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- [00:02]
A
Welcome to the American Society of Hematology Conversations with Blood Authors podcast. This blood podcast is hosted by Dr. James Griffin from the Dana Farber Cancer Institute in Boston. In this episode, he discusses Lipid Nanotubes Unmasked Neutrophils for complement attack with Dr. Christian Gorzalani. And he chats with Dr. Rebecca Karp Leaf regarding immune thrombocytopenia in patients treated with immune checkpoint inhibitors.
- [00:30]
B
Hello everyone, my name is Jim Griffin. I'm one of the blood podcast editors. I'm in the heme malignancies unit at the Dana Farber Cancer Institute. We have two terrific papers to discuss today. The first of these papers is entitled Lipid Nanotubes Unmasked Neutrophils for complement attack by Dr. Christian Gorzalani from the Department of Dermatology, University Medical Center, Hamburg, Uppendorf in Hamburg, Germany.
- [00:59]
C
Dr. Korzolani, thank you for this nice introduction and hello to everyone. Yeah, you asked me to give a short summary of what we did and what is the meaning of our finding. We studied neutrophieds since a couple of years and what we observed a long time ago actually is that neutrophieds come never alone, they always come as a swarm. And what we also see is once they attached to the vascular wall, they. They changed their phenotype. That was the starting point of our research. We tried to understand what happened to the neutrophils once they bound to the vascular wall before they enter the tissue. And we realized that we did some very simple experiments using microphoidics and applying shear stress to the neutrophils. And the first movies were quite boring actually. Then the postdoc working on this lab at that time then added a lipophilic dye and then realize that neutrophils that are exposed to shear stress then release this very long nanotube. So like chewing gum that is pulled out of your mouth. And this length of this nanotubes is really dramatic long, so hundreds of micrometers. So much longer than the need to feel that was the starting point. And then we got really interested in that and we tried to understand is that something that is really happening also in vivo? Is that relevant? Does it have any meaning? And to make a long story short, finally we really realize that this lipid nanotubes connect the neutrophieds to the complement system. Complement system is very complicated. Usually if I have a lecture and telling students about complements, they don't like it. But actually it's very simple. It's very ancient part of the immune system. It's very efficient, very rapid, very powerful, similar to neophytes. And they come together by forming these nanotubes. Usually all our cells are protected against the complement attack and complement is usually there to fight against bacteria or other pathogens. But the nanotubes, once they are getting out of the nitofit, then suddenly they are unmasked, they recognized by the complement opsonized. And then suddenly these nanotubes can alert the immune system. So they can phagocytose by other neutrophils, by monocytes. And we also think that they can be recognized by endothelial cells and mimicking something like, yeah, I would say like a bacteria. And by this they can alert other immune cells. The elegant thing from my personal view is that the formation of nanotubes and the recognition of the nanotubes by the complement system is really fast. So the nanotubes are formed within a few seconds, so 20 seconds roughly. And that is enough for the complement to recognize them. And after a few seconds everything is there and you have a response. And it's a very elegant and fast system without any complicated pathways. To summarize, neutral fields attached to the vascular wall. Formation of nanotubes takes place complement system recognize them and then this is a start point for further inflammation, for alerting ne by cells to activate other parts of the immune system.
- [04:27]
B
Thank you. Just a couple of questions for you. You mentioned in your article that these long nanotubes really hadn't been described before, particularly in their ability to interact with the complement system. You found that both in vitro studies and in vivo studies in mice. Why do you think these were hard to find in the past?
- [04:47]
C
Very good question. We also asked ourselves this question. Two problems. They are, as I said, they have formed within a few seconds and so that's difficult to really find them. So you need to switch on the camera just in the right moment. And they are nano, so they're super tiny. And yeah, you need high resolution microscopy to really see them. So the. Usually the, the diameter of the nanotube is below the optical resolution. And you need, for example, we use step microscopy super resolution to really realize them. So I think that are the limiting points in the past why no one has seen them before.
- [05:27]
B
Do the neutrophils, as they accumulate complement proteins on these nanotubes, do they get subjected to lysis themselves?
- [05:37]
C
We have never seen that. What we think is that once the nanotubes are formed and recognized by the complement, they just disrupt from the neutrophil body. So they are somehow dislocated and just a tiny residual amount of the nanotubes stays with the nutrified but majority of the tube is just disrupted and then part of the circulation.
- [05:59]
B
And it sounds like these nanotubes are important for amplifying the innate immune response to other neutrophils. For example, do they just signal to other neutrophils or are there other cells and other immune cells that become activated by this process?
- [06:16]
C
In our study we focus on neutrophils and we also had to look on monocytes and natural killer cells. We've seen that the response of other nucleus to those nanotubes is really strong. So hundreds of genes have been upregulated using RNA sequencing. But also monocytes react to them. A little bit less strong, but they do react. And we believe so that we are just doing that in the moment that also for example, endothelial cells could recognize those opsonized tubes. At least from our perspectives that makes sense because nucleophites are bound to endothelium produces nanotubes. And we think that these nanotubes can also activate the endothelium, maybe then opening the vascular barrier to facilitate the transmigration. But that is something that we need to do.
- [07:05]
B
Are there any clinical situations where it might be to our advantage to inhibit this process? And if so, how would you do it?
- [07:13]
C
Also very good question. That's also what we currently try to better understand. Neutrophils are involved in many different diseases. We studied tumors, we studied vasculitis and also trauma. And I think especially anthropathysiological disease conditions like vasculitis or trauma, when we have really strong traumatic events and there's an overshooting complement response and it makes sense to quench them to block nanotube formation just to prevent the sepsis like situation that there's too much inflammation. I think that is there are quite many examples where that might make sense. Other question is how to do that. There might be two possibilities that we try to prevent nanotube formation. That's what we currently tries to understand. What kind of molecule we can maybe target to prevent the formation. Maybe more easier is to develop an antibody that somehow recognize than nanotubes or complement degraded nanotubes.
- [08:19]
B
Thank you very much. Terrific paper. We're going to go on to our next presentation. The paper is entitled Immune thrombocytopenia in patients treated with immune checkpoint inhibitors. And the discussion will be led by Rebecca Leif, who's in The Division of Hematology Oncology at the Mass General hospital in Boston.
- [08:40]
A
Dr. Leif, thank you so much. It's really an honor to be selected for this podcast. As hematologists, we see patients who are treated with immune checkpoint inhibitors and have hematologic immune related adverse events from those checkpoint inhibitors. So taking a step back, checkpoint inhibitors like PD1 inhibitors, CTLA4 inhibitors have really revolutionized oncology over the last few years and are now approved for over 20 different tumor types and immune checkpoint inhibitors. They downregulate tumor T cell inhibitory signaling and lead to enhanced T cell anti tumor effects. But they also have these secondary adverse immune side effects. The most common ones we think about are colitis or hypophysitis dermatitis. But hematologic immune related adverse events IRAEs are well described. Immune thrombocytopenia ITP associated with checkpoint inhibitors has been published in small case series and single institution reports. But we really wanted to comprehensively study immune checkpoint inhibitor ICI associated ITP to figure out what the risk factors are for ICI itp, how it's treated most effectively, what the outcomes are and actually really important for clinical care is are we able to rechallenge patients who develop ICI ITP with a checkpoint inhibitor because perhaps they're responding or there are no other antineoplastic options. This was a really grassroots effort. I and the senior author David Leaf, contacted six other large academic cancer centers across the United States and we said, hey, we're doing this project, you want to help out? So people looked at their database from their institution over the last eight years, from 2016 to 2018, 2023 is when we locked the data. It was really when institutions really started using electronic medical records. And we said, get a list of all of the patients at your institution with cancer who have been treated with immune checkpoint inhibitors over this time period. And then by a combination of bioinformatics and looking for keywords as well as manual chart review, we were able to identify from a denominator of over 86,000 patients treated across the US with checkpoint inhibitors to 214 cases of ICI ITP. So again, this was a combination of bioinformatics and then classical hematologists at each site reviewing about 1,000 records to help determine is this truly ICI ITP. And we had various definitions. You can see in the paper platelet count less than 100,000, the patient couldn't have received chemotherapy in the last 30 days and other more plausible causes of thrombocytopenia if the patient had that, they were excluded. So we got 214 patients and we matched them with 428 control patients who received ICIs during the same year but did not develop ICI ITP. What was very interesting is that across these seven sites, the overall incidence of ICI ITP was very similar. So the overall incidence was about 0.25%. And if you looked at the individual sites, there really wasn't much variation in terms of the incidents at each site. We looked at risk factors for development of ICI ITP and that included combination therapy. So patients who are getting more than one class of ICI simultaneously, a stage four cancer, and not surprisingly, a low platelet counts. At baseline, ICI ITP developed at a median of eight weeks from the start of ICI therapy and most patients had an isolated thrombocytopenia. For a few patients, we had data on antiplatelet antibodies and thrombopoietin levels. And in six of eight patients who had antiplatelet antibodies checked, all three autoantibodies were positive and thrombopoietin levels were similar to patients with de novo itp. The most common treatments were glucocorticoids, IVIG and thrombopoietin receptor agonists. Not surprisingly, of course, sort of extrapolating from the data in de novo or primary ITP and response rate was pretty similar to what we see in patients with De Novo ITP. Most patients recovered within about 2 to 2.3 weeks. There was a quick recovery once patients started on treatment and actually some patients just with withholding the ICI, their platelet count recovered spontaneously. 76 out of those 214 patients were rechallenged with ICI and only a third of those patients went on to develop a recurrence of their ICI itp. So that was very helpful to know that not everybody goes on to have a recurrence of this hematologic immune related adverse event. And then the patients who recurred and then were rechallenged again, 73% of them actually had a complex recovery after that second re challenge. So it does look like it's probably in some patients okay, to rechallenge with ICI if that is the treatment of choice. Finally, we did show that ICI ITP and its severity so how low the platelet count was were both independently associated with mortality. The more severe the itp, the higher the cumulative risk of death. In summary, we showed that ICI ITP has an incidence of about 0.2, 5%, which was very consistent. Across seven major cancer centers in the United States. 75% of patients achieved an overall recovery from ICI ITP after their initial episode. Most common treatments were glucocorticoids, IVIG and TPO RAS. Only 30% of patients developed recurrent ICI ITP after they were rechallenged. And ICI ITP and its severity were independently associated with a higher risk of death. So I think it's just important overall that we pay attention to these hematologic ires. As we're treating more and more patients with checkpoint inhibitors, I suspect we will see these more and more and how to manage and how to think about re challenge, I think is very important.
- [15:16]
B
Thank you. Really impressive study, particularly in the scope and the size of the patient population and the details. Very nice study. It sounds like these patients, some of them were on a single immune checkpoint inhibitor and some of them were on a combination therapy. There's several immune checkpoint inhibitors in the patients who were just getting one. Was there any difference between PD1 inhibitors, for example, and CTLA4 inhibitors?
- [15:42]
A
Most people, I'll say not everybody, but most people who are getting CTLA4 inhibitors were getting them in combination with PD1. And then when patients were re challenged, oftentimes that CTLA4 was dropped and they were just continued on a single agent PD1 or PD L1. And it looked like that if you dropped the CTLA4, you had a lower chance of recurrence ICI ITP. So if you again went from combination therapy to single, and that typically included dropping that CTLA 4. And I don't think that's surprising. I think we see more of these IRAEs with the CTLA4 inhibitors.
- [16:19]
B
Generally, how many of the patients had a major bleed in the setting of their itp?
- [16:25]
A
That's a great question. So six patients had a major bleed. An invasive procedure was required to stop the bleed in two of those six patients. And then 16 patients developed a clinically relevant non major bleed. But it didn't really look like the platelet counts per se. The absolute absolute platelet counts was reflective of whether or not somebody was going to have a major bleed or a clinically relevant non major bleed. For instance, the median platelet counts at the time of major bleeding was 43 and the median platelet counts at the time of clinically relevant Non major bleeding was 19. We say in the article that just like with ITP, the absolute platelet count may or may not be an accurate predictor of how likely these patients are to have a bleed.
- [17:12]
B
You mentioned that a significant fraction of the patients were re challenged with inhibitors after they had recovered from the initial bout of itp and they did fairly well. Any thoughts on that? Why would they do so well on rechallenge?
- [17:27]
A
That's an excellent question. Some of these patients were treated with combination therapy and then they were dropped down. I'm not sure if we're sort of somehow changing, manipulating the underlying auto immune milieu. If you get a course of prednisone or if you get some ivig. There were other people who were continued on TPO ras when they were rechallenged. So I think that that might be another option. You know, of course we're always concerned about abrogation of the immune response in these patients. When we're treating them with steroids right off the bat after they get checkpoint inhibitors, they have an irae. We're always hesitant to give them higher doses of steroids. And I think that there's. The TPORAs are actually a very elegant, nice option here. They seem to work very well within ici itp. You don't have that side effect of dampening down the immune response from the checkpoint inhibitor. And I think that you can continue them while you consider Rechallenge as well.
- [18:21]
B
And just one last question. You mentioned that these patients had a lower survival rate than the control population. Were there any specific things that could cause this higher rate of death?
- [18:32]
A
We actually did not collect data on the exact cause of death. Sometimes it's of course difficult to tease out, but we hypothesized that if the platelet count is low, the patients aren't going to be re challenged with any sort of therapy. Sometimes we have these platelet goal posts that need to be met before additional chemotherapy or immunotherapy is given. And then of course, not every patient had a bone marrow biopsy. So we can't for sure say, or we can't with certainty say that no patient actually had myelothesis or cancer involvement of the marrow as a cause of the thrombocytopenia. Again, we tried as hard as we could to adjudicate these patients, but there's still some unknowns.
- [19:09]
B
Thank you very much and I want to thank both of our presenters for great discussions. Really interesting articles. Thanks.
- [19:18]
A
Thank you for listening to this episode of Conversations with Blood Authors. To read the articles, visit bloodjournal.org this episode is copyrighted by the American Society of Hematology.