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Adding RNA-Seq, Ambry Genetics Expects to Reduce VUS Results in Hereditary Cancer Tests

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NEW YORK (GenomeWeb) – Ambry Genetics said this week that it is adding a new feature to its hereditary cancer risk testing menu: a service called +RNAinsight, which adds RNA sequencing of a group of target genes to the company's existing DNA sequencing panel.

The new offering doesn’t change the cost or turnaround time, which is the same as for the company’s DNA-only testing, said Brigette Davis, Ambry's senior VP of research and development.  

For patients whose physicians order +RNAinsight alongside the company’s 34-gene CancerNext DNA panel — or with smaller custom panels if requested — Ambry will now perform parallel RNA testing of 18 genes, chosen based on their propensity to harbor the types of splice variants that RNA can either uncover or help better classify.

The fact that RNA can provide evidence to help classify uncertain genetic variants, or pick up cryptic splice sites that DNA testing would miss altogether, is not a brand new discovery. But implementing RNA testing alongside a DNA panel is a significant shift in clinical practice, and one which Ambry believes could change how many definitive variant calls it is able to return to patients. 

Where RNA aids in variant detection and classification is mainly for splice site variants, Davis explained. DNA variant classification methods can only define or exclude variants as being pathogenic if they fit into a narrow subset. Anything outside of this requires additional evidence to interpret whether the variant has a negative biological effect.

In the past, making these calls for novel variants has certainly been possible, either using RNA assays or other functional study methods, but this has only happened retrospectively and relatively infrequently and inconsistently. In the meantime, the patient obtains a result listing a variant of unknown significance, or VUS.

The move to add RNA sequencing comes from Ambry’s experience doing post-hoc retrospective variant reclassification, which Davis said has been ongoing for about three years. Earlier this year, for example, company researchers shared a study at the American College of Medical Genetics' annual meeting exploring the effects of re-analyzing hereditary cancer variants with the help of supplemental RNA data — including consequences for the medical management of patient or their family members.

In a poster, investigators reported their assessment of 64 suspected splice site variants across 13 hereditary cancer genes, which were identified in clinical patient DNA tests.

Whereas a majority of the variants were considered VUS based on the DNA sequencing data alone, follow-on RNA analysis resulted in a more definitive resolution of nearly 90 percent, depending on the specific genes in question. 

When the group surveyed healthcare providers to find out if and how these reclassifications might affect patients and their families, the responses described profound, significant changes such as referrals to cascade testing if a variant was reclassified as likely pathogenic, or the ability for patients to avoid unnecessary screening and follow up if evidence pushed a classification to likely benign.

The first step in moving to RNA analysis alongside DNA assays was to figure out which genes to include. "We looked at any published literature or reported alterations … that have the potential to be impacted by an RNA assessment, and we [landed on] 18 genes that either had a significant number of variants previously reported or had biology where RNA would be impactful," Davis said.

Also crucial in the company's validation work was setting a definitive cutoff for RNA findings that are conclusive enough to produce a clear classification. "We had to understand how these 18 genes are actually being spliced among a large set of healthy controls [because] there is a lot of RNA splicing variability that happens in the background naturally that isn’t necessarily pathogenic," Davis said. "So we needed to [define] the signal to noise … to know which calls really should be used as evidence toward pathogenicity."

To do this, Ambry tested "several thousand patients," as part of a pilot program and was able to identify a "very nice background threshold for when we can actually make these calls … [when] it is a significant enough splicing impact to use as ACMG-weighted evidence," Davis added. 

Anything under that threshold has to be reported as a VUS. But above it, provided all the other criteria for ACMG classification are in order, the company can classify the variant as pathogenic. A description of the company's validation work is awaiting publication.

The launch this week makes Ambry the first to bring RNA into the hereditary cancer testing process in this way. However, retrospective RNA sequencing, similar to what the company reported at ACMG, has become increasingly popular in the research sphere and is also becoming part of follow-up clinical VUS resolution efforts by some other commercial firms, signaling that some could follow suit and similarly transition to parallel RNA and DNA analyses.

Invitae, for example, stated earlier this year that it has incorporated supplemental RNA testing as part of its "VUS resolution program," analyzing VUS predicted to affect splicing that are identified in any of the company's DNA-based hereditary cancer panels. 

Not every individual seeking a genetic test for hereditary cancer would benefit from RNA sequencing, though. This is both because many variants can still be classified using DNA alone and because the Ambry panel only includes RNA analysis of 18 genes.

But in the ACMG presentation this spring, investigators estimated that roughly one in 50 people receiving DNA testing for hereditary cancer conditions might benefit from RNA-based variant classification.

Announcing its new product this week, Ambry further estimated that +RNAinsight as currently designed could be helpful for about 16,000 of the 700,000 people in the United States who are expected to turn to clinical labs for hereditary cancer testing in a year.

And because of the way this type of genetic testing works, Ambry’s implementation of +RNAinsight won’t just serve new patients but will also impact people who have already been tested, either by Ambry or by other hereditary cancer genetics firms.

During its pilot period, Ambry didn’t contribute any new classifications to the ClinVar shared variant database. But it did update results in its own internal database, recontacting previously tested patients when appropriate. One particular variant classified using RNA during the pilot period, Davis said, has now been updated for 40 other Ambry customers.

Moving forward, the firm will be regularly adding new findings that come from its commercial +RNAinsight test to ClinVar. As a result, individuals tested by other companies may in the future also have VUS variants reclassified as pathogenic or non-pathogenic.

Based on the various frequency projections, and on the pilot program thus far, Davis said it’s apparent that by running RNA sequencing alongside DNA testing commercially, Ambry will likely find itself reclassifying greater numbers of previously-reported VUS, and thus needing to update more patient reports than in past years. She said this shouldn’t impact policies for how the company performs these updates, though.

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