Roberts Robin A, Sabalos Constantine M, LeBlanc Michael L, Martel Ralph R, Frutiger Yvette M, Unger Joseph M, Botros Ihab W, Rounseville Matthew P, Seligmann Bruce E, Miller Thomas P, Grogan Thomas M, Rimsza Lisa M
Department of Pathology, University of Arizona, Tucson, AZ 85724-5043, USA.
Lab Invest. 2007 Oct;87(10):979-97. doi: 10.1038/labinvest.3700665. Epub 2007 Aug 13.
Gene expression profiling (GEP) has identified genes whose expression levels predict patient survival in diffuse large-B-cell lymphoma (DLBCL). Such discovery techniques generally require frozen samples unavailable for most patients. We developed a quantitative nuclease protection assay to measure expression levels of prognostic DLBCL genes using formalin-fixed, paraffin-embedded (FFPE) tissue. FFPE tissue was sectioned, permeabilized, denatured in the presence of specific probes, and hybridized to mRNA in situ. Nuclease subsequently destroyed non-hybridized probe. Alkaline hydrolysis freed mRNA-bound probes from tissue, which were transferred to ArrayPlates for probe capture and chemiluminescent quantification. We validated assay performance using frozen, fresh, and FFPE DLBCL samples, then used 39 archived DLBCL, previously microarray analyzed, to correlate GEP and ArrayPlate results. We compared old (>18 years) with new (<2 months) paraffin blocks made from previously frozen tissue from the original biopsy. ArrayPlate gene expression results were confirmed with immunohistochemistry for BCL2, BCL6, and HLA-DR, showing agreement between mRNA species and the proteins they encode. Assay performance was linear to approximately 1 mg sample/well. RNase and DNase treatments demonstrated assay specificity for RNA detection, both fixed and soluble RNA detection. Comparisons were excellent for lysate vs snap-frozen vs FFPE (R(2)>0.98 for all comparisons). Coefficients of variation for quadruplicates on FFPE were generally <20%. Correlation between new and old paraffin blocks from the same biopsy was good (R(2)=0.71). Comparison of ArrayPlate to Affymetrix and cDNA microarrays showed reasonable correlations. Insufficient power from small sample size prevented successfully correlating results with patient survival, although hazard ratios trended the expected directions. We developed an assay to quantify expression levels of survival prediction genes in DLBCL using FFPE, fresh, or frozen tissue. While this technique cannot replace GEP for discovery, it indicates that expression differences identified by GEP can be replicated on a platform applicable to archived FFPE samples.
基因表达谱分析（GEP）已鉴定出其表达水平可预测弥漫性大B细胞淋巴瘤（DLBCL）患者生存率的基因。此类发现技术通常需要冷冻样本，而大多数患者无法获得此类样本。我们开发了一种定量核酸酶保护测定法，以使用福尔马林固定、石蜡包埋（FFPE）组织来测量预后DLBCL基因的表达水平。将FFPE组织切片、通透化、在特异性探针存在下变性，然后与原位mRNA杂交。核酸酶随后破坏未杂交的探针。碱性水解将与mRNA结合的探针从组织中释放出来，将其转移至ArrayPlates进行探针捕获和化学发光定量。我们使用冷冻、新鲜和FFPE DLBCL样本验证了测定性能，然后使用39例先前进行过微阵列分析的存档DLBCL样本，以关联GEP和ArrayPlate结果。我们比较了由原始活检中先前冷冻组织制成的旧石蜡块（>18年）和新石蜡块（<2个月）。通过对BCL2、BCL6和HLA-DR进行免疫组织化学确认了ArrayPlate基因表达结果，显示mRNA种类与其编码的蛋白质之间具有一致性。测定性能对于每孔约1 mg样本呈线性关系。核糖核酸酶和脱氧核糖核酸酶处理证明了该测定法对RNA检测的特异性，包括固定RNA和可溶性RNA检测。对于裂解物与速冻样本与FFPE样本的比较效果极佳（所有比较的R²>0.98）。FFPE样本一式四份的变异系数通常<20%。同一活检的新旧石蜡块之间的相关性良好（R²=0.71）。将ArrayPlate与Affymetrix和cDNA微阵列进行比较显示出合理的相关性。尽管风险比呈预期趋势，但样本量较小导致的统计效力不足妨碍了将结果与患者生存率成功关联。我们开发了一种测定法，以使用FFPE、新鲜或冷冻组织来定量DLBCL中生存预测基因的表达水平。虽然该技术不能替代用于发现的GEP，但它表明GEP鉴定出的表达差异可以在适用于存档FFPE样本的平台上重现。
