Article Figures & Data
Figures
- Figure 1 Flowchart of the Study
Control cohorts are (i) from in-house HER2-driven control tumors for the RNA-seq, multi-IF, and FISH analyses, (ii) from the COSMIC database19 for the CGHa and the DNA sequencing of CDR2 and CDR2L, or (iii) from a literature-drawn cohort27 for the clinicopathologic comparison. BC = breast cancer; CGHa = comparative genomic hybridization array; FISH = fluorescent in situ hybridization; HER2+ = overexpression of human epidermal growth factor receptor 2; HR− = no overexpression of hormone receptors; PCD = paraneoplastic cerebellar degeneration; w/o = without.
- Figure 2 Genetic, Cytogenetic, Transcriptomic, and Protein Expression of CDR2 and CDR2L in Yo-PCD Breast Cancers
(A) Genetic alterations (mutations and copy number variations) in the CDR2 (in orange) and CDR2L (in blue) locus. Each section of the circle represents 1 Yo-PCD breast cancer (BC); the inner circle shows CDR2L CNV, whereas CDR2 CNV is represented in the intermediate circle. The outer circle gives information on point mutations and fusions on the CDR2 or CDR2L locus. (B.a) Cytogenetic alterations on the CDR2L locus. (B.b) CGHa on Yo-PCD breast tumor: zoom on 17q chromosome holding ERBB2 (HER2) and CDR2L locus amplifications. (B.c) CDR2L-probe FISH in control breast cancer (left) vs Yo-PCD (right). Scale bar: 50 µm. (C) Median boxplots of TPM expression of CRD2L in control breast tumors (left, dark blue) vs Yo-PCD tumors (right, red). CDR2L was found differentially expressed in the RNA-seq analysis. (D.a and D.b) Expression of the CDR2L protein. (D.a) Representative CDR2L IHC staining in control breast tumors (left) vs Yo-PCD tumors (right). Scale bars: 100 µm. (D.b) Semiquantitative evaluation of CDR2L staining intensity in IHC in control breast tumors (left) vs Yo-PCD tumors (right) ranked from 0 for no staining (faded blue) to 3 for cytoplasmic staining with strong intensity (red). Comparison with the χ2 independent test. PCD = paraneoplastic cerebellar degeneration.
- Figure 3 Differentially Expressed Genes and Immune Cell Type Estimates Between Control and Yo-PCD Breast Cancers
(A) Heatmap of the differentially expressed genes (in rows) between Yo-PCD (red) and control (dark blue) samples (in columns). Tumor sites are separated in primary breast cancer (BC; yellow) and metastatic lymph node (light blue). TPM expression values were first transformed into log10(TPM + 0.01) and then transformed into a Z score per gene. Panels B and C show boxplots of the median value and interquartile range (IQR) for the concerned variable of control (on the left, in blue) and Yo-PCD BC samples (on the right, in red); the upper whisker extends from the hinge to the largest value no further than 1.5 × IQR, and the lower whisker extends to the smallest value at most 1.5 × IQR from the hinge; each dot represents the value of a sample. p Values of comparisons between Yo-PCD and control groups using the Wilcoxon rank-sum test are adjusted with the Benjamini and Hochberg method (see eMethods for detail, links.lww.com/NXI/A734) and shown on the top of each couple of boxplots. Yo-PCD samples from metastatic lymph nodes have been removed. (B.a–B.j) Boxplots of MCP-counter estimates for 3 classical cell types in control and Yo-PCD samples. (C.a-b) Boxplots of TPM expression of 2 typical plasma cell markers in control and Yo-PCD samples. PCD = paraneoplastic cerebellar degeneration.
- Figure 4 Multiplex Immunofluorescence and Immune Cell Densities
(A) Representative control and Yo-PCD breast cancer (BC) slides with (from left to right): hematoxylin-phloxine-saffron coloration at low magnification (tumor nests are circled by dotted yellow lines, scale bar: 200 µm), high magnification images illustrating the great density and variety of immune cells infiltrating Yo-PCD BCs as compared to control BCs, and representative multi-immunofluorescence slides of control and Yo-PCD BCs showing the B cells and massive IgG-plasma cells infiltration characterizing Yo-PCD BCs (scale bar: 100 µm). B to D: Represented plots show the median value (top of the gray rectangle) of the concerned variable. Each dot represents the value of a sample. p Values of comparisons between controls and Yo-PCD BCs using the Mann-Whitney method are shown at the top of each panel. B: Absolute counts of T cells/mm2 in controls and Yo-PCD BCs. C: Absolute counts of B cells/mm2 in controls and Yo-PCD BCs. D: Absolute counts of IgG plasma cells/mm2 in controls and Yo-PCD BCs. CT = control tumors; PCD = paraneoplastic cerebellar degeneration.
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