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Non-Islet Origin of Pancreatic Islet Cell Tumors

Molecular Pathogenesis Laboratory, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, and Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Alexander O. Vortmeyer, Molecular Pathogenesis Laboratory, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Building 10, Room 5D37, 10 Center Drive, Bethesda, Maryland 20892. E-mail: vortmeyera{at}ninds.nih.gov.

	Abstract

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The histogenesis of pancreatic islet cell tumors was investigated by morphological identification of putative precursor lesions in pancreatic tissue from patients with multiple endocrine neoplasia type 1 (MEN1), tissue microdissection, and genetic analysis. MEN1 mutation and absence of the MEN1 wild-type allele in different precursor lesions strongly suggest that pancreatic islet cell tumors are derived from the ductal/acinar system but not from pancreatic islet tissue. Pluripotent cells within the exocrine pancreas appear capable of formation into small atypical accumulations of MEN1-deficient cells with both exocrine and endocrine phenotype. The findings suggest presence of multiple developmental aberrations in MEN1 pancreas that potentially serve as precursor material for neuroendocrine tumors.

	Introduction

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MULTIPLE PANCREATIC ISLET cell tumors (neuroendocrine tumors) are a hallmark of multiple endocrine neoplasia type 1 (MEN1). Pancreatic islet cell tumors show marked cytological similarity with pancreatic islets and are therefore believed to originate from the endocrine pancreas. In hereditary tumor syndromes, multiplicity of tumors is frequently accompanied by a variety of pathological changes in the grossly unaffected organ that can only be detected by microscopic examination. For example, patients with germline mutation of the VHL gene not only develop multiple clear cell tumors of the kidney but also numerous simple and complex cysts, many of which require microscopic examination for detection (1). These microscopic lesions, embedded in functional and histologically intact parenchyma, can provide more valuable information about their histogenesis than grossly detectable tumors whose relationship with surrounding parenchyma can be characterized solely by either expansive or infiltrative growth. We have therefore focused our attention on normal pancreatic tissue from patients with MEN1 in which we frequently observed a spectrum of morphological changes ranging from obvious tumor to early neoplastic changes.

In the past, experimental analysis of the origin of neuroendocrine tumors was limited due to the reluctance of both neuroendocrine cells and neuroendocrine tumors to grow in long-term culture systems. In addition, specific growth patterns of tumors cannot be reproduced in cell culture; instead, early morphological lesions must be identified and analyzed using histological preparations. Recent advances in hereditary tumor research, however, have provided us with new tools to approach this question. Firstly, the causative gene for MEN1 has been identified recently (2). Secondly, wild-type allelic deletion has been established recently as a consistent occurrence associated with tumorigenesis (3). Thirdly, tissue microdissection allows for the selective procurement of limited numbers of cells that can be further analyzed with appropriate techniques (4). Therefore, ambiguous small morphological structures can be selectively procured from tissue sections and verified as neoplastic by demonstration of deletion of the MEN1 wild-type allele in addition to germline mutation. With this study, we present evidence that islet cell tumors originate within the pancreatic ductal/acinar system.

	Subjects and Methods

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To demonstrate origin of islet cell tumors from the ductal/acinar system, the following investigational steps were undertaken:

1) Pancreatectomy or partial pancreatectomy specimens from nine patients with MEN1, all of whom have been treated under Institutional Review Board-approved protocols, were fixed in formalin and embedded in paraffin blocks.

2) Neuroendocrine islet cell tumor tissue and tissue from adjacent normal pancreas was subjected to microdissection to procure samples of pure tumor and normal cells. Loss of heterozygosity (LOH) analysis was performed with markers D11S480 and D11S449 flanking the MEN1 locus. Six cases were informative for at least one of the two markers, and all six cases demonstrated loss of the wild-type allele in tumor tissue. Germline mutations were K120X, K119del, R460X, 1132delG, and 1202del2; no germline mutation was identified in one noninformative case. The three noninformative cases were excluded from the study.

3) Using light microscopy, hematoxylin and eosin (HE) slides of all specimens from the six informative cases were carefully searched for atypical areas that were different from regular pancreatic architecture. Atypical areas were morphologically characterized and classified, and the cell count of atypical areas was semiquantitatively assessed (see below).

4) From each informative case, three to five paraffin blocks with the largest number of atypical areas were selected. From each block, five serial sections were taken (sections 1–5). Section 1 was stained with HE and subjected to morphological assessment for the following target structures (Fig. 1):

View larger version (105K): [in this window] [in a new window]   FIG. 1. Representative target structures from different MEN1 pancreata chosen for microdissection and genetic analysis with polymorphic markers flanking the MEN1 locus. Two illustrations are given per target structure. Arrowheads delineate area chosen for microdissection. A1, Area with close association of neuroendocrine and ductal/acinar cells; small arrowheads point to residual acinar cells. A2, Area with close association of neuroendocrine cells and intermediate to large-sized ductal structures; small arrowheads point to residual ductal cells. A3, Islet-like structures with irregular outline and trabecular architecture; small arrowheads point to a few residual acinar cells. A4, Atypical structures resembling acinar nodules; note two regular islets next to acinar nodule in left picture. T, Neuroendocrine islet cell tumor; areas with minimal amounts of fibrosis and vascularization were preferentially chosen for microdissection. I, Pancreatic islet; note marked size differences of pancreatic islets that may occur in MEN1 by comparing large islet (large arrowheads) with small islet (small arrowheads) in upper right corner. For this study, largest islets were chosen from each case for microdissection.

Atypical structure type 2 (A2): Focal areas revealing close association of both atypical cell clusters and pancreatic duct cells outside of the acinar system.

Atypical structure type 3 (A3): Structures of islet size revealing abnormal architectural features including irregular outline, trabecular architecture, and increased intercellular fibrosis.

Atypical structure type 4 (A4): Abnormal clusters of acinar cells, so-called acinar nodules, as identified in two cases. These structures are characterized by well-demarcated clusters of acinar cells with slight irregularities in regard to arrangement and staining quality (5) and have been associated previously with proliferative activity of the endocrine elements of the pancreas (6).

For control purposes, the following target structures also were identified:

T: Structures consistent with neuroendocrine islet cell tumor based on the presence of a neuroendocrine cell tumor mass with lobular or trabecular architecture.

I: Structures morphologically consistent with islets. Islets in MEN1 pancreas may exhibit marked variation in size; this phenomenon gave rise to the controversial concept of islet cell hyperplasia as potential structural hallmarks of subsequent neoplastic growth. From each case, we microdissected at least 10 large islets.

N: Structures morphologically consistent with normal exocrine pancreas.

For microdissection of identified targets (A1–A4, T, I, N), 6-µm-thick serial sections were used (Fig. 2). Twenty to 200 identifiable cells per sample were dissected and placed in 5 µl dissection buffer with proteinase K, as previously described (4). DNA was extracted by incubation at 37 C for 2 d. Proteinase K was inactivated by heating, and 1.5 µl of each sample was used for PCR amplification in the presence of ³²P. For LOH analysis, PCR amplification was performed with polymorphic primers D11S480 and D11S449 flanking the MEN1 locus. Amplification products were separated on 6% polyacrylamide gels.

View larger version (168K): [in this window] [in a new window]   FIG. 2. Microdissection of target structures. Before microdissection, target structures are visualized and subsequently removed from a lightly stained consecutive serial section. The serial section was restained with HE after the microdissection. Arrowheads point to removed structures; A, islet (I); B, A3 structure; C, A4 structure before (left) and after (right) microdissection.

	Results

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After amplification with polymorphic markers flanking the MEN1 locus, the presence of two alleles was consistently detected in microdissected, histologically normal pancreatic tissue samples (N) from informative cases. In contrast, LOH was consistently observed in neuroendocrine islet cell tumor samples (T) (Fig. 3, A, C, and D).

View larger version (58K): [in this window] [in a new window]   FIG. 3. Representative results of genetic analysis of different atypical target structures obtained from different MEN1 pancreata by microdissection using polymorphic primer D11S449. Each lane represents different microdissected sample. A–C, Loss of the wild-type allele is consistently observed in target structures A1, A2, A3, and T. Retention of both alleles is seen in N (normal pancreas) and A4 (acinar nodules); note that among the lesions with allelic deletion, contaminating bands were most frequently seen in A1 composed of a mixture of ductal/acinar cells and proliferating atypical cells. Arrowheads depict site of wild-type allele. D, Representative analysis of different islets obtained from a MEN1 pancreas. Each lane represents a different microdissected sample. Note well-balanced preservation of both alleles in both normal pancreas (N) and pancreatic islets (I). Allelic deletion with some contamination is seen in the three positive control tumor samples (T).

A1 structures, characterized by close association of atypical and ductal/acinar cells, revealed LOH in most cases (Fig. 3, A–C). Typically, the LOH result showed a contamination wild-type band, likely being caused by the presence of wild-type acinar/ductal cells within the sample. A2 structures, areas revealing close association of both atypical cell clusters and larger pancreatic ducts, consistently revealed LOH. The same observations were made after genetic analysis of A3 structures, islet-like structures with abnormal architectural features. In contrast, the presence of balanced alleles was consistently observed in A4 structures, acinar nodules.

By immunohistochemistry, most atypical structures (A1–A4) were negative for markers directed against insulin, pancreatic polypeptide, glucagon, or somatostatin. Occasionally, however, unequivocal positive immunoreactivity was detected for insulin, pancreatic polypeptide, or glucagon (Fig. 4). Serving as internal quality control, pancreatic islets showed the expected immunohistochemical profiles (Fig. 4). Neuroendocrine tumors were either nonsecreting or positive with one of the applied markers.

View larger version (164K): [in this window] [in a new window]   FIG. 4. Immunohistochemistry of atypical lesions. A, Pancreatic polypeptide, most atypical structures (outlined by arrows) are negative for any of the hormonal markers applied; regular islets were positive for pancreatic polypeptide. B, Atypical cells, positive for pancreatic polypeptide, intermixed with acinar cells. C, Glucagon, staining of atypical structures; regular islet staining is outlined by arrows. D, Pancreatic polypeptide, staining of atypical cells within the acinar system. E, Glucagon, staining of atypical cells within acinar system; F, Insulin, scattered staining of cells within atypical architectural structure.

	Discussion

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Although this study shows that small areas of atypical architecture containing MEN1-deficient cells can be detected in grossly unaffected pancreas of MEN1 patients, it cannot provide direct evidence that these structures will eventually develop into clinically relevant neuroendocrine tumors. Microscopic areas with close association of atypical cells and ductal/acinar cells, with close association of atypical cells and duct cells, and with atypical architectural features (designated as A1–A3) are, however, suggested to represent small accumulations of immature MEN1-deficient cells from which tumorigenesis may potentially develop. The high frequency of atypical structures compared with that of grossly visible neuroendocrine tumors, which was observed in some cases, does in fact suggest that most atypical structures will not develop into larger neuroendocrine tumors.

Pancreatic islet cell tumors have been assumed to be derived from pancreatic islets because of their morphological similarity with the neuroendocrine pancreas and due to their ability to secrete a wide variety of hormones. During embryogenesis, however, islet cell development within the pancreas appears to be initiated from primary tubules of epithelial precursor cells (7, 8). This epithelium rapidly proliferates, then subsequently differentiates into both acinar and the various islet-associated cell populations (9, 10, 11, 12).

Recent evidence suggests that embryonic pluripotency may be preserved in adult pancreatic tissue and that functional, endocrine pancreas can be grown in vitro from stem cells isolated from both mouse and human pancreatic ducts. Typically, primary ductal stem cell culture in selective media reveals characteristic epithelial growth, followed by the appearance of round cells budding upward from the epithelial monolayers (13, 14). Further appropriate treatment will induce formation of more compact cell clusters with the potential of maturation into functional islet-like structures containing glucagon-producing -cells, insulin-producing ß-cells, and somatostatin-producing -cells (13, 14). We speculate that this neuroendocrine differentiation process may be altered by early loss of the wild-type MEN1 allele in the setting of MEN1. Similar, pathogenetically different pathways may play a role in persistent neonatal hyperinsulinemic hypoglycemia, which is characterized by microscopic ductuloinsular complexes, suggesting ductular origin of neuroendocrine cell proliferation (15). Partial pancreatectomy in young adult rats induces differentiation into new pancreatic islets from small ductules (16).

With the present study, we provide evidence for a non-islet-cell origin of pancreatic islet cell tumors using a combination of genetic and morphological analysis, suggesting considerable differentiation plasticity not only of embryonal ductular cells but also of cells within the ductular/acinar system that appear committed to neoplastic proliferation after a "second hit" of MEN1. The pluripotency of early neoplasia arising from the pancreatic ductular/acinar system may be analogous to that of MEN1-associated pituitary tumors (17), and similar principles may apply for the tumorigenesis of other tumor suppressor syndromes, e.g. von Hippel-Lindau disease (18). The obvious analogies between the pluripotency of embryonal and early neoplastic cells may provide new insights into the pathogenesis of hereditary and sporadic neoplasms.

	Acknowledgments

 
We thank Dr. Mark Raffeld and Cynthia A. Harris for the excellent immunohistochemistry preparations. We thank all clinicians and surgeons, especially Drs. S. Libutti, S. Marx, R. Jensen, and H. R. Alexander, at the National Institute of Diabetes & Digestive & Kidney Diseases and the National Cancer Institute who provided clinical care for the patients.

	Footnotes

 
Abbreviations: A1–A4, Atypical structure types 1–4; HE, hematoxylin and eosin; I, structures morphologically consistent with islets; LOH, loss of heterozygosity; MEN1, multiple endocrine neoplasia type 1; N, structures morphologically consistent with normal exocrine pancreas; T, structures consistent with neuroendocrine islet cell tumor.

Received September 9, 2003.

Accepted December 30, 2003.

	References

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