Novel multiplex droplet digital PCR assay for scoring PD-L1 in non-small cell lung cancer biopsy specimens

Abstract

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. Materials and methods: 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. Results and conclusions: Using our optimised triplex ddPCR assay, the MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and using a cutoff 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 rebiopsy. Future studies will need to determine diagnostic ddPCR values that are predictive of clinical response to PD-1/PD-L1 immunotherapy.

Integrating endobronchial ultrasound bronchoscopy with molecular testing of immunotherapy biomarkers in non-small cell lung cancer

“…We have developed a multiplex ddPCR assay where absolute MMP9, TIMP3 and PD-L1 transcript copy numbers were determined within a single assay using Taqman primers and the QX200 Droplet Digital PCR System (43). The MMP9:TIMP3 ratio was significantly elevated in NSCLC biopsies and was highly sensitive (99%) for discriminating malignant biopsies.…”

Sensitive molecular testing methods can demonstrate NSCLC driver mutations in malignant pleural effusion despite non-malignant cytology

“…Such specimens may be non-diagnostic or not suitable for molecular testing (20,21), due to small specimen volume, or due to presence of a significant proportion of non-malignant cells (22). Molecular testing even on a single bronchial brushing specimen may significantly enhance diagnostic sensitivity of minimally invasive investigation (23,24), though this remains to be examined in future studies.…”

Harmonization of Molecular Testing for Non-Small Cell Lung Cancer: Emphasis on PD-L1

“…Several studies attempted to investigate in parallel the expression of PD-L1 on the level of RNA and protein in cell cultures and tumor tissues. These smallscale studies provided generally encouraging results indicating that the correlation between PD-L1 RNA and protein level does exist (30)(31)(32)(33)(34)(35)(36).…”

“…In particular, PCR-based RNA expression analysis offers better reproducibility, as it evaluates not the quantity of the gene-specific transcript per se, but the ratio between the RNA messages of the gene-target and gene-referee. Furthermore, PCR tests are usually performed in a semi-automated manner, so they are less labor-consuming as compared to morphology-based analyses (30)(31)(32). However, RNA testing has several limitations.…”