Innovative Approaches for characterisation and treatment of Olfactory Neuroblastoma

Lead Research Organisation: Queen Mary, University of London
Department Name: William Harvey Research Institute

Abstract

Despite recent advances in cancer treatments, outcomes for patients with advanced olfactory neuroblastoma (ONB) remain poor. There is an increasing need to develop novel treatments as alternatives to conventional therapies. The current gold standard treatment is craniofacial surgery followed by radiotherapy and/or chemotherapy, but this is associated with significant long term side effects, particularly in young patients. Early stage disease responds well to treatment, but is associated with a high (60%) recurrence rate, while advanced and metastatic disease has a poor prognosis (5 year survival less than 25% in high grade disease; less than 5% with metastasis at diagnosis).

Cell signalling pathways (protein interaction cascades triggered by biological cues such as hormones) normally act to maintain cell health, but over activity of these pathways is reported in many cancers. Most pathways contain multiple feedback control loops and cross-react with other pathways, hence drugs designed to target a single pathway are usually met with rapid resistance. Two of these pathways 'mTOR/MAPK' and 'SHh" play a role in ONB; therefore combined targeted treatment against both simultaneously is a promising strategy

We are currently establishing specific drug combinations that are highly effective on cells in the lab, using measures of impaired cell health as our outcome. We are also using novel techniques to look at changes in gene expression in ONB, which might suggest alternative targets and prognostic biomarkers. Having established an effective treatment using cells, we will validate this on a mouse model, using bioluminescence techniques to measure tumour size and spread in response to treatment. Our ultimate goal is to establish a suitable treatment combination for human trial stage in ONB.

Technical Summary

Olfactory neuroblastoma (ONB), a rare sinonasal tumour, is advanced at diagnosis in a third of patients, in whom prognosis is poor (disease free survival 1.5y). Paediatric disease is frequently aggressive, and the current gold standard treatment (surgery plus chemoradiotherapy) results in significant long-term sequelae. ONB has a strikingly high recurrence rate of 60%, meaning that alternative treatment options and prognostic biomarkers are urgently required.

The sparse data available on ONB suggests that the mTOR/MAPK and SHh signalling pathways play a role in tumourigenesis. In cancers, cross-talk between pathways provides a mechanism of drug resistance and treatment failure when single agent inhibitors are used against many different tumours. Targeting both pathways therefore makes rational sense.

Aims: i) Establish an inhibitor combination that causes simultaneous pathway inhibition in ONB; ii) RNA seq to identify novel targets and biomarkers; iii) Validate findings on fresh tissue and iv) Reproduce effective treatment in a nude mouse model.

Objectives: Using human ONB cell lines, we have established that inhibitors to both pathways reduce cell viability in a dose and time dependant manner. Comparison of multiple treatment combinations suggests novel S6K1 inhibitors plus growth factor receptor inhibition with SHh inhibitors is highly promising. Further outcome measures of efficacy will include western blot, RT-qPCR, apoptosis and cell cycle analysis. RNA seq (n=4 samples, 4 controls) will examine gene expression data, and significant candidates will be validated in overexpression/siRNA cell line studies and on both fresh and fixed tissue samples. In vivo assessment of the optimised drug combination in nude mice will validate the efficacy and assess tolerability.

Establishment of a safe, effective, targeted, combined therapy could then be proposed for clinical trials, ultimately aiming to treat both primary and recurrent / inoperable disease.

Planned Impact

This project has the potential to impact on a number of different groups:

Patient benefits: Despite receiving current gold standard treatment (craniofacial resection plus radiotherapy plus chemotherapy in selected cases) survival rates for patients with advanced and metastatic disease remain poor. Even for patients who do undergo apparent successful surgical resection, this tumour is associated with significant recurrence rates, and many patients will eventually live with recurrent disease after a disease free interval. Since 33% of cases occur in patients under the age of 40, there is therefore a real need for treatment to:
- Form part of primary treatment with the aim of preventing future recurrence as an adjuvant treatment to surgical excision of the tumour (where possible).
- Prolong survival (and maintain quality of life for as long as possible) by slowing or stopping disease progression in advanced and metastatic disease.
We hope that this study will lead to future introduction of novel treatment options for patients with this recurring condition, and reduce the long term sequelae associated with current treatment options (which do not prevent recurrent disease in many cases). With this in mind, where possible we have chosen drugs already at stage III clinical trial, if not already in clinical practise (ie the safety and side effect profile is already established). This will reduce the time from bench to bedside and ensure that patients benefit directly from the results of this research with minimal delay.

Scientific / Medical community benefit: This work is also of interest to groups studying the same combination of signalling pathways in other tumours, and may suggest novel approaches for cancer where this treatment regimen has not been considered before.
- One of the novel aspects of this project is that we are working with S6K1 inhibitors, which despite great need are not yet in clinical use. Targeting this region has the potential to avoid some of the feedback loops associated with drug resistance to inhibitors that act higher up the signalling pathway. We hope to demonstrate the novel candidates we are working with are both safe and effective, with a view to ultimately establishing first in human trials. Since many cancers and other diseases exhibit aberrations in mTOR signalling, these drugs may be of future widespread importance.
This work is at an early stage, and for novel treatments 5-10 years of trials are needed before the drugs can enter mainstream use. However, aside from the S6K1 inhibitors, the drugs used in this project are already at stage 3 trials if not already in use for another clinical indication. They have established safety margins, and side effect profiles, and it would therefore be possible to instigate human trials of these drugs as treatment for ONB on a much faster timescale.

Commercial sector benefit: The drug development market is a highly competitive one, with some global pharmaceutical companies involved in developing similar agents. This project has potential to support the development of a drug by a UK based company, which may ultimately be used around the world.

NHS benefit: At present ONB patients require lifelong surveillance with annual MRI scan and outpatient endoscopy due to high recurrence rates. A treatment that reduced or removed this recurrence rate would therefore have an economic benefit to the NHS (and also to the patients quality of life).

Personal benefits: On a personal level this project is giving me the chance to improve my understanding of signalling pathways and the different techniques required to investigate them. It will give me a range of transferable skills, and allow me to establish a network of contacts to advise me in my future career as an academic surgeon. My ultimate aim is to translate basic science into therapeutic agents that can be used in the clinical ENT setting.

Publications

10 25 50
 
Title ONB mouse xenograft model 
Description In collaboration with Axis Bioservices we have established a nude mouse xenograft model on ONB, using a human immortalised OB cell lines. Xenograft growth characteristics and metastatic potential established. This will enable us to test promising drug combination (s) in vivo to confirm in vitro work and assess tolerability of combined doses. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact This model will enable us to test inhibitor combinations in vivo and assess tolerability and effectiveness. At present primary cell cultures of ONB are hard to establish, as are patient derived xenografts, and this provides an alternate system with which to study these tumours. 
 
Title Multi Centre ONB database 
Description I have established and continue to maintain a multicentre database of olfactory neuroblastoma patients, summarising their clinical history, treatment of their disease and outcomes to date. This will be compared to other published series to compare our outcomes, and we also aim to establish new guidelines for management of this disease based on the combined finding of this project. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact At present the management of ONB is very variable with different centres following site specific protocols. We aim to provide an evidence based schematic management plan for diagnosis and treatment of this disease aimed at stratifying patients by risk into different treatment algorithms to try and reduce recurrence and metastases rates. 
 
Title RNA seq data 
Description 10 samples 10 controls and 2 human cell lines have been submitted for RNA seq. This has generated the first expression profile data for these tumours, and the database will be interrogated to identify novel targets and disease biomarkers. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact In progress. 
 
Description Aintree Hospital 
Organisation Aintree University Hospital
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We will be using the tissue provided by this department in our research in several ways - including but not limited to RNA seq, primary cell culture, IHC. We will also be using anonymised patient data to generate a database of outcomes with ONB.
Collaborator Contribution Collaboration established to ensure that new cases of ONB will (if they agree) be recruited to our study and permission established to work on older tissue stored under the tissue bank ethics. The team are also helping to track down samples from the wider region.
Impact N/A
Start Year 2017
 
Description BCI collaboration 
Organisation Queen Mary University of London
Department Barts Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with team at BCI to work on attempting to establish PDX mouse models for ONB. Given rareity of this condition, has required training on other tumours to gain required skillset for ONB work.
Collaborator Contribution Provided expertise, advise and in house training and supervision.
Impact N/A
Start Year 2017
 
Description Dr Jenny Worthington Axis Bioservices LTD 
Organisation Axis Bio Services Ltd
Country United Kingdom 
Sector Private 
PI Contribution There is currently no existing animal model for ONB and there is currently a lack of guidelines as to treatment options for patients with recurrent and refractory disease. We have established effective inhibitor combinations in vitro but would like to move forwards and test these combinations in vivo. Initially we plan to do this using a nude mouse xenograft tumour model. We have established effective drug and dose combinations, using multiple outcome measures to assess efficacy. I have also obtained a personal licence to enable me to carry out the animal work required for this part of the project.
Collaborator Contribution Our partner has great experience in establishing tumour xenograft models and also already collaborated with Sentinel oncology working on their trial drugs which form part of this current study. They will be supervising me as I establish and maintain the mouse xenograft model, monitor response to treatment and harvest end organs for analysis.
Impact In process
Start Year 2016
 
Description Dr Owen Dando, Edinburgh University 
Organisation University of Edinburgh
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided novel dataset for analysis and have explored the different RNA seq data outputs generated by this collaboration
Collaborator Contribution Help with analysis of RNA seq data and advice and support regarding next steps with interpretation of data.
Impact N/A
Start Year 2016
 
Description GKT 
Organisation Guy's and St Thomas' NHS Foundation Trust
Country United Kingdom 
Sector Public 
PI Contribution Commencing sampling of tumours and retrospective collection of tissue from tumour bank and histopathology. Retrospective collection of anonymised patient data.
Collaborator Contribution Provision of samples, access to clinical data and on-going collaboration for prospective future tissue sampling.
Impact N/A
Start Year 2017
 
Description Liverpool collaboration 
Organisation The Walton Centre
Country United Kingdom 
Sector Hospitals 
PI Contribution We will be using the tissue provided by this department in our research in several ways - including but not limited to RNA seq, IHC, primary cell culture
Collaborator Contribution Collaboration established to ensure that new cases of ONB will (if they agree) be recruited to our study and permission established to work on older tissue stored under the tissue bank ethics. The team are also helping to track down samples from the wider region.
Impact Nil yet
Start Year 2015
 
Description Royal Marsden Collaboration 
Organisation Royal Marsden NHS Foundation Trust & Institute of Cancer Research
Country United Kingdom 
Sector Hospitals 
PI Contribution I have established an honorary contract with the trust and they are providing clinical data re treatment of patients undergoing surgery and or chemo / radiotherapy for ONB
Collaborator Contribution They have identified patients, helped consent and introduced our study to their departmental team
Impact Multi-disciplinary collaboration with ENT / H&N surgeons, Oncologists, H&N CNS
Start Year 2017
 
Description Terry Roberts, Brunel University 
Organisation Brunel University London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have established techniques for generating primary culture of olfactory neuroblastoma but want to try and generate new immortalised human ONB cell lines, as these tumours are very rare and we are often only able to sample a small amount of fresh tissue which does not give a good yield for experimental practise. In addition we want to luciferase tag these cell lines for future work in a nude mouse xenograft model. We are able to access fresh tumour tissue due to our existing collaborations with head and neck centres and continue to collect fresh material for primary culture whenever possible.
Collaborator Contribution The Brunel team have expertise in immortalisation of primary cells and luciferase tagging of cell lines and we will be working with them to try and achieve these outcomes with primary ONB cells.
Impact Currently in process. We aim to produce an immortalised human ONB cell line, characterise it and luciferase tag it as well as tag the pre-existing lines we currently work on.
Start Year 2017
 
Description UCL 
Organisation University College Hospital
PI Contribution Sharing of some rare samples and anonymised clinical data to increase overall sample size. Potential collaboration on cell culture and mouse model work.
Collaborator Contribution Sharing of some rare samples and anonymised clinical data to increase overall sample size. Potential collaboration on cell culture and mouse model work.
Impact N/A
Start Year 2017
 
Description Aintree presentation 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Summary of ONB situation in the UK, explanation of my project and summary of our clinical data so far, aiming at recruiting more units to join this study
Year(s) Of Engagement Activity 2017
 
Description Presentation at Liverpool Skull Base Meeting 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Thirty minute presentation covering molecular genetics of common skull base tumours, an introduction to ONB and presentation of interim outcome findings.
Year(s) Of Engagement Activity 2017
 
Description Project update - Walton Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact A review of the data we have generated so far and a discussion of future plans including:

Best ways to send samples to the local tumour bank plus collect additional tissue for research to maximise yield from these rare tumours.
Possible QoL review in these patients.
Contact made with new cases and also facilitated introduction to a new team also working on this tumour
Year(s) Of Engagement Activity 2016,2017
 
Description Project update, Imperial College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Study participants or study members
Results and Impact A meeting with key collaborators from the Imperial College team at Charing Cross to review database findings and discuss future plans. This included optimisation of fresh tissue retrieval from theatre with a view to generating an immortalised cell line in due course.

We also discussed ways to optimise tissue collection at the onsite tumour bank, plus sampling of additional specimens including adjacent normal samples in order to maximise sample sets from these rare tumours.

We have also discussed QoL issues surrounding these cases and how to best assess this retrospectively.
Year(s) Of Engagement Activity 2016,2017