Heterogeneity of tumour mutational burden in metastatic NSCLC demonstrated by endobronchial ultrasound sampling.

Introduction: Tumour mutational burden (TMB) is an important emerging biomarker for immune checkpoint inhibitors (ICI). The stability of TMB values across distinct EBUS tumour regions is not well defined in advanced lung cancer patients. Methods: This study included a whole-genome sequencing cohort (n=11, LxG cohort) and a targeted Oncomine TML panel cohort (n=10, SxD cohort), where paired primary and metastatic samples were obtained by endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA). Results: The LxG cohort displayed a strong correlation between the paired primary and metastatic sites, with a median TMB score of 7.70 ± 5.39 and 8.31 ± 5.88 respectively. Evaluation of the SxD cohort demonstrated greater inter-tumoural TMB heterogeneity, where Spearman correlation between the primary and metastatic sites fell short of significance. Whilst median TMB scores were not significantly different between the two sites, 3 out of 10 paired samples were discordant when using a TMB cut-off of 10 mutations per Mb. In addition, PD-L1 copy number and KRAS mutations were assessed, demonstrating the feasibility of performing multiple molecular tests relevant to ICI treatment using a single EBUS sample. We also observed good consistency in PD-L1 copy number and KRAS mutation, where cut-off estimates were consistent across the primary and metastatic sites. Conclusions: Assessment of TMB acquired by EBUS from multiple sites is highly feasible and has the potential to improve accuracy of TMB panels as a companion diagnostic test. We demonstrate similar TMB values across primary and metastatic sites, however 3 out of 10 samples displayed inter-tumoural heterogeneity that would alter clinical management.

Programmed Death-Ligand 1 Copy Number Loss in NSCLC Associates With Reduced Programmed Death-Ligand 1 Tumor Staining and a Cold Immunophenotype.

INTRODUCTION: Programmed death-ligand 1 (PD-L1) copy number gains may be predictive of clinical response to immunotherapy in NSCLC. This study investigated PD-L1 copy number variations in tumor resection and bronchoscopy biopsies and its relationship with PD-L1 tumor cell staining and inflammatory gene expression. METHODS: PD-L1 gene copy number and mRNA expression were evaluated by real-time polymerase chain reaction in surgically resected NSCLC tumor biopsies (n = 87) and control biopsies (n = 20). A second cohort (n = 15) of bronchoscopy-derived tumor biopsies was analyzed, including multiple biopsies from the same patient across different anatomical sites. RESULTS: PD-L1 mRNA levels strongly correlated with PD-L1 tumor staining (r = 0.55, p < 0.0001). Interferon-γ mRNA expression associated with PD-L1 immune cell staining, but not PD-L1 tumor cell staining. In contrast, PD-L1 copy number positively associated PD-L1 tumor staining, but not PD-L1 immune cell staining. PD-L1 copy number analysis detected loss (15 of 87 = 17%) and gain (5 of 87 = 7%) of copy number. Tumors with low PD-L1 copy number expressed significantly reduced levels of inflammatory (interferon-γ, interleukin [IL]-6, IL-1ß, MMP-9) and immunosuppressive (IL-10, transforming growth factor ß) mediators. Analysis of bronchoscopy-derived biopsies revealed low heterogeneity in copy number values across different anatomical sites, in contrast to more variable PD-L1 mRNA expression. CONCLUSIONS: Low PD-L1 copy number tumors display reduced PD-L1 expression, reduced PD-L1 tumor cell staining, and an immunologic cold tumor microenvironment. Because PD-L1 copy number values are highly stable across different tumor regions, its evaluation may represent a robust and complimentary biomarker for predicting response to immunotherapy, where low copy number may predict lack of response.

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

Immunotherapy has transformed treatment of advanced non-small-cell lung cancer (NSCLC) patients leading to remarkable long-term survival benefit. However, only about 20% of advanced NSCLC patients typically respond to immune checkpoint inhibitors (ICIs) that target the PD-1/PD-L1 pathway. The only validated biomarker for ICI therapy is the PD-L1 immunohistochemistry (IHC) test, which is considered an imperfect assay due to several variables including availability and integrity of tumour tissue, variability in staining/scoring techniques and heterogeneity in PD-L1 protein expression within and across tumour biopsies. Herein, we discuss integrating minimally invasive EBUS bronchoscopy procedures with novel molecular approaches to improve accuracy and sensitivity of PD-L1 testing. EBUS guided bronchoscopy facilitates repeated sampling of tumour tissue to increase the probability of detecting PD-L1 positive tumours. Since intra-tumoural PD-L1 (CD274) copy number is reported to be less heterogeneous than PD-L1 protein detection, quantifying PD-L1 transcript levels may increase detection of PD-L1 positive tumours. PD-L1 transcript levels show excellent concordance with PD-L1 IHC scoring and multiplex digital droplet PCR (ddPCR) assays that quantify absolute PD-L1 transcript copy number have been developed. ddPCR can also be automated for high throughput detection of low abundant variants with excellent sensitivity and accuracy to improve the broader application of diagnostic cut-off values. Optimizing diagnostic workflows that integrate optimal EBUS bronchoscopy procedures with emerging molecular ICI biomarker assays may improve the selection criteria for ICI therapy benefit.