Comparison of 5 Immunohistochemical Markers of Hepatocellular Differentiation for the Diagnosis of Hepatocellular Carcinoma

Comparison of 5 Immunohistochemical Markers of Hepatocellular Differentiation for the Diagnosis of Hepatocellular Carcinoma Thuy Nguyen, MD; Daniel Phillips, MD; Dhanpat Jain, MD; Michael Torbenson, MD; Tsung-Teh Wu, MD, PhD; Matthew M. Yeh, MD, PhD; Sanjay Kakar, MD Context. Several immunohistochemical markers are available to establish the diagnosis of hepatocellular carcinoma. Judicious selection is essential to achieve a reliable diagnosis in limited tissue provided by liver biopsy. Objective. To compare the efficacy of 5 hepatocellular markers for the diagnosis of hepatocellular carcinoma across various levels of differentiations. Design. Immunohistochemistry for hepatocyte paraffin antigen 1 (Hep Par 1), polyclonal carcinoembryonic antigen (CEA), glypican-3, arginase-1, and bile salt export pump transporter was performed in 79 hepatocellular carcinomas, yielding 93 observations (13 well-differentiated [14%], 41 moderately differentiated [44%], and 39 poorly differentiated [42%] tumors). Results. Arginase-1 and Hep Par 1 had the highest sensitivity for well-differentiated hepatocellular carcinoma, whereas arginase-1 and glypican-3 had the highest sensitivity for poorly differentiated hepatocellular carcinoma. When staining of more than 50% of the tumor was considered a positive result, arginase-1 remained the most sensitive marker for all differentiations, whereas sensitivity for Hep Par 1 in poorly differentiated hepatocellular carcinoma dropped to 30% and that of glypican-3 in well-differentiated hepatocellular carcinoma was 15%. The addition of Hep Par 1 and/or polyclonal CEA to arginase-1 did not lead to an increase in sensitivity for any differentiation. The combined use of arginase-1 and glypican-3 yielded 100% sensitivity for poorly differentiated hepatocellular carcinoma. Conclusion. Arginase-1 was the most sensitive marker in all differentiations of hepatocellular carcinoma. Glypican-3 had high sensitivity for poorly differentiated cases and its combined use with arginase-1 enabled identification of nearly all cases of poorly differentiated hepatocellular carcinoma. Although bile salt export pump transporter has good overall sensitivity, it has a limited role in establishing hepatocellular differentiation when added to a panel of arginase-1 with either glypican-3 or Hep Par 1. (Arch Pathol Lab Med. 2015;139:1028 1034; doi: 10.5858/arpa.2014-0479-OA) The diagnosis of hepatocellular carcinoma (HCC) and its differentiation from metastatic or nonhepatocellular tumors has important therapeutic implications. This distinction can be challenging if the tumor is poorly differentiated and when limited amounts of tumor are available from needle biopsies. Many immunohistochemical markers are available for establishing the diagnosis, each with its strength and limitations. Hepatocyte paraffin Accepted for publication November 26, 2014. From the Department of Anatomic Pathology, University of California, San Francisco, (Drs Nguyen, Phillips, and Kakar); the Department of Pathology, Yale University School of Medicine, New Haven, Connecticut (Dr Jain); the Department of Pathology, Johns Hopkins, Baltimore, Maryland (Dr Torbenson); the Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota (Dr Wu); the Department of Pathology, University of Washington, Seattle, (Dr Yeh); and the Department of Anatomic Pathology, Veteran Affairs Medical Center, San Francisco (Dr Kakar). The authors have no relevant financial interest in the products or companies described in this article. Reprints: Sanjay Kakar, MD, Department of Anatomic Pathology, University of California, San Francisco, 4150 Clement St, San Francisco, CA 94121 (e-mail: sanjay.kakar@ucsf.edu). antigen 1 (Hep Par 1), which recognizes the urea cycle enzyme carbamoyl phosphate synthetase, 1 and polyclonal antibody directed against carcinoembryonic antigen (p- CEA) are among the most commonly used hepatocellular markers, but they have less than 50% sensitivity for poorly differentiated HCC. 2 5 In addition, Hep Par 1 can be positive in gastric, lung, and esophageal adenocarcinomas 3 and, less commonly, in a wide variety of tumors. 3,4 The canalicular pattern observed with p-cea is considered to be specific for hepatocellular differentiation, but can be difficult to distinguish from membranous or luminal staining patterns seen in adenocarcinomas. 4,6 Other markers, such as CD10 and villin, also yield a canalicular pattern of staining, but suffer from the same limitations. 6,7 Serum levels of a- fetoprotein are routinely used in screening for HCC; however, immunohistochemical staining for a-fetoprotein has 30% to 50% sensitivity and often shows background staining. 3,6,7 In the past few years, glypican-3 (GPC-3) and arginase-1 (Arg-1) have emerged as promising markers of hepatocellular differentiation. The GPC-3 marker is a membraneanchored, heparin-sulfate proteoglycan normally expressed in fetal liver and placenta. Its advantages include negative 1028 Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al

Table 1. results in nonneoplastic liver and high sensitivity in poorly differentiated HCC. 8 11 Arginase-1 is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of arginine to ornithine and urea. Initial studies have shown that Arg-1 is a highly sensitive and specific marker for hepatocellular differentiation. 12 14 Bile salt export pump (BSEP) is a liver-specific ATPbinding cassette transporter encoded by the ABCB11 gene, which is expressed exclusively in the liver canalicular membrane and involved with bile acid transport. 15 The BSEP marker has been shown to be specific and sensitive for hepatocellular differentiation, but its diagnostic utility in HCC has not been explored in detail. 16 The choice of hepatocellular markers used for diagnosis of HCC in liver biopsies varies widely. The choice of the markers requires careful evaluation of the clinical setting and pathologic features, generation of an appropriate differential diagnosis, and framing specific questions to be answered by application of immunohistochemical markers. Indiscriminate or inappropriate use of markers can exhaust the tissue in the block, precluding a definite diagnosis. This study attempted to identify an optimum combination of hepatocellular markers by comparing the sensitivity of 5 immunohistochemical markers (Hep Par 1, p-cea, GPC-3, Arg-1, and BSEP) in well-differentiated, moderately differentiated, and poorly differentiated HCC. MATERIALS AND METHODS The study population comprised 79 cases of HCC from the surgical pathology files of the University of California, San Francisco, Medical Center, the University of Washington Medical Center (Seattle), the Yale-New Haven Hospital (New Haven, Connecticut), the Mayo Clinic (Rochester, Minnesota), and the Johns Hopkins School of Medicine (Baltimore, Maryland). The slides were reviewed to confirm the diagnosis in all cases, and the level of differentiation was assessed based on the 2010 World Health Organization criteria. 17 Well-differentiated HCC showed obvious hepatocellular differentiation with mild cytologic atypia, mildly increased nuclear to cytoplasmic ratios, thin trabeculae (3 cells thick) in most of the tumor, and a frequent pseudoglandular pattern. Moderately differentiated HCC also showed morphologic resemblance to hepatocytes with moderate cytologic atypia and/or thick cell plates (.3 cells thick in most of the tumor). Poorly differentiated HCC showed one or more of the following: little or no resemblance to hepatocytes, marked cytologic atypia, a solid growth pattern, or markedly increased nuclear to cytoplasmic ratios. Formalin-fixed, paraffin-embedded tissue was used to perform immunohistochemistry for Hep Par 1, p-cea, GPC-3, Arg-1, and BSEP (Table 1). The incubation time was 15 minutes for the primary antibody. For Hep Par 1 and pcea, heat-induced, epitoperetrieval pretreatment was performed in a Dako (Carpinteria, California) Pascal pressure cooker for 30 to 45 minutes at 1258C until reaching pressure of 22 to 25 psi in 0.01M citrate buffer (ph 6.0). The slides were then cooled to room temperature, rinsed, and quenched with 3% hydrogen peroxide for 10 minutes. This was followed by incubation with the primary antibody at room Antibodies Used for Immunohistochemistry in the Study Antibody Clone Vendor Dilution Hep Par 1 OCH1E5 Dako, Carpinteria, California 1:200 Polyclonal CEA Polyclonal Dako, Carpinteria, California 1:20 Glypican-3 1G12 BioMosaics, Burlington, Vermont 1:2 Arginase-1 Rabbit polyclonal Sigma-Aldrich, St Louis, Missouri 1:400 BSEP Mouse monoclonal F-6 Santa Cruz Biotechnology, Dallas, Texas 1:200 Abbreviations: BSEP, bile salt export pump; CEA, carcinoembryonic antigen; Hep Par-1, hepatocyte paraffin antigen 1. temperature (Hep Par 1, 30 minutes; pcea, 15 minutes). For detection, a biotin-free, horseradish peroxidase labeled, dextrosebased polymer complex bound to a secondary antibody (K4061, Dako EnVision Plus dual link system-hrp) was used with an incubation time of 15 minutes. Slides were then developed for 10 minutes with 3-3 0 -diaminobenzidine chromogen (DAB þ, K3468, Dako), and counterstained with Gill hematoxylin. For GPC-3 and Arg-1 immunohistochemistry, conventional tissue sections were subjected to heat-induced, epitope-retrieval pretreatment for 20 minutes at 1008C in epitope retrieval solution 1 (ph 6.0; AR9961, Leica Microsystems, Newcastle, United Kingdom), and cooled to room temperature. The Leica Bond Polymer Refine detection kit (DS9800, Leica Microsystems) was used for the blocking and detection steps. The slides were incubated with the primary antibody for 15 minutes, incubated in postprimary reagent (rabbit anti-mouse immunoglobulin G [IgG]) for 8 minutes, followed by incubation in polymer reagent (anti-rabbit poly- HRP-IgG) for 8 minutes. For the blocking step, tissue sections were quenched with peroxide block (3% 4% hydrogen peroxide) for 10 minutes. Slides were then developed for 10 minutes with DAB chromogen, and counterstained with hematoxylin for 1 minute. Immunohistochemistry for BSEP was performed using a monoclonal mouse anti-human antibody BSEP clone F-6 (Santa Cruz Biotechnology, Santa Cruz, California) at 1:200 dilution. The entire process was performed on a Leica BOND III platform, with antigen retrieval using heat-induced epitope retrieval 2. The antibody incubation time was 15 minutes. Slides were then developed for 5 minutes with DAB chromogen. The staining intensity was recorded as 0 to 3 (negative, mild, moderate, and strong), and the extent of staining was recorded into 3 groups based on percentage of tumor showing positive staining (,5%, 5% 50%, and.50%). Cytoplasmic staining was considered positive for Hep Par 1 and GPC-3, whereas nuclear and/or cytoplasmic staining was considered positive for Arg-1. For pcea and BSEP, a canalicular staining pattern was considered positive (Figure 1, A and B); membranous or luminal positivity without canalicular staining was considered negative. For data analysis, cases were considered positive if moderate or strong staining was seen in 5% or more of the tumor cells. All other situations were considered negative. The positive cases were divided into focal positive if moderate or strong staining was seen in 5% to 50% of the tumor and diffuse positive if moderate or strong staining was seen in more than 50% of the tumor cells. The 50% cutoff is not necessarily relevant from a diagnostic standpoint, but was chosen to compare the extent of staining with different markers. The sensitivity for each marker as well as combination of markers for well differentiated, moderately differentiated, and poorly differentiated HCC was determined. RESULTS Clinical Data Seventy-nine cases were examined. The age range was 31 to 83 years (62 men [78%], 17 women [22%]). Cirrhosis was present in 58 (73%) of the cases, whereas 17 HCC cases (22%) arose in noncirrhotic liver (status not known in 4 cases [5%]). Of the 58 cases with cirrhosis, the etiology of the cirrhosis was known in 50 cases (86%): hepatitis C in 35 cases (60%), hepatitis B in 7 cases (12%), hepatitis B and C Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al 1029

Figure 1. A, Well-differentiated hepatocellular carcinoma. B, Canalicular pattern of staining with bile salt export pump (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]). coinfection in 2 cases (3%), alcohol use in 4 cases (7%), hepatitis C and alcohol use in 1 case (2%), and steatohepatitis in 1 case (2%). Immunohistochemistry Of the 79 HCC cases, 14 (18%) showed more than one differentiation pattern; these areas were scored separately yielding 93 observations (13 well differentiated cases [14%], 41 moderately differentiated cases [44%], and 39 poorly differentiated cases [42%]). The results for all antibodies were categorized into focal and diffuse positive cases (Tables 2 and 3, Figures 2, A through E; 3, A through D; and 4, A through D). The Hep Par 1 staining was positive in all welldifferentiated cases (13 of 13; 100%) and most moderately differentiated cases (40 of 41; 98%) (Figures 2, A and C; and 3, A and D), whereas sensitivity in poorly differentiated cases was modest (25 of 39; 64%). Similarly, pcea showed canalicular positivity in most well-differentiated cases (12 of 13; 92%) (Figure 2, E) and most moderately differentiated cases (36 of 41; 88%), but the sensitivity was lower in poorly differentiated HCC (21 of 39; 54%). For both Hep Par 1 and pcea, the numbers were slightly lower for well and moderately differentiated areas when only diffuse positive staining was considered, but fell to below one-third of cases for poorly differentiated areas (Table 3). The Arg-1 staining was positive in all well-differentiated cases (13 of 13; 100%), all moderately differentiated cases (41 of 41; 100%), and most poorly differentiated cases (38 of 39; 97%) (Figures 2, A and B; 3, A and B; and 4, A and B). All positive cases demonstrated cytoplasmic staining, with nuclear positivity also seen in most instances (65 of 79; 82%). The results were similar when only diffusely positive cases were considered (Table 3). The GPC-3 staining had a modest sensitivity in well-differentiated cases (8 of 13; 62%) and a high sensitivity for moderately differentiated cases (33 of 41; 80%) and poorly differentiated HCC (33 of 39; 85%) (Figures 2, E; 3, C; and 4, C). When cases with diffuse staining were considered positive, the results were similar for poorly differentiated HCC, but lower for well and moderately differentiated HCC. Although BSEP results were not available in every case, it yielded 83 observations. It showed canalicular positivity in most well-differentiated cases (12 of 13; 92%) and moderately differentiated cases (37 of 39; 95%) (Figure 1, B), whereas sensitivity was low for poorly differentiated HCC cases (14 of 31; 45%) (Figure 4, D). The numbers were lower for all 3 categories when only cases with diffuse staining were considered positive. Membrane staining with BSEP was seen in 8 of 83 HCCs (10%), which partially obscured the canalicular staining pattern. When a combination of GPC-3 and Arg-1 was evaluated, at least one marker was positive in all poorly differentiated HCCs (Figures 3 and 4). Even when only diffusely positive cases were considered, GPC-3 and/or Arg-1 were positive in 37 of 39 cases (95%) of poorly differentiated HCC (Table 4). The Hep Par 1 marker performed better than p-cea and BSEP in well-differentiated and moderately differentiated cases. However, adding Hep Par 1 to Arg-1 did not improve the sensitivity because Arg-1 was positive in all cases. Hence, with the exception of GPC, addition of other markers to Arg-1 did not improve sensitivity at any differentiation. COMMENT Many tumors can closely mimic HCC, including poorly differentiated cholangiocarcinoma; metastatic, poorly differentiated adenocarcinomas; nonhepatocellular tumors, such as epithelioid angiomyolipoma; and other metastatic tu- Table 2. Immunohistochemical Markers in Hepatocellular Carcinoma With 5% a Staining Considered Positive Differentiation Arginase-1, No. (%) Glypican-3, No. (%) Hep Par-1, No. (%) pcea, No. (%) BSEP, No. (%) Well, n ¼ 13 8 (62) 12 (92) 12 (92) Moderately, n ¼ 41 33 (80) 40 (98) 36 (88) 37 (95) b Poorly, n ¼ 39 38 (97) 33 (85) 25 (64) 21 (54) 14 (45) c Abbreviations: BSEP, bile salt export pump; Hep Par-1, hepatocyte paraffin antigen 1; pcea, polyclonal carcinoembryonic antigen. a Staining in 5% or more of the tumor cells was considered positive. b BSEP results were not available in every case; n ¼ 39. c BSEP results were not available in every case; n ¼ 31. 1030 Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al

Table 3. Immunohistochemical Markers in Hepatocellular Carcinoma With 50%a Staining Considered Positive Differentiation Arginase-1, No. (%) Glypican-3, No. (%) Hep Par-1, No. (%) pcea, No. (%) BSEP, No. (%) Well, n ¼ 13 Moderately, n ¼ 41 Poorly, n ¼ 35 40 (98) 34 (88) 2 (15) 24 (58) 29 (74) 34 (83) 12 (30) 10 (77) 26 (63) 7 (18) 9 (69) 28 (72)b 6 (6)c Abbreviations: BSEP, bile salt export pump; Hep Par-1, hepatocyte paraffin antigen 1; pcea, polyclonal carcinoembryonic antigen. a Staining in 50% or more of the tumor cells was considered positive. b BSEP results were not available in every case; n ¼ 39. c BSEP results were not available in every case; n ¼ 31. Figure 2. Well-differentiated hepatocellular carcinoma (A) showing cytoplasmic staining with arginase-1 (B) and hepatocyte paraffin antigen 1 (C). D, Canalicular pattern of staining with bile salt export pump. E, Staining for glypican-3 is negative (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]; original magnification 3200 [C]; original magnification 3200 [D]; original magnification 3200 [E]). Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al 1031

Figure 3. Moderately differentiated hepatocellular carcinoma (A) showing cytoplasmic staining with arginase-1 (B) and glypican-3 (C), whereas hepatocyte paraffin antigen 1 (Hep Par 1) is negative (D). This was the only moderately differentiated case that was negative for Hep Par 1. Staining for bile salt export pump and polyclonal carcinoembryonic antigen was also negative (not shown) (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]; original magnification 3200 [C]; original magnification 3200 [D]). mors, such as neuroendocrine tumors, renal cell carcinoma, adrenocortical carcinoma, and melanoma. A large armamentarium of immunohistochemical markers is available to help in establishing hepatocellular differentiation in tumors. As a limited amount of tissue is available in needle biopsies, judicious evidence-based use of these markers is imperative. Our results show that Arg-1 is the most sensitive marker across all differentiations of HCC. Several studies have shown the utility of Arg-1 in HCC. Similar to our finding of 98% sensitivity, figures ranging from 84% to 96% have been reported.13,14,18 The high sensitivity of Arg-1 (81% 84%) has also been observed in cytology material.19,20 The high sensitivity of Arg-1 was also observed in poorly differentiated HCC (95% in our study, which is similar to the 90% reported by Yan et al13) as well as in scirrhous HCC.21 In addition, Arg-1 was expressed in all well and moderately differentiated cases, making it an excellent hepatocellular marker for both ends of the differentiation spectrum. In most cases that were Arg-1þ, the staining was diffuse, indicating that this marker will yield similarly high sensitivity in biopsies. The high sensitivity of Arg-1 in a microarray study also indicates that the staining pattern is diffuse in most cases and will have high utility in liver biopsies.22 Arginase-1 is a component of the urea cycle and is found only in the liver. Although no nonhepatocellular 1032 Arch Pathol Lab Med Vol 139, August 2015 tumors were examined in this study, high specificity (.95%) has been reported.13,18 Rare cases of positive staining in adenocarcinomas of the prostate, pancreas, colon, breast, and biliary tree have been reported.13,19 The only exception is a study by Iida et al,23 which reported Arg-1 positivity in 78% of intrahepatic cholangiocarcinomas. However, that result has not been reproduced in other studies and is not supported by our anecdotal experience. Although GPC-3 emerged as a promising hepatocellular marker a few years ago, further experience has shown that it can be expressed in a wide variety of other tumors.10 Even though GPC-3 is not a specific hepatocellular marker, it can be useful because of its high sensitivity in poorly differentiated HCC.9,11 Among the tumors that can resemble HCC, GPC-3 can be rarely positive in cholangiocarcinoma.10,11,21,24 In the present study, GPC-3 was positive in 85% (33 of 39) of poorly differentiated HCC, which is similar to 85% and 89% reported in previous studies.9,11 Use of GPC-3 has also been advocated for distinguishing well-differentiated HCC from hepatocellular adenoma.25 27 Based on our results, the utility of GPC-3 in this setting is limited because it is positive in only 62% (8 of 13) of well-differentiated HCC.28 Even among positive cases, the diffuse staining was seen in only 15% (2 of 13) of the cases, further confirming its limited utility in biopsies. Comparison of 5 Immunohistochemical Markers Nguyen et al

Figure 4. Poorly differentiated hepatocellular carcinoma (A) showing cytoplasmic staining with arginase-1 (B) and glypican-3 (C), whereas bile salt export pump was negative (D). Staining for hepatocyte paraffin antigen 1 and polyclonal carcinoembryonic antigen was also negative (not shown) (hematoxylin-eosin, original magnification 3200 [A]; original magnification 3200 [B]; original magnification 3200 [C]; original magnification 3200 [D]). As previously reported,2 7 Hep Par 1 and pcea are widely used in the diagnosis of HCC, and high sensitivity and specificity has been reported. However, both these markers have poor sensitivity for poorly differentiated and scirrhous HCC.5,9,13,21 Our study also shows similar findings, with sensitivity of Hep Par 1 and pcea for poorly differentiated HCC being 64% (25 of 39) and 54% (21 of 39), respectively. Even among the positive cases, the staining was focal in nearly one-half of the cases, further reducing their utility in biopsies. In addition, Hep Par 1 staining is common in esophageal, gastric, and lung adenocarcinomas2,3 and rare in neuroendocrine, biliary, and other tumors.7,29 Because BSEP is localized to the canalicular membrane, it is an attractive option for establishing hepatocellular differentiation. A preliminary study16 showed that BSEP Table 4. had 100% specificity and 90% sensitivity for HCC, and its use was advocated for the diagnosis of HCC. However, that was a small series, and it is not clear whether any poorly differentiated HCCs were included. In our study, BSEP had high sensitivity for well-differentiated and moderately differentiated HCC, but fell to less than one-half for poorly differentiated cases. If only diffusely positive cases were considered, the sensitivity in poorly differentiated HCC fell even further to 6% (2 of 31). In view of the availability of better markers, such as Arg-1 and GPC-3, for poorly differentiated HCC, and better or equally good markers, such as Hep Par 1 and Arg-1, for moderately differentiated and well-differentiated HCC, our results suggest that the addition of BSEP is not useful for diagnosis. Sensitivity of Different Combinations of Immunohistochemical Markers in Hepatocellular Carcinoma Differentiation Tumor cells staining, % Well, n ¼ 13 Moderately, n ¼ 41 Poorly, n ¼ 39 Hep Par-1þ and/or Glypican-3þ, No. (%) 5 36 (97) 50 40 (98) 34 (87) Hep Par-1þ and/or Arginase-1þ, No. (%) 5 37 (97) 50 40 (98) 34 (88) Glypican-3þ and/or Arginase-1þ, No. (%) 5 39 (100) 50 37 (95) Abbreviation: Hep Par-1, hepatocyte paraffin antigen 1. Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al 1033

Our study indicates that different combinations of hepatocellular markers can be considered based on the differentiation of the tumor. If the morphologic differential diagnosis includes well differentiated or moderately differentiated HCC, Arg-1 is likely to identify most cases. Because Arg-1 can show focal staining in rare cases, and its sensitivity and specificity may decrease with further experience, it is prudent to employ a panel of markers in these situations. If poorly differentiated HCC is a morphologic consideration, a combination of Arg-1 and GPC-3 yields the highest sensitivity with positive staining of at least one marker in all the cases. Even when cases with staining of 50% of cells are considered positive, this combination identifies 95% of poorly differentiated HCCs, indicating that it will be a robust combination in needle biopsies. Along with the data presented in this study, each laboratory should take its practice environment and technical performance of stains into consideration when choosing an optimal combination of markers for specific clinical situations. We thank the University of California, San Francisco, Liver Center for their help in this study (NIH P30 DK026743). References 1. Butler SL, Dong H, Cardona D, et al. The antigen for Hep Par 1 antibody is the urea cycle enzyme carbamoyl phosphate synthetase 1. Lab Invest. 2008; 88(1):78 88. 2. Fan Z, van de Rijn M, Montgomery K, Rouse RV. Hep Par 1 antibody stain for the differential diagnosis of hepatocellular carcinoma: 676 tumors tested using tissue microarrays and conventional tissue sections. Mod Pathol. 2003;16(2): 137 144. 3. Kakar S, Muir T, Murphy LM, Lloyd RV, Burgart LJ. Immunoreactivity of Hep Par 1 in hepatic and extrahepatic tumors and its correlation with albumin in situ hybridization in hepatocellular carcinoma. Am J Clin Pathol. 2003;119(3): 361 366. 4. Morrison C, Marsh W Jr, Frankel WL. A comparison of CD10 to pcea, MOC-31 and hepatocyte for the distinction of malignant tumors in the liver. Mod Pathol. 2002;15(12):1279 1287. 5. Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med. 2007;131(11):1648 1654. 6. Lau SK, Prakash S, Geller SA, Alsabeh R. Comparative immunohistochemical profile of hepatocellular carcinoma, cholangiocarcinoma, and metastatic adenocarcinoma. Hum Pathol. 2002;33(12):1175 1181. 7. Chu PG, Ishizawa S, Wu E, Weiss LM. Hepatocyte antigen as a marker of hepatocellular carcinoma: an immunohistochemical comparison to carcinoembryonic antigen, CD10, and alpha-fetoprotein. Am J Surg Pathol. 2002;26(8): 978 988. 8. Yamauchi N, Watanabe A, Hishinuma M, et al. The glypican 3 oncofetal protein is a promising diagnostic marker for hepatocellular carcinoma. Mod Pathol. 2005;18(12):1591 1598. 9. Shafizadeh N, Ferrell LD, Kakar S. Utility and limitations of glypican-3 expression for the diagnosis of hepatocellular carcinoma at both ends of the differentiation spectrum. Mod Pathol. 2008;21(8):1011 1018. 10. Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM. Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol. 2008;129(6):899 906. 11. Shirakawa H, Kuronuma T, Nishimura Y, et al. Glypican-3 is a useful diagnostic marker for a component of hepatocellular carcinoma in human liver cancer. Int J Oncol. 2009;34(3):649 656. 12. Multhaupt H, Fritz P, Schumacher K. Immunohistochemical localization of arginase in human liver using monoclonal antibodies against human liver arginase. Histochemistry. 1987;87(5):465 470. 13. Yan BC, Gong C, Song J, et al. Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol. 2010; 34(8):1147 1154. 14. Radwan NA, Ahmed NS. The diagnostic value of arginase-1 immunostaining in differentiating hepatocellular carcinoma from metastatic carcinoma and cholangiocarcinoma as compared to HepPar-1. Diagn Pathol. 2012;7:149. doi: 10.1186/1746-1596-7-149. 15. Strautnieks SS, Bull LN, Knisely AS, et al. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat Genet. 1998;20(3):233 238. 16. SM Lagana, H Remotti, RK Moreira. Bile salt export pump (BSEP): a sensitive and specific marker of hepatocytic differentiation in liver tumors [abstract]. Mod Pathol. 2012;25:417A. 17. Bosman FT, Carneiro F, Hruban RH, Theise ND. WHO Classification of Tumours of the Digestive System. 4th ed. Lyon, France: IARC Press; 2010. World Health Organization Classification of Tumours; vol 3. 18. Timek DT, Shi J, Liu H, Lin F. Arginase-1, HepPar-1, and glypican-3 are the most effective panel of markers in distinguishing hepatocellular carcinoma from metastatic tumor on fine-needle aspiration specimens. Am J Clin Pathol. 2012; 138(2):203 210. 19. Fujiwara M, Kwok S, Yano H, Pai RK. Arginase-1 is a more sensitive marker of hepatic differentiation than HepPar-1 and glypican-3 in fine-needle aspiration biopsies. Cancer Cytopathol. 2012;120:230 237. 20. McKnight R, Nassar A, Cohen C, Siddiqui MT. Arginase-1: a novel immunohistochemical marker of hepatocellular differentiation in fine needle aspiration cytology. Cancer Cytopathol. 2012;120(4):223 229. 21. Krings G, Ramachandran R, Jain D, et al. Immunohistochemical pitfalls and the importance of glypican 3 and arginase in the diagnosis of scirrhous hepatocellular carcinoma. Mod Pathol. 2013;26(6):782 791. 22. Ramachandran R BL MI, Chen Y, Kakar S. Evidence-based immunohistochemical panel for the distinction of hepatocellular carcinoma and metastatic carcinoma [abstract]. Mod Pathol. 2010;23:370A. 23. Iida H, Hata M, Kakuno A, Hirano H, et al. Expression of hepatocyte markers in mass-forming peripheral and periductal-infiltrating hilar intrahepatic cholangiocarcinomas. Oncol Lett. 2011;2(6):1041 1046. 24. Wang FH, Yip YC, Zhang M, et al. Diagnostic utility of glypican-3 for hepatocellular carcinoma on liver needle biopsy. J Clin Pathol. 2010;63(7):599 603. 25. Nassar A, Cohen C, Siddiqui MT. Utility of glypican-3 and survivin in differentiating hepatocellular carcinoma from benign and preneoplastic hepatic lesions and metastatic carcinomas in liver fine-needle aspiration biopsies. Diagn Cytopathol. 2009;37(9):629 635. 26. Wang HL, Anatelli F, Zhai QJ, Adley B, Chuang ST, Yang XJ. Glypican-3 as a useful diagnostic marker that distinguishes hepatocellular carcinoma from benign hepatocellular mass lesions. Arch Pathol Lab Med. 2008;132(11):1723 1728. 27. Lagana SM, Salomao M, Bao F, Moreira RK, Lefkowitch JH, Remotti HE. Utility of an immunohistochemical panel consisting of glypican-3, heat-shock protein-70, and glutamine synthetase in the distinction of low-grade hepatocellular carcinoma from hepatocellular adenoma. Appl Immunohistochem Mol Morphol. 2013;21(2):170 176. 28. Shafizadeh N, Kakar S. Diagnosis of well-differentiated hepatocellular lesions: role of immunohistochemistry and other ancillary techniques. Adv Anat Pathol. 2011;18(6):438 445. 29. Minervini MI, Demetris AJ, Lee RG, Carr BI, Madariaga J, Nalesnik MA. Utilization of hepatocyte-specific antibody in the immunocytochemical evaluation of liver tumors. Mod Pathol. 1997;10(7):686 692. 1034 Arch Pathol Lab Med Vol 139, August 2015 Comparison of 5 Immunohistochemical Markers Nguyen et al