My name's Chris Lawrence and I'm the Medical Director of Scientific Affairs at Thermo Fisher Scientific's Transplant Diagnostics division. And, it's a great pleasure to see you all here today. We've got a fantastic program for you. We're going to start by handing you over to Valen Keefer who is our transplant patient advocate at Thermo Fisher Scientific. She's going to say a few words about herself and then introduce each of our speakers in turn.
Please enjoy. Thank you. Hello everyone. My name is Vallen Keefer. I'm grateful to be a patient advocacy consultant for Thermo Fisher Scientific. I'm thrilled to co-moderate today's symposium alongside Dr. Lawrence. It means a lot to have this incredible opportunity to represent our transplant community and bring the patient perspective into today's conversation.
As a dual transplant recipient, I've experienced firsthand that transplantation is not a singular event, or a cure, but a lifelong journey. One that can start with a diagnosis, many years prior to the transplant surgery. I was diagnosed with polycystic kidney disease at 10 years old which led to me needing a kidney transplant at 19 and a liver transplant at 35.
This August, I am so excited to be celebrating my 20-year kidney transplant anniversary, and my 4-year liver transplant anniversary! Thank you. I'm still in awe of it every day. My life has been doubled because of organ donation which just blows my mind. When I received my first transplant, I felt voiceless. I was in the process of switching from pediatric to adult care and there were no resources or support provided to me. When I found my voice after kidney transplant, I learned to be an advocate for myself and others which empowered me to be an active participant in my care, to speak up, and led to the last 18 years of my passion to create resources for others that I wish I'd had.
Because receiving the gift of life equals a lifelong journey of living immunosuppressed, which comes with responsibilities challenges and ongoing maintenance. I am alive today thanks to science, advancements in modern medicine, and the miracle of transplantation. I hope today to be able to illustrate what the promise of cutting-edge science and the use of novel diagnostics means for those of us striving to take great care of our transplants and who hope to lead long and fulfilling lives. And thanks to the work that Dr. Sharma, Dr. Ozzy, and Thermo Fisher Scientific are doing, it is not only improving quality of care, but quality of life for those of us with recipients.
I believe, Thermo Fisher including the patient voice today demonstrates their care and commitment to our amazing community because when the trials and triumphs of transplant recipients are understood I believe it leads to better innovation to improve our quality of life. It means a lot to be here today and I feel really fortunate to join you in hearing two experts in the use of novel diagnostics and transplantation. After they both speak we will open the floor to questions. It is now my pleasure to introduce Dr. Rajiv Sharma,
who is a transplant surgeon at Wayne Health DMC Harper University Hospital in Detroit Michigan. He completed his ASTS transplant surgery fellowship at Northwestern University Chicago and a pediatric transplant surgery fellowship at Children's Hospital of Pittsburgh of UPMC in Pittsburgh, Pennsylvania. He has been a tenure track assistant professor for transplant surgery at University at Buffalo in Buffalo, New York, Director of kidney and pancreas transplant programs at Penn State Hershey medical center in Hershey, PA, and an associate professor of surgery at University of Nevada, Las Vegas, Nevada. His surgical philosophy is centered on delivering best possible care and achieving best possible outcomes for transplant recipients and their families.
Dr. Sharma is well published and has been a recipient of research funding for investigator-initiated studies and clinical trials his clinical research interest is focused on novel diagnostics and therapeutics for antibody-mediated rejection and kidney transplant. His translational research interest is focused on bk virus-specific cell-mediated immune response and immune surveillance and kidney transplant recipients. Please join me in welcoming Dr. Sharma.
Thank you Valen. Thank you, Chris and One Lambda for inviting me to give this talk today. The title of my presentation is Clinical Utility of MMDx: Diagnosing early antibody-mediated rejection. As a disclaimer, the views and opinions expressed in this presentation are mine and do not reflect the official policy or position of One Lambda or any division of Thermo Fisher Scientific this publication is available on the journal website and no grant funding was received for the preparation of this publication so today i'm going to be presenting two cases of early antibody mediated rejection that were diagnosed with the help of donor-derived cell free dna and mmdx and this is the experience that was recently published i would also like to share our unpublished experience with mmdx it's concordance with pathology and utility of mmdx and aki in the post-transplant setting we are using commercially available mmdx testing as a part of our standard of care for second opinion on all our indication biopsies so as all of you know antibody mediated rejection is the predominant cause for late allograft loss after kidney transplant and that there is no fda approved treatment for this condition as yet the standard of care treatment based on expert consensus includes glucocorticoids ivig and plasma exchange with or without rituximab or body zombie but the failure rates are very high so redfield at all they published that there was a 55 rate of two years graft survival for patients with chronic active abmr uh treated with standard of care treatment while 20 for those who receive no treatment.
I'm going to concentrate on the clinical phenotypes so the two commonly recognized clinical phenotypes are acute and chronic where acute generally occurs early more responsive to treatment while chronic occurs late in and is not very responsive to treatment the salient differences between acute and chronic are listed in this table on the right then there is the BANFF classification that has been constantly evolving since it was first introduced with the most recent changes applied in 2019 this is a busy slide just to demonstrate the updates to 2019 BANFF classification. so our understanding of antibody-mediated rejection has been constantly evolving once considered an isolated incident occurring after kidney transplant at a point in time it is now recognized to be a progressive condition that waxes and veins over time and ultimately may lead to chronic allograft damage and allograft loss so if it can be diagnosed early before it has caused significant allograft injury this could revolutionize the treatment of kidney transplant patients with antibody-mediated rejection since early treatment will probably result in better long-term allograft survival MMDx is a central biopsy diagnostic system that uses microarrays to measure mRNA changes with high precision and compare the results to a large reference set using ensembles of up to 100 predefined machine learning derived algorithms or classifiers it interprets the rejection and injury-related transcripts in a biopsy hallowed at all they used a discovery set validation set approach of prospectively collected biopsies to document the rejection associated transcript changes that are either universal TCMR selective or ABMR selective and this table here represents the top 30 mRNA transcripts associated with these archetypes. So coming to the patients our first patient was a 50 year old female who received a deceased donor renal transplant for end-stage renal disease secondary to pkd her cpra was 81 she was on hemodialysis for five years and was in urec donor was a 50 year old male with a kdpi of 74 terminal creatinine was 0.98 donor had hypertension for over 10 years no allograft biopsy so no donor biopsy was available the pump numbers were marginal her past history was significant for cervical cancer chronic diarrhea gi bleed and multiple prior stds she was status post radical hysterectomy with pelvic irradiation she had a frozen pelvis and calcified iliacs but the surgery was uneventful she developed delayed graft function though she received induction with basaleximab and methylprednisolone and maintenance with mmf drolimus and prednisone due to chronic diarrhea she could only tolerate low dose mmf she achieved a baseline serum creatinine of 1.1 at about 2.5 months per stop the baseline donor derived cell free dna at one month was 0.41 the immunosuppression had
to be reduced a few times due to bacteremia secondary to recurrent utis the tacrolimus levels were sub-therapeutic off and on as you can see in this slide the creatinine levels have been pretty steady throughout the course but the tacrolimus levels were all over the place. There was no donor-specific antibody at 11 months post-transplant donor-derived cell-free DNA came back as 1.3 the serum creatinine was still close to baseline though this prompted an indication biopsy due to the elevated donor-derived cell-free DNA there was mild lymphocytic tubulitis limited to the areas of interstitial fibrosis and there was a single glomerulus with focal mesangiolysis which was a finding of undetermined significance MMDx reported early antibody-mediated rejection this created a therapeutic dilemma for us since it was the first time that we had encountered such a clinical situation so we had a long discussion with the patient and after uh patient consent we decided to treat this early antibody-mediated rejection with IVIG the creatinine levels remain stable throughout the course and one month following treatment completion the donor-derived cell-free DNA came down to 0.63 percent here is the patient's allograft biopsy there was no tubular interstitial inflammation, no endothelitis.
Here is the MMDx showing the r4 archetype which is early antibody-mediated rejection. The second patient was a 30 year old female who received a deceased donor renal transplant for end-stage renal disease secondary to chronic gn her cPRA was zero she was on peritoneal dialysis for three years she received a perfect donor kidney with the 20 year old male donor KDP of 9 terminal creatine of 0.9 her post-transplant course was uncomplicated and she achieved a baseline serum creatine of 1 at about 1 month post-transplant she received induction immunosuppression with basiliximab and methylprednisolone and maintenance with MMF diatrolamos and prednisone the tacrolimus levels were mostly therapeutic and there was no DSA for this patient the first donor-derived cell-free DNA was only done at 11 months post-transplant and that was 1.6 percent we did not have a baseline for her because the patient serum creatine was still normal we decided to wait on it for another month and repeat the donor-derived cell-free DNA the repeat value came back as three-point nine percent uh these are the serum creatine and tacrolimus levels over the one year course you can see the creatinine is very steady very stable the technolomous levels are mostly therapeutic so this prompted an allograft biopsy the biopsy demonstrated bodyline acute TCMR along with the PTC one there was occasional mild lymphocytic tubulitis and the interstitium demonstrated mild lymphoplasmacetic inflammation in the unscarred cortex it did not meet the banff criteria for antibody-mediated rejection there was no DSA there was no c4d MMDx reported early antibody-mediated rejection but no TCMR so we treated the patient with methylprednisolone for the TCMR borderline TCMR on the biopsy and IVIG for the early antibody-mediated rejection on the mmdx serum creatine remains stable throughout the treatment course one month following treatment completion the donor-derived cell-free DNA came down to 1.6 percent and at last follow-up, the donor-derived cell-free DNA was less than one here is the allograft biopsy showing uh interstitial inflammation tubulitis peritubular capillaritis no glomerulitis no endothelials. Here is the MMDx showing the r4 archetype which is early antibody-mediated rejection.
Now third case is a patient where I would like to highlight the utility of mmdx in AKI in post-transplant setting so this was a 63-year-old gentleman with end-stage renal disease secondary to diabetes and hypertension who had been on dialysis for eight years he was status post radical prostatectomy with bilateral lymphadenectomy for prostate cancer he had been in uric for eight years his cpra was zero he received a deceased donor renal transplant from a 44 year old donor with a kdp of 70 and a terminal creatinine of almost five uh the surgery was very difficult due to the bladder reconstruction part since the bladder sorry due to the urethra neocystostomy since the bladder was extremely small and had a capacity of less than 50 ml the post-transplant course was complicated by delayed graft function but the patient achieved baseline creatine of 1.1 about three months post-transplant baseline donor-derived cell-free dna at one month post-transplant was 0.3 percent at four months post-transplant he developed bk virus nephropathy for which uh we switched him from tactrolimus to cyclosporin uh mmf dose had to be reduced by 50 uh after making these changes he continued to have a low level bk viremia for about seven months and only cleared bk virus at about 10 months post-transplant there was no dsa but the patient had been on reduced immunosuppression for a long time.
The creatinine progressively increased from 1.1 to 2.1 during this time and the donor-derived cell-free DNA at 10 months when the creatinine was 2 was still 0.15 percent about a month after bk virus clearance there was a sudden bump in creatinine to 9.4 from 2.1 this was exactly one month following bk virus clearance this was a perfect setup for rejection the patient had been on low immunosuppression and we were you know pretty confused is what to do uh with this high creatinine a biopsy was done donor drive cell-free DNA was done which is normal it was still 1.16
urine output was good the ultrasound was normal kidney had great flow this is the trend of the serum creatinine and the cyclosporine and the tacrolimus levels in this patient you can appreciate that sudden bump in creatinine from 2 to 9.4 the cyclosporine levels were maintained between 150 to 200 because of the bk virus nephropathy so the allograft biopsy was done which only showed tubular injury there was no evidence of rejection there was no associated glomerular activity MMDx was also sent on this biopsy and there was no AMBR no TCMR but moderate to severe injury scores here is the biopsy showing the tubular injury and here is the MMDx showing the r1 archetype which is a non-rejecting archetype so the diagnosis in this patient was aki secondary to dehydration so what had actually happened was he had ultra small capacity bladder that could only hold urine for about 60 to 90 minutes even at one year post-transplant the more he drank the more he urinated so he started drinking less and for three weeks preceding the event he was only drinking about one liter of fluids every day and he was still making 2.5 to 3 liters of urine every day so this led to the aki all we did was in patient iv hydration and the serum creatinine came back to the baseline of 1.2 within a week which was better than the 2.1 after bk clearance so in cases like this we found it very useful to have the second opinion of mmdx in addition to the biopsy because when there is a perfect setup for rejection to take place and you have a creative bump like that and the biopsy does not show rejection then it's a difficult decision to make to not treat the patient mmdx in such situations at least for us has proved very helpful as a second opinion so in conclusion the histologic assessment of biopsies has its limitations because of the subjective nature of reporting by pathologists and limited reproducibility between observers while mmdx can assess the t-cell-mediated rejection and antibody-mediated rejection in a reference set of biopsy samples using machine learning-derived classifier algorithms molecular interpretation offers as an objective second opinion that adds to local histology findings thereby increasing clinician confidence with diagnosis and treatment planning so this is essentially the reason that we have been using mmdx on every single biopsy that we do scheduled surveillance with donor-derived cell-free dna and molecular assessment of biopsy with mmdx have made it possible to diagnose a new phenotype which is early antibody mediated rejection this could potentially revolutionize the kidney transplant landscape by enabling early diagnosis and successful treatment of the calmness cause of late kidney allograft failure thereby prolonging allograft survival thank you for your attention Thank you very much Dr Sharma, I'm looking forward to our q a to discuss your presentation more it is now my pleasure to introduce dr Jamil Ozzy, Medical Director vascularized composite aloe transplantation associate director kidney and pancreas transplantation director kidney transplantation fellowship program associate professor Harvard medical school Dr. Ozzie is an associate physician at the renal transplant division at the Brigham and women's hospital director of the kidney transplant fellowship and an associate professor of medicine at Harvard medical school his research focuses on understanding the immune regulatory arm of the immune system and transplantation autoimmunity and cancer with the goal of developing more targeted and safer therapeutic strategies a major focus currently is regulatory t cells and their activation-induced cell death in addition to engineering cell therapies dr Ozzy's laboratory is also exploring multiple genomics and proteomics approaches to develop biomarkers that non-invasively detect rejection in kidney transplant recipients and measure the immune function of immunosuppressed patients please join me in welcoming dr Ozzie. Thank you. Thank you for joining us today. I don't have a patient cases to discuss unfortunately but
we're hoping well with the help of Thermo Fisher we'll be able to do that next year. So let me see here where my presentation is. Very good so um I'll be talking about liquid biopsy and kidney transplant and i believe i mean this is a um the topic that very close to every physician's heart mostly because we all deal with the frustration of not having really good tools in the clinic to manage our patients we don't have good tools to measure the immune function of our patients but also we don't have good tools to measure the health of our our transplanted organs so you know almost 70 years now with transplantation we treat everybody the same we give them all the same immunosuppressants and then we wait for complications to happen we wait for rejection to treat and we wait for you know infection and malignancy to you know realize that we're over immunosuppressing our patients and we adjust accordingly so really there is a there is a urgent need to to develop tools uh to help us managing our patients and before i continue i'll i have some conflict of interest to report um uh regarding the presentation today intellectual properties with exosomedx and accrue health and and some royalties so and i you know i always like to start my my talks especially for the uh trainee in the in the audience you know uh talking about some biology to really uh you know point you know that the transplantation is still in its infancy you know we we um we were born with billion clones of t cells each clone recognized only one antigen and you know the billion clones to cover most of the possible antigen and invaders and what we do currently we you know we suppress in transplant you know we have probably more clones and a large number of clones that mobilize again the kidney but still not the billion clones and what we do currently is we just suppress uh everything and this is what put our patients at risk for uh infections and and malignancies and really what we do in the clinic is as a hand-waving approach to you know balance between uh you know those those different kind of complications um and i think for this audience i don't have to tell you about the limitation of our um you know usual tools we use uh in um in to man to monitor our our uh allograft serum creatinine and cinnamon protein and and and urine protein which are basically um uh has low sensitivity and specificity but they are also late markers uh of rejection so um um and then you know and this really showed the the the uh emergent need to develop better tools um and this becomes even more important when we look at the outcome of of patients who develop acute rejection within the sixth month in fact kidney transplant recipients with early acute rejections you know have higher risk of of of death from cardiovascular disease and cancer and graft failure uh and chronic allograft nephropathy uh long term um and so so we need this ideal biomarker and i'm not going to go you know over the details what you know an ideal biomarker is but you know pointing out that it's very important to have you know tools with high predictive values and when we talk about positive predictive values this is you know the number of true uh positive if the test uh you know we're running is positive and this allows us to really treat based on the on the test and and negative predictive value is the number uh of true negative if the test is negative so if if the test is negative this allows us to say that the the allograft has no rejection and continue monitoring but what's important to point out that really the ppv and the mpv are are really related to the prevalence of the disease so when the prevalence is low like the case of of acute rejection the ppv goes down but the mpv goes up so it's very hard to really develop biomarkers with with both high ppvs and mpvs and in my opinion you know we what we what we need really is a battery of biomarkers to help us our manage our patient some with high ppvs and some with high npvs so and then you know really we owe the advance in the field of biomarker to our understanding of biology so the t cells get activated and the secondary lymphoid organs and and home to the allograft where they cause the injury so really we can look uh at the at this process either in the periphery uh or in the allograft itself or in the case of kidney transplant which is a unique scenario we can look in the urine and you know you know people may argue that you know kidney biopsy is a gold standard but is it really a gold standard and we just heard about the limitation from sampling error to um really inter-observer variability so and this is where the what the mmdx is trying to solve and then the um but you know the blood and the urine obviously are are are a very attractive platform to develop biomarkers but it really was having happening in the periphery does really reflect what's happening in the microenvironment of the of the allograft however when we talk about urine the urine is in the kidney transplants is directly derived from the the allograft and may will carry a very concentrated signature uh from the from the allograft and uh you know this is this is one way to look to look at the explored biomarkers uh in the field currently and and and most of those are in the clinic and and i will talk about some of them um you know we can in the pre-transplant setting we have the donor-specific antibody that i'm sure all of us use and and but they work as a risk susceptibility biomarkers which is which is great it gives us an idea about the risk of our patients uh for developing rejection going on and can be used pre-transplant but also we have a biomarker that now used post transplant and um and and and we are all using them and an example is the cell free dna and the the biology behind that is is nice that you know all cells including you know donor kidneys at baseline will leak continuously leak fragmented dna into the recipient uh blood however you know in the cases of injury you can expect that this leaked dna to to increase so the ratio of donor to recipient dna will uh obviously go up and in a steady state this is up you know approximately um constitute less than one percent the recipient uh the donor dna from the recipient is less than one percent and the the good thing about that that it's a the short half-life of the selfie dna is is about 30 minutes so so theoretically it would trend down quickly after the the the the etiology is corrected um but clearly as you can see this is a marker of injury uh this is not specifically a marker of um of rejection so um and this is the the dart study uh that uh that really launched the uh cell free dna and all of us use it based on the study that showed that um uh uh you know in 107 biopsy that with uh um cell-free dna measured in the blood there was correlation with with rejection uh for the cut off when it was one percent and the the the mpv was 84 and the ppv was 61 and and and based on that you know a lot of clinicians now use uh the cell free dna but you know you always have to remember that there are the the 1a patients with cell rejection 1a were excluded from the study and if they were used probably the performance would have been lower and those are you know patients important to diagnose if we believe that those are early early rejection process and how about the urine so i cannot but mention this you know pioneering pioneering work by sutan tiran group really that launched the the work in unity biomarkers and inspired a lot of us to to you know work on this and this is the uh mrna in the urinary cells and the rationale of measuring mrna uh in the urine is you know a cell cell pallet that we can isolate in the urine they come directly from the allograft and during rejection process some of those cells are cytotoxic cells that will will end up in the urine and we can measure uh transcripts related to rejection in the in the cell palette and and correlate that with rejection and this is what exactly what sutan tiran group did uh and this is a one of the first study that they published and they looked uh at grenzin b and perform and and had a good association with with rejection with a good um uh you know performance and and this was followed by multiple uh studies including this large multi-central trial this is the cto t04 and then that showed they looked at nine transcripts for that are known to be associated with rejection in a very targeted approach and they were able to identify three that correlated with rejection but the question is is always you know uh why this not did not move quickly uh you know to to to the clinic and the reason is is at least in my opinion is the difficulty of maintaining this rna intact in the urine and and the the cell palate the cell that they end up in the urine those are cells that are are are dying and most likely this will lead to uh decay of the rna very quickly and makes it harder to to translate that to a a clinical test and and and really um the limitation of the urine cell palette our convincing uh you know our though that the urine will carry a very concentrated signature from the allograft itself you know made us focus on the urine as a way to uh develop biomarkers and so and this has also ign ignited our interest in extracellular vesicles as potential for developing this test and so what are those EVs. So EVs are are released as part of the normal physiology of cells all cells will will will bud off from their membrane vesicles that will carry protein from the cell RNA from the cell and even DNA and those vesicles work as cargo and it's been shown and this is a you know extensive science going that those uh vesicles can play a major role in intercellular communication by going and fusing in target cells and and exchanging information and and and personally i was interested in how regulatory t-cells use EVs to suppress the microenvironment but then you know and and they can can also be broadly divided into two categories we we talked those are the ectoserums and the exosomes so the ectosomes are vertical that pinch off the surface but the exosome they come from the from endosomal origin and their size is usually significantly smaller than the than the uh micro vesicles so but those evs as i told you carry all these informations from protein to rna to micro microRNA to dna from the parent cells and they will end up in um in biologic fluids such as the urine the blood and the CSF so imagine what a great source of biomarkers to report on the you know the status of the cells where they come from and and um and basically uh this group used a fancy reporter to image exosomes released from cells um okay this video is not working so you have to take my words for that and then this reporter shows the the active release of evs from from those cells and it's been shown that more than 20 000 um you know evs are released uh from cells per day uh from each cell but when you compare that to cell free dna release from sets only released when the cells die similarly to cell pallet you know those are dying cells ending up in the urine so the exosome however is an active process and can continuously inform on the cell status and and can be a perfect platform uh for molecular uh signature and and although so um and now while we're doing a bulk you know analysis of those evs you can imagine a future where the technology will go in isolating evs related to each type of cell and interrogating those evs separately and having a really a perfect platform for the liquid biopsy development this is you know one of the first experiments we did and again i was interested in how t cells you know release ev so we were the evs are too small to be able to uh characterize by flow cytometry so we we basically used to isolate them from the cell culture and then would bind them to beads and then add antibodies to this i know why this is um but anyway so and then we we were able to characterize those evs and this is the first experiment we did where we looked at ev's coming from t cells versus uvs coming from no t cells and we found that cd3 which is the hallmark uh protein of t cells exists on those in those evs uh coming from from t cells but not from non-t cells so you know obviously uh being a clinician and dealing with all this frustration of of of not having really good tools to diagnose rejection we ask the simple question if all cells in the kidney release evs and end up in the urine and now if you have infiltrating immune cells in the kidney the evs coming from immune cells will also end up in the urine and we have technology to to to separate between those coming from immune cells versus non-immune cells we can have a snapshot of what's going on in the kidney and this is you know exactly what we did and okay so also you have to take my my word for that um so and here i was i was showing you hey in fact a an ev from the first patient we recruited to the study you know patient of mind developed cellular rejection we isolated the the evs and we were able to see enriched cd3 derived ev derived from t cells and with electron microscopy we were able to find those cd3 positive evs in the urine and this you know um gave us the you know uh uh the proof of concept that this is a good hypothesis to to carry on and so and this in that regard what we did we started um uh we took t cells we isolated evs uh from the supernatant using ultra centrifugation and and uh and a process that i'm not go over now and we found that those evs coming from t cells have not only cd3 but other markers of t cells and we we used flow cytometry we used elisa but but also we used a another technology that was developed by a colleague of mine and a collaborator um hakoli uh in mass general and and with this what we did here is using a microchips uh that we load it with with urinary evs and we can and then if there is a um if the if those evs express a specific protein it will be retained and through an electrochemical reaction it can uh it can the this this ikea uh can report on the signal and and uh um um and use that as a point of care uh device and this is this this device is smaller than a mouse basically and you know the proof of concept came that you know looking at all these different protein consistently cd3 was the highest expressed in in cellular rejection and and with that we continued the study and tried to correlate cd3 expression in the in this uh and in the rejection uh with uh with those cd3 positive eds but the the question then now you know we decided that yes this is uh this is good but this this has more potential so um and then with all these constituents of the evs in the urine we can develop a high throughput analysis and that's what we did we isolated evs and and we started doing proteomics and genomics and at that point i uh i met johannes cogg who was a postdoc at mgh who really one of the first people started working on in on evs then moved to a company uh called exosomedx and uh to really develop a clinical platform for for ev isolation and development of biomarkers with ev in cancer and and and we i teamed up with uh with johan and we partnered on this project going forward uh mostly to use the clinical platform that will allow us to trans you know whatever we find in discovery we have the highest chance to to translate to the clinic and uh and this is our uh publication in in jason recently and in that we uh we enrolled 175 kidney transplant patients at the time of a clinically indicated renal biopsy from three renal centers we had 219 urine samples and we we included matched durian with uh with with the biopsy uh obviously and and we included tcmr1a1b and and and two but also acute active and chronic active abmr and we exclude in that borderline and and uh bk virus nephropathy um and you know this is the patient characteristics and showing that there was some difference in the the gfr and and the the rate of previous rejection but no difference in the other characteristics and and what we did the the process here it was done on a clinical in a clinical lab to isolate the exosomal rna uh using technology developed by exosome dx for isolation and then with that running a an open array analysis using 600 genes and and after that we and we it included like 21 endogenous control and with that we um uh in the second phase we narrowed down this to 112 gene that we thought uh the most promising and then and then we did this uh you know uh analysis but we didn't do a classical approach of of dividing the sample into two third training and one-third validation but we use this cross-validation technique to generate the rejection probabilities and this is based on our discussion with biostatistician that this approach gives better indication of how well this model will perform on unseen data in the future and this is the signature we identify that can separate rejection from no rejection and then also can this because this is a molecular signature and then you know you can imagine that it allows you to with with higher number of patients with with enough depth to be able to discriminate between different diseases and in that case we were able to differentiate between abmr and tcmr and this is uh you know so the relative quantities of each target gene goes into an algorithm to generate a single score from from zero to one and and this is the waterfall uh plot and you can see the red bars are are the scores from samples that have clinical rejection and the green are from a sample with no rejection and and we then derived this this cutoff for gene signatures uh and and the same we did to separate between abmr and acmr and here if the the red bars is for patients with amr and the uh the green bars are patients from um tcmr so patients below the cutoff are most likely to be a tcmr and this is the roc analysis uh uh that's very promising uh with an auc close to 0.9 uh and this and also this is the signature that discriminate uh rejection from from uh a cellular rejection from antibiotic reduction and that is this is to show you know a high negative predictive value but it kept a decent uh positive predictive value um and the way we envision using this signature is is first we run the first signature to this to differentiate between rejection and no rejection and if there is if it's below the cutoff this mean we can rule out rejection with high certainty and we continue to monitoring patient but if it's above then we can run the second signature to give us an idea if this is a cellular rejection or antibody mediated rejection but this is what i'm excited about and if we take the urine um from the minus 80 we throw it and then we we start isolating rnas and then looking at the the the um and measure those mrna the results are comparable over time at four degrees and this tells you how stable those rnas are um uh in the ear and and this is different because the evs are shielding the the rna from the rnas and and and i think that's the the uh the power of this technology and this is this platform so uh so now you can envision a test where the urine is collected at home and shipped to the lab for for measurements and exactly this is what we do this is a you can see this is from in our lab this is the ice packs in this kit that we sent to patient we asked to put in the fridge for a few hours and put it back in the box with uh with the urine cup and ups can picks up the urine and brings out brings it to our lab for analysis and so we are now in the midst of finalizing our second paper unfortunately you know i didn't have time to present the data uh so now the signature is transferred to a clear lab on rtpcr and the urine test is called exotrue and the signature remains strong with very high npv uh we have over we we have an external validation with 400 urine samples half of them came from protocol biopsies so the the performance for forecast uh rejection is the same as for subclinical rejection and this is something again we're excited about um and um and i cannot help but show you this you know so i this we do basic science uh work on mice and i didn't know what cd74 is but c74 was the highest express gene in our in our signature from evs so we generated mice with crispr and then if you transplant a you know a heart from a white mice into a black mouse they they reject in in seven day very stringent model um but if you trans transplant heart from a bulb c into a into the knockout basically no rejection um and and what is telling me that you know the signature we we found is is biologically significant um and then uh to me was a reassurance that you know the platform is is also working well so many challenges remain uh for by embarking discovery but but in my my opinion things are looking uh very promising with many players in the field and and many biomarkers moving uh for clinical use um and and finally you know i'll have some acknowledgement the lab jean-pierre started this work and then rania continued but really everybody in the lab helped and they are on schedule to collect sample from patients but you know this work really um is the the group at exam the acts that you know i cannot but say great things about them they johann scogg james hurley uh vashist and and brian uh but also many collaborators um mgh from yale uh and and and generous collaboration with uh uh fadi lakis and and camilla to validate our our uh finding in an external uh validation group and obviously all my colleagues at the trc have been been extremely helpful and supportive and thank you thank you so much dr ozzy i'm sitting there and thinking to myself how does this transition into being a patient and something really stuck out to me dr ozzie as you said to have better tools because it's like you're waiting for something to go wrong and that's so interesting because it made me feel like as a patient you get a transplant and you almost have this fear of waiting if something's going to go wrong so how amazing if we could have tools so that we don't have to live in that fear so thank you for that heartfelt sentiment i also just had these thoughts from a patient perspective that we want all of the information we can get to make the most educated decisions if we had just added confidence into the results like i get monthly lab work done i've done it for almost 20 years and there is always a little like this ping whenever the results come through in the email i'm like okay i'm going to open it up i'm instantly going to look at creating an alt and i'm just going to hope for good numbers and then no i'm just going to go for another month and and enjoy the next month until labs again it's like you always just have that connection to your numbers and knowing that they basically dictate your future and the more certainty we can have just the more confidence and comfort i think it would give us as patients on this journey like with biopsies if we can have more certainty that we wouldn't have to think in our minds that maybe we'd need another repeat biopsy to look into it more would be huge i had uh my liver transplant four years ago and about eight months into it my numbers spiked and immediate reaction is fear and they told me i needed a biopsy the anxiety i had around that was almost just i didn't understand why and i was sure you think a biopsy itself isn't a big deal but I was so nervous that day of thinking that a needle was going to go into my liver and my husband said a sentiment that stuck with me. He said, "it's not what you're having done today, but it's the trauma you've endured leading up to this point that is making today what it is. And these emotions that you're having."
So, if we could just have more certainty and confidence and comfort in the results and not have to go through additional testing, it would really impact our lives positively. And I also think that they put me on a high course this high dosage of prednisone for a while just because they thought I might have had rejection. I got the biopsy results, it wasn't rejection, which meant I took all those immunosuppressants that technically I didn't need. And we know that the more immunosuppressants that we take, the more we're at higher risk for infection. And all of those issues, as well.
And the thought of being able to do a urine test at home is mind-blowing to me. I love it because I during COVID participated in an at-home research study. Took my blood at home. Sent it back in. It was so easy! And it was exciting because I felt really active in my care. Like I was doing something! So to think, if I could do that at home, would be amazing. And I think like it would
be neat to think if that's just part of your care and you could have those results and it would be something a lot easier like a sentiment I had to learn to love is following your trend of numbers because I'll get results and the creatine goes up and you're you're instantly like oh something wrong and they're like no we'll wait till next month follow the trend I was like okay I've had to learn that like learn to love that statement but just listening to this sitting here thinking of the young version of me that didn't have education or hope or community and just to listen to what we've learned today and the hope for the future and I'm just so appreciative for all of you and the work that you're doing to make a difference in my life and my future and all of us in the community it's just an honor to just be here today with all of you thank you okay thanks very much for those wonderful talks can I open the floor to any questions. I think we've got some roving microphones there's a question over there. yeah thank you for this question uh great question so obviously um this platform carry those EVs we didn't do the experiment where we isolated of mRNA and and see if they can you know translate but what I can say that those microvesicles and evs carry a lot of proteins and in fact, we have a protein worker and with a protein signature that we're working on but the um uh the uh the the signature from mRNA was very stable and uh you know was very strong so we could not ignore it but I agree that there are potentials of doing proteomic on those eps and we're doing we're pursuing that. I really enjoyed the drugs my question is for Dr. Sharma you had presented first two cases
where you said the mmdx at early avmr but there were no dsa in these patients no there was no dsa did they develop it subsequently no they did not and we just followed with the donor-derived cell free DNA for response to treatment we treated them with IVIG which was the most benign treatment but we were not going to leave them with a diagnosis of early antibody-mediated rejection and no treatment again it's a you know having a hard time conceptualizing that we have antibody-mediated rejection but no antibodies right how many times do we say you know antibody mediated rejection without DSA I would say in my experience at least 15 to 20 percent of the times in my experience and I would say I've treated about 12 patients in last one year and about two of them had no dsa but the biopsy had full-blown uh you know ABMR were there other antibodies no we were we're not testing we don't have the ability to test DMC for 81r and MIC-A MIC-B so we did not test for any of those. Thank you. Thank you. If I can just add to that, that we know that MMDx picks up more mediated rejection than
traditional histopathology and we think there are three possible reasons for this. One is that when we look at the antibody profiles of those patients, some of them have got clear epitope patterns that are below the MFI cutoff so that's a proportion of them is due to not calling the DSA that is there. We think that a proportion of them may have non-HLA antibodies and at One Lambda we're going to test the sera from the trifecta study patients to identify what proportion of patients those are and what antibodies those might be and the third reason is that we think that some of it may be due to NK cell and missing self there's three reasons why we see DSA negative ABMR we think It's a fantastic question. Thank you for those questions. Any other questions from the floor. So but whilst I have a captive audience, I'd just like to let you know that tomorrow we have an extremely busy day in the abstract halls at 3 30 in the afternoon in the Heinz ballroom a we have our collaborators from TSI the inventors of MMDx talking about the correlation between MMDx histology and clinical variables and also the molecular features of late t cell-mediated rejection at 5:30 presentation 337 in room 210. We have a presentation on the molecular
features of chronic lung allograft disease which has parallels with molecular features in kidney biopsies at risk of progression and finally at 7 pm in Heinz hall C and D Colleagues from Alaris are presenting on their urinary metabolomic tests and I encourage you to go and listen to that. It will be really very exciting. And finally, I'd just like to thank all of you for attending and thank our presenters today. Thanks very much.
2023-02-02