Emmanuel Antonarakis: Hello everyone. My name is Emmanuel Antonarakis, and I'm a professor of medicine at the University of Minnesota, Masonic Cancer Center. I'll be speaking about genetic and genomic testing in prostate cancer today.

These are my disclosures.

So today, I'm going to outline some definitions. What's a germline, and what's a somatic mutation? How common are these in prostate cancer? Who should be tested for them and when? And why do these germline or somatic mutations matter? I'm going to end with two real world caveats, and some lessons learned from these caveats.

As many of us know, there's many commercial and in-house platforms to assay the germline DNA and the somatic DNA of our patients; and this can be confusing for many of us, so I'll try to break it down and add some clarity.

The prostate cancer landscape to many of us, including me as a medical oncologist, has become very complicated, so that's a good thing and a bad thing. And one of the interesting aspects of this is that, we now have at least three therapies that target either a genetic alteration or a particular protein in the prostate cancer. So we have olaparib and rucaparib approved, homologous or combination repair deficient, or BRCA1/2 mutated prostate cancers. We have pembrolizumab for the mismatch repair deficient or MSI-high cancers. And of course, we have Lutetium-PSMA, the latest FDA approval, for patients with PSMA-positive metastatic castration-resistant prostate cancer.

But first, let's begin with some definitions. A germline mutation is one which is inherited. It's found in the germ cells. In other words, the sperm or ovum of the parent. And it can be passed to an offspring, and therefore, these are hereditary, and they can be passed off to the offspring. On the other hand, the somatic mutation is found only in the tumor or other somatic tissues. These are acquired after birth, and they cannot be inherited or passed to the offspring.

Now, there are two clinically actionable classes of genetic mutations. The first are the homologous recombination genes. These are genes such as BRCA2 and ATM. And at the somatic level, these mutations are present in about 20 to 25% of metastatic prostate cancers. At the germline level, it turns out that, about 12% of men with advanced to metastatic prostate cancer have an inherited homologous recombination mutation. And the foremost common genes are shown there, BRCA2, ATM, CHEK2, and BRCA1.

Another type of mutation is called the mismatch repair mutation. These are the genes, MSH-2, MSH-6, and LH-1, and PMS-2. And these are the genes that were mutated, caused this microsatellite instability and hypermutation. In prostate cancer this is quite rare, accounting for about 3%, maybe up to 5%, of cases and enriched in the metastatic disease.

Now, let's review the NCCN guidelines. These are similar, although not identical to the AUA guidelines, and other guidelines as well. So first, germline testing on the left. This should currently be recommended for all patients with high-risk, localized, or very high-risk localized prostate cancer, as well as those with node positive disease, in other words, locally advanced. In addition, germline testing should be recommended to low-risk patients, or even very low-risk patients, who either have a personal or family history of cancer, or an Ashkenazi Jewish inheritance, or a family member with a known BRCA1, BRCA2, or mismatch repair mutation.

At the somatic level, tumor genomic testing should be recommended to all patients with metastatic prostate cancer, and should strongly be considered in those also with nodal metastatic disease. And here, we are looking for the homologous recombination mutations. Of course, these are PARP1 inhibitor mutations, as well as the mismatch repair mutations for pembrolizumab eligibility.

Now, the family history has become important, not just the family history of prostate cancer, which we often ask, but it's also important to ask about family history of breast cancer, ovarian cancer, and even pancreatic cancer. Because there are at least four BRCA associated cancer types, only one of which is prostate, and there are three others as well, which can increase the risk of our prostate cancer patient having a germline mutation. So please don't forget to ask about breast, ovarian, and pancreatic cancer history

If a patient is diagnosed himself with a germline mutation, this brings up the point of cascade testing. Meaning that, this patient has a 50% chance of his family members, in other words, siblings, parents, or children, having the same mutation. And this can lead to cascade genetic testing in other family members, to look for the presence or absence of the same germline mutation. So there are family implications if we find a germline mutation in our prostate cancer patient.

These are two recent recommendations of how and what to use for the genomic testing. The left is from a review article that I was privileged to write with two co-authors, and the right is from Wassim Abida up-to-date chapter. Both of these recommendations recommend that a fresh metastatic biopsy is the optimal choice. And both of these also agree that the most inadequate way to test is to do germline only testing.

Now, there are some differences in what is the second best and third best. On the left, we can see that in our review, we thought that the second best was a circulating tumor DNA, while the third best was an archival tumor biopsy, or even a primary tumor. While on the right, Dr. Abida suggested that an archival tumor biopsy was second best after a fresh tumor, followed by circulating tumor DNA as the third choice.

One important thing about circulating tumor DNA is that, the ability to get a positive or a valuable test depends on the PSA level. And what we can see here on the graph on the left is that, the circulating tumor DNA content increases with PSA level, and there appears to be a threshold with a PSA level equal to or above five nanograms per ml, where the chance of finding a valuable tumor DNA with a tumor fraction above 10% becomes high. About 31% of those patients will have a valuable circulating tumor DNA. So in my practice, if a patient has metastatic disease with a PSA of less than five, I think that the yield of a circulating tumor DNA test will be low. While for those with a PSA above five, and especially with a PSA above 20, the ctDNA yield is likely to be much higher, and therefore, giving rise to an invaluable test.

Here's some new data from the Memorial Sloan Kettering Group, trying to answer the question, what proportion of mutations found on tumor testing could in fact be of germline origin? So what this shows is, that there are certain gene mutations that when found in the tumor are virtually never germline mutations. For example, mutations in P53, PTEN, and RB1. When these are found in the tumor, they're virtually always somatic only mutations. However, when you look at other genes like BRCA2, CHEK2, or even APC, which might be surprising, a significant proportion of those mutations, if found in the tumor, can actually reflect a germline mutation. So if someone begins with a tumor only testing, and finds a BRCA2, or a CHEK2, or APC mutation, or any of those with the orange bars, you should strongly consider germline testing afterwards, to disambiguate a germline from a somatic mutation.

And these are some guidelines presented by the same authors, about when to pursue germline testing following tumor only testing. And essentially, if you find a homologous recombination gene mutation in the tumor, or a HOXB13, or certain types of APC mutations, you should reflex to a germline test afterwards. Whereas, if you find a P53, a PTEN, or an RB1 mutation, you may be able to get away with omitting the germline testing. The caveat of course being, that if that patient meets the NCCN guidelines with metastatic disease, node positive disease, or high-risk localized disease, they should undergo germline genetic testing anyway.

We talked about the homologous recombination mutations leading to two FDA approvals of olaparib monotherapy, in the case of 14 gene mutations, and rucaparib monotherapy in the case of BRCA1 and BRCA2 gene mutations. And several additional studies are now looking at combinations of PARP inhibitors plus AR inhibitors, which will be discussed later.

We also have the tumor type agnostic FDA approval of pembrolizumab in 2017, for any cancer with a mismatched repair deficiency or MSI-high phenotype. And as mentioned, this accounts for about three to 4% of prostate cancers.

And this is an example of an early paper, which has been confirmed by other groups, showing that mismatch repair deficient prostate cancers can respond to PD-1 and PD-L1 inhibitors. And in some cases, these responses can be durable.

Now, I'd like to end with two caveats. This is a real patient from my clinic. This report has been de-identified, so there's no identifiable information on it. But as you can see, this patient has a loss of function BRCA1 mutation, pathogenic, but also has a pathogenic MLH1, which is a mismatch repair gene, with an MSI-high status, and a high tumor mutation burden of 20 mutations per megabytes.

So how should this patient be treated? Let's assume for the moment that he has metastatic CRPC. It would seem that a PARP inhibitor could be an option because of the BRCA1 lesion, and also, a pembrolizumab due to the mismatch repair deficiency.

This is a case report that we've published in the JCO Precision Oncology. This patient unfortunately did not respond to PARP inhibitor olaparib, but subsequently, did have a robust 12 month, approximately, response to pembrolizumab. And the lesson here, which is the first caveat, is that a BRCA1 or two mutation in the context of microsatellite instability is usually a passenger mutation, not a driver mutation. And these patients, in most cases, should probably be treated preferentially with the PD-1 inhibitor, rather than a PARP inhibitor.

Second caveat is the issue of clonal hematopoiesis of indeterminate potential, also called CHIP. These are mutations that come from the leukocytes, from the white blood cells, which especially on a circulating tumor DNA assay, could appear to come from the tumor, and could be erroneously classified as a tumor derived mutation, whereas in fact, it's a leukocyte mutation. These mutations increase with age, and are present in about 10% of our prostate cancer population. And interestingly enough, these mutations can occur in the same genes for which PARP inhibitors are approved, such as ATM, CHEK2, and in rare cases, even BRCA2. These mutations are difficult to resolve, but can be identified by sequencing matched circulating tumor DNA and leukocyte DNA, to disambiguate between a clonal hematopoietic mutation versus a tumor deriving mutation.

And this again, is a de-identified report from one of my patients, showing an ATM mutation there, which is marked by a hash sign. And the hash sign says this could be a non-tumor source such as clonal hematopoiesis. And this patient did in fact, have matched leukocyte testing, and that ATM mutation was, in fact, shown to be a CHIP mutation, not a tumor derived mutation. So this patient would not be expected to respond to a PARP inhibitor such as olaparib.

In closing, I'd like to convince you that prostate cancer has a higher rate of actionable germline and somatic mutations, especially in those with metastatic disease. Germline mutations should be tested in all patients with prostate cancer, perhaps with the exception of the low-risk or very low-risk localized, and somatic mutations should be tested in all men with metastatic disease, since there are therapeutic implications.

Homologous repair mutations and mismatch mutations do have therapeutic implications in the context of PARP inhibitors and PD-1 inhibitors, respectively.

And finally, identification of a germline mutation, but not a somatic mutation, does have family counseling and genetic implications for first degree relatives.

Thank you very much for your attention.