Immunohistology of Endocrine and Neuroendocrine Neoplasms

Hormones

An important approach to the diagnosis and classification of ENs and NENs relies on the demonstration of their hormonal content. ^, This goal can be accomplished by the use of antibodies directed against the mature hormones and hormone precursors. An additional approach involves the use of in situ hybridization (ISH; hybridization histochemistry) for the demonstration of specific hormonal messenger RNAs (mRNAs). The latter approach is discussed in detail in several reviews. ^, Virtually all classes of hormones—small peptides, large polypeptide hormones, steroids, amines—and hormone receptors can be visualized in IHC formats. ^, With the advent of microwave-based antigen retrieval methods, the vast majority of these products can be demonstrated in formalin-fixed, paraffin-embedded (FFPE) samples. However, hormonal products by themselves cannot be used as lineage-specific markers. For example, somatostatin is present in the D cells of the pancreatic islets, gastrointestinal (GI) and bronchopulmonary endocrine cells, thymic endocrine cells, and thyroid C cells, and also in their corresponding tumors; therefore the presence of immunoreactive somatostatin by itself does not provide evidence of the site of origin of a metastatic lesion. The discussion of individual hormones is addressed in sections on specific endocrine cell types and their corresponding tumors.

Enzymes

Enzymes that are active in the biosynthesis and processing of hormones are important markers of endocrine cells. Immunoreactivity for aromatic L-amino acid decarboxylase, for example, is widely distributed in neuroendocrine (NE) cells. In contrast, tyrosine hydroxylase (TH), dopamine β-hydroxylase, and phenylethanolamine N -methyl-transferase have a more limited tissue distribution and are confined to known sites of catecholamine biosynthesis. Immunolocalization of these enzymes permits catecholamine synthesizing abilities to be deduced from paraffin sections. The presence of an immunoreactive enzyme, however, does not necessarily imply that the enzyme is present in a functional form.

A variety of endopeptidases and carboxypeptidases required for the formation of biologically active peptides from precursor molecules are present in the trans-Golgi region and in secretory granules of NE cells. They include the prohormone convertases PC1/PC3 and PC2, and carboxypeptidases H and E. ^, The proconvertases are widely distributed in NE cells and their corresponding tumors; whereas other types of endocrine cells—thyroid follicular cells, parathyroid chief cells, adrenal cortical cells, and testis—are negative. NE cells with a neural phenotype (e.g., adrenal medullary cells) contain a predominance of PC2, whereas epithelial NE cells contain a predominance of PC1/PC3. With the exception of parathyroid cells, the presence of PC2 and PC3 correlates with the presence of chromogranins and secretogranins. PC2 and PC1/PC3 are present in normal pituitaries and in pituitary adenomas, adrenocorticotropic hormone (ACTH)–producing adenomas contain a predominance of PC1/PC3, and other adenomas express a predominance of PC2. Both peptidylglycine α-amidating monooxygenase and peptidylamidoglycolate lyase are present in NE secretory granules. These enzymes are responsible for the α amidation of the C-terminal regions of peptide hormones. This function is critical for biological activity of the peptides.

Neuron-specific enolase (NSE) is an additional enzyme that has been studied extensively in NE cells. The staining of NE tumors is unrelated to the cellular content of secretory granules, and even degranulated cells are NSE positive. This enzyme is the most acidic isoenzyme of the glycolytic enzyme enolases, and it is present both in neurons and in NE cells. ^, The enolases are products of three genetic loci that have been designated alpha, beta, and gamma. Nonneuronal enolase (α-α) is present in fetal tissues of different types, in glial cells, and in many non-NE tissues in adults. Muscle enolase is of the β-β type, whereas the neuronal form of enolase has been designated γ-γ. Hybrid enolases are present in megakaryocytes and in a variety of other cell types. NSE (γ-γ) replaces nonneuronal enolase during the migration and differentiation of neurons, and the appearance of this isoenzyme heralds the formation of synapses and electrical excitability. Although many earlier studies used NSE as a marker of NE cells, later studies have indicated that the specificity of this marker is limited. ^,

The protein gene product 9.5 (PGP9.5) is a ubiquitin carboxyterminal hydrolase that plays a role in the catalytic degradation of abnormal denatured proteins. PGP9.5 is present in neurons and nerve fibers and in a variety of NE cells, with the possible exception of those in the normal GI tract. In contrast, carcinoid tumors and a variety of other NE tumors contain PGP9.5. The patterns of staining for NSE and PGP9.5 are generally similar in that positive cells show diffuse cytoplasmic reactivity unrelated to the type of hormone produced or the degree of cellular differentiation. Comparative studies, however, have demonstrated that some NE tumors may be positive for PGP9.5 and negative for NSE, whereas others may be positive for NSE and negative for PGP9.5. Antibodies to PGP9.5 are particularly useful for the demonstration of neurons and cells with neuronal differentiation. It should be recognized that mesenchymal neoplasms and some non-NE tumors of the exocrine pancreas may also be positive for PGP9.5.

Histaminase (diamine oxidase) has been used as a marker for some NE cells and their tumors. This enzyme is present in high concentrations in medullary thyroid carcinomas and has been reported in small cell carcinomas of the lungs and other NENs. High serum levels of histaminase also occur in pregnancy, and IHC studies have revealed the presence of this enzyme in decidual cells. Thyroid peroxidase (TPO) is responsible for the oxidation of thyroidal iodide, and antibodies to the enzyme have been used in IHC formats for the identification of normal and neoplastic thyroid tissue. Enzymes of the biosynthetic pathway of steroid hormones can also be demonstrated effectively by IHC. Among the enzymes that have been localized are P450 _scc (cholesterol side-chain cleavage), 3-β-hydroxysteroid dehydrogenase (3β-HSD), 21-hydroxylase, 17-α-hydroxylase, 11-β-hydroxylase, and 17-β-hydroxysteroid dehydrogenase type 12 (17β-HSD12). To date, relatively few studies have evaluated antibodies to these enzymes as diagnostic reagents. High P450scc (encoded by CYP11A1) expression was detected in a subset of human gonadotroph tumors, and immunolocalization of 17β-HSD12 in invasive ductal carcinoma was associated with poor prognosis and tumor progression.

Deficiency of a DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is more common in pancreatic NE tumors than in carcinoid tumors and is associated with sensitivity to temozolomide. Absence of MGMT identified by IHC may explain the sensitivity of some pancreatic and GI NE tumors to alkylating treatment with increase in progression free survival and overall survival of these patients. ^,

Chromogranins, Secretogranins, and Other Granule Proteins

The chromogranins and secretogranins represent the major constituents of NE secretory granules. Three major chromogranin proteins have been identified and categorized and have been designated chromogranin A , chromogranin B , and secretogranin II , also known as chromogranin C . Additional granins that have been characterized include secretogranin III (1B1075), secretogranin IV (HISL-19 antigen), and secretogranin V (7B2), secretogranin VI (NESP55), secretogranin VII (VGF), and secretogranin VIII (proSAAS). ^,

The chromogranin and secretogranin proteins contain multiple dibasic residues that are sites for endogenous proteolytic processing to smaller peptides. For example, chromogranin A contains 439 amino acids with 10 pairs of amino acids that represent potential cleavage sites by proteases such as the prohormone convertases. Resultant peptides include chromostatin, pancreastatin, parastatin, and vasostatin. Functional roles for these smaller peptides include intracellular hormone-binding functions, inhibitory effects on the secretion of other hormones, and antibacterial and antifungal effects. Many NE cells contain all the major granins, whereas others show distinctive patterns of chromogranin distribution.

The monoclonal antibody LK2H10, developed by Lloyd and Wilson, is directed against chromogranin A and is currently the most commonly used chromogranin antibody. Chromogranins are present within the matrices of secretory granules of NE cells. As a result, tumors with abundant secretory granules demonstrate intense chromogranin immunoreactivity, whereas those with fewer granules are less intensely stained. Numerous studies have demonstrated that chromogranin A represents the single most specific marker of NE differentiation in general use. Antibodies to chromogranin B and secretogranin II are available but are not in general use.

Tissue-specific patterns and ratios of the chromogranin proteins are typically maintained in NE tumors. For example, chromogranin A is the major granin expressed by gastric carcinoids and serotonin-producing carcinoids of the appendix and ileum. In contrast, strong immunoreactivity for chromogranin B and secretogranin II is typical of rectal NE tumors (carcinoids and small cell carcinomas) and of prolactinomas, which lack chromogranin A. ^,

NE secretory protein-55 (NESP-55) is a 241-amino acid polypeptide that is a member of the chromogranin family. It is expressed exclusively in endocrine and neuronal tissue but has a less wide distribution than chromogranin A in human tissues. The reactivity of NESP-55 appears to be restricted to NENs of the pancreas and adrenal medulla, and several studies have indicated that it may be useful in the identification of sites of origin of metastatic NENs. ^,

Synaptophysin And Other Synaptic Vesicle Proteins

Synaptophysin is a calcium-binding glycoprotein (38,000 kDa), which is the most abundant integral membrane protein constituent of synaptic vesicles of neurons. It is also present in a wide spectrum of NE cells and in many of their corresponding tumors. Typically, synaptophysin reactivity is present in a punctate pattern in synaptic regions of neurons and is present diffusely throughout the cytoplasm of NE cells. Ultrastructurally, synaptophysin is present in microvesicles, whereas chromogranin is present in secretory granules. These differences indicate that chromogranins and synaptophysin are complementary generic NE markers. Synaptophysin immunoreactivity, however, is not specific to NE cells, because it is also present in adrenal cortical cells and their tumors.

Synaptic vesicle protein 2 (SVP-2) is present in the central and peripheral nervous systems and in a wide variety of NE cell types. Comparative studies of the distribution of SVP-2, synaptophysin, and chromogranin A in NE tumors have shown excellent agreement, with the exception of hindgut ENs, which showed weak synaptophysin immunoreactivity, no staining for chromogranin A, but strong staining for SVP-2. GI stromal tumors also express SVP-2, suggesting that these tumors may have an NE phenotype.

Vesicular monoamine transporters (VMATs) mediate the transport of amines into vesicles of neurons and endocrine cells. VMAT1 and VMAT2 are differentially expressed by GI ENs with patterns specific for each tumor type. For example, serotonin-producing ENs expressed VMAT1 predominantly, whereas histamine-producing ENs (gastric ENs) expressed VMAT2 almost exclusively. Extensive and intense VMAT2 immunoreactivity was also observed in adrenal pheochromocytomas, chromaffin paragangliomas, and carotid body paragangliomas.

On the other hand, peptide hormone–producing GI tumors (rectal carcinoids) and pancreatic NETs (PanNETs) contained few VMAT1- or VMAT2-positive cells; and only rare VMAT2-positive cells were observed in intestinal enterochromaffin (EC) cell tumors, in pancreatic NE tumors, and in the mixed PDEC/ECL cell carcinoma of the stomach. VMAT2 immunoreactivity was not observed in the series of Rindi and colleagues in gastrin; somatostatin, enteroglucagon/peptide YY tumors of the GI tract, gastric PDECs, adrenocortical growths, parathyroid and lung NE neoplasms, and may be a useful tool for the diagnosis of gastric ECL cell tumors, separating them from all other ENs arising in the gastroduodenal area. Synaptotagmins (p65), which form a large calcium-binding family, are implicated in neurotransmitter release, although synaptotagmin I is the only isoform demonstrated to have a role in vesicle fusion. In the pancreatic islets, synaptotagmins have been co-localized with insulin, but the roles of this family of proteins have not been fully explored as markers of NE tumors. ^,

The vesicle-associated membrane proteins (VAMPs, or synaptobrevins) occur in three isoforms and are proteins that are anchored to the cytoplasmic portion of synaptic membrane vesicles and secretory granules. VAMP2 and VAMP3 are present in pancreatic β cells, but the roles of this family of proteins have not been widely studied as markers of NE tumors. In contrast to synaptophysin and other SVPs, synaptosomal-associated protein, 25 kDa (SNAP-25), and syntaxin are present in the plasma membranes. At present, only a few studies have reported on the application of these markers in diagnostic pathology.

CD57

The CD57 antigen is present on subsets of T cells and natural killer (NK) cells. Antibodies to CD57 also react with Schwann, oligodendroglial, and a variety of NE cells of both neural and epithelial types. Additionally, CD57 positivity is present in prostatic, renal, and cortical thymic epithelial cells. Antibodies to CD57 react with varying proportions of neural tumors, including schwannomas, neurofibromas, neuromas, and granular cell tumors. Among ENs, CD57 has been used most commonly as a marker for NE neoplasms. For example, CD57 is present in 100% of pheochromocytomas, 85% of extraadrenal paragangliomas and NENs of diverse origins, and 50% of small cell bronchogenic carcinomas. However, CD57 is not restricted in its distribution to NE neoplasms, because reactivity is present in more than 95% of papillary thyroid carcinomas (PTCs) and approximately 70% of follicular carcinomas. Nonendocrine neoplasms that are frequently CD57 positive include prostatic carcinomas, thymomas, metanephric adenomas, and a variety of small round blue cell tumors. These results indicate that the use of CD57 antibodies alone is unreliable for the specific identification of NE neoplasms.

Neural Cell Adhesion Molecule (CD56)

The neural cell adhesion molecules (NCAMs) comprise a family of glycoproteins that play critical roles in cell binding, migration, and differentiation. The NCAM family includes three principal moieties that are generated from alternative splicing of RNA from a gene that is a member of the immunoglobulin supergene family. The molecules are modified post-translationally by phosphorylation, glycosylation, and sulfation. The homophilic binding properties of NCAMs are modulated by the differential expression of polysialic acid. Although initial studies indicated that NCAM was restricted in its distribution to the nervous system, more recent studies indicate a considerably wider distribution, including the adrenal medulla and cortex (zona glomerulosa), cardiac muscle, thyroid follicular/epithelium, proximal renal tubular epithelium, nephrogenic rests, metanephric mesenchyme, hepatocytes, gastric parietal cells, and islets of Langerhans. In addition to CD56 expression on NK cells and osteoblasts, several hematopoietic neoplasms, including blastic plasmacytoid dendritic cell neoplasms, plasma cell myelomas, a subset of the T-cell lymphomas, and a subset of acute myeloid leukemias, are known to express CD56. Among solid tumors, both follicular and papillary thyroid carcinomas, as well as renal cell carcinomas (RCCs), Wilms tumors, and hepatocellular carcinomas, are NCAM positive. ^, The Leu-7 antigen, recognized by the human NK antibody 1 (HNK-1) monoclonal antibody, has now been identified as a carbohydrate epitope present on NCAM and a number of other adhesion molecules. Most NE cells and tumors with neurosecretory granules contain both NCAM mRNA and NCAM protein. Antibodies to a long-chain form of polysialic acid (polySia) found on NCAM have been used in studies of normal and neoplastic C cells, and NE neoplasms of the lungs. ^,

Intermediate Filaments

Cytokeratins (CKs) are the major intermediate filaments of endocrine cells, with the exception of steroid-producing cells. These proteins are members of the intermediate filament (10 nm) superfamily of cytoskeletal proteins. They differ from other cytoskeletal filaments on the basis of size and other physical and chemical properties. Microfilaments (5 to 15 nm) contain actin, whereas the 25-nm microtubules contain tubulin. Other types of intermediate filaments present in endocrine cells and their supporting elements include vimentin, glial fibrillary acidic protein (GFAP), and the neurofilament proteins (NFPs). The CKs are the largest and most complex group of intermediate filaments and include a family of at least 30 proteins with molecular weights that range from 40 to 68 kDa. The type II keratins are basic and include eight epithelial proteins, CK1 through CK8. The type I keratins are more acidic and include 11 epithelial keratins, CK9 through CK20. Pairs of basic and acidic keratins are expressed differentially in epithelial cells at different stages of development and differentiation. They can be identified immunohistochemically by using pancytokeratin antibodies that react with epitopes on many different molecular-weight CK species or with chain-specific monoclonal antibodies that recognize one specific CK type. The CKs are distributed in tissue-specific patterns, and primary tumors tend to recapitulate the CK profiles of the cells from which they are derived. ^, In some cases, CK expression patterns tend to be simple, whereas in other cases, complex patterns of expression are apparent. Vimentin (57 kDa) is also expressed together with CKs in many normal and neoplastic endocrine cell types. In steroid-producing cells, vimentin is the major intermediate filament protein, and ablation of vimentin is suggested to result in defective steroidogenesis.

The neurofilaments are composed of heteropolymers of three different subunits with molecular weights of 70, 170, and 195 kDa, which correspond to low (L), medium (M), and high (H) molecular weight subunits. All three neurofilament subunits are phosphorylated in proportion to the molecular weight of each subunit. The neurofilaments represent the major intermediate filaments of mature and developing neurons, paraganglionic cells, and certain normal NE cells. These intermediate filaments are expressed in tumors with evidence of neuronal differentiation and are also present to varying degrees in NE tumors of epithelial type, which also express CKs. Normal epithelial NE-type cells (pancreatic islets, Merkel cells) most commonly lack neurofilament immunoreactivity, whereas their corresponding neoplasms are commonly positive for this marker. Moreover, the pattern of staining in a dot-like area that corresponds to the Golgi region is typical of NENs. The studies of Perez and coworkers have suggested that the differential expression of neurofilament subtypes is related to tumor site, whereas the studies of Schimmack and colleagues demonstrated that internexin α (INA), belonging to class IV of intermediate neurofilaments, is over-expressed in pancreatic NE neoplasms compared with pancreatic adenocarcinomas and normal pancreas (27-fold [ P = .0001], and 9-fold [ P = .02], respectively), and correlated positively with Ki-67 (correlation coefficient [ r ] = 0.5; P < .0001) and chromogranin A ( r = 0.59; P < .0001) distinguishing between primary tumors and metastases.

GFAP (50 kDa) is the major intermediate filament type of fibrous and protoplasmic astrocytes. GFAP is also present in nonmyelinated Schwann cells, supporting cells of the anterior pituitary and paraganglia, and in a variety of carcinomas. Immunoreactivity for GFAP is also present in mixed tumors of the skin and salivary glands, in nerve sheath tumors, and in chordomas.

Transcription Factors

Transcription factors are proteins that bind to regulatory elements in the promoter and enhancer regions of DNA, and either stimulate or inhibit gene expression and protein synthesis. ^, They play critical roles in embryogenesis and development. Transcription factors may be tissue specific, or they may be present in a variety of different tissue types. Many of the so-called tissue-specific transcription factors, however, are not restricted to a single tissue type. For example, thyroid transcription factor 1 (TTF-1) is present both in thyroid follicular ( Fig. 10.1 ) and C cells, and also in the lungs, whereas the adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1) is present in steroid-producing cells and in certain anterior pituitary cell types. Pituitary transcription factor Pit-1 is present in certain cells of the adenohypophysis and is also present in the placenta.

GATA3, a marker useful for detection of breast and urothelial carcinoams, was also reported in gonadotroph tumors and tumors with β-TSH expression of the pituitary, ^, pheochromocytomas, paragangliomas, and neuroblastomas.

The Paired Box (PAX) genes are a family of nine nuclear transcription factors that play a crucial role during human embryogenesis and development. While most become silent in adults, selective reactivation is noted during tissue repair and regeneration and also in various cancers suggesting a process of de-differentiation. PAX2, known to control the development of the central nervous system, thyroid gland, kidney, eye, and female genital tract, has been also identified in glucagon secreting pancreatic cells, whereas PAX5, a transcription factor encoding the B-cell lineage specific activator protein (BSAP) expressed in early, but not late stages of B-cell differentiation, has been also reported in small cell lung carcinomas. PAX8 is a highly sensitive marker for renal, müllerian, thyroid, and thymic carcinomas. Among NENs, PAX6 and PAX8 have been reported as markers of pancreatic neuroendocrine tumors (PanNETs). It is worth mentioning that pathologists should be aware that not all commercially available antibodies against PAX8 stain pancreatic NETs.

Insulin gene enhancer binding protein-1 (islet 1) is a homeobox-gene transcriptional factor expressed in all endocrine pancreatic cells and pancreatic NETs ^, in duodenal and rectal NENs, medullary thyroid carcinomas, and lung small cell neuroendocrine carcinomas.

Insulinoma-associated protein 1 (INSM1), a zinc-finger transcription factor encoded by the insulinoma associated-1 gene located on the short arm of chromosome 2, which has an important role in NE differentiation, ^, has emerged as a general NE marker. In normal adult tissues, INSM1 is expressed in the NE cells of pancreatic islets, adrenal medulla, gastrointestinal enterochromaffin cells, normal bronchial epithelium, and prostate gland. INSM1 has been also identified in a wide range of NENs including lung, pancreas and GI tract, uterine cervix, pheochromocytoma, medullary thyroid carcinoma, pituitary adenoma, and Merkel cell carcinoma. Rosenbaum and coworkers reported INSM1 positivity in 88% of the NENs from various anatomic sites. Rooper and colleagues demonstrated a sensitivity of 96.4% across all grades of thoracic NENs for INSM1, while a panel using traditional markers (synaptophysin, chromogranin, and CD56) had a sensitivity of only 87.4%.

INSM1 is an upstream regulator of Achaete-scute complex-like 1 protein 1 (ASCL1, termed mASH1 in rodents and hASH1 in humans) transcription factor. La Rosa and colleagues reported ASCL1 expression in 82% of lung NE carcinomas (NECs) and 70% of extrapulmonary NECs, while its expression was not detected in any gastroenteropancreatic NET and was found in only a minority of lung carcinoids. ^, Although ASCL1 is not organ specific, it is highly specific for NECs versus lung carcinoids and other non-NE neoplasms.

Somatostatin Receptors

Somatostatin acts via specific receptors that belong to the seven transmembrane G-protein–coupled superfamilies. Somatostatin receptors SST-1 through SST-5 represent the five major subtypes. The inhibitory action of somatostatin on hormone secretion is mediated by SST-2, whereas suppression of cell growth is mediated by SST-1, SST-2, and SST-5; the effects of somatostatin on apoptosis are mediated by SST-2 and SST-3. IHC analysis of somatostatin receptors has been used to gauge the responsiveness of NETs to somatostatin analogs.

Cell-Cycle Markers

Antibodies to cell-cycle markers have been used in endocrine pathology primarily as an adjunct for the distinction of benign and malignant tumors. In general, malignant tumors have a higher labeling index than benign tumors, as assessed with antibodies to Ki-67 (MIB-1). ^, The grading of GI and pancreatic NETs is based on Ki-67 proliferation index and mitotic activity, and while Pelosi and colleagues indicated the value of Ki-67 labeling index in effective separation between carcinoids and small cell carcinomas, Ki-67 index calculation is not yet mandatory for the characterization of lung NENs.

The cyclin-dependent kinase inhibitor p27 is decreased in many malignant endocrine tumors compared with their benign counterparts. In some instances, the combined use of Ki-67 and p27 is more effective than the use of either antibody alone.

Pitfalls of Immunohistochemistry of Endocrine Tumors

Many endocrine cells, including thyroid follicular and adrenal cortical cells, contain high levels of biotin-like activity, which is enhanced following heat-induced epitope retrieval (see Fig. 10.1 ). Because endogenous biotin is often incompletely blocked following standard blocking procedures, the use of polymer-based detection systems is recommended for all studies of endocrine cells and tumors. The use of such a system essentially circumvents the nonspecific background staining observed with avidin-based or streptavidin-based system.

Adenohypophysis

The cell types of the adenohypophysis were categorized originally on the basis of their reactivities with hematoxylin and eosin (H&E) as acidophils, basophils, or chromophobes. With more sophisticated histochemical staining sequences, the three cell types were subdivided further. For example, acidophils were further differentiated into the orange, G-positive, prolactin-positive cells, and the erythrosin-positive growth hormone (GH)–producing cells; whereas basophils could be demonstrated by their periodic acid–Schiff (PAS) positivity. The subsequent development of IHC methods allowed for the distinction of cell types based on their content of specific hormones. The major cell types and their corresponding products include somatotrophs (GH), lactotrophs (prolactin), mammosomatotrophs (GH, prolactin), thyrotrophs (thyroid-stimulating hormone [TSH]), corticotrophs (ACTH, β-endorphin, melanocyte-stimulating hormone), and gonadotrophs (follicle-stimulating hormone [FSH], luteinizing hormone [LH]). The somatotrophs are present predominantly in the lateral wings and account for approximately 50% of the cells of the adenohypophysis. Lactotrophs predominate at the posterolateral edges of the gland and account for 15% to 25% of the cells. The corticotrophs are present primarily in the central mucoid wedge and account for 15% to 20% of the cells. Thyrotrophs account for 5% of the cells and are located in the anteromedial regions of the gland. The gonadotrophs compose approximately 5% of the cell populations and are scattered throughout the anterior lobe.

In addition to the hormone-producing cells, a second cell population is also present (folliculostellate cells/follicular stellate cells/FS) in the normal gland. The latter cells have a dendritic shape, are chromophobe, and typically encircle the hormone-positive cells. The FS cells are positive for S100 protein and are variably positive for GFAP. The FS cells also express Annexin-1, a member of the annexin family of phospholipid- and calcium-binding proteins, and may modulate glucocorticoid feedback loops in the anterior gland or act as antigen-presenting cells.

The 4th edition of the World Health Organization (WHO) classification guidelines for pituitary adenomas integrate molecular aspects and expression of transcription factors in the differentiation of adenohypophyseal cells. Immunohistochemistry retains the central role in diagnosis of the 8 general adenoma categories: somatotroph, lactotroph, thyrotroph, corticotroph, gonadotroph, null cell adenoma, plurihormonal, and double adenoma, and also recognizes subtypes of “high-risk” pituitary adenomas associated with a clinically aggressive behavior: plurihormonal Pit-1–positive adenoma, Crooke’s cell adenoma, silent corticotroph adenoma, lactotroph adenoma in males, and sparsely granulated somatotroph adenoma.

The tumors also have distinctive patterns of reactivity with antibodies to transcription factors and CKs ( Table 10.1 and Figs. 10.2 and 10.3 ). More than 90% of adenomas contain CK8, and in sparsely granulated GH cell adenomas, the staining is globular and corresponds to the presence of fibrous bodies. Perinuclear staining is typical of densely granulated GH cell and mammosomatotroph adenomas, whereas corticotroph cell adenomas exhibit more diffuse cytoplasmic staining for CK8. Approximately 50% of adenomas exhibit keratin immunoreactivity with the AE1/AE3 antibody cocktail, but only 7% and 10% are reactive with CK19 and CK7, respectively ( Fig. 10.4 ). Of note, gonadotroph adenomas and null cell adenomas can be cytokeratin negative and are differentiated from paragangliomas by their lack of tyrosine hydroxylase.

Pituitary adenomas are typically positive for NE markers that include chromogranin (100%), synaptophysin (92%), and NSE (80%). ^{, ,} The hormonal content of these tumors can be demonstrated with monoclonal antibodies to specific anterior pituitary hormones and hormone precursor fragments (see Table 10.1 ). In contrast to their presence in the normal anterior pituitary, S100–positive folliculostellate cells are generally absent from pituitary adenomas. Pisapia and colleagues reported that CD1a, traditionally used to diagnose Langerhans cell histiocytosis, is also immunoreactive in native adenohypophyseal epithelial cells, but not in neoplastic proliferations such as pituitary adenomas. The authors suggested that this could be a helpful discriminatory stain in cases where neoplastic pituitary tumors were in the differential diagnosis ( Fig. 10.5 ). Results of another study showed that protein expression of enhancer of zeste homolog 2 (EZH2), known to have a role in cell cycle regulation, was also found in neoplastic pituitary tumors (163 adenomas, 2 carcinomas), whereas normal adenohypophyseal cells (19 normal) were negative (or positive in only rare single cells), and this difference was significant ( P ≤ .0005).

In addition to immunophenotypic characterization, tumor mitotic count and Ki-67 labeling index with evaluation of the “hot spots”, and correlation with tumor clinical parameters such as size and invasive status (MRI, in-situ intraoperative) is strongly recommended. ^,

Pituitary carcinomas are rare, comprising less than 1% of the pituitary tumors, and their diagnosis rests on the demonstration of involvement of the cerebrospinal fluid and/or systemic metastases. These tumors are typically positive for generic NE markers and for one or more anterior pituitary hormones. ^, The most frequently synthesized hormones are prolactin and ACTH, whereas the production of GH, TSH, and FSH/LH is rare. The distinction of adenomas from carcinomas in the absence of metastases is difficult, if not impossible. Significant differences are found in MIB-1 labeling indices among adenomas, invasive adenomas, and carcinomas, but overlaps in these indices exist, and in some carcinomas, the labeling index is in the range of adenomas. ^, A proliferative index of up to 45% has been detected in metastatic deposits. Thappar and coworkers did not detect any p53 expression in adenomas while all the carcinomas expressed p53, but exceptions have been reported, with undetectable p53 in both primary and metastatic foci. Expression of topoisomerase 2-α, cyclooxygenase-2, galectin3, and VEGF have been reported to be higher in pituitary carcinomas than in adenomas.

Described by Scheithauer and collaborators in 2008, ^, pituitary blastoma is a relatively newly recognized category of primitive neuroendocrine tumors of the pituitary by the 2017 WHO classification Endocrine Tumors. A rare malignant neoplasm, with a median age of 8 months at diagnosis, the tumor is composed of three main elements including Rathke-type epithelial glands with rosette-like formations, small primitive appearing cells with a blastema-like appearance, and larger secretory epithelial cells resembling adenohypophyseal cells. The majority of the cases express adrenocorticotropic hormone and only a few express growth hormone in a subset of their cells. Their proliferative index is variable, ranging from low to high Ki67 indices. Pituitary blastomas belong to the DICER1 syndrome, or pleuropulmonary blastoma familial tumor and dysplasia syndrome, caused by heterozygous germline mutations in the DICER1 gene. ^,

Pineal Gland

Tumors of the pineal gland include parenchymal neoplasms (pineocytoma, pineoblastoma, and pineal parenchymal tumor of intermediate differentiation), papillary tumor of the pineal region, germ-cell neoplasms, gliomas, meningeal tumors, and a variety of other tumor types that include lymphomas and lipomatous tumors. Parenchymal tumors comprise a spectrum of lesions that ranges from the most immature lesion (pineoblastoma) to the well-differentiated pineocytoma. Tumors with intermediate features are referred to as pineal parenchymal tumors of intermediate differentiation (PPT-ID; Fig. 10.6 ).

Most primary tumors of the pineal gland originate from pinealocytes, which represent modified neurons similar to retinal photoreceptor cells. Pineocytomas are typically positive for NSE, synaptophysin, neurofilament proteins, microtubule-associated tau protein, class III β-tubulin, and microtubule-associated protein 2 (MAP-2; Fig. 10.7 ). ^, GFAP and S100 protein are present in 75% and 83% of cases, respectively. Retinal S-antigen, a protein localized in photoreceptor cells, has been demonstrated in 28% of pineocytomas and 50% of pineoblastomas. ^, Most pineoblastomas are negative for NFPs but are typically positive for synaptophysin, and are consistently positive for SMARCB1. ^, In general, neurofilament positivity indicates a better prognosis in pineal parenchymal tumors. The MIB-1 labeling index is variable, being reported 1.58, 16.1, and 23.5 in pineocytomas, PPT-IDs, and pineoblastomas, respectively. Yu and coworkers studied 27 cases of PPT-IDs and found that combining mitotic count and Ki-67 labeling index was prognostically useful, whereas the grade using the WHO classification did not demonstrate significant correlation with patient outcome. In contrast to germ-cell tumors, pineocytomas are negative for placental alkaline phosphatase (PAP), human chorionic gonadotropin (hCG), and α-fetoprotein (AFP).

Cone-rod homebox, CRX, a homeobox transcription factor, has been found to have a strong nuclear expression in more than 95% of retinal and pineal tumors, although not in the majority of tumors considered in the differential diagnosis of lesions of the pineal region, with the exception of medulloblastomas. ^, Intensity of CRX staining did not correlate with the subclassification of pineal tumors studied (four pineocytomas, four PPT-IDs, and five pineoblastomas), and more common than not, distribution of staining was heterogeneous.

Thyroid Follicular Cells and Their Neoplasms

Thyroglobulin, T3 and T4, Thyroid Peroxidase, and Thyroid Transcription Factors 1 and 2

Thyroglobulin (TGB) is a 660-kDa glycoprotein with a sedimentation constant of 19S. Iodoproteins of higher and lower sedimentation constants have also been identified immunohistochemically. Considerable variation is found in TGB staining intensity in normal thyroid gland. The cuboidal to columnar cells of the normal gland consistently exhibit greater degrees of TGB immunoreactivity than the flattened (atrophic) cells of follicles distended with colloid. Hyperplastic cells are typically strongly stained for TGB, whereas cells that line involuted follicles are weakly reactive or negative. Follicular cells both in Graves disease and in the hyperplastic phase of Hashimoto disease are moderately to strongly reactive for TGB. Variation in the staining of colloid is also apparent.

Follicular adenomas are positive for TGB but also show considerable variability in staining intensity based on the functional status of their component cells. As would be expected, hyperfunctional adenomas exhibit strong positivity for TGB, whereas inactive follicular cells, such as those present in dilated follicles, have considerably less reactivity and may be negative. Normofollicular adenomas generally demonstrate moderate immunoreactivity for TGB, whereas adenomas of solid and oncocytic types contain smaller amounts of TGB.

Hyalinizing trabecular tumors are typically positive for TGB and may occasionally exhibit positivity for some NE markers, including chromogranin A and hormonal peptides (neurotensin, endorphins). These tumors have plasma membrane patterns of staining with the monoclonal antibody MIB-1 ; however, this pattern of reactivity occurs only when staining is performed at room temperature rather than at 37°C ( Fig. 10.8 ). The most likely explanation for the plasma membrane pattern of reactivity is that the antibody cross-reacts with an epitope present in the plasma membrane under these conditions.

The frequency of TGB positivity in thyroid carcinomas is dependent on the degree of differentiation and the histologic subtype. In general, poorly differentiated carcinomas contain less TGB than better differentiated tumors. The levels of TGB mRNA are also correspondingly lower in poorly differentiated than in well-differentiated thyroid carcinomas. TGB immunoreactivity is present in more than 95% of papillary carcinomas ( Figs. 10.9 and 10.10 ) and follicular tumors. Because TGB is also expressed in metastatic lesions, stains for this marker are particularly valuable in establishing the origins of metastatic tumors. Immunoreactivity for TGB in differentiated follicular and papillary tumors is generally present in a patchy distribution. Although some cells exhibit diffuse and uniform staining, others have focal, apical, or basal positivity. Some tumor cells may be completely unreactive, and for this reason, the absence of TGB in a small biopsy sample does not completely exclude the possibility of a thyroid origin in a metastatic site. Rarely, TGB immunoreactivity, as demonstrated both with monoclonal antibodies and with polyclonal antisera, has been reported in nonthyroid malignancies.

Poorly differentiated thyroid carcinomas, including those of the insular type, are most often TGB positive, although the extent of cellular staining is generally weak and focal. Undifferentiated (anaplastic) thyroid carcinomas are most commonly negative for TGB. In the series reported by Ordonez and coworkers, approximately 15% of cases of anaplastic carcinoma exhibited TGB immunoreactivity in a small number of cells by using both monoclonal antibodies and polyclonal antisera. Examination of serial sections in these cases failed to reveal evidence of entrapped normal follicular cells or foci of differentiated thyroid carcinoma. Other authors, however, have failed to demonstrate any TGB immunoreactivity in anaplastic carcinomas, except in foci of residual differentiated tumors.

Antibodies to T ₃ and T ₄ have been used less extensively than TGB in studies of thyroid carcinoma. Kawaoi and coworkers reported T ₄ positivity in 95% of papillary carcinomas and 54% of follicular carcinomas, but there was no T ₄ positivity in cases of anaplastic thyroid carcinoma. T ₃ was present in 66% of papillary carcinomas, 81% of follicular carcinomas, and 45% of anaplastic carcinomas. The significance of T ₃ staining in the absence of T ₄ immunoreactivity, however, is unknown. IHC studies have demonstrated that thyroid carcinomas are associated with changes in the quantity and antigenic properties of TPO. However, TPO immunostaining is not sufficiently discriminatory for the differential diagnosis of thyroid malignancies versus benign thyroid lesions.

TTF-1 is a homeodomain-containing transcription factor expressed in the thyroid, diencephalon, and lung. TTF-1 regulates the expression of TPO and Tg (TGB) genes in the thyroid, and in the lungs; it plays a key role in the specific expression of surfactant proteins A, B, and C, and Clara cell secretory protein. TTF-1 immunoreactivity has been reported in 96% of papillary, 100% of follicular, 20% of Hürthle cell, 100% of insular, and 90% of medullary carcinomas ( Fig. 10.11 ). In general, the intensity of TTF-1 staining in C-cell tumors is less than that observed in follicular cell tumors. Undifferentiated (anaplastic) carcinomas, on the other hand, are generally negative. In the lung, this marker has been reported in 72.5% of adenocarcinomas, 10% of squamous carcinomas, 26% of large cell carcinomas, 75% of large cell neuroendocrine carcinomas (NECs), more than 90% of small cell carcinomas, and 100% of alveolar adenomas. In contrast, only a small fraction of adenocarcinomas of nonpulmonary and nonthyroid types are positive for this marker. Expression of TTF1 has been reported in adenocarcinomas of the breast, ovary, endometrium, endocervix, and pancreas, and in colorectal adenocarcinomas, kidney tumors, and malignant mesotheliomas.

In their study of thyroid and pulmonary carcinomas, Kaufmann and Dietel demonstrated reactivity for surfactant protein A in three of seven thyroid carcinomas in a focal pattern. Byrd-Gloster and coworkers reported that TTF-1 is useful in the distinction of pulmonary small cell carcinomas from Merkel cell carcinomas; in their study, 97% of small cell bronchogenic carcinomas were TTF-1 positive, whereas none of 21 Merkel cell tumors exhibited positivity. However, TTF-1 has been reported in some nonpulmonary small cell carcinomas, including those that arise in the prostate, urinary bladder, and uterine cervix. Comperat and collaborators investigated the rate of TTF-1 expression in lung metastases of extrathoracic adenocarcinomas and compared the results of IHC performed with different antibodies. Two different clones of antibodies (8G7G1/1 from Dako, SPT24 from Novocastra) raised against TTF-1 were used on 56 lung-metastatic malignant tumors; 41 from colorectal origin, and also on primary colorectal (90 cases), and primary pulmonary adenocarcinomas (86 cases). Their results suggested that the SPT24 clone seems to have a stronger affinity for TTF-1 protein but may lead to a few positive colorectal adenocarcinomas.

Thyroid transcription factor 2 (TTF-2, FOXE1) and paired box gene 8 (PAX8), which are essential for thyroid organogenesis and differentiation, have also been studied in thyroid tumors. TTF-1 and -2 and PAX8 were expressed in differentiated and poorly differentiated thyroid carcinomas, whereas TTF-1 and -2 were expressed in 18% and 7% of anaplastic carcinomas, respectively. On the other hand, PAX8, as detected with polyclonal antibody from Proteintech, was present in 76% to 79% of anaplastic carcinomas. TTF-2 was negative in all other neoplastic and nonneoplastic tissues, including those of pulmonary origin. Although PAX8 was present in a variety of normal and neoplastic tissues, it was not expressed in pulmonary tumors or normal pulmonary tissue or head and neck squamous cell carcinoma. These findings suggest that PAX8 may be a useful marker for the diagnosis of anaplastic thyroid carcinoma, particularly when the differential diagnosis includes pulmonary or head and neck carcinoma. Several studies showed that PAX8 immunostaining in anaplastic thyroid carcinoma and medullary carcinoma is dependent on the antibody characteristics. When monoclonal MRQ-50 antibody was used, PAX8 expression was found in only 54% of anaplastic thyroid carcinoma and in none of the medullary thyroid carcinomas. Therefore, pathologists should be aware of the details of the PAX8 antibody used.

Intermediate Filaments

An extensive body of literature describes the distribution of intermediate filaments in normal and neoplastic follicular cells ( Table 10.2 ). Broad-spectrum keratin antibodies react with normal and hyperplastic follicular cells, follicular cells in chronic thyroiditis, and virtually all thyroid epithelial malignancies. In contrast, antibodies to high-molecular-weight (HMW) keratins have been reported to react with some follicular cells in 8% of normal thyroid, 44% of hyperplastic glands, and all cases of thyroiditis. HMW keratins were present in 100% of papillary carcinomas, 6% of follicular carcinomas, and 20% of anaplastic carcinomas in one study. Studies reported by Schelfhout and coworkers have demonstrated uniform reactivity for CK19 in 100% of papillary carcinomas ( Fig. 10.12 ). Focal reactivity for CK19 (in <5% of the tumor cells) was present in 80% of follicular carcinomas and 90% of follicular adenomas, whereas 90% of colloid (adenomatous) nodules demonstrated more diffuse positivity in less than 50% of the cells. These data were largely confirmed by Raphael and associates. In general, the extent of CK19 staining in follicular carcinomas is considerably less than that present in papillary carcinomas, except in areas of degenerative changes that may be strongly positive.

TABLE 10.2

Cytokeratin Distribution in Papillary Carcinomas and Follicular Tumors

Cytokeratin Type	Papillary (%)	Follicular (%)
8	100	100
18	100	100
7	100	100
19	98 a	84 a
1, 5, 10, 11/14	97	22
5, 6	68	8
17	40	15
13	30	0
20	26	12
14	11	10
4	2.4	0

a Although CK19 is present in papillary carcinoma and in follicular tumors, the extent of staining is consistently higher in papillary carcinoma.

Raphael et al. demonstrated that normal thyroid strongly expresses the simple epithelial CKs 7 and 18, and to a lesser extent 8 and 19, but not stratified epithelial CKs. The same patterns of staining were present in lymphocytic thyroiditis, but reactivity for CK19 was more intense. Immunoreactivity for CK7, CK8, CK18, and CK19 was present in both papillary and follicular carcinomas, although the extent and intensity of CK19 staining were greater in papillary carcinomas; CK19 was present in all cases of follicular carcinoma, at least focally. The stratified epithelial keratins, CK5/6 and CK13, were present in 66% (27/41) and 34% (14/41) of papillary carcinomas, respectively, but these keratins were absent from other tumor types. Miettinen and associates observed CK19 in all papillary carcinomas and in approximately 50% of follicular carcinomas, whereas CK5/6 was present focally in papillary carcinomas. Kragsterman and coworkers concluded that CK19 is of limited value as a marker for routine histopathologic diagnosis, but that the presence of this marker should raise the suspicion of papillary carcinoma.

Baloch and coworkers examined a large series of papillary carcinomas of both usual types and follicular variants for a spectrum of CKs that included CK5/6/18, CK18, CK10/13, CK20, CK17, and CK19. In this series, all cases of papillary carcinoma, including the follicular variant, were positive for CK19 ( Fig. 10.13 ). The follicular variants showed strong immunoreactivity in areas with nuclear features of papillary carcinoma, whereas the remaining areas had moderate to strong staining. Normal thyroid parenchyma immediately adjacent to the follicular variants was also positive, but normal thyroid tissue adjacent to the conventional papillary carcinomas was negative. The significance of the latter observations is unknown. Follicular adenomas, follicular carcinomas, and hyperplastic nodules were negative for CK19. The reasons for the discrepancies in CK19 immunoreactivity in follicular tumors between this and other series are unknown.

Considerable controversy surrounds the presence of CK19 in hyalinizing trabecular tumors of the thyroid. Fonseca and coworkers reported CK19 in all cases of hyalinizing trabecular tumors and suggested that this tumor represents a peculiar, encapsulated variant of papillary carcinoma. In contrast, Hirokawa and colleagues found minimal to no CK19 in their series of cases.

Liberman and Weidner studied the distribution of HMW CKs as demonstrated with the monoclonal antibody 34βE12 (CK1, CK5, CK10, and CK14) and antibodies to involucrin, a structural protein of the stratum corneum, in a series of papillary and follicular carcinomas. Antibodies to HMW CKs reacted with 91% of papillary carcinomas, including the follicular variant, and 20% of follicular neoplasms (adenomas and carcinomas). In general, the staining pattern in papillary carcinomas was strong and patchy, whereas follicular neoplasms stained weakly. Involucrin was positive in 72.5% of papillary carcinomas and 29% of follicular tumors. It has been suggested that the pattern of staining with 34βE12 might be best explained by the presence of an epitope on CK1 or by an epitope that is not recognized by other monoclonal antibodies to CK5, CK10, and CK14.

CKs are demonstrable in 70% to 75% of anaplastic thyroid carcinomas by using antibodies AE1/AE3, 34βH11, and CAM5.2, and approximately 30% exhibit reactivity with 34βE12 ( Fig. 10.14 ). ^, Poorly differentiated carcinomas exhibit positivity in 100% of cases with broad-spectrum cytokeratin antibodies.

Vimentin is coexpressed with CKs in the vast majority of normal and neoplastic thyroids. In the series of Miettinen and colleagues, follicular and papillary tumors expressed vimentin in more than 50% of the tumor cells. Immunoreactivity for vimentin was generally present in the basal portions of the cells in contrast to the more diffuse cytoplasmic reactivity for CKs, and vimentin immunoreactivity has been reported in 94% of anaplastic thyroid carcinomas.