Immunolocalization and messenger RNA expression of bone morphogenetic protein-6 in human benign and malignant prostatic tissue.

Skeletal metastases are common in advanced prostate cancer, causing considerable morbidity, and they are usually osteoblastic in nature with no clear explanation for this phenomenon. Bone morphogenetic proteins (BMPs) induce bone formation in vivo, and preliminary work showed a possible association between BMPs and prostatic skeletal metastases; differential expression favors BMP-6 as a potential new marker and mediator of osteosclerotic deposit formation. We investigated BMP-6 mRNA and protein expression by in situ hybridization and immunohistochemistry in malignant and benign prostates from 40 men. BMP-6 mRNA expression was detected exclusively in malignant epithelial cells in 20 of 21 patients (95%) with metastases and in 2 of 11 patients (18%) with localized cancer, and it was absent in 8 benign samples. Immunostaining for BMP-6 was predominantly cytoplasmic and was present in all primary tumors with established metastases and in 4 of 11 (36%) organ-confined cancers. In benign prostatic hyperplasia, basal cells and areas of basal cell hyperplasia were positive for BMP-6 by immunohistochemistry. The results suggest a close association between BMP-6 expression in primary malignant prostatic tissue and skeletal metastases. BMP-6 may be responsible, in part, for the osteoblastic changes in metastatic lesions secondary to prostate cancer.

Expression of c-erbB-2 oncoprotein: a prognostic indicator in human breast cancer.

Sections of formalin-fixed, paraffin-embedded tissue from 185 primary breast carcinomas were stained immunohistochemically using a polyclonal antibody against the c-erbB-2 oncoprotein. Positive staining, which is known to correlate with gene amplification, was associated with earlier relapse, shorter postrelapse survival, and shorter overall survival. Lymph node, epidermal growth factor receptor, and estrogen receptor status, tumor size, and histological grade also had prognostic significance but, applying multivariate analysis, only lymph node status was a more important predictor of relapse-free and overall survival than staining for the oncoprotein. Positive staining was correlated with negative estrogen receptor status and high histological grade, but there was no association with either lymph node or epidermal growth factor receptor status or tumor size. Expression of the c-erbB-2 oncoprotein appears to be an important independent indicator of prognosis in human breast cancer.

Determination of the prognostic value of cyclin D1 overexpression in breast cancer.

Cyclin D1 plays a critical role in the timing of the initiation of DNA synthesis in the normal cell cycle of mammalian cells. Deregulated expression of this protein has been seen in a variety of tumours either as a result of gene amplification or chromosomal translocation, in breast cancer and B cell malignancies respectively. In order to determine the role this putative oncoprotein plays in breast cancer, we have applied a new monoclonal antibody, recently produced in our laboratory, in an immunohistochemical study of 93 primary breast carcinomas. We show that approximately 28% of the cases displayed enhanced expression of the cyclin D1 protein. Furthermore, either cyclin D1, cyclin D3, or both, were expressed in 69% of cases, suggesting that overexpression of any one member of this family may relieve cancer cells of their mitogenic stimulatory requirement. In addition, we show that those patients whose breast cancers co-express cyclin D1 with either epidermal growth factor receptor (EGFR) or the retinoblastoma protein (pRB) have a significantly poorer prognosis in comparison to those expressing cyclin D1 alone. Our observations indicate that, in a subset of breast cancers, aberrant cyclin D1 expression is a contributory factor to tumorigenesis and in association with EGFR or pRB expression, identify those tumours which may require more aggressive therapy.

Molecular cloning of a cDNA for human pregnancy-specific beta 1-glycoprotein:homology with human carcinoembryonic antigen and related proteins.

Human pregnancy-specific beta 1-glycoprotein (SP1) plays an essential role in normal pregnancy. It is also a well-characterized oncodevelopmental antigen, expressed aberrantly by all trophoblastic tumors and some other malignant cell types. Here we report the identification of a human placental cDNA encoding the SP1 polypeptide sequence. The coding sequence shows 95% identity at the nucleotide level with a distinct, recently published SP1 cDNA sequence (PSG16). Unexpectedly, the sequence is also highly homologous to the published sequence of human carcinoembryonic antigen (CEA). SP1, CEA and CEA-related nonspecific cross-reacting species thus belong to a group of closely related though antigenically diverse tumor-associated glycoproteins. Comparison of the deduced amino acid sequence of the SP1 cDNA with that of CEA provides insight into the modular nature of these related proteins. This may have implications for the genomic organization and evolution of the CEA gene family.

CHOP-based chemotherapy is as effective as alternating PEEC/CHOP chemotherapy in a randomised trial in high-grade non-Hodgkin's lymphoma. Scotland and Newcastle Lymphoma Group.

The aim of this study was to test whether survival for patients with high-grade non-Hodgkin's lymphoma (NHL) can be improved with a non-cross-resistant regimen as compared to a CHOP-based regimen. This is a multicentre study comprising 325 adult patients, median age 58 years, with high-grade non-Hodgkin's lymphoma: patients of any age and performance status were eligible provided they were able to receive the drugs in the regimens. Patients were randomised to either B-CHOP-M (bleomycin, cyclophosphamide, doxorubicin, vincristine, prednisolone and methotrexate) or PEEC-M (methylprednisolone, vindesine, etoposide, chlorambucil and methotrexate) alternating with B-CHOP-M. At a median follow-up of 9 years, there was no significant difference in overall survival or disease-free survival between the two arms. Toxicities for the two regimens were equivalent. This study confirms that for relatively unselected patients with high-grade non-Hodgkin's lymphoma, an alternating multidrug regimen does not improve upon the results obtained with B-CHOP-M.

Studies on the concentration of pregnancy-associated plasma protein-A during normal and complicated pregnancy.

Pregnancy-associated plasma protein-A was assayed in the blood of 347 women during pregnancy, using a new primary standard of PAPP-A as reference. The protein was assayed by antibody-antigen crossed electrophoresis with the lower limit of confident assay being 9.5 micrograms PAPP-A/ml (13 pmol/ml). PAPP-A was first detected at 14 weeks of gestation; by term it had risen to within the range 20 to 320 micrograms/ml. There was an indication that pregnancies involving a male baby had higher PAPP-A levels in blood than did those involving female babies. In 51 blood samples from 30 patients with gestational diabetes (taken between 28 weeks of pregnancy and term) there was no significant alteration in PAPP-A values compared with controls. In 35 blood samples from 15 patients with insulin-dependent diabetes, levels of PAPP-A were significantly lower than in controls or in gestational diabetes. In 43 blood samples from 35 patients with babies affected with intrauterine growth retardation (between 28 weeks and term), there was no significant difference in PAPP-A levels compared with controls. The effect of insulin on the blood levels of PAPP-A suggests that the concentration of PAPP-A is capable of altering significantly in response to certain physiological changes associated with the control of carbohydrate metabolism.

Oestrogen receptor and oestrogen regulated proteins in human breast cancer: a review.

Breast growth and development is influenced by oestrogens and the growth of many breast cancers is driven by oestrogens, an effect which is utilised in the endocrine treatment of breast cancer. Oestrogens act by binding to the oestrogen receptor, a specific protein which in turn binds to specific regulatory regions of DNA, thereby altering gene expression. The effects of oestrogens may be mediated by growth factors and other substances under oestrogen regulation. Oestrogen receptor status in breast tumours can be determined by cytosolic radioligand binding assays, enzyme linked immunoassay, immunohistochemistry and measurement of messenger RNA levels. Tumour oestrogen receptor content is an established but not absolute predictor of both response to endocrine therapy and prognosis in breast cancer. Paradoxically, a small proportion of apparently oestrogen receptor negative tumours do respond to endocrine therapy, perhaps reflecting expression of low and unmeasurable levels of receptor or tumour heterogeneity with respect to receptor expression. A larger proportion of oestrogen receptor positive tumours unexpectedly fail to respond to endocrine therapy; in these cases it is possible that oestrogen receptor has become dissociated from the transcriptional and translational events which it normally regulates. Determination of levels of expression of substances regulated by oestrogens can provide information regarding the functional integrity of the oestrogen response pathway and such substances include the progesterone receptor, plasminogen activator, cathepsin D and a variety of messenger RNA sequences.

Expression of the glial glutamate transporter EAAT2 in the human CNS: an immunohistochemical study.

Glutamate transporters play an essential role in terminating the excitatory glutamatergic signal at post-synaptic receptors and in protecting neurones from excitotoxic effects, as well as replenishing the neurotransmitter supply at glutamatergic synapses. The distribution and density of glutamate transporters may be important determinants of vulnerability to glutamate-mediated injury. There is emerging evidence that glutamate transporter dysfunction may be present in motor neurone disease (MND). In this study, a monoclonal antibody, suitable for immunohistochemistry (IHC) in human post-mortem tissue, was produced to the human astrocytic glutamate transporter EAAT2 (excitatory amino acid transporter 2). Western blotting of homogenates of human cortical tissue with the EAAT2 antibody produced a discrete band at 66 kDa. Detailed IHC analysis of the expression of the EAAT2 protein in the human CNS was undertaken. EAAT2 was exclusively localised to astrocytes, with preferential expression in the caudate nucleus, nucleus basalis of Meynert, spinal ventral horn, cerebral cortex and hippocampus, but with lower levels of expression throughout many other CNS regions. Motor neurone groups vulnerable to neurodegeneration in MND appeared distinctive in being surrounded by extensive, coarse, strongly immunoreactive perisomatic glial profiles. Motor neurone groups which tend to be spared in MND, such as those present in the oculomotor nucleus, showed a lower expression of EAAT2, with fewer perisomatic profiles. The EAAT2 antibody will provide a useful tool for increasing our understanding of the role of EAAT2 in excitatory neurotransmission in health and disease states.

Plasma pregnancy-associated alpha 2-glycoprotein concentrations in complications of pregnancy and foetal abnormality.

Maternal plasma concentrations of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) were measured in normal pregnancy and in pregnancies complicated by apparent threatened abortion, pre-eclampsia or intrauterine growth retardation (IUGR). alpha 2-PAG levels were significantly decreased in those women who spontaneously aborted and in those with foetal death, but were unaffected in patients who threatened to abort and in whom pregnancy continued successfully. Concentrations of alpha 2-PAG were also unaffected in subjects with mild or severe pre-eclampsia and in those with IUGR. Patients with high alpha-foetoprotein levels associated with foetal abnormality also had normal alpha 2-PAG levels for stage of gestation. The possible immunological implications of these findings are discussed.

Rosette inhibition levels during early human gestation.

The inhibitory effect of antilymphocyte globulin on spontaneous rosette formation by peripheral blood T cells between 10 and 21 weeks gestation was examined. No evidence was obtained from either a cross-sectional or a longitudinal study that increased rosette inhibition levels occur during early human pregnancy. Comparable values were found in oestrogen-treated women. The results are consistent with the view that the intrinsic reactivity of human T lymphocytes is unimpaired during gestation and oestrogen treatment.

Antinuclear antibody-negative systemic lupus erythematosus-how common?

A review of five years' DNA-binding antibody results in a routine service laboratory revealed 38 patients who had high DNA-binding capacity (DNA-bc) but no antinuclear antibodies (ANA). On retrospective case note analysis, 22 patients (58%) were thought to have systemic lupus erythematosus (SLE), although only six (16%) fulfilled the preliminary classification criteria of the American Rheumatism Association (ARA). Our findings indicate that ANA-negative SLE is commoner than generally realised and lead us to recommended the measurement of DNA-bc in every case where clinically appropriate.

Alpha-fetoprotein, alpha-1-antitrypsin, and transferrin in gonadal yolk-sac tumours.

Since gonadal yolk-sac tumour in pure form or as a component of mixed germ cell tumour is in the majority of patients highly malignant, its histological recognition is of great prognostic importance. Yolk-sac tumour may assume various different histological guises, which have hitherto caused considerable terminological confusion; the present paper is aimed at correlating these morphological diversities with biochemical features which are consistent with yolk-sac differentiation. Using an enzyme-bridge immunoperoxidase technique, a series of 16 gonadal germ cell tumours with a yolk-sac component were screened for the presence of alpha-fetoprotein, alpha-1-antitrypsin, and transferrin. These proteins, normally produced by human yolk sac, were demonstrable in all the morphological patterns of yolk-sac tumour we have previously described. Six malignant non-germ cell tumours were submitted to the same investigations, and no evidence of the three protein markers was found in five; one tumour, however, an oat cell carcinoma of the bronchus, stained positively for transferrin.

Detection of pregnancy specific beta1-glycoprotein in formalin-fixed tissues.

Using an enzyme-bridge immunoperoxidase method, pregnancy specific beta1-glycoprotein (PSbetaG) has been demonstrated in the cytoplasm of the trophoblast in several formalin-fixed tissues, namely, implantation sites of ovum, normal placentae, hydatidiform moles, invasive moles, and choriocarcinomata of uterus and testis. It is suggested that this technique may prove helpful in the detection of choriocarcinomatous elements in malignant tumours.

Effects of oestrogen and progestogen on serum levels of alpha(2)-macroglobulin, transferrin, albumin, and IgG.

The levels of four serum proteins, assayed by a radial immunodiffusion technique, have been measured in healthy women who had been given either the oestrogen or progestogen component of a combined oral contraceptive preparation for three weeks. Raised alpha(2)-macroglobulin and transferrin levels were found after oestrogen treatment but albumin and IgG did not significantly alter. In the progestogen-treated group all four proteins remained unchanged. The four proteins have also been assayed at frequent intervals during the normal menstrual cycle. No evidence of cyclical variation was found.

Serum-alpha2-macroglobulin, transferrin, albumin, and IgG levels in preeclampsia.

A radial immunodiffusion technique has been used to measure levels of four serum proteins in preeclampsia with or without proteinuria and in normal pregnant and non-pregnant controls. In preeclampsia unaccompanied by proteinuria, albumin and transferrin levels are similar to those found in the normal pregnant controls, but there are significant falls in alpha(2)-macroglobulin and IgG. When preeclampsia is accompanied by proteinuria there is a marked fall in albumin and an increase in alpha(2)-macroglobulin. Since alpha(2)-macroglobulin has antiplasmin activity it is possible that increased levels of this protein in preeclampsia accompanied by proteinuria contribute to the intravascular coagulation which has been described in this disorder.

Serum protein levels in primary biliary cirrhosis.

Serum levels of albumin, transferrin, alpha(2)-macroglobulin, beta(1)C/beta(1)A, IgA, IgG, and IgM have been determined in 73 patients with primary biliary cirrhosis and in age- and sex-matched controls. A highly significant fall in albumin was demonstrated, and there were highly significant increases in alpha(2)-macroglobulin and all three immunoglobulin levels. Transferrin and beta(1)/Cbeta(1)A levels were unchanged. No significant correlations were found between the titre of antimitochondrial antibody, the duration of symptoms, and any of the serum proteins estimated. A highly significant positive correlation was present between serum albumin and transferrin levels in both patient and control groups.

Serum -macroglobulins in renal disease and preeclampsia.

The serum levels of alpha(2)-macroglobulin and pregnancy-associated globulin (another alpha-macroglobulin) have been measured by means of a radial immunodiffusion technique in (1) renal disease with and without proteinuria, (2) in age- and sex-matched controls, (3) in preeclampsia with and without proteinuria, and (4) in normal pregnant controls. There are significant increases in alpha(2)-macroglobulin and pregnancy-associated globulin in renal disease accompanied by proteinuria but normal levels are found in renal disease without proteinuria. Compared with normal pregnancy, alpha(2)-macroglobulin is significantly raised in preeclampsia with proteinuria but normal in preeclampsia without proteinuria. In contrast, serum pregnancy-associated globulin is significantly reduced in preeclampsia both with and without proteinuria when compared with normal pregnancy.

A novel quantitative immunoassay system for p53 using antibodies selected for optimum designation of p53 status.

AIM: To develop a highly sensitive and specific enzyme linked immunosorbent assay (ELISA) system for analysis of p53 protein in cancer lysates. METHODS: The anti-p53 monoclonal antibodies DO7, 1801, BP53.12, and 421, and anti-p53 polyclonal antiserum CM1 were assessed by immunohistochemistry and western blot analysis to identify those most suitable for determining p53 status of cancer cells. Antibodies with desired characteristics were used to develop a non-competitive sandwich type ELISA system for analysis of p53 expression in cancer cytosols. Using the ELISA, p53 protein concentrations were measured in a small series of breast cancers, and the quantitative values compared with p53 immunohistochemical data of the same cancers. RESULTS: DO7 and 1801 gave the most specific and reliable results on immunohistochemistry and western blot analysis. Using these two antibodies, a non-competitive sandwich type ELISA system was developed to analyse p53 quantitatively. Analysis of the breast cancer series showed a good correlation between immunohistochemistry and the ELISA-tumours were generally positive using both techniques. Discrepancies were noted however: some cancers were immunohistochemically negative but ELISA positive. One explanation for this may be that the ELISA is more sensitive than immunohistochemistry. CONCLUSION: The p53 ELISA system is a non-competitive double monoclonal antibody sandwich method, using DO7 and 1801 which have been shown to be highly specific for p53 protein by immunohistochemistry and western blot analysis. The lower threshold of the assay is 0.1 ng/ml analyte in an enriched recombinant p53 preparation. As p53 is now regarded as a protein associated with prognosis in breast and other cancers, the assay may have clinical applications.

Topoisomerase II alpha and II beta expression in childhood acute lymphoblastic leukaemia: relation to prognostic factors and clinical outcome.

BACKGROUND/AIMS: Many regimens used in the treatment of childhood acute lymphoblastic leukaemia (ALL) include Daunorubicin or Etoposide, which act as topoisomerase poisons. It has been suggested that there may be a relation between topoisomerase expression and response to topoisomerase poisons, based mainly on results from in vitro studies. Therefore, the aim of this study was to investigate this relation in a clinical setting and determine whether topoisomerase II alpha and II beta might be of predictive value in ALL. METHODS: Cellular expression of topoisomerases II alpha and II beta was assessed in 177 cases of ALL by immunohistochemistry using monoclonal antibodies to the two enzymes. The percentages of cell nuclei showing positive staining for topoisomerase II alpha and II beta expression were assessed. RESULTS: Taking the series as a whole, a clear separation of survival curves was seen with the established prognostic markers white blood cell (WBC) count, CD10 status, and sex. However, topoisomerase II alpha and II beta expression showed no relation to survival. No association was found between the topoisomerases and the prognostic markers CD10 and WBC count; however, topoisomerase II alpha expression was found to be related to sex, with expression being lower in girls (p = 0.002). CONCLUSIONS: These results suggest that the response to topoisomerase poisons cannot be predicted by the assessment of topoisomerase II alpha and II beta expression as defined by immunohistochemistry.

A critical comparison of the value of pregnancy-associated alpha2-glycoprotein and carcinoembryonic antigen assays in patients with colorectal cancer.

Absolute serum concentrations of pregnancy-associated alpha2-glycoprotein (alpha2-PAG) and carcinoembryonic antigen (CEA) were compared in 54 patients before and after surgery for colorectal cancer. Preoperatively, elevated levels of alpha2-PAG were found in 32 (59%) and of CEA in 35 (65%). Postoperatively, elevated alpha2-PAG levels were found in 10 of 18 patients (56%) without clinical evidence of recurrence whereas elevated CEA levels were present in three (16%). In patients who developed clinical evidence of tumour recurrence, alpha2-PAG levels were elevated in 8 of 13 (62%) while CEA levels were uniformly abnormal. It is concluded that, in this cross-sectional study, measurement of alpha2-PAG concentrations is less reliable than CEA in the detection of tumour recurrence after apparently curative surgery for colorectal cancer.