Whole transcriptome profiling of adrenocortical tumors using formalin-fixed paraffin-embedded samples

Whole transcriptome profiling is an essential technique in adrenal studies; however, whole transcriptome profiling of adrenal disease using formalin-fixed paraffin-embedded (FFPE) samples has to be further explored. The aim of this study was to evaluate the utility of transcriptome data from FFPE samples of adrenocortical tumors. We performed whole transcriptome profiling of FFPE and fresh frozen samples of adrenocortical carcinoma (ACC, n = 3), aldosterone-producing adenoma (APA, n = 3), and cortisol-producing adenoma (CPA, n = 3), and examined the similarity between the transcriptome data. We further examined whether the transcriptome data of FFPE samples could be used to distinguish tumor types and detect marker genes. The number of read counts was smaller in FFPE samples than in fresh frozen samples (P < 0.01), while the number of genes detected was similar (P = 0.39). The gene expression profiles of FFPE and fresh frozen samples were highly correlated (r = 0.93, P < 0.01). Tumor types could be distinguished by consensus clustering and principal component analysis using transcriptome data from FFPE samples. In the differential expression analysis between ACC and APA-CPA, known marker genes of ACC (e.g., CCNB2, TOP2A, and MAD2L1) were detected in the ACC of FFPE samples. In the differential expression analysis between APA and CPA, known marker genes of APA (e.g., CYP11B2, VSNL1, and KCNJ5) were detected in the APA of FFPE samples. The results indicate that FFPE samples are reliable alternatives to fresh frozen samples for whole transcriptome profiling of adrenocortical tumors.

全转录组分析（whole transcriptome profiling）是肾上腺研究中的关键技术手段，但针对肾上腺疾病、采用福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）样本开展的全转录组分析研究仍有待进一步探索。本研究旨在评估肾上腺皮质肿瘤FFPE样本转录组数据的应用价值。我们针对3例肾上腺皮质癌（adrenocortical carcinoma, ACC）、3例醛固酮生成性腺瘤（aldosterone-producing adenoma, APA）及3例皮质醇生成性腺瘤（cortisol-producing adenoma, CPA）的FFPE与新鲜冷冻样本开展全转录组分析，并比较两类样本转录组数据的相似性。我们进一步验证了FFPE样本的转录组数据是否可用于区分肿瘤类型及筛选标志物基因。结果显示，FFPE样本的测序读段计数低于新鲜冷冻样本（P < 0.01），但二者检测到的基因数量无显著统计学差异（P = 0.39）。FFPE与新鲜冷冻样本的基因表达谱呈现高度相关性（相关系数r=0.93，P < 0.01）。基于FFPE样本的转录组数据，通过一致性聚类与主成分分析可准确区分肿瘤类型。在ACC与APA-CPA的差异表达分析中，FFPE来源的ACC样本可检测到已报道的ACC标志物基因（如CCNB2、TOP2A及MAD2L1）。在APA与CPA的差异表达分析中，FFPE来源的APA样本可检测到已报道的APA标志物基因（如CYP11B2、VSNL1及KCNJ5）。本研究结果表明，在肾上腺皮质肿瘤的全转录组分析中，FFPE样本可作为新鲜冷冻样本的可靠替代样本。