## 5 Biomarkers of Response and/or Resistance to Immune Checkpoint Inhibitors
### Markers of Response and/or Resistance in Tumor Tissue
#### 5.2.2 PD-L1 Expression
[35] in a series of 217 HCC patients showed that tumoral PD-L1 expression was significantly associated with markers of tumor aggressiveness (high serum alpha-fetoprotein [AFP], satellite nodules, macrovascular and microvascular invasion, and poor differentiation). Moreover, high PD-L1 expression on inflammatory cells of the tumor microenvironment correlated with poor prognostic features, such as high AFP levels, macrovascular invasion, poor differentiation, and high PD-1 expression [35]. In 174 patients included in the Check-Mate 040 dose-expansion cohort, PD-L1 expression was associated with a better response to the study drug. In particular, 9/34 patients (26%) with PD-L1 expression on \(\geq\)1% of tumor cells had an objective response to nivolumab compared to 26/140 patients (19%) with PD-L1 expression \(<\)1% [40]. In addition, using fresh and archival tumor samples from dose-escalation and dose-expansion phases of the same study, the median OS was significantly longer in patients with tumor PD-L1 \(\geq\)1% than in those with PD-L1 \(<\)1% (28.1 vs. 16.6 months, \(p=0.03\)) [61]. However, conflicting evidence on the predictive role of PD-L1 expression comes from the phase II KEYNOTE-224 trial, in which pembrolizumab was evaluated in the second-line [41]. In this study the association between the clinical efficacy of treatment and PD-L1 expression, defined as a combined positive score (number of PD-L1 positive tumor or immune cells divided by the total number of viable tumor cells) and as a tumor proportion score (percentage of viable tumor cells with partial or complete staining for PD-L1 relative to all viable cells), was assessed in 52/104 patients. A combined positive score \(>\)1% was associated with higher ORR (\(p=0.021\)) and favorable PFS (\(p=0.026\)), while a tumor proportion score \(>\)1% was not predictive of clinical benefits (\(p=0.088\) and \(p=0.096\), respectively). Moreover, even patients with low or undetectable PD-L1 tumor expression may experience durable clinical benefits when treated with ICIs [105]. Among the reasons potentially explaining these contradictory results on PD-L1 predictivity, the use of different detection assays, inter-assay and/or inter-observer heterogeneity, as well as non-standardized criteria and cut-offs defining positivity, may have played a role [16,106]. Therefore, the evaluation of PD-L1 expression is not a sufficient standalone biomarker for therapeutic decisions in clinic practice. In addition to their expression levels, different isoforms of immune checkpoint inhibitor molecules can also be associated with resistance to immunotherapy. A42 PD-1 is an alternatively spliced isoform of PD-1 lacking a 42-nucleotide region (equivalent to 14 amino acids) in exon 2 [107]. Unlike PD-1, this isoform does not bind to PD-L1 or PD-L2, and is not recognized by PD-1-specific monoclonal antibodies [107]. A42 PD-1 mRNA was found differentially expressed in various immune-related cells (higher expression in monocytes, macrophages and NK cells; lower expression on B cells, CD4\({}^{+}\) or CD8\({}^{+}\) T cells, and dendritic cells) and it could induce the production of several proinflammatory cytokines (TNF\(\alpha\), IL-6, and II-1\(\beta\)) from human peripheral blood mononuclear cells and murine dendritic cells [107]. A recent study evaluated the role of \(\Delta\)42 PD-1 in HCC progression and resistance to nivolumab and pembrolizumab [68]. The authors demonstrated that, compared to PD-1\({}^{+}\) T cells, tumor-infiltrating \(\Delta\)42 PD-1\({}^{+}\) T cells exhibit transcriptomic features of immune exhaustion and correlate positively with HCC severity. In addition, patients treated with anti-PD-1 antibodies (n = 28, nivolumab; n = 5, pembrolizumab) showed effective PD-1 blockade while non-responders displayed increased frequencies of A42 PD-1\({}^{+}\) T cells over time. The A42 PD-1 isoform was not only associated with ICIs resistance, but it could also represent a therapeutic target.