Evaluation of an enzyme immunoassay kit for estrogen receptor measurement--report II

We present evidence to show that monoclonal antibodies to estrogen receptors (ER) in solid phase recognize the secondary estrogen binding sites with moderate to low affinity for estradiol (E2). An excellent quantitative agreement was found in five cytosols between the ER values obtained by the enzyme immunoassay (ER-EIA) and the amount of secondary estrogen binding sites measured by the assay involving dextran-coated charcoal (Clin Chem 1986;32:1496). The immunoreactive protein recognized by the antibody-coated beads, when allowed to react with ER(+) cytosols, is shown to bind [3H]estradiol only when the ligand concentration exceeds 8 nmol/L. Further biochemical and functional characterization of the immunoreactive protein is required to establish similarities/dissimilarities between this protein, high-affinity Type I ER sites, and the secondary sites such as Type II sites.     

Cancer risks associated with 10 inorganic dusts: results from a case-control study in Montreal

A multicancer site, multifactor case-control study was undertaken to generate hypotheses about possible occupational carcinogens. Probing interviews were carried out with eligible cases, comprising all incident cases of 20 types of cancer who were male, aged 35-70 years, and a resident in Montreal. The interview was designed to obtain detailed lifetime job histories and information on potential confounders. Each job history was reviewed by a team of chemists and industrial hygienists who translated it into a history of occupational exposures. These occupational exposures were then analyzed as potential risk factors in relation to the sites of cancer included; 3,726 cases were interviewed. For each site of cancer analyzed, controls were selected from among the other sites in the study. This report concerns the associations between the 12 main types of cancer in our series and 10 inorganic dusts that are found mainly in construction and metal industries. All site-exposure combinations were investigated. After intensive control for confounding, nonadenocarcinoma (NAC) of the lung was associated with long duration-high level exposure to silica (odds ratio [OR] = 1.4), excavation dust (OR = 1.9), concrete dust (OR = 2.5), abrasives dust (OR = 1.4), and alumina (OR = 1.5). It was difficult to disentangle the relative effects of those substances, and confounding among them was a distinct possibility. Although residual confounding by some uncontrolled factors may explain the elevated ORs, the results were compatible with the hypothesis of a nonspecific relation between NAC of the lung and respirable inorganic dusts as a class. Other associations that remained suggestive after in-depth analysis were silica and stomach cancer (OR = 1.2) and concrete dust and lymphoma (OR = 2.9).     

Parathyroid hormone related protein in hypercalcaemia of Hodgkin''s disease.

The role of parathyroid hormone related protein (PTHRP) as a humoral mediator of hypercalcaemia was investigated in a patient with lymphocyte depleted Hodgkin's disease during an episode of hypercalcaemia, using an immunohistochemical staining technique for PTHRP on the tumour tissue and an immunoradiometric (IRMA) assay for PTHRP1-86 on the patient's plasma. The plasma PTHRP was less than 0.23 pmol/l in the range found in normocalcaemic controls, and the immunohistochemical staining was not positive for protein. PTHRP did not have a role in the pathogenesis of hypercalcaemia in this patient.     

Cytokeratin 5/6 in normal human breast: lack of evidence for a stem cell phenotype

In recent studies, B?cker and colleagues described a population of cells in paraffin wax sections of normal human breast that express cytokeratins (CK) 5/6 without expression of CK8/18 or smooth muscle actin (SMA). They proposed that these represent stem cells that give rise to differentiated luminal and myoepithelial cells. The data have been used to generate a model for breast cancer progression and classification with associated implications for management of pre-invasive disease. In this study, the expression of CK5/6, CK8/18, and SMA was investigated using multiple immunofluorescence on matched pairs of paraffin wax-embedded and frozen breast specimens. The staining patterns reported previously in antigen-retrieved paraffin wax-embedded sections were confirmed but no CK5/6-only cells were found in frozen sections of normal breast. There were cells with low levels of CK8/18 expression in frozen sections that may correspond to the CK8/18 'negative' cells seen in paraffin wax sections. This study brings into question the previously described profile of breast 'stem cells' based on CK5/6 staining and hence the breast cancer progression model and classification based on this phenotype.     

Expression of ATM, p53, and the MRE11-Rad50-NBS1 complex in myoepithelial cells from benign and malignant proliferations of the breast

AIMS: To analyse the expression of proteins involved in DNA double strand break detection and repair in the luminal and myoepithelial compartments of benign breast lesions and malignant breast tumours with myoepithelial differentiation. METHODS: Expression of the ataxia telangiectasia (ATM) and p53 proteins was immunohistochemically evaluated in 18 benign and malignant myoepithelial tumours of the breast. Fifteen benign breast lesions with prominent myoepithelial compartment were also evaluated for these proteins, in addition to those in the MRE11-Rad50-NBS1 (MRN) complex, and the expression profiles were compared with those seen in eight independent non-cancer (normal breast) samples and in the surrounding normal tissues of the benign and malignant tumours examined. RESULTS: ATM expression was higher in the myoepithelial compartment of three of 15 benign breast lesions and lower in the luminal compartment of eight of these lesions compared with that found in the corresponding normal tissue compartments. Malignant myoepithelial tumours overexpressed ATM in one of 18 cases. p53 was consistently negative in benign lesions and was overexpressed in eight of 18 malignant tumours. In benign breast lesions, expression of the MRN complex was significantly more reduced in myoepithelial cells (up to 73%) than in luminal cells (up to 40%) (p=0.0005). CONCLUSIONS: Malignant myoepithelial tumours rarely overexpress ATM but are frequently positive for p53. In benign breast lesions, expression of the MRN complex was more frequently reduced in the myoepithelial than in the luminal epithelial compartment, suggesting different DNA repair capabilities in these two cell types.     

Diagnosis of breast cancer using elastic-scattering spectroscopy: preliminary clinical results

We report on the first stages of a clinical study designed to test elastic-scattering spectroscopy, mediated by fiberoptic probes, for three specific clinical applications in breast-tissue diagnosis: (1) a transdermal-needle (interstitial) measurement for instant diagnosis with minimal invasiveness similar to fine-needle aspiration but with sensitivity to a larger tissue volume, (2) a hand-held diagnostic probe for use in assessing tumor/resection margins during open surgery, and (3) use of the same probe for real-time assessment of the "sentinel" node during surgery to determine the presence or absence of tumor (metastatic). Preliminary results from in vivo measurements on 31 women are encouraging. Optical spectra were measured on 72 histology sites in breast tissue, and 54 histology sites in sentinel nodes. Two different artificial intelligence methods of spectral classification were studied. Artificial neural networks yielded sensitivities of 69% and 58%, and specificities of 85% and 93%, for breast tissue and sentinel nodes, respectively. Hierarchical cluster analysis yielded sensitivities of 67% and 91%, and specificities of 79% and 77%, for breast tissue and sentinel nodes, respectively. These values are expected to improve as the data sets continue to grow and more sophisticated data preprocessing is employed. The study will enroll up to 400 patients over the next two years.     

The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours

In a previous study of phyllodes tumours, it has been shown that both the stroma and the epithelium can exhibit distinct molecular changes, suggesting that both are part of the neoplastic process. In view of this finding, it was decided to study stromal-epithelial interactions in these tumours by examining the Wnt-APC-beta-catenin pathway. Beta-catenin and cyclin D1 immunohistochemistry was performed on 119 phyllodes tumours. Eighty-six (72%) showed stromal nuclear beta-catenin localization and in 57% the staining was moderate or strong; however, of the eight malignant tumours in the series, seven showed absent or weak nuclear staining (p<0.025). In no tumour was nuclear beta-catenin staining seen in the epithelial component. Moderate or strong stromal cyclin D1 staining correlated with nuclear stromal beta-catenin staining (p<0.05). Forty-five of the tumours, including two malignant lesions, were screened for beta-catenin exon 3 mutations using SSCP and sequencing, but none was found. Loss of heterozygosity (LOH) of the marker D5S346 was used to infer APC mutation, but only one (benign) tumour showed LOH. Wnt2 and Wnt5a mRNA was localized by in situ hybridization in 13 cases (three malignant) chosen to reflect the different beta-catenin staining patterns. There was an association between strong nuclear beta-catenin staining of stromal cells and epithelial Wnt5a expression (p<0.0015). These data suggest that stromal proliferation in benign phyllodes tumours relies on abnormalities in the Wnt pathway which result not from mutation, but from Wnt5a expression in the epithelium. In the progression to malignancy, the stromal proliferation appears to become independent of the Wnt pathway and, presumably, of the epithelial component of these tumours.     

An analogy between fetal haptoglobin and a potent immunosuppressant in cancer

In spite of the numerous reports indicating the presence of humoral immunosuppressive factors in cancer patients, only a few of these factors have been biochemically identified. Furthermore, their role as effective immunosuppressors in vivo remains to be established. Our laboratory has attempted to isolate and identify the major immunosuppressive factor in the malignant effusions derived from ovarian and lung cancer patients. We have previously demonstrated that the Mr 52,000 immunosuppressive factor isolated from the ascites fluid of an ovarian cancer patient inhibited T-dependent immune responses in vivo and in vitro including the inhibition of E-rosetting. Thus, this immunosuppressive factor was named "suppressive E-receptor" (SER). Our current study demonstrates that this SER factor purified from malignant effusions derived from ovarian, lung, or head and neck cancer patients had a common component which dissociated equally into Mr 38,000-42,000 and 17,000-19,000 moieties on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under vigorous reducing conditions. Electroelution of these two components followed by a limited amino acid sequence determination revealed these two components to have N-terminal amino acid sequences identical to the beta and alpha 2 subunits of normal adult haptoglobin. Immunoelectrophoresis of SER using a polyclonal antiserum to neonatal cord blood demonstrated that SER, unlike normal haptoglobin, has slower electrophoretic mobility than the normal adult haptoglobin. Western blotting analysis of SER separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions failed to recognize a monoclonal antibody directed specifically to SER. However, this monoclonal antibody exclusively reacted with the SER separated by an analytical polyacrylamide gel electrophoresis gel under nondenaturing conditions while normal adult haptoglobins or purified but denatured haptoglobin obtained from the same malignant fluid as SER all failed to react with this antibody. Thus, SER appears to bear an additional epitope(s) that is absent in normal adult haptoglobin. Since the SER as well as the neonatal haptoglobin have at least 100 to 1000-fold more potent immunosuppressive activity than the normal adult haptoglobin, this additional epitope(s) present in SER may be responsible for the potent immunosuppressive property of SER.     

Semi in vitro labelling of red blood cells with99mTc: a comparison with the in vivo labelling

A method ofsemi in vitro labelling of red blood cells (Rbc) is described.Rbc, pre-tinned in the body, were labelledin vitro with99mTc. The labelling efficiency, expressed as the percentage of the administered activity found in the blood, appeared to be higher with this preparation method compared toin vivo labelling ofRbc, respectively 91.3±3.6 and 71.3±3.5 (mean±Sem). There was no difference between both methods in elimination of the label in the urine up to one hour (4.6% of the administered activity).The data here presented, show that thesemi in vitro method provides a preparation with a high cell-bound activity of 96%. Because of a better labelling efficiency, application of thesemi in vitro method results in more circulating activity and a higher left ventricle to background ratio.