1 Introduction
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, which is the third most common cause of cancer-related death and the fifth most common cancer all over the world, followed by lung and stomach cancers.[1] [2] [2,3]
Metastasis is a major cause for the death of HCC patient, with some cases present with metastatic carcinoma before primary liver tumor is found. In metastasis, differentiation of HCC from non-HCC may cause a diagnostic problem, because HCC may show a variety of histologic patterns, mimicking a wide variety of malignant tumors, in addition a number of metastatic tumors may mimic the trabecular, pseudoglandular and solid patterns of HCC. The single routine histopathology can not achieve the diagnosis, so immunostaining was used. Some markers were useful, such as AFP, HepPar-1, and GPC-3, ARG-1, etc.
AFP (α-fetoprotein) is a marker of hepatocellular differentiation, and can express in germ cell tumors (such as yolk sac tumor). AFP is an oncofetal protein produced by liver and yolk sac visceral endoderm.[4]
GPC-3 (glypican-3) is one of the glypican family of glycosyl-phosphatidylinositol- anchored cell surface heparan sulfate proteoglycans. It stains cytoplasm and/or membrane. Some studies have shown that GPC-3 may be a specific tumor marker to diagnose HCC.[5]
HepPar-1 (hepatocyte paraffin-1) is a mitochondrial urea cycle antigen linking mitochondrial antigens from both malignant and nonmalignant hepatocytes. It is a positive marker for hepatocyte differentiation on paraffin-embedded tissue, which has been used to verify hepatic differentiation. It can not differentiate benign from malignant hepatocyte and expresses poorly in HCC with low differentiation. Sometimes it can express in other tumors.[6]
ARG-1 (Arginase-1) is a binuclear manganese metalloenzyme that hydrolyzes arginine to ornithine and urea as a part of the urea cycle, which is specific for hepatocyte. ARG-1 can express in HCC with low differentiation and scirrhous HCC.[7]
Since these markers have also been reported in non-HCC,[8–10] [11]
The aim of this study was to assess retrospectively the diagnostic accuracy of a panel of markers (ARG-1, HepPar-1, GPC-3, and AFP) for the diagnosis of extrahepatic metastatic HCC and to summarize the clinicopathological features of metastatic HCC.
2 Materials and methods
The current study was approved by the institutional review board at Peking University People's Hospital. A total of 195 cases, pathologically diagnosed as distant extrahepatic metastatic HCC were reviewed retrospectively from archived specimens during the period 2001 to 2017, accounting for about 12.2% (195/1592) of HCC diagnosed during the same period in our database. All medical records of relevant clinical, radiological, and laboratory data were collected to analyze. All available histologic and immunostained sections were reviewed according to the 2010 WHO classification.[12]
The immunohistochemical expression of ARG-1, HepPar-1, GPC-3, and AFP in 195 cases was evaluated. The information of antibodies and staining conditions are listed in Table 1 . Paraffin section immunoperoxidase studies were performed manually on 4 μm deparaffinized, formalin-fixed sections. Immunohistochemical studies were performed by the 2-step EnVision procedure. Appropriate positive and negative controls were prepared. The staining result was assessed as negative (<5% of tumor cells stained) or positive. The immunostained slides were independently evaluated by 2 experienced pathologists (DC and LQ)
Table 1: Antibodies used for immunohistochemistry in this study.
In order to verify the effectiveness of ARG-1, HepPar-1, GPC-3, and AFP in differentiating metastatic HCC from non-HCC, 80 cases of metastatic tumors of the liver were also collected, including adenocarcinomas of intestine, biliary duct, breast, kidney, ovary, pancreas, lung, gallblader (58 cases), and other tumors of neuroendocrine carcinoma (NEC), perivascular epithelioid cell tumor (PEComa), hemangiopericytoma (solitary fibrous tumor), gastrointestinal stromal tumor (GIST), osteosarcoma, panceatic solid pseudopapillary tumor, urothelial carcinoma, and squamous cell carcinoma (22 cases).
The SPSS software package version 17.0 for windows was used for all statistical analysis. Data were expressed as numbers and percentages. The data were compared for statistical significance by chi-square (χ2 ) and Fischer exact probability tests, and different significance was considered as P < .05. Effectiveness of the antibodies was evaluated by sensitivity and specificity. The histopathologic diagnosis was considered as the gold standard.
3 Results
In 195 cases, 172 were males, and 23 were females (male-to-female ratio was 7.5:1). The age ranged from 10 to 78 years (mean: 53.0, median: 53.0). The features of the 195 patients were shown in Table 2 . Totally 151 cases had previous history of HCC, in which 49 with history of liver transplantation. Forty-four cases were diagnosed as metastatic HCC at initial presentation, in which 3 cases had metastatic HCC with coexisting intrahepatic masses, 41 cases without known liver primary tumors had no clinical features suggestive of HCC, which were confirmed by the detection of primary HCC subsequently. Their initial presentation was in the form of extrahepatic mass lesion. The symptoms caused by extrahepatic metastasis included pain or fracture caused by bone metastasis, dyspnea caused by multiple lung metastasis, nerve paralysis, and abdominal pain, etc. Totally 133 cases (68.2%) had cirrhosis. The hepatic lesions of 67 cases were focal, of which 41 were solitary (61.2%, 1.5–6 cm in diameter). The focal lesions of 26 cases (38.8%) were multiple (0.5–17 cm). HBV was positive in 152 cases, and 11 were positive for HCV and 1 for HAV. Serum AFP level was elevated in 102 patients (64.6%).
Table 2: Clinicopathological characteristics of 195 extrahepatic metastases .
Bone was the most common site for extrahepatic HCC metastases (111 cases, 57.0%). The vertebrae were the most common site of bone (59 cases, 53.2%), followed by the sacrum (16 cases) (Fig. 1 ), femur (7 cases), pelvis (7 cases), scapula (5 cases), ribs (4 cases), sternum (3 cases), and ilium (3 cases). The sites of the rest cases were the clavicle, humerus, acetabulum (2 cases for each site), and pubis (1 case). Primary or secondary malignant tumors were considered in these cases by clinical and radiological features. (Fig. 2 )
Figure 1: Metastatic HCC of the vertebra. Macroscopic view of a poorly circumscribed tan mass invading the bone. HCC = hepatocellular carcinoma.
Figure 2: A metastatic mass from HCC infiltrating spinous process of first vertebra with lytic lesion (square) in computed tomography (sagittal reconstruction). HCC = hepatocellular carcinoma.
Lung metastasis (41 cases) was the second, followed by lymph node, abdomen, omentum, adrenal gland, soft tissue, pelvic cavity, brain, stomach, intestine, retroperitoneum, diaphragm, ventriculus dexter, kidney, and umbilical region (Table 2 ). A space-occupying lesion or a mass can be seen in radiography, CT scan or ultrasonic examination in these cases.
All metastases demonstrated malignant tumor cells composed of hepatocyte-like cells with mild to severe nuclear atypia and variable mitoses. The cells presented as trabecular, pseudoglandular sinusoidal or solid patterns, some cases showed mixed patterns, just like those of primary HCC (Figs. 3 and 4 ). In some cases, the tumor cells showed granular cytoplasmic positivity for HepPar-1 (Fig. 5 ), GPC-3 (Fig. 6 ), ARG-1 (Fig. 7 ), and/or AFP (Fig. 8 ), which accounted for 83.6%, 89.2%, 82.6%, and 53.8%, respectively.
Figure 3: Moderately differentiated HCC with trabecular growth pattern of 3 or more cells in thickness. The tumor cells have abundant eosinophilic cytoplasm, with moderate atypia (HE staining ×100). HCC = hepatocellular carcinoma.
Figure 4: Specimen of the bone lesion shows histologic features of poorly differentiated HCC with solid and adenoid pattern. The tumor cells show marked atypia, with eosinophilic cytoplasm and an increased nucleus: cytoplasm ratio (HE staining ×200). HCC = hepatocellular carcinoma.
Figure 5: Diffuse HepPar-1 staining (cytoplasmic pattern) on metastatic hepatocellular carcinoma (immunohistochemical staining ×100). HepPar-1 = hepatocyte paraffin-1.
Figure 6: Diffuse GPC-3 staining (cytoplasmic pattern) on metastatic hepatocellular carcinoma (immunohistochemical staining ×200). GPC-3 = glypican-3.
Figure 7: Diffuse ARG-1 staining (both cytoplasmic and nuclear pattern) on metastatic hepatocellular carcinoma (immunohistochemical staining ×100). Arg-1 = arginase-1.
Figure 8: Diffuse AFP staining (cytoplasmic pattern) on metastatic hepatocellular carcinoma (immunohistochemical staining ×200). AFP = α-Fetoprotein.
Of 195 cases, 19 were positive for 1 marker, 22 were positive for 2 markers, 95 were positive for 3 markers, and 59 were positive for 4 markers. When 4 antibodies in the panel were used to differentiate metastatic HCC from other adenocarcinomas, there was no negative case, and at least 1 antibody expressed. There was significant difference between expression in HCC and non-HCC for each antibody (P < .01). (Tables 3 and 4 )
Table 3: Antibodies used in differentiation of hepatocellular carcinoma in extrahepatic metastases .
Table 4: Immunostaining results of hepatocellular carcinoma with different differentiation.
The correlation between expression of makers and different differentiation of HCC was also observed. The ratio of ARG-1, HepPar-1, GPC-3 expression in moderately to well differentiated HCC was higher than that of poorly differentiated HCC (90.5% vs 76.6%, 80.0% vs 74.8%, 92.9% vs 86.5%), whereas it was contrary to AFP (41.7% vs 63.1%). When 4 markers were used, no case was negative in different differentiation, and at least 1 marker can be positive. (Table 4 )
In order to distinguish HCC from adenocarcinoma from other site, CDX2, TTF1, and PSA were also detected in some cases, which were negative except that CDX2 were focal and weak positive in 3 cases.
The diagnostic sensitivity and specificity of ARG-1, GPC-3, HepPar-1, and AFP were calculated based on the combined numbers of metastatic HCC (195 cases) and collected nonhepatocellular adenocarcinomas (58 cases). The sensitivity of ARG-1, GPC-3, HepPar-1, and AFP was 82.6%, 89.2%, 83.6% and 53.8%, and the specificity was 98.3%, 94.8%, 96.2% and 100%, respectively, which were 100% and 96.3% in the panel (Table 5 ). Sensitivity and specificity for ARG-1, GPC-3, HepPar-1 were better, whereas sensitivity for AFP was not ideal, but its specificity was quite good. In 4 markers detected cases, 9.7% were positive for 1 marker, 11.3% for 2 markers, 48.7% for 3 markers, 30.3% for 4 markers, and no case was negative for 4 markers. It indicated that with the markers increased in the panel, the detection ratio was raised.
Table 5: Comparison of ARG-1, HepPar-1, GPC-3, AFP and combination of 4 markers expression between extrahepatic hepatocellular carcinoma and adenocarcinoma from other site.
In 80 cases of intrahepatic metastatic non-HCC, ARG-1, and HepPar-1 showed focal positive in 1 cholangiocarcinoma, HepPar-1 was also focal positive in 1 gall bladder adenocarcinoma, GPC-3 was focal positive in 2 cholangiocarcinoma and 1 ovary high grade serous carcinoma, whereas AFP was negative in all cases. The expression of ARG-1, GPC-3, HepPar-1, and AFP in collected cases other than adenocarcinoma was negative (Table 6 ).
Table 6: Immunostaining results of ARG-1, HepPar-1, GPC-3, and AFP of non-hepatocellular carcinoma tumors∗ .
4 Discussion
The purpose of this study was: to characterize the clinicopathological features of extrahepatic HCC; to verify the specificity and sensitivity of Arg-1 and GPC-3 compared with HepPar-1 and AFP; to recognize the most effective panel of markers to differentiate extrahepatic HCC.
The initial aim of this study was to characterize a relatively large number of patients who presented as extrahepatic metastases of HCC, some of which initially manifested as metastases before the primary HCC were confirmed. Most of such reported cases were described in case reports or case series except for 5 studies[13–16] Table 7 ). The current study included 195 patients from China mainland, most of whom were HBV positive. The number of extrahepatic metastases constitutes about 12.2% of the total number of HCC cases in our hospital, more than those of the literature (5%-6.5%),[13–16] [17] metastases were reported in a larger series.[16] metastases initially diagnosed as HCC were studied in Korea.[14]
Table 7: Comparison of hepatocellular carcinoma initially presented as extrahepatic metastastatic lesions in the literature.
The clinical presentations of patients with extrahepatic metastatic HCC were mostly correlated with the manifestations of the primary tumor and the metastatic presentation were later event. Wu et al[18] [17] [18] [19] metastases was best while that of cases with brain metastases was the worst in both OS and CSS analysis.[18]
The identification of the metastatic lesion before the primary HCC was diagnosed is an important finding, there were 41 cases presented initially with extrahepatic metastases , which were considered as primary or secondary malignant tumor clinically and radiologically, and were confirmed by the presence of primary HCC subsequently. Totally, 151 cases had primary HCC history, 49 of whom had received liver transplantation, and 3 cases had coexisting intrahepatic HCC.
When the patient has a history of primary HCC, it is easy to consider the metastasis as HCC. However, if there is no HCC history and no manifestation indicating HCC in extrahepatic metastasis, the diagnosis is difficult, especially in poorly differentiated tumor. On the basis of histopathology, hepatocyte like cells arranged in trabecular (plate like), pseudoglandular (acinar), solid or mixed patterns. In some instances, lack of typical features of classical HCC causes difficulty in the accurate diagnosis, thereby IHC is employed to identify metastatic HCC. Timek et al[11]
In our study, AFP showed negative in non-HCC, whose specificity is quite good, and is a highly specific marker for HCC (100%), but its sensitivity is 54.2%, lower than those of the other markers. The sensitivity of ARG-1, GPC-3, HepPar-1 was 82.6%, 89.2%, 83.6%, and the specificity was 98.3%, 94.8%, 96.2%, respectively. The specificity of ARG-1 is better than those of GPC-3 and HepPar-1, but its sensitivity is worse than those of GPC-3 and HepPar-1, different from the report of Yan et al[10] [9] [9] metastases . When adenocarcinoma of lung is considered, CK7, TTF1, and NapsinA should be added in the immunohistochemical panel, and CK20, CDX2 for differentiating colorectal carcinoma, villin for stomach, PSA for prostate, CK7, ER, PR, GATA-3 for breast, CK7, ER, WT1, PAX8 for ovary, CK, CD10, Vimentin, PAX8 for renal cell carcinoma, and synaptophysin and/or chromogranin for NET. Timek et al[11] metastases . The high sensitivity and specificity of Arg-1, GPC-3, and HepPar-1 make them the first choice for demonstrating hepatocellular differentiation. We propose increasing AFP in the panel to improve the specificity of differentiation. Choi et al[20]
These data suggest that a panel of ARG-1, GPC-3, HepPar-1, and AFP has a high sensitivity and specificity to differentiate HCC from non-HCC. We recommend the most effective 4 markers as a panel to differentiate HCC from non-HCC in extrahepatic metastases . We can also analyze gene expression and genome to classify tumor origin by molecular methods, especially in poorly differentiated tumor. However, it is infeasible for most cases in routine practice because of the expenses and time.
The unique of this work is trifold: firstly, characterize a relatively large number of patients who presented as ertrahepatic metastases of HCC, some of which initially manifested as metastases before the primary HCC were diagnosed. Also there were more cases who had HCC history, some of whom had received treatment with liver transplantation. Secondly, the present research was about Chinese cases; most of whom were HBV-positive. Extrahepatic metastases of HCC are not rare, and major metastatic organs are the bone, lung, abdomen. Thirdly, an effective imunostaining panel to differentiate HCC from non-HCC in extrahepatic metastases is proposed. It seems that there are no such documented reports in the databases of literature that we have searched.
In summary, the present research emphasizes that metastatic HCC should be put into consideration when evaluating metastatic carcinoma with unclear origin. The most common extrahepatic metastatic sites are bone, lung and abdomen. It is recommended to use a panel of ARG-1, GPC-3, HepPar-1, and AFP to differentiate HCC from non-HCC in extrahepatic metastasis, because of their sensitivity and specificity, especially in poorly differentiated lesions.
Author contributions
Data curation: Dingbao Chen, Batubaiyin Xie.
Formal analysis: Lihua Qian.
Funding acquisition: Zhao Li, Jiye Zhu.
Methodology: Qiujing Song.
Supervision: Jiye Zhu.
Writing – original draft: Dingbao Chen.
Writing – review & editing: Jiye Zhu.
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