Multiplex immunohistochemistry (IHC) is coming to the clinic. That’s the general consensus, according to Dr. Joe Yeong, immunopathologist at the Singapore General Hospital and a pioneer in the field of multiplex IHC. We spoke to Dr. Yeong to learn how he developed his research interests, why multiplex IHC is important in immunopathology research, and future of this tool in the clinic.
Tell us about yourself and your research interests. What drew you to immunopathology?
I’m a research immunopathologist at the Singapore General Hospital Pathology Department. I also have a few labs outside the hospital. One of the key labs is in the Institute of Molecular and Cell Biology at the Singapore Agency of Science, Technology and Research (A*STAR).
While doing my PhD, I studied the tumor microenvironment. After I graduated, I went into the clinical setting and joined the pathology department. At the time, immunotherapy – especially in Asia – was still just starting. We felt that this was an interesting area, and a little bit of a natural progression from studying the tumor microbiology. There are many immune cells there. How do you classify them? How do you understand the significance? All of this becomes relevant to immunotherapy.
One of the key things that drew me into the field was the Opal/Vectra system. I was doing my PhD at the Singapore Immunology Network. PerkinElmer put a demo Vectra machine there for a while and let us play with it. I was one of the ones who got to go play with it and make it work. Very soon after that, I graduated and joined the pathology department. There was one key component really missing there, because when you are in the research setting, you can do all the staining and all these things manually. But in the clinical department, you are not allowed to do manual staining.
We’ve of course increased our productivity, standardized, etc. But if you look at it from the other angles, from the start, we’ve been doing all these things in the clinical department and in the pathology department. It’s a clinical pipeline. The entire staining process of Opal is very similar to the general conventional IHC that is used in the diagnostic setting. So it’s very convenient for our colleagues, especially the oncologists. We actually even do this as a lab developed test. Some of the Opal panels, we perform them as quite a highly standardized test, not only as a research tool.
You’ve been an early adopter of multiplex immunohistochemistry/immunofluorescence (IHC/IF) technologies. Why is multiplex IHC/IF an important tool for immunopathology?
Multiplex IHC gives you the opportunity to actually stain more colors. You can talk about CODEX, or MIBI or CyTOF. People are doing 50 or 60 colors. As a translational group, we are more interested in how to do this in a fashion that can be for clinical tests, or at least for clinical trial monitoring. We care about the costs and the turnaround time. You can spend a lot of money to run one sample, generate big data and analyze it for a long time. There are groups that focus on this, but ours focuses more on the translational part.
The key component is how to make the cost bearable and have an acceptable turnaround time in a translational or clinical setting.
Our focus is the clinical kinds of tests in the market, for example as easy as PD-L1, right? It would be good to have PD-L1 core staining with a tumor marker or immune marker, or even with a macrophage marker. Multiplex IHC definitely gives you a lot of advantages to be able to accurately quantitate the PD-L1 positivity. This becomes very important, because if you want to give the patient anti-PD-1/PD-L1, and other immunotherapies, you obviously need more markers to accurately quantitate this.
Even in a conventional situation, there are already pathologists that, in the past one or two decades, stain two different markers in sequential sections and, in their mind, try to reconstruct it. It’s a no-brainer to use this technology now, especially the Opal/Vectra. It’s been there for years. The recognition, the acceptance levels are high.
It’s also a no-brainer that you want to study more lineage markers. For example, I mean, everybody knows Immunoscore, from HalioDx. It’s very famous. CD3, CD8 – people believe that it is definitely important to score these. And it can also potentially be applied in the clinical setting, for many, many different situations. Why aren’t people doing it? I think the key bottleneck is that you do not want to spend too much tissue on something, and also if the quantification is done by a pathologist, you don’t want to spend more of their time.
But with multiplex IHC, once you optimize it, you have a panel. It can be automated or at least very high throughput. For example, if you’re interested in PD-L1, you stain PD-L1 with some other markers to know if it’s a PD-L1 colocalization. There’s no harm in putting CD3 and CD8 in the patient panel; the readout is almost simultaneous. Then you know the CD3/CD8 quantification, or even spatial information for this patient. All this looks like very easy, basic stuff, but at the translational and clinical level, you need a lot of people and critical mass to push this and then make this something that people believe can be executed in a larger population.
One of the panels that Joe and the team are currently translating
The key component is how to make the cost bearable and have an acceptable turnaround time in a translational or clinical setting. If you show that this is practical and doable in a real-life setting, people will start to adopt it. Instead of asking, “Why?”, they will ask, “Why not?”.
We co-founded this task force last year and now have over 30 members across the world that are particularly focused on the Opal/Vectra system to actually figure out how to do standardization for multiplex IHC.
Why did you choose the Vectra/Opal technology for your multiplex IHC/IF studies?
There are a couple key points. Number one is definitely – especially if we’re talking about Opal system – it is almost no different to the general conventional IHC pipeline, right? The only difference is instead of putting in the brown colored DAB, you put in the fluorescence.
And people can always argue, “You put in fluorescence, you put in TSA, the amplification system is different from DAB,”. But all this can be optimized. As long as you optimize and validate your system, I believe this can be, to some extent, interchangeable.
Our group has published a few papers talking about this as well. Most of our papers actually show the markers side by side. It’s comparing the conventional to the fluorescence and the correlation is quite decent.
There are a few working groups – even under SITC – some task forces and working groups that are purely focusing on this kind of standardization. I’d like to take this opportunity to introduce a worldwide task force that we call the Jedi Council. I co-founded it with Bernie Fox from Providence Oregon, Houssein Sater from the NIH, and Jaime Rodriguez-Canales at AstraZeneca.
We co-founded this task force last year and now have over 30 members across the world that are particularly focused on the Opal/Vectra system to actually figure out how to do standardization for multiplex IHC. How to design controls, how to design panels, that kind of thing.
We believe this system is closer to the clinical and translational setting, especially the Opal staining system. Most of the other available technologies are a little bit on the discovery and research end. I personally don’t see, in the very near future, that any other staining (reagents) system can catch up to the translational setting or get close to the clinical setting. In fact, that might not be their business strategies too.
What is the potential for multiplex IHC/IF? How do you see it being used in the future?
I think if we actually navigate this thing well, the potential is tremendous. There’s a lot of effort in this area, not only from private companies like Akoya. There are more and more researchers, pathologists, oncologists, immunologists. They’re starting to understand this value and they’re trying to push this as well.
I think the general consensus is that multiplex IHC is going to come to the clinic. Once everybody gets involved and there is a systematic mechanism, as you talk to the FDA or talk to regulatory bodies – not necessarily FDA, if it’s a non-USA setting. Sometimes it can be CAP, or doing a lab developed test, which is quite common, especially in Asia.
So again, how to actually convince people and make it doable and practical is the key. But if you settle those things, in the next three to five years, you will see a lot of significant difference in this particular market, and in the translational and clinical area.
What’s next for you? Is there any exciting research coming up in the future?
We are actually working on some COVID-19 autopsy samples. I think in COVID-19 research, even now, most of the studies are focusing on the blood. The tissue levels, spatial information, all these things, I don’t see many papers there – it’s been quite lacking there. But we all know autopsy tissue levels, information is very important, especially for a new disease. I believe these will be impactful findings as well. Hopefully we can share results with the community soon.
Dr. Yeong is part of a task force called the “JEDI Council”, which is working towards multiplex IHC standardization across the world and currently has over 30 members. The task force was co-founded by:
- Dr. Joe Yeong, Research Immunopathologist, Singapore General Hospital Pathology Department
- Prof. Bernard Fox, Molecular & Tumor Immunology Laboratory, Harder Family Endowed Chair in Cancer Research, Providence Oregon
- Asst. Prof. Houssein Sater, Clinician Scientist, NIH
- Dr. Jaime Rodriguez-Canales, Pathologist, AstraZeneca, USA
