. 2024 Feb 17;31:22. doi: 10.1186/s12929-024-01011-y

Table 1.

Representative tissue- and blood-based biomarker results from clinical trials of ICI-based therapy for HCC

Bio-samples	Advantage/ disadvantage	Modality	Key findings
Tissue-based: • Tumour tissues RNA or DNA sequencing • Frozen or FFPE tumor samples	Advantages: Comprehensive multi-omics studies Opportunity to study spatial relationship of tumor and immune cells Disadvantages: Intra-tumoral heterogeneity Difficulty in collecting paired (before- and after-treatment) samples	Immune-related gene expression and signatures (RNA-seq)	• A 4-gene (PD-L1, CD8A, LAG3, and STAT1) signature associated with better response rate and survival (nivolumab; CheckMate 040) [59] • The Gajewski Inflammation signature and IL6-JAK-STAT3 signaling genes were associated with survival benefit (nivolumab; CheckMate 459) [60] • High effector T cell signature, regulatory T cell signature, and myeloid inflammation signature were associated better progression-free and overall survival (atezolizumab + bevacizumab; IMbrave 150) [63]
• A 11-gene INFAP (interferon and antigen presentation) signature predicted response and survival in HCC patients treated with anti-PD-1 monotherapy [61] • A 9-gene exhausted CD8 T-cell signature expression associated with response to ICI therapy. [62]
Whole-exome NGS Target panel NGS	• No consistent association between tumor mutation burden and response or survival [60, 63, 75] • Wild-type CTNNB1 or TERT promoter mutation associate with improved survival [63] • Activating alteration WNT/β-catenin signaling associate with lower response and shorter survival benefits [75]
Epigenetic signature	• Epigenetic regulators (EGRs) score could predict clinical outcome in HCC patients treated with immunotherapy [80] • N6-methyladenosine (m6A) modification-related epigenetic signature as biomarker for response to anti-PD-1 immunotherapy in patients with HCC [81]
Immunohistochemical staining	• Trend of higher objective response rate in tumors with increased PD-L1 expression (nivolumab, atezolizumab + bevacizumab) [59, 63] • Trend of higher objective response rate and better survival in tumors with increased PD-1 + cells (nivolumab) [59] • Tumor cell PD-L1 expression of 1% and greater and less than 1%: no differences in medial overall survival (nivolumab; CheckMate 459) [12] • Increased CD3 and CD8 showed a non-significant trend towards improved OS, and macrophage markers (CD68, CD163) were not associated with OS. [59]
Multiplex IHC staining	• Higher density of infiltrating CD8 + T cells, CD3 + T cells, GZMB + CD3 + T cells in tumors, and MHC class I protein in responders [63] • Higher density of CD8 + LAG3 + cells in the tumors by multiplex immunofluorescence staining in responders (ICI) [74]
Blood-based: • PBMCs • Serum proteins (e.g., cytokines) • Ct- or cf-DNA • Epigenetic signature	Advantages: non-invasive; real-time monitoring Disadvantages: Technical challenges of detailed phenotypical characterization Challenge to link the mechanisms to the local tumor microenvironment	Flow cytometry	• Increase in CD8 + Ki67 + T cells early after treatment associated with higher objective response rate (durvalumab + tremelimumab) [94] • Higher baseline level of PD-1 + CD4 + T cells in patients who responded to the therapy [83] • Lower posttreatment NLR and PLR ratios are associated to better response in nivolumab treated HCC patients [60]
CyTOF and scRNA seq	• Increased CXCR3 + CD8 TEM and APCs are associated to better response to anti-PD-1 ICI treatment [93]
Serum AFP test	• Lower serum AFP of < 400 ng/mL was associated with superior OS in Nivolumab treated HCC patients (Checkmate040) [59] • AFP cutoffs of ≥ 75% decrease and ≤ 10% increase from baseline at 6 weeks were associated with longer OS and PFS [50] • Low AFP (< 100 ng/mL) and low C-reactive protein (< 1 mg/dL) were associated with better survival and treatment outcome (the CRAFITY score) [90–92]
ctDNA or cfDNA sequencing	• Circulating WNT pathway-related mutations were not associated with clinical outcomes in immunotherapy treatment patients [89] • TERT ctDNA mutation predicts shorter OS in HCC patients treated with Atezo/Bev therapy [63]

Bio-samples

Advantage/ disadvantage

Modality

Key findings

Tissue-based:

• Tumour tissues RNA or DNA sequencing

• Frozen or FFPE tumor samples

Advantages:

Comprehensive multi-omics studies

Opportunity to study spatial relationship of tumor and immune cells

Disadvantages:

Intra-tumoral heterogeneity Difficulty in collecting paired (before- and after-treatment) samples

Immune-related gene expression and signatures (RNA-seq)

• A 4-gene (PD-L1, CD8A, LAG3, and STAT1) signature associated with better response rate and survival (nivolumab; CheckMate 040) [59]

• The Gajewski Inflammation signature and IL6-JAK-STAT3 signaling genes were associated with survival benefit (nivolumab; CheckMate 459) [60]

• High effector T cell signature, regulatory T cell signature, and myeloid inflammation signature were associated better progression-free and overall survival (atezolizumab + bevacizumab; IMbrave 150) [63]

• A 11-gene INFAP (interferon and antigen presentation) signature predicted response and survival in HCC patients treated with anti-PD-1 monotherapy [61]

• A 9-gene exhausted CD8 T-cell signature expression associated with response to ICI therapy. [62]

Whole-exome NGS

Target panel NGS

• No consistent association between tumor mutation burden and response or survival [60, 63, 75]

• Wild-type CTNNB1 or TERT promoter mutation associate with improved survival [63]

• Activating alteration WNT/β-catenin signaling associate with lower response and shorter survival benefits [75]

Epigenetic signature

• Epigenetic regulators (EGRs) score could predict clinical outcome in HCC patients treated with immunotherapy [80]

• N6-methyladenosine (m6A) modification-related epigenetic signature as biomarker for response to anti-PD-1 immunotherapy in patients with HCC [81]

Immunohistochemical staining

• Trend of higher objective response rate in tumors with increased PD-L1 expression (nivolumab, atezolizumab + bevacizumab) [59, 63]

• Trend of higher objective response rate and better survival in tumors with increased PD-1 + cells (nivolumab) [59]

• Tumor cell PD-L1 expression of 1% and greater and less than 1%: no differences in medial overall survival (nivolumab; CheckMate 459) [12]

• Increased CD3 and CD8 showed a non-significant trend towards improved OS, and macrophage markers (CD68, CD163) were not associated with OS. [59]

Multiplex IHC staining

• Higher density of infiltrating CD8 + T cells, CD3 + T cells, GZMB + CD3 + T cells in tumors, and MHC class I protein in responders [63]

• Higher density of CD8 + LAG3 + cells in the tumors by multiplex immunofluorescence staining in responders (ICI) [74]

Blood-based:

• PBMCs

• Serum proteins (e.g., cytokines)

• Ct- or cf-DNA

• Epigenetic signature

Advantages:

non-invasive; real-time monitoring

Disadvantages: Technical challenges of detailed phenotypical characterization

Challenge to link the mechanisms to the local tumor microenvironment

Flow cytometry

• Increase in CD8 + Ki67 + T cells early after treatment associated with higher objective response rate (durvalumab + tremelimumab) [94]

• Higher baseline level of PD-1 + CD4 + T cells in patients who responded to the therapy [83]

• Lower posttreatment NLR and PLR ratios are associated to better response in nivolumab treated HCC patients [60]

CyTOF and scRNA seq

• Increased CXCR3 + CD8 TEM and APCs are associated to better response to anti-PD-1 ICI treatment [93]

Serum AFP test

• Lower serum AFP of < 400 ng/mL was associated with superior OS in Nivolumab treated HCC patients (Checkmate040) [59]

• AFP cutoffs of ≥ 75% decrease and ≤ 10% increase from baseline at 6 weeks were associated with longer OS and PFS [50]

• Low AFP (< 100 ng/mL) and low C-reactive protein (< 1 mg/dL) were associated with better survival and treatment outcome (the CRAFITY score) [90–92]

ctDNA or cfDNA sequencing

• Circulating WNT pathway-related mutations were not associated with clinical outcomes in immunotherapy treatment patients [89]

• TERT ctDNA mutation predicts shorter OS in HCC patients treated with Atezo/Bev therapy [63]

HCC, hepatocellular carcinoma; FFPE, formalin-fixed, paraffin-embedded; PBMCs, peripheral blood mononuclear cells; ctDNA, circulating tumor DNA; cfDNA, cell-free DNA; NGS, next-generation sequencing; CyTOF, mass cytometry by time of flight; scRNA seq, single-cell RNA sequencing; AFP, alpha-fetoprotein; NLR, neutrophil–lymphocyte ratio; PLR, platelet–lymphocyte ratio; TEM, effector memory T cells; APCs, antigen-presenting cells; OS, overall survival