Latest / Behind The Knife: The Surgery Podcast / Journal Review in Colorectal Surgery: ctDNA in Colorectal Cancer and Anal Squamous Cell Cancer
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- Journal Review ctDNA in CRC and ASCC_Final === [00:00:00] Welcome back to the Behind the Knife listeners. I welcome our colorectal oncology team back to the field. Today, our topic is circulating tumor DNA in colorectal cancer, as well as anal squamous cell cancer monitoring and, as well as its utility. I'm welcoming back myself, Janet Alvarez, Winnie Zambri, Philip Bauer, and Dr. Jay Joshua Smith. Today, we have our esteemed guests coming from multiple hospitals around the world. I'm very appreciative of everyone's time. First, we have Dr. Paul Ramicor. He is the director of Colorectal and Anal Cancer in the Department of Radiation Oncology, an associate attending radiation oncologist at Memorial Sloan Kettering Cancer Center. Dr. Jeannie Tee, medical oncologist of the Lower GI Unit at the Peter MacCallum Cancer Center. She's also the lower GI research lead, as well as the chair of the Lower GI [00:01:00] Working Party of the GI Cancer Trials. Lastly, we have Dr. Takayuki Yoshino. He is the professor of the Global Center of Research Excellence for Advanced Medicine, also known as G-CORE, at the Keio University School of Medicine. He's the executive advisor to Hospital East Director, as well as the director of the Department of Global Oncology, the chief of the Department of Gastrointestinal Oncology at the National Cancer Center Hospital East Thank you everyone for joining. Now, with the rising rate of early onset colorectal cancer, there's a dire need for prognostic biomarkers as adjuncts to treatment. Circulating tumor DNA has been shown to be a promising marker of molecular residual disease across solid tumor types. ctDNA are small fragments of DNA released by tumor cells in the bloodstream. The amount of circulating tumor DNA found in the blood is related to tumor burden as well as cell turnover. It makes up a small percentage of total [00:02:00] circulating cell-free DNA, which is shed by tumor cells into the blood. Colorectal cancer has been shown to shed high rates of circulating tumor fragments, including cells and methylation markers. This allows for high ctDNA detection rate in colorectal cancer. The proportion of patients with colorectal cancer in whom ctDNA can be detected depends on the extent of the tumor volume and ranges anywhere from fifty percent in those with non-metastatic disease to nearly ninety percent in patients with metastatic disease. Winnie, can you describe a little bit more about ctDNA? Sure. So detection of ctDNA offers a non-invasive approach for evaluating the molecular profile of a patient's tumor. Current research is evaluating the utility of tracking ctDNA at different time points along a patient's treatment course. It has been established as a prognostic biomarker in early localized disease. After curative intent surgery, minimal [00:03:00] residual disease or ctDNA detectability helps us determine recurrence risk and guide future treatment. Phil, can you tell us about the types of ctDNA testing? Yeah, there's currently two approaches that are used for ctDNA assessment in molecular residual disease and colorectal cancer: tumor-informed and tumor-agnostic or plasma-only testing. Tumor-informed testing identifies mutations from a patient's tumor tissue and then designs personalized probes to detect those mutations in plasma Whereas plasma-only approaches do not require tumor tissue and historically were considered less sensitive. However, newer methods using methylation and epigenomic markers have improved their sensitivity. Plasma-only testing may also provide faster turnaround times as it avoids the need for tissue sequencing and custom panel development. This faster assessment can be clinically important in the adjuvant setting where delays in therapy may reduce treatment effectiveness. Along those lines, we would like to discuss a clinical case.[00:04:00] A fifty-two-year-old man is diagnosed with locally advanced rectal adenocarcinoma staged as cT3N1. He underwent total neoadjuvant therapy with chemoradiation followed by surgery. Final pathology showed YN0 disease with negative margins. Postoperatively, imaging showed no evidence of residual disease. His CEA level was also normal. As part of surveillance, a tumor-informed ctDNA assay was obtained four weeks after surgery and was negative. This suggested no molecular residual disease. Dr. T, how would you counsel this patient to proceed? So perhaps let me provide a bit of context by describing clinical trials. So firstly is a DYNAMIC 3 trial, so this is a multicenter randomized phase 2/3 trial in which patients with resected stage three colon cancer underwent a tumor-informed ctDNA testing four to five weeks after surgery, so [00:05:00] one single landmark time point, and then were randomized in a one-to-one ratio to a ctDNA-guided management or to the standard care control management. Clinicians nominate their standard care regimen prior to randomization, and in the ctDNA-guided arm, those with a negative test then received a deescalated less intensive therapy, including a shorter duration of treatment or fewer chemotherapy agent, whereas the positive patient then receive an escalated more intensive chemotherapy regimen, including triplet folfiri. The primary objectives are twofold in this study. One is to demonstrate that we can safely deescalate treatment in ctDNA negative patients compared to standard management, and then to see if we can intensify chemotherapy and improve outcome in the ctDNA positive patients. So the primary endpoints for the [00:06:00] negative patients are three-year recurrence-free survival. For the positive patients are two-year recurrence-free survival. We also included several secondary endpoint, including treatment re-related hospitalization, toxicity, and ctDNA clearance with chemotherapy. So let me go through the ctDNA negative de-escalation cohort. So seventy-three percent of patients tested negative in our study, and a ctDNA-guided de-escalation as demonstrated to reduce chemotherapy exposure by study design, where the main de-escalation strategy was from a doublet oxaliplatin-based chemo to a single agent fluoropyrimidine alone. The oxaliplatin used decreased substantially with ctDNA guidance from eighty-nine percent down to thirty-five percent in the standard of care arm The three-year recurrence-free survival remained high across both [00:07:00] study arms, eighty-eight point one percent versus eighty-five point three percent. Small two point eight percent detriment in recurrence-free survival using the ctDNA guard de-escalation. This study, I have to note that did not meet its pre-specified non-inferiority margin. Uh, nonetheless, the difference are small. We then also did a subgroup analysis looking at clinical low risk disease and versus high risk disease, and we note that in those with clinical low risk disease, so which is relevant to this case, where T one to three and N one disease have a very favorable prognosis at three-year with a three-year RFS exceeding ninety percent in both study arms, so ninety-three in the standard care arm versus ninety-one percent with de-escalation strategy. Obviously, this reduction in chemotherapy intensity led to expectedly signifi-significantly reduction in [00:08:00] treatment-related hospitalization as well as high-grade toxicity of interest. And moving on to the ctDNA positive high-risk group, so twenty-seven percent of patients who tested positive. We found unfortunately intensifying conventional cytotoxic chemotherapy did not improve outcome We also looked at beyond just the positive and negative binary outcome, looked at molecular burden of ctDNA levels after surgery and see if they could further stratify patient's outcome. And indeed, when we divide them into quartiles, we found the patients with the highest tumor burden, molecular burden, had the worst outcome with a three-year RFS of twenty-three percent compared to patient with low level ctDNA. The lowest level had a relatively better outcome, a three-year RFS of seventy-seven percent. And then the question is, if you're ctDNA positive and you receive chemotherapy, what [00:09:00] happened to your ctDNA status following chemotherapy, and does that matter? And we, we looked at ctDNA clearance with the blood taken four to eight weeks after surgery, and we found that about forty percent of patient had persistent ctDNA detectable after three to six months of chemotherapy. And those who had persistent ctDNA detectability, or in other words, they failed to clear their ctDNA with conventional chemotherapy, they have a very poor outcome with a three-year RFS of only fourteen percent. So really, I think to summarize, put this in context, what it w- what it means for, for patients in the clinic, I think we are one step closer to showing that post-op ctDNA negative patient may safely avoid more toxic chemotherapy in reducing treatment burden and toxicity, albeit with a slight detriment in the recurrence-free survival. It probably should be discussed with the patient if they do ask [00:10:00] if they could use ctDNA to guide their therapy. Importantly, what we found was that the positive patient, they have a poor outcome despite standard chemotherapy and remain really a clinical, a major unmet need for patients, where further strategy needs to be investigated further to improve the outcome. So yeah, I think it's suffice to say that post-op ctDNA remains one of the most informative and strongest biomarker we have to date in colorectal cancer and may be helpful in indivi- individualizing treatment intensity. While de-escalation showed promise in reducing toxicity for low-risk patient, escalation strategy have not yet demonstrated clear survival benefit, and this underscore and highlight the need for new therapeutic approaches for this high-risk patients. Perhaps I'll move on to talk about what we'll do for stage two colon cancer. Janet. So we earlier, [00:11:00] prior to DYNAMIC 3, we also did a randomized trial in stage two colon cancer. So this is a phase two study of biomarker-driven adjuvant therapy in stage two colon cancer, where patients are randomized in a two-to-one ratio to have the disease managed by ctDNA monitoring or by conventional clinical pathological criteria. And in the ctDNA-guided arm, those with a positive result received adjuvant chemotherapy, whilst those with a negative result were observed only. And the difference between this trial and the DYNAMIC 3, the stage three trial, is that we collected two blood timepoint at both week four and week seven, and only if both are negative, then the patient observed. If one were positive, they receive adjuvant chemotherapy. And the high level results from this study was that the ctDNA-guided approach was shown to reduce almost half the number of patients receiving [00:12:00] adjuvant chemotherapy without compromising recurrence-free survival. And it appears that the positive patient derived considerable benefit from adjuvant treatment with a three-year RFS of eighty-six percent in the study compared to historical observational study where if the positive patient without further treatment would have a RFS of less than twenty percent. And we similarly saw at ASCO this year the CIRCULATE-German AIRO study where they randomized patient with ctDNA positive for treatment versus observation have similarly shown in the per protocol analysis a significant benefit in terms of recurrence-free survival with the use of chemotherapy in this high-risk group of patient. And importantly, what we also show is that those low-risk ctDNA negative patient who are just observed, so untreated with adjuvant chemotherapy, have a very good outcome with a three-year RFS of ninety-two point [00:13:00] five percent. And in subgroup analysis looking at just the T three disease, their RFS is even better at ninety-five percent, indicating that adjuvant therapy should not be considered for ctDNA negative patient who are stratified as low risk. So I think the DYNAMIC 2 study really support that post-op ctDNA is a potential part for biomarker that could guide adjuvant therapy decision in stage two colon cancer, with benefit shown in the DNA positive patient who receive adjuvant chemotherapy Thanks for that context, Dr. Ti. As you noted, those trials are focused on colon cancer. In this case, we're discussing a patient with rectal cancer who has a first postoperative ctDNA as negative. Can you extrapolate any of that data to this patient's care? I think the concept will be remain similar. In this situation where the patient have had already [00:14:00] received total neoadjuvant, including at least four months of doublet chemotherapy preoperatively, having a negative test after surgery would give me confidence that I should be avoiding further adjuvant chemotherapy. Nonetheless, with the caveat, we know that the landmark ctDNA testing at one single time point may not be perfect, so it doesn't have hundred percent sensitivity. You will get some false negative, particularly in rectal cancer. We do note that can miss some lung metastases based on our prior work, so important to still do serial testing as well as imaging to pick up some of these false negative cases. So in our case, let's say for this patient at his one-year surveillance, his ctDNA becomes newly positive despite normal CT imaging and no associated symptoms. Repeat testing actually confirms persistent ctDNA positivity. Dr. Ti, what steps would you take from here? Oh, [00:15:00] this is one. I always say only do a test if you know what to do with the results. Repeat testings with just CT, I would probably do additional imaging in this scenario, scan, or even an MRI of the liver, really looking for any oligometastatic disease that we can try and salvage. And there's some data now showing that with CT DNA-guided surveillance, you can increase the proportion of patient undergoing oligometastatic-directed therapy, which would hopefully improve outcome for the patient. So further imaging to see if we can find some more occult disease that's missed by CT. If despite that we still show no evidence of-- clinical evidence of disease, then I would be looking for a clinical trial for the patient. There are now multiple different post-adjuvant clinical trial looking at novel therapy to salvage these CTNA positive patient, either [00:16:00] following their standard care treatment, or if they're within a year of surveillance, they become positively eligible for this trial. Now, if there are no clinical trials available, then I think it's just close surveillance for these patients will be important. Well, thank you so much for that fantastic discussion. We're hoping to see this work also further the care of rectal cancer patients as well. Great. Thanks so much to Professor Yoshino, and Tee, and Ramasur for joining us today. What a completely all-star cast that we have in front of our Behind the Knife listeners today, so really excited that they're joining. I'd like to thank Professor Yoshino, you know, I don't know if some of the readers understand it, but TNT, a tumor-neutralizing approach in rectal cancer's not been widely used, but is now being actively studied in the Ensemble trial, which you're a major part in leading. But as you've heard this, the data, and then Professor Tee's work, can you tell us a little [00:17:00] bit about how findings from Galaxy, even the most recent data, are applicable in these scenarios? For example, the scenario where the patient is persistently negative versus the patient who's negative and becomes positive, and then maybe even provide some insight on how many tests are needed in these patients as we're continuing to learn this, and then obviously gaining some insight from your group. Yeah. Thank you very much for the chance to talk about. So firstly, I would love to mention about the GALAXY study. Study is a prospectively conducted large sc-scale registry designed to monitor ctDNA for patient with clinical stage two to four in colorectal cancer who underwent a complete surgical resection. Plasma sample were collected serially up to twenty-four months after surgery, with patient either receiving adjuvant chemotherapy, thirty-eight percent, or observation, sixty-two percent. [00:18:00] Uh, clinically validated, so personalized tumor encode sixteen plex, uh, PCL, NGS, Signatera assay was used for the detection and the quantification of the sample. In this study, primary endpoint was disease-free survival. Secondary endpoint was ctDNA clearance after adjuvant chemotherapy, and exploratory endpoint was molecular re-recurrence analysis. So MRD window was defined as two to ten weeks post-surgery before the start of any adjuvant therapy. Postoperative ctDNA was a strong predictor of DFS in our result in patient with stage two to three rectal cancer treated with approved surgery, supporting its role as a marker of MRD.[00:19:00] The study suggested ctDNA may help personalize adjuvant chemotherapy that show when a MRD positive patient appears to derive substantial benefit from adjuvant chemotherapy, while MRD negative patients show little benefit, suggesting some patient may safely avoid unnecessary chemotherapy, reducing toxicity and the health cost But this sent to a newly positive ctDNA at six months post-surgery associated with significantly shorter DFS, indicating these patient may benefit from the treatment escalation or prolonged therapy. Lung metastases were less likely to produce detectable ctDNA, consistent with prior studies showing lower ctDNA shedding from lung [00:20:00] metastases despite their relatively favorable prognosis. Overall, in this study, the findings support post-operative ctDNA as a strong prognostic biomarker and a promising tool for guiding the elected treatment strategy in colorectal cancer. Serial testing may help identify which patient can benefit from ADT, a high risk for recurrence prior to radiological detection, and which patient are a candidate for treatment escalation, and also until study evaluating treatment strategy for patient who remain ctDNA positive after curative resection but before clinical recurrence develops. And also bigger study, hopefully we will disclose this data next year at ASCO. So this study also investigating [00:21:00] whether adjuvant chemotherapy can sa-safely be omitted in post-operative ctDNA negative patient with high-risk stage two or low-risk stage three colorectal cancer. So I believe that based on the data, when a patient who show, have come out positive during the surveillance period, and Dr. Ti mentioned no clear evidence, but the-- looking at the AALTERA study published early June Nature Medicine this year. So this study suggested substantial benefit of the some drug. Looking at our presentation this year during the ASCO twenty twenty-six suggested two time point, consecutive two time point post-operatively, and eight weeks later, two time point negative, suggesting a likely to no chance of the recurrence, while either one positive, so [00:22:00] the patient should receive adjuvant chemotherapy. But please cautiously think about this result is based on observational study, not interventional study. However, a future investigation should be needed about the how many time point is best for a patient to maximize adjuvant chemotherapy benefit for these patient. Thank you Great, great summary. Thank you so much. I'd like to bring Dr. Ramser into the discussion now and pivot to talking about anal squamous cell carcinoma. So Dr. Ramser, welcome. Can you please describe your findings while monitoring ctDNA levels throughout the treatment of patients with anal squamous cell? Yeah, great. Thanks so much for having me. I really appreciate it. So patients with anal squamous cell carcinoma are typically treated with definitive chemo radiation, and many patients do very well. But one of the major challenges that we see in clinic is that it can take months to know whether a patient's truly [00:23:00] responded, and during that time, patients undergo repeated exams, imaging, and there's a lot of discussion about whether patients are responding or not, and we're really losing valuable time before we can intervene for these patients And that matters because I think the treatment options after failure are somewhat limited. So for patients with persistent or recurrent disease, salvage surgery can be morbid, and outcomes remain challenging. In the setting of metastatic disease, even our best systemic therapies, like the new chemo IO regimens, are generally not curative for the vast majority of patients. So the real opportunity is to identify high-risk patients earlier, before recurrence is obvious clinically or radiographically, when we still have a window to intervene with curative intention. And that's really where circulating tumor DNA becomes exciting. In this study, we evaluate a personalized tumor-informed ctDNA [00:24:00] assay, looking at patients with anal squamous cell carcinoma undergoing definitive intent chemoradiation. It was a real-world two-center cohort with serial blood testing before, during, at end of treatment, and throughout surveillance. And the main finding is that ctDNA behave like a dynamic biomarker of treatment response. This was not just a single blood test at one time point, the kinetics really mattered. So patients who were ctDNA positive at the end of treatment had significantly worse outcomes, with a one-year progression-free survival of only forty-four percent, overall survival of sixty-three percent, and local regional failure of forty percent. So end of treatment ctDNA positively identified a group of patients at very high risk long before we could have prognosticated them using standard clinical or radiographic [00:25:00] measurements. This creates a potential opportunity to act earlier rather than waiting for overt persistence or recurrent disease. And I think this is important because these are the exactly the patients that we need to think about differently. If a patient has persistent molecular evidence of disease after chemoradiation, that's a group that may most likely benefit from additional therapy, such as adjuvant immunotherapy or chemoimmunotherapy, or from other treatment intensification strategies which we're seeing emerging We're now at MSK launching a Phase 2 clinical trial to test this concept prospectively using ctDNA to identify patients at risk after chemoradiation to see if earlier intervention before overt clinical recurrence can improve outcomes and increase curability for these patients. I think the study also showed something equally important on the flip side, and it's that patients who are ctDNA negative before [00:26:00] treatment or who cleared ctDNA early during treatment had excellent outcomes. And here, this raises a very different question. For patients with favorable molecular biology and durable ctDNA clearance, do we need the same intensity of surveillance? Could we eventually personalize follow-up so that high-risk patients are monitored more s- closely, but low-risk patients avoid unnecessary scans, unnecessary flexible sigmoidoscopies, all these procedures that add to anxiety and increase burden on our healthcare system? And I think here the surveillance findings also support this approach, as we do see that some patients who cleared ctDNA ultimately did later become ctDNA positive again. But in all those cases, ctDNA repositivity preceded radiographic or clinical [00:27:00] relapse. So the blood test here was really functioning as an early warning signal, not just telling us who's at risk, but potentially telling us when that risk is becoming clinically relevant, suggesting that we might be able to go or move towards a biologically adapted or ctDNA adapted surveillance follow-up So the larger takeaway is that ctDNA may help us move beyond a one-size-fits-all approach after chemoradiation, gives us a framework for both sides of personalization, identifying molecular high-risk patients who may benefit from treatment escalation, and identifying molecularly favorable patients in whom we can begin to ask whether surveillance can be safely de-intensified. And so in anal cancer or salvage surgery and metastatic disease, treatment remains difficult to cure. The ability to identify those really high-risk patients before clinical transformation provides us with a [00:28:00] window for opportunity. And here I think ctDNA, not as a single test, but as a longitudinal readout of treatment re-response and molecular relapse, is incredibly powerful in this setting. Thank you so much, Dr. Ramasur, for that in-depth description of your trial. All right, so now we'd like to move forward with discussion of future directions if Stell and Dr. Smith can provide us with more information. I think one is, what does the group think as it relates to rectal cancer, for example? We put a rectal cancer case at the beginning as a pro-provocative case study, but where do people think that this is going with rectal cancer? How do we best use it? And where, what are you excited about as you think about the next set of studies that are gonna come out of the rectal cancer space? So I guess that now we treat the DMMR and the PMMR as quite differently, obviously, with the [00:29:00] immunotherapy space being so exciting in the neoadjuvant with, uh, particularly rectal cancer and, and even colon cancer for organ preservation. You know, emerging cumulative data really suggests that early treatment response could predict for CCR and PCR in these patients. So potentially in, you know, even after the first couple of cycles of treatment, you could identify those patients are not gonna do well and potentially intensify immunotherapy or, or look at a, a different way to manage these patients. So that's a useful tool. And the PMR patient, I think The data so far does suggest that it's g- it, it's good response monitoring, but not perfect. Again, these are using, I would call the older generation assay. I would like to really see the performance of these newer generation ultrasensitive assay and see how good they are in predicting CCR. I could see a future being a combination of this, as Dr. [00:30:00] Yoshihara mentioned, with imaging and other even AI path tool to better select our patient. I think lack of response is, I think it's bad news for these patients. It's a real opportunity to try and look at novel therapy, intensified treatment before they go for surgery. I think that's a real case there. And by using CTCAE to select those for op- for, um, or watch and wait, not quite there. I think we need combination of other tools with it this stage. Yeah, great thoughts. Thanks so much. Dr. Armas, what are your thoughts? Yeah, I think those are all really good thoughts, and I think it's important to remind the listeners that not all ctDNA assays are created equally. And what we're seeing is you'll go back and read papers from five years ago and not realize that those are using assays that are g- completely outdated by modern standards, and there's continued evolution of these assays, and there's not a single [00:31:00] assay that wins everything. You have to actually match your assay up to the clinical question that you're answering. And in rectal cancer with non-operative management, you're setting the bar really high because you're not asking now, is there a distant metastatic recurrence where the burden of disease might be higher? But you're asking, can you detect occult cancer cells in a patient's rectum that's been heavily pretreated now with chemotherapy and radiation? And I think there the bar is quite high, but we're seeing these ultrasensitive assays that Dr. T mentioned that are coming out. They're getting down, at least theoretically, to one part per million in best optimized case scenarios, where we're getting a little more excited to put these in and test them and to see how do they stack up against our standard clinical predictors of c- of response, things like endoscopy and MRI. And I'm hopeful, but I'm also biased, that's why I'm on the podcast, that we're gonna [00:32:00] see these be successful in informing our ability to better select patients who have complete responses for non-operative management. And I agree completely. It's likely gonna be a multimodal approach that integrates many things. It's doubtful anything's gonna win all out. But I'm hopeful that we're gonna start to see ctDNA giving us an early response readout like we do in the MR-deficient tumors, as we do. Hopefully, we'll see that in the MMR-proficient tumors, 'cause if we see that, then it's gonna allow us to do interventional studies using MRD to escalate patients who we think aren't quite getting there yet, and that's what I hope for the future, because that would really help us drive more patients to non-operative management through personalization of their T and T journey Yeah, super, super helpful. Before Janet gives us a summary of what we learned a little bit ab- about today, but what trials are you three keeping your eye on [00:33:00] in either the metastatic setting or the primary colon or rectal cancer space? Anything that's really of interest to you as you think about... I think there's certainly some observational data that's important and compelling, but I think where we really are going to learn is in the context of a trial. So any trials that you two are thinking of that you're really keeping your eye on that you think will inform this field moving forward? We mentioned before about surveillance using ctDNA. I know the, um, the European group's done the IMPROVE-IT 2 trial, which is surveil- using ctDNA-guided surveillance, and will be reading out hopefully sometime later this year, showing that using ctDNA-guided surveillance, you can actually reduce CT scan use, use, uh, you do, uh, healthcare costs and so on and so forth. I think that's, I think will be game-changer. I think we follow these patients for a long time, for five years, and a lot of this scanxiety, use of, uh, healthcare resources. So I'm really looking forward to seeing the data from that, [00:34:00] and obviously the VEGA trial as well. I think the difference between VEGA and IMeg3 is that you use serial testing in your study, Dr. Yoshino, whereas we only use a landmark, so that's one of the limitation. And you deescalate to no chemotherapy, which I think will be fantastic if we can stop giving chemotherapy to some patients Yeah. So i- in addition that I guess for elective cancer field, there are many phase three study already started, so to translation research the assessment in the future. So I believe that international collaboration put these studies, so data together, and then we will, we will investigate about the ctDNA power in the elective cancer field future to analyze about the which timing, how many timing over the testing the most beneficial for the patient. So I believe that Japan also be the one with a piece. Australia also, United [00:35:00] States, the European country. Yeah. So such a global collaboration is essential future, I think. Yeah, there's so many great studies out there. I'll say that I really like the ones that we use-- look-- starting to look at MRD as the integral biomarker for response. I just wanna highlight really interesting data that came out of ASCO, and I think Dr. Yoshino already mentioned this, but there was data from his group that showed quite clearly that the earlier you are in your molecular progression, the more likely you were to be cured. So this is something that's always brought up, I think, in, in at least in my tumor boards. Does it matter if we catch them earlier in the disease state, or can we just wait till we'd catch them by CT scan? And can the data starting to emerge that the earlier they are and the lower their tumor burden is and they're-- when they're really molecularly have only molecular residual disease, that the curability of those patients is much higher than patients who have later stage disease, which higher molecular [00:36:00] burden of disease and radiographic disease. And we need that data to start convincing people that this is an integral biomarker that we should be using to guide treatment. Because that's the only way we're gonna convince the skeptics, when we actually show that we're beating out standard CT scans and radiographic scans, and that we're leading to a higher percentage of patients cured. Also brings to the point that the ultrasensitive assay will also be picking up more of these lower level MRD, and therefore more likely to cure these patients, I think. The field is really evolving very rapidly, and we need to generate more clinical data to, to support the utility. Yeah. So current whole exome based, so assay are commercially available, and they cannot identify less than a hundred. So limit of detection. So the refinement of the assay also be important in this field, especially for the promising whole genome based MRD assay. Great. Thank you so [00:37:00] much for participating in our podcast today. It's been a very colorful discussion. I really hope that the Behind the Knife listeners take some time to absorb this information. So let's wrap our session up today with some key takeaways I'm sure our audience can derive that ctDNA is a very powerful prognostic biomarker, especially in colorectal cancer. It can be used to de-detect minimal residual disease after surgery and strongly predicts recurrence risk. CtDNA-guided management can safely reduce unnecessary chemotherapy and lessen toxicity, as seen in the dynamic trial, which Dr. T so kindly described to us, in which ctDNA-negative patients could safely receive less adjuvant chemotherapy without compromising recurrence-free survival or overall survival, and significantly reducing the exposure to oxaliplatin, which we all know has treatment toxicities associated with it. Persistent or recurrent ctDNA positivity can help us identify very high-risk [00:38:00] patients across multiple trials, postoperative or end of treatments, ctDNA positivity with-- associated with markedly worse outcomes and a higher recurrence risk. Persistent ctDNA after chemotherapy strongly predicted relapse, highlighting an unmet need for more effective treatment strategies beyond standard chemotherapy escalation. Serial ctDNA monitoring is more informative than a single time point, as Dr. Yoshino has been hammering. Longitudinal ctDNA testing frequently detected molecular recurrence prior to radiographic recurrence. Patients who initially cleared ctDNA but later converted back to ctDNA-positive status often relapsed clinically, supporting serial monitoring as an early warning sign for recurrence and treatment response assessment. Lastly, ctDNA is adding to the current era of personalized medicine, as all of our guest speakers helped flesh out today. Thank you everyone for joining, and I look forward to the next [00:39:00] podcast