Reduced inducibility of SOCS3 by interferon gamma associates with higher resistance of human breast cancer lines as compared to normal mammary epithelial cells.

The resistance to interferons (IFNs) limits their anticancer therapeutic efficacy. Here we studied the antiproliferative effect of interferon gamma in relation to SOCS3 expression in a panel of breast cancer cell lines and normal mammary epithelial cells. Compared to normal cells most breast cancer lines (7/8) were highly resistant to IFN-gamma. Using Northern blot and real time RT-PCR we investigated transcription of SOCS3 genes. All normal epithelial cells (4/4) showed SOCS3 induction (2-14 fold) while most breast cancer lines did not or weakly activated SOCS3 after the interferon gamma treatment. Among the cancer lines, the MDA-MB-468 cells showed increased sensitivity to IFN-gamma and relatively high level of SOCS3 induction (2-3 fold). Together, there was a good correlation

Conformational changes in p53 analysed using new antibodies to the core DNA binding domain of the protein.

The p53 protein contains a protease resistant core section that binds to DNA in a sequence specific manner and whose crystal structure has been determined. This core is flanked at the N-terminus by the transcriptional transactivation domain and at the C-terminus by sequences involved in the oligomerisation of the protein. Extensive immunochemical analysis of p53 has shown that dominant antigenic sites lie within these N- and C-terminal domains while few antibodies to the central core have been identified. One of these, PAb240, has been extensively characterised as its epitope is cryptic in the native DNA binding core structure but is exposed by denaturation. This epitope is also exposed on many p53 proteins that contain point mutations in the core domain suggesting that these mutations may have a common affect on the structure of the core. To investigate this further we have generated several new antibodies to novel sites on p53 and mapped their epitopes using synthetic peptides. We find that antibodies to two other discrete sites in the core can also, like PAb240, recognize cryptic epitopes and distinguish mutant from wild-type conformations implying that the point mutations found in p53 in human tumours have widespread effects on the folding pattern of the DNA binding domain.

Lack of STAT 1 phosphorylation at TYR 701 by IFNgamma correlates with disease outcome in melanoma patients.

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.

Detection of keratin subtypes in routinely processed cervical tissue: implications for tumour classification and the study of cervix cancer aetiology.

We investigated the expression of keratin subtypes 7, 8, 10, 13, 14, 17, 18 and 19 in the normal cervix, in cervical intraepithelial neoplasia (CIN) lesions and in cervical carcinomas, using a selected panel of monoclonal keratin antibodies, reactive with routinely processed, formalin fixed paraffin embedded tissue fragments. The reaction patterns derived for each keratin antibody were compared with known expression patterns of the various epithelia, previously examined in frozen tissues. Although the reactivity of the antibodies was generally acceptable, considerable modifications to the manufacturers' staining instructions were often necessary. For some antibodies, which were previously thought to be reactive with fresh frozen tissue only, we developed staining protocols rendering them reactive with routinely processed material. As with previous findings in frozen sections we observed increasing expression of keratins 7, 8, 17, 18 and 19 with increasing grade of CIN. In cervical carcinomas the differences in keratin detectability between the main categories were more pronounced than in frozen sections, probably due to fixation and processing. For routine pathology, keratin phenotyping of cervical lesions may be of value in classification. The fact that keratin 7 was detected for the first time in reserve cells, and that this keratin was also found to be expressed in a considerable number of CIN lesions and cervical carcinomas supports the suggestion that reserve cells are a common progenitor cell for these lesions.

Relation of prenephrectomy CD profiles and serum cytokines to the disease outcome and response to IFN-alpha/IL-2 therapy in renal cell carcinoma patients.

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.

Monoclonal antibody against DNA adducts with osmium structural probes.

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.

Malignant melanoma associates with deficient IFN-induced STAT 1 phosphorylation.

STAT 1, a member of signal transducers and transcription activators of STAT family proteins, has been implicated as important mediator of IFN signaling. Functional activation of STAT 1 requires tyrosine and serine phosphorylation. Defects in its expression or activation in response to IFNs were observed in numerous pathological conditions including cancer. To further explore cancer-associated impaired STAT 1 response to IFNs, the inducibility of serine (S 727) and tyrosine (Y 701) phosphorylation by IFN-alpha/-gamma was assessed in 21 melanoma cell lines and in 35 primary cultures derived from melanoma patients. STAT 1 levels and inducibility of its activated phospho-forms were detected by Western analysis using specific polyclonal and monoclonal antibodies. All cell lines as well as patient melanoma samples expressed STAT 1 with variable signal intensity. Significant impaired IFN-induced STAT 1 S 727 phosphorylation was observed in both model systems with average of 77% of non-responders recorded in patient melanoma cells and 76% in melanoma cell lines. Failure of PY 701 induction occurred in patient samples (63% after IFN-alpha and 34% after IFN-gamma induction) and to a lesser degree in cell lines (i.e. response absence to IFN-alpha in 5 and to IFN-gamma in 2 melanoma lines). Our study demonstrates STAT 1 functional abnormalities in melanoma cells. On the basis of detailed analyses of patient melanoma cells with respect to the inducibility of STAT 1 phosphorylation by IFNs, four categories of patients could be distinguished: a) activation on both S 727 and Y 701, b) not inducible response, c) activation on Y 701 but not on S 727, d) heterogeneous response. Clinical study is now in progress to establish the significance of in vitro STAT 1 activation for predicting the response to IFN-based therapy and to explore biological consequences in cases responding in vitro to IFN-induced STAT 1 activation on only one of the critical amino acid residues.

Monoclonal antibodies to membrane components of human breast cancer cell line MDA-MB-231. Production and reactivity with various cells in culture.

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.

A prospective study of lymphocyte responses to phytohemagglutinin in melanoma patients. (lack o prognostic value of correlation with minimal tumor burden).

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.

DNCB and PPD skin tests and prognosis in 152 patients with breast cancer. A prospective 2-year follow-up.

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.

Humoral and cellular immunity in long-term surviving patients with malignant lymphoma.

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.

DNCB and PPD skin testing in breast cancer.

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.

Biochemical and histochemical characteristics of target antigen detected by monoclonal antibody HBCA-12 against a membrane component of human mammary carcinoma cell line.

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.

Evaluation of the LAI test in patients with benign and malignant breast diseases.

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.

Fusion-induced malignancy? A preliminary study. (a challenge to today's common wisdom).

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.

Establishment of cell line derived from human malignant melanoma.

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.

Malignant melanoma associates with Th1/Th2 imbalance that coincides with disease progression and immunotherapy response.

The immunological dysfunction associated with human cancer is well known phenomenon. It comprises number of pathological changes within immune network including imbalance in cytokines of Th1/Th2 origin. The objectives of our study were (i) to evaluate the abnormalities in serum levels of selected cytokines in malignant melanoma patients with regional lymph node metastases as compared to normal values, (ii) to examine the relationship between postoperative cytokine levels and disease progression and (iii) to correlate cytokine profile changes during IFN-alpha therapy with the disease progression and potential therapeutical response. Nine Th1/Th2 related cytokines and sIL-2R were determined in 26 malignant melanoma patients at clinical stage III prior and during adjuvant immunotherapy. Control group consisted of 26 healthy persons. Patients were treated with rIFN-alpha according to EORTC Melanoma group protocol 18952. Cytokines were quantified in patients sera using commercial ELISA kits. Majority of melanoma patients showed significantly lower values of IL-2 and IFN-gamma and pathologically elevated levels of IL-4, IL-6, IL-10 as compared to healthy subjects what indicates disease associated Th1/Th2 imbalance. In addition increased IL-12 and IL-15 values were noted in some patients (54% and 27%, respectively). All patients who manifested early relapse during immunotherapy had significantly lower pretreatment levels of IL-2 and IL-12 than those remaining without progression and probably benefiting from the treatment. Cytokine changes during immunotherapy disclosed that decreases in IL-2 and IL-12 and raises in IL-6 and IL-10 values occurred at least one month prior to relapse. Moreover, the continuous elevation of TNF-alpha and sIL-2R could be observed in patients who remained without progression during 10 months lasting immunotherapy. Our data illustrate that malignant melanoma associates with Th1/Th2 perturbances which are directed towards extended Th2 responses and that measurement of selected cytokines of Th1/Th2 category may be used as an early signal of disease deterioration and for evaluation of immunotherapy response.

p53 protein overexpression associates with growth patterns rather than with metastasizing in operable breast cancer.

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.

Renal cell carcinoma-associated immune impairment that may interfere with the response to cytokine therapy.

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.

Significance of pre-treatment immunological parameters in colorectal cancer patients with unresectable metastases to the liver.

BACKGROUND/AIMS: In this study, we have compared the profiles of peripheral blood lymphocyte (PBL) subsets and serum cytokine levels of healthy individuals with those of patients with unresectable liver metastases from colorectal carcinoma before starting regional chemoimmunotherapy. Since the therapeutic responses are limited only to a subset of patients, we hypothesize that the initial status of immunity and individual immune response to a tumor might be significant to the therapeutic outcome. METHODOLOGY: Cellular and humoral immunological parameters were compared between 10 patients with colorectal cancer metastases to the liver responding and non-responding to regional intra-arterial chemo-immunotherapy, and 5 healty individuals. Analyses included a flow cytometric immunophenotyping of peripheral blood mononuclear cells (CD3, CD4, CD8, CD19, CD25, CD28, CD56, CD57, CD80 and HLA.DR), estimation of serum cytokine levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and other immunological parameters are soluble IL-2 receptor (sIL-2), carcinoembryonic antigen (CEA), gastrointestinal cancer-associated antigen (CA 19-9), and C-reactive acute phase protein (CRP). A significantly lower proportion of CD8 lymphocytes and a trend for decreased CD19, CD28 and CD80 was detected among colorectal cancer patients before liver-directed chemotherapy compared to healthy controls. RESULTS: The cancer patients showed a significantly increased population of peripheral NK cells as detected by both CD56+ and CD57+ phenotypes. Elevated serum levels of CRP, IL-4 and TNF-alpha, sIL-2R, but not IL-2, were also demonstrated in cancer patients as compared to controls. Activated CD25+ lymphocytes correlated negatively with CD28+ lymphocytes (r = -0.68, p < 0.01) and less significantly with CD4+ lymphocytes (r = -0.56, p < 0.05). The CD8+ cytotoxic cell subset might be negatively influenced by serum IL-4 (r = -0.57, p < 0.05). Positive correlation was found between sIL-2R and CRP (r = -0.78, p < 0.01), and between sIL-2R and TNF-alpha (r = 0.64, p < 0.05) serum levels in patients with progressive disease during the course of therapy, the initial proportions of CD4+, CD19+ and CD28+ lymphocytes were significantly lower than those among responders. Among humoral parameters, only sIL-2R showed a marginal correlation with therapeutic response, being more elevated among non-responding patients. Pre-treatment serum levels of CEA and CA 19-9 showed correlation with neither therapeutic response nor with any of the cellular or humoral immunological parameters analyzed. CONCLUSIONS: The results may serve as an initial guideline to open a discussion on the rationale of such a panel of tests, hopefully leading to standardized laboratory pre-selection and monitoring of patients treated with regional chemoimmunotherapy.