International Journal of Surgical Pathology

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The Utility of Immunohistochemistry for Providing Genetic Information on Tumors John K. C. Chan, Yiu-Tung Ip and Wah Cheuk INT J SURG PATHOL 2013 21: 455 DOI: 10.1177/1066896913502529 The online version of this article can be found at: http://ijs.sagepub.com/content/21/5/455

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IJSXXX10.1177/1066896913502529International Journal of Surgical PathologyChan et al

Review Article

The Utility of Immunohistochemistry for Providing Genetic Information on Tumors

International Journal of Surgical Pathology 21(5) 455–475 © The Author(s) 2013 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1066896913502529 ijs.sagepub.com

John K. C. Chan, M.B., B.S.1, Yiu-Tung Ip, M.B., Ch.B.2, and Wah Cheuk, M.B., B.S.1

Abstract With advances in immunohistochemical technology and growing knowledge of the molecular genetics of tumors, immunohistochemistry is playing an increasingly important role in providing genetic information for tumors. Specific chromosomal translocations can be demonstrated through detection of the protein product of one of the genes involved in gene fusion (such as BCL2, cyclin D, and ALK). Some mutations can be detected by (1) aberrant localization of the protein product (such as β-catenin and nucleophosmin), (2) abnormal accumulation of the protein product as a result of stabilization of the protein (such as p53), and (3) mutation-specific antibodies directed against the mutant protein (such as isocitrate dehydrogenase gene R132H mutation, epidermal growth factor receptor gene L858R and exon 19 deletion mutations, and BRAF gene V600E mutation). Gene deletion or loss of function can be demonstrated by the loss of immunostaining for the protein product (such as mismatch repair proteins in microsatellite-unstable tumors, E-cadherin in lobular carcinoma of the breast, and INI1 in rhabdoid tumors, atypical teratoid/rhabdoid tumors, and epithelioid sarcomas). Gene amplification can be demonstrated by overexpression of the protein product (such as HER2 in breast and gastric cancers, and MDM2 or CDK4 in well-differentiated/dedifferentiated liposarcomas). Viruses associated with tumors can be demonstrated directly (such as Epstein-Barr virus latent membrane protein-1 in Hodgkin lymphomas, human herpesvirus 8 in Kaposi sarcomas, and Merkel cell polyomavirus in Merkel cell carcinomas) or by a surrogate marker (such as p16 in human papillomavirus infection). In this review, examples are given to illustrate the principles and pitfalls of these applications. Keywords immunohistochemistry, molecular genetics, tumors, chromosomal translocation, gene mutation, mutation-specific antibody, gene deletion, gene amplification, virus

A New Era in Immunohistochemistry: Providing Genetic Information on Tumors Immunohistochemistry is an indispensable tool for the surgical pathologist. It has wide applications in everyday practice, such as determination of the nature, lineage, or differentiation of normal cells or tumors; distinguishing between benign and malignant neoplasms; assessment of the likely primary site for tumors of uncertain origin; and demonstration of the existence of microorganisms. With the availability of highly effective antigen retrieval techniques, highly sensitive immunohistochemical detection systems, and a wide spectrum of new antibodies and with better understanding of the molecular alterations in tumors, immunohistochemistry is playing an increasingly important role in providing genetic information about tumors.1 In selected circumstances, immunohistochemistry can replace molecular analysis, such as ALK expression indicating the presence of ALK gene translocation. In other

circumstances, immunohistochemistry can complement molecular analysis by serving as a screening tool to triage samples for the latter analysis. Compared with molecular studies, immunohistochemical studies are less expensive and less labor-intensive, can be performed in routine diagnostic laboratories, and can usually be completed in a much shorter time. Types of molecular alterations in tumors that can potentially be detectable by immunohistochemistry include (1) specific chromosomal translocations, (2) specific mutations, (3) gene deletion or loss of function, (4) gene amplification, and (5) viruses associated with tumors (Table 1). In this review, examples are given to illustrate the principles and 1

Queen Elizabeth Hospital, Kowloon, Hong Kong, SAR China Tuen Mun Hospital, Hong Kong, SAR China

2

Corresponding Author: John K. C. Chan, M.B., B.S., Department of Pathology, Queen Elizabeth Hospital, Gascoigne Road, Kowloon, Hong Kong, SAR China. Email: [email protected]


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Table 1. Applicability of Immunohistochemistry for Demonstrating Molecular Alterations in Tumors. Molecular Alterations in Tumors

Principles of Immunohistochemical Staining

Examples

Chromosomal translocation (gene fusion) → Overexpression of protein encoded by one of the genes involved in fusion or a chimeric protein encoded by the 2 genes involved in fusion → Positive immunostaining in tumor cells

• BCL2 translocation in follicular lymphoma → BCL2 staining of follicle centers • CCND1 translocation in mantle cell lymphoma → Cyclin D1 immunoreactivity • ALK translocation in various tumor types → ALK immunoreactivitya • NUT translocation in NUT midline carcinoma → NUT immunoreactivity • ERG translocation in prostate carcinoma → ERG immunoreactivitya

Gene mutation → Aberrant subcellular localization of antigen/protein

• Nuclear translocation of β-catenin (such as colorectal carcinoma, desmoid fibromatosis, and cribriform morular variant of papillary thyroid carcinoma) • Cytoplasmic translocation of nucleophosmin in a subset of acute myeloid leukemia

Gene mutation → Stabilization of protein → Extensive strong immunostaining for the gene product (protein)

• p53 mutation in various tumor types, for example, highgrade serous adenocarcinoma of the ovary

Mutation-specific antibody demonstrates presence of altered protein (resulting from gene mutation); the antibody does not react with the wild-type protein

• Isocitrate dehydrogenase 1 (IDH1) R132H mutation (in gliomas) • EGFR L858R or exon 19 deletion mutation (in pulmonary adenocarcinomas) • BRAF V600E mutation (in various tumor types)

Presence or lack of mutation of certain genes → Overexpression of certain surrogate markers

• Chronic lymphocytic leukemia with unmutated

Inactivating mutation, deletion or promoter hypermethylation of gene → Loss of expression of the encoded protein

• Loss of E-cadherin staining in lobular carcinoma of breast • Loss of INI-1 staining in malignant rhabdoid tumors, atypical teratoid/rhabdoid tumors, and epithelioid sarcomas • Loss of staining for one or more mismatch repair proteins in hereditary nonpolyposis colorectal cancer syndrome and sporadic colorectal carcinoma with microsatellite instability • Loss of staining for parafibromin in parathyroid carcinoma • Loss of staining for retinoblastoma (Rb) protein in spindle cell/pleomorphic lipoma (as a result of partial deletion of 13q, always including 13q14) • Lack of staining for succinate dehydrogenase B (SDHB) in hereditary paragangliomas and in a subset of gastrointestinal stromal tumorsa

Gene amplification

Increase in copy number of a gene → Marked overexpression of the encoded protein

• HER2 amplification in breast or gastric cancer → Strong positive immunostaining for HER2 • Amplification of 12q14-15 in well-differentiated/ dedifferentiated liposarcoma or well-differentiated osteosarcoma → Strong positive staining for MDM2 or CDK4

Virus association

Direct demonstration of virus-associated proteins expressed in tumor cells

• Epstein-Barr virus: EBV-LMP1, EBNA2 (various tumor types) • Human herpesvirus 8 (Kaposi sarcoma and rare cases of lymphoma)

Demonstration of surrogate marker expressed as a result of presence of the virus

• Merkel cell polyomavirus (Merkel cell carcinoma)

Chromosomal translocation Gene mutation

Gene deletion or loss of function

IGH gene → Associated with overexpression of ZAP-70

• High-risk human papillomavirus infection → p16 expression

Abbreviations: EGFR, epidermal growth factor receptor; LMP, latent membrane protein; EBNA, EBV nuclear antigen. a Discussed in article “Newly available antibodies with practical applications in surgical pathology” that will appear in the next issue of the journal.


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Chan et al Table 2. Prerequisites for Successful Use of Immunohistochemistry to Infer the Presence of Gene Fusion in Tumors. •• •• •• ••

The gene fusion should occur at a high frequency in the tumor type, otherwise the diagnostic yield will be too low An appropriate antibody against the protein product of one of the involved genes must be available Restricted expression of the target protein in normal tissues Absence or low frequency of expression of the protein in other tumor types in the differential diagnoses

Figure 1. Immunostaining for BCL2: (A) In reactive lymphoid follicles, the germinal centers are typically BCL2 negative. The scattered positive cells in the germinal centers represent T-cells. (B) In most follicular lymphomas, the germinal centers show BCL2 staining. In this example, the staining is intense.

pitfalls of these applications, mainly from the viewpoint of diagnostic surgical pathology.

Demonstration of Specific Chromosomal Translocations by Immunohistochemistry Some tumor types show distinctive chromosomal translocations (gene fusions), most commonly lymphomas, leukemias, and soft-tissue tumors, although, increasingly, recurrent chromosomal translocations are being discovered in carcinomas. Some, but not all, of the gene fusions can be demonstrated by immunohistochemistry, mainly through demonstration of the protein product of one of the implicated genes. Success in diagnostic application depends on several factors as listed in Table 2.

Examples of Successful Immunohistochemistry Follicular Lymphoma (BCL2). Follicular lymphoma shows the characteristic chromosomal translocation t(14;18) (q32;q21) in a high proportion (70%-90%) of cases.2 The translocation juxtaposes the BCL2 gene on chromosome 18 to the IGH@ gene on chromosome 14, resulting in overexpression of a structurally normal BCL2 protein in

the neoplastic cells. Antibodies against BCL2 are commercially available. Although BCL2 is widely expressed in normal lymphoid cells, including B- and T-cells, it is typically not expressed in the normal germinal centers. Therefore, BCL2 staining of the follicle center compartments can be readily assessed, and positive staining reflects the presence of BCL2 translocation, supporting a diagnosis of follicular lymphoma (Figure 1). False-negative staining for BCL2 can sometimes occur as a result of mutations in the translocated BCL2 gene, and application of additional BCL2 antibodies (such as E17) may help.3 In terms of sensitivity, immunostaining is superior to polymerase chain reaction (PCR) for detection of BCL2 translocation in follicular lymphomas, and is comparable to fluorescence in-situ hybridization (FISH). Mantle Cell Lymphoma (Cyclin D1). The hallmark of mantle cell lymphoma is overexpression of cyclin D1 (encoded by CCND1), found in more than 93% of cases.2 This is readily achieved by immunostaining for cyclin D1 (particularly with the use of a rabbit monoclonal antibody), which is not expressed in normal lymphoid cells (Figure 2). Immunostaining is more sensitive than cytogenetic studies for detection of the cytogenetic abnormality t(11;14) (q13;q32)—positivity rate - 50% to 65%—and PCR for


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Figure 2. Immunostaining for cyclin D1: (A) Bone marrow biopsy shows dense infiltration of small lymphoid cells, consistent with lymphoma. (B) Strong positive staining for cyclin D1 supports a diagnosis of mantle cell lymphoma.

Figure 3. Immunostaining for ALK: (A) In this ALK+ anaplastic large cell lymphoma, the tumor cells show nuclear-cytoplasmic staining, indicating the presence of NPM-ALK gene fusion. (B) In this epithelioid inflammatory myofibroblastic sarcoma, ALK staining occurs in the nuclear membrane, indicating the presence of RANBP2-ALK gene fusion.

CCND1-IGH@ fusion—positivity rate - 25% to 50%. Its sensitivity and specificity are comparable to that of FISH. Cyclin D1 immunoreactivity is rarely seen in other lymphoid neoplasms, except occasional cases of chronic lymphocytic leukemia (predominantly in proliferation centers), some cases of hairy cell leukemia, some cases of multiple myeloma or plasmacytoma, and rare cases of diffuse large-B-cell lymphoma.2 Tumors With ALK Translocation. A variety of tumor types are characterized by chromosomal translocation involving the ALK gene located at 2p13, such as ALK+ anaplastic large

cell lymphoma, ALK+ large-B-cell lymphoma, inflammatory myofibroblastic tumor, and pulmonary adenocarcinoma (5%-7% of cases).2,4-6 The partner genes fused with ALK are highly variable. Because ALK protein is not expressed in normal cells except rare neural cells, positive immunostaining for ALK is a simple method to infer the presence of ALK translocation. In addition, the staining pattern can provide a clue to the likely partner gene, such as nuclear-cytoplasmic staining for nucleophosmin (NPM) and nuclear membrane staining for RANBP2 (Figure 3).4 Thus, ALK immunohistochemistry is much more convenient than PCR for detection of ALK translocation (which


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Chan et al will require multiple reactions to cover the diverse range of partner genes) and is comparable in sensitivity and specificity to the more expensive and more labor-intensive FISH analysis.

Examples of Unsuccessful Immunohistochemistry BCL6 Rearrangement in Large-B-Cell Lymphoma. The BCL6 (3q27) gene is rearranged in about 35% of all cases of diffuse large-B-cell lymphomas, which is not a very high frequency.2 BCL6 protein expression is common in diffuse large-B-cell lymphomas (about 60%), and is not correlated with the presence of BCL6 rearrangement. This is because BCL6 is a germinal center cell marker, which is constitutively expressed at this developmental stage of B lymphoid cells without regard to the status of the BCL6 gene. SS18 (SYT) Rearrangement in Synovial Sarcoma. Synovial sarcoma is characterized by the consistent presence of a distinctive chromosomal translocation t(X;18)(p11;q11), which results in fusion of SS18 (formerly known as SYT) with the SSX1, SSX2, or SSX4 gene. In a study published in 2007, a polyclonal antibody against SYT was explored for its value in immunohistochemical diagnosis of synovial sarcoma.7 Positive staining was seen in 87% of cases of synovial sarcoma but also in 19% of other sarcomas, such as malignant peripheral nerve sheath tumors, solitary fibrous tumors, and extraskeletal mesenchymal chondrosarcomas. This study concluded that immunostaining for SYT can provide an easy, rapid, and widely applicable means of assistance in the diagnosis of synovial sarcoma, with the cautionary note that a positive interpretation should be made only when the staining is strong, nuclear, and present in the majority of cells. However, judging from the not-infrequent SYT immunoreactivity in mimickers of synovial sarcoma, lack of mention of this immunohistochemical marker in the latest (2013) WHO classification of soft-tissue tumors, and the continued efforts to search for other reliable immunohistochemical markers for synovial sarcoma (such as TLE1), SYT immunohistochemistry has failed to serve its purpose of reliably identifying the presence of SYT (SS18) gene fusion.8-11 Either the antibody used is not that specific, or increased expression of SYT can occur in other tumors from mechanisms other than gene fusion. Tumors With TFE3 Translocation. The TFE3 gene located at chromosome Xp11.2, which can be fused with a number of partner genes, is implicated in alveolar soft-part sarcoma (in practically all cases), Xp11 translocation renal cell carcinoma (in all cases, by definition), and epithelioid

perivascular epithelioid cell tumor/PEComa (in a proportion of cases).12,13 Immunostaining with TFE3 antibody gives positive results in the majority of the above-listed tumors.1,14 However, TFE3 is not uncommonly positive in their mimickers, which lack TFE3 gene fusion, such as paragangliomas, adrenal cortical carcinomas, and clear cell renal cell carcinoma, sometimes with diffuse strong nuclear staining.15,16 The lack of success of TFE3 immunostaining for inferring the presence of TFE3 gene fusion is attributable to the following factors: (1) staining with the TFE3 antibody is not always reproducible, being greatly affected by the antigen retrieval technique and automated versus manual staining13; (2) despite lack of staining of normal cells, positive staining is not uncommon in tumors in the differential diagnoses (which may be caused by lack of specificity of the antibody or presence of other mechanisms that can lead to overexpression of TFE3); and (3) negative staining cannot totally rule out TFE3 gene fusion, and thus, immunostaining is not a satisfactory negative screening tool.17,18 A recent study concludes that TFE3 FISH analysis should be the test of choice for establishing the diagnosis of Xp11 translocation renal cell carcinoma.18

Detection of Specific Gene Mutations in Tumors by Immunohistochemistry Many tumor types exhibit specific driver gene mutations, some of which can be demonstrated by immunohistochemistry. The results may have diagnostic or prognostic value.

Aberrant Localization of Signals Reflecting the Presence of Gene Mutation β-Catenin. β-Catenin (CTNNB1) gene mutation occurs in a variety of tumors, such as undifferentiated carcinoma of the thyroid, cribriform-morular variant of papillary thyroid carcinoma, adamantinomatous craniopharyngioma, solidpseudopapillary tumor of the pancreas, and hepatocellular adenoma. β-Catenin is normally localized to the cell membrane, whereas aberrant nuclear localization reflects the presence of mutations in the CTNNB1 gene or other genes of the Wnt signaling pathway (such as the APC gene).1 Examples of the latter are colorectal adenocarcinoma and desmoid fibromatosis. Nuclear immunostaining for β-catenin can aid in diagnosis in the following scenarios: 1. colorectal adenocarcinoma versus adenocarcinoma of the ovary, uterus, or bladder19,20; 2. fibromatosis versus other spindle cell lesions (Figure 4)21;


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Figure 4. Immunostaining for β-catenin: (A) Hypocellular bland-looking spindle cell proliferation consistent with desmoid fibromatosis. (B) Immunostaining shows the presence of nuclear staining for β-catenin. In the right field, the normal endothelial cells of the capillaries do not show nuclear staining.

3. confirming diagnosis of cribriform-morular variant of papillary thyroid carcinoma22; 4. confirming diagnosis of solid-pseudopapillary tumor of the pancreas (for which there are few other specific immunohistochemical markers)23; and 5. classification of hepatocellular adenomas (group 2, with activating mutation in the CTNNB1 gene).24 Nucleophosmin. Mutations in exon 12 of the NPM gene occur in about half of all cases of adult acute myeloid leukemia with normal karyotype and are predictors of favorable prognosis. Immunostaining can provide important information on the status of the NPM gene. In the absence of NPM mutation, immunostaining for NPM is restricted to the nucleus. In the presence of NPM mutation, cytoplasmic immunostaining for NPM is observed.25,26 NPM mutants apparently bind and recruit wild-type NPM into leukemic cell cytoplasm.

Stabilization of Protein as a Result of Gene Mutation: TP53 Mutations of the TP53 gene are common in various types of cancer, mostly as a progression event. However, TP53 mutation can also occur as an early event in some tumor types, such as high-grade serous carcinoma of the female genital tract and ulcerative colitis–associated dysplasia. Thus, its detection may aid in diagnosis or classification. Although the literature on the relationship between TP53 mutation and p53 immunostaining is confusing, there is in fact good correlation if only strong and extensive immunostaining (in >60% of tumor cells) is taken as positive.27 Complete lack of staining for p53 in a tumor, accompanied

by weak to moderate staining of rare admixed normal cells as internal positive controls, also strongly suggests the presence of TP53 mutation, attributable to mutant protein antigenically not recognized by the p53 antibody.27 Immunostaining for p53 can help in the classification of endometrial carcinoma in difficult cases because serous adenocarcinoma and uterine surface carcinoma (also known as endometrial intraepithelial carcinoma) typically show extensive strong staining, whereas endometrioid adenocarcinoma shows negative or weak staining, and clear cell carcinoma often shows patchy moderate staining (Figure 5).28,29 Immunostaining for p53 is essential for identifying “p53 signature,” a putative precursor of serous tubal intraepithelial carcinoma in the fallopian tube.30 p53 signature is defined as a short stretch of normal-looking fallopian tube epithelium, found on immunostaining to be intensely positive for p53 (>12 cells). Serous tubal intraepithelial carcinoma, recognized morphologically by cell stratification, loss of polarity, and nuclear atypia, also frequently shows extensive and intense positive or completely negative staining for p53. The immunostain may help in differential diagnosis with reactive atypia.

Mutation-Specific Antibodies Antibodies have recently been successfully raised against some mutant proteins for immunohistochemistry. These mutation-specific antibodies specifically bind to altered regions of the proteins encoded by the mutated genes but will not bind to the wild-type protein. Hence, positive immunostaining reflects the presence of a specific mutation in the gene of interest. It is likely that more antibodies


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Figure 5. Immunostaining for p53: (A) An endometrial adenocarcinoma with differential diagnosis between serous carcinoma and high-grade endometrioid carcinoma. (B) Extensive strong staining for p53 supports the diagnosis of serous carcinoma. Table 3. Factors Contributing to Success in the Application of Mutation-Specific Antibodies in Surgical Pathology. •• Ability to produce the mutation-specific antibody that is reactive in paraffin sections •• The mutation-specific antibody should not cross-react with the wild-type protein •• The gene mutation should occur with a significant frequency in certain tumor types •• The gene mutation pattern should be limited (such as V600E mutation accounting for the majority of mutations in BRAF)

will become available in the near future for application in surgical pathology. Prerequisites for success in their use are listed in Table 3. Isocitrate Dehydrogenase 1 (IDH1) R132H. Heterozygous point mutations in the IDH1 gene represent the most frequent mutations (about 70%) in grade II or III gliomas, including astrocytomas and oligodendrogliomas, whereas they are rare in other primary brain tumors except secondary glioblastomas.31-33 In more than 90% of cases, IDH1 mutation takes the form of substitution of guanine by adenine at nucleotide 395 (c.395G>A), resulting in substitution of amino acid arginine by histidine at position 132 (R132H). A monoclonal antibody specific for the mutant IDH1-R132H protein (clone H09) is now commercially available.34 Immunostaining using the IDH1-R132H antibody is a simple way to demonstrate the presence of IDH1 gene mutation in gliomas. The main applications include (1) distinguishing between glioma (if positive) from reactive gliosis (negative) and (2) diagnosis of focal or subtle involvement by the glioma, such as biopsies from the tumor edge or posttherapy specimen (Figure 6).35

Mutations in IDH1 and IDH2 genes are frequent in central and periosteal cartilaginous tumors, with the commonest being the IDH1 R132C mutation (not R132H).36 If a mutation-specific antibody becomes available, this might be of great help in distinguishing chondrosarcoma from chondroblastic osteosarcoma. Epidermal Growth Factor Receptor (EGFR) Mutations. Mutations in the EGFR gene are important driver mutations in pulmonary adenocarcinomas, being found in 10% to 20% of cases in Caucasians and about 50% of cases in Asians. L858R mutation and exon 19 deletion mutations account for about 90% of all the mutations.37 Because response to EGFR inhibitor therapy is correlated with the presence of EGFR mutation, the current practice is to determine the EGFR status in the pulmonary carcinoma before initiation of target therapy. Antibodies have been produced against the 2 commonest EGFR mutants: exon 21 L858R and exon 19 E746_ A750 15 bp deletion. Immunostaining with these antibodies shows excellent correlation with molecular findings (Figure 7).38-42 What is more, even some cases with 18 bp deletion in exon 19 show reactivity with the exon 19 E746_A750 15 bp deletion antibody. Thus, an alternative algorithm in the workup of patients with pulmonary adenocarcinoma is to use immunohistochemistry to screen for EGFR mutation, with molecular studies being reserved for cases with negative or equivocal results. Immunohistochemistry has additional advantages over molecular studies in special scenarios, such as decalcified specimens (for which molecular studies often fail) and specimens with scanty tumor cells. Another potential application of the antibodies is for distinguishing primary pulmonary adenocarcinoma from metastatic adenocarcinoma,


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Figure 6. Immunostaining with IDH1-R132H antibody: (A) Biopsy obtained from the edge of a tumor reveals occasional mildly atypical cells, but it is difficult to render a definitive diagnosis of astrocytoma. (B) Immunostaining for IDH1-R132H reveals moderate numbers of positive cells, many of which are impossible to recognize as being neoplastic on routine histological sections. Thus, a diagnosis of astrocytoma can be confirmed.

Figure 7. Pulmonary adenocarcinoma immunostained with mutation-specific antibodies against EGFR. (A) The tumor shows positive staining with exon 19 deletion antibody, indicating the presence of exon 19 deletion mutation. (B) The same tumor showing lack of staining with L858R antibody. Abbreviation: EGFR, epidermal growth factor receptor.

although the sensitivity is much lower than with markers such as TTF-1 and Napsin A.39 BRAF Mutation V600E. BRAF mutation is implicated in diverse tumor types, most commonly papillary thyroid carcinoma, melanocytic nevus, malignant melanoma, lowgrade serous adenocarcinoma, and borderline serous tumor of the ovary, hairy cell leukemia, and Langerhans cell

histiocytosis.43-45 A mutation-specific antibody directed against the commonest mutation V600E (c.1799T>A, resulting in amino acid substitution at position 600 from valine to glutamic acid) in BRAF, which is commercially available, reacts with cells carrying the mutation but not the cells without the mutation. Many studies have shown excellent correlation of immunohistochemical staining with the presence of BRAF mutation.46-53


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Chan et al Table 4. Interpretation of Results of Immunostaining for Mismatch Repair Gene Proteins in Cancers. Immunostaining Results MLH1 − + + +

PMS2

MSH2

MSH6

− − + +

+ + − +

+ + − −

Interpretation: Gene Likely to Be at Fault Somatic MLH1 hypermethylation or germline MLH1 mutation Germline PMS2 mutation or germline MLH1 missense mutationa Germline MSH2 mutation Germline MSH6 mutation

a

About one-third of the mutations in MLH1 are missense mutations that may result in mutant nonfunctional proteins that are antigenically intact.

Gene Deletion or Loss of Function Manifesting as Loss of Immunohistochemical Expression of Protein In some tumors, loss of function of certain key genes is important in tumorigenesis. This can result from inactivating mutation of the gene, deletion, or promoter hypermethylation. Irrespective of the molecular mechanism, the end result is loss of expression of the gene product. This can sometimes be successfully demonstrated by immunohistochemistry in the form of loss of staining for the protein. Interpretation must be made in areas where appropriately stained positive control cells (often lymphocytes or endothelial cells) are present to ascertain validity of the immunostain.

Mismatch Repair Proteins in Microsatellite Unstable Colorectal Cancer Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is characterized by early-onset colorectal cancer and some other cancer types (including endometrial carcinoma), attributable to germline mutation in a mismatch repair gene, such as MLH1, MSH2, MSH6, and PMS2. Mismatch repair gene defect results in a microsatellite instability (MSI) tumor phenotype. However, some sporadic colorectal cancers also exhibit MSI. As a group, colorectal cancers with MSI are associated with a more favorable prognosis than those without MSI. Immunostaining for mismatch repair proteins to look for a loss of nuclear staining in colorectal carcinoma is a simple and accurate method to demonstrate loss of function of the mismatch repair gene. It should be noted that absence of staining for mismatch repair protein in a colorectal cancer does not distinguish between HNPCC and sporadic MSI+ colorectal cancer. Definitive diagnosis of HNPCC requires molecular studies to demonstrate the presence of germline mutation in a mismatch repair gene. In their functional state, mismatch repair proteins form heterodimers, with MLH1 (dominant) pairing with PMS2 and MSH2 (dominant) pairing with MSH6. Abnormalities

in the dominant (obligatory) partner will result in degradation of both components of the dimer. However, when mutation occurs in genes of the secondary proteins (PMS2 or MSH6), there is no concurrent loss of the obligatory partners because function of the secondary proteins may be compensated by other proteins. As a result, mutations of MLH1 or MSH2 often cause concurrent loss of MLH1/ PMS2 or MSH2/MSH6, respectively, whereas mutations of PSM2 or MSH6 often cause isolated loss of PMS2 or MSH6 only (Table 4).54 In the assessment for possible HNPCC, immunohistochemistry has 2 main roles: (1) it can be used as a screening tool to infer the presence of MSI, instead of performing molecular analysis for detection of MSI, and (2) for a tumor shown to exhibit MSI by molecular methods, immunostaining for the various mismatch repair proteins can help in suggesting the gene that is likely to be mutated or hypermethylated, thus directing efforts toward molecular study of that mismatch repair gene (Table 4, Figure 8).54-58 Similar immunohistochemical screening for loss of expression of mismatch repair protein is also applicable for endometrial carcinomas occurring in young patients, in order to detect patients with HNPCC.59-62

E-cadherin in Lobular Carcinoma of Breast A key feature of lobular carcinoma of the breast is loss of cellular cohesion attributable to loss of E-cadherin expression, which can result from a variety of mechanisms: (1) inactivating or truncating mutation in the CDH1 gene, (2) promoter hypermethylation of CDH1, and (3) transcriptional inactivation of CDH1. Thus, a simple immunostain using antibody against E-cadherin can demonstrate the “end result” of different molecular alterations in the gene—loss of staining. The immunostain is helpful for distinguishing invasive lobular carcinoma (negative) from invasive ductal carcinoma (positive), for distinguishing lobular carcinoma in situ (negative) from ductal carcinoma in situ (positive) or nonspecific lobular hyperplasia (positive), and for identifying the pleomorphic variant of lobular carcinoma (Figures 9A and 9B).63


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Figure 8. Colon adenocarcinoma stained for mismatch repair proteins: The carcinoma is shown on the left, and normal mucosa on the right. Positive staining in the normal epithelium and intervening cells (stromal cells and lymphoid cells) indicates that the microscopic field is suitable for assessment of staining in the tumor. (A) Carcinoma shows loss of staining for MLH1. (B) Carcinoma shows loss of staining for PMS2. (C) No loss of staining for MSH2. (D) No loss of staining for MSH6. The findings suggest that MLH1 is the gene at fault, either in the form of somatic hypermethylation or germline mutation.

One pitfall is that up to 16% of lobular carcinomas may stain positive for E-cadherin, probably because of missense mutation with intact antigenic determinant.64 If lobular carcinoma is strongly suspected, additional immunostaining for p120-catenin will provide support for the diagnosis when the tumor cells show diffuse cytoplasmic instead of cell membrane staining (Figures 9C and 9D).64,65 Normally, p120-catenin is bound to the subplasmalemmal portion of the E-cadherin molecule, and thus, immunostaining appears to be in the cell membrane. With a dysfunctional E-cadherin, p120-catenin is released into the cytoplasm, resulting in an aberrant diffuse cytoplasmic staining pattern.

reported that almost all parathyroid carcinomas showed loss of nuclear staining for parafibromin, and parathyroid adenomas were positive except those associated with hyperparathyroidism–jaw tumor syndrome. However, more recent studies have not obtained such clear-cut results—only about half of all parathyroid carcinomas lack staining for parafibromin, whereas some sporadic parathyroid adenomas can also lack staining.66 Furthermore, positive parafibromin staining does not exclude HRPT2 mutation because some tumors with missense mutations may show weak parafibromin expression.67 Thus, parafibromin immunohistochemistry is not a very successful example.

HRPT2 in Parathyroid Carcinoma

INI1 in Rhabdoid and Other Tumors

In parathyroid carcinomas, inactivating mutations in the HRPT2 gene, which encodes parafibromin, are found in more than two-third of cases. These mutations are practically not found in parathyroid adenomas. The original studies

Malignant rhabdoid tumors (renal or extrarenal) and atypical teratoid/rhabdoid tumors of the central nervous system are characterized by biallelic inactivation of the hSNF5/ INI1 gene located on the chromosome 22q11.2, either by


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Figure 9. Lobular carcinoma of breast: (A) Noncohesive cellular growth is the hallmark of lobular carcinoma of breast. (B) On immunostaining for E-cadherin, the tumor cells are typically negative, whereas the residual ducts are positive. The lower field shows lobular carcinoma in situ, which characteristically shows lack of staining of the proliferated cells inside but positive staining of the residual surrounding layer of myoepithelium. (C) An example of lobular carcinoma that shows preserved, albeit weak, staining for E-cadherin. (D) In the same case, immunostaining for p12-catenin reveals abnormal cytoplasmic staining, even though there is still some membranous staining. This indicates a dysfunctional E-cadherin molecule. Note the pure membranous staining of the normal ductal cells in the right lower field.

mutation or deletion. Irrespective of the molecular mechanism of INI1 inactivation, immunostaining for INI1 is lost (Figure 10).1,68-72 Thus, this has practically become the defining feature of these tumor types, for which no other specific immunohistochemical markers are available. Loss of INI1 staining is also a common feature of epithelioid sarcomas and can be utilized to aid in its diagnosis.68,73

Gene Amplification Manifesting as Overexpression of Gene Product Gene amplification usually results in overexpression of the encoded protein. However, immunohistochemical assessment of gene amplification is much more subjective and less

successful compared with that of loss of gene function (for which assessment is on whether there is staining or no staining). This is because there is often a continuum of staining intensities, such that the dividing line to consider an intensity to be indicative of overexpression (and hence implying presence of gene amplification) is somewhat arbitrary. Staining intensity in an individual case can, furthermore, be affected by many technical factors, such as fixation duration, age of the cut section, strength of antigen retrieval, and sensitivity of the immunohistochemical detection system.

HER2 Amplification in Breast Cancer Approximately 20% of all breast carcinomas show amplification of the HER2 gene. HER2 amplification is


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Figure 10. Atypical teratoid/rhabdoid tumor. (A) Atypical teratoid/rhabdoid tumor is often difficult to diagnose because rhabdoid cells may be in the minority. (B) Lack of staining for INI1 supports the diagnosis. Note appropriate staining of the endothelial cells as internal positive control. Inset: FISH studies using probes against ABL (red signal) and BCR (located at the same 22q11.2 locus as INI1, green signal). Almost all nuclei show 2 red signals (normal) and 1 green signal (loss of 1 copy of 22q11.2). Abbreviation: FISH, fluorescence in situ hybridization.

associated with a worse prognosis and relative resistance to endocrine therapy. However, anti-HER2 target therapy (Herceptin, trastuzumab) can improve outcome in these patients. To select breast cancer patients for target therapy, immunostaining for HER2 is the most widely used screening or confirmatory tool because there is good correlation of immunostaining with molecular findings.74 Strong immunostaining for HER2 (score 3) is correlated with HER2 amplification in >90% of cases and is thus considered HER2 positive for purposes of target therapy. Negative or equivocal staining (score 0 and 1) is correlated with a lack of HER2 amplification in >90% of cases and is, thus, considered HER2 negative (Figure 11). Molecular studies are often reserved for cases showing weak staining (score 2).

liposarcomas (Figure 12).1 The sensitivity and specificity of CDK4 are 68% to 86% and 88% to 89%, respectively, and those of MDM2 are 86% to 100% and 59% to 74%, respectively.76,77 Well-differentiated central osteosarcoma and parosteal osteosarcoma are similarly characterized by amplification of 12q14-15.78 Low-grade osteosarcomas show immunostaining for MDM2 in 70% and for CDK4 in 87% of cases. Expression of at least 1 of the 2 markers is seen in 100% and both in 57% of cases. The staining pattern is diffuse in most cases, with moderate or strong intensity. Therefore, immunostaining for MDM2 and CDK4 can serve as a useful adjunct in the difficult differential diagnosis from benign lesions, which are negative for the immunostains.79

12q14-15 Amplification in Well-Differentiated Liposarcoma, Dedifferentiated Liposarcoma, and Low-Grade Osteosarcoma

Immunohistochemical Detection of Virus to Aid in Diagnosis of VirusAssociated Tumors

Well-differentiated liposarcoma (atypical lipomatous tumor) can be difficult to distinguish from lipoma, especially in biopsies. Dedifferentiated liposarcoma is difficult to distinguish from other types of sarcomas. Both types of liposarcomas are cytogenetically characterized by supernumerary rings and giant marker chromosomes formed by multiple copies of the chromosome region 12q14-15. Many different genes are located in this region, such as CDK4 and MDM2.75 Immunostaining for the corresponding overexpressed proteins is a valuable adjunct in the diagnosis of well-differentiated/dedifferentiated

Epstein-Barr Virus Epstein-Barr virus (EBV) is associated with a wide variety of neoplasms, including lymphomas, carcinomas (often with a lymphoepithelial morphology), and mesenchymal tumors, in a variable proportion of cases depending on the tumor type. The EBV-associated genes, and hence their proteins, expressed in these tumors vary with the type of EBV latency (see Table 5).80 Demonstration of EBV may aid in the diagnosis and classification of some of these tumors.


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and less-expensive immunohistochemical test? The most widely available antibody against EBV is one raised against EBV-LMP1. Based on the EBV latency pattern, it would seem that other than Burkitt lymphoma (with type I latency), this immunostain should theoretically detect EBV in all the various types of EBV-associated tumors (Table 5). However, this is not the case. The level of EBV-LMP1 expression in the EBV-associated tumors is not uncommonly below that detectable by conventional immunohistochemistry.81-83 Thus, whereas a positive result supports the presence of EBV, a negative result does not rule this out. The only exception is EBV-associated classical Hodgkin lymphoma, in which EBV-LMP1 is consistently and strongly expressed in the Reed-Sternberg cells (Figure 13A).80 That is, immunostaining for EBV-LMP1 cannot replace in situ hybridization for EBER for consistent demonstration of EBV in tumors, except for classical Hodgkin lymphoma.84,85 How Good Is Immunostaining for EBV Nuclear Antigen 2 (EBNA2)? An antibody directed against EBNA2 that works on paraffin sections is commercially available. This antibody can reliably demonstrate EBV for tumors showing EBV latency type III. One caveat is that some EBV-associated tumors (such as posttransplant lymphoproliferative disorders and EBV+ large-B-cell lymphoma of the elderly) can exhibit either type II or type III EBV latency, and thus, negative staining for EBNA2 does not totally rule out the presence of EBV. Expression of EBNA2 in tumors also has another important meaning: it reflects the presence of underlying immunodeficiency. In most circumstances, there is systemic immunodeficiency (such as HIV infection or posttransplant immunosuppression), but in occasional circumstances, the immunodeficiency is strictly localized to the site of disease, as in diffuse large-B-cell lymphoma associated with chronic inflammation (Figure 13B).80,86

Human Herpesvirus 8 (HHV8) Figure 11. Breast cancer immunostained for HER2: (A) Negative staining, score 0. The inset shows normal FISH pattern, with 2 red signals (2 copies of HER2) in each nucleus. (B) Score 2 staining. (C) Score 3 strong staining. The inset shows clumps of red signals on FISH analysis, indicating amplification of the HER2 gene. Abbreviation: FISH, fluorescence in situ hybridization.

How Good Is Immunostaining for EBV Latent Membrane Protein-1 (LMP1)? The most reliable method to demonstrate EBV in tumors is in situ hybridization for EBV-encoded RNA (EBER). Is it possible to replace this by a simpler

Several types of tumor show a strong association with HHV8, including Kaposi sarcoma, primary effusion lymphoma (including the extracavitary solid variant), large-Bcell lymphoma arising in HHV8-associated multicentric Castleman disease, and HHV8+ germinotropic B-cell lymphoproliferative disorder.87-89 Because immunostaining with an antibody against HHV8 latent nuclear antigen can reliably demonstrate HHV8 in tumors, this can replace molecular studies such as in situ hybridization or PCR for this purpose.90 Besides aid in diagnosis and classification of the abovementioned lymphoma types, HHV8 immunostaining is of great help in the diagnosis of Kaposi sarcoma because


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Figure 12. Dedifferentiated liposarcoma immunostained for CDK4: (A) In the adipose component, the tumor cell nuclei show moderate to strong staining for CDK4. (B) The dedifferentiated component is similarly positive.

Table 5. Relationship of EBV-Associated Tumors With EBV Latency Type. EBV Latency Type

EBV Proteins That Are Characteristically Expressed

Tumor Types Exhibiting the EBV Latency

I

EBNA1

• Burkitt lymphoma

II

EBNA1, EBV-LMP1

• Hodgkin lymphoma • Extranodal NK/T-cell lymphoma • Peripheral T-cell lymphoma associated with EBV (sporadic) • EBV+ diffuse large-B-cell lymphoma of the elderlya • Posttransplant lymphoproliferative disordersa • Primary effusion lymphoma • Nasopharyngeal carcinoma • Lymphoepithelial carcinoma of various sites (such as thymus, salivary gland, and lung) • Gastric adenocarcinoma (some cases) • Inflammatory pseudotumor-like follicular dendritic cell sarcoma

EBNA1, EBV-LMP1, EBNA2

• Posttransplant lymphoproliferative disordersa • Diffuse large-B-cell lymphoma associated with chronic inflammation • Lymphomatoid granulomatosis • EBV+ large-B-cell lymphoma of the central nervous system in immunocompromised host • EBV+ diffuse large-B-cell lymphoma of the elderlya • Smooth muscle tumors occurring in immunocompromised host • Myopericytoma occurring in HIV-positive individuals

III

Abbreviations: EBV, Epstein-Barr virus; LMP, latent membrane protein; EBNA, EBV nuclear antigen. a Lymphoma type that can show either type II or type III latency.

these sarcomas are consistently positive for HHV8, whereas the mimickers are almost invariably negative— only exceptional examples of angiosarcoma have been reported to be HHV8 positive.90-92

Human Papillomavirus (HPV) How Good Are Antibodies Against HPV? HPV is associated with a wide variety of lesions in the skin, upper-aerodigestive tract,


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Figure 13. Immunostaining for EBV-associated proteins in tumors: (A) Classical Hodgkin lymphoma with the Reed-Sternberg cells and variants exhibiting positive staining for EBV-LMP1. The staining shows a granular quality. (B) Diffuse large-B-cell lymphoma associated with chronic inflammation, with the tumor cells showing immunoreactivity for EBNA2. Abbreviations: EBV, Epstein-Barr virus; LMP, latent membrane protein; EBNA, EBV nuclear antigen.

and lower genital tract, ranging from benign lesions (such as condyloma acuminatum, wart, and laryngeal papilloma) to dysplastic lesions (such as cervical and vaginal intraepithelial neoplasia) to carcinomas (such as cervical squamous cell carcinomas and adenocarcinomas and some oropharyngeal squamous cell carcinomas).93 Demonstration of HPV has diagnostic applications, such as supporting the uterine cervical or oropharyngeal origin of a squamous cell carcinoma in the appropriate setting, distinguishing between endocervical adenocarcinoma (HPV+) and endometrial adenocarcinoma (HPV−), and in prognostication for oropharyngeal squamous cell carcinomas (HPV+ subset having a better prognosis). The commercially available antibodies against HPV are often directed against HPV capsid antigens and thus can only detect HPV in the productive phase: that is, the virus is present in the nucleus in an episomal form, replicating to produce complete viral particles (including capsids), a state of HPV found in benign lesions and low-grade dysplastic lesions. Therefore, the antibodies cannot demonstrate HPV that has integrated into the host cell genome—a latent infection characteristically found in high-grade dysplastic lesions and carcinomas. Thus, direct demonstration of integrated HPV requires molecular methods, such as in situ hybridization for HPV DNA or the more sensitive RNAscope HPV assay (in situ hybridization for E6/E7 mRNA),94 although newly available antibodies against HPV E6 protein may be promising. P16 Immunohistochemistry as Surrogate Marker of High-Risk HPV. P16 is a cyclin-dependent kinase inhibitor regulated by RB (retinoblastoma protein). In HPV infection, the HPV oncoproteins E6 and E7 inactivate RB (which

normally inhibits transcription of p16), leading to p16 overexpression.95 Therefore, p16 overexpression can be utilized as a surrogate marker for the presence of high-risk HPV in neoplastic lesions.96-98 However, this is applicable only for anatomical sites where high-risk HPV is present in a high percentage of tumors (such as the lower female genital tract and oropharynx). For sites where the tumors show a low frequency of HPV association (such as the oral cavity, larynx, and nasopharynx), the positive predictive value of a positive p16 immunostain is low.94,99,100 This is because there are mechanisms other than HPV infection that can lead to p16 overexpression, such as in serous carcinomas of the uterus and ovary.101-103 In the lower female genital tract, p16 expression is seen in high-risk HPV-associated squamous lesions, including virtually all cases of high-grade squamous intraepithelial lesions and invasive squamous cell carcinomas and some cases of low-grade squamous intraepithelial lesions (Figures 14A and 14B).95,96,104,105 The normal, inflamed, or atrophic stratified squamous epithelium is negative for p16 or at most shows focal weak staining. p16 is also negative in transitional cell metaplasia and reactive epithelial atypia. Thus, p16 staining is extremely helpful for distinction of squamous intraepithelial neoplasia from its mimickers; negative staining is particularly reassuring because this suggests the absence of high-risk HPV in the lesion.106 p16 Staining occurs in the nucleus and cytoplasm. The lesion is considered positive for p16 when there is strong staining of at least the lower half of the epithelium (noninvasive lesions), although the staining commonly involves the lower twothirds or the full thickness.107 Scattered single or small groups of positive cells are considered p16 negative.105


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Figure 14. Uterine cervix cone biopsy specimen immunostained for p16: (A) Marked detachment of the surface epithelium renders morphological assessment difficult. (B) In a corresponding section immunostained for p16, the strong staining indicates the presence of significant lesion (usually high-grade squamous intraepithelial lesion). (C) In another focus, some endocervical glands are lined by pseudostratified atypical cells, consistent with adenocarcinoma in situ. (D) In a corresponding section immunostained for p16, the adenocarcinoma in situ component is positive, contrasting with the p16-negative normal glands on the left.

Cervical in situ or invasive adenocarcinomas are often positive for high-risk HPV and also show strong immunostaining for p16 (Figures 14C and 14D). Thus, p16 is of value in distinguishing adenocarcinoma in situ from its mimickers, with the caveat that a high proportion of cells can be positive in endometriosis and tuboendometrial metaplasia.106 For head and neck squamous cell carcinomas, p16 positivity is defined by strong staining of >75% of tumor cells or >50% of tumor cells combined with >25% confluent areas108 or strong and uniform p16 staining in all or most cancer cells of basaloid nonkeratinizing/partially keratinizing oropharyngeal carcinoma.109 The immunostain can be of help in confirming the oropharyngeal origin for squamous cell carcinoma occurring in the lung.110

present at a high frequency (approximately 80%) in this tumor type, although the virus is not found in Merkel cell carcinoma with divergent differentiation.111,112 The virus is clonally inserted into the tumor cells, and truncating deletions of the large T antigen gene eliminates viral DNA replication capacity. On the other hand, MCPV is rarely detected in neuroendocrine tumors of various sites.113-117 Monoclonal antibody raised against large T antigen of MCPV is commercially available. The results of immunostaining correlate well with those of molecular analysis, although the sensitivity is slightly lower than that of quantitative PCR.113 Immunostaining for MCPV can help in the diagnosis of Merkel cell carcinoma, especially when CK20 is negative.

Merkel Cell Polyomavirus (MCPV)

Declaration of Conflicting Interests

Recent studies have led to the discovery of a new polyomavirus in Merkel cell carcinoma (MCPV), which is

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.


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Chan et al Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.

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