Analytical pathways for long-read sequencing in neuropathology

Thesis

Rare solid neoplasms of the craniospinal axis are difficult to diagnose due to tumour heterogeneity, and limited understanding of their molecular and genetic drivers has restricted the development of targeted therapies, resulting in reliance on surgery and radiotherapy. Consequently, the prospect of ‘precision oncology’ – defined as the right treatment for the right tumour in the right person at the right time – remains unfulfilled. This thesis explores how advances in long-read Oxford Nanopore Technologies (ONT) sequencing could achieve rapid histomolecular ‘precision diagnostics’ and help dissect the biology of a rare tumour called mesenchymal chondrosarcoma (MCS). The premise of the work is that tissue diagnostics and research in rare neoplasms must be integrated and barriers to access to cutting edge technology removed. This means that novel tissue pathways must be developed and validated in the research setting before they can be implemented as standard of care. Therefore, I set out to examine three related questions:

1. How can we optimally preserve tissue morphology and molecular integrity for rapid ONT diagnostics and research?

2. Can ONT sequencing provide molecular data that allows us to make an integrated histomolecular diagnosis in hours rather than weeks?

3. Can ONT resolve the fusion-gene architecture of MCS at bulk and single cell level to illuminate its biology?

I approached these questions with a combination of histological and emerging ONT wetlab and bioinformatics technologies.

1. I found that a novel approach to rapid tissue freezing without embedding medium or other toxic chemicals is superior to any other tissue preservation technique, as it does not induce any detrimental modifications and still preserves cytoarchitecture.

2. I determined that ONT sequencing of rare neoplasms from both biopsy and postmortem tissues could provide equivalent and, in some cases, superior molecular diagnostic data in a fraction of the time compared to standard-of-care testing.

3. I established pipelines for comparative DNA and RNA ONT long-read sequencing of MCS at bulk and single cell level which allowed me to identify and characterise the pathognomonic HEY1::NCOA2 fusion in its genomic and cellular context.

In conclusion, my thesis establishes the foundations for a novel, rapid, non-toxic and costeffective tissue pathway for near-patient precision oncology for people with rare neoplasms which is suitable for future implementation in the NHS. Further, my work contributes the first bulk and single cell ONT long-read dataset of MCS, an ultra-rare tumour whose biology remains poorly understood.

Authors: Brooks, H

• DOI: 10.5287/ora-kodbmbozj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Mesenchymal Chondrosarcoma; Genomics; Single-cell analysis; Oxford Nanopore Technology; Whole genome sequencing; Single-nuclei long-read sequencing; Molecular biology; Oxford Nanopore Seuqnecing; Neuro-oncology; Rapid brain tumour diagnostics; Long-read sequencing
• Subjects: Neurosciences; Brain--Tumors--Diagnosis; Single-nuclei long-read sequencing; Oncology; Whole genome sequencing; Molecular Biology; Neuropathology