The data of the patients with pathologically diagnosed HCC who underwent OLT at Shanghai General Hospital between August 2002 and July 2016 were collected from China Liver Transplant Registry (https://http://www.cltr.org/) and retrospectively analyzed. Experienced pathologists provided detailed pathological diagnosis and staging of the tumors according to AJCC Cancer Staging Manual sixth edition[15]. Infection-related information, including general and site-specific clinical indices of PTI (Supplementary Table 1), was extracted from the chart record and/or electronic medical record system of our hospital. Definitions of infection regarding the six most frequent causes of PTI (pneumonia, intra-abdominal infection, urinary tract infection, surgical wound infection, intravascular catheter-related infection, and bloodstream infection) were briefly explained in the Attachment 1 and described in detail in the previous literature[16]. Conference was held to reach consensus on the diagnosis of PTI for each patient by a panel of experts including the physicians of intensive care unit (ICU), surgeons, and radiologists. In the present study, we chose to evaluate PTI that occurred within 30 days following OLT mostly during ICU stay, because the recipients had the highest incidence of PTI during this period and the records of patient data were more reliable in the ICU settings[13,17,18].

Chest computed tomography (CT), abdominal lipiodol CT with three-dimensional angiography, and enhanced magnetic resonance imaging (MRI) were routinely performed for diagnosis and assessment of liver cirrhosis and tumor extent. High suspicion of distant tumor metastasis and major vascular invasion explored by imaging were contraindications for OLT. Patients with preoperative imaging diagnosis of liver tumor meeting the Milan criteria[19] (from August 2002 to November 2006), University of California, San Francisco (UCSF) criteria[20] (from December 2006 to May 2008), or Hangzhou Criteria[21] (from June 2008 to date) were included in the waiting list of OLT in our center. Pre-transplant regional therapies employed in our center included trans-catheter arterial chemoembolization, radiofrequency /microwave ablation, and liver resection. Patients with tumors down-staged by regional therapies and fulfilled the selection criteria were also subjected to OLT. Liver transplants at Shanghai General Hospital are performed using standard techniques without the use of venovenous bypass. All patients were admitted to the transplant ICU immediately after OLT.

Cefoperazone/sulbactam (3.0 g intravenous infusion, 2 to 3 doses per day) was used as antibacterial prophylaxis. Caspofungin or micafungin was used as antifungal prophylaxis when indicated by the protocol of our center (Attachment number 2). Ganciclovir or oral valganciclovir was used within three months after OLT as anti-cytomegalovirus (CMV) prophylaxis when proposed by our protocol (Attachment number 3).

Our institution’s postoperative immunosuppression regimen includes induction with basiliximab and a tapered dose of corticosteroids, and maintenance with combination or separate use of corticosteroids, calcineurin inhibitor (cyclosporine or tacrolimus), sirolimus, and mycophenolate mofetil. After discharge, the patients were followed at the outpatient clinic according to the standard protocol of our center as we described previously[5]. Briefly, tumor recurrence was monitored by measurements of plasma AFP and/or abnormal prothrombin and abdominal ultrasonography (every month the first half year, then every 3 months), lipiodol CT (every 3 months for the first year, every 6 months for the second year, and once per year thereafter). MRI, positron emission tomography, or radioisotope bone scan was taken when necessary. No prophylactic measures were taken to prevent tumor relapse after OLT. For the diagnosis of tumor recurrence, a consensus was reached by a panel of experts including the surgeons and radiologists. Treatment of recurrent HCC included trans-catheter arterial chemoembolization, radiofrequency/microwave ablation, systemic chemotherapy, surgical resection, and use of sorafenib. Recurrence-free survival (RFS) was defined as the period of patient survival without any evidence of tumor recurrence from the day of OLT, and detection of tumor relapse after OLT or death without tumor recurrence after OLT was considered as an event. Overall survival (OS) was defined as the period from the day of surgery till patient death, and death after OLT regardless of the cause was considered as an event. Post-recurrence survival (PRS) was calculated from the day when tumor relapse was diagnosed till the day when patient died, and death after tumor recurrence regardless of the cause was regarded as an event. Loss to follow-up or follow-up interruption at the end of the observation was treated as a censoring event.

SPSS software program (version 17; IBM Corporation, Armonk, NY, United States) was used to analyze the data. For continuous variables, data are expressed as medians in the interquartile range (IQR) and were compared using the Mann-Whitney U test. Categorical variables were assessed by the Chi-square test or Fisher’s exact test. Univariate analyses of OS, RFS, and PRS as well as survival curve analysis were performed by the Kaplan-Meier method, and the differences were compared using the log-rank test. The variables with a P-value < 0.1 in univariate analyses were included in the multivariate survival analysis by using a Cox proportional-hazards model. Statistical significance was established when the P-values were < 0.05.

A total of 238 patients with pathologically confirmed HCC who survived more than 30 days after OLT at our center between August 2002 and July 2016 were included in this study. Among these, 53 (22.3%) patients were identified to have PTI and included in the PTI group, and the remaining 185 (77.7%) patients were included in the non-PTI group. Baseline clinicopathological features of the two patient groups were compared and revealed no significant differences (Table 1).

Fifty-three patients had PTI, and the median duration of PTI was 12.0 days (IQR: 7.0 to 21.75 days). The common infection sites included the lungs, abdomen, urinary tract, intravascular catheter, incision, and bloodstream (Supplementary Table 2). The infectious microorganisms included bacterial (Klebsiella pneumoniae, Acinetobacter baumannii, enterococcus faecalis, stenotrophomonas maltophilia, etc.), fungal (Candida albicans, Candida prapsilosis, Aspergillus, etc.) and viral (cytomegalovirus) pathogens. Microbiological cultures were performed for clinical samples including sputum, drainage fluid, blood, catheter segment/tip, mid-stream urine, etc. At least one kind of pathogen was found by microbiological culture in 45 (84.9%) patients (Supplementary Table 3). Microbiological cultures were found negative in 8 (15.1%) patients, among whom 3 were diagnosed with cytomegalovirus infection (Supplementary Table 3). PTI was predominated by bacterial infections followed by fungal and viral infections.

To minimize interference, two patients in the PTI group who died of severe pneumonia at 35 and 55 days after OLT and two patients in the non-PTI group who died of surgical complication at 45 and 50 days after OLT were excluded from survival analyses. The median follow-up was 1117 days (IQR: 439.75 to 2309 days), 70 (29.9%) patients had tumor recurrence, and the median tumor recurrence time was 300.5 days (IQR: 138.25 to 633 days) after OLT. The 1-, 3-, and 5-year OS rates of the study cohort were 86.6%, 69.0%, and 63.6%, and the 1-, 3-, and 5-year RFS rates were 75.7%, 60.0%, and 57.3%, respectively. Frequent tumor recurrence sites were the liver, lungs, bone, brain, and adrenal glands. Among the 80 patients died after OLT, 54 (67.5%) died of tumor recurrence. The 1-, 3-, and 5-year OS rates for the PTI patient group (96.0%, 89.3%, and 74.0%) were significantly higher than those for the non-PTI group (84.0%, 63.4%, and 60.2%, P = 0.033) (Figure 1). Univariate analyses revealed that age (P = 0.093), cirrhotic background (P = 0.004), preoperative alpha fetoprotein (AFP) level (P = 0.002), tumor size (P < 0.001), histological grade (P < 0.001), vascular invasion (VI) (P < 0.001), lymph node involvement (P < 0.001), pathological TNM stage (P < 0.001), and PTI (P = 0.033) were the factors associated with OS, while cirrhotic background (P = 0.020), preoperative AFP level (P = 0.003), tumor size (P < 0.001), histological grade (P < 0.001), VI (P < 0.001), lymph node involvement (P < 0.001), pathological TNM stage (P < 0.001), and PTI (P = 0.063) were the factors associated with RFS (Table 2). Multivariate analysis identified high histological grade (G3-G4) [relative risk (RR) = 1.977, 95% confidential interval (CI): 1.073-3.642 for OS; RR = 1.964, 95%CI: 1.083-3.561 for RFS], VI (RR = 4.237, 95%CI: 2.293-7.827 for OS; RR = 3.702, 95%CI: 1.959-6.999 for RFS), and absence of PTI (RR = 2.584, 95%CI: 1.226-5.449 for OS; RR = 2.683, 95%CI: 1.335-5.390 for RFS) were the independent risk factors for dismal OS and RFS (Table 3). In subgroup analysis, patients with PTI showed significantly better OS than those without PTI in the VI (P = 0.003) subgroup (Figure 2A), whereas PTI did not impact the OS of patients without VI (P = 0.404) (Figure 2B). These findings highlighted that PTI conferred significant survival benefit on patients at a high risk factor for post-OLT death. Furthermore, patients with PTI showed significantly better RFS than those without PTI in the VI (P = 0.003) subgroup (Figure 2C). However, PTI showed no influence on the RFS of the patients without VI (P = 0.251) (Figure 2D). These results indicated that PTI improved the RFS of the patients at a high risk factor for tumor recurrence. Moreover, we evaluated the potential benefit of PTI on the OS and PRS of the patients who suffered post-transplant tumor relapse. As a result, the patients with PTI revealed significantly better OS (P = 0.026) and PRS (P = 0.042) than those without PTI (Figure 3). These data suggested that PTI holds persistent survival benefit on the transplant HCC patients even after tumor relapse.

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Figure 1 Influence of post-transplant infection on overall survival of hepatocellular carcinoma patients treated by orthotopic liver transplantation. The Kaplan-Meier curve shows overall survival of the patients according to the post-transplant infection status. PTI: Post-transplant infection; PRS: Post-recurrence survival; OLT: Orthotopic liver transplantation.

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Figure 2 Influence of post-transplant infection on overall survival and recurrence-free survival of transplant hepatocellular carcinoma patients subgrouped by vascular invasion status. A: Kaplan-Meier overall survival (OS) curve for patients with vascular invasion (VI) classified according to the post-transplant infection (PTI) status (P = 0.003); B: Kaplan-Meier OS curves for patients without VI classified according to the PTI status (P = 0.404); C: Kaplan-Meier recurrence-free survival (RFS) curve for patients with VI classified according to the PTI status (P = 0.003); D: Kaplan-Meier RFS curve for patients without VI classified according to the PTI status (P = 0.251). PTI: Post-transplant infection; OLT: Orthotopic liver transplantation.

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Figure 3 Influence of post-transplant infection on overall survival and post-recurrence survival of transplant hepatocellular carcinoma patients with tumor relapse. A: Kaplan-Meier overall survival curve of patients with recurrent tumors stratified by post-transplant infection (PTI) status (P = 0.026); B: Kaplan-Meier post-recurrence survival curve of patients with recurrent tumors stratified by PTI status (P = 0.042). PTI: Post-transplant infection; OS: Overall survival; OLT: Orthotopic liver transplantation.

Tumor recurrence after orthotopic liver transplantation (OLT) is the stumbling block for long-term survival of the recipients with hepatocellular carcinoma (HCC). Identification of factors or measurements that have influence on overcoming HCC recurrence after OLT is of particular importance to innovate effective treatment strategy. Previous literature demonstrated that postoperative infection suppresses tumor recurrence and improves patient survival in lung cancer and malignant glioma probably via stimulating the immune system. Post-transplant infection (PTI), a common complication after OLT, is deemed to be harmful for the liver transplant recipients from a short-term perspective. Nevertheless, whether PTI inhibits HCC recurrence and prolongs the long-term survival of transplant HCC patients needs to be clarified.

The management of tumor recurrence in transplant HCC patients remains challenging. The results of the present study indicated new insights regarding how to improve outcome of transplant HCC patients, especially those at a high risk for post-OLT death and tumor recurrence.

To investigate the potential influence of PTI on the survival and tumor recurrence of patients with HCC after OLT.

A total of 238 patients with HCC who underwent OLT between August 2002 and July 2016 at our center were retrospectively included and accordingly subdivided into a PTI group (53 patients) and a non-PTI group (185 patients). Univariate analyses, including the differences of overall survival (OS), recurrence-free survival (RFS), and post-recurrence survival (PRS), between the PTI and non-PTI subgroups as well as survival curve analysis were performed by the Kaplan-Meier method, and the differences were compared using the log rank test. The variables with a P-value < 0.1 in univariate analyses were included in the multivariate survival analysis by using a Cox proportional-hazards model.

The 1-, 3-, and 5-year OS and RFS rates of the whole cohort were 86.6%, 69.0%, and 63.6%, and 75.7%, 60.0%, and 57.3%, respectively. The 1-, 3-, and 5-year OS rates for the PTI patient group (96.0%, 89.3%, and 74.0%) were significantly higher than those for the non-PTI group (84.0%, 63.4%, and 60.2%) (P = 0.033). Absence of PTI was an independent risk factor for dismal OS (RR = 2.584, 95%CI: 1.226-5.449) and unfavorable RFS (RR = 2.683, 95%CI: 1.335-5.390). Subgroup analyses revealed that PTI remarkably improved OS (P = 0.003) and RFS (P = 0.003) rates of HCC patients with vascular invasion (IV), but did not impact on OS (P = 0.404) and RFS (P = 0.304) of patients without VI. Among the patients who suffered post-transplant tumor recurrence, the patients with PTI showed significantly better OS (P = 0.026) and PRS (P = 0.042) rates than those without PTI.

PTI improves OS and RFS of transplant HCC patients at a high risk for post-transplant death and tumor recurrence, which may be attributed to suppressive effect of PTI on HCC recurrence.

Future development of bioengineered organisms that could potently activate the innate and adaptive immune system targeting tumor cells may present an attractive strategy to suppress HCC progression and prolong the patient survival after OLT with minimum side effects. Nevertheless, further validity of PTI as a potent tumor suppressor for HCC patients is critically needed in prospective cohorts at other transplant centers, as well as in well-designed experimental animal models.