Growth stimulatory angiotensin II type-1 receptor is upregulated in breast hyperplasia and in situ carcinoma but not in invasive carcinoma.

Two different receptors which bind angiotensin II specifically have been identified in humans and were designated angiotensin II type-1 receptor (AT1) and angiotensin II type-2 receptor (AT2). They only have 34% sequence homology and act through different signalling pathways. AT1 stimulation has been implicated in hypertrophy and hyperplasia in various tissues. In order to study the involvement of AT1 in tissues from controls (n=10) and patients with hyperplasia (n=33), ductal carcinoma in situ (DCIS) (n=23) and invasive carcinoma of the breast (n=25), we tested biopsies and breast-derived cell lines using immunocytochemistry, in situ hybridisation and cell proliferation techniques. The results show specific overexpression of AT1 receptor on the cytoplasmic membrane of cells of hyperplastic lesions with and without atypia and on DCIS of the breast. Evidence for growth stimulation is provided by in vitro experiments showing growth induction by angiotensin II of T47D cells which express the AT1 but not the AT2 receptor. The expression of AT1 on the cell membrane disappears in invasive breast cancer cells suggesting a regulatory pathway which is no longer needed in invasive carcinoma. The specific AT1 expression upregulation might well be an important step in the pathogenesis of hyperplasia of the breast, which is regarded as a precursor lesion for breast cancer.

Pathogenesis of Paget's disease: epidermal heregulin-alpha, motility factor, and the HER receptor family.

BACKGROUND AND METHODS: In Paget's disease of the breast, the epidermis of the nipple is infiltrated by large neoplastic cells of glandular origin. It has been hypothesized that the spread of Paget cells through the nipple epidermis is induced by a motility factor that acts via the HER2/NEU receptor. To test this hypothesis, we characterized and purified a motility factor released by keratinocytes and identified its target receptors in specimens from patients with Paget's disease and in SK-BR-3 breast adenocarcinoma cells, which overexpress HER2/NEU. RESULTS: We isolated the motility factor from keratinocyte-conditioned medium and sequenced tryptic peptides. These sequences were used to identify the motility factor as heregulin-alpha, which is released by skin keratinocytes. Heregulin-alpha induces spreading, motility, and chemotaxis of SK-BR-3 cells, as does motility factor. Motility factor activities of heregulin-alpha are inhibited by monoclonal antibody AB2, directed against the extracellular domain of HER2/NEU, which blocks the binding of heregulin-alpha. We used in situ hybridization to show that normal epidermal cells produce heregulin-alpha messenger RNA and that heregulin receptors, HER3 and/or HER4, as well as their coreceptor HER2/NEU, are expressed by Paget cells. CONCLUSIONS: Heregulin-alpha is a motility factor that is produced and released by normal epidermal keratinocytes and thus plays a key role in the pathogenesis of Paget's disease. Paget cells express heregulin receptors HER2/NEU, as well as HER3 and/or HER4, both of which function as a co-receptor of HER2/NEU. Binding of heregulin-alpha to the receptor complex on Paget cells results in the chemotaxis of these breast cancer cells, which eventually migrate into the overlying nipple epidermis.

Bilateral recurrent adult rhabdomyomas of the pharyngeal wall.

Adult rhabdomyomas of the head and neck are uncommon benign skeletal muscle tumors. Only a few cases occurring in the pharyngeal wall have been described in the world literature. We present a case of recurrent bilateral rhabdomyomas in the pharynx and discuss the clinicopathological features of this lesion, comparing it to those of other neoplasms from which it must be differentiated. To our knowledge, bilaterality of this type of lesion has not been described previously. Although adult rhabdomyomas have a distinct histology, they often are mistaken for a variety of other lesions, particularly granular cell tumor.

Hair removal in 40 hirsute women with an intense laser-like light source.

Until recently, previously applied methods to remove hair have ultimately proven ineffective or resulted in the formation of scars and small wounds. Different methods for removing hair in a more or less permanent way have been used: electrolysis, thermolysis and the blend method. In this study we describe the removal of hair without side-effects by means of non-laser incoherent emitted light, produced by the ILS flashlamp. In a multicenter study we treated 40 women with a median age of 38.6 years with hirsute hair growth of different hair colours on the upper lip and chin. In general 76.7% of the hair was removed within 6 treatments, with an average fluence of 38.7 J/cm2 and a mean wavelength of 585 nm per patient. A correlation was found between the percentage reduction of hairs and the number of treatments and between hair removal and needle epilation before treatment. Furthermore, a correlation was seen between hair reduction and wavelengths of 570 nm and 550 nm. No association was found between hair removal and clinical data of the patients, nor between hair reduction and technical data of the device. This study presents a new alternative for hair removal.

[Monoclonal anti-idiotype antibodies in immunotherapy of ovarian carcinoma (MAb ACA125) and breast carcinoma (MAb ACA14C5)]. / Monoklonale Anti-Idiotypen Antikörper zur Immuntherapie des Ovarialkarzinoms (MAk ACA125) und des Mammakarzinoms (MAk ACA14C5).

Anti-idiotypic antibodies, which imitate a tumor-associated antigen by their variable region, offer an elegant method for the induction of a specific immune response, when used as a surrogate antigen for immunization. We generated anti-idiotypic antibodies imitating 2 different tumor-associated antigens. I. CA125 for ovarian carcinomas and II. 14C5, a tumor-associated cell substrate adhesion molecule on breast cancer cells, whereas the first approach could be introduced in a first clinical trial and the second was evaluated in an immunocompetent animal model. For the induction of an immune response against CA125, 18 patients with advanced ovarian cancer (n = 6) or heavily pretreated recurrences (n = 12) were immunized with the anti-idiotypic antibody MAb ACA125. Patients were treated with 2 mg anti-idiotype antibody every two weeks for 4 injections i.m. and then monthly. 12 of 18 patients demonstrated an anti-anti-idiotypic (Ab3) response, which was to a lower extent also directed against CA125 and 9 of 18 patients developed a CA125 specific cellular immune response by their peripheral blood lymphocytes. Based on this data a follow-up clinical trial in advanced ovarian cancer patients with minimal residual disease in an adjuvant approach after primary therapy was started to evaluate the effect of the immune response on the progression free survival. For immunotherapy of breast cancer, we generated a murine monoclonal anti-idiotypic antibody (MAb ACA14C5), which imitates a cell substrate adhesion molecule on breast cancer cells. The anti-idiotype was introduced in an immunocompetent animal to prove his capability on induction of an immune and tumor response. The results showed a highly significant difference in the tumor growth of the ACA14C5 treated group in contrast to the controls starting the immunization on day 6 after tumor cell application with 10 of 12 animals being cured from their tumor burden. Prophylactic immunization against the invasion antigen of breast cancer by anti-idiotypic antibodies showed protection against increasing tumor burden. However, in the situation of established tumors only minor responses could be detected. Vaccination with anti-idiotypic antibodies comprises an effective method for induction of a specific immune response against non-immunogenic tumor-associated antigens and should be therefore considered in immunological approaches to tumor therapy, where the primary structure and sequence of the antigen, e.g. CA125, is up to now not available.

Cellular uptake and retention measurements of alkylphosphocholines in the SK-BR-3 breast cancer and Molt-4 leukemia cell line using capillary gas chromatography.

The determination of cellular content of octadecylphosphocholine (D-19391) and hexadecylphosphocholine (HePC, D-18506), two anticancer agents of the alkylphosphocholine group, using capillary gas chromatography is described. The compounds' cytotoxicity was first determined by the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium] assay, being indicative for the concentration used in the uptake and retention measurements. D-19391 was added to the SK-BR-3 breast cancer cell line and HePC to the Molt-4 leukemia cell line in concentrations of, respectively, 18.6 and 15.0 microM, during a 36-h incubation period at 37 degrees C, 5% CO2. HePC uptake in the leukemia cells was followed by a 24-h reversibility test in drug-free medium. Subsequently, sample clean-up was performed on a weak cation-exchange column. For the quantitative analysis, HePC was used as internal standard for the D-19391 measurements and vice versa. Derivatization of the samples with trimethylsilylbromide was followed by capillary gas chromatographic analysis. From these data we conclude that our uptake results are quite similar with those of a previous study of HePC cellular uptake in the more resistant Caco-2T colon cancer cell line. Without having investigated the mechanism that underlies the cellular uptake results obtained, our study points to no direct correlation between the compounds' cellular uptake and their cytotoxic effects.

Osteoma cutis in pseudohypoparathyroidism.

Pseudohypoparathyroidism (PHP) is a hereditary disorder characterized by an end-organ resistance for parathormone. PHP can be classified into different types by biochemical and phenotypic characteristics and the level of the defect in the hormone-receptor complex. PHP is described as Albright's hereditary osteodystrophy (AHO) when a specific phenotype is present. We report a case of osteoma cutis in a 30-year-old woman with AHO. Successful treatment was obtained by debriding the lesion followed by split-thickness skin grafting.

Primary pulmonary hypertension with fatal outcome in a young woman and review of the literature.

A 32-year-old female is described, who was admitted with symptoms of severe right heart failure. The most likely diagnosis of pulmonary embolism was excluded. Echocardiography and left-right catheterisation confirmed the diagnosis of primary pulmonary hypertension. A possible mediator in the process of PPH could be the appetite suppressants she had taken for some months after her second pregnancy. Before further pharmacologic tests could be performed the patient died in circulatory collapse. Postmortem pathological examination confirmed the diagnosis of PPH by the presence of narrowed pulmonary arterioles, media hypertrophy, thrombotic lesions and normal surrounding pulmonary parenchyma. The literature on primary pulmonary hypertension is revised with special emphasis on diagnosis and treatment algorithms.

Early prediction of chronic lung disease by tracheal aspirate cytology in ventilated newborns.

UNLABELLED: We assessed the specificity of squamous metaplasia in tracheal aspirates of 69 ventilated newborns (gestational age 25-41 weeks) between days 3 and 7 of life for prediction of chronic lung disease (CLD). CLD was diagnosed when the patient was still requiring ventilation or supplementary oxygen at the postconceptional age of 36 weeks (or postnatal age of 28 days for babies born after 32 weeks gestation) and showed X-ray changes compatible with CLD. In the total population the presence of squamous metaplasia had a sensitivity of 59% and a specificity of 74% for the early diagnosis of CLD. The combination of squamous metaplasia and very low birth weight (VLBW) had a much higher specificity (94%), but a lower sensitivity (45%). Our results show that the presence of squamous metaplasia in VLBW babies during the 1st week of life predicts development of CLD with a specificity of 94% and may be helpful for entering patients into early treatment protocols or trials when a high risk population needs to be identified. As sensitivity of this approach is only 45%, further studies are needed to evaluate the predictive value of the combination of cytology with other markers in tracheal aspirate specimens. CONCLUSION: The presence of squamous metaplasia in tracheal aspirates of VLBW babies between days 3 and 7 of life is significantly associated with the development of chronic lung disease. Simple microscopic evaluation of fresh tracheal aspirates enables us to identify patients at high risk of CLD at a very early stage.

Single cell and tissue specific methods for evaluation of radiation and microgravity effects.

A discussion of different methods to evaluate dose/response and biological effects of ionizing radiation is given. Confocal scanning laser microscopy (CSLM) is presented as a high performing observation method for evaluating different cytological effects. Standard cytochemical techniques can be used to analyse the cell in situ with minimal disturbance of morphology and structure. If a relatively small number of cells are affected by the treatment, the use of confocal microscope observations is fast and has a better resolution than conventional fluorescence microscopy. The optical sectioning capability of the CSLM makes it possible to analyse stacks of cells on detectors up to a depth of 200 micrometer with a resolution of 0.7 micrometer. This is used to analyse single cell electrophoresis results and nuclear track analysis in poly allyl diglycol carbonate (PADC). Consecutive analysis of cells cultivated on PADC, and analysis of nuclear tracks after chemical etched tracks in the PADC, will make it possible to correlate physical dose with direct cellular effects. This is a promising method for single cell analysis and the study of the effects of ionizing radiation at low particle flux density.

Levels of hepatocyte growth factor/scatter factor (HGF/SF) in seminal plasma of patients with andrological diseases.

Hepatocyte growth factor/scatter factor (HGF/SF) has all the characteristics of a molecule suitable for functioning in regulatory networks of motility, such as the spermatogenic epithelium, where spermatogenic cells must migrate between the cells of Sertoli, and it exerts its effect through binding of its high-affinity receptor (c-met). Considering the findings that c-met receptor is expressed in the human testis and on spermatozoa, and that HGF/SF in seminal plasma consists of pro-HGF/SF, mature alphabeta-HGF/SF, and less active forms of HGF/SF, we investigated the concentration and biological activity of HGF/SF in seminal plasma and their correlation with parameters of spermatogenesis to obtain better insight into mechanisms that may be involved in the pathogenesis of male infertility. We also evaluated the potential value of assessment of hepatocyte growth factor concentration and its bioactivity for the diagnosis of certain pathological conditions of male reproduction. We studied the concentration and biological activity of HGF/SF in seminal plasma of normal men and of patients with a range of andrological diseases or conditions by measuring HGF/SF in seminal plasma by enzyme-linked immunosorbent assay and by scatter assay using Madin-Darby canine kidney epithelial cells. We identified three sources of HGF/SF in seminal plasma. In samples from vasectomized men (n = 30; 2.01 ng/ml) and in split ejaculate samples (n = 6; 1e fraction 2.75 ng/ml, 2e fraction 1.62 ng/ml), a prostatic origin can be certified. This HGF/SF has low biological activity (133.3 U/ml). In inflammation of the accessory sex glands (n = 40), a high amount of HGF/SF (3.04 ng/ml) can be generated by white blood cells and has moderate scatter activity (426.7 U/ml). In normozoospermic samples, there is a lower amount of HGF/SF (1.12 ng/ml), with strong scatter activity (1280.0 U/ml). Finally, the clear difference between the low amount of HGF/SF (1.06 ng/ml) with poor scatter activity (106.6 U/ml) in oligozoospermic samples (n = 28) and the high amount of HGF/SF (3.35 ng/ml) with strong scatter activity (853.3 U/ml) in samples from men with azoospermia of primary testicular failure (n = 18) suggests a mainly testicular origin, with different activity in different pathological conditions.

Cytogenetic and molecular analysis of cellular atypical mesoblastic nephroma.

Cytogenetic and molecular analyses were performed on three cellular (atypical) congenital mesoblastic nephromas (CMNs). Two cases had trisomy 11; in one, it was the sole karyotypic abnormality, and the other had additional numerical changes as well as an isochromosome for the long arm of chromosome 1. Markers for the 11p13 and 11p15 loci were present in three copies in these two CMNs. In the third CMN, two apparently normal copies of chromosome 11 were present together with additional numerical and structural chromosome changes. Because loss of heterozygosity was observed for both 11p13 and 11p15 markers, we assume that mitotic recombination occurred. Duplication and loss of imprinting of genes at 11p15 has also been observed frequently in Wilms' tumor. We therefore propose that CMN and Wilms' tumor might share common genetic pathways.

Chondroma of the bladder.

This case report describes a chondroma of the bladder in a 63-year-old woman with clinical complaints of pain in the left fossa iliaca. The lesion was a tumour with a lobulated growth pattern composed of chondrocytes embedded in a chondroid matrix. Neither mitotic figures nor increased cellularity were present. Nuclei were inconspicuous. Immunohistochemical examination showed reactivity for S100 and vimentin.

Ossified retroperitoneal malignant Schwannoma with spinal leptomeningeal metastases.

A malignant retroperitoneal schwannoma in a patient without von Recklinghausen's disease is reported. Ossification in the tumour, shown on CT and MRI in this previously untreated patient is exceptional. MRI demonstration of spinal leptomeningeal metastases supports the hypothesis of haematogenous metastatic spread of systemic malignant tumours to the leptomeningeal spaces.

Jejunal arteriovenous malformation, diagnosed by angiography and treated by embolization and catheter-guided surgery: case report and review of literature.

A case of chronic gastrointestinal hemorrhage caused by a small jejunal arteriovenous malformation (AVM) is reported. Treatment by endovascular embolization was temporarily successful. Subsequently, the patient underwent laparoscopic resection, guided by intraoperative catheter localization with methylene blue. Histopathology confirmed a true AVM. Eighteen months after treatment, the patient is free of symptoms. Literature of jejunal AVMs is reviewed.

Localization of BRCA1 protein at the cellular level.

Based on its amino acid sequence and the existence of three nuclear localization signal (NLS)3 regions, BRCA1 is likely to be a cell cycle-dependent nuclear protein, regulated by cyclin-dependent kinases (cdk) and associated with nuclear proteins such as Rad51 and BARD1, involved in transcription regulation and participating in DNA replication checkpoints. However, many authors have also described a cytoplasmic expression pattern. Moreover, BRCA1 was present not only in a dot like pattern in the nucleus but also associated with a channel-like system of cytoplasm and endoplasmic reticulum invaginating into the nucleus. BRCA1 expression patterns can also be influenced by alternative splice variants and by cell cycle-dependent expression level and localization. Further ultrastructural and confocal studies using C-terminal antibodies, that do not react with C-terminal truncated form of BRCA1 should shed new light upon the exact localization of BRCA1.

Monosomy 22 in a mixed germ cell-sex cord-stromal tumor of the ovary.

We report the cytogenetic findings in a case of mixed germ cell-sex cord-stromal tumor of the ovary in a 5-month-old girl. Monosomy 22 was observed as the sole karyotypic abnormality. This result was confirmed by comparative genomic hybridization, which revealed no additional chromosomal imbalances. This is the first observation of a chromosomal aberration in a mixed germ cell-sex cord-stromal tumor of the ovary. Monosomy 22 has been previously observed in granulosa cell tumors of the ovary. This could suggest a common pathogenetic pathway for both types of tumors.

Amplification units and translocation at chromosome 17q and c-erbB-2 overexpression in the pathogenesis of breast cancer.

Hyperplasia without and with atypia is considered to be a precursor lesion for certain breast carcinomas. The cytogenetic events and the molecular pathology involved in the multistep process from normal to invasive carcinoma are unknown. To characterise the sequence of early genetic abnormalities of chromosome 17q and their biological consequences in the pathogenesis of breast cancer, we performed immunohistochemistry on 451 breast tissues including 180 normal breast specimens, 28 hyperplastic lesions without atypia and 44 with atypia, 100 cases of ductal carcinoma in situ (DCIS) and 99 cases of invasive ductal carcinoma. We correlated the overexpression of the c-ErbB-2 protein, the histological and the recently proposed differentiation classification of DCIS with the extent of DCIS. For fluorescence in situ hybridisation (FISH) analysis, different probes spanning the 17q region including the c-erbB-2 gene locus and those which are found adjacent, were used. Reverse painting and comparative genomic hybridisation (CGH) were performed on several breast cancer cell lines. c-ErbB-2 overexpression was observed in only 29% of DCIS and 23% of invasive carcinomas, but not in hyperplastic and normal tissue. c-ErbB-2 overexpression is correlated with poor differentiation in DCIS but not in invasive carcinoma. In DCIS, there was no correlation with the histological subtype classification. The average extent of DCIS is significantly increased from 13.81 mm in c-ErbB-2 negative cases to 29.37 mm in c-ErbB-2 positive cases. The increase was considered to be a possible consequence of the overexpression and is probably due to the previously described motility enhancing effect of the c-ErbB-2 protein. The histological and differentiation classification of DCIS did not correlate with the extent of disease. Using FISH, amplified genes at 17q12, always including the c-erbB-2 gene, were detected in all cases of DCIS and invasive carcinoma with c-ErbB-2 overexpression. The centromeric region and the NF1 locus, which is located between the centromere and c-erbB-2, were not amplified in any of the DCIS and invasive breast carcinomas, but co-amplification of the myeloperoxidase gene was detected in 3/5 DCIS and 1/5 invasive carcinomas with c-ErbB-2 overexpression. In contrast to c-erbB-2, immunohistochemical overexpression of their respective gene products was not observed. FISH, reverse painting and CGH show similar amplified genes with amplified c-erbB-2 in c-ErbB-2 overexpressing SK-BR-3 and BT474 human breast cancer cells. The amplified genes are part of two different amplicons. Extensive modifications of the 17q chromosomal region, caused by translocation, were also observed in these cell lines. It is concluded that the modifications of chromosome 17q, inducing overexpression of c-ErbB-2 protein, occur at the level of transition from hyperplasia to DCIS. They are preserved in invasive carcinoma with overexpression of c-ErbB-2 protein. This had led to the hypothesis that these modifications at 17q may lead to a larger extent of DCIS.

Generation of a monoclonal antibody directed against a human cell substrate adhesion molecule and the expression of the antigen in human tissues.

Cell substrate adhesion is a prerequisite for invasion and the subsequent formation of metastases. Therefore, we designed monoclonal antibodies (MAbs) against epitopes on the extracellular cell membrane domain of SK-BR-3 cells. One of the antibodies, called MAb 14C5, binds to an extracellular epitope of a plasma membrane antigen of SK-BR-3 and MCF-7 human breast cancer cells. This MAb 14C5 is able to inhibit cell substrate adhesion, not only on culture-treated plastic but also on host tissue, and therefore prevents invasion and metastases. We evaluated the tissue distribution of the 14C5 antigen by immunohistochemistry. The antigen is specifically overexpressed in 64% of invasive ductal adenocarcinomas of the breast (n = 33), in all investigated cases of invasive squamous cell carcinoma (n = 7) and in 40% of basocellular carcinomas of the skin (n = 5). The 14C5 molecule is located on the cell membrane of the carcinoma cells. However, when the tumor is characterized by a highly invasive phenotype, 65% of the cases also show an extensive stromal expression on the fibroblasts between the tumor cells (n = 71). This stromal expression is caused by the presence of the 14C5 antigen on the membrane of the adjacent fibroblasts. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast (n = 15), no expression of the 14C5 antigen occurred. A 90-kDa protein was purified from lysates of human breast cancer cells using a 14C5 MAb Sepharose column and is considered as the antigen recognized by the MAb 14C5.