Bioinformatics and Systems Biology Graduate Program - UC San Diego, 9500 Gilman Dr., La Jolla, CA, 92093, USA.
Moores Cancer Center - UC San Diego Health - 3855 Health Sciences Dr., La Jolla, CA, 92093, USA.
BMC Med Genomics. 2020 Nov 18;13(1):173. doi: 10.1186/s12920-020-00820-y.
Systematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA.
Using test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm) from 3 breast ductal carcinoma in situ (DCIS).
Using low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity.
Alternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.
系统的癌症筛查导致了癌前病变（PML）的检出率增加。由于缺乏可靠的预后标志物，导致了过度治疗，从而给患者带来了潜在的压力，或者治疗不足和可避免的进展。重要的是，大多数突变分析研究依赖于与浸润性癌症同步的 PML，或者在没有结局信息的患者中进行，因此限制了它们在生物标志物发现中的应用。PML 综合突变分析的局限性在很大程度上是由于存在显著的技术和方法学挑战：大多数 PML 标本体积小，固定在福尔马林和石蜡包埋（FFPE）中，并且缺乏匹配的正常 DNA。
使用高度降解的 FFPE 标本中的测试 DNA，评估了多种靶向测序方法，包括 DNA 输入量（3-200ng）、文库制备策略（BE：Blunt-End，SS：Single-Strand，AT：A-Tailing）和靶大小（全外显子组与癌症基因panel）的变化。FFPE 和冷冻镜像标本中高输入量 DNA 的变体分别用于 PML 特异性变体调用训练和测试。然后将得到的方法应用于分析和比较从 3 个乳腺导管原位癌（DCIS）中微切割（平均面积 5mm）的多个区域。
使用低输入量的 FFPE DNA，BE 和 SS 文库在全外显子覆盖率达到 20x 时比 AT 文库分别增加了 4.9%和 3.7%（BE：87%，SS：63%，AT：17%）。与来自冷冻标本的高可信度体细胞突变相比，PML 特异性变体过滤提高了召回率（BE：85%，SS：80%，AT：75%）和精确度（BE：93%，SS：91%，AT：84%），达到了可归因于采样变化的水平。拷贝数改变在所有测试方法中都是一致的，并且仅受捕获探针集设计的影响。应用于从 9 个微切割区域（8 个 PML，1 个正常上皮）中提取的 DNA，该方法表现出相当的性能，说明了该方法足以识别候选驱动事件（GATA3 突变、ERBB2 或 FGFR1 增益、TP53 缺失）和测量病变内遗传异质性。
替代的实验和分析策略提高了从存档的微切割 PML 区域中提取 DNA 测序的准确性，支持了对早期癌症病变的更深入分子特征分析，并在开发基于生物学的预后标志物和精准化疗预防策略方面取得了关键的里程碑。
