August 30, 2008

An Overview of Practical Issues in the Diagnosis of Gastroenteropancreatic Neuroendocrine Pathology

By Chetty, Runjan

* Context.-Although somewhat uncommon, neuroendocrine tumors of the gastrointestinal tract and pancreas have come under scrutiny in recent times. With advances in imaging techniques, more of these tumors are being removed and sent for pathologic evaluation. It is important for the diagnostic pathologist to be aware of recent developments in this field. Objective.-This overview focuses on nomenclature/terminology, classification, practical issues related to recent developments in immunohistochemical markers that aid diagnosis and may relate to prognosis, and molecular advances.

Data Sources.-Currently available literature and personal experience in the field of neuroendocrine pathology.

Conclusions.-The preferred terminology is neuroendocrine/tumor/ carcinoma and it is recommended that the World Health Organization classification be used, taking note of the site variations that may occur. A large number of immunohistochemical markers are available but a core panel that is relevant to the site should be used. Cytokeratin 19 positivity is an independent marker of aggressive behavior in pancreatic neuroendocrine tumors. Gastrointestinal neuroendocrine tumors arise via the CpG island methylator phenotype pathway, whereas their pancreatic counterparts arise as a result of chromosomal instability. The MEN1 gene is implicated in both syndromic and sporadic forms of these tumors.

(Arch Pathol Lab Med. 2008;132:1285-1289)

Endocrine or neuroendocrine cells and their tumors form an enigmatic entity that has caused much confusion through the years among pathologists and clinicians alike.

Despite their relative rarity a huge amount of attention has been paid to this area of pathology and much information regarding histogenesis, morphology, immunohistochemistry, molecular biology, and classification has been generated in the last few years. As such several reviews on various aspects of neuroendocrine pathology and clinical issues have been published recently. This overview focuses on nomenclature/terminology, classification, practical issues related to recent developments in immunohistochemical markers that aid diagnosis and may relate to prognosis, and molecular advances. It is not intended to encompass every aspect but provide a pertinent overview in the aforementioned areas.


The term karzinoid (carcinoid) was introduced by Oberndorfer based on his observation of these peculiar tumors ("little carcinomas") in the small intestine.1

Use of the term carcinoid has become entrenched in the medical literature and has been applied to different entities by pathologists and clinicians. Some pathologists label all tumors with neuroendocrine features carcinoid, whereas some clinicians restrict the use of the term to the so-called carcinoid syndrome resulting from a serotonin-producing tumor.2 It is also known that a carcinoid in one site was not equivalent to a similar tumor in another site within the gastrointestinal (GI) tract. Originally thought to be benign, it is now known that the full histopathologic spectrum from very low-grade to high-grade malignancy can be encountered within this family of tumors.

Should they be called endocrine or neuroendocrine cells/tumors? The cells of the dispersed endocrine system and their tumors that occur in the GI tract share several antigens with nerve elements such as neuron-specific enolase, protein gene product 9.5, chromogranin A, B, and C, and synaptophysin.3 It is for this reason that neuroendocrine is the preferred designation and the term neuroendocrine tumor (NET) is used in this review.

There are at least 15 discrete neuroendocrine cell types distributed throughout the GI tract and these cells are the progenitors of the characteristic tumors that form the rubric of neuroendocrine tumors of the gastroenteropancreatic (GEP) tract.


Given the wide array of cells and sites in which neuroendocrine cells are encountered both within and outside of the GI tract, it is not surprising that a common, unified classification spanning all body sites does not exist. In 2000, Wick4 proposed a generic classification for neuroendocrine neoplasia irrespective of site based on a 3-tiered grading system. The World Health Organization (WHO) classification for NET of the GEP tract categorizes tumors into 3 broad categories5:

1. Well-differentiated neuroendocrine tumor

a. Benign

b. Uncertain malignant potential

2. Well-differentiated neuroendocrine carcinoma-lowgrade malignant

3. Poorly differentiated neuroendocrine carcinoma-highgrade malignant

Categorization is based on tumor size, angioinvasion, extent of organ-specific invasion, proliferation index, functional status/ hormonal syndrome, and metastases to lymph nodes or liver.

However, there are still site-specific variations in the application of this classification and, therefore, the predictors of malignant behavior.

One of the reasons a blanket classification is not readily applicable is because certain neuroendocrine cell types have a specific tissue or site distribution.6 Histamine-producing enterochromaffin-like cells are found only in the stomach; insulin- producing NETs occur only in the pancreas; and gastrin-producing NETs occur in the gastric antrum, upper intestine, and pancreas. On the other hand, serotonin-producing enterochromaffin cells are distributed throughout the GI tract and also in the pancreas, although tumors of these cells are most commonly encountered in the ileum and appendix.

The application of the 3-tier WHO classification system to the various areas of the GEP tract have been laid out succinctly into those occurring in the stomach, duodenum/upper jejunum, appendix, ileum/cecum/colorectum and, finally, the pancreas.2 The reader is encouraged to refer to and use these tables and the categories as the move to more standardized and uniform reporting of GEP NET gains momentum.

In concert with the development of a reproducible classification system (the WHO classification), there has been considerable discussion for the formulation of a TNM staging and grading system under the aegis of the European Neuroendocrine Tumor Society.7,8 Although this is a welcome and important development, it is still to be universally accepted and validated.


The gold standard for diagnosis remains morphologic in the first instance. Gastroenteropancreatic NETs may manifest a diverse range of morphologic appearances from the commonly encountered solid or nested (packeted) pattern (Figure 1) to trabecular/gyriform, glandular, tubular-acinar, and mixed patterns, which are generally encountered in well-differentiated NET. At the other end of the spectrum, large cell neuroendocrine and small cell carcinomas typify the high-grade NET. Less commonly, cystic, papillary (Figure 2), and so-called angiomatoid/angiomatous patterns may also be seen. The cells have readily recognizable cytologic features: round to ovoid cells with eosinophilic, slightly granular cytoplasm and nuclei with a dispersed chromatin pattern ("salt and pepper") and, not infrequently, discernible nucleoli. Ancillary findings such as intracytoplasmic hyaline globules (Figure 3), nuclear pseudoinclusions, and associated amyloidosis are also seen. The presence of calcification is sometimes noted and, when psammomatous (Figure 4) in nature in duodenal NET, is diagnostic of a duodenal somatostatinproducing NET.

Where the diagnosis may not readily be apparent is when there is a cytologic variation. The constituent cells may have abundant granular eosinophilic cytoplasm because of accumulation of mitochondria resulting in an oncocytic/oxyphilic appearance (Figure 5), or the tumor cells may be spindle-shaped, have a clear or finely vacuolated cytoplasm (Figure 6), or be large and pleomorphic sometimes with multiple nuclei (Figure 3) or so-called rhabdoid (Figure 7). The latter cell has a characteristic appearance that is due to collapse of the cytoskeleton (misfolded proteins) with formation of aggresomes, which are perinuclear in location, often displace the nucleus peripherally, and appear as deeply eosinophilic globules in the cytoplasm. Another morphologic variant is a pancreatic NET with ductules (Figure 8). This is not a mixed neuroendocrine-epithelial tumor but a NET with ductules that are either entrapped as the tumor grows into surrounding normal pancreatic tissue or as a result of secondary ductular proliferation occurring as a reactive phenomenon. It is worth mentioning at this juncture that the so-called goblet cell carcinoid of the appendix is a composite or amphicrine carcinoma and not a pure NET, although it is included in many discourses on NET of the GEP tract.


Synoptic reporting is advocated and preferably a format that incorporates all the pertinent histologic and immunohistochemical findings, together with the WHO classification.

It is recommended that the morphologic and functional status be recorded and a possible bottom-line diagnosis for a pancreatic NET is "well-differentiated primary pancreatic neuroendocrine tumor, producing insulin, benign behavior," or "well-differentiated insulin- producing pancreatic neuroendocrine tumor, benign behavior."

It is worth reiterating that immunohistochemically detected peptides do not imply that the patient has clinical symptoms, nor does this finding imply that the tumor is functional. If clinically nonfunctional, these tumors may be designated as "well- differentiated nonfunctioning pancreatic neuroendocrine tumor, composed mainly of glucagon-producing cells, benign behavior." It should be borne in mind that most patients are not evaluated biochemically for the full spectrum of peptide products of NET. Should no dominant hormone be detected immunohistochemically, the tumor is reported simply as "well-differentiated primary pancreatic neuroendocrine tumor, with insulin, glucagon, vasoactive intestinal polypeptide, and somatostatin-producing cells present, benign behavior."

However, it must be remembered that absence of recognizable clinical features may not necessarily reflect true lack of clinical function, and subtle clinical manifestations may be missed.

With regard to GI tract NET, a similar approach is advocated, except a comment on the extent of invasion through the wall of the GI tract may be added, although this feature determines the WHO category that the tumor is placed into and may represent a degree of repetition. In the case of a syndromic NET, a comment on the associated syndrome may be added.


Several markers of neuroendocrine differentiation and more cell- specific markers have emerged recently. The traditional baseline markers that have been the mainstay of NET workup are synaptophysin (a small vesicle-associated marker) and chromogranin (a large secretory granule-associated marker). Added to these are cell- specific markers and/or hormones that are relevant to the site of the NET. For instance, vasoactive monoamine transporter 2 is used for gastric enterochromaffin-like NET; serotonin and substance P for ileal and appendiceal NET; and insulin, glucagon, and pancreatic polypeptide for pancreatic NET. Oftentimes, the functional status and the clinical symptoms of the patient will determine specific markers done in a particular case. Included in the baseline workup of pancreatic NET is Ki-67, as the Ki-67 index separates benign, well-differentiated NET (2%).

Some new and/or lesser known markers that are available include the following:

1. Neuroendocrine secretory protein 55: this protein is a 241- amino acid polypeptide that belongs to the chromogranin family and is thus located within large dense core secretory granules.9 The value of neuroendocrine secretory protein 55 lies in its ability to stain pancreatic NETs (in addition to pheochromocytomas), whereas GI tract NETs are negative.

2. Ghrelin: a 28-amino acid peptide seen in oxyntic glands of the gastric mucosa. However, expression has also been detected in neuroendocrine cells in the pancreas, pituitary, and heart. Ghrelin- producing NETs have been documented in the stomach and intestine.10

3. Somatostatin receptors: somatostatin analogues have been used in the treatment of clinically apparent GEP NET. Five somatostatin receptors have been identified. NETs express several somatostatin receptors, but up to 90% of serotonin- and gastrin-producing NETs of the distal jejunum and ileum, and about 60% of insulin-producing pancreatic NETs, are positive for receptors 2 and 5.11,12

4. CDX-2: a homeobox gene that is essential for intestinal development and differentiation. Approximately 80% of GI tract NETs are CDX-2 positive, especially those occurring in the ileum and appendix. Neuroendocrine tumors occurring in the stomach tend to be CDX-2 negative.

5. Histidine decarboxylase: expression occurs in most neuroendocrine cells, but pancreatic NETs are most frequently positive for this marker.13

6. Xenin: a 25-amino acid peptide that appears to be specific to duodenal neuroendocrine cells. It has been shown that NETs from the duodenum, including nonfunctional, gastrin- and somatostatin- producing tumors show xenin expression.14 In contrast, NETs from other sites in the GEP tract are negative. The utility of this marker may lie, therefore, in determining the site of origin in a NET that would otherwise be classified as "of unknown origin."

7. Cytokeratin 19: a marker that has been shown to have independent prognostic value in pancreatic NET and should form part of the routine immunohistochemical workup of these tumors.15


The advent of high-throughput techniques has meant that the analysis of thousands of genes can be accomplished. The tumor biology of GEP NET is complex without a well-recognized pathway being strongly influential. Instead it is a complex interaction of a myriad of factors that influence growth, differentiation, interaction with tumor environment, and secretion.16 With regard to GEP NET, the most work has been done on pancreatic tumors. In broad terms, 2 mechanisms of tumorigenesis apply: GI tract NETs arise via the CpG island methylator phenotype pathway, whereas their pancreatic counterparts arise as a result of chromosomal instability.17 The microsatellite instability pathway does appear to be implicated to any significant degree in the molecular pathogenesis of GEP NET.

Several studies have shown that the common oncogenes and tumor suppressor genes such as src, ras, myc, fos, jun, p53, and Rb and the DNA mismatch repair genes are not implicated in the molecular pathogenesis of GEP NET.16 Neuroendocrine tumors associated with syndromes are obviously associated with characteristic genetic abnormalities and 4 such syndromes exist: multiple endocrine neoplasia type 1 (MEN1 gene), von Hippel-Lindau disease (VHL gene), neurofibromatosis type 1 (NF1 gene), and tuberous sclerosis (TSC1 and TSC2 genes). The MEN1 gene is involved in 20% to 40% of sporadic NETs as well. The role of NF1, VHL, and TSC1 and TSC2 in sporadic tumors is not well studied.

beta-Catenin and the wnt signaling pathway abnormalities are uncommon in pancreatic NETs and more commonly encountered in GI tract NETs.

Recently it has been suggested that growth factors and their receptors are implicated in the proliferation of NETs, often acting in an autocrine or paracrine fashion. Several growth factors are thought to play a role including transforming growth factor , insulin-like growth factor 1, vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factors.16


1. Oberndorfer S. Karzinoide tumoren des Dunndarms. Frankf Z Pathol. 1907; 1:425-432.

2. Kloppel G, Perren A, Heitz PU. The gastroenteropancreatic neuroendocrine cell system and its tumors: the WHO classification. Ann N Y Acad Sci. 2004; 1014:13-27.

3. Rindi G. Guidelines for the diagnosis and treatment of neuroendocrine gastrointestinal tumours: introduction. Neuroendocrinology. 2004;80:395.

4. Wick MR. Neuroendocrine neoplasia: current concepts. Am J Clin Pathol. 2000;113:331-335.

5. Solcia E, Klo?ppel G, Sobin LH, et al. Histological Typing of Endocrine Tumours. 2nd ed. Berlin, Germany: Springer; 2000. World Health Organization International Histological Classification of Tumours.

6. Rindi G, Kloppel G. Endocrine tumors of the gut and pancreas: tumor biology and classification. Neuroendocrinology. 2004;80(suppl 1):12-15.

7. Rindi G, Kloppel G, Alhman H, et al. TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2006;449:395-401.

8. Rindi G, Kloppel G, Couvelard A, et al. TNM staging of midgut and hindgut (neuro)endocrine tumors: a consensus proposal including a grading system. Virchows Arch. 2007;451:757-762.

9. Srivastava A, Padilla O, Fischer-Colbrie R, Tischler AS, Dayal Y. Neuroendocrine secretory protein-55 (NESP-55) expression discriminates pancreatic endocrine tumors and pheochromocytomas from gastrointestinal and pulmonary carcinoids. Am J Surg Pathol. 2004;28:1371-1378.

10. Papotti M, Cassoni P, Volante M, et al. Ghrelin-producing endocrine tumors of the stomach and intestine. J Clin Endocrinol Metab. 2001;86:5052-5059.

11. Kulaksiz H, Eissele R, Rossler D, et al. Identification of somatostatin receptor subtypes 1, 2A, 3 and 5 in neuroendocrine tumours with subtype specific antibodies. Gut. 2002;50:52-60.

12. Papotti M, Bongiovanni M, Volante M, et al. Expression of somatostatin receptor types 1-5 in 81 cases of gastrointestinal and pancreatic endocrine tumors: a correlative immunohistochemical and reverse-transcriptase polymerase chain reaction analysis. Virchows Arch. 2002;440:461-475.

13. Tanimoto A, Matsuki Y, Tomita T, et al. Histidine decarboxylase expression in pancreatic endocrine cells and related tumors. Pathol Int. 2004;54:408-412.

14. Feurle GE, Anlauk M, Hamscher G, et al. Xenin-immunoreactive cells and extractable xenin in neuroendocrine tumors of the duodenal origin. Gastroenterology. 2002;123:1616-1626.

15. Schmitt A, Anlauf M, Rousson V, et al. WHO 2004 criteria and CK19 are reliable prognostic markers in pancreatic endocrine tumors. Am J Surg Pathol. 2007;31:1677-1682.

16. Grotzinger C. Tumour biology of gastroenteropancreatic neuroendocrine tumors. Neuroendocrinology. 2004;80(suppl 1):8-11.

17. Perren A, Anlauf M, Komminoth P. Molecular profiles of gastroenteropancreatic endocrine tumors. Virchows Arch. 2007;451(suppl 1):S39-S46.

Runjan Chetty, MB,BCh, FRCPath, FRCPC, FCAP, DPhil(Oxon)

Accepted for publication January 30, 2008.

From the Department of Pathology, University Health Network/ University of Toronto, Toronto, Ontario.

The author has no relevant financial interest in the products or companies described in this article.

Reprints: Runjan Chetty, MB BCh, FRCPath, FRCPC, FCAP, DPhil (Oxon), Department of Pathology, The Toronto General Hospital, 200 Elizabeth St, 11th Floor, Eaton Wing, Toronto, Ontario, Canada M5G 2C4 (e-mail: [email protected]).

Copyright College of American Pathologists Aug 2008

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