Genome-Wide Association Study Identifies 4 Novel Risk Loci for Small Intestinal Neuroendocrine Tumors Including a Missense Mutation in LGR5.

BACKGROUND & AIMS: Small intestinal neuroendocrine tumor (SI-NET) is a rare disease, but its incidence has increased over the past 4 decades. Understanding the genetic risk factors underlying SI-NETs can help in disease prevention and may provide clinically beneficial markers for diagnosis. Here the results of the largest genome-wide association study of SI-NETs performed to date with 405 cases and 614,666 controls are reported. METHODS: Samples from 307 patients with SI-NETs and 287,137 controls in the FinnGen study were used for the identification of SI-NET risk-associated genetic variants. The results were also meta-analyzed with summary statistics from the UK Biobank (n = 98 patients with SI-NET and n = 327,529 controls). RESULTS: We identified 6 genome-wide significant (P < 5 × 10-8) loci associated with SI-NET risk, of which 4 (near SEMA6A, LGR5, CDKAL1, and FERMT2) are novel and 2 (near LTA4H-ELK and in KIF16B) have been reported previously. Interestingly, the top hit (rs200138614; P = 1.80 × 10-19) was a missense variant (p.Cys712Phe) in the LGR5 gene, a bona-fide marker of adult intestinal stem cells and a potentiator of canonical WNT signaling. The association was validated in an independent Finnish collection of 70 patients with SI-NETs, as well as in the UK Biobank exome sequence data (n = 92 cases and n = 392,814 controls). Overexpression of LGR5 p.Cys712Phe in intestinal organoids abolished the ability of R-Spondin1 to support organoid growth, indicating that the mutation perturbed R-Spondin-LGR5 signaling. CONCLUSIONS: Our study is the largest genome-wide association study to date on SI-NETs and reported 4 new associated genome-wide association study loci, including a novel missense mutation (rs200138614, p.Cys712Phe) in LGR5, a canonical marker of adult intestinal stem cells.

Diagnosis and staging of small intestinal neuroendocrine tumors with CT enterography and PET with Gallium-68: preoperative risk stratification protocol.

PURPOSE: Pre-operative diagnosis and staging of small intestine neuroendocrine tumors (SI-NETs) remain sub-optimal, with open palpation during surgery still considered the gold standard. This limits a standardized implementation of minimally invasive surgery (MIS). The aim of this single-center retrospective study was to assess a tailored diagnostic work-up to identify candidates at low risk of undetected disease who may benefit from MIS. METHODS: Patients diagnosed with SI-NETs between 2013 and 2022 who underwent contrast-enhanced computed tomography enterography (CTE) and Ga68-DOTATOC-positron emission tomography-CT (68 Ga DOTATATE PET/CT) preoperatively and subsequently underwent open surgical resection were included. Imaging studies were reassessed by two radiologists. Combined use of CTE and 68 Ga DOTATATE PET/CT in determining primary lesion disease burden (number of lesions) and LN disease stage (distal and proximal relative to superior mesenteric vessels) was assessed, using surgical reports and pathology as gold standard. RESULTS: Overall, 56 patients were included. Sensitivity of CTE and 68 Ga DOTATATE PET/CT for at least one primary SI-NET was 100% and 94%, respectively. In the presence of concordance between studies, combined use of CTE and 68 Ga DOTATATE PET/CT for detection of single primary tumors improved specificity to 89% (n = 25/28) with a positive predictive value of 87.5% (n = 21/24). Distal LN disease was identified in 89.2% of cases (n = 33/37). The association of single lesion and distal LN disease was found pre-operatively in 32% of patients (n = 18). CONCLUSION: Combined use of CTE and 68 Ga DOTATATE PET/CT enables identifying low-risk surgical candidates (single SI-NET lesions with distal LN disease).

Mesenteric sparing approach for advanced nodal extent in small intestinal neuroendocrine tumors. Is there a limit to the vascular resection in order to avoid creating a short small bowel syndrome? An anatomic research study.

PURPOSE: By selectively perfusing the first three jejunal arteries (JA), we aim to assess the individual perfusion length of small bowel (SB) and its impact on nodal resection in stage III-up small-intestinal neuroendocrine tumors (SI-NET). METHODS: Our anatomical research protocol implies a midline laparotomy and three measures of the SB length. We then perform a classical anterior approach of the superior mesenteric vessels. We carry on with the complete dissection and checking of the superior mesenteric artery (SMA) in order to identify the first three JA. Then we selectively perfuse each artery with colored latex solutions and measure the length of small bowel perfused respectively. RESULTS: We conducted our protocol on six cadaveric subjects. Mean(SD) SB length was 413(5.7), 535(13.2), 485(15), 353(25.1), 730(17.3) and 525(16° cm respectively from subject one to six. Most JA originated from the left side of the SMA. The first JA originated from its posterior wall in two subjects. Mean(SD) distance of origin of the first three JA was 4.6(1.3)cm, 6(1.1)cm and 7.1(0.9)cm respectively. Mean(SD) diameter of SMA was 10.8(3.3)mm. Mean diameter of the three first JA was 4(1.4)mm, 4(1.5)mm and 5(1.2)mm respectively. Mean(SD) SB length perfused by first and second JA was 224(14.9)cm, 175(8.6)cm, 238.3(7.6)cm, 84.3(5.1)cm, 233.3(5.8)cm and 218.3(10.4)cm respectively from subject one to six. CONCLUSION: We observed a trend suggesting that the first and second JA may sustain a SB length beyond the viable 1.5 m limit, implying the feasibility of stage III-up SI-NET resection with just two JA.

Exposure to nonanoic acid alters small intestinal neuroendocrine tumor phenotype.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NET) are highly differentiated and genetically stable malignant tumors, yet they often present with advanced metastatic spread at the time of diagnosis. In contrast to many other types of malignant tumors, primary SI-NET are often asymptomatic and typically smaller in size compared to adjacent lymph node metastases. This study explores the hypothesis that stimulating the chemosensing olfactory receptor 51E1 (OR51E1) decreases SI-NET proliferation suggesting a mechanism that explains a difference in proliferative rate based on tumor location. METHODS: Clinical data was used to address difference in tumor size depending on location. A SI-NET tissue microarray was used to evaluate expression of OR51E1 and olfactory marker protein (OMP). Primary cultured tumor cells from 5 patients were utilized to determine the effect of OR51E1 agonist nonanoic acid on metabolic activity. The SI-NET cell line GOT1 was used to determine effects of nonanoic acid on the transcriptome as well as long-term effects of nonanoic acid exposure with regards to cell proliferation, serotonin secretion, alterations of the cell-cycle and morphology. RESULTS: Tumor size differed significantly based on location. OR51E1 and OMP were generally expressed in SI-NET. Primary SI-NET cells responded to nonanoic acid with a dose dependent altered metabolic activity and this was replicated in the GOT1 cell line but not in the MCF10A control cell line. Nonanoic acid treatment in GOT1 cells upregulated transcripts related to neuroendocrine differentiation and hormone secretion. Long-term nonanoic acid treatment of GOT1 cells decreased proliferation, induced senescence, and altered cell morphology. CONCLUSION: Our results raise the possibility that exposure of intraluminal metabolites could represent a mechanism determining aspects of the SI-NET tumor phenotype. However, we could not causally link the observed effects of nonanoic acid exposure to the OR51E1 receptor.

Surgical Management of the Primary Tumor in Stage IV Small Intestinal Neuroendocrine Tumors: To Operate or Not to Operate, That Is the Question.

PURPOSE OF REVIEW: Small intestinal neuroendocrine tumors (SI-NET) are rare tumors, often with distant metastases at diagnosis. The objective of this review is to provide an overview of the latest literature regarding surgical management of the primary tumor in stage IV SI-NET. RECENT FINDINGS: Primary tumor resection (PTR) seems to be associated with improved survival in patients with stage IV SI-NET, independent of treatment of distant metastases. A watch and wait approach of the primary tumor increases the risk of needing an emergency resection. PTR improves survival in patients with stage IV SI-NET, decreases the risk of emergency surgery, and should be considered in all patients with stage IV disease and unresectable liver metastasis.

Prognostic differences in grading and metastatic lymph node pattern in patients with small bowel neuroendocrine tumors.

PURPOSE: Neuroendocrine tumors of the small intestine (si-NET) describe a heterogenous group of neoplasms. Based on the Ki67 proliferation index si-NET are divided into G1 (Ki67 < 2%), G2 (Ki67 3-20%) and rarely G3 (Ki67 > 20%) tumors. However, few studies evaluate the impact of tumor grading on prognosis in si-NET. Moreover, si-NET can form distinct lymphatic spread patterns to the mesenteric root, aortocaval lymph nodes, and distant organs. This study aims to identify prognostic factors within the lymphatic spread patterns and grading. METHODS: Demographic, pathological, and surgical data of 208 (90 male, 118 female) individuals with si-NETs treated at Charité University Medicine Berlin between 2010 and 2020 were analyzed retrospectively. RESULTS: A total of 113 (54.5%) specimens were defined as G1 and 93 (44.7%) as G2 tumors. Interestingly, splitting the G2 group in two subgroups: G2 low (Ki67 3-9%) and G2 high (Ki67 10-20%), displayed significant differences in overall survival (OS) (p = 0.008) and progression free survival (PFS) (p = 0.004) between these subgroups. Remission after surgery was less often achieved in patients with higher Ki67 index (> 10%). Lymph node metastases (N +) were present in 174 (83.6%) patients. Patients with isolated locoregional disease showed better PFS and OS in comparison to patients with additional aortocaval and distant lymph node metastases. CONCLUSION: Lymphatic spread pattern influences patient outcome. In G2 tumors, low and high grading shows heterogenous outcome in OS and PFS. Differentiation within this group might impact follow-up, adjuvant treatment, and surgical strategy.

Decrease of 5-hydroxymethylcytosine and TET1 with nuclear exclusion of TET2 in small intestinal neuroendocrine tumors.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from enterochromaffin cells scattered in the intestinal mucosa of the ileum and jejunum. Loss of one copy of chromosome 18 is the most frequent observed aberration in primary tumors and metastases. The aim of this study was to investigate possible involvement of 5-hydroxymethylcytosine (5hmC), TET1 and TET2 in SI-NETs. METHODS: The analysis was conducted using 40 primary tumors and corresponding 47 metastases. The level of 5hmC, TET1 and TET2 was analyzed by DNA immune-dot blot assay and immunohistochemistry. Other methods included a colony forming assay, western blotting analysis, and quantitative bisulfite pyrosequencing analysis. The effect of the exportin-1 nuclear transport machinery inhibitors on cell proliferation and apoptosis was also explored using two SI-NET cell lines. RESULTS: Variable levels of 5hmC and a mosaic staining appearance with a mixture of positive and negative cell nuclei, regardless of cell number and staining strength, was observed overall both in primary tumors and metastases. Similarly aberrant staining pattern was observed for TET1 and TET2. In a number of tumors (15/32) mosaic pattern together with areas of negative staining was also observed for TET1. Abolished expression of TET1 in the tumors did not seem to involve hypermethylation of the TET1 promoter region. Overexpression of TET1 in a colony forming assay supported a function as cell growth regulator. In contrast to 5hmC and TET1, TET2 was also observed in the cytoplasm of all the analyzed SI-NETs regardless of nuclear localization. Treatment of CNDT2.5 and KRJ-I cells with the exportin-1 (XPO1/CRM1) inhibitor, leptomycin B, induced reduction in the cytoplasm and nuclear retention of TET2. Aberrant partitioning of TET2 from the nucleus to the cytoplasm seemed therefore to involve the exportin-1 nuclear transport machinery. Reduced cell proliferation and induction of apoptosis were observed after treatment of CNDT2.5 and KRJ-I cells with leptomycin B or KPT-330 (selinexor). CONCLUSIONS: SI-NETs are epigenetically dysregulated at the level of 5-hydroxymethylcytosine/ TET1/TET2. We suggest that KPT-330/selinexor or future developments should be considered and evaluated for single treatment of patients with SI-NET disease and also in combinations with somatostatin analogues, peptide receptor radiotherapy, or everolimus.

Smoking, alcohol and family history of cancer as risk factors for small intestinal neuroendocrine tumors: a systematic review and meta-analysis.

OBJECTIVES: Risk factors for small intestinal neuroendocrine tumors (SI-NETs) are not well understood. The aim of this systematic literature review was to identify risk factors for SI-NET and to further assess these by meta-analysis. MATERIAL AND METHODS: PubMed and abstracts from the ENETS and NANETS were searched for studies published until May 2015. Eligible studies were selected according to the PRISMA statement. RESULTS: Seven studies evaluating six individual populations were included (study accrual period 1980-2012) in the meta-analysis, involving 765 (range 17-325) cases and 502,282 (range 52-498,376) controls. All studies were case-control by design. The following risk factors were reported in ≥2 studies: family history of any cancer, family history of colorectal cancer, ever alcohol use and ever smoking. The pooled OR was 1.34 (95% CI: 1.12-1.60; p < .01; I2 = 0.0%) for family history of any cancer, 1.43 (95% CI: 1.15-1.79; p < .01; I2 = 0.0%) for family history of colorectal cancer, 1.04 (95% CI: 0.63-1.72; p = .87; I2 = 65.0%) for ever alcohol use and 1.40 (95% CI: 1.06-1.86; p < .05; I2 = 49.3%) for ever smoking. CONCLUSIONS: Family history of any cancer, family history of colorectal cancer and history of ever smoking were associated with an increased risk of SI-NET by meta-analysis. Alcohol consumption was not a significant risk factor for SI-NET. However, the studies reporting smoking and alcohol had a high degree of heterogeneity. Therefore, further studies are needed for clarification of smoking and alcohol as risk factors for the occurrence of SI-NET.

A plausible role for actin gamma smooth muscle 2 (ACTG2) in small intestinal neuroendocrine tumorigenesis.

BACKGROUND: Small intestinal neuroendocrine tumors (SI-NETs) originate from the enterochromaffin cells in the ileum and jejunum. The knowledge about genetic and epigenetic abnormalities is limited. Low mRNA expression levels of actin gamma smooth muscle 2 (ACTG2) have been demonstrated in metastases relative to primary SI-NETs. ACTG2 and microRNA-145 (miR-145) are aberrantly expressed in other cancers and ACTG2 can be induced by miR-145. The aim of this study was to investigate the role of ACTG2 in small intestinal neuroendocrine tumorigenesis. METHODS: Protein expression was analyzed in SI-NETs (n = 24) and in enterochromaffin cells by immunohistochemistry. The cell line CNDT2.5 was treated with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep), the selective EZH2 inhibitor EPZ-6438, or 5-aza-2'-deoxycytidine, a DNA hypomethylating agent. Cells were transfected with ACTG2 expression plasmid or miR-145. Western blotting analysis, quantitative RT-PCR, colony formation- and viability assays were performed. miR-145 expression levels were measured in tumors. RESULTS: Eight primary tumors and two lymph node metastases displayed variable levels of positive staining. Fourteen SI-NETs and normal enterochromaffin cells stained negatively. Overexpression of ACTG2 significantly inhibited CNDT2.5 cell growth. Treatment with DZNep or transfection with miR-145 induced ACTG2 expression (>10-fold), but no effects were detected after treatment with EPZ-6438 or 5-aza-2'-deoxycytidine. DZNep also induced miR-145 expression. SI-NETs expressed relatively low levels of miR-145, with reduced expression in metastases compared to primary tumors. CONCLUSIONS: ACTG2 is expressed in a fraction of SI-NETs, can inhibit cell growth in vitro, and is positively regulated by miR-145. Theoretical therapeutic strategies based on these results are discussed.

Treatment efficacy in a metastatic small intestinal neuroendocrine tumour grade 2 cohort.

Small intestinal neuroendocrine tumours (Si-NET) are often studied as a uniform group. Proliferation index Ki-67 influences prognosis and determines tumour grade. We hypothesized that Si-NET grade 2 (G2) tumours, which have a higher Ki-67 than G1 tumours, might benefit less from established treatments for metastatic disease. We conducted a retrospective cohort study of 212 patients with metastatic Si-NET G2 treated in two Swedish hospitals during 20 years (2000-2019). Median cancer-specific survival on first-line somatostatin analogues (SSA) was 77 months. Median progression-free survival (PFS) was 12.4 months when SSA was given as monotherapy and 19 months for all patients receiving first-line SSA. PFS after SSA dose escalation was 6 months in patients with radiological progression. Treatment efficacies of SSA and peptide receptor radionuclide treatment (PRRT) were studied separately in patients with Ki-67 of 3-5%, 5-10% and 10-20%. For SSA, PFS was significantly shorter at higher Ki-67 levels (31, 18 and 10 months, respectively), while there was only a minor difference in PFS for PRRT (29, 25 and 25 months). Median PFS for sequential treatment with interferon-alpha (IFNα), everolimus and chemotherapy was 6, 5 and 9 months. IFNα seemed to be effective in tumours with low somatostatin-receptor expression. In conclusion, established treatments appeared effective in Si-NET G2, despite their higher proliferation index compared to G1 tumours. However, efficacy of SSA but not PRRT was reduced at higher Ki-67 levels. SSA dose escalation provided limited disease stabilization.

Epigenetic regulation of SST2 expression in small intestinal neuroendocrine tumors.

Background: Somatostatin receptor type 2 (SST2) expression is critical for the diagnosis and treatment of neuroendocrine tumors and is associated with improved patient survival. Recent data suggest that epigenetic changes such as DNA methylation and histone modifications play an important role in regulating SST2 expression and tumorigenesis of NETs. However, there are limited data on the association between epigenetic marks and SST2 expression in small intestinal neuroendocrine tumors (SI-NETs). Methods: Tissue samples from 16 patients diagnosed with SI-NETs and undergoing surgical resection of the primary tumor at Erasmus MC Rotterdam were analysed for SST2 expression levels and epigenetic marks surrounding the SST2 promoter region, i.e. DNA methylation and histone modifications H3K27me3 and H3K9ac. As a control, 13 normal SI-tissue samples were included. Results: The SI-NET samples had high SST2 protein and mRNA expression levels; a median (IQR) of 80% (70-95) SST2-positive cells and 8.2 times elevated SST2 mRNA expression level compared to normal SI-tissue (p=0.0042). In comparison to normal SI-tissue, DNA methylation levels and H3K27me3 levels were significantly lower at five out of the eight targeted CpG positions and at two out of the three examined locations within the SST2 gene promoter region of the SI-NET samples, respectively. No differences in the level of activating histone mark H3K9ac were observed between matched samples. While no correlation was found between histone modification marks and SST2 expression, SST2 mRNA expression levels correlated negatively with DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NETs (p=0.006 and p=0.04, respectively). Conclusion: SI-NETs have lower SST2 promoter methylation levels and lower H3K27me3 methylation levels compared to normal SI-tissue. Moreover, in contrast to the absence of a correlation with SST2 protein expression levels, significant negative correlations were found between SST2 mRNA expression level and the mean level of DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NET tissue. These results indicate that DNA methylation might be involved in regulating SST2 expression. However, the role of histone modifications in SI-NETs remains elusive.

A progeroid syndrome caused by a deep intronic variant in TAPT1 is revealed by RNA/SI-NET sequencing.

Exome sequencing has introduced a paradigm shift for the identification of germline variations responsible for Mendelian diseases. However, non-coding regions, which make up 98% of the genome, cannot be captured. The lack of functional annotation for intronic and intergenic variants makes RNA-seq a powerful companion diagnostic. Here, we illustrate this point by identifying six patients with a recessive Osteogenesis Imperfecta (OI) and neonatal progeria syndrome. By integrating homozygosity mapping and RNA-seq, we delineated a deep intronic TAPT1 mutation (c.1237-52 G>A) that segregated with the disease. Using SI-NET-seq, we document that TAPT1's nascent transcription was not affected in patients' fibroblasts, indicating instead that this variant leads to an alteration of pre-mRNA processing. Predicted to serve as an alternative splicing branchpoint, this mutation enhances TAPT1 exon 12 skipping, creating a protein-null allele. Additionally, our study reveals dysregulation of pathways involved in collagen and extracellular matrix biology in disease-relevant cells. Overall, our work highlights the power of transcriptomic approaches in deciphering the repercussions of non-coding variants, as well as in illuminating the molecular mechanisms of human diseases.

Survival of patients with small bowel neuroendocrine neoplasms in Auckland, Aotearoa New Zealand.

BACKGROUND: Small intestinal Neuroendocrine Neoplasms (SI-NENs) are the most common primary malignancy of the small bowel. The aim of this study is to define the survival of patients with an SI-NEN in Auckland, Aotearoa New Zealand (AoNZ). METHODS: A retrospective study of all patients diagnosed with a jejunal or ileal SI-NEN in the Auckland region between 2000 and 2012 was performed. The New Zealand NETwork! Registry was searched to identify the study cohort. Retrospective data collection was performed to collect stage, survival and follow up data. RESULTS: One hundred and seven patients were included in the study. The mean age of patients was 62.8 years (SD 11.9). The 5 and 10-year disease-specific survival for all patients was 66.1% (95% CI 56.5-75.7%) and 61.8% (95% CI 51.8-71.8%), respectively. Ten-year disease-specific survival was 100% for stage I and II, 74% (95%CI 61.7-84.4%) for stage III and 33.9% (95%CI 16.9-35.6%) for stage IV SI-NEN. Eleven of 40 (27.5%) patients with stage III disease had recurrence and 3 of 7 (42.8%) patients with stage IV disease had recurrence. In patients with stage IV disease, neither primary resection (HR 2.25, 95% CI 0.92-5.5) nor distant resection (HR 1.72, 95% CI 0.63-4.7) were significantly associated with a disease-specific or overall survival benefit. CONCLUSION: This study demonstrates that stage at SI-NEN diagnosis is associated with survival, but resection of the primary or distant metastases in patients with stage IV disease is not. There was no recurrence in patients with stage I or II disease after complete resection.

Evaluation of SSTR2 Expression in SI-NETs and Relation to Overall Survival after PRRT.

(1) Purpose: Small intestinal neuroendocrine tumors (SI-NETs) often present with distant metastases at diagnosis. Peptide receptor radionuclide therapy (PRRT) with radiolabeled somatostatin analogues is a systemic treatment that increases overall survival (OS) in SI-NET patients with stage IV disease. However, the treatment response after PRRT, which targets somatostatin receptor 2 (SSTR2), is variable and predictive factors have not been established. This exploratory study aims to evaluate if SSTR2 expression in SI-NETs could be used to predict OS after PRRT treatment. (2) Methods: Using a previously constructed Tissue Micro Array (TMA) we identified tissue samples from 42 patients that had received PRRT treatment during 2006-2017 at Sahlgrenska University hospital. Immunohistochemical expression of SSTR2, Ki-67 and neuroendocrine markers synaptophysin and Chromogranin A (CgA) were assessed. A retrospective estimation of 177Lu-DOTATATE uptake in 33 patients was performed. Data regarding OS and non-surgical treatment after PRRT were collected. Another subgroup of 34 patients with paired samples from 3 tumor sites (primary tumor, lymph node and liver metastases) was identified in the TMA. The SSTR2 expression was assessed in corresponding tissue samples (n = 102). (3) Results: The patients were grouped into Low SSTR2 or High SSTR2 groups based upon on levels of SSTR2 expression. There was no significant difference in 177Lu-DOTATATE uptake between the groups. The patients in the Low SSTR2 group had significantly longer OS after PRRT than the patients in the High SSTR2 group (p = 0.049). PRRT treated patients with low SSTR2 expression received less additional treatment compared with patients with high SSTR2 expression. SSTR2 expression did not vary between tumor sites but correlated within patients. (4) Conclusion: The results from the present study suggest that retrospective evaluation of SSTR2 expression in resected tumors cannot be used to predict OS after PRRT.

Telomerase activation in small intestinal neuroendocrine tumours is associated with aberrant TERT promoter methylation, but not hot-spot mutations.

Telomere maintenance is a critical requirement for enabling replicative immortality and tumour development. Here, telomerase expression and activity, telomere length (TL) and potential regulatory factors that can underlie telomerase machinery alterations in small intestinal neuroendocrine tumours (SI-NETs) were analyzed. Telomerase activity assessed by TRAP assay was increased in SI-NETs compared to normal ileum (P < 0.001). The telomerase reverse transcriptase gene (TERT) was over-expressed in SI-NETs vs. normal ileal samples (P = 0.01). Furthermore, relative TL assessed by qPCR was found shorter in tumours compared with normal ileum (P = 0.02) and in distant metastasis samples compared to primary tumours and local metastases (P= 0.02). TERT promoter hotspot mutations were not present and TERT copy number gain was only observed in 3/70 tumour samples. TERT or chromosome 18 copy number alterations were not associated with telomerase expression and activity or TL. However, hypermethylation of TERT promoter in Region B - in the proximity of the transcription start site - was inversely correlated with TERT expression and telomerase activity and positively correlated with TL. Global LINE1 methylation was positively correlated with TERT promoter Region B methylation and was inversely correlated with telomerase activity, TERT expression and the upstream Region A methylation. The results show that telomerase activation, TERT expression and shorter telomeres are commonly found in SI-NETs. Aberrant DNA methylation of TERT promoter and of LINE1 can be implicated in abnormal regulation of TERT in SI-NETs.

Improvement of Thermosensitive Liposome Stability by Cerasome Forming Lipid with Si-O-Si Network Structure.

BACKGROUND: Liposomes have been widely used in gene transfection and drug delivery systems due to their excellent biocompatibility and encapsulation ability, especially, the ability to deliver the gene/drug into the cells via the membrane fusion. Thermosensitive liposomes have been proven to be a precise and effective method for cancer treatment in many preclinical studies. But the imperfect crystalline arrangement between grains occurred, resulting in planar defects at the boundaries of membranes, compromising the stability of thermosensitive liposomes. OBJECTIVE: In the present study, we developed a facile method to improve the stability of ordinary thermosensitive liposomes by introducing organic-inorganic hybrid materials with local Si-O-Si net. METHOD: A cerasome forming lipid, N, N-Dihxadecyl-N'- [(3-triethoxysilyl) propyl] urea, was synthesized and then introduced into the thermosensitive lipids to form the composite liposomes (named as cera-liposomes). The effects of the cerasome forming lipid on the cera-liposomes characteristics, including the morphology, drug loading, Zeta potential and stability of vesicles, were investigated. RESULTS: Cera-liposomes were thermosensitive, and they had a loading efficiency over 2 folds more than conventional thermosensitive liposomes. With the enhanced sustain of Si-O-Si, cera-liposomes were able to avoid collapsing and fusing during storage, and had a good resistance to nonionic surfactant. More than 80% drug was still retained after storage of 15 days at room temperature. CONCLUSION: The cerasome forming lipid showed potential in improving the stability of thermosensitive liposomes. This novel kind of cera-liposomes could be a stable and effective drug carrier for anticancer applications.

Global hypomethylation and promoter methylation in small intestinal neuroendocrine tumors: an in vivo and in vitro study.

Aberrant DNA methylation is a feature of human cancer affecting gene expression and tumor phenotype. Here, we quantified promoter methylation of candidate genes and global methylation in 44 small intestinal-neuroendocrine tumors (SI-NETs) from 33 patients by pyrosequencing. Findings were compared with gene expression, patient outcome and known tumor copy number alterations. Promoter methylation was observed for WIF1, RASSF1A, CTNNB1, CXCL14, NKX2-3, P16, LAMA1, and CDH1. By contrast APC, CDH3, HIC1, P14, SMAD2, and SMAD4 only had low levels of methylation. WIF1 methylation was significantly increased (P = 0.001) and WIF1 expression was reduced in SI-NETs vs. normal references (P = 0.003). WIF1, NKX2-3, and CXCL14 expression was reduced in metastases vs. primary tumors (P<0.02). Low expression of RASSF1A and P16 were associated with poor overall survival (P = 0.045 and P = 0.011, respectively). Global methylation determined by pyrosequencing of LINE1 repeats was reduced in tumors vs. normal references, and was associated with loss in chromosome 18. The tumors fell into three clusters with enrichment of WIF1 methylation and LINE1 hypomethylation in Cluster I and RASSF1A and CTNNB1 methylation and loss in 16q in Cluster II. In Cluster III, these alterations were low-abundant and NKX2-3 methylation was low. Similar analyses in the SI-NET cell lines HC45 and CNDT2 showed methylation for CDH1 and WIF1 and/or P16, CXCL14, NKX2-3, LAMA1, and CTNNB1. Treatment with the demethylating agent 5-azacytidine reduced DNA methylation and increased expression of these genes in vitro. In conclusion, promoter methylation of tumor suppressor genes is associated with suppressed gene expression and DNA copy number alterations in SI-NETs, and may be restored in vitro.