DataSheet_2_Integrated spatial analysis of gene mutation and gene expression for understanding tumor diversity in formalin-fixed paraffin-embedded lung adenocarcinoma.xlsx

IntroductionA deeper understanding of intratumoral heterogeneity is essential for prognosis prediction or accurate treatment plan decisions in clinical practice. However, due to the cross-links and degradation of biomolecules within formalin-fixed paraffin-embedded (FFPE) specimens, it is challenging to analyze them. In this study, we aimed to optimize the simultaneous extraction of mRNA and DNA from microdissected FFPE tissues (φ = 100 µm) and apply the method to analyze tumor diversity in lung adenocarcinoma before and after erlotinib administration.
MethodTwo magnetic beads were used for the simultaneous extraction of mRNA and DNA. The decross-linking conditions were evaluated for gene mutation and gene expression analyses of microdissected FFPE tissues. Lung lymph nodes before treatment and lung adenocarcinoma after erlotinib administration were collected from the same patient and were preserved as FFPE specimens for 4 years. Gene expression and gene mutations between histologically classified regions of lung adenocarcinoma (pre-treatment tumor in lung lymph node biopsies and post-treatment tumor, normal lung, tumor stroma, and remission stroma, in resected lung tissue) were compared in a microdissection-based approach.
ResultsUsing the optimized simultaneous extraction of DNA and mRNA and whole-genome amplification, we detected approximately 4,000–10,000 expressed genes and the epidermal growth factor receptor (EGFR) driver gene mutations from microdissected FFPE tissues. We found the differences in the highly expressed cancer-associated genes and the positive rate of EGFR exon 19 deletions among the tumor before and after treatment and tumor stroma, even though they were collected from tumors of the same patient or close regions of the same specimen.
ConclusionOur integrated spatial analysis method would be applied to various FFPE pathology specimens providing area-specific gene expression and gene mutation information.

引言
深入了解肿瘤内异质性，对于临床实践中的预后预测或精准治疗方案决策至关重要。然而，由于福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）标本内的生物分子存在交联与降解，对其开展分析颇具挑战。本研究旨在优化微切割FFPE组织（直径φ=100 μm）中mRNA与DNA的同步提取方法，并将该方法应用于厄洛替尼给药前后肺腺癌的肿瘤异质性分析。

材料与方法
采用两种磁珠同步提取mRNA与DNA。针对微切割FFPE组织的基因突变与基因表达分析，评估了脱交联反应条件。从同一患者体内收集治疗前肺淋巴结组织与厄洛替尼给药后肺腺癌组织，二者均以FFPE标本形式保存了4年。通过微切割技术，对比了肺腺癌不同组织学分类区域的基因表达与基因突变情况，这些区域包括：治疗前肺淋巴结活检肿瘤组织、治疗后肿瘤组织、正常肺组织、肿瘤间质以及切除肺组织中的缓解期间质。

结果
通过优化的DNA与mRNA同步提取及全基因组扩增技术，我们从微切割FFPE组织中检测到约4000~10000个表达基因，以及表皮生长因子受体（epidermal growth factor receptor, EGFR）驱动基因突变。研究发现，尽管这些组织均取自同一患者的肿瘤或同一标本的邻近区域，但二者在癌症相关高表达基因以及EGFR外显子19缺失阳性率上存在差异。

结论
本研究整合的空间分析方法可应用于多种FFPE病理标本，能够提供区域特异性的基因表达与基因突变信息。