Analytical validation of the LungLB test: a 4-color fluorescence in-situ hybridization assay for the evaluation of indeterminate pulmonary nodules.
Michelle L Lutman, Daniel Gramajo-Leventon, Shahram Tahvilian, Lara Baden, Courtney L Gilbert, Michael Trejo, Eric Vail, Michael J Donovan, Benjamin A Katchman, Paul C Pagano
Author Information
Michelle L Lutman: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Daniel Gramajo-Leventon: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Shahram Tahvilian: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Lara Baden: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Courtney L Gilbert: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Michael Trejo: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Eric Vail: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Michael J Donovan: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Benjamin A Katchman: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA.
Paul C Pagano: LungLife AI, Inc., 2545 W. Hillcrest Drive, Suite 140, Thousand Oaks, CA, 91320, USA. ppagano@lunglifeai.com.
BACKGROUND: Evaluation of indeterminate pulmonary nodules (IPNs) often creates a diagnostic conundrum which may delay the early detection of lung cancer. Rare circulating genetically abnormal cells (CGAC) have previously demonstrated utility as a biomarker for discriminating benign from malignant small IPNs in the LungLB assay. CGAC are identified using a unique 4-color fluorescence in-situ hybridization (FISH) assay and are thought to reflect early cell-based events in lung cancer pathogenesis and the anti-tumor immune response. LungLB is a prognostic tool that combines the CGAC biomarker and clinical features to aid in IPN evaluation by improving the stratification of patient risk of malignancy. METHODS: Herein we describe the analytical performance of the LungLB blood test. Analytical validation was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with adaptations for rare cell-based assays. Multiple operators, reagent lots, and assay runs were tested to examine accuracy, precision, reproducibility, and interfering factors. RESULTS: The FISH probes used in the LungLB assay demonstrate 100% sensitivity and specificity for their intended chromosomal loci (3q29, 3p22.1, 10q22.3 and 10cen). LungLB demonstrates analytical sensitivity of 10 CGAC per 10,000 lymphocytes analyzed, 100% analytical specificity, and high linearity (R = 0.9971). Within run measurements across 100 samples demonstrated 96% reproducibility. Interfering factors normally found in blood (lipemia, biotin) and exposure to adverse temperatures (-20ºC or 37ºC) did not interfere with results. Sample stability was validated to 96 hours. CONCLUSION: The analytical performance of LungLB in this validation study successfully demonstrates it is robust and suitable for everyday clinical use.
