Comprehensive genetic evaluation of ductal carcinoma in situ

本项目(第1部分)的目的是在临床标本中确定预测乳腺导管原位癌(DCIS)向浸润性导管癌(IDC)进展的遗传标志。从236个样本的中期分析中，我们证实了主要的基因突变，如PIK3CA、AKT1和TP53，与先前在TCGA数据库中报告的一样被一致地检测到。2020年，我们从临床测序中收集了更多的数据，现在队列中总共包括313个样本(252个来自IDC，61个来自DCIS)。因此，我们正在更新整个队列的分析，并试图识别导致从DCIS进展到IDC的候选基因，即使在不太频繁突变的基因中也是如此。本项目(第二部分)的目的是在该模型中描述乳腺癌进展的分子机制。我们获得了MCF10A和MCF10DCIS细胞系，分别作为乳腺正常上皮和DCIS肿瘤的模型。优化了两种细胞系CRISPR/Cas9筛选的条件。优化包括准确鉴定细胞系的倍增时间，细胞密度和聚丙稀烯浓度等感染条件，以及选择合适的嘌呤霉素/杀菌素浓度来选择感染细胞。

The aim of this project (part 1) was to identify the genetic signature that predict the progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast in clinical specimens. From the interim analysis of 236 specimens, we confirmed that the major genetic mutations such as PIK3CA, AKT1, and TP53 were consistently detected as previously reported in the TCGA dataset. In 2020, we collected more data from clinical sequencing and the cohort now includes total 313 specimens (252 from IDC and 61 from DCIS). Thus, we are updating the analysis of the entire cohort and attempt to identify the candidate genes responsible for progression from DCIS to IDC even in less frequently mutated genes.The aim of this project (part 2) was to describe the molecular mechanism of the breast cancer progression in the model. We obtained cell lines such as MCF10A and MCF10DCIS as models of normal epithelia and DCIS tumor in the breast, respectively. We optimized the conditions for CRISPR/Cas9 screen for both of the cell lines. The optimization included the identification of accurate doubling time of the cell lines, spinfection conditions such as cell density and polybrene concentration and appropriate puromycin/blasticidin concentrations to selected the infected cells.