Stability of miRNA in FFPE tumour samples exhibiting degraded mRNA [Cervix samples series 2]

Background: As degradation of formalin-fixed paraffin-embedded (FFPE) samples limits ability to expression profile, we explored factors predicting success for FFPE profiling and investigated an approach overcoming this limitation. Methods: Bladder (n=141, stored 3-8 years) and cervix (n=160, stored 8-23 years) carcinoma FFPE samples were hybridised to Affymetrix Exon1.0ST arrays. Percent detection above background (%DABG) measured technical success. Biological signal was assessed by distinguishing cervix squamous cell carcinoma (SCC) and adenocarcinoma (AC) using a gene signature. Precursor mir-205 was measured by Exon array and mature miR-205 by qRT-PCR. Eight-old and -young cervix samples were compared using Affymetrix miRNA 2.0 arrays. For comparison, the 'cervix_tumour_cs1_113' (previsously submitted GSM677307) was included and re-analyzed with the samples from the current study (total 161 Cervix samples). Results: RNA quality controls (e.g. RNA integrity number) failed to predict profiling success, but sample age correlated with %DABG in bladder (R2=-0.30, p<0.01) and cervix (R2=-0.69, p<0.01). Biological signal was lost in older samples and neither a signature nor precursor mir-205 separated samples by histology. miR-205 qRT-PCR discriminated SCC from AC, validated by miRNA profiling (26-fold higher in SCC; p=1.10x10-5). Median miRNA probeset expression of eight-old and eight-young cervix samples correlated well (R2=0.95) overcoming the age-related bias of mRNA probesets, suggesting miR-205 stability generalises across microRNA. Conclusions: microRNA profiling overcomes the limitation of degraded FFPE samples 48 Cervix tumour FFPE samples hybridised to exon arrays

背景：福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）样本的降解会限制其表达谱分析能力，本研究探究了可预测FFPE表达谱分析成功的影响因素，并研究了一种可克服该局限性的方法。方法：将存储时长3~8年的141例膀胱癌FFPE样本与存储时长8~23年的160例宫颈癌FFPE样本，与Affymetrix Exon1.0ST芯片进行杂交。以背景以上检测百分比（percent detection above background, %DABG）评估技术成功率。通过基因特征区分宫颈鳞状细胞癌（squamous cell carcinoma, SCC）与腺癌（adenocarcinoma, AC），以此评估生物学信号质量。采用外显子芯片检测前体mir-205，通过实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）检测成熟miR-205。采用Affymetrix miRNA 2.0芯片对存储8年以上与较短的宫颈样本进行比较分析。为便于对比，纳入此前提交的GSM677307样本'cervix_tumour_cs1_113'，并与本研究的样本一同进行重新分析，共计161例宫颈样本。结果：RNA质量控制指标（如RNA完整性数（RNA integrity number, RIN））无法预测表达谱分析的成功率，但样本存储时长与膀胱癌样本的%DABG呈显著负相关（R²=-0.30，p<0.01），宫颈癌样本亦呈类似相关性（R²=-0.69，p<0.01）。存储时间较长的样本会丢失生物学信号，且无论基因特征还是前体mir-205均无法按组织学类型区分样本。通过miR-205的qRT-PCR检测可区分SCC与AC，该结果经miRNA表达谱分析验证：SCC样本中miR-205表达量较AC高26倍（p=1.10×10^-5）。存储8年以上与较短的宫颈样本的miRNA探针组表达中位数相关性极佳（R²=0.95），该结果可克服mRNA探针组受存储时长影响的偏差，提示miR-205的稳定性可推广至整个microRNA群体。结论：microRNA表达谱分析可克服降解FFPE样本的局限性。本研究另有48例宫颈肿瘤FFPE样本与外显子芯片进行了杂交。