First author, publication year Country Patient no. or prognostic analysis no./total no. TNM stage Sample origin Time of sample collection Clinical therapy Detection methods Target genes/variants Cutoff value Experimental no./control no. Positive ratio Endpoint Follow-up duration Confounding factors NOS score Kozak et al., [22 ] Poland 62 IIIC-IV Plasma Pretreatment, posttreatment BRAF inhibitor vemurafenib ddPCR (bio-rad) BRAFV600E NA 48/14 (pretreatment), 18/44 (posttreatment) 48/62 (pretreatment), 18/62 (posttreatment) PFS, OS 55.4 months (median) — 7 Váraljai et al., [23 ] (immune checkpoint inhibition therapy) German 18/33 III-IV Plasma Pretreatment, posttreatment Immune checkpoint inhibitors ddPCR BRAFV600E , NRASQ61 , TERTPROM NA 12/6m (BRAFV600E ) NA PFS NA — 6 Váraljai et al., [23 ] (signaling-targeted therapy) German 33/58 III-IV Plasma Pretreatment, posttreatment MAPK signaling-targeted drugs ddPCR BRAFV600E , NRASQ61 , TERTPROM NA 21/12m (BRAFV600E ), 21/12 (NRASQ61 ) 21/33 (NRASQ61 ) PFS, OS NA — 6 Váraljai et al., [23 ] (validation cohort) German, Belgium 35 III-IV Plasma Pretreatment, posttreatment Pembrolizumab or nivolumab plus ipilimumab ddPCR BRAFV600E , NRASQ61 , TERTPROM NA 20/15m NA PFS NA — 6 Tan et al., [13 ] (MRV) Australia 81/133 III Plasma Pretreatment, posttreatment Operation alone or in combination with adjuvant immune checkpoint inhibitor ddPCR (bio-rad) BRAFV600E/V600K , NRASQ61L/Q61K/Q61R , TP53R248W , KITL576P , TERTC250T/C228T ≥1 copy of mutant DNA detected in both duplicate reactions 21/37 (pretreatment), 13/39 (posttreatment) 21/58 (pretreatment), 13/52 (posttreatment) OS 20 months (median) — 8 Seremet et al., [15 ] Belgium 63/85 III-IV Plasma Pretreatment, posttreatment Anti-PD1 therapy pembrolizumab ddPCR (Bio-Rad) and idylla platform (Biocartis) BRAFV600 , NRASQ61/G12/G13 NA 35/28 28/63 PFS, OS 84 weeks (median) ctDNA, LDH, CRP, number of tumor sites and ECOG variables 7 Forthun et al., [16 ] Norway 26 IV Plasma Pretreatment, posttreatment Bevacizumab treatment ddPCR (Bio-Rad) NRASQ61/G12 , BRAFV600 , TERTC288T/C250T >2 droplets positive for the mutation assay NA 23/26 (NRASQ61/G12 , BRAFV600 ) PFS, OS 6 months ctDNA, LDH 7 Salemi et al., [24 ] Italy 26/28 IV (1 case of unknown) Serum Pretreatment, posttreatment BRAF inhibitors alone or in combination with MEK inhibitors ddPCR (Bio-Rad) BRAFV600E ≥3 FAM/HEX-positive droplets 11/15 11/26 PFS, OS NA — 6 McEvoy et al., [25 ] Australia 32 IV Plasma Pretreatment Targeted therapy and/or immunotherapy ddPCR (Bio-Rad) BRAF, NRAS, KIT NA 23/9 23/32 PFS 64.4 weeks (median) ctDNA, age, sex, ECOG, MTB$, stage 7 Lu et al., [26 ] (chemotherapeutic drug) 12 countriesb 258/338 IIIC-IV Plasma Pretreatment, posttreatment Chemotherapeutic drug dacarbazine ddPCR (Bio-Rad) BRAFV600 ≥10 copies 172/86 172/258 OS 9.5 months (median) — 7 Lu et al., [26 ] (BRAF inhibitor) 12 countriesb 293/337 IIIC-IV Plasma Pretreatment, posttreatment BRAF inhibitor vemurafenib ddPCR (Bio-Rad) BRAFV600 ≥10 copies 188/105 188/293 OS 12.5 months (median) — 7 Lee et al., [14 ] UK 161 II-III Plasma Pretreatment (after operation) Bevacizumab versus placebo ddPCR (Bio-Rad) BRAFV600E , NRASQ61K/Q61L ≥1 copy of mutant DNA detected 19/142 19/161 OS 5 years (median) ctDNA, ECOG, stage 7 Herbreteau et al., [27 ] France 53 IIIC-IV Plasma Pretreatment, posttreatment Nivolumab alone or in combination with ipilimumab dPCR (The QuantStudio 3D digital PCR system) BRAFV600E/V600K , NRASQ61K/Q61R/Q61L/G12D ≥8 mutated copies/mL 30/23 23/53 PFS, OS 6.8 months (median) — 7 Gonzalez-Cao et al., [28 ] Spain 58/66 IV (6 case of unknown) Serum and plasma Pretreatment, posttreatment BRAFi/MEKi, chemotherapy, ipilimumab, palliative PNA probe-based TaqMan assay BRAFV600 NA 38/20, 15/43# 38/58 PFS, OS NA — 6 Lee et al., [29 ] Australia 76 IV Plasma Pretreatment, posttreatment Pembrolizumab or nivolumab monotherapy, or in combination with ipilimumab ddPCR (Bio-Rad) BRAFV600E/V600K/L597Q/L597R/G464E/G466E , NRASQ61H/Q61K/Q61L/Q61R , KITK642E ≥3 positive mutant droplets 36/40 (pretreatment), 58/18 (posttreatment) 40/76 (pretreatment), 18/76 (posttreatment) PFS, OS 17.5 months (median) ctDNA, LDH, disease volume, ECOG and AJCC tumour stage variables 8 Santiago-Walker et al., [30 ] (BREAK-3) 12 countriesb 170/250 III-IV Plasma Pretreatment BRAF inhibitor dabrafenib or DTIC BEAMing technology BRAFV600E/V600K Multiple determination methods 137/33 137/170 PFS, OS 4.9 months (median) — 8 Santiago-Walker et al., [30 ] (BREAK-MB) 6 countriesa 54/172 IV Plasma Pretreatment BRAF inhibitor dabrafenib BEAMing technology BRAFV600E/V600K Multiple determination methods 40/14 40/54 PFS, OS 4 months — 8 Santiago-Walker et al., [30 ] (METRIC) Multi- countriesb 169/322 IIIC-IV Plasma Pretreatment MEK inhibitor trametinib or chemotherapy BEAMing technology BRAFV600E/V600K Multiple determination methods 124/45 124/169 PFS, OS NA — 8 Gray et al., [12 ] (targeted therapy) Australia 29 IV Plasma Pretreatment, posttreatment MAPK inhibiting therapies ddPCR (Bio-Rad) BRAFV600E/V600K , NRASQ61R/K >1 copy per ml 17/12# NA PFS NA ctDNA levels, age, sex, metastatic disease stage and LDH status 6 Gray et al., [12 ] (immunotherapy) Australia 19 IV Plasma Pretreatment, posttreatment Immunotherapies ddPCR (Bio-Rad) BRAFV600E/V600K , NRASQ61R/K >1 copy per ml 15/4# NA PFS NA — 6 Board et al., [17 ] 10 countriesb 45/126 III-IV Serum Pretreatment MEK inhibitor vs TMZ ARMS allele-specific PCR BRAFV600 Only if all three replicates were positive 25/20 25/45 PFS NA — 6 Shinozaki et al., [31 ] (biochemotherapy) USA 20/50 IV Serum Posttreatment, pretreatment Biochemotherapy Real-time PCR BRAFV600E The amount of target mutant DNA contained in 1 µ g/mL MA genomic DNA 8/12 8/20 OS NA — 6
