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Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma
Journal of Hepatology 2000; 33: 241-253 Printed in Denmark All rights reserved Munksgaard Copenhagen
Copyright 8 European Association for the Study of the Liver 2000
JournalofHepatology ISSNO168-8278
Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma Takuya Osada’,
Ikuo Nagashima2,
Nelson H. Tsuno’,
Joji Kitayama’
and Hirokazu
Nagawa’
‘Department of Surgical Oncology, Graduate School of Medicine, University of Tokyo; and 2Second Department of Surgery, Teikyo University, Tokyo, Japan
Background/Aims: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas @WCs). GS expression may he related to tumor recurrence, since GSexpressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply. However, there are no studies concerning the prognostic value of GS expression in patients with HCC. Methods: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier productlimit method and multivariate analysis based on the Cox regression model was performed.
Results: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group). Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=O.O42). Multivariate analysis revealed that GS expression (p=O.O21), as well as Child’s classification (p=O.O05) and portal invasion @=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. Conclusion: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.
H
Glutamine synthetase (GS, EC6.3.1.2), which catalyzes the synthesis of glutamine from glutamate and ammonia, may be such a prognostic factor. Although GS expression in normal liver tissue of mice and humans is restricted to a small subpopulation (~8%) of the hepatocytes, those that surround the terminal hepatic venules (3-5), its expression is upregulated in many human HCCs (6,7). The end product of GS activity, glutamine, participates in a variety of biosynthetic reactions; it is a critical substrate for optimal growth of normal and neoplastic cells in culture; it supports protein synthesis; and it is a major source of energy (8,9). In the absence of exogenous glutamine, human tumor cells with high GS activity proliferate well in vitro compared with those with low GS activity. In addition, as tumors grow in vivo the intracellular glutamine content declines and there is a concomitant increase in GS expression (10). Furthermore, in murine
carcinoma (HCC) is one of the most common types of malignant cancer in the world. Due to great advances in diagnostic techniques such as ultrasonography and computed tomography, the number of resectable cases has increased substantially (1). However, the prognosis for patients with this cancer is poor and recurrence after surgical resection is common (1,2). It is important, therefore, to identify factors that can be used to predict tumor recurrence so that adjuvant therapy can be provided to patients in high-risk groups. EPATOCELLULAR
Received 28 June; revised 16 November; accepted 30 November 1999 Correspondence: Takuya Osada, Department of Surgical Oncology, Graduate School of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel: 81 3 3815 5411. Fax: 81 3 3811 6822. e-mail: [email protected]
Key words: Glutamine synthetase; Hepatocellular carcinoma; Immunohistochemistry; Ki-67, Prognostic factor; Survival analysis.
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hepatocarcinogenesis models, an increase in the frequency of GS-positivity has been observed as foci progress to neoplasms (11). Thus, GS-expressing tumor cells may have a growth advantage due to their independence from the extracellular supply of glutamine, and GS production may be the mechanism by which HCC cells adapt to a nutrient-poor environment. Recently, we reported that the frequency of GS-positivity increases during the progression of human HCC and that GS-positive foci or nodules are occasionally present within GS-negative HCC lesions, indicating that GS expression is acquired during human hepatocarcinogenesis (7). In this study we evaluated the prognostic significance of GS in HCC and studied its relation to proliferation marker (Ki-67) and other clinicopathologic factors.
Patients From 1981 to 1995, 227 patients underwent surgery for HCC at the First Department of Surgery, University of Tokyo Hospital. As multiple HCC nodules may be multicentric in origin and consequently may have different properties, only patients who had a single advanced HCC (adHCC) without extrahepatic tumor spread were included in this study. Since it is difficult to evaluate huge tumors precisely by immunochemical methods, tumors with diameters in excess of 10 cm were excluded. Thus, 73 adHCCs, removed from 73 patients (62 men and 1 I women). were studied All of selected patients had was defined the excision all nodules that were visible and/or demonThe ages the patients ranged from 42 to 80 years (60.328.4 was considered HBV with positive and related including alcohol abuse. found. The from to 189.8 months were performed for first postoperative
Fig. I. Immunohistochemical demonstration of glutamine synthetase (GS) in hepatocellular carcinoma. Sections of live, tissue containing tumorous (T) and non-tumorous portions (N) were ,stained. (A) Homogeneous strong staining for GS. This nodule was included in the high-GS group. (B) A nodule showing weak GS staining. This nodule was included in the low-GS group. Arrowheads indicate the boundary of the nodule. The hepatocytes surrounding the central vein, which always showed intense staining, were used as an internal control. (C) A satellite nodule (S) showed GS staining pattern similar to that of the main tumor (T). (D) A GS-positive focal area (arrowj was seen within a GS-negative HCC nodule. (Inset) High magnification view of the GS-positive focus. (original magmfication: A und D (Inset) ~200. B X20, C and D x30).
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Glutamine synthetase and prognosis in HCC year and every 24 months thereafter. Recurrence was diagnosed by ultrasonography, computed tomography scan, angiography and the serum alpha-fetoprotein (AFP) level. Recurrence of HCC was diagnosed if results of any two of four above follow-up examinations were considered positive. GS .has been reported to serve as a specific marker for tracing cell lineage relationships during hepatocarcinogenesis, so we also studied 10 cases of recurrent HCC in order to estimate the stability of GS expression during metastasis.
Immunohistochemistry Formalin-fixed, paraffin-embedded sections were deparaffinized with xylene and then rehydrated with alcohol. Endogenous peroxidase activity was blocked by soaking the sections in 0.3% H202 for 20 min. The sections were placed in the appropriate 10 mM citrate buffer solution and were autoclaved (12O“C, 20 min) for antigen retrieval (12). Nonspecific binding was blocked by incubation with 10% normal swine serum in phosphate-buffered saline for 30 min at room temperature. Then the sections were incubated with the anti-GS antibody (1:lOOO dilution; Chemicon International Inc., Temecula, CA, USA) or the anti-MIBl antibody (1:50 dilution; Immunotech, Marseille, France) overnight at 4°C. After rinsing, the sections were incubated with the biotinylated secondary antibody (1:200 dilution; Dakopatts, Copenhagen, Denmark) for 30 min at room temperature and then with the avidin-biotin peroxidase complex from the Vector Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Visualization was achieved using 3,3’-diaminobenzidine tetrahydrochloride and H202. Specimens were counterstained with hematoxylin. At least two sections from each tumor were examined when the maximal diameter of the tumor was greater than 5 cm. The intensity of the GS staining was recorded as strong if it was equal to or greater than that of the hepatocytes surrounding the central vein (internal control). Nodules were considered to be strongly GS positive (highGS group) when at least 30% of their area was strongly stained. Nodules were recorded as weakly GS positive when they had strong staining over less than 30% of their area or weak staining over at least 30% of their area. Weakly GS positive nodules and nodules that lacked GS staining were assigned to the low-GS group. The Ki-67 labeling index (Ki-67 LI) was calculated by counting the number of positive nuclei per total number of hepatocytes. At least 1000 random nuclei of hepatocytes in 49 high power optical fields (x400) were evaluated. The procedure was performed three times for the same slide and an average was taken.
statistical analyses were carried out according the Statistical Analysis System (14).
Statistical analysis To compare the clinicopathologic features of the two groups, categorical data were analyzed using the cm-square test, whereas continuous data were evaluated using Student’s t-test. DFS times were calculated by the Kaplan-Meier method (13) and comparisons were made using the log-rank test for univariate analysis. Multivariate analysis was performed using the Cox regression model (14). Test results were considered significant when the p-values were less than 0.05. These
of
Results Glutamine synthetase staining Among 73 adHCC nodules, 26 (35.6%) showed strong GS immunoreactivity, 20 (27.4%) showed weak staining and 27 (37.0%) lacked GS immunoreactivity (Fig. la and lb). Satellite lesions were evaluated and compared with the main tumor in 18 cases. These nodules showed staining patterns similar to those of the main tumors (Fig. lc and Table 1). This result suggests that the expression of GS is relatively stable during synchronous metastasis. In addition, we evaluated 10 recurrent HCC nodules (Table 2). Compared to the parental tumor, the GS staining of the recurrent HCC nodules was similar in 6 cases (60.0%), and was altered in 4 cases (stronger in 3 and weaker in 1). Since half of the recurrent HCC nodules with altered staining were found more than 4 years after the first hepatectomy (4.9 and 10.1 years), they may have been multicentric, rather than metastatic, in origin. Among 7 cases with DFS time shorter than 4 years, 6 cases (85.7%) showed GS-positivity in the recurrent nodules, suggesting the relation of GS expression to metastasis of HCC cells.
TABLE
1
Comparison of glutamine synthetase staining lite lesions of heoatocelltdar carcinoma
Main tumor
Satellite tumor
_ +
+
++ _
++ +
-, no staining;
TABLE
between main and satel-
No. of cases
GS staining
Clinicopathologic parameters The following variables were evaluated:
age, sex, Child’s classification, indocyanine green retention rate (ICG R-15) accompanying cirrhosis, HBsAg, the AFP level, the maximal tumor diameter, degree of cellular differentiation, tumor encapsulation, capsular infiltration, satellite lesions, portal invasion, venous invasion, tumor-free margin, Ki-67 LI and GS expression. Child’s classification was applied to the patients with chronic hepatitis as well as liver cirrhosis, in order to assess the overall functional severity of liver disease. As there are several procedures for treating recurrent HCC (repeatresection, transcatheter arterial embolization and percutaneous ethanol injection therapy) and the choice may influence the prognosis of patients, we analyzed the disease-free survival (DFS) time. DFS time was measured from the date of hepatic resection to the date when recurrent disease was diagnosed or, in the absence of a detectable tumor, to the date of death or the last follow-up examination.
to the procedures
8 5 4 1
+, weak staining;
+ +, strong
staining.
2
Comparison of glutamine synthetase staining between mors and recurrent hepatocellular carcinoma nodules Case
pt.1 pt.2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Pt. 8 Pt. 9 Pt. 10
-, no staining;
tu-
Disease free time
GS staining Primary
Recurrent
_
+ + ++ ++ ++ ++ ++
_ + ++ ++ ++ ++
primary
f, weak staining;
(years) 2.10 2.32 2.45 5.52 10.09 4.90 0.46 1.25 1.64 3.25
+ + strong
staining.
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T. Osada et al.
group compared with the low-GS group. We also found that Child’s classification (p=O.OOS) and portal invasion (p=O.O39) were independent risk factors significantly associated with the DFS time (Table 4).
p = 0.042 E
80-
3 ‘E 60d 8 c=; 40-
Glutamine synthetase parameters
0 d
20, 0
1 2
0
.
I 4
, 6
*
High-GS (~26) I . I I 8 I 12 14 8 10
I 16
Years after Surgery High-GS
26
9
4
2
0
0
0
0
0
Low-GS
47
26
14
6
5
3
1
1
0
Fig. 2. Disease-free survival of the 73 patients with HCC who underwent curative resection. Median follow-up was 17.9 months in high-GS group (n=26) and 27.9 months in low-GS group (n=47). The number of patients still at risk over follow-up time is reported at the bottom of the figure. There was a significant correlation between GS expression and tumor recurrence (p=O.O42) by log rank test.
Survival
analysis
Among 73 patients, 48 (65.8%) experienced tumor recurrence. Recurrence was detected within 24 months of hepatectomy in 20 (41.7%) patients and after the 24th month in 28 (58.3%) patients. No patients died of liver insufficiency without HCC recurrence. The recurrence rate tended to be higher in the high-GS group (20/26 cases, 76.9%) than in the low-GS group (28/47 cases, 59.6%) although the difference was not significant Cp= 0.135). The high-GS group had significantly shorter DFS times compared with the low-GS group (p=O.O42, Fig. 2). Univariate analysis showed that Child’s classification (p=O.O25), portal invasion (p=O.O26) and ICG R-15 (p=O.O39) were also correlated significantly with the DFS time (Table 3). Although patients with large tumors (>5 cm) or with microsatellite nodules tended to have shorter DFS times, these correlations did not reach statistical significance @=0.073 and 0.126, respectively). The high-Ki-67 labeling group (~20%) tended to have shorter DFS time than the low-Ki-67 labeling group (<200/o), although the difference was not statistically significant @=0.224). The other parameters examined were not significantly correlated with the DFS time. Multivariate analysis showed that the high-GS group had significantly shorter DFS times (p=O.O21) than the low-GS group. The estimated relative risk of tumor recurrence was 2.59 for the high-GS patient 250
expression
and clinicopathologic
The correlations between GS expression and clinicopathological parameters are summarized in Table 5. All of the high-GS group lesions were encapsulated; however, 11 of 47 low-GS group lesions (23.4%) lacked fibrous capsules. Thus, tumor encapsulation was significantly related to GS expression @=0.007). HBsAg positivity was inversely related to GS expression. In fact, 16 of 47 low-GS group lesions (34.0%) were HBsAg positive, whereas only 2 of 26 high-GS group lesions (7.7%) were HBsAg positive (p=O.O12). The
TABLE
3
Clinicopathologic variables related to the disease-free-survival after surgical resection in hepatocellular carcinoma
time
p-V&~
Variable
Categories
(cases)
Age (years) Sex Child’s classification ICG R- 15 (%) Cirrhosis HBsAg AFP (rig/ml) Tumor size (cm) Cellular differentiation Encapsulation Capsular infiltration Tumor microsatellite Portal invasion Venous invasion Tumor-free margin Ki-67 LI (“A) GS expression
~60 (37) r.~ 260 (36) Male (62) vs Female (I 1) A (63) c’s B/C (10) 515 (32 vs >15 (41) Absent (30) $1~Present (43) Negative (55) rs Positive (18) 5200 (55) 1’S>200 (18) 550 (57) vs >5.0 (16) Well (19) rs Moderate/Poor (54) Absent (11) 1’s Present (62) Absent (32) 1’s Present (30) Absent (56) 1’s Present (17) Absent (62) ~8sPresent (11) Absent (70) r-y Present (3) Absent (9) 1’s Present (64) s-20 (55) L’S>20 (18) Low-GS (47) 1’s High-GS (26)
0.2712 0.8279 0.0253 0.0394 0.6468 0.1133 0.6729 0.0729 0.5697 0.6136 0.7819 0.1258 0.0261 0.3908 0.7117 0.2240 0.0416
ICG R-15: indocyanine gree retention rate at 15 min; HBsAg: hepatitis B surface antigen; AFP: alpha-fetoprotein; Ki-67 LI: Ki-67 labeling index; GS: glutamine synthetase. p-values are based on Student-r test or chi-square analysis.
TABLE
4
Independent prognostic factors for the disease-free-survival time after surgical resection in hepatocellular carcinoma (multivariate Cox analysis) Variables
Paremeter estimate
Standard error
Risk ratio
p-value
Child’s classification Portal invasion GS expression Ki-67 LI
1.688 1.219 0.952 0.861
0.606 0.591 0.411 0.406
5.411 3.383 2.590 2.366
0.005 0.039 0.021 0.062
GS: glutamine synthetase; Variables with p-value>O.
Ki-67 LI: Ki-67 labeling 1 are not shown.
index.
Glutamine synthetase and prognosis in HCC TABLE 5 Clinicopathologic variables of patients with hepatocellular carcinoma: comparison with regard to glutamine synthetase expression Variable
Low-GS n=41 (%)
High-GS n=26 (%)
p-value
Agea (years)
60.3k8.5
60.3-r-8.5
0.9950
Sex Male Female
38 (80.9) 9 (19.1)
24 (92.3) 2 (7.7)
0.1901
Child’s classification A B or C
40 (85.1) 7 (14.9)
23 (88.5) 3 (11.5)
0.6897
ICG R-15” (%) Cirrhosis HBsAg AFP” (@ml) Tumor sizea (cm)
16.2k10.6 21 (57.4) 16 (34.0) 28445 10997 3.7k2.1
20.626.3 16 (61.5) 2 (7.7) 95?142 3.921.8
0.0590 0.1331 0.0124 0.2081 0.6931
Cellular differentiation Well Moderate or Poor
12 (25.5) 35 (74.5)
7 (26.9) 19 (73.1)
0.8968
Encapsulation Capsular infiltrationb Tumor microsatellite Portal invasion Venous invasion Tumor-free margin Ki-67 LI” (%)
36 (76.6) 18 (50.0) 9 (19.1) 6 (12.8) 2 (4.3) 40 (85.1) 14.2216.7
26 (100) 12 (46.2) 8 (30.8) 5 (19.2) 1 (3.8) 24 (92.3) 11.2211.6
0.0074 0.7649 0.2607 0.4597 0.9328 0.3702 0.4167
ICG R-15: indocyanine green retention rate at 15 min; HBsAg: hepatitis B surface antigen; AFP: alpha-fetoprotein; Ki-67 LI: Ki-67 labeling index. a Results are expressed as the meankstandard deviation. b Frequencies are calculated in cases with tumor encapsulation. p-values are based on Student-r test or chi-square analysis.
high-GS group tended to have higher ICG R-15 scores than the low-GS group although the difference was not significant (p=O.O59). The difference in size between the high-GS and low-GS groups was not significant (p=O.693). Moreover, no correlation was found between GS expression and Ki-67 labeling (high-GS group: 11.2?11.6%, low-GS group: 14.2~16.7%, p= 0.417). None of the other variables examined correlated significantly with strong GS expression.
Discussion GS catalyzes the synthesis of glutamine from glutamate and ammonia and plays a crucial role in ammonia detoxification, nitrogen balance and pH regulation in the mammalian liver (15-l 7). In normal liver tissue GS expression is restricted to the hepatocytes surrounding the central veins (3-5). This characteristic localization of GS, established during fetal and neonatal development (5,18,19), is thought to be due to cell-cell and cell-matrix interactions (20,21) and remains remarkably stable under a variety of physiological and experimental conditions in vivo and in vitro (3,22). Thus, GS-
positive HCCs are believed to be derived from GSpositive hepatocytes (11,23). We have previously reported the up-regulation of GS protein and GS enzyme activity in HCC (7). In particular, (i) the level of GS immunoreactivity increases stepwise from precancerous lesions to early HCCs (24,25) and adHCCs, and (ii) GS-positive foci or nodules are occasionally present within GS-negative HCC lesions. The latter finding was also observed in this study (Fig. Id). These results indicate that the GS-positive phenotype is acquired during the progression of HCC. Furthermore, in murine models of HCC, increased GS immunoreactivity is observed as foci progress to HCCs (23). This finding was suggested to be a direct consequence of the growth advantage of a GS-positive phenotype, rather than the acquisition of this phenotype during progression, because GS-positive lesions are not induced in animals exposed to aflatoxin Br. However, it is clear from our results and our previous report (7) that hepatocytes can acquire GS expression during malignant progression, at least in humans. Thus, it seems inappropriate to regard GS as a specific marker for tracing cell lineage relationships during human hepatocarcinogenesis. It is well known that glutamine, the end product of GS activity, is a critical substrate for cell growth and that it supports protein synthesis and is the major energy source of tumor cells (8,9). On the other hand, the serum glutamine level decreases in patients with advanced cancers of several types (2628), and the glutamine concentration in the parenchyma of livers with HCC is lower in cirrhotic livers than in non-cirrhotic livers (29). Thus, GS-expressing tumor cells may have a growth advantage under conditions of glutamine deficiency because they are independent of the extracellular glutamine supply. Collins et al. (10) reported that glutamine deprivation is greater in large induced tumors than in relatively smaller ones and that the former show GS up-regulation. In our studies, GS-expressing lesions (strongly- and weakly-positive lesions) tended to be larger in size than the lesions that lacked GS although the differences were not significant (4.02k2.05 cm vs 3.43+ 1.90 cm, p=O.222). Therefore, GS-positivity may be acquired by tumor cells in order to adapt to the nutrient-poor environment of the interior of the tumor as it grows. Moreover, a GS-positive phenotype may be advantageous in the establishment of metastatic nodules at secondary sites, because metastatic tumors may be exposed to relatively nutrient-poor conditions when the tumor cells are proliferating rapidly without sufficient neovascularization. Since GS expression is related to growth advantage
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of HCC, it seems reasonable to speculate that GS expression and higher proliferation rate are correlated. Indeed, tumor proliferation rate is currently believed to significantly affect the prognosis of patients with HCC, as well as HCC recurrence after surgery (30,31). Therefore we evaluated the proliferation rate of HCC cells by examining Ki-67 labeling index immunohistochemically. In this study, however, no correlation was found between GS expression and Ki-67 labeling. This result indicates that GS expression is not simply linked to the proliferating activity of HCC. Univariate survival analysis showed that the highGS group had significantly shorter DFS times compared with the low-GS group. Moreover, multivariate analysis showed that GS expression, as well as portal invasion and Child’s classification, was an independent prognostic factor for HCC recurrence. These results indicate that GS plays a significant role in the promotion of metastatic potential in HCC. Portal invasion and Child’s classification have been reported previously to be independent prognostic parameters for HCC recurrence (32-35). In the present study GS expression correlated significantly with tumor encapsulation and a lack of HBsAg. It may be related to the small number of cases in this study. As the present study is retrospective and with the bias of an a priori selection for surgery, further prospective studies are needed to support our data. In conclusion, this is the first report to identify strong GS expression as a significant prognostic factor for human HCC recurrence. There is no doubt that a high recurrence rate is a leading problem in the management of HCC. Evaluation of GS expression may be applicable to the clinical management of HCC to identify patients with poor prognoses.
Acknowledgement The authors thank Dr. Michiie Sakamoto for his critical comments on the paper.
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Copyright 8 European Association for the Study of the Liver 2000
JournalofHepatology ISSNO168-8278
Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma Takuya Osada’,
Ikuo Nagashima2,
Nelson H. Tsuno’,
Joji Kitayama’
and Hirokazu
Nagawa’
‘Department of Surgical Oncology, Graduate School of Medicine, University of Tokyo; and 2Second Department of Surgery, Teikyo University, Tokyo, Japan
Background/Aims: Glutamine synthetase (GS) catalyzes the synthesis of glutamine, a major energy source of cells, and is upregulated in a subset of human hepatocellular carcinomas @WCs). GS expression may he related to tumor recurrence, since GSexpressing tumors have a growth advantage in that they are independent of the extracellular glutamine supply. However, there are no studies concerning the prognostic value of GS expression in patients with HCC. Methods: Seventy-three patients with a single advanced HCC nodule who underwent curative hepatectomy were included in the study. GS expression in the HCC nodules was analyzed immunohistochemically and was compared with clinicopathologic features and the behavior of the tumors. Survival curves were assessed according to the Kaplan-Meier productlimit method and multivariate analysis based on the Cox regression model was performed.
Results: GS expression was strong in 26 cases (35.6%, high-GS group) and weak or absent in 47 cases (64.4%, low-GS group). Univariate analysis showed that the high-GS group had a significantly shorter disease-free survival time than the low-GS group (p=O.O42). Multivariate analysis revealed that GS expression (p=O.O21), as well as Child’s classification (p=O.O05) and portal invasion @=0.039), was a significant and independent prognostic parameter that affected tumor recurrence. Conclusion: The results of this study indicate that GS expression may enhance the metastatic potential in HCC, and GS immunostaining may be helpful in identifying HCC patients at high risk for disease recurrence.
H
Glutamine synthetase (GS, EC6.3.1.2), which catalyzes the synthesis of glutamine from glutamate and ammonia, may be such a prognostic factor. Although GS expression in normal liver tissue of mice and humans is restricted to a small subpopulation (~8%) of the hepatocytes, those that surround the terminal hepatic venules (3-5), its expression is upregulated in many human HCCs (6,7). The end product of GS activity, glutamine, participates in a variety of biosynthetic reactions; it is a critical substrate for optimal growth of normal and neoplastic cells in culture; it supports protein synthesis; and it is a major source of energy (8,9). In the absence of exogenous glutamine, human tumor cells with high GS activity proliferate well in vitro compared with those with low GS activity. In addition, as tumors grow in vivo the intracellular glutamine content declines and there is a concomitant increase in GS expression (10). Furthermore, in murine
carcinoma (HCC) is one of the most common types of malignant cancer in the world. Due to great advances in diagnostic techniques such as ultrasonography and computed tomography, the number of resectable cases has increased substantially (1). However, the prognosis for patients with this cancer is poor and recurrence after surgical resection is common (1,2). It is important, therefore, to identify factors that can be used to predict tumor recurrence so that adjuvant therapy can be provided to patients in high-risk groups. EPATOCELLULAR
Received 28 June; revised 16 November; accepted 30 November 1999 Correspondence: Takuya Osada, Department of Surgical Oncology, Graduate School of Medicine, University of Tokyo, 7-3-l Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel: 81 3 3815 5411. Fax: 81 3 3811 6822. e-mail: [email protected]
Key words: Glutamine synthetase; Hepatocellular carcinoma; Immunohistochemistry; Ki-67, Prognostic factor; Survival analysis.
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hepatocarcinogenesis models, an increase in the frequency of GS-positivity has been observed as foci progress to neoplasms (11). Thus, GS-expressing tumor cells may have a growth advantage due to their independence from the extracellular supply of glutamine, and GS production may be the mechanism by which HCC cells adapt to a nutrient-poor environment. Recently, we reported that the frequency of GS-positivity increases during the progression of human HCC and that GS-positive foci or nodules are occasionally present within GS-negative HCC lesions, indicating that GS expression is acquired during human hepatocarcinogenesis (7). In this study we evaluated the prognostic significance of GS in HCC and studied its relation to proliferation marker (Ki-67) and other clinicopathologic factors.
Patients From 1981 to 1995, 227 patients underwent surgery for HCC at the First Department of Surgery, University of Tokyo Hospital. As multiple HCC nodules may be multicentric in origin and consequently may have different properties, only patients who had a single advanced HCC (adHCC) without extrahepatic tumor spread were included in this study. Since it is difficult to evaluate huge tumors precisely by immunochemical methods, tumors with diameters in excess of 10 cm were excluded. Thus, 73 adHCCs, removed from 73 patients (62 men and 1 I women). were studied All of selected patients had was defined the excision all nodules that were visible and/or demonThe ages the patients ranged from 42 to 80 years (60.328.4 was considered HBV with positive and related including alcohol abuse. found. The from to 189.8 months were performed for first postoperative
Fig. I. Immunohistochemical demonstration of glutamine synthetase (GS) in hepatocellular carcinoma. Sections of live, tissue containing tumorous (T) and non-tumorous portions (N) were ,stained. (A) Homogeneous strong staining for GS. This nodule was included in the high-GS group. (B) A nodule showing weak GS staining. This nodule was included in the low-GS group. Arrowheads indicate the boundary of the nodule. The hepatocytes surrounding the central vein, which always showed intense staining, were used as an internal control. (C) A satellite nodule (S) showed GS staining pattern similar to that of the main tumor (T). (D) A GS-positive focal area (arrowj was seen within a GS-negative HCC nodule. (Inset) High magnification view of the GS-positive focus. (original magmfication: A und D (Inset) ~200. B X20, C and D x30).
248
Glutamine synthetase and prognosis in HCC year and every 24 months thereafter. Recurrence was diagnosed by ultrasonography, computed tomography scan, angiography and the serum alpha-fetoprotein (AFP) level. Recurrence of HCC was diagnosed if results of any two of four above follow-up examinations were considered positive. GS .has been reported to serve as a specific marker for tracing cell lineage relationships during hepatocarcinogenesis, so we also studied 10 cases of recurrent HCC in order to estimate the stability of GS expression during metastasis.
Immunohistochemistry Formalin-fixed, paraffin-embedded sections were deparaffinized with xylene and then rehydrated with alcohol. Endogenous peroxidase activity was blocked by soaking the sections in 0.3% H202 for 20 min. The sections were placed in the appropriate 10 mM citrate buffer solution and were autoclaved (12O“C, 20 min) for antigen retrieval (12). Nonspecific binding was blocked by incubation with 10% normal swine serum in phosphate-buffered saline for 30 min at room temperature. Then the sections were incubated with the anti-GS antibody (1:lOOO dilution; Chemicon International Inc., Temecula, CA, USA) or the anti-MIBl antibody (1:50 dilution; Immunotech, Marseille, France) overnight at 4°C. After rinsing, the sections were incubated with the biotinylated secondary antibody (1:200 dilution; Dakopatts, Copenhagen, Denmark) for 30 min at room temperature and then with the avidin-biotin peroxidase complex from the Vector Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s instructions. Visualization was achieved using 3,3’-diaminobenzidine tetrahydrochloride and H202. Specimens were counterstained with hematoxylin. At least two sections from each tumor were examined when the maximal diameter of the tumor was greater than 5 cm. The intensity of the GS staining was recorded as strong if it was equal to or greater than that of the hepatocytes surrounding the central vein (internal control). Nodules were considered to be strongly GS positive (highGS group) when at least 30% of their area was strongly stained. Nodules were recorded as weakly GS positive when they had strong staining over less than 30% of their area or weak staining over at least 30% of their area. Weakly GS positive nodules and nodules that lacked GS staining were assigned to the low-GS group. The Ki-67 labeling index (Ki-67 LI) was calculated by counting the number of positive nuclei per total number of hepatocytes. At least 1000 random nuclei of hepatocytes in 49 high power optical fields (x400) were evaluated. The procedure was performed three times for the same slide and an average was taken.
statistical analyses were carried out according the Statistical Analysis System (14).
Statistical analysis To compare the clinicopathologic features of the two groups, categorical data were analyzed using the cm-square test, whereas continuous data were evaluated using Student’s t-test. DFS times were calculated by the Kaplan-Meier method (13) and comparisons were made using the log-rank test for univariate analysis. Multivariate analysis was performed using the Cox regression model (14). Test results were considered significant when the p-values were less than 0.05. These
of
Results Glutamine synthetase staining Among 73 adHCC nodules, 26 (35.6%) showed strong GS immunoreactivity, 20 (27.4%) showed weak staining and 27 (37.0%) lacked GS immunoreactivity (Fig. la and lb). Satellite lesions were evaluated and compared with the main tumor in 18 cases. These nodules showed staining patterns similar to those of the main tumors (Fig. lc and Table 1). This result suggests that the expression of GS is relatively stable during synchronous metastasis. In addition, we evaluated 10 recurrent HCC nodules (Table 2). Compared to the parental tumor, the GS staining of the recurrent HCC nodules was similar in 6 cases (60.0%), and was altered in 4 cases (stronger in 3 and weaker in 1). Since half of the recurrent HCC nodules with altered staining were found more than 4 years after the first hepatectomy (4.9 and 10.1 years), they may have been multicentric, rather than metastatic, in origin. Among 7 cases with DFS time shorter than 4 years, 6 cases (85.7%) showed GS-positivity in the recurrent nodules, suggesting the relation of GS expression to metastasis of HCC cells.
TABLE
1
Comparison of glutamine synthetase staining lite lesions of heoatocelltdar carcinoma
Main tumor
Satellite tumor
_ +
+
++ _
++ +
-, no staining;
TABLE
between main and satel-
No. of cases
GS staining
Clinicopathologic parameters The following variables were evaluated:
age, sex, Child’s classification, indocyanine green retention rate (ICG R-15) accompanying cirrhosis, HBsAg, the AFP level, the maximal tumor diameter, degree of cellular differentiation, tumor encapsulation, capsular infiltration, satellite lesions, portal invasion, venous invasion, tumor-free margin, Ki-67 LI and GS expression. Child’s classification was applied to the patients with chronic hepatitis as well as liver cirrhosis, in order to assess the overall functional severity of liver disease. As there are several procedures for treating recurrent HCC (repeatresection, transcatheter arterial embolization and percutaneous ethanol injection therapy) and the choice may influence the prognosis of patients, we analyzed the disease-free survival (DFS) time. DFS time was measured from the date of hepatic resection to the date when recurrent disease was diagnosed or, in the absence of a detectable tumor, to the date of death or the last follow-up examination.
to the procedures
8 5 4 1
+, weak staining;
+ +, strong
staining.
2
Comparison of glutamine synthetase staining between mors and recurrent hepatocellular carcinoma nodules Case
pt.1 pt.2 Pt. 3 Pt. 4 Pt. 5 Pt. 6 Pt. 7 Pt. 8 Pt. 9 Pt. 10
-, no staining;
tu-
Disease free time
GS staining Primary
Recurrent
_
+ + ++ ++ ++ ++ ++
_ + ++ ++ ++ ++
primary
f, weak staining;
(years) 2.10 2.32 2.45 5.52 10.09 4.90 0.46 1.25 1.64 3.25
+ + strong
staining.
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T. Osada et al.
group compared with the low-GS group. We also found that Child’s classification (p=O.OOS) and portal invasion (p=O.O39) were independent risk factors significantly associated with the DFS time (Table 4).
p = 0.042 E
80-
3 ‘E 60d 8 c=; 40-
Glutamine synthetase parameters
0 d
20, 0
1 2
0
.
I 4
, 6
*
High-GS (~26) I . I I 8 I 12 14 8 10
I 16
Years after Surgery High-GS
26
9
4
2
0
0
0
0
0
Low-GS
47
26
14
6
5
3
1
1
0
Fig. 2. Disease-free survival of the 73 patients with HCC who underwent curative resection. Median follow-up was 17.9 months in high-GS group (n=26) and 27.9 months in low-GS group (n=47). The number of patients still at risk over follow-up time is reported at the bottom of the figure. There was a significant correlation between GS expression and tumor recurrence (p=O.O42) by log rank test.
Survival
analysis
Among 73 patients, 48 (65.8%) experienced tumor recurrence. Recurrence was detected within 24 months of hepatectomy in 20 (41.7%) patients and after the 24th month in 28 (58.3%) patients. No patients died of liver insufficiency without HCC recurrence. The recurrence rate tended to be higher in the high-GS group (20/26 cases, 76.9%) than in the low-GS group (28/47 cases, 59.6%) although the difference was not significant Cp= 0.135). The high-GS group had significantly shorter DFS times compared with the low-GS group (p=O.O42, Fig. 2). Univariate analysis showed that Child’s classification (p=O.O25), portal invasion (p=O.O26) and ICG R-15 (p=O.O39) were also correlated significantly with the DFS time (Table 3). Although patients with large tumors (>5 cm) or with microsatellite nodules tended to have shorter DFS times, these correlations did not reach statistical significance @=0.073 and 0.126, respectively). The high-Ki-67 labeling group (~20%) tended to have shorter DFS time than the low-Ki-67 labeling group (<200/o), although the difference was not statistically significant @=0.224). The other parameters examined were not significantly correlated with the DFS time. Multivariate analysis showed that the high-GS group had significantly shorter DFS times (p=O.O21) than the low-GS group. The estimated relative risk of tumor recurrence was 2.59 for the high-GS patient 250
expression
and clinicopathologic
The correlations between GS expression and clinicopathological parameters are summarized in Table 5. All of the high-GS group lesions were encapsulated; however, 11 of 47 low-GS group lesions (23.4%) lacked fibrous capsules. Thus, tumor encapsulation was significantly related to GS expression @=0.007). HBsAg positivity was inversely related to GS expression. In fact, 16 of 47 low-GS group lesions (34.0%) were HBsAg positive, whereas only 2 of 26 high-GS group lesions (7.7%) were HBsAg positive (p=O.O12). The
TABLE
3
Clinicopathologic variables related to the disease-free-survival after surgical resection in hepatocellular carcinoma
time
p-V&~
Variable
Categories
(cases)
Age (years) Sex Child’s classification ICG R- 15 (%) Cirrhosis HBsAg AFP (rig/ml) Tumor size (cm) Cellular differentiation Encapsulation Capsular infiltration Tumor microsatellite Portal invasion Venous invasion Tumor-free margin Ki-67 LI (“A) GS expression
~60 (37) r.~ 260 (36) Male (62) vs Female (I 1) A (63) c’s B/C (10) 515 (32 vs >15 (41) Absent (30) $1~Present (43) Negative (55) rs Positive (18) 5200 (55) 1’S>200 (18) 550 (57) vs >5.0 (16) Well (19) rs Moderate/Poor (54) Absent (11) 1’s Present (62) Absent (32) 1’s Present (30) Absent (56) 1’s Present (17) Absent (62) ~8sPresent (11) Absent (70) r-y Present (3) Absent (9) 1’s Present (64) s-20 (55) L’S>20 (18) Low-GS (47) 1’s High-GS (26)
0.2712 0.8279 0.0253 0.0394 0.6468 0.1133 0.6729 0.0729 0.5697 0.6136 0.7819 0.1258 0.0261 0.3908 0.7117 0.2240 0.0416
ICG R-15: indocyanine gree retention rate at 15 min; HBsAg: hepatitis B surface antigen; AFP: alpha-fetoprotein; Ki-67 LI: Ki-67 labeling index; GS: glutamine synthetase. p-values are based on Student-r test or chi-square analysis.
TABLE
4
Independent prognostic factors for the disease-free-survival time after surgical resection in hepatocellular carcinoma (multivariate Cox analysis) Variables
Paremeter estimate
Standard error
Risk ratio
p-value
Child’s classification Portal invasion GS expression Ki-67 LI
1.688 1.219 0.952 0.861
0.606 0.591 0.411 0.406
5.411 3.383 2.590 2.366
0.005 0.039 0.021 0.062
GS: glutamine synthetase; Variables with p-value>O.
Ki-67 LI: Ki-67 labeling 1 are not shown.
index.
Glutamine synthetase and prognosis in HCC TABLE 5 Clinicopathologic variables of patients with hepatocellular carcinoma: comparison with regard to glutamine synthetase expression Variable
Low-GS n=41 (%)
High-GS n=26 (%)
p-value
Agea (years)
60.3k8.5
60.3-r-8.5
0.9950
Sex Male Female
38 (80.9) 9 (19.1)
24 (92.3) 2 (7.7)
0.1901
Child’s classification A B or C
40 (85.1) 7 (14.9)
23 (88.5) 3 (11.5)
0.6897
ICG R-15” (%) Cirrhosis HBsAg AFP” (@ml) Tumor sizea (cm)
16.2k10.6 21 (57.4) 16 (34.0) 28445 10997 3.7k2.1
20.626.3 16 (61.5) 2 (7.7) 95?142 3.921.8
0.0590 0.1331 0.0124 0.2081 0.6931
Cellular differentiation Well Moderate or Poor
12 (25.5) 35 (74.5)
7 (26.9) 19 (73.1)
0.8968
Encapsulation Capsular infiltrationb Tumor microsatellite Portal invasion Venous invasion Tumor-free margin Ki-67 LI” (%)
36 (76.6) 18 (50.0) 9 (19.1) 6 (12.8) 2 (4.3) 40 (85.1) 14.2216.7
26 (100) 12 (46.2) 8 (30.8) 5 (19.2) 1 (3.8) 24 (92.3) 11.2211.6
0.0074 0.7649 0.2607 0.4597 0.9328 0.3702 0.4167
ICG R-15: indocyanine green retention rate at 15 min; HBsAg: hepatitis B surface antigen; AFP: alpha-fetoprotein; Ki-67 LI: Ki-67 labeling index. a Results are expressed as the meankstandard deviation. b Frequencies are calculated in cases with tumor encapsulation. p-values are based on Student-r test or chi-square analysis.
high-GS group tended to have higher ICG R-15 scores than the low-GS group although the difference was not significant (p=O.O59). The difference in size between the high-GS and low-GS groups was not significant (p=O.693). Moreover, no correlation was found between GS expression and Ki-67 labeling (high-GS group: 11.2?11.6%, low-GS group: 14.2~16.7%, p= 0.417). None of the other variables examined correlated significantly with strong GS expression.
Discussion GS catalyzes the synthesis of glutamine from glutamate and ammonia and plays a crucial role in ammonia detoxification, nitrogen balance and pH regulation in the mammalian liver (15-l 7). In normal liver tissue GS expression is restricted to the hepatocytes surrounding the central veins (3-5). This characteristic localization of GS, established during fetal and neonatal development (5,18,19), is thought to be due to cell-cell and cell-matrix interactions (20,21) and remains remarkably stable under a variety of physiological and experimental conditions in vivo and in vitro (3,22). Thus, GS-
positive HCCs are believed to be derived from GSpositive hepatocytes (11,23). We have previously reported the up-regulation of GS protein and GS enzyme activity in HCC (7). In particular, (i) the level of GS immunoreactivity increases stepwise from precancerous lesions to early HCCs (24,25) and adHCCs, and (ii) GS-positive foci or nodules are occasionally present within GS-negative HCC lesions. The latter finding was also observed in this study (Fig. Id). These results indicate that the GS-positive phenotype is acquired during the progression of HCC. Furthermore, in murine models of HCC, increased GS immunoreactivity is observed as foci progress to HCCs (23). This finding was suggested to be a direct consequence of the growth advantage of a GS-positive phenotype, rather than the acquisition of this phenotype during progression, because GS-positive lesions are not induced in animals exposed to aflatoxin Br. However, it is clear from our results and our previous report (7) that hepatocytes can acquire GS expression during malignant progression, at least in humans. Thus, it seems inappropriate to regard GS as a specific marker for tracing cell lineage relationships during human hepatocarcinogenesis. It is well known that glutamine, the end product of GS activity, is a critical substrate for cell growth and that it supports protein synthesis and is the major energy source of tumor cells (8,9). On the other hand, the serum glutamine level decreases in patients with advanced cancers of several types (2628), and the glutamine concentration in the parenchyma of livers with HCC is lower in cirrhotic livers than in non-cirrhotic livers (29). Thus, GS-expressing tumor cells may have a growth advantage under conditions of glutamine deficiency because they are independent of the extracellular glutamine supply. Collins et al. (10) reported that glutamine deprivation is greater in large induced tumors than in relatively smaller ones and that the former show GS up-regulation. In our studies, GS-expressing lesions (strongly- and weakly-positive lesions) tended to be larger in size than the lesions that lacked GS although the differences were not significant (4.02k2.05 cm vs 3.43+ 1.90 cm, p=O.222). Therefore, GS-positivity may be acquired by tumor cells in order to adapt to the nutrient-poor environment of the interior of the tumor as it grows. Moreover, a GS-positive phenotype may be advantageous in the establishment of metastatic nodules at secondary sites, because metastatic tumors may be exposed to relatively nutrient-poor conditions when the tumor cells are proliferating rapidly without sufficient neovascularization. Since GS expression is related to growth advantage
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of HCC, it seems reasonable to speculate that GS expression and higher proliferation rate are correlated. Indeed, tumor proliferation rate is currently believed to significantly affect the prognosis of patients with HCC, as well as HCC recurrence after surgery (30,31). Therefore we evaluated the proliferation rate of HCC cells by examining Ki-67 labeling index immunohistochemically. In this study, however, no correlation was found between GS expression and Ki-67 labeling. This result indicates that GS expression is not simply linked to the proliferating activity of HCC. Univariate survival analysis showed that the highGS group had significantly shorter DFS times compared with the low-GS group. Moreover, multivariate analysis showed that GS expression, as well as portal invasion and Child’s classification, was an independent prognostic factor for HCC recurrence. These results indicate that GS plays a significant role in the promotion of metastatic potential in HCC. Portal invasion and Child’s classification have been reported previously to be independent prognostic parameters for HCC recurrence (32-35). In the present study GS expression correlated significantly with tumor encapsulation and a lack of HBsAg. It may be related to the small number of cases in this study. As the present study is retrospective and with the bias of an a priori selection for surgery, further prospective studies are needed to support our data. In conclusion, this is the first report to identify strong GS expression as a significant prognostic factor for human HCC recurrence. There is no doubt that a high recurrence rate is a leading problem in the management of HCC. Evaluation of GS expression may be applicable to the clinical management of HCC to identify patients with poor prognoses.
Acknowledgement The authors thank Dr. Michiie Sakamoto for his critical comments on the paper.
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