Table 2.
Studies on Use of Cell-Free DNA for Hepatocellular Carcinoma Detection
| Study | cfDNA property | HCC patients | Control | Comparator, AFP cut-off value, ng/mL | Findings: sensitivity/specificity, AUC |
|---|---|---|---|---|---|
| Amount or integrity | |||||
| Chen et al,60 2013 | Total amount | N = 39 Stage: multifocal disease in 13% Treatment: NR |
45 healthy | NS | Sensitivity: cfDNA: 56.4% AFP: 53.8% cfDNA + AFP: 71.8% (P < .05 for 3-way comparison) cfDNA + AFP + α-L-fucosidase vs cfDNA + AFP: 89.7% (P < .05) |
| El-Shazly et al,63 2009 | Total amount, integrity | N = 25 Stage: 12%/32%/48%/8% TNM I/II/III/IV Treatment: NR |
25 CLD, 15 healthy | 20 | HCC vs CLD comparison: Total DNA amount: 72%/68%, AUC, 0.57 DNA integrity: 88%/92%, AUC, 0.75 |
| Huang et al,68 2012 | Total amount | N = 72 Stage: 24%/76% TNM e I+II/III+IV Treatment: NR |
37 CLD, 41 healthy | 400 | Total cfDNA amount: HCC vs CLD: 60%/78%, AUC, 0.71 HCC vs healthy: 90%/90%, AUC, 0.95 Total DNA + AFP (HCC vs healthy): 95%/94%, AUC, 0.97 |
| Huang et al,66 2016 | cfDNA integrity | N = 53 Stage: 24%/76% TNM I+M/III+IV Treatment: 100% resection |
15 CLD, 22 healthy | 20 | cfDNA integrity: 43.4%/100%/0.71 AFP: 50.9%/100%/0.61 cfDNA integrity + AFP: 79.2%/100%/0.85 |
| Iizuka et al,71 2006 | Total amount | N = 52 Stage: 46%/37%/17% TNM I/II/III Treatment: 100% resection |
30 CLD, 16 healthy | AFP: 10.2 DCP: 29.5 ng/mL |
AFP: 69.2%/72.7% DCP: 73.1%/75.0% cfDNA amount: 69%/93% (P < .05 vs both AFP and DCP) |
| Marchio et al,80 2018 | Total amount, TP53 R249S mutation by digital droplet PCR | N = 149 Stage/treatment: NR | 164 CLD, 49 healthy | 10 | AUC: Proportion of droplets with TP53 R249S: 0.83 AFP: 0.81 (P > .05 vs TP53 R249S droplets) cfDNA amount: 0.59 |
| Piciocchi et al,82 2013 | Total amount | N = 66 Stage: 59% within Milan criteria Treatment: NR |
76 CLD | 14 | cfDNA: 91%/43%, AUC, 0.69 AFP: 45%/83%, AUC, 0.64 (P > .05) |
| Ren et al,84 2006 | Total amount, chromosome 8p allelic imbalance (D8S258 or D8S264) | N = 79 Stage: 62%/38% TNM I+II/III+IV Treatment: NR |
20 CLD, 20 healthy | 20 | Total amount: HCC vs healthy, 52%/95%, AUC, 0.80 Allelic imbalance at D8S258: sensitivity, 57% for TNM stage III/IV and 22% for stage I/II High cfDNA concentration + allelic imbalance: abnormal in 8 of 24 with low AFP |
| Yan et al,93 2018 | Total amount | N = 24 Stage: 58%/33%/8% BCLC A/B/C Treatment: NR |
62 CLD | 80.5 | cfDNA amount: 62.5%/93.6%, AUC, 0.82 AFP alone: AUC, 0.67 cfDNA amount + AFP + age: 87%/100%, AUC, 0.98 (P < .05) |
| Mutations | |||||
| An et al,56 2019 | cfDNA mutations | N = 26 Stage: 77%/23% TNM I/II+III Treatment: 100% resection |
20 CLD | NS | AUC of different tests: cfDNA concentration: 0.917 Mutation number: 0.878 ctDNA concentration (cfDNA concentration times variant allele frequency): 0.871 Maximal variant allele frequency: 0.802 AFP: 0.783 |
| Cai et al,57 2019 | Fraction of single-nucleotide or copy number variants | N = 34 Stage: NR Treatment: 100% resection |
N/A | NR | ctDNA: sensitivity, 100% AFP: sensitivity, 56% AFP-L3%: sensitivity, 50% DCP: sensitivity, 82% |
| Igetei et al,69 2008 | TP53 R249S mutation | N = 85 Stage/treatment: NR |
77 healthy | 400 | cfDNA TP53 R249S mutation: Overall: 7.6%/100% Patients with HCC and AFP measurements: 16.7% overall, 20% without increased AFP (P > .05) |
| Liao et al,78 2016 | TERT, CTNNB1, or TP53 mutation | N = 41 Stage: 42% ≥5 cm, 27% multiple tumors, 61% vascular invasion Treatment: 100% resection |
10 healthy | 20 | cfDNA mutations: sensitivity, 23% vs 13% in high vs low AFP (P = .70) Specificity, 90% |
| Qu et al,83 2019 | Integrated hepatitis B virus DNA and mutations in TP53, CTNNB1, AXIN1, and TERT promoter Age, sex, AFP, and DCP also included |
Training: N = 65 Validation: N = 24 |
Training: 70 CLD Validation: 307 CLD |
None | Training cohort: patients with (+) AFP or ultrasound 93%/93%, AUC, 0.93 Both the cfDNA and protein markers contributed predictive power Validation cohort: 331 patients with (−) AFP and ultrasound screening sensitivity, 100%, specificity only 4 of 24 |
| Xiong et al,90 2019 | Mutations in TP53, ARID1A, FLCN, SETD2, PTEN, BUB1B, CTNNB1, JAK1, AXIN1, EPS15, or CACNA2D4 | N = 37 Stage: NR Treatment: 100% resection |
6 healthy | 400 | cfDNA mutations: Overall: 65%/100% Low AFP: 73%/100% High AFP: 53%/100% |
| Xu et al,91 2015 | Copy number variation of 1q, 7q, or 19q forward strand or 1p, 9q, or 14q reverse strand | N = 31 Stage: mean maximum tumor size, 5.7 cm (SD, 3.1 cm) Treatment: 100% resection |
8 CLD | 10 | Copy number variation score: All HCC: 83.9%/100% HCC <5 cm maximum tumor size: 68.8%/100% HCC <3 cm maximum tumor size: 57.1%/100% Low AFP: sensitivity, 7 of 10 |
| Methylation/epigenetics | |||||
| Cai et al,57 2019 | 5hmC modifications in cfDNA | N = 1204 Stage: 12%/36%/25%/13% BCLC 0/A/B/C Treatment: NR |
392 CLD, 958 healthy | 20 | Early stage HCC vs CLD: 5hmC-based score: 82.7%/67.4%, AUC, 0.87 in training set, 0.85 in validation set AFP: 44.8%/76.1%, AUC, 0.79 in training set, 0.69 in validation set |
| Chan et al,59 2008 | RASSF1A methylation | N = 63 Stage: NS Treatment: 100% resection |
63 CLD, 50 healthy | 20 |
RASSF1A methylation detected in: 93% HCC (50% among normal AFP) 58% CLD 8% healthy controls |
| Chu et al,61 2004 | P16 methylation | N = 46 Stage/treatment: NR |
23 CLD | 20 |
P16 methylation: Overall cohort: 48%/83% Normal AFP HCC: sensitivity, 50% |
| Dou et al,62 2016 | CDH1, DNMT3b, or ESR1 promoter methylation | N = 183 Stage/treatment: NR |
173 CLD, 50 healthy | NS | Methylation frequency: HCC: CDH1 31%, DNMT3b 41%, ESR1 30% CLD: <10% for all 3 genes Healthy controls: 0% AUC for methylation of any gene, 0.75 (0.70–0.80) AUC for AFP, 0.62 (0.55–0.68) |
| Han et al,64 2014 | TGR5 promoter methylation | N = 160 TNM stage: 59%/41% I+II/III+IV Treatment: NR |
88 CLD, 45 healthy | 200 | HCC vs CLD: TGR5 methylation + AFP: 68.1%/78.4% AFP alone: 30.6%/92.1% TGR5 alone: 48.1%/86.3% |
| Hu et al,65 2017 | UBE2Q1 methylation | N = 80 TNM stage: 43%/57% I+II/III+IV Treatment: NR |
80 CLD, 20 healthy | 200 |
UBE2Q1 methylation + AFP: 53.8%/87.5%, AUC, 0.76 UBE2Q1 methylation alone: 66%/58%, AUC, 0.62 AFP alone: 53.8%/87.5%, AUC, 0.67 |
| Huang et al,67 2014 | INK4A methylation | N = 66 Stage: 24%/23%/27%/26% TNM I/II/III/IV Treatment: NR |
43 CLD | 200 |
INK4A methylation + AFP: sensitivity, 80.3% (P < .05 vs AFP alone) AFP alone: sensitivity, 45.5% INK4A methylation alone: sensitivity, 74.2% |
| Iizuka et al,70 2011 | SPINT2 and SRD5A2 methylation | N = 220 Stage: 15%/34%/30%/21% TNM I/II/III/IV Treatment: NR |
202 CLD | AFP: 20 DCP: 40 mAU/mL |
Methylation of SPINT2 and SRD5A2, AFP, and DCP: 82%/82% AUC, 0.72 for ≤5 cm HCC and 0.89 for >5 cm HCC AFP alone: 57.4%/85.7% DCP alone: 60.2%/89.3% |
| Ji et al,72 2014 | MT1M and MT1G methylation | N = 121 Stage: 53%/47% TNM I+II/III+IV Treatment: 100% noncurative |
37 CLD, 31 healthy | 20 |
MT1M methylation: 49% HCC, 5% CLD, 7% healthy controls MT1G methylation: 70% HCC, 16% CLD, 13% healthy controls MT1M or MT1G methylation: HCC vs CLD: 90%/81%, AUC, 0.86 HCC vs healthy: 91%/84%, AUC, NR AFP alone: HCC vs healthy: 56.0%/62.1% |
| Kanekiyo et al,73 2015 | RASSF1A, CCND2, CFTR, SPINT2, SRD5A2, and/or BASP1 methylation | N = 125 Stage: 46%/54% TNM I+II/III+IV Treatment: 100% resection |
N/A | AFP: 20 DCP: 40 ng/mL |
Serum methylation score: Positive in 41% vs 48% of high vs low AFP Positive in 42% vs 46% of high vs low DCP (P > .05 for both) |
| Kisiel et al,74 2018 | Methylation score: HOXA1, EMX1, ECE1, AK055957, PFKP, CLEC11A | N = 116 Stage: 4%/44%/15%/30%/7% BCLC 0/A/B/C/D Treatment: NR |
80 CLD, 98 healthy | 10 | Methylation score: HCC vs cirrhosis: 95%/86%, AUC, 0.93–no improvement after adding AFP HCC vs healthy: 95%/95% Sensitivity based on cancer stage: 75% (BCLC stage 0), 93% (stage A/B), and 100% (stage C/D) |
| Kuo et al,75 2014 | HOXA9 methylation | N = 40 Stage/treatment: NR |
34 healthy | 10 |
HOXA9 methylation: 73%/97% HOXA9 methylation + AFP: 95%/97% |
| Li et al,76 2014 | IGFBP7 promoter methylation | N = 136 Stage: 51%/49% TNM I+II/III+IV Treatment: NR |
46 CLD, 35 healthy | 20 |
IGFBP7 promoter methylation + AFP: 85%/41% (P < .05 for both sensitivity and specificity vs AFP) IGFBP7 methylation alone: 65%/83% AFP alone: 57%/52% |
| Li et al,77 2018 | IGFBP7 promoter methylation | N = 155 Stage: 63%/37% TNM I+II/III+IV Treatment: 100% resection |
60 CLD, 20 healthy | 200 | Serum IGFBP7 promoter methylation: sensitivity, 69% vs 67% in high vs low AFP (P = .81) |
| Lu et al,79 2017 | Methylation score: APC, COX2, RASSF1A, miR-203 | N = 203 Stage: 89%/11% TNM I+II/III+IV Treatment: 100% resection |
104 CLD, 50 healthy | 20 | HCC vs controls: 84%/83%, AUC, 0.87 Sensitivity, 75% of patients with low AFP |
| Oussalah et al,81 2018 | SEPT9 methylation | Derivation cohort (France): N = 51, BCLC 25%/39%/31%/2% A/B/C/D Validation cohort (Germany): N = 47, BCLC stage 39%/22%/15%/24% A/B/C/D |
Derivation cohort: 135 CLD Validation cohort: 56 CLD |
NS |
SEPT9 methylation: 85%/91%, AUC, 0.96 AFP alone: AUC, 0.85 (P = .002 vs SEPT9 methylation) |
| Sun et al,85 2013 | TFPI2 methylation | N = 43 Stage: 40%/19%/33%/9% TNM I/II/III/IV Treatment: NR |
24 CLD, 26 healthy | 400 |
TFPI2 methylation or AFP alone: similar sensitivity (47% vs 54%) TFPI2 methylation + AFP: sensitivity, 61% (P < .05) |
| Tangkijvanich et al,86 2007 | LINE-1 hypomethylation | N = 85 Stage: 82% >5 cm, 36% multiple tumors Treatment: various |
93 CLD, 30 healthy | 400 | LINE-1 hypomethylation: sensitivity in high vs low AFP (61.7% vs 52.6%), P > .05 |
| Wang et al,87 2006 | GSTP1 methylation | N = 32 Stage: NR Treatment: 100% resection |
8 CLD | N/A | Methylated cfDNA GSTP: sensitivity, 50% (61% among patients with tissue GSTP1 methylation), specificity, 100% Methylation detected in 4 of 9 patients with low AFP |
| Wei et al,88 2018 | SOCS3 promoter methylation | N = 119 Stage: 34% tumor >5 cm, 17% >1 tumor, 17% portal vein involvement Treatment: 100% resection |
157 CLD, 50 healthy | 400 |
SOCS3 cfDNA methylation: Overall: 28.6%/95% High vs low AFP (52.9% vs 10.3%), P < .001 |
| Wen et al,89 2015 | Methylation score: RGS10, ST8SIA6, RUNX2, VIM, CACNA1C, TBX2, SOX9 (5’ end), NEDD4L (intron), ALX3, ZNF683 (3’ end), KCNQ4 (i), ERG, PTPN18 (intron), SYN2, LINC00682 (3’ end), CPLX1 (intron), FLJ42709, UBD (3’ end), SNX10 (3’ end), TRPS1 (intron) | N = 36 Stage: 36%/25%/22%/17% TNM I/II/III/IV Treatment: NR |
17 CLD, 38 healthy | 20 | Two cfDNA methylation scores, either score positive: Training set: 93%/91% Validation set: 100%/80% Combined cohort: 94%/89% Sensitivity, 100% in patients with low AFP (N = 10) |
| Xu et al,92 2017 | Methylation score: cg10428836, cg26668608, cg25754195, cg05205842, cg11606215, cg24067911, cg18196829, cg23211949, cg17213048, cg25459300 |
N = 1098 Stage: 16%/16%/52%/12% TNM I/II/III/IV Treatment: NR |
835 healthy | 25 | cfDNA levels: Training set: 85.7%/94.3%, AUC, 0.97 Validation set: 83.3%/90.5%, AUC, 0.94 AFP: AUC, 0.82 (P < .05 vs cfDNA) |
| Yeo et al,94 2005 | RASSF1A methylation | N = 40 Stage: 30% ≥5 cm Treatment: 100% resection |
10 healthy | 20 |
RASSF1A methylation Overall: 43%/100% Low AFP: sensitivity, 36% |
NOTE. Test characteristics are reported either as sensitivity (%)/specificity (%), area under the receiver operating characteristic curve; sensitivity (%)/specificity (%); or as individual parameters.
5hmC, 5-hydroxymethylcytosine; AFP, α-fetoprotein; AUC, area under the receiver operating characteristic curve; BCLC, Barcelona Clinic Liver Cancer; CLD, chronic liver disease; cfDNA, cell-free DNA; ctDNA, circulating tumor DNA; DCP, des-carboxyprothrombin; HCC, hepatocellular carcinoma; NR, not reported; PCR, polymerase chain reaction.