Effect of paclitaxel (Taxol) on CA 125 expression and release by ovarian cancer cell lines.

Two ovarian cancer cell lines, OVC432 and the newly established CVU4I, were used to study the effect of Taxal on cell growth and simultaneous CA 125 antigen expression. Growth of both cell lines was effectively inhibited by drug concentrations of 0.1 microM and higher. Complete inhibition of cell growth may result from high concentrations of Cremophor EL present in the Taxol formulation. Immunohistochemical analysis demonstrated that both cell lines retained the CA 125 expression on the cell surface during exposure to paclitaxel. This was reflected in a constant statistically significant correlation between cell numbers and CA 125 concentrations found in cell lysates. CA 125 levels in the culture medium showed a significant relation to cell numbers and, consequently, to the response of the cell line to the administered anticancer drug. It may be concluded from this study that CA 125 seems to be a reliable tumor marker in monitoring tumor response during paclitaxel treatment.

Cytokeratin 18 expression in squamous cell carcinoma of the head and neck.

Cytokeratin (CK) expression was studied in squamous cell carcinomas of different subsites in the head and neck by using cryostat sections from 27 head and neck squamous cell carcinomas (HNSCCs) and 6 cell lines established from HNSCC. All tissues were analyzed immunohistochemically with a panel of monospecific anti-keratin monoclonal antibodies. Most carcinomas recapitulated the expression pattern of keratins present in the basal layer of normal epithelium from the site of tumor origin. Regional differences in the expression of simple-epithelial type of keratins in stratified (pseudostratified) epithelia were to a large extent repeated in corresponding carcinomas. In the present study, localization of various keratins were surveyed and CK 18 specific monoclonal antibodies were specifically used to distinguish SCCs of the larynx or hypopharynx from SCCs of the oral cavity. CK 18 staining of almost all tumor cells was detected in 11 of 12 SCCs of the larynx and hypopharynx, but was only detected sporadically in 3 of 9 SCCs of the oral cavity. The present results show that CK 18 typing might be useful for distinguishing sites of origin of various HNSCCs. Findings also indicate that CK 18 expression in SCC might be modulated by microenvironmental factors.

Tenascin expression in basal cell carcinoma.

Tenascin (hexabrachion, cytotactin) is an extracellular matrix glycoprotein whose expression is strongly increased in hyperproliferative skin diseases, as shown by immunohistochemistry with polyclonal sera. In this study we describe a new monoclonal antibody (T2H5) against human tenascin. The specificity of T2H5 was validated by sequential immunoprecipitation of tenascin with polyclonal sera. T2H5 was used to analyse the presence of tenascin in basal cell carcinoma. Using Western blotting, at least two forms of tenascin were found, with approximate molecular weights of 210 and 300 kDa. In cultured human skin fibroblasts only the high molecular weight form was found.

Carcinoma-associated monoclonal antibodies in head and neck carcinoma. Immunohistochemistry and biodistribution of monoclonal antibodies 175F4 and 175F11.

Monoclonal antibodies (mAbs) 175F4 (IgG1) and 175F11 (IgG2a), originally raised against the human mammary carcinoma cell line (ZR-75-1), react with a carcinoma-associated antigen in both adenocarcinomas and squamous cell carcinomas of different origins. Immunohistochemically, the mAbs exhibited reactivity with 42 out of 43 squamous cell carcinomas of the head and neck. Normal squamous epithelia were also reactive with the antibodies in the basal and suprabasal cell layer. 111In 175F4 F(ab')2 fragments localized a squamous cell carcinoma xenograft (HT-6) in nude mice with low blood pool activity, indicating a potential clinical usefulness for staging of squamous cell carcinomas in the head and neck.

Metastatic ovarian or colonic cancer: a clinical challenge.

Clinical problems arise when histology is unable to differentiate between an ovarian carcinoma infiltrating into the rectosigmoid region and a colonic cancer with ovarian metastases. To evaluate the discriminative value of immunohistochemistry we studied four groups: (A) ovarian carcinoma (n = 21), (B) ovarian carcinoma with sigmoid stenosis (n = 18), (C) colonic carcinoma (n = 20) and (D) a group in which the differential diagnosis was a problem (n = 19). Paraffin sections stained with a panel of monoclonal antibodies revealed specific patterns: in group A and B a negative Parlam-4 and positive OC-125; in group C the opposite; in group D the 'colonic' pattern in 15 cases, and the 'ovarian' pattern in only 2. The clinical diagnosis in group D during follow-up was ovarian carcinoma in 7, colonic carcinoma in 8, double tumour in 1 and still unknown in 3. This was based on high levels of serum tumour markers such as carcinoembryonic antigen (n = 5) and CA-125 (n = 4), laparotomy (n = 4), autopsy (n = 1), barium enema and/or endoscopy (n = 5). The response to chemotherapy in group D was extremely poor.

Fetal mouse alveolar type II cells in culture express several type II cell characteristics found in vivo, together with major histocompatibility antigens.

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.

Keratin subtypes in carcinomas of the uterine cervix: implications for histogenesis and differential diagnosis.

Normal epithelia and carcinomas of the human uterine cervix were studied by monoclonal antibodies chain specific for cytokeratins 4, 8, 10, 13, 14, 18, and 19. Most cells in 13 examined squamous carcinomas revealed a cytokeratin phenotype detected in ectocervical basal cells and endocervical subcolumnar reserve cells: 8+, 14+, 18+, 19+, 4-, 10-, 13-. We propose that these two cell types are closely related or identical and that squamous carcinoma of the cervix originates in this cell type. In more differentiated tumor cells cytokeratins 4, 10, and 13, which are present in suprabasal layers of the normal ectocervical epithelium, were coexpressed with basal cell cytokeratins. Thus, contrary to previous beliefs, all cytokeratins detected in carcinomas were also present in normal epithelium of uterine cervix. The cytokeratin profile of cervical adenocarcinomas corresponded to that of columnar endocervical cells (8+, 18+, 19+), although two of the three adenocarcinomas also expressed cytokeratin 4, which in the normal endocervix was detected in scattered single columnar cells only. The new monoclonal antibody DE-K14, specific for cytokeratin 14, proved a specific marker of subcolumnar reserve cells in the endocervix. It was also the only one that reacted with all cervical squamous carcinomas but with none of the cervical adenocarcinomas and, as such, has a potential value for pathological differential diagnosis of cervical tumors.

New monoclonal antibodies recognizing epidermal differentiation-associated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10 expression in carcinoma of the vulva.

Two monoclonal antibodies (MAb) specific for differentiation-related epidermal keratins have been developed. They represent specific molecular probes for different stages of epidermal differentiation. Antibody DE-K10 is chain-specific for cytokeratin polypeptide no. 10 (56.5 kD) expressed in all suprabasal layers of the epidermis. Antibody DE-SCK is specific for modified stratum corneum keratins and thus represents a marker for the terminal step of epidermal differentiation. Since the epitopes identified by both antibodies are preserved in formalin-fixed, paraffin-embedded tissue sections, these antibodies can be used for retrospective studies of differentiation in various pathological processes. We have used antibody DE-K10 to study the cytokeratin 10 expression in 26 stage II or III vulvar squamous cell carcinomas. Preliminary data suggest an increased risk of recurrence in cytokeratin 10 negative tumours.

Monoclonal antibodies to epithelial sialomucins recognize epitopes at different cellular sites in adenolymphomas of the parotid gland.

Fourteen monoclonal antibodies (MAbs) to epithelial sialomucins were studied for their immunohistochemical reactivity on serial sections of 14 formalin-fixed and paraffin-embedded adenolymphomas of the parotid gland. Two types of reactivity were observed, suggesting different cellular distribution of the corresponding epitopes. Most antibodies reacted with the luminal membrane of the columnar tumor cells (type-A reaction). The other reaction (type B) was observed with the membrane of basal epithelial cells. The antibodies could be ranked according to their tendency to show type-A and/or type-B reactions. MAb Cal was the only one with a pure type-A reaction. A strong tendency to type-A reactivity (with traces of type-B reactions) was observed for the antibodies HMFG-2, M8, E29 and NCRC-II. Several antibodies gave good type-B reactions in addition to strong type-A reactivity (MAbs 126E7, 115G2, 115D8, 140C1, F36/22, 139H2). MAb DF3 showed equally strong reactions with both cell types. A clear-cut preference of the reactions with basal cells was seen with the antibodies HMFG-1 and 115F5. This subclassification of the antibodies is in accordance with epitope mapping data, obtained by conventional blocking studies reported in the literature.

Immunohistochemical localization of the epithelial marker MAM-6 in invasive malignancies and highly dysplastic adenomas of the large intestine.

MAM-6 antigen is detectable in a large variety of formalin fixed and paraffin embedded normal and neoplastic tissues throughout the body with some exceptions. One such exception is the large intestine in which normal mucosa has been found negative for MAM-6, while colorectal carcinomas express the antigen in almost all cases. We examined the expression of MAM-6 in 147 benign epithelial tumors (adenomas) of the colorectum and compared this with the detectability of the antigen in 123 colorectal cancers and in 108 non-neoplastic mucosa specimens. The tissue samples were subjected to an indirect immunoperoxidase assay applying the MoAb 115D8 which recognizes the epitope "a" of MAM-6. A majority of the carcinomas (99.2%) and of the adenomas (61.4%) exhibited reactivity for MAM-6 "a", whereas only 8 of the 108 non-neoplastic biopsy specimens (= 7.4%) exhibited faint focal staining. In adenomas, positive reactions were only seen in highly dysplastic tissue areas, in part reflecting carcinomata in situ. The reactivity in four non-neoplastic tissue samples could be explained by shedding of the antigen from carcinomas into glandular structures of the mucosa. Two out of 32 cases exhibiting severe colitis were also focally positive. The detectability of MAM-6 "a" in routinely processed specimens from the large intestine is therefore considered as being closely associated with malignant and premalignant processes. According to a limited study with the antibodies 139H2 and 140C1, this holds true for two additional MAM-6 epitopes. MAM-6 might be considered as a marker of severe (premalignant) dysplasia in adenomas of the large intestine. Further studies are needed to clarify if there is a biological (possibly prognostic) difference between MAM-6 positive and negative high epithelial atypia in these adenomas.

Studies on antibodies against feline leukaemia virus (FeLV) in cat sera and rabbit anti-FeLV sera: cross reaction and differences.

The indirect immunoferritin technique (IFT) that enables us to distinguish clearly whether an antibody reacts with a virus particle or only with the cell membrane, was used to study 25 cat sera and one rabbit anti-feline leukaemia virus (FeLV) serum using FL-74 cells as target. (1) All sera contained antibodies against FeLV even though 11 of the cats were viraemic at the same time; (2) from the effect of glutaraldehyde fixation of the FL-74 cells on the reaction with cat sera and the results of blocking experiments, it could be concluded that cat sera and rabbit anti-FeLV sera react partly with different antigenic specificities of FeLV, partly with the same antigens; and (3) the indirect membrane immunofluorescence test using FL-74 cells as target is not a good test to detect the presence of antibodies against feline oncornavirus-associated cell membrane antigen (FOCMA) because FL-74 cells produce a large quantity of FeLV and the fluorescence measured could be from antibodies against FeLV.

Virus-like particles in bovine sera for tissue culture.

Virus-like particles were found in nine different bovine sera for tissue culture from commercial suppliers. These particles were spherical with an overall diam. between 70 and 95 nm. After negative staining, surface projections of about 11 to 12 nm were clearly seen. One of the nine sera was positive in an Ouchterlony test with antisera against bovine viral diarrhoea virus.

Antibody-induced modulation and shedding of mammary tumor virus antigens on the surfaces of GR ascites leukemia cells as compared with normal antigens.

The distribution, antibody-induced redistribution, and shedding of murine mammary tumor virus (MuMTV) antigens and the surfaces of GR mouse ascites leukemia (GRSL) cells were studied by the immunoferritin technique and compared with the same activities of thy 1.2 and H-2.8 antigens. MuMTV antigens were redistributed easily and then largely shed from the cell surface; in contrast, H-2.8 antigen moved easily and probably was partially released from the plasms membrane and Thy 1.2 antigen moved slowly and was some what interiorized. The complement-dependent cytotoxicity test was used to study the possibility of antigenic modulation for these cell-surface antigens from the surface of the GRSL cells could be modulated by preincubation with anti-MuMTV serum, in contrast to H-2.8 and Thy 1.2 antigens. The results obtained with the immunoferritin technique and the cytotoxicity test correlated well and sug-ested that the shedding of MuMTV antigens from the cell surfaces may occur in vivo, providing the tumor a way to escape from the immune defense of the host. Thy 1.2 and H-2.8 antigens were present on the envolope of B and C particles, which suggested that these viruses do not select a Thy 1.2 or H-2.8-negative area of the GRSL cell surface as amaturation site.

Immunologic, virologic, and genetic aspects of mammary tumor virus-induced cell-surface antigens: presence of these antigens and the Thy 1.2 antigen on murine mammary gland and tumor cells.

The distribution of the normal differentiation antigen Thy 1 and the mammary tumor virus (MTV)-induced antigens or antigen complexes MLm and MLr were studied in mouse mammary gland cells, mammary tumor cells, and other cell types, by use of ascites leukemia cells of the GR mouse strain as target cells in the cytotoxicity test. The Thy 1.2 antigen was detected by an AKR antiserum to C3Hf thymocytes. MLm was shown by a homologous C57BL antiserum to GRSL2 leukemia (absorbed in vivo in GR mice); MLr was detected by a rabbit heterologous antiserum (absorbed in vivo in C57BL or GR mice and in vitro with BALB/c milk) prepared against Tween 80- and ether-treated purified B particles. Sera from Sprague-Dawley rats bearing murine leukemia virus (MuLV)-producing syngeneic tumors were not cytotoxic or only slightly cytotoxic for GR leukemias transplanted in vivo, which indicated that MuLV-induced antigens were absent or present in very low quantity in such leukemias. The MLr and MLn antigens or antigen complexes were possibly identical to the mammary leukemia (ML) antigen, since they could be detected not only on GR but also on DBA/2 leukemia cells and since their distribution was exactly the same as that of MTV. Both the MLr and MLm antigens were present in purified B particles, and antigenic activity were present in purified B particles, and antigenic activity was enhanced by destruction of the purified virus particles. The antigens were about eightfold enriched in a preparation of B-particle envelopes, as shown by quantitative cytotoxicity absorption (CYTA) tests. Purified nucleoid fractions of B particles were only lightly positive for the antigen, probably due to envelope contamination. One dominant gene was responsible for the expression of MLr, as shown by CYTA tests with mammary glands of individual animals of segregating crosses between the GR strain with high mammary cancer incidence and strains with low incidence. This gene was closely linked with or was possibly identical to 1) the gene for cytoplasmic MTV gs antigen expression as seen by fixed cell immunofluorescence, and 2) the gene causing mammary tumors in the GR mouse strain.

Quantitative estimation of mouse mammary tumor virus (MTV) antigens by radioimmunoassay;.

A radioimmunoassay for the antigens of the mouse mammary tumour virus has been developed. -125Iodine-labelling of intact virus [derived from mammary tumours of (C3H TIMES 020)F1 mice] followed by ether extraction and separation of the ether and water layers resulted in a separation of the labelled material into two main groups of antigenic components. The intact labelled material from each group was separated from viral debris and other possible contaminants by affinity chromatography. The antigens of the ether phase were proved to belong mainly to the viral nucleoid whereas the water phase contained mainly envelope antigens. No type-specific antigens could be demonstrated in either of the phases. Radioimmunological assessment of plasma revealed that in the plasma of MTV-S- and MTV-P-positive animals viral antigens were only measurable when palpable mammary tumours were present, whereas in MTV-L-positive tumour-bearing animals some negative samples were found. In milk of individual nontumour-bearing mice a close correlation was found between the expression of viral antigens and the generally accepted presence of virus in the strain of mice. In milk, viral antigen levels tend to increase with parity with a possible decrease after a finite number of pregnancies. In a pilot study the presence of MTV antigens could also be demonstrated in epididymis extracts of male GRS/A mice. Genetical analysis of the low-virulent MTV-L by radioimmunoassay of milk is in progress.