Carcinoma: Report of Two Cases with Immunohistochemical Detection of Ezrin,
and Ultrastructural and K-ras Mutation Analysis
Phui-Ly Liew
1, Ming-Te Huang
2, Sey-En Lin
3, Hsin-An Chen
2, Chih-Hsiung Wu
2, Wei-Hwa Lee
1 * 1Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan2Division of General Surgery, Department of Surgery, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan 3Department of Pathology, Taipei Medical University Hospital, Taipei Medical University, Taipei, Taiwan
a r t i c l e i n f o
Article history: Received: Jan 20, 2012 Revised: Feb 6, 2012 Accepted: Mar 20, 2012 KEY WORDS: ezrin; K-ras mutation; liver;malignantfibrous histiocytoma; ultrastructural
We reported two primary malignantfibrous histiocytoma (MFH) cases (cases 1 and 2) strikingly simu-lating hepatocellular carcinoma with immunohistochemical (IHC) stains, and ultrastructural and K-ras mutation analysis. Cases 1 and 2 both included only men, aged 65 and 72 years, respectively. Case 1 presented with abdominal pain, fever, and bilateral legs edema. Case 2 was found to have a liver tumor during routine heath examination. Computed tomography scan showed hepatic mass withfine tumor vessels. Case 1 had one 16 15 13 cm3liver mass with tumor rupture in the right lobe, and case 2 had
one 3 2.5 1 cm3liver tumor in segment 6. Microscopically, the tumors were composed of spindle,
pleomorphic, and multinucleated giant cells arranged in storiform, sheet-like, and fascicular patterns. Strong immunoreactivity for CD68 and vimentin in tumor cells in both cases was noticed. Ezrin immunoexpression in tumor tissue from case 1 was diffusely strong; in contrast, case 2 showed sparse and weak ezrin expression in tumor cells. Ultrastructurally, cells withfibroblasts and histiocytic char-acteristics were present in both cases. K-ras gene analysis of exon 2codon 12 or 13 showed no mutation. This is thefirst report describing K-ras gene analysis of liver MFH. We emphasize that ezrin immuno-reactivity may be correlated with poorer prognosis.
CopyrightÓ 2012, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
1. Introduction
In Taiwan, hepatocellular carcinoma (HCC) is the most common liver tumor, which is associated with hepatitis virus infection and liver cirrhosis. Primary sarcomas of the liver are rare neoplasms.1 Primary hepatic malignant fibrous histiocytoma (MFH) is extremely rare,2e4and only 49 cases have been reported to date in the literature.5e27
Ezrin is a novel cytoskeleton linker protein that is involved in regulating cellular growth and metastatic potential of various types of malignancies.28e30 Point mutation of the K-ras oncogene is a frequent event in many gastrointestinal carcinomas.31,32However, little is known about the occurrence of this mutation in primary hepatic MFH.
In this report, we present two cases of primary hepatic MFH. We investigated extensive immunohistochemical (IHC) stains, electron
microscopic studies, and K-ras mutation analysis to describe the possible histological prognostic factor in hepatic MFH.
2. Case reports 2.1. Case 1
A 65-year-old male patient suffered from abdominal pain, fever, and bilateral legs edema for 1 month. No history of previous operation, alcohol consumption, or smoking was found. Laboratory data showed slightly increased glutamic-oxaloacetic transaminase (GOT) (50 IU/L; normal, 5e40 IU/L), carbohydrate antigen 19-9 (64.40 U/ mL; normal, <37.00 U/mL), and anemia (hemoglobin ¼ 7.3 g/dL; normal, 14e18 g/dL). Serologic anti-hepatitis C virus (anti-HCV) antibody and hepatitis B virus (HBV) surface antigen were positive. Serum alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were within normal limits.
Preoperative abdominal computed tomography (CT) showed a heterogeneously enhancing mass (16 15 13 cm3) in the right hepatic lobe, with suspicious invasion of right portal vein and tumor rupture (Figure 1). Under the impression of huge HCC with suspected rupture, the patient underwent extended right * Corresponding author. Wei-Hwa Lee, No. 291, Jhongjheng Road, Jhonghe
District, New Taipei City 23561, Taiwan.
E-mail: W.-H. Lee <[email protected]>
1878-3317/$ e see front matter Copyright Ó 2012, Taipei Medical University. Published by Elsevier Taiwan LLC. All rights reserved. doi:10.1016/j.jecm.2012.04.011
lobectomy of the liver, omentectomy, and cholecystectomy. Written informed consent was obtained.
The patient complained of right shoulder and neck pain 1½ month after operation. Abdominal and pelvis CT scan showed numerous variable-sized heterogeneously enhanced masses at right resection margin of liver with diffuse peritoneal seeding. Bilateral metastatic lung masses with right pleural effusion and massive ascites were also noted. The patient expired 2 months after surgery.
2.2. Case 2
A 72-year-old male patient was incidentally found to have a hepatic tumor during routine medical examination. The patient had no history of previous operation, alcohol consumption, or smoking. Laboratory data showed anemia with hemoglobin of 12.4 g/dL,
a decreased platelet count of 72 103/
m
L (normal, 130e400 103/
m
L), slightly increased GOT (63 IU/L), glutamic-pyruvic trans-aminase (60 IU/L; normal, 5e40 IU/L), and Indocyanine green retention test (ICG) 16.0% (normal, <10%). Serologic anti-HCV antibody and HBV surface antigen were negative. The AFP level was within normal limit.Preoperative CT scan showed a hypervascular liver mass (3 2.5 1 cm3) in segment 6 of liver with patent portal vein (Figure 2). Under the impression of HCC, the patient underwent laparoscopic segmentectomy of segment 6. Written informed consent was obtained. The postoperative course was smooth. 2.3. Macroscopic and light microscopicfindings
These two tumors were solitary and well circumscribed, but unencapsulated whitish to yellow tan mass with areas of hemor-rhage and necrosis (Figures 3A and B). Rupture of tumor is present Figure 1 CT scan of case 1. A lobulated mass (16 15 13 cm3) occupying segments 4,
5, and 8 of liver, with an initial peripheral and gradualfill-in enhancement pattern, and central areas of necrosis were found. Tumor had ruptured at its lateral margin, with blood clot in right perihepatic space. CT¼ computed tomography.
Figure 2 CT scan of case 2 showed a solitary mass (3 2.5 1 cm3) with irregular
margin in segment 6 of liver, with a peripheral enhancement pattern and central area of necrosis. CT¼ computed tomography.
in case 1. Histopathologicalfindings of the hepatic tumors from cases 1 and 2 (Figures 4A and B) were similar. Proliferation of spindle, pleomorphic, and multinucleated giant cells arranged in storiform, sheet, and/or fascicle patterns was noticed. In both cases, marked nuclear pleomorphism, prominent mitoses (five in 10 fields of high-power view), and foci of inflammatory cellular infiltration were noticed. The tumor stages of case 1 and case 2 were stage IVB and stage I, respectively.
2.4. Immunohistochemistryfindings
IHC staining was performed on paraffin-embedded tissue sections (Table 1). Positive control for ezrin (small bowel mucosa) and negative controls by omitting primary antibodies were performed.
Staining intensity was assessed by an arbitrary value of 0 (no staining, <5%), 1 (weak staining, 5e33%), 2 (moderate staining, 33e66%), or 3 (strong staining, >66%).
Tumor cells in both cases were positive for vimentin and CD68. Cellular proliferative indexes (Ki-67 stain) were 75% and 80% in cases 1 and 2, respectively. Faint immunoreactivity (<5% of tumor cell) was found for smooth muscle actin and epidermal growth factor receptor (EGFR) in both cases. Ezrin immunoexpression showed diffuse and strong cytoplasmic and/or membranous staining in case 1 (Figure 5A). In contrast, the immunoreactivity against ezrin in tumor cells of case 2 was weak and showed a scattered distribution (Figure 5B). The other immunostains were negative in both cases.
2.5. Electron microscopyfindings
Tumor tissue sections were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, progressively dehydrated in graded acetones, and then embedded in epoxy resin. After staining with uranyl acetate and lead citrate, ultrathin sections were analyzed with a transmission electron microscope (Hitachi H-600). In our cases, predominantly fibroblast-like cells with well-developed rough endoplasmic reticulum and some histiocyte-like cells with lyso-somes and Golgi apparatus were observed (Figures 6A and B).
2.6. K-ras mutation analysis
Mutation analysis of the K-ras oncogene (exon 2 codon 12 or 13) was performed using the polymerase chain reaction (PCR)-depen-dent preferential homoduplex formation assay. The K-ras gene was amplified by PCR with a forward primer (huKRAS2 ex2F: 50-GAA TGG TCC TGC ACC AGT AA-30) and reverse primer (huKRAS2 ex2R: 50-GTG TGA CAT GTT CTA ATA TAG TCA-30). All sequencing reactions used ABI standard reagents and protocols for the ABI 3100 auto-mated DNA sequencer. The K-ras mutation analysis failed to demonstrate any single-point mutation in both cases (Figure 7).
3. Discussion
Primary hepatic MFH is a rare entity.3,4It is a disease of adult patients (mean age: 57.6 years) without sex predilection (male:female ratio is 18:16). Most reported cases were solitary lesions, with tumor size ranging from 5.5 to 20 cm (average 12 cm). To the best of our knowledge, the size of the tumor of case 2 (3 2.5 1 cm3) in our present report was the smallest in hepatic MFH. The diagnosis of primary hepatic MFH is often delayed because of nonspecific clinical symptoms.
Figure 4 Microscopic features of hepatic MFH in (A) case 1 and (B) case 2. The storiformepleomorphic growth patterns were similar in both cases (hematoxylin and eosin staining, original magnification in A and B, 100). MFH ¼ malignant fibrous histiocytoma.
Table 1 Primary antibodies used in the two cases of primary hepatic malignant fibrous histiocytoma
Antibody Manufacturer Clone Dilution
Actin DAKO 1A4 1/400
AFP DAKO Polyclonal 1/250
ALK-1 Novocastra Polyclonal 1/500
b-Catenin DAKO Polyclonal 1/200 Bcl-2 Novocastra 3.1 Predilute Calretinin Novocastra 5A5 1/100 CD1a Novocastra MTB-1 Predilute CD21 Cell Marque Polyclonal 1/200
CD23 Biocare 1B12 1/200 CD31 DAKO JC70A 1/200 CD34 DAKO QBEnd10 1/200 CD56 DAKO Polyclonal 1/100 CD68 DAKO PG-M1 Predilute CD117 DAKO C-kit 1/200
CEA Zymed Polyclonal 1/1000
Cytokeratin DAKO Polyclonal 1/400 Cytokeratin 7 DAKO OV-TL 12/30 1/200 Cytokeratin 19 DAKO RCK108 1/200
Desmin DAKO D33 1/100
EGFR Novocastra EGFR.25 1/100 Epithelial membrane antigen DAKO E29 1/200 Estrogen receptor Novocastra 6F11 1/200 Ezrin Epitomics EP886Y 1/250
HMB-45 DAKO HMB-45 1/500
Hep Par-1 DAKO OCH1E5 1/100
Ki-67 DAKO MIB-1 1/100
Progesterone receptor Novocastra 16 1/200 p53 Novocastra DO-7 Predilute S-100 DAKO Polyclonal 1/200 Thyroid transcription factor 1 DAKO 8G7G3/1 1/200
Vimentin DAKO V9 1/400
AFP¼ alpha-fetoprotein; CEA ¼ carcinoembryonic antigen; EGFR ¼ epidermal growth factor receptor.
Most hepatic MFHs demonstrate a well-defined mass with a complex pattern on radiology image studies.10,19Marginal tumor staining without a central feeding artery is a characteristic feature of MFH on hepatic angiography. However, no specific radiologic features are diagnostic of hepatic MFH. The conclusive diagnosis of hepatic MFH relies on histological examination only.
Histopathological section of hepatic MFH was usually well cir-cumscribed, without encapsulation, and with firm consistency. About 32% of tumors presented with local invasion, most commonly into diaphragm and lung. Histologically, the tumor consisted of bundles of spindle cells arranged in storiform and fascicle patterns, intermingled with bizarre and multinucleated giant cells. Most tumor cells expressed vimentin and CD68 IHC stains. The absence of antigens for epithelial tumors of the liver and the lack of expression of CD45 suggest the diagnosis of primary hepatic MFH.25
Primary hepatic MFH is a diagnosis of exclusion. A history of viral hepatitis infection, an elevated serum AFP level, a rich blood supply from hepatic artery, and a frequent portal vein invasion on imaging are the clinical factors that favor the diagnosis of HCC in Taiwan. A previous report showed that cirrhosis has never been
reported in hepatic MFH.33However, liver cirrhosis was found in case 1 of our present report, which was probably related to hepa-titis virus infection. Therefore, the preoperative diagnostic chal-lenge of case 1 was more difficult. Intrahepatic cholangiocarcinoma is the second primary epithelial neoplasm that needs to be included in the differential diagnosis. Cytokeratin 7 IHC stain is useful to exclude intrahepatic cholangiocarcinoma.
HCCs vary architecturally and cytologically. Special variants of HCC includefibrolamellar carcinoma, scirrhous HCC, undifferenti-ated carcinoma, lymphoepithelioma-like carcinoma, and sarco-matoid HCC. Immunohistochemically, HCC is characterized by cytoplasmic positivity with antibodies to carbamoyl phosphate synthetase-1 (HepPar1). Canalicular patterns with IHC staining of polyclonal antibodies to CEA or antibody to CD10 may be seen. Besides, HCC is also positive for AFP and pan-cytokeratin, but usually negative for cytokeratins 19 and 20 and epithelial membrane antigen. Occasionally, HCC is partially or fully comprised of malignant spindle cells, and may be difficult to distinguish from various sarcomas. Some sarcomatoid HCC with undifferentiation may show storiform pattern resembling MFH, especially in case 1 of our recent report. Although in case 1, the tumor was arising from an Figure 5 Ezrin immunoexpressions in tumor cells from (A) case 1 and (B) case 2. Immunohistochemical staining with hematoxylin counterstained was shown (original magni-fication in A and B, 400).
Figure 6 Representative electron microscopic images of the tumor from (A) case 1 and (B) case 2. Fibroblast-like cells with prominent cytoplasmic rough endoplasmic reticulum and histiocyte-like cells with prominent lysosomes were noticed (original magnification in A, 5000; in B, 12,000).
HBV- and HCV-positive cirrhotic liver, we were convinced that this was not a sarcomatoid HCC because we could notfind areas of more typical HCC by extensive gross and histological sampling. In addi-tion, thorough IHC stains (up to 32 antibodies) were performed and the results were not compatible with sarcomatoid HCC.
Metastatic MFH from other sites or invasion from retroperito-neal MFH should be considered. Liver metastases from extrahepatic MFH usually occur until late stages. The primary MFH becomes large and, therefore, the primary location could easily be discov-ered. In our two cases, no tumors were found in any other organs on radioimaging examinations or intraoperative gross examination. The fourth differential diagnosis of primary mesenchymal neoplasms of the liver includes angiosarcoma, dedifferentiated liposarcoma, rhabdomyosarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, and fibrosarcoma. The immu-noexpression of certain specific lineage markers is useful. We could use S100 for malignant peripheral nerve sheath tumor and lip-osarcoma; CD31 and CD34 for angilip-osarcoma; and desmin and actin for skeletal and smooth muscle tumors, respectively. Inflammatory pseudotumor, a rare entity, is thefifth disease that mimics primary hepatic MFH. In microscopic examination, the lack of significant nuclear pleomorphism and the presence of a large number of inflammatory cells throughout the background should help differ-entiate it from primary hepatic MFH.
Many patients survived less than 1 year, which was associated with large tumor size, late clinical stage, and a high ezrin expres-sion score.25,26,28,29Our observation of higher ezrin immunoreac-tivity score associated with poorer prognosis is consistent with previous reports. Akatsu et al32first reported K-ras mutation in pancreatic MFH. However, similar genetic analysis showed no mutation.31In soft tissue MFH, Wilke et al34and Bohle et al35found ras point mutation at codon 12. Our patients carried K-ras wild-type sequences, which clearly differed from the most frequent malig-nant tumor of digestive organs.
In conclusion, primary hepatic MFH has certain characteristic clinicopathologic features that differentiate it from other more common neoplasms in the liver. Ezrin immunoexpression was probably correlated with poorer outcomes in patients with primary hepatic MFH.
Conflict of interest None.
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