Ann Surg Oncol DOI 10.1245/s10434-014-3786-3
ORIGINAL ARTICLE – HEPATOBILIARY TUMORS
Clinicopathological and Prognostic Significance of Preoperative Serum Zinc Status in Patients with Hepatocellular Carcinoma After Initial Hepatectomy Katsunori Imai, MD, PhD1, Toru Beppu, MD, PhD1,2, Takanobu Yamao, MD1, Hirohisa Okabe, MD, PhD1, Hiromitsu Hayashi, MD, PhD1, Hidetoshi Nitta, MD, PhD1, Daisuke Hashimoto, MD, PhD1, Kosuke Mima, MD, PhD1, Shigeki Nakagawa, MD, PhD1, Keita Sakamoto, MD1, Akira Chikamoto, MD1, Takatoshi Ishiko, MD, PhD1, and Hideo Baba, MD, PhD1 1
Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan; Department of Multidisciplinary Treatment for Gastroenterological Cancer, Kumamoto University Hospital, Kumamoto, Japan 2
ABSTRACT Background. The purpose of this study was to determine the relationship between serum zinc (Zn) level and clinicopathological profiles in patients with hepatocellular carcinoma (HCC). Furthermore, we investigate the influence of serum Zn level on the long-term prognosis after hepatectomy. Methods. A retrospective analysis was conducted of 310 patients who underwent initial hepatectomy for HCC. The correlation between serum Zn level and preoperative liver functional indicator, pathological features, including the degree of hepatic fibrosis, and perioperative outcome were evaluated. The patients were divided into two groups (serum Zn B 65 mg/dL; n = 71 and Zn [ 65 mg/dL; n = 239), and their long-term prognosis were compared. Results. There were strong correlations between preoperative serum Zn level and serum albumin (r = 0.47, P \ 0.0001), branched-chain amino acids (r = 0.28, P \ 0.0001), and hyaluronic acid level (r = -0.33, P \ 0.0001). The patients with low Zn group were characterized as hepatitis C virus (HCV) infection, impaired liver function, background hepatic fibrosis, and
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3786-3) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 23 March 2014 H. Baba, MD, PhD e-mail: [email protected]
pathological vascular invasion. The disease-free and overall survivals in the low Zn group were significantly lower than those in the high Zn group, especially in subgroup with HCV-related liver disease (P = 0.041 and 0.0004, respectively). Multivariate analyses showed that serum Zn level was an independent prognostic factor for overall survival (hazard ratio 1.79, P = 0.038). Conclusions. Serum Zn level reflects liver function and is useful for predicting the hepatic fibrosis before surgery. The prognosis of patients with HCC was found to be associated with preoperative serum Zn level, especially in patients with HCV-related chronic liver disease.
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide.1 Most patients with HCC have chronic liver disease that includes cirrhosis. It is well known that the long-term outcome of HCC patients is influenced by several factors related to both the extent of the tumor invasion and the parenchymal liver function.2 Zinc (Zn) is an essential trace element in the human body that has crucial roles in physical growth and development, immune system function, reproductive health, sensory function, and neurobehavioral development.3 A deficiency of Zn results in various pathological disorders, including taste disorder, dermatitis, wound healing disturbances, and testicular dysfunction. It is known that Zn deficiency is associated with liver diseases, including chronic hepatitis and liver cirrhosis.4–7 A previous report demonstrated that the serum Zn status was inversely correlated with the liver function and the severity
K. Imai et al.
of hepatic fibrosis in patients with hepatitis C virus (HCV)related chronic liver disease.5 In addition, it has been reported that Zn supplementation enhanced the response to interferon therapy and improved the degree of liver damage and liver pathology, and reduced the incidence of HCC.8–10 To our knowledge, no study has examined the precise relationship between the serum Zn status and the prognosis in HCC patients who underwent hepatectomy. In the current study, we evaluated the serum Zn level in patients who underwent initial hepatectomy for HCC and examined the relationship between the serum Zn level and the clinical profile, liver function indicators, and pathological hepatic fibrosis. Furthermore, we also investigated the effects of the serum Zn status on HCC recurrence and survival after initial hepatectomy. PATIENTS AND METHODS Patients From January 2004 to December 2012, a total of 351 patients with HCC underwent curative hepatectomy as an initial treatment at the Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University (Kumamoto, Japan). Forty patients were excluded because their preoperative serum Zn level had not been determined. The remaining 310 patients were enrolled in this study. All patients underwent routine laboratory and liver function tests before treatment, including measurements of the levels of bilirubin, albumin, hyaluronic acid, Zn, iron (Fe), the branchedchain amino acids (BCAA) to tyrosine ratio (BTR), the indocyanine retention rate at 15 minutes (ICG-R15) and 99mTcgalactosyl human serum albumin (GSA) scintigraphy also was performed. All clinicopathological data, including the serum Zn level, were collected in a prospective database. This study was approved by the institutional ethics committee of Kumamoto University Hospital and was performed in accordance with the 1975 Declaration of Helsinki. Written, informed consent was obtained from all patients before treatment. Hepatectomy The type of hepatectomy was selected based on the tumor location, extent of the tumor invasion, parenchymal liver function, and the patients’ general condition, as described previously.11,12 If the liver function allowed, anatomical resection was employed. In patients with insufficient liver functional reserve, limited resection was performed. Postoperative morbidities were graded using the validated classification criteria as described by Dindo et al. and was defined as any complication of Grade IIIA or higher.13 Postoperative liver dysfunction was defined as a total bilirubin level C50 lmol/L and/or a prothrombin time index \50 %.14
Histological Study The final diagnosis of HCC was confirmed pathologically in the resected specimens. A histological examination of the resected specimens was performed by pathologists who did not know the outcome of the patients. The severity of liver disease, including the liver fibrosis stage and hepatitis activity grade, also was evaluated according to the criteria proposed by the New Inuyama Classification.15 Follow-up After treatment, all of the patients underwent regular follow-up examinations of their serum alpha-fetoprotein (AFP), lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) and des-c-carboxy prothrombin (DCP) levels, and ultrasonography (US) and enhanced computed tomography (CT) or magnetic resonance imaging (MRI) studies were undertaken every 2–4 months to detect any intrahepatic recurrence or distant metastasis, as described previously.11,16 When tumor recurrence was confined to the remnant liver, various treatment modalities were selected, including repeat hepatectomy, radiofrequency ablation, transcatheter arterial chemoembolization, chemotherapy with sorafenib, or a combination of these methods. Statistical Analysis Continuous variables were expressed as the mean ± standard deviation (SD) and were compared using the Mann– Whitney U test. Categorical variables were compared using the v2 test or Fisher’s exact test. The relationships between Zn and other clinical and demographic data were examined using Spearman’s rank correlation coefficient. The disease-free survival (DFS) and overall survival (OS) after hepatectomy were computed using the Kaplan–Meier method and were calculated using the log-rank test. A Cox regression analysis was utilized for the multivariate analysis. Only variables deemed to be significant in the univariate analysis were included in the subsequent multivariate analysis. All statistical analyses were performed using the JMP software program (SAS institute, Cary, NC). Values of P \ 0.05 were considered to be statistically significant. RESULTS Patient Characteristics Among the 310 patients, there were 244 males and 66 females (mean age, 66.8 ± 9.3 years; range, 33–84 years). The majority of patients had chronic viral infection with hepatitis B (25.5 %) or C (48.4 %). A total of 306 patients
Prognostic Significance of Zinc in HCC TABLE 1 Correlation between the serum Zn level and clinical and demographic parameters r Age
P value
-0.10
0.080
Albumin
0.47
\0.0001
Platelet count
0.12
0.0028
BTR
0.18
0.0018
BCAA
0.28
\0.0001
Tyrosine
-0.11
0.054
Hyaluronic acid Fe
-0.33 -0.07
\0.0001 0.22
ICG-R15
-0.21
0.0003
0.21
0.0003
0.14
0.012
99m
Tc-GSA LHL15
AFP DCP
0.11
0.045
Length of operation
0.10
0.086
Blood loss
0.070
0.22
Tumor size
-0.071
0.21
Tumor number
-0.097
0.088
Zn zinc, BTR branched-chain amino acids (BCAA) to tyrosine ratio, ICG-R15 indocyanine green retention rate at 15 minutes, 99mTc-GSA 99m Tc-galactocyl human serum albumin, LHL15 the ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, DCP des-c-carboxy prothrombin
(98.7 %) had well-preserved liver function (Child–Pugh class A), whereas 4 patients (1.3 %) had Child–Pugh class B functional status. The mean serum Zn level was 75.8 ± 14.1 mg/dL (range, 43–145). The mean tumor size was 44.8 ± 33.5 mm (range, 8–230), and the mean number of tumors was 1.4 ± 0.7 (range, 1–5). Of these patients, 132 (42.9 %) had pathological vascular invasion, and 114 (37 %) had pathologically proven liver cirrhosis, which was pathologically determined to be of grade F4 using the New Inuyama Classification. The 5-year DFS and OS were 32.8 and 70.4 %, respectively. The mean follow-up period was 40 months after initial hepatectomy (range, 1.0– 115.0 months). Correlation between the Serum Zn Level and the Clinical and Demographic Data The correlations between the serum Zn level and several clinical and demographic parameters are shown in Table 1. There were significant correlations between the preoperative serum Zn level and the albumin (r = 0.47, P \ 0.0001), BCAA (r = 0.28, P \ 0.0001), and hyaluronic acid (r = -0.33, P \ 0.0001) levels (Supplementary Fig. 1). The platelet count (r = 0.12, P = 0.028), BTR (r = 0.18, P = 0.0018), ICG-R15 (r = -0.21, P = 0.0003), and the ratio of the liver to heart-plus-liver radioactivity at 15 min (LHL15) as determined by 99mTc-GSA (r = 0.21,
P = 0.0003), were also significantly correlated with the preoperative Zn level. There were also weak correlations between the serum Zn level and tumor markers, including AFP (r = 0.14, P = 0.012) and DCP (r = 0.11, P = 0.045). There was no correlation between the serum Zn level and the patient age, serum tyrosine level or serum Fe level, the length of the operation, blood loss, tumor size, or tumor number. Clinicopathological Features of the Patients according to the Serum Zn Status Because the lower limit of the normal range of the serum Zn level in our institute was 65 mg/dL, we divided the patients into two groups based on their serum Zn status: B65 mg/dL (n = 71) and [65 mg/dL (n = 239). The clinicopathological characteristics of the two groups are summarized in Table 2. HCV infection was significantly more common in the low Zn group (P = 0.0007). Patients in the low Zn group had higher serum bilirubin levels, lower serum albumin levels and platelet counts, lower BTR (lower serum BCAA and lower serum tyrosine), higher serum hyaluronic acid level, and higher ICG-R15 and lower LHL15 values. The tumor and surgery-related factors, including the stage, tumor markers, tumor number and size, gross type, tumor differentiation, surgical procedure, length of the operation, and blood loss, were not significantly different between the groups. The postoperative complications, 90-day mortality, postoperative liver dysfunction rate, recurrence pattern, and in-hospital stay also were not significantly different between the groups. Correlation between the Serum Zn Status and Hepatic Fibrosis The correlations between the serum Zn level and liver fibrosis stage are shown in Supplementary Fig. 2. The fibrosis stage of the liver tended to be higher in the low Zn group than in the high Zn group (Supplementary Fig. 2a), and pathologically proven liver cirrhosis (F4) was significantly more common in the low Zn group than in the high Zn group (54.3 vs. 31.9 %, P = 0.0005, Supplementary Fig. 2b). The serum Zn level in the cirrhotic patients (F4) was significantly lower than that in the noncirrhotic patients (F0-3) (73.0 ± 14.1 vs. 77.5 ± 13.8 mg/dL, P = 0.0035; Supplementary Fig. 2c). Results of the Disease-free and Overall Survival Analyses According to the Serum Zn Status The cumulative DFS and OS according to the serum Zn status are shown in Fig. 1. The cumulative DFS rate of the patients in the low Zn group was significantly lower than that of the patients in the high Zn group (5-year DFS rate:
K. Imai et al. TABLE 2 Demographic and clinical characteristics of patients divided according to the serum Zn status Zn B 65 (n = 71)
Zn [ 65 (n = 239)
P value
Gender (male/female)
58/13
186/53
0.62
Age
66.5 ± 9.3
67.8 ± 9.6
0.19
HBs-Ag positive (%)
10 (14.1%)
69 (28.9%)
0.013
HCV-Ab positive (%)
47 (66.2%)
103 (43.1%)
0.0007
Total bilirubin (mg/dL)
0.76 ± 0.31
0.86 ± 0.32
0.0046 \0.0001
Albumin (g/dL)
3.71 ± 0.4
4.08 ± 0.4
Platelet count (/lL)
13.9 ± 5.3
16.0 ± 6.9
0.018
BTR
5.16 ± 2.19
5.94 ± 1.94
\0.0001
BCAA (nmol/mL)
408.9 ± 78.0
457.0 ± 94.9
Tyrosine (nmol/mL)
86.3 ± 23.6
80.3 ± 19.1
Hyaluronic acid (ng/mL) Fe (lg/dL)
244.1 ± 223.2 115.8 ± 60.8
118.8 ± 117.9 115.3 ± 49.7
ICG-R15 (%)
15.9 ± 7.45
12.9 ± 7.2
0.0012
99m
0.89 ± 0.04
0.92 ± 0.03
0.0002 0.98
Tc-GSA LHL15
0.0002 0.033 \0.0001 0.27
AFP (ng/mL)
4123.8 ± 29028.2
3889.0 ± 20193.1
AFP-L3 [ 10 (%)
17 (23.9%)
68 (28.5%)
0.55
DCP (mAU/mL)
1357.2 ± 3705.7
5354.3 ± 20518.1
0.18
Child-Pugh classification (A/B)
69/2
237/2
0.23
Stage (I/II/III/IV)
8/39/21/3
25/125/71/18
0.89
Surgical procedure (anatomical/non-anatomical)
45/26
153/86
0.99
Length of operation (min)
386.4 ± 114.6
408.6 ± 102.9
0.07
Blood loss (g)
489.1 ± 602.5
490.0 ± 432.7
0.33
Tumor size (mm)
41.1 ± 29.3
45.9 ± 34.6
0.54
Tumor number
1.5 ± 0.7
1.4 ± 0.7
0.45
Gross type (SD/non-SD)
21/50
88/151
0.32
Tumor differentiation (well/mod-poor)
27/44
81/157
0.57
Vascular invasion positive (%) Stage of fibrosis (F1/F2/F3/F4)
22 (31.4%) 0/10/9/13/38
110 (46.2%) 7/53/60/42/76
0.028 0.0052
Complication(s) (%)
17 (23.9%)
47 (19.7%)
0.5
Postoperative liver dysfunction (%)
3 (4.2%)
5 (2.1%)
0.39
90-day mortality (%)
2 (2.8%)
3 (1.3%)
0.32
In-hospital stay (days)
30.9 ± 84.4
19.8 ± 23.2
0.41
Recurrence site (intrahepatic/extrahepatic/combine)
42/1/0
102/9/4
0.2
15/27
43/63
0.71
42
102
Liver (solitary/multiple) Liver only Liver ? lung
0
1
Liver ? bone
0
1
Liver ? lymph node
0
1
Liver ? brain
0
1
Lung only
1
5
Lung ? brain
0
1
Lymph node Lymph node ? bone
0 0
2 1
HBs-Ag hepatitis B surface antigen, HCV-Ab anti-hepatitis C antibody, BTR branched-chain amino acids (BCAA) to tyrosine ratio, Zn zinc, Fe iron, ICG-R15 indocyanine green retention rate at 15 min, 99mTc-GSA 99mTc-galactocyl human serum albumin, LHL15 ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, AFP-L3 lens culinaris agglutinin-reactive fraction of alpha-fetoprotein, DCP desc-carboxy prothrombin, SD single nodular
Prognostic Significance of Zinc in HCC
Disease-free survival rate
0.8 P=0.018
0.6 0.4
Zn>65
0.2
Zn≤65
0.8 Zn>65
0.6 0.4
Zn≤65
P=0.0007
0.2 0.0
0.0 0 Patients at risk Zn ≤ 65 Zn > 65
Overall survival rate
b 1.0
a 1.0
71 239
2 4 6 Years after hepatectomy 25 102
11 49
2 17
8
0
2
71 239
46 162
Patients at risk 0 4
Zn ≤ 65 Zn > 65
4 6 Years after hepatectomy 23 90
8 34
8 2 7
FIG. 1 Cumulative disease-free (a) and overall survival (b) after hepatectomy according to serum Zn status
21.6 vs. 36.1 %, P = 0.018; Fig. 1a), and the cumulative OS rate of the patients in the low Zn group also was significantly lower than that of the patients in the high Zn group (5-year OS rate: 59.4 vs. 73.6 %, P = 0.0007; Fig. 1b). Table 3 shows the results of the univariate and multivariate analyses of the prognostic factors for the DFS and OS. In the univariate analysis, the serum albumin (P = 0.001), hyaluronic acid (P \ 0.0001), Zn (P = 0.023), and DCP (P \ 0.0001) levels, the 99mTcGSA LHL15 (P = 0.033), blood loss (P = 0.0054), tumor size (P = 0.0012), and the tumor number (P \ 0.0001) were significant predictors of the DFS. The multivariate analysis revealed that the serum hyaluronic acid level (hazard ratio (HR) 1.96, P = 0.0004), serum DCP level (HR 1.86, P = 0.0014), and the tumor number (HR 2.05, P \ 0.0001) were the independent prognostic factors for the DFS. As for OS, in the univariate analysis, the serum albumin (P \ 0.0001), hyaluronic acid (P = 0.0018), Zn (P = 0.0015), and DCP (P = 0.0001) levels, the blood loss (P = 0.0039), tumor size (P \ 0.0001), tumor number (P = 0.0049), gross type (P = 0.0093), and vascular invasion (P = 0.021) were significant predictors of the OS, and the multivariate analysis revealed that the serum albumin (HR 2.16, P = 0.0038), Zn (HR 1.79, P = 0.038), and DCP (HR 1.73, P = 0.047) levels, tumor number (HR 1.7, P = 0.041), and gross type (HR 1.76, P = 0.029) were the independent prognostic factors for the OS. Subsequently, we performed the subgroup survival analyses based on the presence or absence of HCV-related liver disease in Fig. 2. In the analysis of the patients with HCV-related liver disease, the cumulative DFS rate of the patients in the low Zn group was significantly lower than that of the patients in the high Zn group (5-year DFS rate: 13.5 vs. 36.2 %, P = 0.041; Fig. 2a), and the cumulative OS rate of the patients in the low Zn group also was
significantly lower than that of the patients in the high Zn group (5-year OS rate: 55.5 vs. 78.5 %, P = 0.0004; Fig. 2b). In contrast, in the analysis of the patients without HCV-related liver disease, neither the DFS nor OS showed any significant differences between the groups (5-year DFS rate: 35.0 vs. 36.2 %, P = 0.27; Fig. 2c; and 5-year OS rate: 67.3 vs. 70.8 %, P = 0.55; Fig. 2d). A multivariate analysis of the patients with HCV-related liver disease demonstrated that the patients’ age (HR 2.09, P = 0.0025), hyaluronic acid level (HR 1.94, P = 0.012), serum DCP level (HR 1.96, P = 0.0094), and tumor number (HR 2.03, P = 0.0052) were independent prognostic factors for the DFS; the patients’ age (HR 2.63, P = 0.0048), serum Zn level (HR 2.55, P = 0.009), and DCP level (HR 2.03, P = 0.042) were independent prognostic factors for the OS (Supplementary Table 1). On the other hand, a multivariate analysis of the patients without HCV-related liver disease demonstrated that the serum hyaluronic acid level (HR 2.14, P = 0.0029), DCP level (HR 2.06, P = 0.012), tumor number (HR 2.06, P = 0.007), and the gross tumor type (HR 2.34, P = 0.0012) were the independent prognostic factors for the DFS, and the serum albumin level (HR 2.57, P = 0.012) and gross tumor type (HR 2.85, P = 0.005) were independent prognostic factors for the OS (Supplementary Table 2). DISCUSSION It is known that the serum Zn level decreases in patients with liver disease in parallel with the progression of the disease stage.5,16–18 Moriyama et al. reported that the serum Zn level correlated significantly with the serum albumin level, platelet count, and several markers reflecting hepatic fibrosis, including the serum hyaluronic acid level, in patients with HCV-related liver disease.5 In the current study, the serum Zn level was found to be
K. Imai et al. TABLE 3 Results of the univariate and multivariate analyses of the disease-free and overall survival after hepatectomy Disease-free survival
Overall survival
Univariate analysis HR (95% CI)
P value
Multivariate analysis
Univariate analysis
HR (95% CI)
HR (95% CI)
P value
Multivariate analysis P value
HR (95% CI)
P value
2.16 (1.28–3.70)
0.0038
Gender Male
1.23 (0.84–1.86)
0.31
1.00 (0.61–2.00)
0.99
1.29 (0.94–1.78)
0.11
1.58 (0.99–2.53)
0.054
0.89 (0.61–1.26)
0.52
0.79 (0.45–1.34)
0.4
1.11 (0.81–1.52)
0.51
1.32 (0.83–2.12)
0.23
0.98
0.88 (0.49–1.48)
0.63
2.89 (1.79–4.77)
\0.0001
Female Age (years) [68 B68 HBs-Ag Positive Negative HCV-Ab Positive Negative Total bilirubin (mg/dL) [1.0
1.00 (0.70–1.40)
B1.0 Albumin (g/dL) B4.0
1.7 (1.24–2.32)
0.0010
1.36 (0.97–1.92)
0.074
[4.0 Platelet count (/lL) B10
0.99 (0.64–1.46)
0.95
1.11 (0.58–1.95)
0.74
1.15 (0.84–1.58)
0.40
1.33 (0.82–2.17)
0.24
[10 BTR B0.55 [0.55 Hyaluronic acid (ng/mL) [100
2.44 (1.81–3.5)
\0.0001
1.96 (1.35–2.86)
0.0004
2.13 (1.32–3.51)
0.0018
1.40 (0.79–2.5)
0.25
1.52 (1.06–2.14)
0.023
1.18 (0.79–1.73)
0.41
2.23 (1.37–3.56)
0.0015
1.79 (1.03–3.08)
0.038
1.58 (0.93–2.53)
0.085
1.68 (0.74–3.33)
0.20
1.27 (0.92–1.76)
0.14
1.46 (0.91–2.39)
0.12
1.41 (1.03–1.95)
0.033
1.18 (0.74–1.90)
0.49
1.01 (0.71–1.42)
0.96
1.24 (0.73–2.02)
0.41
1.07 (0.74–1.51)
0.72
1.51 (0.90–2.47)
0.12
B100 Zn (mg/dL) B65 [65 Fe (lg/dL) B65 [65 ICG-R15 (%) [12 B12 99m
Tc-GSA LHL15
B0.92
1.05 (0.73–1.50)
0.80
[0.92 AFP (ng/mL) [100 B100 AFP-L3 (%) [10 B10
Prognostic Significance of Zinc in HCC TABLE 3 continued Disease-free survival
Overall survival
Univariate analysis
Multivariate analysis
Univariate analysis HR (95% CI)
HR (95% CI)
P value
HR (95% CI)
2.06 (1.48–2.83)
\0.0001
1.86 (1.27–2.71)
1.14 (0.83–1.58)
P value
Multivariate analysis P value
HR (95% CI)
P value
1.73 (1.01–2.99)
0.047
DCP (mAU/mL) [400
0.0014
2.53 (1.59–4.03)
0.0001
0.43
1.58 (0.96–2.71)
0.074
1.31 (0.96–1.79)
0.089
1.26 (0.79–2.00)
0.33
1.56 (1.14–2.14)
0.0054
1.30 (0.92–1.85)
0.14
1.63 (1.02–2.63)
0.039
1.15 (0.69–1.95)
0.6
1.67 (1.23–2.29)
0.0012
1.27 (0.87–1.87)
0.22
2.67 (1.64–4.46)
\0.0001
1.69 (0.94–3.08)
0.08
2.11 (1.52–2.89)
\0.0001
2.05 (1.45–2.87)
\0.0001
2.00 (1.24–3.18)
0.0049
1.7 (1.02–2.79)
0.041
1.33 (0.95–1.81)
0.092
1.86 (1.17–2.96)
0.0093
0.96 (0.69–1.33)
0.81
0.86 (0.51–1.40)
0.55
1.32 (0.97–1.81)
0.081
1.73 (1.08–2.78)
0.021
1.24 (0.90–1.70)
0.19
1.11 (0.68–1.78)
0.66
1.17 (0.79–1.69)
0.42
1.33 (0.77–2.21)
0.30
B400 Surgical procedure Anatomical Nonanatomical Length of operation (min) [400 B400 Blood loss (g) [380 B380 Tumor size (mm) [3.5 B3.5 Tumor number [1 B1 Gross type Not SD SD
1.76 (1.06–2.9)
0.029
1.58 (0.95–2.67)
0.080
Tumor differentiation Well Moderately/poor Vascular invasion Positive Negative Staging of fibrosis 4 0–3 Complication(s) Present Absent HR hazard ratio, CI confidence interval, HBs-Ag hepatitis B surface antigen, HCV-Ab antihepatitis C antibody, BTR branched-chain amino acids (BCAA) to tyrosine ratio, Zn zinc, Fe iron, ICG-R15 indocyanine green retention rate at 15 min, 99mTc-GSA 99mTc-galactocyl human serum albumin, LHL15 ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, AFP-L3 lens culinaris agglutinin-reactive fraction of alpha-fetoprotein, DCP des-c-carboxy prothrombin, SD single nodular
significantly correlated with parameters that reflect the hepatic fibrosis and liver function. In particular, the serum Zn level showed a strong correlation with the serum albumin, BCAA, and hyaluronic acid level (Table 1). In addition, this study demonstrated that pathologically proven liver cirrhosis (F4) was significantly more common in the patients with a low Zn level than with a high Zn level (Supplementary Fig. 1). These results suggest that the serum Zn level is one of the useful markers for assessing
the extent of liver damage and the degree of hepatic fibrosis before hepatectomy in patients with HCC. The present study showed that the DFS and OS in the low Zn group were significantly worse than those in the high Zn group (Fig. 1). However, the multivariate analyses demonstrated that the serum Zn level was an independent prognostic factor only for the OS (Table 3). The clinical and demographic characteristics of the low Zn group included more frequent HCV-related liver disease, poor
K. Imai et al.
1.0 Disease-free survival rate
b
HCV positive
0.8
P=0.041
0.6
Zn>65
0.4
Zn≤65
0.2 0.0
P=0.0004
0.8 0.6
Zn>65 Zn≤65
0.4 0.2 0.0
Patients at risk Zn ≤ 65 47 Zn > 65 103
2 4 6 Years after hepatectomy
8
18 43
0 2
c
9 22
2 9
Patients at risk
P=0.27 Zn>65
0.4
Zn≤65
0.2
8
31 70
2 2
0.0
16 36
5 14
HCV negative
1.0
0.8 0.6
Zn ≤ 65 47 Zn > 65 103
2 4 6 Years after hepatectomy
d
HCV negative
1.0
0
Overall survival rate
0
Disease-free survival rate
HCV positive
1.0 Overall survival rate
a
0.8
P=0.55
0.6
Zn≤65
Zn>65
0.4 0.2 0.0
0
Patients at risk Zn ≤ 65 24 Zn > 65 136
2
4
6
8
Years after hepatectomy
8 62
6 28
1 9
0
Patients at risk 0 3
Zn ≤ 65 24 Zn > 65 136
2
4
6
8
Years after hepatectomy
16 92
8 55
4 21
0 6
FIG. 2 Results of the cumulative disease-free survival analyses based on the presence or absence of HCV-related liver disease. Cumulative disease-free (a) and overall (b) survival rates in patients
with HCV-related liver disease. Cumulative disease-free (c) and overall (d) survival in patients without HCV-related liver disease
liver functional reserve, and the presence of hepatic fibrosis (Table 2). The tumor and surgery-related factors, except for vascular invasion, were not significantly different between the groups. These results suggest that the serum Zn level is associated with the background liver function, and the prognostic impact of the preoperative serum Zn level only on the OS thus may reflect this point. The serum Zn level has been previously reported not to reflect tumor-related factors, such as the tumor number or size, in HCC patients.5 The present study also showed no correlation between the serum Zn level and tumor number or size. However, there were weak correlations between the serum Zn level and the levels of tumor markers, including AFP and DCP. Furthermore, pathological vascular invasion was found more frequently in the low Zn group than in the high Zn group (Table 2). Previous studies reported association between Zn and several cancers, including colorectal cancer, prostate cancer, esophageal cancer, and breast cancer.17–21 It is known that Zn exerts anticarcinogenic
effects by stabilizing the structure of deoxyribonucleic acids, ribonucleic acids, and the ribosomes, and protects cells from free-radical injury.22 It also was reported that Zn supplementation improves the anticancer activity of monocytes expressing transmembrane tumor necrosis factor-a.23 Considering all these findings, it is highly possible that the Zn might have some kind of influences on the occurrence or development of HCC. Many previous studies have mentioned the correlation between the Zn level and liver function or pathology, and the effectiveness of Zn supplementation therapy has been demonstrated in patients with HCV-related chronic liver disease.5,9,10,24,25 Therefore, we performed the subgroup analyses based on the presence or absence of HCV-related liver disease. As a result, we found that the DFS and OS were significantly worse in the low Zn group than in the high Zn group in the subgroup of patients with HCVrelated liver disease. However, there was no significant difference in the subgroups of patients without HCV-
Prognostic Significance of Zinc in HCC
related liver disease (Fig. 2). Although the precise reason for this is unclear, this difference might have been due to the biological features of HCV infection. It is well recognized that chronic HCV infection eventually leads to the production of reactive oxygen species in the liver.26 The oxidative stress induced by HCV infection consequently facilitates iron storage in the liver. On the other hand, Zn play a crucial role in antioxidative actions and confers protective effects on hepatocytes.27 Zn has an inhibitory effect on iron-dependent radical reactions and on lipid peroxidation, and subsequently alleviates hepatic cell injury.28 Yasue et al. reported that Zn was a negative regulator of HCV RNA replication.29 Tellinghuisen et al. reported that HCV NS5A protein, which is an integral part of the viral replication, is a Zn metalloprotein, and that Zn coordination is likely required for NS5A function during HCV replication.30 Furthermore, it also has been reported that the concomitant use of agents containing Zn leads to an increase in the rate of eradication of HCV in interferon therapy.31 These findings suggest that the presence of HCV somehow influences the serum Zn status, and the prognostic impact of Zn for patients with HCC might be limited to HCV-related disease. CONCLUSIONS The serum Zn level reflects the liver function and is useful for predicting the extent of hepatic fibrosis before surgery. Furthermore, the prognosis of patients with HCC was found to be associated with the preoperative serum Zn level, especially in patients with HCV-related chronic liver disease. CONFLICT OF INTEREST of interest to declare.
None of the authors has any conflict
REFERENCES 1. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer. 2001;94:153-6. 2. The Liver Cancer Study Group of Japan (1994) Predictive factors for long-term prognosis after partial hepatectomy for patients with hepatocellular carcinoma in Japan. Cancer 74:2772–2780. 3. Brown KH, Rivera JA, Bhutta Z, et al. International Zinc Nutrition Consultative Group (IZiNCG) technical document #1. Assessment of the risk of zinc deficiency in populations and options for its control. Food Nutr Bull. 2004;25:S99-203. 4. Vallee BL, Wacker WE, Bartholomay AF, Hoch FL. Zinc metabolism in hepatic dysfunction. II. Correlation of metabolic patterns with biochemical findings. N Engl J Med. 1957;257:1055-65. 5. Moriyama M, Matsumura H, Fukushima A, et al. Clinical significance of evaluation of serum zinc concentrations in C-viral chronic liver disease. Dig Dis Sci. 2006;51:1967-77. 6. Stamoulis I, Kouraklis G, Theocharis S. Zinc and the liver: an active interaction. Dig Dis Sci. 2007;52:1595-12.
7. Bode JC, Hanisch P, Henning H, Koenig W, Richter FW, Bode C. Hepatic zinc content in patients with various stages of alcoholic liver disease and in patients with chronic active and chronic persistent hepatitis. Hepatology. 1988;8:1605-9. 8. Takagi H, Nagamine T, Abe T, et al. Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C. J Viral Hepat. 2001;8:367-71. 9. Matsumura H, Nirei K, Nakamura H, et al. Zinc supplementation therapy improves the outcome of patients with chronic hepatitis C. J Clin Biochem Nutr. 2012;51:178-84. 10. Matsuoka S, Matsumura H, Nakamura H, et al. Zinc supplementation improves the outcome of chronic hepatitis C and liver cirrhosis. J Clin Biochem Nutr. 2009;45:292-303. 11. Imai K, Beppu T, Chikamoto A, et al. Comparison between hepatic resection and radiofrequency ablation as first-line treatment for solitary small-sized hepatocellular carcinoma of 3 cm or less. Hepatol Res. 2013;43:853-64. 12. Beppu T, Ishiko T, Chikamoto A, et al. Liver hanging maneuver decreases blood loss and operative time in a right-side hepatectomy. Hepatogastroenterology. 2012;59:542-5. 13. Dindo D, Demartines N, Clavien PA. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004; 240: 205-213. 14. Balzan S, Belghiti J, Farges O, et al. The ‘‘50–50 criteria’’ on postoperative day 5: an accurate predictor of liver failure and death after hepatectomy. Ann Surg. 2005;242:824–8, discussion 828–9. 15. Ichida F, Tsuji T, Omata M. New classification; new criteria for histological assessment of chronic hepatitis. Int Hepatol Commun. 1996;6:112-9. 16. Imai K, Beppu T, Nakayama Y, et al. Preoperative prediction of poorly differentiated components in small-sized hepatocellular carcinoma for safe local ablation therapy. J Surg Oncol. 2009; 100:121-6. 17. Qiao L, Feng Y. Intakes of heme iron and zinc and colorectal cancer incidence: a meta-analysis of prospective studies. Cancer Causes Control. 2013;24:1175-83. 18. Epstein MM, Kasperzyk JL, Andren O, et al. Dietary zinc and prostate cancer survival in a Swedish cohort. Am J Clin Nutr. 2011;93:586-93. 19. Feng P, Li TL, Guan ZX, Franklin RB, Costello LC. Direct effect of zinc on mitochondrial apoptogenesis in prostate cells. Prostate. 2002;52:311-8. 20. Goyal MM, Kalwar AK, Vyas RK, Bhati A. A study of serum zinc, selenium and copper levels in carcinoma of esophagus patients. Indian J Clin Biochem. 2006;21:208-10. 21. Grattan BJ, Freake HC. Zinc and cancer: implications for LIV-1 in breast cancer. Nutrients. 2012;4:648-75. 22. Wu T, Sempos CT, Freudenheim JL, Muti P, Smit E. Serum iron, copper and zinc concentrations and risk of cancer mortality in US adults. Ann Epidemiol. 2004;14:195-201. 23. Meksawan K, Sermsri U, Chanvorachote P. Zinc supplementation improves anticancer activity of monocytes in type-2 diabetic patients with metabolic syndrome. Anticancer Res. 2014;34:295-9. 24. Takahashi M, Saito H, Higashimoto M, Hibi T. Possible inhibitory effect of oral zinc supplementation on hepatic fibrosis through downregulation of TIMP-1: A pilot study. Hepatol Res. 2007;37:405-9. 25. Ishikawa T. Can zinc enhance response interferon therapy for patients with HCV-related liver disease? World J Gastroenterol. 2012;18:3196-200. 26. Okuda M, Li K, Beard MR, et al. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology. 2002;122:366-75. 27. Powell SR. The antioxidant properties of zinc. J Nutr. 2000; 130(5S Suppl):1447S–54S.
K. Imai et al. 28. Himoto T, Yoneyama H, Deguch A, et al. Insulin resistance derived from zinc deficiency in non-diabetic patients with chronic hepatitis C. Exp Ther Med. 2010;1:707-11. 29. Yuasa K, Naganuma A, Sato K, et al. Zinc is a negative regulator of hepatitis C virus RNA replication. Liver Int. 2006;26:1111-8. 30. Tellinghuisen TL, Paulson MS, Rice CM. The NS5A protein of bovine viral diarrhea virus contains an essential zinc-binding site
similar to that of the hepatitis C virus NS5A protein. J Virol. 2006;80:7450-8. 31. Coon JT, Ernst E. Complementary and alternative therapies in the treatment of chronic hepatitis C: a systematic review. J Hepatol. 2004;40:491-500.
ORIGINAL ARTICLE – HEPATOBILIARY TUMORS
Clinicopathological and Prognostic Significance of Preoperative Serum Zinc Status in Patients with Hepatocellular Carcinoma After Initial Hepatectomy Katsunori Imai, MD, PhD1, Toru Beppu, MD, PhD1,2, Takanobu Yamao, MD1, Hirohisa Okabe, MD, PhD1, Hiromitsu Hayashi, MD, PhD1, Hidetoshi Nitta, MD, PhD1, Daisuke Hashimoto, MD, PhD1, Kosuke Mima, MD, PhD1, Shigeki Nakagawa, MD, PhD1, Keita Sakamoto, MD1, Akira Chikamoto, MD1, Takatoshi Ishiko, MD, PhD1, and Hideo Baba, MD, PhD1 1
Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University, Kumamoto, Japan; Department of Multidisciplinary Treatment for Gastroenterological Cancer, Kumamoto University Hospital, Kumamoto, Japan 2
ABSTRACT Background. The purpose of this study was to determine the relationship between serum zinc (Zn) level and clinicopathological profiles in patients with hepatocellular carcinoma (HCC). Furthermore, we investigate the influence of serum Zn level on the long-term prognosis after hepatectomy. Methods. A retrospective analysis was conducted of 310 patients who underwent initial hepatectomy for HCC. The correlation between serum Zn level and preoperative liver functional indicator, pathological features, including the degree of hepatic fibrosis, and perioperative outcome were evaluated. The patients were divided into two groups (serum Zn B 65 mg/dL; n = 71 and Zn [ 65 mg/dL; n = 239), and their long-term prognosis were compared. Results. There were strong correlations between preoperative serum Zn level and serum albumin (r = 0.47, P \ 0.0001), branched-chain amino acids (r = 0.28, P \ 0.0001), and hyaluronic acid level (r = -0.33, P \ 0.0001). The patients with low Zn group were characterized as hepatitis C virus (HCV) infection, impaired liver function, background hepatic fibrosis, and
Electronic supplementary material The online version of this article (doi:10.1245/s10434-014-3786-3) contains supplementary material, which is available to authorized users. Ó Society of Surgical Oncology 2014 First Received: 23 March 2014 H. Baba, MD, PhD e-mail: [email protected]
pathological vascular invasion. The disease-free and overall survivals in the low Zn group were significantly lower than those in the high Zn group, especially in subgroup with HCV-related liver disease (P = 0.041 and 0.0004, respectively). Multivariate analyses showed that serum Zn level was an independent prognostic factor for overall survival (hazard ratio 1.79, P = 0.038). Conclusions. Serum Zn level reflects liver function and is useful for predicting the hepatic fibrosis before surgery. The prognosis of patients with HCC was found to be associated with preoperative serum Zn level, especially in patients with HCV-related chronic liver disease.
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide.1 Most patients with HCC have chronic liver disease that includes cirrhosis. It is well known that the long-term outcome of HCC patients is influenced by several factors related to both the extent of the tumor invasion and the parenchymal liver function.2 Zinc (Zn) is an essential trace element in the human body that has crucial roles in physical growth and development, immune system function, reproductive health, sensory function, and neurobehavioral development.3 A deficiency of Zn results in various pathological disorders, including taste disorder, dermatitis, wound healing disturbances, and testicular dysfunction. It is known that Zn deficiency is associated with liver diseases, including chronic hepatitis and liver cirrhosis.4–7 A previous report demonstrated that the serum Zn status was inversely correlated with the liver function and the severity
K. Imai et al.
of hepatic fibrosis in patients with hepatitis C virus (HCV)related chronic liver disease.5 In addition, it has been reported that Zn supplementation enhanced the response to interferon therapy and improved the degree of liver damage and liver pathology, and reduced the incidence of HCC.8–10 To our knowledge, no study has examined the precise relationship between the serum Zn status and the prognosis in HCC patients who underwent hepatectomy. In the current study, we evaluated the serum Zn level in patients who underwent initial hepatectomy for HCC and examined the relationship between the serum Zn level and the clinical profile, liver function indicators, and pathological hepatic fibrosis. Furthermore, we also investigated the effects of the serum Zn status on HCC recurrence and survival after initial hepatectomy. PATIENTS AND METHODS Patients From January 2004 to December 2012, a total of 351 patients with HCC underwent curative hepatectomy as an initial treatment at the Department of Gastroenterological Surgery, Graduate School of Life Sciences, Kumamoto University (Kumamoto, Japan). Forty patients were excluded because their preoperative serum Zn level had not been determined. The remaining 310 patients were enrolled in this study. All patients underwent routine laboratory and liver function tests before treatment, including measurements of the levels of bilirubin, albumin, hyaluronic acid, Zn, iron (Fe), the branchedchain amino acids (BCAA) to tyrosine ratio (BTR), the indocyanine retention rate at 15 minutes (ICG-R15) and 99mTcgalactosyl human serum albumin (GSA) scintigraphy also was performed. All clinicopathological data, including the serum Zn level, were collected in a prospective database. This study was approved by the institutional ethics committee of Kumamoto University Hospital and was performed in accordance with the 1975 Declaration of Helsinki. Written, informed consent was obtained from all patients before treatment. Hepatectomy The type of hepatectomy was selected based on the tumor location, extent of the tumor invasion, parenchymal liver function, and the patients’ general condition, as described previously.11,12 If the liver function allowed, anatomical resection was employed. In patients with insufficient liver functional reserve, limited resection was performed. Postoperative morbidities were graded using the validated classification criteria as described by Dindo et al. and was defined as any complication of Grade IIIA or higher.13 Postoperative liver dysfunction was defined as a total bilirubin level C50 lmol/L and/or a prothrombin time index \50 %.14
Histological Study The final diagnosis of HCC was confirmed pathologically in the resected specimens. A histological examination of the resected specimens was performed by pathologists who did not know the outcome of the patients. The severity of liver disease, including the liver fibrosis stage and hepatitis activity grade, also was evaluated according to the criteria proposed by the New Inuyama Classification.15 Follow-up After treatment, all of the patients underwent regular follow-up examinations of their serum alpha-fetoprotein (AFP), lens culinaris agglutinin-reactive fraction of AFP (AFP-L3) and des-c-carboxy prothrombin (DCP) levels, and ultrasonography (US) and enhanced computed tomography (CT) or magnetic resonance imaging (MRI) studies were undertaken every 2–4 months to detect any intrahepatic recurrence or distant metastasis, as described previously.11,16 When tumor recurrence was confined to the remnant liver, various treatment modalities were selected, including repeat hepatectomy, radiofrequency ablation, transcatheter arterial chemoembolization, chemotherapy with sorafenib, or a combination of these methods. Statistical Analysis Continuous variables were expressed as the mean ± standard deviation (SD) and were compared using the Mann– Whitney U test. Categorical variables were compared using the v2 test or Fisher’s exact test. The relationships between Zn and other clinical and demographic data were examined using Spearman’s rank correlation coefficient. The disease-free survival (DFS) and overall survival (OS) after hepatectomy were computed using the Kaplan–Meier method and were calculated using the log-rank test. A Cox regression analysis was utilized for the multivariate analysis. Only variables deemed to be significant in the univariate analysis were included in the subsequent multivariate analysis. All statistical analyses were performed using the JMP software program (SAS institute, Cary, NC). Values of P \ 0.05 were considered to be statistically significant. RESULTS Patient Characteristics Among the 310 patients, there were 244 males and 66 females (mean age, 66.8 ± 9.3 years; range, 33–84 years). The majority of patients had chronic viral infection with hepatitis B (25.5 %) or C (48.4 %). A total of 306 patients
Prognostic Significance of Zinc in HCC TABLE 1 Correlation between the serum Zn level and clinical and demographic parameters r Age
P value
-0.10
0.080
Albumin
0.47
\0.0001
Platelet count
0.12
0.0028
BTR
0.18
0.0018
BCAA
0.28
\0.0001
Tyrosine
-0.11
0.054
Hyaluronic acid Fe
-0.33 -0.07
\0.0001 0.22
ICG-R15
-0.21
0.0003
0.21
0.0003
0.14
0.012
99m
Tc-GSA LHL15
AFP DCP
0.11
0.045
Length of operation
0.10
0.086
Blood loss
0.070
0.22
Tumor size
-0.071
0.21
Tumor number
-0.097
0.088
Zn zinc, BTR branched-chain amino acids (BCAA) to tyrosine ratio, ICG-R15 indocyanine green retention rate at 15 minutes, 99mTc-GSA 99m Tc-galactocyl human serum albumin, LHL15 the ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, DCP des-c-carboxy prothrombin
(98.7 %) had well-preserved liver function (Child–Pugh class A), whereas 4 patients (1.3 %) had Child–Pugh class B functional status. The mean serum Zn level was 75.8 ± 14.1 mg/dL (range, 43–145). The mean tumor size was 44.8 ± 33.5 mm (range, 8–230), and the mean number of tumors was 1.4 ± 0.7 (range, 1–5). Of these patients, 132 (42.9 %) had pathological vascular invasion, and 114 (37 %) had pathologically proven liver cirrhosis, which was pathologically determined to be of grade F4 using the New Inuyama Classification. The 5-year DFS and OS were 32.8 and 70.4 %, respectively. The mean follow-up period was 40 months after initial hepatectomy (range, 1.0– 115.0 months). Correlation between the Serum Zn Level and the Clinical and Demographic Data The correlations between the serum Zn level and several clinical and demographic parameters are shown in Table 1. There were significant correlations between the preoperative serum Zn level and the albumin (r = 0.47, P \ 0.0001), BCAA (r = 0.28, P \ 0.0001), and hyaluronic acid (r = -0.33, P \ 0.0001) levels (Supplementary Fig. 1). The platelet count (r = 0.12, P = 0.028), BTR (r = 0.18, P = 0.0018), ICG-R15 (r = -0.21, P = 0.0003), and the ratio of the liver to heart-plus-liver radioactivity at 15 min (LHL15) as determined by 99mTc-GSA (r = 0.21,
P = 0.0003), were also significantly correlated with the preoperative Zn level. There were also weak correlations between the serum Zn level and tumor markers, including AFP (r = 0.14, P = 0.012) and DCP (r = 0.11, P = 0.045). There was no correlation between the serum Zn level and the patient age, serum tyrosine level or serum Fe level, the length of the operation, blood loss, tumor size, or tumor number. Clinicopathological Features of the Patients according to the Serum Zn Status Because the lower limit of the normal range of the serum Zn level in our institute was 65 mg/dL, we divided the patients into two groups based on their serum Zn status: B65 mg/dL (n = 71) and [65 mg/dL (n = 239). The clinicopathological characteristics of the two groups are summarized in Table 2. HCV infection was significantly more common in the low Zn group (P = 0.0007). Patients in the low Zn group had higher serum bilirubin levels, lower serum albumin levels and platelet counts, lower BTR (lower serum BCAA and lower serum tyrosine), higher serum hyaluronic acid level, and higher ICG-R15 and lower LHL15 values. The tumor and surgery-related factors, including the stage, tumor markers, tumor number and size, gross type, tumor differentiation, surgical procedure, length of the operation, and blood loss, were not significantly different between the groups. The postoperative complications, 90-day mortality, postoperative liver dysfunction rate, recurrence pattern, and in-hospital stay also were not significantly different between the groups. Correlation between the Serum Zn Status and Hepatic Fibrosis The correlations between the serum Zn level and liver fibrosis stage are shown in Supplementary Fig. 2. The fibrosis stage of the liver tended to be higher in the low Zn group than in the high Zn group (Supplementary Fig. 2a), and pathologically proven liver cirrhosis (F4) was significantly more common in the low Zn group than in the high Zn group (54.3 vs. 31.9 %, P = 0.0005, Supplementary Fig. 2b). The serum Zn level in the cirrhotic patients (F4) was significantly lower than that in the noncirrhotic patients (F0-3) (73.0 ± 14.1 vs. 77.5 ± 13.8 mg/dL, P = 0.0035; Supplementary Fig. 2c). Results of the Disease-free and Overall Survival Analyses According to the Serum Zn Status The cumulative DFS and OS according to the serum Zn status are shown in Fig. 1. The cumulative DFS rate of the patients in the low Zn group was significantly lower than that of the patients in the high Zn group (5-year DFS rate:
K. Imai et al. TABLE 2 Demographic and clinical characteristics of patients divided according to the serum Zn status Zn B 65 (n = 71)
Zn [ 65 (n = 239)
P value
Gender (male/female)
58/13
186/53
0.62
Age
66.5 ± 9.3
67.8 ± 9.6
0.19
HBs-Ag positive (%)
10 (14.1%)
69 (28.9%)
0.013
HCV-Ab positive (%)
47 (66.2%)
103 (43.1%)
0.0007
Total bilirubin (mg/dL)
0.76 ± 0.31
0.86 ± 0.32
0.0046 \0.0001
Albumin (g/dL)
3.71 ± 0.4
4.08 ± 0.4
Platelet count (/lL)
13.9 ± 5.3
16.0 ± 6.9
0.018
BTR
5.16 ± 2.19
5.94 ± 1.94
\0.0001
BCAA (nmol/mL)
408.9 ± 78.0
457.0 ± 94.9
Tyrosine (nmol/mL)
86.3 ± 23.6
80.3 ± 19.1
Hyaluronic acid (ng/mL) Fe (lg/dL)
244.1 ± 223.2 115.8 ± 60.8
118.8 ± 117.9 115.3 ± 49.7
ICG-R15 (%)
15.9 ± 7.45
12.9 ± 7.2
0.0012
99m
0.89 ± 0.04
0.92 ± 0.03
0.0002 0.98
Tc-GSA LHL15
0.0002 0.033 \0.0001 0.27
AFP (ng/mL)
4123.8 ± 29028.2
3889.0 ± 20193.1
AFP-L3 [ 10 (%)
17 (23.9%)
68 (28.5%)
0.55
DCP (mAU/mL)
1357.2 ± 3705.7
5354.3 ± 20518.1
0.18
Child-Pugh classification (A/B)
69/2
237/2
0.23
Stage (I/II/III/IV)
8/39/21/3
25/125/71/18
0.89
Surgical procedure (anatomical/non-anatomical)
45/26
153/86
0.99
Length of operation (min)
386.4 ± 114.6
408.6 ± 102.9
0.07
Blood loss (g)
489.1 ± 602.5
490.0 ± 432.7
0.33
Tumor size (mm)
41.1 ± 29.3
45.9 ± 34.6
0.54
Tumor number
1.5 ± 0.7
1.4 ± 0.7
0.45
Gross type (SD/non-SD)
21/50
88/151
0.32
Tumor differentiation (well/mod-poor)
27/44
81/157
0.57
Vascular invasion positive (%) Stage of fibrosis (F1/F2/F3/F4)
22 (31.4%) 0/10/9/13/38
110 (46.2%) 7/53/60/42/76
0.028 0.0052
Complication(s) (%)
17 (23.9%)
47 (19.7%)
0.5
Postoperative liver dysfunction (%)
3 (4.2%)
5 (2.1%)
0.39
90-day mortality (%)
2 (2.8%)
3 (1.3%)
0.32
In-hospital stay (days)
30.9 ± 84.4
19.8 ± 23.2
0.41
Recurrence site (intrahepatic/extrahepatic/combine)
42/1/0
102/9/4
0.2
15/27
43/63
0.71
42
102
Liver (solitary/multiple) Liver only Liver ? lung
0
1
Liver ? bone
0
1
Liver ? lymph node
0
1
Liver ? brain
0
1
Lung only
1
5
Lung ? brain
0
1
Lymph node Lymph node ? bone
0 0
2 1
HBs-Ag hepatitis B surface antigen, HCV-Ab anti-hepatitis C antibody, BTR branched-chain amino acids (BCAA) to tyrosine ratio, Zn zinc, Fe iron, ICG-R15 indocyanine green retention rate at 15 min, 99mTc-GSA 99mTc-galactocyl human serum albumin, LHL15 ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, AFP-L3 lens culinaris agglutinin-reactive fraction of alpha-fetoprotein, DCP desc-carboxy prothrombin, SD single nodular
Prognostic Significance of Zinc in HCC
Disease-free survival rate
0.8 P=0.018
0.6 0.4
Zn>65
0.2
Zn≤65
0.8 Zn>65
0.6 0.4
Zn≤65
P=0.0007
0.2 0.0
0.0 0 Patients at risk Zn ≤ 65 Zn > 65
Overall survival rate
b 1.0
a 1.0
71 239
2 4 6 Years after hepatectomy 25 102
11 49
2 17
8
0
2
71 239
46 162
Patients at risk 0 4
Zn ≤ 65 Zn > 65
4 6 Years after hepatectomy 23 90
8 34
8 2 7
FIG. 1 Cumulative disease-free (a) and overall survival (b) after hepatectomy according to serum Zn status
21.6 vs. 36.1 %, P = 0.018; Fig. 1a), and the cumulative OS rate of the patients in the low Zn group also was significantly lower than that of the patients in the high Zn group (5-year OS rate: 59.4 vs. 73.6 %, P = 0.0007; Fig. 1b). Table 3 shows the results of the univariate and multivariate analyses of the prognostic factors for the DFS and OS. In the univariate analysis, the serum albumin (P = 0.001), hyaluronic acid (P \ 0.0001), Zn (P = 0.023), and DCP (P \ 0.0001) levels, the 99mTcGSA LHL15 (P = 0.033), blood loss (P = 0.0054), tumor size (P = 0.0012), and the tumor number (P \ 0.0001) were significant predictors of the DFS. The multivariate analysis revealed that the serum hyaluronic acid level (hazard ratio (HR) 1.96, P = 0.0004), serum DCP level (HR 1.86, P = 0.0014), and the tumor number (HR 2.05, P \ 0.0001) were the independent prognostic factors for the DFS. As for OS, in the univariate analysis, the serum albumin (P \ 0.0001), hyaluronic acid (P = 0.0018), Zn (P = 0.0015), and DCP (P = 0.0001) levels, the blood loss (P = 0.0039), tumor size (P \ 0.0001), tumor number (P = 0.0049), gross type (P = 0.0093), and vascular invasion (P = 0.021) were significant predictors of the OS, and the multivariate analysis revealed that the serum albumin (HR 2.16, P = 0.0038), Zn (HR 1.79, P = 0.038), and DCP (HR 1.73, P = 0.047) levels, tumor number (HR 1.7, P = 0.041), and gross type (HR 1.76, P = 0.029) were the independent prognostic factors for the OS. Subsequently, we performed the subgroup survival analyses based on the presence or absence of HCV-related liver disease in Fig. 2. In the analysis of the patients with HCV-related liver disease, the cumulative DFS rate of the patients in the low Zn group was significantly lower than that of the patients in the high Zn group (5-year DFS rate: 13.5 vs. 36.2 %, P = 0.041; Fig. 2a), and the cumulative OS rate of the patients in the low Zn group also was
significantly lower than that of the patients in the high Zn group (5-year OS rate: 55.5 vs. 78.5 %, P = 0.0004; Fig. 2b). In contrast, in the analysis of the patients without HCV-related liver disease, neither the DFS nor OS showed any significant differences between the groups (5-year DFS rate: 35.0 vs. 36.2 %, P = 0.27; Fig. 2c; and 5-year OS rate: 67.3 vs. 70.8 %, P = 0.55; Fig. 2d). A multivariate analysis of the patients with HCV-related liver disease demonstrated that the patients’ age (HR 2.09, P = 0.0025), hyaluronic acid level (HR 1.94, P = 0.012), serum DCP level (HR 1.96, P = 0.0094), and tumor number (HR 2.03, P = 0.0052) were independent prognostic factors for the DFS; the patients’ age (HR 2.63, P = 0.0048), serum Zn level (HR 2.55, P = 0.009), and DCP level (HR 2.03, P = 0.042) were independent prognostic factors for the OS (Supplementary Table 1). On the other hand, a multivariate analysis of the patients without HCV-related liver disease demonstrated that the serum hyaluronic acid level (HR 2.14, P = 0.0029), DCP level (HR 2.06, P = 0.012), tumor number (HR 2.06, P = 0.007), and the gross tumor type (HR 2.34, P = 0.0012) were the independent prognostic factors for the DFS, and the serum albumin level (HR 2.57, P = 0.012) and gross tumor type (HR 2.85, P = 0.005) were independent prognostic factors for the OS (Supplementary Table 2). DISCUSSION It is known that the serum Zn level decreases in patients with liver disease in parallel with the progression of the disease stage.5,16–18 Moriyama et al. reported that the serum Zn level correlated significantly with the serum albumin level, platelet count, and several markers reflecting hepatic fibrosis, including the serum hyaluronic acid level, in patients with HCV-related liver disease.5 In the current study, the serum Zn level was found to be
K. Imai et al. TABLE 3 Results of the univariate and multivariate analyses of the disease-free and overall survival after hepatectomy Disease-free survival
Overall survival
Univariate analysis HR (95% CI)
P value
Multivariate analysis
Univariate analysis
HR (95% CI)
HR (95% CI)
P value
Multivariate analysis P value
HR (95% CI)
P value
2.16 (1.28–3.70)
0.0038
Gender Male
1.23 (0.84–1.86)
0.31
1.00 (0.61–2.00)
0.99
1.29 (0.94–1.78)
0.11
1.58 (0.99–2.53)
0.054
0.89 (0.61–1.26)
0.52
0.79 (0.45–1.34)
0.4
1.11 (0.81–1.52)
0.51
1.32 (0.83–2.12)
0.23
0.98
0.88 (0.49–1.48)
0.63
2.89 (1.79–4.77)
\0.0001
Female Age (years) [68 B68 HBs-Ag Positive Negative HCV-Ab Positive Negative Total bilirubin (mg/dL) [1.0
1.00 (0.70–1.40)
B1.0 Albumin (g/dL) B4.0
1.7 (1.24–2.32)
0.0010
1.36 (0.97–1.92)
0.074
[4.0 Platelet count (/lL) B10
0.99 (0.64–1.46)
0.95
1.11 (0.58–1.95)
0.74
1.15 (0.84–1.58)
0.40
1.33 (0.82–2.17)
0.24
[10 BTR B0.55 [0.55 Hyaluronic acid (ng/mL) [100
2.44 (1.81–3.5)
\0.0001
1.96 (1.35–2.86)
0.0004
2.13 (1.32–3.51)
0.0018
1.40 (0.79–2.5)
0.25
1.52 (1.06–2.14)
0.023
1.18 (0.79–1.73)
0.41
2.23 (1.37–3.56)
0.0015
1.79 (1.03–3.08)
0.038
1.58 (0.93–2.53)
0.085
1.68 (0.74–3.33)
0.20
1.27 (0.92–1.76)
0.14
1.46 (0.91–2.39)
0.12
1.41 (1.03–1.95)
0.033
1.18 (0.74–1.90)
0.49
1.01 (0.71–1.42)
0.96
1.24 (0.73–2.02)
0.41
1.07 (0.74–1.51)
0.72
1.51 (0.90–2.47)
0.12
B100 Zn (mg/dL) B65 [65 Fe (lg/dL) B65 [65 ICG-R15 (%) [12 B12 99m
Tc-GSA LHL15
B0.92
1.05 (0.73–1.50)
0.80
[0.92 AFP (ng/mL) [100 B100 AFP-L3 (%) [10 B10
Prognostic Significance of Zinc in HCC TABLE 3 continued Disease-free survival
Overall survival
Univariate analysis
Multivariate analysis
Univariate analysis HR (95% CI)
HR (95% CI)
P value
HR (95% CI)
2.06 (1.48–2.83)
\0.0001
1.86 (1.27–2.71)
1.14 (0.83–1.58)
P value
Multivariate analysis P value
HR (95% CI)
P value
1.73 (1.01–2.99)
0.047
DCP (mAU/mL) [400
0.0014
2.53 (1.59–4.03)
0.0001
0.43
1.58 (0.96–2.71)
0.074
1.31 (0.96–1.79)
0.089
1.26 (0.79–2.00)
0.33
1.56 (1.14–2.14)
0.0054
1.30 (0.92–1.85)
0.14
1.63 (1.02–2.63)
0.039
1.15 (0.69–1.95)
0.6
1.67 (1.23–2.29)
0.0012
1.27 (0.87–1.87)
0.22
2.67 (1.64–4.46)
\0.0001
1.69 (0.94–3.08)
0.08
2.11 (1.52–2.89)
\0.0001
2.05 (1.45–2.87)
\0.0001
2.00 (1.24–3.18)
0.0049
1.7 (1.02–2.79)
0.041
1.33 (0.95–1.81)
0.092
1.86 (1.17–2.96)
0.0093
0.96 (0.69–1.33)
0.81
0.86 (0.51–1.40)
0.55
1.32 (0.97–1.81)
0.081
1.73 (1.08–2.78)
0.021
1.24 (0.90–1.70)
0.19
1.11 (0.68–1.78)
0.66
1.17 (0.79–1.69)
0.42
1.33 (0.77–2.21)
0.30
B400 Surgical procedure Anatomical Nonanatomical Length of operation (min) [400 B400 Blood loss (g) [380 B380 Tumor size (mm) [3.5 B3.5 Tumor number [1 B1 Gross type Not SD SD
1.76 (1.06–2.9)
0.029
1.58 (0.95–2.67)
0.080
Tumor differentiation Well Moderately/poor Vascular invasion Positive Negative Staging of fibrosis 4 0–3 Complication(s) Present Absent HR hazard ratio, CI confidence interval, HBs-Ag hepatitis B surface antigen, HCV-Ab antihepatitis C antibody, BTR branched-chain amino acids (BCAA) to tyrosine ratio, Zn zinc, Fe iron, ICG-R15 indocyanine green retention rate at 15 min, 99mTc-GSA 99mTc-galactocyl human serum albumin, LHL15 ratio of the liver to the heart-plus-liver radioactivity at 15 min, AFP alpha-fetoprotein, AFP-L3 lens culinaris agglutinin-reactive fraction of alpha-fetoprotein, DCP des-c-carboxy prothrombin, SD single nodular
significantly correlated with parameters that reflect the hepatic fibrosis and liver function. In particular, the serum Zn level showed a strong correlation with the serum albumin, BCAA, and hyaluronic acid level (Table 1). In addition, this study demonstrated that pathologically proven liver cirrhosis (F4) was significantly more common in the patients with a low Zn level than with a high Zn level (Supplementary Fig. 1). These results suggest that the serum Zn level is one of the useful markers for assessing
the extent of liver damage and the degree of hepatic fibrosis before hepatectomy in patients with HCC. The present study showed that the DFS and OS in the low Zn group were significantly worse than those in the high Zn group (Fig. 1). However, the multivariate analyses demonstrated that the serum Zn level was an independent prognostic factor only for the OS (Table 3). The clinical and demographic characteristics of the low Zn group included more frequent HCV-related liver disease, poor
K. Imai et al.
1.0 Disease-free survival rate
b
HCV positive
0.8
P=0.041
0.6
Zn>65
0.4
Zn≤65
0.2 0.0
P=0.0004
0.8 0.6
Zn>65 Zn≤65
0.4 0.2 0.0
Patients at risk Zn ≤ 65 47 Zn > 65 103
2 4 6 Years after hepatectomy
8
18 43
0 2
c
9 22
2 9
Patients at risk
P=0.27 Zn>65
0.4
Zn≤65
0.2
8
31 70
2 2
0.0
16 36
5 14
HCV negative
1.0
0.8 0.6
Zn ≤ 65 47 Zn > 65 103
2 4 6 Years after hepatectomy
d
HCV negative
1.0
0
Overall survival rate
0
Disease-free survival rate
HCV positive
1.0 Overall survival rate
a
0.8
P=0.55
0.6
Zn≤65
Zn>65
0.4 0.2 0.0
0
Patients at risk Zn ≤ 65 24 Zn > 65 136
2
4
6
8
Years after hepatectomy
8 62
6 28
1 9
0
Patients at risk 0 3
Zn ≤ 65 24 Zn > 65 136
2
4
6
8
Years after hepatectomy
16 92
8 55
4 21
0 6
FIG. 2 Results of the cumulative disease-free survival analyses based on the presence or absence of HCV-related liver disease. Cumulative disease-free (a) and overall (b) survival rates in patients
with HCV-related liver disease. Cumulative disease-free (c) and overall (d) survival in patients without HCV-related liver disease
liver functional reserve, and the presence of hepatic fibrosis (Table 2). The tumor and surgery-related factors, except for vascular invasion, were not significantly different between the groups. These results suggest that the serum Zn level is associated with the background liver function, and the prognostic impact of the preoperative serum Zn level only on the OS thus may reflect this point. The serum Zn level has been previously reported not to reflect tumor-related factors, such as the tumor number or size, in HCC patients.5 The present study also showed no correlation between the serum Zn level and tumor number or size. However, there were weak correlations between the serum Zn level and the levels of tumor markers, including AFP and DCP. Furthermore, pathological vascular invasion was found more frequently in the low Zn group than in the high Zn group (Table 2). Previous studies reported association between Zn and several cancers, including colorectal cancer, prostate cancer, esophageal cancer, and breast cancer.17–21 It is known that Zn exerts anticarcinogenic
effects by stabilizing the structure of deoxyribonucleic acids, ribonucleic acids, and the ribosomes, and protects cells from free-radical injury.22 It also was reported that Zn supplementation improves the anticancer activity of monocytes expressing transmembrane tumor necrosis factor-a.23 Considering all these findings, it is highly possible that the Zn might have some kind of influences on the occurrence or development of HCC. Many previous studies have mentioned the correlation between the Zn level and liver function or pathology, and the effectiveness of Zn supplementation therapy has been demonstrated in patients with HCV-related chronic liver disease.5,9,10,24,25 Therefore, we performed the subgroup analyses based on the presence or absence of HCV-related liver disease. As a result, we found that the DFS and OS were significantly worse in the low Zn group than in the high Zn group in the subgroup of patients with HCVrelated liver disease. However, there was no significant difference in the subgroups of patients without HCV-
Prognostic Significance of Zinc in HCC
related liver disease (Fig. 2). Although the precise reason for this is unclear, this difference might have been due to the biological features of HCV infection. It is well recognized that chronic HCV infection eventually leads to the production of reactive oxygen species in the liver.26 The oxidative stress induced by HCV infection consequently facilitates iron storage in the liver. On the other hand, Zn play a crucial role in antioxidative actions and confers protective effects on hepatocytes.27 Zn has an inhibitory effect on iron-dependent radical reactions and on lipid peroxidation, and subsequently alleviates hepatic cell injury.28 Yasue et al. reported that Zn was a negative regulator of HCV RNA replication.29 Tellinghuisen et al. reported that HCV NS5A protein, which is an integral part of the viral replication, is a Zn metalloprotein, and that Zn coordination is likely required for NS5A function during HCV replication.30 Furthermore, it also has been reported that the concomitant use of agents containing Zn leads to an increase in the rate of eradication of HCV in interferon therapy.31 These findings suggest that the presence of HCV somehow influences the serum Zn status, and the prognostic impact of Zn for patients with HCC might be limited to HCV-related disease. CONCLUSIONS The serum Zn level reflects the liver function and is useful for predicting the extent of hepatic fibrosis before surgery. Furthermore, the prognosis of patients with HCC was found to be associated with the preoperative serum Zn level, especially in patients with HCV-related chronic liver disease. CONFLICT OF INTEREST of interest to declare.
None of the authors has any conflict
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