HER2 gene amplification in patients with breast cancer with equivocal IHC results.

AIMS: Equivocal human epidermal growth factor receptor 2 protein (HER2) (2+) immunohistochemistry (IHC) is subject to significant interobserver variation and poses a challenge in obtaining a definitive positive or negative test result. This equivocal test result group accounts for approximately 15% of all tumours, and for optimal guidance of HER2 targeted therapy, a further analysis of quantification of gene copy number and amplification status is needed for patients with early or metastatic breast cancer. METHODS: 553 breast-cancer specimens with equivocal HER2 IHC(2+) test results were collected and subsequently centrally retested by chromogenic in situ hybridisation (CISH), and HER2 gene copy numbers per tumour cell nucleus were determined. RESULTS: Using CISH, 77 of 553 equivocal HER2 IHC(2+) test result cases (13.9% of total) showed high levels of HER2 gene amplification (≥10.0 gene copies per nucleus), and 41 of 553 (7.4% of total) showed low-level HER2 gene amplification (6.0-9.9 gene copies per nucleus). In 73.6% of cases, no amplification of the HER2 gene was shown, and in only 4.9% of cases was an equivocal test result by CISH observed (4.0-5.9 gene copies per nucleus). CONCLUSIONS: Testing by CISH of all equivocal HER2 IHC(2+) test result provides a definitive guidance in HER2 targeted therapy in 95.1% of cases. A significant proportion (21.3%) of patients with equivocal IHC(2+) test results show amplification of the HER2 gene.

Dynamic culturing of smooth muscle cells in tubular poly(trimethylene carbonate) scaffolds for vascular tissue engineering.

Porous, tubular, flexible, and elastic poly(trimethylene carbonate) (PTMC) scaffolds (length 8 cm and inner diameter 3 mm) for vascular tissue engineering were prepared by means of a dip-coating and particulate leaching procedure. Using NaCl as porogen, scaffolds with an average pore size of 110 µm and a porosity of 85% were obtained. Before leaching the salt, the structures were made creep-resistant by means of crosslinking at 25 kGy gamma irradiation. To increase the efficiency of cell seeding, the scaffolds were provided with a microporous outer layer of 0.2 mm with an average pore size of 28 µm and a porosity of 65% (total wall thickness 1 mm). Human smooth muscle cells (SMCs) were seeded in these scaffolds with an efficiency of 43%, as determined after 24 h cell adhesion. SMCs were cultured in the scaffolds up to 14 days under stationary conditions or under pulsatile flow conditions in a bioreactor (pressure 70-130 mmHg, 69 pulsations/min, and average wall shear rate 320 s(-1)). Although SMCs proliferated under both conditions, cell numbers were three to five times higher in case of dynamic culturing. This was qualitatively confirmed by means of histology. Also, in terms of mechanical properties, the dynamically cultured constructs performed better than the statically cultured constructs. After culturing for 14 days, the maximum tensile strengths of the constructs, determined in the radial direction, had increased from 0.16 MPa (unseeded scaffold) to 0.48 MPa (dynamic culturing) and 0.38 MPa (static culturing). The results of this study indicate that a potentially useful medial layer for tissue-engineered vascular grafts can be prepared by dynamic culturing of human SMCs seeded in porous tubular poly(trimethylene carbonate) scaffolds.

A nonresponding small cell lung carcinoma.

We present a case with limited disease small cell lung carcinoma with persisting disease and a troublesome syndrome of inappropriate antidiuretic hormone, despite concurrent chemoradiotherapy and second-line chemotherapy. To gain local control, a lobectomy was performed.

Endochondral bone tissue engineering using embryonic stem cells.

Embryonic stem cells can provide an unlimited supply of pluripotent cells for tissue engineering applications. Bone tissue engineering by directly differentiating ES cells (ESCs) into osteoblasts has been unsuccessful so far. Therefore, we investigated an alternative approach, based on the process of endochondral ossification. A cartilage matrix was formed in vitro by mouse ESCs seeded on a scaffold. When these cartilage tissue-engineered constructs (CTECs) were implanted s.c., the cartilage matured, became hypertrophic, calcified, and was ultimately replaced by bone tissue in the course of 21 days. Bone aligning hypertrophic cartilage was observed frequently. Using various chondrogenic differentiation periods in vitro, we demonstrated that a cartilage matrix is required for bone formation by ESCs. Chondrogenic differentiation of mesenchymal stem cells and articular chondrocytes showed that a cartilage matrix alone was not sufficient to drive endochondral bone formation. Moreover, when CTECs were implanted orthotopically into critical-size cranial defects in rats, efficient bone formation was observed. We report previously undescribed ESC-based bone tissue engineering under controlled reproducible conditions. Furthermore, our data indicate that ESCs can also be used as a model system to study endochondral bone formation.

Biological characterisation of vascular grafts cultured in a bioreactor.

In this study, the development is described of a tissue-engineered construct mimicking the structure of a natural blood vessel. Smooth muscle cells (SMC) were cultured under pulsatile flow conditions in porous tubular scaffolds composed of crosslinked type I insoluble collagen and insoluble elastin. Under these dynamic culture conditions, average wall shear rate, systolic and diastolic pressures and pressure wave-forms comparable to conditions in the human carotid artery were obtained. Culturing of SMC in tubular scaffolds under dynamic conditions resulted in enhanced tissue formation compared to static conditions. Higher SMC numbers, a more homogeneous distribution of SMC throughout the scaffolds and higher collagen mRNA expression levels were found when cells were cultured under dynamic compared to static conditions. mRNA expression levels of markers of proliferation and apoptosis showed that the higher cell numbers in the scaffolds cultured under dynamic conditions can be explained by increased cell proliferation but not by decreased apoptosis. Glucose consumption and lactate formation by the cells showed that cell metabolism was more aerobic under dynamic compared to static conditions. Lining of the dynamically cultured constructs with a luminal monolayer of endothelial cells might result in vessels suitable for in vivo applications.

Tissue engineering of blood vessels: characterization of smooth-muscle cells for culturing on collagen-and-elastin-based scaffolds.

Tissue engineering offers the opportunity to develop vascular scaffolds that mimic the morphology of natural arteries. We have developed a porous three-dimensional scaffold consisting of fibres of collagen and elastin interspersed together. Scaffolds were obtained by freeze-drying a suspension of insoluble type I collagen and insoluble elastin. In order to improve the stability of the obtained matrices, they were cross-linked by two different methods. A water-soluble carbodi-imide, alone or in combination with a diamine, was used for this purpose: zero- or non-zero-length cross-links were obtained. The occurrence of cross-linking was verified by monitoring the thermal behaviour and the free-amino-group contents of the scaffolds before and after cross-linking. Smooth-muscle cells (SMCs) were cultured for different periods of time and their ability to grow and proliferate was investigated. SMCs were isolated from human umbilical and saphenous veins, and the purity of the cultures obtained was verified by staining with a specific monoclonal antibody (mAb). Cultured cells were also identified by mAbs against muscle actin and vimentin. After 14 days, a confluent layer of SMCs was obtained on non-cross-linked scaffolds. As for the cross-linked samples, no differences in cell attachment and proliferation were observed between scaffolds cross-linked using the two different methods. Cells cultured on the scaffolds were identified with an anti-(alpha-smooth-muscle actin) mAb. The orientation of SMCs resembled that of the fibres of collagen and elastin. In this way, it may be possible to develop tubular porous scaffolds resembling the morphological characteristics of native blood vessels.

Association of the tetraspanin CD151 with the laminin-binding integrins alpha3beta1, alpha6beta1, alpha6beta4 and alpha7beta1 in cells in culture and in vivo.

CD151 is a cell surface protein that belongs to the tetraspanin superfamily. It forms complexes with the laminin-binding integrins alpha3beta1, alpha6beta1 and alpha6beta4 and is codistributed with these integrins in many tissues at sites of cell-matrix interactions. In this study we show that CD151 can also form stable complexes with the laminin-binding integrin alpha7beta1. The strength of this interaction is comparable to that between CD151 and alpha3beta1. Complexes of alpha3beta1, alpha6beta1 and alpha7beta1 with CD151 are equally well formed with all splice variants of the alpha3, alpha6 and alpha7 subunits, and complex formation is not affected by mutations that prevent the cleavage of the integrin alpha6 subunit. Like the expression of alpha3beta1 and alpha6beta1, expression of alpha7beta1 in K562 cells results in increased levels of CD151 at its surface. Two non-integrin laminin receptors, dystroglycan and the polypeptide on which the Lutheran blood group antigens are expressed, are also often colocalized with CD151, but no association with CD151-alpha3beta1 complexes was found with biochemical analysis. The anti-CD151 antibody TS151R detects an epitope at a site at which CD151 interacts with integrins, and therefore it cannot react with CD151 when it is bound to an integrin. Comparison of the straining patterns produced by TS151R with that by of an anti-CD151 antibody recognizing an epitope outside the binding site (P48) revealed that most tissues expressing one or more laminin-binding integrins reacted with P48 but not with TS151R. However, smooth muscle cells that express alpha7beta1 and renal tubular epithelial cells that express alpha6beta1 were stained equally well by TS151R and P48. These results suggest that the interactions between CD151 and laminin-binding integrins are subject to cell-type-specific regulation.