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Application of Immunohistochemistry in Gastrointestinal and Liver Neoplasms :New Markers and Evolving Practice.

Immunohistochemistry (IHC) is commonly used in the diagnosis of gastrointestinal (GI) and liver neoplasms to facilitate accurate tumor classification. (1-4) There are two practical goals: One is to confirm a tumor diagnosis by excluding morphologic mimickers or to identify the most reasonable tissue or organ of origin in cases of metastatic carcinoma of unknown primary. (5,6) The other is to provide meaningful prognostic information and even predict responsiveness to standard chemotherapy or novel molecular targeted therapy. (7-10) In the last 5 to 6 years, several new sensitive and specific markers with proven diagnostic value have become available for pathologists to better address these goals. The purpose of this review is to discuss practical applications of these new markers in the context of common problematic issues encountered in the routine diagnosis of

GI tract and liver tumors. We limited our scope to include mainly tumors of epithelial cell origin and will not emphasize mesenchymal tumors, with the exception of GI stromal tumors (GISTs) and tumors of lymphoid origin.

It is noteworthy that assessment of prognostic and predictive markers by IHC in GI tumors has become increasingly popular. (7) Microsatellite instability (MSI) in colorectal carcinoma (CRC), (11,12) human epithelial growth factor receptor 2 (HER2) in gastric and gastroesophageal junction (GEJ) adenocarcinoma, (8-10) and even Ki-67 in neuroendocrine tumors (NETs) (13,14) are some familiar examples. Currently, not only are pathologists depending on IHC results for diagnosis, but our clinical colleagues have also become interested in knowing the results before they start to formulate the best management plan for individual patients. In a sense, this has begun a new trend to transform IHC from a traditional qualitative assay most useful in distinguishing tumor types to a quantitative clinical test whose result is essential for clinical decision making. With rapid advances in our understanding of molecular and genetic mechanisms of carcinogenesis and a continuous push for personalized cancer therapy, it is highly likely that this trend will rapidly progress. As practicing pathologists, it is our clinical responsibility to be vigilant in overseeing all aspects of IHC to ensure test precision and result accuracy.


The most common primary tumors of epithelial origin in liver are hepatocellular carcinoma (HCC) (15) and cholangiocarcinoma (CCA) (16) in adults, and hepatoblastoma (17) in young children. Hepatocellular adenoma (HCA) is a relatively rare benign tumor and must be distinguished from HCC and other benign nonneoplastic hepatic lesions. (18) Metastatic carcinomas are common in the liver. Differentiating them from primary liver tumors can pose a real diagnostic challenge for pathologists.


Based on classic morphologic features, most HCCs can be easily recognized on hematoxylin-eosin sections. However, many types of benign or malignant tumors may share morphologic similarities. Some of the most notorious mimickers of HCC include adrenocortical carcinoma, renal cell carcinoma, clear cell sarcoma, melanoma, large cell neuroendocrine carcinoma, and angiomyolipoma. (19-21) In addition, poorly differentiated HCC and some HCC variants may be difficult to discern based on morphology alone. (22) In these circumstances, IHC is not only helpful but necessary for accurate diagnosis. In our experience, the most effective IHC approach to address these problematic issues is to positively confirm hepatocellular differentiation in the tumor cells in conjunction with various tumor-specific markers (covered in other review articles in this 2-part special IHC series of articles) to exclude possible mimickers. Table 1 lists a recommended panel of IHC markers along with their reported sensitivities and specificities for diagnosing HCC. The top three markers are preferable because they show a cytoplasmic expression pattern that is easily recognizable, even in small biopsies with disrupted architecture or in cytologic specimens. The other markers are useful supplements to increase sensitivity and specificity. Within the panel, arginase 1 (ARG1) (19,23-27) is a newly described marker with promising performance.


ARG1 is a binuclear manganese metalloenzyme involved in the urea cycle. It catalyzes the hydrolysis of arginine to ornithine and urea. (28) Recent studies have clearly demonstrated that it is a great marker for hepatocellular differentiation. (19) Compared with hepatocyte paraffin 1 (HepPar1) and glypican 3 (GPC3), it shows better sensitivity and specificity. (23-27) It is also positive in hepatoblastoma. So far, only rare cases of non-HCC carcinomas have been reported to be immunoreactive to ARG1. (19,26,27) To the best of our knowledge, there has not been any report of ARG1 expression in hepatoid carcinoma, a rare tumor from extrahepatic sites (mostly stomach, pancreas, and uterus) that shows hepatocellular differentiation. (29-33) In our experience, ARG1 is best used together with either HepPar1 or GPC3 to increase diagnostic sensitivity and specificity for HCC. Figure 1, a and b, show staining patterns of HepPar1 and ARG1 in a needle biopsy specimen of HCC. When all 3 markers are used simultaneously, they can identify almost all HCCs, including rare variants. (22) However, caution is still warranted when the combination of these markers is used to differentiate metastatic carcinomas from HCC, particularly hepatoid carcinoma. In difficult scenarios, a distinction between primary HCC and metastasis has to be made on clinical grounds based on a patient's history and an imaging survey of most suspicious sites.


Scirrhous HCC is a rare morphologic variant, composed of less than 5% of HCCs. (34) It is characterized by marked stromal fibrosis, subcapsular location, multinodularity, and changes in clear cells, with preserved intratumoral portal tracts. It also shows an unusual IHC phenotype. Most of the tumors are negative for HepPar1 and positive for cytokeratin 7 (CK7), CK19, and epithelial cell adhesion molecule (EPCAM), making it difficult to distinguish from intrahepatic CCA (IHCCA) and metastatic adenocarcinoma. It is supposed to have a better prognosis than IHCCA. (35) Recently, Krings et al (22) studied 20 such tumors and found that 85% were positive for ARG1 and 79% were positive for GPC3, whereas only 26% were positive for HepPar1; 53% of the tumors were also positive for CK7, and 26% were positive for CK19.

Fibrolamellar HCC is also a variant of HCC with a distinct morphology and characteristic clinicopathologic features. (36) Unlike most conventional HCCs, the tumor cells are often positive for CK7 and embedded in the dense fibrosis of a noncirrhotic liver. (37,38) Sometimes it can be challenging to distinguish fibrolamellar HCC from metastatic tumor, epithelioid hemangioendothelioma, or IHCCA, particularly in small-needle biopsy specimens. (38) It is helpful to confirm the diagnosis by demonstrating expression of hepatocellular differential markers. Recently, it was found that most tumors also show immunoreactivity to CD68 in an antibody-dependent fashion. (39) The positive rate is higher when the KP-1 clone is used, compared with other clones. (3) This unique finding is also useful in practice to differentiate fibrolamellar HCC from conventional HCC, which is important for prognostic indication. (36)


Hepatoblastoma is the most common primary liver neoplasm in children younger than 5 years. (17) Morphologically, it is usually composed of embryonal and fetal hepatocyte-like cells with or without a mesenchymal component, (40) although in rare cases a purely fetal-type hepatoblastoma may be difficult to distinguish from HCC. A recent study of immunophenotypes of the tumor revealed that high-mortality group AT hook 2 (HMGA2) is usually positive in hepatoblastoma cells, as are GPC3 and [beta]-catenin. (41) Interestingly, although HMGA2 appears to be positive in all components, GPC3 is more likely to be positive in the fetal component, and [beta]-catenin positivity is mainly seen in the embryonal component. (41) The same study also showed that HMGA2 was positive in about 40% of HCCs developed in patients younger than 30 years and was seldom positive in HCCs from older patients. (41,42) The clinical significance of this finding is still elusive. In our experience, ARG1 is also positive in hepatoblastoma cells, particularly in the fetal component. Figure 1, c and d, show examples of HepPar1 and ARG1 staining in a case of hepatoblastoma.


Hepatocellular adenoma is a relatively rare benign tumor of the liver. (18) Recent studies have suggested a molecular classification system that identifies 4 major subtypes (43,44): hepatocyte nuclear factor 1 alpha (HNF1a)-mutated HCA (30%-35%); b-catenin-mutated HCA (10%-15%); inflammatory HCA (50%); and unclassified (roughly 10%). Within inflammatory HCAs, 10% of tumors may also be [beta]-catenin mutated. Identification of [beta]-catenin-mutated HCA is important because these tumors show a strong association with risk of HCC. Although some unique morphologic features may be found in certain subtypes, a set of 4 or 5 IHC markers is needed for accurate classification. (18,44) A summary of these markers and their staining patterns in HCA subtypes is listed in Table 2.

Differentiating inflammatory HCA from focal nodular hyperplasia, a nonneoplastic condition related to aberrant vascular proliferation, sometimes can be challenging on a small biopsy. It has been noted that focal nodular hyperplasia usually shows a unique geographic pattern of glutamine synthetase stain and is consistently negative for serum amyloid A protein (SAA) and C-reactive protein (CRP). (17,44,45) On the contrary, inflammatory HCA is usually negative for glutamine synthetase (diffuse positivity seen in tumors with [beta]-catenin activation) and positive for SAA and CRP. A caveat is that the staining patterns may be difficult to interpret when lesional tissue is small or when not enough normal liver tissue is present to compare.

Another practical challenge in diagnosing HCA is to differentiate it from well-differentiated HCC. (46) A number of studies have suggested panels of IHC markers to help in this distinction. (46,47) Table 3 lists some of the most commonly used IHC markers for this purpose. In addition, reticulin stain is also helpful in revealing the abnormal trabecular pattern in HCC. Some pathologists like to use endothelial markers, such as CD34, to illustrate thickened trabeculae. In our experience, these markers generally have low sensitivity or specificity for HCC, and one has to be cautious not to rely solely on them for the diagnosis. It is noteworthy that the term well-differentiated hepatocellular neoplasm of uncertain malignant potential (HUMP) has been proposed by some experts to describe HCA-like lesions with atypical features. (48,49) However, at present this is just an evolving concept. There has been no consensus on any specific diagnostic criteria.


The practical challenge in the diagnosis of IHCCA is to distinguish it from metastatic adenocarcinomas from various sites. In most cases this can be resolved quite effectively with the help of IHC and a detailed clinical history. A list of tumor-specific markers that are commonly used for this distinction is summarized in Table 4. However, differentiating IHCCA from metastatic pancreatic ductal adenocarcinomas or adenocarcinoma from the upper GI tract can be extremely difficult, if not impossible. Even IHC can offer little help because of the lack of tissue-specific markers due to the close relationship of these anatomic sites in the embryonic and fetal development process. Interestingly, a recent study by Lok et al (50) showed that a panel of nonconventional markers (placental S100 [S100P], von Hippel-Lindau tumor suppressor [pVHL], mucin 5AC [MUC5AC], and CK17) may offer more help than anyone would anticipate. They studied the staining patterns in 41 IHCCAs and 60 pancreatic ductal adenocarcinomas and identified a specific pattern ([S100P.sup.-]/[pVHL.sup.+]/[MUC5AC.sup.-]/ [CK17.sup.-]) essentially indicative of IHCC, whereas two other patterns ([S100P.sup.+]/[pVHL.sup.-]/[MUC5AC.sup.+]/[CK17.sup.+] and [S100P.sup.+]/ pVHL-/MUC5AC-/[CK17.sup.+]) were more suggestive of pancreatic ductal adenocarcinoma. The IHCCA-specific pattern picked up almost 60% (24 of 41) of tumors tested. These results are quite intriguing. However, the number of cases examined was still very small. A thorough validation in a large number of cases is necessary to establish its diagnostic utility.


The GI tract is a large organ system with complex tissue composition. Histologically, the most common primary tumors of epithelial origin are adenocarcinoma and NETs. (51) Some tumors may show dual differentiation, particularly when they exhibit poorly differentiated or undifferentiated morphology. Immunohistochemistry is helpful and often necessary to confirm the diagnosis. In addition, more and more frequently, IHC detection of Ki-67 is routinely performed in NETs to calculate proliferative index and to predict the aggressiveness of these tumors. (52) Other IHC applications focusing on assessment of predictive or prognostic markers in GI tumors have also been gaining popularity. (7-12) Currently, these tests include HER2 in gastric and GEJ adenocarcinomas, and MSI in CRCs, with the most recent development of the mutation-specific IHC marker vraf murine sarcoma viral oncogene homolog B (BRAF) V600E. (53-55) Nevertheless, in daily practice a major application of IHC remains to help resolve problematic diagnostic issues, such as distinguishing a possible metastatic carcinoma from an adjacent organ system or, rarely, a carcinoma of unknown primary involving the GI tract. In this regard, IHC markers highly sensitive and specific for GI tumors are potentially of help. A set of IHC markers is routinely used to facilitate the distinction. These include CK7, CK20, villin, caudal type homeobox 2 (CDX2), mucin core proteins (MUCs), and others. (56) Recently, two new markers were added to the list, and their enhanced ability to further improve diagnostic accuracy has been gradually appreciated.


Cadherin 17 (CDH17) is also known as liver-intestine cadherin because it was originally discovered as a novel calcium-dependent cell adhesion molecule expressed in the liver and intestine of rats. (57,58) In humans its distribution is actually limited to the duodenum, jejunum, ileum, colon, and part of the pancreatic duct. It is believed to function as an intestinal peptide transporter. (58,59) Its clinical utility in diagnosing GI tumors was only recognized recently. (60-62) The combined data indicate that positive CDH17 immunoreactivity is most commonly seen in colorectal adenocarcinomas (up to 96%) and a significant portion of gastric, pancreatic, and biliary adenocarcinomas (25%-50%). It is rarely found in adenocarcinomas from outside of GI tract (1%-10%). Interestingly, although CDH17 is transcriptionally regulated by CDX2, some authors found it to be slightly more sensitive and specific than CDX2 in identifying colorectal adenocarcinomas. (60,62) Recently, we also studied CDH17 immunoreactivity in a large number of tumors derived from various organ systems. (63) Not only did our data further confirm the reported findings, they also demonstrated its usefulness in diagnosing CRC variant with poorly differentiated or undifferentiated morphology, such as medullary carcinoma, which characteristically lacks expression of conventional intestinal differential markers, such as CK20 and CDX2. CDH17 has also been reported as a sensitive marker for intestinal metaplasia, and thus helpful for histologic diagnosis of early Barrett esophagus. (64-66) Recently, CDH17 has also been studied as a potential prognostic marker in GI and pancreatobiliary carcinomas; however, its clinical implication has not been well established. (67,68)


Special AT-rich sequence binding protein 2 (SATB2) belongs to a family of nuclear matrix-associated transcription factors that function as epigenetic regulators of gene expression in a tissue-specific manner. (69-72) Studies have shown that SATB2 carries out a wide spectrum of biologic functions. It is a transcriptional activator of immunoglobulin i expression. (70) It also regulates neuronal and osteoblast differentiation. (69) Haploinsufficiency of the SATB2 gene is associated with cleft palate syndrome in humans. (71) However, the role of SATB2 in the GI tract is still elusive. Recently, Magnusson et al (73) found that SATB2 immunoreactivity was restricted to the glandular lining cells of the human lower GI tract, including appendix, colon, and rectum, and a subset of neuronal cells in the cerebral cortex and hippocampus. Some lymphocytes and cells lining the seminiferous ducts and epididymis also showed weak to moderate immunoreactivity. All other tissue types tested were negative. They also studied SATB2 expression in a large number of human carcinomas. Positive nuclear stain was found in 1336 of 1558 primary colon adenocarcinomas (86%) and 205 of 252 metastatic carcinomas of the colon (81%). (73) Within the noncolonic carcinomas, weak positive immunoreactivity was found in 6 of 147 breast adenocarcinomas, 3 of 53 lung adenocarcinomas, 5 of 153 ovarian carcinomas, 1 of 15 cholangiocarcinomas, and 5 of 9 sinonasal carcinomas. Upper GI carcinomas and pancreatic adenocarcinomas were usually negative for SATB2. (73) We recently also studied SATB2 expression in a large number of tumors derived from various organs. (63) Our results confirm that SATB2 is a highly sensitive and specific marker for adenocarcinomas of the colon and rectum, with a diagnostic sensitivity of 97% (121 of 125 cases) in CRCs. Interestingly, we also found that SATB2 immunoreactivity was seen in a significant number of medullary carcinomas of the colon.

In addition to diagnostic utility, recent reports also indicate a prognostic value of SATB2 in CRCs. (74,75) High SATB2 expression was associated with good prognosis in colon cancer and might modulate sensitivity to chemotherapy and radiation, whereas reduced expression of SATB2 in colorectal adenocarcinomas was found to be associated with poor prognosis, including tumor invasion, lymph node metastasis, and distant metastasis.


Compared with CDH17 and CDX2, SATB2 immunoreactivity is much more selective for CRC and is rarely seen in carcinomas of the esophagus and stomach. Whether SATB2 can also help to differentiate a primary small intestinal adenocarcinoma from metastatic CRC involving the small intestine has not been formally tested. This may be a practical diagnostic challenge, and currently no specific IHC markers can offer resolution. (56,76) We recently examined SATB2 immunoreactivity in 5 primary small intestinal adenocarcinomas and found that 3 were negative. The other 2 tumors with positive immunoreactivity were from the distal ileum, where the normal small intestinal mucosa adjacent to tumors was also positive for SATB2. In contrast, all 4 metastatic colonic carcinomas involving the small intestine showed positive immunoreactivity to SATB2. These preliminary results are intriguing and suggest that SATB2 may be useful in distinguishing adenocarcinomas from the small and large intestine. However, a couple of caveats warrant caution. First, the differential capacity is dependent on whether or not SATB2 immunoreactivity is present in the normal small intestinal mucosa in the same region where the tumor occurs. Second, SATB2 expression may be down-regulated or completely lost in some colonic adenocarcinomas with aggressive behavior; a comparison with the expression profile of the original tumor may be recommended.


Medullary carcinoma of the colon is a distinct variant of colonic adenocarcinoma. (77,78) It usually occurs in elderly patients, more frequently in women than men (2:1 ratio), and often presents as a large mass in the right colon, especially in the cecum. Histologically, the tumor is characteristic for a number of features, including poorly differentiated or undifferentiated morphology, pushing border invasion, markedly increased intratumoral lymphocytosis, and peritumoral Crohn-like lymphoid reaction. Molecular pathogenically, almost all tumors are MSI with deficiency in mismatch repair proteins, predominantly a lack of MutL homolog 1 (MLH1)/postmeiotic segregation increased 2 (PMS2) expression due to MLH1 promoter hypermethylation. (79,80) Like most MSI colon adenocarcinomas, despite high tumor stages and grades, the prognosis in terms of lymph node and distant metastasis is more favorable compared with microsatellite-stable poorly differentiated adenocarcinomas or neuroendocrine carcinomas of the large intestine. Immunohistochemically, the tumor is also unique. Unlike conventional colorectal adenocarcino mas, medullary carcinoma of the colon frequently lacks CDX2 and CK20 expression. (81-83) Instead, it may express markers not commonly associated with CRCs, such as CK7 and calretinin. (83) Given these unusual features, an accurate diagnosis of this tumor can be quite challenging, particularly in a metastatic setting.

Recently we studied the diagnostic utility of SATB2 and CDH17 in a cohort of 18 medullary carcinomas of the colon. (63) We found that CDH17 was positive in 16 of 18 cases (89%), and SATB2 was positive in 16 of 18 cases (89%). Interestingly, the 2 CDH17-negative cases were positive for SATB2, and the 2 SATB2-negative cases were positive for CDH17. Nearly all positive cases showed a diffuse and strong staining pattern (Figure 2). In stark contrast, only 5 of 18 cases (25%) were focally positive for CK20, and 5 of 18 cases (27%) were convincingly positive for CDX2. Our data demonstrate the usefulness of CDH17 and SATB2 in diagnosing medullary carcinoma of the colon. They also suggest that the combined use of the two markers may increase diagnostic sensitivity. Figure 2, a and b, demonstrate staining patterns of SATB2 and CDH17 in a medullary carcinoma of the colon, respectively.


The GI tract is a common site for NETs to develop. Most GI NETs are well differentiated, and their biologic behavior is primarily dependent on anatomic location, tumor size, and mitotic counts or Ki-67 proliferative index. (13) Clinically, it is not uncommon to encounter metastases to lymph nodes and liver even before the primary NET is big enough to be recognized by conventional imaging studies. It is therefore highly desirable to have GI-specific markers to help diagnose and differentiate other NETs, such as those from pancreas or lung. On the other hand, primary high-grade neuroendocrine carcinomas of the GI tract are relatively rare. (84) When such a tumor is encountered, it is important to exclude the possibility of metastasis from a small cell carcinoma of the lung, where these tumors most commonly occur. The diagnostic utility of IHC in distinguishing NETs from various organ systems has been extensively studied. (85,86) However, very little is known about the usefulness of SATB2 and CDH17 in this setting. One report demonstrated that CDH17 immunoreactivity was present in 100% of well-differentiated NETs of the small intestine and appendix, whereas CDX2 immunoreactivity was present only in 74% and 90% of cases, respectively. A minority of pancreatic endocrine tumors (3 of 26 cases; 12%) and bronchial carcinoid tumors (12 of 50 cases; 24%) were also immunoreactive to CDH17 but were all negative for CDX2. (62) These findings suggest that CDH17, compared with CDX2, is more sensitive but less specific in well-differentiated GI NETs. There has not been any published study on SATB2 expression in NETs. However, data presented in a poster from the 2013 United States and Canadian Academy of Pathology meeting showed that SATB2 immunoreactivity was seen in most well-differentiated NETs of hindgut origin. (87)

We recently examined CDH17 and SATB2 immunoreactivity in 158 well-differentiated NETs from various anatomic sites (data not shown) and found that both markers are highly selective for GI NETs. Figure 3 shows examples of CDH17 and SATB2 immunostaining patterns in a well-differentiated NET of the rectum metastasizing to liver. Although our preliminary data on CDH17 immunoreactivity are similar to the published results, the data on SATB2 immunoreactivity are more interesting. We found that positive SATB2 immunoreactivity is seen in most NETs from the appendix, colon, and rectum but is rarely seen in NETs from the stomach, duodenum, pancreas, or lung. Taken together, in our experience both CDH17 and SATB2 are potentially useful markers for diagnosing GI NETs, and SATB2 seems more specific for NETs from the lower GI tract. In practice, one should consider using these new markers together with CDX2 to maximize sensitivity and specificity for GI NETs.


Succinate dehydrogenase (SDH)-deficient GIST has been recognized as a unique variant with characteristic clinicopathologic features. (88-90) It tends to occur in young female patients, with an exclusively gastric location. In fact, a great majority of pediatric GISTs and all GISTs that occur in Carney triad and Carney-Stratakis syndrome are found to be in this category. Histologically, they show a predominantly epithelioid morphology and often a plexiform growth pattern. They have a tendency to be associated with multifocal or metachronous disease. They usually show an indolent biologic behavior and may not follow the risk stratification rule (based on size and mitosis) for conventional GISTs. From a clinical perspective, it is important to recognize this variant because of its absence of KIT (CD117) or platelet-derived growth factor receptor mutations and its primary resistance to imatinib therapy. The genetic pathogenesis of these tumors is complex because of the fact that multiple subtypes of SDH may be affected. (88) However, from a diagnostic perspective only one specific IHC assay for succinate dehydrogenase B (SDHB) is necessary and sufficient to identify all SDH-deficient GISTs. (88,89) The complete absence or a significant reduction of SDHB immunoreactivity in tumor cells essentially confirms the diagnosis. (91)


HER2 is an important driver of tumorigenesis in several solid tumors. (92) For many years, anti-HER2 targeted therapy has been effective in the treatment of breast carcinomas with HER2 gene amplification. (93) A recent clinical trial also demonstrated that trastuzumab, an anti-HER2 agent, could prolong survival in patients with HER2-positive gastric or GEJ adenocarcinoma. (94) This has started a new era of HER2 testing in these GI tumors. A number of excellent review articles were published in the last 2 years to provide comprehensive background knowledge and up-to-date practical guidance on HER2 testing and scoring in gastric and GEJ carcinomas. (8-10) To avoid repetition here, we simply point out a couple of the most important practical issues that may help to ensure accurate and consistent HER2 testing results. First, specific training is required before a pathologist can embark on HER2 testing in gastric and GEJ cancer. (8) This should be required regardless of his or her previous experience with HER2 testing in breast cancer, because significant differences in staining pattern and scoring criteria exist between the two tumors. Secondly, due to HER2 heterogeneity in gastric and GEJ adenocarcinomas, it is preferred to perform the test on resection specimens whenever possible; when an in situ hybridization test is performed to examine gene amplification in IHC 2+ samples, it is most helpful to use IHC-stained sections as a guide to locate the same area of interest for counting the in situ hybridization signals. The overall HER2-positive rate in diffuse-type gastric or GEJ adenocarcinoma is lower (5%) compared with intestinal type (up to 30%). (8-10,95) It may be worth the effort to perform in situ hybridization to confirm an IHC 3+ result, particularly if a signet ring cell carcinoma is involved. We have noticed a peculiar false-positive circumferential membranous staining pattern in some of the signet ring cell carcinomas tested. With regard to in situ hybridization interpretation, HER2 copy number equal to or greater than 6 should be considered as positive for amplification regardless of HER2/chromosome enumeration probe 17 ratio. (8)


BRAF represents one of the most frequently mutated protein kinase genes in human tumors. (96) It is found in melanoma, papillary thyroid carcinoma, ovarian serous tumors, CRCs, gliomas, hepatobiliary carcinomas, and hairy cell leukemias. (97-101) In CRC, the most common mutation is BRAF V600E. Currently, the mutation is tested in CRC mainly for two purposes. BRAF V600E mutation in MSI CRCs can virtually exclude Lynch syndrome, and mutation-positive tumors are resistant to anti-epithelial growth factor receptor therapy. (53,54) Although it has been shown that BRAF V600E mutation predicts a poor prognosis in right-sided microsatellite-stable CRC, this prognostic indication has not been widely explored clinically. (102)

Traditionally, BRAF mutation is detected by DNA sequencing or polymerase chain reaction-based mutation detection methods. Recently, antibodies specific to BRAF V600E have been developed, and their use in IHC on formalin-fixed, paraffin-embedded tumor tissue has become popular. (53-55,97,98,100-103) Currently, 2 antibodies are commercially available. VE1 is the most commonly used antibody clone and is recommended because of its high sensitivity and specificity. (104)

At present, BRAF V600E IHC has not been widely used in diagnostic laboratories. However, several recent publications have shown promising results using clinical samples. (53-55,97,98,100-104) It seems that a new trend of using IHC as screening test for BRAF V600E mutation in CRC has evolved. In fact, some authors have proposed incorporating BRAF V600E IHC into the current algorithm for universal screening of CRC for Lynch syndrome. (53-55,100-103)

However, from a technical perspective, BRAF V600E IHC still has some issues to be ironed out before it can be deployed to routine clinical labs. Since its debut there have been some negative comments in the literature regarding the sensitivity and specificity of the assay for clinical use. (105) This concern no longer exists in the most recently published data, which indicate that BRAF V600E IHC is a sensitive and reliable assay, generating results that correlated well with those from molecular detection methods. (53,54,101-103) It is most likely that optimizing the staining protocol has contributed to the recent improvement of the assays performance. Nevertheless, it is our experience that even with an ideal staining protocol, the cytoplasmic staining intensity in BRAF V600E-mutated tumor samples may still vary significantly, ranging from weak to strong. Background nonspecific stain, particularly a peculiar nuclear staining, is common in normal mucosa as well as nonmutated tumor cells. These variables may cause significant problems in accurately interpreting the staining results. Therefore, specific training for pathologists and an appropriate quality assurance program with prospective validation are absolutely needed before the assay can be widely used.

Please Note: Illustration(s) are not available due to copyright restrictions.


As the new era of genomic medicine unfolds, personalized cancer therapy will become a reality. This will require a new standard of diagnostic accuracy and precision. Immunohistochemistry as an integral component of the practice of diagnostic pathology will continuously evolve to help us meet the new challenge. As practicing pathologists, it is our responsibility to lead this evolution and to ensure the best practice of IHC for patient care.


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Zongming Eric Chen, MD, PhD; Fan Lin, MD, PhD

Accepted for publication May 29, 2014.

From the Department of Laboratory Medicine, Geisinger Medical Center, Danville, Pennsylvania.

The authors have no relevant financial interest in the products or companies described in this article.

Reprints: Zongming Eric Chen, MD, PhD, Department of Laboratory Medicine, MC 01-31, Geisinger Medical Center, 100 N Academy Ave, Danville, PA 17822 (e-mail:

Caption: Figure 1. a and b, Hepatocellular carcinoma. a, Hepatocyte paraffin 1 (HepPar1) staining. b, Arginase 1 (ARG1) staining. c and d, Hepatoblastoma. c, HepPar1 staining. d, ARG1 staining (original magnifications X100 [a and b] and X200 [c and d]).

Caption: Figure 2. Medullary carcinoma of the colon. a, Special AT-rich sequence binding protein 2 staining. b, Cadherin 17 staining (original magnification X200).

Caption: Figure 3. Metastatic, well-differentiated neuroendocrine tumor from rectum. a, Cadherin 17 staining. b, Special AT-rich sequence binding protein 2 staining (original magnification X200).
Table 1. A Panel of Immunomarkers Helpful in
Confirming Hepatocellular Differentiation

Markers   Staining Pattern   Sensitivity, %   Specificity, %

ARG1      Cytoplasmic            80-95            95-100
            and nuclear
HepPar1   Cytoplasmic            70-80             ~80
GPC3      Cytoplasmic            50-80             ~95
pCEA      Canalicular              90              l00
CD10      Canalicular             n/a              n/a
CD34      Sinusoidal              n/a              n/a

Abbreviations: ARG1, arginase 1; GPC3, glypican 3; HepPar1,
hepatocyte paraffin 1; n/a, not available; pCEA, polyclonal
carcinoem-bryonic antigen.

Table 2. Immunohistochemistry Markers and Staining Patterns in
Different Types of Hepatocellular Adenoma

                             LFABP     SAA/CRP

HNF1 [alpha] inactivation   Negative   Negative
[beta]-Catenin activated    Positive   Negative
Inflammatory                Positive   Positive
Unclassified                Positive   Negative
FNH                         Positive   Negative
Normal liver                Positive   Negative


HNF1 [alpha] inactivation   Central vein
[beta]-Catenin activated    Diffusely positive
Inflammatory                Central vein
Unclassified                Central vein
FNH                         Irregular anastomosing
Normal liver                Central vein


HNF1 [alpha] inactivation   Membranous
[beta]-Catenin activated    Nuclear
Inflammatory                Membranous or nuclear
Unclassified                Membranous
FNH                         Membranous
Normal liver                Membranous

Abbreviations: CRP, C-reactive protein; FNH, focal nodular
hyperplasia; GS, glutamine synthetase; HNF1 a, hepatocyte nuclear
factor la; LFABP, liver fatty acid binding protein; SAA, serum
amyloid-associated protein.

Table 3. Immunohistochemistry Markers to
Differentiate Well-Differentiated Hepatocellular
Carcinoma From Hepatocellular Adenoma

Markers   Malignant    Benign         Sensitivity,   Specificity,
                                           %              %

GPC3      Positive     Negative          40-60          95-100
HSP70     Positive     Negative          40-60          95-100
GS        Diffuse      Patchy/focal       ~80            ~50
PCNA or   High         Low                ~90            ~60

Abbreviations: GPC3, glypican 3; GS, glutamine synthetase; HSP70,
heat shock protein 70; PCNA, proliferative cell nuclear antigen.

Table 4. Immunohistochemistry Markers to Differentiate
Intrahepatic Cholangiocarcinoma (IHCCA) From Metastases (a)

Markers    IHCCA    Lung-A    PDA     Upper GI   Colon   Breast

CK7          +        +        +         +         -       +
CK20         -        -        -       + or -      +       -
GATA3        -        -       -/+        -         -       +
ER           -        -        -         -         -       +
TTF1         -        +        -         -         -       -
Napsin A     -        +        -         -         -       -
SATB2        -        -        -       - or +      +       -
pVHL         +        -        -         -         -       -
CDH17       +/-       -       +/-        +         +       -
DPC4       - or +     +      - or +      +         +       +
CK17       + or -     -      + or -      -         -       -

Markers    Bladder

CK7           +
GATA3         +
ER            -
TTF1          -
Napsin A      -
SATB2         -
pVHL          -
CDH17        -/+
DPC4          +
CK17         -/+

Abbreviations: Bladder, urothelial carcinoma; CDH17, cadherin 17; CK,
cytokeratin; DPC4, SMAD family member 4; ER, estrogen receptor; GATA3,
GATA-binding protein 3; GI, gastrointestinal; Lung-A, lung
adenocarcinoma; PDA, pancreatic ductal adenocarcinoma; pVHL, von
Hippel-Lindau tumor suppressor; SATB2, special AT-rich sequence
binding protein 2; TTF1, thyroid transcription factor 1.

(a) "+" indicates that usually more than 75% of cases are
positive; "-," less than 5% of cases are positive; "+ or -," usually
more than 50% of cases are positive; and "- or +," less than 50% of
cases are positive.
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Title Annotation:Special Issue--An Update in Immunohistochemistry (Part II)
Author:Chen, Zongming Eric; Lin, Fan
Publication:Archives of Pathology & Laboratory Medicine
Date:Jan 1, 2015
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