Discovery of biomarkers in malignant pleural mesothelioma

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive cancer that occurs in the pleura, a thin membrane that lines the lungs and the chest cavity. It is associated with exposure to asbestos. MPM is considered a rare cancer; however, it has one of the poorest survival rates of any cancer in the world, with the median survival ranging from a few months to a couple of years. There is currently an urgent clinical need for biomarkers that can help with early diagnosis, improve prognostication, and help develop targeted therapies for MPM.

In the first study, the expression of BIN1, IDO1 and PD-L1, and the presence of T-cells (CD3+), B-cells (CD20+) and macrophages (CD68+), was evaluated by immunohistochemistry in tumour samples from 67 patients who underwent surgical resection between 1992 and 2007. IDO1 was positive for 20 patients (30%) and BIN1 expression was high for 35 patients (50%). Survival analyses revealed that high BIN1 expression was associated with better overall survival (median: 12 vs 6 months for high and low BIN1 respectively, p=0.03). However, neither IDO1 expression, nor the numbers of lymphocytes and macrophages were significantly associated with overall survival. Multivariate analysis showed BIN1 remained an independent prognostic indicator (HR 0.44; 95% CI: 0.23-0.83, p=0.01). There was a significant correlation between PD-L1 and CD3 in stroma cells (p=0.02), CD3+ in tumour cells (p=0.01) and CD68+ in tumour cells (p=0.004). This study is the first to demonstrate that high BIN1 expression is a favourable prognostic biomarker in MPM.

In the second study, the efficacy and toxicity of pembrolizumab, an anti-PD-1 checkpoint inhibitor antibody, was evaluated in a real-world Australian population. Clinical information was collected retrospectively and tumour biomarkers including PD-L1, BAP1 and tumour infiltrating lymphocytes (TILs; CD3+) were examined for their predictive potential using archival formalin-fixed paraffin-embedded tissues. Several clinical factors were also analysed for their potential to predict outcomes from pembrolizumab. The study demonstrated that single agent pembrolizumab is well tolerated by patients and is effective in a small proportion of patients, with an overall response rate of 18%. Traditional performance status assessment was the most robust predictor for patient outcome, with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 associated with better overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Clinical factors that predicted for longer survival included the lack of corticosteroids pre-treatment, non-elevated absolute platelet count, onset of immune-related adverse events and PD-L1 expression. TILs in the tumour were significantly associated with overall response rate, suggesting it may help select patients who are likely to respond to pembrolizumab. BAP1 loss was not significantly associated with any efficacy outcomes. Our study included 98 patients, representing the largest retrospective study of outcomes in patients receiving pembrolizumab outside of a clinical trial to date.

The final study involved laboratory research to investigate the role of tumour-derived extracellular vesicles (EV) as potential biomarkers in MPM. EV are 30-1000 nm membranous particles secreted by all cells (including cancerous cells) and carry various cargo containing proteins, lipids, and genetic materials from one cell to another. The availability and accessibility of EV in all body fluids make them a great candidate for liquid biopsies. EV was isolated from five MPM cell lines, an immortalized mesothelial cell line, and patient plasma samples. Three main subtypes of EV (exosomes, microvesicles and large oncosomes) were characterised using nanoparticle tracking analysis, flow cytometry, western blotting, transmission electron microscopy and proteomics. Major differences were found in the cargo between the different EV subtypes in all MPM cell lines. This study demonstrated, for the first time, the presence of PD-L1 and mesothelin in distinct subtypes of EV, opening a novel pathway to non-invasive or minimally invasive biopsies for the identification of biomarkers in MPM.

In summary, this discovery project has identified multiple candidate biomarkers for MPM and laid the foundation for the development of future novel therapeutic targets that may significantly improve the clinical management of MPM.