Tumor-informed circulating tumor DNA assay for surveillance post-liver transplantation in patients with hepatocellular and cholangiocarcinoma

Abstract

Background

Liver transplantation (LT) for primary liver cancers achieves excellent patient outcomes, but a minority recur with poor prognosis. Survival may be improved by earlier recurrence detection. This study aims to evaluate the feasibility and performance of a personalized tumor-informed assay utilizing circulating tumor DNA (ctDNA) from peripheral blood for surveillance after LT in patients with hepatocellular carcinoma (HCC) or cholangiocarcinoma (CCA).

Methods

Here, we test whether a personalized tumor-informed assay utilizing ctDNA from peripheral blood informs post-LT surveillance. Personalized ctDNA assays were employed for surveillance in 38 LT recipients, alongside standard-of-care imaging and peripheral tumor biomarkers [alpha-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9)].

Results

Recurrence was detected radiologically in 6 patients, with positive ctDNA assays in 3 and negative/insufficient to process (ITP) in 3. Nine ITP ctDNA assays were due to insufficient primary tumor tissue. In 31 patients without ITP ctDNA tests, the sensitivity and specificity of ctDNA were 75% [95% confidence interval (CI): 19–99%] and 100% (95% CI: 87–100%). Standard-of-care tumor biomarkers had sensitivity and specificity of 75% (95% CI: 19–99%) and 93% (95% CI: 76–99%), respectively (P>0.99 and P=0.16; McNemar χ²). Only 1 patient had ctDNA positive prior to imaging-based diagnosis.

Conclusions

This study corroborates the feasibility of ctDNA assays for recurrence surveillance in LT recipients. The results imply that ctDNA assays show promise in confirming recurrence and minimizing the need for invasive biopsy. However, additional prospective studies are needed to confirm ctDNA test utility in surveillance protocols.

Highlight box.

Key findings

• This study evaluated a personalized tumor-informed circulating tumor DNA (ctDNA) assay for surveillance after liver transplantation (LT) in 38 patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Recurrence was detected in 6 patients, with ctDNA positive in 3 and negative or insufficient to process (ITP) in 3. Sensitivity of ctDNA was 75% and specificity was 100%, compared to 75% sensitivity and 93% specificity for standard biomarkers like alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9). Only one patient had ctDNA positivity before imaging confirmed recurrence.

What is known and what is new?

• Post-LT surveillance for HCC and CCA typically relies on imaging and tumor markers like AFP and CA19-9, which have limited sensitivity and specificity. ctDNA, a non-invasive biomarker, has shown promise in other cancers but lacks robust data in LT settings.

• This study is the first to prospectively assess serial ctDNA testing post-LT, demonstrating its feasibility and high specificity, though with comparable sensitivity to existing methods.

What is the implication, and what should change now?

• The high specificity of ctDNA suggests it could confirm recurrence, potentially reducing invasive biopsies when imaging or biomarkers are inconclusive. However, its limited sensitivity and infrequent early detection indicate it should complement, not replace, current surveillance. Larger prospective studies are needed to refine its role, optimize testing frequency, and address ITP rates, particularly in patients with prior locoregional therapy.

Introduction

Primary liver malignancies have rising incidence and mortality rates in the United States and globally (1,2). Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common types. With best palliative management alone, these have 5-year overall survival (OS) probability of under 5% (3,4). Multi-modal therapy including liver transplantation (LT) offers excellent treatment for both localized HCC (5) and CCA (6,7), as per the National Comprehensive Cancer Network (NCCN) and American Association for the Study of Liver Diseases (AASLD) guidelines (8-11), with 5-year OS probabilities of greater than 50%. However, post-LT 5-year recurrence rates are reported at greater than 15% for HCC and 40% for CCA patients. Recurrence is associated with poor prognosis and is the most common cause of death in these patients. Patients who recur have a median post-recurrence survival of under 1 year, due in part to limited treatment options in immunosuppressed LT recipients (12-15).

There is limited evidence on the role, modality, and frequency of surveillance post-LT for HCC and CCA recurrence. The NCCN, AASLD, and International Liver Transplantation Society (ILTS) (16,17) guidelines recommend circulating tumor markers, alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA19-9), and multiphasic cross-sectional imaging [i.e., computed tomography (CT) or magnetic resonance imaging (MRI)] every 3–6 months for 2 years then every 6 months for up to 5 years. One retrospective, multicenter study reported that greater use of surveillance imaging was associated with improved post-recurrence survival, though the threshold for “high frequency” was only 3 imaging studies in the first 24 months post-LT (17-19). More so, currently recommended circulatory markers, including AFP and CA19-9, have well-noted limitations in both sensitivity and specificity (20-22).

Circulating tumor DNA (ctDNA) in peripheral blood has been studied as a non-invasive biomarker for recurrence post-curative intent therapy in multiple cancers, especially breast (23) and colorectal (24-27). Further, ctDNA detection may inform the use of adjuvant and palliative systemic therapies (25,28), but not local therapies that require confirmatory imaging (29). In HCC and CCA, the current evidence for ctDNA is limited to retrospective series which only suggest a role in informing prognosis (30). Thus, integration of ctDNA testing into peri-LT care is not currently recommended by guidelines for primary liver cancers (8-11,31,32). The purpose of this study was to evaluate the feasibility of a personalized, tumor-informed ctDNA assay for post-LT recurrence surveillance in HCC and CCA patients, and to evaluate its performance relative to standard-of-care modalities. We present this article in accordance with the STARD reporting checklist (available at https://jgo.amegroups.com/article/view/10.21037/jgo-24-791/rc).

Methods

Patient population

Patients undergoing LT between April 2018 and February 2023 at a single center were identified prospectively. The inclusion criterion was histologic diagnosis of primary liver cancer. Clinical criteria including demographics, pathology, treatment dates, time to recurrence, and time to last follow-up were extracted from the electronic medical records. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the Houston Methodist Hospital (No. PRO00035386). Informed consent was waived due to the use of de-identified data collected as part of routine clinical care.

Standard of care surveillance testing

Multiphasic cross-sectional imaging, AFP, and CA19-9 were obtained every 3 months for 2 years then every 6 months up to 5 years post-LT.

Tumor-informed ctDNA testing

Tumor-informed ctDNA testing was ordered post-transplant at the discretion of each patient’s treatment teams. The intention was to obtain these tests every 3 months, analogous to standard-of-care testing.

The cancer diagnosis was confirmed by histopathological examination of the explanted liver. Representative formalin-fixed, paraffin-embedded (FFPE) tumor tissue from the explanted liver underwent whole exome sequencing. From this, a set of up to 16 somatic, clonal single nucleotide variants (SNVs) were selected per patient to design a personalized, tumor-informed ctDNA assay [Signatera^TM, bespoke multiplex (m) polymerase chain reaction (PCR)-next-generation sequencing], as previously described (24). Peripheral blood samples were collected from patients following LT. Peripheral blood samples with ≥2 SNVs detected were defined as ctDNA-positive. All biological samples were processed at Natera, Inc. (Austin, Texas, USA), following Clinical Laboratory Improvement Amendments (CLIA)-validated standard operating procedures. Timing of blood draws was intended to be performed every 3 months for 2 years then every 6 months for up to 5 years post-LT.

Outcomes

True recurrence was defined as any new lesion on CT or MRI with appropriate features, as reported by a board-certified clinical radiologist, and subsequently confirmed clinically by a multi-disciplinary team, with or without tissue confirmation. Positive AFP and CA19-9 were defined as an abnormally elevated level above 8.3 and 35 ng/mL, respectively, in the absence of other possible causes (e.g., biliary obstruction). AFP and CA19-9 values were tracked serially post-transplant. For patients with positive ctDNA tests, the post-transplant AFP and CA19-9 values up to the date of the positive ctDNA blood draw were considered. For patients with serially negative ctDNA tests, post-transplant values were considered up to the latest date of ctDNA blood draw. Positive ctDNA assay results were defined by the manufacturer. Test outcomes were categorized as successful (negative or positive) and insufficient to process (ITP). Primary outcomes were test parameters for ctDNA, AFP, and/or CA19-9. Secondary outcomes included observed ctDNA testing frequency, turnaround time, and ITP assay rate.

Statistical analysis

Patient characteristics were summarized according to test outcomes. All data were presented as median and interquartile range (IQR) for continuous variables, and number and % for categorical variables. Fisher’s exact test for categorical variables and Kruskal-Wallis test for continuous variables were used to compare between the test successful and ITP. Sensitivity, specificity, positive and negative predictive values were calculated and presented as percentage [95% confidence interval (CI)] (33). Statistical significance of differences in sensitivity and specificity between AFP, CA19-9, and ctDNA were determined by McNemar’s test. All analyses were performed in R (R Foundation for Statistical Computing, Vienna, Austria). Statistical significance was defined as two-tailed P<0.05 for all tests.

Results

Cohort

During the study period, 38 patients with primary liver cancer underwent LT followed by surveillance with ctDNA at a single institution (Table 1, Figure 1). No patients were excluded from downstream analyses. At the time of transplant, the median age was 63 years (IQR, 58–66 years), body mass index (BMI) 28.2 kg/m² (IQR, 25.6–35.6 kg/m²), and laboratory Model for End-Stage Liver Disease (MELD) score of 14 (IQR, 9–30). Most patients were male (n=28, 73.7%), White (19, 50%), had chronic viral infections (12, 31.6%) or metabolic dysfunction-associated steatohepatitis (MASH) (12, 31.6%), and presented from home for LT (27, 71.1%). The median waitlist time was 284 days (IQR, 126–465 days). Thirty-one (81.6%) patients underwent pre-LT therapy, including loco-regional (29, 76.3%) and/or systemic (9, 23.7%). One (2.6%) had pre-LT hepatectomy. All patients underwent LT, with 3 (7.9%) receiving simultaneous kidney transplants. Eight (21.1%) patients received adjuvant systemic therapy. This included 4 patients with CCA and 1 with mixed tumor who received 5FU or gemcitabine for eight cycles, and 3 with HCC who received sorafenib or lenvatinib for 6 months. This center’s practice is to give adjuvant therapy to all CCA and mixed tumor patients with 5-fluorouracil (5-FU) or gemcitabine, informed by the BILCAP trial (34), and to HCC patients with findings on final pathology concerning for high recurrence risk, including greater than 3 nodules, maximum size greater than 5 cm, and lympho-vascular invasion. Median recurrence-free survival (RFS) was 583 days (IQR, 368–931 days), and OS was 728 days (IQR, 411–1,087 days) (Figure 2A,2B). Six (15.8%) patients had recurrences. Sites of initial recurrence included the liver allograft (n=3), lung (n=2), and bone (n=2). Three (7.9%) patients died, 2 of malignancy and 1 of respiratory failure.

Table 1. Clinical, pathologic, and outcome variables of patients undergoing ctDNA surveillance for primary liver cancer recurrence after liver transplantation.

Variable	n	Total	Test outcome		P
			ITP (n=7)	Successful (n=31)
Recipient clinical variables
Age at transplant (years)	38	63.0 (58.0, 66.0)	66.0 (57.5, 66.0)	63.0 (58.0, 65.0)	0.61
Sex	38				0.75
Female		10 (26.3)	1 (14.3)	9 (29.0)
Male		28 (73.7)	6 (85.7)	22 (71.0)
Race/ethnicity	38				0.13
White		19 (50.0)	4 (57.1)	15 (48.4)
Hispanic		15 (39.5)	1 (14.3)	14 (45.2)
Black		4 (10.5)	2 (28.6)	2 (6.5)
BMI at transplant (kg/m2)	37	28.2 (25.6, 35.6)	29.9 (24.3, 32.2)	28.2 (26.3, 35.7)	0.61
Laboratory MELD at transplant	38	14.0 (9.0, 30.0)	9.0 (8.5, 10.0)	18.0 (9.0, 33.5)	0.04
Primary tumor type	38				0.09
Cholangiocarcinoma		4 (10.5)	1 (14.3)	3 (9.7)
Hepatocellular carcinoma		33 (86.8)	5 (71.4)	28 (90.3)
Hepatocellular-cholangiocarcinoma		1 (2.6)	1 (14.3)	0 (0.0)
Etiology of liver disease
Alcohol-associated liver disease	38	7 (18.4)	0 (0.0)	7 (22.6)	0.33
MASH		12 (31.6)	2 (28.6)	10 (32.3)
Viral (HCV, HBV)		12 (31.6)	4 (57.1)	8 (25.8)
Other		7 (18.4)	1 (14.3)	6 (19.4)
Time on waitlist (days)	38	284.0 (126.0, 465.0)	293.0 (206.0, 565.0)	278.0 (72.0, 464.5)	0.30
Medical condition at transplant	38				0.17
Home		27 (71.1)	7 (100.0)	20 (64.5)
Hospital, not in ICU		4 (10.5)	0 (0.0)	4 (12.9)
In ICU		7 (18.4)	0 (0.0)	7 (22.6)
Simultaneous liver-kidney transplant	38	3 (7.9)	0 (0.0)	3 (9.7)	0.94
AFP at transplant (ng/mL)	27	3.2 (2.5, 8.4)	2.2 (2.2, 2.5)	4.1 (2.5, 10.1)	NA
CA19-9 at transplant (U/mL)	7	53.0 (32.0, 287.5)	487.0 (487.0, 487.0)	52.5 (12.0, 88.0)	NA
CEA at transplant (ng/mL)	10	5.1 (1.8, 12.2)	1.5 (1.5, 1.5)	5.1 (4.1, 12.2)	NA
Adjunctive therapy
Pre-transplant hepatectomy	38	1 (2.6)	1 (14.3)	0 (0.0)	0.41
Pre-transplant LRT	38	29 (76.3)	7 (100.0)	22 (71.0)	0.03*
Pre-transplant systemic therapy	38	9 (23.7)	2 (28.6)	7 (22.6)	>0.99
Peri-transplant Whipple procedure	38	1 (2.6)	1 (14.3)	0 (0.0)	0.41
Adjuvant systemic therapy	38	8 (21.1)	3 (42.9)	5 (16.1)	0.29
Palliative systemic therapy	38	2 (5.3)	1 (14.3)	1 (3.2)	0.81
Explant pathology
Tumor focality	38				0.22
Solitary		22 (57.9)	6 (85.7)	16 (51.6)
Multifocal		16 (42.1)	1 (14.3)	15 (48.4)
Number of tumor nodules	38	1.0 (1.0, 3.0)	1.0 (1.0, 1.0)	2.0 (1.0, 3.5)	0.19
Maximum tumor size (cm)	37	2.9 (1.8, 3.6)	2.8 (2.5, 3.6)	2.9 (1.8, 3.5)	0.89
Lympho-vascular invasion present	38	8 (21.1)	2 (28.6)	6 (19.4)	0.98
Perineural invasion present	38	5 (13.2)	1 (14.3)	4 (12.9)	>0.99
R1 margins	38	2 (5.3)	1 (14.3)	1 (3.2)	0.81
Response to neoadjuvant therapy (percentage necrosis)	25	65.0 (30.0, 85.0)	99.0 (35.0, 100.0)	55.0 (30.0, 75.0)	0.21
Cirrhosis present	38	36 (94.7)	6 (85.7)	30 (96.8)	0.81
Outcomes
Hospital length of stay (days)	37	15.0 (10.0, 19.0)	10.0 (9.5, 16.5)	15.0 (11.0, 20.0)	0.38
Experienced recurrence	38	6 (15.8)	2 (28.6)	4 (12.9)	0.65
Recurrence-free survival (days)	38	583.0 (368.0, 931.0)	638.0 (366.0, 1,259.0)	577.0 (390.0, 920.0)	0.63
Location of recurrence
Local	38	3 (7.9)	1 (14.3)	2 (6.5)	>0.99
Regional	38	1 (2.6)	0 (0.0)	1 (3.2)	>0.99
Distant	38	3 (7.9)	1 (14.3)	2 (6.5)	>0.99
Recurrence site
Liver allograft	38	3 (7.9)	1 (14.3)	2 (6.5)	>0.99
Lung	38	2 (5.3)	1 (14.3)	1 (3.2)	0.81
Bone	38	2 (5.3)	0 (0.0)	2 (6.5)	>0.99
Patient status	38				>0.99
Alive		34 (89.5)	6 (85.7)	28 (90.3)
Dead		4 (10.5)	1 (14.3)	3 (9.7)
Patient survival (days)	38	728.0 (411.0, 1,087.0)	638.0 (442.0, 1,302.5)	744.0 (426.0, 988.0)	0.68
Cause of death	3				>0.99
Cancer		2 (66.7)	1 (100.0)	1 (50.0)
Respiratory		1 (33.3)	0 (0.0)	1 (50.0)

Data are presented as median (IQR) or n (%). *, P<0.05. AFP, alpha fetoprotein; BMI, body mass index; CA19-9, carbohydrate antigen 19-9; CEA, carcinoembryonic antigen; ctDNA, circulating tumor DNA; HBV, hepatitis B virus; HCV, hepatitis C virus; ICU, intensive care unit; IQR, interquartile range; ITP, insufficient to process; LRT, locoregional therapy; MASH, metabolic dysfunction-associated steatohepatitis; MELD, Model for End-Stage Liver Disease; NA, not available.

Figure 1.

Overview of study cohort. (A) Study cohort including 100 ctDNA assays performed on 38 primary liver cancer patients post-liver transplantation. (B) Point-and-segment plot depicting the natural history of each patient, including recurrence status (far left), tumor type (middle left) and dates of liver transplant, recurrence, death/censor, and ctDNA reporting dates, normalized to transplant date. CCA, cholangiocarcinoma; ctDNA, circulating tumor DNA; Fail, failed; HCC, hepatocellular carcinoma; ITP, insufficient to process; Neg, negative; OLT, orthotopic liver allotransplantation; Pos, positive.

Figure 2.

Post-transplant survival. (A) Overall and (B) recurrence-free survival for the entire cohort. The dotted lines indicate the 95% confidence interval for the survival curves.

Tumor types included 33 HCC, 4 CCA, and 1 mixed HCC-CCA. At transplant, the median AFP was 2.8 ng/mL (IQR, 2.4–8.4 ng/mL) in HCC patients, and the median CA19-9 was 287.5 U/mL (IQR, 70–611 U/mL) in CCA patients. On explant pathology, the median number of tumor nodules was 1 (IQR, 1–3) and the maximum tumor size was 2.9 cm (IQR, 1.8–3.7 cm). There were 3 HCC (9.1% of 33) beyond Milan criteria (35). Lymphovascular invasion (LVI) was present in 8 (21.1%), perineural invasion (PNI) in 5 (13.2%), and background liver cirrhosis in 36 (94.7%). One (25% of 4) patient with CCA had lymph node metastases and 2 (50%) had R1 margins, requiring either margin revision or Whipple surgery. The median treatment effect, measured as histologic percent tumor necrosis, was 62.5% (IQR, 30–87.5%). Recipient and donor features stratified by tumor type are available in Table S1.

ctDNA test features

During post-LT surveillance, 100 ctDNA assays were ordered between 9/17/2020 and 5/11/2023 (Figure 3). The median time from LT to first test ordering was 117.5 days (IQR, 78–467 days), to first blood collection 148 days (IQR, 88–467 days), and first reporting 175 days (IQR, 122–607 days). The median time between test orders was 135.6 days (IQR, 78–189.3 days). The median number of tests per patient was 3 (range, 1–7). The time from ordering to collection increased significantly with each serial assay (P<0.001, Kruskal-Wallis), while the time from collection to reporting significantly decreased from the first to subsequent assays (P<0.001, Kruskal-Wallis), resulting in no significant difference in serial time from ordering to reporting (P=0.06, Kruskal-Wallis).

Figure 3.

Circulating tumor DNA assay metrics. (A) Barplot depicting time from first order to first blood collection (dark blue) and first ctDNA report (light blue) per patient, normalized to first order date. (B) Boxplots of times from ctDNA test order to blood collection for first and subsequent assays. (C) Boxplots of times from blood collection to ctDNA result report for first and subsequent assays. (D) Boxplots of times from ctDNA test order to result report for first and subsequent assays. ctDNA, circulating tumor DNA.

ctDNA assay logistics

Of the 100 ctDNA assays, there were 6 positive results, 85 negative, and 9 ITP. The number of positive tests per patient ranged from 0–3, negative tests 0–7, and ITP tests 0–2. ITP test assay was significantly associated with patients who received neoadjuvant loco-regional therapy and whose explanted tumors were of lower T stage and poorer differentiation (Tables 1,2; all P<0.05). The causes for ITP assays included insufficient tumor tissue (n=8, 21.1%) and improper blood handling/storage (n=1, 2.6%). The 9 tests with ITP results were performed on 8 patients. Of those 8 patients, 6 had only 1 test (i.e., they were not retested) and 2 were retested. Of the 2 retested patients, 1 had a second “ITP” test result, and 1 had 3 subsequent “negative” tests. Seven patients did not receive test results, only ITP assays (Table 2).

Table 2. ctDNA test features for the pooled cohort.

Variable	N	Values
Test results per patient	38
Positive		3 (7.9)
Negative		28 (73.7)
ITP		7 (18.4)
Reason for test ITP	38
Insufficient tumor tissue		8 (21.1)
Improper blood storage/handling		1 (2.6)
Number of ctDNA tests per patient	38	3.0 (1.0, 3.8)
Number of positive ctDNA tests per patient	38	1.0 (1.0, 1.0)
Number of negative ctDNA tests per patient	38	3.5 (2.0, 4.0)
Time between test orders (days)	26	135.6 (78.0, 189.3)
Time between blood collections (days)	26	148.8 (96.3, 193.0)
First ctDNA tests
Time from transplant to test order (days)	38	117.5 (78.0, 467.0)
Time from transplant to blood collection (days)	38	148.0 (88.0, 467.0)
Time from transplant to first test report (days)	38	175.0 (122.0, 607.0)
Time from test order to blood collection, (days)	38	0.0 (0.0, 21.0)
Time from blood collection to report (days)	38	33.5 (22.0, 41.0)
Time from test order to report (days)	38	41.0 (34.0, 64.0)
Subsequent ctDNA tests
Time from test order to blood collection (days)	26	18.5 (10.0, 48.0)
Time from blood collection to report (days)	26	9.0 (8.0, 10.0)
Time from test order to report (days)	26	31.8 (19.0, 57.0)

Data are presented as median (IQR) or n (%). ctDNA, circulating tumor DNA; IQR, interquartile range; ITP, insufficient to process.

ctDNA test sensitivity, specificity, and predictive values

In the 29 HCC patients without recurrence, there were 79 negative and 4 ITP assays. In the 4 HCC patients with recurrence, there were 3 positive tests in 1 patient, 1 negative test in 1 patient, and 2 ITP tests in 2 patients. In the 2 CCA patients without recurrence, there were 4 negative and 2 ITP tests. In the 2 CCA patients with recurrence, there was 1 positive test prior to and 1 positive test post-imaging-based recurrence in 1 patient, and 1 positive test and 1 negative test post-recurrence in another patient. In the 1 mixed tumor patient, there was 1 ITP test.

Ignoring the ITP tests (n=31 patients, Table 3), ctDNA test parameters include sensitivity 75% (95% CI: 19–99%), specificity 100% (95% CI: 87–100%), negative predictive value 96.4% (95% CI: 82–100%) and positive predictive value 100% (95% CI: 29–100%). For standard-of-care biomarkers in the same 31 patients, namely AFP for HCC or CA19-9 for CA19-9 patients, test parameters included sensitivity 75% (95% CI: 19–99%), specificity 93% (95% CI: 76–99%), negative predictive value 96% (95% CI: 80–100%) and positive predictive value 60% (95% CI: 15–95%). There was no significant difference in either sensitivity or specificity between standard and ctDNA biomarkers (P>0.99, P=0.16, respectively), though ctDNA specificity was non-significantly greater. When considering ITP tests as negative results (n=38 patients; Table 3), statistical significances of sensitivity and specificity differences remained the same. When considering HCC patients alone, the test features and statistical significances of sensitivity and specificity differences also remained the same (Table S2). As the most positive ctDNA tests (5/6) were ordered after imaging-based tumor recurrence, the time from assay positivity to clinical diagnosis could not be estimated.

Table 3. Biomarker test parameters for circulating tumor DNA versus traditional primary liver cancer biomarkers.

Variables	Detection method, % [95% CI]					P value†
	Sensitivity	Specificity	PPV	NPV		Sensitivity	Specificity
Cholangiocarcinoma only
Excluding patients with non-complete ctDNA tests						0.32	0.32
ctDNA	100 [16–100]	100 [3–100]	100 [3–100]	100 [16–100]
CA19-9	50 [1–99]	0 [0–97]	0 [0–97]	50 [1–99]
Non-complete tests = negative results						0.32	0.32
ctDNA	100 [16–100]	100 [16–100]	100 [16–100]	100 [16–100]
CA19-9	50 [1–99]	50 [1–99]	50 [1–99]	50 [1–99]
Hepatocellular carcinoma only
Excluding patients with non-complete ctDNA tests						0.32	0.32
ctDNA	50 [1–99]	100 [87–100]	96 [81–100]	100 [3–100]
AFP	100 [16–100]	96 [80–100]	100 [86–100]	67 [9–99]
Non-complete tests = negative results						0.08	0.32
ctDNA	25 [1–81]	100 [88–100]	91 [75–98]	100 [3–100]
AFP	100 [40–100]	97 [82–100]	100 [88–100]	80 [28–99]
All tumor types
Excluding patients with non-complete ctDNA tests						>0.99	0.16
ctDNA	75 [19–99]	100 [87–100]	100 [29–100]	96 [82–100]
AFP/CA19-9	75 [19–99]	93 [76–99]	60 [15–95]	96 [80–100]
Non-complete tests = negative results						0.32	0.16
ctDNA	50 [12–88]	100 [89–100]	100 [29–100]	91 [77–98]
AFP/CA19-9	83 [36–100]	94 [79–99]	71 [29–96]	97 [83–100]
Including patients with non-complete ctDNA tests
ctDNA and AFP/CA19-9	100 [54–100]	94 [79–99]	75 [35–97]	100 [88–100]

^†, P values reflect differences between ctDNA and AFP/CA-19-9 tests. AFP, alpha-fetoprotein; CA19-9, carbohydrate antigen 19-9; CI, confidence interval; ctDNA, circulating tumor DNA; NPV, negative predictive value; PPV, positive predictive value.

When results of ctDNA, AFP, and CA19-9 were considered jointly, test parameters included sensitivity 100% (95% CI: 54–100%), specificity 94% (95% CI: 79–99%), positive predictive value 75% (95% CI: 35–97%), and negative predictive value 100% (95% CI: 88–100%).

Discussion

This study presents a clinical cohort of 38 patients with primary liver cancers prospectively undergoing post-LT tumor-informed ctDNA testing in serial peripheral blood samples. Approximately 10% of ctDNA assays were ITP, often due to insufficient solid tumor tissue availability, which was associated with receipt of pre-LT locoregional therapy. Specificity and positive predictive value were greater than sensitivity and negative predictive value. The specificity of ctDNA was non-significantly improved relative to conventional peripheral blood biomarkers (AFP, CA19-9). To our knowledge, this is the first study to implement serial ctDNA prospectively post-LT for cancer recurrence surveillance.

Data on the utility of peripheral blood ctDNA assays includes numerous studies on post-resection prognosis for primary liver cancers and recurrence surveillance in other solid tumors, including breast and colorectal cancers. However, most studies are retrospective and use differing methodologies. There is very little data specific to primary liver cancers and LT (30). Two abstracts from China presented at the 2022 American Society for Clinical Oncology (ASCO) Annual Meeting retrospectively explored the utility of ctDNA testing peri-LT for HCC (36,37). The first by Huang et al. evaluated pre- and post-LT ctDNA in 74 HCC patients (36). The second study by Jiang et al., in 45 patients, included only pre-LT ctDNA (37). In both, ctDNA detection was associated with recurrence but not with OS. Thus, the clinical translation of ctDNA assays in post-LT cancer surveillance remains in question. We did not identify a difference in sensitivity and specificity of the ctDNA assay over standard-of-care methods of detecting recurrence. Often (5/6 patients), recurrences were detected sooner by imaging than by ctDNA. Even in the 1 patient where ctDNA positivity preceded other tests, it led to confirmatory imaging rather than treatment. Given its non-significantly greater specificity than standard biomarkers, ctDNA may better serve as a confirmatory test for situations of diagnostic uncertainty in standard-of-care surveillance testing results. This would fulfill the terminology of “liquid biopsy”, potentially usurping the role of invasive solid tissue biopsies. To develop its potential utility in this role, trials allowing diagnosis based on ctDNA-positivity are needed (29).

This study demonstrates the feasibility of using longitudinal ctDNA testing post-LT in patients with HCC or CCA. Testing frequency was approximately every 4 months, in line with recommendations for standard screening every 3–6 months (8). The observed test ITP rate is higher than that reported in other solid tumors, reflecting the high frequency and effectiveness of pre-LT locoregional therapies in this clinical context. The increasing time interval from ordering to blood collection with serial testing observed here also raises concerns for patient compliance, which would ultimately limit its role as a surveillance test over the recommended 5-year post-LT period. Interestingly, high failure rate and low compliance were not reported in a prospective series of peripheral blood multi-analyte testing as pan-cancer screening, suggesting a different adoption of ctDNA in pre- versus post-treatment patients (38).

This study was limited by its relatively small sample size, low frequency of recurrences, short follow-up, and single-center nature. The sample size was inherently limited by the various histologies of primary liver cancers and their different peri-operative adjunctive therapies. This study focused on determining ctDNA test parameters for post-transplant recurrence in the pooled cohort. Factors including test timing relative to transplant and number of tests performed per patient could not be considered given sample size limitations. Analyses considering these factors would require a larger sample size. Nonetheless, we feel that our results are meaningful to clinicians practicing in this space, where single center patient sizes are typically on the scale included here or smaller. The infrequent recurrences prevented the association of ctDNA positivity with the anatomic site of recurrence. Primary liver cancers can recur after our 2-year follow-up period when the frequency of standard-of-care surveillance decreases. Paradoxically, less frequent imaging may accentuate the utility of ctDNA as a screening modality by introducing lead-time bias (27). Nonetheless, this is the first prospective study of peripheral blood tumor-informed ctDNA testing post-LT and can inform future studies.

Conclusions

Our study informs the application of peripheral blood tumor-informed ctDNA assays in surveillance post-LT for primary liver cancer. The high specificity and low sensitivity suggest a confirmatory or diagnostic role may supplant invasive solid tissue biopsy when abnormal conventional modalities suggest recurrence and show the need for more investigations in the surveillance setting. Additional studies are required to determine the best way to utilize these tests before clinical adoption in this context.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Institutional Review Board of the Houston Methodist Hospital (No. PRO00035386). Informed consent was waived due to the use of de-identified data collected as part of routine clinical care.

Data Sharing Statement

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DOI: 10.21037/jgo-24-791