Table 1.
Summary of ctDNA detection and prognostic significance in PDAC.
| Study | Biomarker | Source media | Sampling volume | Sampling point | Method of ctDNA detection | Detection technique | Cohort | Extracted cfDNA yield | % ctDNA detection (resectable cases) | % ctDNA detection (unresectable cases) | % ctDNA detection (total cohort) | Conclusions for OS | Conclusions for PFS |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Singh et al., 2020 | Prognostic | Plasma | – | – | Methylation | Real-time SYBR Green PCR (SPARC, UCHL1, NPTX2, PENK) | n = 65 | – | – | – | Methylation indices for all 4 genes higher in PDAC cases, compared to healthy individuals. | Higher ctDNA methylation indices for SPARC and NPTX2 associated with a poorer OS. | – |
| Strijker et al., 2020 | Prognostic | Plasma | 4 mL plasma | Pre-treatment | Mutation | Targeted NGS (34 amplicons panel covering KRAS, TP53, NRAS, SMAD4, CDKN2A, PIK3CA, GNAS, BRAF) | n = 58 | Median 3.2 ng/μL (range 0.58–23) | – | 44.80% | 44.80% | Median OS 3.2 months (95% CI 1.6–4.9) vs 8.4 (95% CI 1.6–15.1) months (detection vs undetectable ctDNA, respectively) (0 = 0.005). | – |
| Sugimori et al., 2020 | Prognostic, predictive | Serum | 2–3 mL serum | Pre-treatment + during treatment | Mutation | dPCR (KRAS codon 12/13) | n = 45 | – | – | 51% (baseline) | 51% (baseline) | – | Median PFS 248.5 vs 50 days (p < 0.001) for consistent detection vs absence of mutant KRAS ctDNA following chemotherapeutic treatment. |
| Bernard et al., 2019 | Predictive, prognostic | Plasma | 1 mL plasma | Pre-treatment + during treatment | Mutation | ddPCR (multiplex assay: KRAS G12D, G12V, G12R, G12C, G12S, G12A, G13D) | n = 194 | – | 34% (baseline) | 53% (baseline) | 44% (baseline) | Presence of ctDNA associated with shorter OS (HR, 2.36; 95% CI, 1.16–4.79; p = 0.018), with median OS of 258 vs 440 days (detection vs no detection, respectively). On multivariate analysis, ctDNA detection was a significant predictor of poorer OS in combination with CA19-9 >300 U/mL at pre-treatment baseline sampling (HR, 6.37; 95% CI, 2.36–17.24; p = 0.0003). | Presence of ctDNA associated with significantly shorter PFS (log-rank test: HR, 1.93; 95% CI, 1.15–3.22; p = 0.012). Median PFS of 118 vs 321 days (detection vs no detection). |
| Eissa et al., 2019 | Diagnostic | Plasma | 2 mL plasma | Pre-surgery | Methylation | Quantitative methylation-specific PCR (ADAMTS1, BNC1) | n = 39 | – | 97% | 100% | Methylation of either gene in combination panel: 97.3% (sensitivity) + 91.6% (specificity). Individual genes: ADAMTS1 87.2% (sensitivity) + 95.8% (specificity), BNC1 64.1% (sensitivity) + 93.7% (specificity). | – | – |
| Gall et al., 2019 | Prognostic | Plasma | – | Pre-surgery | Mutation | Targeted NGS | n = 16 | Mean 63.67 ± 24.37 ng/μL | 12.50% | – | 12.50% | – | – |
| Groot et al., 2019 | Prognostic | Plasma | 40 mL whole blood | Pre-surgery + post-surgery | Mutation | ddPCR (KRAS G12D, G12V, G12R, Q61H) | n = 59 | – | 49% (pre-surgery) | – | 49% (pre-surgery) | Pre-operative ctDNA detection vs absence: median OS 14 months vs median OS not reached (p < 0.001). Post-operative ctDNA detection vs absence: median OS of 17 months vs median OS not yet reached at 30 months (p = 0.011). | Median PFS 8 months vs 19 months (p < 0.001) for pre-surgery ctDNA detection vs absence. Median PFS of 5 months vs 15 months (p < 0.001) for post-surgical ctDNA detection vs. absence. Post-surgical ctDNA detection predicted clinical recurrence (sensitivity 90% (95% CI 74–98%), specificity 88% (95% CI 62–98%)) with median lead time of 84 days. |
| Lee et al., 2019 | Pharmacodynamic, prognostic | Plasma | 3.5 mL plasma | Pre-surgery + post-surgery | Mutation | Safe-SeqS (KRAS) | n = 42 | – | 62% (pre-surgery), 37% (post-surgery) | – | 62% (pre-surgery), 37% (post-surgery) | Pre-operative ctDNA detection associated with shorter OS at median follow-up (38.4 months): HR 4.1; p = 0.015. Post-operative ctDNA detection associated with shorter OS: HR 4.0; p = 0.003. | Pre-surgery ctDNA detection associated with significantly shorter PFS at median follow-up (38.4 months): HR 4.1; p = 0.002. ctDNA detection was significant predictor of disease recurrence (HR 6.3; 95% CI 2.4–16.2; p ≤ 0.0001) and death (HR 7.5; 95% CI 2.1–27.7; p = 0.002) during multivariate analyses. |
| Liu et al., 2019 | Diagnostic | Plasma | 5–10 mL plasma | Pre-treatment | Mutation/Fragment size | Targeted NGS (62-gene panel) | n = 80 | Median 16.2 ng/mL (range 9.3–25.9) | 88% | 95% | 90% | On multivariate analysis, mutant KRAS copy number was a significant predicter of poorer OS (HR: 3.3, 95% CI: 1.1–10.6; p = 0.037)). | – |
| Mohan et al., 2019 | Prognostic | Plasma | – | Pre-treatment | Mutation/Copy number | Targeted NGS (641-gene panel) | n = 55 | – | – | 62.5% (locally-advanced), 87% (metastatic) | 76% | Combined presence of KRAS ctDNA mutations and KRAS copy number gain associated with poorer overall prognosis (median survival 2.5 months, log-rank p-value < 0.0001). | – |
| Patel et al., 2019 | Pharmacodynamic, prognostic | Plasma | 10 mL whole blood | Pre-treatment/post-treatment | Mutation | Targeted NGS (54–73 gene panel) | n = 112 | 5–30 ng total yield | – | – | 70% | In univariate analysis, presence of KRAS mutations in ctDNA and percentage ctDNA abundance (≥0.6%) associated with poorer OS. | – |
| Pratt et al., 2019 | Diagnostic | Plasma | 1–2 mL plasma | Pre-treatment + post-treatment | Mutation | ddPCR | n = 7 | Median 3–49 ng/mL | – | 86% | 86% | – | – |
| Wang et al., 2019 | Diagnostic | Plasma | 1–4 mL whole blood | Pre-treatment | Mutation | ddPCR (KRAS codon 12, 13) | n = 95 | – | – | – | 47.40% | – | – |
| Watanabe et al., 2019 | Pharmacodynamic | Plasma | 2 mL plasma | Pre-treatment + post-treatment | Mutation | ddPCR (KRAS G12D, G12V, G12R, Q61H) | n = 78 | – | 48.70% | 71.80% | 62.80% | Post-operative emergence of KRAS mutant ctDNA (HR = 54.5, 95% CI: 6.64–447.6, p < 0.001 significant factor for poorer OS. Emergence of KRAS mutant ctDNA (HR = 10.4, 95% CI: 2.95–37.0, p < 0.001) was only significant factor for OS in unresectable patients. | Emergence of KRAS mutant ctDNA within 6 months of chemotherapy significantly associated with poorer PFS (median PFS: 14.9 months versus 4.8 months). |
| Wei et el. 2019 | Predictive | Plasma | – | Pre-treatment + post-treatment | Mutation | Targeted NGS (560-gene panel) | n = 38 | Median 28.4 ng/mL | – | – | 66% | Poorer prognosis observed in patients with ctDNA MAF >1.5%, compared to patients with <1.5% ctDNA MAF. | – |
| Berger et al., 2018 | Pharmacodynamic, predictive | Plasma | 2 mL plasma | Pre-treatment + post-treatment | Mutation | Targeted NGS (TP53, SMAD4, CDKN2A, KRAS, APC, ATM, FBXW7) and ddPCR | n = 20 | – | – | 80% | 80% | – | Combined ctDNA MAFs of KRAS and TP53 during treatment were significantly correlated with PFS (Spearman, r = −0.8609, p = 0.0013). |
| Cohen et al., 2018 | Diagnostic | Plasma | 7.5 mL plasma | Pre-surgery | Mutation + proteins | Targeted NGS (16-gene panel) | n = 93 | Median 7.54 ng/mL | – | – | 72% (mutations + proteins) | – | – |
| Hellwig et al., 2018 | – | Plasma | 8 mL plasma | – | Mutation/Fragment size | ddPCR, targeted NGS (128-gene panel) | n = 2 PDAC | 20.1 ± 14.5 ng/mL (yield across PDAC, colorectal, melanoma cohorts) | – | – | 100% | – | – |
| Kim et al., 2018 | Prognostic | Plasma | 1 mL plasma | Pre-treatment + post-treatment | Mutation | ddPCR (multiplex assay: KRAS G12D, G12V, G12R, G12C, G12S, G12A, G13D) | n = 77 | Median 427 ng/mL | 69% | 83% (locally-advanced), 86% (metastatic) | 78% | Low (≤41.5%) vs high ((>41.5%) KRAS ctDNA MAF associated with OS: 13 vs 8 months. Mutant KRAS ctDNA concentration identified as a prognostic factor for OS (HR 1.97, 95%CI 1.05–3.67). | High (>41.5%) vs low (<41.5%) KRAS ctDNA MAF associated with shorter PFS: 12.6 vs 4.7 months. Mutant KRAS ctDNA concentration identified as a prognostic factor for PFS (HR 2.08, 95%CI 1.20–3.63). |
| Kruger et al., 2018 | Predictive, prognostic | Plasma | – | Pre-treatment + during treatment | Mutation + proteins | dPCR (KRAS) | n = 54 | – | – | 67% | 67% | Presence of KRAS mutant ctDNA and higher pre-treatment levels of CA19-9, CEA and CYFRA 21-1 were significantly correlated with a poorer OS. | Serial measurement of KRAS mutant ctDNA during follow-up was superior to protein-based markers for detection of tumour progression: sensitivity (83%), specificity (100%). |
| Lapin et al., 2018 | Prognostic | Plasma | 4 mL (1–2 mL for 8 patients) | Pre-treatment + post-treatment | Fragment size analysis | Fragment size analysis | n = 61 | Locally advanced: median 3.26 ng/mL (range 1.16–7.98); Metastatic: median 6.58 ng/mL (range 0.53–1911.63) | – | – | cfDNA fragment size: healthy controls (median 176.5bp, range 168–185bp), locally-advanced PDAC (median 170bp, range 167–173bp), metastatic (median 167bp, range 148–180bp). Fragment sizes significantly larger in healthy controls vs locally-advanced (p = 0.001)/metastatic (p < 0.001) PDAC. | Short pre-treatment cfDNA fragment sizes (≤167 bp) were associated with poorer OS (4.6 months vs 10.5 months; log-rank p = 0.001). Pre-treatment cfDNA levels were independent predicter of poorer OS (HR = 2.236, p = 0.028). | Pre-treatment cfDNA levels were independent predicter of shorter PFS (HR = 3.049, p = 0.005). |
| Lin et al., 2018 | Predictive | Plasma | 2 mL plasma | Pre-treatment | Mutation | ddPCR (KRAS) | n = 65 | – | – | – | 80% | ctDNA detection vs absence: median OS 11.4 months vs 14.3 months (P < 0.001). On multivariate analysis, ctDNA presence identified as independent prognostic factor associated with poorer OS (HR = 3.1, 95% CI: 1.6–4.9, p < 0.001). | – |
| Mouliere et al., 2018 | – | Plasma | 2 mL plasma | Pre-treatment + post-treatment | Mutation/Fragment size | Fragment size analysis, sWGS, TAM-Seq, WES | n = 7 PDAC | – | – | – | 17% (across low-ctDNA cancers: glioma, renal, bladder, and pancreatic) | – | – |
| Nakano et al., 2018 | Predictive, prognostic | Serum | 1–4 mL serum | Pre-surgery + post-surgery | Mutation | PNA clamp PCR (KRAS codons 12, 13) | n = 45 | – | 24.4% (pre-surgery), 44.4% (post-surgery) | – | 24.4% (pre-surgery), 44.4% (post-surgery) | Change in KRAS mutation dynamics (pre-surgery wild-type to post-surgery mutant) significantly associated with poorer OS (HR 9.42, 95%CI 2.02–44.04, p = 0.004)). | – |
| Park et al., 2018 | Diagnostic, predictive | Plasma | 2–5 mL plasma | Pre-treatment + post-treatment | Mutation | Targeted NGS (83-gene panel) | n = 17 | – | – | – | 88.20% | – | – |
| Perets et al., 2018 | Pharmacodynamic, predictive | Plasma | – | During treatment | Mutation | Targeted NGS (KRAS exon 2) | n = 17 | – | – | 29.40% | 29.40% | Mutant KRAS ctDNA detection vs absence: 8 vs. 37.5 months. | – |
| Riviere et al., 2018 | Pharmacodynamic | Plasma | – | – | Mutation | Targeted NGS (68-gene panel, Guardant360) | n = 25 | – | – | – | 64% (known mutations), 100% (all mutations) | – | – |
| Shroff et al., 2018 | Predictive | Plasma | – | Pre-treatment | Mutation | Targeted NGS (62-gene panel, Foundation Medicine) | n = 16 | – | – | 69% | 69% | – | – |
| Adamo et al., 2017 | Prognostic | Plasma | – | Pre-treatment | Mutation | ddPCR, targeted NGS (50 gene-panel)) | n = 26 | Median 585 ng/mL (range 120–4180) | 17% | 40% | 35% | Presence of KRAS mutant ctDNA vs absence: 60 vs. 197 days. KRAS mutant ctDNA identified as prognostic factor for OS (HR 2.89 95%CI 1.2–7.3). | – |
| Ako et al., 2017 | Prognostic | Serum, plasma (paired) | 1 mL | Post-treatment | Mutation | ddPCR (KRAS G12D, G12V, G12R) | n = 40 | 17.9 ng/mL (plasma), 129 ng/mL (serum) | – | – | 48% (serum), 48% (plasma) | Presence of KRAS G12V alleles in serum or plasma ctDNA associated with poorer OS (p < 0.01). | – |
| Allenson et al., 2017 | Diagnostic | Plasma | 0.9–1.5 mL plasma | Pre-surgery + post-surgery | Mutation | ddPCR (multiplex assay: KRAS G12D, G12V, G12R, G12C, G12S, G12A, G13D) | n = 52 | – | 45.50% | 58% | 50% | Presence of KRAS mutant ctDNA vs absence (metastatic patients): 115 days vs. 506 days OS (p = 0.107). | – |
| Cohen et al., 2017 | Diagnostic | Plasma | 7.5 mL plasma | Pre-surgery | Mutation + proteins | Targeted NGS | n = 221 | Median 5.92 ng/mL (range 0.51–121.81) | 30% | – | 30% | ctDNA detection using combination assay was independent predictor of OS (HR = 1.76, 95% CI 1.10–2.84, p = 0.018, multivariate analyses). | – |
| Del Re et al., 2017 | Pharmacodynamic | Plasma | 3 mL plasma | Pre-treatment + during treatment | Mutation | ddPCR (KRAS (G12D, G12V, G12R, G13D)) | n = 27 | – | – | 70% (baseline) | 70% (baseline) | Increase vs reduction in KRAS mutant ctDNA abundance at day 15 follow-up (median OS 6.5 vs 11.5 months, p = 0.009). | Increase vs stability/reduction in KRAS mutant ctDNA abundance at day 15 follow-up (median PFS 2.5 vs 7.5 months, p = 0.03). |
| Henriksen et al., 2017 | Prognostic | Plasma | 500μL plasma | Pre-treatment | Methylation | Methylation-specific PCR (28-gene panel) | n = 95 | – | – | – | – | Decreased 6-month, 1-year and 2-year OS observed for patients with 0–10 hypermethylated genes in ctDNA (73% (95% CI; 61%–82%), 56% (95% CI; 43%–66%). | – |
| Pishvaian et al., 2017 | Prognostic | Plasma | 20 mL whole blood | Post-treatment | Mutation | Targeted NGS (68-gene panel, Guardant360) | n = 34 (n = 26 ctDNA analysis) | – | – | – | 73% | ctDNA detection vs absence: 11/24 deaths vs. 1/10 deaths (log-rank p = 0.045). | – |
| Song et al., 2017 | Diagnostic | Plasma | – | Pre-treatment + post-treatment | 5hmC analysis | 5hmC sequencing | n = 7 | – | – | – | Up-regulation and down-regulation of 5hmC genes (ZFP36L1, DCXR, GPR21, SLC19A3) in PDAC, compared to healthy controls. | – | – |
| Van Laethem et al., 2017 | Predictive | Plasma | – | During treatment | Mutation | dPCR (KRAS) | n = 60 | – | – | 65% | 65% | Presence vs absence of KRAS mutant ctDNA: median OS 6.6 months vs 18.2 months, respectively. | Presence vs absence of KRAS mutant ctDNA: median PFS 5.3 months vs 8.8 months, respectively. |
| Vietsch et al., 2017 | Pharmacodynamic, predictive | Plasma | 200μL plasma | Pre-surgery + post-surgery | Mutation | Targeted NGS | n = 5 | – | 100% (range 5–12 mutations in 14/56 genes assessed) | – | 100% | – | |
| Berger et al., 2016 | Diagnostic | Plasma | 2 mL plasma | – | Mutation | ddPCR (GNAS GNAS R201C, R201H, KRAS G12D/G12V) | n = 24 | Median 4.22 ng/μL ± 2.501 | – | 25% (GNAS), 42% (KRAS) | 25% (GNAS), 42% (KRAS) | – | – |
| Brychta et al., 2016 | Diagnostic | Plasma | 2 mL plasma | Pre-surgery | Mutation | Chip-based dPCR (KRAS codon 12) | n = 50 | Median 43.9 ng/mL (range 4.14–250) | 35% | – | 35% | – | – |
| Hadano et al., 2016 | Prognostic | Plasma | 1 mL plasma | Pre-surgery | Mutation | ddPCR (G12D, G12V, G12R) | n = 105 | – | – | – | 31% | Presence vs absence of KRAS mutant ctDNA: 13.6 vs. 27.6 months. Presence vs absence of KRAS mutant ctDNA identified as prognostic factor for OS (HR 3.2, 95%CI 1.8–5.4). | Presence vs absence of KRAS mutant ctDNA: 6.1 vs. 16.1 months PFS. |
| Henriksen et al., 2016 | Diagnostic | Plasma | 500μL plasma | Pre-treatment | Methylation | Methylation-specific PCR (28-gene panel) | n = 95 | Median 11.60 ng/mL (range 0.60–957.17) | – | – | Mean number of methylated genes in PDAC cases (8.41 (95% CI 7.62–9.20)) significantly higher than in acute/chronic pancreatitis controls (4.74 (95% CI 4.40–5.08)) (p < 0.001). Combined model (age >65 + hypermethylation frequencies of BMP3, RASSF1A, BNC1, MESTv2, TFPI2, APC, SFRP1 and SFRP2): 76% (sensitivity), 83% (specificity) for PDAC. | – | – |
| Pietrasz et al., 2016 | Prognostic | Plasma | 2 mL plasma | Pre-treatment | Mutation | ddPCR (KRAS G12D, V, R), targeted NGS | n = 135 | Mean 92 ± 201 ng/mL (resectable 52.5 ± 79.5, unresectable 105.8 ± 227.25) | 19% | 48% | 41% | Presence vs absence of ctDNA: (unresectable patients) 6.5 vs. 19 months OS (log-rank p < 0.001); (resectable patients) 19.3 vs. 32.2 months (p = 0.027). ctDNA detection identified as prognostic factor for OS (HR 1.96, 95%CI 1.2–3.2). | Presence vs absence of ctDNA: (resectable patients): 4.6 vs. 17.6 months (log-rank p = 0.03). |
| Takai et al., 2015 | Predictive | Plasma | 2 mL plasma | Pre-treatment | Mutation | ddPCR (KRAS G12D/V/R and G13D), targeted NGS (60-gene panel) | n = 259 | Median 20.13 ng/2 mL plasma (stage IV 21.65, stage I-III 17.59) | 8.30% | 47% | 32% | Presence of KRAS mutant ctDNA identified as prognostic factor associated with poorer OS (HR 3.04). | – |
| Zill et al., 2015 | Pharmacodynamic | Plasma | 1 mL plasma | Pre-treatment + during treatment | Mutation | Targeted NGS (54-gene panel) | n = 18 | – | – | 89% | 89% | – | – |
| Bettegowda et al., 2014 | Diagnostic | Plasma | 2 mL plasma | Pre-treatment | Mutation | dPCR, PCR/ligation, Safe-SeqS | n = 155 | – | ≥49% (localised) | >80% (metastatic) | ≥49% (localised), >80% (metastatic) | – | – |
Abbreviations: 5hmC, 5-hydroxymethylcytosine; CP, chronic pancreatitis; ddPCR, droplet digital PCR; NGS, next-generation sequencing; Safe-SeqS, Safe Sequencing System; TAM-Seq, tagged amplicon deep sequencing; sWGS, shallow whole genome sequencing; WES, whole exome sequencing.