case_images: PMC11748356_01
case_images: Histomorphologic evaluation of the CT-guided liver biopsy of the metastatic renal medullary carcinoma. A, H&E stained section showing atypical epithelioid cells with abundant eosinophilic cytoplasm arranged in glands and embedded in a desmoplastic stroma. B, PAX8 shows strong, diffuse nuclear positivity within the neoplastic cells. C, INI1 shows high background staining, but demonstrates loss of nuclear expression, similar to the external control, within the tumor cells. D, A recent kidney biopsy performed confirms the absence of nuclear INI1 expression in the primary tumor cells with appropriate internal positive control E, H3K27me3 stain highlighting intact nuclear expression in both neoplastic cells and background stromal cells
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case_images: Fig1
case_images: Biopsy of the liver lesion revealed RMC with immunohistochemical stains showing strong expression of PAX8 (Fig. 1A-B). INI-1 was noted to be lost, though there was high background staining (Fig. 1C). INI-1 staining was performed on a later kidney biopsy which showed clearcut evidence of loss of protein expression (Fig. 1D). Furthermore, though not meeting quality control threshold, RNA-seq data revealed a SMARCB1::SREBF2-ASI fusion in both the liver and brain specimens with identical breakpoints (left breakpoint: chr22:23803408:+, right breakpoint: chr22:41833358:-) (Supplemental Fig. 1). This metastatic lesion, which harbored morphologic features classic for RMC, showed retained expression of H3K27me3 (Fig. 1E). Given the unusual nature of this case, we performed comprehensive immunofluorescence using CODEX as previously described to gain a better understanding of the biology of this rare brain metastasis and to assess its evolution from the prior liver metastasis (Supplemental Figs. 2-5 and Tables 1 and 2). More broadly, the tumor cells in the cerebellar metastasis showed focal expression for PanCK (3.5%) and CD56 (13.9%), with a small fraction expressing synaptophysin (1.8%) (Supplemental Fig. 4 and Table 1). In contrast, while similar studies performed on the liver metastasis also showed that the tumor cells expressed PAX8, the percentage of cells expressing other renal markers was markedly higher than in the brain lesion with CA9 expression measured at 77.0% and CD10 expression at 29.5% (Supplemental Fig. 2 and Table 1). Of interest, only a small percentage of tumor cells expressed PD-L1 (< 1%) whereas 11.4% of the immune cells showed expression (Supplemental Fig. 4 and Table 1). PD-L1 expression was higher in the liver metastasis tumor cells vis-a-vis the cerebellar tumor cells and was estimated at 20% (Supplemental Fig. 3 and Table 1).
case_images: Morphologic appearance of the cerebellar lesion following H&E staining. The undifferentiated tumor shows variable histologic features including A, adenoid structures, B, large atypical cells with prominent nucleoli and perinuclear clearing, C, loosely arranged cells in a myxoid matrix, D, densely cellular areas with high mitotic activity, E, focal clusters of epithelioid cells with abundant eosinophilic cytoplasm, and F, rare cells with abundant eosinophilic cytoplasm imparting a vaguely rhabdoid appearance
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case_images: Fig2
case_images: In some areas, the tumor cells were embedded in a myxoid matrix with some foci showing adenoid-type features (Fig. 2A), some demonstrating large, atypical cells with prominent nucleoli and slight perinuclear clearing (Fig. 2B), and others more loosely arranged (Fig. 2C). Scattered regions illustrated more cellular tumor with conspicuous mitotic activity (Fig. 2D). Although the majority of the tumor demonstrated an undifferentiated appearance, focal regions of clusters of more epithelioid-appearing cells with eosinophilic cytoplasm with cell dropout were noted (Fig. 2E) and some areas with a vaguely rhabdoid appearance were also seen (Fig. 2F). Given the unusual nature of this case, we performed comprehensive immunofluorescence using CODEX as previously described to gain a better understanding of the biology of this rare brain metastasis and to assess its evolution from the prior liver metastasis (Supplemental Figs. 2-5 and Tables 1 and 2). In contrast, while similar studies performed on the liver metastasis also showed that the tumor cells expressed PAX8, the percentage of cells expressing other renal markers was markedly higher than in the brain lesion with CA9 expression measured at 77.0% and CD10 expression at 29.5% (Supplemental Fig. 2 and Table 1). M2 macrophages were the predominant phenotype and were mostly found in regions of desmoplasia and high microvessel density (Supplemental Fig. 3 and Table 2). M1 macrophages were mostly seen in the dense regions of the tumor. Overall, the liver metastasis exhibited lower proliferation rate by Ki-67 (38.3% versus 51.8%), higher density of both CD8 + T lymphocytes (495.5 cells per mm3 versus 93.7 cells per mm3) and CD4 + FOXP3 + T lymphocytes (37.4 cells per mm3 versus 9.5 cells per mm3), as well as higher tumor cell diversity when compared with the brain metastasis (Supplemental Fig. 3A, 4 A and Table 2).
case_images: Immunohistochemical studies performed on the cerebellar lesion. A, PAX8 is largely negative within the undifferentiated tumor cells but shows nuclear positivity in the regions with epithelioid morphology. B, GFAP shows diffuse positivity within the tumor. C, Cam5.2 highlights the epithelioid structures. D, INI1 expression is lost within the tumor cells with internal positive control showing retained expression in background non-neoplastic tissue. E, H3K27M stain is negative. F, H3K27me3 stain shows loss of nuclear expression within the tumor cells with internal positive control demonstrating intact expression within the non-neoplastic cells
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case_images: Fig3
case_images: Immunohistochemical stains showed the tumor cells were overwhelmingly negative for PAX8; however, the scant clusters of epithelioid cells appeared strongly PAX8 positive (Fig. 3A). GFAP was diffusely positive in the tumor cells and Cam5.2 highlighted scattered cells, including the epithelioid foci (Fig. 3B, C). INI1 expression was lost within the tumor cells with appropriate internal positive control (Fig. 3D). A stain for a mutant histone protein, H3K27M, was negative in the tumor cells (Fig. 3E); however, expression of H3K27me3 was lost in the vast majority of the tumor with satisfactory internal positive control (Fig. 3F). Given the unusual nature of this case, we performed comprehensive immunofluorescence using CODEX as previously described to gain a better understanding of the biology of this rare brain metastasis and to assess its evolution from the prior liver metastasis (Supplemental Figs. 2-5 and Tables 1 and 2). Notably, immunohistochemistry for PAX8 was mostly negative (Fig. 3A) suggesting that the signal amplification from the fluorescence-based detection in CODEX was able to detect lower levels of PAX8 expression in the cerebellar metastasis that were not visible using conventional immunohistochemical techniques. M2 macrophages were the predominant phenotype and were mostly found in regions of desmoplasia and high microvessel density (Supplemental Fig. 3 and Table 2). M1 macrophages were mostly seen in the dense regions of the tumor. PD-L1 expression was higher in the liver metastasis tumor cells vis-a-vis the cerebellar tumor cells and was estimated at 20% (Supplemental Fig. 3 and Table 1). Overall, the liver metastasis exhibited lower proliferation rate by Ki-67 (38.3% versus 51.8%), higher density of both CD8 + T lymphocytes (495.5 cells per mm3 versus 93.7 cells per mm3) and CD4 + FOXP3 + T lymphocytes (37.4 cells per mm3 versus 9.5 cells per mm3), as well as higher tumor cell diversity when compared with the brain metastasis (Supplemental Fig. 3A, 4 A and Table 2).
case_images: Molecular sequencing of the RMC liver and brain metastases. A, Detailed copy number alteration overview of the brain metastasis sample showed partial loss of one copy of chromosome 22 including the SMARCB1 gene encoding INI1. B, RNA expression of INI1 in each of the RMC liver and brain metastasis samples (denoted by the dashed red line in each panel) compared with the distribution of INI1 RNA expression in glioblastoma and clear cell renal cell carcinoma (KIRC) samples from the Cancer Genome Atlas (TCGA). C, PPP2R5E::KLC1 fusion identified in both liver and brain metastasis. Annotation was performed using the AGFusion package. D, Integrative genome viewer (IGV) showing that the PPP2R5E::KLC1 fusion fully corresponds with nucleotide precision in both the liver and brain metastasis samples
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case_images: Fig4
case_images: Both tumors showed downregulation of INI1 (Fig. 4A-B), as expected given the INI1 loss observed by immunohistochemistry in both tumors. Additionally, both tumors demonstrated the presence of a unique fusion, PPP2R5E::KLC1 (Fig. 4C-D). The left breakpoint for PPP2R5E was found in chr14:63453689 per the Homo sapiens genome assembly GRCh38 (hg38), which is within the coding sequence (CDS) of exon 2 (per NM_001282181.3), whereas the right breakpoint for KLC1 was in chr14:103654564 which is the 5' untranslated region (5'UTR) (Fig. 4C). Therefore, the detection of this identical, likely passenger, molecular alteration in both liver and brain tumor tissues strongly suggested a common origin from the same primary lesion (Fig. 4D). Given the unusual nature of this case, we performed comprehensive immunofluorescence using CODEX as previously described to gain a better understanding of the biology of this rare brain metastasis and to assess its evolution from the prior liver metastasis (Supplemental Figs. 2-5 and Tables 1 and 2). These assays revealed the tumor cells in the cerebellar lesion showed expression of PAX8 along with focal expression of CA9 (23.7%) and CD10 (4.4%) (Supplemental Fig. 4). More broadly, the tumor cells in the cerebellar metastasis showed focal expression for PanCK (3.5%) and CD56 (13.9%), with a small fraction expressing synaptophysin (1.8%) (Supplemental Fig. 4 and Table 1). Of interest, only a small percentage of tumor cells expressed PD-L1 (< 1%) whereas 11.4% of the immune cells showed expression (Supplemental Fig. 4 and Table 1). Overall, the liver metastasis exhibited lower proliferation rate by Ki-67 (38.3% versus 51.8%), higher density of both CD8 + T lymphocytes (495.5 cells per mm3 versus 93.7 cells per mm3) and CD4 + FOXP3 + T lymphocytes (37.4 cells per mm3 versus 9.5 cells per mm3), as well as higher tumor cell diversity when compared with the brain metastasis (Supplemental Fig. 3A, 4 A and Table 2).
case_images: Principal component analysis (PCA) plots of RNA-seq expression in the liver and brain metastasis samples compared with gene expression from the Cancer Genome Atlas (TCGA) of clear cell renal cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP), sarcoma, astrocytoma, glioblastoma and oligodendroglioma samples
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case_images: Fig5
case_images: This analysis placed the liver tumor closest to the kidney cancer samples whereas the brain tumor was closest to glioblastoma samples (Fig. 5). Given the unusual nature of this case, we performed comprehensive immunofluorescence using CODEX as previously described to gain a better understanding of the biology of this rare brain metastasis and to assess its evolution from the prior liver metastasis (Supplemental Figs. 2-5 and Tables 1 and 2). Assessment of the tumor microenvironment of the brain metastasis revealed a mixture of inflammatory cells with a concentration of both M1 and M2 macrophages clustering both at the border of the tumor as well as within the tumor proper (Supplemental Fig. 5).