Detecting Early Testicular Cancer Relapse with Plasma MicroRNA Analysis - Lucia Nappi
July 5, 2023
Zach Klaassen interviews Lucia Nappi about her research on the role of microRNAs in early-stage testicular cancer. MicroRNAs are small RNA fragments crucial for the regulation of oncogenes and tumor suppressors, with certain microRNAs notably overexpressed in testicular cancer. Dr. Nappi's team analyzed the microRNA-371 in plasma samples from stage I testicular cancer patients, finding that patients with a positive microRNA-371 test all experienced a relapse. The results point towards the potential use of microRNA-371 as a predictive biomarker for relapse in early-stage testicular cancer, opening the door for precision medicine in this field. Dr. Nappi believes that this discovery has the potential to prevent both over- and undertreatment in testicular cancer care, demonstrating the continual room for improvement and discovery in cancer research.
Biographies:
Lucia Nappi, MD, PhD, Medical Oncologist, Vancouver Prostate Centre, Vancouver, Canada
Zachary Klaassen, MD, MSc, Urologic Oncologist, Assistant Professor Surgery/Urology at the Medical College of Georgia at Augusta University, Georgia Cancer Center, Augusta, GA
Biographies:
Lucia Nappi, MD, PhD, Medical Oncologist, Vancouver Prostate Centre, Vancouver, Canada
Zachary Klaassen, MD, MSc, Urologic Oncologist, Assistant Professor Surgery/Urology at the Medical College of Georgia at Augusta University, Georgia Cancer Center, Augusta, GA
Read the Full Video Transcript
Zach Klaassen: Hi, my name is Dr. Zach Klaassen. I'm a urologic oncologist at the Georgia Cancer Center in Augusta, Georgia. We are live at ASCO 2023 in Chicago, and I'm pleased to be joined with Dr. Lucia Nappi from the University of British Columbia, Assistant Professor, thanks so much for joining us today.
Lucia Nappi: Thank you very much for the invitation.
Zach Klaassen: This is a very exciting topic. You presented some amazing data at the oral abstract session today. So just walk us through miRNA in general, and how it sort of came in evolution in testis cancer.
Lucia Nappi: MicroRNAs are small fragments of RNAs, very small, only 22 to 23 nucleotides. And they're not coding for any genes, but they're very important for transcription regulation of onco-suppressors and the oncogenes. They were firstly described in tissue of germ cell tumors, from both pediatric and adult germ cell tumors. And there is a cluster of microRNAs, not just one microRNA.
Zach Klaassen: Right.
Lucia Nappi: It's a family of microRNAs that is over-expressed in a seminal mind, non-seminoma.
Zach Klaassen: Right.
Lucia Nappi: It's not expressed in teratoma, or in healthy testicular tissue, and were described in the early 2000 in the tissue. But the revolution happened when Matt Murray from the University of Cambridge in the UK started to detect these microRNAs in the blood of patients with germ cell tumors. He started with pediatric germ cell tumor patients, and then the whole field has developed around that discovery.
So right now, we know that we can detect these microRNAs easily in the peripheral blood. And we have been focused on one specific microRNA, which is called microRNA-371a-3p, or microRNA-371, which seems to have the highest occurrence. So sensitivity and specificity for germ cell malignancy.
Zach Klaassen: Excellent. And so, a lot of the work up until this point has been mostly in advanced, post-chemo. And your data today started talking about really early on with surveillance. So what was sort of the objective of your guys' study that you presented today?
Lucia Nappi: We know from many studies, most of them are retrospective.
Zach Klaassen: Sure.
Lucia Nappi: Few of them are perspective. Us and the German group published in JC in 2019. The biggest prospective studies. And you're right, most of these data have been produced in patients with more advanced disease. So metastatic germ cell tumors, prior to chemotherapy, or, especially the German group, described it in patients with testicular cancer prior to the orchiectomy.
Zach Klaassen: Right.
Lucia Nappi: So in both situations, we were quite sure that there was testicular cancer.
Zach Klaassen: Sure.
Lucia Nappi: Our study that I presented today is different, because we wanted to see if the very high occurrence of this biomarker, that we described in more advanced disease, was applicable to early stage disease. In other words, if we can use these peripheral biomarkers to detect early-stage disease in testicular cancer.
Zach Klaassen: That's great. So from your study, just walk us through the methods. I know this was University of British Columbia, but there's a lot of sites in BC that have sort of filtered down. Correct?
Lucia Nappi: Correct. So in terms of patient's enrollment and biospecimens availability, we have this beautiful GU BioBank in British Columbia. So British Columbia has a centralized kind of health system and healthcare. Meaning, that all the patients with testicular cancer are seen in a few places, or part of the BC cancer. And all these places are connected with the rural hospitals, because the problem is big. Right?
Zach Klaassen: Yeah.
Lucia Nappi: And we can get blood and the clinical annotation for all the patients who actually consent to this BioBank. So we got almost like every patient with testicular cancer is offered to be involved in this BioBank. And most of them signed the consent form. So we have probably blood and clinical annotation for almost every patient with particular cancer in British Columbia.
Zach Klaassen: So this study had, I was thinking, 101 patients. And just sort of walk through how these patients were identified, how many non-seminoma, seminoma, et cetera.
Lucia Nappi: So when we planned to do this study, to answer it to the question, can we adapt these microRNAs you want to detect to predictory labs in early-stage germ cell tumors. So we went through the BioBank, and there were almost 300 patients with testis cancer banked, but only 101 had the inclusion criteria for this study.
And we were targeting patients with clinical stage I germ cell tumors, both seminoma or known seminoma, with available blood samples collected after the orchiectomy, and followed with active surveillance.
Zach Klaassen: Right.
Lucia Nappi: So they were the inclusion criteria for this study. And essentially, of these 101 patients that had these characteristics, 68 of these patients had post-orchiectomy blood samples available. Meaning, samples were collected within three months of their orchiectomy, when none of these patients had evidence of clinical relapse. But later on, some of these patients had their relapse. So that is, although we analyzed the 101 patients, I think the 68 patients is the most informative of this study.
Zach Klaassen: Because it's that tight window they're on. They're going to be on surveillance and you got them early.
Lucia Nappi: Correct.
Zach Klaassen: Yeah. So, some very exciting results. So just walk us through some of the highlights that you want to share with the audience.
Lucia Nappi: We analyzed the microRNA-371 in the plasma of patients with stage I testicular cancer. And I want to highlight, that's plasma, not serum, because a lot of the other studies used the serum collected in serum separator tubes.
Zach Klaassen: Yes.
Lucia Nappi: So we use plasma collecting Streck tubes, which I think, does have an impact, in terms of stabilizing the microRNAs.
Zach Klaassen: Right.
Lucia Nappi: And we use realtime PCR to evaluate the expression of the microRNA-371. So the occurrence of the whole cohort, so the 101 patients, was 0.82. And the median follow-up was long, it was more than 40 months.
Zach Klaassen: Yeah, that's great.
Lucia Nappi: Which exceeds the expected median time of relapse in patients with stage I.
Zach Klaassen: Especially on the surveillance patients. Yeah.
Lucia Nappi: Correct.
Zach Klaassen: Yeah.
Lucia Nappi: And we didn't have any false positive results. So this means, that both the specificity and the positive predictive value were 100%.
Zach Klaassen: It's incredible.
Lucia Nappi: And the negative predictive value was around 83% in the whole cohort. The sensitivity was around 60%. So we were very happy to see that the positive predictive value and the specificity were the same that we observed in patients with more advanced disease, but the sensitivity was lower.
Zach Klaassen: Yeah. And I think, one thing I took out too is that, the patients that had a positive miRNA, within three months before any clinical evidence, they were able to predict that they were going to occur.
Lucia Nappi: Correct. So I think that's probably the most important data of the whole study, because we analyze this very clean kind of patient population. 68 patients with blood collecting within three months, no evidence of relapse whatsoever, and then we followed these patients up. And if they had the post-orchiectomy positive microRNA, they all relapsed.
Zach Klaassen: Yeah.
Lucia Nappi: So they relapsed. And the range was between four months and 24 months. The median time of relapse was four months. But all of them had a relapse of the tumor.
Zach Klaassen: And I think this is exciting, because you're in Vancouver, I turn in Toronto, we're big fans of active surveillance for these patients.
Lucia Nappi: Yes.
Zach Klaassen: But it's almost, if we see these patients in the clinic, when this becomes available, we can say, "If this is positive, we should treat you with something now." Because it's going to come, based on the data that you guys have.
Lucia Nappi: I completely agree. I think that's probably the most clinically relevant data.
Zach Klaassen: Yeah.
Lucia Nappi: And obviously, the missing piece of this story is like, okay, what do I do this with these patients-
Zach Klaassen: Right.
Lucia Nappi: ... with the positive microRNA, but without clinical evidence. So normal tumor markers, no CT scan evidence. So how do we consider these patients are as advanced as the clinical stage I-S, for example.
Zach Klaassen: Right.
Lucia Nappi: Or they are less advanced, so we could consider surgery for example. So less invasive or less morbid or toxic approach for these patients.
Zach Klaassen: And the question too is, if we're thinking on the chemo side of things, let's say, do we treat them as a preventative clinical stage I, or do we think they're going to be a II-A?
Lucia Nappi: Correct.
Zach Klaassen: Great questions.
Lucia Nappi: So we don't know yet. For this clinical utility question, there have been studies planned mainly with surgery, like using the microRNA-371 to allocate patients to either surveillance if it's negative, or surgery, primary RPLND if it's positive.
Zach Klaassen: Nice primary RPLND. Yeah, that's a good point. Tell us about the SWOG 1823 study. I know you're one of the co-PIs on that. This is going to be exciting, because it's going to kind of validate some of the work you're talking about. Correct?
Lucia Nappi: Yes. So S1823 is a cohort longitudinal study. It's been sponsored by SWOG, and by the Canadian Cancer Trials Group in Canada. So it's opened in the US through SWOG and NCORP.
Zach Klaassen: That's great.
Lucia Nappi: And in Canada, through the CCTG. So we opened the study in July 2020, and we plan to enroll 956 patients, and 744 patients have been enrolled so far.
Zach Klaassen: That's incredible. In just about three years.
Lucia Nappi: Yeah.
Zach Klaassen: That's great.
Lucia Nappi: And we had a high-risk group, patients with metastatic disease. We closed that group. We enrolled 145 patients in that group. And now we are still enrolling patients with the clinical stage I, and stage II-A on surveillance.
Zach Klaassen: Yes.
Lucia Nappi: Patients who get adjuvant therapy are also eligible, just because we wanted to check the microRNA post-orchiectomy.
Zach Klaassen: Sure.
Lucia Nappi: So the study's extremely easy and straightforward. I think that's why it enrolled so well.
Zach Klaassen: That's excellent.
Lucia Nappi: We just need additional blood work during the surveillance of these patients. And everyone has a blood sample collected post-orchiectomy.
Zach Klaassen: Yeah.
Lucia Nappi: And then during the follow-up, and until two years, so we stopped collecting blood samples at two years mark. Because after 24 months, the relapse in clinical stage I-
Zach Klaassen: Is so low.
Lucia Nappi: ... is extremely rare. Yeah.
Zach Klaassen: So the hope from this trial is, we learn more about the feasibility. We get some more information in all these different parts of the disease process. And hopefully, getting towards approval. Right? I mean, this is the ultimate goal.
Lucia Nappi: Yes, it is the ultimate goal. We have, obviously, to see the data from 1823. There are also other studies that are evaluating microRNA-371. It's slightly different design than S1823, yes. But you're right. If we will validate in this very large cohort of patients, the very high occurrence of microRNA-371, we could start to use it for our patients. So integrate that in our clinical practice, to have better and more accurate diagnosis of germ cell tumors.
Zach Klaassen: It's incredibly exciting. Give us a couple of take home messages for our audience. It's been a great discussion.
Lucia Nappi: I think this biomarker has a very high chance to change our clinical practice, and to make actually, our care for these very young patients with testicular cancer more accurate. So it has the potential to reduce suboptimal treatment.
Zach Klaassen: Sure.
Lucia Nappi: Meaning, like overtreatment or undertreatment.
Zach Klaassen: Precision medicine, right?
Lucia Nappi: Correct.
Zach Klaassen: Yeah.
Lucia Nappi: And the second one is that, although we thought that we knew everything about testicular cancer, because we knew how to cure these patients, I think this study, and in general, what is happening in germ cell tumors with this new biomarker, actually highlights that, in science there is always something new to discover and room for improvement.
Zach Klaassen: Absolutely. Great discussion. Thanks so much for joining us today, Dr. Nappi.
Lucia Nappi: Thanks.
Zach Klaassen: Hi, my name is Dr. Zach Klaassen. I'm a urologic oncologist at the Georgia Cancer Center in Augusta, Georgia. We are live at ASCO 2023 in Chicago, and I'm pleased to be joined with Dr. Lucia Nappi from the University of British Columbia, Assistant Professor, thanks so much for joining us today.
Lucia Nappi: Thank you very much for the invitation.
Zach Klaassen: This is a very exciting topic. You presented some amazing data at the oral abstract session today. So just walk us through miRNA in general, and how it sort of came in evolution in testis cancer.
Lucia Nappi: MicroRNAs are small fragments of RNAs, very small, only 22 to 23 nucleotides. And they're not coding for any genes, but they're very important for transcription regulation of onco-suppressors and the oncogenes. They were firstly described in tissue of germ cell tumors, from both pediatric and adult germ cell tumors. And there is a cluster of microRNAs, not just one microRNA.
Zach Klaassen: Right.
Lucia Nappi: It's a family of microRNAs that is over-expressed in a seminal mind, non-seminoma.
Zach Klaassen: Right.
Lucia Nappi: It's not expressed in teratoma, or in healthy testicular tissue, and were described in the early 2000 in the tissue. But the revolution happened when Matt Murray from the University of Cambridge in the UK started to detect these microRNAs in the blood of patients with germ cell tumors. He started with pediatric germ cell tumor patients, and then the whole field has developed around that discovery.
So right now, we know that we can detect these microRNAs easily in the peripheral blood. And we have been focused on one specific microRNA, which is called microRNA-371a-3p, or microRNA-371, which seems to have the highest occurrence. So sensitivity and specificity for germ cell malignancy.
Zach Klaassen: Excellent. And so, a lot of the work up until this point has been mostly in advanced, post-chemo. And your data today started talking about really early on with surveillance. So what was sort of the objective of your guys' study that you presented today?
Lucia Nappi: We know from many studies, most of them are retrospective.
Zach Klaassen: Sure.
Lucia Nappi: Few of them are perspective. Us and the German group published in JC in 2019. The biggest prospective studies. And you're right, most of these data have been produced in patients with more advanced disease. So metastatic germ cell tumors, prior to chemotherapy, or, especially the German group, described it in patients with testicular cancer prior to the orchiectomy.
Zach Klaassen: Right.
Lucia Nappi: So in both situations, we were quite sure that there was testicular cancer.
Zach Klaassen: Sure.
Lucia Nappi: Our study that I presented today is different, because we wanted to see if the very high occurrence of this biomarker, that we described in more advanced disease, was applicable to early stage disease. In other words, if we can use these peripheral biomarkers to detect early-stage disease in testicular cancer.
Zach Klaassen: That's great. So from your study, just walk us through the methods. I know this was University of British Columbia, but there's a lot of sites in BC that have sort of filtered down. Correct?
Lucia Nappi: Correct. So in terms of patient's enrollment and biospecimens availability, we have this beautiful GU BioBank in British Columbia. So British Columbia has a centralized kind of health system and healthcare. Meaning, that all the patients with testicular cancer are seen in a few places, or part of the BC cancer. And all these places are connected with the rural hospitals, because the problem is big. Right?
Zach Klaassen: Yeah.
Lucia Nappi: And we can get blood and the clinical annotation for all the patients who actually consent to this BioBank. So we got almost like every patient with testicular cancer is offered to be involved in this BioBank. And most of them signed the consent form. So we have probably blood and clinical annotation for almost every patient with particular cancer in British Columbia.
Zach Klaassen: So this study had, I was thinking, 101 patients. And just sort of walk through how these patients were identified, how many non-seminoma, seminoma, et cetera.
Lucia Nappi: So when we planned to do this study, to answer it to the question, can we adapt these microRNAs you want to detect to predictory labs in early-stage germ cell tumors. So we went through the BioBank, and there were almost 300 patients with testis cancer banked, but only 101 had the inclusion criteria for this study.
And we were targeting patients with clinical stage I germ cell tumors, both seminoma or known seminoma, with available blood samples collected after the orchiectomy, and followed with active surveillance.
Zach Klaassen: Right.
Lucia Nappi: So they were the inclusion criteria for this study. And essentially, of these 101 patients that had these characteristics, 68 of these patients had post-orchiectomy blood samples available. Meaning, samples were collected within three months of their orchiectomy, when none of these patients had evidence of clinical relapse. But later on, some of these patients had their relapse. So that is, although we analyzed the 101 patients, I think the 68 patients is the most informative of this study.
Zach Klaassen: Because it's that tight window they're on. They're going to be on surveillance and you got them early.
Lucia Nappi: Correct.
Zach Klaassen: Yeah. So, some very exciting results. So just walk us through some of the highlights that you want to share with the audience.
Lucia Nappi: We analyzed the microRNA-371 in the plasma of patients with stage I testicular cancer. And I want to highlight, that's plasma, not serum, because a lot of the other studies used the serum collected in serum separator tubes.
Zach Klaassen: Yes.
Lucia Nappi: So we use plasma collecting Streck tubes, which I think, does have an impact, in terms of stabilizing the microRNAs.
Zach Klaassen: Right.
Lucia Nappi: And we use realtime PCR to evaluate the expression of the microRNA-371. So the occurrence of the whole cohort, so the 101 patients, was 0.82. And the median follow-up was long, it was more than 40 months.
Zach Klaassen: Yeah, that's great.
Lucia Nappi: Which exceeds the expected median time of relapse in patients with stage I.
Zach Klaassen: Especially on the surveillance patients. Yeah.
Lucia Nappi: Correct.
Zach Klaassen: Yeah.
Lucia Nappi: And we didn't have any false positive results. So this means, that both the specificity and the positive predictive value were 100%.
Zach Klaassen: It's incredible.
Lucia Nappi: And the negative predictive value was around 83% in the whole cohort. The sensitivity was around 60%. So we were very happy to see that the positive predictive value and the specificity were the same that we observed in patients with more advanced disease, but the sensitivity was lower.
Zach Klaassen: Yeah. And I think, one thing I took out too is that, the patients that had a positive miRNA, within three months before any clinical evidence, they were able to predict that they were going to occur.
Lucia Nappi: Correct. So I think that's probably the most important data of the whole study, because we analyze this very clean kind of patient population. 68 patients with blood collecting within three months, no evidence of relapse whatsoever, and then we followed these patients up. And if they had the post-orchiectomy positive microRNA, they all relapsed.
Zach Klaassen: Yeah.
Lucia Nappi: So they relapsed. And the range was between four months and 24 months. The median time of relapse was four months. But all of them had a relapse of the tumor.
Zach Klaassen: And I think this is exciting, because you're in Vancouver, I turn in Toronto, we're big fans of active surveillance for these patients.
Lucia Nappi: Yes.
Zach Klaassen: But it's almost, if we see these patients in the clinic, when this becomes available, we can say, "If this is positive, we should treat you with something now." Because it's going to come, based on the data that you guys have.
Lucia Nappi: I completely agree. I think that's probably the most clinically relevant data.
Zach Klaassen: Yeah.
Lucia Nappi: And obviously, the missing piece of this story is like, okay, what do I do this with these patients-
Zach Klaassen: Right.
Lucia Nappi: ... with the positive microRNA, but without clinical evidence. So normal tumor markers, no CT scan evidence. So how do we consider these patients are as advanced as the clinical stage I-S, for example.
Zach Klaassen: Right.
Lucia Nappi: Or they are less advanced, so we could consider surgery for example. So less invasive or less morbid or toxic approach for these patients.
Zach Klaassen: And the question too is, if we're thinking on the chemo side of things, let's say, do we treat them as a preventative clinical stage I, or do we think they're going to be a II-A?
Lucia Nappi: Correct.
Zach Klaassen: Great questions.
Lucia Nappi: So we don't know yet. For this clinical utility question, there have been studies planned mainly with surgery, like using the microRNA-371 to allocate patients to either surveillance if it's negative, or surgery, primary RPLND if it's positive.
Zach Klaassen: Nice primary RPLND. Yeah, that's a good point. Tell us about the SWOG 1823 study. I know you're one of the co-PIs on that. This is going to be exciting, because it's going to kind of validate some of the work you're talking about. Correct?
Lucia Nappi: Yes. So S1823 is a cohort longitudinal study. It's been sponsored by SWOG, and by the Canadian Cancer Trials Group in Canada. So it's opened in the US through SWOG and NCORP.
Zach Klaassen: That's great.
Lucia Nappi: And in Canada, through the CCTG. So we opened the study in July 2020, and we plan to enroll 956 patients, and 744 patients have been enrolled so far.
Zach Klaassen: That's incredible. In just about three years.
Lucia Nappi: Yeah.
Zach Klaassen: That's great.
Lucia Nappi: And we had a high-risk group, patients with metastatic disease. We closed that group. We enrolled 145 patients in that group. And now we are still enrolling patients with the clinical stage I, and stage II-A on surveillance.
Zach Klaassen: Yes.
Lucia Nappi: Patients who get adjuvant therapy are also eligible, just because we wanted to check the microRNA post-orchiectomy.
Zach Klaassen: Sure.
Lucia Nappi: So the study's extremely easy and straightforward. I think that's why it enrolled so well.
Zach Klaassen: That's excellent.
Lucia Nappi: We just need additional blood work during the surveillance of these patients. And everyone has a blood sample collected post-orchiectomy.
Zach Klaassen: Yeah.
Lucia Nappi: And then during the follow-up, and until two years, so we stopped collecting blood samples at two years mark. Because after 24 months, the relapse in clinical stage I-
Zach Klaassen: Is so low.
Lucia Nappi: ... is extremely rare. Yeah.
Zach Klaassen: So the hope from this trial is, we learn more about the feasibility. We get some more information in all these different parts of the disease process. And hopefully, getting towards approval. Right? I mean, this is the ultimate goal.
Lucia Nappi: Yes, it is the ultimate goal. We have, obviously, to see the data from 1823. There are also other studies that are evaluating microRNA-371. It's slightly different design than S1823, yes. But you're right. If we will validate in this very large cohort of patients, the very high occurrence of microRNA-371, we could start to use it for our patients. So integrate that in our clinical practice, to have better and more accurate diagnosis of germ cell tumors.
Zach Klaassen: It's incredibly exciting. Give us a couple of take home messages for our audience. It's been a great discussion.
Lucia Nappi: I think this biomarker has a very high chance to change our clinical practice, and to make actually, our care for these very young patients with testicular cancer more accurate. So it has the potential to reduce suboptimal treatment.
Zach Klaassen: Sure.
Lucia Nappi: Meaning, like overtreatment or undertreatment.
Zach Klaassen: Precision medicine, right?
Lucia Nappi: Correct.
Zach Klaassen: Yeah.
Lucia Nappi: And the second one is that, although we thought that we knew everything about testicular cancer, because we knew how to cure these patients, I think this study, and in general, what is happening in germ cell tumors with this new biomarker, actually highlights that, in science there is always something new to discover and room for improvement.
Zach Klaassen: Absolutely. Great discussion. Thanks so much for joining us today, Dr. Nappi.
Lucia Nappi: Thanks.