Immunotherapy against programmed death-1/programmed death ligand 1 in hepatocellular carcinoma: Importance of molecular variations, cellular heterogeneity, and cancer stem cells

Table 1 Programmed death-1/programmed death ligand 1 nucleotide variations and their clinical relevance in liver disease

	Variants	Relevance	Ref.
PD-1	rs7421861	Increases cancer risk, especially in NAFLD-HCC	[127,140]
	rs2227981	Reduces cancer risk	[127]
	rs42386439	Low HBV viral load	[129]
	rs36084323	Worse disease progression in chronic HBV, low TNF-α and IFN-γ levels	[15,131,132]
	rs10204525	Disease progression in chronic HBV, high TNF-α and IFN-γ levels, longer overall survival	[131,133-135]
	rs11568821	Reduces cancer risk	[23,127,136]
PD-L1	rs4143815	Increases cancer risk	[141,143]
	rs17718883	Reduces cancer risk	[142]
	rs10815225	Increases cancer risk	[143]
	rs2297136	Increases cancer risk	[142]
	rs2890658	Not associated with cancer risk	[142]
PD-1 and PD-L1	PD-1 rs11568821 and PD-L1 rs4143815	Liver transplantation setting	[148]

PD-1: Programmed death-1; PD-L1: Programmed death ligand 1; NAFLD: Non-alcoholic fatty liver disease; HCC: Hepatocellular carcinoma; HBV: Hepatitis B virus; TNF-α: Tumor necrosis factor α; IFN-γ: Interferon-.

Table 2 Major molecular classifications of hepatocellular carcinoma

Ref.	Subclass	Marker	Main methods
Yamashita et al[158]	Type A-D	EpCAM, AFP	cDNA microarray, IHC
Lee et al[157,159]	Cluster A and B, and 3 distinct phenotypes	Sets of survival genes, AFP	Integrated cDNA microarray in human specimens and animal models
Boyault et al[160]	Groups G1-G6	Tumor heterozygosity, gene mutations, methylation, and HBV DNA	cDNA microarray, IHC, qRT-PCR
Hoshida et al[161]	Subclasses S1-S3	Cellular differentiation, serum AFP, signaling pathways	Meta-analysis of gene expression profiles datasets, IHC
Sia et al[163]	Immune classes active and adaptive	Immune response genes	cDNA microarray, IHC, TCGA analysis
Zhang et al[164]	Immunophenotypic subtypes 1-3	Immune response genes and antitumoral immunity	Whole-exome and RNA sequencing, mass spectrometry-based proteomics and metabolomics, CyTOF, single-cell analysis

HBV: Hepatitis B virus; IHC: Immunohistochemistry; TCGA: The Cancer Genome Atlas; AFP: Alpha-fetoprotein; qRT-PCR: Quantitative real-time polymerase chain reaction.

Table 3 Association between programmed death-1 expression and cancer stem cell markers

Malignancy	Stemness/CSC markers	Method	Relevance	Ref.
HCC	CD133+ interaction with lymphatic endothelial cells	Co-culture	Upregulates PD-L1	[196]
HCC	CK-19, SALL-4	IHC	Positively associated with PD-L1	[194]
HCC	EpCAM	IHC	Negatively associated with PD-L1	[194]
Breast cancer	CD44hiCD24lo	Flow cytometry	High PD-L1 expression	[182]
Breast cancer	EpCAM+CD44hi CD24lo	Flow cytometry	High PD-L1 expression	[184]
Breast cancer	EpCAM+CD90hi	Flow cytometry	High PD-L1 expression	[184]
Breast cancer	EpCAM+CD44hiCD24lo	Flow cytometry, IF	High PD-L1 expression; nuclear PD-L1	[185]
Breast cancer	Stemness score	mRNA from TCGA	Significant correlation to PD-L1	[185]
Breast cancer	ALDH+, CD44hi	Flow cytometry	High PD-L1 expression	[183]
Colorectal cancer	CD44hiCD133hi	Flow cytometry	High PD-L1 expression	[182]
Colorectal cancer	CD133+CD44+	Flow cytometry	High PD-L1 expression	[189]
Ovarian cancer	ALDH	qRT-PCR, IF	High PD-L1 expression	[188]
HNSCC	CD44+	Flow cytometry, qRT-PCR, RNA in situ hybridization	High PD-L1 expression	[191]
Lung adenocarcinoma	CD44	mRNA in TIMER datasets	Positively associated with PD-L1	[186]
NSCLC	ALDH	mRNA from TCGA, IHC	Negatively associated with PD-L1	[192]
Cholangiocarcinoma	ALDH	Sorting of PD-L1 cells	High ALDH+ in PD-L1lo	[193]
Pancreatic cancer	CD44+CD133+	IF	Positively associated with PD-L1	[190]

PD-1: Programmed death-1; PD-L1: Programmed death ligand 1; IHC: Immunohistochemistry; TCGA: The Cancer Genome Atlas; qRT-PCR: Quantitative real-time polymerase chain reaction; HNSCC: Head and neck squamous cell carcinoma; NSCLC: Non-small cell lung carcinoma; HCC: Hepatocellular carcinoma.