Read the Full Video Transcript

Emmanuel Antonarakis: Thank you, Neal. I've been working in prostate cancer for 12 years, and this is my first time at a LUGPA meeting so, hopefully, it won't be the last. I will be sharing the stage with my esteemed colleagues, doctors Fabrizio, Morris, and Hafron, and I'll be beginning with some remarks for about 25 minutes, and then we are going to bring it home with some real cases from clinical practice. So, these are my disclosures. So, in the next 20, 25 minutes, I'm going to start with some definitions, what is a germline mutation? What is a somatic mutation? We will talk about how common they are in prostate cancer and then who should be tested for them and when, and why do they matter?  And then I'm going to walk you through a genomics report, a next-generation sequencing report and I'm going to cover two caveats that I think are relevant for this audience and for GU oncologists and urologists.

So, all these different vendors, many of which are out in the hall have led to a lot of confusion, which one do I order? Which one of these is a germline test versus a somatic test? Which one of these covers both the germline and the somatic? And it can be confusing and of course, then we've got these liquid biopsies where you get a somatic test from a blood sample. So, it can be confusing, and I will try to deconvolute some of the main principles.

But the reason that we are interested in these is that the landscape of prostate cancer is becoming more exciting, yet more complicated.  And we are beginning like some other cancers to have now a few genetically targeted therapies where the treatment is relevant only in the context of a genetic mutation or a genomic signature. And those are overlaid on the top right with those explosive red explosive signs there, PARP inhibitors for BRCA1 and BRCA2 cancers and pembrolizumab for MSI-high. Of course, that doesn't apply only to prostate cancer or urological cancers, it goes beyond that.

So, let's start with some definitions. So, a germline mutation, what is that? It's a genetic change in the parental germ cell, either the sperm or the ovum that then becomes incorporated into the DNA of every cell in the body of the offspring of that parent.  And variant identified in the germline can be passed from one generation to another and therefore it is hereditary or inherited. On the other hand, a somatic mutation occurs in a somatic cell and occurs after the time of conception. And it occurs in any cell of the body, not just limited to a cancer cell, but any cell in the body except for the germ cells. So, a liver cell is a somatic cell, a skin cell is a somatic cell, a leukocyte is a somatic cell and a cancer cell is also a somatic cell. Somatic mutations are not passed from one generation to another because they do not affect the germ cells and they are therefore not inherited.  In the context of cancer, they are found only in the tumor and not in the germ cells.

So, I'm going to review the prevalence of these mutations, and first I'll talk about the homologous recombination deficiency mutations, which are the ones that lead to PARP inhibitor sensitivity. And if you take a metastatic prostate cancer biopsy, you will find these in about 20% to 25% of patients so, about a quarter of patients will have one or more of these homologous recombination mutations. And the most common gene by far is BRCA2 followed actually by ATM and interestingly in prostate cancer, unlike breast or ovarian cancers, the BRCA1/BRCA2 prevalence is not 50, 50 like it is in ovarian cancer, let's say it's about a 10 to one shift favoring BRCA2.  So, in this disease for reasons that are unclear to me, BRCA1 is relatively rare compared to BRCA2.

What about the germline? So, if you take a blood sample and look at germline DNA from a metastatic prostate cancer patient, they have a 12% chance of having one or more of these homologous repair mutations and the pie chart shows the relative distribution. So, three-quarters of the 12% are in one of those four genes, BRCA2, ATM, CHEK2, or BRCA1. So, somatic about 20% to 25% prevalence, germline about 10% to 12% so, not so rare.

And then the other one is the mismatch repair genes.  And this was the science publication looking at 12,000 cancers of all different types and looking at the prevalence of mismatch repair deficiency. And in prostate cancer, unfortunately, it's only 2% to 3% so, while this is immediately actionable in the context of pembro, these mutations are quite rare, but we do see them from time to time and I believe that one of the examples at the end will be relevant to this.

This slide is now out of date because we have version 1.2022 that was just published of the NCCN guidelines. And if you look at the left, this basically says that germline genetic testing should be recommended to anyone with localized high-risk, very high-risk, or regional disease, which means lymph node-positive or anyone with low-risk and very low-risk who has one of those below characteristics, either Ashkenazi Jewish ancestry, or a family history of breast or colon cancers, or a positive family history of cancer.

So, if you look at that box on the left, this is almost all the patients that we see in the urology clinic with the exception, let's say of a very low-risk patient with none of those other family histories. So, it's a very large group. The somatic testing recommendations are a little bit more restricted because right now the actionability only applies to metastatic patients. So, at this point in time, somatic testing is not recommended for a very high-risk localized patient let's say, who is obviously going to have treatment with curative intent, but if there is metastatic disease including lymph node metastases, then somatic testing becomes relevant because we have the two indications for PARP inhibitors and PD-1 inhibitors.

And one of the caveats is that when we're asking about family history, we should go beyond the family history of prostate cancer or GU cancers. And when I was first starting my career, I really wouldn't ask about breast or ovarian cancer or pancreatic cancer. And by doing so, you miss a lot of relevant signals where a man comes in and says, no, there's no prostate cancer in my family. And if you stop there, you would never know that his mother and two sisters had breast cancer in their 30s. So, when we ask about the family history, even if we're urologists or urological oncologists, we should always ask about all cancers because a lot of these genes, predispose to other non-urologic cancers in the family members.

Germline testing has implications for the family, which are called cascade implications. And this basically means that if you discover a germline mutation in your patient with prostate cancer, 50% of his first-degree relatives by chance would have the same mutation. So, that means a sibling of either sex, a parent of either sex, and a child of either sex would have the same mutation. And a second-degree relative would have a 25% chance, a third-degree relative would have a 12.5% chance. So, this means that when you find one of these mutations, you can't just stop with your patient, you have to recommend genetic counseling for his whole family, given the implications for other family members.

One of the questions that always comes up is when it comes to somatic testing, what should you go after first? And I put up two potential paradigms here, which are not 100% in agreement with each other and that is to show that there is not an agreement in the field about how to do this best. If you look at these two, the first, which is a review article that I helped to write for European Urology, and the second of which is the up-to-date entry by Wassim Abida.  Both recommendations agree that a fresh biopsy is the number one choice, but we all know how difficult that can be, especially if the only lesion is in the bone and both recommendations agree that doing blood or saliva only in other words, germline only is the least favorable choice.

But there is disagreement around the two bullet points in the middle which is, if fresh tissue is not available, what is the second-best? In my opinion, I thought circulating tumor DNA was my second best choice. But the up-to-date guideline actually suggests archival tissue and recommends ctDNA as the third choice. But the ultimate teaching from this slide is that blood or saliva alone is not enough, it's really not adequate because you're missing the somatic half and a fresh biopsy although it's optimal may not always be feasible.

And I'm not going to show the primary data for these, but just to make everyone in the audience aware that last May within a five-day interval actually, we had two FDA approvals of Olaparib and Rucaparib respectively.  Olaparib has been approved for a broad panel of 14 genes, and they are listed at the bottom so, they're a little bit hard to see, but BRCA1 and BRCA2 are listed in there, including ATM, BARD1, BRIP1, CDK12, CHEK2 et cetera. And these patients do not have to have received a taxane agent because the trial that led to this approval did not mandate a taxane, but they had to have received either abi or enza. And the Rucaparib indication is a bit more restrictive. First of all, it's restricted to only the BRCA1 and BRCA2 genes, not the other 14. And it is a post AR therapy and a post-taxane indication so, these patients are third line and beyond mCRPC patients.  And it's hard to imagine that four years have passed since the tumor type agnostic pan-cancer approval of pembrolizumab, has been four and a half years, actually and has again, we talked about the 3% prevalence of this phenomenon in prostate cancer.

There are some things that can increase the chance of having a mismatch repair deficiency and one of them is as obvious as the Gleason score. So, in this study that we did at John's Hopkins before I left, we showed that people that have primary Gleason pattern five, in other words, five plus five or five plus four disease have an 8% prevalence of mismatch repair. So, it's still rare but the prevalence does go up two or threefold. And there's another paper that I'm not showing here, that if you have variant histology such as intraductal carcinoma or pure ductal carcinoma of the prostate, those patients are also enriched. So, if you ever see variant histology or a very high Gleason score, those patients are enriched for mismatch repair deficiency.

And this was the first publication showing that these patients can respond to PD-1 inhibitors.  The surprising and slightly depressing fact is that in this paper of 11 patients, five of the 11 had PSA responses but only two of the five had a response lasting more than a year, as you can see the time scale on the bottom is weeks. So, if you look at 52 weeks, only two patients are getting past that. So, this is very different from colorectal cancer, let's say where the MSI-high colorectal cancer patients when they respond, they typically respond for an average of 24 months which means that something is very different about the prostate cancer microenvironment, which is very immune-suppressive, even when you have all these neoantigens.

So, now I'm going to take a practical approach and go through some of the key features of a so-called next-generation sequencing report. And just to start with what type of information can you expect to find on an NGS report? And these bullet points apply to really almost all the companies that offer these. So, the patient information, the cancer type, the tissue source, and then is it from a biopsy or is it from a circulating tumor DNA that has implications when you interpret that? And then there's a list of mutations or alterations. Almost all of these reports now include the microsatellite status, unstable versus stable. And they report the tumor mutation burden, which is basically an estimate that is reported as a number of mutations per megabase. And then they try to help us by putting in the companion diagnostic claims. So, if you have a BRCA2 patient, the company will usually say, there's a companion diagnostic claim for olaparib or rucaparib, so they can help us.

The VUS page is the variant of unknown significance page and this is something that most people ignore.  Sometimes you can find interesting stuff in there for example, in prostate cancer, we know that SPOP mutations make the tumors more androgen addicted, and therefore sensitive to hormone therapy. SPOP mutations almost never occur on the front page, they are almost always on the VUS page. So, as we learn more and more about the clinical implications of these genes, the companies may not necessarily think of them as pathogenic but on a disease by disease basis we might find something on that VUS page that is actionable.

So, if I was giving this to a non-urological audience, I would point out that there are four histology agnostic indications now. Two of them are for pembrolizumab for MSI-high and then separately for TMB-high, which is defined as 10 or more mutations per megabase. And then, although we do not see NTRK1, two, and three fusions in prostate cancer that often, definitely less than 1% of the time, there are two drugs for that as well. So, we are beginning to see for the first time, the FDA approving drugs, not based on the origin of the disease but based on some biomarker or genomic feature. And I think that will continue to expand over time.

So, all of these reports that I'm going to show you have been de-identified so there is no patient information on these. So, this is a real patient with prostate cancer, metastatic CRPC, who had a biopsy of their lymph node. And what I'm showing here is that this is an MSI-high patient. When I look at this report, I try to find what I call the trifecta and there are three things that I look for when I see an MSI-high. First is the microsatellite status itself. The second is the TMB. If it's a trim MSI-high, you expect to see a high TMB. So in this case, you can see that the TMB is 44 mutations per megabase, by the way, that's really, really high for prostate cancer. Only 3% of prostate cancers have TMB above 20 so, that's sky-high. And then the third thing is if you look on the gene list, it's always nice to see a loss of function mutation in one of the mismatch repair genes.

So, third from the bottom is MSH2 V722 frameshift 11 so, that's a loss of function, MSH2 mutation. So, when you see all three, a loss of function, mismatch repair mutation, and a high TMB, and the microsatellite instability, you begin to believe that this is a true pembrolizumab sensitive tumor. Oftentimes you get tricked and you see one of the three or two of the three, and then you are kind of stuck because you sort of want to give pembrolizumab, but you don't know if that patient is going to respond. And in this case, I made it easy because the person has all three, but sometimes it's not the case.

The other thing that is always very helpful, and some of the NGS companies do this like Caris but others do not, is you can do immunohistochemistry for the four mismatch repair proteins, and actually, many pathology labs are doing this now routinely in clear certified settings. And in this case, you can see all four of the mismatch repair proteins are intact so, this is a patient where you would not expect a mismatch repair deficiency. So, in that previous example, I would've expected MSH2 to be lost at the protein level if you did immunohistochemistry, And in fact, it was.

This is another scenario that we don't see very often in prostate cancer, I had to dig through a lot of reports to find an example. This is an example of a patient that has a TMB of 10 mutations per megabase in the absence of microsatellite instability and in the absence of a mismatch repair mutation.  So, as you can see there on the bottom, Foundation Medicine tries to help us by saying that Keytruda or pembrolizumab is FDA approved because of the TMB of 10. But, I have to say it's really unclear in the rare patients with prostate cancer that have a TMB of 10 or above in the absence of mismatch repair deficiency if those patients actually respond to pembrolizumab, and that's something that I have not yet seen any publications on. What is the efficacy of pembro in the MS-stable but TMB high patients? So, this is an unmet need that I think the field needs to more clearly elucidate.

I want to go through some special considerations and some caveats here and I won't read through this because I'm going to go through them one by one. One of the things that have always annoyed me is when the allele fraction is not mentioned in the report. So, this is an example of a clear loss of function, BRCA2 mutation. But if I asked most people in the audience, if I said to you, is this a germline or a somatic? Most people wouldn't know. It turns out that the [inaudible Siri 00:20:20]Ser1982 frameshift 22 is the most common Ashkenazi founder germline mutation, which occurs in 4% of all Ashkenazi people.

If the allele frequency had been reported on this and the allele frequency was 49%, many of us would be thinking germline because a germline mutation is present in every cell and is present in one of the two alleles. So, the allele frequency should be somewhere between 45% and 55%. And so, one of my plugs, if any of our company sponsors are here, is please put as much information as you can on the reports, because it does help clinicians. And I happen to know this was a germline mutation but others may not have known that and it really does help. If you ever get a mutation like this, your reflex should be to order a germline test, to absolutely prove or disprove what is the origin of the mutation, is it germline or somatic?

Now, there are some companies, I'm not trying to pick favorites but there are some companies that do report the allele frequency.  So Tempus, which happens to be based in Chicago does mention this. So, the FBX011 gene, I have no idea what that is, but the reason I'm showing this is that it has a 68.7% allele fraction so, that's helpful to me. It shows me that the majority of cancer cells have that. And if it was a therapeutic target that might help me to determine what percentage of the cancer cells express that mutation. The other thing is that some companies are collecting paired blood, white blood cell samples, leukocyte, and they can report both somatic and germline on the same report. And Tempus is one such example where if you order a Tempus test, they will send the patient a saliva kit and also organize a blood draw. And what they do is they do the germline DNA from a leukocyte and the somatic DNA from the tumor, and then they report both.  So, in this case, there were no germline mutations found, but if you did see a BRCA2 at the top for example, if it was in the germline, they would have reported it in the same test.

And then just taking a page from ovarian cancer. So, in ovarian cancer, there's an FDA approval as a companion diagnostic for this so-called loss of heterozygosity score, which basically is a surrogate for homologous recombination dysfunction. So, in ovarian cancer, if the loss of heterozygosity score is more than 16%, meaning that more than 16% of the genome shows loss of heterozygosity, then this actually has a companion diagnostic claim for rucaparib. And in this case, which is an ovarian patient, you can see they also have a BRCA1 mutation. So, that explains why they have a high LOH score.

But I think we have to be very careful when we get a report like this.  This is from a prostate cancer patient, and I don't think this was a responsible thing of Caris to report, and they reported this and it said gLOH high, and then next to it, it says therapy association, olaparib. And this was concerning to me because this is a prostate cancer patient, not an ovarian cancer patient. And there is no FDA-approved companion diagnostic claim for olaparib, and this patient does not have a BRCA1 or BRCA2 mutation, but they have a high gLOH score. So, I would encourage the companies to be very careful about what they report and only to report if there's a true companion diagnostic claim because a clinician reading this might order olaparib and that might not work. It is possible that a high gLOH score does predict PARP sensitivity, but we haven't proven that.

And I think we have to be responsible in the way that we report these things because busy clinicians that are seeing 20 patients in the day, if I got this report and I was spending 10 minutes with a patient, I might get excited in prescribing olaparib, and it might not be the right thing to do. And it turns out that the gLOH scores in prostate cancer are very, very different from those in ovarian, breast, and pancreatic cancer. And the thresholds that we use in breast and ovarian of about 16% don't apply in prostate cancer, the thresholds should be more like 8%. So there really should be a disease by disease thresholds that are studied.

And then finally, some of the platforms are now beginning to do RNA sequencing in addition to DNA sequencing. And the example here is that you can detect splicing variants, which are not DNA level mutations but are alterations in the transcript.  And these RNA seek analyses are also actually the best way to find fusion genes, where one gene is fused to another. And the example here is an ARV seven detection, which may help you in terms of not prescribing an AR targeting therapy.

Now, this is a caveat that I've shared with Neil before and he asked me to showcase this to a broader audience. This is a real patient from my clinic, and I made a mistake on this patient and I wanted others to learn from the mistake. And the mistake came from my excitement over the BRCA1 mutation, which is clearly a loss of function, frameshift mutation, but what I've failed to pay attention to was the MLH1, which is a mismatch repair, and the microsatellite status, which came back as intermediate. But the TMB was relatively high for a prostate cancer patient. So, when I saw this, I assumed that the BRCA1 gene was driving his cancer. And as you can see, this patient had no response whatsoever to olaparib.  He took it for about three months and his PSA continued going up.

And then after I went back to the report and I was scratching my head, like why didn't this patient respond to olaparib? I then noticed that the BRCA1 mutation was probably a passenger and was a secondary event caused by the mismatch repair deficiency and not a driver event. And when I went back to Foundation Medicine and I asked them to look at their database and see, how often does this happen? This happens 14% of the time where you have a concurrence of BRCA1 and BRCA2 plus MSI-high. And in those cases, the MSI is the driving event and not the BRCA mutation. So, this person had a one-year response to pembro eventually progressing, but that's an important caveat.

And then the second caveat is this new term that we're beginning to hear, clonal hematopoiesis of indeterminate potential or CHIP.  And what this is, is mutations in the leukocytes, not the cancer cells, which can confound the results of an NGS report. So, in this publication, the authors showed that about 10% of the time, when you find an HRR mutation in an NGS report, it actually comes not from the cancer cell, but from the white blood cell. And we know that as people age, both men and women, these clonal hematopoiesis alterations can accumulate in white blood cells. And it turns out that these mutations can affect the ATM, the BRCA2, and the CHEK2 gene, all three of which are listed in the FDA approval of olaparib. And the only way you can really tell where the mutation is coming from is if you do a paired leukocyte DNA analysis, which very few of these companies are doing right now, and very few academic centers are doing, frankly.

So, this is an example, again, one of my patients, that his ATM mutation is pathogenic, but you can see there's a little hash mark next to it. And if you look at the small print at the bottom, it says, variations in this gene may be derived from clonal hematopoiesis CH. And when I saw this, we actually got a blood sample from the patient, of course, it's an extra step, and it's not something you can do in busy clinical practice. And we actually found that this ATM mutation was in fact coming from a leukocyte. And so, this patient was older, he was in his mid-80s. And so, that ATM mutation would not sensitize his cancer to a PARP inhibitor. And this is scary on the one hand and on the other hand, there's no imminent solution to the problem because many of these NGS companies, perhaps with the exception of Tempus are not sequencing paired leukocytes, it's just an extra step, it's an extra expense. It doubles the cost of the assay, and it's inconvenient for the patient to have to do a blood test and a biopsy.

So, we're going to end with some cases, but first my conclusions. Prostate cancer has a high rate of germline mutations, about 10%, and somatic mutations 20%. Germline mutations should be tested in most patients except perhaps for the very low-risk or low-risk localized patients.  A somatic analysis should be done in all metastatic patients. And then at this point in time, we have two actionable reasons to look for these, the first of which is PARP inhibitors for HRR mutations and the second is PD-1 inhibitors for MMR mutations. And of course, with a germline mutation, your responsibility doesn't end there, you have to send the patient for genetic counseling and make sure that his family members do not have the same mutation. And this is the way that the future is going to start looking for prostate cancer, where we get the genetic analysis, both the germline and the somatic, and then we can put patients into these different bins and then treat them according to their genomic status.  And I think we will be seeing a lot more of this in the future.

David Morris: Thank you very much. I think I speak for the three of us up here, that just when we think we've figured this out, a talk like that scares us into the reality that we are a long way from understanding it. So, I'll start just with, I want to hand it off because I could spend the whole 20 minutes myself. So, really the first one is where the rubber meets the road. When we've got patients in our clinic, some of them have germline tests that we've already run for the high-risk localized, some don't and are metastatic patients with us.

So, I guess when you mentioned fresh biopsies being preferred, I guess the first question on here is for me, I have several patients who have germline negative tests from three, four years ago. Is it still acceptable in that situation? A lot of us have recent archival tissue, I know a lot of the trials relied on archival tissue, it's very easy for us to order archival tissue. And do you feel that is doing a disservice to the patient if we can do it, and if there is no result, then move on to a fresh biopsy? I'd just like you to comment on that.

Emmanuel Antonarakis: Yeah. If that particular patient has not had a biochemical recurrence or metastatic disease, there's no reason to get somatic testing from a clinical perspective so, you can skip it. If they have recurred or metastasized, it turns out that the answer to that question depends on what you are looking for. And in the case of the homologous recombination mutations like BRCA2, the vast, vast, vast majority of those are so-called truncal mutations, which means that they arise in the primary tumor and they don't arise as a selective pressure of therapy. Now, on the other hand, mutations like P53, mutations like RB1, which are currently not actionable, do accumulate over time and under selective pressures. So, for the context of the FDA-approved therapies in 2021, you could very easily rely on the archival tissue for finding the two mutation types that are actionable.

Jason Hafron: Yeah. Thanks, Emmanuel, every time you speak or a paper you write, I always learn something new, really impressive, one of our greatest thought leaders in urology and in prostate cancer today. What comes up a lot in the clinic is you have a metastatic patient, you've done your germline, you've done your somatic testing, you start them on the first line of therapy, is there any value for repeat testing as they progress through their lines of therapy? Is there value to longitudinal testing or is it once the germline's not going to change, but is there going to be much longitudinal change, and is there value for retesting these patients as they progress through the therapies?

Emmanuel Antonarakis: Well, there is academic value for sure. It pains me to say this, I think the clinical value right now is a little bit hard to justify. The BRCA mutations as I mentioned, will almost never become acquired, on the other hand, the mismatch repair mutations, about a third of the time, especially MSH2 and MSH6 can be acquired after the primary clone.  And furthermore, the tumor mutation burden increases over time. So, even if you have a patient with an MSH2 mutation, let's say, if you do an analysis of the primary tumor versus a castration-resistant met in the same patient, three different time points, you'll find the MSH2 in all three cases, but the TMB will go up because tumor mutation burden increases every time a cell replicates. So, the more cell replications, the greater the TMB.

So, one potential scenario, and again, I don't know how common this is, is you may have a patient who starts off with a TMB of eight and you've exhausted all the other therapies and you really want to give him pembro based on the TMB of 10. And if you check a second one or a third one, he might sneak above that artificial threshold. I don't know if that's the greatest reason. Now over time, we are learning a lot about mechanisms of resistance to PARP inhibitors, for example. And one of the questions that come up is, we've all been using platinum agents in the clinic for these patients without a specific FDA approval, but there is a series of mutations that occur after someone has received a PARP inhibitor that is called the reversion mutations, where the BRCA two-gene, for example, it boggles the mind.

It goes from mutant back to wild type. And by going from mutant back to wild type, that cancer cell can survive the PARP inhibitor. And this can happen even when the first mutation is a germline mutation, which boggles the mind even more. And so, I can sometimes envision a scenario where I've used all the FDA-approved drugs, I find a BRCA2, I put the patient on a PARP inhibitor, then they progress a year later. And I'm wondering, should I treat this patient with single-agent carboplatin? And if I do that test and I don't find one of those reversions, that I may consider platinum for example, but in general, these are sort of case by case. And I would say for the most part, although great for academic interest, there may not be a very big clinical rationale to do serial testing in this day and age.  That may change in three years.

Neal Shore: So, can I ask you just a quick question based upon Jason's question about getting serial biopsies per lines of therapy.  Maybe just touch on this acronym that's out there now for a lot of our colleagues, maybe explain it, the whole notion of MRD in circulating tumor cells and just trying to get a handle on that. I know it's a little bit more information on bladder cancer but maybe even so in the prostate, what are your thoughts on MRD?

Emmanuel Antonarakis: Right. Minimal Residual Disease or MRD? So, this has been used in diseases such as leukemia to track how many cancer cells are still left behind after some therapy that either has curative intent or cytoreductive intent. And as we know in bladder cancer and some of the adjuvant studies with immunotherapy, whether or not there is or isn't any circulating tumor DNA left after a period of therapy itself could be prognostic. So, this is a little bit different way of thinking about it, Neil, it's not that we are using the circulating tumor DNA to find any particular mutation, we're just looking at the sheer presence or absence of ctDNA as a marker of disease burden.

One of the things we have to remember is yes, there have been publications showing that if you use a systemic therapy and the circulating tumor DNA goes from detectable to undetectable, the person is going to do better than if it remains detectable. But, unlike the bladder data that you mentioned, we do have PSA and PSA is a surrogate for disease burden, and I'm not convinced that an expensive test such as ctDNA, although it's cool, it's great if you can do that in the academic setting, I'm not sure how many patients are going to be, let's say PSA undetectable, but ctDNA detectable that's possible. If that was the case, that would be a great utility for it.

Michael Fabrizio: So, just to finish up, we're going to have a couple of cases here just to stress the importance of why you need to be familiar with this, and that was once again a great talk. So, this is a 69-year-old gentleman who presented with metastatic prostate cancer. He had a history of Lynch syndrome, and we can maybe touch base on that.  He had a colon resection in his history, family history of breast cancer, and colon cancer, as you would expect, they have family histories and multiple malignancies. He had a biopsy in 2017 with fairly aggressive disease, Gleason nine, eight, and nine, T2A, PSA 11.  He had radiation therapy in two years of androgen deprivation in North Carolina, and then presented to my clinic with a biochemical recurrence, as soon as he stopped his ADT therapy and subsequently his CT/Bone scan was negative.  He did have an F-18 scan, which showed really intense uptake in his iliac bone, right ischium highly suspicious.

Of course, started ADT right out of the gate and also underwent genetic testing and was found to have microsatellite instability, and that you would, of course, associate probably with this autosomal dominant condition with the mismatch repair. We started him on Erleada and also he received radiation therapy to his ischium and his pelvis. He also received Provenge. He continues to progress now, his bone scan showed numerous areas, and based on the genetic study, he was now referred to one of our local groups for Keytruda infusion which hopefully in the next few months we will be doing ourselves. But I think this case emphasizes the importance of really understanding the disease process and making sure the patient is eligible for any therapy.

I don't know if our panel has any discussion or-

Emmanuel Antonarakis: Are you going to show the report or is that it? 

Michael Fabrizio: So, this is the report. I don't know if you'd like to comment on it?

Emmanuel Antonarakis: Right. Well, I'll give you guys all the caveats here. So, MSH2 is clearly one of the four Lynch syndrome genes. So, Lynch syndrome is the inherited mismatch repair deficiency also called HNPCC, hereditary nonpolyposis colorectal cancer. And the four cancers that are linked to that are colon, bladder, endometrial, and prostate. And this is clearly a pathogenic mutation because it's a deletion so, it wipes out a bunch of exons. So, is it pathogenic? Yes. Is it in the germline? Yes. The caveat here is that unfortunately about a quarter of prostate cancers that occur in patients like these are sporadic cancers and they have nothing to do with the MSH2.

So, the teaching point here is if you have a germline mutation in any of these genes, it doesn't mean that by necessity the cancer is going to be caused by that. There is a way to find out. And the way to find out is to get a tumor biopsy, any tumor biopsy from that patient and you look for two things. First, you look for loss of heterozygosity of the second allele, because remember the mechanism of action of these tumors is Knudson's "two-hit" hypothesis. And Knudson's hypothesis, Alfred Knudson was that the first mutation is inherited, that's the first hit and then you lose the other copy of the gene from the other chromosome in the tumor only, that's the second hit. So, you can look for the loss of heterozygosity.

The second sort of defacto way is, is the tumor hypermutated or not. If you did a somatic analysis and the TMB came back at three, even without knowing what the second allele is doing, frankly, even if the second allele is lost, if that's not resulting in hypermutation, it won't result in pembro sensitivity. And please let me know what happens to this patient. Unfortunately, I hope I'm wrong, I would predict that he won't respond and the reason is he's already had Provenge, and there have been some case reports that the true MSH-high patients can have dramatic responses to Provenge and this patient from what you told me blew right through Provenge?

Michael Fabrizio: Right.

Emmanuel Antonarakis: So, I hope I'm wrong on that, please close the loop once you give him the pembro.

Michael Fabrizio: Thank you. That's great. And then this is the second patient. An 80-year-old physician who had his radical prostatectomy here in Chicago in 2003, underwent salvage radiation therapy, his PSA went to 0.2 at that time, and had ADT for three years. In May of 2011, you can see here that his PSA bounced around, he was started on other therapeutics, at that time, his imaging had no lymphadenopathy, but a suspicious area in the T-spine and rib. And then subsequently he presented to our clinic for follow-up. He started Erleada, an MRI showed bone marrow replacement, pretty expansive tumor burden, he had palliative radiation, Provenge, and PSA 11. Now, no one had done genetic testing so, we did genetic testing and it was found to be BRCA2 positive, ARV7 was negative.

He had progressive metastasis, his PSA rising and he was entered into the TRITON trial, and Rubraca was the therapeutic choice of course, in that trial. The patient had significant anemia, interestingly, his hemoglobin was 12.3 when we started when he came to my office with his wife who was also a former nurse and he looked like Casper the ghost.  His hemoglobin was 4.5. We diagnosed it by looking at him, but he thought he was a little anemic, but didn't really want to report it. So, but subsequently, he did well, transfused and resumed therapy at a lower dose, and now he is progressing. And this is his genetic marker study, any commentary there?

Emmanuel Antonarakis: Yeah. So, you can tell immediately this is from a circulating tumor DNA, not from a biopsy. And the reason I can tell that is, so it turns out that Foundation Medicine reports the allele fraction for ctDNA, but not for tumor biopsies so, that's how I know this is a ctDNA test. And the second thing that you can see immediately is that the R2520 is a germline mutation. And I don't know if you've done a germline test to confirm it. I hope you do one because that 48% allele frequency is going to be germline. But interestingly, he's got another mutation in BRCA2, Q1998, which is 3% allele frequency and that is almost certainly the second hit.

If it was on the same allele, the MAF would be around 49%. So, this is inferred to be a biallelic BRCA2 mutation. So, this is the guy that you would expect to have the greatest tumor response to a PARP inhibitor. Unfortunately, it's also the guy that you would potentially expect to have the greatest toxicity from a PARP inhibitor because all of the cells in his body have the BRCA2 mutation, including his blood cells. And so, the precursors for his red blood cells will also be more susceptible to aplasia from the PARP inhibitor so, maybe that's one of the reasons that his hemoglobin was so suppressed.

By the way, about 15% of people with PARP inhibitors do require a blood transfusion. So, grade three to grade four anemia, we do see, and it needs to be addressed. That HRAS mutation is almost certainly on the same allele as the second BRCA2 mutation, because it's the same allele frequency.  In prostate cancer, I would never use any of those three agents that are shown there because we just do not have the data, but this is a great case and obviously, if this person has living first-degree relatives, they should undergo genetic counseling.

Michael Fabrizio: Well, thank you for that great explanation. Truly humbling. I think the three of us here will, I think, most of the messaging here is we should send most of our genetic patients there so, thank you so much.