Ordóñez, Nelson G. MD
Arginase is a member of the ureohydrolase family of enzymes. It catalyzes the final step of the urea cycle which detoxifies ammonia in mammals. Specifically, arginase hydrolyzes l-arginine to l-ornithine and urea. In mammals, 2 arginase isoenzymes that are encoded by distinct genes and which are designated arginase-1 and arginase-2 exist. They catalyze the same biochemical reaction but differ in cellular expression, regulation, and localization.1,2 Arginase-1 is cytosolic and is primarily expressed in hepatocytes, whereas arginase-2 is expressed as a mitochondrial protein in a variety of tissues, predominantly in the kidney, small intestine, prostate, and lactating mammary gland.2,3 Arginase-1, also known as liver-type arginase and EC188.8.131.52, exists as a homotrimeric protein and contains a binuclear manganese cluster as part of the catalytic site cleft.4 It is comprised of 322 amino acids, has a molecular weight of ∼35 kDa, and is encoded by the ARG1 gene located on chromosome 6q23. This gene consists of 8 exons and spans about 11 kb of genomic DNA. Mutation of the ARG1 gene results in argininemia, an autosomal recessive disease characterized by hyperammonemia. In normal liver, arginase-1 is expressed in hepatocytes, but not in bile duct epithelial cells, Kupffer cells, or vascular endothelial cells.5 Arginase-1 has also been reported to be expressed in neutrophils and macrophages, but not in other normal cells or tissues.5–7
ARGINASE-1 AS A MARKER OF HEPATOCELLULAR DIFFERENTIATION
The histologic distinction of hepatocellular neoplasms, particularly hepatocellular carcinomas, from other tumors involving the liver can be very challenging, especially in needle core biopsies and fine-needle aspiration specimens. Over the past 2 decades, a variety of immunohistochemical markers that can facilitate the differential diagnosis between hepatocellular carcinomas and those neoplasms with which they may be confused have become available, but their sensitivity and/or specificity for hepatic differentiation has, in general, proven to be low. Among these markers is Hep Par 1 (hepatocyte paraffin 1), also known as hepatocyte-specific antigen, a mouse monoclonal antibody that was developed using formalin-fixed tissue from a failed liver allograft as immunogen.8 Because it is highly sensitive for reacting with normal and neoplastic hepatocytes, Hep Par 1 is regarded as being one of the most useful immunohistochemical markers for assisting in the diagnosis of hepatocellular carcinomas. Recent investigations have identified the antigen with which the Hep Par 1 antibody reacts as carbamoyl phosphate synthetase 1, a ligase enzyme located in the mitochondria of normal liver that is involved in the production of urea.9 Because arginase-1, another enzyme involved in the urea cycle, was also found to be expressed in the normal liver with a high degree of specificity,10 Yan et al5 investigated its potential utility as an immunohistochemical marker for hepatic differentiation. In that investigation, which was published in 2010 and which used a commercially obtained anti-arginase-1 polyclonal antibody on tissue microarrays, the authors demonstrated expression in 145 (96%) of 151 hepatocellular carcinomas, as well as in all (100%) hepatic adenomas, focal nodular hyperplasias, and dysplastic nodules investigated. The sensitivities reported for arginase-1 in well, moderately, and poorly differentiated hepatocellular carcinomas were 100%, 96.2%, and 85.7%, respectively, whereas in comparison, Hep Par 1 was demonstrated in 100%, 83%, and 46.4% of these tumors, respectively. Among the nonhepatocellular tumors, arginase-1 positivity was reported in 1 (17%) of 6 intrahepatic cholangiocarcinomas and 1 (7%) of 15 prostatic adenocarcinomas, but in none of the other tumors investigated, including those which can potentially mimic hepatocellular carcinomas (ie, renal cell carcinomas, neuroendocrine carcinomas, gastric adenocarcinomas, adrenal cortical carcinomas, and melanomas). The conclusion of the study by Yan and colleagues was that arginase-1 was a highly sensitive and specific marker of hepatocellular carcinomas, which could be used to distinguish between these tumors and metastatic tumors to the liver. Following this investigation, several other studies on the value of arginase-1 immunostaining in the diagnosis of hepatocellular carcinomas have been published.11–15 The percentage of arginase-1 expression reported in these tumors has ranged from 81% to 96% of the cases.5,11–15 In a combined review of 511 cases from 6 published studies, 466 (91%) were reported to be arginase-1 positive.5,11–15 Arginase-1 has also been reported to be commonly expressed in hepatic adenomas (100%),5,12 as well as in focal nodular hyperplasias, and macroregenerative and dysplastic nodules.5 In Table 1 are summarized the results of published studies on arginase-1 expression in hepatocellular tumors.5,11–15
ARGINASE-1 EXPRESSION IN NONHEPATOCELLULAR TUMORS
Although the number of published studies on the expression of arginase-1 in nonhepatocellular tumors is limited, the results of these investigations indicate that this marker is highly sensitive and specific for hepatocellular differentiation. Among nonhepatocellular tumors, arginase-1 positivity was reported in 3 (20%) of 15 pancreatic adenocarcinoma cases investigated by Fujiwara et al11 using fine-needle aspiration biopsy material and in 1 of 2 cases studied by Radwan and Ahmed13 on whole sections, whereas all 50 such cases investigated by Timek et al14 using tissue microarray were negative for this marker. Individual cases of cholangiocarcinomas5,13 and adenocarcinomas of the breast,11 prostate,5 colon,11 and endometrium11 have also been reported to be positive. In most of these cases, however, the staining was described as being weak and focal. All adenocarcinomas of other sites that were investigated, including those of the lung, thyroid, esophagus, stomach, kidney, and ovary, squamous cell carcinomas of the lung, and neuroendocrine carcinomas, have been arginase-1 negative.5,11–14 In Table 2 are summarized the results of published studies on the expression of arginase-1 in nonhepatocellular tumors.5,11–15
COMPARISON OF ARGINASE-1 WITH COMMONLY USED HEPATOCELLULAR CARCINOMA-ASSOCIATED MARKERS
Hep Par 1 and glypican 3 are currently considered to be the best hepatocellular carcinoma–associated markers that can be used to assist in the diagnosis of these tumors. In normal liver, Hep Par 1 is strongly expressed in hepatocytes, but it is absent in bile duct epithelium. The percentage of Hep Par 1 positivity reported in hepatocellular carcinomas has ranged from 69% to 100% of the cases.15–17 In a combined review of 1646 cases from 27 published studies, 1395 (85%) hepatocellular carcinomas were reported to be Hep Par 1 positive.5,8,11–35 Despite its high sensitivity for hepatocellular carcinomas, only a small percentage (14% to 57%) of poorly differentiated tumors express Hep Par 15,11–14 (Table 3). In a combined review of 56 poorly differentiated hepatocellular carcinomas from 5 published studies, only 21 (37.5%) were reported to be Hep Par 1 positive.5,11–14 Scirrhous hepatocellular carcinomas have also been reported to be frequently negative for Hep Par 1 (>50%).36,37 This is in contrast to arginase-1 in which 34 (61%) of 56 poorly differentiated hepatocellular carcinomas investigated in the 5 previously mentioned studies5,11–14 and 11 (85%) of 13 scirrhous hepatocellular carcinomas15 were reported to express this marker. In addition to hepatocellular tumors, Hep Par 1 expression has been reported in a variety of other neoplasms, most often in adenocarcinomas of the stomach (12% to 47%),5,14,23,27,30,32 esophagus (∼27%),14 colon (4% to 12%),14,27,30,32 pancreas (2% to 10%),14,23,30,32 gallbladder (∼25%),30 and lung (6% to 24%),5,14,23,27,30 cholangiocarcinomas (7% to 30%),23,27,33 and adrenal cortical carcinomas (∼20%).27,30 Adenocarcinomas of the breast, prostate, ovary and salivary gland, renal cell carcinomas, and mesotheliomas are usually negative.11,14,23,26,27,30,32 Yolk sac tumors and carcinomas originating in the gastrointestinal tract and pancreas exhibiting hepatoid morphology have been reported to be frequently positive for Hep Par 1.31,38,39 Because current information indicates that arginase-1 is rarely expressed in nonhepatocellular tumors, this marker, when compared with Hep Par 1, is not only more specific, but it is also more sensitive for hepatocellular differentiation. In a combined review of 6 comparative studies, arginase-1 positivity was demonstrated in 428 (91%) of 471 hepatocellular carcinomas, whereas 359 (75%) of 477 such cases were found to express Hep Par 1 (Table 3).5,11–14
Glypican 3 is one of the most recent hepatocellular carcinoma–associated markers that has been found to be useful for assisting in the diagnosis of these tumors. It is a membrane-bound heparan sulfate proteoglycan that is normally expressed in trophoblasts and a wide variety of fetal tissues, but its expression is limited in normal adult tissues. In a recent immunohistochemical study in which a wide range of normal tissues were investigated, glypican 3 expression was demonstrated only in gastric glands, kidney tubules, and testicular germ cells, but not in any of the other tissues investigated, including the liver.40 Among hepatocellular tumors, glypican 3 is commonly expressed in hepatoblastomas and hepatocellular carcinomas. In a review of 74 hepatoblastomas from 3 published studies, all (100%) of these tumors were reported to be glypican 3 positive.41–43 The percentage of glypican 3 positivity reported in hepatocellular carcinomas has ranged from 39% to 100% of the cases.34,40,44–46 In a combined review of 2431 cases from 23 published series, 1689 (69%) were reported to express glypican 3.11,34,35,40,41,43–60 This marker is not expressed in hepatocellular adenomas,34,41,43,47,49,52–55,60 but it has been reported, although very rarely, in focal nodular hyperplasia.41 In a review of 99 cases of focal nodular hyperplasia from 8 published studies, only 1 (1%) was reported to be focally and weakly positive for glypican 3.34,41,43,47,52,53,55,57 Dysplastic liver nodules, especially those with high-grade dysplasia, have been reported to express glypican 3. In a combined review of 78 liver nodules with low-grade dysplasia from 7 published series and 154 liver nodules with high-grade dysplasia from 9 published series, 5 (6%) and 27 (18%) of the cases, respectively, were found to be glypican 3 positive.40,41,43,47–50,54,58 In all of these cases, however, the staining was described as being focal and/or weak. In addition to hepatocellular carcinomas, glypican 3 has been reported to be expressed in a wide variety of other neoplasms, including all subtypes of ovarian carcinomas, most frequently clear cell carcinomas (17% to 64%),61–63 renal cell carcinomas, including clear cell (2% to 5%), papillary (4% to 26%), and chromophobe (20% to 80%),40,64 adenocarcinomas of the stomach (9% to 20%),40,65 lung (7% to 10%),40,59,66 and breast (∼10% to 20%),11,40 cholangiocarcinomas (2% to 10%),40,43,47 squamous cell carcinomas of the esophagus (25%),67 lung (13% to 55%),59,66,68 and larynx (13%),40 melanomas (∼30%),40,69 and some germ cell tumors, particularly yolk sac tumors (100%)42,52,70–72 and choriocarcinomas (∼85%).42,70,71 When compared with arginase-1, glypican 3 is, in general, less sensitive for hepatocellular carcinomas (∼70% vs. 90%) (Table 4). However, because the latter marker is often absent in benign hepatocellular lesions, it has the advantage over arginase-1 in that it may assist in distinguishing benign from malignant hepatocellular lesions in those instances in which the differential diagnosis is difficult. Since, in contrast to glypican 3, arginase-1 is rarely expressed in nonhepatocellular tumors, its specificity for distinguishing between hepatocellular carcinomas and nonhepatocellular neoplasms is much higher than that of glypican 3.
At present, several anti-arginase-1 antibodies that can be used on formalin-fixed, paraffin-embedded tissue specimens are commercially available. These include several rabbit and goat polyclonal antibodies, as well as the 4E6, CL0186, and 5F7 mouse monoclonal antibodies and the SP156 and EPR6672(B) rabbit monoclonal antibodies. Of these, the rabbit polyclonal antibodies HPA003595 from Sigma-Aldrich5,11,12 and H-52 from Santa Cruz Biotechnology (sc-20150),13 and the EPR6672(B) rabbit monoclonal antibody14 are the ones that have been used most often in published studies on arginase-1 expression in tumors. The staining pattern for arginase-1 is cytoplasmic (Fig. 1).
Because arginase-1 is a highly specific marker of hepatocellular differentiation, immunostaining for this marker can be useful in the differential diagnosis between hepatocellular carcinomas and those malignancies with which they may be confused. When compared with other hepatocellular carcinoma–associated markers that are frequently expressed in hepatocellular carcinomas, arginase-1 has, in general, a higher sensitivity and specificity for these tumors than Hep Par 1 or glypican 3, which are the markers that are at present most often recommended as being useful for assisting in the diagnosis of these tumors. Since arginase-1 is expressed in both hepatocellular carcinomas and benign hepatocellular lesions, including hepatocellular adenomas, focal nodular hyperplasias, and dysplastic nodules, immunostaining for this marker has no utility for distinguishing between these lesions.
The author thanks Janet Quiñones for technical assistance and Kim-Anh Vu for assistance with digital images.
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