Simon Schimmack

The diversity and commonalities of gastroenteropancreatic neuroendocrine tumors

BackgroundRecent data demonstrate that the incidence of gastroenteropancreatic neuroendocrine tumors (GEP-NETs) has increased exponentially (overall ~500%) over the last three decades, thus refuting the erroneous concept of rarity. GEP-NETs comprise 2% of all malignancies and in terms of prevalence, are the second commonest gastrointestinal malignancy after colorectal cancer. Diagnosis is usually late since there is no biochemical screening test and symptoms are protean and overlooked. As a consequence, 60–80% exhibit metastases with a consequent suboptimal outcome.DiscussionThe gastrointestinal tract and pancreas exhibit ~17 different neuroendocrine cell types, but neither the cell of origin nor the biological basis of GEP-NETs is understood. This review examines GEP-NETs from the cellular and molecular perspective and addresses the distinct patterns of functional tumor biology pertinent to clinicians. Although grouped as a neoplastic entity (NETs), each lesion is derived from distinct cell precursors, produces specific bioactive products, exhibits distinct chromosomal abnormalities and somatic mutation events and has uniquely dissimilar clinical presentations. GEP-NETs demonstrate very different survival rates reflecting the intrinsic differences in malignant potential and variations in proliferative regulation. Apart from the identification of the inhibitory role of the somatostatin receptors, there is limited biological knowledge of the key regulators of proliferation and hence a paucity of successful targeted therapeutic agents. IGF-I, TGFβ and a variety of tyrosine kinases have been postulated as key regulatory elements; rigorous data is still required to define predictably effective and rational therapeutic strategy in an individual tumor. A critical issue in the clinical management of GEP-NETs is the need to appreciate both the neuroendocrine commonalities of the disease as well as the unique characteristics of each tumor. The further acquisition of a detailed biological and molecular appreciation of GEP-NETs is vital to the development of effective management strategy.

Chronic Pancreatitis Is Associated With Disease-Specific Regulatory T-Cell Responses

BACKGROUND & AIMS Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. METHODS We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-gamma, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. RESULTS Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-gamma secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10(+)IFN-gamma(-)FoxP3(+) regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-gamma, suggesting pancreatitis-specific activity of regulatory T cells in situ. CONCLUSIONS Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

The archaic distinction between functioning and nonfunctioning neuroendocrine neoplasms is no longer clinically relevant

BackgroundNeuroendocrine neoplasms (NENs) are increasing in incidence and prevalence. This reflects greater clinical awareness, effective imaging, and increasing pathological diagnostic recognition. Although the identification and treatment of clinical neuroendocrine syndromes are established, there is confusion when a NEN has no discernible clinical symptoms.DiscussionNonfunctional tumors are usually diagnosed incidentally and at a later stage largely because either they do not secrete a bioactive product or do so, but in a form that is either inactive or in quantities that have no discernible effect. Nevertheless, the histopathology is indistinguishable from functional NENs, and tumors exhibit somatostatin receptor expression, and positive immunohistochemistry for neuroendocrine cell markers (CgA, NSE/synaptophysin). Similarly, their rates of growth and metastatic behavior are, like other NENs, predictably based on staging and grading (mitotic rate and Ki67 expression). Both types are diagnosed biochemically (CgA) and by imaging in an identical fashion with computed tomography, magnetic resonance imaging, somatostatin receptor scintigraphy, and endoscopic ultrasound. NENs, irrespective of function or bioactive secretory profile, respond with equal efficacy to the same regimen of surgery or antitumor drugs (e.g., somatostatin analogs with or without tyrosine kinase inhibitors/antiangiogenics or cytotoxics) depending on grade. Given the efficacy of somatostatin analogs in increasing progression free survival, nonfunctional NENs should be managed identically to symptomatic NENs. The consideration of NENs as functional or nonfunctional is an archaic clinical concept that should be discarded since the tumors are indistinguishable at a cellular, biological, and morphological level. All current evidences indicate that their diagnosis and treatment should follow the same common principles.

Quality of life is not influenced by the extent of surgery in patients with benign goiter.

IntroductionThe surgical approach to benign goiter is becoming increasingly radical due to the risk of recurrent goiter. The aim of this prospective study was to evaluate the impact of surgery on health-related quality of life (HRQoL) of patients with benign goiter.Material and methodsHRQoL data from 115 patients with benign goiter were analyzed. Thirty-three patients (group 1) had a hemithyroidectomy. Sixty-five patients (group 2) had a so-called Dunhill operation (hemithyroidectomy + near-total thyroidectomy of the opposite side), and in 17 patients, a total resection of the goiter was performed. The validated HRQoL instrument, the EuroQol-5D, was applied to measure the health-related quality of life.ResultsWith an overall complication rate of 10% and no permanent recurrent laryngeal nerve palsy, it was shown that surgery for benign goiter is safe. In the validated HRQoL questionnaire (EQ-5D), no significant variance could be found between different surgical procedures such as thyroidectomy, hemithyroidectomy, or Dunhill procedure. Further, no significant differences in QoL were found in EQ-5D questionnaire compared to normal population.ConclusionThyroid surgery can be done safely and without impairment of life quality, regardless of the extent of the operation.

A mechanistic role for the chromatin modulator, NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases

BackgroundThe chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57Kip2 (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57Kip2.ResultsNAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic (mammalian) target of rapamycin pathway proteins and their phosphorylation (p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57Kip2 promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57Kip2 (transcript and protein) was upregulated. For methylation of the p57Kip2 promoter, NAP1L1 bound directly to the promoter (−164 to +21, chromatin immunoprecipitation). In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57Kip2 (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed.ConclusionNAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of p57Kip2 promoter methylation.

Anti-angiogenic activity of VXM01, an oral T-cell vaccine against VEGF receptor 2, in patients with advanced pancreatic cancer: A randomized, placebo-controlled, phase 1 trial.

VEGFR-2 is expressed on tumor vasculature and a target for anti-angiogenic intervention. VXM01 is a first in kind orally applied tumor vaccine based on live, attenuated Salmonella bacteria carrying an expression plasmid, encoding VEGFR-2. We here studied the safety, tolerability, T effector (Teff), T regulatory (Treg) and humoral responses to VEGFR2 and anti-angiogenic effects in advanced pancreatic cancer patients in a randomized, dose escalation phase I clinical trial. Results of the first 3 mo observation period are reported. Locally advanced or metastatic, pancreatic cancer patients were enrolled. In five escalating dose groups, 30 patients received VXM01 and 15 placebo on days 1, 3, 5, and 7. Treatment was well tolerated at all dose levels. No dose-limiting toxicities were observed. Salmonella excretion and salmonella-specific humoral immune responses occurred in the two highest dose groups. VEGFR2 specific Teff, but not Treg responses were overall increased in vaccinated patients. We furthermore observed a significant reduction of tumor perfusion after 38 d in vaccinated patients together with increased levels of serum biomarkers indicative of anti-angiogenic activity, VEGF-A, and collagen IV. Vaccine specific Teff responses significantly correlated with reductions of tumor perfusion and high levels of preexisting VEGFR2-specific Teff while those showing no antiangiogenic activity had low levels of preexisting VEGFR2 specific Teff, showed a transient early increase of VEGFR2-specific Treg and reduced levels of VEGFR2-specific Teff at later time points – pointing to the possibility that early anti-angiogenic activity might be based at least in part on specific reactivation of preexisting memory T cells.

The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms.

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.

Chromogranin A and its fragments as regulators of small intestinal neuroendocrine neoplasm proliferation.

Introduction Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation. Methods Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines. Results Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05). Conclusion Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.

Serotonin and the 5-HT7 receptor: The link between hepatocytes, IGF-1 and small intestinal neuroendocrine tumors

Platelet‐derived serotonin (5‐HT) is involved in liver regeneration. The liver is also the metastatic site for malignant enterochromaffin (EC) cell “carcinoid” (neuroendocrine) neoplasms, the principal cellular source of 5‐HT. We hypothesized that 5‐HT produced by metastatic EC cells played a role in the hepatic tumor‐microenvironment principally via 5‐HT7 receptor‐mediated activation of hepatocyte IGF‐1 synthesis and secretion. Using isolated rat hepatocytes, we evaluated 5‐HT7 receptor expression (using PCR, sequencing and western blot). ELISA, cell transfection and western blots delineated 5‐HT‐mediated signaling pathways (pCREB, AKT and ERK). IGF‐1 synthesis/secretion was evaluated using QPCR and ELISA. IGF‐1 was tested on small intestinal neuroendocrine neoplasm proliferation, while IGF‐1 production and 5‐HT7 expression were examined in an in vivo SCID metastasis model. Our results demonstrated evidence for a functional 5‐HT7 receptor. 5‐HT activated cAMP/PKA activity, pCREB (130–205%, P < 0.05) and pERK/pAKT (1.2–1.75, P < 0.05). Signaling was reversed by the 5‐HT7 receptor antagonist SB269970. IGF‐1 significantly stimulated proliferation of two small intestinal neuroendocrine neoplasm cell lines (EC50: 7–70 pg/mL) and could be reversed by the small molecule inhibitor BMS‐754807. IGF‐1 and 5‐HT were elevated (40–300×) in peri‐tumoral hepatic tissue in nude mice, while 5‐HT7 was increased fourfold compared to sham‐operated animals. We conclude that hepatocytes express a cAMP‐coupled 5‐HT7 receptor, which, at elevated 5‐HT concentrations that occur in liver metastases, signals via CREB/AKT and is linked to IGF‐1 synthesis and secretion. Because IGF‐1 regulates NEN proliferation, identification of a role for 5‐HT7 in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine‐producing mid‐gut tumors.

Stathmin in pancreatic neuroendocrine neoplasms: a marker of proliferation and PI3K signaling

Chromosome 1p35-36, which encodes tumor suppressors and mitotic checkpoint control genes, is commonly altered in human malignancies. One gene at this locus, stathmin 1 (STMN1), is involved in cell cycle progression and metastasis. We hypothesized that increased STMN1 expression may play a role in pancreatic neuroendocrine neoplasm (pNEN) malignancy. We investigated stathmin copy number variation, mRNA, and protein expression using PCR-Taqman Copy Number Assays, Q-PCR, Western blot, and immunohistochemistry. A mechanistic role for stathmin in proliferation was assessed in the BON cell line under growth-restrictive conditions and siRNA silencing. Furthermore, its role in PI3K signaling pathway activation was evaluated using pharmacological inhibitors. mRNA (p = 0.0001) and protein (p < 0.05) were overexpressed in pNENs. Expression was associated with pNEN tumor extension (p < 0.05), size (p < 0.01), and Ki67 expression (p < 0.01). Serum depletion decreased Ki67 expression (p < 0.01) as well as Ser38 phosphorylation (p < 0.05) in BON cells. STMN1 knockdown (siRNA) decreased proliferation (p < 0.05), and PI3K inhibitors directly inhibited proliferation via stathmin inactivation (dephosphorylation p < 0.01). We identified that stathmin was overexpressed and associated with pathological parameters in pancreatic NENs. We postulate that STMN1 overexpression and phosphorylation result in a loss of cell cycle mitotic checkpoint control and may render tumors amenable to PI3K inhibitory therapy.