Characterisation of a rare neuroendocrine tumour, olfactory neuroblastoma, to establish novel

Solomon Muna, Queen Mary University of London

 

Aims

Olfactory neuroblastoma (ONB) is rare but aggressive malignant neuroendocrine tumour, which arises from the nasal vault. It can secrete arginine vasopressin, ACTH or cathecholamines. Current treatments are mainly surgery and radiotherapy for early primary disease and chemotherapy for advanced and recurrent disease. However, these treatment modalities have limited effects and no disease-specific therapy exists.

The aim of our project is to identify potential targets for treatment as well as biomarkers for advanced disease. We have collected 65 ONB samples from 4 large UK centres specialising in ONB surgery. Ten of the freshly collected tumour samples (five high grade and five low grade samples according to the Hyams classification) and ten normal olfactory mucosa samples (controls) as well as two human ONB cell lines underwent RNA sequencing (RNAseq).

The aim of the current study is:

1. To perform in depth analyses of RNAseq data to identify genes/pathways specifically differentially expressed in ONB and in high versus low grade adenomas.
2. To validate selected genes by RT-qPCR followed by immunostaining on FFPE tumour samples, ONB cell lines and control tissues.

 

Evaluation

Significant progress has been made with:

• The analysis of the RNAseq data. Using clustering analysis as well as ingenuity pathway analysis, and based on published data, TENM1 and PRAME have been selected, initially, as molecules of interest for further analysis.
• Variant calling from RNAseq data. This is ongoing and variants found from the initial set of molecules (TP53, IDH2, ARID1A, MUC16, KMT2, NUMA1 and SMARC) are being correlated with published data.
• Validation of RNAseq expression of Teneurin Transmembrane protein 1 (TENM1) and preferentially expressed Antigene in Melanoma (PRAME) performed by qPCR. Confirming increased expression of both genes in high grade tumours. Overexpression and knock down experiments as well as migration and invasion assays are being performed. Validation of PRAME and TEMN1 expression in primary ONB cells and cell lines has been performed by immunocytochemistry.
• Tissue microarray (TMA) creation with a uniquely high number of cases of this relatively rare disease, which currently being used to validate the above findings at protein level by immunostaining for TENM1, PRAME as well as markers of vascular invasion and immune infiltration. 
• Assessment of our cohort for recently established novel categorisation of ONBs. 

The difficulties encountered include:

• The challenge of getting somatic mutation data from RNAseq analysis. Even with the help of bioinformaticians it has been an arduous process. 
• The challenge of working with ONB cell lines as they are difficult to maintain in culture, and viability can reduce significantly with any treatment. They are also difficult to transfect.
• The challenge of optimising experiments on fresh primary ONB tissue as these tumours have low incidence.
• Funding constraints limiting the type and number of experiments. 

 

Grant awarded: £1,966.85