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Aberrant CDX2 expression in hepatocellular carcinomas: an important diagnostic pitfall
Aberrant CDX2 expression in Hepatocellular Carcinomas: An Important Diagnostic Pitfall. Sejal S. Shah, Tsung-Teh Wu, Michael S. Torbenson, Vishal S. Chandan PII: DOI: Reference:
S0046-8177(17)30018-7 doi: 10.1016/j.humpath.2016.12.029 YHUPA 4111
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
18 October 2016 17 December 2016 28 December 2016
Please cite this article as: Shah Sejal S., Wu Tsung-Teh, Torbenson Michael S., Chandan Vishal S., Aberrant CDX2 expression in Hepatocellular Carcinomas: An Important Diagnostic Pitfall., Human Pathology (2017), doi: 10.1016/j.humpath.2016.12.029
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Aberrant CDX2 expression in Hepatocellular Carcinomas: An Important Diagnostic Pitfall.
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Sejal S. Shah, M.D., Tsung-Teh Wu, M.D., Ph.D., Michael S. Torbenson, M.D., and Vishal S. Chandan, M.D.
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Division of Anatomic Pathology, Mayo Clinic, Rochester, MN.
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Address correspondence to: Vishal S. Chandan, M.D. Department of Pathology
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Mayo Clinic 200 First Street SW
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Rochester, MN 55905 Tel: 507-284-0711
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Fax: 507-284-1599
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Email: [email protected]
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Conflict of interest: None. All authors listed above have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Each author also certifies that this material or similar material has not been and will not be submitted to or published in any other publication. Disclosure of funding: None. Running head: CDX2 expression in Hepatocellular Carcinoma
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ABSTRACT
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CDX2 is a sensitive and specific marker of intestinal differentiation. It is routinely used in
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surgical pathology, as its expression within a tumor favors an origin within the gastrointestinal tract. We had anecdotally encountered occasional hepatocellular carcinomas (HCC) that were
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CDX2 positive. CDX2 expression in HCC has not yet been reported but it has also not been
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examined in detail. Therefore, we evaluated CDX2 expression in a large number of resected HCCs. Full tumor sections from 172 resected HCCs and 6 resected fibrolamellar carcinomas
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(FLC) were stained for CDX2. Nine (5.2%) of 172 HCCs were positive for CDX2 while all 6 FLCs were negative. CDX2 expression in HCCs was more commonly seen in poorly
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differentiated tumors (5 of 16 cases, 31%) than well and moderately differentiated tumors (4 of 156 cases, 2.5%), p value = 0.0004. No other statistically significant correlations were observed
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(p > 0.05). Results of our study show that a small subset (5%) of HCCs can be CDX2 positive.
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Awareness of this phenomenon is important as CDX2 expression in a liver tumor does not
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completely exclude a diagnosis of HCC.
Keywords: CDX2, hepatocellular carcinoma, fibrolamellar carcinoma, liver, metastasis.
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INTRODUCTION
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CDX2 is a nuclear transcription factor encoded by the caudal homeobox family and it plays a
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critical role in embryologic development of the gastrointestinal tract and intestinal differentiation (1-3). CDX2 immunostain is widely used in surgical pathology, as expression of CDX2 is
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retained in carcinomas arising from or related to intestinal cells. Positive CDX2 expression
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within a tumor is considered a sensitive and specific marker for intestinal differentiation (4). In addition to colorectal adenocarcinomas, CDX2 expression can also be seen within
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adenocarcinomas arising in stomach, duodenum, esophagus, pancreas and biliary tract (4, 5). Neuroendocrine tumors arising within the intestinal tract can also be CDX2 positive (6-9). CDX2
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expression has also been reported in tumors of extra-gastrointestinal tract origin showing mucinous or intestinal morphological features, such as those arising within the urinary bladder,
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lung, sinonasal tract, cervix and ovary (4, 10-15). The columnar cell variant of papillary thyroid
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carcinomas have also recently been shown to be CDX2 positive (16, 17). In North America and Europe, metastatic tumors to the liver are the most common
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malignant hepatic neoplasms. Adenocarcinomas and neuroendocrine tumors from the gastrointestinal tract frequently metastasize to the liver. Hepatocellular carcinoma is the most common primary hepatic malignancy (18). Distinguishing hepatocellular carcinoma from other tumors including cholangiocarcinomas, metastatic adenocarcinomas or neuroendocrine tumors is clinically very important, as their treatment and prognosis are different. Knowing the patient’s prior history of a primary tumor outside the liver can be helpful in distinguishing hepatocellular carcinoma from a metastasis, especially when dealing with liver biopsies. However, sometimes this clinical information is not available and many times the patient also may not have a prior known history of malignancy. Well differentiated hepatocellular carcinoma usually can be 3
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readily diagnosed by typical morphological features on hematoxylin and eosin stain and hence
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distinguished from adenocarcinoma. However, well differentiated neuroendocrine tumors can
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sometimes mimic hepatocellular carcinoma on morphology. When dealing with poorly differentiated tumors, immunohistochemistry plays an important role in distinguishing
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hepatocellular carcinoma from metastatic adenocarcinoma or neuroendocrine carcinoma.
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Immunohistochemistry can also be helpful in identifying a primary site of origin for a tumor that has metastasized to the liver. In this context, CDX2 is frequently used in hepatic tumor
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pathology, as it is considered to be a sensitive and specific marker for carcinomas arising from the gastrointestinal tract. CDX2 positivity has not yet been reported in hepatocellular carcinoma
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(4, 19, 20), making it a potentially useful marker for excluding hepatocellular carcinoma when CDX2 expression is seen within a tumor involving the liver. However, we have anecdotally
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encountered occasional hepatocellular carcinomas that are CDX2 positive, including cases
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submitted for consultation with a probable diagnosis of metastatic adenocarcinoma from the gastrointestinal tract, where the referring pathologist had excluded the diagnosis of
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hepatocellular carcinoma because the liver tumor was CDX2 positive. Therefore, we evaluated a large number of previously diagnosed hepatocellular carcinomas for CDX2 expression. The aims of this study were to evaluate the CDX2 expression in hepatocellular carcinoma, to access the frequency and significance of CDX2 in hepatocellular carcinomas and to verify if CDX2 expression in a liver tumor is useful in the exclusion of hepatocellular carcinoma.
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MATERIALS AND METHODS
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Case selection and Histological Review
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Full tumor sections from 172 resected hepatocellular carcinomas and 6 resected fibrolamellar carcinomas between years 1999 and 2010 were selected for this study from previously diagnosed
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in-house archival material. Institutional Review Board approved this study. Relevant clinical
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information was obtained from the patient’s medical records. Only resected cases were included to ensure the diagnosis of hepatocellular carcinoma. Only unequivocal hepatocellular and
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fibrolamellar carcinomas were selected for this study. Combined hepatocellularcholangiocarcinomas were not included. All the cases included in this study were confirmed by
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re-review of routine morphological features on hematoxylin and eosin sections such as >3 cell thick plates, presence of cytologic atypia, loss of reticulin framework and relevant use of
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ancillary immunohistochemical markers of hepatocellular differentiation when necessary,
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including HepPar-1, glypican-3, arginase-1, as well as albumin in-situ hybridization. The hepatocellular carcinomas were graded into well-differentiated, moderately differentiated or
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poorly differentiated as per the WHO grading system (21). Immunohistochemistry
Immunostains were performed on formalin-fixed paraffin embedded tissue using steam retrieval and established methodologies. CDX2 immunostain (EPR2764Y, rabbit monoclonal, Cell Marque, dilution 1:200) was performed in all cases on full tumor sections. Only moderate to strong intensity of nuclear staining within the neoplastic cells was considered positive. The CDX2 immunostain was graded as negative (no positive cells), 1+ (<10% positive cells), 2+ (1125% positive cells), 3+ (26-50% positive cells) or 4+ (>51% positive cells). The expression of CDX2 was correlated with clinicopathological features. 5
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Statistical Analysis
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difference was considered significant if the p-value was ≤ 0.05.
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Data from different groups was compared using Fisher’s exact and Chi-Square tests, and the
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RESULTS
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Clinical and Histological Features
Age, gender and other clinical information on all hepatocellular carcinomas are summarized in
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Table 1. There were 114 men and 58 women (age range, 34-88 years; median age, 63 years). The background chronic liver disease was hepatitis B virus in 22 cases (13%), hepatitis C virus in 25
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cases (14%), both hepatitis B and C viruses in 3 cases (2%), alcoholic liver disease in 5 cases (3%), primary biliary cirrhosis in 3 cases (2%), non -alcoholic liver disease and autoimmune
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hepatitis in 2 cases each (1%), and hereditary hemochromatosis in 1 case (1%). In 109 cases
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(63%) there was no known chronic liver disease. 57 (33%) of these hepatocellular carcinomas arose within a cirrhotic liver. 51 (30%) were well-differentiated, 105 (61%) were moderately-
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differentiated and 16 (9%) were poorly-differentiated tumors. All the tumors were conventional hepatocellular carcinomas. The six fibrolamellar carcinomas showed typical morphology and all were confirmed by FISH and/or RT-PCR to have the classic DNAJB1-PRKACA fusion product (22). There were 3 men and 3 women (age range, 21-48 years; median age, 32 years). None of these 6 tumors had any known background chronic liver disease and none arose within a cirrhotic liver. All these 6 fibrolamellar carcinomas were moderately-differentiated.
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CDX2 expression in hepatocellular and fibrolamellar carcinomas
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The details of CDX2 expression are summarized in Table 1. Nine (5.2%) of 172 hepatocellular
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carcinomas were positive for CDX2. Of these 9 tumors, 1 (11%) was well differentiated, 3 (33%) were moderately differentiated and 5 (56%) were poorly differentiated (Figures 1A and 2A). The
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background liver showed cirrhosis in 5 (56%) of these 9 cases and the background liver disease
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was hepatitis C in 4 (45%), hepatitis B in 1 (10%) and unknown in 4 (45%) cases. The extent of nuclear staining for CDX2 was scored as 1+ in 5 cases (1 well differentiated, 3 moderately
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differentiated and 1 poorly differentiated), 2+ in 2 cases (both poorly differentiated) and 3+ in 2 cases (both poorly differentiated). The staining pattern in 7 cases was notable for discrete
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clusters of malignant cells with areas of intervening negative tumor cells, whereas 2 cases showed scattered positive cells within the tumor (Figures 1B and 2B). Arginase-1 immunostain
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was positive in all 9 cases (Figures 1C and 2C), including the foci within the tumors that were
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CDX2 positive. Glypican-3 immunostain was performed in 5 cases and it was positive in all 5 cases (Figure 1D), including the foci within the tumors that showed CDX2 expression. CK7,
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CK19 and CK20 immunostains were performed in 6 cases and all three immunostains were negative within these 6 cases, including the foci that were CDX2 positive. The background nonneoplastic liver parenchyma was negative for CDX2 in all cases when present on the stained sections. CDX2 expression in hepatocellular carcinomas was more commonly seen in poorly differentiated tumors (5 of 16 cases, 31%) than in well and moderately differentiated tumors (4 of 156 cases, 2.5%), p value = 0.0004. No other statistically significant correlations were observed (p > 0.05) between CDX2 expression in hepatocellular carcinomas and age, gender, background liver disease and cirrhosis. All six fibrolamellar carcinomas were CDX2 negative.
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DISCUSSION
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CDX2, a homeobox nuclear transcription factor, is considered a sensitive and specific
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marker of intestinal differentiation because of its perceived restrictive pattern of expression within the gastrointestinal tract. In addition to colorectal tumors, adenocarcinomas arising within
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the esophagus, stomach and pancreatobiliary tract are also frequently CDX2 positive. The liver is
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frequently involved by metastatic carcinoma and hence, CDX2 is a widely used immunohistochemical marker in surgical pathology to identify the site of origin when dealing
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with metastatic carcinoma of unknown primary within the liver. CDX2 can also be positive in some extra-gastrointestinal tumors, especially in tumors showing intestinal or intestinal-like
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phenotypes (23). Therefore, CDX2 positivity is not entirely specific for neoplasms arising in the gastrointestinal tract, but its expression is at least considered an indication of intestinal-type
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differentiation.
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CDX2 expression in hepatocellular carcinomas has not yet been reported. Werling et al studied 12 hepatocellular carcinomas and none were CDX2 positive (4). Similar results were
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reported in another study by Moskaluk et al, in which all 12 hepatocellular carcinomas were negative for CDX2 (20). A third study on 31 hepatocellular carcinomas using tissue microarray also showed no CDX2 expression (19). However, these studies included only a small number of hepatocellular carcinomas. We sought to systematically evaluate CDX2 expression in hepatocellular carcinomas, as we had anecdotally encountered occasional hepatocellular carcinomas that were CDX2 positive. Interpretation of the positive staining was also compounded by the lack of a comprehensive evaluation of CDX2 staining in hepatocellular carcinomas within the literature. Our study is the largest to study CDX2 expression in hepatocellular carcinomas using full tumor sections and is 8
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the first to report CDX2 positivity in hepatocellular carcinomas. We have shown CDX2
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positivity in 9 (5.2%) of 172 hepatocellular carcinomas. Of these 9 cases that were positive, 5
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showed 1+ staining (<10% positive cells), 2 showed 2+ staining (11-25% positive cells) and 2 were 3+ (26-50% positive cells).
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Colonic adenocarcinomas usually show strong and diffuse CDX2 expression, however,
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CDX2 expression in adenocarcinomas arising within the pancreatobiliary tract and upper gastrointestinal tract can often show focal positivity for CDX2 (4, 20, 24). Hence focal staining
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for CDX2 in a liver tumor could be misinterpreted as indication of a metastasis from the gastrointestinal tract. However, the results of our study show that a subset (up to 5%) of
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hepatocellular carcinomas can be positive for CDX2. The main significance of aberrant CDX2 expression in hepatocellular carcinoma is for its
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potential for causing diagnostic confusion. Whether or not this becomes problematic is highly
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dependent on the clinical and morphological features of any given case, the differential diagnosis generated by the pathologist and the immunohistochemical approach taken on the case. Aberrant
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CDX2 expression in hepatocellular carcinoma may not be an issue in well to moderately differentiated hepatocellular carcinomas, in which a diagnosis is possible without ancillary immunostains such as CDX2. Poorly differentiated tumors involving the liver usually require immunohistochemical workup that may include CDX2 to distinguish metastatic poorly differentiated adenocarcinomas from poorly differentiated hepatocellular carcinomas. Five of the 13 (31%) poorly differentiated hepatocellular carcinomas in our study were CDX2 positive, indicating that a significant proportion of poorly differentiated hepatocellular carcinomas are CDX2 positive. Only one third of the hepatocellular carcinomas (57 of the 172 cases; 33%) in our study arose within a cirrhotic liver. This rate of underlying cirrhosis for hepatocellular 9
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carcinomas in our study is lower than that reported in the literature (which varies from 50% to
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90%) and the reason for this difference is not entirely clear. However, the lack of underlying
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cirrhosis increases the probability of a tumor within the liver to be a metastasis, as tumors in cirrhotic livers are less likely to be metastases. Hence, awareness of CDX2 expression in a subset
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of hepatocellular carcinomas is critical for reducing the potential for misdiagnosis of these
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tumors as non-hepatocellular carcinomas.
ACAT1 and ACAT2 are two ACAT (acyl-CoA:cholesterol acyltransferase) genes in
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humans that play an important role in cellular cholesterol homoeostasis (25). ACAT2 is mainly expressed in intestinal cells (26). CDX2 is known to govern and stimulate ACAT2 expression in
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human intestinal cells (27). A proportion of hepatocellular carcinomas have also been shown to exhibit elevated ACAT2 expression (27) and hence we speculate that this might correlate with
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CDX2 expression in a subset of hepatocellular carcinoma. CDX2 has also been shown to
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regulate the expression of Cadherin-17 (CDH17), a member of the non-classical cadherin family in colorectal cancer and stomach adenocarcinomas (28-30). Similar association between CDX2
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and CDH17 has also been shown in hepatocellular carcinoma and overexpression on CDH17 in hepatocellular carcinoma is associated with poor clinical outcomes (31). In summary, although CDX2 remains a highly sensitive and specific marker of intestinal origin in tumor pathology, this study demonstrates that a subset of hepatocellular carcinomas (5%) can be CDX2 positive. A significant proportion of poorly differentiated hepatocellular carcinomas (31%) are CDX2 positive. Awareness of this phenomenon is important, as CDX2 expression in a liver tumor does not completely exclude hepatocellular carcinoma. Appropriate immunohistochemical workup for hepatocellular carcinoma with immunostains such as arginase1, HepPar-1 and glypican-3 which are related to hepatocellular lineage as well as other 10
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immunostains such as CK7 and CK20 should be pursued in CDX2 positive liver tumors,
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particularly in poorly differentiated tumors, to avoid the possibility of missing a diagnosis of
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hepatocellular carcinoma.
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FIGURE LEGENDS
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FIGURE 1. Poorly differentiated hepatocellular carcinoma. A, H&E stain showing tumor cells
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with eosinophilic cytoplasm and large pleomorphic nuclei (200x). B, CDX2 showing nuclear expression (200x). C, Arginase-1 immunostain is positive within the tumor (200x). D, Glypican-
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3 immunostain is also positive within the tumor (200x).
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FIGURE 2. Poorly differentiated hepatocellular carcinoma. A, H&E stain showing large polygonal tumor cells with deep eosinophilic cytoplasm and few mitotic figures (200x). B,
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CDX2 is positive in small clusters of tumor cells (200x). C, Arginase-1 immunostain is positive
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within the tumor cells (100x).
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Fig. 1A
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TABLES
Age (yr) ≤40 >40
2 (22%) 7 (78%)
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Gender Male Female
CDX2 negative n = 163 (94.8%)
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CDX2 positive n = 9 (5.2%)
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Differentiation Well differentiated Moderately differentiated Poorly differentiated
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1
108 (66%) 55 (34%) 0.50
1 (10%) 4 (45%) 0 0
21 (13%) 21 (13%) 3 (2%) 2 (1%)
0 0 0 0 4 (45%)
5 (3%) 2 (1%) 1 (1%) 3 (2%) 105 (64%)
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Background liver disease Hepatitis B virus Hepatitis C virus Hepatitis B and C viruses Non-alcoholic steatohepatitis Alcoholic liver disease Autoimmune hepatitis Hereditary hemochromatosis Primary biliary cirrhosis Unknown
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6 (67%) 3 (33%)
p value
13 (8%) 150 (92%)
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Characteristics
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Table 1. CDX2 expression in hepatocellular carcinomas.
0.0004a 1 (11%) 3 (33%) 5 (56%)
50 (31%) 102 (63%) 11 (6%)
Background cirrhosis 0.16 Present 5 (56%) 52 (32%) Absent 4 (44%) 111 (68%) HCC, hepatocellular carcinoma; a p value was calculated by combining well differentiated and moderately differentiated HCC versus poorly differentiated HCC
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S0046-8177(17)30018-7 doi: 10.1016/j.humpath.2016.12.029 YHUPA 4111
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
18 October 2016 17 December 2016 28 December 2016
Please cite this article as: Shah Sejal S., Wu Tsung-Teh, Torbenson Michael S., Chandan Vishal S., Aberrant CDX2 expression in Hepatocellular Carcinomas: An Important Diagnostic Pitfall., Human Pathology (2017), doi: 10.1016/j.humpath.2016.12.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Aberrant CDX2 expression in Hepatocellular Carcinomas: An Important Diagnostic Pitfall.
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Sejal S. Shah, M.D., Tsung-Teh Wu, M.D., Ph.D., Michael S. Torbenson, M.D., and Vishal S. Chandan, M.D.
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Division of Anatomic Pathology, Mayo Clinic, Rochester, MN.
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Address correspondence to: Vishal S. Chandan, M.D. Department of Pathology
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Mayo Clinic 200 First Street SW
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Rochester, MN 55905 Tel: 507-284-0711
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Fax: 507-284-1599
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Email: [email protected]
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Conflict of interest: None. All authors listed above have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Each author also certifies that this material or similar material has not been and will not be submitted to or published in any other publication. Disclosure of funding: None. Running head: CDX2 expression in Hepatocellular Carcinoma
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ABSTRACT
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CDX2 is a sensitive and specific marker of intestinal differentiation. It is routinely used in
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surgical pathology, as its expression within a tumor favors an origin within the gastrointestinal tract. We had anecdotally encountered occasional hepatocellular carcinomas (HCC) that were
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CDX2 positive. CDX2 expression in HCC has not yet been reported but it has also not been
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examined in detail. Therefore, we evaluated CDX2 expression in a large number of resected HCCs. Full tumor sections from 172 resected HCCs and 6 resected fibrolamellar carcinomas
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(FLC) were stained for CDX2. Nine (5.2%) of 172 HCCs were positive for CDX2 while all 6 FLCs were negative. CDX2 expression in HCCs was more commonly seen in poorly
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differentiated tumors (5 of 16 cases, 31%) than well and moderately differentiated tumors (4 of 156 cases, 2.5%), p value = 0.0004. No other statistically significant correlations were observed
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(p > 0.05). Results of our study show that a small subset (5%) of HCCs can be CDX2 positive.
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Awareness of this phenomenon is important as CDX2 expression in a liver tumor does not
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completely exclude a diagnosis of HCC.
Keywords: CDX2, hepatocellular carcinoma, fibrolamellar carcinoma, liver, metastasis.
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INTRODUCTION
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CDX2 is a nuclear transcription factor encoded by the caudal homeobox family and it plays a
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critical role in embryologic development of the gastrointestinal tract and intestinal differentiation (1-3). CDX2 immunostain is widely used in surgical pathology, as expression of CDX2 is
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retained in carcinomas arising from or related to intestinal cells. Positive CDX2 expression
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within a tumor is considered a sensitive and specific marker for intestinal differentiation (4). In addition to colorectal adenocarcinomas, CDX2 expression can also be seen within
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adenocarcinomas arising in stomach, duodenum, esophagus, pancreas and biliary tract (4, 5). Neuroendocrine tumors arising within the intestinal tract can also be CDX2 positive (6-9). CDX2
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expression has also been reported in tumors of extra-gastrointestinal tract origin showing mucinous or intestinal morphological features, such as those arising within the urinary bladder,
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lung, sinonasal tract, cervix and ovary (4, 10-15). The columnar cell variant of papillary thyroid
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carcinomas have also recently been shown to be CDX2 positive (16, 17). In North America and Europe, metastatic tumors to the liver are the most common
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malignant hepatic neoplasms. Adenocarcinomas and neuroendocrine tumors from the gastrointestinal tract frequently metastasize to the liver. Hepatocellular carcinoma is the most common primary hepatic malignancy (18). Distinguishing hepatocellular carcinoma from other tumors including cholangiocarcinomas, metastatic adenocarcinomas or neuroendocrine tumors is clinically very important, as their treatment and prognosis are different. Knowing the patient’s prior history of a primary tumor outside the liver can be helpful in distinguishing hepatocellular carcinoma from a metastasis, especially when dealing with liver biopsies. However, sometimes this clinical information is not available and many times the patient also may not have a prior known history of malignancy. Well differentiated hepatocellular carcinoma usually can be 3
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readily diagnosed by typical morphological features on hematoxylin and eosin stain and hence
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distinguished from adenocarcinoma. However, well differentiated neuroendocrine tumors can
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sometimes mimic hepatocellular carcinoma on morphology. When dealing with poorly differentiated tumors, immunohistochemistry plays an important role in distinguishing
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hepatocellular carcinoma from metastatic adenocarcinoma or neuroendocrine carcinoma.
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Immunohistochemistry can also be helpful in identifying a primary site of origin for a tumor that has metastasized to the liver. In this context, CDX2 is frequently used in hepatic tumor
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pathology, as it is considered to be a sensitive and specific marker for carcinomas arising from the gastrointestinal tract. CDX2 positivity has not yet been reported in hepatocellular carcinoma
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(4, 19, 20), making it a potentially useful marker for excluding hepatocellular carcinoma when CDX2 expression is seen within a tumor involving the liver. However, we have anecdotally
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encountered occasional hepatocellular carcinomas that are CDX2 positive, including cases
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submitted for consultation with a probable diagnosis of metastatic adenocarcinoma from the gastrointestinal tract, where the referring pathologist had excluded the diagnosis of
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hepatocellular carcinoma because the liver tumor was CDX2 positive. Therefore, we evaluated a large number of previously diagnosed hepatocellular carcinomas for CDX2 expression. The aims of this study were to evaluate the CDX2 expression in hepatocellular carcinoma, to access the frequency and significance of CDX2 in hepatocellular carcinomas and to verify if CDX2 expression in a liver tumor is useful in the exclusion of hepatocellular carcinoma.
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MATERIALS AND METHODS
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Case selection and Histological Review
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Full tumor sections from 172 resected hepatocellular carcinomas and 6 resected fibrolamellar carcinomas between years 1999 and 2010 were selected for this study from previously diagnosed
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in-house archival material. Institutional Review Board approved this study. Relevant clinical
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information was obtained from the patient’s medical records. Only resected cases were included to ensure the diagnosis of hepatocellular carcinoma. Only unequivocal hepatocellular and
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fibrolamellar carcinomas were selected for this study. Combined hepatocellularcholangiocarcinomas were not included. All the cases included in this study were confirmed by
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re-review of routine morphological features on hematoxylin and eosin sections such as >3 cell thick plates, presence of cytologic atypia, loss of reticulin framework and relevant use of
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ancillary immunohistochemical markers of hepatocellular differentiation when necessary,
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including HepPar-1, glypican-3, arginase-1, as well as albumin in-situ hybridization. The hepatocellular carcinomas were graded into well-differentiated, moderately differentiated or
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poorly differentiated as per the WHO grading system (21). Immunohistochemistry
Immunostains were performed on formalin-fixed paraffin embedded tissue using steam retrieval and established methodologies. CDX2 immunostain (EPR2764Y, rabbit monoclonal, Cell Marque, dilution 1:200) was performed in all cases on full tumor sections. Only moderate to strong intensity of nuclear staining within the neoplastic cells was considered positive. The CDX2 immunostain was graded as negative (no positive cells), 1+ (<10% positive cells), 2+ (1125% positive cells), 3+ (26-50% positive cells) or 4+ (>51% positive cells). The expression of CDX2 was correlated with clinicopathological features. 5
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Statistical Analysis
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difference was considered significant if the p-value was ≤ 0.05.
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Data from different groups was compared using Fisher’s exact and Chi-Square tests, and the
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RESULTS
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Clinical and Histological Features
Age, gender and other clinical information on all hepatocellular carcinomas are summarized in
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Table 1. There were 114 men and 58 women (age range, 34-88 years; median age, 63 years). The background chronic liver disease was hepatitis B virus in 22 cases (13%), hepatitis C virus in 25
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cases (14%), both hepatitis B and C viruses in 3 cases (2%), alcoholic liver disease in 5 cases (3%), primary biliary cirrhosis in 3 cases (2%), non -alcoholic liver disease and autoimmune
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hepatitis in 2 cases each (1%), and hereditary hemochromatosis in 1 case (1%). In 109 cases
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(63%) there was no known chronic liver disease. 57 (33%) of these hepatocellular carcinomas arose within a cirrhotic liver. 51 (30%) were well-differentiated, 105 (61%) were moderately-
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differentiated and 16 (9%) were poorly-differentiated tumors. All the tumors were conventional hepatocellular carcinomas. The six fibrolamellar carcinomas showed typical morphology and all were confirmed by FISH and/or RT-PCR to have the classic DNAJB1-PRKACA fusion product (22). There were 3 men and 3 women (age range, 21-48 years; median age, 32 years). None of these 6 tumors had any known background chronic liver disease and none arose within a cirrhotic liver. All these 6 fibrolamellar carcinomas were moderately-differentiated.
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CDX2 expression in hepatocellular and fibrolamellar carcinomas
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The details of CDX2 expression are summarized in Table 1. Nine (5.2%) of 172 hepatocellular
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carcinomas were positive for CDX2. Of these 9 tumors, 1 (11%) was well differentiated, 3 (33%) were moderately differentiated and 5 (56%) were poorly differentiated (Figures 1A and 2A). The
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background liver showed cirrhosis in 5 (56%) of these 9 cases and the background liver disease
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was hepatitis C in 4 (45%), hepatitis B in 1 (10%) and unknown in 4 (45%) cases. The extent of nuclear staining for CDX2 was scored as 1+ in 5 cases (1 well differentiated, 3 moderately
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differentiated and 1 poorly differentiated), 2+ in 2 cases (both poorly differentiated) and 3+ in 2 cases (both poorly differentiated). The staining pattern in 7 cases was notable for discrete
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clusters of malignant cells with areas of intervening negative tumor cells, whereas 2 cases showed scattered positive cells within the tumor (Figures 1B and 2B). Arginase-1 immunostain
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was positive in all 9 cases (Figures 1C and 2C), including the foci within the tumors that were
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CDX2 positive. Glypican-3 immunostain was performed in 5 cases and it was positive in all 5 cases (Figure 1D), including the foci within the tumors that showed CDX2 expression. CK7,
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CK19 and CK20 immunostains were performed in 6 cases and all three immunostains were negative within these 6 cases, including the foci that were CDX2 positive. The background nonneoplastic liver parenchyma was negative for CDX2 in all cases when present on the stained sections. CDX2 expression in hepatocellular carcinomas was more commonly seen in poorly differentiated tumors (5 of 16 cases, 31%) than in well and moderately differentiated tumors (4 of 156 cases, 2.5%), p value = 0.0004. No other statistically significant correlations were observed (p > 0.05) between CDX2 expression in hepatocellular carcinomas and age, gender, background liver disease and cirrhosis. All six fibrolamellar carcinomas were CDX2 negative.
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DISCUSSION
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CDX2, a homeobox nuclear transcription factor, is considered a sensitive and specific
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marker of intestinal differentiation because of its perceived restrictive pattern of expression within the gastrointestinal tract. In addition to colorectal tumors, adenocarcinomas arising within
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the esophagus, stomach and pancreatobiliary tract are also frequently CDX2 positive. The liver is
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frequently involved by metastatic carcinoma and hence, CDX2 is a widely used immunohistochemical marker in surgical pathology to identify the site of origin when dealing
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with metastatic carcinoma of unknown primary within the liver. CDX2 can also be positive in some extra-gastrointestinal tumors, especially in tumors showing intestinal or intestinal-like
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phenotypes (23). Therefore, CDX2 positivity is not entirely specific for neoplasms arising in the gastrointestinal tract, but its expression is at least considered an indication of intestinal-type
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differentiation.
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CDX2 expression in hepatocellular carcinomas has not yet been reported. Werling et al studied 12 hepatocellular carcinomas and none were CDX2 positive (4). Similar results were
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reported in another study by Moskaluk et al, in which all 12 hepatocellular carcinomas were negative for CDX2 (20). A third study on 31 hepatocellular carcinomas using tissue microarray also showed no CDX2 expression (19). However, these studies included only a small number of hepatocellular carcinomas. We sought to systematically evaluate CDX2 expression in hepatocellular carcinomas, as we had anecdotally encountered occasional hepatocellular carcinomas that were CDX2 positive. Interpretation of the positive staining was also compounded by the lack of a comprehensive evaluation of CDX2 staining in hepatocellular carcinomas within the literature. Our study is the largest to study CDX2 expression in hepatocellular carcinomas using full tumor sections and is 8
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the first to report CDX2 positivity in hepatocellular carcinomas. We have shown CDX2
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positivity in 9 (5.2%) of 172 hepatocellular carcinomas. Of these 9 cases that were positive, 5
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showed 1+ staining (<10% positive cells), 2 showed 2+ staining (11-25% positive cells) and 2 were 3+ (26-50% positive cells).
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Colonic adenocarcinomas usually show strong and diffuse CDX2 expression, however,
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CDX2 expression in adenocarcinomas arising within the pancreatobiliary tract and upper gastrointestinal tract can often show focal positivity for CDX2 (4, 20, 24). Hence focal staining
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for CDX2 in a liver tumor could be misinterpreted as indication of a metastasis from the gastrointestinal tract. However, the results of our study show that a subset (up to 5%) of
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hepatocellular carcinomas can be positive for CDX2. The main significance of aberrant CDX2 expression in hepatocellular carcinoma is for its
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potential for causing diagnostic confusion. Whether or not this becomes problematic is highly
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dependent on the clinical and morphological features of any given case, the differential diagnosis generated by the pathologist and the immunohistochemical approach taken on the case. Aberrant
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CDX2 expression in hepatocellular carcinoma may not be an issue in well to moderately differentiated hepatocellular carcinomas, in which a diagnosis is possible without ancillary immunostains such as CDX2. Poorly differentiated tumors involving the liver usually require immunohistochemical workup that may include CDX2 to distinguish metastatic poorly differentiated adenocarcinomas from poorly differentiated hepatocellular carcinomas. Five of the 13 (31%) poorly differentiated hepatocellular carcinomas in our study were CDX2 positive, indicating that a significant proportion of poorly differentiated hepatocellular carcinomas are CDX2 positive. Only one third of the hepatocellular carcinomas (57 of the 172 cases; 33%) in our study arose within a cirrhotic liver. This rate of underlying cirrhosis for hepatocellular 9
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carcinomas in our study is lower than that reported in the literature (which varies from 50% to
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90%) and the reason for this difference is not entirely clear. However, the lack of underlying
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cirrhosis increases the probability of a tumor within the liver to be a metastasis, as tumors in cirrhotic livers are less likely to be metastases. Hence, awareness of CDX2 expression in a subset
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of hepatocellular carcinomas is critical for reducing the potential for misdiagnosis of these
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tumors as non-hepatocellular carcinomas.
ACAT1 and ACAT2 are two ACAT (acyl-CoA:cholesterol acyltransferase) genes in
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humans that play an important role in cellular cholesterol homoeostasis (25). ACAT2 is mainly expressed in intestinal cells (26). CDX2 is known to govern and stimulate ACAT2 expression in
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human intestinal cells (27). A proportion of hepatocellular carcinomas have also been shown to exhibit elevated ACAT2 expression (27) and hence we speculate that this might correlate with
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CDX2 expression in a subset of hepatocellular carcinoma. CDX2 has also been shown to
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regulate the expression of Cadherin-17 (CDH17), a member of the non-classical cadherin family in colorectal cancer and stomach adenocarcinomas (28-30). Similar association between CDX2
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and CDH17 has also been shown in hepatocellular carcinoma and overexpression on CDH17 in hepatocellular carcinoma is associated with poor clinical outcomes (31). In summary, although CDX2 remains a highly sensitive and specific marker of intestinal origin in tumor pathology, this study demonstrates that a subset of hepatocellular carcinomas (5%) can be CDX2 positive. A significant proportion of poorly differentiated hepatocellular carcinomas (31%) are CDX2 positive. Awareness of this phenomenon is important, as CDX2 expression in a liver tumor does not completely exclude hepatocellular carcinoma. Appropriate immunohistochemical workup for hepatocellular carcinoma with immunostains such as arginase1, HepPar-1 and glypican-3 which are related to hepatocellular lineage as well as other 10
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immunostains such as CK7 and CK20 should be pursued in CDX2 positive liver tumors,
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particularly in poorly differentiated tumors, to avoid the possibility of missing a diagnosis of
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hepatocellular carcinoma.
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FIGURE LEGENDS
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FIGURE 1. Poorly differentiated hepatocellular carcinoma. A, H&E stain showing tumor cells
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with eosinophilic cytoplasm and large pleomorphic nuclei (200x). B, CDX2 showing nuclear expression (200x). C, Arginase-1 immunostain is positive within the tumor (200x). D, Glypican-
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3 immunostain is also positive within the tumor (200x).
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FIGURE 2. Poorly differentiated hepatocellular carcinoma. A, H&E stain showing large polygonal tumor cells with deep eosinophilic cytoplasm and few mitotic figures (200x). B,
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CDX2 is positive in small clusters of tumor cells (200x). C, Arginase-1 immunostain is positive
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TABLES
Age (yr) ≤40 >40
2 (22%) 7 (78%)
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Gender Male Female
CDX2 negative n = 163 (94.8%)
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CDX2 positive n = 9 (5.2%)
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Differentiation Well differentiated Moderately differentiated Poorly differentiated
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108 (66%) 55 (34%) 0.50
1 (10%) 4 (45%) 0 0
21 (13%) 21 (13%) 3 (2%) 2 (1%)
0 0 0 0 4 (45%)
5 (3%) 2 (1%) 1 (1%) 3 (2%) 105 (64%)
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Background liver disease Hepatitis B virus Hepatitis C virus Hepatitis B and C viruses Non-alcoholic steatohepatitis Alcoholic liver disease Autoimmune hepatitis Hereditary hemochromatosis Primary biliary cirrhosis Unknown
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6 (67%) 3 (33%)
p value
13 (8%) 150 (92%)
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Characteristics
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Table 1. CDX2 expression in hepatocellular carcinomas.
0.0004a 1 (11%) 3 (33%) 5 (56%)
50 (31%) 102 (63%) 11 (6%)
Background cirrhosis 0.16 Present 5 (56%) 52 (32%) Absent 4 (44%) 111 (68%) HCC, hepatocellular carcinoma; a p value was calculated by combining well differentiated and moderately differentiated HCC versus poorly differentiated HCC
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