PTH-171 Evaluation of TP53 mutations on barrett-s epithelium FFPE specimens with a novel next generation sequencing platform highlights a discordance with P53 immunohistochemistry: analysis of the barrett-s epithelium genome investigation study (begins)
PTH-171 Evaluation of TP53 mutations on barrett-s epithelium FFPE specimens with a novel next generation sequencing platform highlights a discordance with P53 immunohistochemistry: analysis of the barrett-s epithelium genome investigation study (begins)
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PTH-171 使用新型下一代测序平台评估巴雷特上皮 FFPE 标本上的 TP53 突变，突出显示与 P53 免疫组织化学的不一致：巴雷特上皮基因组调查研究的分析（开始）
DOI：
10.1136/gutjnl-2015-309861.1059
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发表时间：
2015
期刊：
影响因子：
24.5
通讯作者：
Butt M
中科院分区：
文献类型：
--
作者：
Butt M
遗传因素在Barrett上皮（BE）易感性和食管腺癌（OA）进展中起着至关重要的作用。在冷冻组织上使用下一代测序（NGS）方法，在BE中以阶段特异性方式鉴定了TP 53突变（1）。保存BE的标准方法是福尔马林固定和石蜡包埋（FFPE）。微小FFPE标本的NGS具有挑战性。方法对37例微小FFPE BE活检标本进行测序和免疫组化染色，其中包括非异型增生BE（NDBE; n = 7）、低度异型增生（LGD; n = 7）、高度异型增生（HGD; n = 9）、粘膜内癌（IMC; n = 6）和侵袭性OA（IOA; n = 8）。我们使用内部高通量多路复用方法，使用NGS对从FFPE组织提取的DNA进行靶向突变筛选，并使用我们独特的隐马尔可夫分析方法分析文件。使用克隆D 07的自动p53 IHC被用来关联检测到的TP 53突变与组织expression.ResultsDNA提取的变化从2-50 ng，包括一些高度降解的DNA材料。TP 53基因突变率为40.5%（15/37），其中NDBE为28.6%（2/7），LGD为71.4%（5/7），HGD为44.4%（4/9），IMC为33.3%（2/6），IOA为25%（2/8）。有趣的是，只有40%（6/15）的病例同时显示TP 53突变和p53畸变（过度表达或不表达），60%（9/15）有突变但无蛋白质畸变，45%（5/11）有p53畸变但无突变异常。数据质量极佳，所有病例的覆盖深度至少为x800（平均覆盖x1200（图1A））。然而，p53染色水平与突变数据无关（图1B）。结论这些结果表明，使用突变分析和当前p53 IHC方法检测TP 53之间存在不一致性。此外，我们确定了NDBE中TP 53突变的患者，这可能会阻止进展为OA。确定早期基因组畸变驱动OA的发展，BE持有的关键，准确的风险分层BE患者到更积极的治疗策略或频繁的监测regimes.Disclosure的利益无声明。2014;46（8）：837-43
IntroductionGenetic factors play a crucial role in the predisposition to Barrett’s epithelium (BE) and progression to oesophageal adenocarcinoma (OA). Using next generation sequencing (NGS) methods on frozen tissue, TP53 mutations have been identified in a stage specific manor in BE (1). The standard method of preserving BE is formalin fixing and paraffin embedding (FFPE). NGS on tiny FFPE specimens is challenging. We examined the prevalence of TP53 genomic aberrations and p53 expression in tiny FFPE BE biopsies using an adapted NGS platform.Method37 cases including non-dysplastic BE (NDBE; n = 7), low grade dysplasia (LGD; n = 7), high grade dysplasia (HGD; n = 9), intramucosal cancer (IMC; n = 6) and invasive OA (IOA; n = 8) were sequenced and immunostained. We used an inhouse, high throughput multiplexing method for targeted mutational screening using NGS on DNA extracted from FFPE tissue and analysed the files with our unique hidden Markov analytical method. Automated p53 IHC using clone D07 was used to correlate detected TP53 mutations with tissue expression.ResultsDNA extracted varied from 2–50 ng, including some highly degraded DNA material. TP53 was mutated in 40.5% (15/37) of cases; 28.6% of NDBE (2/7), 71.4% of LGD (5/7), 44.4% of HGD (4/9), 33.3% of IMC (2/6) and 25% of IOA (2/8). Interestingly, only 40% (6/15) of cases showed both TP53 mutations and p53 aberrations (over- or absence of expression), 60% (9/15) had mutations but no protein aberration, and 45% (5/11) had p53 aberration but no mutational abnormality. The data quality was excellent with all cases having a depth of coverage of at least x800 (mean coverage x1200 (Figure 1A). The level of p53 staining did not however correlate with mutation data (Figure 1B).ConclusionThese results show discordance between detection of TP53 using mutation analysis and current p53 IHC methods. Furthermore, we identified patients with TP53 mutations in NDBE, which may pre-empt progression to OA. Identifying early genomic aberrations driving the development of OA from BE holds the key to accurately risk stratifying BE patients into more aggressive treatment strategies or frequent surveillance regimes.Disclosure of interestNone Declared.ReferenceWeaveret al.Nat Genet. 2014;46(8):837-43