Mark Kidd

BACKGROUND: Although gastroenteropancreatic neuroendocrine neoplasms (GEP‐NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki‐67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP‐NEN biomarker. METHODS: Functional assays were undertaken to investigate the mechanistic role of INA in the pancreatic BON cell line. Expression levels of INA were investigated in 50 pancreatic NENs (43 primaries, 7 metastases), 43 small intestinal NENs (25 primaries, 18 metastases), normal pancreas (n = 10), small intestinal mucosa (n = 16), normal enterochromaffin (EC) cells (n = 9), mouse xenografts (n = 4) and NEN cell lines (n = 6) using quantitative polymerase chain reaction, Western blot, and immunostaining analyses. RESULTS: In BON cells, decreased levels of INA messenger RNA and protein were associated with the inhibition of both proliferation and mitogen‐activated protein kinase (MAPK) signaling. INA was not expressed in normal neuroendocrine cells but was overexpressed (from 2‐fold to 42‐fold) in NEN cell lines and murine xenografts. In pancreatic NENs, INA was overexpressed compared with pancreatic adenocarcinomas and normal pancreas (27‐fold [P = .0001], and 9‐fold [P = .02], respectively). INA transcripts were correlated positively with Ki‐67 (correlation coefficient [r ] = 0.5; P < .0001) and chromogranin A (r = 0.59; P < .0001). INA distinguished between primary tumors and metastases (P = .02), and its expression was correlated with tumor size, infiltration, and grade (P < .05). CONCLUSIONS: INA is a novel NEN biomarker, and its expression was associated with MAPK signaling and proliferation. In clinical samples, elevated INA was correlated with Ki‐67 and identified malignancy. INA may provide additional biologic information relevant to delineation of both pancreatic NEN tumor phenotypes and clinical behavior. Cancer 2011. © 2011 American Cancer Society.

BACKGROUND: Microsatellite instability (MSI) with concomitant mutations in the coding region of transforming growth factor‐beta type II receptor (TGFβRII) results in an aberrant growth‐regulatory phenotype in colorectal carcinomas. The authors postulated that a similar mechanism occurred during the malignant evolution of small bowel carcinoid tumors.METHODS: Mutational analysis of two coding regions in the TGFβRII gene associated with MSI and BAT‐26 within intron 5 of the mismatch repair gene, hMSH2, was undertaken in small bowel carcinoids (n = 14), lymph node metasasis (n = 1) and liver metastases (n = 5). Quantitative PCR analysis [TAQMAN, Applied Biosystems, Foster City, CA] was then undertaken to examine gene alterations in mismatch repair genes (hMLH1 and hMSH2) in small bowel carcinoids (n = 7) and matched normal mucosa (n = 5). Staining was then analyzed using quantitative tissue array profiling (AQUA analysis) in a small bowel EC carcinoid tissue microarray (n = 55 tumors) with immunostaining against TGFβRII and MSH2.RESULTS: Mutational examination of the TFGβRII gene and BAT‐26 demonstrated that MSI was not present in any carcinoid material. Q RT‐PCR analysis demonstrated statistically significant increased message levels of hMSH2 but not hMLH1 in carcinoid tumors. Quantitative analysis of membrane TGFβRII immunostaining using AQUA demonstrated that TGFβRII expression was down‐regulated (P < 0.0002) in thirty‐three primary small bowel carcinoids that exhibited lymph node and liver metastases compared to normal mucosa. AQUA analysis of nuclear MSH2 immunostaining demonstrated no differences for MSH2 between normal tissue and carcinoid tumor metastasis. Small bowel carcinoids characterized by variable expression of TGFβRII, did not exhibit MSI and had no differences in MSH2 expression.CONCLUSIONS: The molecular events leading to the formation of carcinoid tumors in the small bowel were different from those resulting in epithelial carcinomas. The usually slow‐growing and relatively nonaggressive carcinoid tumors had variable expression of TGFβRII but were associated with the retention of mismatch repair protein function and a microsatellite‐stable phenotype. Cancer 2005. © 2004 American Cancer Society.

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8–16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.