School of Health & Biomedical Sciences, RMIT University, Bundoora, Victoria, Australia.
Department of Anatomical Pathology, St Vincent's Hospital, Melbourne, Victoria, Australia.
Lung Cancer. 2019 Aug;134:233-237. doi: 10.1016/j.lungcan.2019.06.029. Epub 2019 Jul 2.
Immune checkpoint inhibitors have become integrated into the clinical management of non-small cell lung cancer (NSCLC). Using RTqPCR, we have previously identified a gene expression panel that detected presence of malignant cells (MMP9:TIMP3 ratio) and quantified PD-L1 transcript levels in small biopsy specimens. However, RTqPCR has diagnostic limitations as it does not generate absolute copy number and is not readily multiplexed. To address this, we have developed a multiplex droplet digital PCR (ddPCR) assay.
Biopsies obtained from NSCLC patients (n = 48 adenocarcinoma and n = 40 squamous cell carcinoma) and control lung biopsy specimens (n = 20) were analysed. Absolute MMP9, TIMP3 and PD-L1 transcript copy numbers were determined within a single assay by multiplex ddPCR using Taqman primers and the QX200 Droplet Digital PCR System.
Using our optimised triplex ddPCR assay, the MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and using a cut-off of >0.028, was 99% (95% CI; 80.5-94.5) sensitive and 80% specific for identifying malignant biopsies. The PD-L1:TIMP3 ratio significantly associated with PD-L1 tumour cell immunohistochemistry staining (r = 0.539, p < 0.0001) and was significantly higher in biopsies with >50% PD-L1 tumour cell staining (p < 0.0001). In summary, a major advantage of our workflow is that it can accurately quantify PD-L1 tumour levels and provide sufficient nucleic acid for screening additional targetable mutations such as EGFR, ALK and ROS1 from a single small biopsy, thereby potentially avoiding the need for re-biopsy. Future studies will need to determine diagnostic ddPCR values that are predictive of clinical response to PD-1/PD-L1 immunotherapy.
免疫检查点抑制剂已成为非小细胞肺癌（NSCLC）临床治疗的一部分。我们之前使用 RTqPCR 确定了一个基因表达谱，该谱可检测到恶性细胞的存在（MMP9:TIMP3 比值）并定量小活检标本中的 PD-L1 转录本水平。然而，由于 RTqPCR 不能生成绝对拷贝数且不易多重化，因此它具有诊断局限性。为了解决这个问题，我们开发了一种多重数字 PCR（ddPCR）检测方法。
分析了 NSCLC 患者（n=48 例腺癌和 n=40 例鳞状细胞癌）和对照肺活检标本（n=20）获得的活检。通过使用 Taqman 引物和 QX200 微滴数字 PCR 系统在单个检测中，通过多重 ddPCR 确定 MMP9、TIMP3 和 PD-L1 转录本的绝对拷贝数。
使用我们优化的三重 ddPCR 检测方法，NSCLC 活检中的 MMP9:TIMP3 比值显著升高，使用>0.028 的 cutoff 值，其对恶性活检的敏感性为 99%（95%CI；80.5-94.5），特异性为 80%。PD-L1:TIMP3 比值与 PD-L1 肿瘤细胞免疫组化染色显著相关（r=0.539，p<0.0001），且在>50%PD-L1 肿瘤细胞染色的活检中显著更高（p<0.0001）。总之，我们的工作流程的一个主要优势是它可以准确地定量 PD-L1 肿瘤水平，并从单个小活检中提供足够的核酸，用于筛选其他可靶向的突变，如 EGFR、ALK 和 ROS1，从而可能避免重新活检的需要。未来的研究需要确定 ddPCR 诊断值，以预测 PD-1/PD-L1 免疫治疗的临床反应。
