Mixed Neuroendocrine Non-Neuroendocrine Neoplasm Arising in the Ectopic Gastric Mucosa of Esophagus

Esophageal neuroendocrine neoplasms are extremely rare, and their prognosis is poor. Mixed neuroendocrine non-neuroendocrine neoplasms (MiNENs) are even more rare and are defined as tumors consisting of neuroendocrine carcinoma and either adenocarcinoma or squamous cell carcinoma. We report a rare case featuring endoscopic submucosal dissection (ESD) for an esophageal MiNEN, arising from the ectopic gastric mucosa in the lower thoracic esophagus. A 92-year-old male patient was referred to this hospital for investigation of an esophageal tumor. An endoscopic examination revealed a 10 mm elevated lesion, with 8 mm flat areas on the anal side, within the ectopic gastric mucosa located in the lower thoracic esophagus. ESD was carried out, and a histopathological examination revealed a tubular adenocarcinoma composed of differentiated neuroendocrine cells. Immunohistochemical staining was positive for synaptophysin and negative for chromogranin A. The labeling index of Ki-67 was more than 80%. Based on these results, we diagnosed the lesion as an esophageal MiNEN, arising in the ectopic gastric mucosa of the esophagus. The patient remains alive, without recurrence of cancer, 24 months after ESD

E-NPP3 controls plasmacytoid dendritic cell numbers in the small intestine

<div><p>Extracellular adenosine 5’-triphosphate (ATP) performs multiple functions including activation and induction of apoptosis of many cell types. The ATP-hydrolyzing ectoenzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 3 (E-NPP3) regulates ATP-dependent chronic allergic responses by mast cells and basophils. However, E-NPP3 is also highly expressed on epithelial cells of the small intestine. In this study, we showed that E-NPP3 controls plasmacytoid dendritic cell (pDC) numbers in the intestine through regulation of intestinal extracellular ATP. In <i>Enpp3</i>^-/- mice, ATP concentrations were increased in the intestinal lumen. pDC numbers were remarkably decreased in the small intestinal lamina propria and Peyer’s patches. Intestinal pDCs of <i>Enpp3</i>^-/- mice showed enhanced cell death as characterized by increases in annexin V binding and expression of cleaved caspase-3. pDCs were highly sensitive to ATP-induced cell death compared with conventional DCs. ATP-induced cell death was abrogated in <i>P2rx7</i>^-/- pDCs. Accordingly, the number of intestinal pDCs was restored in <i>Enpp3</i>^-/- <i>P2rx7</i>^-/- mice. These findings demonstrate that E-NPP3 regulates ATP concentration and thereby prevents the decrease of pDCs in the small intestine.</p></div

P2X7-dependent induction of pDC apoptosis in vivo.

<p><b>(A, B)</b> Frequencies of annexin V-positive (A) and active caspase-3-positive (B) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> mice (n = 7 per groups in a, and n = 5 per groups in b). Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01 <b>(c)</b> Frequency and number of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>P2rx7</i>^<i>-/-</i>, and <i>Enpp3</i>^<i>-/-</i> <i>P2rx7</i>^<i>-/-</i> (n = 13 per groups) mice. Representative dot plots are shown (left) and the means ± SD of the percentages of pDCs are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Enhancement of ATP-induced apoptosis of pDCs.

<p><b>(A, B)</b> Cells isolated from MLNs were treated with the indicated concentrations of ATP for 3 h. Annexin V-positive cells (A) and active caspase-3-positive cells (B) among PDCA-1⁺ CD11c^med pDCs and PDCA-1^- CD11c^high cDCs were analyzed by flow cytometry. Representative histograms are shown (left) and the means ± SD (n = 3) of the percentages of annexin V-positive (A) and active caspase-3-positive cells (B) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. <b>(C)</b> Frequency of active caspase-3-positive cells among CD45⁺ PDCA-1⁺ CD11c^med pDCs and and PDCA-1^- CD11c^high cDCs from the PPs and SILP of mice injected with 300 μl PBS (n = 6) or 1 mM ATP-γS (n = 5) into their small intestinal lumen. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, NS: not significant.</p

Expression of <i>Enpp3</i> in small intestinal epithelia.

<p><b>(A)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in the indicated tissues (n = 3). <b>(B)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells and lamina propria in four parts of the small intestine. A smaller number represents a more proximal portion of the intestine (n = 4). <b>(C)</b> Quantitative RT-PCR analysis of <i>Enpp3</i> mRNA expression in epithelial cells of the small intestine in specific-pathogen free (SPF) and germ-free (GF) mice (n = 3). <b>(D)</b> Immunohistochemical analysis of the small intestine. E-NPP3 (red) and DAPI (blue). Swiss roll frozen sections were stained with the anti-mouse E-NPP3 antibody. A smaller number represents more proximal portion of the intestine. Scale bars, 100 μm. <b>(E)</b> ATP concentrations in luminal contents of the small intestine of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 6 per groups). *<i>p</i> < 0.05. <b>(F)</b> ATP concentration in the proximal portion of the small intestinal lumen from wild-type, <i>Enpp3</i>^<i>-/-</i>, <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice. All data are mean values ± SD (n = 4 per groups). *<i>p</i> < 0.05, NS: not significant. <b>(G)</b> ATP concentration in the proximal portion of the small intestinal lumen from <i>Kit</i>^{<i>W-sh/W-sh</i>} and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice with or without adoptive transfer of wild-type or <i>Enpp3</i>^<i>-/-</i> bone marrow-derived mast cells. All data are mean values ± SD (n = 5 for mast cells transferred mice groups and <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group, and n = 3 for non transferred <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} mice group). *<i>p</i> < 0.05, NS: not significant.</p

Decrease in the number of intestinal pDCs in <i>Enpp3</i>^<i>-/-</i> mice.

<p><b>(A</b>–<b>C)</b> Frequency and number of CD45⁺ PDCA-1⁺ CD11c^med pDCs (A, C) and CD45⁺ PDCA-1^- CD11c ^high cDCs (B, C) in the Peyer’s patches (PPs), small intestinal lamina propria (SILP), bone marrow (BM), and spleen (SPL) of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. All data are mean values ± SD (n = 7 for PP and SILP, and n = 6 for SPL and BM). *<i>p</i> < 0.05, NS: not significant. <b>(D)</b> Frequency of CD45⁺ PDCA-1⁺ CD11c^med pDCs in the PPs and SILP of wild-type (n = 6), <i>Enpp3</i>^<i>-/-</i> (n = 8), <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 7), and <i>Enpp3</i>^<i>-/-</i> <i>Kit</i>^{<i>W-sh/W-sh</i>} (n = 6) mice. All data are mean values ± SD. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant. <b>(E, F)</b> Frequency of annexin V-positive (E) and active caspase-3-positive (F) cells gated on CD45⁺ PDCA-1⁺ CD11c^med pDCs from the PPs and SILP of wild-type and <i>Enpp3</i>^<i>-/-</i> mice. Representative histograms are shown (left) and the means ± SD of the percentages of positive cells (right) are shown (n = 6 in e, and n = 5 in f). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(G)</b> Frequency of PDCA-1⁺ CD11c^med pDCs in the PPs and SILP from antibiotic-treated wild-type (n = 11) and <i>Enpp3</i>^<i>-/-</i> (n = 12) mice as well as untreated wild-type (n = 10) and <i>Enpp3</i>^<i>-/-</i> (n = 10) mice. Data are the means ± SD of the percentages of pDCs. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, NS: not significant.</p

P2X7-dependent induction of pDC apoptosis in vitro.

<p><b>(A)</b> Expression of P2X receptors in pDCs or cDCs of MLN was analyzed by quantitative RT-PCR. Data are means ± SD (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(B)</b> mRNA expression of <i>P2rx4</i> and <i>P2rx7</i> in pDCs and cDCs of SILP, PP, bone marrow (BM) and spleen (SPL) was analyzed by quantitative RT-PCR. Data are means ± SD (n = 3). *<i>p</i> < 0.05, **<i>p</i> < 0.01. <b>(C, D)</b> Isolated MLN pDCs of wild-type and <i>P2rx7</i>^<i>-/-</i> mice were treated with the indicated concentrations of ATP for 3 h. Annexin V-positive cells (C) and active caspase-3-positive cells (D) were analyzed by flow cytometry. Representative histograms are shown (left) and the means ± SD of the percentages of annexin V-positive (C) and active caspase-3-positive cells (D) (n = 4) are shown (right). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p