Volume 123, Issue 5, Pages 1616-1626 (November 2002) Xenin-immunoreactive cells and extractable xenin in neuroendocrine tumors of duodenal origin  Gerhard E. Feurle, Martin Anlauf, Gerd Hamscher, Rudolf Arnold, Günter Klöppel, Eberhard Weihe  Gastroenterology  Volume 123, Issue 5, Pages 1616-1626 (November 2002) DOI: 10.1053/gast.2002.36590 Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 1 Distribution and morphology of xenin-immunoreactive cells in 2 different types of duodenal gastrinomas. (A, B) A duodenal microadenoma (no. 2 of Table 2) with xenin-immunoreactive cells showing a trabecular and tubular growth pattern (A). The cellular distribution is shown with higher magnification in (B). (C, D) Another duodenal gastrinoma (no. 1 of Table 2) with a solid growth pattern (C). Higher magnification is shown in (D). This histological section reveals a high regional density of xenin-immunoreactive tumor cells (scale bars: A, C = 200 μm; B, D = 40 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 2 Adjacent sections of a duodenal gastrinoma (no. 5 of Table 2) showing xenin-immunoreactive cells in a diffuse distribution pattern in a subpopulation of tumor cells (A), whereas gastrin immunoreactivity is present in the majority of cells (B). Peri-/infranuclear granular pattern of xenin immunoreactivity in endocrine cells is depicted in the high-power magnification inset of (A). Preabsorption of the xenin antibody with 25 μmol xenin peptide (XN + 25 μmol XN) abolishes the immunoreactions with the xenin antibody (C). The antiserum against neurotensin did not stain any endocrine cells in this tumor (D) (scale bars: A–D = 40 μm; high-power magnification in A = 10 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 3 Pancreatic gastrinoma (no. 56 of Table 3). Adjacent sections showing an absence of xenin (XN) immunoreactivity from tumor tissue (A) and an abundance of gastrin-immunoreactive (GA) cells (B) (scale bars: 100 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 4 Adjacent sections through an invasive duodenal gastrinoma (A–C; no. 14 of Table 2) and an invasive duodenal somatostatinoma (D–F; no. 18 of Table 2) showing coexpression of xenin and chromogranin A in both types of endocrine tumors. Arrowheads in (A–C) indicate the cellular segregation of xenin- and gastrin-immunoreactive tumor cells. (A and D) Antiserum 2815/3 against xenin (XN). (B) Antiserum A 568 against gastrin (GA). (E) Antiserum PEPA 38 against somatostatin (SOM). (C and F) Antiserum LK2 H10 against CgA (scale bar: A–C = 200 μm; D–F = 100 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 5 High-resolution confocal laser microscopy in an identical section through a small tumor nodule of a neuroendocrine duodenal gastrinoma (no. 13 of Table 2) shows cellular and subcellular colocalization of xenin and CgA in a single endocrine cell. Other neuroendocrine cells are labeled by CgA but are negative for xenin (XN). (A) Antiserum 2815/3 against xenin; (B) antiserum LK2 H10 against CgA; (C) co-staining for xenin and CgA (scale bar: 10 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 6 Adjacent sections of normal duodenal mucosa (A, B) showing coexpression of xenin (XN) (A) and GIP (B) in endocrine cells of Brunner's glands. In contrast, endocrine tumor cells reveal a xenin-positive (C) but GIP-negative (D) phenotype (C, D; no. 13 in Table 2) (scale bars: A, B = 50 μm; C, D = 100 μm). Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 7 Xenin-immunoreactive high-pressure liquid chromatography elution profile from a μ-Bondapak C18 high-pressure liquid chromatography column of an extracted duodenal gastrinoma (no. 13 of Table 4). The xenin-immunoreactive peak appears at exactly the time of synthetic xenin 25; the metabolites xenin 16 and xenin 9 are identified. Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 8 Quantification of immunoreactive xenin 25 from high-pressure liquid chromatography extracted from frozen and untreated tissue. The xenin content of 3 duodenal gastrinomas (Table 4) is higher than that of normal human gastric mucosa (n = 5). MEN, multiple endocrine neoplasia. Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions

Fig. 9 Content of immunoreactive xenin 25 after acid extraction in various endocrine foregut and midgut tumors with (P) and without (A) pepsin predigestion of the acidified tissue. Duodenal gastrinomas 13, 28, and 29 (Table 4) were extracted after thawing and refreezing 1 year earlier. There is measurable xenin 25 immunoreactivity when extracted without pepsin, although less than in the previous extraction. The addition of pepsin augments the amount of extractable xenin. In pancreatic gastrinomas 31, 32, and 34, insulinoma 35 (Table 4), and the 5 bronchial carcinoids, xenin 25 immunoreactivity was extractable only after predigestion with pepsin. Gastroenterology 2002 123, 1616-1626DOI: (10.1053/gast.2002.36590) Copyright © 2002 American Gastroenterological Association Terms and Conditions