Utilizing miRNA as a Liquid Biomarker for Malignancy of Small Testicular Masses

We spoke with Julian Chavarriaga, MD, Princess Margaret Cancer Centre, on his study on the use of miRNA as a liquid biomarker to detect malignancy in testicular masses.

Dr. Chavarriaga details the unreliability of certain tumor markers, and how the study’s researchers addressed potential variability introduced by utilizing different miRNA extraction methods across multiple research facilities.

Results of the study were presented at the 2024 European Association of Urology Congress.

Why are serum tumor markers and imaging methods often unreliable in their predictive capabilities?

Dr. Chavarriaga: We mainly rely on ultrasound for diagnosing small testicular masses, and it’s quite accurate in determining if a mass is a tumor or not. However, it’s not very reliable in determining if a tumor is malignant or not. Multiple imaging methods, including contrast-enhanced ultrasound, elastography, and MRI have been used, but none of these improve the sensitivity and specificity of imaging – we can’t determine if a small testicular mass is benign or malignant just by using current imaging practices.

Testicular markers are not very sensitive or specific for detecting germ cell tumors. When combined, the accuracy of all three markers is usually about 60% so they’re not very accurate and even less accurate in predicting the histology of small testicular masses.

What was the goal of this study, and what were its outcomes?

Dr. Chavarriaga: Our main goal was to determine if microRNA-371a-3p could predict the presence of germ cell tumors in small testis masses. We also wanted to explore if there was any association between multiple microRNAs that we tested for, including microRNAs 371, 372, 373 and 367.

Were there any challenges encountered during your study? How were they addressed?

Dr. Chavarriaga: The main challenge involved our use of multiple databases from our institution at University Health Network (UHN). It’s hard to find a large enough sample size of patients with small testis masses. We had a few to begin with that were taken from data used from our infertility center, but they’re not very common to see in practice.

The main setback was trying to find enough patients; we also used a very unique interlab collaboration. We worked with researchers in Portugal, Vancouver, as well as our own lab here at UHN. There was a small setback involving a shipment to Portugal, but it was all resolved.

How did you address the potential variability introduced by using different miRNA extraction methods across multiple research facilities?

Dr. Chavarriaga: We wanted to compare different pipelines, and didn’t want to have only one pipeline for microRNA extraction. Since we utilized 3 labs, we had different pipelines; one lab used serum, while the other one used plasma.

For example, in Portugal, we utilized Dr. João Lobo’s lab, were he used serum and real-time polymerase chain reaction (PCR) for extracting the microRNA. In Vancouver, which is Dr. Lucia Nappi’s lab, she used plasma along with real-time PCR for extracting the microRNAs. At UHN, we used serum, as well as a digital droplet PCR for extracting microRNA. We used completely different pipelines, but that was part of the goal of our project. We wanted to see if they worked the same, along with what the predictive capabilities of each pipeline were.

How do the findings of this study contribute to the current understanding and management of incidental STMs?

Dr. Chavarriaga: As far as we know, we’ve presented the largest series of small testicular masses – I don’t think anyone has analyzed microRNAs in a large series such as this one. In our lab in Portugal, the area under the curve was actually quite good, at 0.73. This was certainly better than all serum tumor markers combined, including AFP, ACG and LDH.

In our Vancouver lab, through the use of real-time PCR on plasma, they reported an area under the curve of almost 93.2%, which is actually quite remarkable. It’s better than anything we’ve seen before for small testis masses, so the result was very good.

At UHN, our area under the curve wasn’t as good as the other two labs, at 0.53. One of the possible reasons that this may have happened is that at UHN’s lab, they only use exosomal or microvesicle microRNAs, instead of total microRNAs. The other two labs extracted total microRNAs, so they were extracted from a limited pool of microRNAs. Since they were using digital droplet PCR, they were not doing any pre-amplification, which could explain the difference in the area under the curve.

In a study by Dr. Lobo, he compared GCT digital droplet PCR with real-time PCR, and found virtually no difference; they performed practically the same. However, our results were not similar – we believe it’s probably because of the exosomal microRNA that was used in our UHN lab.

What further research is needed to ensure the predictive capabilities of miRNA for germ cell tumors?

Dr. Chavarriaga: For germ cell tumors, there have been many trials in different stages of the disease. For small testis masses, we need prospective data, so we are currently running a prospective trial at UHN with the same collaborators from Portugal and UHN in Vancouver.

We’re recruiting patients with small testis masses who come to our clinic, and will offer them either active surveillance or orchiectomy, but they all get blood and serum extracted before surveillance or orchiectomy.

If they go for surveillance, they will come for a follow-up every 3 months where we will take blood samples for analysis. Right now, we’re working on gathering prospective data. I believe we have recruited 35 patients so far in our ongoing trial.