William Jeck：纳米孔测序和快速融合测试 - 分子病理学中的“杀手级应用”
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William Jeck, of the Massachusetts General Hospital Department of Pathology, described his team’s use of nanopore sequencing as a ”killer app” for molecular pathology: the rapid detection of oncogenic gene fusions in clinical samples.
William demonstrated the challenges of quickly diagnosing and treating acute leukemia and sarcoma by outlining a clinical case for each. The first was of a man who was diagnosed with acute promyelocytic leukemia following cytogenic testing, 48 hours after referral to the emergency room; by this time, a treatment which specifically targets the PML-RARA translocation responsible for acute promyelocytic leukemia had been pre-emptively started. William asked: can we identify the presence or absence of the PML-RARA translocation faster than this? This would enable more rapid diagnosis-to-treatment, and prevent time wasted on an incorrect treatment. The second case was that of a man whose diagnosis of atypical Ewing sarcoma took 16 days and several methods of testing: immunohistochemistry analysis (identified unclassified round cell sarcoma, but did not allow a more precise diagnosis), targeted testing for CIC translocation (returned negative after one week) and EWSR break-apart FISH analysis (positive for rearrangement). William asked: can we detect soft tissue-related fusions in the same timeframe as immunohistochemistry testing?
William highlighted how “Nanopore sequencing fits perfectly with the needs of clinical fusion detection”: the MinION fits easily into hospital laboratories which are stretched for space, where real-time sequencing has potential to provide rapid diagnosis at a cost within the range for a clinical test. He noted that the long-read output “covers fusion breakpoints… and then some”, with throughput generating more than enough coverage for fusion calling.
The team developed a pipeline to test for gene fusions in clinical samples using Anchored Multiplex PCR (AMP) and nanopore sequencing. RNA was extracted, reverse transcribed and ligated to adapters; this was then amplified via AMP, using a nested PCR with both adapter-specific and gene-specific primers to enrich for the regions of interest. The enriched samples were then amplified with Oxford Nanopore’s barcoded primers and sequenced in multiplex on the MinION device. Reads were aligned via BWA-MEM to a synthetic transcript reference library constructed from existing transcript annotations (William noted here that he would like to use minimap2 in future); the resulting dataset allowed for both the identification and quantification of fusions. The pipeline was first used to sequence an erythroleukemia cell line, in which it successfully identified a BCR-ABL1 fusion, with fusion reads being generated within seconds of starting sequencing – William described how he “stopped the run within ten minutes because I knew we had a success” and was then able to reuse the Flow Cell. He demonstrated how the long nanopore reads resolved long-range exon structure across the fusion.
The workflow was then put to the test on clinical samples: a PML-RARA fusion was identified in a leukemia patient sample, whilst a HAS2-PLAG1 fusion was identified in an FFPE lipoblastoma patient specimen. A blinded test of the fusion-calling workflow was then performed, using 11 fresh hematologic specimens and 5 FFPE sarcoma specimens. From these, 12 true positives and 3 true negatives were identified. Following four false-negative calls, with two due to the low fraction of tumor DNA present in a qPCR validation sample used (representing less than would generally be seen in a clinical sample), the team then investigated the sensitivity of the test. Samples containing a varying fraction of tumor DNA were sequenced; fusion genes were identified in samples containing down to a fraction of 5% tumour DNA.
William concluded that nanopore sequencing of AMP-prepared libraries successfully identifies oncogenic gene fusions, rapidly and with high sensitivity, in both fresh and FFPE samples; he highlighted that this demonstrates the technology’s potential for use as a rapid clinical diagnostic test, given suitable validation for this purpose. Lastly, William revealed that he had recently sequenced, to his knowledge, the first clinical sample to be run on a Flongle Flow Cell; he was able to identify an ELBR-FLI1 gene fusion, showing its potential as a single-sample, affordable diagnostic tool.