RNA-Seq leads to prognosis predictions in rare bone cancer

Although previous studies have identified candidate prognostic or therapeutic targets using Next Generation Sequencing (NGS) technologies, this is the first report describing RNA expression profiling of patient-derived paired ES primary cell cultures and ES-CSCs.

For around one in three patients diagnosed with Ewing sarcoma in just one part of the body – known as localised cancer – current treatments are ineffective. The new research, funded by the Bone Cancer Research Trust and the Ewing’s Sarcoma Research Trust, opens up the possibility of offering these patients additional treatment that could increase their chance of survival.

The research team from the University of Leeds were able to identify the particular cancer cells – known as cancer stem-like cells – in Ewing sarcoma that are resistant to chemotherapy and responsible for recurrence of the disease. In the laboratory, the researchers found a protein called neurexin-1 at much higher levels in the cancer stem-like cells.

They then looked at survival data. Patients whose tumours had higher levels of this protein were more likely to see the disease return and less likely to survive.

In Ewing sarcoma, chemotherapy, radiotherapy and surgery increase long term survival in between 55 and 65% of patients who are diagnosed with localised cancer. However, the other patients see their disease progress and suffer a relapse. For these patients, the outcomes are as poor as those diagnosed when the cancer has already spread. 

Lead researcher Sue Burchill, Professor of Cancer Research in the School of Medicine, believes these patients could benefit from additional treatment.

She said: “The initial clinical impact from this research is the ability to identify those patients with localised disease who experience the poor outcomes normally associated with patients who have disease in multiple parts of the body. If doctors can use high levels of neurexin-1 to identify these patients at an early stage and treat them with a stronger combination of drugs, this could increase survival for some patients.”

Working with surgeons from the Royal Orthopaedic Hospital in Birmingham and the Translational and Clinical Research Institute at the University of Newcastle, the team were able to use samples of tumour tissue from Ewing sarcoma patients and grow the cells in the laboratory.

Dangerous cells

Dr Elizabeth Roundhill, Research Fellow in Leeds’ School of Medicine, explained: “Tumours are a mix of different cells which interact with each other and with cells of the surrounding environment to grow and spread. However, cancer stem-like cells are different: they can divide and multiply independently of other cells in the tumour and can initiate new tumour spread and growth. These are the particularly dangerous cells we wanted to find.”

From each sample, the team took approximately 1,000 single cells to identify those that were able to grow and were resistant to current chemotherapy. Using state-of-the-art RNA sequencing, the researchers were able to see that neurexin-1 was expressed at high levels in these cancer stem-like cells.

Highly expressed cell surface proteins and validation of differentially expressed mRNAs between ES and ES-CSCs

Fig. 2

(A) Interactions between proteins predicted to have high levels of cell surface expression in patient-derived ES cells and ES-CSCs and low expression in normal tissues (www.humanproteinatlas.org), generated using the protein-protein interaction tool STRING database (http://string-db.org) and visualised using Cytoscape v3.7.1 (www.cytoscape.org). (B) Butterfly plot of mRNAs with signficant (adjusted p < 0.001) differential expression in patient-derived parental ES and ES-CSCs. Black circle = all differentially expressed genes with an adjusted p value < 0.001. Dotted line = 2 fold increase (fold change of > 2) or 2 fold decrease (fold change of < 2) in mRNA level in ES-CSCs compared to ES cell cultures. Target mRNAs identified for validation by RT-qPCR are labelled; blue and red circles. Grey circle = currently unannotated genes. Quantification of target mRNAs using RT-qPCR in patient-derived primary ES and ES-CSCs; (C) neurexin-1 (NRXN1), (D) ELFN2, (E) SLC38A11, (F) CCDC190, (G) TLR4 and (H) PIEZO2. Median gene expression is reported as 2-ΔΔCt (results shown as median ± SD). RNA expression was compared between populations using a non-parametric Mann-Whitney two-tailed t-test

The team are already working with colleagues in Spain, Italy and Switzerland on a large study comparing neurexin-1 with other possible markers, to validate the best way to identify patients who need treatment that is more intensive. They hope to have the results of this study later this year. If the study confirms their findings, the next stage would be to use neurexin-1 to select patients for treatment in a clinical trial.

Dr Zoe Davison, Head of Research, Information and Support at the Bone Cancer Research Trust, said: “This research marks an incredibly significant find in understanding and predicting which patients may not respond to the standard chemotherapy treatment for Ewing sarcoma. By understanding this, it may lead to new and more effective ways to treat these patients. We are delighted that we have been able to fund this vital work on multiple occasions, which demonstrates the importance of consistent funding to enable research to progress and succeed.”

SourceUniversity of Leeds

Roundhill EA, Chicon-Bosch M, Jeys L, Parry M, Rankin KS, Droop A, Burchill SA. (2021) RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma. Cell Oncol (Dordr) [Epub ahead of print]. [article]

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