Manju George 0:00
Hello everyone. Welcome to DocTalks. Today, we have Dr Pashtoon Kasi with us, and he will be talking aboutneoadjuvant, Bot/Bal in resectable mismatch proficient colorectal cancer. But before that, I want to ask Dr Kasi how he got involved in doing work on colorectal cancer. I’m not going to introduce Dr Kasi more, because for people from COLONTOWN, he’s a familiar figure. And the only thing I want to say is that if you’re interested in learning about what’s happening in the CRC world, I would recommend that you follow Dr Kasi on Twitter. So welcome, Dr Kasi.

Dr. Pashtoon Kasi 0:38
Oh, thank you again for the kind words, Manju. So in terms of how I started in oncology and GI, specifically in colorectal cancer, I think for me, the journey has been punctuated by the experiences during residency and fellowship and the mentors and the colleagues you encounter that inspire you to seek a particular pathway. Mayo Clinic is home to a lot of innovation when it comes to the role of GI oncology and colorectal cancer. Some of the regimens that we talk about, some of the figures who have shaped the whole field are folks that you get to walk the same hallway with, especially during the fellowship at Mayo Clinic that allowed me to focus on GI cancers and then colorectal cancer specifically moving forward.

Manju George 1:34
Okay, thank you very much. I also want to say that your poster was one of the most trafficked ones at GI ASCO, so we are really excited to hear about your work

Dr. Pashtoon Kasi 1:43
Yeah, when I went to put up the poster at 6:15am before I could even put up the poster, there were people asking me about the poster. So and then, from six to nine, and then there was a brief break for the Bespoke CtDNA oral presentation, and there were people still walking upstairs with me. And then when I came back at 10:30 we were still talking till like 2:30 till they forced us to get out of the area. So again, it’s a good problem. I lost my voice later that evening.

Manju George 2:19
I can imagine. Okay. Thank you so much for doing this for us.

Dr. Pashtoon Kasi 2:25
No, thanks for having me. Yeah. So do you want me to discuss some of the high level updates and the about the trial in the background next?

Manju George 2:35
Yes, yes, please.

Dr. Pashtoon Kasi 2:37
So you know some of you might have seen some of the discussion, but I kind of wanted to high level describe, all the relevant findings to date. I have some slides, and I also then have the poster that we can dive into. But in terms of background there’s a lot of things going on the world of colorectal cancer in the last few years. Two things that are in particular, of note, that allowed us to develop this particular concept was, number 1), there’s been a neoadjuvant paradigm shift. So the term neoadjuvant means that treatment that you would do before somebody’s surgery happens. People are familiar with the term adjuvant chemo, which is kind of like the adjuvant or mop up chemo that you do to eliminate somebody’s cancer. The idea of doing that treatment beforehand is something that is already considered the standard for patients with rectal cancer, where you want to do your chemotherapy and or radiation if necessary, and the final plan for surgery is the last step. In fact, people have gone from neoadjuvant to something called TNT, which stands for Total Neodjuvant Therapy for rectal cancer. So that’s how quickly that has changed for patients with rectal cancer. In fact, all the trials moving forward are focusing on TNT as a strategy, not just neoadjuvant, but in general, for not just for colorectal cancer, but for all cancers, neoadjuvant studies, there are many things that why would one would want to do the systemic therapy part?

Dr. Pashtoon Kasi 4:11
First, a lot of our surgeons who like to operate, they’ve actually been in the proponent to help us understand that maybe them operating right away is not always the best strategy. There is suggestion that they might be perturbing the micro environment of the cancer by doing surgery first. There could be reasons to affecting your immune system in an adverse way, where they’re taking a lot of lymph nodes and things out at the time of surgery, you might be affecting drug delivery or the immune response that your body itself may be mounting. There’s also suggestion that maybe keeping the primary or the mothership where everything started would be helpful, that be part of the treatment plan, as you introduce, especially something like immunotherapy, and also just the whole stress of surgery itself. Even though patients are getting minimally invasive surgery or robotic surgery, especially for colon cancer, and literally going home the next day. Those are all things that are happening every day in our hospitals and other hospitals that are Centers for Excellence for colorectal cancer. But also you have to realize the body is still surviving that insult or injury, so to speak. So immune changes in a negative way could also be in the post operative phase. So for all these reasons, there’s suggestion or idea that maybe doing some treatment beforehand might help increase the chances of success, and some of the parallels have been happening in other tumor types. Melanoma or skin cancer, is one of the tumor types where a lot of research has been focusing on the vaccine, immunotherapy, before and after surgery, and they’ve been striking differences of the exact same treatment done in a clinical trial, randomized fashion, where outcomes were significantly better if the same treatment was done before surgery, as opposed to doing surgery first and thereafter.

Dr. Pashtoon Kasi 6:17
In the world of colorectal cancer, one trial that a lot of colleagues and patient advocate as well as caregivers, are familiar about is the story of the MSI high, or mismatch repair deficient colon and rectal cancer. And we’ve noted that in the metastatic setting, while a third of patients with MSI high cancers, even with drugs like pembrolizumab, and at the actual GI oncology conferences a couple of days ago, at ASCO, they also presented data with Ipi-Nivo, with updates. So there are striking differences, but there’s still a proportion of patients who don’t respond, and it’s anywhere from a third to at least, 20% if not more. We’re still trying to figure out why that case is, but when Dr. Chalabi and their group moved the treatment with their NICHE-! study to neoadjuvant setting. Nearly everybody responded. So I guess there is definitely some pretext to why neoadjuvant would be a good strategy. From a clinical trial standpoint and from a scientific standpoint, neo adjuvant — they call like them as the window of opportunity, meaning that you could still do whatever you’re trying to do, but get a quick readout as opposed to doing calls with hundreds of patients, I think, with novel clinical trial designs and how things are evolving, this is something that is being challenged, that we need trials that take years to mature, that take hundreds of patients & effort to realize that something is not working. With novel precision medicine based focused design, and in this case, the Precision Medicine being the setting itself, you can probably get away with smaller numbers, especially if the difference is significant.

Dr. Pashtoon Kasi 7:58
You don’t need huge trials to tease out those minor differences depending on the therapy you’re dealing with. So that was one reason why we thought that neoadjuvant would be a great fit. But the other parallel update that has happened in the last couple of years while patients with the so called cold tumors, or mismatch repair proficient tumors or microsatellite stable tumors, all meaning the same, which is the fair majority, 95% in the metastatic setting, and anywhere from 80 to 90% in the non metastatic setting are the MSS cancers that unfortunately don’t respond to immunotherapy. In the last few years, we’ve seen this regimen called the Bot Bal, or Botensilimab, combined with Balstilimab showing the first hint of a promise of novel immunotherapy that works in patients with cold tumors. One interesting finding that shaped the change in the trial moving forward is they noticed that folks who had active liver metastases, and the definition of active liver metastases was that, it’s not like you could never have had liver metastases. It’s just that at the time when you enroll in the study, they had to be resected, ablated, removed, or radiated at least six months ago, because the first 17 are patients that they treated, the ones who were benefiting the most were the ones who didn’t have liver metastases at the time.

Dr. Pashtoon Kasi 9:28
So moving forward the phase II study, and then the randomized phase II that has been completed third quarter of last year, just eagerly awaiting readout, there was a huge proportion of patients who did not have liver metastases at the time have benefited. So that’s how this regimen came to note in terms of patients with colorectal cancer. So keeping these two big advances in mind with the neoadjuvant paradigm shift, and then this regimen Bot/Bal, we had proposed as an investigator initiated trial to Agenus, the company who makes this drug–how about if we could consider the same combination, but in the neoadjuvant setting. We also looked at designing this in terms of as a parallel to the NICHE study that was published in Nature Medicine. Also there’s been an update to that presentation at ASCO, with over 29 patients treated with the immunotherapy that is approved for MSI high in melanoma, the ipilimumab or nivolumab, or ipi- nivo for short. So the idea was to compare and contrast how would this combination Bot/Bal fare in the setting of mismatch repair proficient and deficient tumors in the neoadjuvant setting, we designed it to kind of keep safety in mind.

Dr. Pashtoon Kasi 10:56
Its the last thing you want to do is in a person who can get colon cancer surgery tomorrow, next week, curative intent, is to jeopardize the surgery. Because we cannot cure patients with colon cancer for the most part, without surgery and or chemotherapy for the ones who need it, who are high risk. So the main objective of our trial was safety. So one of the things that we wanted to make sure was any surgery that got delayed would be considered a negative and the study would have to be shut down if two or more patients in the 12 patients that we wanted to treat would lead to a delay or cancelation of their surgery. So even a delay beyond 12 weeks was considered as what we call in our scientific world, like a dose limiting toxicity or something that would want us to stop the trial prematurely. So that was the main goal. And of course, we wanted to make sure that surgery happens safely as well, so any kind of serious adverse events or toxicity happening in the perioperative period even up to 90 days or 100 days after surgery, because immutherapy is a like a vaccine, it stays in your system.

Dr. Pashtoon Kasi 12:00
So could it even impact somebody’s clinical care beyond the first few months was the main end point, and so we really find combed any kind of side effects that were attributed to treatment and or any side effects period, that happened during the build up to the surgery as well as in the post operative period. But at the same time, we, of course, wanted to see the efficacy question as well. And we on purpose, had MSI high cancer patients as well, because if immunotherapy is already known to work there in the subset that have mismatched efficient tumors, we should have proof that it’s working in those patients as well. But more so, the interest and focus was on the cold tumors, which is mismatch proficient tumors. And at the time when we were designing the study, I would say anything more than a couple of patients responding would have been still promising for further investigation, because pretty much the bar has been close to zero in the metastatic setting and some hint of signal in the early stage setting. So any kind of responses for us would have been considered promising.

Dr. Pashtoon Kasi 13:13
Just to give you an idea of the dose, you know, the actual doses that have been studied have been the up to 150 milligrams of bot, which is a multi immune activator. Some folks, while can argue that this is the next generation, or the second generation, CTLA-4 antibody, with the mechanisms that we were able to find in our trial, as well as some of the correlative work. It’s not simplistically, just another ipilimumab. It is different, which is why even the toxicity that we are seeing is different, and also the efficacy that we’re seeing is different in also in parallels in other cold tumors like sarcoma and other tumor types. The fact that this is working is is very promising. So we only chose half of the recommended dose, or the lowest possible dose that we thought would be effective, and also just one dose of it, as opposed to ongoing immunotherapy, which is every six weeks, up to four times in some of the studies. So just to kind of give you an idea, this was the lowest possible dose that one could have thought of or conceived to be given in this study. As shown here in this cartoon as well these are kind of like don’t eat any signals or signals that prompt the immune cells to stay away from the cancer that some of these immune checkpoint blockade as the name checkpoint suggests is to kind of break those interactions so that the immune cells are able to recognize, expand and activate, and also try to kill the cancer. But there are other mechanisms that are it seems like there are some immune cells that are the good immune cells that are killing the cancer. But then there are some immune cells that can dampen the response, these are Tregs or bad immune cells. So it seems like it’s not as simple as just activating your immune system, but kind of the diversity that’s expanded is of value to note. And then which of the cells are expanded more than the other, the ratio of it in terms of circuitry, is important to recognize.

Dr. Pashtoon Kasi 15:22
So jumping onto the results, and the waterfall plot, where things hanging down is a good sign, and every single line is a patient and their response, which was actually the surgical response, meaning how much of the cancer was alive or dead when the pathologist looked under the microscope. Just to kind of give you an idea, I mean, this was also a learning experience for me as well. The pathologist do look under the microscope, and they have a surgical sample, but they don’t necessarily look at every single looking corner of the specimen. They, of course, examine the lymph nodes, and they examine how much tumor is left. But in general, not every single section of a huge specimen is looked at. But in this particular trial, our surgical pathologist, Dr Erica song is kind of worked hand in hand as a co- PI on the study, and also one of the senior author on our paper. Is also important, because without the pathologist, and of course, it goes without saying, without the colorectal surgeons who see these patients, this trial would not have seen the light of day. And so these are in some ways conservative numbers, because even in some cases where there were just a couple of cells floating around in a dead mucin pool, like this person and MSI high on the farthest right, who had a 98% responsepretty much the whole entire ginormous tumor was all dead, but just a small cluster of cells were present. So we made sure that we reported all of this so that we, in any way, the last thing you want to do is overestimate something and lead to a wrong signal. So these are, in fact, conservative numbers. But it’s hard to fake no cancer being present at all, or the cancer disappearing. And what was very fascinating as an experience, and very rewarding as an experience for us was, the it was a subjectivity, and the adjectives that our pathology colleagues started describing at some of these tumor board meetings, where every week here at Cornell, on every Thursday night, we review every single patient’s case, every new patient gets discussed, and that’s true for all academic and community centers as well. It’s good practice to be making decisions as a team, but then that’s also a meeting where we look into the microscope together as to what did the pathologist find? How aggressive was the tumor? What did it look like? What was the mutation testing? And also in patients who got any chemotherapy or immunotherapy, what was the pattern response? Did you see immune cells around the cancer? How much of the cancer was alive or dead? But the way they were describing from the very first patient that got this as a treatment was what did the patient get? What did you give the patient? And we’ve never seen immune response like this much, or the exuberant nature of the response, or the striking area of immune cells, or tons of inflammation and necrosis, meaning dying of the cancer cells. There were things that even the first two patients that were the cold tumors that had over 90% and 85% kill, the patients here that are on the third and fourth that prompted us to write a whole publication that was in the Oncogene journal. And we can share the links afterwards, and it’s available online, free to read because it has some striking images that we had pre and post, even just the first two patients. We wanted to get the word out, because for things to work and also to work as quickly, remember, these patients could have gotten surgery as early as three weeks from starting the treatment. That’s very important to recognize that if you can have just three weeks of a treatment causing this much kill of the cancer, that is unprecedented in many ways. In fact, a lot of patients as early as 21 days had surgery.

Dr. Pashtoon Kasi 19:32
We wanted to keep this trial pragmatic and practical meaning that right now in the United States, a patient like I said, with colon cancer is often seen by the surgeon first, and with the surgery team as well as the patient and caregiver alike, the immediate plan is to get the tumor out of your body as soon as possible. Now again, I would do the same if this was me or my family member, but at the same time. It’s good to realize that this didn’t start this week or last week, this month or this year. Some of the polyp to adenoma to carcinoma sequence can take three to five years to develop. That’s why, if somebody has a colonoscopy that’s clean, the next one is not due to, like, five years or so. So that’s because we know that this is how things develop. But we still have to remember there’s the psychosocial piece. So the surgery happening ASAP on the calendar whenever the surgeon can get an operating date, which could be tomorrow, it could be next week. So most patients, if not all of them, actually already came with a surgery date in hand, and we’re told that I’m getting my surgery on February 4. The surgeon asked me to do the pre op testing and the COVID testing, and I’m also here to talk to you, because I hear you have a trial. So keep that in mind we wanted to keep it as pragmatic as possible, so that all we were asking patients is you’re getting a surgery on February 4, a couple of weeks away from now. We have this immunotherapy that is not yet proven or tested in the neoadjuvant setting, but may have value, especially for those with advanced cancers, with chemo and surgery, may or may not even cure all of them, is to consider this trial. So we were only pushing out surgery by a week or so at the most, so that was something that was acceptable to all the patients that the trial was offered. So it also shows the interest in the patient and caregivers as well who participated in the study, and the trust that they showed to consider something which is not necessarily the norm.

Dr. Pashtoon Kasi 19:48
And like I said, as the trial progressed, was also very fascinating was patients and their caregivers, also the surgeons were themselves asking, can we postpone the surgery by another couple of weeks? Because maybe we can get more more shrinkage and kill all the cancer? So that’s how you might see, I’ll show you in the poster as well, that there were patients who had surgery a little later. Wasn’t because of any side effects or anything. In a positive way it was because the signal was getting more profound as we waited even a few days longer, that prompted the surgeons and the teams taking care of the patient on a week by week basis to maybe purposely allow for things to be delayed some.

Dr. Pashtoon Kasi 22:24
As shown here in dark green, are the MSS, or cold tumors. You can see from cancers entirely disappearing. The one of the first patients who is a 100% is somebody who had so much bulky tumor that even one of my colleagues who saw me the next day, who where the patient was starting the trial, was a little apprehensive about a person who was having some symptoms and bulky tumor to consider a trial that you know potentially may or may not work. So that patient’s symptoms and pain and things improving, and then the cancer being down to nothing it was very rewarding. The second patient was a 100% is somebody who actually had rectal cancer. So it’s important that while this trial and NICHE-1 looked at colon cancer, and a lot of the mismatch deficient studies from Dr. Cercek from Sloan and other teams have looked at Dostarlimab in rectal cancer where, of course, living with a permanent ostomy and can be a life altering experience from a quality of life perspective. So we did allow rectal cancers to enroll as well, as long as there was a plan for surgery as next and no chemotherapy or radiation. Because, of course, the ones who are candidates for chemotherapy and radiation, maybe in the future, in different trials, but for the context of this trial high rectal cancers or rectal cancer, where there’s a plan for surgery and no chemotherapy or radiation, are pretty much like colon cancer. So they were allowed to enroll.

Dr. Pashtoon Kasi 23:47
So we had one, mismatch deficient MSI high rectal cancer that had a 100% response. The second, mismatch repair proficient MSS rectal cancer had a very low rectal cancer, it would have been something called an ultra low LAR, something that could have risked an ostomy as well, that the type of surgery that was done had changed and ended up in a complete response, as well. We had an 85% and also we had a 10% response. And also, as we understood some of the micro metastatic disease in the pattern response, just trying to give you an idea, as shown here in the cartoon on the right hand side, we also noticed that what was striking was even if the cancer was present, it was kind of at the luminal or the tip of the colon. So if you can imagine this cancer reaching through the walls of the colon and the lymph node that’s involving, typically when we’re killing the cancer with chemotherapy or radiation, even if it’s eradicating let’s say approximately 90% of the cancer here is dead when it’s dead and scattered in different parts, and there could be cells floating around that are not bigger than we’ve seen, because we never really fail locally. For the most part, things are getting so much better with surgery and radiation, with sophisticated software and stuff. It’s more so the question of systemic recurrence or the cells kind of being present in the soil, just like grass with pollen or grass with weed, or in terms of things spreading elsewhere. But with immunotherapy, the responses that we see, we call it like inside out, versus like root up or kind of like serosa to mucosa, meaning, even if there was cancer, the immune cells, like a wave, were destroying them in a way where, kind of like the trash was just at the door for pickup. So even if there was leftover cancer, 75% alive, 90% alive, as long as it was in the specimen that is already out of the patient’s body in the trashcan honestly, from my standpoint, it’s great to have more kill, but at the same time, as long as we didn’t leave any cancer cells behind, that’s more important, because for MSS cold tumors, they’ll get their surgery anyways. It’s not like we’re planning to avoid surgery here, but the fact that you can eradicate micrometastatic disease, that was something very fascinating and novel, which was part of the like I said, this paper is available online on Oncogene, and also some cool immunofluorescent analyzes looking at immune cells that we did with this company called Rarecyte Inc, which required very minimal tissue to be sent to them. But you can see pre and post shown here in this golden is the cancer itself and around the golden cancer is the immune cells, different kinds. CD4, CD8, good cells, bad cells. Foxp3 it’s like a swarm of bees that you can see between pre and post, just a few weeks apart. The swarm of bees around the cancer is such exuberant increase, and even within the same patient, parts of the body that had some cancer left behind, versus part of the body that had more cancer, you could see the activity of immune cells like a busy office versus an office after 5pm as you could see, the differences between the type of immune cells, how active they were pre and post. So it was something that was very striking.

Dr. Pashtoon Kasi 27:24
While arbitrarily, you can argue that for major response, anything less than 50% is considered maybe not a success, and anything that 10% or less is considered “a failure”. I would argue, if even for the 0% and 10% since we saw some inflammation, and if we eradicated the micrometastatic disease, and the person still had surgery and everything is removed with the goal being cure, then even these might be not necessarily failures, in my opinion. So I would say that every single case has been a valuable insight since even the patients were at 50% 25%, when we checked their cancer cells or DNA after surgery with a blood draw to see if there was any leftover cancer, in any of these patients at more than 30 time points over six months, none of them had a recurrence. So the fact that we have such a strong black and white signal kind of speaks to the mechanism of action here, and also our understanding, a lot of these crude measures of response were invented back in the day with the esophagus cancer getting chemotherapy/radiation. So these numbers may need to be revised. In fact, a new study published in lung cancer, for example, question some of these arbitrary cut offs. Who said 90% is the cut off or 50% is good? We kind of use the real world where we come up with these arbitrary numbers of six months or three months or 50% is good, or the glass half full or half empty. But they actually showed that it’s a continuous measure that every 10% increase, or as a continuous variable, the more shrinkage you were getting that was equating to the being cancer free odds years down the line. So this is kind of changing the understanding, so to speak, of what we understand with with what’s happening in these patients.

Dr. Pashtoon Kasi 29:18
And so the study, by the way, is expanded based on these results. Iwanted to kind of highlight some of the key conclusions first, and then I’ll show some details regarding the poster as well. So we found it to be a safe and active regimen. Again, going back to the safety that we didn’t discuss yet, there was only one person, and again, that’s the same person who had the 100% response who had diarrhea, that’s from inflammation of the colon called colitis, that can be managed proactively with what we call steroid sparing drugs, or as the name suggests, you don’t need to use steroids to hamper the immune system or dampen the immune system. And ifrecognized early can allow the side effects to be nipped in the bud. That patient had the diarrhea on a Friday. It’s always the Friday afternoon when you hear about these events, and he still was able to get surgery the following Thursday, so the surgery was not delayed. And in fact, that’s the same person who also had the 100% response. So if you talk to some of these immunologists and oncologists who’ve been treating and have had a lot of experience with immunotherapy of the years, of course, we don’t want you to have severe side effects, but even in the days of melanoma, back in the day, having some rash or having some diarrhea or having some thyroid inflammation, it was one of those things that patients, caregivers and oncologists were looking for, and there would be more worried if there was none of it, because how do you know? There’s no direct of knowing if somebody’s immune system is activated, not activated, some of these side effects, symptoms, what they were going by and not just this particular regimen. Across the board, having any immune adverse events is very different than coming off of the trial let’s say if you had a serious adverse event for chemotherapy or target therapy, where as soon as you stop the treatment, pretty much the cancer is going to do what it was doing before. With immunotherapy, it is very different. I’ve had patients with vaccines or different kinds of immunotherapy that didn’t even get more than one dose and had to be taken off because of whatever adverse events. It’s the tale of patients who are alive and alive years out where the word cure is being used, that is happening even in the patients who there’s no specific number of doses, in my opinion, and no specific duration that you need to get immunotherapy as long as it’s working, it’s a it’s a gift that keeps giving.

Dr. Pashtoon Kasi 31:43
And because of the down staging we saw and the responses we saw in MSI high, the goal that we’re going to ask in the next set of patients is, and that was also something that we actually changed last minute again, because we also heard from our surgeons that in a good way that they were not inclined to operate on the next patient who was MSI high who got immunotherapy, because they never find cancer, and often everything responds so briskly, just like a marshmallow melting, that sometimes it’s actually it’s harder for them to operate because there’s a lot of scar tissue and so much robust infiltration from an immune standpoint. In fact, even at the discussion section at the ASCO, somebody asked about strictures developing. And we’ve had some similar experiences as well with just even pembrolizumab used in advanced setting, where the surgery was only done, because in a good way, there was so much response that actually there was a narrowing that had developed as an outcome of that. So maybe in those cases, we have less reliance on surgery and no surgery at all, or give that decision to the patient and caregivers and the surgeon. If they wanted to go for surgery and everything was dead, that will still be a success. If they didn’t want to go for surgery and just had no cancer worsening, that should still be considered a success. So we’ll allow for that as a composite or as an option, to give and empower the patient, so to speak, and the caregiver to make that decision. And then for the ones who are going for surgery, we’ll look at how much of it is dead as the key outcome variable. But going back to the MSS or cold tumor story, if we were able to get this much shrinkage with just three to four weeks of therapy, the idea is what would happen if we let the immunotherapy brew longer?

Dr. Pashtoon Kasi 33:25
We have some idea from the NICHE-3 study that was recently presented from Dr. Chalabi and colleagues, where there were deeper responses with the immunotherapy, nivo, with LAG-3. And while some folks were discussing if LAG-3 is the one that’s causing this, I think the big difference between that and NICHE-1 is also the fact that they use the monthly four week dosing of NIVO, so the average time of surgery was longer. So that just the fact that immunotherapy was allowed to simplistically bloom for longer, in my opinion, that’s what led to more responses. So we’re going to formally investigate without changing the doses of the Bot, just one dose still and keeping safety in mind, we’re going to allow for patients to have double the duration of the same regimen in MSS moving forward, and compare and contrast the results with the results what we have here.

Dr. Pashtoon Kasi 34:18
So with that let me just quickly, just show a little bit of the the poster as well, which had some additional data that is available online. But one of the things that we found, as shown here in the poster is shown here in the red are the patients who had CT DNA positivity, who through different platforms, again, this was commercially available tests, robust, deep analysis in-house that we’re doing with some of the scientists here, which had more insights, but none of the patients in the post op setting that have been tested, some tested as much as four times on both plasma only assays, as well as tumor informed assays, have remained negative to date. And if you look at the mutations, for the next 12 patients that might be seen in any cancer center, this is a mix of anybody and everybody. We had a patient who was HER2 positive, KRAS mutant, KRAS wild type, P53 mutant, Wnt mutations, MSI high. We had young folks were germline Lynch syndrome. We had older folks who had the somatic MSI high, from as young as 26 years of age to as old as 78. We only had four Caucasians in our trial, which is quite the opposite of the story of all the trials that are conducted in the United States, where sometimes, even in hundreds of patients, you barely find one African American enrolled in the trial. That’s important for immune therapy. That’s not just about equity and diversity, but also there isdifferent types of HLA and immune features that distinguish us apart. Again, not just race, even males, females, we had six of each. Same thing, for example, we saw was, there were five people, four out of five, all young females who had this thing called early immune activation, where they had fevers as high as like, 102 103, 101 but no signs of infection. And the symptoms of the flu, like symptoms that go along with it, that again, with just some proactive Tylenol or Alleve or naproxen. If necessary, in some patients, just the low dose, 20 milligrams of prednisone, just for a couple of days to kind of nip it in the bud, was something that overcame that. Again, goes to show that there are biological differences in your immune system as well, that have to be kept in mind from, male female biology, as well as different races. So we had Southeast Asians, we had African Americans, we had Hispanic Mexicans, we had Middle Eastern we also had Caucasian. So we hadpeople mix of older, younger. So in a good way, there’s no signal to differentiate who is the one who had more response and where the one who is this fast. Because we had folks older, whichever race, who also had the response, and younger, whatever age. Because, of course, we are noting there’s also the difference in biology between young onset, colon versus older onset. And you know the plenary at ASCO-GI, for those of you who attended or heard was in fact, on the rise of cancers in young adults from Dr Kimmie Ng. That was the plenary. So it’s important to note that biology differences might be there between young and older onset. So the fact that we saw no differences so far on all the crude analysis that we’ve done to date, nothing from a CPS or PDL-1. In fact, just a few hours ago, I got results from all the patients before and after PDL-1. And in every single patient, the PDL one went through the roof so and a lot of these patients had the so called cold tumors with not much immune inflammation. So there’s not much to tell or tease apart. We’ll have more additional results from Rarecyte to go over. And as you can see in this line plot, you can see all kinds of immune cells, CD20, B cell, T cells, that went up. But then if you look at the cells that are, what we call immune proliferation, versus the ones that are that dampen the immune cells, the ratio of that was going down, we should explain some of the responses that we are seeing. So I know there are a lot of questions, so I think I’ll probably just stop there and we can maybe look at the questions, and then based on the questions, we can revisit things on the poster, or the talk.

Manju George 38:58
Okay, sounds good. Thank you very much, Dr Kasi, so shall we start from the top of the questions?

Dr. Pashtoon Kasi 39:05
Yeah, yeah.

Manju George 49:42
So the first question is, how do Bot/Bal findings apply to unresectable cases? And then I’ve heard on COLONTOWN that liver Mets disqualify one from receiving Bot/ Bal. Is this accurate?

Dr. Pashtoon Kasi 52:04
Yeah, it’s a great question. We saw a lot of CD20 cells going up as well. And I think in the comments it’s also mentioned that it was a tumor up stage as well. I think the issue, as I said, is as opposed to rectal cancer, where you can pretty much, it’s not 100%, but it’s really good, in terms of MRI pre op staging versus post op staging. Of course, there is discrepancy, but colon cancer is a mess. Colon cancer does not get MRIs. Colon cancer is diagnosed on CT scan, and if the lumen is collapsed, you may not even be able to assess how big the tumor is. And lymph nodes are always tricky to evaluate on a CT scan. So overall, the work from European colleagues show that colon cancers are under staged when it comes to the MSS variety, and overstaged for the MSI, because in the MSI, the lymph node may just be swollen because of your immune system activation. So they could be misinterpreted as stage III, and then post op, they may be stage II. So overall, we saw pretty much clearance for them. For the most part, I think the anybody who waited at least a month to get their surgery, we saw like that inside out where the lymph nodes were negative. There was no lymphovascular invasion, no perineural invasion. And now the tumors were beyond T1, T2 so I think that the N1, N2 patients, if they had waited a few weeks longer, which we will test in the upcoming cohort, we would have seen the activity go down.