Data from: Reporting tumor molecular heterogeneity in histopathological diagnosis

Background: 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. Aim: To evaluate the diagnostic potential of targeted
multigene next-generation sequencing (TM-NGS) in characterizing
gastrointestinal cancer molecular heterogeneity. Methods: 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. Results: 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. Conclusions: 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.

背景：随着靶向治疗的发展，肿瘤分子异质性检测已成为临床与科研领域的核心议题。此类检测需满足全面性、时效性要求，并基于常规获取的肿瘤样本开展。
目的：评估靶向多基因下一代测序（targeted multigene next-generation sequencing, TM-NGS）在表征胃肠道肿瘤分子异质性中的诊断应用潜力。
方法：本研究纳入35例胃肠道肿瘤样本，涵盖肠型胃癌、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、胆管癌及胰腺实性假乳头状瘤各5例；采用基于Ion Torrent半导体测序技术的TM-NGS，对46个癌症相关基因的突变状态进行检测。此外，以1株壶腹癌细胞系及1例肝癌肉瘤样本评估该检测方法的灵敏度。同时，采用桑格测序（Sanger sequencing）对TP53、PIK3CA、KRAS及BRAF的突变结果进行验证。
结果：TM-NGS在匹配的新鲜冷冻组织与福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织中获得了一致的检测结果；其突变检测限为：新鲜冷冻高分子量DNA样本为1%，FFPE部分降解DNA样本为2%。所有受试肿瘤均检出至少1个体细胞突变，其中20例（57%）肿瘤检出多基因变异。7例肿瘤的不同突变等位基因频率存在显著差异，提示存在瘤内分子异质性；针对部分样本开展p53与Smad4免疫组化检测后，证实了该结论，且与突变分析结果高度一致。
结论：TM-NGS可从微量DNA样本中检测并定量多基因变异，向实现整合形态学、免疫表型与多基因突变分析的下一代组织病理学诊断迈出了关键一步；此类基于常规处理样本的检测，对于个体化癌症治疗具有不可或缺的重要价值。