Table 2.

Advantages and disadvantages of the liquid biomarkers with potential use in immunotherapy response monitoring.

Liquid Biomarker	Advantages	Disadvantages
ctDNA 1	-Ready access to genetic material of the primary tumour -Dynamic information over the course of treatment -Predictive value in quantification of absolute levels and alternate source for TMB calculation	-Limits of detection: allele fraction and variant calling pipelines may produce many false negatives and/or false positives -Lack of standardization of thresholds
CTCs 2	-Provide reflection of tumour status and tumour heterogeneity -Dynamic information over the course of treatment -Predictive value in quantification of absolute cell counts, examination of cell membrane markers expressed, and omic characterisation	-Disparity in methods used for isolation and enrichment -Identification and isolation methods require high sensitivity -False negatives -Limited and fragile population -Low yield of genetic material
EVs 3	-Many different types available from different sources -Stable, can efficiently preserve contents -Can provide protein and lipidic markers, and genetic material	-Contained genetic material is very limited -High heterogeneity makes it difficult to distinguish EVs of tumoral origin
Tc counts 4	-Standard, reliable methods of isolation. -Easy identification and count through flow cytometry -Dynamic information over the course of treatment	-Lack of standardization of thresholds -Levels greatly vary from patient to patient -Contradictions when considering marker PD-1
Th counts 5	-Standard, reliable methods of isolation. -Easy identification and count through flow cytometry -Dynamic information over the course of treatment	-Lack of standardization of thresholds -Levels greatly vary from patient to patient
Tregs counts 6	-Standard, reliable methods of isolation. -Easy identification and count through flow cytometry	-Lack of standardization of thresholds -Levels greatly vary from patient to patient -Requires multiple markers for high sensitivity of detection
TCR determination 7	-Segment of DNA of interest is short, well defined, and has been explored in detail -Multiple protocols and kits available to amplify region of interest.	-Requires analysis of many individual cells to be significant -Careful selection of specific population of interest is required
Transcriptomic analysis of T cells	-High yield of genetic material from the high amount of T cells available in peripheral blood. -Dynamic information over the course of treatment -Activation of different pathways may be tracked throughout treatment.	-Lack of reproducibility—multitude of expression signatures that vary between experiments -Disparity in methods used to identify significant expression signatures
IFN-γ expression 8	-Specific, well-defined marker -High availability -Standardised, targeted methods of quantification	-Expressed by many cell types. Not specific to response. Cells of interest must be selected
sPD-L1 9	-Great focus in immunotherapy -Specific, easily targetable marker -Direct, less invasive alternative to PD-L1 expression in primary tumour	-Lack of standardization of thresholds -Has same accuracy limitations as conventional PD-L1 assay
LDH 10	-Cheap, easy method of enzyme quantification	-Lack of standardization of thresholds
Microbiome	-Many novel potential markers available to study	-Still in very early stages of study -Microbiota that determine response to immunotherapy widely vary between different types of cancer -Lack of standardization, no fixed markers across studies

¹ Circulating tumour DNA. ² Circulating tumour cells. ³ Extracellular vesicles. ⁴ T-cell counts. ⁵ T helper-cell counts. ⁶ T regulatory cell counts. ⁷ T-cell receptor repertoire. ⁸ Interferon gamma expression. ⁹ Soluble PD-L1. ¹⁰ Lactate dehydrogenase.