*Purpose: Peritumoral neutrophil (PMN) infiltration has been associated with poor clinical outcomes in various tumors; however, its role in hepatocellular carcinoma (HCC) remains understudied. Moreover, novel methods to analyze tumor microenvironments are needed in HCC, an immune responsive tumor. We sought to use quantitative multiplex immunofluorescence (qmIF) in HCC to better study the immune milieu and detect cellular predictors of clinical outcomes.

*Methods: Adult patients without hepatitis viral infection and with available clinical follow up from a single center cohort of 645 consecutive patients who underwent liver transplantation (LT) for HCC between 1998 and 2018 were included in a pilot analysis. The expression of MPO (PMNs), CD3 (T cells), CD8 (CTL), CD68 (Macrophages), Hep-Par1 (Hepatocytes) and HLA-DR was assessed on each tissue sample using qmIF. Tumor sections were pre-selected by a pathologist and visualized using Vectra (Akoya). Using an artificial intelligence-capable software (Inform, Akoya) and RStudio, cell densities were calculated by dividing over the total number of nucleated cells as defined by DAPI staining (Nuclei).

*Results: A preliminary cohort of 18 patient samples was stained and tissue integrity allowed for data to be interpreted in 13 patients, 5 of whom recurred at a median of 2.4 years while the other 8 remained recurrence-free at a median of 12 years post-LT. The etiologies of liver disease were comparable among the two groups and included: NASH (6 patients), Alcoholic liver disease (3 patients), cryptogenic cirrhosis (2 patients), autoimmune (1 patient) and primary biliary cirrhosis (1 patient). We found that MPO+ PMN densities are significantly higher in patients who suffered from post-LT HCC recurrence as compared to those who remain recurrence-free, and that this difference is primarily driven by PMNs located within the peritumoral stroma. (Median [IQR] 2.46 [1.99 – 2.92] vs 1.23 [0.723 -1.78], p=0.019). Moreover, CD3 lymphocytes intratumoral and peritumoral densities were not correlated with recurrence, nor was the tissue-derived neutrophil-to-lymphocyte ratio.

*Conclusions: Higher densities of peritumoral PMNs are associated with post-LT HCC recurrence in this preliminary cohort of 13 patients. This work, the first reported application of qmIF in HCC, is ongoing and will be validated within a larger cohort of patients.

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