Gregory W. Charville

Keratin-positive giant cell–rich tumor (KPGCT) is a newly described bone and soft tissue tumor. The tumor is characterized by scattered keratin-positive cells and the presence of HMGA2::NCOR2 fusions. It is not known if the HMGA2::NCOR2 fusion is located in the keratin-positive cells, and little is known about how KPGCT develops. KPGCT shares some histologic features with tenosynovial giant cell tumor (TGCT), a soft tissue tumor with CSF1 rearrangements. Single-nuclei RNA sequencing and Xenium spatial transcriptomics were used to elucidate the most likely mechanisms driving KPGCT and compare KPGCT to TGCT. We identified a population of cells coexpressing keratin, HMGA2 , and CSF1 as the likely neoplastic cells; these cells constitute the minority of the cells in the tumor. These cells express no synovial markers, confirming KPGCT as a distinct entity, separate from TGCT. The bulk of the tumor consists of CSF1R -expressing macrophages and multinucleated giant cells (MNGCs), suggesting an important role for CSF1–CSF1R signaling. In addition, we find that the likely neoplastic cell population shows activation of the hippo signaling pathway, a possible central pathway in KPGCT tumorigenesis. The CSF1–CSF1R axis likely stimulates the migration and proliferation of macrophages, which form the majority of cells in the tumor, as well as their differentiation into MNGCs. These results provide a rationale for the use of CSF1R inhibitors, which have already shown efficacy in TGCT, as a therapy for KPGCT.

Recurrent USP6 gene rearrangements underlie a subset of mesenchymal neoplasms, including nodular fasciitis and aneurysmal bone cyst. These rearrangements place USP6 under the regulatory control of another gene's constitutively active promoter, resulting in USP6 overexpression. Recently, it has been demonstrated in a limited number of cases that detection of USP6 overexpression by RNA chromogenic in situ hybridization (CISH) can serve as a surrogate marker for USP6 -rearranged tumors. To further assess the diagnostic utility of USP6 RNA CISH, we evaluated its application in 1210 bone and soft tissue tumors, using a combination of whole tissue sections and tissue microarrays. USP6 overexpression, defined as RNA CISH staining in >1% of lesional cells, was observed in 92% (36/39) of nodular fasciitis cases and in 85% (17/20) of primary aneurysmal bone cysts. All examples of nodular fasciitis (15/15) and primary aneurysmal bone cyst (8/8) with USP6 fusion confirmed by molecular genetic testing were positive for overexpression. Other tumors staining positive for USP6 included cellular fibroma of tendon sheath (3/3, 100%) and myositis ossificans (3/3, 100%). Although USP6 RNA CISH proved to be highly specific for USP6 -rearranged lesions when assessed in 1145 negative control cases reflecting 53 other tumor types, positivity was rarely seen in isolated cases of angiosarcoma (1/16, 6%), malignant peripheral nerve sheath tumor (1/80, 1%), and carcinosarcoma (1/7, 14%). Taken together, these findings indicate that USP6 RNA CISH represents a useful diagnostic tool; careful interpretation remains critical to its successful implementation, given that isolated examples of non- USP6 -rearranged tumors rarely stain positive.

This Figure illustrates spatial segregation of IL4I1+ vs FOLR2+ macrophages in BC and CRC tumor metastasis to the LN, and across eight different tumor types.

This Figure shows protein marker expression across different cell types detected in CODEX experiment, macrophage distance to the closest tumor cell, macrophage distribution across different tissue niches, and tissue niche distribution across different anatomical locations.

This Figure is a visual summary of this work’s findings and shows the spatial distribution of distinct macrophage subsets in the tumor microenvironment and the clinical significance of IL4I1+ and SPP1+ macrophages in breast and colon tumors.

Thus Figure shows spatial enrichment of NLRP3+ macrophages with neutrophils in Crohn’s Disease, and immunostimulatory and immunosuppressive protein marker expression in NLRP3+ vs SPP1+ macrophages.