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.

Interleukin-1 is a motility factor for human breast carcinoma cells in vitro: additive effect with interleukin-6.

Interleukin-1 beta (Il-1 beta) and interleukin-1 alpha (Il-1 alpha) were shown to act as motility factors for the human breast carcinoma cell lines SK-BR-3 and ZR-75-1 in vitro. Both cytokines induced transition from the stationary to the motile phenotype (spreading). Il-1 beta stimulated translocation, shape change and random migration (chemokinesis) of SK-BR-3 cells as demonstrated by time-lapse video recordings and by a modified Boyden chamber assay. Interleukin-6 (Il-6) stimulated spreading of the SK-BR-3 cells; an additive effect with Il-1 beta on spreading and fast plasma membrane movements was evidenced. In the SK-BR-3 cell line, the signal transduction of Il-1 beta and Il-6 differed, since only the effect of Il-6 on spreading was sensitive to pertussis toxin. Both Il-1 beta and Il-6 required protein synthesis to stimulate spreading, since cycloheximide inhibited the effect of the cytokines. Induction of an autocrine loop of Il-6 in the SK-BR-3 cells by Il-1 beta was unlikely, since after stimulation with Il-1 beta, no induction of Il-6 activity was measured, nor was inhibition of stimulated spreading seen in the presence of an antiserum against Il-6. Addition of Il-8 or of an antiserum against Il-8 did not affect spreading. We concluded that Il-1 and Il-6 could act as motility factors for human breast carcinoma cells, in both an independent and an additive way.(ABSTRACT TRUNCATED AT 250 WORDS)

The p185erbB2 protein is localized on cell organelles involved in cell motility.

In a previous paper, we have shown that the c-erbB-2-encoded protein p185erbB2 is localized on the brush border of the proximal tubule kidney cells. In invasive duct cell carcinomas, the labeling was most obvious on the villous plasma membrane protrusions. From these observations the hypothesis was raised that p185erbB2 could play a role in motility. To test this hypothesis, we quantified its distribution on the microvilli and plasma membrane protrusions and on the straight parts of the cell membrane after immunoelectron microscopy. These findings were compared with the localization on p185erbB2 overexpressing SK-BR-3 human breast cancer cells before and after stimulation of motility by treatment with conditioned medium from COLO-16 cells (CM), which is also able to induce chemotaxis of these cells in a Boyden chamber assay. In the invasive duct cell carcinomas, the number of gold particles was nine times higher at the plasma membrane protrusions than at the straight parts of the cell membrane. In untreated SK-BR-3 cells, p185erbB2 was similarly concentrated on the membrane of small microvilli and plasma membrane protrusions. Treatment of SK-BR-3 cells with CM leads to cell spreading, enlargement of the microvilli, formation of pseudopodia and chemotaxis. Aggregation of p185erbB2 at the plasma membrane protrusions and pseudopodia is observed on immunofluorescence light microscopy. The concentration of p185erbB2 is several times higher on these membrane extensions than on the straight parts after immunogold labeling. It is concluded that p185erbB2 is localized on cell organelles involved in motility, and it is suggested that the molecule plays a role in cell movement, providing the capacity of tumor cells to spread and metastasize.

Sensitive and reliable detection of genomic imbalances in human neuroblastomas using comparative genomic hybridisation analysis.

Deletions of the short arm of chromosome 1, extra copies of chromosome 17q and MYCN amplification are the most frequently encountered genetic changes in neuroblastomas. Standard techniques for detection of one or more of these genetic changes are karyotyping, FISH analysis and LOH analysis by Southern blot or PCR. Each of these techniques has its own particular limitations. More recently, comparative genomic hybridisation (CGH) was introduced for detection of genomic imbalances including deletions, duplications and gene amplification. We evaluated the sensitivity and reliability of CGH for detection of the most frequently encountered genetic changes in neuroblastoma. For this purpose a panel of well-characterised neuroblastoma cell lines as well as a series of 11 primary neuroblastomas was analysed. Our results show that CGH is a valuable tool for the genetic characterisation of neuroblastomas, both for the detection of frequently occurring genomic imbalances and for the identification of previously unnoticed genetic changes.

Crohn's disease with adenocarcinoma and dysplasia. Macroscopical, histological, and immunohistochemical aspects of two cases.

We present two cases of small-bowel adenocarcinoma and dysplasia in patients with longstanding Crohn's disease. In one case, the dysplasia and cancer were exclusively located in the terminal ileum, whereas in the other case, several cancers were found from the ileum toward the transverse colon. In both cases, we found a clinically unsuspected Dukes C1 mucinous adenocarcinoma together with large foci of polypoid villous dysplasia or with multifocal high-grade dysplasia and intramucosal carcinoma. Immunohistochemical staining for carcinoembryonic antigen (CEA) revealed a different staining pattern in various diseased areas. The intensity of CEA staining paralleled the histologic degrees of dysplasia and neoplasia. Cytokeratin expression was disturbed in inflamed mucosa, and it was more pronounced in high-grade dysplasia and invasive carcinoma. We conclude that the presence of dysplasia in an intestinal biopsy of a patient with Crohn's disease should arouse the pathologist's suspicion of carcinoma and force him or her to take multiple sections from strictures and polypoid lesions, especially since the clinical symptoms of a carcinoma may be obscured by the symptoms of inflammatory bowel disease. Immunohistochemical staining with CEA and cytokeratin are useful in the objectivation of dysplasia.

The neu-oncogene: more than a prognostic indicator?

Overexpression of the neu-protein, evidenced as membrane staining by immunohistochemistry, is detected in approximately 20% of invasive duct cell carcinomas, in approximately 50% of in situ duct cell carcinomas, and in almost 100% of cases of Paget's disease. Apart from a growth stimulatory effect, the molecule plays an important role in cell motility of tumor cells by the activity of a motility factor, which acts as a specific ligand for the neu-protein. The motility factor induces chemotaxis of neu-overexpressing breast cancer cells. The motility function of the neu-protein may lead to an increased metastatic potential of neu-overexpressing breast tumors. Also in Paget's disease of the breast, a motility factor secreted by epidermal keratinocytes attracts the neu-overexpressing Paget's cells by chemotaxis and leads to invasion of the epidermis by the tumor cells.

neu overexpression correlates with extent of disease in large cell ductal carcinoma in situ of the breast.

In a retrospective study of ductal carcinoma in situ (DCIS) of the breast, the expression of the neu oncogene was determined immunohistochemically in 76 women treated by local excision or mastectomy. The histopathological features, including the extent of the lesion, histological subtype, cell type, and number of mitoses, were related to neu overexpression. Immunopositivity was found only in DCIS of large cell type, where it correlated with extent of disease but not with mitotic rate. Our findings, together with previous experimental evidence, suggest that this relationship is a consequence of the effect of the neu protein on cell motility.

The neu-protein and breast cancer.

The neu-protein is overexpressed in about 20% of invasive duct cell carcinomas of the breast. The only reliable sign for neu-overexpression by immunohistochemistry is membrane staining. Its overexpression is correlated with decreased overall survival and disease free survival due to increased metastatic activity of neu-overexpressing tumour cells. This increased metastatic potential is a consequence of the motility enhancing activity of the neu-protein, which is exclusively expressed on pseudopodia, and to a lesser extent of its growth stimulating effect. From a clinical point of view, the assessment of neu-overexpression in breast cancer might become a useful tool in the future treatment of patients by chemotherapy, since patients whose tumour shows neu-overexpression benefit from higher doses of chemotherapy. The molecule plays a key role in the pathogenesis of Paget's disease of the breast. A chemotactic factor which is secreted by epidermal keratinocytes attracts the Paget cells to spread into the epidermis and acts via the neu-protein. In ductal carcinoma in situ, the combination of neu-overexpression and large cell type is highly correlated with extent of disease and therefore neu-overexpression might be a predictive marker for recurrence of disease after tumour resection.

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.

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.

Multiple polysomies in nasal polyps in children.

Karyotyping of a nasal polyp from a 10-year-old girl revealed multiple numerical chromosome changes. Extra copies of chromosome 5, 7, 8, 12, 16, 17, 18, 19, 20, and 21 were present. Only one cytogenetically abnormal nasal polyp of a child has been reported in the literature. The karyotypes of the reported and the present case are remarkably similar. The significance of this finding in a nonneoplastic proliferation is discussed.

Chromosome aberrations in fibrous dysplasia.

We report the cytogenetic findings of two cases of fibrous dysplasia, one occurring in the tibia, the other in the sphenoid. Both cases exhibited only one chromosome change: a t(6;11)(q15;p15) in the first case, a derivative chromosome 2 in the second. The previous cytogenetic report on fibrous dysplasia revealed only numerical changes. The significance of these inconsistent chromosomal findings in fibrous dysplasia is unclear.

i(12p) in a malignant ovarian tumor.

We have found one or more copies of i(12p) in an ovarian germ cell tumor, histologically a yolk sac tumor. This chromosome marker is characteristically associated with germ cell tumors in males. This report indicates that further investigation is necessary to establish the role of the i(12p) marker in the pathogenesis of germ cell tumors also in females.

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.

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.

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.

CEA and HMFG in hyperplastic and malignant lesions of the breast.

In order to evaluate a possible transition from proliferative lesions of the breast to ductal carcinoma in situ (DCIS), an immunohistochemical retrospective analysis was carried out. Twelve patients with hyperplasia without atypia, 11 patients with hyperplastic lesions with atypia, 21 patients with DCIS and 24 patients with invasive carcinoma were studied. The expression of carcino-embryonic antigen (CEA), a dedifferentiation marker, was investigated, applying one monoclonal antibody (Amersham). The expression of human milk fat globulin (HMFG), a differentiation marker, was studied by means of three monoclonal antibodies. The results reveal that no CEA activity can be demonstrated in normal and hyperplastic breast tissue, either with or without atypia. The monoclonal antibody was positive in 48% of DCIS and 50% of the invasive carcinomas. We failed to observe a correlation between the presence of CEA and the differentiation of the tumor. The application of this antiserum adds no substantial information about the phenotypic alterations during the progression from atypical hyperplasia to DCIS. HMFG was present in benign as well as in malignant breast lesions. Therefore, we conclude that HMFG is not useful for the evaluation of the phenotypic change from atypical hyperplasia to malignancy. However, as pointed out by others, it can be used in a diagnostic panel of different antibodies in the distinction between epithelial and non-epithelial lesions.

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.

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.