 1998 and 2007. Slides and histological sections were obtained from the archives of Ege University Hospital.
Results:  FNA smears were reviewed for the following cytomorphological features: background, cellularity, architecture, nuclear pleomorphism and the presence of SRCs. The background was bloody in eight cases, necrotic in one, and clean in one. There was no mucinous material in any of the cases. Cellularity was prominent in five cases (hypercellular), moderate in three (cellular) and low in two (hypocellular). Loosely cohesive groups of tumour cells of varying size were observed in all cases. A plasmacytoid appearance to some of the tumour cells was seen in all cases and discohesive tumour cells were present in eight. Nuclear pleomorphism was high in six cases and moderate in four. SRCs were observed in seven of the ten cases. Two of these seven cases also had a tubular pattern and one had tumour giant cells.
Conclusions:  FNAC should be evaluated carefully regarding the presence of SRCs when cells with a plasmacytoid appearance are observed in either hyper- or hypocellular smears. The presence of single SRCs in FNACs with hypercellularity, high nuclear grade and tubular formation or tumour giant cells may be a clue in favour of ductal carcinoma. The presence of single SRCs in FNACs with hypocellularity and mild to moderate nuclear grade may be suggestive of lobular carcinoma. However, larger studies would be needed to establish the predictive value of the presence of SRCs on FNAC.     

Uridine labeling of human lymphocytes: Differential uptake by T and B cells

Human peripheral blood lymphocytes were labeled with [³H]uridine and separated into immunoglobulin (Ig)-positive-enriched and Ig-negative populations by rosetting and Ficoll sedimentation. The Ig-negative (T cell-rich) fraction was found to be more heavily labeled than the B cell-enriched population, in agreement with previous results in rats. Combined autoradiography and rosetting confirmed the differential uridine labeling of T and B cells. Incorporation of cytidine and adenosine by T and B cell-enriched populations showed similar but less dramatic differential labeling.     

GD3-reactive antibodies can inhibit the lysis of autologous tumor cells by tumor-infiltrating lymphocytes

Summary GD3 is expressed in high concentrations on melanoma cells and may serve as a useful target antigen for mAb-mediated immunotherapy. Monoclonal antibodies (mAbs) against GD3 stimulate cell-mediated immune responses against tumor cells in vitro and this activity may contribute to antitumor effects in patients with melanoma treated with GD3-reactive mAbs. In the present study the effects of GD3-reactive mAbs on autologous tumor cell lysis by a human melanoma-derived tumor-infiltrating lymphocyte (TIL) population were examined. Unlike results reported for other GD3⁺ T cells isolated from melanoma patients, the tumor-specific lytic activity of the TIL line was inhibited by incubation with mAbs against GD3. Other melanoma-reactive mAbs, including those against GD2 and the high-molecular-weight melanoma-associated Ag, had no effect on the TIL lytic activity. Overall, these results indicate that mAbs against GD3 may have different effects on T cell/tumor cell interactions.     

Structure of the genes encoding the CD19 antigen of human and mouse B lymphocytes

CD19 is a B lymphocyte cell-surface marker that is expressed early during pre-B-cell differentiation with expression persisting until terminal differentiation into palsma cells. CD19 is a member of Ig gnee superfamily with two extreacellular Ig-like domains separated amino acid cytoplasmic domain. In this study, Southern blot analysis revelaed that the human and mouse CD19 genes were compact single copy genes. Both the human and mouse CD19 genes were isolated and the nucleotide sequences flanking each exon were determined. Both genes were composed of 15 exons and spanned 8 kilobases (kb) of DNA in human and 6 kb in mouse. The positions of exon-intron boundaries were identical between human and mouse and correlated with the putative functional domains of the CD19 protein. The 200 bp region 5 of the putative translation initiation AUG codon as well conserved in sequence between human and mouse and contained potential trasncription regulatory elements. In addition, the 3 untranslated regions (UT) of the CD19 genes following the termination codon were conservedf in sequence. The high level conservation of nucleotide sequences between species in all exons and 5 and 3 UT suggests that expression of the CD19 gene may be regulated in a similar fashion in human and mouse.The nucleotide sequence database reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers: human CD19 gnee, M62544 to M62550; mouse CD19 gene, M62551 to M62553.     

The proliferative response of human lymphocytes to antigen is suppressed preferentially by lymphocytes precultured with the same antigen.

Human blood lymphocytes activated in vitro with antigen to which the donor is reactive are capable of suppressing the secondary proliferative response of autochthonous fresh cells to antigen. Both antigen-specific and antigen-nonspecific suppression can be detected in each experiment. These suppressor cells act by decreasing the number of lymphocytes entering the proliferative response rather than by slowing or otherwise inhibiting ongoing proliferation. The suppressor cells must be added soon after fresh cells are stimulated with antigen to be effective, but the suppressor cells themselves need not proliferate to exert their effect. Suppressor cells are optimally effective when added in numbers equal to those of the responding population, but still exert a significant effect at one-eighth that number.     

The consumption of seaweed as a protective factor in the etiology of breast cancer: proof of principle

Daily consumption of seaweed has been proposed as a factor in explaining lower postmenopausal breast cancer (BC) incidence and mortality rates in Japan. This clinical trial assessed the impact of introducing seaweed- to non-seaweed-consuming American postmenopausal women. Fifteen healthy postmenopausal women were recruited for a 3-month single-blinded placebo controlled clinical trial; five had no history of BC (controls) and ten were BC survivors. Participants ingested ten capsules daily (5 g day^?1) of placebo for 4 weeks, seaweed (Undaria) for 4 weeks, then placebo for another 4 weeks. Blood and urine samples were collected after each treatment period. Urinary human urokinase-type plasminogen activator receptor concentrations (uPAR) were analyzed by ELISA, and urine and serum were analyzed for protein expression using surface-enhanced laser desorption/ionization–time-of-flight mass spectrometry (SELDI-TOF-MS). Urinary creatinine standardized uPAR (in pg mL μg^?1 creatinine) changed significantly between groups, decreasing by about half following seaweed supplementation (placebo 1, 1.5 (95 % CI, 0.9–2.1) and seaweed, 0.9 (95 % CI, 0.6–1.1) while placebo 2 returned to pre-seaweed concentration (1.7 (95 % CI, 1.2-2.2); p?=?0.01, ANOVA). One SELDI-TOF-MS-identified urinary protein (m/z 9,776) showed a similar reversible decrease with seaweed and is reported to be associated with cell attachment. One serum protein (m/z 8,928) reversibly increased with seaweed and may be the immunostimulatory complement activation C3a des-arginine. uPAR is higher among postmenopausal women generally, and for BC patients, it is associated with unfavorable BC prognosis. By lowering uPAR, dietary seaweed may help explain lower BC incidence and mortality among postmenopausal women in Japan.     

Kinetics of activation antigen expression by in vitro-stimulated human T lymphocytes

In this study a panel of monoclonal antibodies was used to investigate the kinetics of the appearance of activation-linked surface determinants as well as cytoplasmic and nuclear determinants in human T cells following lectin stimulation. Well known activation markers, such as Ia/DR, transferrin receptor, IL-2 receptor, T10, and gp24, were compared and investigated together with the T13 structure, recently found in this laboratory. T13, not demonstrable on resting T cells, could be seen within 24 hr after lectin stimulation. Kinetics of the appearance were similar to IL-2 receptor and transferrin receptor expression. Ia/DR synthesis was investigated separately for each polypeptide and the cytoplasmic invariant gamma-chain expression could be demonstrated for the first time with a gamma-chain-specific monoclonal antibody VIC-Y1. Moreover, gamma-chain synthesis seems to precede alpha- and beta-chain occurrence in human T cells. In addition, data from quantitative studies on antigenic densities are presented.     

Epigenotyping in peripheral blood cell DNA and breast cancer risk: a proof of principle study

Background

Epigenetic changes are emerging as one of the most important events in carcinogenesis. Two alterations in the pattern of DNA methylation in breast cancer (BC) have been previously reported; active estrogen receptor-α (ER-α) is associated with decreased methylation of ER-α target (ERT) genes, and polycomb group target (PCGT) genes are more likely than other genes to have promoter DNA hypermethylation in cancer. However, whether DNA methylation in normal unrelated cells is associated with BC risk and whether these imprints can be related to factors which can be modified by the environment, is unclear.

Methodology/Principal Findings

Using quantitative methylation analysis in a case-control study (n = 1,083) we found that DNA methylation of peripheral blood cell DNA provides good prediction of BC risk. We also report that invasive ductal and invasive lobular BC is characterized by two different sets of genes, the latter particular by genes involved in the differentiation of the mesenchyme (PITX2, TITF1, GDNF and MYOD1). Finally we demonstrate that only ERT genes predict ER positive BC; lack of peripheral blood cell DNA methylation of ZNF217 predicted BC independent of age and family history (odds ratio 1.49; 95% confidence interval 1.12–1.97; P = 0.006) and was associated with ER-α bioactivity in the corresponding serum.

Conclusion/Significance

This first large-scale epigenotyping study demonstrates that DNA methylation may serve as a link between the environment and the genome. Factors that can be modulated by the environment (like estrogens) leave an imprint in the DNA of cells that are unrelated to the target organ and indicate the predisposition to develop a cancer. Further research will need to demonstrate whether DNA methylation profiles will be able to serve as a new tool to predict the risk of developing chronic diseases with sufficient accuracy to guide preventive measures.     

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.     

Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.

An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.