Microscopic vascular invasion by hepatocellular carcinoma in liver transplant patients

Corresponding Author:

Brian I Carr
E-mail: brianicarr@hotmail.com

Abstract

Background: A characteristic of Hepatocellular Carcinoma (HCC) is to invade the portal venous system in the liver as a means of spread within the liver and systemically. The ensuing Portal Vein Thrombosis (PVT) is a poor prognosis parameter and often diagnosed radiologically pre-treatment. More limited Microvascular Portal Invasion (microPVI) is typically diagnosed on examination of tumors removed after treatment by resection or transplant. The biological characteristics and subsets of PVI are incompletely characterized. Aims: To examine HCC patients with and without microPVI to understand the clinical relationships to other tumor and clinical characteristics and to survival. Methods: A cohort of 270 liver transplant patients with HCC without macroscopic PVT that were available to us was examined. Patients with (165) and without (105) microPVI were compared for survival and clinical features. Results: The mean survival of patients with and without microPVI was significantly different: 86.6 versus 110.5 months, p=0.007. The microPVI+ patients differed from microPVI- patients in having a significantly larger number of tumor nodules, tumor size and higher serum levels of both Alpha-Fetoprotein (AFP) and almost significant for higher Gamma-Glutamyl Transpeptidase (GGT, p=0.053). Survival in microPVI+ patients related significantly to serum GGT (p=0.006) but not to AFP levels. The incidence of microPVI increased with increase in tumor size and survival decreased significantly with increase in tumor size for microPVI patients. Increase in tumor size was also associated with significantly higher serum GGT levels in patients who were microPVI+, but not in those who were microPVI. Furthermore, patients with microPVI who had prolonged survival significantly differed from those with shorter survival in respect only to tumor size and serum GGT levels. Conclusion: These findings draw attention to a group of patients with microPVI who have long survival and to the usefulness of serum GGT levels in their evaluation and prognosis.

Keywords

HCC, portal vein invasion, survival

Abbreviations

HCC: Hepatocellular Carcinoma; MTD: Maximum Tumor Diameter; AFP: Alpha-Fetoprotein; GGT: Gamma Glutamyl Transpeptidase; ALKP: Alkaline Phosphatase; AST: Aspartate Amino Transferase; ROC: Receiver Operating Characteristic Curve; OS: Overall Survival; PVT: Portal Vein Thrombosis (Macroscopic); DCP: Des Gamma Carboxy-Prothrombin; MRI: Magnetic Resonance Imaging; CT: Computerized Axial Tomography

Introduction

Microscopic Portal Vein Invasion (Micro- PVI) by Hepatocellular Carcinoma (HCC) is currently only diagnosed on tissue histological examination (in contrast to macroscopic portal vein thrombosis, which is diagnosed radiologically). This can be via biopsy, but more typically by examination of HCC tissues after surgical resection or Liver Transplantation (LT). MicroPVI is also considered to be an independent poor prognostic indicator following both hepatic resection for HCC [1-9] and liver transplantation [10]. Several risk factors for microPVI have been reported, and include tumor size and especially serum DCP levels [10-19], as well as AFP levels [14,18]. Several attempts have been made into classifying the degree of microPVI, based on numbers of invaded vessels and numbers of invading cells [20-22]. Altogether, MicroPVI has been considered to be a major mechanism for intra-hepatic spread of HCC [23].

The current work was undertaken to investigate, in a large series of patients after liver transplant for HCC, the factors associated with microPVI in this group of patients and the prognostic factors that might be involved. We found a correlation between presence of microPVI and Maximum Tumor Diameter (MTD) and also found a significant association between serum GGT levels and survival in microPVI patients.

Methods

Patients who underwent LT for HCC at our Liver Transplantation Institute without macroscopic portal vein thrombosis were the subjects of this study. The data were collected prospectively and were analyzed retrospectively. This study has been approved by Inonu University Institutional Review Board (Approval no: 2018/1-9).

The clinical parameters and tumor characteristics of 270 HCC patients who underwent LT were reviewed according to vascular invasion based on explant pathology report. AFP cut off 200 ng/ml and GGT cut off 100 IU/ml. These cutoffs were found by analysis of ROC and were significantly associated with survival post liver transplant [24]. Categorical (qualitative) variables were expressed as count and percentage and were compared using univariate analysis methods (Pearson chi-square test, continuity corrected chi-square test or Fisher’s exact test where appropriate). Multivariate binary logistic regression was performed for odds ratio estimations. As recommended by Hosmer and Leme show the variables with p value less than 0.25 in univariate analysis were included in multivariate analysis. Continuous (quantitative) variables were summarized by median, minimum, maximum values and compared using Mann-Whitney U test. Kaplan-Meier survival estimate was used to determine overall survival of the patients. Follow-up period was defined as the interval between LT until the date of last visit to the outpatient department for living patients or until the date of patient death. Statistical tests were considered significant when the corresponding p value was less than 5%. All statistical analyses were performed using IBM SPSS Statistics for Windows version 25.0 (New York, USA).

Results

The 270 patients who were transplanted for HCC were dichotomized into 165 patients with microscopic vascular invasion (microPVI +ve) and 105 patients without microscopic vascular (microPVI -ve). The survival of the 2 groups was significantly different, 2660 versus 3315 days (86.6 versus 110.5 months), p=0.007 (TABLE 1 AND FIGURE 1) accompanying cumulative survival graph). The cumulative proportion of surviving patients was also less for microPVI +ve compared to microPVI -ve patients at 1, 3, 5 and 10 years (TABLE 2).

TABLE 1. Survival of HCC patients with microscopic vascular invasion (PVI+) versus (PVI), by the Kaplan-Meier method.

TABLE 2. Cumulative proportion of surviving patients.

FIGURE 1. Graphical representation of survival of HCC patients with microscopic vascular invasion (PVI+) versus (PVI), by the Kaplan-Meier method.

The associated clinical characteristics of the 2 groups were also compared (TABLE 3) The tumor characteristics comprising MTD, tumor number and serum AFP levels were all significantly different between the 2 groups and worse for the microPVI +ve patients: MTD 4.1 vs. 2.4 cm, p<0.001; median tumor number was 2 vs. 1, p<0.001; serum AFP levels were 26.3 vs. 11.15, p<0.001. PVI +ve patients also had significantly higher serum AST, ALT and almost significant GGT levels (81 vs. 60, p=0.053) than microPVI -ve patients. Parameters with p value <0.2 were then included in a subsequent multivariate analysis model (TABLE 3). This multivariate analysis showed Odds Ratios (OR) for microPVT of significant parameters: MTD (OR: 1.427, p<0.001) and tumor number (OR: 1.266, p<0.001).

TABLE 3. Univariate and multivariate analysis of baseline parameter values and tumor characteristics of HCC patients with microscopic vascular invasion (microPVI+) versus without it (microPVI-).

MicroPVI +ve patients were then dichotomized according to high or low serum AFP levels, but no significant survival differences were found (TABLE 4). However, when the micro- PVI +ve patients were dichotomized according to high or low serum GGT levels, using GGT 100 IU/ml for cutoff as determined by previous ROC curves [24], the 2 microPVI +ve patient groups were found to have significantly different survival (1981.56 vs. 2866.59 days, p=0.006) by Kaplan Meier analysis (TABLE 5) and associated cumulative survival (FIGURE 2).

FIGURE 2. Graphical representation of survival of patients with Microscopic Portal Vein Invasion (PVI +ve) by the Kaplan-Meier method, dichotomized by serum GGT levels.

TABLE 4. Survival of patients with microscopic portal vein invasion (PVI+ve) by the Kaplan-Meier method, dichotomized by serum AFP levels.

TABLE 5. Survival of patients with Microscopic Portal Vein Invasion (PVI +ve) by the Kaplan-Meier method, dichotomized by serum GGT levels.

The patients were then ordered according to Maximum Tumor Diameter (MTD). TABLE 6 shows an incremental increase in the percent of patients having microPVI +ve, as MTD increased. Patients with <3 cm MTD tumors had 24% microPVI +ve rate; patients with 3-6 cm MTD had 47.7% microPVI +ve rate; while patients with MTD >6 cm tumors had a 61.5% microPVI +ve rate. There was a significantly different decrease in survival rate as the MTD increased for the microPVI +ve patients, but not for the microPVI -ve patients (TABLE 7). Interestingly, as MTD increased, there was a statistically significant increase in serum GGT levels for the microPVI +ve patients (p<0.001), but not for the microPVI -ve patients with similar MTD (TABLE 8).

TABLE 6. Percent of patients with microPVI (+) by MTD group.

TABLE 7. Survival for different MTD groups, by the Kaplan-Meier method, according to microPVI status.

Table 8. Comparison of microPVI (+) and microPVI (-) patients in relation to baseline serum GGT levels (IU/ml) in each MTD group.

Inspection of the Kaplan-Meier survival curve associated with (TABLE 1), showed that for microPVI +ve patients, there was an inflex ion point, suggesting the presence of shorter and longer survivors. The clinical characteristics of these 2 microPVI +ve groups of patients were compared (TABLE 9) and longer survivors were found to have significantly lower serum GGT levels and also significantly smaller tumors.

TABLE 9. Comparison of clinical characteristics between long (≥ 2000 days) vs. short survivors (<2000 days) post liver transplantation, all with microscopic portal vein invasion (microPVI+).

Discussion

Currently, microPVI+ is diagnosed after biopsy, most frequently, on pathological examination of a specimen obtained post resection or transplantation. That is the reason for focusing on these transplanted patients, since the diagnosis is usually made unambiguously, although only after treatment. Several questions therefore arise, including the prognostic significance, the possibility of non-surgical diagnosis, and the reasons for its association with larger MTD and higher levels of serum Des Gamma Carboxy Prothrombin (DCP) and the possibility of its prevention.

Most reports of the presence of microPVI +ve patients indicate a worse prognosis than those patients who do not have microPVI or macroPVT [1-15]. We found similarly a worse prognosis in patients with microPVI than in patients without it (TABLE 1). However, the worse prognosis also may depend in part on the degree of microPVI+ [8,25] and has been reported to be present in between 15 and 57% of HCC specimens [25].

Several reports are available on pre-operative and non-surgical, radiological diagnosis of microPVI+, including incomplete HCC capsule and typical dynamic HCC pattern on contrast- enhanced MRI scan [26-30]. Furthermore, molecular markers for microPVI+ have also been recently reported [23,31].

Many reports showed a significant association between increase in MTD and microPVI+ [11-14,32]. We also found a significant relation on multivariate analysis, as well as an increased percent of patients with microPVI+ with increase in MTD (TABLE 6). There has been little published investigation that we are aware of, as to the reasons for increase in incidence of microPVI with increasing MTD. Perhaps it is due to presence of constant microPVI+ per unit of tumor mass, so that as MTD increases so does the probability of diagnosing microPVI+. Possibly, as the tumor enlarges, there is a change in biology (as happens with tumor angiogenesis), so that increase in tumor size and microPVI+ are both inextricably involved in the mechanisms of increasing tumor aggressiveness, and thus of decrease in survival. The clinical pathology results of TABLE 1 give some support to this idea, as the patients with microPVI+ compared to microPVI- have significantly higher levels of serum AFP, as well as liver inflammation and damage parameters AST, ALT and almost, GGT.

Perhaps the most useful serum marker for microPVI+ and PVT is an increase in levels of the HCC marker des-gamma carboxy prothrombin or DCP [12,13,15,18,19,32]. Unfortunately, access to the DCP assay was not available to us. The reasons for the strong association of microPVI and PVT with high DCP levels in HCC patients have not been thoroughly investigated. However, vitamin K, which corrects the biochemical defect that causes DCP production by HCCs, also inhibits HCC migration [33- 35].

The main novel findings in this work are the relationships of GGT in microPVI+ patients. Although it was not statistically significant in multivariate analysis, possibly due to small patient numbers, serum GGT was significantly associated with microPVI+ as compared with microPVI- patients (TABLE 1) and it was a significant factor for survival in microPVI+ patients (TABLE 5). Serum GGT levels were also associated with survival in microPVI+ patients, but not in microPVI- patients (TABLE 8, FIGURES 3 AND 4), was the only significant poor prognosis factor, together with MTD, when long versus short survival patients with micro- PVI+ were compared (TABLE 9). The role of GGT in HCC survival has been shown previously and is associated with parameters of HCC aggressiveness [36,37] and its HCC expression may also offer a growth advantage [38] and it has previously been shown to be significantly associated with presence of PVT [39]. Thus, GGT levels might be usefully investigated in future analyses for both their association and their prognostic significance in patients with both microPVI+ and with PVT.

FIGURE 3. Graphical representation of comparison of microPVI (+) patients in relation to baseline serum GGT levels (IU/ml) in each MTD group.

FIGURE 4. Graphical representation of comparison of microPVI (-) patients in relation to baseline serum GGT levels (IU/ml) in each MTD group.

Conclusion

These findings draw attention to a group of patients with microPVI who have long survival and to the usefulness of serum GGT levels in their evaluation and prognosis.

Funding

This work is supported in part by NIH grant CA 82723 (B.I.C)

Conflicts of Interest

The authors declare that there are no conflicts of interest regarding the publication of this article.

References

1. Liu J, Zhu Q, Li Y, et al. Microvascular invasion and positive HB e antigen are associated with poorer survival after hepatectomy of early hepatocellular carcinoma: A retrospective cohort study. Clin Res Hepatol Gastroenterol. 42: 330-338 (2018).
2. Sparrelid E, Del Chiaro M. Microvascular invasion in hepatitis B virus-related hepatocellular carcinoma: another step toward preoperative evaluation? JAMA Surg. 151: 364 (2016).
3. Du M, Chen L, Zhao J, et al. Microvascular Invasion (MVI) is a poorer prognostic predictor for small hepatocellular carcinoma. BMC Cancer. 14: 38 (2014).
4. Lim KC, Chow PK, Allen JC, et al. Microvascular invasion is a better predictor of tumor recurrence and overall survival following surgical resection for hepatocellular carcinoma compared to the Milan criteria. Ann Surg. 254: 108-113 (2011).
5. Barreto SG, Brooke-Smith M, Dolan P, et al. Cirrhosis and microvascular invasion predict outcomes in hepatocellular carcinoma. ANZ J Surg. 83: 331-335 (2013).
6. Hirokawa F, Hayashi M, Asakuma M, et al. Risk factors and patterns of early recurrence after curative hepatectomy for hepatocellular carcinoma. Surg Oncol. 25: 24-29 (2016).
7. Kim H, Park MS, Park YN, et al. Preoperative radiologic and postoperative pathologic risk factors for early intra-hepatic recurrence in hepatocellular carcinoma patients who underwent curative resection. Yonsei Med J. 50: 789-795 (2009).
8. Shirabe K, Kajiyama K, Harimoto N, et al. Prognosis of hepatocellular carcinoma accompanied by microscopic portal vein invasion. World J Gastroenterol. 15: 2632-2637 (2009).
9. Tsai TJ, Chau GY, Lui WY, et al. Clinical significance of microscopic tumor venous invasion in patients with resectable hepatocellular carcinoma. Surgery. 127: 603-608 (2000).
10. Kaihara S, Kiuchi T, Ueda M, et al. Living-donor liver transplantation for hepatocellular carcinoma. Transplantation. 75: 37-40 (2003).
11. Okamura Y, Sugiura T, Ito T, et al. The predictors of microscopic vessel invasion differ between primary hepatocellular carcinoma and hepatocellular carcinoma with a treatment history. World J Surg. 42: 3694-3704 (2018).
12. Shimada S, Kamiyama T, Yokoo H, et al. Clinicopathological characteristics of hepatocellular carcinoma with microscopic portal venous invasion and the role of anatomical liver resection in these cases. World J Surg. 41: 2087-2094 (2017).
13. Eguchi S, Takatsuki M, Hidaka M, et al. Predictor for histological microvascular invasion of hepatocellular carcinoma: a lesson from 229 consecutive cases of curative liver resection. World J Surg. 34: 1034-1038 (2010).
14. Sakata J, Shirai Y, Wakai T, et al. Preoperative predictors of vascular invasion in hepatocellular carcinoma. Eur J Surg Oncol. 34: 900-905 (2008).
15. Masuda T, Beppu T, Okabe H, et al. Predictive factors of pathological vascular invasion in hepatocellular carcinoma within 3 cm and three nodules without radiological vascular invasion. Hepatol Res. 46: 985-991 (2016).
16. Poté N, Cauchy F, Albuquerque M, et al. Performance of PIVKA-II for early hepatocellular carcinoma diagnosis and prediction of microvascular invasion. J Hepatol. 62: 848-854 (2015).
17. Wu J, Xiang Z, Bai L, et al. Diagnostic value of serum PIVKA-II levels for BCLC early hepatocellular carcinoma and correlation with HBV DNA. Cancer Biomark. 23: 235-242 (2018).
18. Miyaaki H, Nakashima O, Kurogi M, et al. Lens culinaris agglutinin-reactive alpha-fetoprotein and protein induced by vitamin K absence II are potential indicators of a poor prognosis: a histopathological study of surgically resected hepatocellular carcinoma. J Gastroenterol. 42: 962-968 (2007).
19. Koike Y, Shiratori Y, Sato S, et al. Des-gamma-carboxy prothrombin as a useful predisposing factor for the development of portal venous invasion in patients with hepatocellular carcinoma: a prospective analysis of 227 patients. Cancer. 91: 561-569 (2001).
20. Rodríguez-Perálvarez M, Luong TV, Andreana L, et al. A systematic review of microvascular invasion in hepatocellular carcinoma: diagnostic and prognostic variability. Ann Surg Oncol. 20: 325-339 (2013).
21. Roayaie S, Blume IN, Thung SN, et al. A system of classifying microvascular invasion to predict outcome after resection in patients with hepatocellular carcinoma. Gastroenterology. 137: 850-855 (2009).
22. Zhao H, Chen C, Fu X, et al. Prognostic value of a novel risk classification of microvascular invasion in patients with hepatocellular carcinoma after resection. Oncotarget. 8: 5474-5486 (2017).
23. Iguchi T, Shirabe K, Aishima S, et al. New pathologic stratification of microvascular invasion in hepatocellular carcinoma: predicting prognosis after living-donor liver transplantation. Transplantation. 99: 1236-1242 (2015).
24. Ince V, Akbulut S, Otan E et al. Liver transplantation for hepatocellular carcinoma: Malatya experience and proposals for expanded criteria. Int J GI Ca. (2020) (in press).
25. Zhao H, Chen C, Fu X, et al. Prognostic value of a novel risk classification of microvascular invasion in patients with hepatocellular carcinoma after resection. Oncotarget. 8: 5474-5486 (2017).
26. Ariizumi S, Kitagawa K, Kotera Y, et al. A non-smooth tumor margin in the hepatobiliary phase of gadoxetic acid disodium (Gd-EOB-DTPA) enhanced magnetic resonance imaging predicts microscopic portal vein invasion, intrahepatic metastasis, and early recurrence after hepatectomy in patients with hepatocellular carcinoma. J Hepatobiliary Pancreat Sci. 18: 575-585 (2011).
27. Lei Z, Li J, Wu D, et al. Nomogram for preoperative estimation of microvascular invasion risk in hepatitis b virus-related hepatocellular carcinoma within the Milan criteria. JAMA Surg. 151: 356-363 (2016).
28. Ahn SY, Lee JM, Joo I, et al. Prediction of microvascular invasion of hepatocellular carcinoma using gadoxetic acid-enhanced MR and (18) F-FDG PET/CT. Abdom Imaging. 40: 843-851 (2015).
29. Unal E, Idilman IS, Akata D, et al. Microvascular invasion in hepatocellular carcinoma. Diagn Interv Radiology. 22: 125-132 (2016).
30. Tanaka S, Mogushi K, Yasen M, et al. Gene-expression phenotypes for vascular invasiveness of hepatocellular carcinomas. Surgery. 147: 405-414 (2010).
31. Ono A, Fujimoto A, Yamamoto Y, et al. Circulating tumor DNA. Analysis for liver cancers and its usefulness as a liquid biopsy. Cell Mol Gastroenterol Hepatol. 1: 516-534 (2015).
32. Poté N, Cauchy F, Albuquerque M, et al. Performance of PIVKA-II for early hepatocellular carcinoma diagnosis and prediction of microvascular invasion. J Hepatol. 62: 848-854 (2015).
33. Carr BI, Wang Z, Wang M, et al. Differential effects of vitamin K1 on AFP and DCP levels in patients with unresectable HCC and in HCC cell lines. Dig Dis Sci. 56: 1876-1883 (2011).
34. Ma M, Qu XJ, Mu GY, et al. Vitamin K2 inhibits the growth of hepatocellular carcinoma via decrease of des-gamma-carboxy prothrombin. Chemotherapy. 55: 28-35 (2009).
35. D'Alessandro R, Refolo MG, Lippolis C, et al. Strong enhancement by IGF1-R antagonists of hepatocellular carcinoma cell migration inhibition by Sorafenib and/or vitamin K1. Cell Oncol (Dordr). 41: 283-296 (2018).
36. Carr BI, Guerra V. A hepatocellular carcinoma aggressiveness index and its relationship to liver enzyme levels. Oncology. 90: 215-220 (2016).
37. Fu SJ, Zhao Q, Ji F, et al. Elevated preoperative serum gamma-glutamyl transpeptidase predicts poor prognosis for hepatocellular carcinoma after liver transplantation. Sci Rep. 6: 28835 (2016).
38. Hanigan M. Expression of gamma-glutamyl transpeptidase provides tumor cells with a selective growth advantage at physiologic concentrations of cyst(e)ine. Carcinogenesis. 16: 181-185 (1995).
39. Lu WP, Dong JH, Huang ZQ, et al. Clinical related factors of portal vein tumor thrombosis in patients with hepatocellular carcinoma: a logistic regression analysis. Zhonghua Wai Ke Za Zhi. 46: 733-736 (2008).