Postradiation Cutaneous Angiosarcoma After Treatment of Breast Carcinoma is Characterized by MYC Amplification in Contrast to Atypical Vascular Lesions After Radiotherapy and Control Cases

Clinicopathological, Immunohistochemical and Molecular Analysis of 66 Cases

T Mentzel; H U Schildhaus; G Palmedo; R Büttner; H Kutzner


Mod Pathol. 2012;25(1):75-85. 

In This Article

Materials and Methods

The cases were retrieved from the routine files of the Department of Pathology, University of Bonn, the Dermatopathologie Bodensee, Friedrichshafen, and from referral files of three of the authors (TM, HUS, and HK), and clinical and follow-up information were obtained from the referring pathologists. As control cases classical hemangiomas and cases of postradiation changes without significant vascular proliferation were chosen. In addition, some cases of spontaneous angiosarcoma were added for comparison. The tissue was fixed in 4% buffered formalin in all cases, routinely processed and embedded in paraffin and 2–4 μm thick sections were stained with hematoxylin and eosin. In addition, representative sections were stained immunohistochemically by the labeled streptavidin biotin technique using commercially available antibodies; antigen retrieval was used for all antibodies. Stainings for CD 31 (clone JC70A, dilution 1:100, DAKO, Hamburg, Germany), α-smooth muscle actin (1A4, 1:500, DAKO), Ki-67 (SP6, 1:300, Roche Diagnostics, Mannheim, Germany), MYC (Y69, 1:100, Biomol, Hamburg, Germany), and prox-1 (polyclonal, 1:200, RELIATech, Wolfenbüttel, Germany) were available in most cases. Appropriate positive and negative controls were used. Fluorescence in situ hybridization was performed on 3 μM sections of formalin-fixed, paraffin-embedded tissue after deparaffinization with xylene, dehydration by graded ethanol and air-drying. All tissue sections were pretreated and digested as recommended by the probe supplier. Digestion times were optimized on a case-by-case basis. After pretreatment and digestion steps, sections were washed in 2 × SSC, pH 7.0, for 2 min at 45 °C. The MYC probe (Vysis LSI C-MYC, Abbott, Wiesbaden, Germany) was applied to the sections and the coverslides were sealed with rubber cement, heat-denatured for 10 min and hybridized at 37 °C for 16 h or overnight. Co-hybridization with the centromeric enumeration probe (CEP8, D8Z2, Abbott) was carried out in a subset of cases. Stringent washing was performed as recommended by the probe supplier. All sections were counterstained with DAPI II in mounting medium (125 ng/ml, Abbott). A minimum of 45 non-overlapping intact nuclei were assessed for the presence of amplifications; an amplification was defined as multiple clustered signals (> 8 signals) according to the previously described findings.[6] FISH slides were evaluated by two experienced investigators (GP, HUS) using appropriate microscopes and filter sets. After initial investigation, all specimens were cross-validated by GP and HUS in a consensus meeting. For photographic documentation, nuclei with the appropriate signals were digitally photographed with a monochrome RT3 CCD camera (Diagnostic Instruments, Sterling Heights, USA) connected to a Zeiss Axioplan 2 microscope (Zeiss, Oberkochen, Germany) using a HBO103 lamp and the appropriate filters (spectrum orange and 4′,6-diamino-2-phenylindole) for the two fluorescence dyes (Zeiss). Reconstruction into a single superimposed image with blue and orange pseudocolors was accomplished using the SPOT software (Visitron Systems, Puchheim, Germany).


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