Analysis of available microarray mRNA expression data was conducted using the Oncomine web platform (Life Technologies) (5). The Steidl Lymphoma, Brune Lymphoma and the Basso Lymphoma datasets were used (6–8). In the Steidl study 130 HL samples were analyzed using Human Genome U133 Plus 2.0 Array (19 574 genes, 54 675 reporters). To determine the cut-off for high and low VEGFA gene expression levels the software Cut-off Finder was used (9). In the Brune study 42 malignant lymphoma samples and 25 normal B-cell samples of various types were analyzed using as well the Human Genome U133 Plus 2.0 Array. In the Basso study 336 B-cell samples (normal, tumor, cell line, and cell line perturbation samples) were analyzed using the Affymetrix U95Av2 Array (8,603 genes, 12,651 reporters). Data were analyzed using GraphPad Prism 6.04 (GraphPad Software Inc., La Jolla, USA). Preparing FFPE tissue blocks Hodgkin and other lymphoma cases from all age groups that were routinely diagnosed between 2001 and 2014 were identified within the files of the Lymph Node Registry Kiel. Lymphoma biopsy specimens were routinely fixed in 10% buffered formalin, embedded in paraffin (FFPE), cut into 3-5 µm sections and mounted on coated glass slides for conventional immunohistochemistry. Preparing Tissue Micro Arrays Cases with sufficient FFPE tissue were arranged in a tissue microarray (TMA) with duplicate cores 0.6 mm in diameter for each lymphoma. TMA and whole tissue sections were subsequently subjected to immunohistochemistry. Immunohistochemistry on FFPE sections
Analysis of available microarray mRNA expression data was conducted using the Oncomine web platform (Life Technologies) (5). The Steidl Lymphoma, Brune Lymphoma and the Basso Lymphoma datasets were used (6–8). In the Steidl study 130 HL samples were analyzed using Human Genome U133 Plus 2.0 Array (19 574 genes, 54 675 reporters). To determine the cut-off for high and low VEGFA gene expression levels the software Cut-off Finder was used (9). In the Brune study 42 malignant lymphoma samples and 25 normal B-cell samples of various types were analyzed using as well the Human Genome U133 Plus 2.0 Array. In the Basso study 336 B-cell samples (normal, tumor, cell line, and cell line perturbation samples) were analyzed using the Affymetrix U95Av2 Array (8,603 genes, 12,651 reporters). Data were analyzed using GraphPad Prism 6.04 (GraphPad Software Inc., La Jolla, USA). Preparing FFPE tissue blocks Hodgkin and other lymphoma cases from all age groups that were routinely diagnosed between 2001 and 2014 were identified within the files of the Lymph Node Registry Kiel. Lymphoma biopsy specimens were routinely fixed in 10% buffered formalin, embedded in paraffin (FFPE), cut into 3-5 µm sections and mounted on coated glass slides for conventional immunohistochemistry. Preparing Tissue Micro Arrays Cases with sufficient FFPE tissue were arranged in a tissue microarray (TMA) with duplicate cores 0.6 mm in diameter for each lymphoma. TMA and whole tissue sections were subsequently subjected to immunohistochemistry. Immunohistochemistry on FFPE sections