Table 1.
Summary of included studies on the association between circulating cell-free (tumor) DNA and prognosis.
| Author, Year | Biomarkers | Assay | n | Tumor Stage | % Detectable Presurgery | Correlation with Stage | Outcome |
|---|---|---|---|---|---|---|---|
| Cell-Free DNA | |||||||
| Czeiger et al., 2016 [20] | Cell-free DNA | SYBR Gold fluorometry | 38 | I: n = 5 | 100% detectable | Higher levels in stage IV | mHR for DFS = 6.03 (95% CI 1.87–19.41) |
| II: n = 20 | 49% above cutoff of 800 ng/mL |
mHR for OS = 3.53 (95% CI 1.46–8.55) | |||||
| III: n = 7 | |||||||
| IV: n = 5 | |||||||
| Guadalajara et al., 2008 [21] | Cell-free DNA | Spectrophotometry (NanoDrop) | 73 | I: n = 17 | Not reported. | Higher levels in stage IV | No significant correlation between cfDNA concentration and development of metastases or mortality. Trend toward worse prognosis for patients with cfDNA concentration >60 ng/μL |
| II: n = 25 | |||||||
| III: n = 19 | |||||||
| IV: n = 11 | |||||||
| Benign: n = 1 | |||||||
| Fleming et al., 2020 [22] | Cell-free DNA | Spectrophotometry (NanoDrop) | 20 | I–II: n = 9 | Not reported | Not reported | Slightly higher cfDNA levels in patient with a recurrence compared to non-recurrence patients |
| III: n = 11 | |||||||
| Zhong et al., 2020 [23] | Cell-free DNA | qPCR | 60 | I–II: n = 26 | Not reported. | Yes | cfDNA concentration was an independent risk factor for PFS in both univariate and multivariate regression analysis |
| III–IV: n = 34 | |||||||
| Somatic alterations | |||||||
| Wang et al., 2004 [24] | APC, KRAS, TP53 | PCR-SSCP tumor-naive, serum |
104 | I: n = 7 II: n = 49 III: n = 39 IV: n = 9 |
0.46 | Non-significant trend | 75% vs. 9.5% recurrences (p < 0.001) |
| Lecomte et al., 2002 [25] | KRAS (codon 12, 13) Also: cfDNA, p16 methylation |
PCR tumor-informed, plasma |
58 | I: n = 8 II: n = 21 III: n = 16 IV: n = 13 |
cfDNA: 43% KRAS2: 45% p16: 68% Overall: 68% (stage I–III) |
No | Significant worse RFS for ctDNA+ stage I–III patients: 2 y RFS of 66% (95% CI 36–84%) vs. 100%. mHR for OS in stage I–IV = 13 (95% CI 1.5–112). |
| Shin et al., 2017 [26] | KRAS | Sequenom MassARRAY + modified ultrahigh-sensitivity assay tumor-naive, plasma |
160 | I–II: n = 19 III: n = 35 IV: n = 106 |
17% in stage I–III | Correlation with heavier tumor burden | 89% vs. 78% recurrences in stage I–III patients. Lower PFS (17 vs. 21 months), but not significant |
| Reinert et al., 2016 [27] |
Patient-specific somatic structural variants | dPCR tumor-informed, plasma |
11 | I: n = 1 II: n = 5 III: n = 2 IV: n = 3 |
0.73 | Non-significant trend | ctDNA+: 5/8 rec ctDNA−: 1/3 rec |
| Scholer et al., 2017 [28] | Patient-specific somatic structural variants and SNVs | dPCR tumor-informed, plasma |
27 | I: n = 5 II: n = 8 III: n = 8 IV: n = 6 |
0.74 | Yes | 8/10 ctDNA+ in stage I–III patients with relapse 6/11 ctDNA+ in stage I–III patients without relapse |
| Thomsen et al., 2017 [29] | RAS, BRAF | dPCR tumor-informed, serum |
294 | I: n = 40 II: n = 151 III: n = 103 |
0.42 | Yes |
RAS: mHR for DFS = 2.18 (95% CI 1.26–3.77). mHR for OS = 2.30 (95% CI 1.27–4.15). BRAF and pMMR: mHR for DFS = 3.61 (95% CI 1.70–7.67). mHR for OS = 3.45 (95% CI 1.52–7.85). |
| Tarazona et al., 2019 [30] | 29 cancer-related genes | dPCR tumor-informed, plasma |
94 | I: n = 14 II: n = 41 III: n = 39 |
0.64 | Lower levels in stage I | No relation between ctDNA and outcome: uHR for DFS = 0.93 (95% CI: 0.33–2.69) |
| Nakamura et al., 2021 [31] | KRAS (codon 12, 13) | dPCR tumor-naive, plasma |
180 | I–III: n = 154 IV: n = 26 |
33% (30% in stage I–III) | Non-significant trend | Increased recurrence risk for ctDNA+ patients (27% vs. 3%). mHR for RFS = 2.18 (95% CI 1.02–4.61) |
| Reinert et al., 2019 [12] | Patient-specific mutations | Multiplex PCR-based NGS tumor-informed, plasma |
125 | I: n = 5 II: n = 39 III: n = 81 |
0.89 | Lower levels in stage I | No significant association between ctDNA and outcome |
| Allegretti et al., 2020 [32] | 15 cancer-related genes | Targeted NGS + dPCR tumor-naive, plasma |
39 | I: n = 9 II: n = 14 III: n = 11 NR: n = 5 |
0.44 | Weak, non-significant trend | 3/10 recurrences in follow-up patients. 3 recurrences: 100% ctDNA+ before surgery. 7 non-recurrences: 4/7 ctDNA+ before surgery |
| Phallen et al., 2017 [33] | 58 cancer-related genes | Targeted NGS tumor-naive, plasma |
42 | I: n = 8 II: n = 9 III: n = 10 IV: n = 15 |
0.83 | Lower levels in stage I | uHR for PFS/OS = 1.13 (95% CI 1.03–1.24) in stage I–III mHR for PFS = 36.3 (95% CI 2.8–471.1) in stage I–IV |
| Suzuki et al., 2020 [34] | 52 cancer-related genes | Targeted NGS tumor-naive, plasma |
154 | I: n = 29 II: n = 64 III: n = 50 IV: n = 11 |
0.73 | Non-significant trend | 4 recurrences in CRC patients with detectable ctDNA before surgery. MAF heat plot does not discriminate between recurrence and non-recurrence patients |
| Methylation | |||||||
| Matthaios et al., 2016 [35] | APC, RASSF1A methylation | PCR | 155 | I–III: n = 88 IV: n = 67 |
APC: 33% RASSF1A: 25% |
Yes |
APC: OS 27 vs. 81 months RASSF1A: OS 46 vs. 71 months (p < 0.001) |
| Xue et al., 2017 [36] | Cystathionine-beta-synthase (CBS) hypomethylation | PCR | 95 | I: n = 10 II: n = 22 III: n = 38 IV: n = 15 |
0.64 | Yes | RR of RFP = 1.54 (95% CI 1.18–3.02) RR of OS = 1.35 (95% CI 1.09–2.41) |
| Rasmussen et al., 2018 [37] | 30 gene promotor regions | PCR | 193 | I–III: n = 159 IV: n = 34 |
NR | Non-significant trend | Signification association between OS and >4 methylated regions RARB or RASSF1A: mHR for OS = 2.53 (95% CI 1.60–3.90) |
| Lin et al., 2015 [38] | TWIST1, FLI1, AGBL4 | qPCR (Sequenom MassArray) | 353 | I: n = 42 II: n = 140 III: n = 108 IV: n = 63 |
≥1 meth: 93% AGBL4: 65% FLI1: 66% TWIST1: 70% |
No | No significant association between (number of) methylated genes and DFS |
| Wallner et al., 2006 [39] | TMEFF2, HLTF, hMLH1 | qPCR | 77 | I: n = 10 II: n = 24 III: n = 24 IV: n = 15 |
HLTF: 22% HPP1: 12% hMLH1: 23% |
Yes | TMEFF2 or HLTF: mRRD = 3.4 (95% CI 1.4–8.1) |
| Herbst et al., 2009 [40] | HLTF, TMEFF2 | qPCR | 106 | I: n = 13 II: n = 39 III: n = 54 |
HLTF: 12% TMEFF2: 6% |
No | HLTF: mRRR = 2.5 (95% CI 1.1–5.6). Significant worse RFS (p = 0.014). |
| Liu et al., 2016 [41] | SST, MAL, TAC1, SEPT9, EYA4, CRABP1, NELL1 | qPCR | 165 | I: n = 26 II: n = 62 III: n = 62 IV: n = 15 |
0.5 | NR |
mSST: mHR for DFS = 2.60 (95% CI 1.37–4.94) mSST: mHR for CSD = 1.96 (95% CI 1.06–3.62) |
| Bedin et al., 2017 [42] | SFRP1, OSMR Also: total amount cfDNA |
qPCR | 114 | I: n = 38 II: n = 29 III: n = 32 IV: n = 15 |
0.67 | Methylation: No cfDNA: Higher levels in stage III/IV |
Methylation: no significant association with DFS/OS cfDNA: high level associated with poor prognosis. mHR for OS ALU83 = HR 2.71 (95%CI 1.22–6.02), mHR for OS ALU244 = 2.40 (95% CI 1.11–5.19). |
| Song et al., 2018 [43] | SEPT9 | qPCR | 120 | I: n = 14 II: n = 40 III: n = 45 IV: n = 21 |
0.87 | Yes | uHR for OS = HR 2.51 (95% CI 1.03–6.12) |
| Constâncio et al., 2019 [44] | APC, FOXA1, GSTP1, HOXD3, RARβ2, RASSF1A, SEPT9, SOX17 | qPCR | 100 | I–II: n = 39 III: n = 43 IV: n = 18 |
SEPT9: 8% SOX17: 11% |
Higher levels in stage IV | Significant association between RARβ2, SEPT9 and SOX17 and DSM |
| Leon Arellano et al., 2020 [45] | SEPT9 | qPCR | 10 | II: n = 4 III: n = 3 IV: n = 3 |
0.8 | NR | No significant association with recurrence ctDNA+: 1/5 recurrence ctDNA−: 0/2 recurrence |
| Jin et al., 2021 [46] | SEPT9 | qPCR | 82 | I: n = 5 II: n = 30 III: n = 40 IV: n = 7 |
0.89 | Higher levels for stage III and IV | No significant association with recurrence |
| Luo et al., 2020 [47] | Diagnostic score (cd-score) including 9 methylation markers | Targeted NGS + dPCR | 801 | I: n = 38 II: n = 139 III: n = 209 IV: n = 406 |
0.88 | Higher levels for stage III and IV | mHR for OS = 2.24 (SE 0.11) |
PCR: polymerase chain reaction. dPCR: digital PCR. ddPCR: digital droplet PCR. NGS: next-generation sequencing. qPCR: quantitative PCR. MASA: mutant allele-specific amplification. SSCP: single-strand conformation polymorphism. mHR: hazard ratio in multivariate analysis. uHR: hazard ratio in univariate analysis. DFS: disease-free survival. OS: overall survival. PFS: progression-free survival. RFS: recurrence-free survival. RR: relative risk. mRRD: RR of death in multivariate analysis. RFP: recurrence-free probability. MAF: mutant allele fraction. pMMR: proficient mismatch repair. DSM: disease-specific mortality. CSD: cancer-specific death.