Combining Immunohistochemistry and Gene Expression Analysis to Refine Scoring

Description

Assessing Tumor-Infiltrating Lymphocytes in Breast Cancer: Combining Immunohistochemistry and Gene Expression Analysis to Refine Scoring

Hanne Locy1, Stefaan Verhulst2, Kirsten De Ridder1, Magali Verdonck1, Wilfried Cools3, Wim Waelput4, Stefanie Brock4, Louise Cras4, Ann Schiettecatte5, Jan Jonckheere5, Annick Luppens5, Claudia Housen5, Amina Harraq5, Carlo Heirman7, Leo A. van Grunsven2, Marina Cools7, Marian Vanhoeij6, Kris Thielemans1, Karine Breckpot1

1 Laboratory for Molecular and Cellular Therapy, Department of Biomedical Sciences, Vrije Universiteit Brussel, Laarbeeklaan 103/E, B-1090 Brussels, Belgium, Karine Breckpot, [email protected], +32-2-477 45 66.
2 Liver Cell Biology Research Group, Department of Biomedical Sciences, Vrije Universiteit Brussel, Laarbeeklaan 103/D, B-1090 Brussels, Belgium.
3 Interfaculty Center Data processing and Statistics, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
4 Department of Anatomo-Pathology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
5 Department of Radiology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
6 Department of Surgery, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium.
7 eTheRNA immunotherapies, Galileilaan 19, B-2845 Niel, Belgium.


ABSTRACT:
Scoring tumor-infiltrating lymphocytes (TILs) in breast cancer has gained increasing attention, as TILs have prognostic value in several breast cancer types. We evaluated the intra- and inter-rater variability when scoring TILs by visual inspection of hematoxylin and eosin stained tissue sections. We further addressed whether immunohistochemistry (IHC), staining sections for immune cell surface markers CD45, CD3, CD4 and CD8, and nanoString nCounter® gene expression analysis (GEA) could refine TIL-scoring. Formalin-fixed paraffin-embedded and fresh-frozen core needle biopsies of twelve female and treatment naive breast cancer patients were first included. Scoring of TILs was performed twice by three independent pathologists with a wash-out period of three days. Increasing intra- and inter-rater variability was observed with higher TIL-numbers. The highest reproducibility was observed on tissue sections stained for CD3 and CD8. The latter TIL-scores correlated well with the TIL-scores obtained through nanoString nCounter® GEA. The latter also revealed genes that are positively and negatively related to both TIL-scores. These findings indicate that integration of IHC and GEA is a valuable strategy to refine TIL-scoring. We used this workflow to perform a midpoint analysis of breast cancer samples collected in an ongoing phase I clinical trial (NCT03788083). Herein, TriMix mRNA, an immune-activating agent, was injected at escalating doses in tumors of early-stage breast cancer patients (three patients/cohort). Mild but manageable adverse events were observed across all treatment cohorts (incl. placebo). Comparison of pre- and post-treatment samples in IHC showed a significant though subtle increase in CD3+ TILs. GEA corroborated that changes were subtle and indicated a significant change in macrophages, though with little changes on the individual gene level. Lessons learned from this work will help refine the workflow for TIL-analysis in breast cancer samples and will advance the design of immunotherapies purposed to stimulate anti-breast cancer immunity.

Period	20 Sep 2022 → 21 Sep 2022
Event title	7th Global Insight Conference on Breast Cancer (GICBC-2022)
Event type	Conference
Location	Barcelona, Spain
Degree of Recognition	International