Variants of Uncertain Significance – RNA to the Rescue

Nothing has plagued genetic testing for cancer predisposition more than the Variant of Uncertain Significance (VUS). After 23 years since BRCA testing became commercially available, surveys continue to reveal misinformation surrounding the VUS and/or how often the VUS is explained to patients incorrectly. Consider, too, that patients are much more likely to harbor a VUS than a pathogenic variant (a.k.a. deleterious mutation), so it’s anything but a rare occurrence.

Then, just when we thought Myriad had worked out the majority of VUS issues surrounding BRCA-1 and BRCA-2 testing, Next Generation Sequencing hit the clinic in 2013, and we were doing multi-gene panels, with VUS rates as high as 40%. “Plan on it,” we began telling our patients.

Out of frustration that clinicians were not “getting it” when it came to the VUS, word was issued from on high to simplify things – that is, consider the VUS as a “negative” result. Or, “clinically negative.” Or, more realistically: “No clinically actionable results, so don’t change your plans with the proband and don’t test unaffected relatives. And, stay tuned for updates.”

But if you do enough testing, and if you get a little too comfy with the VUS, you’ll get burned by thinking they’re all going to be negative. Yes, the vast majority of updates later on from the testing laboratory will be downgrades to benign polymorphisms, but every now and then, you’ll get an upgrade to “likely pathogenic” or “pathogenic” status.

It’s easier to dismiss the VUS when it occurs in one of those genes that barely made the A-list in the first place. When dealing with these “lesser” genes, it’s not unusual for unaffected patients to carry a higher risk for breast cancer using standard risk models than the risk imparted by “gene-positivity.”

But what about the VUS that occurs in a modest-risk or high-risk gene? This is where I rely on a genetics team that analyzes every VUS. With a medical geneticist (Marsha Pratt, MD), board-certified genetic counselor (Kate Small, MS, LCGC), and nurse practitioner (Jennifer Lee, APRN-CNP), our policy is to review the evidence for calling a variant as having uncertain significance when it involves a major gene. And what an eye-opener it is when we go to one of the various resources like ClinVar and discover that most labs call the same variant “likely pathogenic” instead of a VUS. Indeed, it is not unusual for 10 different sources to be completely split – half calling a VUS, while the other half call the same variant “likely pathogenic.” Determining whether a variant is significant or not is exceedingly complex, and it’s not only the patient who is often bewildered but also the testing lab and clinical staff.

New development – Another layer is being added to the process of working out the clinical impact of the VUS – RNA sequencing. By analyzing (post-transcription) RNA, one can more accurately separate the wheat from the chaff. More assurance will be offered that today’s VUS can be managed as a “negative” (you likely won’t know that RNA made the difference unless you read the fine print). That said, some patients currently being labeled as having a VUS will be classified as “likely pathogenic” or “pathogenic.” RNA testing works both ways – today’s VUS could be tomorrow’s deleterious mutation.

So, what about the thousands of patients we’ve tested over the years? Will we need to go back and re-test everyone who had a VUS? We did it when BART was introduced for rare rearrangements in the BRCA genes, then again with multi-gene panels, so that many of my long-term patients have undergone 3 genetic tests over the years.

As for the impact of RNA testing, we’ll need to watch for some guidelines here when it comes to those patients who completed testing in the past. It might be that only certain VUS results in the more powerful genes will need to be re-analyzed. Will we be rummaging through 23 years of records? What about those patients with a VUS that needs re-evaluation who have been lost to follow-up?

In the meantime, it is unsettling to see how many of the VUS cases are actually pathogenic upon RNA testing, after we’ve been telling patients for years that: “Your result shows a variant of uncertain significance, which is essentially negative, from a practical standpoint, that is, I mean, it could be altered in the future, but for now, well, we don’t do anything differently, and your kids or siblings do not need to be tested, so you can rest easy and just because I’m rambling doesn’t make the VUS any more suspicious than we think it is, which is, of course…essentially negative.”

Myriad Genetics has been chipping away at the VUS problem in the two BRCA genes since Day One in 1996. So, when Next Gen Sequencing (and a Supreme Court decision) opened the door to testing multiple genes in multiple labs, everyone pooled their data in order to address the VUS rate in the many genes being tested. Myriad was not so pleased about 20 years of labor being tossed out the window into the laps of their competitors, so they held their VUS cards close to the chest and did not share their massive database for BRCA1 and BRCA2. But the other labs began pooling BRCA data right away, culminating in the “BRCA Exchange,” announced January 2019 and partly funded by the NCI. The BRCA exchange is composed of information from existing databases (Breast Information Core, ClinVar, and the Leiden Open Variation Database), as well as population databases from clinicians, clinical laboratories, and researchers worldwide. At last count, the BRCA Exchange addressed 20,000 unique variants in comparison to Myriad’s claim of data on 17,000 variants. I’m not sure anyone knows to what degree there is overlap.

Despite all this refining effort, our geneticist, Dr. Marsha Pratt, presented a case a few weeks ago where a BRCA-2 VUS (confirmed as such by all sources) tracked perfectly with early-onset breast cancers and ovarian cancers in a family. Ordinarily, one would have stopped testing the family after the first affected patient tested “negative” in the form of a VUS. But Dr. Pratt was very much aware of the vagaries of labeling a VUS, so she tested another affected family member, then another and another, while at the same time, testing the older family members who had escaped a diagnosis of cancer. The so-called VUS tracked perfectly with the ovarian and early-onset breast cancer patients. After she notified the testing laboratory, this rare VUS was shifted immediately into the “pathogenic” classification, based on this one family. Ergo, the VUS problem is alive and well, 23 years after the start date for commercial testing, even in the “old” BRCA genes. Before this family came to see us, patients with this variant were called VUS and treated as “negative.” From now on, the identical variant will be called “likely pathogenic,” a world of difference.

In light of cases like this, what about the longstanding effort to train clinicians to stop over-reacting to the VUS designation, to the point that many of us can become comfortably dismissive? I don’t know the answer. All I can say is the VUS is a lurking enemy that still must be dealt with, and it’s one of the reasons I prefer working with a team of genetics specialists. (Another reason is that this team handles all tumor predispositions, not just breast cancer.) RNA sequencing won’t get rid of the VUS problem entirely, but it’s a huge step in the right direction.

POINT OF POSSIBLE INTEREST: What’s a former breast surgeon doing in this genetics space anyway? Well, the groundwork was laid when I witnessed a BRCA-positive disclosure in the research setting of linkage analysis circa 1991. I was struck by the fact that the young woman’s questions were 100% breast-related (screening, preventive mastectomies, reconstruction), and the PhD geneticist could not provide answers. I realized at that moment that genetic testing for adult-onset cancers was going to be another situation where interdisciplinary care would be critical.

Then, in 1994, two years before genetic testing began, I served as host to visiting professor Mark Skolnick, PhD who had just sequenced the BRCA-1 gene in an exciting (and controversial) race involving 12 different teams. Coverage in newspapers and magazines (TIME pic below) had generated celebrity status for Dr. Skolnick and hope for all. For all we knew at the time, the BRCA-1 gene might explain the majority of breast cancers, with or without family history (turns out, it did not).

Will Rogers -- 1995 -- Skolnick visits

During his 2-day stay in OKC, he discussed the new company he was starting called Myriad Genetics, predicting that someday testing would be so commonplace that it would be part of routine primary care. It seemed to me that clinicians were going to have to follow developments here very closely, and I was skeptical that genetic testing would become simplified to the point that it would be “just another blood test.”

At the time, I held an endowed chair at my alma mater, and I requested the formation of a task force to study the implications of genetic-based medicine. There was no response from the university hierarchy (all the way to the top), so I obtained a $100K grant from the local Presbyterian Health Foundation that allowed me to send a nurse to Fox Chase for special training as well as meeting all the requirements of excellence in genetic testing as outlined by several genetics organizations and the American Cancer Society.

When Day One came in 1996, launching this new era in medicine, I drew first blood as the only physician in Oklahoma listed on the Good Housekeeping Seal of Approval that was published by the American Cancer Society. The research geneticist, a PhD in Tulsa, was also on the list, so there were two approved sites in Oklahoma for BRCA testing. Those early years were wild and woolly (anonymous testing, high level of fear and distrust, etc.), but to watch the progress in this arena has been a rewarding experience.

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