Thoracic malignancies, in particular non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM), are a major cause of morbidity and mortality in both developed and developing nations. A barrier to improving the clinical management of patients with thoracic malignancies is a lack of clinically applicable diagnostic, predictive and prognostic biomarkers. The overall aim of my thesis was to study biomarkers (predictive and prognostic for NSCLC and diagnostic for MPM), with the expectation that the incorporation of the biomarkers into clinical practice will translate into better outcomes for patients with NSCLC and MPM.
A molecular biomarker that has dramatically altered the management of patients with advanced stage, NSCLC is activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene. However, the role of screening for EGFR mutations in surgically resected NSCLC is less well understood. A limitation to the universal screening for EGFR mutations of NSCLC tumours is that the gold standard technique for EGFR mutation detection (DNA sequencing) has a relatively low limit of detection. The validation of an EGFR mutation test that can accurately detect mutations in tumour tissue and low-tumour content cytological specimens (e.g. serum) would be beneficial to all lung cancer patients. I performed a retrospective study using lung tumour DNA from 522 surgically resected stage I–IV NSCLCs and matched serum DNA from a subset of 64 subjects and analysed them for EGFR mutations using High-Resolution Melt (HRM) analysis and Mutation Enrichment PCR (ME-PCR), two EGFR mutation tests with high limits of mutation detection. I found that in the subset of 97 subjects examined by all three methods, compared to DNA Sanger sequencing, ME-PCR had a sensitivity of 100% and specificity of 99% while HRM had 100% sensitivity and specificity. Among the subjects with TNM pathological stage I, II and IIIA, there was no difference in survival according to EGFR mutation status (Kaplan-Meier log rank analysis, p = 0.951).
Early stage NSCLC is an indication for performing curative intent surgical resection. However, despite undergoing surgical resection, 40% of patients will have a recurrence of their primary tumour. Currently, there are no biomarkers available to identify patients at risk of recurrence. In order to identify a prognostic molecular biomarker for patients with surgically resected early stage NSCLC (TNM pathological stage I, II and IIIA), I used data from array comparative genomic hybridization (aCGH) experiments from the two most common histological subtypes of NSCLC, Squamous Cell Carcinoma (SCC) and Adenocarcinoma (ADC) to identify genes affected by copy number alterations and which are associated with recurrence. The study cohort consisted of a training set of 56 primary lung SCCs (24 with recurrence and 32 with non-recurrence) and 66 primary lung ADCs (32 with recurrence and 34 with non-recurrence). In the primary lung SCC tumours, the copy number loss of the SOCS6 gene within 18q22.3 was associated with recurrence and this finding was technically and biologically replicated. SOCS6 copy number loss correlated with reduced mRNA expression in the study samples. Decreased SOCS6 qPCR copy number (‘SOCS6 loss’) was associated with worse survival compared to subjects without ‘SOCS6 loss’ (Kaplan-Meier log rank analysis, p = 0.009) and the worse survival was present even after correction of TNM stage. This finding was replicated in the test set subjects as well. A similar biomarker could not be identified in primary lung ADCs. While the copy number loss of 16q21 occurred more frequently in the recurrence ADC tumours, there were no differences in the individual genes contained within these regions.
The majority of patients with MPM present with a pleural effusion. However establishing the diagnosis in patients with pleural effusions can sometimes be difficult. The sensitivity of pleural fluid cytology is 30% for mesothelioma. There is a need for a biomarker that can complement cytology in the diagnosis of malignant pleural effusions (MPE) and MPM. The cell-free DNA (cfDNA) integrity index, measured as the ratio of longer to shorter DNA fragments, has been shown to be higher in the blood of patients with solid organ malignancies compared to normal individuals. I studied matched pleural fluid and serum samples from 75 subjects with pleural effusions. Pleural fluid and serum cfDNA ALU DNA repeats (115 bp, 247 bp and 247 bp/115 bp ratio [cfDNA integrity index]) were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were also quantified using ELISA. The pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs 0.8; p <0.001). The combination of cytology and pleural fluid DNA integrity index in combination provided 81% sensitivity and 87% specificity in identifying MPEs. In the detection of the mesothelioma subset, the pleural fluid DNA integrity index had a sensitivity of 75% and specificity of 90%. This biomarker could guide the decision to undertake more definitive testing, i.e. thoracoscopy, in establishing the diagnosis of MPM.