Promising Novel Biomarkers for Hepatocellular Carcinoma: Diagnostic and Prognostic Insights

Figures & data

Table 1 Phases of Cancer Biomarker Development for Early Detection of HCC

Phase	Study Design	Method	Goal	Outcome Measure
1	Preclinical exploratory	Compare biomarker expression in tumor vs non-tumor tissue	Find biomarker	TPR (sensitivity), FPR (1 − specificity), AUROC
2	Clinical, case-controlled, at the time of diagnosis	Compare biomarker expression in subjects with vs without tumor by clinical assays	Evaluate clinical assay	TPR, FPR, AUROC after HCC diagnosis Comparison with AFP ± ultrasound is recommended
3	Retrospective longitudinal, before and at the time of diagnosis	Comparing biomarker expression in subjects who have versus have not developed cancer before and at the time of cancer diagnosis (multiple timelines), ideally PRoBE	Evaluate the ability of biomarkers to detect cancer before clinical diagnosis	TPR, FPR, AUROC before and after HCC diagnosis Comparison with AFP ± ultrasound is recommended
4	Prospective screening	Definitive diagnostic tests are performed at the time of screening biomarker positive. Compare results of definitive diagnostic test vs biomarker at the time of biomarker positivity	Evaluate the number and nature (stage) of cancer detected by biomarker screening	Early detection rate (benefit)*, false referral rate (harm)* Comparison with AFP ± ultrasound is recommended
5	Randomized controlled trial (RCT)	RCT with therapeutic intervention as needed with the positive screening test	Evaluate the reduction in mortality by biomarker screening	Mortality benefits from all causes, HCC-related mortality benefits, overdiagnosis and overtreatment (harm), cost and quality of life

Notes: *Detection rate: Positive by biomarker and disease positive following definitive tests/Screening cases. False referral rate: Negative by definitive test/Screening positive by biomarker.^11,12

Abbreviations: AFP, alpha-fetoprotein; AUROC, area under the receiver operating characteristics curve; FPR, false-positive rate; HCC, hepatocellular carcinoma; PRoBE, prospective-specimen collection, retrospective-blinded-evaluation; TPR, true-positive rate.

Table 2 Summary of Proposed Prognostic and Predictive Biomarkers for TKIs and Anti-Angiogenesis Agents by Biomarker Studies from Major Clinical Trials

Treatment	Trial (NCT) Trial Design; Biomarker Study Design	Line of Tx	Biomarkers	Assays	No. in Original Trial; No. in Biomarker Study (%)*	Rationale	Multivariate Analysis	Validation	Authors^Ref
Sorafenib vs Placebo	SHARP (NCT00105443) Phase 3, RCT Post-hoc Exploratory	1st	Plasma ANG2, VEGF, s-c KIT, HGF	Plasma, ELISA	602 491 (81.6%)	Baseline ANG2 and VEGF levels independently predict OS (Prognostic) Sorafenib showed a trend toward OS (p=0.081) and TTP (0.052) benefit compared to placebo in high s-c KIT (Predictive – sorafenib) Sorafenib showed a trend toward OS (p=0.073) benefit compared to placebo in low HGF (Predictive – sorafenib)	Yes	No validation	Llovet et al^Citation69
	SHARP (NCT00105443) + Asia-Pacific (NCT00492752) Phase 3 RCTs Post-hoc Exploratory	1st	NLR, AFP	Serum, AFP, neutrophil, lymphocyte	828 827 (99.9%)	High AFP (>200 ng/mL) and NLR (> median) independently predict poor OS (Prognostic) Sorafenib OS benefit was only observed in low NLR (≤ median) but not in high NLR (> median) (Predictive – sorafenib)	Yes	No validation	Bruix et al^Citation71
Lenvatinib vs Sorafenib	REFLECT (NCT01761266) Phase 3 RCT Post-hoc Exploratory	1st	Serum VEGF, ANG2, FGF21	Serum, ELISA	954 407 (42.7%)	Baseline high ANG2, FGF21, and VEGF were correlated with poor OS (Prognostic) Lenvatinib showed OS benefit compared to sorafenib in high FGF21 but no OS benefit in low FGF21 (Predictive – lenvatinib)	Yes	No validation	Finn et al^Citation85
			Gene expression profiling, VEGF, FGF	Gene panel	954 58 (6.1%)	VEGF/FGF-enriched groups showed longer OS compared to intermediate VEGF/FGF in lenvatinib VEGF/FGF-enriched groups showed shorter OS compared to intermediate VEGF/FGF in sorafenib	No	No validation
Ramucirumab vs Placebo	REACH2 (NCT02435433) Biomarker-driven Phase 3 RCT Pre-specified	Later	AFP ≥400 ng/mL	Serum, AFP	292 292 (100.0%)	Ramucirumab showed improved OS and PFS compared to placebo in this biomarker-selected (AFP ≥400 ng/mL) RCT (Prognostic, Predictive – ramucirumab)	Not applicable	No validation	Zhu et al^Citation10
	REACH (NCT01140347) + REACH2 (NCT02435433) Phase 3 RCTs Post-hoc Exploratory		NLR AFP >1000 ng/mL	Serum, AFP, neutrophil, lymphocyte	857 542 (63.2%)	Baseline high AFP (≥1000 ng/mL) and NLR independently predict poor OS (Prognostic)	Yes	No validation	Llovet et al^Citation86
Regorafenib vs Placebo	RESORCE (NCT01774344) Phase 3 RCT Post-hoc Exploratory	Later	Plasma AFP, c-MET, ANG1, Cystatin-B, LAP TGF-β1, LOX-1, MIP-1α	Multiplex immunoassay	573 499 (87.1%)	High baseline AFP and c-MET were related to shorter OS independently of treatment (Prognostic) Decreased baseline plasma ANG1, cystatin-B, LAP TGF-β1, LOX-1, and MIP-1α were related to longer OS with regorafenib (Predictive – regorafenib)	Yes	No validation	Teufel et al^Citation87
			miRNA	miRNA PCR	573 349 (60.9%)	9 plasma miRNAs (miR-15B, 30A, 107, 122, 125B, 200A, 320b, 374B, 645) were associated with OS in regorafenib (Predictive – regorafenib)	Yes	No validation
Atezo-Bev vs Atezo	GO30140 (NCT02715531) Phase 1b, RCT Post-hoc Exploratory	1st	RNA	RNA sequencing	119 91 (76.5%)	High expression of VEGF receptor 2 (KDR), Treg (CCR8, BATF, CTSC, TNFRSF4, FOXP3, TNFRSF18, IKZF2, IL-2RA), myeloid inflammation (CXCL1, CXCL2, CXCL3, CXCL8, IL-6, PTGS1), and Teff (CXCL9, PRF1, GZMB) signatures were associated with improved PFS in atezo-bev compared to atezo (Predictive – bevacizumab)	Yes	KDR, Treg were validated by a small population of 14 pts	Zhu et al^Citation88
			Tumor vessel density by CD31	Multiplex IHC panel	119 67 (56.3%)	High tissue blood vessel density by CD31 was associated with longer PFS in atezo-bev compared to atezo (Predictive – bevacizumab)	Yes	No validation
Cabozantinib vs Placebo	CELESTIAL (NCT01908426) Phase 3 RCT Post-hoc Exploratory	Later	Plasma ANG2, GAS6, HGF, IGF-1, IL-8, MET	Luminex (immunoassay)	707 674 (95.3%)	Low baseline levels of ANG2, GAS6, HGF, IL-8, MET were related to favorable survival (Prognostic) High baseline levels of IGF-1 were related to favorable survival (Prognostic)	Yes	No validation	Rimassa et al^Citation89

Note: *(No. in original trial/No. in biomarker study) ×100.

Abbreviations: atezo, atezolizumab; bev, bevacizumab; NLR, neutrophil to lymphocyte ratio; No., number; OS, overall survival; PFS, progression-free survival; RCT, randomized controlled trial; Teff, effector T cell; TKI, tyrosine kinase inhibitor; TTP, time to progression; Tx, treatment.

Table 3 Summary of Proposed Prognostic and Predictive Biomarkers for CPIs by Biomarker Studies from Major Clinical Trials

Biomarker	Assay	Trial (NCT) Trial Design; Biomarker Study Design	Treatment	Line of Treatment	No. in Original Trial; No. in Biomarker Study (%)*	Rationale	Multivariate Analysis	Validation	Authors^Ref
Tumor PD-L1	IHC	CheckMate 040 (NCT01658878) Phase 1/2, Non-comparative Prespecified (2nd endpoint)	Nivolumab	1st or later	214 174 (81.3%)	PD-L1 status did not have an apparent effect on ORR (PD-L1 was a secondary endpoint)	Not applicable	Not applicable	El-Khoueiry et al^Citation90
		CheckMate 040 (NCT01658878) Phase 1/2, Non-comparative Post-hoc Exploratory	Nivolumab	1st or later	214 195 (91.1%)	PD-L1 expression ≥1% was associated with improved OS ORR was higher in PD-L1 ≥1% vs PD-L1 <1 (28% vs 16%)	No	No validation	Sangro et al^Citation91
		CheckMate 040 (NCT01658878) Phase 1/2 RCT Prespecified (2nd endpoint)	Nivolumab + Ipilimumab	Later	148 145 (98.0%)	PD-L1 status did not have an apparent effect on ORR (PD-L1 was a secondary endpoint)	Not applicable	Not applicable	Yau et al^Citation92
		CheckMate 459 (NCT02576509) Phase 3 RCT Prespecified (2nd endpoint)	Nivolumab vs Sorafenib	1st	743 730 (98.3%)	PD-L1 status did not have an apparent effect on ORR, PFS, or OS (PD-L1 was a secondary endpoint)	Not applicable	Not applicable	Yau et al^Citation93
		KEYNOTE-224 (NCT02702414) Phase 2, Non-randomized Prespecified exploratory endpoint	Pembrolizumab	Later	104 52 (50.0%)	Tumor PD-L1 expression alone was not significantly associated with ORR and PFS Combined PD-L1 (tumor cells, macrophages, and lymphocytes) was associated with favorable ORR and PFS	Yes	No validation	Zhu et al^Citation94
CD3^+, CD8+ TILs	IHC	CheckMate 040 (NCT01658878) Phase 1/2, Non-comparative Post-hoc Exploratory	Nivolumab	1st or later	214 195 (91.1%)	CD3 expression was related to better ORR CD3 and CD8 expression in TIL showed a trend toward improved OS (p=0.08)	No	No validation	Sangro et al^Citation91
		IMbrave150 (NCT03434379) Phase 3, RCT Post-hoc Exploratory	Atezo-bev vs Sorafenib	1st	501 177 (35.3%)	High CD8 showed a trend toward longer PFS (p=0.053) and significantly longer OS (p=0.001) in atezo-bev compared to sorafenib (Predictive – atezo-bev)	Yes	High CD8 was discovered by GO30140-A cohort and validated by IMbrave150 cohort	Zhu et al^Citation88
PD-1^+ TIL Treg (CD4, FOXP3) TAM (CD68, CD163)	IHC	CheckMate 040 (NCT01658878) Phase 1/2, Non-comparative Post-hoc Exploratory	Nivolumab	1st or later	214 195 (91.1%)	PD-1 expression showed a trend toward increased OS (p=0.05) Treg (CD4, FOXP3) was not associated with response or OS TAM (CD68, CD163) was not associated with response or OS	No	No validation	Sangro et al^Citation91
NLR, PLR	Serum, neutrophil, lymphocyte					Lower baseline serum NLR and PLR were related to better OS	No	No validation	Sangro et al^Citation91
GEP	RNA sequencing	CheckMate 040 (NCT01658878) Phase 1/2, Non-comparative Post-hoc Exploratory	Nivolumab	1st or later	214 37 (17.3%)	Inflammatory signature improved nivolumab ORR and OS Interferon-gamma and T-cell exhaustion signatures were associated with ORR and OS	No	No validation	Sangro et al^Citation91
		IMbrave150 (NCT03434379) Phase 3, RCT Post-hoc Exploratory	Atezo-bev vs Sorafenib	1st	501177 (35.3%)	High ABRS, CD274, and Teff signatures were associated with better OS and PFS with atezo-bev compared to sorafenib (Predictive – atezo-bev) Low Treg/Teff, AFP, and GPC3 expression was associated with better PFS and OS with atezo-bev compared to sorafenib (Predictive – atezo-bev)	Yes	ABRS, CD274, and Teff were discovered by GO30140-A cohort and validated by IMbrave150 cohort	Zhu et al^Citation88
		CheckMate 459 (NCT02576509) Phase 3, RCT Post-hoc Exploratory	Nivolumab vs Sorafenib	1st	743 469 (63.1%)	High Gajewski inflammation gene signature was related to improved ORR, PFS, and OS with nivolumab but not sorafenib (Predictive – nivolumab) Expression of inflammatory response and IL6-JAK-STAT3 signaling gene sets showed improved OS with nivolumab but not sorafenib (Predictive – nivolumab)	Unknown	No validation	Neely et al^Citation95
IGF-1	Serum, IGF-1	IMbrave150 (NCT03434379) Phase 3, RCT Post-hoc Exploratory	Atezo-bev vs Sorafenib	1st	501 371 (74.1%)	High baseline IGF-1 level was associated with improved OS in both arms (Prognostic) Decreased IGF-1 during treatments showed poor OS (Surrogate, Prognostic)	Yes	No validation	Kaseb et al^Citation96
AFP	Serum, AFP	IMbrave150 (NCT03434379) Phase 3, RCT + GO30140 (NCT02715531) Phase 1b, RCT Post-hoc Exploratory	Atezo-bev vs Sorafenib Atezo-bev	1st	605 208 (34.4%)	AFP level changes during treatment correlated with PFS and OS (Surrogate, Prognostic)	Yes	Identified by GO30140-A cohort and validated by IMbrave150 cohort	Zhu et al^Citation97

Note: *(No. in original trial/No. in biomarker study) ×100.

Abbreviations: atezo, atezolizumab; bev, bevacizumab; CKI, checkpoint inhibitor; GEP, gene expression profiling; IHC, immunohistochemistry; NLR, neutrophil to lymphocyte ratio; No., number; ORR, overall response rate; OS, overall survival; PFS, progression-free survival; PLR, platelet to lymphocyte ratio; RCT, randomized controlled trial; TAM, tumor-associated macrophage; Teff, effector T cell; TIL, tumor-infiltrating lymphocyte; Treg, regulatory T cell.

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