| Abstract|| |
Background: Hepatocyte Paraffin 1 (Hep Par 1) was being extensively used to recognize the hepatocellular carcinomas, until recognition of its expression in tumors without hepatocellular differentiation. Aims and Objectives: The aim of this study was to analyze if Hep Par 1 stain can serve as a specific marker of the small intestinal (SI) adenocarcinomas, versus other gastrointestinal tract (GIT) primary tumors. Materials and Methods: In this retrospective cross-sectional study, normal GIT mucosa (n - 60), corresponding adenocarcinomas (n - 60) and nodal metastatic foci (n - 60) from the same patients, including 10 cases each from the esophagus, stomach, SI periampullary region, colon, rectum, and gall bladder were included. H-score was calculated by multiplying the stain distribution and intensity scores. The H-scores were compared with other clinical and histological parameters. Results: While normal SI mucosa showed diffuse strong Hep Par 1 staining, normal esophageal and gastric epitheliums were negative and normal colon, rectal, and biliary epithelium showed weak focal positivity. Adenocarcinomas from all these sites, however, showed Hep Par 1 expression, irrespective of the tumor type, site or origin, and tumor stage. The corresponding metastatic sites also showed variable Hep Par 1 positivity, without any site specificity. Conclusion: Hep Par 1 stain cannot help to determine the exact site of origin of primary GIT tumors. Its expression in adenocarcinomas across the GIT and their metastatic foci proves that it cannot be regarded as a marker of SI differentiation, especially in malignancy.
Keywords: Gastrointestinal tract adenocarcinomas, Hepatocyte Paraffin 1, immunohistochemical panel, normal gastrointestinal mucosa, unknown gastrointestinal primary
|How to cite this article:|
Yadav R, Chopra S, Garg A, Gupta B, Kinra P, Gupta SD, Das P. Does Hepatocyte Paraffin 1 expression stand a role in determining the site origin of an adenocarcinoma from unknown gastrointestinal primary?. Indian J Pathol Microbiol 2016;59:474-80
|How to cite this URL:|
Yadav R, Chopra S, Garg A, Gupta B, Kinra P, Gupta SD, Das P. Does Hepatocyte Paraffin 1 expression stand a role in determining the site origin of an adenocarcinoma from unknown gastrointestinal primary?. Indian J Pathol Microbiol [serial online] 2016 [cited 2020 Apr 2];59:474-80. Available from: http://www.ijpmonline.org/text.asp?2016/59/4/474/191781
| Introduction|| |
Hepatocyte Paraffin 1 (Hep Par 1) is a marker of hepatocyte mitochondrial antigen and was described as a highly sensitive (sensitivity 92%) and specific (specificity 90%) marker for hepatocyte differentiation. , It was given a great value in identifying the hepatocellular carcinomas (HCC), until it was gradually recognized to be expressed in tumors without hepatocellular differentiation along with reports regarding its inconsistent expression pattern across the different grades of HCCs.  It remained at the center of discussions since 1996, as reflected by as many as 84 published papers in the English literature regarding the utilities of this stain. It was so because this stain was relied on mostly for differentiating the primary and metastatic hepatic lesions, especially when they were multifocal and radiological features were not typical of HCC. ,,,,,,,,, Simultaneously, large number of other antibodies, including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), various cytokeratins (CK 7, 8, 18, 19, and 20), ferritin, albumin, fibrinogen, and alpha 1 antitrypsin, were also tried to determine definite hepatocyte differentiation. All of these markers fall short of popularity, due to their limited sensitivity and specificities. , Gradually, it was also identified as a marker of the small intestinal (SI) goblet cell differentiation, followed by its known expression in other nonintestinal tumors such as lung, prostate, and adrenal carcinomas. 
In this scenario, it seemed an important proposition to analyze the Hep Par 1 expression pattern across the different parts of the gastrointestinal tract (GIT) mucosa, corresponding malignancies arising from these primary sites and in metastatic foci. We were interested to get a comprehensive picture of Hep Par 1 staining patterns along the whole GIT, because in clinical pratice, identifying the probable site of origin of a metastatic tumor especially in liver, lung and lymphonodes are the onus on histopathologists, when radiological and biochemical finidngs do not give substantial evidence. Along with CK7, CK20 stains, Hep Par 1 stain is also commonly used in the panel, as the marker of hepatocyte differentiation. As dobted, if not certain, giving Hep Par 1 positivity too much priority may misguide oncologists, increase overall treatment cost and delay targeted management.
| Materials and Methods|| |
Cases and controls
A total of sixty cases of adenocarcinoma, which had lymph node metastases, were included retrospectively in the study with ten cases each from the esophagus, stomach, periampullary region, colon, rectum, and gall bladder. Normal mucosa away from the tumor and sections from the metastatic foci in lymph node were used to study the comparative expression pattern of Hep Par 1 in normal mucosa, primary tumors, and in the metastatic foci. Institutional ethical clearance was taken.
Histological analysis was performed by two histopathologists with experience in GIT pathology and was blinded about the detail. Histological parameters examined were as follows: histological tumor grade, lymphovascular emboli, perineural tumor invasion, extent of tumor infiltration, presence or absence of mucin, resection margin status, pathologic stage, lymph node tumor metastasis, and perinodal soft tissue deposits. Relevant demographic data of the patients were collected. Normal mucosa from esophagus and stomach were examined for the presence of intestinal metaplasia. Adenocarcinomas were graded as per the WHO histological grading system.
Tissue sections (4-5 μm) cut from representative paraffin blocks were deparaffinized in xylene and rehydrated through graded alcohol. Endogenous peroxidase was blocked by using 4% hydrogen peroxide. Sections were processed by conventional microwave heating in 10 mMol/L sodium citrate buffer (pH 6.0) for 30 min for antigen retrieval. The sections were then incubated with primary antibody (Hep Par 1 [Clone OCH1E5], Spring Bioscience, CA, USA; dilution 1:100) at 4°C, for overnight. Subsequently, sections were incubated with anti-mouse immunoglobulin in phosphate buffer saline containing carrier protein and 15 mM sodium azide at room temperature for 30 min. After washing, the reaction product was developed with 3,3'-diaminobenzidine and counterstained with hematoxylin. Appropriate positive and negative controls were used as per manufacturer's guidance.
Evaluation of immunoreactivity
Immunoreactivity was judged independently by two pathologists who were blinded to the clinical data and pathological diagnoses. Immunoreactivity was semi-quantitatively evaluated on the basis of staining intensity and distribution using the H-score.
H-score was calculated as follows = intensity score × percentage of positive cells. The intensity score was defined as 0: negative; 1: weak; 2: moderate; or 3: strong. The total score ranged from 0 to 300. A score of >5 was considered as positive. This cutoff was selected arbitrarily, considering the minimum accountable area positivity as 5%, and minimum intensity score as 1. Area of positivity <5% was taken as negative.
The statistical comparisons were performed using STATA software, version 12 (TX, USA). Pearson's Chi-square, Fisher's exact, Kruskal-Wallis, and Wilcoxon rank sum tests were applied. The value of P < 0.05 was considered statistically significant.
| Results|| |
Age of the patients ranged from 11 to 80 years with a median of 48.5 years and a mean of 50.3 years. The study group comprised 41 males and 19 females.
The tumor size varied from 0.8 to14 cm with a median of 4 cm and a mean of 4.7 cm. All the sixty tumors were diagnosed as adenocarcinomas on histopathological examination. Out of all, 53 (88.3%) had low histological grade (G1 and G2), whereas 7 (11.7%) had high histological grade (G3). Among these, three gastric, one esophageal, one colonic, one rectal, and one gall bladder adenocarcinomas showed poor differentiation with majority of areas lacking acinar differentiation. All the low histological grade tumors revealed unequivocal evidence of glandular differentiation. Twenty-four (40%) tumors showed mucin production and presence of signet ring cells. Thirty (50%) tumors showed lymphovascular emboli and 27 (45%) revealed perineural invasion. All the tumors showed transmural infiltration. The total lymph node yield varied from 2 to 44 lymph nodes, least in the rectal and gall bladder excision specimens. The number of positive lymph nodes (lymph nodes with tumor metastasis) varied from 1 to 40. Peri-nodal soft tissue tumor deposits were seen in 41 (68.3%) cases. Resection margin was positive in 11 (18.3%) cases. Nine periampullary, one esophageal and one gall bladder tumors were staged as Stage II. One periampullary, one colonic, and two gall bladder tumors were Stage IV. The rest of the tumor was Stage III.
Immunohistochemical expression patterns of Hepatocyte Paraffin 1
0Out of sixty cases, the normal esophageal and gastric mucosa did not show staining for Hep Par 1 [Table 1]. Hep Par 1 expression was, however, noted in the zone of intestinal metaplasia in the stomach [[Figure 1] - arrows]. Normal SI mucosa from cases of 10 periampullary tumors showed diffuse and strong staining. Normal colonic mucosa showed weak focal expression in five cases, whereas normal rectal mucosa showed weak focal expression in three cases. Only three gall bladder cases showed weak staining of normal mucosa.
|Figure 1: (a and b) Photomicrographs show negative Hep Par 1 staining in esophageal and gastric mucosa (×100). (c and d) Diffuse and strong expression in small intestinal mucosa and focal, low intensity positive in colonic mucosa (c, ×100; d, ×200). (d and f) Rectal mucosa and gall bladder epithelium were negative (×100)|
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|Table 1: Mean Hepatocyte Paraffin 1 H - scores in the normal mucosa, primary adenocarcinoma, and corresponding metastatic foci in gastrointestinal tract|
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Forty-one (68.3%) adenocarcinomas showed Hep Par 1 expression out of which 9 were esophageal, 8 gastric, 5 periampullary, 5 colonic, 7 rectal, and 7 from gall bladder [[Figure 2] - arrows]. Of these, percentage of positive cells varied from 2% to 100% with a mean of 26.4%. H-score ranged from 2 to 300 with a mean of 61.5 [Table 1]. Interestingly, this differential expression was not in accordance to the histological tumor grade or tumor type [Table 2].
|Figure 2: Esophageal (a), gastric signet ring cell (b), periampullary (c), colonic (d), rectal (e) and gall bladder (f) adenocarcinomas showed variable Hep Par 1 positivity (a-f; ×100)|
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|Table 2: Correlation of Hepatocyte Paraffin 1 expression in tumor and metastatic foci with patient demography and histological variables|
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Metastatic foci in lymph node
Twenty-nine (48.3%) metastatic adenocarcinomas in lymph node showed Hep Par 1 immunopositivity, of which 6 were esophageal, 6 gastric, 3 periampullary, 5 colonic, 4 rectal, and 5 from gall bladder adenocarcinomas [[Figure 3] - arrows]. Among these, the percentage of positive tumor cells varied from 2% to 95% with a mean of 19.6%. H-score varied from 4 to 285, with a mean of 44.5. Another interesting fact was that the Hep Par 1 expression pattern in the metastatic foci was not proportionate with the expression in primary tumors [Table 1].
|Figure 3: The metastatic foci in lymph nodes from primary adenocarcinomas of esophagus (a), stomach (b), small intestine (c), colon (d), rectum (e) and gall bladder (f) also showed variable Hep Par 1 immunopositivity (a-f; ×100)|
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Comparison of Hepatocyte Paraffin 1 expression between the normal mucosa, primary tumor, and metastases at different sites
Normal esophageal and gastric mucosas were completely negative for Hep Par 1, whereas the majority of the tumors and metastases arising from these sites showed Hep Par 1 expression. In contrast to strong expression in normal periampullary mucosal biopsies, in both periampullary carcinomas and metastatic tumors, Hep Par 1 expression was significantly low [Table 1]. In comparison to Hep Par-1 expression in gastric carcinomas, in metastatic sites, also expression was significantly lower [Table 1]. In colon, rectal, and gall bladder, normal mucosa showed weak positivity in a few cases as compared to higher H-score in the tumors and metastatic foci originating from these sites. The mean H-scores in the primary tumor and metastatic foci of these sites, although were not similar, the differences were not statistically significant [Table 1]. When the mean H-scores in normal mucosal biopsies of both upper GI (esophagus and stomach) and lower GI (colonic and rectal), including the gallbladder and periampullary regions were compared, Hep Par 1 expression was found to be significantly higher in the latter. However, Hep Par 1 expression was not significantly different either in the primary tumors or in the metastatic foci arising from different sites included in this study [Table 1].
Correlation of Hepatocyte Paraffin 1 expression at different sites with clinical and histological parameters
The expression pattern of Hep Par 1 was correlated with patient's demographic data as well as different histological parameters, both in the primary and metastatic tumors [Table 2]. Its expression did not differ according to the patient's age, tumor size, histological tumor grade, positive or negative resection margin status, perinodal soft tissue deposits and evidence of mucin production and perineural tumor spread status. Expression in males was high (both the primary tumor [P - 0.01] and metastases [P - 0.02]) and higher H-score was related to frequent lymphovascular tumor embolization (P - 0.04) and more numbers of nodal metastasis (positive nodes ≥ 5, [P - 0.03]).
| Discussion|| |
Histological prediction of the possible primary site is an important clinical question from the oncologists, in case of a metastatic tumor of unknown primary. It is known that a major portion of the metastatic malignancies is possibly from GIT primary, lung tumors, or adnexal tumors in females. While saying that, histological appearance of the metastatic tumor site is not reliable to predict the possible primary site; hence, often histopathologists resort to a wide panel of immunohistochemical (IHC) stains. Apart from CK7 and CK20, inclusion of Hep Par 1 stain is also common in such panel, which is used to determine a primary hepatic origin. Other IHC markers, such as AFP, polyclonal CEA, and alpha1 antitrypsin, which have been used for this purpose are known to be not specific. , However, expression of Hep Par 1 has been described in SI tumors and in many other nonintestinal tumors in microarray-based analyses. GIT tumors with hepatoid features were also found to express Hep Par 1 avidly. ,,
The target of Hep Par 1 antibody is the mitochondrial enzyme carbamoyl phosphate synthetase 1 (CPS 1), responsible for initiation of the urea cycle.  Chu et al. found Hep Par 1 granular cytoplasmic immunoreactivity in 92% of HCCs and correlated its differential expression with tumor nuclear grades. , However, gradually it was found that Hep Par 1 stain also comes positive in SI adenocarcinoma, skin, respiratory, and urological tumors, though most of these staining was weak and inconsistent. ,,,,,,,, This is also not clear, if Hep Par 1 expression is seen only in the normal SI mucosa and adenocarcinomas, or is also expressed in other areas of GIT and corresponding adenocarcinomas. This study was conducted to get a comprehensive picture of Hep Par 1 expression along different sites of GIT mucosa, corresponding adenocarcinomas and in metastatic foci.
In this study, normal esophageal and gastric mucosa did not show Hep Par 1 expression, except the focal expression in foci of incomplete intestinal metaplasia of gastric epithelium. In SI (periampullary mucosa), its expression was strong, diffuse, and consistent. It was identified that the specificity and sensitivity of this stain for detecting SI metaplasia is higher than that of keratin 7, 20 or MUC2. , Jeung et al. in their study had identified Hep Par 1 expression in columnar cell metaplasia, even in the absence of goblet cells, They concluded that Hep Par 1 can be regarded as a marker of evolving SI metaplasia. 
In adenocarcinomas arising from the esophagus, stomach, SI, LI, and bile ducts all show Hep Par 1 expression, without any significant difference of expression among these sites. Strong Hep Par 1 expression was also seen in a majority of esophageal and gastric adenocarcinomas, by Kakar et al.  There are other studies, who reported, higher Hep Par 1 expression in gastric signet ring cell adenocarcinomas and in hepatoid adenocarcinomas. , However, in this study, Hep Par 1 expression was found irrespective of histological tumor types and histological grades. While saying so, we also want to highlight the patchy Hep Par 1 expression pattern in the esophageal adenocarcinomas, than its expression in other GIT primary tumors [Table 1]. An important observation was that although, in patients with SI primary, Hep Par 1 was strong and diffuse; in the metastatic (nodal) site, the expression was focal with low intensity positivity, in comparison to their primary sites [Table 1]. This might be explained by the findings of a study by Cardona et al.,  that CPS 1, which is the antigenic target of Hep Par 1 stain, although present abundantly in normal SI, is depleted in metastatic site.  CPS 1 is involved in producing arginine from dietary proline or glutamine.  Gulluoglu et al. noticed a higher expression pattern of Hep Par 1 in intestinal type periampullary cancers than in the pancreatobiliary type carcinomas, suggesting a possible role of Hep Par 1 in differentiating these two histological tumor types.  In the metastatic sites also, Hep Par 1 expression was irrespective of their primary site of origin.
Hep Par 1 expression in normal colon and rectal mucosa was also noted in a few cases; however, the intensity was weaker than its expression in normal SI. Fifty percent of the primary colonic and > 50% of the rectal tumors as well as their metastatic counterparts showed patchy low-intensity expression of Hep Par 1 [Table 1]. Villari et al. also demonstrated Hep Par 1 expression in 50% of the colon tumors,  while, the others have reported very low expression or no expression. , Her Par 1 expression also has been described in a subset of dysplastic polyp which transformed into malignancy.  Abu-Zeid and Farid found a decreasing grade of Hep Par 1 staining in colon carcinoma, adenomas with high-grade dysplasia and in low-grade dysplasias. In the mentioned study, Hep Par 1 expression was inversely correlated with tumor type, grade, lymph node metastasis, and AJCC stage, but not with the depth of invasion or lymphovascular invasion.  We did not identify any significant correlation of Hep Par 1 expression when compared to different surgical and histological parameters. However, the Hep Par 1 positive cases were associated with higher number of lymph node metastasis and lymphovascular emboli. In cholangiocarcinomas, Hep Par 1 expression was focal in a few cases (<5%); ,, whereas others had reported relatively more expression (16%).  As described in literatures, we also identified rare positivity of Hep Par 1 in periampullary adenocarcinomas, in contrast to the normal periampullary mucosa and mild focal positivity in cholangiocarcinomas. ,
Hence, to conclude, Hep Par 1, a marker of normal SI differentiation, is not helpful to differentiate adenocarcinomas arising from different parts of GIT, and hence should not be included in the diagnostic panel of metastatic tumors of unknown primary, to determine a possible primary site of origin. In metastatic foci, the Hep Par 1 expression is also not proportional to its expression patterns in primary GIT tumor foci.
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Conflicts of interest
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| References|| |
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;26:978-88.
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;16:137-44.
Wee A. Diagnostic utility of immunohistochemistry in hepatocellular carcinoma, its variants and their mimics. Appl Immunohistochem Mol Morphol 2006;14:266-72.
Lee HS, Kim WH, Kang GH. Hepatocyte expressions in hepatocellular carcinomas, gastrointestinal neoplasms, and non-neoplastic gastrointestinal mucosa: Its role as a diagnostic marker. J Korean Med Sci 2003;18:842-8.
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;10:686-92.
Lamps LW, Folpe AL. The diagnostic value of Hepatocyte paraffin antibody 1 in differentiating hepatocellular neoplasms from nonhepatic tumors: A review. Adv Anat Pathol 2003;10:39-43.
Wennerberg AE, Nalesnik MA, Coleman WB. Hepatocyte paraffin 1: A monoclonal antibody that reacts with hepatocytes and can be used for differential diagnosis of hepatic tumors. Am J Pathol 1993;143:1050-4.
Lugli A, Tornillo L, Mirlacher M, Bundi M, Sauter G, Terracciano LM. Hepatocyte paraffin 1 expression in human normal and neoplastic tissues: Tissue microarray analysis on 3,940 tissue samples. Am J Clin Pathol 2004;122:721-7.
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;119:361-6.
Thamboo TP, Wee A. Hep Par 1 expression in carcinoma of the cervix: Implications for diagnosis and prognosis. J Clin Pathol 2004;57:48-53.
Nemolato S, Ravarino A, Fanni D, Coni P, Felice ED, Senes G, et al
. Hepatocyte paraffin 1 immunoreactivity in early colon carcinogenesis. Gastroenterol Res 2009;2:277-81.
Chu PG, Jiang Z, Weiss LM. Hepatocyte antigen as a marker of intestinal metaplasia. Am J Surg Pathol 2003;27:952-9.
Butler SL, Dong H, Cardona D, Jia M, Zheng R, Zhu H, et al.
The antigen for Hep Par 1 antibody is the urea cycle enzyme carbamoyl phosphate synthetase 1. Lab Invest 2008;88:78-88.
Kumagai I, Masuda T, Sato S, Ishikawa K. Immunoreactivity to monoclonal antibody, Hep Par 1, in human hepatocellular carcinomas according to histopathological grade and histological pattern. Hepatol Res 2001;20:312-9.
Sugiki T, Yamamoto M, Aruga A, Takasaki K, Nakano M. Immunohistological evaluation of single small hepatocellular carcinoma with negative staining of monoclonal antibody Hepatocyte paraffin 1. J Surg Oncol 2004;88:104-7.
Ocak GA, Yildirim G, Elpek GO. Hepatocyte antigen expression in subtypes of intestinal metaplasia of the stomach. West Indian Med J 2012;61:659-64.
Jeung JA, Coran JJ, Liu C, Cardona DM. Hepatocyte paraffin 1 antigen as a biomarker for early diagnosis of Barrett esophagus. Am J Clin Pathol 2012;137:111-20.
Maitra A, Murakata LA, Albores-Saavedra J. Immunoreactivity for Hepatocyte paraffin 1 antibody in hepatoid adenocarcinomas of the gastrointestinal tract. Am J Clin Pathol 2001;115:689-94.
Cardona DM, Zhang X, Liu C. Loss of carbamoyl phosphate synthetase I in small-intestinal adenocarcinoma. Am J Clin Pathol 2009;132:877-82.
Gulluoglu MG, Karayigit E, Ozden I, Kapran Y, Dizdaroglu F. Does HepPar-1 immunoexpression have a role in differential diagnosis of periampullary cancer? Pathology 2008;40:35-41.
Villari D, Caruso R, Grosso M, Vitarelli E, Righi M, Barresi G. Hep Par 1 in gastric and bowel carcinomas: An immunohistochemical study. Pathology 2002;34:423-6.
Köhler ES, Sankaranarayanan S, van Ginneken CJ, van Dijk P, Vermeulen JL, Ruijter JM, et al.
The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and catabolizing enzymes. BMC Dev Biol 2008;8:107.
Abu-Zeid RM, Farid RM. Role of Hepatocyte paraffin 1 antigen in the course of colorectal carcinogenesis. Int J Physiol Pathophysiol Pharmacol 2013;5:177-83.
Leong AS, Sormunen RT, Tsui WM, Liew CT. Hep Par 1 and selected antibodies in the immunohistological distinction of hepatocellular carcinoma from cholangiocarcinoma, combined tumours and metastatic carcinoma. Histopathology 1998;33:318-24.
Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]