Introduction
There are many broad-spectrum neuroendocrine markers used in diagnostic pathology to characterize neuroendocrine tumors. The two most common markers include chromogranin and synaptophysin. To use these and other broad-spectrum markers in diagnostic pathology effectively, the advantages as well as the limitations of each marker should be fully appreciated.

General Neuroendocrine Marker

Chromogranin/Secretogranin
The chromogranin/secretogranin (Cg/Sg) family is composed of several acidic proteins present in the secretory granules of neuroendocrine cells. The 3 major Cg/Sg proteins are currently designated as chromogranin (Cg) A and B and secretogranin II (Sg II). Others include SgIII, SgIV, and SgV (or 7B2). The distribution of chromogranin A has been studied extensively in human tumors.^4-7  It is present in most neuroendocrine cells and neoplasms. However, most neoplasms with only a few endocrine secretory granules, such as small-cell carcinomas of the lung and Merkel cell carcinomas, do not react strongly with chromogranin A antibodies.^4-7  Because of their widespread distribution and high degree of specificity, Cg/Sg are excellent markers for neuroendocrine cells and neoplasms.^8,9 

Although Cg AB is a highly specific neuroendocrine marker, it may have limited sensitivity with some tumors. For example, hindgut carcinoids have limited immunoreactivity for Cg A, with only 20-50% of cases positive in a recent series.¹⁰  Similarly, pituitary prolactinomas are often negative for Cg A. Because hindgut carcinoids and prolactinomas often express Cg B, using antibodies against Cg B or a cocktail of Cg A and B usually increases the sensitivity for detecting neuroendocrine cells with Cg/Sg antibodies.

Synaptophysin
Synaptophysin, a 38KD protein molecule, is a component of the membrane of presynaptic vesicles. It is widely distributed in neurons and neuroendocrine cells and their neoplasms, and is a good broad-spectrum neuroendocrine marker.¹¹  Unlike the Cg/Sg proteins that are well preserved in formalin fixed tissues, ethanol fixation provides optimal preservation of the synaptophysin antigen. However, most of the anti-synaptophysin monoclonal antibodies work well in formalin fixed sections. Synaptophysin is localized to vesicles in neuroendocrine cells of tumors, but immunostaining is present diffusely in the cytoplasm.

Proconvertases
The proconvertases (PC) are recently described enzymes that process propeptides into active peptides within cells.^12,13  Some of these, including PC1/PC3 and PC2, are highly specific for neuroendocrine cells and tumors and can be used as specific neuroendocrine markers. Others, such as PC4, are present in the testes, whereas PC5/6 is more prevalent in the gastrointestinal tract and adrenal.

Neuron-Specific Enolase (NSE)
The enzyme is a very sensitive, but not too specific, marker for neuroendocrine cells and tumors. It is commonly found in neurons, peripheral nerves, and neuroendocrine cells.^14,15  Some non-neuroendocrine cells and neoplasms also react with antisera against NSE, NSE should be used only with other broad-spectrum markers of neuroendocrine cells in the diagnosis of neuroendocrine tumors because of its relative lack of specificity. It is used infrequently in our laboratory.

Bombesin/Gastrin-Releasing Peptide and Leu-7 (HNK-1)
Bombesin is a tetradecapeptide originally isolated from amphibian skin. It is present in many endocrine cells as well as in central and peripheral neurons.¹⁶  GRP, the proposed mammalian analogue of bombesin, has been found in many lung and gastrointestinal endocrine tumor and can be used as a broad-spectrum marker for many endocrine neoplasms.¹⁷ 

Leu-7 (HNK-1), a monoclonal antibody that was produced against a T-cell leukemia cell line, recognizes natural killer cells in blood and lymphoid tissues. It also reacts with small-cell carcinomas of the lung as well as with pheochromocytomas and other neuroendocrine neoplasms.^18,19 

PGP9.5
PGP9.5 is a soluble protein that was originally isolated from brain. It is good general marker for neuronal and neuroendocrine tissues.^20,21  Interestingly, about half of melanomas stain for PGP9.5, whereas most melanocytic tumors are usually negative for Cg/Sg and for synaptophysin. PGP is a cytoplasmic soluble protein that frequently co-localizes in normal and neoplastic neuroendocrine tissues.

Neural Cell Adhesion Molecule (NCAM)
NCAM is a member of the family of membrane-bound glycoproteins present mainly in brain and muscle.^22,23  It is involved in neuron-neuron and nerve-muscle interactions. The distribution of NCAM in neuroendocrine tissues and tumors was first reported by Jin et al.²³  They found a widespread distribution of NCAM. NCAM is present in about 20% of non-small cell lung carcinomas and is often associated with a poor prognosis (24). Small cell lung carcinomas frequently stain with NCAM antibodies .^25,26 

Peptidylglycine a-amidating monooxygene (PAM)
Amidation is an important step in the maturation of some neuropeptides (27-30). The enzyme peptidylglycine a-amidating monooxygene (PAM) catalyzes the posttranslational modification of many neuropeptides. It consists of 2 enzymes that convert peptidylglycine substrates into a-amidating products and glyoxylate. The PAM proteins are usually released along with their peptide products during exocytosis, whereas membrane bound PAM remains associated with the cell (27). Several studies have examined PAM expression in neuroendocrine cells .^30-33  A recent study by Scopsi et al found PAM in all neuroendocrine cell types.³⁴  They found a close correlation between PAM expression and at least 1 of the 3 principal granin proteins (CgA, CgB, or SgII).³⁰ 

Synaptic Proteins and Neuroendocrine-Specific Protein (NSP)-reticulons
A series of proteins involved in neurotransmitter secretion have also been associated with neuroendocrine cells and tumors. These synaptic proteins include SNAP-25 and Rab3A.^35-42 

SNAP-25 was originally identifies as a neuron-specific protein associated with the plasma membrane of the presynoptic nerve terminal. It was shown to the part of the putative docking complex that is implicated in membrane fusion. A homologous protein SNAP-23 is expressed ubiquitously in human non-neuronal tissues, including endocrine organs.

Rab3A is a small GTP-binding protein of the rab family that is expressed mainly in neurons and neuroendocrine cells.^39-42  Rab3A is thought to be an important control system for exocytosis in neuron and neuroendocrine cells. A recent study showed increased SNAP-25 immunoreactivity in most PRL-cell adenomas and in GH-cell adenomas, suggesting that this protein is involved in the mechanism of exocytosis in neoplasms derived from these cell types.⁴² 

NSP-reticulons are endoplasmic reticulum-associated protein complexes consisting of 2 closely related protein constituents, NSP-A and NSP-C.^43-46  In a recent report, the expression of NSP-reticulons NSP-A and NSP-C was examined in lung carcinomas. NSP-A and NSP-C were reactive with most carcinoid tumor and small cell lung carcinomas. There was a high concordance between expression of NSPA and NSP-C in neuroendocrine tumors. These investigators noted that NSP-A was more sensitive than synaptophysin, chromogranin A, Leu 7, and neurofilament proteins in detecting neuroendocrine differentiation in non-small cell lung carcinomas.⁴⁶  They also observed that NSP-A expression showed a stronger correlation with conventional neuroendocrine markers than NCAM.

Myosin XVA
– Myosins constitute a large family of proteins. Recent studies have shown that myosin XVA was highly expressed in the pituitary with lower levels in other neuroendocrine cells and tumors.^47-49 

Transcription Factors in NE Tumors
Transcription factors are important proteins in gene regulation. They bind to the upstream regulatory elements of the gene in the promoter and enhancer regions and can stimulate or inhibit gene function. They are usually critical factors during embryogenesis and general development. Examples of transcription factors that can function as NE markers include Pit-1, Prop-1 and Ad4BP/SF-1 in the pituitary ^{50, 51} and thyroid transcription 1 and 2 in the thyroid including medullary thyroid carcinoma and in the lungs.

NE Markers in Medullary thyroid Carcinomas
Calcitonin is the commonly used marker for MTC and is positive in more than 95% of cases. Rare case may be negative, but the mRNA for calcitonin can be detected by ISH.⁵²  Many other markers studied in MTC include chromogranins, carcinoembryonic antigen (CEA) human chorionic gonadotropins and thyroglobulin.³  The latter may be present in entrapped follicles or in mixed follicular/papillary and medullary thyroid carcinomas. Polysialic acid has been detected in 100% of MTC. Komminoth et al reported immunoreactivity for polysia primary C-cell hyperplasia, but not in normal C-cells or secondary C-cell hyperplasia.⁵³ 

Adrenal Medulla and Paraganglia
Most pheochromocytomas and paragangliomas are negative for cytokeratins although positive cases have been reported. Most of these tumors are positive for NSE (100%) ;PGP9.5, chromogranin (95%) and synaptophysin (100%). Ectopic production of hormones such as corticotropic hormone or corticotropic hormone-releasing hormone can be detected in pheochromocytomas and paragangliomas.

Pancreatic Endocrine Tumors
Pancreatic endocrine tumors are usually positive for cytokeratins in more than 90% of cases.³  Low molecular weight keratins such as CAM5.2 are more sensitive for neuroendocrine tumors rather than the keratin cocktails such as AE1/AE3 or molecular weight keratin.

Specific types of pancreatic endocrine tumors can be characterized by the hormone they produce such as insulin, glucagon, somatostatin, pancreatic polypeptide, gastrin and vasoactive intestinal polypeptide. Most insulin producing tumors are benign while most tumors that produce ACTH or calcitonin are usually malignant.

Pulmonary Endocrine Tumors
These tumors include typical and atypical carcinoids and large and small cell carcinomas. Most (about 85%) are reactive with cytokeratin. Chromogranins are usually positive in carcinoids, atypical carcinoids, and large-cell neuroendocrine carcinomas, while only about 50% of small cell carcinomas are positive. However, other broad-spectrum neuroendocrine markers are positive in small-cell carcinomas .³ 

Ectopic Production of NE Markers
Many carcinomas at various sites produce broad-spectrum neuroendocrine markers or specific hormone. These include tumors of the cervix, breast, prostate, thymus, larynx, skin, and other sites. Broad-spectrum markers as well as specific markers for hormones can be used in the diagnosis depending on the clinical sign and symptoms.

In Situ Hybridization

General Applications
The localization of mRNA for specific peptides and other neuroendocrine markers by ISH is another practical approach to analyzing hormone expression.^54-57  Some neuroendocrine neoplasms may contain mostly mRNA but very little translated protein products, so detection of the mRNA within cells can help to characterize neuroendocrine tumors. Advantages of ISH over immunohistochemistry include distinguishing between nonspecific uptake of protein by neuroendocrine tumor cells and de novo synthesis (as the presence of the mRNA indicates de novo synthesis) and detecting gene expression by mRNA within cells even when the protein products are not stored in the cells. Discordance between protein storage and mRNA content has been shown for chromogranin A in small cell carcinoma of the lung.⁵⁴  Studies of gene expression in endocrine tumors by ISH for chromogranin A and B, thyroglobulin, parathyroid hormone, calcitonin, estrogen receptor proteins, and other targets have contributed to our understanding of the biology and pathophysiology of many endocrine disorders.

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