Immunoelectron microscopic localization of DF 3 cancer-associated antigen in human breast cancer.

Breast cancer cells are characterized by frequent occurrence of the DF 3 breast-cancer associated antigen in the cytoplasmic area. This study elucidated the details of this cytoplasmic reactivity by immunoelectron microscopy. In infiltrating breast carcinomas, the DF 3 antigen was localized in rough endoplasmic reticulum (rER), perinuclear space, Golgi complex, membrane-bound granules and along the outer surface of cell membrane. The antigen was also accumulated in the cytosol. In contrast to this, benign lesions showed localization of the DF 3 antigen mainly along the outer surface of apical cell membrane and rarely in rER or cytosol. The present study indicated that the DF 3 antigen was of secretory type and commonly localized on the outer surface of apical cell membrane in both malignant and benign lesions. Carcinoma tissues were further characterized by frequent occurrence of immunoreactivity related to the process of antigen synthesis. The diffuse cytosolic reactivity may be associated with disordered transportation of the antigen in the cytoplasm.     

A monoclonal antibody directed against a human cell membrane antigen prevents cell substrate adhesion and tumor invasion.

It was the aim of this study to design mouse monoclonal antibodies (MAbs) that can inhibit the invasion of breast cancer cells in the host tissue. Therefore, MAbs were raised against epitopes on the extracellular domain of SK-BR-3 human breast cancer cells, and biological assays were performed to test the capability of the MAbs to inhibit cell substrate adhesion. MAb 14C5 bound an extracellular plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells and inhibited the cell substrate adhesion of these cells in vitro. The MAb delayed the adhesion of MCF-7 and SK-BR-3 cells on precultured embryonic heart fragments (PHFS). It inhibited the destruction of the PHF by MCF-7 cells and the invasion of the PHF by SK-BR-3 cells. The MAb reacted with an epitope on the cell membrane of in situ and invasive ductal carcinomas of the breast in immunohistochemistry. Poorly differentiated, highly invasive ductal carcinomas show extensive staining of long plasma membrane extensions. Normal multilayered epithelia, normal connective tissue, and tumors derived from these tissues as well as normal breast tissue were negative. From both cell lines a protein complex consisting of two subunits with molecular weight of 50 and 90 kd, respectively, was immunoprecipitated. It is concluded that the 14C5 antigen plays a role in cell substrate adhesion and subsequently also in invasion of breast cancer cells. The 14C5 MAb was able to inhibit cell substrate adhesion and invasion in vitro of breast cancer cells.     

Immunoperoxidase studies by monoclonal antibody B72.3 applied to breast aspirates: diagnostic considerations

The purpose of this study was to determine the reactivity of monoclonal antibody (MAb) B72.3 when applied directly to aspiration biopsy cytology (ABC) of the breast in the following conditions: (1) infiltration lobular carcinoma; (2) fibrocystic disease; (3) fibroadenoma; and (4) apocrine cysts. Nine of ten aspirates from infiltrating lobular carcinoma were positive in these assays, while 21 of 22 benign cases reacted negatively. The single false-positive benign aspirate manifested a staining pattern characteristic of apocrine cells. This study demonstrates that the MAb B72.3 can be employed as a potentially valuable diagnostic adjunct. It can be used on stained aspirates to assist in the interpretation of ABC from breast lesions.     

Monoclonal antibody specific for histone H1 phosphorylated by cyclin-dependent kinases: a novel immunohistochemical probe of proliferation and neoplasia.

Monoclonal antibody 12D11 (MAb 12D11) has been shown to bind histone H1 isolated from human placenta and other tissues but not histone H1 that has been digested with bacterial alkaline phosphatase. We show here that phosphorylation of phosphatase-treated histone H1 with cyclin dependent-kinase (CDK) restores binding by MAb 12D11. We conclude that MAb 12D11 selectively binds histone H1 that has been phosphorylated by CDKs, and we have investigated the use of MAb 12D11 as an immunohistochemical probe of CDK activity in situ. Previous immunofluorescence studies have revealed strong nuclear staining by MAb 12D11 in proliferating cultured cells and the absence of staining in terminally differentiated cells. Immunohistochemical staining of frozen and formalin-fixed, paraffin-embedded sections of benign tissues with MAb 12D11 was nuclear and confined to recognized foci of cell proliferation. In lymphoid germinal centers, MAb 12D11 preferentially stained large lymphoid cells with a relative lack of staining in small cleaved cells, contrasting with a lack of cell size discrimination observed with the monoclonal antibody proliferation probe, MIB-1. Tumor tissues displayed strong albeit heterogeneous staining of malignant cells by MAb 12D11, with little or no staining observed in surrounding nonneoplastic stromal cells. Differential staining by MAb 12D11 of invasive and in situ carcinoma suggest applications in prognostication. MAb 12D11 may also be useful in identification of tumors more likely to respond to therapeutic CDK inhibitors.     

Monoclonal antibody CIBCHTB1 defining an epitope on carcinoembryonic antigen (CEA)

Carcinoembryonic antigen (CEA) is an oncofoetal protein first identified by Gold and Freedman (1965) in colorectal cancer. It is a cell surface tumor marker which has been characterised as a heterogenous group of glycoproteins. It is also present in a variety of benign and non-neoplastic diseases like ulcerative colitis, Adenomatous polyp, Liver cirrhosis and other cancers like GI tract tumors, Cancer of the breast, lung, ovary, pancreas, prostate, hepatoma etc. Elevated CEA levels serve as clinical tool in the diagnosis, monitoring, detecting early any recurrence or metastasis and in prognostication for confirmed colorectal cancers. In order to develop an Enzyme Immuno Assay and immunocytochemical assay for CEA, an MAb designated as CIBCHTB1 has been generated using CEA isolated from a cell line HT115, human adenocarcinoma of the colon, as immunogen by the conventional Hybridoma technology. This MAb of IgG1 isotope was selected by screening of culture supernatents by ELISA and then by its high binding affinity with HT115 cells as revealed by flowcytometric analysis. By ABC method of immunocytochemical assay, this Ab exhibited strong staining of cells in frozen tissue sections of normal colon and malignant colorectal lesions and various other types of human cancers. This MAb has useful application to study the expression of CEA in human cancers. Serum CEA levels of patients with colorectal cancers and other CEA producing cancers and controls determined by EIA using this MAb were in good correlation with the results obtained using commercial kit. The diagnostic potential of this Mab in the management of colorectal cancers is discussed.     

Wide spectrum screening keratin as a marker of metaplastic spindle cell carcinoma of the breast: an immunohistochemical study of 24 patients

Wide spectrum screening keratin as a marker of metaplastic spindle cell carcinoma of the breast: an immunohistochemical study of 24 patients
Aims : Metaplastic spindle cell carcinomas may be difficult to distinguish histologically from other spindle cell lesions in the breast. Variable staining with cytokeratin immunomarkers has been reported for metaplastic carcinomas. We evaluated the diagnostic utility of anti-cytokeratin polyclonal antibody, wide spectrum screening keratin, to assess spindle cell breast lesions.
Methods and results : Twenty-four patients with spindle cell breast carcinoma and 31 patients with benign or malignant spindle cell tumours were studied using a panel of antibodies directed against multiple cytokeratins (AE1/AE3, CAM5.2, wide spectrum screening keratin), epithelial membrane antigen (EMA), and vimentin. Sites of origin for the 31 controls included breast, bone, and soft tissue. All but one (95.8%) metaplastic carcinomas stained positively with wide spectrum screening keratin. Only rare or focal immunoreactivity was observed with AE1/AE3 in four cases; however, sensitivity of AE1/AE3 was improved in 13 cases using steam EDTA as an antigen retrieval technique. Three cases were immunoreactive with CAM5.2 and eight cases were immunoreactive with EMA. All control cases lacked immunoreactivity with the cytokeratin panel and EMA. The spindle cells in the metaplastic breast tumours (88%) and in the controls (97%) stained with vimentin.
Conclusions : Wide spectrum screening keratin may be the most useful and convenient antibody in differentiating metaplastic spindle cell carcinoma from other spindle cell lesions in the breast.     

Characterization of monoclonal antibody CIBCNSH3 generated to the human EGF receptor

Monoclonal antibody CIBCNSH3 of IgG1 isotype has been generated against human epidermal growth factor receptor (EGFR) using MDA MB 468 breast carcinoma cell line as immunogen. Earlier studies have revealed that this MAb blocked growth factor-receptor interaction and thus inhibited cell proliferation and tumor growth. In the present paper, this MAb has been extensively characterized to evaluate its application in the study of human cancers. The results were compared with those obtained using a control MAb ICR 62 specific to EGFR. Competitive assay showed that this MAb bound to an epitope in the extracellular domain of the EGFR to which MAb ICR 62 also bound. This MAb immunoprecipitated the 170 kD glycoprotein. The specificity was further confirmed by the formation of a single discrete band in western blot analysis. By flow cytometric analysis this monoclonal antibody revealed high binding affinity with MDA MB 468 cells. By immunocytochemical assay, out of 35 breast tumors studied, 40% were found to exhibit strong cell membrane staining and in the case of 25 oral cancers studied, 56% were strong positive. High expression of EGFR was observed in MDA MB 468 cells and HN 5 cells. These studies clearly indicate that MAb CIBCNSH3 might prove useful to identify tumors with high level of expression of EGFR associated with poor prognosis.     

Immunocytochemical distribution of a breast carcinoma associated glycoprotein identified by monoclonal antibodies.

A glycoprotein, BCA-225 (Mr 225,000-250,000), has been identified in cells and spent medium of clone 11 T47D breast carcinoma cells by three murine monoclonal antibodies, CU18, CU26, and CU46. The antigen was localized in paraffin sections of 167/178 (94%) Bouin's-fixed human breast carcinoma tissues and few other carcinomas (1/8 lung [squamous], 4/4 uterine cervix) in an intracellular pattern, whereas an apical or glycocalyx distribution was seen in several normal tissues, benign lesions, and malignant tumors. Although the immunocytochemical staining patterns observed with these antibodies have many similarities to those described with other previously reported monoclonal antibodies, notable differences include the lack of reactivity of CU18, CU26, and CU46 with lactating mammary gland and with gastrointestinal malignancies. BCA-225 binds to wheat germ lectin, not to concanavalin A, but monoclonal antibody binding does not appear to involve the carbohydrate component of the molecule. The frequency of the immunocytochemical detection of BCA-225 in breast carcinomas and its restricted distribution in other human tissues suggest considerable clinical potential for this antigen and its corresponding monoclonal antibodies.     

Lectin receptors in the breast and its tumors

Distribution of lectin receptors of peanut, soy-bean, wheat ovary and concanavalin A was studied in normal and tumor human breast tissue. It is established, that in dysplasia and benign neoplasms there is a shift of lectin receptors from the apical or basal pole of acinar epithelium and ducts to the whole plasmolemma surface. There appears a mild affinity of the cytoplasm to the lectins. In carcinoma homogeneous staining of tumour cell cytoplasm and plasmolemma increase. The number of lectin receptors in the cells from low differentiated tumours decreases, a major part of the cells losing the ability of binding to lectins. Malignancy is associated with an increase in mosaic staining. The earliest signs of malignancy can be revealed with the peanut lectin.     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

Myoepithelial cells in the differential diagnosis of complex benign and malignant breast lesions: an immunohistochemical study.

The distinction between sclerosing adenosis, radial scars, noninvasive carcinomas occurring in sclerosing adenosis, and invasive carcinoma can be difficult. The identification of a myoepithelial (ME) cell layer is helpful in establishing a diagnosis of complex benign breast proliferation as well as intraepithelial neoplasia in sclerosing adenosis. We reviewed pathologic material from patients with tubular carcinoma (23) and complex breast proliferations (28), including sclerosing adenosis (12), radial scars (9), sclerosing adenosis with intraepithelial neoplasia (5), and sclerosing adenosis with atypical apocrine metaplasia (2). Immunoperoxidase stains on formalin-fixed, paraffin-embedded tissue using a muscle actin-specific antibody of clone HHF35 and high molecular weight cytokeratin of clone 34 beta E12 (HMW keratin) were performed to identify myoepithelial cells. Muscle actin was uniformly reliable in staining ME cells, as well as other actin-containing cells such as myofibroblasts and vascular smooth muscle. HMW keratin was less reliable, being poorly sensitive and less specific than muscle actin for labeling of ME cells. ME cells were readily identified at the periphery of ductules in all complex benign breast lesions. The presence of ME cells distinguished intraepithelial neoplasia involving sclerosing adenosis from invasive carcinomas. Well differentiated invasive carcinoma forming tubular structures lacked a ME cell layer.     

干细胞样细胞在乳腺良恶性病变中的数量与分布

目的 探讨乳腺上皮干细胞样细胞(stem-like cells,SIC)在乳腺部分良恶性病变中的数量及分布.方法 使用免疫组化双染法检测了89例乳腺良性病变及36例乳腺浸润性导管癌(invasive ductal carcinoma,IDC)中SLC的密度及分布部位.结果 ①SLC在良性病变中主要分布于乳腺腺管的腔面顶层上皮和肌上皮细胞之间;②在实质增生区SLC密度多于纤维腺瘤及实质减少区(P<0.05);③MUC-/ESA 细胞在IDC中数量差异较大.结论 SLC的数量及分布随病变不同而异.     

The value of monoclonal antibody B72.3 for the diagnosis of breast carcinoma: experience with the first commercially available source.

One hundred cases of invasive breast carcinoma were studied using the commercially available monoclonal antibody Anti-Human Tumor-Associated Glycoprotein-72 (MAb B72.3, Biomedical Technologies Inc, Stoughton, MA) prediluted at 8.5 micrograms/mL. Forty-three cases displayed positive reactivity with this antibody. Intensity and distribution of positive staining varied among the tumor cells. Twenty-two cases had 1% or less reactive cells, while eight cases contained 40% or more positive tumor cells. Apical cell membrane and diffuse cytoplasmic staining were present. In fifteen cases intracytoplasmic lumina and extra-cellular secretory material were highlighted by positive staining. Thirty-five cases had benign breast tissue adjacent to the tumor. Benign ductal and lobular epithelial cells were nonreactive except for two cases in which small foci of apocrine metaplasia were positive. Reactivity with MAb B72.3 was not dependent upon histologic grade, nuclear grade, nodal status, or patient age. Excluding the lower number of positively stained cases, our findings were similar to other MAb B72.3 investigations. The number of positively stained cases and the intensity of the positivity were increased by using MAb B72.3 at 5.0 micrograms/mL with overnight incubation, or by using MAb B72.3 at 40.0 micrograms/mL with 2 hours of incubation. Our findings confirm that MAb B72.3 shows reliable reactivity with breast carcinoma that is sensitive to antibody concentration and incubation time without loss of specificity for tumor cells. Our results are also consistent with the view that MAb B72.3 probably detects epithelial membrane-related antigens in breast carcinoma, as do several other antibodies.     

Expression of gap junction proteins connexin 26 and connexin 43 in normal human breast and in breast tumours

Gap junctional intercellular communication (GJIC) has been proposed as a cellular mechanism for tumour suppression and there is experimental evidence in support of this. If aberrant GJIC contributes to the formation of human breast tumours, one might expect that the connexins (gap junction proteins) expressed by epithelial cells in normal human breast would be down-regulated in tumour epithelial cells, or that tumour cells might show aberrant expression of other connexin family members. This study examines the immunocytochemical expression of connexins 26 (Cx26) and 43 (Cx43) in normal human breast, 11 benign breast lesions, two special-type carcinomas, and 27 invasive carcinomas of no special histological type (NST). Cx26 generally was not expressed at detectable levels in normal human breast, but punctate Cx43 immunostaining of the myoepithelial cells was found. Cx43 staining of the myoepithelium was also a feature of the benign lesions and ductal carcinoma in situ (DCIS). In general, the epithelial cells of benign lesions failed to stain for either connexin. Similarly, a lobular carcinoma did not express Cx26 or Cx43, but there was punctate Cx43 in the epithelial cells of a mucoid carcinoma. Cx26 was up-regulated in the carcinoma cells of 15 of the 27 invasive NST carcinomas, although the staining was usually cytoplasmic and heterogeneous. Cx43 was expressed by stromal cells, possibly myofibroblasts, in all NST carcinomas. Furthermore, there was heterogeneous Cx43 expression in the carcinoma cells of 14 of the 27 NST carcinomas and the staining was often intercellular and punctate, characteristic of functional connexins. Up-regulation of Cx26 and/or Cx43 in the carcinoma cells of over two-thirds of invasive lesions of NST is not necessarily inconsistent with a tumour suppressor role for GJIC. However, the role of gap junctions in the formation and progression of solid human tumours is likely to be more complex than indicated from experimental systems. © 1998 John Wiley & Sons, Ltd.     

Nuclear translocation of monoclonal antibody directed against cell-surface carbohydrate Y determinant.

Monoclonal antibody (MAb) Br 15-6A directed against the carbohydrate Y determinant expressed on tumor cells was found to be internalized and translocated to the nucleus of SK Br 5 breast carcinoma and SW 1116 and SW 707 colorectal carcinoma cells. Intracellular localization of MAb Br 15-6A was determined by cell fraction and by indirect immunofluorescence staining. Internalization of MAb Br 15-6A seems to be mediated by a specific cell surface protein of M(r) 108,000 in colorectal carcinoma cells and M(r) 92,000-96,000 in breast carcinoma cells. The MAb Br 15-6A precipitates an 88,000 M(r) chromatin protein and appears to be bound specifically to two EcoRI-digested and two HincII-chromatin fragments. Another MAb against the Y determinant (MAb Br 55.2) recognizes the same antigens as MAb Br 15-6A, but is not internalized.     

Axillary apocrine carcinoma with benign apocrine tumours: a case report involving a pathological and immunohistochemical study and review of the literature

BACKGROUND: Apocrine carcinoma is rare and often occurs in the axilla. This is the second apocrine carcinoma arising in bilateral axillae with associated apocrine hyperplasia to be reported. AIMS/METHODS: Because benign apocrine tumours may be precursors of cancer, this case was investigated immunohistochemically and histologically, and a literature (English and Japanese) review undertaken of cases with coexistent malignant and benign apocrine tumours in the axilla to elucidate the relation between apocrine carcinoma and benign apocrine tumours. RESULTS: Only four cases of axillary apocrine carcinoma with benign apocrine tumours were identified in the literature. In each case, benign apocrine hyperplasia was situated within and surrounding the adenocarcinomatous nests. Staining for epithelial membrane antigen revealed three patterns: (1) poorly differentiated tumour cells showing strong cytoplasmic staining; (2) combined luminal surface and cytoplasmic staining of glandular cells; and (3) a strongly positive lineal staining pattern at the luminal membrane surface, comprising one or two apocrine hyperplastic secretory cells. The basal lesions of apocrine hyperplasia were strongly positive for alpha smooth muscle actin, whereas the periphery of adenomatous lesions showed weaker positive staining, even though the periphery of adenocarcinomatous lesions was negative. CONCLUSIONS: All five apocrine carcinomas with benign apocrine tumours occurred in elderly Japanese men who had bilateral benign apocrine tumours even if affected by unilateral axillary apocrine carcinoma. The immunohistochemical results support the notion that apocrine hyperplasia is a precursor of cancer and that apocrine carcinoma, adenoma, and hyperplasia may be successive steps in the linear progression to carcinoma.     

Monoclonal antibodies against NIH 3T3 cells transformed by human thyroid carcinoma DNA

First cycle transformants of NIH 3T3 cells transfected with metastatic human thyroid carcinoma DNA were used as immunogen to obtain monoclonal antibodies (MAbs) against normal and transformation-related antigens. The transformed cell line (M33) was shown to contain Alu sequences. Two MAbs were selected on the basis of their differential reactivity toward untreated NIH 3T3 cells or the transformed M33 cell line. By immunofluorescence, immunoelectronmicroscopy and biochemical analysis, the first MAb (MTr1) was demonstrated to recognize an epitope on cytoskeletal filaments of proliferating murine fibroblasts. Similar MTr1-labelled filaments were also found to accumulate into cytoplast-like structures spontaneously produced by M33 cells. The characterization by immunofluorescence of MTr2, the second MAb, indicates that it recognizes a specific human antigen associated with normal thyroid epithelial cells and differentiated thyroid tumors.     

Characterization of a CD11c-reactive monoclonal antibody (HC1/1) obtained by immunizing with phorbol ester differentiated U937 cells

A mouse monoclonal antibody (HC1/1) specific for a differentiation marker of human monocytes and granulocytes has been generated by using as immunogen the monocytic cell line U937 differentiated with phorbol esters. This differentiation antigen has been characterized as the p150/95 member of the CD11 family of glycoproteins by cellular distribution studies, immunoprecipitation and competition experiments with MAb 3.9 specific for the CD11c antigen. Immuno-alkaline phosphatase staining of normal tissue sections with the HC1/1 MAb demonstrated that the CD11c antigen is a useful macrophage marker. Furthermore, the MAb HC1/1 stains specifically populations of macrophages on skin and lymph node sections from different pathologies such as Sarcoidosis, Granuloma annulare, Lepromatous leprosy and Toxoplasmosis.     

Distribution of ferritin, transferrin and lactoferrin in breast carcinoma tissue.

An immunoperoxidase staining technique was used for detecting three major iron binding proteins (ferritin, transferrin and lactoferrin) in 40 breast carcinoma cases and six benign breast proliferative lesions. Ferritin staining was detected mainly in connectival stroma and in histiocytes surrounding neoplastic cells. Few and faint ferritin positivities were also detected in neoplastic cells of 20 carcinoma cases. Transferrin was found inconsistently in myoepithelial cells surrounding normal ductules, or around neoplastic ducts of ductal in situ carcinoma. In eight carcinoma cases, transferrin staining was also positive in neoplastic cells. Lactoferrin was detected only in normal breast epithelial cells and in benign breast proliferative lesions. These immunohistochemical findings may suggest that raised serum ferritin concentrations in breast carcinoma patients might be attributed to stromal reaction rather than to tumour synthesis. Transferrin staining of neoplastic cells in these carcinoma cases appears to be very intriguing, particularly since transferrin is considered an obligate requirement for growing cells, and transferrin receptors have been demonstrated only in dividing cells. On the basis of the immunohistochemical data, lactoferrin might be used as a pointer to benign lesions.     

Diagnostic utility of CD10 in differentiating hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration biopsy (FNAB) of the liver

The differential diagnosis between hepatocellular carcinoma (HCC) and metastatic carcinoma, especially in moderate-poorly differentiated (MPD) HCC and poorly differentiated carcinoma, can be challenging in fine-needle aspiration biopsy (FNAB) of the liver. Recent studies demonstrate that canalicular staining for CD10 appears to be a highly specific marker for hepatocytic differentiation. The objective of this study was to test the utility of CD10 in differentiating HCC from metastatic carcinoma in FNAB of the liver. Formalin-fixed, paraffin-embedded cell blocks of 55 cases (22 HCC, 23 metastases, and 10 benign hepatic lesions) of FNAB of the liver were immunostained using monoclonal antibody against CD10, with microwave oven antigen retrieval, followed by a standard ABC method. Nineteen (86%) of 22 HCC cases were positive for CD10 with a canalicular staining pattern. Among them, 9 (82%) of 11 well-differentiated (WD) HCC and 10 (91%) of 11 MPD HCC were positive for CD10. Three (13%) of 23 metastatic carcinomas were positive for CD10, demonstrating a contrasting cytoplasmic and membranous staining pattern. The three positive cases were metastatic renal cell carcinoma (RCC), choriocarcinoma, and adenocarcinoma of the lung. All 10 cases of benign hepatic lesions showed positivity for CD10 with a canalicular and focal membranous staining pattern. In conclusion, CD10 appears to be a useful marker in discriminating between HCC and metastatic carcinoma when applied to FNAB of the liver. CD10 does not provide discrimination between WD HCC and benign hepatocytes.