参考基因是临床测试的重要组成部分，例如液滴数字聚合酶链反应（ddPCR），它们可以测量基因工程细胞中整合载体的拷贝数和临床细胞疗法中重编程细胞中质粒的丢失。应该注意选择参考基因，因为已经发现在不同个体间的基因组片段中，可能存在数千个拷贝数的变异。此外，在肿瘤和其他增生性疾病情况下，同一个人的一部分基因组在患病和健康人的细胞之间的拷贝数也可能大相径庭。本研究的目的是鉴定可以用于转导的嵌合抗原受体T细胞的拷贝数变异分析和使用ddPCR进行诱导多能干细胞中的质粒丢失分析的参考基因。我们使用癌症基因组图谱（TCGA）评估候选参考基因。如果TCGA发现候选基因在癌症中的拷贝数方差较低，则使用ddPCR测量来自健康受试者、癌症细胞系和急性淋巴细胞白血病、淋巴瘤、多发性骨髓瘤和人乳头瘤病毒相关癌症患者的潜在参考基因的拷贝数。除了我们在拷贝数分析中使用的rPP30基因外，TCGA还评估了另外三个候选参考基因。分析结果表明，我们设施目前用细胞疗法治疗的所有癌细胞类型中，这四个基因区域（AGO1，AP3B1，MKL2和rPP30）都没有被扩增或删除。 ddPCR测量的基因AP3B1、AGO1、rPP30和MKL2的拷贝数在健康受试者的细胞中相似。我们发现AGO1在部分临床样本中具有拷贝数变化，而在使用基因工程T细胞疗法治疗的患有癌细胞的患者细胞中，ddPCR测量的AP3B1、MKL2和rPP30基因的拷贝数相似。根据我们目前的结果，AP3B1、MKL2和rPP30三个基因适合用作测量具有急性白血病、淋巴瘤、多发性骨髓瘤和人乳头瘤病毒相关癌症的患者中嵌合抗原受体T细胞的向量拷贝数的参考基因。我们将继续对我们未来的样本进行AGO1的评估。

Reference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people. The purpose of this study was to identify reference genes that could be used for copy number variation analysis of transduced chimeric antigen receptor T cells and for plasmid loss analysis in induced pluripotent stem cells using ddPCR.We used The Cancer Genome Atlas (TCGA) to evaluate candidate reference genes. If TCGA found a candidate gene to have low copy number variance in cancer, ddPCR was used to measure the copy numbers of the potential reference gene in cells from healthy subjects, cancer cell lines and patients with acute lymphocytic leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers.In addition to the rPP30 gene, which we have has been using in our copy number assays, three other candidate reference genes were evaluated using TCGA, and this analysis found that none of the four gene regions (AGO1, AP3B1, MKL2 and rPP30) were amplified or deleted in all of the cancer cell types that are currently being treated with cellular therapies by our facility. The number of copies of the genes AP3B1, AGO1, rPP30 and MKL2 measured by ddPCR was similar among cells from healthy subjects. We found that AGO1 had copy number alteration in some of the clinical samples, and the number of copies of the genes AP3B1, MKL2 and rPP30 measured by ddPCR was similar among cells from patients with the cancer cell types that are currently being treated with genetically engineered T-cell therapies by our facility.Based on our current results, the three genes, AP3B1, MKL2 and rPP30, are suitable for use as reference genes for assays measuring vector copy number in chimeric antigen receptor T cells produced from patients with acute leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers. We will continue to evaluate AGO1 on our future samples.Published by Elsevier Inc.