TY - JOUR
T1 - Impact of Region-of-Interest Size on Immune Profiling Using Multiplex Immunofluorescence Tyramide Signal Amplification for Paraffin-Embedded Tumor Tissues
AU - Sun, Baohua
AU - Laberiano-Fernández, Caddie
AU - Salazar-Alejo, Ruth
AU - Zhang, Jiexin
AU - Rendon, Jose Luis Solorzano
AU - Lee, Jack
AU - Soto, Luisa Maren Solis
AU - Wistuba, Ignacio Ivan
AU - Parra, Edwin Roger
N1 - Funding Information:
The pathology laboratory from the Translational Molecular Pathology Department, thanks their members, Mei Jiang, Barbara Mino, Wei Lu, Auriole Tamegnon, Ou Shi, Saxon Rodriguez, Khaja Khan, Steven Powell, Heladio Iburgen, Jianling Zhou, Salome A McAllen, and Lakshmi Kakarala, who contribute daily to quality mIF and IHC staining. We thank the pathologist team from the digital pathology laboratory at Translational Molecular Pathology Department that works with image analysis and our data analysts, Renganayaki Krishna Pandurengan and Shanyu Zhang. Editorial support was provided by Tamara Locke and Ann Sutton from the Research Medical Library at The University of Texas MD Anderson Cancer Center.
Funding Information:
This study was supported in part by the scientific and financial support for the CIMAC-CIDC Network provided through the National Cancer Institute (NCI) Cooperative Agreement U24CA224285 of the MD Anderson Cancer Center CIMAC and for the Translational Molecular Pathology Immunoprofiling Laboratory, National Institutes of Health/NCI through Cancer Center Support Grant P30CA016672 (used the Institutional Tissue Bank), and SPORE Grant 5P50CA070907-18, by the Cancer Prevention and Research Institute of Texas through MIRA RP160668.
Publisher Copyright:
© 2023 S. Karger AG. All rights reserved.
PY - 2023/1/1
Y1 - 2023/1/1
N2 - Introduction: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. Methods: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. Results: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. Conclusion: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.
AB - Introduction: Representative regions of interest (ROIs) analysis from the whole slide images (WSI) are currently being used to study immune markers by multiplex immunofluorescence (mIF) and single immunohistochemistry (IHC). However, the amount of area needed to be analyzed to be representative of the entire tumor in a WSI has not been defined. Methods: We labeled tumor-associated immune cells by mIF and single IHC in separate cohorts of non-small cell lung cancer (NSCLC) samples and we analyzed them as whole tumor area as well as using different number of ROIs to know how much area will be need to represent the entire tumor area. Results: For mIF using the InForm software and ROI of 0.33 mm2 each, we observed that the cell density data from five randomly selected ROIs is enough to achieve, in 90% of our samples, more than 0.9 of Spearman correlation coefficient and for single IHC using ScanScope tool box from Aperio and ROIs of 1 mm2 each, we found that the correlation value of more than 0.9 was achieved using 5 ROIs in a similar cohort. Additionally, we also observed that each cell phenotype in mIF influence differently the correlation between the areas analyzed by the ROIs and the WSI. Tumor tissue with high intratumor epithelial and immune cells phenotype, quality, and spatial distribution heterogeneity need more area analyzed to represent better the whole tumor area. Conclusion: We found that at minimum 1.65 mm2 area is enough to represent the entire tumor areas in most of our NSCLC samples using mIF.
KW - Immunohistochemistry
KW - Multiplex immunofluorescence
KW - Regions of interest
KW - Spearman correlation coefficient
KW - Whole slide images
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U2 - 10.1159/000523751
DO - 10.1159/000523751
M3 - Article
C2 - 35609532
AN - SCOPUS:85131320161
SN - 1015-2008
VL - 90
SP - 1
EP - 12
JO - Pathobiology
JF - Pathobiology
IS - 1
ER -