The endocrine pancreas is a complex and important gland that is essential for fuel metabolism and
metabolic homeostasis. Morphologic investigations play an important role in the study of the normal and
pathological endocrine pancreas. Endocrine tumors of the pancreas represent a group of hormone-producing
neoplasms that can give rise to a wide variety of clinical manifestations. They range from small benign
incidental findings to functional hormone-secreting neoplasms to aggressive malignancies.
Recent advances in molecular biologic techniques have brought a new approach to the field
of investigative and diagnostic pathology. It has become possible to gain insight into the cellular and
subcellular defects that form the basis for a variety of diseases. It has also become apparent that both
heritable and sporadic neoplasms are genetic disorders with discrete cellular defects. In general, a
single genetic defect or mutation is not sufficient to initiate tumorigenesis but rather these contribute
in a cumulative fashion to the transformation and/or proliferative process. For each cell type, a unique
combination of genetic hits results in the pathologic phenotype. In some instances the mutation is
oncogenic, i.e it involves a gene whose gain of function is associated with dysregulated synthesis of a
normal product; eg. ras and myc. In other
situations, it is characterized by loss of tumor suppressor gene (TSG) activity , i.e. it involves a gene
whose product normally restrains cell proliferation eg. Rb, p53. Usually it is the combination of gain
of oncogenic function coupled with loss of tumor suppressor action which permits autonomous cell
proliferation. Other factors including angiogenesis, telomerase activity, gain of survival signals, and
metastatic potential all contribute to the ultimate pathologic phenotype that we recognize as carcinoma.
While many of operative mechanisms responsible for human neoplasia probably apply to neuroendocrine
tumorigenesis, considerably less is known about the molecular events resulting in these lesions.
The morphologic analysis of endocrine pancreatic lesions requires the combination of
histology and immunohistochemistry at the minimum(1-3). It is important to characterize
pancreatic tumors as endocrine in nature and to distinguish neoplasms from pseudoneoplastic conditions.
Endocrine tumors generally are well delineated but unencapsulated lesions that have a characteristic
histopathology. They are composed of small nests, trabecula or sheets of epithelial cells in a highly
vascular stroma. They occasionally form gland-like structures. The stroma may, in some instances, form
amyloid. The tumor cells usually have poorly-defined cell borders, abundant cytoplasm that may contain
eosinophilic, amphophilic or basophilic granules. Characteristically the nuclei of tumor cells are
bland; nuclear pleomorphism that defines malignancy in other epithelial tumors is not a reliable
indicator of aggressive behavior. Poorly differentiated carcinomas have less cytoplasm, lack granularity
and have larger hyperchromatic nuclei. The differential diagnoses include non-endocrine tumors, such as
solid and cystic neoplasms, and non-neoplastic proliferative lesions of pancreatic endocrine cells:
nesiodioblastosis and pseudoneoplastic proliferation of endocrine cells in chronic pancreatitis.
Determination of the endocrine nature of pancreatic tumors relies on immunolocalization of
common markers of neuroendocrine differentiation. They stain for synaptophysin, a 38 Kd molecular weight
molecule that is associated with synaptic vesicles of neurons and neuroendocrine cells. Most contain
chromogranins, proteins associated with secretory granules. There are two families of chromogranins, A
and B; to appropriately classify these lesions one needs to identify both chromogranins. In the
pancreas, the most numerous cells are B cells that produce insulin and these are negative for
chromogranin A, the only chromogranin recognized by most widely used antibodies applied for
immunohistochemistry. Moreover, chromogranin immunoreactivity is directly related to the number of
secretory granules that may be scarce in poorly differentiated tumors. Other markers of neuroendocrine
differentiation include CD57 (Leu 7), neural cell adhesion molecule (NCAM; CD56), neuron specific enolase
(NSE), and Protein Gene Product 9.5 (PGP 9.5) that stain variable subpopulations of endocrine lesions and
some, such as NCAM and NSE stain some non-endocrine tumors as well.
The structure-function correlations of these lesions are best defined by immunoreactivity
for peptide hormones. The hormones expressed by normal pancreatic endocrine elements show a highly
characteristic pattern of expression. Pancreatic A cells express glucagon and have a normal distribution
around the periphery of the bands of the normal islet. Insulin is localized to B cells that comprise the
majority of cells that fill the bands of the normal islets. Somatostatin is produced by D cells that are
scattered in the islets between the A and B cells. Pancreatic polypeptide is localized to PP cells that
are randomly found within islets but are more numerous in the head of pancreas and specifically in the
posterior portion. This distribution of hormones is lost in abnormal islets and in tumors. Other
hormones may be expressed, reflecting either fetal regression (e.g. gastrin) or true ectopia of hormone
expression (e.g. expression of GRH, ACTH). The wide spectrum of clinical symptomatology associated with
these lesions is attributed to the numerous peptide hormones that can be elaborated by the cells of the
diffuse endocrine system.
The ultrastructure of these lesions is usually highly characteristic. Tumor cells have
well developed rough endoplasmic reticulum, prominent Golgi complexes and membrane-bound secretory
granules that store hormones for secretion in response to stimulation. The development of these
organelles varies with cell differentiation and hormonal activity; the numbers of secretory granules
reflect the balance between synthesis, storage and secretion. The morphology of secretory granules is
reflective of cell type and hormone content.
The current approach to the classification of these lesions (4) is based on
three principles: (i) the diagnosis should be based on the microscopic features of the lesion but must
incorporate useful immunohistochemical data, (ii) tumors should be distinguished according to the
differentiated cell type and site of origin, and (iii) tumors should be subdivided based on biological
behaviour, into benign, low grade malignant and high grade malignant lesions; the latter include poorly
differentiated endocrine carcinoma and the so-called "small cell" or "oat cell" carcinomas. The
determination of malignant potential is based on architectural and cytologic features including
invasion. The location and hormone content of individual lesions is of importance, especially in the
classification of tumors of histological low grade malignancy, the "well differentiated endocrine
carcinoma" where these additional pieces of information aid in predicting the likelihood of metastatic
Ploidy and Karyotype Studies of Neuroendocrine Tumors
Although DNA image and flow cytometry have shown a relationship between a high DNA index and
a poor prognostic outcome, examination of a limited number of endocrine pancreatic tumors has failed to
show any karyotypic abnormalities in these tumors (5).
Oncogenes and Neuroendocrine Tumorigenesis
Proto-oncogenes are normal cellular genes that play an essential role in the
proliferation and differentiation of normal cells. They function at each step of signal transduction
pathways as growth factors (eg. c-sis), membrane receptors (eg. c-erb-B, c-neu, c-fms),
GTP binding proteins (eg. ras family) and nuclear proteins (eg. c-myc, c-fos). Proto-oncogenes may be activated by
point mutations, translocations or by increased expression. Genetic alteration in these genes leads to
sustained activation of the gene product in the absence of the normal control mechanisms. Activated
oncogenes have been associated with a large number of human tumors, for example N-myc amplification in neuroblastomas and K-ras point
mutations in colonic carcinomas.
Ras proteins are involved in transducing signals from the
cell surface to a number of ligand-receptor complexes. The commonest mutational sites alter the
GTP-binding domain (codons 12/13) or more rarely the GTPase domain (codon 61). Point mutations of all 3
ras genes (H-, K- and N-) are thought to be common in endocrine tumors such
as thyroid tumors (6) but not neuroendocrine tumors.
G-proteins are heterotrimeric membrane-anchored peptides that play a central role in
transducing signals from the cell surface ligand-receptor complexes to the downstream effectors. The a
-subunit dissociates from the ß- and gamma-subunits of Gs when GTP displaces its bound GDP, stimulates
adenylyl cylase to produce cyclic AMP from ATP. Cyclic AMP (cAMP) in turn activates c-AMP-dependent
protein kinases, increases intracellular calcium transport, and may potentiate the effect of activated
inositol phospholipid-dependent protein kinases. The weak intrinsic GTPase activity of Gsa and the
action of GTPase activating peptides (GAP) dissociates GTP from Gs a and terminates the response.
Additionally, the multiple structural and functional isoforms of adenylyl cyclase underscore the
complexity of this redundant system of signal transduction coupling and provides some insight into the
array of potential sites of somatic mutations which could alter both cell division and hormone
production. Indeed one of the earlier and most exciting molecular defects to be described in endocrine
tumors involved the single point mutations in two critical domains of the Gs a subunit of GTPase codon
201 where Arg is switched to a Cys or codon 227 where Gln is replaced with Arg (7).
Substitutions at these codons (the gsp mutation) activate adenylyl cyclase
by inhibiting the hydrolysis of GTP and thereby maintaining Gs a in a constitutively activated state.
Marked elevation in mRNA levels of the Gs a gene have been documented in insulin-secreting pancreatic
Over-expression of genes which act as inhibitors of the apoptotic process may also act as
oncogenes. In some studies, the oncogenes c-myc, bcl-2, c-erb B-2, and c-jun have been shown to be
frequently expressed in human gastroenteropancreatic tumors. The expression of these oncogenes may
represent pathogenic events in the generation, malignant transformation, and progression of
gastroenteropancreatic endocrine tumors.
Tumor Suppressor Genes in Endocrine Tumorigenesis
Products of tumor suppressor genes (TSG) act as sequestering agents for transcription
factors, thereby, modulating physiologic growth by arresting cell division in the G1 phase.
This delay may allow for repair of genomic damage or may trigger apoptotic cell death. Deletion or
reduced expression of TSGs appear to be a commonly shared mechanism in human tumorigenesis.
Multiple Endocrine Neoplasia (MEN) type 1 is an autosomal dominant disorder characterized by multiple
tumors of endocrine tissues, including pancreas. In patients with MEN-1, loss of heterozygosity in
tumors was mapped to 11q13. The MEN-1 tumor suppressor gene was cloned at this site; patients in
kindreds with MEN-1 have been shown to have inherited inactivating mutations of one allele with loss of
heterozygosity (LOH) of the normal allele in tumors. MEN-1 gene mutations have also been found in a
significant number of sporadic tumors of the dispersed endocrine system, primarily gastroenteropancreatic
endocrine tumors (8-10) including 44% of sporadic gastrinomas and 19% of insulinomas
(11). This region of 11q13 contains several other genes that are also known to be associated
with tumorigenesis and may be implicated in the development of tumors that show a high frequency of LOH
in this region.
Von Hippel-Lindau disease is an autosomal dominant disorder characterized by the
development of tumors in multiple organs. The pancreas is affected in up to 75% of patients, but the
majority have benign cysts or microcystic adenomas. The endocrine pancreas is affected in 12-17% of
patients who develop multiple endocrine tumors (12). The VHL gene on chromosome 3p encodes a
213 amino acid protein that is a TSG. Patients with VHL inherit one mutant gene and their pancreatic
tumors show loss of the normal allele, proving the role of the VHL gene in their development
(12). Although sporadic pancreatic endocrine tumors show significant LOH of chromosome 3p
(13), somatic mutations of the VHL gene have not been identified.
Patients with von Recklinghausen's disease (neurofibromatosis type 1, NF-1) develop
somatostatin-producing endocrine tumors of the duodenum but rarely have pancreatic endocrine lesions.
The reason for this distribution is not clear (14). There is little known about the biology
of NF1 in sporadic pancreatic tumorigenesis.
The p16ink4a/CDK2A gene has been implicated in nonendocrine tumorigenesis
where this TSG is frequently altered by deletion, mutation or methylation. Mutation or promoter
methylation of the p16 gene has been reported in a significant proportion of sporadic pancreatic
endocrine tumors, predominantly gastrinomas (15,16).
The p53 protein plays a role in cell cycle regulation; mutations, deletions, or
rearrangements of the p53 gene are among the commonest genetic mutations in human neoplasms. Although
p53 mutations may play a role in the pathogenesis of some endocrine tumors of the appendix, they have not
been reported in pancreatic endocrine neoplasia.
Case reports implicate deletions and possible rearrangements of the Rb gene in
insulin-producing carcinomas (17), deletions of the Wilms' tumor and transformation suppressor
gene krev-1 in an insulinoma (18). The APC gene on chromosome
5q21 has been implicated by association with sporadic pancreatic endocrine tumors (19) but
there is no evidence for a casual relationship. A putative tumor suppressor gene on chromosome 1 may
predict prognosis in pancreatic endocrine carcinomas (20,21). Other significant genomic
mutations in human pancreatic endocrine tumorigenesis include chromosome 10 or 17
monosomy(22), and loss of the adhesion molecule DCC (deleted in colonic carcinoma)
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