Clinical Analysis of the Risk Factors of Early and Late Phase Recurrence after Surgical Excision of Hepatocellular Carcinoma: Experience of One Center with 345 Patients

    

    

    Figure 8: Cumulative recurrence-free survival curve for early phase
recurrence (Patients were divided into high-risk and low-risk group) (log-rank
test, χ2 value = 33.57, P‹0.001).
    

Establishing the mathematics model for late phase tumor recurrence

Cox’s multivariate proportional hazard model analysis identified that serum GGT level, tumor lesion number, preoperative CHE level and background liver cirrhosis status were the independent risk factors for late phase recurrence. On the basis of the results of Cox’s multivariate proportional hazard model analysis, the mathematics model for late phase tumor recurrence wash (t)=h0(t)exp(1.360 *tumor lesion number-2.869*background liver cirrhosis status +0.049*CHE+0.08*GGT).

Discussion

Hepatectomy can simultaneously remove the main tumor lesion and the surrounding tissue that may contain microscopically lesions that cannot be detected by conventional imaging techniques. And hepatectomy can be carried out almost without any waiting time. The two advantages mentioned above make hepatectomy the first choice treatment for most respectable HCC. Although the prognosis for most HCC patients has improved a lot over the last several decades, tumor recurrence remains the most common cause of treatment failure and even the deaths of HCC patients [1,2,6,9,17,20,22]. It has been reported in several studies that early phase recurrences are mainly due to intrahepatic dissemination and therefore are associated with aggressive pathological tumor factors such as vascular invasion, tumor diameter, multiplicity of tumor lesions, incomplete tumor capsule and tumor cell dissemination during the surgery [6,9,10,21,27,30,38]. It has been postulated that the factors mentioned above are very much likely to lead to intrahepatic metastasis and subsequently to early phase tumor recurrences [2,5,9,12,19,24,46]. Many experts believe early phase recurrences are associated with invasive nature of the tumor cells [1-5,15,26,34,42,46]. However, some other researchers found that liver status plays a much more important role in the tumor recurrence [5,12,26,34,45]. Thus, the precise mechanism of tumor recurrence still remains controversial.

In our study, we divided the tumor recurrences into two categories according to the recurrence time distribution pattern. Recurrences within two years after the surgery were defined as early phase recurrences and recurrences beyond two years after the surgery were defined as late phase recurrences. Moreover, it has been suggested by many experts in HCC treatment that different phases of recurrences are associated with different kinds of risk factors [1,7,9,18,25,39,42,46]. These findings may in part explain why many previous studies came to conflicting conclusions, since these studies did not distinguish early phase recurrences from late phase recurrences. Poon et al, were the first to distinguish early recurrences from late phase recurrences and find different phases of recurrences were correlated with different kinds of risk factors [2,9,14,20,27,36,42,46]. So they furthermore suggested that early phase recurrences mainly originated from intrahepatic metastases and late phase recurrences actually represented new tumor formation. However, the exact time that separates early phase recurrences from late phase recurrences is still controversial. Poon et al, suggested one year, which is different from our the two years we concluded from the recurrence time distribution pattern [2,9,14,20,27,36,42,46]. This difference might be due to different patient features. Imaura et al, also proposed that two years should be the demarcation time, although their research determined different risk factors from ours [5,14,17,18,23,46]. (The HCC patients in their research are HCVpositive, whereas most patients in our research are HBV-positive).

Our research found 4 risk factors associated with early phase recurrence, namely tumor lesion number, serum GGT level, high preoperative fibrinogen level and intraoperative blood transfusion. The identification of portal vein and IVC (inferior vena cava) cancerous thrombus as an adverse independent risk factors indicates that the main route by which early phase recurrence occurs is spreading through the portal vein. Several previous researches have pointed out that preoperative vascular invasion or even portal vein and IVC cancerous thrombus often heralds fulminant recurrence and extremely terrible prognosis [1-4,7,14,27,33,46].

Tumor lesion multiplicity is another adverse risk factor associated with early phase tumor recurrence and the possible explanation is that tumor lesion multiplicity reflects microscopic vascular invasion. Some other studies reported that serum AFP level, pTNM score, histological differentiation and tumor diameter are useful prognostic factors to predict early phase tumor recurrence [1- 6,10,19,21,37,44,46]. However, in our research, none of the factors mentioned above were proven to be significantly associated with early phase tumor recurrence. Contrary to the results of other studies, none of the surgery-related factors were identified to contribute to early phase recurrence. To sum up, the vascular invasion behavior of tumor cells is believed to be the most important mechanism how the early phase tumor recurrence occurs.

Besides the factors that reflect tumor cell vascular invasion nature, some other factors which include serum GGT level, preoperative fibrinogen level and intraoperative blood transfusion are also identified to be independent adverse risk factors of early phase tumor recurrence. The following three passages will be dedicated to discussing how these three factors are related to early phase tumor recurrence.

GGT, also known as gama glutamyl transpeptidase, is widely distributed through the human body tissues. The kidney tissue has the most abundant GGT content, followed by pancreas, liver and heart tissue [5,11,22,36,44]. Elevated serum GGT level is most often seen in hepatobiliary diseases. In HCC patients, serum GGT level is often several times above the upper limit. In our study, GGT is proven to be negatively related to early phase tumor recurrence. The GGT gene hypomethylation status of the HCC malignancy is the possible reason why GGT level is significantly elevated in most HCC patients [24,30,44]. Both the malignant cells in the tumor and adjacent inflamed liver tissue are responsible for the significantly elevated serum GGT level [24,30,41,44]. So significantly elevated serum GGT level can in part reflect the invasive nature of the tumor cells of HCC patients. So we can also relate elevated serum GGT level to the early phase tumor recurrence after surgical removal of all the visible HCC lesions. GGT has different isoforms which include F(fetus) type, P (placenta) type and H (HCC malignant cell ) type [44]. Of all these three types of GGT, HS-GGT is most valuable in the diagnosis of HCC [44]. Apart from aiding in the diagnosis of HCC, GGT especially HSGGT can be used to predicting tumor recurrence [44].

As one of the most important technologies promoting the development of surgical treatment, blood transfusion plays an irreplaceable role in the treatment of HCC. Opelz et al, found that blood transfusion before kidney transplantation could prolong the graft survival time through suppression of the immune system of the recipients [21,32,35]. Subsequent researches concluded that patients receiving blood transfusion especially massive blood transfusion were much more likely to experience tumor recurrence and to have a worse prognosis (breast cancer lung cancer and renal cell carcinoma and so on) [21,32,35]. Despite the discoveries mentioned above, the precise mechanism of this phenomenon still remains unknown. The following studies revealed that blood transfusion inhibits the immune system of the patients, such as the reactivity of the lymphocytes, NK cells (natural killer) and macrophages [21,32,35]. Blood transfusion makes the system produce all kinds of inhibitory factors enabling the tumor cells to escape from the attacks of the immune system and therefore to maintain able to grow and spread to other sites of the body. The fact that massive blood transfusion promotes tumor growth and recurrence also applies to HCC. Many retrospective studies of independent risk factors associated with HCC tumor growth and recurrence found that massive blood transfusion were significantly adversely correlated with recurrence and worse prognosis of HCC [21,32,35]. The development of modern surgical instruments, the perioperative management and the comprehensive treatment of HCC patients make the prognosis of the HCC patients much better than that of several decades ago [1-6,14,21,37,46]. However, the prognosis of HCC patients remains relatively poorer than other malignant diseases. And tumor recurrence remains the main factor affecting the long-term survival rates of HCC patients [1-9,15,26,33,46]. Intraoperative massive blood transfusion increases the recurrence rate and adversely affected the long-term prognosis of HCC patients. Hepatobiliary surgeons must do their best to improve their surgical skills and make use of the modern surgical instruments to reduce the unnecessary or uncalled-for blood transfusion to improve the prognosis of HCC patients to the maximum extend.

Many kinds of malignant tumors are with elevated serum fibrinogen and elevated level of fibrinogen is associated with tumor recurrence, vascular invasion and metastasis [6,14,25,37,44]. HCC is not exceptional. The liver plays a vital role in maintaining the normal function of the blood coagulation system. First, the liver syntheses many kinds of coagulation, anticoagulation and fibrinolytic factors. Second, the liver is responsible for eliminating the activated coagulation factors, plasminogen activator and fibrinogen degradation products. So, the liver is vital in keeping the balance between the coagulation system and the anticoagulation system. The coagulation state of the HCC patients is varied among the patients while most patients who have malignant tumors are usually in hypercoagulative state. The reason for the phenomenon mentioned above is that HCC patients usually have cirrhosis background. Some patients are in hypercoagulative state because they have multiple tumor lesions or lesions invading the vascular system which produce too much procoagulation factors while the background cirrhosis is not so severe. Some other patients are in hypo coagulation state because of the too severe cirrhosis background. As one of the factors involved in the coagulation system, fibrinogen level shows the same pattern as the hypercaogulation state. Normally, patients with liver diseases usually has low levels of fibrinogen, since the ability to produce fibrinogen of the patients with liver diseases is compromised. So, the tumor stage and the liver function are two main factors affecting the levels of fibrinogen. Fan et al found that HCC HepG2 produced more fibrinogen than normal cells [4,8,13,22,47]. They also concluded that the levels of fibrinogen of the patients with vascular invasion or cancerous thrombus were much higher than those of the patients without vascular or cancerous thrombus [4,8,13,22,47]. That the level of fibrinogen is significantly positively associated with tumor stage was also confirmed [4,8,13,22,47].`The fibrinogen level of stage III and IV patients is significantly higher than that of stage I and II patients when the patients have the same liver function status [4,8,13,22,47]. Pajumbo et al, found mice without fibrinogen expression are immune to tumor recurrence and metastasis, which was consistent with many clinical researches [4,8,13,22,47]. Possible reasons why fibrinogen is associated with tumor recurrence and metastasis are as follows: (1) fibriogen molecule serves as a bridge of tumor cells, platelet cells and endothelial cells to facilitate the recurrence and metastasis of tumor cells, (2) fibrinogen molecule helps the positioning and attachment of the malignant microembolism in the targeted organ, which is vital to tumor recurrence and metastasis, (3) fibriogen molecules serve as the temporary mesenchymal tissue that provide the tumor cells with nutrients and oxygen, (4) the microcomplex of fibrinogen molecules, tumor cells and platelets protect the tumor cells from attacks of the immune system [4,8,13,22,47]. In summary, serum fibrinogen level can be used as one of the markers predicting the tumor development, local invasion and metastasis.

Contrary to early phase tumor recurrence, late phase tumor recurrence is associated with different risk factors. Tumor lesion number, background liver cirrhosis status, serum CHE level and GGT were found to be independent risk factors for HCC late phase recurrence. As discussed above, tumor lesion multiplicity is one risk factor for early phase recurrence, but we can also explain that tumor multiplicity implies that the liver is severely damaged and is prone to form new tumor lesions in the diseased tissue. It has been confirmed by many previous studies that late phase HCC recurrence after surgery is mainly due to intrahepatic carcinogenesis de novo and the microenvironment is the basis of tumor late phase recurrence [1-5,19,25,37,45,46]. Although liver cirrhosis status was found to be adversely affect tumor late phase recurrence, Li Bo et al, did not confirm liver cirrhosis as an independent risk factor for late phase tumor recurrence [46]. But their research concluded that continuous variables such as albumin level and ICGR15 (indocyanine green retention after 15 minutes) could more accurately reflect the damage degree of remnant liver tissue than categorical variables such as liver cirrhosis status [46]. ICGR15 test have been adopted by many surgeons and the hospital we work with has just initiated ICGR15 test. So we could have analyzed tumor late phase recurrence more accurately and objectively if we had adopted ICGR15 test to reflect liver function reserve instead of liver cirrhosis status. Although many surgeons believed antiviral therapy preoperatively and postoperatively may reduce the recurrence risk, we did not confirm this conclusion, however. It may be due to the fact that many patients lack the data of HBV infection and antiviral therapy. Li Bo et al, as well as many other surgeons concluded that perioperative antiviral therapy, no matter preoperatively or postoperatively, might reduce tumor late phase recurrence to a rather great extend [5,17,27,45,46]. We adopted HBVDNA to reflect HBV infection status and analyzed the relationship between preoperative antiviral therapy and tumor late phase recurrence. However, the result of our research was not so hopeful as other studies. Although we have not confirmed that perioperative antiviral therapy reduces the tumor recurrence risk, we still recommend that antiviral therapy be initiated perioperatively given many other experts confirmed the finding. Serum GGT level significantly increases either when tumor vascular invasion occurs or remnant liver damage is severe. We can explain why serum GGT level is significantly associated with tumor late recurrence since elevated GGT level could be related to cirrhotic liver background.

CHE, also known as cholinesterase, has two types, namely true CHE and false CHE. True CHE is mainly located within the neuromuscular junction and responsible for the breakdown of acetylcholine while false CHE is widely distributed all over the whole body (mainly the brain, serum, liver and intestine and kidney). The CHE in the serum is mainly produced by the liver. Most of the HCC patients have decreased CHE level, since most patients have the cirrhosis background (the liver produces most of the CHE in the serum). Our study found decreased CHE level was significantly associated with late phase tumor recurrence and the possible mechanisms are as follows. The liver produces most of the CHE in the serum and thus serum CHE level is one marker of cirrhosis and liver function reserve. Low serum concentration of CHE is indicative of liver microenvironment damage and thus is associated with late phase recurrence. Zhao et al found in their study that most of the molecules of the cholinergic system were expressed in HCC tumor tissue and the expression of CHE was significantly lower than the adjacent normal liver tissue or even the cirrhotic tissue [43]. It was also discovered in the experiment that CHE molecules could inhibit the growth and cloning of SMMC-7221 and HepG2 cells and Ache is the direct acting molecule of the inhibiting procedure [43]. They also confirmed that the possible molecular signal transduction pathway involved was inhibition of the MAPK-PI3K/Akt [43]. So the low serum CHE level is indicative of low expression of CHE in the tumor cells and cirrhotic tissue and thus is significantly associated with tumor late phase recurrence [43].

In summary, our study finds that intrahepatic metastasis from the primary tumor contributes to the early phase tumor recurrence after the surgery and the carcinogenesis de novo from the cirrhotic liver is the main reason why late phase tumor recurrence occurs. So it is understandable that early phase tumor is mainly associated with tumor-related factors and late phase tumor recurrence is chiefly associated with factors reflecting the severity of the damage of the remnant liver tissue. The recurrence and metastasis of the tumor can be avoided or delayed at least through the following means: to limit the extend of the excising and adopt sophisticated surgical skills to reduce the blood loss and the blood transfusion as far as possible, to adopt comprehensive treatment protocols for the patients with vascular invasion or cancerous thrombus, to control the longstanding infection of the HCC patients to preserve the liver function reserve of the remnant liver lobes. And furthermore, patients who are at high risk of recurrence, no matter early phase or late phase, should be closely monitored and receive corresponding treatment. Through all the efforts mentioned above, we can hope to increase the recurrence-free survival time of the patients as far as possible.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (No. 81470870, 81570593), Sci-tech Research Development Program of Guangzhou city (No. 2014Y2-00200, 201604020001, 201508020262, 201400000001-3, 201607010024), National 13^th Five-Year Science and Technology Plan Major Projects of China (2017ZX10203205-006-001); Guangdong Key Laboratory of Liver Disease Research (2017B030314027).

References

1. Kim JH, Choi MS, Lee H. Clinical features and prognosis of hepatocellular carcinoma in young patients from a hepatitis B-endemic area. J Gastroenterol Hepatol. 2010; 21: 588-594.
2. Lam VW, Ng KK, Chok KS. Risk factors and prognostic factors of local recurrence after radiofrequency ablation of hepatocellular carcinoma. J Am Coll Surg. 2011; 207: 20-29.
3. Clark HP, Carson WF, Kavanagh PV. Staging and current treatment of hepatocellular carcinoma. Radiographics. 2009; 25: S3-23.
4. Schwartz M, Roayaie S, Konstadoulakis M. Strategies for the management of hepatocellular carcinoma. Nat Clin Pract Oncol. 2012; 4: 424-432.
5. Llovet JM, Schwartz M, Mazzaferro V. Resection and liver transplantation for hepatocellular carcinoma. Semin Liver Dis. 2013; 25: 181-200.
6. Yi NJ, Suh KS, Kim T, Kim J. Current role of surgery in treatment of early stage hepatocellular carcinoma: resection versus liver transplantation. Oncology. 2013; 75: 124-128.
7. Fan ST, Lo CM, Liu CL. Hepatectomy for hepatocellular carcinoma: toward zero hospital deaths. Ann Surg. 2009; 229: 322-330.
8. Lau WY, Lai EC. Hepatocellular carcinoma: current management and recent advances. Hepatobiliary Pancreat Dis Int. 2011; 7: 237-257.
9. Poon RT, Fan ST, Lo CM. Long-term survival and pattern of recurrence after resection of small hepatocellular carcinoma in patients with preserved liver function: implications for a strategy of salvage transplantation. Ann Surg. 2002; 235: 373-382.
10. Tsai TJ, Chau GY, Lui WY, Tsay SH, King KL, Loong CC, et al. Clinical significance of microscopic tumor venous invasion in patients with respectable hepatocellular carcinoma. Surgery. 2010; 127: 603-608.
11. Cha C, Fong Y, Jarnagin WR, Blumgart LH, DeMatteo RP. Predictors and patterns of recurrence after resection of hepatocellular carcinoma. J Am Coll Surg. 2009; 197: 753-758.
12. Nagano Y, Shimada H, Takeda K, Ueda M, Matsuo K, Tanaka K, et al. Predictive factors of microvascular invasion in patients with hepatocellular carcinoma larger than 5 cm. World J Surg. 2008; 32: 2218-2222.
13. Sumie S, Kuromatsu R, Okuda K, Ando E, Takata A, Fukushima N, et al. Microvascular invasion in patients with hepatocellular carcinoma and its predictable clinicopatho-logical factors. Ann Surg Oncol. 2010; 15: 1375- 1382.
14. Yamamoto J, Kosuge T, Takayama T, Shimada K, Yamasaki S, Ozaki H, et al. Recurrence of hepatocellular carcinoma after surgery. Br J Surg. 2005; 83: 1219-1222.
15. Nagasue N, Uchida M, Makino Y, Takemoto Y, Yamanoi A, Hayashi T, et al. Incidence and factors associated with intrahepatic recurrence following resection of hepatocellular carcinoma. Gastroenterology. 2004; 105: 488- 494.
16. Eguchi S, Kanematsu T, Arii S, Okazaki M, Okita K, Omata M, et al. Comparison of the outcomes between an anatomical subsegmentectomy and a non-anatomical minor hepatectomy for single hepatocellular carcinomas based on a Japanese nationwide survey. Surgery. 2011; 143: 469-475.
17. Hasegawa K, Kokudo N, Imamura H, Matsuyama Y, Aoki T, Minagawa M, et al. Prognostic impact of anatomic resection for hepatocellular carcinoma. Ann Surg. 2009; 242: 252-259.
18. Hasegawa K, Kokudo N. Surgical treatment of hepatocellular carcinoma. Surg Today. 2012; 39: 833-843.
19. Du ZG, Li B, Wei YG, Yin J, Feng X, Chen X. A new scoring system for assessment of liver function after successful hepatectomy in patients with hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int. 2011; 10: 265-269.
20. Sakka SG, Meier-Hellmann A. Non-invasive liver function monitoring by indocyanine green plasma disappearance rate in critically ill patients. Int J Intensive Care. 2008; 9: 66-72.
21. Shindoh J, Hasegawa K, Inoue Y, Ishizawa T, Nagata R, Aoki T, et al. Risk factors of post-operative recurrence and adequate surgical approach to improve long-term outcomes of hepatocellular carcinoma. HPB (Oxford). 2013; 15: 31-39.
22. Bruix J, Hessheimer AJ, Forner A, Boix L, Vilana R, Llovet JM. New aspects of diagnosis and therapy of hepatocellular carcinoma. Oncogene. 2010; 25: 3848-3856.
23. Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer. 1988; 7: 462-503.
24. Poon RT, Fan ST, Ng IO, Lo CM, Liu CL, Wong J. Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma. Cancer. 2006; 89: 500-507.
25. Imamura H, Matsuyama Y, Tanaka E, Ohkubo T, Hasegawa K, Miyagawa S, et al. Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol. 2007; 38: 200-207.
26. Poon RT, Fan ST, Ng IO, Lo CM, Liu CL, Wong J. Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma. Cancer. 2006; 89: 500-507.
27. Ikai I, Arii S, Kojiro M, Ichida T, Makuuchi M, Matsuyama Y, et al. Reevaluation of prognostic factors for survival after liver resection in patients with hepatocellular carcinoma in a Japanese nationwide survey. Cancer. 2005; 101: 796-802.
28. Wu CC, Cheng SB, Ho WM, Chen JT, Liu TJ, P’eng FK. Liver resection for hepatocellular carcinoma in patients with cirrhosis. Br J Surg. 2005; 92: 348- 355.
29. Sakata J, Shirai Y, Wakai T, Kaneko K, Nagahashi M, Hatakeyama K. Preoperative predictors of vascular invasion in hepatocellular carcinoma. Eur J Surg Oncol. 2008; 34: 900-905.
30. Kim H, Park MS, Park YN, Kim H, Kim KS, Choi JS, 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. 2009; 50: 789-795.
31. Tarao K, Ohkawa S, Shimizu A, Harada M, Nakamura Y, Ito Y, et al. Significance of hepatocellular proliferation in the development of hepatocellular carcinoma from anti-hepatitis C virus-positive cirrhotic patients. Cancer. 1994; 73: 1149- 1154.
32. Ruà S, Comino A, Fruttero A, Torchio P, Bouzari H, Taraglio S, et al. Flow cytometric DNA analysis of cirrhotic liver cells in patients with hepatocellular carcinoma can provide a new prognostic factor. Cancer. 1996; 78: 1195- 1202.
33. Morris-Stiff G, Gomez D, de Liguori Carino N, Prasad KR. Surgical management of hepatocellular carcinoma: is the jury still out? Surg Oncol. 2009; 18: 298-321.
34. Bigourdan JM, Jaeck D, Meyer N, Meyer C, Oussoultzoglou E, Bachellier P, et al. Small hepatocellular carcinoma in Child A cirrhotic patients: hepatic resection versus transplantation. Liver Transpl. 2003; 9: 513-520.
35. Kumada T, Nakano S, Takeda I, Sugiyama K, Osada T, Kiriyama S, et al. Patterns of recurrence after initial treatment in patients with small hepatocellular carcinoma. Hepatology. 1997; 25: 87-92.
36. Wakai T, Shirai Y, Yokoyama N, Nagakura S, Hatakeyama K. Hepatitis viral status affects the pattern of intrahepatic recurrence after resection for hepatocellular carcinoma. Eur J Surg Oncol. 2003; 29: 266-271.
37. Portolani N, Coniglio A, Ghidoni S, Giovanelli M, Benetti A, Tiberio GA, et al. Early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications. Ann Surg. 2006; 243: 229-235.
38. Du Y, Su T, Ding Y, Cao G. Effects of antiviral therapy on the recurrence of hepatocellular carcinoma after curative resection or liver transplantation. Hepat Mon. 2012; 12: e6031.
39. Yang T, Lu JH, Zhai J, Lin C, Yang GS, Zhao RH, et al. High viral load is associated with poor overall and recurrence-free survival of hepatitis B virusrelated hepatocellular carcinoma after curative resection: a prospective cohort study. Eur J Surg Oncol. 2012; 38: 683-691.
40. Lok AS. Does antiviral therapy prevent recurrence of hepatitis B virus-related hepatocellular carcinoma after curative liver resection? JAMA. 2012; 308: 1922-1924.
41. Shah SA, Greig PD, Gallinger S. Factors associated with recurrence after resection for hepatoceluular carcinoma [J] Am surg. 2006; 202: 275-283.
42. Poon RT, Fan ST, Lo CM, Liu CL, Wong J. Intrahepatic recurrence after curative resection of hepatocellular carcinoma: long-term results of treatment and prognostic factors. Ann Surg. 1999; 229: 216–222.
43. Zhao JY, Gu JR, He XH, Wan DF. The study on the function and the molecular mechanism of Ache in hepatic carcinogenesis. Hepatology. 2010; 187: 195-199.
44. Yao FD, Dong ZZ, Gu QQ, Yao M. Specific diagnosis of gamma-glutamyl transferase subfraction and its genotyping for hepatocellular carcinoma. Surg Oncol. 2009; 18: 298-321.
45. Di-Peng Qu, Lian-Yue Yang, Geng-Wen Huang, Yi-Ming Tao, Xiang Ding, Zhi-Gang Chang. Clinical analysis of the risk factors for recurrence of hepatocellular carcinoma and its relationship with hepatitis virus B infection. World J Gstroenterol. 2005; 11: 2061-2066.
46. Zheng-Gui Du, Yong-Gang Wei, Ke-Fei Chen, Bo Li. Risk factors associated with early and late recurrence after curative resection of hepatocellular carcinoma: a single institutions experience with 345 consecutive patients. Hepatobiliary PANCREAT Dis Int. 2014; 13: 153-161.
47. Zhao-Xia Duan, Lin-Hua Yang. Research of coagulation, fibrinolysis and matrix metalloproteinase system with cancer metastases and thrombosis. Oncology. 2008; 10: 423-430.