INCREASE OF CD4+ CELLS AS A POTENTIAL BIOMARKER TO PREDICT THE DEVELOPMENT OF HEPATOCELULLAR CARCINOMA IN PATIENTS WITH NON-ALCOHOLIC STEATOHEPATITIS.

RODRIGUEZ EM; SIMON PETER; CALAFAT, P; KURPIS M; BOONSTRA, ANDRE; VOGEL, ARNDT; BALDERRAMO DC; ROMAGNOLI, PA

San Luis

Congreso; Reunion Anual Sociedad Argentina de Inmunologia 2023; 2023

Sociedad Argentina de Inmunologia

Hepatocellular carcinoma (HCC) is the fourth-leading cause of cancer deaths worldwide. This high mortality rate, observed particularly in low-income countries, is related to late diagnosis. On the other hand, the fastest growing cause of HCC is the Non-Alcoholic Fatty Liver Disease (NAFLD) that is found in 25% of the world population and encompasses a spectrum of diseases including Non-Alcoholic Steatohepatitis (NASH) characterized by steatosis, inflammation, hepatocellular damage, and fibrosis that increase the risk of developing HCC. Despite the growing evidence that innate and adaptive immune mechanisms are critical components in the NAFLD progression, the composition of the immune microenvironment in the NASH liver preceding HCC is still unclear. Objective:To study the phenotype and spatial disposition of immune cells infiltrating the liver of NASH patients to identify biomarkers that can predict the development of HCC. Methods: NASH patients were retrospectively selected by having or not subsequently developed HCC according to clinical and pathological reports from Hospital Privado (Argentina), Hospital Universitario Santa Fe Bogota (Colombia), and Erasmus MC (The Netherlands) and two groups were defined: NASH pre-HCC (n=11) and NASH control biopsies (n=13). Formalin-fixed paraffin-embedded (FFPE) non-tumoral liver biopsies were obtained from each patient and a multiplex immunohistochemistry (IHC) was performed to stain for CD4, CD8, PD1, PD-L1, FoxP3, and CXCR6 markers using the Opal 7-Color Manual IHC Kit. The slides were scanned using the multispectral scanner Phenoimager Fusion (Akoya Biosciences) and the images were analyzed using the InForm Advanced Image Analysis Software. The images were obtained and quantitatively analyzed selecting 5 to 10 areas within each tissue where inflammatory infiltrate was observed. Images were spectrally unmixed and each cell was segmented and phenotyped to get individual cell data. ResultsThe phenotypic analysis from a total of 291908 different immune cells using markers such as CD8+, CD8+ CXCR6+, CD4+ FoxP3+ (Tregs), and CD4+ PD1+ showed that the cell density (cells/mm2) was not significantly different between groups. Interestingly, CD4+ cell density was higher in NASH pre-HCC patients compared to the NASH control group (p=0.039). We also performed a Nearest-neighbor analysis to measure the distance between the different cell phenotypes within the tissue but we did not observe differences. Conclusion Multiplex immunohistochemistry allows the study in depth the composition and spatial distribution of immune cells within liver biopsies of NASH patients that precede HCC development. In this study, we detected the presence of an inflammatory infiltrate with a higher number of CD4+ cells in patients with NASH who later developed HCC. This finding could have an impact on clinical decision-making and could help to reduce the number of patients who evolve to HCC by predicting their risk of cancer development.