Robin Huiras (00:01):

Hello. Welcome to today's installment of “Diagnostics and Practice,” where we discuss the latest diagnostic offerings for Mayo Clinic Labs. I'm Robin Huiras, a senior marketing specialist with Mayo Clinic Labs and strong proponent of advanced laboratory testing to improve patient’s lives. I'm really excited to be here today with our two guests to talk about Mayo Clinic Laboratories’ new innovative whole transcriptome RNA sequencing assay. This is a robust test that looks for gene fusions in solid tumors, including central nervous system tumors and sarcomas, in order to help establish the cancer's molecular profile. Dr. Kevin Halling and Dr. Hussam Al Kateb are both co-directors of the Clinical Molecular Genomics Laboratory in the Division of Laboratory Genetics and Mayo Clinic's Department of Laboratory Medicine and Pathology. Thanks to you both for joining us today.

Kevin Halling (00:53):

Thanks. Thank you. Glad to be here.

Robin Huiras (00:55):

Now, before we jump into today's conversation, I'm hoping you can share with our listeners a little bit about your experience with molecular genetic testing at Mayo Clinic.

Kevin Halling (01:05):

So this is Dr. Halling. I'm a co-director of the Genomics Laboratory. My main interest over my career has been in developing and implementing molecular diagnostic assays that are helpful for cancer patients. I've been on staff since 2000.

Hussam Al Kateb (01:19):

And my name is Hussam Al Kateb. I also serve as a co-director of the Clinical Genomic Laboratories at Mayo Clinic. And in the past 15 years of my clinical practice, I've been focusing on also developing diverse clinical molecular assays for cancer patients, with particular interest in the comprehensive genomic profiling assays for cancer patients.

Robin Huiras (01:45):

Thanks for sharing that information. The expertise you have in this space really does highlight the knowledge that supports test development at Mayo Clinic. Now let's dig in.

Whole transcriptome RNA sequencing is an emerging testing technology that detects gene fusions in tumors. Now, gene fusions are common in tumors, some types more than others, and when detected, gene fusions can help guide diagnosis, prognosis, and treatment decisions for patients. Dr. Halling, maybe you can kick off our conversation today by providing our listeners with a brief background and overview of this new assay.

Kevin Halling (02:19):

So this is a test that's been in development for approximately four years. The test was needed because our current targeted gene fusion assays for sarcoma and brain tumors were missing genes of importance and did not offer gene expression analysis. The main offering that we're going to be offering has a test code of MCSRP, and it covers 1,445 genes. This test is going to replace three tests that went live about six years ago, including our SARCP sarcoma targeted gene fusion panel, our NONCR neuro-onc gene rearrangement panel, and our NTRK gene fusion panel. This will be the most comprehensive test on the market, and it shouldn't need to be updated for a long time. I also want to mention that at Mayo Clinical Laboratories, in addition to bringing up the larger whole transcriptome RNA sequencing panel, we've also upgraded our targeted sarcoma and neuro-onc DNA NextGen sequencing panels, which previously included our smaller targeted RNA-seq panels. So these are going to be updated now to include the whole transcriptome RNA-seq. Those panels are the MCSRP panel, which is 31-gene sarcoma DNA mutation panel, and our NONCP 87-gene DNA mutation panel. So whole transcriptome RNA-seq is going to be added to both of those.

Robin Huiras (03:46):

These tests really seem to be quite an advancement from our previous sequencing tests. Now, you mentioned sarcomas and brain tumors, but can our larger panel be used for other cancer types?

Kevin Halling (03:56):

Yes. For example, it could be used for salivary gland tumors, which frequently have gene fusions, also lung cancers and other tumor types that have fusions. We have not validated it yet for hematologic tumors, but we're working on that. Currently, the main utility is for sarcomas and neuro-oncology tumors, and that's because many of these tumors are classified by the presence of certain gene fusions.

Examples being EWSR1::FLI1 and Ewing sarcoma, SS18 and SSX fusions, and synovial sarcoma and other examples. And we've also validated the test for cytology specimens.

Robin Huiras (04:36):

Thanks, Dr. Halling. Thanks for really elaborating on the test utility and validation. It's great to know that these tests actually have such broad applicability and even better to hear that it can be performed on cytology specimens, which can sometimes be the only option for certain cancer types.

Hussam Al Kateb (04:52):

So MCL, as you know, is one of a few labs that are able to validate cytology and specimen types. As a reference laboratory, we receive samples from around the world. This gives us the unique opportunity to encounter cases that many other laboratories may not see. The broad exposure allowed us to include cytology specimens in our validation as well as a wide spectrum of both rare and common fusions.

Robin Huiras (05:22):

Thanks, Dr. Al Kateb. It's really great that the wide array of patient samples we receive here at Mayo Clinic Labs allows us to bring up such unique assays. But I'm curious to know, how is whole transcriptome RNA sequencing different or better than targeted RNA sequencing as well as DNA sequencing when it comes to detecting gene fusions?

Hussam Al Kateb (05:44):

That's great, but let me break down this important question into two parts. The first question is why whole transcriptome sequencing is superior to targeted panels for gene fusion detection? And the second one is, why RNA is used for fusion detection rather than DNA? So starting with the first question, targeted assays such as our SARCP and NEUFUS panels, which this assay will replace, were designed to interrogate specific genes or portion of genes based on the knowledge available at that time for their development.

However, new gene infusions and novel breakpoints involving known fusion partners continue to be discovered on a frequent basis. Because the targeted panels only cover the predefined regions. So novel events may not be detected. In our practice. We have encountered several such examples, including a case of endometrial carcinoma in which the SARCP assay failed to detect the fusion involving a well-established oncogene called ALK.

It turned out that the fusion break points occurred within a region of ALK that is not covered by the SARCP design. In contrast, the fusion was successfully identified using another assay that interrogated the entire ALK transcript.

And for the second part of the question, which is why RNA is used for fusion detection rather than DNA. While many fusions can be indeed detected using DNA-based assays, RNA offers several key advantages for fusion detection. First, although there are some complementarity between the two approaches, for example, some fusions may be detectable only in DNA or only in RNA. RNA-based fusion detection is generally more efficient and more accurate. One major reason for that is that fusion points often occur within large introns, which can be difficult to capture using short read DNA sequencing. In contrast, RNA undergoes splicing, and this event removes intronic regions and leaves behind short, clean exon-exon junctions that make fusions easier to identify with high confidence.

Another advantage for RNA sequencing is its ability to detect novel and unknown fusion partners because RNA-based methodologies identify new exon-exon junctions directly from the transcript data, they can reveal fusions even when the genomic breakpoints or partner genes were not previously known. DNA-based methodology, by comparison, often rely on targeted capture of known breakpoints regions, limiting their ability to identify unexpected rearrangements. That said, DNA-based fusion detection can also be advantageous in certain situations, such as when RNA quality is poor, for example, a degraded FFPE tissue, or when a fusion is not transcribed or expressed at very low levels, making it undetectable by RNA sequencing.

Robin Huiras (09:15):

Thank you so much for breaking that down for us, Dr. Al Kateb. That's some really great information on why this particular test is more useful than other methods, but I'm wondering what is it about Mayo Clinic Labs’ whole transcriptome RNA sequencing offering that sets it apart? I mean, it's very large, so I'm wondering, is it capturing more fusions than other tests on the market?

Kevin Halling (09:41):

Yes. It's one of the largest, if not the largest panels commercially available. As mentioned previously, we're covering 1,445 genes. Other labs frequently cover only a couple hundred genes. In addition to adding additional genes beyond what we currently cover, we've improved the assay by spiking in a large number of additional probes to cover areas of the transcriptome that were underrepresented with existing commercially available reagents. So when you put that all together, it should really be the best assay out on the market. And having done all this boosts our ability to detect challenging fusions.

Robin Huiras (10:22):

Thanks, Dr. Halling. It really does sound like this test is pushing the envelope on what's possible with genetic tumor testing. Now I'm curious, is there a general ordering scenario for this test? Would it be ordered alongside or after other tests, such as PDL1, for instance, or immunohistochemistry? Dr. Al Kateb, do you want to take this one?

Hussam Al Kateb (10:43):

Sure. So at Mayo Clinic, we have the privilege of offering a wide range of molecular tests, both DNA-based and RNA-based. Each is designed to answer a specific clinical question. Sometimes one test is enough, but in other cases, combining both types is necessary to fully understand that tumor.

For example, in brain tumors, our DNA test, NONCM, looks for important gene mutations, while our RNA test actually detects gene fusions. So for some tumors like polymorphous, low-grade neuroepithelial tumor of the young, or referred to as PLENTY, this type of tumor can have either a BRAF mutation, which is found by DNA testing, or it can be also caused by FGFR2 or (FGFR)3 fusions. These are only found by RNA testing. So, in such cases, both tests are needed to make an accurate diagnosis.

And another example would be when a novel fusion involving ALK gene is detected, and we are uncertain about the clinical significance of this fusion. So, by confirming ALK overexpression by IHC, this would be very useful for the clinicians.

Robin Huiras (12:09):

Thanks, Dr. Al Kateb. Now, would there ever be a scenario where it would make sense to only order whole transcriptome RNA sequencing? Dr. Halling?

Kevin Halling (12:18):

Yes. For example, if the pathologist is fairly certain that they're dealing with a fusion-positive tumor, for example, something that looks like a Ewing sarcoma or a synovial sarcoma under the microscope, in that scenario, it'd be reasonable to start with whole transcriptome RNA sequencing alone, because that might provide the answers they need without DNA testing. However, it's important to remember that DNA sequencing can detect alterations that RNA sequencing cannot, as alluded to by Dr. Al Kateb, for instance, DNA sequencing can pick up single nucleotide variants, indels and copy number changes, and RNA-seq is not designed to do that, and so the two tests are complementary.

In many cases, the DNA testing will identify mutations that are diagnostically or therapeutically useful that RNA-Seq does not identify. Additionally, for fusion-positive tumors such as Ewing sarcoma with an EWSR1:FLI1 fusion, DNA testing may find additional alterations that may have clinical significance, for example, P53 mutations, which would have additional prognostic significance. The DNA plus RNA sequencing approach aligns with updated guidelines for integrated molecular reporting established by the World Health Organization and the National Comprehensive Cancer Network, and also the College of American Pathologists.

Robin Huiras (13:40):

Wow. It sounds like the best approach to capture all of the relevant alterations really is a comprehensive approach.

Hussam Al Kateb (13:46):

Yes. I have always believed in taking a more comprehensive approach to molecular testing. So, from my experience with running paired tumor, normal whole exome, whole transcriptome sequencing, I've seen firsthand how these methods uncover much more information than targeted assays ever could. When you combine both DNA and RNA analysis, you really boost the diagnostic yield, and you get much more clear and much more complete pictures of what's happening in the tumor.

Robin Huiras (14:21):

Thanks, Dr. Al Kateb. Providing as complete a picture as possible for a patient's tumor really does support better treatment outcomes. Now I'm wondering if we can pivot a little bit and talk about the sample type and sample volume. Can you explain what is needed for both the tissue and the cytology specimens? Dr. Al Kateb?

Hussam Al Kateb (14:41):

We have validated the test on both FFPE tissues and cytology specimens. In terms of the requirements for nucleic acid input, I can say that 10 years ago we needed around one microgram of DNA. That is 1,000 nanograms just to run a 150-gene panel. The technology has come a long way since then. Today we can perform whole exome or even whole genome sequencing with as little as 100 nanograms of DNA. So, for this test, for RNA sequencing test, we validated the test to 15 nanograms of input RNA, and our data show that even we can still get reliable results with input as low as 30 nanograms.

Robin Huiras (15:32):

Wow, that's really incredible. It's amazing how quickly these testing technologies advance. Now, one thing we haven't touched on yet is the sensitivity and specificity of the test. I'm curious, does the size of the test and number of genes affect the sensitivity of the test and or impact the specificity of the test?

Kevin Halling (15:54):

Yes. We should see at least a moderate increase in the sensitivity. So if you compare our test to the existing SARCP and NEUFUS test, which have about a hundred genes on each of those, if you did a head-to-head comparison, we should increase the sensitivity because we're covering every last exon of those genes, whereas the current assays just target certain exons.

So, we should see an increase in the sensitivity for that reason. But in addition, we're going to see an increase in sensitivity because we have so many more genes on this panel compared to the current panels. So, putting it all together, yes, we should see an increased sensitivity. We do know that occasionally we can miss some fusions with our current targeted panels because certain exons are not represented in our validation. We've shown that the sensitivity and specificity for our whole transcriptome RNA-seq test is 99% sensitivity with a specificity of 96% and an overall accuracy of 98%.

Robin Huiras (16:57):

Thanks, Dr. Halling. That's really great to hear. 99% sensitivity with a 96% specificity on a 1000-plus gene test is just really tremendous performance. Now, can you provide our listeners just a little bit of context by sharing how these rates compare to DNA sequencing or FISH testing, for example?

Kevin Halling (17:19):

So, as I mentioned previously, for a whole transcriptome RNA-seq and DNA next-gen sequencing, these detect different types of alterations and they're complementary. Consequently, it would be sort of an apples and oranges comparison to compare sensitivity for the two assays because they are complementary.

However, when we compare a whole transcriptome RNA-seq to FISH, one should realize that FISH is another way to enable detection of gene fusions and also gene rearrangements that do not lead to a gene fusion. For example, FISH can detect gene rearrangements in certain hematologic malignancies, such as Burkitt’s lymphoma, that are undetectable by RNA-seq. RNA-seq requires that there be a fusion transcript to be able to detect a fusion.

But for tumors where there is a gene fusion, RNA-seq has the advantage over FISH of testing for many gene fusions simultaneously, whereas FISH can only test for a single fusion at a time. And consequently, RNA-seq would have much greater sensitivity for rearrangements that lead to a gene fusion, whereas something like FISH or another method would be required to detect rearrangements that do not lead to a gene fusion.

Another advantage of RNA-seq over FISH is that RNA-seq can tell you both gene partners that are involved in a fusion, whereas FISH can only identify one partner.

Finally, there are occasions where FISH is advantageous over RNA-seq when you're dealing with very small specimens with not very many cells. So, FISH may be able to work on those when RNA-seq might not be able to, because you can't get enough RNA.

Robin Huiras (18:55):

Thanks for that nice explanation of the detection differences between FISH testing and RNA sequencing, Dr. Halling. Those insights are really helpful and so clarifying, and I have just one final question. Are there other advantages to our whole transcriptome RNA sequencing assay that we haven't discussed? Dr. Al Kateb, you want to take this one?

Hussam Al Kateb (19:15):

Yeah. I would say this is a test that can identify rare, novel, or unexpected fusions that might be missed by targeted panels. It also can be applied to multiple tumor types without redesigning the assay for each new fusion of interest. Particularly, it's helpful in rare and unusual tumors where the expected fusions may not be known. And finally, it helps with identifying actionable fusions that can guide diagnosis/prognosis of the disease in addition to targeted therapy selection and enrollment in clinical trials.

Robin Huiras (19:56):

Thanks, Dr. Al Kateb. It's really great to hear that these test results might open doors for patients that would otherwise remain closed, and really providing patients and the physicians with answers that pave the way for better outcomes is the point of what we do here at Mayo Clinic Labs.

Hussam Al Kateb (20:12):

Yes. Could not agree more.

Kevin Halling (20:13):

Yeah, I agree, too. And we're very excited about this upcoming test offering because we know it's going to improve patient care, and that's what we get excited about doing every day, and so very excited to see this going forward.

Robin Huiras (20:27):

Thanks, Dr. Halling, and thank you too, Dr. Al Kateb, for your time and such an interesting discussion. I learned so much and hope our listeners did as well.

Hussam Al Kateb (20:35):

Thank you, Robin.

Kevin Halling (20:36):

Thanks.

Robin Huiras (20:37):

And thanks to our listeners for joining us today. We hope you enjoyed our discussion and will tune in again for the next installment of “Diagnostics and Practice.”