Preoperative Immune Cells Infiltration in the Tumor Microenvironment as a Prediction and Prognostic Factor for Postoperative Recurrence in Hepatocellular Carcinoma

Abstract

Background: Surgical resection remains the mainstay for the treatment of hepatocellular carcinoma (HCC). However, the postoperative recurrence considerably undermines the prognosis of patients receiving hepatectomy. The tumor immune microenvironment (TIME) of HCC has been shown to play a vital rule during the progression of HCC, but if and how the preestablished TIME could influence the postoperative recurrence of HCC is not yet elucidated clearly.

Method: We profiled the immune infiltration of three bulk RNA sequencing datasets and tissue array using ssGSEA and multiplex Immunohistochemistry. The immune populations associated with recurrence were identified by Kaplan-Meier (KM) and COX analysis. We classified patients into immune hot and cold subtypes with consensus clustering, which was validated by five independent immune infiltration algorithms. Based on the immune cluster, a recurrence related immune gene (RRIG) signature was constructed and its correlation with antitumor immunity was identified. The RRIG was additionally characterized by single cell RNA sequencing.

Result: The abundance of CD8 T cell and T helper cell in preoperative TIME was associated with longer recurrence free survival (RFS). Patients classified into the 'immune cold' subtype were prone to postoperative early recurrence with shorter RFS. The RRIG signature derived from immune cluster not only served as an independent risk factor for HCC recurrence, but also corelated with immune infiltration, immune cluster and better responses towards immunotherapy. Among the RRIG, we identified FYN as novel biomarkers for anti-tumor immunity and postoperative HCC recurrence. FYN are downregulated in HCC and recurrent patients and demonstrates specific expression on NK cells and CD8+ T cells as revealed by single cell transcriptome sequencing and in-house tissue array. The FYN expression in CD8+ cells was proved to be an independent prognostic marker.ConclusionPatients with 'immune cold' primary tumors are prone to postoperative recurrence. CD8+ cells-expressing FYN was identified as a novel biomarker correlating with robust anti-cancer immunity and the prognosis of HCC patients.

Interpretation: This work for the first time proved and validated that preestablished immune microenvironment in primary tumor, rather than the para-tumor liver tissue, has prognostic values in predicting the onset of postoperative HCC recurrence.

Funding Information: This research was partially supported by the Research Council of the University of Hong Kong (project codes: 104004092 and 104004460), the Wong's donation (project code: 200006276), a donation from the Gaia Family Trust of New Zealand (project code: 200007008), the Research Grants Committee (RGC) of Hong Kong, HKSAR (Project Codes: 740608, 766211, 17152116 and 17121419), the Health and Medical Research Fund (Project code: 15162961 and 16172751), the Enhanced new staff start up fund (Project code: 204610519) and the Pre-emptive retention fund (Project code: 202007002).

Declaration of Interests: Authors declare no conflict of interests.

Ethics Approval Statement: This study protocol was reviewed and approved by the Ethics Committee of Shanghai Outdo Biotech Company, approval number YB M-05-02. All patients gave written informed consent according to Outdo Biotech.

Keywords: Hepatocellular carcinoma, Postoperative Recurrence, Immune Microenvironment, Immunotherapy, FYN