Multiple neuroendocrine tumors of the pancreas in von Hippel-Lindau disease patients: histopathological and molecular genetic analysis

Abstract

Although pancreatic neuroendocrine tumors (NETs) in von Hippel-Lindau (VHL) disease have been reported, their pathological features have not been characterized. In addition, it is unknown whether alterations of the VHL gene are responsible for pancreatic NET development. To evaluate NETs in VHL patients, we performed histopathological analysis of 30 pancreatic tumors in 14 patients. In addition, DNA from NETs and normal pancreatic tissue from 6 patients with documented germ-line VHL gene mutations was studied for allelic deletions of the second copy of the VHL gene by fluorescence in situ hybridization and polymerase chain reaction-based single-strand conformational polymorphism analysis. Morphologically, the tumors were characterized by solid, trabecular, and/or glandular architecture and prominent stromal collagen bands. Sixty percent of the tumors revealed at least focally clear-cell cytology. All tumors were positive for panendocrine immunohistochemistry markers (chromogranin A and/or synaptophysin); 35% of NETs demonstrated focal positivity for pancreatic polypeptide, somatostatin, insulin, and/or glucagon; and no immunostaining for pancreatic and gastrointestinal hormones was observed in 65% of tumors. Dense core neurosecretory granules were evident by electron microscopic examination, and the clear cells additionally revealed abundant intracytoplasmic lipid. All NETs that were subjected to genetic analysis showed allelic loss of the second copy of the VHL gene. We conclude that multiple, nonfunctional pancreatic NETs occur in VHL patients. Stromal collagen bands and clear-cell morphology are important histological features of VHL-associated NETs. The presence of allelic deletions of the VHL gene in pancreatic NETs provides direct molecular evidence for a role of the gene in their tumorigenesis and establishes NET as an independent tumor type of VHL disease.

Figure 1.

Gross photographs of two VHL-associated pancreatic NETs. A: A 3-cm solid, well-circumscribed, tan-gray tumor (tumor 1) with prominent collagen stroma removed from patient 1 by partial pancreatectomy. B: A 1-cm NET (tumor 29) removed from patient 14 by enucleation. The tumor displays a prominent yellow color secondary to abundant lipid content.

Figure 2.

Histology and electron microscopy of representative VHL-associated pancreatic NETs. A: Tumor 19 (patient 9) shows trabecular architecture and small NET cells with eosinophilic cytoplasm (H&E, ×400). B: Tumor 18 (patient 8) shows solid architecture, small vessels, and cells with prominent clear cytoplasm (H&E, ×400). C: Tumor 14 (patient 6) demonstrates nests of tumor cells with clear cytoplasm and focal nuclear atypia surrounded by stromal collagen bands (H&E, ×630). D: Electron micrograph of a “clear” cell from tumor 18 (patient 8); prominent lipid globules and myelin figures and small dense core granules (arrows). Magnification, ×8900.

Figure 3.

Immunohistochemistry results in representative VHL NETs. A: Positive synaptophysin stain in tumor 19 (patient 9) (×400). B: Positive S100 stain in tumor 19 (patient 9) (×400). C: Focally positive pancreatic polypeptide stain in tumor 11 (patient 5) (×400). D: Focally positive insulin stain in tumor 14 (patient 6) (×400).

Figure 4.

A: Detection of VHL gene (3p25.5) LOH by PCR-SSCP analysis in tumor 1 (T₁; patient 1) and tumor 9 (T₂; patient 4) using a single nucleotide polymorphic marker (104/105) upstream of the coding region of the VHL gene. Arrowheads: Loss of the lower allele is detected in lane T₁ in patient 1, and loss of the upper allele is detected in lane T₂ in patient 4, containing DNA from pure populations of microdissected NET cells as compared with matched normal DNA (lanes N₁ and N₂, respectively) procured from the adjacent pancreas. B, C, and D: VHL gene deletion detected by FISH in interphase touch preparations of three VHL NETs. Green signal: α-Satellite centromeric marker specific for chromosome 3; red signal: chromosome 3p25 P1 probe containing the VHL gene. Both centromeric probes are retained in tumor cells and are seen in normal somatic cells (green signals appear as dots or as a “blush” when out of focus). B: Tumor 1 (patient 1) showing allelic deletion of the VHL gene in two tumor cells one red signal as compared with a normal somatic cell with two red signals (bottom). C: Allelic deletion of the VHL gene in NET cells (tumor 17, patient 7; one red signal. D: Allelic deletion of the VHL gene in NET cells (tumor 20, patient 10; one red signal).