RT-qPCR for mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue.

Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (), paired-like homeobox 2b () and doublecortin () mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated ( model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the model, and transcripts were detected in uncontaminated testicular tissues, whereas mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for following contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, mRNA expression was not observed, but high expression levels of and mRNA were detected, which were similar to expression detected in the model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.