Impact of microvascular invasion on clinical outcomes in hepatocellular carcinoma patients following liver transplantation.

1. Introduction
Hepatocellular carcinoma (HCC) is currently the sixth most common cancer in the world and the third most fatal cancer.[1] Hepatitis B virus and hepatitis C virus infection play an important role in the occurrence of liver cancer. As one of the countries with the highest hepatitis B infection rate, China has the largest number of hepatitis B patients in the world, and the incidence of liver cancer has remained high. HCC has had a great impact on the public health of Chinese people. With the development of medical technology, the treatment effect of many cancers has improved significantly, but HCC is still a difficult problem in cancer. Not only is the incidence rate high, but the overall 5-year survival rate of liver cancer patients is reported to be only 12% to 18%.[1] Despite advances, surgical resection and liver transplantation offer the only potential for cure, yet recurrence remains a daunting obstacle.[2] HCC patients meeting the Milan Criteria could achieve good outcomes with 5-year survival rate more than 70%.[3] However, 20% to 57.8% of patients experience tumor recurrence within 5 years.[4–6] This persistent threat underscores the urgent need for better strategies to detect, prevent, and treat recurrence, making HCC a relentless adversary in the journey towards improved patient outcomes.
Microvascular invasion (MVI) is defined as tumor cells invading into micro vessels of the surrounding hepatic tissue and visible only through microscopy.[7,8] Multiple studies have approved that MVI is one of the most important factors associated with the poor survival and recurrence of HCC patients after surgical resection.[9,10] The 5-year survival rate and PFS rate of HCC patients with MVI were significantly lower than patients without MVI.[11,12] There were several studies that constructed the nomogram to predict the presence or absence of MVI using preoperative imaging and laboratory test results.[13–15] The accurate prediction of MVI before could help surgeons to make more suitable surgical plans as some studies have demonstrated that anatomical resection could achieve better OS and PFS for HCC patients with MVI.[16–18] However, for HCC patients underwent liver transplantation, the influence of MVI on patient prognosis was still controversial.[19–21] Neither the accepted Milan criteria nor the UCSF criteria mention the effect of MVI on patient outcomes. In this study, we collected the clinical and pathological data of HCC patients underwent liver transplantation and analyzed the influence of MVI on patient survival and recurrence.

2. Materials and methods

2.1. Clinical samples
The clinical data of 130 hepatocellular carcinoma (HCC) patients underwent classic orthotopic liver transplantation from January 2011 to October 2024 in our department were collected. All patients have signed the informed consent to participate in this project and this project was approved by the Ethics Committee of First People’s Hospital of Foshan (IRB approval number: FSYYY-EC-SOP-009-02.01-A01). Inclusion criteria: pathological diagnosis was HCC (American Joint Committee on Cancer criteria); Between 18 and 80 years old. Exclusion criteria: Incomplete clinical or pathological data; perioperative death; combined with other malignant tumor.

2.2. Follow-up
Patients were followed up regularly after the surgery. Patients would have physical examinations, tumor marker tests, biochemistry tests, blood routine and image examinations (ultrasound, CT or MRI) every month after surgery in the outpatient clinic and every 3 months 1 year later. Overall survival (OS) was defined as the date of surgery to the death of the patient or the date of the last follow-up, and progression-free survival (PFS) was defined as the surgery to the recurrence or metastasis of the patient. The follow-up time was until February 28, 2025.

2.3. Statistic analysis and sample size calculation
The sample size calculation was based on the 3-year survival rates of patients after surgery. According to preliminary experimental data, the survival rates were 0.3 for the MVI-positive group and 0.6 for the MVI-negative group. With a significance level (alpha) of 0.05 and beta value of 0.2, the sample size ratio between groups was 0.5. Using the formula to calculate sample size needed to compare 2 proportions, the calculated sample size was 29 cases for the experimental group and 58 cases for the control group, totaling 87 cases. Considering a 20% dropout rate, the total required sample size was determined to be 110 cases.
T test and Chi-square test were used to compare the differences between basic characteristics of 2 groups. The overall survival of 2 groups was analyzed using the Kaplan–Meier method (Log-rank test). Univariate and multivariate Cox regression were used for the analysis of prognostic factors. The survival curves were drawn using GraphPad Prism 8 for Windows Version 8.0.2. All statistical analysis were performed using SPSS IBM Statistics 25.0.

3. Results
There were 130 patients included in this study. The mean age of these patients was 51.49 ± 8.431 years (range, 31-67 years) and 118 (90.8%) patients were male. Among them, 101 (77.7%) patients had HBV infection and the AFP level of 29 (22.3%) patients were higher than 400 ng/ml. The average Child-Pugh score was 7.86 ± 2.174 and the average model for end-stage liver disease (MELD) score was 13.39 ± 7.089. There were 70 patients that have underwent pretransplant treatment including transcatheter arterial chemoembolization (TACE) or radiofrequency ablation (RFA). According to the postoperative pathological results, the maximal tumor diameter was 4.452 ± 3.142cm and 66 (50.77%) patients had single tumor lesion. There were 26 (20.00%) stage III-IV patients according to the American Joint Committee on Cancer version 8.
MVI was present in 39 (30.00%) patients. Among these patients, 17 had grade 2 MVI and 22 patients had grade 1 MVI. Table 1 showed the basic clinical and pathological characteristics of patients according to the existence of MVI. According to the difference analysis of relevant parameters between the 2 groups, patients with MVI have higher AFP levels(P = .001), larger tumor size(P < .001) and more advanced tumor stage(P < .001) than patients without MVI. Moreover, we also performed the correlation analysis between MVI and clinical indicators (Table S1, Supplemental Digital Content, https://links.lww.com/MD/R231). The result showed that MVI positive was associated with AFP level higher than 400 ng/ml, tumor diameter > 5cm and advanced tumor stage (III-IV).
According to the result of survival analysis (Fig. 1A), the overall survival of patients with MVI was significantly worse than patients without MVI (P = .0486). The 1-, 3-, and 5-year survival rates (69.23%, 30.77%, and 28.21%, respectively) in the MVI-positive group were significantly lower than those in the MVI-negative group (80.22%, 59.34%, and 38.46%, respectively).
We further performed the univariate and multivariate Cox regression analysis to study whether MVI were independent prognostic factor of patient overall survival. The result of univariate analysis (Table 2) showed that AFP level(P = .002), maximal tumor diameter(P = .006), tumor number(P = .024), tumor stage(P = .001) and MVI(P = .026) were influence factors of patient postoperative survival. According to the result of multivariate COX regression (Table 2), AFP level(P = .046) was prognostic factor of patient survival. According to Cox univariate analysis, AFP level, MELD score, tumor diameter, tumor number, tumor stage and MVI were influencing factors for postoperative PFS in patients with liver transplantation (P < .05). In multivariate analysis, AFP level was independent influencing factors for postoperative PFS in patients with liver transplantation (P = .013, Table 3).
The result of progression-free survival analysis also showed that patients with MVI are more likely to relapse after surgery(P = .0309) (Fig. 1B). The 1-, 3-, and 5-year PFS rates (58.97%, 23.08%, and 12.82%, respectively) in the MVI-positive group were significantly lower than those in the MVI-negative group (69.23%, 38.46%, and 20.88%, respectively).
In addition, among the 130 patients, a total of 69 patients met the Milan criteria. Among these patients, 10 of whom had MVI (14.49%), and 3 of these 10 patients (30.00%) had postoperative recurrence (recurrence times were 7,13.8,9.8 month), 2 of whom died of recurrence and metastasis at 18 and 16 months after surgery. Among 59 patients without MVI, only 4 patients (6.78%) had recurrence after surgery (recurrence times were 8,49,29,29 month). Among them, only 1 patient died of recurrence and metastasis at 17 months after the surgery. For patients within the Milan criteria, the postoperative OS and PFS were also significantly worse in the MVI positive group (Fig. 2A and 2B).

4. Discussion
HCC is one of the diseases that threaten human life and health the most in the world. Its incidence rate has been on the rise in recent years, and the mortality rate has remained high.[1] Liver transplantation is one of the most effective methods for the treatment of HCC, and it is also the only method that can cure both liver cancer and liver cirrhosis at the same time. Liver transplantation brings hope to HCC patients, but it does not mean that patients will no longer suffer from this disease even after the entire diseased liver was removed. The recurrence rate of HCC after transplantation was very high.[4–6] In this study, among the 130 HCC patients underwent liver transplantation, there were 27 patients underwent tumor recurrence or metastasis. We found that patients with MVI have higher proportion of early recurrence which was consistent with the more malignant tumor biology of MVI positive patients. Several literatures have explored the clinical influencing factors of MVI in HCC patients. Through literature search, we found that the occurrence of MVI in HCC patients was mainly correlated with the AFP level, tumor maximal diameter and tumor stage,[22–24] which was consistent with the findings of this study.
According to the literature, factors associated with the recurrence of patients after liver transplantation includes histological differentiation grade, tumor stage, serum AFP level and MVI.[25,26] However, studies on the effect of MVI on the survival of liver transplant patients are few and controversial.[27] Nitta et al reported that the 5-year OS rate and PFS rate of MVI-positive patients (60.9% and 51.4%) were lower than MVI-negative patients (89.9% and 80.6%).[19] However, Chan et al reported that the 5-year OS rate of HCC patients after liver transplantation was 85.7%, and was not affected by the presence or absence of MVI (88.2% vs. 85.1%).[21] There were also studies reported that the survival of patient was not influenced by the presence of MVI if the tumor size was <2 cm.[20] In this study, by reviewing the clinicopathological data of 130 HCC patients underwent liver transplantation in our hospital, we analyzed the effect of MVI on the survival and recurrence of patients and provided clinical evidence for the follow-up related research.
The reported incidence rate of MVI in HCC patients was 15% to 57.1%.[12] The proportion of MVI-positive HCC patients in this study was 24.4%, which was consistent with the data reported in the literature. The relatively low incidence of MVI in this study may be related to the generally earlier stage of the tumors in the included patients. It was reported that the proportion of MVI positive is mainly related to the diameter of the tumor. HCC with larger tumor size have higher probability of MVI positive.[28] The mean maximum diameter of MVI-positive tumors in this study was larger than that of MVI-negative patients, and the difference was statistically significant. In this paper, through correlation analysis, we found that MVI positivity was related to AFP, tumor diameter and tumor stage. These factors are all prognostic factors that have been reported in the literature and are closely related to patient survival after transplantation.[25]
MVI was acknowledged as tumor cells invading into a portal vein, hepatic vein, or large capsular vessel of surrounding hepatic tissues, which was only visible under microscopy.[8] Sumie et al had proposed the sub-classification of MVI into 3 grades, including NVI (no vascular invasion), mild MVI (1–5 vessels invaded), and severe MVI (more than 5 vessels invaded).[29] Kang et al had reported that MVI should be divided microvessel invasion (MI) and microscopic portal vein invasion (MPVI), and MPVI patients were reported to have poorer postoperative survival than MI patients.[9] In this paper, the grouping was based on the presence or absence of microvascular invasion, and there was no further subgroup analysis based on the degree of MVI, mainly because the sample size was not large enough.[30] This study found that MVI was an independent influencing factor of postoperative survival for HCC patients underwent liver transplantation, and MVI positive patients had a a lower 1, 3, 5-year survival rate after surgery. In addition, the 1, 3, 5-year PFS rate of MVI-positive patients were also significantly lower than that of the MVI-negative group, which was consistent with the previous study.[19]
At present, the worldwide accepted standard for recipient selection for liver cancer patients with liver transplantation is the Milan criteria.[31] It is believed that patients who meet the Milan criteria can achieve a good prognosis after surgery.[3] However, in clinical practice, we have found that some patients still appear in the early postoperative period recurrence phenomenon. This study found that among patients who met the Milan criteria, patients with MVI still had a worse prognosis than other patients and had a higher recurrence rate after surgery. This suggests that MVI may be an important reason for the poor prognosis of some patients who meet the Milan criteria, which deserves attention. However, due to the small sample size of this study, there may be bias, and large-scale clinical studies are still needed to further confirm the effect of MVI on the prognosis of liver cancer patients who meet the Milan criteria.
In order to further improve the postoperative survival of HCC patients after liver transplant, some researchers have proposed some predictive model for predicting the presence or absence of MVI through preoperative imaging examinations and laboratory indicators.[32,33] Zhang et al have recently reported a predictive model combining Gd-EOB-DTPA MRI and biochemical indicators for MVI prediction before surgery that could achieve the sensitivity and specificity of 94.7% and 93.2%, respectively.[34] For patients predicted to have MVI with a higher risk of recurrence, we should monitor the patient’s condition more closely and we could use immunotherapy, targeted therapy, adjuvant therapy or other therapies to further improve patient’s prognosis. Recently, have reported that bridging loco-regional therapy with Y-90 trans-arterial radio-embolization could reduce the incidence of MVI after liver transplantation and could improve patient’s prognosis and reduce recurrence.[35] It is feasible to reduce the risk of MVI recurrence after transplantation through various treatment methods, but further confirmation by large-scale clinical studies is still needed in the future. In addition, since the current prediction models still have certain defects, the accuracy of prediction needs to be further improved.
This study have the following limatations. Firstly, the single-center, retrospective design renders the findings vulnerable to selection and ascertainment biases; the cohort may not reflect the broader spectrum of HCC patients referred for transplantation elsewhere, and unmeasured confounders could distort the observed association between MVI and survival. Secondly, The sample size was not large enough and lacks the statistical power needed to yield precise or generalizable effect estimates, and the number of patients with MVI positive was small, so further subgroup analysis could not be performed based on MVI grading. Thirdly, the 13-year research period introduces temporal heterogeneity, as advances in imaging, peri-operative care, and immunosuppression over this span may confound the observed link between microvascular invasion and post-transplant outcomes. Prospective, multi-center validation with harmonized pathology protocols and granular MVI quantification is therefore needed before revising current transplant selection algorithms.

5. Conclusions
In conclusion, HCC patients with MVI have a worse prognosis after liver transplantation than patients without MVI. MVI was also correlated with the recurrence of patients after transplantation. Patients with MVI are more likely to have recurrence after surgery, and the PFS time is shorter. In addition, even for patients who met the Milan criteria, patients with MVI had a higher rate of postoperative recurrence and a worse prognosis.

Author contributions
Conceptualization: Huan-Wei Chen.
Funding acquisition: Huan-Wei Chen.
Investigation: Jing Deng, Ying Liu.
Methodology: Qing Yan.
Project administration: Qing Yan, Jing Deng, Ying Liu, Feng-Jie Wang.
Resources: Ying Liu, Feng-Jie Wang.
Supervision: Huan-Wei Chen.
Writing – original draft: Qing Yan, Jing Deng.
Writing – review & editing: Qing Yan, Jing Deng, Ying Liu, Feng-Jie Wang, Huan-Wei Chen.

Supplementary Material