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.