The evolving role of microRNAs in testicular cancer management

Pandit is a research scholar at UC San Diego School of Medicine in La Jolla. Giannikou is an academic program management officer at UC San Diego School of Medicine. Smitham is a staff research associate at the UC San Diego School of Medicine Department of Pharmacology. Murray is director of medical genetics and genomics at the UC San Diego Center for Advanced Laboratory Medicine. Bagrodia is an associate professor of urology at UC San Diego School of Medicine.

"As we move closer to the clinical application of miRNAs, validation in clinical trials and performance evaluation across diverse patient populations and disease stages is paramount," the authors write.

Testicular cancer is the most common malignancy among men aged 20 to 40 years.¹ Although traditional serum tumor markers such as lactate dehydrogenase, α-fetoprotein, and human chorionic gonadotropin have long been used to diagnose and monitor testicular cancer, their low sensitivity and specificity limit their use.² In recent years, microRNAs (miRNAs) have emerged as a promising noninvasive biomarker, offering a higher degree of diagnostic accuracy and potentially opening new avenues for personalized patient management.^3,4 This brief review captures the evolving role of miRNAs, focusing on their clinical relevance and implications as well as future directions for more effective targeted therapeutic interventions.

MiRNAs: An Emerging Diagnostic Tool

MiRNAs are small, noncoding RNA molecules (20-24 nucleotides in length) that regulate gene expression at the posttranscriptional level. In the context of cancer development, they can function as oncogenes or tumor suppressors, resulting in tumorigenesis and cancer progression.⁵ Due to their distinct expression profiles, several miRNAs have emerged in testicular cancer, including miR-371a-3p, miR-372-3p, miR-373-3p, and miR-367-3p, collectively often referred to as the miR-371-373 cluster.³ MiR-371a-3p has garnered the most attention because of its validation through multiple independent studies. A prospective, multi-institutional study by Dieckmann et al revealed sensitivity of 90% and a specificity of 94% with an area under the curve (AUC) of 0.97 for miR-371a-3p, significantly outperforming traditional tumor markers. They also found that biomarker levels were significantly correlated with tumor size, clinical stage, and response to treatment.⁶ Other studies have also validated these findings, demonstrating higher sensitivity ranges between 93% and 96%, specificity of 100%, and a similar AUC.^7,8

Role in Response Monitoring and Detection of Relapse

Beyond diagnosis, miR-371a-3p is also promising for monitoring treatment response and disease progression. In a study assessing 151 clinical stage I patients, although miR-371a-3p levels immediately after orchiectomy were not significantly associated with relapse, 94% of patients who eventually experienced relapse showed elevated levels at the time of recurrence.⁹ Another study evaluating 33 patients similarly found that miRNA levels immediately after orchiectomy were not predictive for relapse; however, all the patients who experienced relapse demonstrated increasing levels during follow-up, preceding detection by standard serum markers and imaging modalities.¹⁰ Thus, although miR-371a-3p hasn’t been shown to predict relapses after orchiectomy and guide decisions for surveillance vs adjuvant therapy, it has shown promise in detecting relapses earlier than standard investigations. Validation in larger, prospective cohorts could lead to miRNAs replacing imaging modalities, leading to decreased exposure to ionizing radiation.

Leão et al conducted a study evaluating miR-371a-3p as a biomarker for chemotherapy response in 82 patients with nonseminomatous germ cell tumor (NSGCT). They found that marker levels decreased significantly in patients responding to chemotherapy.¹¹ Another study evaluated the association between miR levels and clinical outcomes in 109 patients with metastatic testicular cancer. They found that miR-371a-3p levels at the start of chemotherapy were significantly higher in patients who later developed a recurrence. On the other hand, miRNA levels decreased within the first week of chemotherapy for patients who achieved remission and remained low during follow-up.¹² In a study by Mego et al, elevated plasma levels of miR-371a-3p in chemotherapy-naive patients were associated with poorer progression-free and overall survival compared to patients with negative plasma levels.¹³ This suggests the tremendous potential for clinical utility of miRNAs in early relapse detection and clinical outcomes prediction, as early intervention based on miRNA levels could lead to timely treatment adjustments.

Use of miRNAs in Detecting Residual Masses

A clinical dilemma in managing metastatic testicular cancer is the selection of patients who require a postchemotherapy retroperitoneal lymph node dissection (pcRPLND). Although pcRPLND is indicated in residual masses harboring viable tumors and teratomas, it could be avoided for patients with necrosis only, which is present in about 50% of pcRPLND specimens.¹⁴ This presents another exciting avenue for evaluating miRNAs in reliably differentiating viable tumors/teratomas from necrosis.

It was found that miR-371a-3p had a sensitivity and a negative predictive value of 100% for detecting viable tumors in masses less than 3 cm for patients with NSGCT.¹¹ Although these results are encouraging for differentiating viable tumors, detecting teratomas before RPLND remains challenging.¹⁵ Multiple groups have evaluated the utility of miR-375 in detecting teratomas, including ours where we reported that serum miR-375-3p and miR-375-5p demonstrated poor sensitivities and specificities in the detection of teratomas at pcRPLND.¹⁶

The utility of miRNAs in detecting viable seminomas has also been assessed. A study evaluating 23 patients with postchemotherapy residual masses found that normal serum levels reliably indicated the absence of viable seminomas after RPLND while elevated levels predicted the presence of viable disease.¹⁷ However, another study found that although miR-371a-3p performed reasonably well in detecting viable seminomas in chemotherapy-naive patients, its performance in the postchemotherapy setting was modest, with a specificity of 60%.¹⁸ Given these inconsistent results, further studies are required in larger cohorts to validate performance in detecting viable seminomas on pcRPLND.

Transition of miRNA Testing from Research to Clinical Labs

The transition of miRNA testing from research labs to Clinical Laboratory Improvement Amendments (CLIA)–certified labs is a crucial step in ensuring the reproducibility and reliability of this biomarker in clinical settings. To ensure consistent results across different clinical labs, it is essential to establish standardized workflows for sample extraction, use of reagents, and assay reproducibility. Furthermore, ensuring consistent thresholding and handling indeterminate results are important steps in implementing this molecular assay.

In our previous work on miR-371a-3p in a cohort of 32 patients in a research laboratory, we compared raw Cq and normalized values to previous quantitative polymerase chain reaction assays and validated interlaboratory concordance. We also introduced an indeterminate range (Cq, 28-35) and found that rerunning these samples improved assay accuracy from 0.84 to 0.92.¹⁹ We recommend that CLIA protocols adopt raw Cq value-based thresholding instead of using normalized values and also utilize an analytic pipeline that retests indeterminate samples.

Future Directions

As we move closer to the clinical application of miRNAs, validation in clinical trials and performance evaluation across diverse patient populations and disease stages is paramount. Equally important is further work on standardization, optimization, and validation of assays before clinical use. By addressing these challenges, miRNAs can revolutionize testicular cancer management by offering personalized and precise approaches to early diagnosis, surveillance, and detection of relapse, reducing the need for invasive procedures.

REFERENCES

1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68(1):7-30. doi:10.3322/caac.21442

2. Egan J, Salari K. Biomarkers in testicular cancer: classic tumor markers and beyond. Urol Clin North Am. 2023;50(1):133-143. doi:10.1016/j.ucl.2022.09.002

3. Piao J, Lafin JT, Scarpini CG, et al. A multi-institutional pooled analysis demonstrates that circulating miR-371a-3p alone is sufficient for testicular malignant germ cell tumor diagnosis. Clin Genitourin Cancer. 2021;19(6):469-479. doi:10.1016/j.clgc.2021.08.006

4. Seales CL, Puri D, Yodkhunnatham N, et al. Advancing GCT management: a review of miR-371a-3p and other miRNAs in comparison to traditional serum tumor markers. Cancers (Basel). 2024;16(7):1379. doi:10.3390/cancers16071379

5. Saliminejad K, Khorram Khorshid HR, Soleymani Fard S, Ghaffari SH. An overview of microRNAs: biology, functions, therapeutics, and analysis methods. J Cell Physiol. 2019;234(5):5451-5465. doi:10.1002/jcp.27486

6. Dieckmann KP, Radtke A, Geczi L, et al. Serum levels of microRNA-371a-3p (M371 test) as a new biomarker of testicular germ cell tumors: results of a prospective multicentric study. J Clin Oncol. 2019;37(16):1412-1423. doi:10.1200/JCO.18.01480

7. Badia RR, Abe D, Wong D, et al. Real-world application of pre-orchiectomy miR-371a-3p test in testicular germ cell tumor management. J Urol. 2021;205(1):137-144. doi:10.1097/JU.0000000000001337

8. Nappi L, Thi M, Lum A, et al. Developing a highly specific biomarker for germ cell malignancies: plasma miR371 expression across the germ cell malignancy spectrum. J Clin Oncol. 2019;37(33):3090-3098. doi:10.1200/JCO.18.02057

9.Lobo J, Leão R, Gillis AJM, et al. Utility of serum miR-371a-3p in predicting relapse on surveillance in patients with clinical stage I testicular germ cell cancer. Eur Urol Oncol. 2021;4(3):483-491. doi:10.1016/j.euo.2020.11.004

10. Fankhauser CD, Christiansen AJ, Rothermundt C, et al. Detection of recurrences using serum miR-371a-3p during active surveillance in men with stage I testicular germ cell tumours. Br J Cancer. 2022;126(8):1140-1144. doi:10.1038/s41416-021-01643-z

11. Leão R, van Agthoven T, Figueiredo A, et al. Serum miRNA predicts viable disease after chemotherapy in patients with testicular nonseminoma germ cell tumor. J Urol. 2018;200(1):126-135. doi:10.1016/j.juro.2018.02.068

12. Rosas Plaza X, van Agthoven T, Meijer C, et al. miR-371a-3p, miR-373-3p and miR-367-3p as serum biomarkers in metastatic testicular germ cell cancers before, during and after chemotherapy. Cells. 2019;8(10):1221. doi:10.3390/cells8101221

13. Mego M, van Agthoven T, Gronesova P, et al. Clinical utility of plasma miR‐371a‐3p in germ cell tumors. J Cell Mol Med. 2019;23(2):1128-1136. doi:10.1111/jcmm.14013

14. Daneshmand S, Albers P, Fosså SD, et al. Contemporary management of postchemotherapy testis cancer. Eur Urol. 2012;62(5):867-876. doi:10.1016/j.eururo.2012.08.014

15. Yodkhunnatham N, Pandit K, Puri D, Yuen KL, Bagrodia A. MicroRNAs in testicular germ cell tumors: the teratoma challenge. Int J Mol Sci. 2024;25(4):2156. doi:10.3390/ijms25042156

16. Lafin JT, Kenigsberg AP, Meng X, et al. Serum small RNA sequencing and miR-375 assay do not identify the presence of pure teratoma at postchemotherapy retroperitoneal lymph node dissection. Eur Urol Open Sci. 2021;26:83-87. doi:10.1016/j.euros.2021.02.003

17. Dieckmann KP, Klemke M, Grobelny F, et al. Serum levels of microRNA-371a-3p (M371) can predict absence or presence of vital disease in residual masses after chemotherapy of metastatic seminoma. Front Oncol. 2022;12:889624. doi:10.3389/fonc.2022.889624

18. Konneh B, Lafin JT, Howard J, et al. Evaluation of miR-371a-3p to predict viable germ cell tumor in patients with pure seminoma receiving retroperitoneal lymph node dissection. Andrology. 2023;11(4):634-640. doi:10.1111/andr.13317

19. Lafin JT, Scarpini CG, Amini A, et al. Refining the serum miR-371a-3p test for viable germ cell tumor detection. Sci Rep. 2023;13(1):10558. doi:10.1038/s41598-023-37271-1