TECHNICAL ADVANCE A Novel Approach to Detect Programed Death Ligand 1 (PD-L1) Status and Multiple Tumor Mutations Using a Single NoneSmall-Cell Lung Cancer (NSCLC) Bronchoscopy Specimen Amanda Vannitamby,* Shona Hendry, y Tanvi Makadia, z Janine Danks,* John Slavin, y Louis Irving, x Daniel Steinfort, x and Steven Bozinovski* From the School of Health & Biomedical Sciences,* and the College of Science, Engineering and Health, z Royal Melbourne Institute of Technology University, Bundoora; the Department of Anatomical Pathology, y St. Vincent’s Hospital, Melbourne; and the Department of Respiratory Medicine, x Royal Melbourne Hospital, Melbourne, Victoria, Australia CME Accreditation Statement: This activity (“JMD 2019 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the accreditation requirements and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint providership of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity (“JMD 2019 CME Program in Molecular Diagnostics”) for a maximum of 18.0 AMA PRA Category 1 Credit(s) ä . Physicians should claim only credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Accepted for publication October 12, 2018. Address correspondence to Steven Bozinovski, Ph.D., RMIT University, PO Box 71, School of Health & Biomedical Sciences, Building 223, Level 2, Office 13A, Bundoora, Victoria 3083, Australia. E-mail: steven. bozinovski@rmit.edu.au. Multiple biomarkers are under evaluation to guide the use of immune checkpoint inhibitors in nonesmall- cell lung cancer (NSCLC), including programed death ligand 1 (PD-L1) tumor cell staining. We have developed a new approach that accurately quantifies PD-L1 status and identifies multiple mutations by using a single bronchoscopy specimen. A novel molecular marker was identified to detect the presence of malignant cells in radial endobronchial ultrasound bronchial brushings from NSCLC (n Z 15) and benign (n Z 13) nodules by quantitative real-time RT-PCR (RT-qPCR). The MMP9:TIMP3 transcript ratio was significantly increased in NSCLC and using receiver operating characteristic curve analysis accurately discriminated malignant and benign bronchoscopy specimens (area under the curve Z 0.98; 95% CI, 0.93e1; P < 0.0001). Utilizing the same specimens, PD-L1 expression and multiple oncogenic mutations were detected by RT-qPCR and next- generation sequencing. A second archive of snap-frozen squamous cell carcinoma (n Z 40) and control (n Z 20) biopsies with matching formalin-fixed, paraffin-embedded slides were used to compare PD-L1 status by immunohistochemistry and RT-qPCR. The biopsy cohort confirmed that the MMP-9:TIMP3 ratio was predictive of malignancy and demonstrated that PD-L1 transcript expression was concordant with PD-L1 tumor cell membrane staining in NSCLC (Spearman r Z 0.636, P < 0.0001). This rapid molecular approach can detect malignant cells and using the same single bronchoscopy specimen can generate high-quality unfixed nucleic acid that accurately quantify PD-L1 status and identify multiple oncogenic mutations. (J Mol Diagn 2019, 21: 186e197; https://doi.org/10.1016/j.jmoldx.2018.10.001) Radial probe endobronchial ultrasound (EBUS) is a mini- mally invasive procedure that improves localization of pe- ripheral pulmonary lesions. Cytology samples of the suspect lesion site collected by radial EBUS bronchoscopy provides good diagnostic sensitivity estimated at 73%, 1 whereby histology does not definitely diagnose malignancy in approximately 20% cases. 2,3 When this procedure is com- bined with rapid on-site examination (ROSE) of cytologic samples, shorter procedure times are achieved because of Supported by the National Health and Medical Research Council and the Australian Research Council. Disclosures: None declared. Copyright ª 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0). https://doi.org/10.1016/j.jmoldx.2018.10.001 jmd.amjpathol.org The Journal of Molecular Diagnostics, Vol. 21, No. 2, March 2019 2019 JMD CME Program