Fig. 4

Findings of Azan staining and immunohistochemical staining of the axillary lymph node. a Azan staining showed that the metastatic tumor lesion was collagen rich (× 40). b Immunohistochemical staining using an antibody for chromogranin A showed negative staining in the metastatic tumor lesion as well as in the primary lesion. c The tumor cells also showed negative staining for synaptophysin. d The primary tumor cells showed positive staining for CDX2 in the nucleus (× 200). e The tumor cells in the lymph node also showed positive staining for CDX2 (× 200). f, g The tumor cells in the lymph node were negative for osteoblast marker CD56 (f) and breast cancer marker CK7 (g). h Cells positive for the macrophage cell marker CD68 were observed around the osseous lesion. i CD68-positive cells were also seen in the primary tumor. j The tumor cells showed BMP-2 overexpression in the lymph node (× 400). k, l BMP-2 expression was weakly positive in the cell membrane on the lumen side of the primary tumor (k: × 400) and adjacent normal mucosa (l: × 400). m, n Osteonectin expression was strongly positive (m: × 200) and osteocalcin expression was weakly positive (n: × 200) in the tumor cells. o, p The tumor cells expressed cytokeratin (AE1/AE3) (o: × 200) and TGF-β1 (p: × 200; *HO tissue). In the HO lesion, cytokeratin-positive osteocytes were observed (o: arrows). q, r Gli2 (q: × 200) and pSmad2/3 (r: × 200) showed overexpression in the nucleus of the tumor cells (*HO tissue). The upper right of q and r are magnified views (× 400). s The tumor cells showed no nestin expression (× 200). t Fibroblasts surrounding the tumor cells showed alpha-smooth muscle actin (α-SMA) expression (× 100)