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MGH Details Method for Targeted RNA-seq Fusion and Snapshot NGS Assays


NEW YORK (GenomeWeb) – Researchers from Massachusetts General Hospital have described a method behind two clinical cancer assays — a targeted RNA-seq fusion assay and a hotspot mutation assay — each of which the team has now used to assess more than 1,000 patients.

The method for both assays relies on anchored multiplex PCR (AMP) coupled with next-generation sequencing and was described recently in Nature Medicine.

Long Le, an assistant professor of pathology at MGH, told GenomeWeb that Enzymatics has commercialized the AMP technology. MGH had exclusively licensed the IP to startup ArcherDx, which Enzymatics subsequently purchased for $50 million last year.

MGH currently uses the AMP technology in an RNA-seq clinical assay that looks at fusions involving ALK, RET, and ROS1 in all its lung cancer patients, Le said.

In addition, Le said, MGH has also now converted a routine cancer genotyping assay known as Snapshot from a genotyping capillary electrophoresis assay that evaluated 90 hotspot mutations to a NGS-based assay using the AMP technology. As of April, all incoming cancer patients to MGH receive the Snapshot NGS test, Le said.

The key to detecting gene fusions with the AMP protocol, Le said, is the use of one anchored end for targeting gene specific primers while allowing the other end to be amplified using a common adapter primer, which allows fusions involving a particular gene to be targeted with no prior knowledge of its partner gene, he said.

For its fusion panel, MGH evaluated rearrangements that involved ALK, RET, and ROS1, which are known targets that predict response to the drug crizotinib in lung cancer patients.

In the study, the group also evaluated a larger rearrangement panel that included 14 additional genes and tested it on 115 glioblastoma samples, identifying two novel fusions that were confirmed.

They also found five novel fusions with potential therapeutic implications in lung cancer samples, one in a cholangiocarcinoma sample, and one in a thyroid carcinoma.

Le said that although anchored PCR is not a new technique, what is different about the lab's protocol is that it is enhanced to achieve high levels of multiplexing and specificity.

MGH performs its clinical sequencing assays on the Illumina MiSeq system, but Le said the protocol would also be amenable to sequencing on other instruments such as Thermo Fisher's Ion Torrent PGM.

In the study, the team demonstrated multiplexing of more than 300 amplicons per tube, but Le said that in the lab, the researchers have demonstrated they can multiplex 2,000 targets in one reaction. "We haven't determined the ceiling," he said.

AMP involves a two-step process, Le said. In an initial PCR first reaction, a gene-specific primer and a common adapter primer are used, Le said. It's "not like regular PCR where you are flanking two sides of a target," he said. Rather, "you are anchoring one known targeted end, but the other is random." If the researchers stopped after that first round of PCR, specificity would be about 50 percent, Le said. A second round of PCR uses a gene-specific primer that is 3' downstream of the first gene-specific primer along with a common adapter primer. The combination of the two PCR reactions is essentially a nested PCR approach, Le explained, and drives specificity to 90 percent, which is on par with hybrid capture.

Tagging the second gene-specific primer with NGS adapter sequences also "functionalizes" the library for next-gen sequencing, Le said.

Turnaround time for library construction is just over one day, and total turnaround time for sample to answer is about three days using the Illumina MiSeq sequencer.

In the Nature Medicine study, the team validated the fusion assay on 319 formalin-fixed paraffin-embedded samples showing 100 percent sensitivity and specificity compared to reference assays. Looking at 986 clinical samples, the team showed it could be used as a discovery tool and identified novel gene fusions: RHGEF2-NTRK1 and CHTOP-NTRK1 in glioblastoma; MSN-ROS1, TRIM4-BRAF, VAMP2-NRG1, TPM3-NTRK1, and RUFY2-RET in lung cancer; FGFR2-CREB5 in cholangiocarcinoma; and PPL-NTRK1 in thyroid carcinoma.

In addition, they also showed that the technology could be used to identify point mutations and evaluated assay performance of both the 96-amplicon hotspot Snapshot assay and also a 626-amplicon assay that analyzed the coding regions of 18 tumor suppressor genes.

Looking at previously genotyped samples, the Snapshot NGS assay identified all expected SNVs, insertions, and deletions. Next, they evaluated the AMP technology using a 168-target cancer panel on 63 samples and demonstrated concordance with genotyping.

Moving forward, Le said the lab is currently validating two of the kits Enzymatics has commercialized for clinical use — a fusion panel for hematological malignancies and a sarcoma fusion panel.

In addition, he said the lab would like to expand on its current fusion panel and also on its Snapshot NGS panel to include targets that have been reported in the last year.