Huge congratulations to Dr Marc Ooft, Consultant Histopathologist, King’s College Hospital – the recipient of the 2020 NCUK Research Grant. The grant will fund a study entitled ‘Improving patient stratification through epigenetic and tumour microenvironment analysis of the 100,000 Genomes project Neuroendocrine tumour cohort.’
The 2020 grant was awarded at our 18th National Conference Online, and is supported by NCUK, including a generous donation of £10,000 from Mr P Moxham
This year’s award went to:
The title of their project is Development and characterisation of organoid (gastroid) cultures from low grade gastric neuroendocrine tumours.
It would be helpful to be able to culture cells from gastric NETs in the laboratory to provide platforms that permit analyses of tumour behaviour as well as in vitrotumour responses to various potential treatments. This approach might in due course lead to personalisation of treatment approaches for patients with gastric NETs.
Until recently, it has not been possible to culture primary normal human gastrointestinal cells and the successful in vitro culture of NET cells has also been somewhat of a ‘holy grail’ among the NET research community for many decades. However, Hans Clevers and colleagues recently developed techniques for the successful culture of normal gastrointestinal epithelial cells as so-called ‘organoids’. The crucial breakthrough was an understanding of the cocktail of growth factors that are required to maintain cellular growth and the stem cell niche. These organoid culture techniques have also recently been employed to culture pancreatic neuroendocrine tumour cells. We have shown that gastroid cultures can be successfully grown in our lab from endoscopic biopsies and that they respond in vitro to treatment with gastrin. However, to our knowledge, nobody has yet reported (or attempted) to develop organoid cultures from gastric NETs.
The aim of this project is to develop and characterise primary cultures from low grade type 1 and type 3 gastric NETs as well as from their adjacent unaffected mucosa. After culturing these gastroids, we will characterise them at the cellular and transcriptomic level using immunohistochemistry and RNASeq and the results will be compared with the same characteristics within the native NET/unaffected gastric mucosa from which they were initially derived. These assessments will estimate the degree of cellular and genetic similarity between gastroids and their originating tissue and whether these characteristics are maintained in long term culture. Finally we will investigate the responses of gastric NET cultures to the CCK-2 receptor antagonist netazepide and the somatostatin analogue octreotide.
By the end of the project we will hopefully establish the extent to which gastroids derived from NETs and their adjacent normal/atrophic gastric epithelium retain the genetic and cellular characteristics of the tissues from which they were derived. We will also establish a bank of cryopreserved gastroids. If we confirm that these gastroids are representative of their native tumours, this will provide an invaluable resource for future experiments.
We are pleased to announce the 2018 recipients of the UKINETs and NET Patient Foundation, which were awarded at the UKINETS Annual Conference on 9th December.
Aileen Waddle Grant
Dr Mariam Jamal-Hanjani
What is the molecular evolution of neuroendocrine lung tumours, and can genomic tumour characteristics predict patient outcome?
Primary pulmonary neuroendocrine tumours make up 20-25% of all lung cancer diagnoses and on a spectrum of malignant behaviour, ranging from the low grade classical carcinoid tumours up to the high grade, and notoriously aggressive, small cell lung carcinoma. The evolutionary relationships between neuroendocrine tumours of different grades remain poorly understood.
Design: The TRACERx study (TRAcking non-small cell lung Cancer Evolution
through therapy (Rx)) is a prospective cohort study across multiple UK centres which explores the genomic evolution of lung cancers. Tissue and blood samples have been taken from a cohort of 20 consented patients with pulmonary neuroendocrine tumours which have been banked through TRACERx, but excluded from the main arm of the study. Multi-region sampling and sequencing was performed on fresh frozen tissue from each tumour. Histological assessment of each tumour region was performed, including description of architecture, morphology and tumour infiltrating lymphocyte (TIL) score. Germline samples derived from pre-operative blood were taken from each patient for comparative analysis. The data were analysed using the established TRACERx bioinformatics pipeline.
Results: Phylogenetic trees were generated, illustrating genomic divergence for each tumour in our cohort and identifying points at which specific known driver mutations were acquired by the tumour. Differences were found between tumour grades and subtypes across a number of parameters, including somatic copy number alterations (SCNA), clonal and subclonal tumour mutational burden and acquisition of driver mutations.
Findings were correlated with histological subtype and clinical data to give a uniquely rounded perspective on the evolutionary behaviour of these tumours.
Conclusion: Genomic differences are identifiable between different members of the primary pulmonary neuroendocrine tumour family, with high grade tumours showing greater whole genome instability indices and phylogenetic divergence than low grade tumours. Repeated acquisition of specific driver mutations is found in respective tumour subtypes.
NET Patient Foundation Grant
Dr Kate Lines
Can conjugating the bromodomain inhibitor JQ1 to the somatostatin analogues pasireotide or octreotide provide a novel targeted epigenetic therapy for pancreatic neuroendocrine tumours?
This project focused on evaluating the efficacy of a novel somatostain-JQ1 analogue for the treatment of pancreatic neuroendocrine tumours (PNETs). Epigenetic inhibitors may offer a novel class of anti-cancer drugs, as we have previously demonstrated that JQ1, a bromo and extra terminal domain inhibitor (BETi) that prevents binding of the BET family of proteins to acetylated histone residues, significantly decreased proliferation and increased apoptosis of a human PNET cell line, and PNETs in a pancreatic cell specific Men1knockout mouse model. However, JQ1 may lack specificity as it is effective against multiple tumours. This could be overcome by targeting JQ1 specifically to PNET cells. Neuroendocrine tissues, including PNETs express somatostatin receptors (SSTRs), and analogues that bind these receptors, including octreotide are routinely used in the clinic.In our study, we have therefore synthesised a novel molecule that is a conjugate of an orally active derivative of JQ1, OTX-015, that is currently undergoing clinical trials for leukaemia and glioblastoma, to the somatostatin analogue octreotide, via a protease degradable linker. We have undertaken initial dose escalation studies in conditional Men1knockout mice that we have shown develop SSTR 1, 2, 3 and 5 expressing PNETs from 6 months of age. These studies indicate that the conjugate molecule is well tolerated in concentrations up to 50mg/kg, injected intraperitoneally, which is comparable to the concentration of JQ1 used in previous in vivo mouse studies. Furthermore, preliminary results indicate that conjugate treatment reduces proliferation of the PNETs in the Men1knockout mouse, compared to vehicle treated mice, without reducing proliferation of cells within the spleen. Thus, we have developed a novel NET-targeted BETi that is now available for preclinical in vitro and in vivo evaluation.
Congratulations to Christodoulos Pipinikas, the recipient of our 2016 award, which will be funding this exciting project:
Using an integrated approach combining data generated through the use of different, advanced molecular tools, we have previously demonstrated that neuroendocrine tumours of the pancreas and gastrointestinal tract are highly epigenetically dysregulated and have identified several altered biological pathways and genes that may form the basis for the development of novel therapeutic targets. In addition, our group has identified specific molecular disease subtypes associated with a significant impact on patients’ survival, indicating that these may benefit from different treatments. Using a similar approach, we would like to extend our understanding of the key molecular events involved in the development and progression of bronchopulmonary neuroendocrine tumours (BP-NETs). BP-NETs represent a significant disease burden with socioeconomic extensions due to their increased incidence and decreased 5-year survival rates. BP-NETs comprise approximately 20-25% of all lung cancers and represent a spectrum of tumours arising from neuroendocrine cells of the bronchopulmonary epithelium. Tumour classification into the correct histological sub-group is strongly predictive of patients’ prognosis but relies on few, difficult to reproduce pathological parameters which are often affected by a high inter-observer variability. Therefore, the aim of this project is to identify novel and accurate molecular tools in order to improve the classification of these tumours. This in turn will provide better prognostic information and help with choosing more appropriate treatments. In addition, we aim to study the underlying pathogenic mechanisms involved in disease development and progression through the use of large-scale integrated omics analyses.
Jorge Barriuso who works with Prof. Juan Valle at the Christie Hospital was awarded £30,000. The aim of the project is to identify predictive genetic markers of exceptional response to targeted therapies, therefore allowing treatment stratification. Samples from the Christie NET biobank would be used for this pilot study.
This year the £30,000 grant will be funded solely by the NET Patient foundation, the details and timeline for applying will be posted on the UKINETs and NET Patient Foundation websites in July 2016. The successful application will be invited to present their project and results at the UKINETs annual meeting.
Dr Davlinder Mandair is a trainee gastroenterologist who is currently undertaking a PhD under the supervision of Prof Tim Meyer, Prof Martin Caplin and Dr Chrissie Thirlwell at the UCL Cancer Institute in London.
The project builds on previous work conducted in Prof Meyer’s Lab which demonstrated, for the first time, that circulating tumour cells (CTCs) were detectable in the blood of patients with a range of NETs, and that their presence indicated an adverse prognosis. Technology has now advanced to the extent that detailed molecular characterisation can be conducted on single cells and this project will compare the genetic changes seen in primary tumour tissue with that of CTCs and also with cfDNA (pieces of DNA that arise from the tumour and can be found circulating in the blood stream).
If we can show that CTCs and cfDNA does indeed accurately represent the tumour, then we will be able to track how the tumour evolves over time and during treatment. This may allow clinicians treating patients with NETs to select the appropriate therapy and to anticipate the emergence of resistance. As cancer therapy becomes increasingly personalised, it is important that we can undertake this type of analysis so that patients can benefit fully from the new drugs that target specific cancer pathways.
Prof Ramage is a Consultant Physician in Gastroenterology and Hepatology, and lead clinician for the Neuroendocrine Tumour Service at King’s College Hospital NHS Foundation Trust.
The project studied the NETs that were identified in the Colorectal NET and the Bowel Cancer Screening Programme (BCSP). The aim was to understand how these were identified, how they were investigated and how they were treated.
The Bowel Cancer screening programme has screened 1 million people using faecal occult blood (FOB -blood in the stool) testing and 17,500 colonoscopies have been performed. Overall, 7.8% ( women) and 11.6% (men) of the colonoscopies detected a colorectal (CR) cancer. However the data regarding the NET tumours that were identified has not been analysed.
The incidence of CR NET in the general population is thought to be 1.3 per 100,000. The incidence in patients with positive FOB is unknown but CR cancer incidence is 46 per 100,000 in England and thus NET incidence might be 46/1.3 times less likely to be found than CR cancer. This would be 35.4 times less likely which would equate to 1772/ 35.4= 50 cases. It is also possible that some ileal carcinoids will have been found during colonoscopy, and these are twice as common as CR NET.
Professor Mark Prichard led a team aiming to understand the cause of genetic changes that result in neuroendocrine tumours of the stomach. Prof Prichard is Professor of Gastroenterology at the Institute of Translational Medicine, University of Liverpool.
Neuroendocrine tumours (NETs) of the stomach occur most commonly in patients who have pernicious anaemia. In this condition, patients can no longer produce stomach acid. This means that they cannot absorb vitamin B12 from their food and this leads to the development of anaemia.
Patients with pernicious anaemia also produce too much of a hormone called gastrin and in these individuals high levels of gastrin can usually be detected in a fasting blood sample. In people without pernicious anaemia, gastrin stimulates the stomach to produce acid. However, patients with pernicious anaemia cannot produce stomach acid, and therefore, gastrin stimulates another cell type in the stomach (called the ECL cell) instead. Gastrin makes these ECL cells divide and grow more quickly than normal and in some cases this can lead to the development of NETs.
If these so called ‘type I gastric NETs’ are small (less than 1cm in diameter), patients usually require no treatment. Doctors normally recommend that such tumours are regularly monitored by 1-2 yearly camera tests (endoscopies). However type I gastric NETs that measure more than 1-2cm in size may cause problems and they are therefore usually removed (by endoscopy or surgery).
We have recently conducted a clinical trial in eight patients with type I gastric NETs to see whether a new tablet called Netazepide can be used instead of surgery to make these tumours shrink. Netazepide is a gastrin receptor antagonist, meaning that it is a drug that blocks the effects of gastrin. We showed that netazepide caused some type I gastric NETS to shrink in size and importantly this medication caused no significant side effects. Further longer trials involving larger numbers of patients are needed to assess how effective this new treatment really is, but netazepide may turn out to be an effective alternative to the more invasive surgical treatment options for this type of tumour.
We plan to use the current grant funding from the NET patient foundation and TransNETS to analyse how the expression of several thousand genes changed in the stomachs of our eight trial patients during and after netazepide treatment. Biopsy samples are already available for these studies. The results of this research may eventually enable us to develop tests to monitor whether netazepide treatment is effective in an individual patient and may also help us to decide whether we should add in treatment with an additional second drug to make netazepide therapy more effective.