Renin-Angiotensin Inhibitor, Captopril, Attenuates Growth of Patient-Derived Colorectal Liver Metastasis Organoids.

The recurrence of colorectal liver metastasis (CRLM) following liver resection is common; approximately 40% of patients will experience tumor recurrence post-surgery. Renin-angiotensin inhibitors (RASis) have been shown to attenuate the growth and progression of CRLM in pre-clinical models following liver resection. This study examined the efficacy of the RASi captopril on patient-derived colorectal liver metastasis organoids. Patient-derived organoids (PDOs) were established using fresh samples of colorectal liver metastasis from appropriately consented patients undergoing liver resection. To mimic the regenerating liver post-CRLM liver resection, PDOs were cultured under hepatocyte regeneration conditions in vitro. CRLM PDOs were established from three patients' parent tissue. CRLM PDOs and parent tissue expressed markers of colorectal cancer, CDX2 and CK20, consistently. Furthermore, CRLM PDOs treated with captopril showed a dose dependent reduction in their expansion in vitro. In conclusion, CRLM PDOs recapitulate in vivo disease and displayed a dose-dependent response to treatment with captopril. RASis may be an additional viable treatment for patients with CRLM.

Optimal Timing of Perioperative Chemoprophylaxis in Patients With High Thromboembolic Risk Undergoing Major Abdominal Surgery: A Multicenter Cohort Study.

OBJECTIVE: To determine whether early (before skin closure) versus postoperative chemoprophylaxis affects the incidence of venous thromboembolism (VTE) and bleeding following major abdominal surgery, in a high thromboembolic risk population. BACKGROUND: Major abdominal surgery incurs both VTE and bleeding risks. Patients with high preoperative VTE risk derive the most benefit from chemoprophylaxis, but carry an increased risk of bleeding. The optimal window for chemoprophylaxis in the perioperative period, whereby both VTE and bleeding risks are minimized, is unknown. METHODS: Analysis of pooled data from 5 multicenter studies including only high thromboembolic risk (Caprini score >4) patients. Clinical VTE was defined as radiographically proven symptomatic disease <30 days postsurgery. Major bleeding was defined as the need for blood transfusion, reintervention, or >20 g/L fall in hemoglobin. RESULTS: From 5501 cases, chemoprophylaxis was initiated early in 1752 (31.8%) patients and postoperatively in 3749 (68.2%) patients. Baseline characteristics were similar between study groups. The incidence of clinical VTE was not associated with chemoprophylaxis timing [early 0.7% vs. postop 0.7%, odds ratio (OR): 1.11, 95% confidence interval (CI): 0.60-2.15, P =0.730]. Contrastingly, compared with postoperative chemoprophylaxis, early usage increased the risk of all bleeding (5.1% vs. 2.6%, OR: 2.04, 95% CI: 1.52-2.73, P <0.001) major bleeding (3.6% vs. 1.8%, OR: 1.99, 95% CI: 1.40-2.81, P <0.001), and reintervention (2.0% vs. 1.0%, OR: 2.10, 95% CI: 1.32-3.35, P =0.003). Early chemoprophylaxis independently predicted postoperative bleeding (OR: 1.71, 95% CI: 1.25-2.34, P <0.001), but not VTE. CONCLUSIONS: In high VTE risk patients undergoing major abdominal surgery, chemoprophylaxis commenced postoperatively reduces bleeding risk without affecting clinical VTE risk.

Low Levels of Hepatocyte-Specific Methylation in Cell-Free DNA Are a Strong Negative Predictor for Acute T Cell-Mediated Rejection Requiring Treatment Following Liver Transplantation.

Graft-derived cell-free DNA (gdcfDNA) quantification is a promising, minimally invasive tool for detecting acute T cell-mediated rejection (ATCMR) following liver transplantation (LT). We investigated the utility of measuring hepatocyte-specific methylation in cfDNA (HS-cfDNA) to quantify gdcfDNA, examining its accuracy in detecting ATCMR in a prospective, cross-sectional study. Blood was collected from LT recipients immediately prior to graft biopsy for suspected rejection. HS-cfDNA was quantified using droplet-digital polymerase chain reaction. Prebiopsy liver function tests (LFTs) and HS-cfDNA levels were correlated with biopsy results and the primary outcome of treated biopsy-proven acute rejection (tBPAR). A total of 51 patients were recruited; 37 had evidence of rejection on biopsy and 20 required treatment. As much as 11 patients needed inpatient treatment for rejection. HS-cfDNA significantly outperformed LFTs in identifying patients with tBPAR, particularly those needing inpatient treatment (area under the curve, 73.0%; 95% confidence interval, 55.4%-90.6%; P = 0.01). At a threshold of <33.5% of the total cfDNA fraction, HS-cfDNA had a specificity of 97%, correctly excluding tBPAR in 30/31 patients. Quantifying graft-specific methylation in cfDNA has a major advantage over previous gdcfDNA techniques: it does not require genotyping/sequencing, lending it greater feasibility for translation into transplantation care. Low levels of HS-cfDNA were a strong negative predictor for tBPAR (negative predictive value, 86%) and may have a future role in triaging patients prior to invasive graft biopsies.

Captopril, a Renin-Angiotensin System Inhibitor, Attenuates Tumour Progression in the Regenerating Liver Following Partial Hepatectomy.

(1) Liver regeneration following partial hepatectomy for colorectal liver metastasis (CRLM) has been linked to tumour recurrence. Inhibition of the renin−angiotensin system (RASi) attenuates CRLM growth in the non-regenerating liver. This study investigates whether RASi exerts an antitumour effect within the regenerating liver following partial hepatectomy for CRLM and examines RASi-induced changes in the tumour immune microenvironment; (2) CRLM in mice was induced via intrasplenic injection of mouse colorectal tumour cells, followed by splenectomy on Day 0. Mice were treated with RASi captopril (250 mg/kg/day), or saline (control) from Day 4 to Day 16 (endpoint) and underwent 70% partial hepatectomy on Day 7. Liver and tumour samples were characterised by flow cytometry and immunofluorescence; (3) captopril treatment reduced tumour burden in mice following partial hepatectomy (p < 0.01). Captopril treatment reduced populations of myeloid-derived suppressor cells (MDSCs) (CD11b+Ly6CHi p < 0.05, CD11b+Ly6CLo p < 0.01) and increased PD-1 expression on infiltrating hepatic tissue-resident memory (TRM)-like CD8+ (p < 0.001) and double-negative (CD4-CD8-; p < 0.001) T cells; (4) RASi reduced CRLM growth in the regenerating liver and altered immune cell composition by reducing populations of immunosuppressive MDSCs and boosting populations of PD-1+ hepatic TRMs. Thus, RASi should be explored as an adjunct therapy for patients undergoing partial hepatectomy for CRLM.

Hepatectomy after bile duct injury: a systematic review.

BACKGROUND: Bile duct injury (BDI) after cholecystectomy can lead to recurrent cholangitis, even after biliary reconstruction. This necessitates hepatectomy in a minority of patients. A systematic review was conducted, summarizing the pattern of biliary injury sustained in this group and their outcomes after hepatectomy. METHODS: A literature search included the MEDLINE, EMBASE, PubMed and Cochrane libraries. Retrospective cohort studies describing outcomes for hepatectomy after BDI, and the nature of the antecedent BDI, published between 1999 and 2019, were selected. RESULTS: Eight articles described a cohort of 2110 patients with BDI. Of these, 84 underwent hepatectomy. Complex vasculo-biliary injuries had been sustained in most cases. The mean time to hepatectomy was between 26 and 224 months after BDI. A right hepatectomy was performed in 67-89% of cases. Post hepatectomy, intra-abdominal infection (range 0-50%) and bile leaks (range 0-45%) occurred variably. Mortality occurred in three series. Nineteen percent of patients (16 of 84) developed recurrent symptoms at follow up. CONCLUSION: Hepatectomy after bile duct injury is an uncommon procedure and represents a salvage strategy when vasculo-biliary injury happens. Liver resection leads to resolution of symptoms in the majority of the cases however postoperative bile leaks and intra-abdominal infection are common.

A Synthetic DNA Construct to Evaluate the Recovery Efficiency of Cell-Free DNA Extraction and Bisulfite Modification.

BACKGROUND: Despite improvements in the genetic and epigenetic analysis of cell-free DNA (cfDNA), there has been limited focus on assessing the preanalytical variables of recovery efficiency following cfDNA extraction and bisulfite modification. Quantification of recovery efficiency after these steps can facilitate quality assurance and improve reliability when comparing serial samples. METHODS: We developed an exogenous DNA Construct to Evaluate the Recovery Efficiency of cfDNA extraction and BISulfite modification (CEREBIS) after cfDNA extraction and/or subsequent bisulfite modification from plasma. The strategic placement of cytosine bases in the 180 bp CEREBIS enabled PCR amplification of the construct by a single primer set both after plasma DNA extraction and following subsequent bisulfite modification. RESULTS: Plasma samples derived from 8 organ transplant donors and 6 serial plasma samples derived from a liver transplant recipient were spiked with a known number of copies of CEREBIS. Recovery of CEREBIS after cfDNA extraction and bisulfite modification was quantified with high analytical accuracy by droplet digital PCR. The use of CEREBIS and quantification of its recovery was useful in identifying problematic extractions. Furthermore, its use was shown to be invaluable towards improving the reliability of the analysis of serial samples. CONCLUSIONS: CEREBIS can be used as a spike-in control to address the preanalytical variable of recovery efficiency both after cfDNA extraction from plasma and following bisulfite modification. Our approach can be readily implemented and its application may have significant benefits, especially in settings where longitudinal quantification of cfDNA for disease monitoring is necessary.

Elevated levels of circulating mitochondrial DNA predict early allograft dysfunction in patients following liver transplantation.

BACKGROUND AND AIM: The role of circulating mitochondrial DNA (cmtDNA) in transplantation remains to be elucidated. cmtDNA may be released into the circulation as a consequence of liver injury; yet recent work also suggests a causative role for cmtDNA leading to hepatocellular injury. We hypothesized that elevated cmtDNA would be associated with adverse events after liver transplantation (LT) and conducted an observational cohort study. METHODS: Twenty-one patients were enrolled prospectively prior to LT. RESULTS: Postoperative complications were observed in 47.6% (n = 10). Seven patients (33.3%) had early allograft dysfunction (EAD), and six patients (28.5%) experienced acute cellular rejection within 6 months of LT. cmtDNA levels were significantly elevated in all recipients after LT compared with healthy controls and preoperative samples (1 361 937 copies/mL [IQR 586 781-3 399 687] after LT; 545 531 copies/mL [IQR 238 562-1 381 015] before LT; and 194 562 copies/mL [IQR 182 359-231 515] in healthy controls) and returned to normal levels by 5 days after transplantation. cmtDNA levels were particularly elevated in those who developed EAD in the early postoperative period (P < 0.001). In all patients, there was initially a strong overall positive correlation between cmtDNA and plasma hepatocellular enzyme levels (P < 0.05). However, the patients with EAD demonstrated a second peak in cmtDNA at postoperative day 7, which did not correlate with liver function tests. CONCLUSIONS: The early release of plasma cmtDNA is strongly associated with hepatocellular damage; however, the late surge in cmtDNA in patients with EAD appeared to be independent of hepatocellular injury as measured by conventional tests.

A systematic review of small for size syndrome after major hepatectomy and liver transplantation.

BACKGROUND: Major hepatectomy (MH) and particular types of liver transplantation (LT) (reduced size graft, living-donor and split-liver transplantation) lead to a reduction in liver mass. As the portal venous return remains the same it results in a reciprocal and proportionate rise in portal venous pressure potentially resulting in small for size syndrome (SFSS). The aim of this study was to review the incidence, diagnosis and management of SFSS amongst recipients of LT and MH. METHODS: A systematic review was performed in accordance with the 2010 Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines. The following terms were used to search PubMed, Embase and Cochrane Library in July 2019: ("major hepatectomy" or "liver resection" or "liver transplantation") AND ("small for size syndrome" or "post hepatectomy liver failure"). The primary outcome was a diagnosis of SFSS. RESULTS: Twenty-four articles met the inclusion criteria and could be included in this review. In total 2728 patients were included of whom 316 (12%) patients met criteria for SFSS or post hepatectomy liver failure (PHLF). Of these, 31 (10%) fulfilled criteria for PHLF following MH. 8 of these patients developed intractable ascites alongside elevated portal venous pressure following MH indicative of SFSS. CONCLUSION: SFSS is under-recognised following major hepatectomy and should be considered as an underlying cause of PHLF. Surgical and pharmacological therapies are available to reduce portal congestion and reverse SFSS.

Searching for the link; mechanisms underlying liver regeneration and recurrence of colorectal liver metastasis post partial hepatectomy.

Despite excellent treatment of primary colorectal cancer, the majority of deaths occur as a result of metastasis to the liver. Recent population studies have estimated that one quarter of patients with colorectal cancer will incur synchronous or metachronous colorectal liver metastasis. However, only one quarter of these patients will be eligible for potentially curative resection. Tumor recurrence occurs in reportedly 60% of patients undergoing hepatic resection, and the majority of intrahepatic recurrence occurs within the first 6 months of surgery. The livers innate ability to restore its homeostatic size, and volume facilitates major hepatic resection that currently offers the only chance of cure to patients with extensive hepatic metastases. Experimental and clinical evidence supports the notion that following partial hepatectomy, liver regeneration (LR) paradoxically drives tumor progression and increases the risk of recurrence. It is becoming increasingly clear that the processes that drive liver organogenesis, regeneration, and tumor progression are inextricably linked. This presents a major hurdle in the management of colorectal liver metastasis and other hepatic malignancies because therapies that reduce the risk of recurrence without hampering LR are sought. The processes and pathways underlying these phenomena are multiple, complex, and cross-communicate. In this review, we will summarize the common mechanisms contributing to both LR and tumor recurrence.

Outcomes of central hepatectomy versus extended hepatectomy.

BACKGROUND: Central hepatectomy (CH) is more difficult than extended hepatectomy (EH) and is associated with greater morbidity. In this modern era of liver management with aims to prevent post-hepatectomy liver failure (PHLF), there is a need to assess outcomes of CH as a parenchyma-sparing procedure for centrally located liver tumors. METHODS: A total of 178 major liver resections performed by specialist surgeons from two Australian tertiary institutions between June 2009 and March 2017 were reviewed. Eleven patients had CH and 24 had EH over this study period. Indications and perioperative outcomes were compared between the groups. RESULTS: The main indication for performing CH was colorectal liver metastases. There was no perioperative mortality in the CH group and four (16.7%) in the EH group (P = 0.285). No group differences were found in median operative time [CH vs. EH: 450 min (290-840) vs. 523 min (310-860), P = 0.328], intraoperative blood loss [850 mL (400-1500) vs. 650 mL (100-2000), P = 0.746] or patients requiring intraoperative blood transfusion [1 (9.1%) vs. 7 (30.4%), P = 0.227]. There was a trend towards fewer hepatectomy-specific complications in the CH group [3 (27.3%) vs. 13 (54.2%), P = 0.167], including PHLF (CH vs. EH: 0 vs. 29.2%, P = 0.072). Median length of stay was similar between groups [CH vs. EH: 9 days (5-23) vs. 12 days (4-85), P = 0.244]. CONCLUSIONS: CH has equivalent postoperative outcomes to EH. There is a trend towards fewer hepatectomy-specific complications, including PHLF. In appropriate patients, CH may be considered as a safe parenchyma-sparing alternative to EH.

Probe-Free Digital PCR Quantitative Methodology to Measure Donor-Specific Cell-Free DNA after Solid-Organ Transplantation.

BACKGROUND: Donor-specific cell-free DNA (dscfDNA) is increasingly being considered as a noninvasive biomarker to monitor graft health and diagnose graft rejection after solid-organ transplantation. However, current approaches used to measure dscfDNA can be costly and/or laborious. A probe-free droplet digital PCR (ddPCR) methodology using small deletion/insertion polymorphisms (DIPs) was developed to circumvent these limitations without compromising the quantification of dscfDNA. This method was called PHABRE-PCR (Primer to Hybridize across an Allelic BREakpoint-PCR). The strategic placement of one primer to hybridize across an allelic breakpoint ensured highly specific PCR amplification, which then enabled the absolute quantification of donor-specific alleles by probe-free ddPCR. METHODS: dscfDNA was serially measured in 3 liver transplant recipients. Donor and recipient genomic DNA was first genotyped against a panel of DIPs to identify donor-specific alleles. Alleles that differentiated donor-specific from recipient-specific DNA were then selected to quantify dscfDNA in the recipient plasma. RESULTS: Lack of amplification of nontargeted alleles confirmed that PHABRE-PCR was highly specific. In recipients who underwent transplantation, dscfDNA was increased at day 3, but decreased and plateaued at a low concentration by 2 weeks in the 2 recipients who did not develop any complications. In the third transplant recipient, a marked increase of dscfDNA coincided with an episode of graft rejection. CONCLUSIONS: PHABRE-PCR was able to quantify dscfDNA with high analytical specificity and sensitivity. The implementation of a DIP-based approach permits surveillance of dscfDNA as a potential measure of graft health after solid-organ transplantation.

Comparison of 3 Methodologies for Genotyping of Small Deletion and Insertion Polymorphisms.

BACKGROUND: The quantification of genomic chimerism is increasingly recognized for its clinical significance after transplantation. Before the measurement of chimerism, accurate genotyping of genetic polymorphisms for informative alleles that can distinguish donor DNA from recipient DNA is essential. The ease of allelic discrimination of small deletion and insertion polymorphisms (DIPs) makes DIPs attractive markers to track chimerism. Current methodologies for the genotyping of DIPs are largely based on "open-tube" approaches. "Closed-tube" approaches involving no or minimal post-PCR handling are preferred. We compared 3 distinct methodologies to determine an optimal platform for DIP genotyping. METHODS: Genomic DNA from 19 normal individuals was genotyped for 6 small biallelic DIPs using high-resolution melting analysis (HRMA), probe-free droplet digital PCR (ddPCR), and microfluidic electrophoresis of PCR products. For HRMA, 3 different platforms were compared. RESULTS: Our newly developed probe-free ddPCR approach allowed the genotype of each DIP to be determined by fluorescence intensity based on amplicon size. Microfluidic electrophoresis also allowed genotypes to be determined by amplicon size. HRMA assays allowed the genotype of each DIP to be determined by melting profile. Genotyping results were concordant between the 3 methodologies. HRMA was the most readily performed methodology and was robust across 3 separate HRMA-capable platforms. CONCLUSIONS: We demonstrated the effectiveness of probe-free ddPCR to accurately genotype small biallelic DIPs. Nevertheless, HRMA proved to be the optimal approach for genotyping small DIPs because closed-tube approaches are preferred owing to rapid and less laborious workflows and least risk of PCR contamination.

Metabolic response to preoperative chemotherapy predicts prognosis for patients undergoing surgical resection of colorectal cancer metastatic to the liver.

BACKGROUND: Biological characteristics of colorectal cancer liver metastases (CRCLM) are increasingly recognized as major determinants of patient outcome. The purpose of this study was to evaluate the prognostic value of metabolic response to preoperative chemotherapy as quantified by (18)F-FDG positron emission tomography (PET) for patients undergoing liver resection of CRCLM. METHODS: All patients (n = 80) who had staging PET before liver resection for CRCLM at Austin Health in Melbourne between 2004 and 2011 were included. Thirty-seven patients had PET and CT imaging before and after preoperative chemotherapy. Semiquantitative PET parameters-maximum standardized uptake variable (SUVmax), metabolic tumour volume (MTV), and total glycolytic volume (TGV)-were derived. Metabolic response was determined by the proportional change in PET parameters (∆SUVmax, ∆MTV, ∆TGV). Prognostic scores, CT RECIST response, and tumour regression grading (TRG) were also assessed. Correlation to recurrence-free (RFS) and overall survival (OS) was assessed using Kaplan-Meier survival and multivariate analysis. RESULTS: Semiquantitative parameters on staging PET before chemotherapy were not predictive of prognosis, whereas all parameters after chemotherapy were prognostic for RFS and OS. Only ∆SUVmax was predictive of RFS and OS on multivariate analysis. Patients with metabolically responsive tumours had an OS of 86 % at 3 years vs. 38 % with nonresponsive or progressive tumours (p = 0.003). RECIST and TRG did not predict outcome. CONCLUSIONS: Tumour metabolic response to preoperative chemotherapy as quantified by PET is predictive of prognosis in patients undergoing resection of CRCLM. Assessing metabolic response uniquely characterizes tumour biology, which may allow future optimization of patient and treatment selection.

Pressurized pulse irrigation with saline reduces surgical-site infections following major hepatobiliary and pancreatic surgery: randomized controlled trial.

BACKGROUND: Surgical site infections (SSI) are a significant cause of postoperative morbidity. Pressurized pulse irrigation of subcutaneous tissues may lower infection rates by aiding in the debridement of necrotic tissue and reducing bacterial counts compared to simply pouring saline into the wound. METHODS: A total of 128 patients undergoing laparotomy extending beyond 2 h were randomized to treatment of wounds by pressurized pulse lavage irrigation (<15 psi) with 2 L normal saline (pulse irrigation group), or to standard irrigation with 2 L normal saline poured into the wound, immediately prior to skin closure (standard group). Only elective cases were included, and all cases were performed within a specialized hepatobiliary and pancreatic surgery unit. RESULTS: There were 62 patients managed by standard irrigation and 68 were managed by pulse irrigation. The groups were comparable in most aspects. Overall there were 16 (13 %) SSI. Significantly fewer SSI occurred in the pulse irrigation group [4 (6 %) vs. 12 (19 %); p = 0.032]. On multivariate analysis, the use of pulse irrigation was the only factor associated with a reduction in SSI with an odds ratio (OR) of 0.3 [95 % confidence interval (95 % CI) 0.1-0.8; p = 0.031]. In contrast, hospital length of stay of greater than 14 days was associated with increased infections with an OR of 7.6 (95 % CI 2.4-24.9; p = 0.001). CONCLUSIONS: Pulse irrigation of laparotomy wounds in operations exceeding 2 h duration reduced SSI after major hepatobiliary pancreatic surgery. (Australian New Zealand Clinical Trials Registry, ACTRN12612000170820).

Lymphatics and colorectal liver metastases: the case for sentinel node mapping.

BACKGROUND: Hepatic resection remains the treatment of choice for patients with colorectal liver metastases (CLM). Indications for hepatic resection have been extended to include extrahepatic lymph node groups, resulting in increased survival benefits. The identification of specific lymph pathways and involved nodes is necessary to support the development of guidelines for a more focused approach to the management of this disease. The feasibility of sentinel node mapping should be investigated to define specific lymphatic groups involved in CLM. METHODS: Scientific papers published from 1950 to 2012 were sought and extracted from the MEDLINE, PubMed and University of Melbourne databases. RESULTS: Several studies have reported microscopic lymph node involvement in 10-15% of patients undergoing hepatic resection for CLM in which no macroscopic involvement was evident. In retrospect, over 80% of lymphadenectomies are proven unnecessary. Traditional imaging modalities have limited predictive value in detecting lymph node involvement. Sentinel node mapping has proved an extremely accurate tool in detecting lymph node involvement and can identify patients in whom lymphadenectomy may be beneficial. CONCLUSIONS: Current imaging techniques are inadequate to detect microscopic lymph node involvement in patients with resectable CLM. The use of sentinel node mapping is proposed to identify nodal groups involved and provide management strategies.

The hospital costs of complications following major abdominal surgery: a retrospective cohort study.

OBJECTIVE: Postoperative complications following major abdominal surgeries is a pressing concern for hospital care and health economics. Given the paucity of available cost data for patients undergoing major abdominal surgery, we evaluated the number and the severity of postoperative complications following major abdominal surgeries and calculated the costs borne by a single centre university hospital within an Australian healthcare system. RESULTS: The overall incidence of postoperative complications for 1790 adult patients undergoing major abdominal surgeries (i.e., colonic, liver, small bowel resections and Whipple procedures) between January 2013 and June 2018 was 75.2%. Of these complications, 56.9% were minor (Clavien-Dindo (CVD) Grades I or II) and 15.5% were major (CVD Grades III or IV). As the severity of complications increased, median adjusted total hospital costs rose significantly, with a median (interquartile range [IQR]) of AUD 29,519.70 (IQR 21,828.80-40,527.90) in CVD Grade II versus AUD 50,702.40 (IQR 35,866.00-69,296.80) in CVD Grade III (p <.001). Further, developing one, two or three complications resulted in significantly increased hospital costs by AUD 2618.30 (13.3% increase), AUD 3605.50 (16.2% increase) and AUD 3173.00 (12.3% increase) (p <.0001), respectively, with an exponential spike in costs incurred by patients who developed more than three complications (AUD 23,719.70; 81.7% increase; p < 0001).

Graft-derived cfDNA Monitoring in Plasma and Bile During Normothermic Machine Perfusion in Liver Transplantation Is Feasible and a Potential Tool for Assessing Graft Viability.

BACKGROUND: Ex vivo normothermic machine perfusion (NMP) is an organ preservation technique that enables an extended assessment of graft suitability before liver transplantation (LT). Established monitoring protocols used during NMP vary significantly in their assessment of transplant suitability when applied to the same grafts. Graft-derived cell-free DNA (gdcfDNA) analysis is an emerging tool for monitoring graft health post-transplantation. We investigated the feasibility of monitoring gdcfDNA during NMP for LT in a proof-of-concept, observational study. METHODS: Serial plasma and bile samples were collected during NMP for 10 consecutive grafts, at 15 min post-machine reperfusion and then 2-h intervals. Digital polymerase chain reaction was used to quantify gdcfDNA at each time point. RESULTS: Five grafts were suitable for LT, there were no cases of primary nonfunction or death in the recipients. gdcfDNA was quantified in all bile and plasma samples (n > 100). In plasma, gdcfDNA concentrations climbed post-machine reperfusion until 4.25 h (median 2.25 h = 15.98 × 10 6 copies/mL, 4.25 h = 40.21 × 10 6 copies/mL). gdcfDNA levels then diverged significantly when comparing the viable and non-viable graft groups (6.25 h, median viable: 117.15 × 10 6 copies/mL versus non-viable: 16.72 × 10 6 copies/mL, P = 0.01). These opposing trends correlated in each graft and in all cases with the viable/non-viable outcome. There was a trend of gradual decline in bile gdcfDNA from viable grafts post-machine reperfusion; discarded grafts showed more variable patterns of release. CONCLUSIONS: gdcfDNA analysis during NMP is a feasible and potential tool to inform viability assessment during NMP for LT. Bile gdcfDNA monitoring offers the prospect of an objective means to assess the degree of biliary injury associated with organ procurement.