A Novel Approach to Detect Programed Death Ligand 1 (PD-L1) Status and Multiple Tumor Mutations Using a Single Non–Small-Cell Lung Cancer (NSCLC) Bronchoscopy Specimen

Vannitamby, Amanda; Hendry, Shona; Makadia, Tanvi H.; Danks, Janine A.; Slavin, John; Irving, Louis; Steinfort, Daniel P.; Bozinovski, Steven

doi:10.1016/j.jmoldx.2018.10.001

Cited by 11 publications

(24 citation statements)

References 26 publications

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“…We have recently taken an alternative approach to determine PD-L1 expression in small biopsy specimens using quantitative reverse transcription PCR (RTqPCR) [3]. Utilising minimally-invasive bronchoscopy and archived biopsy specimens, we identified the MMP9:TIMP3 mRNA ratio as a molecular marker for presence of malignant cells in NSCLC [3]. We also quantified PD-L1 gene expression by RTqPCR and demonstrated excellent concordance with the Ventana PD-L1 (SP263) Assay [3].…”

Section: Introductionmentioning

confidence: 89%

“…Matching snap frozen and FFPE specimens were provided by the Victorian Cancer Biobank with ethics approval (Ethics ID: SEHAPP 09-17) as previously detailed [3]. The specimens were obtained from surgically resected tumour tissue from patients with stage IA-IIIA NSCLC (n = 88) at the Royal Melbourne Hospital pathology department and included adenocarcinoma (n = 48) and squamous cell carcinoma (n = 40) subtypes.…”

Section: Patient Characteristicsmentioning

confidence: 99%

“…A 1-2 cm sample of tumour tissue excess to diagnostic requirements was obtained from the fresh resection specimen and bisected with one piece snap frozen and one piece formalin fixed and paraffin embedded as per standard laboratory protocols. Control tissue resection specimens (n = 20) were obtained from patients without malignant disease (n = 10) and adjacent tumour-free region of adenocarcinoma patients (n = 10), as previously described [3].…”

Section: Patient Characteristicsmentioning

confidence: 99%

“…Hence, the assessment of PD-L1 expression is dependent on how much FFPE tumour tissue is left and the integrity of this fixed tissue specimen. We have recently taken an alternative approach to determine PD-L1 expression in small biopsy specimens using quantitative reverse transcription PCR (RTqPCR) [3]. Utilising minimally-invasive bronchoscopy and archived biopsy specimens, we identified the MMP9:TIMP3 mRNA ratio as a molecular marker for presence of malignant cells in NSCLC [3].…”

Section: Introductionmentioning

confidence: 99%

“…Utilising minimally-invasive bronchoscopy and archived biopsy specimens, we identified the MMP9:TIMP3 mRNA ratio as a molecular marker for presence of malignant cells in NSCLC [3]. We also quantified PD-L1 gene expression by RTqPCR and demonstrated excellent concordance with the Ventana PD-L1 (SP263) Assay [3]. In this study, we have significantly advanced our approach by https://doi.org/10.1016/j.lungcan.2019.06.029 developing a droplet digital PCR (ddPCR) assay to accurately and rapidly assess multiple biomarkers within the same specimen.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Introductionmentioning

confidence: 89%

Section: Patient Characteristicsmentioning

confidence: 99%

Section: Patient Characteristicsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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