194P - Achieving Reproducible Maturation Staging of Tertiary Lymphoid Structures: from Imaging Mass Cytometry Data to Pathology Applications

Background

In cases of persistent inflammation, the migration of immune cells follow the organogenesis of secondary lymphoid organs (SLO) within the organs, leading to the formation of tertiary lymphoid structures (TLS). These structures have become important as diagnostic and prognostic markers in chronic diseases and cancers. However, there is ongoing debate regarding how to assess the maturation of TLS in tissue samples, both in terms of determining the stages themselves and achieving agreement among pathologists. Based on the composition and organization of TLS revealed through imaging mass cytometry (IMC), we aimed to propose a reproducible classification system consisting of three maturation stages for TLS, which can be applied in pathology practice.

Methods

We used formalin-fixed and paraffin-embedded gastric samples (45 tissues with 134 TLS) and colonic samples (50 tissues with 159 TLS), representing inflammatory, cancerous, and control conditions. These samples were subjected to IMC analysis using a panel of 39 markers. Additionally, we sought correlations between markers that reflect the maturation of TLS (composition and architecture). The two IMC-markers with the strongest correlation with TLS maturation were selected to transfer the TLS maturation staging from IMC to dual-staining immunohistochemistry (IHC). This transfer was done to investigate the level of agreement among pathologists when classifying TLS using a set of 60 tissue IHC images.

Results

Using IMC, we were able to characterize the maturation of TLS and propose three distinct stages: Early TLS, Primary Follicle-Like TLS, and Secondary Follicle-Like TLS, identified as IHC CD21-CD23-, CD21+CD23-, and CD21+CD23+, respectively. The analysis of the 60 dual CD21-CD23 IHC images by three pathologists showed a high level of inter-pathologist agreement in classifying TLS into the three maturation stages (Cohen’s Kappa values: > 0.8).

Conclusions

With a strong correlation to cellular and architectural changes during TLS maturation, the dual CD21-CD23 IHC marker allows for the staging of TLS in tissue samples with a high level of inter-pathologist agreement, which holds potential for prognostic and predictive purposes.

Legal entity responsible for the study

M. Le Rochais.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.