Introduction

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide [1]. Although it is common in Asia and Africa, the incidence of HCC has increased rapidly in the USA [2]. Because of technical improvements in hepatic resection and earlier diagnosis, the overall and disease-free survival rates after resection of HCC have improved significantly in recent decades [3]. However, long-term survival remains unsatisfactory because of a high incidence of recurrence. Even after curative resection, the postoperative recurrence rate remains high, and most recurrences tend to occur in remnant liver.

The tumor number [4–7] and macrovascular invasion [4, 7] are poor predictive factors after resection in HCC patients. The latest TNM classification for HCC from UICC/AJCC also emphasizes that the number of nodes and vascular invasion are the crucial factors for classification and prognosis of HCC [8]. Although several previous studies have reported the outcomes and prognostic factors after hepatectomy in patients with single HCC [7, 9, 10], they were performed in patients who had confounding factors, such as noncurative resection [7, 9] and macrovascular invasion [7, 9, 10]. So far, no study has focused on patients with single HCC without macrovascular invasion, and the long-term outcome and prognostic factors of these patients remain unknown.

Tumor vessel invasion can be divided into macroscopic and microscopic or only microscopic. Macroscopic vessel invasion is an important prognostic factor after surgical resection of HCC [4, 7]. Although several studies have suggested that the presence of MVI is an independent factor predictive of poor survival after resection of HCC [11–14], the prognostic significance of MVI after curative resection of patients with solitary HCC without macrovascular invasion is still unclear.

We retrospectively reviewed 260 patients with single HCC without macrovascular invasion. The purpose of this study was to evaluate the prognostic value of clinical and histopathologic variables, especially MVI, after curative resection.

Materials and methods

Patients

From December 2004 to December 2007, 491 consecutive patients with macroscopically single HCC underwent hepatectomy in the Hepatobiliary Department of the Sun Yat-sen University Cancer Center. The diagnosis of HCC was confirmed by histopathologic examination. Patient inclusion criteria for this study were as follows: (1) macroscopically single HCC; (2) no evidence of macrovascular invasion as evaluated by two independent, experienced imaging physicians who examined the preoperative imaging and resected specimens; (3) underwent curative hepatic resection; (4) received no previous therapy. Curative resection of single HCC without macrovascular invasion was performed as described. First, the tumor was resected. Second, negative surgical margins were confirmed by histologic examination. Third, the absence of extrahepatic metastasis was confirmed. Fourth, the absence of a residual tumor was confirmed by dynamic contrast-enhanced computerized tomography (CT) or ultrasonography 3–5 weeks postsurgery. Of these 491 patients, 293 patients met these criteria. Thirty-three of the 293 patients were excluded from the study because detailed records regarding survival and/or cancer recurrence were not available, and the remaining 260 patients were enrolled in this study.

Clinicopathologic variables

We obtained the following clinicopathologic information from chart review: gender, age, ALT, AST, albumin, total bilirubin, prothrombin time, hepatitis B serology, HCV-Ab, cirrhosis, AFP, tumor size, tumor capsule, Edmondson–Steiner grade, microvascular invasion, resection margin, blood loss, blood transfusion, and hepatectomy procedure. Tumor size was based on the largest diameter of the tumor specimen. The width of the surgical margin was measured as the smallest distance from the tumor edge to the resection line.

The histologic grade (I–IV) was determined by using the criteria of Edmondson and Steiner and was based on the areas showing the highest grade. Microvascular invasion was defined as the presence of clusters of cancer cells floating in the vascular space lined by endothelial cells on histopathologic examination of the resected specimens. The presence of MVI was assessed by two independent pathologists.

Patient follow-up

After discharge, ultrasonography or contrast-enhanced dynamic CT was performed monthly in the first 2–3 months after surgery, then every 2–3 months in the first year, and every 3–6 months thereafter. When tumor recurrence or metastasis was suspected, further investigations, including magnetic resonance imaging (MRI), hepatic angiography, and biopsies, were performed. Telephone calls were performed when necessary. The follow-up data were regularly updated in the database for each patient. Follow-up data were obtained from the patient’s database for this study. Follow-up ended on September 1, 2012 or the date of the patient’s death. By the end of follow-up, none of the 260 patients were lost. The median follow-up of the 260 patients was 60.90 months (range 5.40–93.87 months).

Statistical analysis

Overall survival (OS) was measured from the date of hepatectomy to death or the most recent follow-up time. Disease-free survival (DFS) was measured from the date of hepatic resection to the date of the first diagnosis of tumor recurrence or the most recent follow-up time. Survival rates were calculated by using the Kaplan–Meier method. Survival rates were compared by the log-rank test for univariate analysis. All of the variables that were significant in univariate analysis were then entered into a Cox proportional hazards model for multivariate analysis by using stepwise selection to identify independent variables correlated with disease-free survival and overall survival. Statistical analysis was performed by using SPSS 16.0 for Windows. p values < 0.05 were considered statistically significant.

Results

Clinicopathologic characteristics

There were 35 women and 225 men. The median age was 49 years (range 15–76, mean 49.15 ± 11.68). All 260 patients in this study were stage I according to the 7th NCCN TNM classification for HCC. Table 1 summarizes the clinicopathologic characteristics of the 260 patients with single HCC without macrovascular invasion.

Table 1 Clinicopathologic characteristics of the 320 curatively resected patients with single hepatocellular carcinoma (HCC) without macrovascular invasion and univariate analysis of survival after hepatectomy according to clinicopathologic factors
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Predictors of overall survival

No patients died within 1 month after the operation. The 1-, 3-, and 5-year overall survival rates for this series of patients were 96.54, 83.46, and 74.01 %, respectively (Fig. 1).

Fig. 1
figure 1

Survival curve for patients with single hepatocellular carcinoma (HCC) without macrovascular invasion who underwent curative resection between 2004 and 2007 (n = 260 patients)

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The results of univariate analysis for survival rate after curative resection of single HCC without macrovascular invasion are summarized in Table 1. According to univariate analysis, the presence of microvascular invasion (p = 0.015) was a significant prognostic factor associated with poor prognosis (Fig. 2), while resection margin ≥ 1 cm, tumor size > 5 cm, and AFP elevation were not significant (Table 1). The overall survival rate tended to be higher in patients who underwent curative resection with resection margin ≥ 1 cm (77.46 % at 5 years) than in those who with resection margin < 1 cm (66.94 % at 5 years), although this difference did not reach statistical significance (p = 0.0504). To further evaluate the relationship between tumor size, AFP level and overall survival in patients with single HCC without macrovascular invasion, we divided these patients into groups according to the different cutoff values and compared overall survival rates between these groups. We defined 3 groups according to tumor size: group I (n = 158), tumor size ≤ 5 cm; group II (n = 115), tumor size between 5 and 10 cm; group III (n = 47), tumor size > 10 cm. We found no difference in overall survival between these three groups (p = 0.202). We defined low-AFP, intermediate-AFP, and high-AFP groups by using 100, 400, and 1000 ng/ml as cutoff values, respectively. We found that AFP level was not associated with survival in this population (low AFP: p = 0.784; intermediate AFP: p = 0.796; high AFP: p = 0.157).

Fig. 2
figure 2

Overall survival curve for patients with and without microvascular invasion (MVI). The overall survival rate of the MVI(−) group was significantly better than that of the MVI(+) group (p = 0.015)

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The results of a multivariate analysis model including microvascular invasion and resection margin indicated that only microvascular invasion was a significant predictive factor of survival after curative resection for single HCC without macrovascular invasion (Table 2). The overall survival rates at 1, 3, and 5 years for patients with and without MVI were 88.46, 71.15, 63.30 and 98.56, 86.54, 76.70 %, respectively (Table 1).

Table 2 Independent predictors for overall survival identified by multivariate analysis
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Predictors of disease-free survival

During follow-up, 122 patients were diagnosed as having HCC recurrence, and 138 patients had no evidence of recurrence. The overall recurrence rate was 46.92 %. At the date of the first diagnosis of tumor recurrence, 105 patients had developed intrahepatic recurrence only, 15 patients had developed extrahepatic recurrence only, and 2 patients had developed both intrahepatic and extrahepatic recurrence. The disease-free survival rates for this series of patients at 1, 3, and 5 years were 79.62, 62.69, and 56.01 %, respectively (Fig. 3).

Fig. 3
figure 3

Disease-free survival curve for patients with single hepatocellular carcinoma (HCC) without macrovascular invasion who underwent curative resection between 2004 and 2007 (n = 260 patients)

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Univariate analysis identified two significant prognostic factors for disease-free survival: gender (p = 0.031) and microvascular invasion (p = 0.031) (Figs. 4, 5). The cumulative disease-free survival rate tended to be higher in patients who underwent curative resection with resection margin ≥ 1 cm (59.68 % at 5 years) than in those with resection margin < 1 cm (48.51 % at 5 years), although this difference did not reach statistical significance (p = 0.090). Multivariate analysis including microvascular invasion, gender, and resection margin indicated that only microvascular invasion was a significant predictor of disease-free survival for this series of patients (Fig. 4). The disease-free survival rates at 1, 3, and 5 years for patients with and without MVI were 65.38, 51.92, 45.93 and 83.17, 65.38, 58.53 %, respectively (Table 1).

Fig. 4
figure 4

Disease-free survival according to gender. The disease-free survival rate of males was significantly better than that of females (p = 0.031)

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Fig. 5
figure 5

Disease-free survival according to microvascular invasion (MVI). The disease-free survival rate of the MVI(−) group was significantly better than that of the MVI(+) group (p = 0.031)

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Discussion

Hepatocellular carcinoma has a tendency to invade the intrahepatic vasculature, such as the portal veins (PVs) and the hepatic veins (HVs). PV involvement is more common than HV involvement [15]. In patients with HCC, tumor vascular invasion can be divided into macroscopic and microscopic. Macroscopic vascular invasion, such as a tumor thrombus in the portal vein portal and/or hepatic vein, has been identified as one of the most important prognostic factors affecting survival or recurrence after surgical resection of HCC [16, 17]. The extent of the tumor thrombus in the portal vein and/or hepatic vein is correlated with overall survival after treatment with partial hepatectomy [18, 19].

Although some studies have suggested that the presence of MVI is an independent predictor of poor survival after resection of HCC, the clinical significance of microvascular invasion for patients with single HCC without macrovascular invasion remains unclear. MVI occurrence rates are between 23 and 29 % in single HCC [12, 20]. Shirabe et al. [20] investigated 218 HCC patients without any extrahepatic metastases and vascular invasion detected during preoperative evaluation. Among the 218 patients, 146 patients had single HCC. They found that 42 of the 146 single HCC patients had MVI. Cho et al. [12] reported that among 230 HCC patients with single HCC less than 10 cm in diameter, 53 patients had MVI. Our results are similar, as we found that 22.8 % patients with single HCC without macrovascular invasion had MVI, which suggests that even in early-stage HCC patients, the MVI rate is relatively high. MVI is often an independent factor for overall survival [11, 13, 14, 21] and disease-free survival [12, 13, 21]. In a report of 408 HCC patients within the Milan criteria who underwent partial hepatectomy, Fan et al. [14] identified the absence of microvascular invasion as a favorable predictor for overall survival. Cho et al. [12] found that the presence of microvascular invasion was a significant prognostic factor for disease-free survival in both single large HCC (> 5–10 cm in diameter) patients and single small HCC (≤ 5 cm) patients. Sumie [21] investigated 110 HCC patients without macroscopic vascular invasion who underwent curative resection. In their study, MVI was identified as an independent risk factor for both recurrence-free survival and disease-free survival. Similar findings have been reported by other authors. We also identified the presence of MVI as an independent predictor for both poor survival and disease-free survival after curative resection of HCC. The overall survival rate and disease-free survival rate were significantly worse for patients with MVI than those without MVI. These findings indicate that microscopic venous invasion may play an important role in short-term survival and cancer cell spreading in HCC patients after curative resection. The presence of vascular invasion has been considered direct evidence of intrahepatic metastasis. Therefore, it is important to detect whether MVI is present in the resected specimen of patients with single HCC without macrovascular invasion because of its importance in predicting both overall survival and disease-free survival after curative resection. MVI(+) HCC patients need careful follow-up, and these patients may be good candidates for adjuvant chemotherapy. Although some investigators have reported that postoperative TACE could improve the prognosis of HCC patients with macroscopic portal vein tumor thrombi, its efficacy in MVI(+) HCC patients is still controversial. So far, no report has focused on its efficacy in MVI(+) patients with single HCC without macroscopic vascular invasion. In a future study, we will further analyze this tissue to improve the prognosis of these HCC patients (Table 3).

Table 3 Independent predictors for disease-free survival identified by multivariate analysis
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The prognostic significance of resection margin in postoperative survival and disease-free survival remains controversial in HCC. Some investigators have found that the extent of the liver resection margin was an independent prognostic factor for overall survival [22, 23] and disease-free survival [22–24]. However, other investigators have found that the extent of liver resection margin did not influence postoperative overall survival [25, 26] or disease-free survival [25–28]. In the present study, we found that the resection margin did not influence postoperative overall survival or disease-free survival in patients with single HCC without macrovascular invasion who underwent curative resection. Although both the cumulative overall survival rate and disease-free survival rate tended to be higher in patients who underwent curative resection with resection margin ≥ 1 cm than those with resection margin < 1 cm, this difference did not reach statistical significance (p = 0.0504; p = 0.090, respectively). These results imply that resection margin ≥ 1 cm has little beneficial effect in prolonging both overall survival rate and disease-free survival rate for patients with single HCC without macrovascular invasion who undergo curative resection. This observation might have 2 explanations. First, tumor recurrence could be classified as intrahepatic metastasis and multicentric occurrence, according to its origin. Multicentric recurrence could occur anywhere in the liver remnant and might not be prevented by a wide resection margin. Among the 15 paired samples from 15 HCC patients who underwent re-resection, Poon et al. [29] demonstrated that 8 of 9 (89 %) resected early recurrent (≤ 1 year) tumors were intrahepatic metastases, whereas all 6 (100 %) resected late recurrent (> 1 year) tumors were classified as multicentric occurrences. In our study, 57 of the 122 patients with recurrent HCC developed late intrahepatic recurrence (> 1 year) that may have mainly originated from multicentric occurrences. Second, HCC has a propensity to disseminate by means of vascular invasion, and intrahepatic metastasis is likely to be present beyond 1 cm in most patients before operation. Lai et al. [30] investigated 23 resected liver specimens by serial sectioning followed by histologic examination, and they found that 20 of the 23 specimens had either microsatellites or histologic venous permeation. Among these 20 specimens, either microsatellites or histologic venous permeation extended beyond 1 cm from the resection margin in 17 specimens. In their study, 9 specimens had single HCC without a gross tumor thrombus. Six of the 9 specimens had either venous permeation or microsatellites, and either microsatellites or histologic venous permeation was beyond 1 cm from the resection margin in 4 of these 6 specimens. However, a 2-cm margin improved the potential for a cure in macroscopically solitary HCC without vascular invasion. Our previous study showed that micrometastases extended beyond the 1-cm margin in 28 (24.8 %) patients but beyond the 2-cm margin in only nine (8.0 %) patients with macroscopically solitary tumors without vascular invasion [15]. Thus, a wide resection margin (2 cm) should be recommended for the patient to survive the operation.

Tumor size is not an indicator of poor prognosis in HCC patients with single tumors after hepatic resection [7, 9, 10]. Yang et al. [9] reported that solitary large HCC patients had similar overall survival and disease-free survival to small HCC patients. The latest American Joint Commission on Cancer (AJCC) staging classification for HCC also classified single HCC without vascular invasion as T1, irrespective of its size. The present study is consistent with the previous studies showing that tumor size did not affect the postoperative outcomes in patients with single HCC. Although AFP level is the most effective marker for HCC diagnosis and recurrent surveillance, the prognostic value of AFP has been controversial. While some studies suggested that AFP was associated with survival after resection [10, 31], other studies found that AFP was not a predictor of survival. In the present study, AFP level did not have prognostic value for overall survival or disease-free survival.

There are some limitations of this study. First, it was a retrospective study. Second, we examined only the resected specimens for MVI. Whether MVI was present in the liver remnant is not known. Lastly, sampling may have affected the pathologists’ diagnosis of MVI, and inadequate sampling may have caused false-negative detection.

In conclusion, in patients with single HCC without macrovascular invasion, we found that the presence of MVI was a poor prognostic factor for both overall survival and disease-free survival after curative resection. These findings suggest that MVI can be employed to estimate both overall survival and disease-free survival after curative resection in patients with single HCC without macrovascular invasion. Patients with MVI may be good candidates for adjuvant chemotherapy. Further studies to analyze whether adjuvant chemotherapy can improve the survival of these patients are necessary.