Search

STAT 1, a member of signal transducer and transcription activator family has been implicated as key downstream mediator of interferon (IFN) signaling. Its functional activation requires phosphorylation at Tyr 701 and Ser 727 residues. Various STAT abnormalities have been found in cancer cells but their relation to oncogenesis, tumor behavior and disease outcome remains mostly unknown. We have examined the inducibility of STAT 1 phosphorylation by IFN alpha/gamma in primary cultures established from melanoma lymph node metastases at first progression and correlated our results with disease outcome and overall survival. Forty-four patients at clinical stage I-III at initial diagnosis entered the study. STAT 1 inducibility of phosphorylation by IFNs was assessed in melanoma cell lysates by means of standard immunoprecipitation and Western blotting using polyclonal and monoclonal antibodies. Lack of STAT 1 phosphorylation at Ser 727 after either IFN was recorded in 75% of patients, however, no correlations with disease evolution could be proved. In contrast, STAT 1 phosphorylation response at Tyr 701 after IFNalpha occurred in 13 (29.5%) and after IFNgamma in 32 (73%) patients. Inducibility of STAT 1 activation at Tyr 701 but not at Ser 727 driven by IFNgamma but not by IFNalpha significantly and unfavorably [corrected] influenced disease- free interval and overall survival. In conclusion, these results show that the absence of IFNgamma inducibility of STAT 1 phosphorylation at Tyr 701 positively correlates with disease outcome in malignant melanoma patients and may represent new independent prognostic marker.

STAT 1, a member of latent cytoplasmic proteins, plays a pivotal role in mediating biological effects of interferons. Its transducing, DNA binding and transcriptional activity require phosphorylation at both Tyr 701 (Y 701) and Ser 727 (S 727) residues. Deficient phosphorylation or constitutive activation of the STAT 1 protein were observed in some human malignancies. Using immunoprecipitation and Western blots performed with lysates made of melanoma cells derived from patients with clinical stage II/III and employing specific anti-STAT 1 PS 727/PY 701 immunoprobes, we show that STAT 1 activation response induced by IFN-alpha/-gamma is significantly impaired. On average, three quarters of patients were lacking phosphorylation at S 727. STAT 1 PY 701 was not inducible by IFN-alpha in 63% and by IFN-gamma in 34% of samples. However, these STAT 1 activation defects showed no correlation with the disease outcome and immunotherapy response as indicated by progression-free survival profiles in patients treated with IFN-alpha2b.

Immune parameters, including cytokine levels and CD profiles were determined in 78 renal cell carcinoma patients (RCC) prior to nephrectomy. The values were correlated with the outcome of disease and response to cytokine-based treatment during a 3-year follow-up. Significantly lower frequency of progressions and higher proportion of survivors were recorded in 24 treated patients compared to 43 untreated ones (22.9% vs. 53.5% and 82.9% vs. 55.8%) illustrating the beneficial effect of immunotherapy on the course of RCC at localized stage. RCC-related immune changes are demonstrated by reduced proportion of CD19+, CD28+, HLA-DR+, CD19+/80+ and CD8+/28+ subsets, by increased serum levels of IL-6, sIL-2R, CRP and by impaired production of IL-2 and TNF-alpha released by in vitro stimulated PBMC. Only increased CRP, IL-6 serum values, decreased CD8+ and increased CD122+ were significantly related to patients' prognosis. Comparisons of preoperative CD profiles and cytokine values with the response to IL-2/IFN-alpha based therapy disclosed significant correlation in only CD80+ and CD19+/80+ subsets. Treated patients who relapsed during the 3-year follow-up exhibited at the diagnosis significantly reduced proportion of CD80+ and CD19+/80+ cells (CD80+ means - 0.79 vs. 1.69 and CD19+/80+ means - 0.32 vs. 0.61) comparing to those surviving disease-free. In addition initial proportion of CD3+, CD8+ and CD19+ cells was reduced in treated patients who manifested progression but statistical difference from those remaining disease-free was not proved.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

This prospective study was carried out to explore cytokine-related immune alterations in 69 renal cell carcinoma patients (RCC) and to look for changes which might potentially serve as a reliable predictors of response to cytokine-based therapy. Interleukin-2 (IL-2), its soluble receptor (sIL-2R) and tumor necrosis factor (TNF-alpha) levels produced in vitro by PHA activated and intact mononuclear cells (PBMC) were determined. Concentrations of IL-2, IL-4, IL-6, sIL-2R, TNF-alpha and CRP were measured in sera. Cytokine level was evaluated by enzyme-linked immunoadsorbent assay (ELISA) and CRP was determined by means of turbidimetric method. All measurements were performed in patients without any prior treatment. PHA activated PBMC of RCC patients were significantly defective in producing IL-2 and TNF-alpha comparing to controls (p < 0.03 and p < 0.001). The difference of sIL-2R was noted in metastatic stage only (p < 0.03). Unstimulated PBMC manifested decrease in IL-2 (p < 0.03) and increased level of TNF-alpha in advanced disease (p < 0.02). This impairment reflected tumor size and differentiation stage. Serum concentrations of IL-2, sIL-2R and TNF-alpha were within normal range. However, in relation to the clinical stage, significantly increased serum IL-2 was noted in combined Stage I and II as compared to controls (p = 0.012). IL-6 and CRP showed markedly elevated levels with a significancy which allowed to distinguish samples from metastatic patients. In conclusion careful comparisons of these data with clinical course of cytokine treated patients will disclose which of those tests may possess predictive power in the individual patients who are likely to respond to cytokine-based treatment.

We have analyzed p53 protein expression in 121 primary breast cancer biopsies by immunohistochemistry using the monoclonal antibody DO-1 and polyclonal serum CM-1. p53 protein overexpression has correlated in our study with mitotic activity (p=0.001), nuclear atypia (p=0.002), less favorable histological type of tumor and in a lesser extent with tumor size. The inverse, but highly significant, correlation (p=0.007) has been observed with lymph node involvement. There was also a trend for higher p53 positivity among DNA aneuploid tumors as compared with DNA diploid cases, but this was not significant. Our study suggests that p53, at least in some patients, may not be directly involved in the process of metastatic progression in breast cancer. Preliminary data would suggest that the detection of p53 protein overexpression could be a useful additional prognostic parameter in breast cancer.

Five fusions between mouse embryonic cells and syngeneic adult peritoneal macrophages were performed. The resulting hybrids as well as both parental cells (6 cultures of embryonal cells and 6 cultures of adult macrophages) were grown in vitro under the same culture conditions. All populations of explanted macrophages died during the second month in primary culture and five populations of cultured embryonic cells were lost within six months under in vitro conditions as well. One embryonic cell line survived and acquired transformed and/or malignant phenotype: When inoculated into either newborn or adult syngeneic mice, progressive growth of tumors with 100% take (6/6), histologically classified as poorly differentiated fibrosarcoma with areas of metaplastic bone and osteoid, was observed. Two out of five wild hybrid strains died within six months of cell culture. The resulting three hybrid cultures adapted themselves to in vitro conditions and finally permanent lines were established with all features of transformed phenotype in vitro and with the capacity to grow as undifferentiated fibrosarcomas with 100% take (6/6) when inoculated into syngeneic mice either s.c. or i.p. Cytogenetic studies were performed and phenotypic characteristics of these lines were explored as well. Biological assays performed for the presence of oncogenic viruses were negative and none of the malignant cell lines showed positive staining with the monoclonal antibody specific for large T-antigen. It is suggested that cell fusion of two normal partners may switch on the cascade of abnormal processes which may culminate in neoplastic conversion. Cell fusion might play also a significant role in the so called "spontaneous" transformation.

Humoral and cellular immunity was followed in a group of long-term surviving patients with malignant lymphoma, viz. serum immunoglobulins quantitatively, T lymphocytes by the E rosette and B lymphocytes by the EAC rosette methods. A single examination of this group was compared with data from a control group of normal humans and a smaller group of patients with malignant lymphoma at an advanced stage. A significant increase of IgA (3.73 g/l onthe average) was noted in the surviving group as against both the controls (mean 2.31 g/l in the healthy and 1.23 g/l in the patients with advanced disease). IgM values were found to be significantly decreased in the latter patients (mean 0.85 g/l) in comparison with both the other groups. The rosette tests yielded lower absolute values of E rosettes in the same patients with advanced lymphoma (mean 725/ml) as against the healthy subjects (mean 1525/ml) and the long surviving patients (mean 1277/ml). The absolute value of active E rosettes in the two groups of patients was lower (741 and 580/ml) than that in the healthy group (mean 1067/ml). A percentage determination of rosette values proved to be of low statistical significance.

Hybridoma clones were established by fusing spleen cells from mice hyperimmunized with human breast cancer cells of MDA-MB-231 line with murine myeloma cells P3-X63-Ag8-653. Ten permanent hybridomas were stabilized. The monoclonal antibodies of three of them, i.e. HBCA-6, HBCA-4 and HBCA-12 were tested against 20 various established cell lines. The most restricted binding properties showed HBCA-12 antibody which reacted positively only with two types of target cells. The cross-reactivity of HBCA-12 with human breast cancer cell line MDA-MB-231 and human myeloma derived cells ARH-77 is discussed in view of the pertinent target structure, i.e. differentiation antigens, allospecific antigens, hormone receptors and shared tumor associated antigens. It was shown that the target structure for HBCA-12 is localized on the cell surface.

Tumor "specific" immune recognition was assayed in 125 patients with various breast diseases including breast cancer of clinical stage I and II, 22 patients with other malignancies and 64 healthy persons employing leukocyte adherence inhibition test (LAI). In the group of breast cancer patients (BC) there were 81% of positive responders (52/65) with a mean nonadherence index (NAI) value 67.4. Sensitization to extract derived from breast cancer was detected in 38.3% (23/60) of patients with benign breast diseases (BBD). The mean NAI value was significantly lower comparing to NAI value of BC patients (34.8 vs. 67.4) but exceeded the upper limit of normal values. The most frequent positive responders of BBD group were found in patients with proliferative mastopathy (11/17). Our study brought further evidence that BC patients and in a lesser degree BBD patients are sensitized to some antigen(s) contained in selected breast tumor extracts. However, high proportion of false positive results in healthy persons (14.1%) and mainly considerable number of positive responders in BBD patients represent a major limitation for clinical diagnostic usefulness of the LAI assay.

Monoclonal antibody HBCA-12 obtained by hybridoma procedure after immunization with human mammary adenocarcinoma cell line MDA-MB-231 immunoprecipitated a cell surface sialoglycoprotein gp80 (apparent molecular weight 80 000) from MDA-MB-231 cells and a glycoprotein gp78 from human myeloma cell line ARH 77. A protein of a similar electrophoretic mobility was immunoprecipitated also from 35S-methionine metabolically radiolabeled human melanoma cell line VUP 1. The expression of the antigen recognized by HBCA-12 monoclonal antibody could be detected neither on PHA-induced nor on EBV-transformed peripheral blood mononuclear cells from healthy donors.

DNCB and PPD skin testing was performed in 152 breast cancer patients. Bates' instruction with a plea for uniformity was used (Cancer, 43, 1979, 2306). Majority of patients were tested while being diagnosed and prior to the treatment. There were no differences in the reactivity within early operable breast cancer patients (Stage I and II) with respect to nodal involvement. Patients with N1 reacted in the same manner as those with N0. The reactivity of patients with locoregionally advanced disease (Stage III) was similar to that of Stage I and II patients. Significantly lower responsiveness was found in Stage IV patients, the depressed response to DNCB being more pronounced than to PPD.

Establishment of a mouse-mouse hybridoma and partial characterization of IgM monoclonal antibody (M-04) identifying cytoplasmic filamentous structures is described. Immunofluorescence performed on a panel of various cultured human cell types as well as on frozen sections of normal and tumour tissues revealed specificity of M-04 antibody for cells of epithelial origin. Using MCF-7 cell line as a model, staining patterns of microtubules, microfilaments and M-04-target filaments in untreated cells were compared with those pretreated with Colcemid and Cytochalasin B. From both differential staining of various cell types and the results of drug studies it is concluded that monoclonal antibody M-04 binds to intermediate filaments of cytokeratin type. Furthermore, restricted expression of M-04 target determinant among epithelial tissues is suggested from the lack of reaction in stratified skin epithelium.

Clinical findings in 63 patients with malignant melanoma were compared with the results of PHA-induced lymphocyte transformation tests. The patients had been followed up for 1 to 60 months (average 16 months) and repeatedly tested at regular intervals. Reactivity of lymphocytes in stage I patients was not altered if compared with that of controls. The frequency of lowered values increased in more advanced stages. Only in stage II patients a significantly impaired PHA-lymphocyte transformation was demonstrated in comparison with controls. In stage I and II patients, there was no significant difference in lymphocyte transformation between those with the presence of tumor and those without clinically evident tumor. Prognostic value of PHA transformation tests as regards the appearance of distant metastases within two years after the first examination, could not be proven in patients at initial stages of melanoma.

Two monoclonal antibodies, DA7 and DC10, were obtained from fusions of mouse myeloma cells with splenic lymphocytes from mice immunized with human breast cancer cells of PMC 42 line. The indirect immunofluorescence studies performed on established tumor cell lines together with immunoperoxidase staining of normal human tissues showed that the components reacting with the antibodies were cytokeratins. Positive reaction was noted in all epithelia derived cultured cells and in all simple epithelial tissues known to express keratin 18. Immunoblotting performed on various cytoskeletal preparations demonstrated strong staining of a single band with a mobility corresponding to that of cytokeratin 18 (45 kD). The negative immunoperoxidase reaction found in different epithelial tissues of seven animal species suggests that both antibodies are specific for human keratin 18. It was shown that DA7 and DC10 antibodies exhibited strong reaction in paraffin embedded tissues fixed in either methacarn or standard formalin. These characteristics predetermine both antibodies as suitable reagents for the specialized histopathological work.

Leukocyte adherence inhibition (LAI) assay was employed to detect cell-mediated immune response in breast cancer patients of clinical stages I and II. Twenty-one breast cancer extracts were screened in 232 breast cancer patients and 343 healthy persons. The activity of individual extracts differed remarkably. Overall, LAI response was 58.6% (136/232) correct and 18.9% (65/343) false positive. Fifteen extracts were not suitable from the clinical point of view. Only two extracts gave a constantly high rate of tumor-specific LAI-positive values in breast cancer patients, ie, 83.3% (25/30) and low percentage of false positive results in controls, ie, 4% (2/50). A further study dealt with the possible relation between the activity of extracts (expressed in percentage of positive LAI results in the tumor group) and histology of primary tumor of patients whose tumor tissue was used for extraction. This relationship could not be proven statistically. We investigated also whether the percentage of correct LAI responses in breast cancer patients could be affected by histological agreement or disagreement (grading and node involvement) between primary tumor of extract donor and primary tumor of patients tested. No significant relationship was found in this respect.

A new human cell line of malignant melanoma (MJM) was established with the use of the in vitro fragment technique. It has been maintained over 34 months of continuous cultivation. Three types of cells can be recognized by light microscope. The epitheloid elements predominate, less frequent are fibroblastoid and giant multinuclear cells. The pigment production is not macroscopically visible. Over 60 per cent of analyzed metabphases showed hyperdiploid number of chromosomes, the rest was mostly tetra and hexaploid. No marker chromosomes were detected. The growth studies indicate the MJM cells have 63-hr doubling time. Cytochemistry revealed positive pigment or propigment granules in 36 per cent of cells. Ultrastructural studies did not detect melanin granules but some particles resembling atypical premelanosomes and melanosomes were recognized in some sections.

A panel of 17 monoclonal antibodies (MAbs) recognizing various keratin polypeptides has been used to define their binding on non-epithelial elements in 28 bone-marrow samples and 14 lymph nodes, in order to establish their limitations for use as a possible tool for immunodiagnosis of carcinoma spread. Immunocytochemical studies have shown that only 8 antibodies consistently exhibited no false-positive staining of marrow cells. All the remaining MAbs labelled (mostly in a non-specific manner) a few cells of marrow samples derived from patients with either haematological disorders or malignant lymphomas. Fine granules and droplet-like cytoplasmic inclusions were predominant patterns of positive reactions. Homogeneous cytoplasmic staining reminiscent of specific keratin immunolabelling was occasionally seen as well. The positive cells could be also identified in some lymph nodes free of tumour infiltration. All antibodies visualized cytoplasmic droplets in scattered cells of lymph nodes taken from a patient with non-Hodgkin lymphoma. This type of positivity was mostly associated with positive histochemical reactions for iron. Quite significant was the detection of fibrillar positivity in the extrafollicular reticular cells in all nodes examined. Such a specific type of staining was exclusively induced by antibodies directed against epitopes of keratin 8 and 18, whereas those MAbs recognizing keratin 7 and 19 always gave negative results. Our data indicate that caution is required when such MAbs, considered as markers of specific cell types, are being used as an immunodiagnostic tool to identify single carcinoma cells. A series of criteria, including morphological ones, must be utilized in order to obtain meaningful results.

The relationship of pretreatment immunologic status in terms of skin tests to prognosis within stages was studied in 152 breast cancer patients. DNCB and PPD testing was used. As for DNCB, no relationship was found at early stages of the disease. In locoregionally advanced disease, patients with stronger test grades had longer disease-free intervals. In case of distant dissemination significant difference in reactivity with respect to survival was found: short survivors were more frequently nonresponders or mild responders. Anergy was, however, more frequent in patients with general ill health and therefore this test does not provide an important additional prognostic information as compared to that given by conventional clinical findings. As for PPD, no relationship between reactivity to this antigen and prognosis at any stage of the disease was found.