Grèze Victoria, Kanold Justyna, Chambon Fanny, Halle Pascale, Gremeau Anne-Sophie, Rives Nathalie, Rouel Nadège, Pereira Bruno, Tchirkov Andrei, Brugnon Florence
Service Hématologie Oncologie Pédiatrique, CHU Clermont-Ferrand, France.
Université Clermont Auvergne, INSERM-CIC 1405, Unité CRECHE, CHU Clermont-Ferrand, F-63000 Clermont-Ferrand, France.
Oncol Lett. 2017 Jul;14(1):860-866. doi: 10.3892/ol.2017.6238. Epub 2017 May 24.
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.
神经母细胞瘤（NB）是儿童最常见的颅外实体瘤类型，在幼儿中发病率很高。对于儿童癌症幸存者而言，保留生殖潜能是生活质量的一个重要因素。优化睾丸组织中NB微小残留病（MRD）的检测对于评估恶性细胞重新植入的风险至关重要。本研究的第一步是评估逆转录定量聚合酶链反应（RT-qPCR）检测酪氨酸羟化酶（TH）、配对样同源盒2b（PHOX2B）和双皮质素（DCX）mRNA在非阻塞性无精子症（NOA）患者经越来越多的IMR-32和SK-N-SH NB细胞污染（污染模型）的冻融睾丸组织中的表达准确性。睾丸组织通过慢速或快速冷冻进行冷冻。第二步是确定这些标志物在4名青春期前男性（2名患有IV期NB和2名患有非NB恶性肿瘤）的睾丸样本中的表达水平。通过慢速或快速冷冻的睾丸样本中提取RNA的产量相似。在污染模型中，未受污染的睾丸组织中检测到TH和PHOX2B转录本，而未检测到DCX mRNA。用于污染的NB细胞数量与TH转录本水平之间存在强正相关。对于IMR-32和SK-N-SH NB细胞系，在被10个肿瘤细胞污染后，TH的检测特异性和灵敏度率均为100%。在有或无NB的青春期前男性的睾丸样本中，未观察到DCX mRNA表达，但检测到TH和PHOX2B mRNA的高表达水平，这与在污染模型中检测到的表达相似。在用于NB MRD研究的血液和骨髓中的标志物中，通过RT-qPCR检测TH转录本可能为需要保留生育能力的男性的睾丸组织中的NB细胞提供准确评估。
