Early CTLA4 increase in CD45+ blood cells: an emerging biomarker of atezolizumab–bevacizumab resistance and worse survival in advanced hepatocarcinoma

Background: Advanced hepatocellular carcinoma (HCC) has a dismal prognosis; however, the introduction of atezolizumabebevacizumab combination has improved overall survival and novel immune checkpoint inhibitors are entering the clinics. Despite more therapeutic options being available, no biomarker guides treatment choice. Indeed, tissue-based analyses and complex analytical procedures hinder clinical translation. We explored the informativeness of a simple, non-invasive, repeatable cytofluorimetric assay on peripheral blood to predict response and survival in HCC patients treated with atezolizumabebevacizumab. Materials and methods: Twenty-five cirrhotic patients, 50 HCC patients undergoing atezolizumabebevacizumab and an independent validation cohort of 25 HCC patients were subjected to a cytofluorimetric study of peripheral white blood cells (WBCs) to assess baseline programmed death-ligand 1-positive (PD-L1þ) and cytotoxic T-lymphocyte antigen 4positive (CTLA4þ) cell percentage in the different populations and their early on-treatment variations. Immunophenotypes were evaluated against treatment response. RNA sequencing followed by RTePCR validation were used to elucidate the molecular correlates of immunophenotypic observations. Results: PD-L1þ cell percentage did not predict response either at baseline or when evaluating treatment-induced early changes. Conversely, the percentage of CTLA4þ lymphocytes at baseline showed a predictive significance (35.37 in responders versus 31.5 in non-responders, P ¼ 0.03). More interestingly, the early CTLA4þ changes during treatment in lymphocytes (responders 0.95 versus non-responders 1.08, P ¼ 0.05), monocytes (responders 0.95 versus non-responders 1.04, P ¼ 0.03), granulocytes (responders 0.94 versus non-responders 1.14, P ¼ 0.001) and, even stronger, the early CTLA4þ percentage change in the whole WBCs displayed a predictive significance in terms of time to progression (TTP) (P < 0.0001) and overall survival (OS) (P ¼ 0.005). The immunophenotypic findings correlated with transcriptional modulation of CTLA4 target genes and genes involved in immune response. Conclusions: A repeatable, easy, non-invasive blood test predicts response to immunotherapy in patients with HCC, both in terms of TTP and OS. CTLA4þ cell percentage increase in non-responders suggests a possible resistance mechanism which deserves attention as a druggable target. Key words: immunotherapy, CTLA4, PD-L1, biomarkers