Does gamma-glutamyltransferase correlate with liver tumor burden in neuroendocrine tumors?

PURPOSE: In patients with neuroendocrine tumors (NETs) and liver metastases, increased gamma-glutamyltransferase (GGT) is commonly assumed as an indicator for progressive disease. To date, however, empirical data are lacking. This study aimed to investigate associations between GGT and liver tumor burden. In longitudinal analyses, associations of GGT and radiographic responses of liver metastases under therapy were investigated. METHODS: The cross-sectional sample consisted of 104 patients who were treated at the University Medical Center Hamburg-Eppendorf from 2008 to 2021 (mean age 62.3 ± 12.6 years, 58.7% male). GGT and liver imaging were identified in a time range of 3 months. Radiologic reassessments were performed to estimate liver tumor burden. In a separate longitudinal sample (n = 15), the course of GGT levels under chemotherapy was analyzed. Data were retrospectively analyzed with a univariate ANOVA, linear regression analyses, and Wilcoxon tests. RESULTS: Of 104 cross-sectionally analyzed patients, 54 (51.9%) showed a GGT elevation. GGT levels and liver tumor burden were positively correlated (p < 0.001), independently from age, gender, primary tumor location, grading, and cholestasis. Notably, GGT increase was associated with a liver tumor burden of >50%. In the longitudinal sample, 10 of 11 patients with progressive disease showed increasing GGT, whereas 4 of 4 patients with regressive disease showed declining GGT. CONCLUSION: Our findings indicate that GGT is associated with liver tumor burden. Over the course of therapy, GGT appears to change in line with radiographic responses. Further longitudinal studies with larger sample sizes are required to define GGT as a reliable marker for tumor response.

Cancer-Associated Fibroblasts Induce Proliferation and Therapeutic Resistance to Everolimus in Neuroendocrine Tumors through STAT3 Activation.

INTRODUCTION: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. METHODS: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. RESULTS: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. CONCLUSION: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

Differential Effects of Somatostatin, Octreotide, and Lanreotide on Neuroendocrine Differentiation and Proliferation in Established and Primary NET Cell Lines: Possible Crosstalk with TGF-ß Signaling.

GEP-NETs are heterogeneous tumors originating from the pancreas (panNET) or the intestinal tract. Only a few patients with NETs are amenable to curative tumor resection, and for most patients, only palliative treatments to successfully control the disease or manage symptoms remain, such as with synthetic somatostatin (SST) analogs (SSAs), such as octreotide (OCT) or lanreotide (LAN). However, even cells expressing low levels of SST receptors (SSTRs) may exhibit significant responses to OCT, which suggests the possibility that SSAs signal through alternative mechanisms, e.g., transforming growth factor (TGF)-ß. This signaling mode has been demonstrated in the established panNET line BON but not yet in other permanent (i.e., QGP) or primary (i.e., NT-3) panNET-derived cells. Here, we performed qPCR, immunoblot analyses, and cell counting assays to assess the effects of SST, OCT, LAN, and TGF-ß1 on neuroendocrine marker expression and cell proliferation in NT-3, QGP, and BON cells. SST and SSAs were found to regulate a set of neuroendocrine genes in all three cell lines, with the effects of SST, mainly LAN, often differing from those of OCT. However, unlike NT-3 cells, BON cells failed to respond to OCT with growth arrest but paradoxically exhibited a growth-stimulatory effect after treatment with LAN. As previously shown for BON, NT-3 cells responded to TGF-ß1 treatment with induction of expression of SST and SSTR2/5. Of note, the ability of NT-3 cells to respond to TGF-ß1 with upregulation of the established TGF-ß target gene SERPINE1 depended on cellular adherence to a collagen-coated matrix. Moreover, when applied to NT-3 cells for an extended period, i.e., 14 days, TGF-ß1 induced growth suppression as shown earlier for BON cells. Finally, next-generation sequencing-based identification of microRNAs (miRNAs) in BON and NT-3 revealed that SST and OCT impact positively or negatively on the regulation of specific miRNAs. Our results suggest that primary panNET cells, such as NT-3, respond similarly as BON cells to SST, SSA, and TGF-ß treatment and thus provide circumstantial evidence that crosstalk of SST and TGF-ß signaling is not confined to BON cells but is a general feature of panNETs.

Novel preclinical gastroenteropancreatic neuroendocrine neoplasia models demonstrate the feasibility of mutation-based targeted therapy.

PURPOSE: Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) form a rare and remarkably heterogeneous group of tumors. Therefore, establishing personalized therapies is eminently challenging. To achieve progress in preclinical drug development, there is an urgent need for relevant tumor models. METHODS: We successfully established three gastroenteropancreatic neuroendocrine tumor (GEP-NET) cell lines (NT-18P, NT-18LM, NT-36) and two gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) cell lines (NT-32 and NT-38). We performed a comprehensive characterization of morphology, NET differentiation, proliferation and intracellular signaling pathways of these five cell lines and, in addition, of the NT-3 GEP-NET cell line. Additionally, we conducted panel sequencing to identify genomic alterations suitable for mutation-based targeted therapy. RESULTS: We found that the GEP-NEN cell lines exhibit a stable neuroendocrine phenotype. Functional kinome profiling revealed a higher activity of serine/threonine kinases (STK) as well as protein tyrosine kinases (PTK) in the GEP-NET cell lines NT-3 and NT-18LM compared to the GEP-NEC cell lines NT-32 and NT-38. Panel sequencing revealed a mutation in Death Domain Associated Protein (DAXX), sensitizing NT-18LM to the Ataxia telangiectasia and Rad3 related (ATR) inhibitor Berzosertib, and a mutation in AT-Rich Interaction Domain 1A (ARID1A), sensitizing NT-38 to the Aurora kinase A inhibitor Alisertib. Small interfering RNA-mediated knock down of DAXX in the DAXX wild type cell line NT-3 sensitized these cells to Berzosertib. CONCLUSIONS: The newly established GEP-NET and GEP-NEC cell lines represent comprehensive preclinical in vitro models suitable to decipher GEP-NEN biology and pathogenesis. Additionally, we present the first results of a GEP-NEN-specific mutation-based targeted therapy. These findings open up new potentialities for personalized therapies in GEP-NEN.

Possible tumour cell reimplantation during curative endoscopic therapy of superficial Barrett's carcinoma.

BACKGROUND AND AIMS: Endoscopic resection has been established as curative therapy for superficial cancer arising from Barrett's oesophagus (BE); recurrences are very rare. Based on a case series with unusual and massive early recurrences, we analyse the issue of tumour cell reimplantation. METHODS: This hypothesis was developed on the basis of two out of seven patients treated by circumferential (n=6) or nearly circumferential (n=1) en bloc and R0 endoscopic resection of T1 neoplastic BE. Subsequently, a prospective histocytological analysis of endoscope channels and accessories was performed in 2 phases (cytohistological analysis; test for cell viability) in 22 different oesophageal carcinoma patients undergoing endoscopy. Finally, cultures from two oesophageal adenocarcinoma cell lines were incubated with different triamcinolone concentrations (0.625-10 mg/mL); cell growth was determined on a Multiwell plate reader. RESULTS: Cancer regrowth in the two suspicious cases (male, 78/71 years) occurred 7 and 1 months, respectively, after curative tumour resection. Subsequent surgery showed advanced tumours (T2) with lymph node metastases; one patient died. On cytohistological examinations of channels and accessories, suspicious/neoplastic cells were found in 4/10 superficial and in all 5 advanced cancers. Further analyses in seven further advanced adenocarcinoma cases showed viable cells in two channel washing specimens. Finally, cell culture experiments demonstrated enhanced tumour cell growth by triamcinolone after 24 hours compared with controls. CONCLUSIONS: Tumour cell reimplanation from contaminated endoscopes and accessories is a possible cause of local recurrence after curative endoscopic therapy for superficial Barrett carcinoma; also, corticosteroid injection could have promoted tumour regrowth in these cases.

Cdk5 drives formation of heterogeneous pancreatic neuroendocrine tumors.

Pancreatic neuroendocrine tumors (PanNETs) are a heterogeneous population of neoplasms that arise from hormone-secreting islet cells of the pancreas and have increased markedly in incidence over the past four decades. Non-functional PanNETs, which occur more frequently than hormone-secreting tumors, are often not diagnosed until later stages of tumor development and have poorer prognoses. Development of successful therapeutics for PanNETs has been slow, partially due to a lack of diverse animal models for pre-clinical testing. Here, we report development of an inducible, conditional mouse model of PanNETs by using a bi-transgenic system for regulated expression of the aberrant activator of Cdk5, p25, specifically in ß-islet cells. This model produces a heterogeneous population of PanNETs that includes a subgroup of well-differentiated, non-functional tumors. Production of these tumors demonstrates the causative potential of aberrantly active Cdk5 for generation of PanNETs. Further, we show that human PanNETs express Cdk5 pathway components, are dependent on Cdk5 for growth, and share genetic and transcriptional overlap with the INS-p25OE model. The utility of this model is enhanced by the ability to form tumor-derived allografts. This new model of PanNETs will facilitate molecular delineation of Cdk5-dependent PanNETs and the development of new targeted therapeutics.

High Serum Levels of Wnt Signaling Antagonist Dickkopf-Related Protein 1 Are Associated with Impaired Overall Survival and Recurrence in Esophageal Cancer Patients.

Dickkopf-related protein 1 (DKK1), an antagonist of the canonical Wnt pathway, has received tremendous attention over the past years as its dysregulation is said to be critically involved in a wide variety of gastrointestinal cancers. However, the potential clinical implications of DKK1 remain poorly understood. Although multimodal treatment options have been implemented over the past years, esophageal cancer (EC) patients still suffer from poor five-year overall survival rates ranging from 15% to 25%. Especially prognostic factors and biomarkers for risk stratification are lacking to choose the most beneficial treatment out of the emerging landscape of different treatment options. In this study, we analyzed the serum DKK1 (S-DKK1) levels of 91 EC patients prior to surgery in a single center study at the University Medical Center Hamburg-Eppendorf by enzyme-linked immunosorbent assay. High levels of S-DKK1 could be especially observed in patients suffering from esophageal adenocarcinoma which may promote the hypothesis of a crucial role of DKK1 in inflammation. S-DKK1 levels of ≥5800 pg/mL were shown to be associated with unfavorable five-year survival rates and the presence of CTCs. Interestingly, significantly lower S-DKK1 levels were detected in patients after neoadjuvant treatment, implying that S-DKK1 may serve as a useful biomarker for treatment monitoring. Multivariate analysis identified S-DKK1 as an independent prognostic marker with respect to overall survival in EC patients with a hazard ratio of 2.23. In conclusion, our data implicate a negative prognostic role of DKK1 with respect to the clinical outcome in EC patients. Further prospective studies should be conducted to implement S-DKK1 into the clinical routine for risk stratification and treatment monitoring.

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

Homogeneous MMR Deficiency Throughout the Entire Tumor Mass Occurs in a Subset of Colorectal Neuroendocrine Carcinomas.

Neuroendocrine neoplasms comprise a heterogeneous group of tumors, categorized into neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) depending on tumor differentiation. NECs and high-grade NETs (G3) confer a poor prognosis, demanding novel treatment strategies such as immune checkpoint inhibition in tumors with microsatellite instability (MSI). To study any possible intratumoral heterogeneity of MSI, a tissue microarray (TMA) containing 199 NETs and 40 NECs was constructed to screen for MSI using immunohistochemistry (IHC) for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. Four cases suspicious for MSI were identified. Validation of MSI by repeated IHC on large sections and polymerase chain reaction (PCR)-based analysis using the "Bethesda Panel" confirmed MSI in 3 cecal NECs. One pancreatic NET G3 with MSI-compatible TMA results was MMR intact on large section IHC and microsatellite stable (MSS). The remaining 235 tumors exhibited intact MMR. Protein loss of MLH1/PMS2 was found in two and MSH6 loss in one cancer with MSI. Large section IHC on all available tumor-containing tissue blocks in NECs with MSI did not identify aberrant tumor areas with intact MMR. Our data indicate that MSI is common in colorectal NECs (3 out of 10) but highly infrequent in neuroendocrine neoplasms from many other sites. The lack of intratumoral heterogeneity of MMR deficiency suggests early development of MSI during tumorigenesis in a subset of colorectal NECs and indicates that microsatellite status obtained from small biopsies may be representative for the entire cancer mass.

Metastasis of pancreatic cancer: An uninflamed liver micromilieu controls cell growth and cancer stem cell properties by oxidative phosphorylation in pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed when liver metastases already emerged. We recently demonstrated that hepatic stromal cells determine the dormancy status along with cancer stem cell (CSC) properties of pancreatic ductal epithelial cells (PDECs) during metastasis. This study investigated the influence of the hepatic microenvironment - and its inflammatory status - on metabolic alterations and how these impact cell growth and CSC-characteristics of PDECs. Coculture with hepatic stellate cells (HSCs), simulating a physiological liver stroma, but not with hepatic myofibroblasts (HMFs) representing liver inflammation promoted expression of Succinate Dehydrogenase subunit B (SDHB) and an oxidative metabolism along with a quiescent phenotype in PDECs. SiRNA-mediated SDHB knockdown increased cell growth and CSC-properties. Moreover, liver micrometastases of tumor bearing KPC mice strongly expressed SDHB while expression of the CSC-marker Nestin was exclusively found in macrometastases. Consistently, RNA-sequencing and in silico modeling revealed significantly altered metabolic fluxes and enhanced SDH activity predominantly in premalignant PDECs in the presence of HSC compared to HMF. Overall, these data emphasize that the hepatic microenvironment determines the metabolism of disseminated PDECs thereby controlling cell growth and CSC-properties during liver metastasis.

Liver metastasis of pancreatic cancer: the hepatic microenvironment impacts differentiation and self-renewal capacity of pancreatic ductal epithelial cells.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced stages with the liver as the main site of metastases. The hepatic microenvironment has been shown to determine outgrowth of liver metastases. Cancer stem cells (CSCs) are essential for initiation and maintenance of tumors and acquisition of CSC-properties has been linked to Epithelial-Mesenchymal-Transition. Thus, this study aimed at elucidating whether and how the hepatic microenvironment impacts stemness and differentiation of disseminated pancreatic ductal epithelial cells (PDECs). Culture of premalignant H6c7-kras and malignant Panc1 PDECs together with hepatocytes and hepatic stellate cells (HSC) promoted self-renewal capacity of both PDEC lines. This was indicated by higher colony formation compared to cells cocultured with hepatocytes and hepatic myofibroblasts. Different Panc1 colony types derived from an HSC-enriched coculture were expanded and characterized revealing that holoclones exhibited an enhanced colony formation ability, elevated and exclusive expression of the CSC-marker Nestin and a more pronounced mesenchymal phenotype compared to paraclones. Moreover, Panc1 holoclone cells showed an increased tumorigenic potential in vivo leading to formation of undifferentiated tumors in 7/10 animals, while inoculation of paraclone cells only led to formation of tumors in 2/10 animals being smaller in number and size. Holoclone tumors were characterized by elevated expression of mesenchymal markers, complete loss of E-cadherin expression and high expression of Nestin. Finally, Etanercept-mediated TNF-α blocking partly reversed the mesenchymal CSC-phenotype of Panc1 holoclone cells. Overall, these data provide evidence that the hepatic microenvironment determines stemness and differentiation of PDECs, thereby substantially contributing to liver metastases of PDAC.

Diabetes as risk factor for pancreatic cancer: Hyperglycemia promotes epithelial-mesenchymal-transition and stem cell properties in pancreatic ductal epithelial cells.

Type 2 diabetes mellitus (T2DM) is associated with hyperglycemia and a risk to develop pancreatic ductal adenocarcinoma (PDAC), one of the most fatal malignancies. Cancer stem cells (CSC) are essential for initiation and maintenance of tumors, and acquisition of CSC-features is linked to epithelial-mesenchymal-transition (EMT). The present study investigated whether hyperglycemia promotes EMT and CSC-features in premalignant and malignant pancreatic ductal epithelial cells (PDEC). Under normoglycemia (5 mM d-glucose), Panc1 PDAC cells but not premalignant H6c7-kras cells exhibited a mesenchymal phenotype along with pronounced colony formation. While hyperglycemia (25 mM d-glucose) did not impact the mesenchymal phenotype of Panc1 cells, CSC-properties were aggravated exemplified by increased Nanog expression and Nanog-dependent formation of holo- and meroclones. In H6c7-kras cells, high glucose increased secretion of Transforming-Growth-Factor-beta1 (TGF-ß1) as well as TGF-ß1 signaling, and in a TGF-ß1-dependent manner reduced E-cadherin expression, increased Nestin expression and number of meroclones. Finally, reduced E-cadherin expression was detected in pancreatic ducts of hyperglycemic but not normoglycemic mice. These data suggest that hyperglycemia promotes the acquisition of mesenchymal and CSC-properties in PDEC by activating TGF-ß signaling and might explain how T2DM facilitates pancreatic tumorigenesis.

The hepatic microenvironment essentially determines tumor cell dormancy and metastatic outgrowth of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when liver metastases already emerged. This study elucidated the impact of hepatic stromal cells on growth behavior of premalignant and malignant pancreatic ductal epithelial cells (PDECs). Liver sections of tumor-bearing KPC mice comprised micrometastases displaying low proliferation located in an unobtrusive hepatic microenvironment whereas macrometastases containing more proliferating cells were surrounded by hepatic myofibroblasts (HMFs). In an age-related syngeneic PDAC mouse model livers with signs of age-related inflammation exhibited significantly more proliferating disseminated tumor cells (DTCs) and micrometastases despite comparable primary tumor growth and DTC numbers. Hepatic stellate cells (HSC), representing a physiologic liver stroma, promoted an IL-8 mediated quiescence-associated phenotype (QAP) of PDECs in coculture. QAP included flattened cell morphology, Ki67-negativity and reduced proliferation, elevated senescence-associated ß galactosidase activity and diminished p-Erk/p-p38-ratio. In contrast, proliferation of PDECs was enhanced by VEGF in the presence of HMF. Switching the micromilieu from HSC to HMF or blocking VEGF reversed QAP in PDECs. This study demonstrates how HSCs induce and maintain a reversible QAP in disseminated PDAC cells, while inflammatory HMFs foster QAP reversal and metastatic outgrowth. Overall, the importance of the hepatic microenvironment in induction and reversal of dormancy during PDAC metastasis is emphasized.