Autofluorescence (safe) bronchoscopy and p21/ki-67 immunostaining related to carcinogenesis.

Archival biopsy materials from 20 randomly selected asymptomatic volunteers from the Czech uranium miners (CZ UM) risk group (n=98) were examined for p21 and ki-67 immunostatning. There were 16 areas with normal respiratory epithelium and 22 areas with bronchial intra-epithelial neoplasia (IEN). Normal and IEN areas were identified by autofluorescence (System Autofluorescence Endoscopy, SAFE-1000) and monitored during 1998-2002. The majority of specimens from areas with normal autofluorescence intensity with ciliated columnar bronchial epithelium showed strong predominantly cytoplasmic p21 positivity. The SAFE monitoring divided areas of decreased autofluorescence intensity with early stage IEN lesions into two groups. Persistent lesions (P)-showing a spectrum of p21 cytoplasmic staining ranging from negative or isolated negativity to weak or moderate positivity combined with higher proliferative capacity proved by ki-67 nuclear staining. Disappearing lesions (D)-showing strong cytoplasmic p21 positivity and negative ki-67 staining. The IEN lesions were classified into three groups based on p21/ki-67 immunostaining: proliferative lesions at risk (R) with low or without p21 plasma immunostaining combined with high ki-67 nuclear reactivity; ambiguous lesions (A) including cases combining strong p21 cytoplasmic positivity with high ki-67 nuclear reactivity or p21 cytoplasmic negativity with ki-67 negativity staining patterns; the quiescent lesion group (Q) was characterized by strong p21 cytoplasmic positivity and negative ki-67 immunostaining.

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.

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.

DNA flow cytometry: a predictor of a high-risk group in cervical cancer.

We studied the relationship between the 5-year disease-free interval or the occurrence of distant metastases, and the flow cytometric nuclear DNA content in a group of 55 patients treated by radiation for squamous cell carcinoma of the uterine cervix, stages Ib-IIIb (FIGO). The diploid DNA content was associated with a better prognosis and a lower incidence of distant metastases, while aneuploid tumors tended to be prognostically unfavorable and had distant metastases more often. We consider the flow cytometric nuclear DNA content a prospective prognostic parameter in squamous cell carcinoma of the uterine cervix treated by radiotherapy.

Phylloid breast tumors and three steroid hormone receptors.

The concentrations of three steroid hormones (estrogen, progesteron and 1,25-dihydroxycholecalciferol) receptors (ER, PgR, DR) in tissue cytosol were analyzed in a group of 17 breast phylloid tumors. Comparison with breast carcinoma tissue samples (n = 37) did not reveal significant differences in average values of ER, PgR, and DR. Comparison with another control set of 30 samples of dysplastic tissue of the mammary gland showed significant differences only in PgR values. Only 18% of phylloid tumor samples contained levels above cut-of-line of all three receptors (ER, PgR, DR-5,10,10 resp. fmol/ mg protein). The most frequent combination was ER+PgR+DR-(41%). As far as we know, DR in phylloid breast tumors have never been examined before. In approximately 60% of our samples we found the expression of DR, in 36% the estimated values were above 10 fmol/mg protein. Cells of the tissue not expressing DR seem to belong to a special phenotype. We found no ER+PgR- or ER-PgR-combinations in them. The group which expresses DR is characterized by a higher dispersion of PgR values.

The intermediate filaments and prognostically oriented morphological classification in ductal breast carcinoma.

The expression of cytokeratins 7, 8, 14, 18, 19 and vimentin was examined in 100 cases of ductal invasive breast carcinomas. While the predominantly diffuse immunohistological positivity of simple epithelia cytokeratins 7 (in 93), 8 (in 100), 18 (in 100) and 19 (in 97) cases represents a constant feature of these tumors, cytokeratin 14 was detected in only 36 cases which were mostly of low grade and in a focal pattern. Vimentin positivity was found in 53 intermediate and high grade tumors and, again the pattern was also rarely diffuse. The ductal carcinomas can be grouped into four classes according to vimentin and cytokeratin 14 immunoreactivity. This grouping correlates well with tumor grade and with simple histological classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories, as proposed here. The types ofductal carcinomas can be sorted into prognostically different subgroups, according to ICD-O morphologic terminology and commonly adopted results of morphologic and prognostic studies.

[The prognosis of ductal invasive breast carcinoma in histopathology]. / Prognóza duktálního invazivního karcinomu prsu v histologii.

A simple classification of ductal breast carcinoma, consisting of the low, intermediate and high malignancy categories is proposed. The tumour subtypes, categorized according to WHO classification, were sorted into prognostically different subgroups, taking the modified Bloom's grading score and previously published histological studies into account. The low grade category includes papillary, cribriform, tubular, colloid and typical medullary (with the lymphoid stroma) carcinomas. The intermediate group consists of ductal not otherwise specified and mixed ductal/lobular tumours. The high grade category includes atypical medullary, comedo and mucin producing carcinomas. This classification is fully compatible with the commonly adopted ICD-O (1990) morphologic terminology. It should simplify the histological reporting of breast cancer so that the results were as reproducible as from the working formula of malignant lymphomas.

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.

[Cytokeratin expression in spinocellular carcinoma of the uterine cervix]. / Exprese cytokeratinu u spinocelulárního karcinomu delozního cípku.

Expression of cytokeratins 7, 8, 10, 14, 18 and 19 was studied in 48 cases of advanced (stage II and III) squamous cell carcinomas of the uterine cervix. Despite of the degree of differentiation, the expression of simple epithelia cytokeratins 8 (72.9%) and 19 (97.9%) was high. A subset of eight (16.6%) predominantly poorly differentiated tumours had the expression restricted to simple epithelia pattern (cytokeratin 7 and/or 8 and 18 and/or 19). In twenty cases (41.7%) the cytokeratin 14 was added to this pattern, representing an intermediate differentiation level, while the other twenty tumours, usually exhibiting more pronounced squamous differentiation had the most complete cytokeratin pattern including class 10. This grouping was of no prognostic significance but might represent a valuable tool in the classification of cervical squamous cell carcinoma.

Biochemical analysis of breast cyst fluid as a possible predictor of breast carcinoma development.

The occurrence of breast cancer in patients with gross cystic disease is 2-5 times higher as compared to control group of women. During 3 years, 183 cyst fluid samples were analyzed in 129 females, in 30 patients of them the samples were analysed repeatedly. The distribution of the Na+/K+ ratio, considered as the measure of cancer risk, was found to be bimodal. In repeated analyses the type I cyst fluid markedly predominated (Na+/K+ < or = 4.0). A direct dependence on this ratio was found in the concentration of glucose, albumin, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA) and its specific form, TPS; an indirect dependence was found for the level of uric acid, phosphates, alkaline phosphatase (ALP) and alpha-amylase (AMS). The predominance of apocrine metaplasia cells released into the cyst fluid is characteristic of type I cysts. A definitive assessment of significance of these parameters will be enabled by a long-term follow-up of the disease in the respective patients.

Immunochemical analysis of the p53 oncoprotein in matched primary and metastatic human tumours.

There is much interest in the range of genetic aberrations which occur in human malignancies. An immunohistochemical study has been carried out to investigate the consistency of expression of abnormally accumulated p53 protein in paired samples of archival primary and metastatic carcinomas. The staining of methacarn-fixed tissue from 136 matched pairs of mammary carcinoma and 20 cancers from other sites was completed using antibody CM-1 and DO1 in a sensitive peroxidase-conjugated streptavidin-biotin technique. The majority of tumour cells were positive in 25% and the tumours were negative in 17% of the primary carcinomas; staining was heterogeneous in the remaining cases. Staining was identical in 180/186 (96%) metastatic lesions. An ELISA assay carried out on 12 matched pairs of the tumour specimens demonstrated that altered conformation of the aberrant p53 protein present in a primary lesion was maintained in its metastasis. These data indicate that alterations in the p53 gene result in a relatively stable phenotype and that progression of disease is not usually accompanied by either further mutation or loss of the mutant allele.

[DNA flow cytometry: perspective prognostic parameters in carcinoma of the uterine cervix]. / DNA flow cytometrie: perspektivní prognostický parametr karcinomu delozního cípku.

We studied the relation between the 5-year disease free interval and the flow cytometric DNA content in a group of 55 patients treated by radiation for squamous cell carcinoma of the uterine cervix, stages Ib-IIIb (FIGO). The diploid DNA content was associated with a better prognosis, while prognostically unfavourable tumours tended to be aneuploid. The relation was statistically significant in the whole group (p = 0.016), in stage II disease (p = 0.003) and in the subgroup formed by the combination of stages I and II (p = 0.000). In stage III we did not prove the relation. Analysis of the survival function revealed also a better prognosis of diploid tumours (p = 0.041) in the whole group. The division into clearly diploid and non-diploid tumours seems to be more suitable for evaluation (p = 0.012). The difference between the prognostically favourable and unfavourable groups is expressed more clearly. We consider the flow cytometric DNA content a perspective prognostic parameter in squamous cell carcinoma of the uterine cervix. Its significance is apparent especially in patients treated by radiotherapy, because the size of the tumour cannot be assessed reliably in these cases.

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.

[Partial mastectomy in the treatment of breast cancer]. / Parciální mastektomie v terapii rakoviny mlécné zlázy.

In the submitted paper the authors summarize findings on conservative surgery in the treatment of breast cancer. Based on data in the literature and their own experience with surgical treatment of breast cancer of all stages and a detailed analysis of a group of 52 conservatively treated patients, the authors discuss indications for this treatment which must respect staging, typing and grading of tumours as well as attitudes of patients. In particular in tumours classified as T1, NO, MO (as well as T2, NO, MO up to 3 cm) with a non-central localization it is possible by using conservative surgery to avoid somatic mutilation and psychic sequelae of radical procedures. When an individual approach is used and the correct indication is selected, partial mastectomy with dissection of the axilla and postoperative radiotherapy is equivalent to therapeutic results of radical operation.

Aberrant expression of the p53 oncoprotein is a common feature of a wide spectrum of human malignancies.

Accumulation of the p53 protein was analysed in 212 human malignant lesions. Immunohistochemical staining with new polyclonal (CM-1) and monoclonal antibodies (BP 53-12 and BP53-24) to p53 on methacarn-fixed paraffin sections showed positive staining in 161 (76%). The positive tumours were found across a wide range of human malignancies including breast, colon, stomach, bladder and testis carcinomas, soft-tissue sarcomas and melanomas. The staining was always confined to the malignant lesion. Immunoprecipitation and quantitative ELISA assays established that the positive staining was associated with accumulation of the protein and that the protein was frequently in a mutant conformation. Accumulation of mutant p53 protein is therefore a common feature of human malignant disease.

p53 protein alterations in human testicular cancer including pre-invasive intratubular germ-cell neoplasia.

Expression of the p53 oncoprotein was examined in a wide range of primary human testicular germ-cell tumours using a new mouse monoclonal antibody (MAb) BP53-11 raised and characterized in this study, in parallel with a polyclonal rabbit antiserum CM-1. Immunohistochemistry on paraffin sections showed positive nuclear reaction in at least a fraction of malignant cells in 90 (84%) out of 107 cases studied. Aberrant accumulation of the p53 protein was found among testicular tumours of all major histological types, although generally a higher percentage of positive cases and a higher proportion of p53 over-expressing nuclei within individual lesions was observed in embryonal carcinomas when compared with seminomas. The typical heterogeneous staining pattern characteristic of histological specimens was also found in a cultured cell line derived from a human embryonal carcinoma. In contrast to immunohistochemically undetectable levels in normal testes and morphologically normal tissue areas in the tumour-bearing testes, the accumulation of the p53 protein was clearly identified in a high proportion (59% of cases) of the pre-invasive lesions with positive atypical intratubular germ cells often found in the tissue adjacent to invasive tumours. Altered expression of the p53 protein is therefore a unifying feature of the majority of invasive male germ-cell tumours and the change resulting in high levels of p53 appears to be a relatively early step in the human testicular cancer pathogenesis.

A series of 14 new monoclonal antibodies to keratins: characterization and value in diagnostic histopathology.

A series of 14 new mouse monoclonal antibodies (MAbs) to keratins is described and the data suggesting their potential value in the differential diagnosis of human tumours are reported. The specificities of individual MAbs of the 'C-series' presented here range from monospecificity for keratin No. 7 (MAbs C-18, C-35, C-62, and C-68), keratin No. 8 (MAbs C-15, C-43, and C-15), and keratin No. 18 (MAbs C-04 and C-08) up to the broadly reacting 'pan-keratin' MAb C-11, with the target epitopes of the remaining four MAbs being shared by different pairs of keratin polypeptides. The results of the biochemical characterization of the MAbs, together with their immunohistochemical staining patterns on frozen as well as on paraffin sections of normal human tissues, suggest that they represent a significant contribution to the growing list of anti-keratin MAbs applicable in both research and routine diagnostic pathology. The immunohistochemical examination of a wide range of human neoplasms with the new MAbs not only confirmed their value in making distinctions between carcinomas, on the one hand, and lymphomas, and gliomas, on the other, but also verified the possibility of more subtle subdivisions within the group of adenocarcinomas and their metastases. Furthermore, the identification of small subsets of breast carcinomas with decreased levels or apparent loss of the keratin No. 7 polypeptide and some cases of stomach carcinoma with apparently induced expression of this keratin suggests that such 'exceptions' must be considered when using keratin spectra as one of the criteria in differential diagnosis.

Do some malignant melanoma cells share antigens with the myeloid monocyte lineage?

Melanoma cells freshly isolated from 63 advanced primary lesions and 103 metastases were analyzed by staining with monoclonal antibodies MEM 28 directed against a 200 kDa antigen present on all leukocytes and tissue macrophages (CD 45), MEM 18 directed against a monocyte antigen of 53 kDa, anti CD 14--Immunotech, Marseille and 3.9 directed against a 150 kDa antigen expressed on monocytes and to even greater degree on most tissue macrophages (CD 11 c). All antibodies showed variable reactivity with melanoma cells, percentage of positive tumor cells ranged from 0 to 70.