PMC8217650_01
Case 1 (A) H&E image, showing metaplastic carcinoma with chondroid differentiation (B) PD-L1 by the Ventana PD-L1 SP263 assay (C-F). Radiographic changes in Case 1 from (C) week 0, (D) 12 weeks, (E) 16 weeks, and (F) 24 weeks. Images C1 to F1 showing regression of the dominant right lung mass, then regrowth. Images C2 to F2 showing growth of an initially non-target left lung nodule. Images (G-I) show RNA expression heatmaps with modified z-scores of expression vs. non-metaplastic TNBC cases in pathways of interest for metaplastic breast cancer (G) RAS-MEK-ERK, (H) PI3K-AKT-mTOR (I) TP53. Genes with DNA mutations are outlined in red.
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Imaging at 12 weeks showed an overall partial response, though with mixed findings showing significant shrinkage of her dominant tumor, but enlargement of a left lung lesion ( Figures 1C-F ). In a comparison of RNA expression, there were no marked differences in expression within the TP53 or the RAS/MRK/ERK pathways, but PDK1 appeared less expressed within the PI3K pathway compared to other cases ( Figures 1G-I ).
PMC8217650_02
Case 2 (A) H&E image, showing metaplastic squamous carcinoma (B) PD-L1 by the Ventana PD-L1 SP263 assay (C-E). Radiographic changes from (C) week 0, (D) 12 weeks, and (E) 24 weeks. A mixed, but overall partial response by RECIST criteria is noted initially (D) followed by progression (E). Images (F-H) show RNA expression heatmaps with modified z-scores of expression vs. non-metaplastic TNBC cases in pathways of interest for metaplastic breast cancer (F) RAS-MEK-ERK, (G) PI3K-AKT-mTOR (H) TP53. Genes with DNA mutations are outlined in red.
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Follow up CT scans at 12 weeks showed a partial response, with an overall shrinking of multiple lung nodules, while also noting growth of other smaller nodules ( Figures 2C-E ). Higher expression was seen within the RAS/MEK/ERK pathway ( Figures 2F-H ).
Immune cell counts by mIF. Total immune cell counts for metaplastic Cases 2, 4, and 5 plotted with non-metaplastic (cases identified as 'NM') TNBC from the same clinical trial in a violin plot. No clear difference is noted between the metaplastic cases and non-metaplastic cases (A) Helper T-cells (B) Cytotoxic T-cells (C) Macrophages (D) Regulatory T-cells.
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CD3+ Helper T-cell counts were near median values, with both CD8+ Cytotoxic T-cell and CD163+ Macrophages lower than non-metaplastic cases ( Table 3 ,  Figure 6 ).
PMC8217650_03
Case 3 (A) H&E image, showing mixed metaplastic squamous carcinoma and pleomorphic invasive lobular carcinoma (B) PD-L1 by the Ventana PD-L1 SP263 assay (C-E). Lesions at baseline (C) initially appeared worsened at 4 weeks (D), then demonstrated a complete clinical response by week 14 (E).
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At 1 month follow up she had worsening skin lesions, but nivolumab was continued with the possibility of a flare reaction causing the exam findings rather than disease progression. 2 months into treatment skin lesions appeared to be crusting over, and at 4 months appeared to have a complete response ( Figures 3C-E ).
PMC8217650_04
Case 4 (A) H&E image, showing metaplastic squamous carcinoma (B) PD-L1 by the Ventana PD-L1 SP263 assay. Images (C-E) show RNA expression heatmaps with modified z-scores of expression vs. non-metaplastic TNBC cases in pathways of interest for metaplastic breast cancer (C) RAS-MEK-ERK, (D) PI3K-AKT-mTOR (E) TP53. Genes with DNA mutations are outlined in red.
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Strong expression was seen in the RAS/MRK/ERK pathway, particularly of JUN and FOS ( Figures 4C-E ).
Immune cell counts by mIF. Total immune cell counts for metaplastic Cases 2, 4, and 5 plotted with non-metaplastic (cases identified as 'NM') TNBC from the same clinical trial in a violin plot. No clear difference is noted between the metaplastic cases and non-metaplastic cases (A) Helper T-cells (B) Cytotoxic T-cells (C) Macrophages (D) Regulatory T-cells.
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CD3+ Helper T-cell counts were near median values, with both CD8+ Cytotoxic T-cell and CD163+ Macrophages lower than non-metaplastic cases ( Table 3 ,  Figure 6 ).
PMC8217650_05
Case 5 (A) H&E image, showing metaplastic squamous carcinoma (B) PD-L1 by the Ventana PD-L1 SP263 assay. Images (C-E) show RNA expression heatmaps with modified z-scores of expression vs. non-metaplastic TNBC cases in pathways of interest for metaplastic breast cancer (C) RAS-MEK-ERK, (D) PI3K-AKT-mTOR (E) TP53. Genes with DNA mutations are outlined in red.
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In the PI3K pathway, PIK3CA and PIK3R1 were relatively lower, but no pattern of reduced expression was noted in the rest of the pathway ( Figures 5C-E ).
Immune cell counts by mIF. Total immune cell counts for metaplastic Cases 2, 4, and 5 plotted with non-metaplastic (cases identified as 'NM') TNBC from the same clinical trial in a violin plot. No clear difference is noted between the metaplastic cases and non-metaplastic cases (A) Helper T-cells (B) Cytotoxic T-cells (C) Macrophages (D) Regulatory T-cells.
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CD3+ Helper T-cell counts were near median values, with both CD8+ Cytotoxic T-cell and CD163+ Macrophages lower than non-metaplastic cases ( Table 3 ,  Figure 6 ).