Three other recipients in the longitudinal cohort were diagnosed with cholestatic pathologies that were associated with notably abnormal cholestatic LFTs. Two were conservatively managed, and one required surgical reconstruction of the biliary anastomosis (Table 2). dscfDNA in all 3 of these recipients was within the median of the uneventful group (Figure 4).
Overall, the recipients with tBPAR demonstrated distinctively higher dscfDNA compared with the uneventful subgroup and cholestasis subgroup (Figure S2, SDC, http://links.lww.com/TXD/A212, and Table S1, SDC, http://links.lww.com/TXD/A212).
Notably, LFTs showed greater overlap between the subgroups (Figure S2, SDC, http://links.lww.com/TXD/A212). ALT and bilirubin did not differentiate the 3 different subgroups (Table S1, SDC, http://links.lww.com/TXD/A212). Alkaline phosphatase (ALP; at days 7, 14, and 28) and gamma-glutamyl transferase (GGT; at days 7 and 14) differentiated the uneventful subgroup from recipients with cholestasis but did not differentiate between recipients with cholestasis or tBPAR (Table S1, SDC, http://links.lww.com/TXD/A212).
Cohort B: Cross-sectional Evaluation of Diagnostic Accuracy of dscfDNA for tBPAR
The average recipient age was 55.3 ± 12 years (Table 3). The causes leading to LT were similar to that of cohort A. The MELD score was 22 ± 7. Almost 50% of the cohort had an underlying hepatocellular carcinoma before transplantation. The average organ donor age was 42 ± 17 years. Eighteen of the recipients received grafts from donation after brain death organ donors, and 2 of the recipients received grafts from organ donors after circulatory death. One of the recipients received a split LT. The average donor risk index was 1.5 ± 0.4.
The average age of transplantation of the recipient was 50.1 ± 11.6 years. The average operative time was 477 ± 118 minutes. The cold and warm ischemic times were 398 ± 99 and 42 ± 7 minutes, respectively. The average maximum ALT was 1266 ± 1963 U/L. The average LOS was 15 ± 8 days, and the ICU LOS was 3 ± 2 days. The duration from LT to biopsy was 1641 ± 1357 days.
No significant differences were identified among the clinical variables of the 3 subgroups. In this cohort, dscfDNA was analyzed on 20 blood samples (1 sample/recipient) (Figure 5).
dscfDNA Identified tBPAR With Superior Performance Compared With LFTs
Posttransplant recipients may present at the outpatient clinic with abnormal LFTs that require further investigations. These include imaging, endoscopy, or liver biopsies. We assessed the value of dscfDNA in discriminating recipients with abnormal LFTs and tBPAR (n = 6) from the recipients with abnormal LFTs and without tBPAR (n = 10) in this context. The findings showed that higher dscfDNA was significantly associated with tBPAR (Figure S3, SDC, http://links.lww.com/TXD/A212). On the other hand, ALT, ALP, GGT, and bilirubin were not indicative of tBPAR (Figure S3, SDC, http://links.lww.com/TXD/A212).
Among the recipients who underwent liver biopsies, dscfDNA accurately identified recipients with tBPAR and recipients without tBPAR (Table 4; Figure S4, SDC, http://links.lww.com/TXD/A212). The diagnostic performance based on the area under the curve of dscfDNA (96.7%; confidence interval [CI], 88.5%-100%) was superior to ALT (64.2%; CI, 31.5-96.8), ALP (48.3%; CI, 15.0%-81.6%), GGT (55.8%; CI, 24.9%-86.8%), and bilirubin (38.3%; CI, 9.5%-67.1%). At a threshold of 898 copies/mL, dscfDNA had a clinical sensitivity of 83.3% (CI, 35.9%-99.6%) and clinical specificity of 100% (87.7%–100%).
The analysis was extended to determine the value of dscfDNA in discriminating recipients with tBPAR (n = 6) from a group which included both patients without tBPAR and those who were clinically well (n = 28 total) with normal LFTs. dscfDNA, ALT, and GGT were significantly higher in recipients with tBPAR, when compared with those recipients without tBPAR and those who were clinically well (Figure S3, SDC, http://links.lww.com/TXD/A212).
Receiver operating characteristics analysis demonstrated that dscfDNA was superior to LFTs in discriminating recipients with tBPAR from those without tBPAR and those who were clinically well (Table 5; Figure S5, SDC, http://links.lww.com/TXD/A212). At a threshold of 898 copies/mL, dscfDNA had a clinical sensitivity of 83.3% (CI, 35.9%-99.6%) and clinical specificity of 100% (87.7%–100%).
Applying the dscfDNA Threshold for Surveillance of tBPAR Early After LT
The threshold of 898 copies/mL was applied to the longitudinal cohort (cohort A). Due to the high median dscfDNA of the uneventful subgroup at days 3 (1936 copies/mL) and 7 (1015 copies/mL), the diagnostic performance is of limited value. The threshold was most reliable from day 14 onward. Notably, from day 14 onward, all 3 of the recipients with cholestasis had dscfDNA that were below the tBPAR threshold of 898 copies/mL (Table 2).
At day 14, all 3 of the recipients with tBPAR had dscfDNA levels that were above the threshold (Table 2). For recipients 1 and 2, dscfDNA was measured during the decay phase after the treatment for the episode of acute rejection was instituted. Recipient 1 was diagnosed with tBPAR on day 7. One week after the treatment, the dscfDNA was 952 copies/mL. Although the dscfDNA was marginally above the threshold, the levels continued to decline below the threshold at subsequent time points after successful treatment. Recipient 2 was diagnosed with tBPAR on day 9. Five days after the treatment, the dscfDNA remained at markedly elevated levels of 1821 copies/mL. The levels continued to decline below the threshold after successful treatment. Recipient 3 was diagnosed with tBPAR on day 14. For this recipient, dscfDNA was markedly elevated at 10 769 copies/mL and then declined below the threshold after successful treatment at subsequent time points.
There is a clinical need for accurate blood-based tests to diagnose acute rejection after solid-organ transplantation. Several large studies have established the clinical utility of dscfDNA for acute rejection after heart, lung, and kidney transplantation.10-12,24,32,33 Reports pertaining to the role of dscfDNA in LT remain limited. At the time of writing, only a few small studies (≤17 recipients)16,19,25,34 and one large prospective study (115 recipients) have been published to date13.
In this study, we demonstrated that dscfDNA was indicative of acute rejection after LT. Uncomplicated clinical progress was associated with a stereotypic decrease in dscfDNA (Figure 2). Furthermore, serial monitoring of dscfDNA identified the recipients with tBPAR and facilitated the assessment of response to antirejection therapy (Figure 3). Importantly, we also showed that dscfDNA was superior to LFTs in identifying recipients with tBPAR (Tables 4 and 5).
Our findings were consistent with previous dscfDNA studies in LT13,16,19,25,34 and independently affirmed that dscfDNA is of clinical value for acute rejection after LT. There were, however, 2 key aspects of our study that differed to the other published studies.
First, the more clinically relevant endpoint of tBPAR was adopted. Mild histological rejection is often not treated, and maintenance immunosuppression is not modified.35,36 Many recipients with mild rejection will improve spontaneously without adverse clinical outcomes. To evaluate the value of dscfDNA in identifying clinically relevant episodes of acute rejection that required treatment, the endpoint of tBPAR (a standard in high-quality LT trials37-39) was thus employed in our study.
Second, unlike majority of the other published methodologies,19,20,22 our ddPCR methodology measured dscfDNA by absolute quantification (ie, copies/mL of dscfDNA) as compared to relative abundance (ie, percentage of dscfDNA: donor-specific DNA divided by the sum of donor-specific and recipient-specific DNA).
Measurement by relative abundance internally controls for sample processing variables (ie, DNA extraction yields). The disadvantage in doing so is that numerous factors such as exercise,40 infection,16 poor collection techniques,41 and poor sample processing42 increase recipient-specific DNA. These factors can confound relative abundance measurements and hence reduce the clinical sensitivity of detecting an event. Absolute quantification of donor alleles was thus adopted in this study to maximize clinical sensitivity. Attributed to this difference, direct comparison of healthy dscfDNA threshold (ie, 10% with the study by Schütz et al13) was not possible.
Despite the disparities in study endpoint and assay methodologies, our results supported the notion that the performance of dscfDNA is superior to LFTs in diagnosing recipients with acute rejection. This finding is readily explained by the unique and inherent physiology of dscfDNA.11,13 Because each cell has 2 haploid copies of the genome, the death of a cell derived from the donor organ will release 2 haploid copies of the donor-specific genome into the blood circulation. The amount of donor-specific DNA that is quantifiable in the plasma directly correlates to the degree of cell death that occurs during acute rejection.
On the other hand, LFTs are intrinsically different. The levels of bilirubin, ALP, GGT, and ALP are highly dependent on complex cellular interactions of transcriptional activity, translational processes, biochemical modifications, clearance, membrane permeability, and enzymatic leakage. These factors hence limit both the sensitivity and specificity of LFTs.
On the basis of our reported findings, we considered that dscfDNA may improve several aspects of clinical management after LT. First and foremost, dscfDNA could be used as a liquid biopsy for the surveillance of tBPAR in recipients after LT. In recipients with dscfDNA that surpass the threshold (ie, >898 copies/mL), antirejection therapy may be instituted. The size of our study precluded the formal assessment of the relationship between dscfDNA, rejection severity, and the type of antirejection therapy that was required to treat the rejection episode. Larger multicenter studies are necessary to further evaluate and refine the diagnostic and treatment thresholds of dscfDNA.
Second, dscfDNA could be used to guide investigational decisions early (ie, within the first 2 wk) after LT. Numerous perioperative variable such as donor organ quality, ischemic reperfusion injury, ischemic times, and transfusion of blood products could increase dscfDNA.43,44 These factors compromise and limit the clinical utility of dscfDNA to diagnose tBPAR. However, we considered that the serial monitoring of dscfDNA within the first 2 weeks after LT may be useful to determine the choice of further diagnostic tests when investigating recipients with abnormal cholestatic LFTs (a common finding early after LT).
For instance, the clinician may opt to perform a liver biopsy to confirm acute rejection in a recipient with abnormal cholestatic LFTs and abnormal dscfDNA (ie, twice the median of the uneventful subgroup). In a separate clinical scenario, the clinician may elect to watch and wait and not perform a liver biopsy in a recipient with abnormal cholestatic LFTs and normal dscfDNA. The analysis of dscfDNA could, hence, be used to determine the group of recipients who may benefit the most from undergoing tissue biopsies.
Third, serial monitoring of dscfDNA could complement LFTs in monitoring antirejection therapy response following the diagnosis and treatment of acute rejection. Consistent with studies in both liver and other types of solid-organ transplantation,10,11,13 dscfDNA normalized with successful treatment of acute rejection (Figure 3). Serial monitoring of dscfDNA has particular relevance in those patients where LFTs are slow to normalize after commencement of treatment of acute rejection. It may obviate the need for repeat liver biopsies to assess response. Owing to the intrinsic characteristics of dscfDNA, close monitoring of dscfDNA in conjunction to LFTs may offer valuable information pertaining to both graft integrity and treatment responses.
We recognize that our study has several limitations. The sample size is small, and this precluded formal multivariate analyses. Nevertheless, we considered that this sample size was sufficient to demonstrate the clinical utility of dscfDNA and also test the feasibility of our novel methodology to effectively measure dscfDNA within a clinically relevant turnaround time. Although only 40 recipients were presented, this is the second largest dscfDNA study in LT at the time of writing to reinforce the clinical value of dscfDNA in LT.
Importantly, protocol biopsies were not performed in our study to correlate dscfDNA with histopathological outcomes. While protocol biopsies may identify recipients with subclinical rejection, the risks (ie, pain, bleeding, infection) outweighed the benefits (ie, identifying clinically significant outcomes that require treatment). Similar to mild rejection, subclinical rejection may spontaneously improve without further treatment, and this suggests that, if truly present, the episode of rejection would have negligible impact on clinical outcomes. The use of biopsy-proven acute rejection requiring treatment (tBPAR) as an endpoint, as discussed above, was considered to address this limitation.
In conclusion, our findings support the recent study showing that dscfDNA provides an independent clinical value for sensitive detection of acute rejection after LT.13 Herein, we used a highly feasible and readily performed probe-free ddPCR methodology with clinically relevant turnaround times to measure dscfDNA in 2 separate cohorts of recipients after LT. Despite differences in endpoint and assay platforms, our findings provide more evidence supporting the value of dscfDNA for acute rejection in LT.
We acknowledge Boris Wong, Fang Shen, Tom Witkowski, Ashan Musafer, and Ramyar Molania for their technical assistance and the staff of the Liver Transplantation Unit of Victoria and DonateLife for their continuous support of this project.
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