Musella Valeria, Callari Maurizio, Di Buduo Eleonora, Scuro Manuela, Dugo Matteo, Miodini Patrizia, Bianchini Giampaolo, Paolini Biagio, Gianni Luca, Daidone Maria Grazia, Cappelletti Vera
Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Department of Pathology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
PLoS One. 2015 Apr 6;10(4):e0123194. doi: 10.1371/journal.pone.0123194. eCollection 2015.
To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.
为了从临床试验收集的样本中获取基因表达谱，我们开展了一项初步研究，以评估对福尔马林固定、石蜡包埋标本进行分析时的可行性并估计样本损耗率。分别使用Illumina HT-12芯片和Ref-8芯片对10对匹配的新鲜冷冻和固定乳腺癌样本进行了分析。使用Ref 8芯片获得的图谱在技术上和生物学上均不可靠，因为它们未能在雌激素受体阳性和阴性样本之间产生预期的区分。在固定样本上使用Affymetrix HG-U133 2.0 Plus芯片并结合基于定量聚合酶链反应的样本预评估步骤，尽管有效检出率较低（M = 21%±5），但在生物学可靠性方面结果令人满意。与Illumina DASL WG平台相比，Affymetrix的数据显示出更宽的四分位间距（1.32对0.57，P<2.2 E-16）和更大的倍数变化。Affymetrix芯片用于对60例固定乳腺癌样本进行初步研究。通过在工作流程中纳入样本预评估步骤，从技术和生物学意义的角度来看，预计会产生良好结果的样本中有96%（44/46）实际上被评为令人满意。我们的基因表达谱与免疫组织化学数据高度一致，能够重现乳腺癌分子亚型，并验证了在冷冻样本中得出的雌激素受体状态分类器。因此，该方法适用于对临床试验中收集的福尔马林固定石蜡包埋样本进行分析，前提是在阵列杂交之前（样本预评估）和之后都进行质量控制。
