Immunoreactivity of Hep Par 1 in Hepatic and Extrahepatic Tumors and Its Correlation With Albumin In Situ Hybridization in Hepatocellular Carcinoma
Sanjay Kakar, MD, Trudie Muir, MD, Linda M. Murphy, HT(ASCP), Ricardo V. Lloyd, MD, PhD, and Lawrence J. Burgart, MD
Key Words: Hep Par 1; Immunohistochemistry; Albumin; In situ hybridization; Hepatocellular carcinoma; Adenocarcinoma; Cholangiocarcinoma
DOI: 10.1309/8L872RPHEJRKF5JJ
Abstract
We evaluated the expression of Hep Par 1 (hepatocyte paraffin 1 monoclonal antibody) in 42 hepatocellular carcinomas (HCCs), 25 cholangiocarcinomas, 18 tumors metastatic to the liver, and 87 primary extrahepatic tumors. Albumin in situ hybridization (ISH) was performed in the HCC cases. Of 42 cases of HCC, 39 (93%) were positive for Hep Par1. All cases of cholangiocarcinoma, renal cell carcinoma, adrenocortical carcinoma, and islet cell tumors were negative; 1 case each of primary urinary bladder (n = 10) and pancreatic (n = 10) adenocarcinoma, and 3 of 11 cases of primary pulmonary adenocarcinoma showed focal positivity; 7 of 10 gastric and 6 of 8 esophageal adenocarcinomas were strongly positive. Albumin ISH was positive in 39 (93%) HCC cases. All cases of HCC were positive for Hep Par 1 or albumin ISH. Hep Par 1 immunoreactivity has high sensitivity in the diagnosis of HCC. Strong positive staining also occurs in gastroesophageal adenocarcinomas. Cholangiocarcinoma and carcinomas from most other sites are negative for Hep Par 1. Hep Par 1 immunoreactivity shows high correlation with albumin ISH; their combined use for diagnosis of HCC had a sensitivity of 100% in this population.
It can be difficult to distinguish hepatocellular carcinoma (HCC) from cholangiocarcinoma and metastatic tumors by light microscopy alone. Metastatic tumors, like renal cell carci- noma, adrenocortical tumors, carcinoid, malignant melanoma, and poorly differentiated adenocarcinoma, can closely mimic HCC and pose diagnostic problems. Several immunohisto- chemical markers such as polyclonal carcinoembryonic antigen, various cytokeratins, and alpha fetoprotein have been used for this distinction. However, these immunostains have limitations in terms of specificity and sensitivity. The sensi- tivity ranges from 17% to 61.5% for alpha fetoprotein1-3 and from 47% to 71% for polyclonal carcinoembryonic antigen.1,4,5
Detection of albumin messenger RNA (mRNA) by in situ hybridization (ISH) in neoplastic and nonneoplastic tissues is a useful method for demonstrating hepatocellular differentiation as albumin is synthesized only in the hepato- cytes.6,7 This method has high sensitivity (93%-96%) and specificity (close to 100%), but can be positive in extrahep- atic germ cell tumors with hepatoid features7 and rare cases of clear cell carcinoma of the ovary.8
Wennerberg et al9 developed a new monoclonal antibody designated hepatocyte paraffin 1 (Hep Par 1) using tissue from a formalin-fixed failed allograft liver. Initial reports have shown that immunohistochemical analysis using the Hep Par 1 antibody may be a highly sensitive and specific marker of hepatocellular differentiation.1,9-11 However, the staining characteristics of Hep Par 1 in a large variety of neoplasms have not been well studied.
The aim of the present study was to evaluate the expres- sion of Hep Par 1 in hepatic and extrahepatic tumors and to correlate the results of Hep Par1 with albumin in situ hybridization in HCCs.
Materials and Methods
Cases
The study cases were selected based on a search in the Mayo Clinic pathology database from January 1998 through December 2001. The study was approved by the institutional review board at Mayo Clinic, Rochester, MN.
Primary Hepatic Tumors
We studied 42 cases of hepatocellular carcinoma (40 resections, 2 biopsies) and 25 cases of resected cholangio- carcinoma. The HCC cases included clear cell (n = 7) and fibrolamellar (n = 9) variants. The remaining 26 were conventional HCC. The HCC cases were graded from I through IV based on Edmondson-Steiner grading scheme.12 Grades I and II were regarded as low grade, while grades III and IV were regarded as high grade. Of the 42 HCC cases, 6 cases were categorized as high grade (grade III) and the remaining 36 as low grade (grades I and II). There were no grade IV tumors. All high-grade tumors had conventional histologic features.
Metastatic Hepatic Tumors
This group included 18 metastatic tumors to the liver: renal cell carcinoma, 2; pancreatic carcinoma, 1; pulmonary adenocarcinoma, 1; melanoma, 1; endometrial carcinoma, 1; urinary bladder adenocarcinoma, 2; ovarian adenocarcinoma, 1; neuroendocrine tumors, 6 (islet cell tumor, carcinoid, and neuroendocrine carcinoma, 2 each), breast ductal adenocar- cinoma, 1; adrenocortical carcinoma, 1; and epithelioid sarcoma, 1.
Other Primary Tumors
This group included 87 primary extrahepatic tumors (renal cell carcinoma, 10; adrenocortical carcinoma, 8; urinary bladder adenocarcinoma, 10; pancreatic islet cell tumors, 10; pancreatic adenocarcinoma, 10; colonic adeno- carcinoma, 10; gastric adenocarcinoma, 10; esophageal adenocarcinoma, 8; and pulmonary adenocarcinoma, 11).
Immunohistochemical Analysis
Immunohistochemical staining was performed on 5-um, formalin-fixed, paraffin-embedded sections using the Hep Par 1 antibody at 1:50 dilution (DAKO, Carpinteria, CA). Antigen retrieval was performed in all cases by steam heating the slides in a 1-mmol/L solution of EDTA (pH 8.0) for 30 minutes. After blocking of endogenous biotin, staining was performed using an automated immunostainer (DAKO), followed by detection by using a streptavidin-biotin detec- tion system (DAKO Envision Plus). Positive and negative control sections were used for each assay. The results were
recorded as positive or negative. Among the positive cases, the percentage of positive cells was noted, and the staining was categorized as weak or strong.
Albumin ISH
ISH was performed on all cases of HCC using 5-um, formalin-fixed, paraffin-embedded sections with appropriate negative and positive control sections. Albumin mRNA oligonucleotide probes synthesized at the Biochemistry and Molecular Biology department at the Mayo Clinic were used according to a previously described method.7 Staining with a polyadenylated probe (Vector Laboratories, Burlingame, CA) was done in all cases to check the reactivity of mRNA. The results were scored as positive or negative.
Results
Immunohistochemical Analysis for Hep Par 1
Primary Hepatic Tumors
Of the 42 HCC cases, 39 (93%) were positive for Hep Par 1 ITable 11. All 7 cases of clear cell HCC were positive. Of the 3 negative cases, 2 were high grade and 1 was a fibro- lamellar variant. In 35 (90%) of 39 positive cases, more than 90% of tumor cells were positive IImage 11. In 1 case each, 80% and 30% of tumor cells were positive, and in 2 cases, and 10% were positive IImage 21. All 3 Hep Par 1-negative cases were positive for albumin ISH. All 25 cases of cholan- giocarcinoma showed negative results (Table 1).
Metastatic Hepatic Tumors
Of the metastatic tumors, focal (10%), strong positive staining was seen in 1 (of 2) cases of urinary bladder adeno- carcinoma. All other metastatic tumors were negative.
Primary Extrahepatic Tumors
All cases of renal cell carcinoma, adrenocortical carci- noma, islet cell tumor, and colonic adenocarcinoma were negative. One case of urinary bladder adenocarcinoma showed focal strong positive (<10%) staining; the remaining 9 were negative. One case of pancreatic adenocarcinoma showed focal weak positive (<5%) staining; the remaining 9 cases were negative. Of 11 pulmonary adenocarcinomas, 3 (27%) showed strong positive reactions, as did 7 (70%) of 10 gastric and 6 (75%) of 8 esophageal adenocarcinomas IImage 31.
Albumin In Situ hybridization
Positive results were obtained in 39 (93%) of 42 HCC cases with albumin ISH ITable 21. All 7 cases of clear cell HCC were positive. Of the 3 negative cases, there was 1 case
ITable 11 Hep Par 1 Immunoreactivity in Hepatic and Extrahepatic Tumors
| Tumor | No. (%) of Hep Par 1-Positive Cases | Staining Characteristics |
|---|---|---|
| Hepatocellular carcinoma (n = 42) | 39 (93) | Strong, diffuse staining in >80% of tumor cells (n = 36); strong, focal staining in <30% of tumor cells (n = 3) |
| Cholangiocarcinoma (n = 25) | 0 (0) | 0 |
| Renal cell carcinoma (n = 10) | 0 (0) | 0 |
| Adrenocortical carcinoma (n = 8) | 0 (0) | 0 |
| Islet cell tumor (n = 10) | 0 (0) | 0 |
| Adenocarcinoma | ||
| Pancreas (n = 10) | 1 (10) | Weak, focal staining in <5% of tumor cells |
| Colon (n = 10) | 0 (0) | 0 |
| Lung (n = 11) | 3 (27) | Strong, focal staining in 10%-30% of tumor cells |
| Stomach (n = 10) | 7 (70) | Strong, diffuse staining in >80% of tumor cells (n = 5); strong, focal staining in 10%-20% of tumor cells (n = 2) |
| Esophagus (n = 8) | 6 (75) | Strong, diffuse staining in >80% of tumor cells (n = 4); strong, focal staining in 10%-20% of tumor cells (n = 2) |
| Urinary bladder (n = 10) | 1 (10) | Strong, focal staining in 10% of tumor cells |
A
B
C
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each of poorly differentiated, fibrolamellar, and conventional HCC. All 3 albumin ISH-negative cases showed positive immunoreactivity for Hep Par 1. Analysis of polyadenylated mRNA was positive in all cases, indicating adequate preser- vation of mRNA.
Discussion
Hep Par 1 is a recently described monoclonal antibody that is highly sensitive and specific for hepatocellular
differentiation. The antibody was developed in mice by using tissue from a formalin-fixed failed liver allograft.9 It is suitable for use in formalin-fixed material and reacts with both benign and malignant hepatocytes, yielding a granular cytoplasmic staining pattern. The target antigen is not known but is thought to be localized to the mitochondria.1,9
Several studies have demonstrated the efficacy of Hep Par1 in the diagnosis of HCC.1,9-11 In the original study, 37 (97%) of 38 HCCs were reported to be positive.9 However, 4 cases in that study showed rare positive cells, and if these cases are considered negative, the sensitivity drops to 87%.
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ITable 21
| Type of Hepatocellular Carcinoma | Hep Par 1-Positive Cases | Albumin ISH-Positive Cases | Combined Positive Cases |
|---|---|---|---|
| Conventional (n = 26) | 24 (92) | 24 (92) | 26 (100) |
| Clear cell (n = 7) | 7 (100) | 7 (100) | 7 (100) |
| Fibrolamellar (n = 9) | 8 (89) | 8 (89) | 9 (100) |
| Total (n = 42) | 39 (93) | 39 (93) | 42 (100) |
Hep Par 1, hepatocyte paraffin 1 monoclonal antibody; ISH, in situ hybridization. * Data are given as number (percentage).
Other studies1,10 have reported sensitivities ranging from 82% to 93.7%, which is comparable to our result of 93%. Although Hep Par 1 staining can be patchy, the present study shows that it is an uncommon finding. In our series, more than 80% of tumor cells showed strong immunoreactivity in 36 of 39 positive cases. Of the 3 cases that were negative, 1 was fibrolamellar HCC and 2 were high grade tumors. All fibrolamellar HCCs reported in the literature so far have been positive for Hep Par 1.1,9,10 Leong et al10 reported a poorly differentiated HCC that was Hep Par 1-negative. Expression of Hep Par 1 may be a function of differentiation leading to negative results in some high-grade tumors. However, the cause of occasional negative results in conven- tional and fibrolamellar HCC is not known.
Albumin ISH is a recently developed technique that has proven to be highly specific and sensitive for the diagnosis of HCC.6-8 The combined use of Hep Par 1 and albumin ISH for the diagnosis of HCC has not been studied previously. Our results show that both techniques have the same high sensitivity (93%) for the diagnosis of HCC. Positive results with these assays have a high correlation. These assays complement each other, as all 3 cases negative with Hep Par 1 were positive for albumin ISH, and vice versa. All cases of clear cell HCC, which often can be mistaken for metastatic clear cell tumors, were positive with both Hep Par 1 and albumin ISH. All cases of conventional, high-grade, and fibrolamellar HCC were identified successfully by the combined use of these two techniques.
It is imperative to know the expression of Hep Par 1 in a wide variety of extrahepatic neoplasms before it can be applied successfully for the diagnosis of HCC. Our results demonstrate that tumors that can be confused histologically with HCC, including cholangiocarcinoma, renal cell carci- noma, adrenocortical carcinoma, pancreatic islet cell tumor, and malignant melanoma, are negative for Hep Par 1. Occa- sional cases of cholangiocarcinoma have been reported to show weak and focal positive reactions.9,10 Negative results with Hep Par 1 were obtained with 5 renal cell carcinomas and 14 nonpulmonary neuroendocrine tumors by Wenner- berg et al9 and in 13 neuroendocrine tumors by Minervini et
al.1 Reactivity in adrenocortical tumors has not been reported. One case each of pancreatic and urinary bladder adenocarcinoma showed focal (<10%) staining, while colonic adenocarcinomas were negative in our series. Rare positive staining has been noted previously in pancreatic adenocarcinomas, but all reported cases of colonic adenocar- cinoma have been negative.9
Surprisingly, strong positive Hep Par 1 staining is seen in the majority of esophageal and gastric adenocarcinomas. The positive reaction in esophageal and gastric tumors is strong and diffuse and includes both intestinal type and diffuse (signet-ring cell) cancers. In the original Hep Par 1 study, 30% of the gastric adenocarcinomas were positive.9 Positive Hep Par 1 results have been obtained in hepatoid adenocarcinomas of the stomach.13 Esophageal squamous cell carcinomas have been reported to be negative for Hep Par 1,9 but esophageal adenocarcinomas have not been studied previously. Similarly, the strong but focal positive reaction in 27% (3/11) of the pulmonary adenocarcinomas in our study has not been reported previously. All 5 pulmonary adenocarcinomas in the original Hep Par 1 study were nega- tive.9 These findings suggest that Hep Par 1 staining may be useful for identifying the source of primary tumor in metastatic adenocarcinoma. It often is difficult to distinguish pancreaticobiliary, gastroesophageal, and lung primary sites based on morphologic features and cytokeratin profiles of the metastatic tumor. Positive Hep Par 1 staining in metastatic adenocarcinoma would favor a gastric, esophageal, or lung primary site. The results of immunohis- tochemical and ISH studies must be integrated with clinical and radiologic data to arrive at the final diagnosis.
Hep Par1 immunoreactivity has high sensitivity in the diagnosis of HCC. It correlates highly with albumin ISH, and their combined use is complementary in the detection of HCC, with sensitivity approaching 100%. Cholangiocarci- noma, renal cell carcinoma, adrenocortical carcinoma, islet cell tumors, malignant melanoma, pancreatic adenocarci- noma, and colonic adenocarcinoma are negative for Hep Par 1. The strong positive staining in a majority of gastric and esophageal adenocarcinomas and the occasional positive
reaction of pulmonary and urinary bladder adenocarcinoma should be kept in mind while evaluating tumors metastatic to the liver. In addition to identifying HCC, Hep Par 1 may help sort out the site of adenocarcinoma metastatic to the liver. A positive result would favor a gastroesophageal or lung primary site and make pancreaticobiliary and colon primary sites unlikely.
From the Department of Pathology, Mayo Clinic, Rochester, MN.
Address reprint requests to Dr Burgart: Dept of Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
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