Webinar: Combined RNA ISH and IHC

Judy O’Rourke: Hello, everyone, and welcome to today's live broadcast application of combined RNA in situ hybridization and multiplex immunohistochemistry to unravel tumor heterogeneity in prostate cancer presented by Nallasivam Palanisamy, PhD, and Courtney Anderson, PhD. We are excited to bring you this educational web seminar presented by LabRoots and sponsored by Novus Biologicals, a Bio-Techne brand.

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I am Judy O’Rourke of LabRoots, and I'll be your moderator for today's event. This webinar has been approved for continuing educational credits. Please click on the CE button at the bottom left corner and follow the process to receive your credits. Before we begin, I would like to remind everyone that this event is interactive. We encourage you to participate by submitting as many questions as you want at any time you want during the presentation. Just click on the green Q&A button located in the lower left of the presentation window, and type your question into the box that appears on the screen. We'll answer as many questions as we have time for at the end of the presentation. And also, please note that you'll be viewing the presentation in the slide window. To enlarge the window, just click on the screen icon located on the lower right. And finally, if you have trouble seeing or hearing the presentation, please click on the support button at the top right of the presentation window or use the Q&A button to let us know that you're having a problem. Thank you.

Now I would like to introduce Dr. Anderson. Dr. Anderson is a senior scientist in R&D at Advanced Cell Diagnostics in Newark, California. In her current role, she manages numerous projects with collaborators from around the globe to demonstrate new and exciting applications of the RNAscope and BaseScope technologies and their ability to detect RNA biomarkers with single-molecule sensitivity and single-cell resolution with morphological context. Prior to joining ACD, Dr. Anderson completed her postdoctoral studies in metabolic biology at the University of California Berkeley. She received a PhD in molecular and developmental biology from the University of California San Francisco. Please join me in welcoming Dr. Anderson. I will now turn the presentation over to her.

Courtney Anderson, PhD: Thank you very much, Judy, for that introduction and thank you all for joining us. Today, I will give an overview of the RNAscope in situ hybridization technology and show how you can combine it with immunohistochemistry, which I will refer to in this talk as dual ISH/IHC. The first key feature of the RNAscope technology is the unique probe design. So first, this is a basic overview of the technology. What we see here is the permeabilization of the cells – it's a slide-based assay so we have permeabilization of the cells or tissues on the slide – followed by hybridization of the target probes to the RNA. We also have amplification of the signal following probe hybridization. And then visualization of the staining with morphological context. Because we have single-molecule resolution, this can be quantified by single-cell expression.

Now, this is just a brief overview of the probe design. The basic probe is designed or shown as a double Z where the base of the Z is the target-specific binding site that targets approximately 50 base regions. And the top of the double Z is where the preamplifier binding site is present. Now, a typical RNAscope probe consists of approximately 20 ZZ pairs that allows us to target 1,000 base pair region.

So, just to go into a little bit more detail, the first I'm going to show here is the signal amplification. As I mentioned, we have the double Z probe, which binds to the target sequence. Upon binding to the sequence, a preamplifier then binds to the top of the double Z pair. Then multiple amplifiers can then bind to each preamplifier. And then, each preamplifier can then bind multiple labeled probes. These labeled probes can either be a chromogenic enzyme or a fluorophore. This single-amplification strategy allows for visualization of target RNAs as a single dot, where each dot represents an individual RNA molecule that can be quantified. Lastly, with the RNAscope assay, background is eliminated because the signal is dependent on two Zs binding next to each other on the target sequence. If both Zs do not bind, then the preamplifier cannot form a stable hybridization to the top of the Z, and the amplification tree does not get built. Consequently, no amplification of nonspecific hybridization occurs, generating little to no background signal. The combination of the probe design and the amplification system ensures a high signal to noise ratio.

Provided that the target is expressed in the sample and the protocol is followed as recommended, the performance of the RNAscope assay is guaranteed. Because tissue samples can be highly variable, proper sample preparation and the right controls are essential to an RNAscope experiment. Tissue samples must be properly fixed and prepared so that there is good quality RNA in the sample and no background due to poor fixation. ACD provides guidelines on how tissues should be fixed for optimal performance of the RNAscope assay.

Shown here on this slide are serial sections from a human lung cancer sample on which we performed the manual red chromogenic assay. In this first panel, we see our negative control probe dapB, which is a bacterial gene that is nonexpressed in animal tissues. As observed in this sample, we see no signal from the dapB probe. In this middle panel is a section from the same sample that was stained with our positive control probe for the housekeeping gene PPIB. As observed with this sample, you want to see fairly uniform detection with the positive control probe indicating good quality RNA. Certain tissues may express the positive control probe at higher or lower levels but, in general, you want to see expression throughout the entire sample.

With the results from these two probes, we can then have confidence in the test data that we see here in the right panel. As an example, I am showing a section from the same lung cancer sample probed for the immune checkpoint marker programmed death ligand 1 or PD-L1. PD-L1 exhibits a wide range of expression in tumor tissues. In this particular human lung cancer sample, we observe robust punctate dots with the PD-L1 probe indicating expression of PD-L1 in this tumor sample. The controls show us that there is good quality RNA throughout the tissue sample, and that there is little to no background. So, we can be confident that the localized pattern of expression we observe for PD-L1 is, in fact, the correct results.

The RNAscope assay is ideal for many applications, including the detection of any gene expression, long non-coding RNAs for which antibody detection is not possible, molecules that are challenging to develop antibodies against such as GPCRs, and identifying the cellular source of secreted proteins. The RNAscope assay can also be used to validate high-throughput data, identify colocalization of multiple genes, and visualize gene expression with morphological and spacial resolution. The assay has been applied in a wide range of research areas, including neuroscience, stem cell and developmental biology, infectious diseases, and cancer, in addition to drug discovery and development in the biopharma arena.

Now, I'd like to show you how you can combine the RNAscope ISH assay with IHC. Two complementary assays that – when combined – can allow you to gain more information on gene expression. Despite the fact that ISH and IHC target different molecules, they can be used as complementary assays rather than mutually exclusive assays. They can each provide valuable information that compliment one another. For example, one can identify the origin of secreted proteins by using ISH to identify the cells that produced the protein and IHC to identify where the secreted protein is located. One can also characterize complex tissue structures by using IHC to identify the cell type and ISH to detect gene expression inside these cells. Lastly, one can dissect the regulation of gene expression by using ISH and IHC to differentiate between gene regulation at the transcriptional level and at the translational or posttranslational level.

Here is an overview comparing the two detection methods. While the RNAscope ISH assay detects RNA using DNA oligonucleotide probes, IHC detects protein using an antibody. Both assays can visualize gene expression with morphological context at the single-cell level using either a chromogenic or fluorescent signal. However, the RNAscope assay can detect gene expression with single-molecule resolution, allowing for quantification of gene expression in situ. Because of the similar workflows between ISH and IHC – including sample fixation, pretreatment, probe hybridization, signal detection, and data analysis, as well as the unique benefits of each assay described earlier – ISH and IHC are ideal to combine into one workflow.

Here is an overview of the dual RNAscope ISH/IHC workflow, in which the RNAscope ISH assay is performed first followed by the IHC assay. First, samples are pretreated followed by hybridization of the RNAscope probes and then amplification of the RNAscope signal. The IHC assay is performed next starting with the blocking step then primary and secondary antibody incubation and lastly detection of the signal, which can be chromogenic or fluorescent.

So, we do have some recommendations to ensure success with your dual ISH/IHC assay. First, all dual ISH/IHC protocols require optimization. In general, it is recommended to combine a working RNAscope protocol with a working IHC protocol. In addition, you should work with antibodies and a protocol that are known and already established with your tissue samples. Second, it is advisable to perform ISH first followed by IHC. Third, is it also advisable to optimize the IHC assay separately using the RNAscope pretreatment reagent to ensure that your target protein can still be detected following RNAscope pretreatment. And lastly, the dual ISH/IHC assay works better for highly expressed proteins due to protease treatment that is used during the ISH protocol.

Now I'd like to show you a few examples of the dual RNAscope ISH/IHC assay. Shown here on this slide is a non-small cell lung carcinoma FFPE section that was probed for CD8A mRNA (in green) and CD274, also known as PD-L1 (in red), using this RNAscope assay. This was then followed by IHC for CD8 protein (in white) on the same section. And what we can see here – shown at higher magnification on the right – is that the CD8 mRNA signal (in green) colocalizes with the CD8 protein signal (in white), demonstrating the colocalization of CD8 mRNA and proteins.

In another non-small cell lung carcinoma FFPE section, we probed for the immune checkpoint markers IDO1 mRNA (in green) and PD-L1 mRNA (in red) using the RNAscope ISH assay. We then performed IHC for CD8 protein (in white) on the same section. The combined assays indicate no colocalization of the three molecules, suggesting mutually exclusive coexpression of PD-L1, IDO1, and CD8 in different cells. This image demonstrates how the dual RNAscope ISH/IHC assay can provide further understanding of immune function in the tumor microenvironment.

And lastly, here are some more images depicting the mRNA expression pattern of multiple immune checkpoint markers by RNAscope ISH followed by IHC for the CD8 protein. From all of these images, we can appreciate infiltration of CD8+ cells visualized by IHC into tumor regions that are marked by PD-L1 mRNA. Furthermore, the combination of an additional checkpoint or immune marker provides insight to further characterize the immune response involved in the tumor microenvironment.

For more information on the dual RNAscope ISH assay, including protocol recommendations, please visit us on the web or contact us at the addresses shown here. And now, I'd like to turn the presentation over to Dr. Palanisamy.

O’Rourke: Thank you, Dr. Anderson. Yes, next we will hear from Dr. Palanisamy. Dr. Palanisamy is an associate scientist at the Henry Ford Health System and holds an adjunct faculty appointment with the Michigan Center for Translational Pathology at the University of Michigan. His research interests are in the discovery of cancer biomarkers in lymphomas and solid cancers. During his postdoctoral research at the Memorial Sloan Kettering Cancer Center, Dr. Palanisamy's work contributed to the discovery of recurrent gene fusions in follicular and diffuse large B-cell lymphomas. He was the founding director of R&D at Cancer Genetics where he introduced novel approaches to develop diagnostic reagents targeting chromosomal translocations in cancer. Dr. Palanisamy pioneered the application of next generation sequencing technology for transcriptome sequencing and discovered novel "druggable" gene fusions in prostate cancer, gastric cancer, and melanoma. His current research aims to understand the molecular basis of tumor heterogeneity in solid cancers, with particular focus on prostate cancer and its impact in early diagnosis, response to treatment, and clinical outcome. I will now turn the presentation over to Dr. Palanisamy.

Nallasivam Palanisamy, PhD: Hello, everyone. I hope everyone will hear me well. Thank you for the introduction, and also I would like to thank Courtney Anderson for giving an excellent review of the technology and its initial application. So what I would like to do is I'm going to talk about the application of the RNA in situ hybridization – so hereafter I am going to call it RNA ISH – combined with the immunohistochemistry to study the tumor heterogeneity in prostate cancer. But I would like to mention that the technique and the application that I am presenting is not limited only to prostate cancer. So I think any investigators, who are interested in using this technology, as Dr. Anderson mentioned, you know, you have to optimize this protocol in your research needs before using it. But I think at the end of the presentation you'll be able to understand the power of this RNA in situ hybridization combined with immunohistochemistry to assess the expression of individual molecular markers in the different cancer type.

So here, what I'm going to do is the objectives of my presentation is first, because of the essence of time I may not be able to go into the details of each methods, but I will briefly give an overview of what I have done using this technology. So before that, I want to actually put a disclaimer that I'm not actually receiving any funding or any support from ACD Bio; I am just sharing my experience in using this technology for the last seven years in my research, and I have published extensively using this technology. So with that disclaimer, I would like to move on with my presentation.

So the objective of my presentation is to show the comparative analysis of how powerful the RNA in situ hybridization is comparable to immunohistochemistry. Because as you all know, everybody is familiar with immunohistochemistry, but I want in the one slide I would just to show you the power of the comparable quality of the RNA in situ hybridization just by measuring protein at the RNA level. Then I will show you the single-plex combined immunohistochemistry and RNA in situ hybridization in the prostate cancer model, as Dr. Anderson she already showed how she can use it. But I am showing an example how I use it on the prostate cancer. Then, I will show you the duplex or the multiplex approach where we can actually combine both RNA ISH and the immunohistochemistry under the duplex mode so that we will be able to detect up to four markers in a single hybridization. And I will show you, lastly, on two slides, show how I have actually used this technology with a combined multiplex RNA ISH/IHC to study the tumor heterogeneity in prostate cancer.

So in this slide, as you know, that prostate cancer is a well characterized cancer model where many recurrent molecular aberrations have been identified in this distinct subset of prostate cancer. As you can see in the outer line, there are many genes that are listed that are undergoing mutations. But, in the inner circle, if you see it, there are many kind of genes that are listed – particularly the genes that are undergoing translocations or, in other words, gene fusions like the ETS family genes, including the ERG, ETV1, ETV4, ETV5, and RAF1. These are the well characterized recurrent gene fusions in prostate cancer. So, given this enormity of the large number of markers, I have selected only the ones that are most prevalent like the ERG gene fusions present in about 30% of the prostate cancer like SPINK1 in 10% and ETV1 in 5 or 10% of the cases. So I selected only the major molecular markers and applied this RNA ISH and combined IHC technology to study their expression pattern in the multifocal heterogenous prostate cancer.

For the purpose of this presentation, I have selected only four genes – these are the ETS family genes, the ERG, ETV1, ETV4, and ETV5 – and the boxes represents the number of exons in each gene; and the black horizontal bar shows the exact location of the probes where we have designed the RNA in situ hybridization probe. Although one may think that, you know, you can actually construct a plasmid and you can actually make antisense probe by enzymatic reactions, but those type of probes will give more like nonspecific and off-target hybridizations that will compromise the quality of the results. So I would encourage anyone who are interested in applying this RNA in situ hybridization technology work with the ACD Bio technical staff; they'll be able to help you to design a well characterized and a well optimized highly sensitive and specific probe, so that will give you completely clean results.

So here, as you can see, these boxes are the percent of the specific regions where we have actually identified the qualified probes. Like for the ERG, these probes are coming from the 3’ untranslated region; then for the ETV4 and most genes called be used like that. And some like for the ETV5 only the last two exons where we have well-characterized probes. So think it's important that we have to work with the ACD Bio to have this probe design done, so that we will get good quality results. So using these four markers, I will show you how we actually applied this technology.

So here, in this slide, I'm showing an example to show the comparability of the RNA in situ hybridization with immunohistochemistry. So here I'm using the ERG gene as a model because the ERG gene is the most commonly rearranged gene in prostate cancer, in about 50% of the prostate cancer. So here, in this example, on the left side, I'm showing you the tissue section that is hybridized with ERG antibody by immunohistochemistry where you see the specific expression of the protein only in the tumor area and nothing in the stromal area. So what we did was we took a section from the same patient and performed RNA in situ hybridization. As you can see, the punctate dots are actually represent only in the tumor areas and nothing in the stromal area or in the benign tissues. So which tells you the power of this technology and the sensitivity and the specificity of the probes that will recognize only the tumor and the protein that is expressed there. So this is because I don't have much time to show more examples, but I think this is more convincing to show how comparable the RNA in situ hybridization in place of IHC. Because this is very useful when you have a gene for which we don't have a good antibody; I think the best alternative is the RNA in situ hybridization where you can perform this on these tissues and there too, under bright field, you know, you can see that.

In the next slide, this is, again, to show the specificity and the sensitivity of these probes designed by the ACD Bio. So here I'm showing an example of the ETV1, ETV4, and ETV5 probes that is hybridized on a tissue that is positive for only one gene. So for example, in the top panel, which is an ETV1 positive case, which was hybridized with both ETV1, ETV4, and ETV5 probe, and we see specific hybridization on the ETV1 section. And similarly, in the bottom panel, you see the sample is positive for ETV4, and we find a specific hybridization only for the ETV4, but the ETV1 and ETV5 probe did not give any hybridization.

So although this ETS family genes have a high sequence homology, the specific design of the probe that helps to avoid all the nonspecific hybridization or nonspecific signal to give you the high sensitivity and specificity of this probe. So this is, again, important that, you know, we did all these initial kind of quality control validations before actually adopting this technology in my laboratory. And all these data are published in this paper. In the essence of time, I will not be able to show you the complete validation data, but anyone who is interested to learn more about the sensitivity of this probe, so please read this paper where we have clearly demonstrated the application of the RNA in situ hybridization for the deduction of the ETS gene fusions in prostate cancer.

So in this slide, I'm going to show you a little more the application of this RNA in situ hybridization in different type of tissues. It is not only on kind of FFPE tissues like a radical prostatectomy or the larger tissues; where here I've tried to show that this technology can be applied to needle biopsy sample where the sample availability is a major limitation. So especially when you take a biopsy, you don't have abundant material. So you are limited with one or two slides. And if you want to screen like three or four markers with the one or two slides, I think you cannot do these assays in a kind of single-plex board. So it is important that we have developed this multiplex, and it is working across different tissue types.

So here, as you know, the ETS gene fusions led to the first ever molecular classification of the prostate cancer but we have the ETV1, ETV4, ETV5 and ERG then SPINK1 and the RAF gene fusions. They identify account for about 50 to 60% of the prostate cancer. And there are specific drugs targeting these gene fusions have been developed, although, you know, these drugs are not really available for prime time clinical use. And I hope, eventually, this will lead to the clinical use. But right now, many drugs in preclinical studies they have clearly established the potential of this drug targeting the specific molecule. So in this situation, it is important that we need to have a very sensitive assay that you be able to assess the presence of this molecular markers in different tissues.

So in this slide, I'm showing you one poster. Although this is busy, you may not be able to read the whole thing. But this is to emphasize this is the first clinical trial that was performed at the University of Michigan using the ETS gene fusion status. Okay, we are not using the radical prostatectomy tissues or anything else. It is only the bone mets and the lymph node biopsy. So imagine that the amount of material that is available is very limited. And moreover, the bone mets biopsy – because it is also coming from the bone – the tissue has to go through decalcification and other complex process that will compromise the quality of the tissues. So with that, it'll be very difficult to perform like FISH or any other methods. And also, the availability of the small tumor tissue that limits our power to evaluate all these molecular markers, you know.

So that's why we developed that combined IHC and RNA ISH so that we can screen more than one marker in a single experiment. So this is to show the application of this technology. So here in this slide, I'm showing you the systematic evaluation of the molecular markers in prostate cancer. So one example is identified with cancer you can profile first with the ERG because of the higher incidence or higher prevalence of these markers that can be performed with IHC because we have good antibody available. The next day you can do SPINK1. If the sample is negative for ERG, we can do SPINK1, that also can be performed by IHC. But next if the samples of negative for ERG and SPINK1 if you want to screen by ETV1, ETV4, ETV5, there is no specific antibody available. So one has to do either PCR or FISH. So there is no way you can evaluate the presence of these molecular markers at the tissue level. So, given the importance of these molecular markers and the development of specific inhibitors and potential application of these markers in clinical trials and eventually for clinical use, I think it is important to develop more robust sensitive and specific test for evaluating these molecular markers.

So here, in this slide, I'm showing you all these markers like ERG, SPINK1, PTEN marker. Since we have good antibody for ERG, PTEN, and SPINK1, we can perform this IHC either in a single-plex or duplex model. But whereas the ETV1, ETV4, and ETV5, since we don't have a good antibody, we have to perform only RNA if you want to see the expression of the genes at that tissue level, although we can do PCR and other methods. But in order to assess at the tissue level, you have to do RNA ISH. And similarly, there are other markers like this noncoding RNA like PCA3, PCAT1, Schlap1, and KLKP1. These are the well characterized molecular markers in prostate cancer. These are the noncoding RNA with the important functions in the tumor development. So, these markers – because they don't make proteins – it is very difficult to develop antibodies. So have to do evaluation only with RNA ISH. So, given the importance of these molecular markers in prostate cancer, it is essential that we have to develop a sensitive and a robust technology where we can develop very sensitive assay.

So here, in this slide, I want to show you the application of these RNA in situ hybridization on tissue microarray. So here, in such a large tissue microarray – this is a prostate cancer tumor array – we use the ETV1 RNA ISH probe that we clearly show that we were able to recognize only about the three cases that are positive with the RNA signal; whereas, you don't find any nonspecific or off-target hybridization on any other cases involved, which clearly tells the power of this technology and the sensitivity of this approach, which is comparable to immunohistochemistry. And the next, this is a tissue from patient-derived xenograft tissue of a metastatic prostate cancer. So this tumor is grown in a mouse where this tumor is positive for ETV1. So which we performed the RNA in situ hybridization, we were able to show the specific hybridization only in the human tumor tissue, and there is no cross-hybridization with a mouse or stroma or anything like that. So we just had to specifically get hybridization that one can appreciate.

So next, since we have these molecular markers and we have this combined IHC and RNA ISH, so we want to apply this technology to understand the tumor heterogeneity in prostate cancer. Because it is the tumor heterogeneity is a major problem not only in prostate cancer; it is the well-known problem with other solid cancers, as well. The tumor heterogeneity is the main reason that it's actually very difficult to make accurate diagnosis and also to come up with a better prognosis for each tumor. Since prostate cancer is a multifocal disease with independent tumors actually developed at different stages of development, it's important to understand the significance of smaller secondary tumors and the relationship between the extent of molecular changes and the metastatic potential of the tumors. We need to understand what are the driving molecular aberrations in each tumor foci in multifocal cancer. All these multifocal tumors are identical or different, so that is a fundamental question that we want to understand by using the well established markers.

So here I'm going to show you an example how we have recognized the intertumor heterogeneity in a radical prostatectomy sample. Here I am showing an example. This is a small tumor foci from a radical prostatectomy sample; the anterior region is shown in the hematoxylin and eosin staining the whole area containing tumor. And by FISH, initially, we can form that this tumor is positive for ETV1. So by using our ETV1 RNA ISH probe, we are able to perform this hybridization, and we see that the anterior tumor area is actually hybridizing the ETV1 red color signal. But within this tumor area, we found there are two areas that are completely negative for the ETV1. Still, it is tumor, it is cancer, but there is no specific hybridization. Initially, we thought that it could be some technical artifact because of some bubble or something like that. We repeated this experiment and we conform that still this area was negative.

So that prompted, out of curiosity, what I did was we hybridized another section with the family gene – that is the ERG – by immunohistochemistry. What I found was the area that is negative for ETV1 is positive for ERG. So which clearly demonstrates the interactive heterogeneity where under the histological examination although they appear as cancerous with the same kind of stage and grade, but they carry independent driver molecular aberrations, which clearly tells there is intratumor heterogeneity. So this we can do both by RNA ISH and IHCs. So initially we did this on a kind of single-plex mode and two separate slides.

So next I will show you, this is another example to show the tumor heterogeneity. In the second case where we recognized there is a high grade tumor that is positive for the ERG immunohistochemistry. And immediately adjacent to that there is a high Gleason grade cancer that is positive for ETV1. So right next to that, each one, each tumor foci is actually independent and there is no overlap in the presence of these molecular markers. These markers are mutually exclusive in each tumor foci, and they carry two driver molecular aberrations. Which clearly recognizes that each tumor foci that is developing in prostate cancer may be doing by different molecular aberrations. So this is a well recognized kind of example to show the tumor heterogeneity at the molecular level.

So given the importance of these molecular markers, the potential therapeutic inhibitors, and with the combined technology of IHC and RNA ISH, and also given the limitations in the availability of the tumor materials, we want to actually develop a more robust multiplex assay where we will be able to screen more than one marker in a single hybridization experiment. So here I am going to show you the dual IHC and the dual RNA ISH where we can screen all of these four markers on a single slide in a single hybridization method. So here, as I told you before, the ETV1 and ETV4 we can detect only by RNA ISH; and ERG and SPINK1 we can direct by IHC.

So there I'm showing you this method. Each molecule can be recognized with the different colors, as indicated here. You can see it on the tissues. So as Dr. Anderson mentioned, it is important that you perform the RNA in situ hybridization first then followed by the immunohistochemistry. We realize it's important that if you do the opposite way you will compromise the quality of the RNA, so that is potential that doing that way you may lose the RNA. So it's better to do the RNAs first and do the immunohistochemistry on the second day. So it is important to note that you don't have to do an antigen retrieval for the second time for the immunohistochemistry. So what about the pretreatment that was done for the RNA ISH? There is more than sufficient for doing the ISAs; there is no need for a second step for antigen retrieval for IHC. So, after the RNA detections, you can go with the IHC primary antibody incubated overnight or whatever the time that is needed then go for the IHC detection. So you will be able to see these signals very clearly.

So next this is another example how we are going to recognize this tumor heterogeneity. So with this, I will go to the application of the multiplex. So here, in a single tumor foci, as you can see on the left side, only one portion of the tumor is actually positive for ERG, that is a brown stain. And immediately adjacent to that, you are seeing the area that is completely negative for ERG, but then we did the ETV1 the other area is positive for ETV1. So as you can recognize, there is no kind of tumor boundary or anything like that in terms of lesion grade, I don't think that it is any much different. But still, you'll be able to appreciate the difference in the presence of different molecular markers driving the growth of these tumors and the different levels. So this is a well recognized tumor heterogeneity in radical prostatectomy samples.

So as I mentioned before, this technique RNA ISH can be applied in all different type of tissues. So we have worked and we have spent a lot of time optimizing the protocols because we may not be able to use the same conditions that is used on the radical prostatectomy on the TMA on needle biopsy. So it is important that each laboratory spend enough time to optimize this protocol on different type of tissues. So here, we have a well worked up protocol if anyone is interested; please email me or contact me, and I will be happy to share the protocol. I will work with you to establish this protocol in your lab. So here we clearly show that we can actually perform RNA in situ hybridization on needle biopsy on the radical prostatectomy tissues and also on the tissue microarray.

So here is another example that we performed the RNA in situ hybridization on a needle biopsy sample where a tumor foci is actually strongly positive for ETV4 and with the area that are not tumor or stroma where you don't see any background signal or any nonspecific hybridization. So this is a very specific hybridization. And it is important to note that we can recognize the tumor heterogeneity that I talked about in the radical prostatectomy samples you will be able to recognize this tumor heterogeneity on the needle biopsy also. Suppose if you have two independent tumor foci separated by a stroma or benign tissue. Here in this example I'm showing you there are two foci, which are very close to each other but there is kind of in between there is benign tissue. But one tumor foci we see that there's positive stain for the SPINK1 (that is blue), and immediately adjacent foci is positive for ETV1. So you can recognize even this tumor heterogeneity on the needle biopsy sample, as well. So this has some very important diagnostic implications, as well.

So this is the last slide that I want to show you the application of this duplex pattern where you can actually combine both a dual IHC and a dual RNA ISH. So this is a tissue microarray where we were actually fortunate to have tumors that are actually positive for four different markers. We have ERG positive samples, with ETV1, ETV4, and SPINK1 positive sample. The one thing important that I want to mention that in prostate cancer these molecular markers will be present only in the tumor samples. There is no endogenous or background level of expression of any of these genes in the benign tissue or the exclusion negative samples. Because the ETS genes ERG or ETV1 or ETV4 are expressed only after the chromosome translocation, only when there is a gene fusion you will see the expression of these genes.

Another important point that I would like to make is that these markers are mutually exclusive, but you don't find like two markers present in the same tumor foci. As I told you when I showed you in the tumor heterogenic examples, where independent tumor foci are the one driven by different molecular aberrations. So here, in this example, I clearly show that four different tumors that are positive, four different markers like the red color that is positive for the top one, that is positive for ETV4. And right next to that, that tumor is positive for SPINK1. And the middle one that is positive for ERG that is recognized by the green color. And the bottom right that is a tumor that is positive for ETV1 that is recognized by the ETV1 signal. So each one, you know, you can clearly separate these markers with the respective distinct colors and everything you can visualize under a bright field. This is such a very clear example where you can use this combined dual IHC and a dual RNA ISH to detect up to four different molecular markers.

So here, because in prostate cancer these molecular markers are actually expressed in a mutually exclusive manner, but it may not be the same case in other cancer as well. I supposed if anyone is interested in applying this technology in a multiplex approach, to study or to evaluate that is more than one markers expressed in the same tumor then each one has to develop their own assay. So I suppose if these tumors and these markers are expressed like one is nuclear and the other one is cytoplasmic, still you will be able to use it even if they are expressed in the same tissue. So you want to be careful in designing your experiments in such a way that these markers have a distinct pattern of expression either nuclear or cytoplasmic or the mutually exclusive kind. So in that way, you will be able to actually evaluate on these molecular markers in an easy way.

So because of the limitation in the availability of the chromogens, I think right now we have only these four marker evaluation at this point, and they continue to work to develop even adding more colors than this. So right now, the other important point that I want to mention is this 4-color assay but what combined IHC and RNA ISH can be performed both by manual method and also by automatic method. So you don't have to think about the need for having an automated instrument in your lab. Even if you don't have an automated instrument, still you will be able to do it in a manual method. So right now, in my laboratory, all these experiments are done by manual procedures, although we have the automated instruments. We have actually optimized the protocols to run it on the machine. So there also you can do both dual IHC and the dual RNA ISH on the automated method, as well.

So you're not limited with the availability of any instrument or anything like that. If anyone is interested, you know, even without the instrument, you can do it by manual method. And if you have an instrument, you can do it in the automated method. For the application of the automated method please contact the ACD Bio; they will be able to give you all the information and help you set up this procedure. And if anyone is interested to set up this manual procedure, I will be happy to share my experience and our protocol so that they can easily adapt this method in your laboratory.

I think with that, I will stop and I want to acknowledge the people who are involved in this study. My office coordinator, Shannon Carskadon, who is the one who performed all these experiments – IHC and RNA ISH experiments. And Dr. Nilesh Gupta, Sean Williamson, and Dhanajay Chitale, who are our pathologists at the Henry Ford Health System who helped me in actually providing the tissues and evaluating the tumor samples and also the staining patterns. And Dr. Mani Menon, who's our chairman, who is the leading surgeon in the urology department who performs all the radical prostatectomy, which leads to the kind of tissue resources for our research. And this work is supported by funding from the Department of Defense grant to me and also start-up funding from the Henry Ford Health System. And last, I want to thank Advanced Cell Diagnostics for working with me really on a collaborative basis by meeting with all my needs in my research. So whenever I contact them, they are very cordial and very helpful and accommodating in actually designing kind of different probes, and I really appreciate their help in my research. So I think with that I will stop my presentation, and I will be happy to answer any questions from the audience. Thank you very much.

O'Rourke: Thank you both for your informative presentations. It's time for Q&A. If you have a question you'd like to ask Dr. Anderson and/or Dr. Palanisamy, please do so now. Just click on the green Q&A button at the lower left of the presentation window, type your question into the box that appears on your screen, and click on the send button. We'll answer as many of your questions as we have time for. Okay, let's get started. Our first question is for Courtney. Does each dot represent one mRNA or one ZZ pair?

Anderson: Sure. So, each dot actually represents a single mRNA transcript.

O'Rourke: Can I use the RNAscope technology for short RNA sequences like microRNA, or is it only for longer RNA molecules?

Anderson: So, the RNAscope technology cannot be used to detect microRNAs. We can go as short as a 50-nucelotide sequence.

O'Rourke: This one's for you, too. Does dual RNAscope and IHC work better with fluorescent labels or with colorimetric labels?

Anderson: They work with both labels. So again, you'd want to optimize the IHC first, but you can certainly use both either chromogenic or fluorescent.

O'Rourke: This one's for you, too. Why not do the RNA ISH second since it requires protease treatment? Is the RNA too degraded?

Anderson: So, in-house we have tested the protocol, and we found, in our hands, that it worked best to perform the RNAscope assay first followed by the IHC. So, that's what we recommend.

O'Rourke: Okay. Another one for you. Are these combined assays optimized for automated staining. And if so, which platforms were tested? Thank you.

Anderson: Sure, yes, you can perform this automated. We have optimized it on the Leica BOND RX platform.

O'Rourke: Which IHC should be favorable after the RNA ISH process – I mean direct IHC or indirect IHC?

Anderson: Okay. So, really you can use either. The only thing we recommend is that you have a working IHC protocol first. So whatever works best for you in your hands. If the direct IHC works or the indirect IHC, as long as that is working optimally with the RNAscope pretreatment, you can use either one.

O'Rourke: Okay. And, Nalla, this next one's for you. Can I ask which instrument you are using for the automated staining?

Palanisamy: Right now we have the Ventana Discovery XT.

O'Rourke: Okay. And this next one is for you, too. You mentioned briefly that the RNAscope protocol treatment preempts the requirement for heat antigen retrieval later on during IHC. Is this true for any and all Abs that require antigen retrieval?

Palanisamy: So I think this has to be optimized for individual gene targets. But right now, based on our experience, for the molecules that we worked up, so that's what we learned that, you know, we don't have to do a second antigen retrieval. Because initially we thought that we have to do it; when we did it, that compromised the quality of our result that affected the intensity of the signal and some of the expression level was kind of low. So, what we learned was even without the second antigen retrieval step we can get away with that, and we got kind of high-quality results, at least for the genes that we are working on. So, this may not be the kind of same thing when people are working on other genes, as well, they may need antigen retrieval second. So I think that's why it is Dr. Anderson and myself we want to emphasize that if anyone is adapting this technology it is important that you have to individually optimize this protocol both for IHC and RNA ISH for the gene target that they're working on. Then they can actually combine it. Before combining it, I would highly recommend that each kind of gene target has to be tested in a single-plex mode first.

O'Rourke: Courtney, the next one's for you. When ISH is done first then IHC, is there a problem with RNAscope probe binding to the target RNA during IHC washes?

Anderson: No. So, the RNAscope protocol’s all completed first, so the signal has already been detected, so then you would proceed with the IHC. So, you don't have to worry about the probe binding; the signal has already been detected for RNAscope.

O'Rourke: Okay, this next one is for either of you. How do you preserve the RNA quality for ISH?

Palanisamy: So, there is no particular method for preserving the RNA, but what I recommend is always make sure that if you have a tumor in a paraffin block, don't cut the slides upfront. So cut the slides only when you need it. So it is not recommended that you cut the slides and keep it in the room temperature and things like that. So, I would recommend use the slides that are cut within one to three months. They are ideal, without compromising the quality you'll get good results. Any slides that are cut and kept at room temperature for more than like three months definitely because after six months I would not recommend using those slides. So if you have a tumor block, please preserve the block in a temperature controlled condition, so whenever you need just go ahead and cut the slides and I would recommend using fresh tissue sections rather than kind of archived and slides cut long time ago and kept at room temperature. So I think it may work if the slides are actually cut and kept in like minus 80 or in a cold temperature. It might work, but it is up to the individuals to have tested at least a couple of pilot kind of experiments. If there is only kind of material available, I would run some pilot experiment to see if the RNA is still preserved. Otherwise, I would always recommend to use fresh tissues within the one to three months old.

O'Rourke: Okay, this next one is for both of you. Nalla, you might want to jump in first. What is the maximum thickness of the sections that can be done for dual ISH IHC?

Palanisamy: Right now in all of our experiments, we are using the FFPE sections cut at 3 micron thickness. I don't even go for 5 microns, we always use 3 microns.

Anderson: I'll say at ACD, we've tested at 5 microns, so that's all that we've optimized in.

O'Rourke: The next one is, Courtney, how can you quantify the mRNA signal?

Anderson: Sure. So, since the dots represents a single mRNA transcript, you can count the dots. So one could be done manually. ACD has an RNAscope scoring system where a score of 0 to 4 based on number of dots per cell. You also can use a digital imaging software system such as HALO from Indica Labs or ImageJ, software platforms like that that will actually count the number of dots in a particular region or in a specific cell.

O'Rourke: Courtney, this one is for you. Does the fluorescence of FISH affect if we proceed with IHC thereafter?

Anderson: No, we haven't seen any change in the fluorescent signal if you do ISH first followed by IHC.

O'Rourke: Okay, here's another one for you. Discuss if one can use FFPE tissue with ISH. How do you optimize?

Anderson: Yes, you absolutely can use FFPE tissue with in situ hybridization, specifically the RNAscope assay. We first recommend, again, following our fixation protocol for the tissue, which is 16 to 32 hours in 10% neutral buffered formalin. And then, you would want to embed. And as Nalla mentioned, you want to keep your blocks stored properly and use freshly cut sections for the assay. And then, following that, we have a pretty standardized protocol for the actual RNAscope assay.

O'Rourke: Okay, Nalla, this next one is for you. Are all four colors on the same sample?

Palanisamy: No, not all the assay mentioned before these markers are mutually exclusive; all four markers are not expressed on the same tissue. But it is possible because since here I'm showing only one tumor foci on a multifocal tumor, if you evaluate at different foci it is possible that you can see different markers and different tumor foci. But in this example in the last slide that I showed you where all the four colors there are around four different tumors, they are not on the same tumors. Since we are evaluating only one tumor and these markers are mutually exclusive, more than one marker is not expressed in a given tumor sample. But, if you take a multifocal cancer in a given patient, each tumor foci may express more than one marker, yes.

O'Rourke: Courtney, this one's for you. Have you tried the dual ISH/IHC using focal polymers for detection in both assays?

Anderson: Yes, we actually have here. We've tested PerkinElmer dyes, and they work with up to 4-plex.

O'Rourke: Next one is for either of you. If you had to test the efficiency of conditional knockout – let's say specific deletion of a single exon – is it possible to design the ZZ probes within that single exon, assuming the exon is larger than 100 base pairs? If not, what would your recommendation be?

Palanisamy: I think it is possible. I remember I think ACD Bio they have already developed such probes to detect kind of spliced variants in lung cancer. I think – if I remember correctly – it's called the MET gene, where there is only one single exon deletion that leaves the splice variant. So yes, that can be designed using…I think we can actually design probes from a single exon, I think, depending upon the sequence and other things. I think Courtney can add more on that.

Anderson: Yes, we actually have a new assay called BaseScope where we can get down to as short as a 1 ZZ pair probe that can span the exon junction, so we can detect either a rearrangement of the deleted exon and the newly rearranged exon junction. Or, if you prefer, we can also design probes that do fit within that deleted exon. Depending on the sequence length, our probe design team can work with you to find the best probe design that would work for your experiment.

O'Rourke: Okay, this next one is for Nalla. Can you detect the percentage of cells expressing androgen receptor and those other receptors, for example, in stem cells within a single tumor?

Palanisamy: Yes, we have actually used the ACD Bio probe, particularly for the androgen receptor splice variant, the ARB7, which is associated with the treatment resistance. So we have optimized the ARB7-specific probe, and it is working consistently well, yes.

O'Rourke: Okay. Courtney, next one's for you. How do you control probe specificity?

Anderson: Sure. So, we have a two-step method, one during the probe design. Our probe design team will screen against all known sequences to confirm that we are truly detecting the sequence of interest. And also, the double Z design requires that the two Zs bind in tandem right next to each other in order for the preamplifier to bind and the amplification signal to occur. So, if you don't have both of these binding, you can't get any amplified signals.

O'Rourke: Okay. Let's see. This one is for Nalla or Courtney. Can you distinguish normal splice variants coexpression with wild-type MA in single cells and tissue?

Palanisamy: I don't have any kind of experience in doing such experiments. So as long as we have the probes that is specifically able to detect the splice variants and the wild-type, and if we can detect them with the two different chromogen, yes, it is possible to do it on a single cell, yes.

Anderson: Yes, if this is a sequence, that would be sequences that are longer than 300 base pairs, that would be RNAscope would be applicable, and you can do the multiplexing to detect both.

O'Rourke: Okay. We are almost out of time, so this will have to be our last question and, Courtney, this one will be for you. Does PFA or ZN fixative, will this affect the RNA quality?

Anderson: Yes, they all work fine. So you can use any of these fixatives and the RNA signal will still be preserved for RNAscope assay.

O'Rourke: Well, I would like to, once again, thank Dr. Anderson and Dr. Palanisamy for their presentations and answering those many questions. Do you have any final comments for us?

Palanisamy: Well, I would like to say that if anyone is interested in adapting this technology if they are new, if they want to establish procedure in the lab, please feel free to contact me. I'll be happy to share the protocol because there is nothing proprietary from my side. I'll be happy to work with any investigators who want to run this method in their lab. I think someone else asked my email address. So, you may want to provide that. I saw one question somebody is asking for my email address. So please, make sure that everybody, all the contact, they get it.

Anderson: I just wanted to say also if anyone has specific questions regarding the actual protocol or would like help from support, you can contact us.

O'Rourke: Well, thank you both, again, and I'd also like to thank our sponsor, Novus Biologicals, for making today's educational webcast possible. This webinar has been approved for continuing educational credit. Please click on the CE button at the bottom left corner and follow the process to receive your credits. And before we go, I want to let everyone know that today's webcast will be available for On Demand viewing through September 21st, 2017. You'll receive an email from LabRoots letting you know when this webcast will be available for replay. Please share that announcement with your colleagues who may have missed today's live event. That's all for now. Thank you for joining us. We hope to see you again soon. Goodbye.