Table 1.

Circulating tumor DNA studies in HCC and transplantation cases to evaluate minimal residual disease, cancer recurrence, and transplant rejection.

Study	Number of HCC Patients	Technique	Biomarkers	Outcomes
Liao et al., 2016 [104]	41	Illumina miSeq NGS	Hotspot mutations of TP53, TERT, and CTNNB1	-Tumor-associated mutations found in 8/41 (19.5%) of plasma samples. -ctDNA with mutations more commonly found in patients who suffered from vascular invasion (p = 0.041) and predicted a shorter recurrence-free survival (89 days) versus 365 for patients with no mutations in cfDNA from plasma. -There is no relationship between the presence of tumor-associated mutations and the concentration of ctDNA (p = 0.818).
Cai et al., 2019 [105]	34	Target sequencing and low-coverage whole-genome sequencing	ctDNA (harboring copy number variants [CNV] or single-nucleotide variants [SNV])	-CNVs and SNVs detected in plasma correlated with the patients’ tumor burden. -Comprehensive ctDNA mutation profiles correlated well with imaging results in accurately assessing patients’ tumor burden. -ctDNA detection could discover tumor recurrence and minimal residual disease before MRI imaging by a median of 4.6 months and outperformed other clinical biomarkers AFP, AFP-L3%, and des-gamma-carboxy prothrombin (DCP). -ctDNA detection can effectively predict patients’ prognostic outcomes for relapse-free survival (p = 0.0001) and overall survival (p = 0.0001).
Oh et al., 2019 [106]	151 HCC, 14 healthy controls	Low-depth whole-genome sequencing	CNV, VEGF amplification	-cfDNA concentrations were significantly higher in patients with HCC than healthy controls (0.71 vs 0.34 ng/µL, p < 0.0001). -High concentration of cfDNA was associated with HCC patients who did not achieve disease control, had a worse time to progression and overall survival. -VEGFA ratio was not significantly associated with sorafenib treatment outcomes.
Jiao et al., 2018 [107]	218 HCC, 81 cirrhotic	Droplet digital PCR (ddPCR)	TERT C228T and C250T promoter mutations	-TERT promoter mutations are detectable in plasma cfDNA in similar prevalence (47.4%) to those recorded in The Cancer Genome Atlas (44.4%). -Long-term imaging surveillance and cfDNA TERT promoter mutation assessment in patients with cirrhosis may act as potential early biomarkers for HCC.
Oversoe et al., 2020 [108]	95 HCC, 45 liver cirrhotic	ddPCR	TERT C228T mutation	-The TERT promoter mutation was detected in 44% of HCC and none in non-HCC patients. -TERT mutations found in cfDNA as opposed to tumor tissue were associated with increased mortality. -A positive correlation was found with TERT mutations found in the plasma with advanced TNM staging and vascular invasion.
Kim et al., 2020 [109]	107 HCC	Target deep sequencing, ddPCR	Deep sequencing of SNVs in 69 genes	-At least one SNV was found in 55.9% of all patients and the four most frequently observed SNVs found in ctDNA of HCC patients were MLH1 (13%), STK11 (13%), PTEN (9%), and CTNNB1 (4%). -The presence of the MLH1 SNV, in combination with increased ctDNA, predicted poor overall survival among 107 patients.
Hirai et al., 2021 [110]	130 HCC	ddPCR	TERT promoter mutation	-54.6% of HCC patients were positive for TERT promoter mutations, and the presence of these mutations was associated with large intrahepatic tumor size and high des-gamma carboxyprothrombin levels. -The presence of TERT promoter mutations in ctDNA is associated with short survival and may be a valuable biomarker for predicting the prognosis of patients with advanced HCC.
Shen et al., 2020 [111]	895 HCC	ddPCR, genomic sequencing	TP53 mutations (recurrent missense mutations R100L, V157F, A159P, and R249S)	-The R249s TP53 mutation encompassed 60.28% of all TP53 mutations. -The overexpression of the R249S TP53 mutation was associated with more malignant phenotypes, worsened overall survival, and progression-free survival than other recurrent TP53 missense mutations found in ctDNA from HCC patients.
Chan et al., 2013 [112]	26	Massively parallel bisulfite sequencing	Methylation density (MD) per 1 million base pairs (Mb) [bin], copy number aberrations (CNA)	-Tumor-associated CNAs and hypomethylation patterns can be detected in plasma as indicators for cancer detection and monitoring. -In plasma samples of HCC patients, a median of 34.1% of bins showed hypomethylation versus 0% of bins in healthy controls. -Serial analysis of plasma samples in an HCC patient found that elevation in hypomethylation and CNA percentages was associated with poor prognosis. -In one HCC patient, at the pre-operational stage, the % of bins showing hypomethylation and CNAs was 64.3% and 57%, respectively. Three days post-operation, the % of bins showing hypomethylation and CNAs was 75.5% and 11.7%, respectively. Two months post-operation, there was a continued increase in hypomethylation/CNAs, which was associated with cancer recurrence and the development of multiple lung metastases. -In another HCC patient, at post-operation, the hypomethylation and CNA levels were both 6.3%. Three- and 12-months post-resection, both parameters were undetectable which associated with clinical remission.
Tie et al., 2021 [125]	54 patients with colorectal cancer liver metastasis (CRLM)	Safe-sequencing (Safe-SeqS) assay	Somatic mutations in 15 genes recurrently mutated in CRC (SMAD4, TP53, AKT1, APC, BRAF, CTNNB1, ERBB3, FBXW7, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, RNF43, and POLE).	-Plasma samples from patients with resectable CRLM, including pre-and post-surgical samples, serial samples from pre-and post-operative chemotherapy, and serial samples in follow-up. -ctDNA was detectable in 85% of patients prior to treatment and 24% of patient’s post-surgery -End-of-treatment (surgery +/- adjuvant chemotherapy) ctDNA detection was associated with a 5-year recurrence-free survival of 0% compared to 75.6% for patients with undetectable end-of-treatment ctDNA. -Serial evaluation of ctDNA post-treatment may be an effective biomarker for HCC recurrence.
Hann et al., 2017 [113]	10 HCC	Bisulfite treatment of DNA and quantitative PCR, magnetic resonance imaging (MRI)	TP53 249T mutations and aberrant methylation of RASSF1A and GSTP1 genes	-Urine samples were collected prospectively from HCC patients after curative treatment, during follow-up visits. Urine DNA markers were compared to standard diagnostic methods (alpha-fetoprotein [AFP], MRI) for diagnosis of HCC recurrence. -MRI identified recurrence in 50% of HCC patients, and for 40% of recurrent patients in the study, urine DNA markers were elevated in urine samples nine months before MRI confirmation. -Urine cfDNA testing may be a highly sensitive, non-invasive tool to detect HCC recurrence post-treatment.
Wang et al., 2020 [114]	81 HCC	ddPCR	Four hotspot mutations: TP53-rs28934571 (c.747G > T), TRETrs1242535815 (c.1-124C > T), CTNNB1-rs121913412 (c.121A > G), and CTNNB1-rs121913407 (c.133T > C)	-70.4% (57/81) had detectable ctDNA before hepatectomy. -The positive pre-operative ctDNA status was related to large tumor size (p = 0.001), multiple tumor lesions (p = 0.001), microvascular invasion, advanced BCLC stages (p < 0.001), shorter disease-free survival (p < 0.001), and overall survival (p < 0.001). -Patients with an increased mutant allele frequency had more incidences of microvascular invasion (p = 0.016) and post-operative recurrence (p < 0.001).
Ono et al., 2015 [116]	46 HCC	Whole exome sequencing, PCR	ctDNA, a-fetoprotein (AFP), and des-g-carboxy prothrombin (DCP)	-ctDNA was detected in 7/46 patients before surgery and the levels increased in associated with disease progression. -Cancer recurrence and extrahepatic metastasis were significantly worse in the ctDNA-positive group versus that ctDNA-negative group (p = 0.0102 and p = 0.0386). -Multivariate analysis revealed that ctDNA (OR 6.10; 95% CI, 1.11–33.33, p = 0.038) is an independent predictor of microscopic vascular invasion of the portal vein.
Long et al., 2020 [115]	82 HCC patients	Fluorometric Qubit dsDNA BR assay kit	Cell-free dsDNA	-82 HCC patients underwent liver surgery and post-operative blood samples were collected. -cfDNA low and high groups had median recurrence times of 19.5 months and 14 months, respectively (p = 0.023). -Multivariate analysis revealed that post-operative cfDNA, tumor number, and microvascular invasion (p < 0.05) were independent risk factors for recurrence in operable HCC.
Lo et al., 1998 [124]	8 female transplant patients	PCR, gel electrophoresis	Y-chromosome specific genetic sequences	-In six female liver-transplant recipients with male donors, plasma and cellular chimerism were found in six (100%) and five (83%) patients, respectively.
Lehmann-Werman et al., 2018 [117]	18 transplant patients	Bisulfite conversion, PCR, and massively parallel sequencing	3 genomic loci, adjacent to the ITIH4, IGF2R, and VTN genes, which were unmethylated in the liver compared with other tissues and cell types.	-Elevations of hepatocyte-specific cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and in healthy individuals after partial hepatectomy. -Patients with sepsis also had high levels of hepatocyte-specific cfDNA that also correlated with elevations in liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT).
Ng et al., 2019 [118]	2 transplant patients diagnosed with propionic acidemia	Amplification refractory mutation system PCR (ARMS-PCR)	Graft-derived cell-free DNA (Gcf-DNA), liver enzymes (alanine transaminase [ALT], aspartate transaminase [AST])	-5 mL whole blood specimens were collected at six specific time points (day 0, 1, 7, 14, 30, 60). -Gcf-DNA levels were the highest on day 1 post-transplantation due to ischemia and reperfusion injury and then declined from day 7–60 due to recovery. -The levels of Gcf-DNA and liver function enzymes had similar change-tendency curves. -The ARMS-PCR method can detect Gcf-DNA without knowledge of donor information.
Macher et al., 2016 [119]	17 transplant patients	RT-PCR	Rh gene	-Rh gene found in circulating DNA was quantified by RT-PCR, at day 0 of liver transplantation and during the stay at the intensive care unit. -Patients with no complications and patients that accepted the liver transplant but had other medical complications had low levels of RH gene at follow-up, with elevations of the gene that were associated with clinical complications. -Patients that had liver transplant rejection had an associated increase in the Rh gene in cfDNA.
Beck et al., 2013 [120]	Stable liver (n = 10), heart (n = 8), and kidney (n = 9) transplant recipients, and seven additional patients directly after transplantation	ddPCR	SNPs from graft-derived cell-free DNA (GcfDNA)	-The GcfDNA in stable transplant patients was 6.8% (liver), 2.5% (kidney), and 3.4% (heart). -On the day of a liver transplant, the GcfDNA was approximately 90% and by day 10, it was 15% in complication-free liver transplant recipients. -In 2 patients with biopsy-proven rejection, GcfDNA increased to >60%.
Schutz et al., 2017 [121]	115 liver transplant patients	ddPCR	SNP loci with known high population minor allelic frequency	-GcfDNA was increased >50% on post-operation day 1, likely from ischemia/reperfusion injury, and rapidly declined in patients without graft injury within 7–10 days to a median <10%, where it remained for one year. -Liver function tests (LFTs) had a low overall correlation (r = 0.28–0.62) with GcfDNA. -The diagnostic sensitivity and specificity were 90.3% (95% CI 74.2–98%) and 92.9% (95% CI 89.3–95.6%), respectively for GcfDNA at a threshold value of 10%.
Ng et al., 2019 [122]	11 liver transplant recipients	Y-chromosome capture methodology and sequencing read lengths	GcfDNA was defined by DNA fragment sizes (105–145 bp, 160–170 bp). The ratio of short fragments/long fragments (S/L) were calculated.	-High S/L ratio was associated with an early trend toward graft injury when compared to routine liver function enzymes (ALT/AST) and GcfDNA. -The high S/L ratio was significantly associated with ALT (p < 0.0001) and AST (p < 0.0001) during flow-blown rejection. -Size profiles of GcfDNA in patient’s post-liver transplant may be a potential biomarker to monitor graft function.
Goh et al., 2019 [126]	20 liver transplant recipients	ddPCR	Deletion/insertion polymorphisms	-Post-transplant donor-specific cell-free DNA (dscfDNA) was measured in the plasma of transplant recipients. -dscfDNA was serially measured at days 3, 7, 14, 28, and 42. -There was an exponential decrease in dscfDNA in patients who underwent a LT without complications. -DscfDNA was higher in patients with biopsy-proven acute rejection compared to those without rejection. -The area under the receiver operator curve of DscfDNA was higher than that of routine LFTs for acute rejection (DscfDNA: 98.8% with 95% CI 95.8–100%, ALT: 85.7%).
Ng et al., 2018 [123]	Two liver transplant recipients	PCR of Y-chromosome specific genes	ALT, AST, and GcfDNA	-The trend of GcfDNA levels was comparable to routine LFTs to evaluate graft injury. -Limitations of this study were the small sample size and the results apply only to donor-recipient-sex-mismatch pairs.