Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) germ cell tumor (GCT) pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28–35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to (a) utilize threshold-based approaches using raw Cq values, (b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and (c) to re-run any sample with an indeterminate result.

424 Background: Conventional serum tumor markers (STMs) are noted to have poor specificity in detecting seminomatous germ cell tumors (SGCTs). Recent publications have suggested that miR-371a-3p may offer improved performance for these cases. However, miR-371a-3p’s performance in detecting pure seminoma at retroperitoneal lymph node dissection (RPLND) has yet to be elucidated in any publicly available study. This lack of data is in part due to the rare occurrence of such cases, and it represents a distinct path for exploring miR-371a-3p’s clinical utility. Methods: Pre-surgical serum samples were collected prospectively from patients prior to RPLND. Within the 15-patient cohort, 6 were assigned as ‘Control’ and 9 were assigned as ‘Seminoma’ based on pathological confirmation of viable GCT. Out of all 15 patients, 10 were chemotherapy naïve prior to RPLND. Post-chemotherapy patients made up 5/6 ‘Control’ patients. MiR-371a-3p levels were quantified by RT-qPCR. Using 2−∆∆Cq method, miR-371a-3p expression was first normalized to the miR-30b-5p reference gene and then calculated relative to the mean target expression across a collection of RNA extracts from healthy male donors’ serum. The Cq values were statistically evaluated to obtain performance characteristics (sensitivity and specificity). Results: Although assay results revealed no significant difference in miR-371a-3p expression between groups, median relative expression in the seminoma group trended higher than the control group (Rq = 3705 and 241 respectively, p = 0.2844). Out of all chemotherapy naïve patients, 9/10 had viable GCT at RPLND whereas 7/10 showed elevated miR-371a-3p. Among the five post-chemotherapy patients, 0/5 had viable GCT at RPLND and 2/5 had elevated miR-371a-3p. The assay provided 7/9 true positive designations in the seminoma group and 4/6 true negatives in the control group. The two false positive results were from post-chemotherapy patients. Of the two optimal thresholds calculated by Youden’s J statistic, the lower threshold of 28.62 (78% sensitivity ad 67% specificity) was selected because it was more in line with previously published data. ROC analysis provided an AUC of 0.704 (95% confidence interval: 0.43-0.98, p = 0.1949). STMs performance was poorer at 67% sensitivity and 17% specificity. Conclusions: Performance metrics for miR-371a-3p exceed those of STMs but were substantially lower than previous reports that evaluated performance in pre-orchiectomy and primary RPLND settings. A possible explanation for this disparity is that miR-371a-3p’s performance is hindered in the post-chemotherapy RPLND setting. However, any strong conclusions from these results are limited by the small sample size which is partly due to the rarity of this clinical scenario. These results suggest that pure seminoma at RPLND may be a key clinical context wherein the miRNA assay may require further refinement.

414 Background: Patients with clinical stage IIA (CS IIA) testicular cancer often present a diagnostic and management dilemma. Clinical guidelines recommend primary retroperitoneal lymph node dissection (RPLND), chemotherapy, or radiotherapy with excellent recurrence- and disease-specific survival (RFS, DSS) rates. Given the favorable prognosis and young age of men with CS IIA disease, efforts should be made to limit treatment burden while maintaining favorable oncologic outcomes. Here we present a risk-adapted, multi-disciplinary model in the management of CS IIA testicular cancer. Methods: This was a retrospective study of men undergoing primary RPLND for CS IIA testicular cancer from 6/1/2015 to 2/28/2021. Each patient underwent radical orchiectomy with a histologic diagnosis of testicular cancer. Staging was performed with cross-sectional imaging and serum tumor markers. The case and management options were presented in a multi-disciplinary setting with the preference for re-imaging in 6-8 weeks and performance of RPLND so long as no involution of nodes or development of distant metastases. Node positivity at RPLND and clinical recurrence were evaluated. Pearson’s correlations and logistic regression were performed to evaluate the relationship between time from staging imaging to surgery and the outcomes of interest. Results: Nineteen men with CS IIA testicular cancer underwent primary RPLND. Median age at RPLND was 28 years. There were 7 (36.8%) men with pure seminoma, 11 (57.9%) with non-seminomatous germ tumor (NSGCT), and 1 (5.3%) with burnt-out primary on orchiectomy. Median largest node size was 1.2 cm (IQR 1.0-1.4 cm) on staging imaging. Final RPLND pathology revealed 2 benign (10.5%), 11 with pure seminoma (57.9%), and 6 with NSGCT elements (31.6%). The median number of positive lymph nodes on final pathology was 2 with median largest node size of 2.0 cm (IQR 1.25-3.00 cm). 9 patients (47.4%) upgraded to pN2. Median follow-up was 15 months. There were two clinical recurrences, yielding a median RFS of 9 months. Pearson correlation and univariate regression revealed no significant association between time from imaging to RPLND and upstaging from cN1 to pN2, recurrence, or recurrence-free survival. Conclusions: This study demonstrates a multidisciplinary approach selecting patients that are best managed with RPLND, supporting the oncologic safety of this risk-adapted model in the management of CS IIA testicular cancer. The pN0 rate of 10.5% is lower than historical rates in CS IIA disease and may be related to selecting out patients more appropriate for active surveillance. Similarly the 10% recurrence rate is lower than large surgical series. We are limited by lack of the denominator of patients that developed metastases at interval imaging that subsequently received systemic therapy.

Circulating miR-371a-3p has excellent performance in the detection of viable (non-teratoma) GCT pre-orchiectomy; however, its ability to detect occult disease is understudied. To refine the serum miR-371a-3p assay in the minimal residual disease setting we compared performance of raw (Cq) and normalized (∆Cq, RQ) values from prior assays, and validated interlaboratory concordance by aliquot swapping. Revised assay performance was determined in a cohort of 32 patients suspected of occult retroperitoneal disease. Assay superiority was determined by comparing resulting receiver-operator characteristic (ROC) curves using the Delong method. Pairwise t-tests were used to test for interlaboratory concordance. Performance was comparable when thresholding based on raw Cq vs. normalized values. Interlaboratory concordance of miR-371a-3p was high, but reference genes miR-30b-5p and cel-miR-39-3p were discordant. Introduction of an indeterminate range of Cq 28–35 with a repeat run for any indeterminate improved assay accuracy from 0.84 to 0.92 in a group of patients suspected of occult GCT. We recommend that serum miR-371a-3p test protocols are updated to a) utilize threshold-based approaches using raw Cq values, b) continue to include an endogenous (e.g., miR-30b-5p) and exogenous non-human spike-in (e.g., cel-miR-39-3p) microRNA for quality control, and c) to re-run any sample with an indeterminate result.

No abstract