Microrna and retroperitoneal lymph node dissection : advances in testicular cancer treatment
Authors: Thor Anna 
Published: January 2025

<p dir="ltr">BACKGROUND<br>Testicular germ cell tumor (TGCT) is the most common malignancy in young men, with an excellent five-year survival rate over 95%. Metastatic TGCTs can be cured by either chemotherapy, radiation or retroperitoneal lymph node dissection (RPLND), either solitary or in combination. Current research focuses on refining treatments and minimizing side effects. Over the past decade, the novel biomarker microRNA 371a-3p has demonstrated promising performance with high sensitivity and specificity for germ cell tumors (GCT), however, excluding teratomas. RPLND is a complex surgical procedure associated with certain morbidity, requiring substantial surgical experience for optimal outcomes. A global trend is emerging toward surgery as primary treatment for select patients with limited disease burden. The collaboration within the Swedish and Norwegian Testicular Cancer Group (SWENOTECA) enables populationbased studies on a relatively rare condition.<br><br>AIMS<br>Paper I, to evaluate miR-371a-3p levels in TGCT patients undergoing orchiectomy and in healthy blood donors using digital droplet PCR (ddPCR).<br>Paper II, to assess miR-371a-3p levels in GCT patients undergoing RPLND using ddPCR and to evaluate the marker’s ability to predict viable cancer.<br>Paper III, to report the outcomes for the first 62 patients in Sweden and Norway with low-stage, low-volume metastatic seminoma receiving RPLND as first-line treatment.<br>Paper IV, to assess post-operative outcomes in a population-based cohort of metastatic GCT patients who underwent RPLND over a five-year period, in the context of centralization and wide-ranging adoption of robot-assisted procedures.<br><br>METHODS<br>Paper I is first part of the binational prospective multicenter study SWENOTECA-MIR. 180 TGCT patients and 50 healthy blood donors were analyzed for miR-371a-3p before and after orchiectomy, using ddPCR. The performance of ddPCR was compared to the quantitative PCR (qPCR) made by others to detect miR-371a-3p. Results were stratified by tumor subtype, tumor size, and clinical stage (CS), and levels of miR-371a-3p were statistically analyzed across the groups. Performance of the miR-371a-3p test was compared to conventional tumor markers.<br><br>Paper II is the third part of the SWENOTECA-MIR study. 114 patients (86 nonseminomas, 28 seminomas) underwent open or robot-assisted RPLND from 2017 to 2022. miR-371a-3p levels were analyzed in pre- and post-operative samples using ddPCR. The cohort was categorized into primary and postchemotherapy RPLND groups and further subdivided into seminomas and nonseminomas. Statistical comparisons were made between pre- and postoperative miR-371a-3p levels, with optimism-corrected performance metrics assessed against conventional serum tumor biomarkers.<br><br>Paper III describes the outcomes of a prospective population-based cohort. 62 seminoma patients from Norway and Sweden were operated between 2019 and 2022. Patients with lymphadenopathy £ 3 cm, primary CS IIA/B or CS I with relapse, underwent uni- or bilateral template RPLND, either open or robotassisted. Outcome measures included surgical complications according to the Clavien-Dindo classification, and Kaplan-Meier survival estimates for 24-month progression-free survival (PFS) and overall survival (OS).<br><br>Paper IV is a prospective, population-based, observational multicenter study including all GCT patients who underwent RPLND in Sweden from 2018 to 2022. The cohort comprised 217 patients, with 175 nonseminomas and 42 seminomas. Unilateral and bilateral primary (P) RPLND and post-chemotherapy (PC) RPLND were performed, either open or robot-assisted. Primary outcomes included intra- and post-operative complications, loss of antegrade ejaculation, and histopathological findings of viable cancer or teratoma.<br><br>RESULTS<br>In Paper I, ddPCR demonstrated high performance in detecting miR-371a-3p, with a sensitivity of 89% for the entire cohort. Sensitivities for CS I seminomas and CS I nonseminomas were 87% and 89%, respectively. Sensitivity for CS I–IV seminomas was 89%, and for CS I–IV nonseminomas, it was 90%. The specificity for the cohort was 100%, with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 55%. In comparison, AFP demonstrated 52% sensitivity and b-HCG 51%, in nonseminomas. Linear regression indicated an association between tumor size and miR-371a-3p levels across the entire cohort (R² = 0.159, p < 0.001). Levels of miR-371a-3p decreased significantly following orchiectomy in nearly all patients, with the change being less evident in metastatic patients.<br><br>In Paper II, all seminoma patients (n = 24) undergoing primary RPLND had normal conventional markers, with six having received adjuvant treatment prior to surgery. miR-371a-3p demonstrated a sensitivity of 74%, specificity of 100%, PPV of 100%, and NPV of 21% for detecting viable tumor. miR-371a-3p levels decreased significantly after surgery (p = 0.001). In the nonseminoma group (n = 18) undergoing primary RPLND, 22% had elevated conventional markers, and three patients had received prior adjuvant treatment. For primary nonseminoma patients, miR-371a-3p demonstrated a sensitivity of 34%, specificity of 88%, PPV of 67%, and NPV of 62%. No significant association was observed between stage or prior adjuvant treatment and miR-371a-3p outcome. In the postchemotherapy group (n = 72), miR-371a-3p sensitivity was 9%, and it dropped to 0% when seminoma patients (n = 4) were excluded. Teratomas and benign histology yielded essentially negative results.<br><br>In Paper III, 33 patients (53%) had CS I with relapse during surveillance, six patients (10%) had CS I with relapse after adjuvant chemotherapy, and 23 patients (37%) had initial CS IIA/B disease. Post-operative analysis confirmed metastatic seminoma in 58 patients (94%), with a median largest diameter of 18 mm (IQR: 13–24). Robot-assisted RPLND was performed in 40 patients (65%). Clavien-Dindo III complications occurred in three patients (5%), and no grade ³ IV complications were observed. Eighteen patients (29%) received adjuvant chemotherapy after surgery. The median follow-up was 23 months (IQR: 16–30), with recurrence occurring in six patients (10%) after a median of 8 months (IQR: 4–14). PFS was 90% (95% CI: 0.86–1), and OS was 100% at 24 months.<br><br>In Paper IV, the cohort experienced intra-operative complications in 8% of unilateral and 0% of bilateral P-RPLND procedures, with renal injury being the most common event. For PC-RPLND, the rates were 0% for unilateral and 8% for bilateral templates. Post-operative complications were more frequent with bilateral templates (P-RPLND: 40% vs. 26%, p = 0.3; PC-RPLND: 49% vs. 18%, p = 0.0), with Clavien-Dindo ≥ IIIb complications in 2% of P-RPLND cases and 3% of PC-RPLND cases. Loss of antegrade ejaculation was more common after bilateral templates (P-RPLND: 60% vs. 31%, p = 0.07; PC-RPLND: 53% vs. 38%, p = 0.09). Viable cancer was found in 95% of seminomas and 52% of nonseminomas for primary procedures, while nonseminoma PC-RPLND showed 11% viable cancer, 50% teratoma, and 39% benign nodes. Robot-assisted procedures were not associated with higher rates of intra-operative or post-operative complications, nor increased loss of antegrade ejaculation. Patients who underwent robotic procedures had shorter hospital stays, and conversions to open surgery occurred in 10%.<br><br>CONCLUSIONS<br>Paper I: miR-371a-3p using ddPCR showed 89% sensitivity and 100% specificity in TGCTs, outperforming conventional biomarkers. It decreased postorchiectomy, with less reduction seen in metastatic patients.<br><br>Paper II: miR-371a-3p had superior performance over conventional markers in seminomas undergoing primary RPLND (74% sensitivity, 100% specificity) but was less effective in nonseminomas and post-chemotherapy patients.<br><br>Paper III: Primary RPLND appears to be a safe and effective treatment for selected metastatic seminomas, offering low complication and relapse rates, and potentially reducing long-term risks compared to conventional chemotherapy and radiotherapy.<br><br>Paper IV: Centralized RPLND procedures are associated with low complication rates, with robotic surgery further improving certain outcomes. In PC-RPLND, rates of teratoma and viable cancer increased, with fewer benign findings. Careful patient selection and outcome monitoring are crucial.<br><br><b>List of scientific papers</b><br><br>I. Serum miR371 in testicular germ cell cancer before and after orchiectomy, assessed by digital-droplet PCR in a prospective study Myklebust MP, Thor A, Rosenlund B, Gjengstø P, Karlsdottir A, Brydøy M, Bercea B S, Olsen C, Johnson I, Berg M I, Langberg C W, Andreassen K E, Kjellman A, Haugnes H, Dahl O Scientific Reports, 2021 Aug 2: 11:15582. <a href="https://doi.org/10.1038/s41598-021-94812-2" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41598-021-94812-2</a><br><br>II. miR-371a-3p Predicting Viable Tumor in Patients Undergoing Retroperitoneal Lymph Node Dissection for Metastatic Testicular Cancer: the SWENOTECA-MIR study Thor A, Myklebust MP, Grenabo Bergdahl A, Lundgren P-O, Skokic V, Almås B, Haugnes H, Tandstad T, Akre O, Cohn-Cedermark G, Dahl O, Kjellman A Journal of Urology, 2024 Nov; 212(5):720-730. <a href="https://doi.org/10.1097/JU.0000000000004164" target="_blank">https://doi.org/10.1097/JU.0000000000004164</a><br><br>III. Primary Retroperitoneal Lymph Node Dissection as Treatment for Low-volume Metastatic Seminoma in a Population-based Cohort: the Swedish Norwegian Testicular Cancer Group Experience Thor A, Negaard H, Grenabo Bergdahl A, Almås B, Melsen Larsen S, Lundgren P-O, Gerdtsson A, Halvorsen D, Johannsdottir B, Jansson A K, Hellström M, Wahlqvist R, Langberg C W, Hedlund A, Akre O, Glimelius I, Ståhl O, Haugnes H, Cohn-Cedermark G, Kjellman A/ Tandstad T European Urology Open Science, 2024 Jun 11:65:13-19. <a href="https://doi.org/10.1016/j.euros.2024.05.006" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.euros.2024.05.006</a><br><br>IV. Complications and clinical outcomes of retroperitoneal lymph node dissection in a centralized population-based cohort: insights from the SWENOTECA group Thor A, Grenabo Bergdahl A, Abniki A, Almås B, Melsen Larsen S, Gerdtsson A, Habberstad A, Halvorsen D, Lundgren P-O, Akre O, Cohn-Cedermark G, Kjellman A. 2024 [Manuscript] </p><p dir="ltr"><br></p>