Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer

Abstract Background The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic...

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Main Authors: Katherine Sanchez, Isaac Kim, Brie Chun, Joanna Pucilowska, William L. Redmond, Walter J. Urba, Maritza Martel, Yaping Wu, Mary Campbell, Zhaoyu Sun, Gary Grunkemeier, Shu Ching Chang, Brady Bernard, David B. Page
Format: Article
Language:English
Published: BMC 2021-01-01
Series:Breast Cancer Research
Subjects:
Online Access:https://doi.org/10.1186/s13058-020-01378-4
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spelling doaj-15073214387d495795317299a2e6cda42021-04-02T19:56:35ZengBMCBreast Cancer Research1465-542X2021-01-0123111510.1186/s13058-020-01378-4Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancerKatherine Sanchez0Isaac Kim1Brie Chun2Joanna Pucilowska3William L. Redmond4Walter J. Urba5Maritza Martel6Yaping Wu7Mary Campbell8Zhaoyu Sun9Gary Grunkemeier10Shu Ching Chang11Brady Bernard12David B. Page13Earle A. Chiles Research InstituteEarle A. Chiles Research InstituteEarle A. Chiles Research InstituteEarle A. Chiles Research InstituteEarle A. Chiles Research InstituteEarle A. Chiles Research InstituteDepartment of Pathology, Providence Portland Medical CenterEarle A. Chiles Research InstituteDepartment of Pathology, Providence Portland Medical CenterEarle A. Chiles Research InstituteMedical Data Research Center, Providence Health & ServicesMedical Data Research Center, Providence Health & ServicesEarle A. Chiles Research InstituteEarle A. Chiles Research InstituteAbstract Background The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents. Methods Serial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power. Results mIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p < .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25➔n = 13) to detect the observed increases in sTIL/PD-L1. Conclusion mIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials. Trial registration NCT02950259 .https://doi.org/10.1186/s13058-020-01378-4ImmunotherapyIRX-2Multiplex immunofluorescenceEarly-stage breast cancerPD-L1sTIL
collection DOAJ
language English
format Article
sources DOAJ
author Katherine Sanchez
Isaac Kim
Brie Chun
Joanna Pucilowska
William L. Redmond
Walter J. Urba
Maritza Martel
Yaping Wu
Mary Campbell
Zhaoyu Sun
Gary Grunkemeier
Shu Ching Chang
Brady Bernard
David B. Page
spellingShingle Katherine Sanchez
Isaac Kim
Brie Chun
Joanna Pucilowska
William L. Redmond
Walter J. Urba
Maritza Martel
Yaping Wu
Mary Campbell
Zhaoyu Sun
Gary Grunkemeier
Shu Ching Chang
Brady Bernard
David B. Page
Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
Breast Cancer Research
Immunotherapy
IRX-2
Multiplex immunofluorescence
Early-stage breast cancer
PD-L1
sTIL
author_facet Katherine Sanchez
Isaac Kim
Brie Chun
Joanna Pucilowska
William L. Redmond
Walter J. Urba
Maritza Martel
Yaping Wu
Mary Campbell
Zhaoyu Sun
Gary Grunkemeier
Shu Ching Chang
Brady Bernard
David B. Page
author_sort Katherine Sanchez
title Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
title_short Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
title_full Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
title_fullStr Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
title_full_unstemmed Multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and PD-L1 in early-stage breast cancer
title_sort multiplex immunofluorescence to measure dynamic changes in tumor-infiltrating lymphocytes and pd-l1 in early-stage breast cancer
publisher BMC
series Breast Cancer Research
issn 1465-542X
publishDate 2021-01-01
description Abstract Background The H&E stromal tumor-infiltrating lymphocyte (sTIL) score and programmed death ligand 1 (PD-L1) SP142 immunohistochemistry assay are prognostic and predictive in early-stage breast cancer, but are operator-dependent and may have insufficient precision to characterize dynamic changes in sTILs/PD-L1 in the context of clinical research. We illustrate how multiplex immunofluorescence (mIF) combined with statistical modeling can be used to precisely estimate dynamic changes in sTIL score, PD-L1 expression, and other immune variables from a single paraffin-embedded slide, thus enabling comprehensive characterization of activity of novel immunotherapy agents. Methods Serial tissue was obtained from a recent clinical trial evaluating loco-regional cytokine delivery as a strategy to promote immune cell infiltration and activation in breast tumors. Pre-treatment biopsies and post-treatment tumor resections were analyzed by mIF (PerkinElmer Vectra) using an antibody panel that characterized tumor cells (cytokeratin-positive), immune cells (CD3, CD8, CD163, FoxP3), and PD-L1 expression. mIF estimates of sTIL score and PD-L1 expression were compared to the H&E/SP142 clinical assays. Hierarchical linear modeling was utilized to compare pre- and post-treatment immune cell expression, account for correlation of time-dependent measurement, variation across high-powered magnification views within each subject, and variation between subjects. Simulation methods (Monte Carlo, bootstrapping) were used to evaluate the impact of model and tissue sample size on statistical power. Results mIF estimates of sTIL and PD-L1 expression were strongly correlated with their respective clinical assays (p < .001). Hierarchical linear modeling resulted in more precise estimates of treatment-related increases in sTIL, PD-L1, and other metrics such as CD8+ tumor nest infiltration. Statistical precision was dependent on adequate tissue sampling, with at least 15 high-powered fields recommended per specimen. Compared to conventional t-testing of means, hierarchical linear modeling was associated with substantial reductions in enrollment size required (n = 25➔n = 13) to detect the observed increases in sTIL/PD-L1. Conclusion mIF is useful for quantifying treatment-related dynamic changes in sTILs/PD-L1 and is concordant with clinical assays, but with greater precision. Hierarchical linear modeling can mitigate the effects of intratumoral heterogeneity on immune cell count estimations, allowing for more efficient detection of treatment-related pharmocodynamic effects in the context of clinical trials. Trial registration NCT02950259 .
topic Immunotherapy
IRX-2
Multiplex immunofluorescence
Early-stage breast cancer
PD-L1
sTIL
url https://doi.org/10.1186/s13058-020-01378-4
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