Abstract PD5-10: Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort
Authors: Szekely B , Bareche Y , Van den Eynden G , Salgado R , Buisseret L , Garaud S , Willard-Gallo K , Hatzis C , Szasz M , Kulka J , Larsimont D , Sotiriou C , Pusztai L , Desmedt C 
Published: May 2019

Abstract
Introduction
While immune infiltrates have already been extensively characterized in primary tumors (P), data on breast cancer metastases (M) remains limited. To this end we quantified and qualified the immune cells in a unique cohort of multiple matched P and M samples selected from an institutional breast cancer autopsy cohort.
Patients and methods
Twenty-three patients were selected from an institutional autopsy program (Semmelweis University, Budapest, Hungary) based on matched P and M sample availability (124 samples). All samples were centrally characterized for estrogen (ER), progesterone (PR) and HER2 receptors. The primary molecular subtypes were as follows: 9 ER+/PR+/HER2-, 8 triple negative and 6 HER2+. Ten patients relapsed ≤1 year after diagnosis and were further referred to as “early relapsers”, as opposed to the remaining qualified as “late relapsers”. Immunohistochemistry (IHC) was carried out against CD3/CD20 and CD4/CD8 in 21 patients (119 samples). Tumor infiltrating lymphocytes (TILs) were assessed on hematoxylin and eosin (H&E) and CD3-stained slides. Gene expression data were generated using the NanoString nCounter assay (PanCancer Immune Profiling Panel) for 11 patients (35 samples) and analyzedusing the R package NanoStringQCPro. The scores from published immune gene signatures were calculated as a weighted sum of the expressions of their genes. All samples were analyzed for 22 immune cell subtypes relative abundance using CIBERSORT.
Results
TILs assessed on H&E and CD3-stained slides were weakly correlated (Rho= 0.38, p&lt;.001). TIL levels as well as the number of tertiary lymphoid structures (TLS) were significantly lower in Ms as compared to Ps (p&lt;.001). Among the different metastatic sites, the lung was more infiltrated when considering CD3+ and CD4+ cells (p=.01 and .02, respectively). We further observed significantly higher levels of TILs, CD3+, CD4+ and CD8+ cells in the Ms but not in the Ps from late relapsers as opposed to those from early relapsers. Gene expression analyses further confirmed these observations as several immune gene signatures displayed significantly higher scores in the Ms from late compared to early relapsers. An unsupervised analysis identified 13 genes significantly differentially expressed between Ps and Ms: CSF1R, CXCL14, CYBB, IL21R, IL2RB, TNF and TNFSF15 were upregulated in Ps while BCL2L1, C7, HSD11B1, and PSMB7 were upregulated in Ms. The matched P/M CIBERSORT analyses revealed a distinct composition of immune cell types between P and M of a same patient. Apart from a potential increase in M0 macrophages, no common trait was observed in immune cell composition between the Ms from the different patients.
Conclusion
This is to the best of our knowledge the first study characterizing the immune infiltration in patients with multiple matched P and M samples. The results suggest that Ms have not only a globally lower immune infiltration as compared to Ps, but also a different immune composition. Additionally, Ms from late relapsers are more infiltrated as compared to early relapsers. The present data also uncovers not only important inter-patient but also intra-patient immune heterogeneity, which should be taken into consideration for optimal treatment decision.
Citation Format: Szekely B, Bareche Y, Van den Eynden G, Salgado R, Buisseret L, Garaud S, Willard-Gallo K, Hatzis C, Szasz M, Kulka J, Larsimont D, Sotiriou C, Pusztai L, Desmedt C. Immune characterization of matched primary and multiple metastatic samples issued from an institutional autopsy cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-10.