Chromogenic in situ hybridization for Her-2/neu-oncogene in breast cancer: comparison of a new dual-colour chromogenic in situ hybridization with immunohistochemistry and fluorescence in situ hybridization.

AIMS: Her-2/neu testing is used as a marker for Herceptin therapy. The aim was to investigate new dual-colour chromogenic in situ hybridization (CISH), in a large number of breast carcinomas (n = 205) with DNA-specific dual-colour probes (ZytoVision, Bremerhaven, Germany) and to compare the results with immunohistochemistry (n = 205) and fluorescence in situ hybridization (FISH) (n = 129). METHODS AND RESULTS: Paraffin-embedded tissue of 205 patients was used. After immunohistochemistry with a focus on immunohistochemically uncertain cases, Her-2/neu amplification using dual-colour CISH (ZytoVision) was analysed. Validation by FISH was performed. The results were: immunohistochemistry, 27.8% with strong expression, 53.7% with uncertain overexpression and 18.5% with no expression; FISH, 25.6% amplified and 74.4% negative; CISH, 35.6% amplified, 62.9% negative and 1.5% not evaluable. Comparison of immunohistochemistry with CISH: CISH negative in 100% with immunohistochemistry 0/1+, amplified in 82.5% with immunohistochemistry 3+; 5.9% contradictory results: 4.4% immunohistochemistry 3+ and negative by CISH, 1.5% negative in immunohistochemistry but amplified by CISH; FISH (129 cases), 8.5% contradictory results to immunohistochemistry, 6.2% immunohistochemistry 3+ and negative by FISH, 2.3% negative by immunohistochemistry and amplified by FISH; comparison of CISH and FISH, 94.6% same results, 3.9% different ones, 1.6% CISH not analysable. CONCLUSIONS: CISH, using dual-colour probes (ZytoVision) is as good as FISH for Her-2/neu analysis. The few discrepant results are likely to be caused by polysomy or tumour heterogeneity.

Technique and proof of patency of microsurgical lympho-lymphonodular anastomoses: a study in the rat model.

BACKGROUND: Lymphedemas due to local lymphatic blocks can be treated by microsurgical transplantation or transposition of lymphatic vessels. Here, the anastomoses are usually made end-to-end between lymphatics, but occasionally appropriate lymphatic recipient vessels are missing. In such cases, reconstructing lymph drainage by connection to a lymph node could be another technical option. The purpose of this study was to examine the patency rate of such lympho-lymphonodular anastomoses in an experimental animal model. METHODS: Male Sprague-Dawley rats were anesthetized, and the retroperitoneum was exposed. Patent blue dye was injected into the left foot to stain lymphatic structures. In group A (n = 8), the left lumbar trunk was cut centrally, the distal part was turned over to the right lumbar lymph node, and a microsurgical lympho-lymphonodular anastomosis was performed. In group B (n = 8), the left lumbar trunk was cut. After 8 weeks, the lumbar region was surgically re-explored, and the lymphatic drainage was examined by injection of Patent blue dye into the left lumbar lymph node. RESULTS: In 8/8 animals of group A, patent transposed lymphatics were found. The patency of the anastomosis was proven directly by observation of blue dye transit and indirectly by observation of blue staining of the right lumbar lymph node. In 6/8 animals of group B, no lymphatic connection to the right lumbar lymphatic system was observed. CONCLUSIONS: This is the first report of the microsurgical technique and the proof of patency of lympho-lymphonodular anastomoses. The novel animal model for testing the patency of transposed lymphatics is discussed.

Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH).

Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest); three cases showed a gene amplification (one of them with failing overexpression by HercepTest); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study.