BETA - What is immune status? What does it mean to profile the bone marrow (immmune) microenvironment?

In this Health Tree University lesson, we will discuss how the bone marrow microenvironment is studied. Practically, profiling means to study the characteristics and how the bone marrow microenvironment is in a specific snapshot of a patient. So we talk a lot about different cell types, immune cells, myeloid cells, macrophages, so profiling really is a way to test and see how many cells of different types are there, what are their characteristics, what's their function, and what which type of cytokines, which type of genes they are expressing. How is the bone marrow microenvironment profiled or examined? We have many ways that we can do that with. There are traditional ways and more advanced ways that are still at the research levels. Previously we used to profile, and we still do that, we used to profile the immune microenvironment with something called flow cytometry, and there's something a bit more advanced right now called CytOF. Basically, the way that is done is we get those cells and we culture them for a little bit in test tubes with different antibodies, and those antibodies actually go to those cells and latch on to specific targets, and each antibody has like a signal that lights up on its end, so imagine that each cell will be bound to one or more different antibodies depending on the different proteins it's expressing. Each different protein will have a different antibody, and each antibody will have a different And then after that happens, we allow those cells to pass through a machine called a flow cytometer, and that machine will be able to tell us how many cells express which markers. So this way we'll be able to tell, oh, we have this amount of T cells, this amount of B cells, this amount of NK cells, macrophages, neutrophils, because each type of those cells have different markers that they express on their surface, so depending which antibodies actually latched on those cells, when they pass through the machine, each one will give a different signal, and the machine will then be able to tell us, okay, you have this many of each type of cells. So what we do is when we do a bone marrow biopsy, which all of our patients are very familiar with, and it's an unfortunate procedure that we have to do, we take the fluid part of the bone marrow and we do some processing on it and we incubate those cells with those antibodies and then we run it through the machine, and that's how we know how much of each immune cell is present in their bone marrow, and this is basically profiling of the bone marrow microenvironment. What helpful information can flow cytometry yield? Historically, flow cytometry, that is a technique that allows us to practically look and quantify and tells you a percentage of specific bone marrow population is what has been used to study the bone marrow microenvironment. And for instance, if you have a population of lymphoid cells, they are not all the same. So for people like me, I'm a physician scientist, so I study a lot of these changes in the response to therapy, knowing that a patient has 100% or 80% of lymphoid cells is not as relevant as knowing what actually these 80% of cells are and what they're doing and why they're there. For instance, we know that patients with myeloma tend to have a lot of immune-suppressive cells, so they tend to have higher expression of a subset of T cells called T regulatory cells that has practically an inhibitory function towards the antitumoral immunity. They tend to have less natural killer cells that are activated, and they tend to have less of these active cytotoxic T cells and more of exhaustive T cells. So all of this is practically profiling. It's done in very sophisticated ways using specific markers, like we're putting flags to different cell populations and moving them around and saying, hey, you are exhaustive T cells, you are B cells, and kind of go from there. Besides flow cytometry, are there other ways to examine the bone marrow microenvironment? Otherwise, more easily, and this can be done practically by every pathology lab, we can use techniques such as immunohistochemistry, and that's a very basic technique that's been going on for several years, like 50 years, and kind of based just on that, it can tell us the expression of specific markers on myeloma cells or T cells or B cells. So for instance, we know that myeloma cells usually are CD138 positive. That's what sometimes you see on your report of a bone marrow biopsy. But then we can look at a little bit of different markers. We can check for CD38, that is the target of isotoxin and benzeratumuma. We can look for BCMA expression. We can look for CD56 expression, that's kind of like a barometer marker on plasma cells. So all this information is relevant for us to understand a little bit of biology with the hope to kind of translate that, then to kind of like predicting who will respond to therapy. So for instance, in my lab, we saw that the expression of CD56 on myeloma cells can promote and increase the response to deratumuma. So these are kind of like small things that we are looking at like patient samples, we did some studies in the lab. So all these types of profiling are really what bring us to practically connect science profile of the bone marrow microenvironment to actual patients. What newer techniques are being explored to profile the bone marrow microenvironment? More newer version of this is single cell sequencing, which is a totally different process. It's still at the research level, we don't do that clinically yet. Flow cytometry is done clinically, like at the clinical setting. Single cell sequencing is a slightly different method, where we actually are able to pick each cell individually and be able to put it, encapsulate it in like a bubble and lyse the cell break the cell apart, release the RNA content of it. RNA is basically what the cell changes the DNA from. So the DNA changes to RNA, and this helps the cell produce proteins. So each cell is encapsulated in a bubble, it gets lysed. So all the RNA is released. And this RNA is bound to pieces of other RNA that we provide. And those, each one has a specific marker on them that tells us that everything that was in that bubble comes from that cell only. And this happens to thousands or even millions of cells individually. And then this gets run in a sequencer, what we call a sequencer, and it gives us a lot of sequencing data out. It's way more complicated. And then a bioinformatician has to go all over the data and analyze it. And then we'll be able to know how many cells we have from each type. But not only that, the extra information that the flow doesn't provide is what each cell is actually expressing as well. So the genes they're expressing, the proteins they're expressing, what makes them different from other cells. So we don't just look at groups of cells in general, we look at every single cell, what it's expressing, what it's producing, and that gives us way more data than the traditional flow. And this is where technology is heading. It's still at the research level, it still needs more and more validation, but we're slowly getting there. Why is immune profiling important to the myeloma patient? Immune profiling is really important to the myeloma patient. After all, you've got a B-cell clone sitting in your bone marrow that has already made you immunosuppressed. It's kind of your B-cell function. It's not good. You can profile T-cells. You can do that with flow cytometry. You can do it in the laboratory. At some level, the profile of your T-cells can govern your response to these new immunotherapies. The other side of immune profiling is, what does the therapy do to your immune system long term? If there's a significant immune paresis, and maybe you could catch COVID and have serious or pick up regular bug infections, then that's a problem. And so before we fall headlong into using bispecifics and CAR-Ts on everybody, I think we need to look carefully at what the impact of these therapies are on the immune system so that we don't misplace something and then people come to harm. As well as looking at efficacy, always be very safe with medicines and choose things that are highly active but well tolerated. And the anti-CD38 antibodies have been one of these choices where it seems they combine well with other therapies, and we haven't seen a lot of side effect profile from them. So I'm hopeful that's where we go with the bispecific antibodies, but I think we need to be watching for the immune system and the long term immune impacts and secondary infections.