Synaptophysin

One to two representative formalin fixed paraffin tissue blocks containing tumor from each case were retrieved to generate 4 µm thick unstained slides for immunohistochemical staining for SATB2 (dilution 1:100, clone EPNCIR 130A, Abcam, Cambridge, MA) and CDX2 (prediluted, clone EPR2764Y, Cell Marque, Rocklin, CA) on a Ventana Benchmark automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to standard protocols with appropriate positive and negative controls. Chromogranin (prediluted, clone LK2H10, Ventana, Tucson, AZ) and synaptophysin (predilute, polyclonal, Cell Marque, Rocklin, CA) were also performed for AdexGCCs using a Ventana Benchmark automated immunostainer.
Only nuclear staining was considered positive for SATB2 and CDX2. The staining was reviewed in a consensus manner (CY, LZ, DC) with a multiheaded microscope. The results were scored in a semi-quantitative pattern as 0 (< 1% cells), 1+ (1-25%), 2+ (26-50%), 3+ (51-75%), 4+ (76-100%). The percentage of positive cells was visually estimated with 5% increment.

Four micrometer sections of formalin-fixed paraffin-embedded tissue were stained with hematoxylin and eosin. Immunostaining and detection were performed on a Leica Bond III automated immunostainer using primary antibodies, clones, dilutions and sources, respectively: synaptophysin (polyclonal; 1:100, Cell-Marque), SOX10 (EP268, 1:250 Cell-Marque), CD99 (EPR3097Y, 1:100, Cell-Marque), H3K27Me3 (C36B11, 1:50, Cell Signaling Technology). synaptophysin was done to investigate the presence of neuroendocrine differentiation—SOX10 for nerve sheath/melanocytic differentiation and CD99 as a sensitive, but not specific, stain for ES.