Department of Plant Biotechnology and Bioinformatics, Department of Information Technology, IDLab, imec, iGent Toren, Ghent, Belgium.
Department of Human Structure and Repair, Ghent University Hospital, Ghent, Belgium.
BMC Med Genomics. 2020 Jul 6;13(1):94. doi: 10.1186/s12920-020-00746-5.
Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor.
Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants.
We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH.
This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studies.
研究级新鲜冷冻（FF）DNA 材料尚未在临床实践中常规收集。然而，许多医院都收集和存储福尔马林固定石蜡包埋（FFPE）肿瘤样本。因此，如果包括 FFPE 样本，整个基因组癌症队列研究的样本量可以大大增加，尽管存在人工制品可能会使变异检测变得模糊。为了评估 FFPE 材料是否可用于队列研究，我们对从同一肿瘤中提取的匹配 FF 和 FFPE 全基因组序列（WGS）样本上调用的体细胞 SNV 进行了深入比较。
使用四个变体调用器（即 Strelka2、Mutect2、VarScan2 和 Shimmer）在转移性前列腺肿瘤的匹配 FF 和 FFPE WGS 样本上调用体细胞变体。使用这些调用器识别的变体，我们开发了一种启发式方法，以最大程度地提高 FF 和其 FFPE 对应物在灵敏度和精度方面的重叠。然后，我们在九个匹配的原发性样本上验证了该变体调用方法。最后，通过比较克隆和亚克隆体细胞变体的重叠，评估差异的一部分可归因于肿瘤内异质性（ITH）。
我们首先比较了转移性前列腺肿瘤的 FF 和 FFPE 样本之间的变体，结果表明，FF 中约有 50%的变体在 FFPE 样本中被回收，不同的调用者之间存在明显差异。使用简单的启发式方法组合不同调用者的变体，可以提高变体调用的精度和灵敏度。在另外九个额外的匹配 FF-FFPE 样本上验证启发式方法，导致平均 F1 得分为 0.58，并且优于任何单个调用者。此外，我们可以证明，FF 和 FFPE 样本之间的差异部分可归因于 ITH。
这项研究表明，当使用正确的变体调用策略时，高精确度和灵敏度下可以从 FFPE 样本中恢复大多数克隆 SNV。这些结果表明，源自 FFPE 材料 WGS 的体细胞变体可用于队列研究。
