PMC9850095_01
Roentgenographic, histopathological and blood examination findings of the patient with PMT (A) Deficiency of mineralization in the epiphysis of the bilateral proximal tibias and distal femurs on admission. (B) Radiolucent lesion with endosteal scalloping and marginal sclerosing in the left fibular shaft. A red arrowhead indicates the tumor-like lesion. (C) The cavity of the tumor filled with beta-tricalcium phosphate (beta-TCP) after resection. (D) Good bone formation observed at 21 months after surgery. (E) Hematoxylin and eosin staining of the resected specimen. Irregularly deposited osteoid and osteoblast-like tumor cells were scattered between the osteoid. Black arrowheads indicate reactive osteoclastic giant cells (original magnification, x400). (F) Immunohistochemical staining of FGF23. Positive staining was observed in the cytoplasm of the osteoblast-like tumor cells (original magnification, x800). (G, H) Transition of serum FGF23 (G) and phosphorus (H) level after excision of the tumor. Normal ranges of serum FGF23 and phosphorus level are <50 pg/mL and 3-4.7 mg/dL, respectively.
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Roentgenographic examination revealed a deficiency of mineralization like that seen in rickets patients in the epiphysis of the bilateral proximal tibias and distal femurs ( Figure 1A ), and a radiolucent lesion with endosteal scalloping and marginal sclerosing in the left fibula ( Figure 1B ). We resected the tumor en bloc by preserving the periosteum of the fibula after confirming its benignity by intraoperative frozen section diagnosis, and the cavity of the tumor was filled with beta-tricalcium phosphate (beta-TCP) ( Figure 1C  and  Supplemental Figure 1 ). Roentgenographic examination revealed absorption of the beta-TCP and bone formation and union of the fibular shaft ( Figure 1D ). Histopathological examination of the resected tumor revealed irregularly deposited osteoid and osteoblast-like tumor cells scattered between the osteoid and reactive osteoclastic giant cells ( Figure 1E  and  Supplemental Figure 2 ). In immunohistochemistry, expression of FGF23 was shown in the cytoplasm of the osteoblast-like tumor cells ( Figure 1F ), CD56 was diffusely positive on the cell membrane of the tumor cells and SATB2 was diffusely positive on the tumor cells ( Supplemental Figure 2 ). After resecting the tumor, the serum FGF23 level started to decrease immediately and normalized within 3 hours ( Figure 1G ). The increase in serum phosphorus level was slightly delayed as compared with that of the FGF23 level, and was observed 6 days after the operation ( Figure 1H ).
Fusion protein NIPBL-BEND2 and functional analyses (A) Structure of the NIPBL and BEND2. Boxes indicate exons, the gray parts of boxes indicate untranslated region (UTR) and the dotted line indicates genomic breakpoints. The fusion transcript contained exons 1-6 of NIPBL and exons 7-14 of FGFR1. (B) Structure of predicted NIPBL-BEND2 fusion protein. Numbers indicate amino acid residues. (C) Cell counts of mock- or NIPBL-BEND2- transfected HEK293T cells at 48hr. n=3 each. The plus signs indicate mean values. (D) MTS assay of mock- or NIPBL-BEND2- transfected MG63 cells at 48hr. n=5 each. The plus signs indicate mean values. (E) Gene set enrichment analysis comparing the expression profiles obtained from mock- and NIPBL-BEND2- transfected HEK293T cells. The genes in the HALLMARK_MYC_TARGET_V1 gene set were upregulated in NIPBL-BEND2-transfected cells. NES, normalized enrichment score; FDR, false discovery rate.
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Histopathological examination of the resected tumor revealed irregularly deposited osteoid and osteoblast-like tumor cells scattered between the osteoid and reactive osteoclastic giant cells ( Figure 1E  and  Supplemental Figure 2 ). In immunohistochemistry, expression of FGF23 was shown in the cytoplasm of the osteoblast-like tumor cells ( Figure 1F ), CD56 was diffusely positive on the cell membrane of the tumor cells and SATB2 was diffusely positive on the tumor cells ( Supplemental Figure 2 ). We performed RNA sequencing using a resected specimen from the patient and identified a novel in-frame fusion involving NIPBL (encoding Nipped-B gene product and fungal Scc2-type sister chromatid cohesion proteins) and BEND2 (encoding a protein which has two BEN domains in the C-terminus) ( Figure 2A ). The fusion protein contained the phosphorylation site derived from NIPBL and the BEN domain derived from BEND2 ( Figure 2B ). The NIPBL-BEND2 transfected cells showed faster proliferation at 48 hours after transfection (p = 0.001 and 0.003, Student's t-test, respectively). ( Figures 2C, D ). HEK293T cells identified a significant enrichment of MYC-target genes, consistent with faster proliferation ( Figure 2E ). However, the expression of FGF23 (log2 fold change; 0.031) and FGFR1 (log 2 fold change; 1.59) was not changed significantly by the transfection in addition to the KL/KLB, SPP1, SFRP4, and MEPE ( Supplemental Data 1 ), even though the tumor mRNA showed relatively high FPKM in these genes ( Supplemental Table 3 ).