Table 1.

Summary of the main evidences from trials on liquid biopsy in non-small cell lung cancer (NSCLC) patients receiving immunotherapy.

Method	Authors	Sample Size	ICI(s)	Cutoff(s)	Results	Annotations
ctDNA	Gandara, et al. [20]	n = 853	atezolizumab	TMB ≥ 16	bTMB indipendently predicts PFS benefit PFS outcomes best in pts with combined high bTMB and PD-L1 expression	tTMB and bTMB are positively correlated bTMB is not associated with high PD-L1 expression
	Wang et al. [21]	n = 98	anti-PD1/PD-L1	TMB > 6	High bTMB associates with better PFS and ORR	This study validates the NCC-GP150 panel for ctDNA-based bTMB measure
	Goldberg et al. [22]	n = 28	anti-PD1/PD-L1	ctDNA drop ≥ 50% from baseline	ctDNA response associates with superior PFS and OS	ctDNA is an early marker of clinical benefit Pts with ctDNA responses are more likely to have longer duration of treatment benefit
	Ijima et al. [23]	n = 14	nivolumab	VAF ≥ 2%	Early (i.e., first 2 weeks) changes in ctDNA levels predict treatment benefit	ctDNA is detected more frequently in pts with high tumor burden
	Raja et al. [24]	n = 73	durvalumab	dVAF < 0	dVAF associates with ORR, longer DOR and improved PFS and OS dVAF is an early marker for clinical benefit	dVAF does not significantly correlate with PD-L1 status
	Guibert et al. [25]	n = 86	nivolumab, pembrolizumab	dVAF < 30% and 50%	Pts with any decrease of ctDNA AF at one month have longer PFS and increased DOR	“High immune score” associated with better PFS PD-L1 expression less predictive of response than ctDNA profiling
	Socinski et al. [26]	n = 152	atezolizumab	TMB ≥ 16	High bTMB has numerical benefit for PFS and OS	Decreased serum CRP over 6 weeks predicts PFS and OS benefit
CTCs	Tamminga et al. [27]	n = 63	anti-PD1/PD-L1, anti-PD1 + anti-CTLA4	CTC ≥ 1 tdEV ≥ 18	CTC is an independent predictive factor for durable tumor response rates	tdEV are not associated with response, but with worse PFS and OS
	Castello et al. [28]	n = 35	anti-PD1/PD-L1, anti-PD1 + anti-CTLA4	CTC ≥ 1	CTC count at 8 weeks is an independent predictor for PFS and OS Combination of mean CTC and median MTV at 8 weeks associates with PFS and OS	CTC correlates with tumor burden CTC count variation is associated with tumor metabolic response at 18FDG-PET/CT scan
	Nicolazzo et al. [29]	n = 24	nivolumab	PD-L1(+) CTC ≥ 1	PD-L1(+) CTCs at 6 months of treatment correlates with PD	Presence of CTCs and expression of PD-L1 are associated with poor outcomes
	Guibert et al. [30]	n = 96	nivolumab	PD-L1(+) CTC ≥ 1	Higher baseline PD-L1(+) CTC associates with poor PFS	No correlation between tissue and CTC PD-L1 expression
	Dhar et al. [31]	n = 22	nivolumab, avelumab	>1.32 CTCs/mL PD-L1(+) CTC ≥ 2	High PD-L1(+) CTC associates with better DCR	Combination of PD-L1 positivity on tumor tissue and on CTCs as a potential biomarker for clinical benefit
	Kulasinghe et al. [32]	n = 33	anti-PD1/PD-L1	>1 CTC/3.75 mL	Presence of CTCs at baseline is not associated with PFS, nor is CTC PD-L1 expression status	Different role of CTCs in HNC and NSCLC
miRNAs/exosomes	Del Re et al. [33]	n = 8	nivolumab, pembrolizumab	PD-L1 miRNA copies	Exosomal PD-L1 expression associates with response	Concordant results among pts with NSCLC and melanoma
	Halvorsen et al. [34]	n = 20	nivolumab	NA	7miRNA signature associates with survival	Validation in an independent cohort is needed
	Costantini et al. [35]	n = 43	nivolumab	sPD-L1 > 33.97 pg/mL	High sPD-L1 and its increase associates with worse PFS and OS Pts with clinical benefit had a down expression in miRNA-320b and -375	sPD-L2, sIL-2 and sIFN-γ associate with serious irAEs

Abbreviations: 18FDG-PET/CT, 18f-fluorodeoxyglucose positron emission tomography/computed tomography scan; bTMB, blood tumor mutational burden; CRP, C-reactive protein; CTC, circulating tumor cells; ctDNA, circulating tumor DNA; CTLA-4, cytotoxic T-lymphocyte antigen 4; DCR, disease control rate; DOR, duration of response; dVAF, change in mean variant allelic frequency; HNC, head and neck cancer; ICIa, immune checkpoint inhibitors; irAEs, immune-related adverse events; miRNAs, microRNAs; MTV, metabolic tumor volume; NA, not applicable; NSCLC, non-small cell lung cancer; ORR, objective response rate; OS, overall survival; PD, progressive disease; PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; PD-L2, programmed cell death ligand 2; PFS, progression free survival; pts, patients; sIFN-, soluble interferon gamma; sIL-s, soluble interleukin 2; sPD-L1/2, soluble programmed cell death ligand 1/2; tdEV, tumor-derived extracellular vesicles; VAF, variant allelic frequency.