## 5 Biomarkers of Response and/or Resistance to Immune Checkpoint Inhibitors
### Circulating Markers of Response and/or Resistance  
In order to have a broad application in routine clinical practice, biomarkers should be assessed as less invasively as possible. Non-invasive biomarkers could be extremely useful for diagnostic or prognostic purposes. Therefore, the identification of whole blood-derived or serum-derived biomarkers through liquid biopsy is of great interest. In different tumor types, several blood-derived markers have been associated with the clinical outcomesof ICI treatment. In particular, total lymphocyte count, monocyte count, T cell clonality, neutrophil-to-lymphocyte ratio (NLR), relative eosinophil count, circulating Treg cell levels, circulating monocytes or MDSCs, cytokine levels (IL-6, IL-8 and IL-10), and lactate dehydrogenase (LDH) activity have been evaluated as potential biomarkers [16; 120; 121; 122]. Other potential predictive biomarkers associated with ICIs response may be identified through genomic analyses in blood-derived circulating-free DNA (cfDNA) [121]. In a study including 69 patients with 23 different cancer types (including HCC), the number of mutations detected in cfDNA was positively associated with ICIs' response and survival [123]. However, limited data on cfDNA-derived biomarkers are currently available in patients with HCC receiving ICIs. ICIs are monoclonal antibodies that can be recognized as non-self by the immune system, thus triggering the production of antidrug antibodies (ADAs). As for other malignancies, the ADAs relevance in HCC patients has aroused great interest. ADAs, reducing the drug bioavailability, decrease anti-tumor activity with detrimental effects on clinical outcomes. Therefore, the development of ADAs represents a potential mechanism for primary and acquired resistance. The probability of developing ADAs varies according the specific type of the drug administered, ranging from 54% with atezolizumab, to 10% with nivolumab, or 1.5% with pembrolizumab [3]. In the IMbrave150 trial, 28% of patients developed ADAs during treatment and less clinical benefit from the combination atezo+beva was demonstrated in ADAs positive compared to ADAs negative patients after 6 weeks of treatment (HR 0.93, 95% CI, 0.57-1.53, vs. HR 0.39, 95% CI, 0.26-0.60, respectively). This is probably due to a higher clearance of the drug in patients who developed ADAs [3]. In the HIMALAYA trial, among patients under immunotherapy, 1.7% developed neutralizing ADAs against durvalumab and 4.4% against tremelimumab [9]. However, the impact of ADAs development on outcomes was not reported. In a prospective cohort study enrolling 174 patients with HCC treated with atezo+beva (61 in the discovery cohort and 113 in the validation cohort), those with a robust ADA response (\(\geq\)1000 ng/mL) after 3 weeks of therapy had a worse PFS and OS compared to the counterpart [70]. Moreover, compared with patients with low ADA levels, patients with high ADAs showed reduced serum atezolizumab concentration, impaired CD8+ T cell proliferation and had a decreased IFN\(\gamma\) and TNF\(\alpha\) production from CD8+ T cell. All these data would confirm the potential role of ADAs as predictors of resistance to immunotherapy. AFP is the most widely used circulating biomarker to define HCC aggressiveness. Its measurement has been studied as a tool for HCC early detection as well as to predict prognosis and response to locoregional and systemic therapies [124]. Baseline serum AFP may help in identifying those patients who will benefit most from molecular target therapies such as the VEGFR inhibitor ramucirumab, which demonstrated its effectiveness only in patients with AFP \(\geq\) 400 ng/mL [125]. An AFP level drop during immunotherapy (the \"AFP response\") has been investigated as a potential prognostic predictor. However, in different studies, the time point for AFP measurement during treatment (i.e., ranging from 1 week to 3 months) and the extent of AFP response to be considered relevant (i.e., a reduction of 20-50% compared with the baseline value) were chosen arbitrarily [126].