Neal Shore: I'm here today with my good friend and colleague, Badri Konety, Badri is the Chairman of Urology at University of Minnesota, and Badri has been a pioneer, researcher in biomarker development, particularly in bladder cancer. He and I had a great privilege to work together on this particular study and paper assessing Cxbladder, and we've worked together on other biomarkers, and I think it's great, I'd love to have Badri run through the paper that we had published, he's the lead author that was published recently in a European urology, so Badri thanks so much.

Badri Konety: Not at all, my pleasure, Neal. Great to be with you and go over some of this data that we put together. I think it's really exciting and I have a particular interest in making sure people are aware of this. Just as a preamble, Cxbladder is a, I would say, a next generation of bladder markers, and we've been trying to develop bladder markers to better identify bladder cancer using urine specimens for the better part of three or four decades now. And each decade or two we come up with the next generation, initially, it was just proteins and it was cell-based and now we're getting genetics and then we're getting genetics combined with the phenotypic information, which is what Cxbladder is. There have been other papers published about Cxbladder as a biomarker, and it has shown great promise in adjudicating patients who have hematuria and also in monitoring patients who have a known diagnosis of bladder cancer.

This particular paper, we used data from all the studies to really examine the value of Cxbladder in adjudicating a typical cytology, and some cases equivocal cystoscopy. Oftentimes, we do see patients who have this read on the cytology and we're not sure what to do next or what is the likelihood they do have cancer.

I do want to take this opportunity to acknowledge all my co-authors, without whose contributions in the study wouldn't have been possible, as well as Pacific Edge that has come up with Cxbladder. It's been a brilliant technology that we are going to be able to use, I use it quite a bit in my practice based on specific indications.

Now, one of the things we know about urine cytology is it's not easy to get, you have to send out the specimen somewhere and somebody in the lab will read it out and send it. So if you're in practice and you don't have quick and easy access to a cytopathologist who is an expert in reading urine samples, it's not an easy test to get an immediate answer on. Often times the read can be very confusing, you've got a read of atypical, atypical suspicious, suspicious. And when you get these kinds of reads, it's unclear what they mean. Do they mean that this patient has cancer or doesn't have cancer? Should you work them up or can you just ignore it?

And if you get this kind of a read in somebody who's never had a diagnosis, you don't want to miss cancer, so that's a problem. So when we get these kinds of reads, oftentimes it becomes a diagnostic dilemma for physicians, and the default position is to submit them to a complete workup, cystoscopy, bladder barbotage, retrograde pyelograms, maybe even ureteroscopy, which are all have their own attendant cost and complications, and oftentimes you will turn up with nothing and then you're sort of not sure, you may even biopsy the bladder as well as the upper tracts, which yield have nothing.

So the idea is, can we somehow figure out a way to reduce all these unnecessary workup in patients who are going to turn out to be negative on that kind of a workup anyways. So this marker Cxbladder in a nutshell, we think can really help, we'll show you how based on the data in this paper.

In this study, which really was used samples taken from four different studies done on three different continents, we had a total of about 1700 plus patients, 1774 patients. And after excluding some of those and whom we didn't have a cytology result or the Cxbladder results, we came down with 852 samples that had both a Cxbladder and a cytology result in a cystoscopy. So we wanted to make sure we had all of these just so that we understood what the ultimate yield or benefit of Cxbladder would be. So after these 852 samples, we had 153 of them or 18% were classified as atypical, and that was really the key target population for the study.

Now, as I mentioned before, there's a lot of different ways to read urine cytology and classify it differently, the most recent classification system is called the Paris system, which was devised in 2016. In that system, really the whole idea is to identify only high-grade urothelial carcinoma, so the presumption is if you miss some low-grade ones, it doesn't matter as much.

So the newer systems are trying to reduce the number of atypical calls, but still, there's about 20% of all urine cytology that'll be read as atypical or suspicious or some other read other than a confirmatory read like negative or positive. And you're obliged to work up all of these, particularly if the readings atypical or suspicious.

So in this study, just to give you a quick overview of the data that was used, we used data from 371 samples that were obtained in one of the US studies. Second US study had about a thousand samples, a third study done in Australia had 485 samples, in New Zealand, there was a study with 178 samples. So we had a total of 2000 plus samples from 1,784 patients and we excluded a whole bunch of samples, we did not have concomitant cytology in Cxbladder leading to about 852 samples from 775 patients that I mentioned before.

Of these 852 samples, 436 samples were obtained from patients who came in with hematuria and underwent a Cxbladder test. 416 samples were obtained from patients who had recurrent bladder cancer, or a history of bladder cancer and were being monitored for recurrence. And in all of these patients, in these 852 samples, we had local cytology that was done, and the report was available and the reason we use local cytology rather than a central single cytology read, was because we wanted to really make a real-world comparison because that's what happens normally is you have arbitrary cytology reads done by the locally available expert.

One of the things to point out is Cxbladder is really not just one test, there are three formats for the test, there's a Cxbladder T or Cxbladder triage, which is really the first test that you would use in somebody presenting with Just hematuria to really rule in or rule out the need for additional testing or further workup. CX-D or Cxbladder detect is a test that is used as a second-level test in somebody who has a Cxbladder T-test that's positive, once you have a positive test, now you want to try to find out if this patient has a high-grade or low-grade cancer and that's what the Cxbladder detect will allow you to do, a positive test indicates greater likelihood of having high-grade cancer.

A Cxbladder M or CXBM is a format of the Cxbladder test that's really meant for patients who have a prior diagnosis of bladder cancer and are currently being monitored or under surveillance for recurrence. The difference between these three tests are in the algorithm that's used to predict the likelihood of cancer. There's five gene panel that's assessed using the mRNA from the cells in the urine, they check for five different genes, and then they combine the genetic expression data along with the phenotypic data, including history of prior tumor or lack thereof, and patient characteristics and they put all of this together into a mathematical algorithm that spits out a predictive value or the probability that this patient will have bladder cancer or not, and that those are the the format is the same except the different variables put into the prediction models are different based on the type of test, whether it's CXT or CXBD or CXPM.

These tables actually really give you an idea of the negative predictive value and sensitivity of Cxbladder as well as cytology. The Cxbladder test is really geared towards attaining a high negative predictive value, which is particularly useful if you're evaluating patients with gross hematuria because you don't want to submit everybody to unnecessary workup, but you also don't want to miss patients with bladder cancer. So in a test that has a high negative predictive value, if the test is negative, you can safely assume that the patient does not have cancer.

So in the 852 samples tested with both Cxbladder and cytology, the negative predictive value of Cxbladder was 97.4%, cytology was 92.6% and we had 302 patients with a negative result on Cxbladder, and with cytology there were 801 patients with a negative result. Now when you actually looked at the number of tumors detected in patients who had these negative results, Cxbladder only missed eight tumors, meaning they had a false negative result in only about 8.5% of the cases. Where cytology missed 59 of these tumors resulting in a false negative rate of 63%, so, we had about 94 of the 852 samples were associated with cancer, so clearly cytology missed a lot more cancers than Cxbladder did. There's almost a seven-fold, actually a little more than a seven-fold difference between the two tests. So that's quite a powerful vast gap between the two tests.

Now when you, again, look at the true negative and the false negative rate, you can clearly see that cytology ruled out about two and a half times more patients. So clearly when cytology was positive, it was more likely to be a true positive. So Cxbladder is more likely to be a, you may have a slightly higher false-positive rate, but the true negative rate was much higher for Cxbladder. And that's something to keep in mind.

Now when we look specifically at subgroups of patients, subgroup of patients who were undergoing the testing for hematuria, that is, they had a Cxbladder triage, and a Cxbladder detect test. We look to see the number and percentage of tumors that are missed in samples with a negative result and the negative predictive value in the subset of patients who had hematuria, that's 436 samples, we obtained a negative predictive value of 97.6% for Cxbladder T, but when you combine the Cxbladder T and a Cxbladder D it still retained the high negative predictive value was 97.8% and cytology was still low, was 93.9%. So it was lower than the other two tests.

The patients who had negative results in whom a tumor was present, meaning that they were falsely negative. So Cxbladder T missed only four patients, or 8.9% and all of them were low-grade tumors. Whereas the combination of Cxbladder T and CXBD, that combination missed seven of the patients or 16%, four of which were low-grade tumors and there were two low-grade tumors and one high-grade tumors missed by the CXBD test. However, cytology miss 25 of these tumors, these are in patients presented with hematuria or it missed about half the tumors, so 56% of the tumors were missed, 14 of these cytologies were read as atypical, six with low-grade and seven with high-grade and one with mixed, and then 11 patients were completely negative on cytology. So a total of 25 patients, 11 had a negative cytology, 14 at atypical cytology and all of them had some kind of tumor.

When you look at the subset of patients who specifically had atypical cytology, we had 153 patients, 150 samples that were read as a typical, and these samples that had a typical cytology, Cxbladder was negative in 47 of these samples and none of them were found to have urothelial carcinoma, and subsequent workup. If the Cxbladder was positive, which was the case in 26 of these samples, they had urothelial carcinoma as well.

However, 80 samples that were Cxbladder positive did not have urothelial carcinoma. So the positive predictive value was a little bit low, but the negative predictive value was extremely high, it missed none of the urothelial carcinoma, which is really an excellent thing. Of the tumors that were found in the patients with atypical cytology results, 13 were low-grade and urine cytology is well known to miss low-grade tumors, 12 were high-grade and one was mixed grade tumor, bit of high and bit of low-grade. 31% of the patients or 47 samples of atypical cytology were appropriately ruled out by Cxbladder with no false-negative results. So if Cxbladder is negative in somebody with an atypical cytology, you can at least based on these data, we can be pretty darn sure that they're not going to show up with a tumor on workup. Of course, we have to keep in mind this is one study with a reasonably good sample size, but it needs to be duplicated.

So to summarize, in this study, Cxbladder was able to correctly adjudicate atypical cytology results and rule out the diagnosis of urothelial carcinoma in 31% of the samples, thereby sparing these patients from unnecessary invasive workup. In 14 of these cases with atypical cytology, the Cystoscopy was also equivocal, so now you're stuck in these patients, cytology is atypical, cystoscopy is also equivocal and two of these patients actually had urothelial carcinoma and both were high-risk tumors, one with a high-grade T1 and the other with CIS, and both of these tested positive for Cxbladder.

So this was really very helpful because in this case with atypical cytology and unequivocal cystoscopy, it was hard to figure out what to make of this and Cxbladder was able to provide the answer, particularly in these two patients with high-risk tumors. This really provides a strong argument for using Cxbladder instead of cytology as the accompanying test to a cystoscopy. This will avoid the atypical cytology results and potentially minimize the unnecessary workup in some of these patients, particularly those undergoing hematuria workup.

These data don't really try to address the role of central cytology with the expert read because we use local cytology, but this is the real world situation, so it's not a centralized cytology reads are not feasible in most practice settings.

Again to reiterate the results, Cxbladder correctly adjudicated all the atypical cytology samples and identified all the true negatives correctly so had almost a 100% negative predictive value. It also identified two tumors in patients who had atypical cytology and Equivocal Cystoscopy.

So in conclusion, Cxbladder clearly appears to outperform urine cytology for identifying patients with Urothelial Carcinoma overall, it missed fewer cancers, only about 8% of cancers as opposed to a much larger percentage with urine cytology. It clearly adjudicated all the atypical cytology patients and helps in patients with atypical cytology, and Equivocal Cystoscopy. I think there is a significant utility that is gained from including Cxbladder in the evaluation of patients with Urothelial Carcinoma, especially as a potentially the second level test in patients who have an atypical cytology, at the very least you could use it in that context. And the AUA guidelines on non less invasive for bladder cancer do indicate that urinary markers could potentially be of value in interpreting the likelihood of cancer in patients with atypical urine cytology, or in those who are undergoing intravascular BCG therapy to assess response.

So that may be one potential setting in which something like Cxbladder could be used in adjudicating atypical reads on cytology or it could also be used in place of cytology as a companion to cystoscopy, to really help identify more of the tumors and also decrease the false-negative rate.

So just to go over the main points of our study again, and to give you some take home messages, Cxbladder significantly outperformed urine cytology for identifying patients with urothelial carcinoma, it correctly adjudicated all atypical urine cytology reads, and also atypical cytology with the equivocal cystoscopy. Cxbladder and cystoscopy were concordant for all positive cases previously categorized as atypical by local cytology, including all positive urothelial carcinomas that were read as atypical, and cytology and were equivocal on cystoscopy.

I think there's significant utility in using Cxbladder in evaluation of patients both in the context of hematuria, but also for surveillance of those who have a prior history of urothelial carcinoma. This would allow us to avoid unnecessary cystoscopies potentially in about 35% of patients. In my opinion, Cxbladder can be used either as a reflux to cytology as a reflex test to cytology, particularly in case of atypical reads, or potentially even as a replacement for cytology to alleviate the diagnostic dilemma posed by the variable cytology reads.

Neal Shore: Well, that was a masterful review of the work that we did for this paper in European Urology, Badri, and thanks to your leadership on it. I think you did a really wonderful job of outlining the nomenclature for the Cxbladder test to detect, triage the monitor, and how to use it and where its value is in terms of its negative predictive value, it's adjudication for atypical cytology. And a big thing that I think is important, at least for all of us is we recognize that in so many of our patients who have asymptomatic microscopic hematuria, or if we're monitoring them for recurrence of low-grade bladder cancer, we would like to avoid unnecessary cystoscopies. No patient wants to be cyctoscoped if it can be avoided.

So you really clearly outlined the correct or the appropriate patients who can benefit from this advancement in urinary biomarkers. And as you also said, we look forward to additional studies that you and I are both involved with. So thank you so much, it was a great presentation and I hope the audience finds it of value as well as the paper.

Badri Konety: I do too, I think this is going to be a new age in urinary marker testing, these are all very sophisticated tests so I'm very excited, and I really find it extremely useful in my practice, I use for the appropriate patient, it's very helpful.

Neal Shore: Yeah, same here. I use it routinely and I use it exactly the way you've outlined it in the presentation, so thanks very much and hope it was a value for our audience.

Badri Konety: Yeah, thank you, and yes, I hope so too.