The potential of nanopore cell-free RNA sequencing for earlier cancer detection

Presenting a plenary at London Calling 2024, Daniel Kim (University of California, Santa Cruz, USA) set the stage for how his lab is advancing RNA liquid biopsy technology for potential early cancer detection. He explained how a single millilitre of human blood contains billions of extracellular vesicles (EVs) secreted by cells from across the body and encapsulating the most basic building blocks of life — proteins, lipids, RNA and DNA. Daniel and his colleagues are utilising nanopore sequencing for EV-protected cell-free RNA (cfRNA) liquid biopsy research to investigate the ‘vast RNA landscape’ and its potential in earlier disease diagnosis, treatment, and monitoring. He highlighted the power of these minimally invasive systemic methods to reveal a wealth of data for complex diseases, such as cancer, that would otherwise require multiple and invasive tissue biopsies.

Daniel explained that ‘existing DNA-based methods could potentially miss the earliest stages of cancer’. He emphasised that early diagnosis of cancer could enable earlier treatments and eventually lead to decreased mortality. While 75% of the human genome is transcribed, surprisingly only 1–2% of the human genome encodes for protein-coding transcripts; thus, the vast majority of transcribed RNA sequences are non-coding. It is this non-coding majority — the ‘RNA dark matter of the genome’ — that Daniel and his colleagues are leveraging for early cancer detection research.

Mutations in the RAS family of genes are frequent drivers of human cancers. In an in vitro study, Daniel and his team observed that introduction of a mutant KRAS gene into a lung cell line led to upregulation of non-coding RNAs originating from repeat elements, which are then preferentially secreted in EVs. This finding led them to investigate whether this phenomenon could also be observed in human cancer research samples.

Daniel introduced their COMPLETE-seq RNA liquid biopsy workflow, enabling ‘comprehensive profiling of the cell-free RNA transcriptome’. In this research workflow, cfRNA is isolated from EVs in blood and sequenced using long nanopore reads and a short-read technology. Harnessing the repeat element data generated by the workflow and a machine learning model, they were able to identify ‘many more features with which to potentially diagnose disease’; this disease classification model ‘works a lot better than just looking at … protein-coding genes’.

The team used full-length cDNA nanopore sequencing on a MinION device to characterise cfRNAs from pancreatic cancer and control plasma research samples. The results challenged the dogma that cfRNAs are short and degraded: the long nanopore reads revealed the presence of full-length, polyadenylated, capped RNA molecules spanning up to 1 kb in length and included long non-coding RNA (lncRNA). To Daniel’s knowledge, this data represented ‘the first time we’ve really been able to identify the true length of these cell-free RNAs’. Further analysis of the data revealed novel transcripts that were specific to the pancreatic cancer research samples. Daniel described how he and his team are ‘really excited about this novel RNA isoform discovery that’s enabled by this platform’, and which ultimately may represent biomarkers that could be leveraged in the future for early diagnosis of disease.

The team also used the workflow to study liver and oesophageal cancer. Daniel described how each cancer had ‘their own distinct cell-free RNA signatures’, with much originating from non-coding and repeat RNAs. Addition of this cfRNA nanopore data to their machine learning model resulted in ‘much better performance’ than when only using data from protein-coding regions. Notably, a differential cfRNA signature was especially pronounced in oesophageal cancer research samples, so the researchers decided to further investigate the potential of nanopore technology for early detection of this disease. Daniel noted the poor five-year survival rate of just 6% associated with late-stage detection of oesophageal cancer¹, further underlining the importance of their continued studies.

They used a PromethION 48 to sequence cfRNAs in research samples from individuals with oesophageal cancer, the pre-malignant condition Barrett’s oesophagus, and controls. The high-depth sequencing revealed ‘about 287,000 novel, unannotated cell-free RNA transcripts’. Had they looked only at protein-coding regions, Daniel explained, they would have only found around 10,000 transcripts. He emphasised the potential of this novel data to enable ‘better, early detection of … not only Barrett’s oesophagus but also oesophageal cancer’. Furthermore, the transcripts from the cancer research samples were significantly shorter than the controls, suggesting that fragmentomics-based methods could hold promise in the future.

Finally, Daniel presented how the team leveraged oesophageal cancer sequencing data from The Cancer Genome Atlas Program (TCGA) and normal/healthy samples in the large GTEX Consortium cohort to probe for cancer-specific oesophageal RNA signatures. This revealed ‘hundreds of these repeat-derived transcripts that are significantly and specifically enriched in oesophageal cancer’. Using long nanopore reads, they were able to gain isoform-level information. In one example of a ‘highly, specifically upregulated’ lncRNA, Daniel showed how the nanopore data revealed three different isoforms of the transcript in a single oesophageal cancer research sample — ‘we’re able to now see the true nature of these cell-free RNAs’.

‘We’re really excited about the tremendous potential for novel, cell-free RNA biomarker discovery, especially RNAs that are highly specific to a given disease’

Concluding his talk, Daniel emphasised the future potential of nanopore cDNA sequencing as the ‘only way’ to gain such a systemic view of health and disease. As well as their research with the PromethION enabling ‘very deep profiling of these plasma samples to really get a better understanding of just what’s happening in the context of disease’, Daniel highlighted the ‘potential of using [the] handheld MinION device to provide more equitable access in … remote or resource-limited settings where we could potentially bring this … cutting edge, potentially life-saving RNA liquid biopsy technology to individuals around the world’.