Sam Chang: Good morning everyone. My name is Sam Chang. I'm a urologist in Nashville, Tennessee. We are honored to have, actually, two physicians from Barcelona, Spain, Dr. Andrea Gallioli and Dr. Angelo Territo, who will be presenting their work on DNA methylation markers for actually upper tract urothelial carcinoma, an area that has really gotten not a lot of attention but I think will become increasingly an area focus of research. Thank you both for joining us today and look forward to your presentation.

Andrea Gallioli: Thank you.

Angelo Territo: Thank you for the introduction.

Andrea Gallioli: It is a pleasure for me to speak about DNA manipulation urine biomarkers test in the diagnosis of upper tract urothelial cancer as we did a single center prospective clinical trial in Fundació Puigvert under the leadership of Dr. Breta. As we know, upper tract urothelial carcinoma may be treated with a kidney sparing surgery if it is a low-risk tumor or if it is a high-risk tumor but in the distal ureter. The problem is that the diagnosis is challenging. 35% of biopsies may be inconclusive. There is a 30% of upgrading in the nephrectomy specimen and urinary cytology sensitivity for a grade is only 56% in voided urine and 71% in situ. While CT scan cannot provide us the grading and its sensitivity to characterize the lesion, meaning the number of lesions, the correct size is only 47%. Ureteroscopy may change a treatment choice in 30% of cases. We have two open issues. First of all, the problem of correct diagnosis and second, the probable of surveillance after kidney-sparing surgery.

This is why we decided to evaluate the accuracy of a urine biomarker test in the upper tract to compare it with the current standard and to show its applicability in specific clinical scenario and related cost-effectiveness. The question would be, is the biomarker test easier, better, and faster and cheaper than our current standard to evaluate? We first of all put as primary endpoint to the overall acuity of the biomarker for upper tract and bladder urinary samples and the secondary objective we compare the biomarker with urinary cytology in both selective and bladder urinary samples and tested the performance of these biomarker according to tumor characteristics. The biomarker was chosen was a Bladder EpiCheck. This is a panel of 15 informative DNA manipulation biomarkers, which is actually evaluated with a PCR amplification and with a score which is called EpiScore that come from zero to 100 and it is positive if it is more or equal than 60.

As you can see in the bladder cancer it worked very well with the sensitivity of up to 92% and the negative predictive value of 99% in no pTa low-grade bladder cancer. We decided to use EpiCheck because we know that upper tract urothelial carcinoma and bladder cancer are disparate twins actually. That this is a biomarker that has got a panel. It's not that one single analysis that we are doing. It is a high analytical sensitivity. It requires a small amount of urine and there is no interference with hemoglobin, red or white cells or ethanol in the urine. It's easy to storage, and the EpiScore can be converted to a binary result but is also a continuous variable.

We decided to start a prospective study which was performed between 2019 and 2020. As you can see, these were the outcomes and the results were published in the Journal of Urology by our team. As you can see in the results, the bladder EpiCheck had a sensitivity of 83% and specificity of 81% in the upper tract with a very high negative predictive value, which was up to 80%. In the bladder it dropped a little bit as was expected to 55% with a specificity that was maintained to 81%. Problem is that here the negative predictive value dropped to 55%. As you can see, once we compare EpiCheck to the cytology, EpiCheck outperformed cytology in any aspect. Here you can see a table regarding the performance of EpiCheck and cytology in selective upper tract samples and specifically in high-grade or CIS tumors, the sensitivity of EpiCheck was 96% with a negative predictive value of 97% while cytology at a sensitivity of only 71% and then negative predictive value of 86%.

If we have a look into our data from the bladder samples, while as we have said there is of course there is a dropping in the sensitivity of EpiCheck, but we have to acknowledge that cytology had the sensitivity of only 33%. Importantly, in In high-grade or CIS disease, EpiCheck had sensitivity of 71% with the negative predictive value of 88% against 59% and 87% for cytology. Our conclusion is that EpiCheck could potentially change the assessment of upper tract urothelial cancer, decreasing the number of unnecessary procedures without compromising the diagnostic effectiveness. Clinical scenarios that could be employed are follow-up after conservative management and this is probably the most fit clinical scenario for this biomarker or the first observation. Primary tumors in order to understand if we can skip the ureteroscopy and go directly to a nephrectomy if this tumor is actually a high-risk one. Limitation of the study included the heterogeneous population, small sample size and no cost effective analysis. Thank you.

Sam Chang: Great. Thank you so much for that presentation. EpiCheck is not available in the United States as you know, but the results are definitely promising. Why do you think for the bladder analysis there seems to be a decrease in both the sensitivity and specificity as opposed to the upper tract evaluation?

Angelo Territo: Is a good question. Good to remark. I think one of the big problem that we can identify in managing this kind of marker is that the manipulation of the bladder sample could be still a problem. The other thing that you should consider is that as you can see in the literature, there are still a lot of concerns in terms of using biomarkers in bladder. The reason why, despite the many investigation in several papers on bladder cancer with the goals to identify bladder biomarkers, so far nobody of this was introduced in the clinical decision making process. Reason why maybe there is something that is so far we don't understand. We don't know. We didn't identify a reason why. I think the research on this topic should be still considered ongoing. On the abstract, maybe the results are different. This is our analysis. Unicenter study obviously in order to spread this data and to allow to introduce this data in the clinical activity we may need for external validation maybe or better multicenter study in order to demonstrate in a multicenter fashion as this data are not only related for a single center study.

Andrea Gallioli: Well, and then I think that actually you can add that there is always a problem when we speak about other samples about to find an upper tract tumor. First of all, we know that sometimes the urinary tract may be excluded, so maybe the vast majority of the urine from that kidney are not coming to the bladder and that there is a problem of dilution because the other urinary tract is not involved in the neoplastic process. I think that this is something that we know and it is the same for cytology and probably will be the same for any biomarker.

Sam Chang: I was really impressed with the impact of the ability to differentiate for CIS lesions, which obviously for diagnostic purposes can be difficult to biopsy, difficult to visualize those types of things. Were there other clinical characteristics of the upper tract that made the test more likely to be successful? Were larger tumors, multifocal tumors, renal pelvis tumors, were there any other factors clinically that seemed to make this test more accurate knowing it was a very small sample?

Andrea Gallioli: Yeah. Yeah. I know it was a very small sample. I have to say that we didn't find something specifically. I was thinking that probably in the distal, if the lesion was in the distal ureter, something would have changed. Actually we couldn't see the difference in this kind of situation. I would not say that there was a specific difference depending on the dimension or something else that wasn't degrading. It was just degrading that could affect the performance of the test.

Sam Chang: How are you now using this test now? I think some of the proposed kind of uses to decrease the number of interventions and that type of thing is incredibly exciting and appealing, especially for patients. Just as you said that we're trying to do nephron sparing. How are you and your colleagues using this test in those patients with upper tract cancer?

Andrea Gallioli: I would consider two scenarios. First of all the follow-up. I think that here we can have a very interesting changing because we could decide to use it in situ or to use a bladder sample depending on the risk of recurrence. If we know that for example we are doing an imperative conservative treatment in the patient with a high-grade disease, I would consider to do a follow-up with a simple catheterization and 10 milliliters of urine. Then we have got EpiCheck and we know if there is a recurrence of not provided that we will do also a CT scan. On the other end, we could decide to or at least evaluate to use it in the bladder sample. Considering that for high-grade and CIS disease, the sensitivity was almost 75% also in the bladder samples. We could miss potentially some low-grade disease.

It's also true that if we complement it with a CT scan, we could have the right diagnosis and perform the ureteroscopy just when it is necessary. Other thing is to use it in that scenarios when there is a first observation tumor that you know that the patient for example could not be selected for a conservative treatment but there is a suspicious of cancer. In those cases you can use the EpiCheck and if the EpiCheck is positive, go directly to the nephrectomy. Otherwise, if you need to have a biopsy or a cytology just to be sure that it is a pure ureter carcinoma for example if you need to do a chemotherapy, while in those cases we still are depending on the ureteroscopy.

Sam Chang: With your important work, where are you guys going to go next in terms of research? You mentioned multi institution, you mentioned broadening. Where next in terms of evaluation? I think the use of urine biomarkers, evaluation, urine biomarkers as you two know is becoming increasingly a popular area of research. Where are you guys going to go next in terms of either using EpiCheck more, different situations, multi institution? Where next is most exciting for you two?

Angelo Territo: Oh, this is important consideration so far. I guess that one thing should be emphasized. It is biomarker EpiCheck is an additional tool in andrological armamentarium of urologists and reason why in all the situations describe it and by Andrea Gallioli. You have an additional tool to make a decision in order to get a diagnosis in case of suspicious lesion or whatever. There is a discussion on conservative versus radical treatment in patients where there is not an imperative indication because imperative indication are other thing to consider, other patients with the different features. Next step of course should be firstly to increase the population.

We know that upper tract urothelial cancer is a rare disease in our hospital. We have a large experience on this topic since we have between 80 and 90 cases per year, including the second look in inspection of the EpiCheck, whatever there was a conservative treatment by laser for example. Reason why we were able in the last year to investigate and publish our results on other issues of the upper tract urothelial cancer in terms of biopsies, in terms of the performance, the yield, diagnostic yield and the limits of the CT scan, compare it with the ureteroscopy.

Firstly, increasing the number of cases in a single-center fashion could be the next space in order to confirm this data already published. As I told you before, a multi-center fashion study including different European or extra European center could be the right way to go in order to better demonstrate the results and to confirm these results in an international scenario. By doing this, I think that we were able in the next future to introduce this EpiCheck, this additional tool in the daily activity in particular in the center where there is an nice high volume for this kind of disease that as I would like again to highlight, it is rare disease and nowadays we believe that such cases, such diseases should be treated possibly when a conservative approach is offered in the referral centers.

Sam Chang: Well thank you very much Dr. Gallioli and Dr. Territo. This work on biomarkers, I think underscores the increasing kind of importance of all the points that you've emphasized in terms of evaluation, diagnosis, and importantly perhaps decreasing the kind of morbidity associated with some of our continued evaluation of these patients. Thank you so much for spending some time with us and thank you so much for the research and we look forward to future publications as well. Thanks again.

Andrea Gallioli: Thank you.

Angelo Territo: Thank you very much. Thank you very much and thanks for giving for this invitation. We'll keep in touch and collaboration is always very welcome for us.