Abstract

Emerging data indicate that genomic alterations can shape immune cell composition in early breast cancer. However, there is a need for complementary imaging and sequencing methods for the quantitative assessment of combined somatic copy number alteration (SCNA) and immune profiling in pathological samples. Here, we tested the feasibility of three approaches—CUTseq, for high-throughput low-input SCNA profiling, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) for quantitative immuno-profiling- in archival formalin-fixed paraffin-embedded (FFPE) tissue samples from patients enrolled in the randomized SBG-2004-1 phase II trial. CUTseq was able to reproducibly identify amplification and deletion events with a resolution of 100 kb using only 6 ng of DNA extracted from FFPE tissue and pooling together 77 samples into the same sequencing library. In the same samples, mfIHC revealed that CD4 + T-cells and CD68 + macrophages were the most abundant immune cells and they mostly expressed PD-L1 and PD-1. Combined analysis showed that the SCNA burden was inversely associated with lymphocytic infiltration. Our results set the basis for further applications of CUTseq, mfIHC and DIA to larger cohorts of early breast cancer patients.

Highlights

  • The substantial proportion of patients with breast cancer (BC) that do not respond to immunotherapy by checkpoint inhibition underscores the importance of identifying reliable predictive biomarkers

  • The only prospectively validated marker remains Programmed Death Ligand 1 (PD-L1) protein expression, which predicts benefit to immunotherapy in metastatic triple-negative BC, albeit with contradictory results depending on the chemotherapy backbone[1,2]

  • We sought to demonstrate the applicability of CUTseq, multiplexed fluorescent immunohistochemistry (mfIHC) and digital-image analysis (DIA) to formalin-fixed paraffin-embedded (FFPE) BC tissue sections from patients enrolled in the Scandinavian Breast Group (SBG) 2004-1 phase II early BC trial and to portray immuno-genomic correlates detected in these samples

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Summary

1234567890():,; INTRODUCTION

The substantial proportion of patients with breast cancer (BC) that do not respond to immunotherapy by checkpoint inhibition underscores the importance of identifying reliable predictive biomarkers. Other factors that describe or determine tumor-host interactions such as tumor-infiltrating lymphocytes (TILs), gene expression immune signatures and tumor mutational burden (TMB) represent markers of response to chemotherapy[4,5,6,7], with emerging data supporting prediction of benefit from immunotherapy[8,9]. In parallel to sequencing technologies for profiling aneuploidy/SCNAs, emerging automated digital imaging analysis (DIA) technologies, such as multiplexed fluorescent immunohistochemistry (mfIHC) and automated TIL enumeration allow robust identification and quantitation of multiple immune markers even in FFPE tumor tissue sections[17]. We sought to demonstrate the applicability of CUTseq, mfIHC and DIA to FFPE BC tissue sections from patients enrolled in the Scandinavian Breast Group (SBG) 2004-1 phase II early BC trial and to portray immuno-genomic correlates detected in these samples

RESULTS
DISCUSSION
Zerdes et al 5
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CODE AVAILABILITY

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