The RNAscope IHC Breakthrough: When Clarity Matters

Hello, and welcome to this round table discussion hosted by Leica Biosystems. Today we are privileged to have two distinguished experts with us, Dr. Rob Monroe, Vice President and Chief Scientific Officer at Danaher Diagnostics and Chief Medical Officer at Leica Biosystems and Jordan Laser, Senior Director of Clinical and Medical Affairs at Advanced Cell Diagnostics, a Bio-Techne company.

They will both introduce themselves shortly. But first, why are we here? In situ hybridization has progressed in recent years, paving the way for extraordinary insights into the molecular underpinnings of disease with a critical spatial component. The purpose of our gathering today is to delve into and highlight a next-generation RNA in situ hybridization technology in diagnostics, RNAScope. We look forward to highlighting RNAScope detection through image examples, and along the way discuss how RNAScope ISH can help solve classically observed problems in immunohistochemistry and even traditional ISH staining while continuing to shape trends in testing, regulatory guidelines, and publications. With that, we'll move to introductions. Dr. Monroe, we'll start with you.

Thanks very much, Jack. I'm Rob Monroe, and it's a pleasure to be here with all of you today. As Jack mentioned, I currently serve as Chief Scientific Officer of Oncology for Danaher's Diagnostic Platform, as well as Chief Medical Officer for Leica Biosystems, which is a member of the Danaher family of companies. By way of background, I'm a pathologist by training. I have board certification in anatomic clinical and cytopathology and have been working in the diagnostics industry for about 15 years. Prior to joining Danaher, I was part of the team at Advanced Cell Diagnostics that developed the RNAScope technology specifically for the Leica BOND platform, and have co-authored several publications on the technology ending. Over to you, Jordan.

Yes, thank you Rob. Jordan Laser. I am also a pathologist by training. I'm board certified in anatomic pathology, clinical pathology, and molecular genetic pathology. And as Jack mentioned, I am the Senior Director for Clinical and Medical Affairs at Bio-techne and really happy to be here today so we can discuss some of the advancements in situ hybridization.

Yeah, thank you both. The longstanding partnership between ACD and Leica Biosystems brings RNAScope and automation together on the BOND platform, and continues to gain momentum in the testing landscape because of its ability to provide high sensitivity and specificity in RNA detection, even in challenging samples. We're excited to get started. To begin our discussion, we'll discuss traditional in situ hybridization and its history, Dr. Monroe, we'll start with you. Give us an idea of where ISH sits within the testing landscape in the pathology lab.

Absolutely, Jack. In situ hybridization or ISH, as I'll refer to it, is very much a complimentary technique to immunohistochemistry or IHC but rather than using antibodies to detect proteins, like you do in IHC, ISH leverages DNA probes to detect either DNA or RNA targets and this is done through hybridization of those DNA probes to complimentary sequences, either in RNA or DNA. DNA ISH in particular is used to assess chromosomal alterations. These can include translocations like the one involving chromosomes 9 and 11 that lead to the BCR-ABL gene fusion. Another application of DNA ISH is to assess copy number and the most common example of A DNA ISH assay to detect copy number alterations is the HER2 ISH assay. As you know, HER2 could be amplified in, in breast as well as other cancers. On the other hand, RNA ISH uses somewhat shorter probes that target RNA, rather than DNA molecules in the cell. It's typically used to assess gene expression, which, is closely correlated or a surrogate for protein expression that could be measured again by IHC. Then the question arises of, well, why would someone choose to use RNA ISH for detection of expression of a biomarker versus IHC to just look at the protein directly? Well, there's a number of instances, where it's actually advantageous
to look at the RNA versus protein. One of these instances is the case where proteins don't lend themselves to the creation of good antibodies. An example of this is the E6 or E7 oncoprotein of HPV, both of which don't lend themselves to the creation of good antibodies. In this case, RNA ISH is a great alternative to IHC to determine expression of that particular biomarker.

Another example is where RNA ISH can be advantageous to IHC is the case where RNAs don't encode proteins themselves but nonetheless can serve as a biomarker for a given process like an infectious disease. In this case, a ribosomal RNAs serve as a good example and could be targeted with RNA ISH to distinguish different infectious agents but that couldn't be done with IHC because these are not these ribosomal RNAs are not translated into proteins.

Thank you, Dr. Monroe. ISH seems to have quite a history in the pathology laboratory. Dr. Laser, take us through a high level history of ISH in diagnostics. Where has it traditionally solved problems where other assays have fallen short?

Yeah. Thank you Jack. Happy to provide some critical background here and give a high level history of in situ hybridization over the decades. As I said, it really does go back decades. In 1970s Joseph Gall and Mary-Lou Pardue were using radioactive copies of ribosomal DNA sequences to detect the complimentary DNA sequences in frog eggs. While it sound may sound a little esoteric, this really was the beginning of molecular cytogenetics. We were able to now first identify the position of specific DNA sequences within the chromosome. Of course, time went on and we obviously had advancements in this technology and in the 1980s, it was a shift away, radioactive detection towards fluorescence, and we were able to benefit with
that from an increase in sensitivity. And we also saw in the 1980s this technique in situ hybridization being used to diagnose chromosomal abnormalities and a wide range of genetic disorders.

Now, in 1981, RNA ISH was identified, and it enabled us to be able to both locate and understand the expression of RNA within the spatial context of tissues. This provided really deep insights into expression, localization, and regulation of RNA.

In the 1990s, there was a real expansion in clinical and research applications leveraging situ hybridization. FISH or fluorescence situ hybridization gave way at least partially to some newer techniques, namely SISH or silver in situ hybridization or CISH, chromogenic in situ hybridization. With RNA ISH, there were really improvements in the probe design and the signal detection, which really enabled a much greater expansion of both sensitivity and specificity. And because of all these advancements over all these years, in situ hybridization became widely used in cancer diagnostics, constitutional testing, and prenatal testing.

In 2011, RNAScope was identified or discovered, and it really is an RNA ISH technology that leverages a really unique RNA probe design and with a backbone of branch DNA signal amplification, which provides really exquisitely high sensitivity and specificity down to the detection of single RNA molecules within cells.

This was a real advancement in unlocking low abundance RNA detection, something that previous technologies were unable to do. As a result, there's been a wide range of, adoption in laboratories for a wide range of meaningful applications. I think that's a high level history brings us to where we are today, and, I think sets a perfect foundation for the rest of our discussion.

Yeah, absolutely. Thank you, Dr. Laser. It seems like in situ hybridization has evolved over time and continues to be an important tool for laboratorians. Dr. Monroe, now let's walk through traditional RNA and situ hybridization technology. Are there inherent challenges to this traditional technology as shown in the image?

Absolutely Jack. I'm going to quickly go through traditional ISH technology as depicted in the image here. In traditional ISH, single-stranded probes are labeled, with a molecule called digoxigenin which is shown in the aqua color. These probes can be subsequently detected, by anti-digoxigenin antibodies. Then in turn, secondary antibodies conjugated to horseradish peroxidase or HRP. HRP can then act on the substrate diaminobenzidine or DAB, as many of you know of this compound. It can produce then a brown chromogen at the target site.

This technique works quite well for some targets, particularly those that are expressed at high levels. This technique also has several disadvantages. The main disadvantage, of this so-called direct labeling technique is that the method produces only weak signals, in the absence of high levels of target RNA expression. This doesn't allow for significant amplification of the signal. You can only load so much to digoxigenin into these probes, so that limits, the amplification and the ability to detect lower expressive targets.

Another disadvantage of this traditional ISH technique is that the technique has the potential to produce relatively high levels of background staining and that's to the nonspecific binding of the ISH probes to sequences with only partial homology, to the actual target probes. This can make interpretation and distinguishing the true hybridization signal, from the background very challenging.

Great. Thank you, Dr. Monroe. I think we can see similarities here with IHC and probably many in the audience are at least, somewhat familiar with traditional ISH in their workflow, or having sent out for this test at some point. Now that we've talked about the traditional uses of in situ hybridization, let's discuss how the technology behind the assay has progressed. 

Some in the pathology lab may think that RNA ISH has one meaning, and all ISH assays are basically the same. Let's ask the experts. Is RNA ISH all the same? Has there been progression in the technology over the years? Dr. Laser, what are your thoughts?

Yeah, thank you. I'm glad you called that out right off the bat. I sometimes feel there's a general misperception that in situ hybridization is this monolith, it's just one technique, it's an older technique. There has been some really significant advancements over the years, as you just heard the kind of the high level history of some of those advancements and just to summarize them right from a methodological perspective, we matured from radioactive detection to fluorescent to chromogenic. Our probe sizes have gone from really large to much smaller. They've been able, as a result of the them being smaller, we're actually able to interrogate the genome or whatever we're looking at a much higher resolution.

We've also seen that shift from DNA hybridization to RNA hybridization as well. And again, all of those advancements lending itself to a much greater sensitivity and specificity. I also mentioned there are more recent advancements in RNAs with the development of RNAScope.
And it really combined the utilization of much smaller probes that are really critical in terms of enabling formalin-fixed paraffin-embedded RNA in situ hybridization.

As I mentioned, there's this massive signal amplification technique that brings forward that exquisite sensitivity and unparalleled specificity. We do have an image I'd love to walk everyone through, kind of the design and the innovation of RNAScope. The key of the innovation is in this double Z probe design that you see on the lower left hand side.

The bottom of the Z is where the complementary sequence exists to the target sequence. The RNA molecule that we want to identify. Of course you can't tell on this image, but that binding area that at the bottom of the Z is actually quite small. It's double digit base pairs. This really enables us to have that high degree of specificity, but also enables us to be able to detect RNA, which is even partially degraded as we frequently experience in formalin-fixed paraffin-embedded tissue.

The middle of the Z is a linker and then as we go to the top of the Z, you'll see that this represents the pre-amplifier binding site.
I like to use the analogy here of a tree in terms of this branch DNA signal amplification. You can consider the top of both Zs and I want to stress that both of those Z probes must bind in the right location, in order to create the total binding site for the pre-amplifier molecule.

You can consider the top of Z as the soil where the root of the tree and the trunk of the tree, which is the pre amplifier, will therefore bind in a subsequent hybridization step. Now, as we continue moving along towards the right hand side of the image, you'll see in that that third ZZ pair, there's an additional hybridization step where now we're starting to hybridize on the branches of the tree.

Here you'll see this small complimentary sequence to the trunk. The amplifiers are then representing the branches, and I'm sure you can predict where I'm going next, but the very next hybridization step includes the hybridization of a labeled probe. This probe can be labeled with either a chromogenic enzyme or a fluorophore in fluorescent applications. You could see that there are any labeled probes that can bind to a single amplifier branch.

You can see where this massive signal amplification starts to occur. There are many labeled probes to each amplifier. There are many amplifiers to each pre-amplifier. While I didn't say this at the beginning, because I didn't want to be overly confusing, but when we did design RNAScope probes, we do not usually design a single ZZ pair for the detection. It's typically around 20 ZZ pairs for each of these targets. You can see this massive signal amplification, 20 ZZ pairs, all of them have pre amplifiers. All of those pre amplifiers have multiple amplifiers. All of those amplifiers have multiple labeled probes that are bound to them. This is what unlocks that exquisite sensitivity with that detection all the way down to single RNA molecules in that spatial context.

A lot of that was building up to the really advancements in the sensitivity but I also want to touch upon the specificity as well. In these designs, and Rob had mentioned it well in the traditional ISH methodologies, those probes tend to be quite large. And because they were quite large, they were less susceptible to mismatches, DNA or base pair mismatches, and so they would bind anyway. That contributes to a meaningful amount of background signal. Because of the advancements in RNAScope and these double Z probes and how really small the binding site is to the target sequence, it is much more susceptible to mismatches.

When we have those mismatches, either one or both of the Z probes won't bind. And without that co-localization and binding of both Z probes, you never get to generate the soil for where the pre-amplifier, could actually bind to.

This is an innovative design that provides that exquisite sensitivity to that massive signal amplification. It is very susceptible to basepair mismatch, which result in non-binding events and exquisite specificity as well. This is where the innovation is in RNAScope and where some of the events have occurred over the years.

Great, thank you Dr. Laser. I really like the analogy of the soil and the trees. This technology has really expanded on what traditional ISH can do and built on that history to move the technology forward. Hopefully our audience can appreciate the improvements over a traditional ISH provided by RNAScope. What better way to understand RNAScope detection than by reviewing examples of RNAScope staining?

Dr. Monroe and Dr. Laser will walk us through two examples in the next couple of slides. As a reminder to the audience, the pathologist views are their own and are for the purpose of discussing the RNAScope technology principles. RNAScope detection is a general purpose reagent or GPR in the US and is not intended for in vitro diagnostic use. For any attendees outside of the US, RNAScope detection is marketed by Leica Biosystems as IVD in Europe, and the HR HPV 18 probe is marketed by ACD as IVD in Europe. Regulatory status can vary by country, so please check with your local Leica representative about your current regulatory status on this assay. With that, we will move to the first image, and I'll leave it to Dr. Monroe to start walking us through this RNAScope image.

Thanks, Jack. This photo micrograph that you see here shows an oropharyngeal squamous cell carcinoma that's been probed or stained with an RNAScope ISH probe for high risk HPV or human papilloma virus. High risk HPV is a great example of a biomarker that could be very challenging to accurately detect with traditional RNA ISH technology and that's because of the high background staining that's observed due to the cross hybridization of these high risk HPV sequences with low risk HPV sequences, as well as other genomic sequences.

As Dr. Laser described, RNAScope for high risk HPV and other targets can overcome these challenges with this exquisite and powerful double Z technology that has the capacity to both suppress the background staining while simultaneously amplifying the true signal of several hundred fold.

In this example, you can appreciate the staining of the malignant squamous carcinoma cells that are surrounded by a relatively dense lymphocytic infiltrate. The squamous cells are the ones with larger nuclei that also show the brown punctate staining with the RNAScope probes. You can appreciate the punctate dots and clusters that are representative of HPV RNA molecules located primarily in the cytoplasm but also in the nucleus, which is representative of the transcription as well as that subsequent export of RNA in an HPV-infected cell.

The next slide highlights a case that's negative, again, a similar type of squamous cell carcinoma. In this case that does not express high risk HPV. You can see how clean the background is in the absence of HPV involvement in this type of squamous cell carcinoma. As many of you know squamous cell carcinomas of the head and neck are typically subdivided into two main types, the HPV-related and the non-HPV-related, which typically results from environmental causes like smoking and alcohol.

The next slide really highlights the difference where you can see the very clean background in the HPV negatives or unrelated squamous cell carcinoma on the right relative to the HPV related squamous cell carcinoma on the left where you can appreciate the high expression of the HPV oncoproteins E6 and E7, which are the targets of that high risk HPV RNAScope probes. With that, I'll hand it back to Jack and Dr. Laser for a review of the next example of RNAScope.

Thank you, Dr. Monroe. Dr. Laser will review this next case.

First I wanted to start by providing a high level background. We're going be looking at both IG or immunoglobulin Kappa and immunoglobulin Lambda as RNAScope stains. Again, a little background on both of them. Many of you already know this, but mature B cells express immunoglobulins or antibodies. In order to do so they generally undergo a gene rearrangement in response to some antigen being presented to them and their goal in life is to create the best antibody possible. B cells, the couple of chances of making the best antibody, those chances are actually the light change Kappa and Lambda. Laboratories can leverage this biology to identify monoclonal B cell populations. In a normal state, we expect to see a mixture of polyclonal B cells, meaning a mixture of Kappa-expressing and Lambda-expressing B cells.

In disease states as lymphoma, this polyclonal or normal mixture of Kappa Lambda goes away because it's overridden by a monoclonal population, which will either express Kappa or Lambda. What are we looking at here? Here we have a sheet of mostly lymphocytes and these are mature B cells. To orient the audience this image is stained using RNAScope ISH for IG Lambda. What do we see? Lots of brown. All this brown is really an indication of expression of immunoglobulin lambda, and I do want to call out that there are two types of staining patterns that we see here.

You see this both on lambda. First, you see these scattered really dark cells in the image. These are plasma cells that we see in the background and the reason why they're so dark is they're chockfull of messenger RNA molecules for IG Lambda, right? Their job in life is to produce a massive amount of antibodies. It makes total sense that they are chockfull of this messenger RNA. In fact, RNAScope generates so much signal from these, the plasma cells tend to be really dark brown or almost black. That's probably the minority of the signal you see here. The more dominant pattern is this more granular cytoplasmic staining that you see throughout the rest of the slide. It really outlines the nucleus here. This really scant cytoplasm that is associated with lymphocytes. The holes there are nucleus and the staining, as you'll see the cytoplasm fold with this granular dotlike staining pattern. These represent mature immunoglobulin expressing B cells of this tissue. Here is already highly suspicious that there's a monoclonal population going on here that is expressing or restricted to immunoglobulin lambda.

If we were to stain this with a classic ISH technique or technology with its somewhat limited sensitivity, I'm very confident we would see the plasma cells, those dark staining cells. I would expect to see them because again, they have this massive expression of immunoglobulin lambda, but the rest of the staining pattern we see in the background would very likely be absent or very difficult to interpret with traditional ISH, especially when we know that traditional ISH has a meaningful background staining to begin with. Separating that out would be challenging in that situation. However, this is not an image of classic in situ hybridization, but it is RNAScope and it demonstrates the exquisite sensitivity of how easily we are able to visualize the mature B cells that are expressing immunoglobulin Lambda here and the plasma cells.

If we go to the next slide it is very common for laboratories of all types, when they're evaluating clonality of B cells, they typically stain in both IG Lambda and IG Kappa. This slide is the same exact issue section except stain for IG Kappa opposed to IG Lambda by RNAScope. As you can see, just like in situation that we had just shown with HPV testing, there's absolutely no signal here. There's no background, and again, demonstrates the incredible specificity of RNAScope as a result of that double Z probe design.

This absolutely no background is in contrast to what we would typically see in other methodologies such as immunohistochemistry for IG Kappa and IG Lambda, which as these are secreted proteins, there generally is a significant background stain. But also, as mentioned, the traditional is methodologies would also demonstrate some background which we do not see here.

The next slide is a great visualization where we can see these two images side-by-side. Same tissue section looking for IG Lambda and IG Kappa in the assessment of whether a monoclonal population exists or does not exist in these B cells. You can see the very strong staining of the plasma cells, the uniform staining of the B cells, which should clearly support monoclonal immunoglobulin expressing B cell process that's going on in these images.

Thank you Dr. Laser and Dr. Monroe for walking us through the images and telling us a little about the RNAScope probes as we go. I'm always convinced, and note the striking difference when you can see the negatives as you went through about the specificity of these probes, I hope the audience can appreciate that as well. While the potential for RNAScope is exciting, some in the audience may be thinking that implementation of a new test seems daunting. Dr. Laser, for pathologists who haven't used the RNAScope technology before, who may be listening, what are the initial challenges with reading the slide?

Yeah, thank you. If it's all right, I'll immediately turn that around because how RNAScope was designed, we were really thoughtful in terms of mitigating those challenges upfront in the design itself. The most striking thing that you may notice is the brown on blue staining pattern, and this brown on blue, the brown signal is a DAB chromogen, and the blue background counter stain is hematoxylin. This is exactly the same color profile, in fact, the same chemistry that you see with immunohistochemistry. This color scheme is something everyone should be expecting and something that you're quite familiar with from the beginning.

The signal is slightly different. The RNAScope signal tends to be these discreet dots or clusters rather than the more diffuse or block-like signal that we see with immunohistochemistry. In moderate-to-high-expressing targets, you could easily visualize this at low power. Because of these discreet dotlike signal, particularly in lower expressing targets may require scanning to go through to see if you can identify the signal. But again, for the most targets that are either moderate or highly expressed, it's easily visualized at low power.

Each of these dots actually represent a single RNA molecule. Sometimes these then the targets are expressed to a certain degree and spatially where these molecules located, you may get some clustering of the dots where, they're just overlapping dots that form these clusters. You could obviously carry that all the way through to the extreme, like we just saw in the plasma cells, where the dots coalesce to clusters and the clusters can coalesce to overt staining of the entire cell like we saw in those plasma cells. While they are dots and RNAScope is largely at this point a qualitative assessment, it's useful for the comparison of expression versus not expression or varying degrees of expression, high levels of an expression versus lower levels of expression.

That said, as these dots and clusters are the signal, it does lend itself to quantitation. As we look to the future, digital pathology takes a hold in a wide range of laboratories. We can see a future where quantitation maybe another advantage of RNAScope. I want to stress that the majority of the applications today are largely qualitative in assessment.

Great. Thank you Dr. Laser. Now, Dr. Monroe, when we think about the anatomic pathology ecosystem, how does RNAScope fit within that? Again, thinking about pathologists who may not have used RNAScope technology before, who may be listening, or the technologists and directors of the lab who may be listening, how can they fit the RNAScope technology into their current ecosystem? How does it fit generally into the AP lab?

The answer at a very high level is that it fits very seamlessly into anatomic pathology labs and workflows. RNAScope has a similar workflow to IHC and so the whole upfront preparation of slides for RNAScope staining is essentially identical to preparing slides for IHC. The staining process itself is automated on the Leica BOND instruments, very similar to the automation of IHC. It's simply a procedure where you load the slides, select the protocol, and then walk away.

The probes and the staining reagents themselves are related but different from those that are used for IHC. They're available in the same reagent trades, that you may be familiar with, if you're familiar with the Leica BOND staining platform. They allow for the laboratories to put in or utilize whichever RNAScope probe they'd like along with the detection reagent.

What about the validation process? That question comes up not infrequently and it's no different from IHC. I would suggest consulting the College of American Pathology guidelines for recommendations on validation of new ISH or IHC stains. To summarize, onboarding RNAScope is very similar to onboarding a new IHC antibody on the BOND platform and the entire staining protocol is fully automated like it is for IHC, which makes RNAScope very straightforward to adopt and get up and running in most any anatomic pathology lab with a BOND platform.

Excellent. Thank you very much. Thanks to both of you. As Dr. Laser pointed out, as we look to the future and continue to look for our partnership between Leica Biosystems and ACD to remain strong and continue to develop assays together. Our final topic here is the current guidelines regarding RNA ISH testing, as this type of testing continues to increase in prevalence and significance. Dr. Monroe briefly mentioned, the guidelines in its last answer. Dr. Monroe, as we think about guidelines specifically, how is the utilization of ISH continuing to evolve in standard guidelines? How and when you think about the future then, what do you think those guidelines will continue to look to ISH to solve?

In addition to the broad guidelines for implementing ISH, in a laboratory there are some guidelines that specifically call out RNA ISH or ISH technology for the characterization of head and neck cancers. The guidelines specifically do this are the National Comprehensive Cancer Network or NCCN guidelines, along with the College of American Pathology or CAP guidelines. In the NCCN guidelines that were published in 2021, HPV testing is recommended for all squamous cell carcinomas that are found in the head and neck region or so-called head and neck squamous cell carcinomas.

The NCCN guidelines state that HPV testing could be done using a variety of techniques including in situ hybridization, along with P16 immunohistochemistry, with P16 being a surrogate marker for HPV detection, that's been used for many years as a surrogate given some of the challenges prior to RNAScope in looking directly at HPV sequences. Interestingly, the NCCN guidelines note that the specificity for HPV is highest for ISH, and that's related to the fact that P16 immunohistochemistry can be positive in the absence, at least in a relatively small percentage, but nonetheless, a significant percentage of cases that are negative for true HPV infection or involvement.

Those are the NCCN guidelines. Moving on to the CAP guidelines that were published in 2018, again for head and neck cancers, the recommendation is for pathologists to perform high-risk HPV testing, the oropharyngeal tumors, essentially the throat cancers with P16 IHC, but that additional HPV specific testing, which would include HPV RNA ISH, can be done at the discretion of the pathologist or the treating clinician.

The history of these recommendations is that P16 has traditionally been more accessible to more labs given the availability of those antibodies with the RNAScope and RNA ISH technology, being relatively new at the time those guidelines originally came out.

Interestingly, the CAP guidelines also state that for metastatic squamous cell carcinomas where the primary site is unknown or in the case of a multi-site tumor that involves the oropharynx, that pathologists should perform P16 IHC followed by additional high risk HPV testing including HPV RNA ISH on the P16=positive cases to confirm the HPV status. The reason for this recommendation for confirmatory testing is that HPV ISH is more specific for HPV-related cancers, given that P16 can be positive in tumors that are not related to HPV, particularly in squamous cell carcinomas that arise outside of the oral pharynx.

To summarize, RNAScope and in particular HPV ISH, is now mentioned and included in guidelines around the testing for head and neck squamous cell carcinomas. We expect as the literature and the accessibility of the technology continues to increase in the coming years, that this inclusion in guidelines and that the strength of the recommendations will increase.

Excellent overview. Thank you, Dr. Monroe. Dr. Laser, what thoughts would you add especially looking at the current landscape and into the future?

Yeah, so we've obviously had the opportunity in this session to look at Kappa Lambda and discuss HPV testing, but there really are a lot of other applications that laboratories are using today.

Just to kind of take that step back, there are over 11,000 publications that have utilized RNAScope in the literature today, and we have over 50,000 catalogued covering the vast majority of the human genome and exome or exome more specifically, and the other species as well. I want to go through this waterfall of some publications that we had to see and demonstrate the utility that researchers and laboratories have identified with RNAScope.

You can see the top two are focusing on HPV and head and neck cancer and how it's becoming part of routine clinical practice to do so, and how it is either supplementary or as Dr. Monroe mentioned, with the update of the CAP guidelines, where HPV specific testing is becoming more useful, if not required.

You can see the next one below discussing B-cell clonality. To highlight some of the key words in this title is how ultrasensitive RNAScope, or RNA in situ hybridization is for kappa and lambda and really compact superior performance to other methodologies that are used in laboratories today, such as flow cytometry. It extends, as I said, beyond Kappa and Lambda and HPV test, and you can see that they're on the bottom left hand side. These authors shown that RNA scope for albumin has been helpful in assessing hepatocellular carcinomas or intra hepatic cholangiocarcinomas and differentiating them from secondary tumors or metastatic lesions of other origins, which is sometimes a challenge to do so without RNAScope as this technology.

You could also see if you scoot over to the bottom right hand side, this is a publication that leveraged RNA scope for MDM2, which is a really useful in the evaluation of atypical lipomas tumors. Gene amplification of MDM2 is not only the pathogenesis, but a hallmark in terms of a tool in terms of assessing if this is an atypical lipomas tumor or not. RNAScope enables to keep this type of testing in an anatomic pathology laboratory as opposed to having to leverage other technologies such as FISH, which require different sets of microscope, expertise, and environment as well.

Of course, there's a very long list of markers described in the literature that can be used in a wide range of applications. Just to mention a few here that are not on this waterfall. Recently discovered Trim 63 is an RNAScope target that is useful in the identification of translocation associated renal cell carcinomas, as well as RNAScope happens to perform beautifully at infectious agents such as EBV, CMV, and HSV in the context of tissues. Of course, that represents only a small window into the current use of RNAScope, and I am excited that the future is currently still being written.

Dr. Monroe mentioned the inclusion to the current gap guidelines for oropharyngeal squamous cell carcinoma. This is specifically HPV testing by RNAScope. The pending updates that we're expecting any day, given the open comment period, we are expecting more favorable or more stringent or advocacy for using HPV-specific testing in addition to P16 IHC. I alluded to very quickly before, we're really looking into the future as digital pathology continues to take hold in laboratories, RNAScope could be one of the tools to help us usher in the next phase of quantitative pathology. We can start using this to be able to not just qualitatively look to see if something's expressed or not, or if it's highly expressed or not, but we can now start to count this signal and make some interesting biological insights leveraging this technology and digital pathology as well.

Extending that all the way out to the extreme, this could be in terms of ushering in this new era of quantitative pathology, partnerships with pharma, et cetera, can result in other utilize utilization techniques such as developing diagnostics or companion diagnostics in those settings. We've gone through the long history of in situ hybridization. I think we're in a wonderful space where we are in terms of RNAScope and RNA and in situ hybridization, and I'm really excited to see where we go into the future as we just continue to expand our applications.

Great. Thank you Dr. Laser, and thank you both for this discussion. Thanks to our audience as well. We hope it has been insightful to dive into RNA scope automation and understand how ISH has progressed the image reviews, especially highlight the technology and the impact it can make on spatial detection of RNA. The guideline review and this highlight of publications is insightful for us as we continue to look to the future.

As Dr. Laser mentioned, the partnership between Leica Biosystems and ACD to automate RNA scope continues to enhance the workflow of the lab and ultimately to improve patient care. Thanks to all of you for being here.