Comprehensive immune profiling and analysis of significance of S100A8-positive immune cells in breast cancer = 유방암 조직의 포괄적인 면역 프로파일링과 S100A8 양성 면역세포의 의의 분석

다국어 초록 (Multilingual Abstract)

Background: Immune microenvironment differs according to hormone receptor (HR) status or tumor progression stage. Its importance is ever-increasing as drugs targeting tumor immunity came up as a game changer in cancer therapeutics. Myeloid-derived suppressor cells (MDSCs) are one of the key players in tumor immunity but not well studied in human tissue because of its phenotypic complexity. They are known to promote tumor progression via various immune-mediated and non-immune-mediated mechanisms.
Methods: Differential expression of immune-related genes was evaluated via comprehensive immune profiling according to unsupervised clustering and HR status, and a target gene with significant fold change, possibly MDSC-associated gene, was searched for further analysis. The difference in its expression was validated using tissue microarray of 700 cases of human breast cancer. Its clinicopathological significance and relationship between other tumor infiltrating lymphocytes (TILs) including CD4+, CD8+ and FOXP3+ TILs or PD-L1+ immune cells (ICs) were investigated.
Results: In immune profiling analysis, S100A8, a known MDSC marker, showed the most striking fold change in the cluster 2 in unsupervised clustering and HR status dependent grouping. In immunohistochemistry (IHC), S100A8 was stained in both tumor cells and ICs in breast cancer samples. Infiltration of S100A8+ ICs was associated with aggressive clinicopathological features and poor clinical outcome. They were already present in pre-invasive stage of breast cancer and were associated with increased infiltration of CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs. Prognostic impact of S100A8+ ICs was stronger in HR-positive, PD-L1+ IC-negative, CD8+ TIL-low and FOXP3+ TIL-low subgroups. When stained with various IHC markers, S100A8+ ICs were mostly revealed as CD33+ CD15- CD163+ cells which are thought to be monocytic-MDSCs derived macrophages.
Conclusion: S100A8 is a key molecule that differentiates distinct immune clusters and also highly expressed in HR-negative breast cancers. S100A8 is usually expressed in monocytic-MDSCs derived macrophages and contributes to breast cancer progression from pre-invasive stage. S100A8+ ICs were associated with increase in CD4+, CD8+, and FOXP3+ TILs and PD-L1+ ICs but prognostic impact of S100A8+ ICs was stronger in less immunogenic condition.

국문 초록 (Abstract)

서론: 유방암의 면역 미세환경은 호르몬 수용체 발현이나 종양의 진행 단계에 따라 다양한 모습을 보인다. 종양 면역을 대상으로 하는 면역치료제의 효과가 입증됨으로서 유방암 면역에 대한 연구의 중요성은 날로 커지고 있다. 골수유래 면역억제세포는 종양 면역에 중요한 기능을 담당하고 있으나 그 표현형이 복잡하기 때문에 인체 조직에서 연구가 어려운 분야이다. 이 세포들은 면역과 관련된 기전 뿐만 아니라 면역과 관련되지 않은 기전을 통하여서도 다양하게 암의 진행을 촉진시킨다.
방법: 포괄적인 면역 프로파일링을 이용하여 비지도 군집분석과 호르몬 수용체 발현에 따른 면역 관련 유전자들의 발현 차이를 평가하였다. 추가 분석을 위하여 유의미한 발현량 차이를 보이는 유전자로 나타난 골수유래 면역억제세포 관련 유전자를 선택하였다. 700명의 유방암 환자 조직으로 만들어진 조직 마이크로어레이에서 해당 단백질 발현의 차이를 확인하였다. 이 단백질 발현이 가지는 임상병리학적 중요성과 다른 종양 침윤 림프구 (CD4, CD8, FOXP3 양성 림프구), PD-L1 양성 면역세포와의 관계를 분석하였다.
결과: 면역 프로파일링 자료를 비지도 군집분석에서 확인된 ‘군집 2’와 다른 군집들의 비교 및 호르몬 수용체 발현에 따라서 분석한 결과 골수유래 면역억제세포 표지자로 알려진 S100A8이 가장 큰 발현량 차이를 보였다. 면역조직화학염색 결과 S100A8은 유방암 조직의 종양세포와 면역세포 모두에서 염색되었다. 특히 S100A8 양성 면역세포의 침윤은 나쁜 임상병리학적 특성, 나쁜 예후와 관련이 있었다. 이들은 유방암의 침윤 전 단계부터 존재하였고 CD4, CD8, FOXP3 양성 종양 침윤 림프구, PD-L1 양성 면역세포 침윤의 증가와 연관을 보였다. S100A8 양성 면역세포의 예후적 중요성은 호르몬 수용체 양성인 경우, PD-L1 양성 면역세포가 없는 경우, CD8, FOXP3 양성 림프구가 적은 경우에 더 두드러지게 나타났다. 다양한 면역조직화학검사 결과 S100A8 양성 면역세포는 대부분 CD33 양성, CD15 음성, CD163 양성 표현형을 보여 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포로 생각되었다.
결론: S100A8은 특정 면역 군집을 구분하는 중요한 단백이며 또한 호르몬 음성 유방암에서 증가하였다. 인간 유방암 조직에서 S100A8은 주로 단핵구 계열 골수유래 면역억제세포에서 기원한 대식세포에서 발현되며 침윤 전 단계부터 존재하여 유방암의 진행을 촉진하는 역할을 할 것으로 생각된다. S100A8 양성 면역세포는 CD4, CD8, FOXP3 양성 종양 침윤 림프구와 PD-L1 양성 면역세포의 증가와 연관이 있었으나 그 예후적 중요성은 면역성이 낮은 조건에서 더 두드러지게 나타났다.

목차 (Table of Contents)

• Abstract i
• Table of Contents iii
• List of Tables iv
• List of Figures vi
• List of Supplementary Tables viii
• List of Abbreviations ix
• Introduction 1
• Materials and Methods 4
• Results 11
• Discussion 42
• Conclusion 50
• Bibliography 55
• Abstract in Korean 60

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