Orion™: 14-plex clinical sample imaging of liver cancer modalities using one-step staining and imaging

Multiplexed protein imaging and the H&E that pathology reads are normally taken from two different sections; this AACR 2024 poster works one FFPE liver section carrying metastatic adenocarcinoma through a 14-plex immunofluorescence stain applied in a single round and then an H&E, imaging both modalities on the Orion instrument.

Presented by RareCyte.

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• Both imaging modalities come off one section: a 14-plex immunofluorescence stain in a single round, then H&E on that same slide. The FFPE liver section was de-paraffinized, antigen-retrieved and autofluorescence-quenched, stained with a panel of ArgoFluor-conjugated antibodies in one staining process, imaged whole-slide at 20X on the Orion instrument in one imaging round and written out as OME-TIFF; it was then de-coverslipped in aqueous solution, H&E-stained and imaged again in brightfield on that same instrument. The 14 stained channels are Hoechst, Albumin, CD31, CD4, CD68, CD3e, CD8a, CD163, CD20, FOLR2, Ki-67, Pan-CK, PD-1 and PD-L1, with tissue autofluorescence isolated as an additional channel on top of those 14.
• The section is liver; the tumor in it is not liver cancer. The lesion is described as metastatic moderately-differentiated adenocarcinoma with a high Ki-67 proliferation index and central dirty necrosis, morphologically consistent with a primary tumor from the colon, and the H&E shows liver parenchyma extensively replaced by metastatic tumor nodules, with normal liver cells surrounded by lymphocytic infiltrate and neoplastic glands surrounded by necrotic areas infiltrated by histiocytes. The panel is built for that question: the tissue's own autofluorescence picks out the hepatocytes (grey) still standing, Pan-CK (cyan) outlines the neoplastic glands and Ki-67 (magenta) marks the metastatic epithelial cells that are proliferating. This is one section shown as a demonstration — no cohort and no cell counts are reported, RareCyte instruments are for research use, and while H&E is a clinical stain read on a patient sample, the poster makes no diagnostic claim.
• The immune microenvironment sorts into 3 regions, and the macrophage phenotype flips between them. The histopathological pattern is divided into 3 categories: tumor with dirty necrosis, whose glands hold mainly CD68+CD163- macrophage aggregates and rare T lymphocytes; desmoplastic stroma, densely populated with the opposite CD68-CD163+ macrophages plus scattered CD4+ helper and CD8+ cytotoxic T cells and very rare B cells; and the invasive front, predominantly aggregates of CD4+ T helper cells with high PD-1 expression. In one region CD4 positive cells co-express PD-1 while FOLR2 positive macrophages co-express PD-L1, and FOLR2/CD68/CD163 triple-positive macrophages resolve alongside FOLR2-negative double-positive and CD68 single-positive ones. The functional roles the poster attributes to these cells are its authors' reading of one stained section: the images show where the cells sit, not what they do.