How Emerging Technologies Are Filling The Gaps And Positively Impacting Post-Transplant Care

How Emerging Technologies Are Filling The Gaps And Positively Impacting Post-Transplant Care

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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 07:58

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