multiplexed-fluorescent-immunohistochemical-staining-imaging-analysis

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

National University of Singapore

The molecular biology of primary nodal T- and NK-cell lymphoma and its relationship with extranodal NK/T-cell lymphoma, nasal type is poorly understood. In this study, we assessed the relationship between nodal and extranodal Epstein-Barr virus-positive T/NK-cell lymphomas using gene expression profiling and copy number aberration analyses. We performed gene expression profiling and copy number aberration analysis on 66 cases of Epstein-Barr virus-associated T/NK-cell lymphoma from nodal and extranodal sites, and correlated the molecular signatures with clinicopathological features. Three distinct molecular clusters were identified with one enriched for nodal presentation and loss of 14q11.2 (TCRA loci). T/NK-cell lymphomas with a nodal presentation (nodal-group) were significantly associated with older age, lack of nasal involvement, and T-cell lineage compared to those with an extranodal presentation (extranodal-group). On multivariate analysis, nodal presentation was an independent factor associated with short survival. Comparing the molecular signatures of the nodal and extranodal groups it was seen that the former was characterized by upregulation of PD-L1 and T-cell-related genes, including CD2 and CD8, and downregulation of CD56, consistent with the CD8/CD56-immunophenotype. PD-L1 and CD2 protein expression levels were validated using multiplexed immunofluorescence. Interestingly, nodal group lymphomas were associated with 14q11.2 loss which correlated with loss of TCR loci and T-cell origin. Overall, our results suggest that T/NK-cell lymphoma with nodal presentation is distinct and deserves to be classified separately from T/NK-cell lymphoma with extranodal presentation. Upregulation of PD-L1 indicates that it may be possible to use anti-PD1 immunotherapy in this distinctive entity. In addition, loss of 14q11.2 may be a potentially useful diagnostic marker of T-cell lineage.

Epstein-Barr virus-associated primary nodal T/NK-cell lymphoma shows a distinct molecular signature and copy number changes.

Immunohistochemistry (IHC) provides clinically useful information on protein expression in cancer cells. However, quantification of colocalizing signals using conventional IHC and visual scores is challenging. Here we describe the application of quantitative immunofluorescence in angioimmunoblastic T-cell lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity that impedes IHC interpretation and quantification. A multiplexed immunofluorescence (IF) panel comprising T- and B-lymphocyte markers along with T-follicular helper (T) markers was validated for appropriate cellular localization in sections of benign tonsillar tissue and tested in two samples of AITL, using a Vectra microscope for spectral imaging and InForm software for analysis. We measured the percentage positivity of the T markers, BCL6 and PD1, in AITL CD4-positive cells to be approximately 26% and 45%, with 12% coexpressing both markers. The pattern is similar to CD4 cells within the germinal center of normal tonsils and clearly distinct from extragerminal CD4 cells. This study demonstrates the feasibility of automated and quantitative imaging of a multiplexed panel of cellular markers in formalin-fixed, paraffin-embedded tissue sections of a cellularly heterogenous lymphoma. Multiplexed IF allows the simultaneous scoring of markers in malignant and immune cell populations and could potentially increase accuracy for establishment of diagnostic thresholds.

Quantitative Analysis of a Multiplexed Immunofluorescence Panel in T-Cell Lymphoma.

DNA alterations have been extensively reported in multiple myeloma (MM); however, they cannot yet fully explain all the biological and molecular abnormalities in MM, which remains to this day an incurable disease with eventual emergence of refractory disease. Recent years have seen abnormalities at the RNA levels being reported to possess potential biological relevance in cancers. ADAR1-mediated A-to-I editing is an important posttranscriptional mechanism in human physiology, and the biological implication of its abnormality, especially at the global level, is underexplored in MM. In this study, we define the biological implications of A-to-I editing and how it contributes to MM pathogenesis. Here, we identified that the MM transcriptome is aberrantly hyperedited because of the overexpression of ADAR1. These events were associated with patients' survival independent of 1q21 amplifications and could affect patients' responsiveness to different treatment regimes. Our functional assays established ADAR1 to be oncogenic, driving cellular growth and proliferation in an editing-dependent manner. In addition, we identified NEIL1 (base-excision repair gene) as an essential and a ubiquitously edited ADAR1 target in MM. The recoded NEIL1 protein showed defective oxidative damage repair capacity and loss-of-function properties. Collectively, our data demonstrated that ADAR1-mediated A-to-I editing is both clinically and biologically relevant in MM. These data unraveled novel insights into MM molecular pathogenesis at the global RNA level.

Shuangyi Fan

Department of Pathology,
Yong Loo Lin School of Medicine,
Department of Pathology, Yong Loo Lin School of Medicine

National University of Singapore

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Shuangyi Fan

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pathology,Yong Loo Lin School of Medicine,Department of Pathology, Yong Loo Lin School of Medicine

National University of Singapore

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Department of Pathology,
Yong Loo Lin School of Medicine,
Department of Pathology, Yong Loo Lin School of Medicine