An immunohistochemically positive E-cadherin status is not always predictive for a good prognosis in human breast cancer.

BACKGROUND: in primary breast cancers dichotomic classification of E-cadherin expression, according to an arbitrary cutoff, may be inadequate and lead to loss of prognostic significance or contrasting prognostic indications. We aimed to assess the prognostic value of high and low E-cadherin levels in a consecutive case series (204 cases) of unilateral node-negative non-lobular breast cancer patients with a 8-year median follow-up and that did not receive any adjuvant therapy after surgery. METHODS: expression of E-cadherin was investigated by immunohistochemistry and assessed according to conventional score (0, 1+, 2+, 3+). Multiple correspondence analysis was used to visualise associations of both categorical and continuous variables. The impact of E-cadherin expression on patients outcome was evaluated in terms of event-free survival curves by the Kaplan-Meier method and proportional hazard Cox model. RESULTS: respect to intermediate E-cadherin expression values (2+), high (3+) or low (0 to 1+) E-cadherin expression levels had a negative prognostic impact. In fact, both patients with a low-to-nil (score 0 to 1+) expression level of E-cadherin and patients with a high E-cadherin expression level (score 3+) demonstrated an increased risk of failure (respectively, hazard ratio (HR)=1.71, confidence interval (CI)=0.72-4.06 and HR=4.22, CI=1.406-12.66) and an interesting association with young age. CONCLUSIONS: the findings support the evidence that high expression values of E-cadherin are not predictive for a good prognosis and may help to explain conflicting evidence on the prognostic impact of E-cadherin in breast cancer when assessed on dichotomic basis.

Concerning the localization of steroids in centrioles and basal bodies by immunofluorescence.

Specific steroid antibodies, by the immunofluorescence technique, regularly reveal fluorescent centrioles and cilia-bearing basal bodies in target and nontarget cells. Although the precise identity of the immunoreactive steroid substance has not yet been established, it seems noteworthy that exogenous steroids can be vitally concentrated by centrioles, perhaps by exchange with steroids already present at this level. This unexpected localization suggests that steroids may affect cell growth and differentiation in some way different from the two-step receptor mechanism.

High incidence of ependymomas induced by BK virus, a human papovavirus: brief communication.

Ependymomas were produced in 44 of 50 Syrian golden hamsters and in 9 of 31 outbred Swiss mice inoculated intracerebrally with high-titer, purified BK virus (BKV). Tumors contained a T-antigen that reacted with BKV-specific T-antibody in immunofluorescence and complement-fixation tests. A proportion of tumor-bearing animals had antibodies to BKV T-antigen in their sera. BKV could be rescued from two tumor cell lines by Sendal virus-mediated fusion with Vero cells. A low, or lack of, oncogenic activity was displayed by BKV inoculated sc, ip, or iv.

Receptor and centriole pathways of steroid action in normal and neoplastic cells.

An experimental system designed for tracing the estradiol kinetics in target cells by specific antibodies has been applied to human breast cancer. Several major defects of the estradiol receptor mechanism have been demonstrated. The detected changes (lack of cytoplasmic receptors, impaired nuclear transfer of estradiol:receptor complexes, and abnormal nuclear retention of these complexes) have been demonstrated in most human breast cancers that appear to be composed of hormone-dependent and autonomous mixed-cell populations. These abnormalities could be the biological background for the overall or partial unresponsiveness of breast cancer to endocrine management. The participation of steroids in the regulation of centriole activities is taken into account since immunoreactive steroids are traceable by UV and electron microscopy at the level of this cell organelle by steroid antibodies. Moreover, the presence of steroids in the pericentriolar material correlates well with the modulating appearance and activity of the centriole throughout the cell cycle. A new centriole pathway is suggested by which steroid hormones can regulate cell proliferation.

Immunocytochemical demonstration of estrogen receptors by monoclonal antibodies in human breast cancer: correlation with estrogen receptor assay by dextran-coated charcoal method.

Immunocytochemical demonstration of estrogen receptors in 115 human breast cancer specimens was performed using mouse monoclonal antibodies against estrogen receptor and avidin-biotin as the displaying system. The antibody indicated a highly heterogeneous endowment of neoplastic cells with estrogen receptor at both nuclear and cytoplasmic levels. The percentage of labeled cells within each tumor specimen was recorded to compare this immunocytochemical assay with the biochemical assay of estrogen receptors by the dextran-coated charcoal method. A significant correlation was observed between these two assays. The present results show that estrogen receptors can be confidently demonstrated at the single cell level, thus providing additional information to quantitative biochemical assays. Their prognostic and therapeutic predictive powers may be usefully integrated, particularly in view of the heterogeneous distribution of receptors among cancer cells.

Suppression of tumorigenesis by the breast cancer cell line MCF-7 following transfer of a normal human chromosome 11.

Breast cancer development is associated with several genetic abnormalities. Loss of heterozygosity in the short arm of chromosome 11 has been observed in 30% of tumors. We found homozygosity at five chromosome 11 polymorphic loci in genomic DNA of the MCF-7 breast carcinoma cell line, suggesting a possible loss of one chromosome 11. We have studied the transformed and tumorigenic phenotypes of MCF-7 cells following introduction of a normal human chromosome 11 via microcell fusion. MCF-7/H11 cell hybrids, containing chromosome 11, showed in vitro characteristics similar to the parental cell line. However, tumorigenicity in athymic mice was completely suppressed. Since tumor formation by MCF-7 cells is estrogen dependent, we have analysed the expression of the estrogen receptor and of the estrogen-activated gene pS2. No difference was detected between the parental MCF-7 cells and the derived chromosome 11 cell hybrids, indicating that the mechanism of MCF-7 tumor suppression by chromosome 11-associated functions does not directly involve the estrogen/estrogen receptor molecular pathway.

Chemotherapy versus tamoxifen versus chemotherapy plus tamoxifen in node-positive, estrogen receptor-positive breast cancer patients: results of a multicentric Italian study. Breast Cancer Adjuvant Chemo-Hormone Therapy Cooperative Group.

Between November 1, 1983 and June 30, 1987, 510 node-positive, estrogen receptor (ER)-positive breast cancer patients have been randomly allocated to receive either chemotherapy (six intravenous [IV] cyclophosphamide, methotrexate, and fluorouracil [CMF] courses followed by four IV epirubicin courses) or 5 years of tamoxifen treatment or a combination of both therapies. After a median follow-up of 40 months, patients receiving the combined treatment achieved the best results, and those treated with chemotherapy alone achieved the worst, the difference being particularly evident in postmenopausal women. However, while the concurrent use of chemotherapy and tamoxifen did improve the results achieved by chemotherapy alone, particularly in postmenopausal women and in those with four or more involved nodes, it did not significantly improve the results achieved by tamoxifen alone, particularly in patients with higher ER tumor concentrations. Side effects were more numerous and more severe in patients receiving chemotherapy (with or without tamoxifen). Our findings, although still preliminary, confirm that tamoxifen should be the treatment of choice for postmenopausal breast cancer patients with node-positive, ER-positive tumors. In addition, the findings suggest that tamoxifen may represent a safe alternative to chemotherapy (at least to the cytotoxic regimen we used) for younger women, provided they have ER-positive tumors. In patients with ER-positive tumors, the addition of chemotherapy to tamoxifen does not seem to improve significantly the effectiveness of tamoxifen alone.

Chemotherapy versus tamoxifen versus chemotherapy plus tamoxifen in node-positive, oestrogen-receptor positive breast cancer patients. An update at 7 years of the 1st GROCTA (Breast Cancer Adjuvant Chemo-Hormone Therapy Cooperative Group) trial.

504 evaluable node positive oestrogen receptor (ER) positive breast cancer patients were randomly allocated to receive either 5 years tamoxifen treatment or chemotherapy [six courses of cyclophosphamide, methotrexate and 5-fluorouracil (CMF) followed by 4 courses of epirubicin] or a combination of both treatments. At a median follow-up of 5 years tamoxifen appeared to be more effective than chemotherapy, the difference being highly significant in postmenopausal women. The addition of chemotherapy to tamoxifen was not able to significantly improve the results achieved by tamoxifen alone, irrespective of menopausal status. Trends were similar even after stratification for the number of involved nodes. The protective effect of tamoxifen in terms of reduction of the odds of death increased with time and no rebound phenomena on recurrence or death has occurred so far after the completion of tamoxifen treatment. Overall, the prognostic value of number of involved nodes and of progesterone receptor (PgR) status was confirmed by multivariate analysis. However, the predictive value of PgR was lost in patients receiving tamoxifen alone. Similarly, the degree of ER positivity was not predictive of the response to tamoxifen. Tamoxifen treatment should still be regarded as the gold standard for postmenopausal ER positive patients. In younger women the antioestrogen proved to be safe and at least as effective as chemotherapy. However, the analysis of the annual risks suggests that the concurrent or the sequential use of chemotherapy and tamoxifen might represent a more appropriate treatment for this patient subset, particularly for those with four or more involved nodes. Different cut-offs of ER and PgR assays from those we have arbitrarily employed in the present analysis should probably be used to select more properly the patients who can benefit from endocrine therapy.

Estrogen receptor cytochemistry by fluorescent estrogen.

A recently synthesized fluorescein-labeled estrogen (17FE, 1-(N)-fluoresceinyl-estrone-thiosemicarbazone) interacts with estrogen-target cells like the native hormone and visualizes the uptake, transport, and distribution of estrogen in intact target cells. Moreover, estrogen binding sites are traced by 17FE in cryostat sections of estrogen target tissues as well. Cell and tissue 17FE binding sites fulfill the accepted criteria for specific estrogen receptors (finite binding capacity, high affinity, steroid and tissue specificity). This fluorescent probe allows estrogen receptors to be studied in a wide variety of cell and tissue preparations under varying conditions of physiologic and pathophysiologic interest.

MIB-1 proliferative activity in invasive breast cancer measured by image analysis.

AIMS: To determine cell proliferation in infiltrating breast carcinomas. METHODS: Using the MIB-1 monoclonal antibody, the proliferation index was measured in paraffin wax sections of 871 breast cancers. The MIB-1 proliferation index was compared with other markers of disease progression: size, lymph node status, histotype, oestrogen and progesterone receptor status, expression of p53 and Neu, and DNA ploidy. All parameters were measured using image analysis. In 347 tumours, the MIB-1 and Ki-67 proliferation indexes were compared. Follow up data were available for 170 cases (median 66.5 months). RESULTS: Of the tumours, 314 (36%) had a high proliferation index. The MIB-1 proliferation index was correlated directly with size, nodal status, overexpression of p53 and Neu, and the DNA index; and inversely with oestrogen and progesterone receptor status. The correlation between MIB-1 and Ki-67 proliferation indexes was statistically significant. In patients with pT1 tumours, a low proliferation index correlated with a longer relapse-free interval and overall survival; node negative patients with a low proliferation index had a longer overall survival. CONCLUSIONS: The MIB-1 proliferation index is a reliable, practical and useful method of measuring proliferative activity and is an important predictor of clinical behaviour.

Linkage of biopsy, cancer, and population records aimed at the estimation of family risks in neoplasia: a pilot study.

STUDY OBJECTIVE: The aim was to link individual demographic and medical records into sibships to obtain the sibling distribution of biopsies and cancers, and thereby calculate heritability and recurrence risks in families, thus aiding early diagnosis and prevention of cancers. DESIGN: The 157,823 individual records of the inhabitants of the town of Ferrara in Italy were automatically linked into 106,821 sibships. A 10% sample (10,842 sibships) was then extracted from the distribution of sibships and tabulated, for linkage to medical records. PATIENTS: The biopsy records at the Institute of Pathological Anatomy of the University of Ferrara were manually linked to cancer records and then to sibships. It was possible to construct the distribution of 2062 biopsies and of 829 cancers in sibships. RESULTS: From the distribution of biopsies and tumours in sibships, it was possible to estimate the incidence of tumours in the population (0.052) and in siblings of affected (0.083), and to apply to such distributions current methods for the estimate of heritability (h2 = 0.246) and of recurrence risks of tumours in sibships, age independent. CONCLUSIONS: The study shows that the procedure resulting in the estimation of incidences and recurrence risks for tumours could be completely automated, and extended to whole populations and homogeneous subgroups in post industrial cultures.

Quality control for biomarker determination in oncology: the experience of the Italian Network for Quality Assessment of Tumor Biomarkers (INQAT).

Biomarker analysis and evaluation in oncology is the product of a number of processes (including managerial, technical and interpretation steps) which need to be monitored and controlled to prevent and correct errors and guarantee a satisfactory level of quality. Several biomarkers have recently moved to clinical validation studies and successively to clinical practice without any definition of standard procedures and/or quality control (QC) schemes necessary to guarantee the reproducibility of the laboratory information. In Italy several national scientific societies and single researchers have activated -- often on a pilot level -- specific external quality assessment protocols, thereby potentially jeopardizing the clinical reality even further. In view of the seriousness of the problem, in 1998 the Italian Ministry of Health sponsored a National Survey Project to coordinate and standardize the procedures and to develop QC programs for the analysis of cancer biomarkers of potential clinical relevance. Twelve QC programs focused on biomarkers and concerning morphological, immunohistochemical, biochemical, molecular, and immunoenzymatic assays were coordinated and implemented. Specifically, external QC programs for the analytical phase of immunohistochemical p53, Bcl-2, c-erb-2/neu/HER2, and microvessel density determination, of morphological evaluation of tumor differentiation grade, and of molecular p53 analysis were activated for the first time within the project. Several hundreds of Italian laboratories took part in these QC programs, the results of which are available on the web site of the Network (www.cqlaboncologico.it). Financial support from the Italian Government and the National Research Council (CNR) will guarantee the pursuit of activities that will be extended to new biomarkers, to preanalytical phases of the assays, and to revision of the criteria of clinical usefulness for evaluating the cost/benefit ratio.

Biophenotypes of breast carcinoma in situ defined by image analysis of biological parameters.

In 50 in situ breast cancers an immunohistochemical study, evaluating estrogen (ER) and progesterone (PR) receptors, Proliferation Index (PI), c-erbB-2/Neu and p53 expression was performed. According to histopathological diagnosis, cases were classified as follows: 14 comedo, 8 solid, 5 micropapillary, 6 lobular, 3 papillary, 1 apocrine and 12 mixed in situ carcinomas. The quantitation of immunohistochemical results was obtained with an image analysis computerized system (CAS 200) with a lesion-field method; tumors were subdivided in fields (1177) histologically homogeneous, with 40 x microscopic objective. For ER, PR, Neu and p53, 10% of the positive area was used as cut-off value; 13% was used for PI. Cribriform and lobular types showed a higher positivity for ER (92.1% and 95.5% of the fields); cribriform and papillary a higher for PR (92.6% and 93.9%). Comedo variant demonstrated the higher PI (52.7%), Neu and p53 expression (67.7% and 43%). A cluster analysis performed on 608 fields, defined two groups according to biological homogeneous criteria. The results obtained identify the different biophenotypes of in situ carcinomas, suggesting the possibility of multiple cancerogenetic ways with a different weight of biological events.

Estrogen receptors and post-receptor markers in human breast cancer: a reappraisal.

Several major defects in the estrogen receptor pathway have been evidenced in most human breast cancers by an immunofluorescence tracing of estradiol receptor complexes at the single cell level. Endogenous peroxidase seems a reliable postreceptor marker for estrogen-sensitive breast cancer cells. Since almost all human breast cancers appear to include both hormone-sensitive and autonomous cell populations, a combined use of endocrine and cytotoxic regimens is urged. The hormonal regulation of tumor growth parameters could be exploited in order to achieve a maximum recruitment of synchronized tumor cells at risk to chemotherapy.

Cancer incidence and mortality in the province of Ferrara 1989-1990.

BACKGROUND: Mortality data have clearly highlighted the province of Ferrara as an area with a particular distribution of tumors strictly related with environmental factors. METHODS: The project of a tumor registry has been planned for a better description of cancer incidence and for a deeper insight into etiologic factors, considering the typical features of the province from geographic and occupational points of view. RESULTS: This study presents the registration results of the first 2 years, in order to verify the quality level of data recruitment and to confirm that observed in previous studies. The population covered by the registry was 151,968 males and 165,835 females, with high representation of the elderly. In this period 2,087 tumors in men and 1,778 in women were observed. Lung cancer reaches one of the highest levels in Italy, according to that observed in Lombardy and Veneto regions and the northern Adriatic coast. Incidence and mortality are, however, significantly higher than in other Emilia-Romagna areas, as pointed out by the registries of Parma, Modena and Forii. Colon cancer also presents high frequencies in comparison with neighboring areas, whereas non-Hodgkin lymphomas reach the highest level in Italy. Gastric tumors, although well represented in males and females, show lower levels than the high-risk neighboring Romagna region. In women, a low incidence of cervix uteri-tumors and high levels of breast cancer have also been observed. CONCLUSIONS: The distribution of such neoplasms and the differences observed among neighboring areas deserve further analytical studies, with the aim of a better reading of cancer onset and diffusion. The quality of data obtained (about 70% of histocytologic confirmations, and 5% of "final" death-certificate-only cases), appears to reach satisfying levels, considering the starting phase of the registry.

Clinical usefulness of estrogen receptor immunocytochemistry in human breast cancer.

We assessed the reliability of the immunocytochemical assay of estrogen receptor (ER-ICA) as a marker of clinical outcome. Relapse-free interval (RFI) and overall survival (OS) according to ER-ICA status were retrospectively evaluated on a series of 210 patients who had undergone surgery for primary breast cancer between January 1985 and December 1988. ER assay by the dextran-coated charcoal method (DCC) was also performed in 189 tumors. A significant positive correlation was found between the DCC and ER-ICA assays, with an overall agreement of 79%. ER-ICA status showed a prognostic predictive power with respect to OS and RFI in the whole series of patients and in the subset of node-positive patients. It was also a marker of outcome with respect to OS in the subsets of node-negative patients and patients with tumors < or = 2 cm in diameter. Moreover, the predictive value of the ER-ICA assay was higher than that of the DCC assay in the present study. These findings emphasize the clinical usefulness of the ER-ICA assay as a measure for prognosis.

Combined and sequential expression of p53, Rb, Ras and Bcl-2 in bronchial preneoplastic lesions.

AIMS AND BACKGROUND: Several simple molecular abnormalities have been detected in bronchial preneoplastic lesions, but the simultaneous presence of these alterations has been scarcely investigated. METHODS: We studied, by an immunohistochemical method, the expression of p53, Rb, Ras and Bcl-2 in 65 samples from surgical specimens and diagnostic biopsies selected for the presence of preneoplastic changes in the bronchial epithelium. To perform an analysis of the combined expression of all markers in the same areas, we accurately mapped every consecutive section on which immunohistochemical reactions were performed, subdividing each specimen into 25x microscopic fields, which allowed good topographical mapping. RESULTS: It was found that the frequency of p53-positive and Rb-negative microscopic fields was directly related to the morphological grading of lesions. On the other hand, Ras expression characterized high-grade lesions not showing squamous differentiation (non-squamous Cis). Regarding Bcl-2 expression, only slight differences in positivity distribution were found between the different lesions. More interesting was the parallel evaluation of all markers in the same areas: one of the main patterns, found to be correlated with the severity of histopathological features, was characterized by combined p53 hyperexpression/Rb hypoexpression; furthermore, when Ras and Bcl-2 hyperexpression were superimposed to the above pattern, the former mainly characterized non-squamous Cis, while the latter was present only in high-grade squamous lesions. However, the most frequently encountered pattern did not show any alteration of the studied markers, suggesting that other mechanisms could be involved in bronchial carcinogenesis. CONCLUSIONS: The detection of combined molecular abnormalities in bronchial preneoplasia could clarify the steps involved in lung carcinogenesis; furthermore, a simple and inexpensive method, such as immunohistochemistry, could be routinely applied also to cytologic specimens in order to detect those lesions, or patients, that are prone to progression towards lung cancer.

Immunohistochemical expression of c-erbB-2 in human breast cancer by monoclonal antibody: correlation with lymph node and ER status.

c-erbB-2 Protein expression was investigated in a series of fifty primary breast cancers by means of a specific monoclonal antibody and immunocytochemistry. Specific staining was observed at the plasma membrane level of neoplastic cells, according to the reported localization of c-erbB-2 protein. Sixty-four percent of tumors scored positive, with a variable amount of stained cells. The rate of protein expression was found to exceed the reported gene amplification. No relationship was observed between c-erbB-2 protein staining and age, menopausal status or histologic subtypes. An inverse association was found between c'erbB-2 protein staining and estrogen receptor content of tumors, assayed by immunocytochemistry. A positive relationship was observed between c-erbB-2 protein expression and presence of axillary node metastasis. These findings suggest that c-erbB-2 protein expression is a marker of tumor aggressiveness and that its prognostic power deserves further investigation both in node-positive and node-negative patients.

Fine needle aspiration cytology of thyroid gland diseases.

From 1982 to 1987, 2,433 lesions of the thyroid gland in 1,796 patients were examined by fine needle aspiration (FNA). Cytopathology classified 66.91% of the aspirates as benign, 10.76% as thyroiditis, 4.89% as suspected (unspecified) neoplasia, 1.31% as positive for malignancy and 16.11% (392) as unsatisfactory. The histologic diagnoses in 257 cases were compared with cytologic diagnoses to determine the accuracy of FNA cytology of thyroid lesions, yielding a sensitivity of 71.43%, a specificity of 100% and an accuracy of 95.09%. This data strongly supports thyroid FNA as an important preoperative diagnostic tool. Follicular carcinomas were difficult to cytologically differentiate from nonmalignant follicular neoplasms, and papillary thyroid carcinomas less than 2 cm in diameter in elderly patients were frequently misdiagnosed or diagnosed only as "suspect lesion."

Immunocytochemical detection of estrogen receptors by staining with monoclonal antibodies on cytologic specimens of human breast cancer.

A study was undertaken to test the possibility of determining the estrogen receptor (ER) content in human breast cancers by staining with commercial specific monoclonal antibodies (MAbs) on cytologic specimens (touch imprints and fine needle aspirates). The aspirates were suspended in a cell culture medium and cytocentrifuged onto slides to preserve their morphologic characteristics and to allow a proper immunocytochemical staining for ERs. MAb staining for ER was also performed on the respective surgical samples. The staining of cytologic samples for ER showed 100% specificity and 95% sensitivity in comparison to the staining of the histologic samples. Moreover, comparison of the percentage of stained cells in the cytologic specimens to the ER content in the respective surgical specimens, as assayed by the dextran-coated charcoal method, showed the MAb staining of cytologic samples to have 94% specificity and 100% sensitivity. These results support the reliability of MAb staining for ERs in cytologic samples and suggest that it could be the assay of choice in particular clinical settings in the evaluation of primary and recurrent breast cancers.