Reporting tumor molecular heterogeneity in histopathological diagnosis.
Reporting tumor molecular heterogeneity in histopathological diagnosis.
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DOI：
10.1371/journal.pone.0104979
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Scarpa A
中科院分区：
文献类型：
--
作者：
Mafficini A;Amato E;Fassan M;Simbolo M;Antonello D;Vicentini C;Scardoni M;Bersani S;Gottardi M;Rusev B;Malpeli G;Corbo V;Barbi S;Sikora KO;Lawlor RT;Tortora G;Scarpa A
随着靶向治疗的出现，检测分子肿瘤的异质性变得至关重要。检测分析应全面、及时，并以常规的肿瘤样本为基础。目的探讨靶向多基因下一代测序技术(TM-NGS)在胃肠道肿瘤分子异质性诊断中的应用价值。应用基于离子激流半导体的TM-NGS技术，对35例胃肠道肿瘤，其中肠型胃癌、胰腺导管腺癌、胰腺导管内乳头状黏液性肿瘤、壶腹癌、肝细胞癌、胆管癌、胰腺实性假乳头状肿瘤各5例进行了46个癌相关基因的突变检测。一个Vater癌细胞株的壶腹和一个肝癌肉瘤用于评估检测的敏感性。通过常规Sanger测序验证了TP53、PIK3CA、KRAS和BRAF突变。TM-NGS对匹配的新鲜冷冻和福尔马林固定石蜡包埋(FFPE)组织的检测结果重叠，对新鲜冷冻的高分子量DNA的突变检测限为1%，对FFPE部分降解的DNA的突变检测限为2%。在所有被测试的肿瘤中，至少观察到一个体细胞突变；在20/35(57%)个肿瘤中检测到多个改变。7例癌组织中存在不同突变的等位基因频率存在显著差异，提示肿瘤内存在分子异质性；通过免疫组织化学P53和Smad4免疫组织化学方法在所选样本上证实了这一点，这与突变分析相一致。TM-NGS能够从有限的DNA中检测和量化多个基因的变化，向下一代组织病理学诊断又迈进了一步，该诊断集成了常规处理组织的形态、免疫表型和多基因突变分析，对于个性化癌症治疗至关重要。
Detection of molecular tumor heterogeneity has become of paramount importance with the advent of targeted therapies. Analysis for detection should be comprehensive, timely and based on routinely available tumor samples. To evaluate the diagnostic potential of targeted multigene next-generation sequencing (TM-NGS) in characterizing gastrointestinal cancer molecular heterogeneity. 35 gastrointestinal tract tumors, five of each intestinal type gastric carcinomas, pancreatic ductal adenocarcinomas, pancreatic intraductal papillary mucinous neoplasms, ampulla of Vater carcinomas, hepatocellular carcinomas, cholangiocarcinomas, pancreatic solid pseudopapillary tumors were assessed for mutations in 46 cancer-associated genes, using Ion Torrent semiconductor-based TM-NGS. One ampulla of Vater carcinoma cell line and one hepatic carcinosarcoma served to assess assay sensitivity. TP53, PIK3CA, KRAS, and BRAF mutations were validated by conventional Sanger sequencing. TM-NGS yielded overlapping results on matched fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissues, with a mutation detection limit of 1% for fresh-frozen high molecular weight DNA and 2% for FFPE partially degraded DNA. At least one somatic mutation was observed in all tumors tested; multiple alterations were detected in 20/35 (57%) tumors. Seven cancers displayed significant differences in allelic frequencies for distinct mutations, indicating the presence of intratumor molecular heterogeneity; this was confirmed on selected samples by immunohistochemistry of p53 and Smad4, showing concordance with mutational analysis. TM-NGS is able to detect and quantitate multiple gene alterations from limited amounts of DNA, moving one step closer to a next-generation histopathologic diagnosis that integrates morphologic, immunophenotypic, and multigene mutational analysis on routinely processed tissues, essential for personalized cancer therapy.
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