Current State of Liquid Biopsies in Prostate Cancer - Gert Attard

(Length of Interview: 13 min)

Charles Ryan and Gert Attard discuss what is currently being done with regards to serum analysis, plasma analysis, liquid biopsies, circulating tumor cells, in the field of prostate cancer.   

Biographies:

Professor Gerhardt Attard MD Ph.D. FRCP Prof Attard is a John Black Charitable Foundation Endowed Chair in Urological Cancer Research at University College London. He holds an advanced Cancer Research UK Clinician Scientist award and is Team Leader of the Treatment Resistance Group at the UCL Cancer Institute.

Charles J. Ryan, MD

 
Read The Full Video Transcript

Dr. Charles Ryan: I'm joined by Gerhardt Attard, who is the John Black Charitable Foundation endowed chair for urological cancer research and recently took this position at University College of London. Congratulations on your new position. We're going to talk today about plasma analysis, liquid biopsies and what's going on with that in respect to prostate cancer. 

So before we get into the real detailed science, Gerhardt, tell us a little bit about the current state of liquid biopsies. Can we get them on patients. What are we currently able to do with regards to serum analysis, plasma analysis, liquid biopsies, CTCs and prostate cancer. 

Dr. Gerhardt Attard: As you know, Chuck, I spend most of my time, my research time thinking about liquid biopsies, but I would not yet use a liquid biopsy test to change one of my patient's practice. I think we still need to put a lot more work into analytical validation and clinical qualification. 

But that said, there's good reason for all the excitement surrounding liquid biopsies. And to take a step back and take a broad overview, there's multiple elements we can extract from blood. The two that have given most data so far, that most work has been into so far in cancer patients, are circulating tumor cells and plasma cell free DNA. And there's merits and pros and cons to the use of both. And as we've discussed, I've invested a lot of time in analysis of plasma DNA, which is DNA we extract from the compartment of blood that does not contain cells. So, the tube is spun twice and we remove the upper plasma compartment, and that contains enough DNA for us to generate libraries and perform next generation sequencing. The benefit of plasma DNA versus cellular analysis is that the ratio of tumor to normal is often higher. 

Dr. Charles Ryan: Mm-hmm (affirmative). 

Dr. Gerhardt Attard: So when we're looking at CTC analysis, really looking for relatively few cells amongst millions of leukocytes. Of course, there's great merit and value in extracting and capturing those cells, but that's a major effort in itself. While the plasma DNA, we can extract the DNA, and that's what my lab's been doing, we extract the DNA, we sequence it and we're able to detect tumor mutations as an allele frequency at an abundance as low as 1%. Even if only 1% of that DNA is tumor in origin, we can detect the mutation. And indeed samples from all progressing metastatic CRPC patients, we detect tumor mutations, and in the majority we also detect copy number gains and losses. That, of course, is valued because that gives us molecular information from a liquid biopsy test that can be done relatively economically and can also importantly be done on stored samples. Again, one strength of plasma analysis is that we can collect the samples, spin them and keep them in the freezer. And now multiple trials in prostate cancer and multiple cancer types, but certainly prostate cancer, have collected plasma, which is going to allow us to clinically qualify and test the assays we're developing. 

Dr. Charles Ryan: Are there older studies where plasma was collected where we now have very mature data where you can go back and do this analysis or is it not stable enough to do that?

Dr. Gerhardt Attard: It is stable enough, but I don't think we knew enough five or ten years ago, so of course- 

Dr. Charles Ryan: To collect the samples. 

Dr. Gerhardt Attard: In fact, this morning we were COUGAR 302 that you ran, and I guess we didn't collect plasma back then, but ten years ago when you were designing the trial, that's not something we...we didn't have this data at hand. 

More recent trials, the majority of trials initiated in the past four to five years have started to collect plasma, and it's not just the sponsors that need to be brought in, also needs all the investigators across multiple centers. 

Dr. Charles Ryan: Right. 

Dr. Gerhardt Attard: And certainly in the trials I've been running, for four or five years we have been very aggressive and very careful with collection of plasma, so that data is coming through. It's starting to get maturity, mature data that will allow those correlations with outcome that are key to clinically qualifying the assays. 

Dr. Charles Ryan: Now, I understand there's a critical issue about the sensitivity of the testing based on the state of the disease or the line of therapy that the patient's receiving, such that we may not be able to take data from a person whose getting second line cabazitaxel and say that the same type of a frequency of a mutation would occur in somebody receiving first line abiraterone. Tell us a little bit about that. 

Dr. Gerhardt Attard: When interpreting liquid biopsy data, and this is a case for any modality, CTCs, plasma DNA, one also always needs to factor for tumor fraction. And as I said at the start, that fraction can vary from 1% to more than 90%, and clearly as men go through multiple lines of treatment and the disease volume increases, we do have patients who have micrograms of DNA, large amounts of DNA with a fraction in excess of 90%, but the majority of men who are pre-line one or pre-line two, MCRPC, the fraction is 1% to 10%. And clearly an earlier disease is a lower median. So when reporting a result, one always needs to take that into account. The lower the fraction, the higher the chance of missing a mutation that's actually there, and it's certainly more difficult to assess copy number changes, and especially deletions. 

So several groups and several studies have used cutoffs of around 30% to 35%, for example, to call it deletion, and that's technically very robust and sensible, but it, of course, reduces the population one can study in an earlier setting. 

Dr. Charles Ryan: Right. 

Dr. Gerhardt Attard: And to answer your question, when you compare prevalence in an early versus a late setting, differences can occur, not because the tumor has changed but because the fraction is very different. 

Dr. Charles Ryan: We for years, with regards to copy number, amplification of the antigen receptor for example, I remember we published a study with autopsy samples that it was prevalent in 75% of the cases. And the earlier data was saying it was prevalent in 25% based on tissue analysis. Do you think that the...Just to make sure I understand, that the prevalence of amplification is maybe not going up as we originally thought, but the amount of tissue that we can analyze is getting better, or is it that it's actually amplification as a major component of the tissue compartment is actually going up as the disease progresses?

Dr. Gerhardt Attard: Yes. I think you mentioned the classic example of a genomic change that occurs as a result of treatment resistance. 

Dr. Charles Ryan: Yes. 

Dr. Gerhardt Attard: And whether it is de novo occurrence or selection, it's clearly selection of AR amplified clones-

Dr. Charles Ryan: Yeah, yeah. 

Dr. Gerhardt Attard: With hormonal treatment. And as patients go through multiple lines of hormonal treatment, the prevalence of air amplification increases, and as you said in the autopsy series, it's not 100%, but it's a high proportion. 

Dr. Charles Ryan: Right. 

Dr. Gerhardt Attard: 50% to 75%. It's much harder to define that prevalence in the first line MCRPC setting. 

Dr. Charles Ryan: Mm-hmm (affirmative). 

Dr. Gerhardt Attard: Because of the bias we've just discussed, so both when using tissue biopsies that may have less tumor in them and certainly with plasma where the fraction is lower in the line one setting. We detect our amplification in 10% to 15% pre abi-enza and as you said, compared to 75% after multidrug resistant disease, I think the prevalence is increasing, but we may be under-reporting as well pre-line one. Now, despite that, in those 15% to 20% where we detect in their amplification, we know those men do badly. 

Dr. Charles Ryan: Mm-hmm (affirmative). 

Dr. Gerhardt Attard: And that's some of the data I presented at the EAU and previously, that very robustly now we have shown that when we test for AR copy number in a plasma sample 30 days before starting abi or enza, the men who have AR copy number gain...AR amplification as defined by copy number cutoff of 2, do badly. Their progression free survival, their response rate, their overall survival is significantly shorter. 

Dr. Charles Ryan: Mm-hmm (affirmative). 

Dr. Gerhardt Attard: And there could be a bias there because we're more likely to detect gain in the ones who have a higher fraction. Even when we control for fraction and we look at different states of the disease, it's been repeatedly qualified now in three different sets so far and we're presenting a poster on the fourth set in the poster session at the ASCO meeting later today. We see that association, a very strong association for worse outcome in the MCRCP setting for men who have AR copy number gain. 

Dr. Charles Ryan: One of the issues that I've confronted now as I've been getting more genomic sequencing, is the multiplicity of abnormalities that you can see. So it's nice to isolate AR amplification. It makes sense that there's going to be a potentially worse outcome with these patients. 

My next question is, are these patients who are also more likely to have P53 loss, P10 loss, TMPRSS rearrangements. I am sort of beginning to think about these in three and four dimensions, if that's possible. And what we haven't seen really, and these are all very common things, P10 loss, P53, is the majority of patients, but I haven't seen how these things quite interact in terms of their prognostic ability yet, and I certainly haven't seen that from plasma analysis. I presume that's the type of thing you’re thinking as well, and that there's work going on to try to look at the various contributions of those abnormalities. 

Dr. Gerhardt Attard: Yes. We do not see an association between AR copy number gain and any other specific genomic change. So, the common ones you mention, P53, P10, ERG gene rearrangements, they occur at a uniform prevalence in both the gained and the non-gained, and that we've seen in our plasma analysis. It was also seen in the tissue analysis in CRPC cohorts, multiple now, but starting off with the Stand Up to Cancer set published in Cell a few years ago. 

Dr. Charles Ryan: Yeah. 

Dr. Gerhardt Attard: The distribution of all other genomic changes sat equally in both the AR gain and the non-AR gain. So I haven't...I guess I don't have enough patients yet to split into multiple different groups. 

Dr. Charles Ryan: Right. 

Dr. Gerhardt Attard: We've been quite careful to test questions we're only powered to do. 

Dr. Charles Ryan: Right. 

Dr. Gerhardt Attard: And when it comes to AR, those men clearly do worse and we're not seeing an association with any other genomic change. 

Dr. Charles Ryan: So that's an important point, in that it appears to be an isolated, maybe probable driver as opposed to a bystander effect. Right? We think this is the driver and we've known this for a long time. 

Dr. Gerhardt Attard: I agree. But we're not testing all the other layers. We're not testing methylation. 

Dr. Charles Ryan: Yeah, right. 

Dr. Gerhardt Attard: We're not testing all the transcriptional changes and, in fact, how AR gain interacts with AR-V7 remains unexplored. 

Dr. Charles Ryan: Yeah. 

Dr. Gerhardt Attard: And we have not really explored it in detail because of the different assays used for testing AR-V7. There is a presentation by Andy Armstrong on Monday, who Andy certainly has a very powerful cohort where AR-V7 has been tested in multiple ways plasma is being collect, and that could start to give us an indication of how this AR gain interacts with AR-V7 transcripts, et cetera. 

Dr. Charles Ryan: Yeah, yeah, yeah. 

Dr. Gerhardt Attard: But clearly there are multiple layers that we have yet to dissect. 

Dr. Charles Ryan: Right. And I believe they're looking at AR-V7 using the CTC based assay, and that's a whole separate thing because, as you note, the cells are relatively uncommon and there are many patients who just may not have adequate cells for us to look at. 

Dr. Gerhardt Attard: Correct. 

Dr. Charles Ryan: I want to congratulate you on your success and your current work and your current data and your current move, and your new position. You are really leading the field I think in helping us to understand what we can do with plasma analysis and the genetics that we can follow in real time in our patients with prostate cancer. It's always great to talk to you. 

Dr. Gerhardt Attard: Thank you. 

Dr. Charles Ryan: Thanks for joining us. 

Dr. Gerhardt Attard: Thank you.
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