Although gastric neuroendocrine neoplasias (gNEN) are an orphan disease, their incidence is rising. The heterogeneous clinical course powers the ongoing discussion of the most appropriate classification system and management. Prognostic relevance of proposed classifications was retrospectively analysed in 142 patients from a single tertiary referral centre. Baseline, management and survival data were acquired for statistical analyses. The distribution according to the clinicopathological typification was gNEN-1 (n = 86/60.6%), gNEN-2 (n = 7/4.9%), gNEN-3 (n = 24/16.9%) and gNEN-4 (n = 25/17.6%), while hypergastrinemia-associated gNEN-1 and -2 were all low-grade tumours (NET-G1/2), formerly termed sporadic gNEN-3 could be subdivided into gNEN-3 with grade 1 or 2 and gNEN-4 with grade 3 (NEC-G3). During follow-up 36 patients died (25%). The mean overall survival (OS) of all gNEN was 14.2 years. The OS differed statistically significant across all subgroups with either classification system. According to UICC 2017 TNM classification, OS differed for early and advanced stages, while WHO grading indicated poorer prognosis for NEC-G3. Cox regression analysis confirmed the independent prognostic validity of either classification system for survival. Particularly careful analysis of the clinical course of gNEN-1 (ECLomas, gastric carcinoids) confirmed their mostly benign, but recurrent and extremely slowly progressive behaviour with low risk of metastasis (7%) and an efficient long-term control by repetitive endoscopic procedures. Our study provides evidence for the validity of current classifications focusing on typing, grading and staging. These are crucial tools for risk stratification, especially to differentiate gNEN-1 as well as sporadic gNET and gNEC (gNEN-3 vs -4).
S Felder, H Jann, R Arsenic, T Denecke, V Prasad, B Knappe-Drzikova, S Maasberg, B Wiedenmann, M Pavel, A Pascher, and U F Pape
Emanuel Christ, Kwadwo Antwi, Melpomeni Fani, and Damian Wild
Receptors for the incretin glucagon-like peptide-1 (GLP-1R) have been found overexpressed in selected types of human tumors and may, therefore, play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Targeting GLP-1R with indium-111, technetium-99m or gallium-68-labeled exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new non-invasive technique has the potential to replace the invasive localization of insulinomas by selective arterial stimulation and venous sampling. In contrast to benign insulinomas, malignant insulin-secreting neuroendocrine tumors express GLP-1R in only one-third of the cases, while they more often express the somatostatin subtype 2 receptors. Importantly, one of the two receptors appears to be always overexpressed. In special cases of endogenous hyperinsulinemic hypoglycemia (EHH), that is, in the context of MEN-1 or adult nesidioblastosis GLP-1R imaging is useful whereas in postprandial hypoglycemia in the context of bariatric surgery, GLP-1R imaging is probably not helpful. This review focuses on the potential use of GLP-1R imaging in the differential diagnosis of EHH.
Kristen K Rumer, Miriam D Post, Rhea S Larivee, Martina Zink, Jill Uyenishi, Anita Kramer, Deanna Teoh, Kevin Bogart, and Virginia D Winn
Sialic acid immunoglobulin-like lectin (Siglec)-6 is a transmembrane receptor that binds leptin. Leptin is an obesity-associated peptide hormone overexpressed in gestational trophoblastic disease (GTD). GTD encompasses several placental abnormalities that range from benign to malignant. Among GTD, molar placentas are characterized by excess proliferation, whereas gestational trophoblastic neoplasias (GTN) have characteristically aggressive invasion. We hypothesized that in GTD, Siglec-6 expression would increase with disease severity and that Siglec-6 and leptin would promote proliferation, inhibit apoptosis and/or promote invasion. Siglec-6 expression patterns were evaluated with particular attention to the diagnostic utility of Siglec-6 in GTD (controls: normal placentas (n=32), hydropic abortus placentas (n=7), non-GTD reproductive tract cancers (n=2); GTD: partial moles (PM; n=11), complete moles (n=24), GTN (n=6)). In normal placentas, Siglec-6 expression dramatically decreased after 8 weeks gestation. Complete molar placentas had significantly higher Siglec-6 expression than controls, but expression was not significantly different from PM. In GTN, Siglec-6 expression was low. These data suggest that Siglec-6 may have diagnostic utility for distinguishing complete moles from normal and hydropic abortus placentas. Functional studies in choriocarcinoma-derived BeWO cells demonstrated a complex interplay between Siglec-6 expression and leptin exposure. In cells lacking Siglec-6, leptin treatment promoted invasion, likely through interaction with LepR leptin receptor, without affecting proliferation or apoptosis. Siglec-6 expression promoted proliferation in a leptin-dependent manner, but protected cells from apoptosis and promoted invasion in a leptin-independent manner. We propose that Siglec-6 and leptin play a role in the aberrant properties characteristic of GTD, namely excess proliferation and invasion.
Toyoshi Endo and Tetsuro Kobayashi
A variant located on 14q13.3 nearest to thyroid transcription factor-1 (TTF1) predisposes individuals to thyroid cancer, but whether this variant is related to the RET/PTC rearrangement associated with human papillary thyroid carcinomas (PTCs) is unknown. The aims of this study were to investigate the effects of RET/PTC1 on the expression of thyroid-specific genes in thyrocytes and their relationship with malignant transformation of the thyrocytes. In the absence or presence of TSH, an extracellular signal-regulated kinase was phosphorylated in FRTL5 cells that stably expressed RET/PTC1, and these cells grew independently of TSH. FRTL (RET/PTC1) cells produced 566% more thyroglobulin mRNA and 474% more Na+/I− symporter mRNA than did the control FRTL (pcDNA) cells. FRTL (RET/PTC1) cells expressed 468% more Ttf1 mRNA than did FRTL (pcDNA) cells, but these two cell types did not differ significantly with respect to Pax8 or Ttf2 mRNA levels. When FRTL (RET/PTC1) cells and FRTL (pcDNA), cells were injected into each of nine nude mice, each mouse developed a single tumor at the site of FRTL (RET/PTC1) cell injection; in contrast, tumor formation never occurred at sites of FRTL (cDNA) cells injection. Tumors resulting from FRTL (RET/PTC1) cells retained 125I-uptake activity; moreover, the cells invaded into surrounding skeletal muscle. When overexpression of Ttf1 in FRTL (RET/PTC1) cells was silenced, the cells completely lost their tumorigenic potential. Exogenous TTF1 cDNA enhanced the tumorigenicity of BHP18-21v cells, human PTC cells that express RET/PTC1, in nude mice. These results indicated that concurrent overexpression of RET/PTC1 and TTF1 confers tumorigenicity to FRTL5 and BHP18-21v cells in nude mice.
Tobias Hofving, Yvonne Arvidsson, Bilal Almobarak, Linda Inge, Roswitha Pfragner, Marta Persson, Göran Stenman, Erik Kristiansson, Viktor Johanson, and Ola Nilsson
Experimental models of neuroendocrine tumour disease are scarce, and no comprehensive characterisation of existing gastroenteropancreatic neuroendocrine tumour (GEPNET) cell lines has been reported. In this study, we aimed to define the molecular characteristics and therapeutic sensitivity of these cell lines. We therefore performed immunophenotyping, copy number profiling, whole-exome sequencing and a large-scale inhibitor screening of seven GEPNET cell lines. Four cell lines, GOT1, P-STS, BON-1 and QGP-1, displayed a neuroendocrine phenotype while three others, KRJ-I, L-STS and H-STS, did not. Instead, these three cell lines were identified as lymphoblastoid. Characterisation of remaining authentic GEPNET cell lines by copy number profiling showed that GOT1, among other chromosomal alterations, harboured losses on chromosome 18 encompassing the SMAD4 gene, while P-STS had a loss on 11q. BON-1 had a homozygous loss of CDKN2A and CDKN2B, and QGP-1 harboured amplifications of MDM2 and HMGA2. Whole-exome sequencing revealed both disease-characteristic mutations (e.g. ATRX mutation in QGP-1) and, for patient tumours, rare genetic events (e.g. TP53 mutation in P-STS, BON-1 and QGP-1). A large-scale inhibitor screening showed that cell lines from pancreatic NETs to a greater extent, when compared to small intestinal NETs, were sensitive to inhibitors of MEK. Similarly, neuroendocrine NET cells originating from the small intestine were considerably more sensitive to a group of HDAC inhibitors. Taken together, our results provide a comprehensive characterisation of GEPNET cell lines, demonstrate their relevance as neuroendocrine tumour models and explore their therapeutic sensitivity to a broad range of inhibitors.
Rodrigo A Toledo, Roxanne Hatakana, Delmar M Lourenço Jr, Susan C Lindsey, Cleber P Camacho, Marcio Almeida, José V Lima Jr, Tomoko Sekiya, Elena Garralda, Michel S Naslavsky, Guilherme L Yamamoto, Monize Lazar, Osorio Meirelles, Tiago J P Sobreira, Maria Lucia Lebrao, Yeda A O Duarte, John Blangero, Mayana Zatz, Janete M Cerutti, Rui M B Maciel, and Sergio P A Toledo
Accurate interpretation of germline mutations of the rearranged during transfection (RET) proto-oncogene is vital for the proper recommendation of preventive thyroidectomy in medullary thyroid carcinoma (MTC)-prone carriers. To gain information regarding the most disputed variant of RET, ATA-A Y791F, we sequenced blood DNA samples from a cohort of 2904 cancer-free elderly individuals (1261 via Sanger sequencing and 1643 via whole-exome/genome sequencing). We also accessed the exome sequences of an additional 8069 individuals from non-cancer-related laboratories and public databanks as well as genetic results from the Catalogue of Somatic Mutations in Cancer (COSMIC) project. The mean allelic frequency observed in the controls was 0.0031, with higher occurrences in Central European populations (0.006/0.008). The prevalence of RET Y791F in the control databases was extremely high compared with the 40 known RET pathogenic mutations (P=0.00003), while no somatic occurrence has been reported in tumours. In this study, we report new, unrelated Brazilian individuals with germline RET Y791F-only: two tumour-free elderly controls; two individuals with sporadic MTC whose Y791F-carrying relatives did not show any evidence of tumours; and a 74-year-old phaeochromocytoma patient without MTC. Furthermore, we showed that the co-occurrence of Y791F with the strong RET C634Y mutation explains the aggressive MTC phenotypes observed in a large affected family that was initially reported as Y791F-only. Our literature review revealed that limited analyses have led to the misclassification of RET Y791F as a probable pathogenic variant and, consequently, to the occurrence of unnecessary thyroidectomies. The current study will have a substantial clinical influence, as it reveals, in a comprehensive manner, that RET Y791F only shows no association with MTC susceptibility.
Nimrod B Kiss, Andreas Muth, Adam Andreasson, C Christofer Juhlin, Janos Geli, Martin Bäckdahl, Anders Höög, Bo Wängberg, Ola Nilsson, Håkan Ahlman, and Catharina Larsson
Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.
Rajeev Mishra, Subhash Haldar, Surabhi Suchanti, and Neil A Bhowmick
Genomic changes that drive cancer initiation and progression contribute to the co-evolution of the adjacent stroma. The nature of the stromal reprogramming involves differential DNA methylation patterns and levels that change in response to the tumor and systemic therapeutic intervention. Epigenetic reprogramming in carcinoma-associated fibroblasts are robust biomarkers for cancer progression and have a transcriptional impact that support cancer epithelial progression in a paracrine manner. For prostate cancer, promoter hypermethylation and silencing of the RasGAP, RASAL3 that resulted in the activation of Ras signaling in carcinoma-associated fibroblasts. Stromal Ras activity initiated a process of macropinocytosis that provided prostate cancer epithelia with abundant glutamine for metabolic conversion to fuel its proliferation and a signal to transdifferentiate into a neuroendocrine phenotype. This epigenetic oncogenic metabolic/signaling axis seemed to be further potentiated by androgen receptor signaling antagonists and contributed to therapeutic resistance. Intervention of stromal signaling may complement conventional therapies targeting the cancer cell.
Jonathan R Strosberg, James C Yao, Emilio Bajetta, Mounir Aout, Bert Bakker, John D Hainsworth, Philippe B Ruszniewski, Eric Van Cutsem, Kjell Öberg, and Marianne E Pavel
Somatostatin analogues (SSA) have demonstrated antiproliferative activity in addition to efficacy for carcinoid symptom control in functional neuroendocrine tumors (NET). A post hoc analysis of the placebo arm of the RAD001 In Advanced Neuroendocrine Tumors-2 (RADIANT-2) study was conducted to assess the efficacy of octreotide long-acting repeatable (LAR) on progression-free survival (PFS) and overall survival (OS) estimated using the Kaplan–Meier method. Out of 213 patients randomized to placebo plus octreotide LAR in RADIANT-2, 196 patients with foregut, midgut, or hindgut NET were considered for present analysis. Of these, 41 patients were SSA-treatment naïve and 155 had received SSA therapy before study entry. For SSA-naïve patients, median PFS by adjudicated central review was 13.6 (95% CI 8.2–22.7) months. For SSA-naïve patients with midgut NET (n=24), median PFS was 22.2 (95% CI 8.3–29.5) months. For patients who had received SSA previously, the median PFS was 11.1 (95% CI 8.4–14.3) months. Among the SSA-pretreated patients who had midgut NET (n=119), the median PFS was 12.0 (95% CI 8.4–19.3) months. Median OS was 35.8 (95% CI 32.5–48.9) months for patients in the placebo plus octreotide LAR arm; 50.6 (36.4 – not reached) months for SSA-naïve patients and 33.5 (95% CI 27.5–44.7) months for those who had received prior SSA. This post hoc analysis of the placebo arm of the large phase 3 RADIANT-2 study provides data on PFS and OS among patients with progressive NET treated with octreotide therapy.
Tobias Hofving, Viktor Sandblom, Yvonne Arvidsson, Emman Shubbar, Gülay Altiparmak, John Swanpalmer, Bilal Almobarak, Anna-Karin Elf, Viktor Johanson, Erik Elias, Erik Kristiansson, Eva Forssell-Aronsson, and Ola Nilsson
177Lu-octreotate is an FDA-approved radionuclide therapy for patients with gastroenteropancreatic neuroendocrine tumours (NETs) expressing somatostatin receptors. The 177Lu-octreotate therapy has shown promising results in clinical trials by prolonging progression-free survival, but complete responses are still uncommon. The aim of this study was to improve the 177Lu-octreotate therapy by means of combination therapy. To identify radiosensitising inhibitors, two cell lines, GOT1 and P-STS, derived from small intestinal neuroendocrine tumours (SINETs), were screened with 1224 inhibitors alone or in combination with external radiation. The screening revealed that inhibitors of Hsp90 can potentiate the tumour cell-killing effect of radiation in a synergistic fashion (GOT1; false discovery rate <3.2 × 10−11). The potential for Hsp90 inhibitor ganetespib to enhance the anti-tumour effect of 177Lu-octreotate in an in vivo setting was studied in the somatostatin receptor-expressing GOT1 xenograft model. The combination led to a larger decrease in tumour volume relative to monotherapies and the tumour-reducing effect was shown to be synergistic. Using patient-derived tumour cells from eight metastatic SINETs, we could show that ganetespib enhanced the effect of 177Lu-octreotate therapy for all investigated patient tumours. Levels of Hsp90 protein expression were evaluated in 767 SINETs from 379 patients. We found that Hsp90 expression was upregulated in tumour cells relative to tumour stroma in the vast majority of SINETs. We conclude that Hsp90 inhibitors enhance the tumour-killing effect of 177Lu-octreotate therapy synergistically in SINET tumour models and suggest that this potentially promising combination should be further evaluated.