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Researchers Validate ArcherDx Targeted Sequencing Approach for Sarcoma Gene Fusions

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NEW YORK (GenomeWeb) – Researchers at Leiden University Medical Center have used a genotyping assay and next-generation sequencing workflow developed by ArcherDx to detect gene fusions in sarcoma patients.

The team believes that the assay could eventually be developed into a multiplex diagnostic tool to diagnose rare forms of sarcoma.

Sarcomas are rare cancers that grow in a patient's connective tissue, such as bones, muscles, tendons, nerves, and fat. Symptoms may not be apparent, since the only signs of sarcomas are lumps on a person's skin.

In addition, it has been demonstrated that recurrent gene translocations are prevalent in these cancers. Although researchers currently use a variety of techniques to detect translocations, they often use fluorescence in situ hybridization (FISH) in routine diagnostic settings. Despite its proven uses, the tool's drawbacks include false-negative results when there are a high percentage of nontumor cells, atypical FISH signals, and the dependency of probe availability.

"Regarding sarcomas and gene fusions, if a patient has a particular gene fusion, it's easier to perform a diagnosis. However, it must be done in the background of histology and patient specifics," said Anne-Marie Cleton-Jansen, a study author and associate pathology professor at Leiden University Medical Center.

In a validation study published earlier this week in The Journal of Molecular Diagnostics, Cleton and her team used ArcherDx's anchored multiplex PCR (AMP) FusionPlex tool on 26 genes linked to sarcoma. They then compared the results to those obtained with FISH, RT-PCR, and specific immunohistochemistry.

The team collected 81 soft tissue or bone marrow samples — eight freshly frozen and 73 FFPE — that either had a fusion-positive or unknown translocation status. To detect gene translocations, they isolated DNA and RNA from the tissue samples, then prepared sequencing libraries of target-enriched cDNA using the FusionPlex Sarcoma kit. This involved performing reverse transcription and real-time quantitative PCR to determine sample quality, then conducting two rounds of low-cycle PCR with universal and gene-specific primers. The team then analyzed the sequencing data with ArcherDx's software to detect gene fusions. While the assay needs less than "10 nanograms of nucleic acid per sample and about 2.5 hours of hands-on time," the overall workflow requires about a week to produce results.

According to Cleton, the team used secondary molecular techniques on 58 out of the total 81 samples to directly compare with the FusionPlex platform.  The researchers used commercial FISH probes to detect translocations involving EWSR1, FUS, ALK, SS18, and USP6, COL1A1 and PDGFB. In addition, the team used TaqMan probes to detect the most common fusion transcripts in Ewing and synovial sarcomas. The team performed immunohistochemistry for CAMTA1, TFE3, STAT6, ALK, ETV4, and CCNB3 mutations.

Cleton and her team then directly compared the AMP-based targeted outcomes to results from the secondary molecular techniques.

The researchers saw that NGS yielded an average of 2.4 million read numbers per sample, with an average of 64 percent RNA reads. They found that 48 cases showed a fusion with AMP-based targeted NGS. In the remaining 33 cases, they did not find any gene fusion mutations.In addition, NGS results concorded with FISH or RT-PCR in 29 of the cases. They included eight cases of Ewing sarcomas, with seven demonstrating an EWSRI-FLI1 fusion. In situations with discrepant results, the team pursued further analysis with additional FISH probes and RT-PCR primers.

However, in 19 of the 48 fusion-positive cases, the team could not validate the NGS data using other molecular techniques. In 12 cases, the team could not evaluate aberrant protein expression that indicated a translocation.

In seven samples, the team could not produce a specific immunohistochemical evaluation. However, the researchers noted that these cases had histologic and immunohistochemical features that were characteristic for the tumors. The team therefore confirmed the proposed diagnoses by identifying other gene fusions, including HEY-NCOA2, JAZF1-SUZ12, PAX3-FOXO1, and HAS2-PLAG1.  

In two cases of nodular fasciitis (a form of benign soft tissue lesion), the team identified two novelfusion partners for USP6, including SEC31 and COL1A1. Using FISH, the team then confirmed a break in USP6 with 11 and 26 split signals for SEC31 and COL1A1 in 200 nuclei respectively.

"One of the great things about using the [FusionPlex Sarcoma] kit is that you only need to know one of the translocation partners," Cleton explained. "You can then identify the second partner by next-generation sequencing."

Because targeted NGS allows detection of fusions in RNA expression, the authors believe that the positive results may suggest functional importance in tumor development. However, the team could not find any correlation between the number of split-apart signals and fusion reads.

The authors noted that the AMP-based targeted NGS assay struggled to find gene fusions outside selectively captured regions on the genome. The team could not detect any specific fusion with ESWRI, despite demonstrating convincing ESWRI breakage with FISH probes.

In contrast, the team found that both RT-PCR and FISH were easy to operate, inexpensive, and had high clinical accuracy. When using RT-PCR to make DNA, however, Cleton said that researchers needed to ensure they had selected the correct translocation areas on the genome, as they might have to repeat the process if they identified the wrong fusion type.

In addition, Cleton pointed out that her team encountered a massive bottleneck while analyzing the sequencing results.  

"ArcherDx's software could only analyze one sample in the one to three hour cycle," Cleton explained.  She noted that the issue "delay[ed] the time to results for the extracted genetic samples."

The authors also pointed out that the AMP-based targeted NGS had a 14 percent failure rate of detecting gene fusions, potentially caused by "decalcification, [degradation], and variability in tissue and fixation conditions." They saw that "in poor quality samples, FISH may be successfully performed, whereas targeted NGS may fail."

However, the authors highlighted that "none of the molecular assays used in the study was able to provide a 100 percent certainty with respect to false-positive and false-negative results," indicating that each tool had its own major strengths and weaknesses.

"Before we had this technology — where we can look at a lot of different gene fusions in one experiment — we had to guess which probe to use to identify the right fusion, either with PCR or FISH, Cleton explained. "But with ArcherDx's tool, we can [test] all the important translocations in [a single] experiment."

ArcherDx VP of Assay Development and Scientific Operations Ryan Walters explained that the firm has been optimizing the assay to efficiently capture RNA molecules for sarcoma detection. While Cleton's team used Thermo Fisher's Ion Proton system in the validation study, ArcherDx's kit is also compatible with Illumina's MiSeqDx instrument.  

Walters added that ArcherDx is constantly adding new genes with help from Cleton's team to improve the assay's accuracy.

Cleton said that the assay cost her team about $800 to run a single cartridge on the FusionPlex platform, but she also noted that the final sequencing cost could be upwards of $1,500 due to additional factors. However, Cleton believes that the tool can act as the best resource to accurately guide downstream treatment for sarcoma patients.

However, Walters declined to comment on whether the firm plans to commercialize the assay for full diagnostic use, rather than maintaining its research-use-only status.

"We think that RNA is the best molecule to target for simple [sarcoma] detection, as opposed to a [complicated] DNA hybrid capture panel, or FISH," Walters explained. "This serial liquid transfer [process], from one tube to the next, makes it easy for labs to pick up and run [on samples]."

Walters said that the firm plans to further develop FusionPlex's customization and flexibility.

"If one hospital wants to combine sarcoma assays with a solid tumor assay, that's something we would be able to provide within a matter of weeks," he explained.

As it aims to improve the technology, ArcherDx is currently suing Qiagen over alleged infringement of a patent covering parts of the AMP-assay.

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