CD30 expression is frequently decreased in relapsed classic Hodgkin lymphoma after anti-CD30 CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cells anti-CD30 is an innovative therapeutic option that has been used to treat cases of refractory/relapsed (R/R) classic Hodgkin lymphoma (CHL). Limited data are available regarding the CD30 expression status of patients who relapsed after this therapy. This is the first study to show decreased CD30 expression in R/R CHL in patients (n = 5) who underwent CAR T-cell therapy in our institution between 2018 and 2022. Although conventional immunohistochemical assays showed decreased CD30 expression in neoplastic cells in all cases (8/8) the tyramide amplification assay and RNAScope in situ hybridisation detected CD30 expression at different levels in 100% (n = 8/8) and 75% (n = 3/4), respectively. Hence, our findings document that certain levels of CD30 expression are retained by the neoplastic cells. This is not only of biological interest but also diagnostically important, as detection of CD30 is an essential factor in establishing a diagnosis of CHL.

Exploring the expression of Adenosine Pathway-Related Markers CD73 and CD39 in Colorectal and Pancreatic Carcinomas Characterized by Multiplex Immunofluorescence: A Pilot Study.

INTRODUCTION: Generating high levels of immunosuppressive adenosine in the tumor microenvironment contributes to cancer immune evasion. CD39 and CD73 hydrolyze adenosine triphosphate into adenosine; thus, efforts have been made to target this pathway for cancer immunotherapy. Our objective was optimizing a multiplex immunofluorescence (mIF) panel to explore the role of CD39 and CD73 within the tumor microenvironment. MATERIALS AND METHODS: In three-time points, a small cohort (n=8 ) of colorectal and pancreatic adenocarcinomas were automated staining using an mIF panel against CK, CD3, CD8, CD20, CD39, CD73 and CD68 to compare them with individual markers immunohistochemistry (IHC) for internal panel validation. Densities of immune cells and distances from different tumor-associated immune cells to tumor cells were exploratory assessment and compared with clinicopathologic variables and outcomes. RESULTS: Comparing the three-time points and individual IHC staining results, we demonstrated high reproducibility of the mIF panel. CD39 and CD73 expression was low in malignant cells; the exploratory analysis showed higher densities of CD39 expression by various cells, predominantly stromal cells, followed by T cells, macrophages, and B cells. No expression of CD73 by B cells or macrophages was detected. Distance analysis revealed proximity of cytotoxic T cells, macrophages, and T cells expressing CD39 to malignant cells, suggesting a close regulatory signal driven by this adenosine marker. CONCLUSIONS: We optimized an mIF panel for detection of markers in the adenosine pathway, an emerging clinically relevant pathway. The densities and spatial distribution demonstrated that this pathway may modulate aspects of the tumor immune microenvironment.

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples.

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.