PMC8570369_01
Histopathology, timeline, and radiologic findings. (A) Hematoxylin and eosin staining (left panel) followed by molecular characterization of patient breast carcinoma (MIB2, mindbomb E3 ubiquitin protein ligase 2; ER, estrogen receptor; PR, progesterone receptor; HER2, Erb-b2 receptor tyrosine kinase 2). The carcinoma was rated as E-cadherein and MIB2 expressing, ER/PR negative, and HER2 positive. (B) Timeline of diagnosis, symptoms, imaging, electrodiagnostic studies, and treatment. Imaging corresponding to 27 weeks is indicated in the first subpanel one of (C). (C) MRI of the spine without gadolinium at 27 months showed a dorsally predominant T2 hyperintense signal spanning T5-T10. At 33 months there was some improvement with the T2 hyperintense signal now spanning T7-T10 which was not contrast enhancing (additional views and brain MRI in Supplementary Figure 1). All times are relative to the initial diagnosis of cancer, neurological symptoms began 2 months after the cancer diagnosis.
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Pathology had demonstrated an aggressive neoplasm with high nuclear grade and extensive perineoplastic lymphocytic infiltration [Figure 1A, WHO 2019 classification "Invasive carcinoma of no special type," T2N0, human epidermal growth factor receptor (HER2) positive, estrogen receptor, and progesterone receptor negative]. Five months after her diagnosis, she underwent bilateral mastectomy, demonstrating no residual disease (Figure 1B). When treatment frequency was reduced to every 3 months, her symptoms worsened, prompting return to the monthly IVIg (Figure 1B). Repeat imaging of the brain, and first-time MRI of the cervical and thoracic spine without gadolinium were performed 27 months after her cancer diagnosis (25 months after onset of neurological symptoms), revealing a longitudinally extensive, dorsally predominant T2 hyperintensity from T5 to T10 (Figure 1C, Supplementary Figure 1).
Identification of anti-CDR2L and anti-CDR2 antibodies and PNM CSF tissue-based assay. (A) Dot plot of individual PhIP-seq-identified peptides that were enriched in both technical replicates at least 10-fold above reference samples (R1 and R2, replicates 1 and 2, respectively). The most enriched peptide in each replicate mapped to CDR2L (blue points). A CDR2 peptide (yellow) was also identified. PhIP-Seq data for all other enriched peptides (black) are available in Supplementary Table 2. (B) CDR2L and CDR2 proteins and relevant epitopes. The intensity of red shading is proportional to the enrichment of that peptide by PNM CSF and the rpK are in red above each of the 3 detected CDR2L peptides and the single enriched CDR2 peptide. The dotted regions (3 rightmost) indicate the peptides that were enriched by PCD CSF whereby density of the dots represents the degree of enrichment. The immunogens for CDR2L and CDR2 commercial antibodies used for immunostaining are indicated by horizontal lines. A sequence alignment for the first 49 amino acids for CDR2L and CDR2 is shown [green, identical amino acids (AA); yellow, chemically similar AA; orange, minimally similar AA; and red, dissimilar AA]. The sequence alignment for the first 49 amino acids of human, monkey, and murine CDR2L are shown. Red rectangles indicate AA that differ from human. (C) Epifluorescent and confocal imaging of murine brain tissue immunostained with PNM CSF at 1:4 or PCD CSF at 1:25. cb, cerebellum; CA3, cornu Ammonis 3 of the hippocampus. Scale bars = 1,000 mum for whole brain sagittal images, 50 mum for epifluorescent hippocampal and cerebellar images, and 10 mum for confocal images. (D) Epifluorescent images immunostaining of human cerebellum by PNM and PCD CSF at 1:4 and 1:25, respectively. Arrows indicate Purkinje cells (PCs). In the upper left set of images, PNM CSF failed to immunostain CDR2L immunostained PCs (unfilled arrows). On the lower right, anti-CDR2 failed to immunostain PCs immunostained by PCD CSF (unfilled arrows). The asterisk indicates a blood vessel immunostained by anti-CDR2. Scale bars = 50 mum.
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Unexpectedly, the N-terminal peptide for CDR2L, the major antigen in anti-Yo paraneopalstic syndrome, was the most enriched peptide by PhIP-Seq in both technical replicates (Figure 2A). Two additional CDR2L peptides were also detected but less enriched, as was the N-terminal peptide of CDR2 (Figure 2B). Peptides to other known paraneoplastic autoantigens were not enriched (Figure 2A and Supplementary Table 2). As a positive control, we used CSF from a previously reported case of clinically diagnosed anti-Yo paraneoplastic cerebellar degeneration (PCD CSF) that enriched multiple CDR2L peptides by PhIP-Seq [patient 03, ] (Figure 2B). At a 1:25 dilution, control PCD CSF robustly immunostained cerebellar Purkinje cells as well as sparse cerebral and hippocampal neurons (Figure 2C and Supplementary Figure 2). In contrast, CSF from our patient with paraneoplastic myelopathy (PNM CSF) did not immunostain Purkinje cells when performed in technical triplicate and assessed by epifluorescence microscopy at a 1:4 dilution, consistent with the negative clinical testing (Figure 2C). Likewise, PNM CSF did not immunostain murine Purkinje cells even after antigen retrieval (Figure 2C). However, in one replicate PNM CSF did immunostain hippocampal neurons in a pattern similar to PCD CSF, albeit more sparsely (Figure 2C). We further characterized immunopositive replicates by confocal microscopy and found that immunostained hippocampal neurons were morphologically similar to those immunostained by PCD CSF, and that previously undetectable Purkinje cells were also faintly immunostained (Figure 2C). In contrast, hippocampal CDR2 expression was limited to arteriolar smooth muscle cells, consistent with CDR2 expression as annotated in the Human Protein Atlas (Supplementary Figure 2; http://www.proteinatlas.org). Mouse and human CDR2L are 93% similar by amino acid sequence, however some of the sequence differences are within the peptide that was most enriched by PNM CSF by PhIP-Seq (Figure 2B). As with mouse, PNM CSF at a 1:4 dilution did not appreciably immunostain human Purkinje cells while PCD CSF was strongly reactive at a 1:25 dilution and colocalized with CDR2L but not CDR2 (Figure 2D).
Validation of anti-Yo antibodies in PNM CSF. (A) HEK293T cells were transfected with FIP1L1-FLAG, CDR2L-FLAG, or CDR2-FLAG. Lysates were separated by SDS-PAGE and resulting immunoblots were probed with PNM CSF (green, 1:250) and counterstained with an anti-Human IgG 800CW secondary antibody (green). The membrane was the probed with anti-FLAG and anti-rabbit 680RD secondary antibody (red). PNM CSF IgG recognized recombinant CDR2L and CDR2 but not FIP1L1. (B) HEK293T cells were transfected with CDR2-FLAG and immunostained with PNM CSF at 1:10, PCD CSF at 1:50 (both green) and an anti-FLAG antibody (red). Scale bars are 10 mum. (C) HEK293T cells were transfected with CDR2L-FLAG and immunostained with PNM CSF at 1:10, PCD CSF at 1:50 (both green) and an anti-FLAG antibody (red). Scale bars are 10 mum. (D) 3,3'-Diaminobenzidine (DAB) immunohistochemistry of serial sections of the patient's breast carcinoma tissue for CDR2L (left) and HER2 (right). Black arrowheads highlight a corresponding area of DAB staining on serial sections. Scale bars = 200 mum. (E) Dual chromophore immunohistochemistry for CDR2L (purple HRP, horse radish peroxidase) and HER (yellow AP, alkaline phosphatase) on control cerebellum (top), control HER2 breast tissue (middle), and the patient's breast carcinoma (bottom). The purple arrowhead indicates a CDR2L+/HER2- Purkinje cell in the top panel. The yellow arrowheads indicate CDR2L-/HER2+ cells in control breast carcinoma (middle) and the patient's breast carcinoma (bottom). The brick red arrow indicates CDR2L+/HER2+ cells in the patient's breast carcinoma (bottom). Red results from the co-localization of the yellow and purple chromogens. Scale bars = 100 mum.
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PNM CSF at a 1:250 dilution and a commercial anti-FLAG antibody. PNM CSF IgG bound CDR2L and CDR2, but not untransfected HEK293T cell lysate or FIL1LP1-FLAG (end point dilution of 1:1,000 for PNM CSF not shown) (Figure 3A). CBA. HEK 293T cells were mock transfected, transfected with CDR2L-FLAG, or transfected with CDR2-FLAG, and immunostained with either PNM CSF (1:10) or PCD CSF as a positive control (1:50). Consistent with our immunoblot results, both PNM and PCD CSF immunostained CDR2L and CDR2-overexpressing HEK 293T CBAs (end point dilution of 1:100 for PNM CSF not shown) (Figures 3B,C). CDR2L is expressed in up to 100% of ovarian carcinomas from patients with anti-Yo PCD and is overexpressed relative to control ovarian carcinoma tissue. Consistent with these molecular associations, we found that CDR2L and HER2 were expressed in the same anatomic regions when comparing serial sections of the patient's breast carcinoma tissue (Figure 3D). We found that CDR2L expression in our patient's breast carcinoma colocalized with, and was restricted to, HER2+ carcinoma cells (Figure 3E).
PNM CSF, PCDF CSF, CDR2L and CDR2 immunostaining of spinal cord. (A) Cervical (cerv), thoracolumbar (thor/lum), sacrococcygeal (sac/cocc) murine spinal cord was immunostained with PNM CSF (1:4) or PCD CSF (1:25). The rostral (r) and caudal (c) ends are on the right and left of each image, respectively. PNM CSF did not immunostain murine spinal tissue above background (asterisk = tissue artifact also seen in the negative control). PCD CSF immunostained cells along the longitudinal axis of the spinal cord (filled arrows). The red rectangle indicates the approximate region of images shown in (B). Scale bars = 2,000 mum. (B) Coimmunostaining of murine spinal cord with PCD CSF (1:25) and anti-CDR2L or anti-CDR2. PCD CSF and anti-CDR2L immunostained the cytoplasm of large cells and colocalized with cytoplasmic anti=CDR2L immunostaining (white arrowheads). However, anti-CDR2L also immunostained nuclei (filled red arrows) but PCD CSF did not (unfilled red arrowheads). Scale bars = 200 mum. (C) Coimmunostaining of human spinal cord with PCD CSF. PNM CSF failed to immunostain human spinal cord (not shown). PCD CSF immunostained sparse cells that were also CDR2L positive (top row, white arrowheads). In the lower row, PCD CSF cells are costained by anti-CDR2L (white arrowheads). In the lower row, only some cells immunostained by PCD CSF were also immunostained by anti-CDR2 (filled arrow heads, co-stained cells; unfilled, PCD CSF exclusive cells). Scale bars = 20 mum.
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In contrast, PCD CSF immunostained large cells along the longitudinal axis of the murine spinal cord (Figure 4A). In contrast, CDR2L immunostaining was both cytoplasmic and nuclear; however, all PCD CSF-immunostained cells were co-stained with CDR2L and vice versa (Figure 4B). In contrast, the anti-CDR2 antibody was poorly immunoreactive to neural cells but again strongly stained arteriolar smooth muscle cells (Figure 4B). In the human spinal cord, PCD CSF immunostaining also colocalized with anti-CDR2L more so than with anti-CDR2 immunostaining (Figure 4C).