Kojima Kensuke, April Craig, Canasto-Chibuque Claudia, Chen Xintong, Deshmukh Manjeet, Venkatesh Anu, Tan Poh Seng, Kobayashi Masahiro, Kumada Hiromitsu, Fan Jian-Bing, Hoshida Yujin
Liver Cancer Program, Tisch Cancer Institute, Division of Liver Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York, United States of America.
Illumina, Inc., San Diego, California, United States of America.
PLoS One. 2014 Jan 30;9(1):e86961. doi: 10.1371/journal.pone.0086961. eCollection 2014.
Archived tissues from previously completed prospective trials represent invaluable resource for biomarker development. However, such specimens are often stored as sections on glass slides, in which RNA is severely degraded due to prolonged air exposure. We evaluated whether a proportion of archived sectioned formalin-fixed paraffin-embedded (AS-FFPE) tissues yield transcriptome profiles comparable to freshly cut (FC) FFPE tissues, which can be used for retrospective class prediction analysis.
Genome-wide transcriptome profiles of 6 to 7-year-old AS-FFPE tissue sections (generated from 5 to 16-year-old blocks) of 83 hepatocellular carcinoma (HCC) and 47 liver cirrhosis samples were generated by using whole-genome DASL assay (Illumina) and digital transcript counting (nCounter) assay (NanoString), and gene signature-based prediction of HCC subclasses and prognosis was compared with previously generated FC-FFPE profiles from the same tissue blocks.
RNA quality and assay reproducibility of AS-FFPE RNA were comparable to intermediate to poor quality FC-FFPE samples (RNA Integrity Number: up to 2.50, R-square for technical replicates: up to 0.93). Analyzable transcriptome profiles were obtained in 64 (77%) HCC and 36 (77%) cirrhosis samples. Statistically more confident predictions based on random resampling-based method (nearest template prediction) were obtained in 37 (58%) HCC and 13 (36%) cirrhosis samples. Predictions made in FC-FFPE profiles were reproduced in 36 (97%) HCC and 11 (85%) cirrhosis AS-FFPE profiles. nCounter assay was tested in 24 cirrhosis samples, which yielded confident prediction in 15 samples (63%), of which 10 samples (67%) showed concordant predictions with FC-FFPE profiles.
AS-FFPE tissues yielded poorer quality RNA and transcriptome profiles compared to FC-FFPE tissues. Statistically more confident class prediction was feasible in 37 of 83 HCC samples and 13 of 47 cirrhosis samples. These results suggest that AS-FFPE tissues can be regarded as a resource for retrospective transcriptome-based class prediction analysis when they are the only available materials.
来自先前完成的前瞻性试验的存档组织是生物标志物开发的宝贵资源。然而，此类标本通常以玻片切片的形式保存，其中的RNA由于长时间暴露于空气中而严重降解。我们评估了一定比例的存档切片福尔马林固定石蜡包埋（AS-FFPE）组织能否产生与新鲜切割（FC）FFPE组织相当的转录组图谱，后者可用于回顾性分类预测分析。
使用全基因组DASL分析（Illumina）和数字转录本计数（nCounter）分析（NanoString），生成了83例肝细胞癌（HCC）和47例肝硬化样本的6至7岁AS-FFPE组织切片（由5至16岁的组织块制成）的全基因组转录组图谱，并将基于基因特征的HCC亚类和预后预测与先前从相同组织块生成的FC-FFPE图谱进行比较。
AS-FFPE RNA的质量和分析重现性与质量中等至较差的FC-FFPE样本相当（RNA完整性数值：最高2.50，技术重复的R平方：最高0.93）。在64例（77%）HCC和36例（77%）肝硬化样本中获得了可分析的转录组图谱。基于随机重采样法（最近模板预测）在37例（58%）HCC和13例（36%）肝硬化样本中获得了统计学上更可靠的预测。FC-FFPE图谱中的预测在36例（97%）HCC和11例（85%）肝硬化AS-FFPE图谱中得到了重现。在24例肝硬化样本中对nCounter分析进行了测试，其中15例（63%）样本获得了可靠的预测，其中10例（67%）样本与FC-FFPE图谱显示出一致的预测。
与FC-FFPE组织相比，AS-FFPE组织产生的RNA质量和转录组图谱较差。在83例HCC样本中的37例和47例肝硬化样本中的13例中，统计学上更可靠的分类预测是可行的。这些结果表明，当AS-FFPE组织是唯一可用材料时，可将其视为基于转录组的回顾性分类预测分析的一种资源。
