Harnessing the chronic inflammatory environment in non-small cell lung cancer for biomarker discovery

Vannitamby, A 2019, Harnessing the chronic inflammatory environment in non-small cell lung cancer for biomarker discovery, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Harnessing the chronic inflammatory environment in non-small cell lung cancer for biomarker discovery
Author(s) Vannitamby, A
Year 2019
Abstract Lung cancer is the leading cause of cancer-related deaths and has one of the lowest overall survival rates. The high mortality rate can be attributed to the fact that a majority of lung cancer patients are diagnosed with advanced disease. Low-dose Computed Tomography (LDCT) screening of high-risk individuals is emerging as a useful tool for the early detection of lung cancer. Subsequent bronchoscopic sampling of the suspect site is required for a tissue-based diagnosis. Almost 40% of patients that are unsuitable for surgical resection are diagnosed solely on bronchoscopy-derived cytology specimens without additional biopsy material. These formalin-fixed specimens need to provide sufficient amounts of material to confirm diagnosis, subtype and stage as well as enable molecular testing to guide treatment-based decisions. Effective treatments options for patients with metastatic disease are limited. However, the recent development of targeted therapies against specific genetic alterations are improving clinical outcomes in a subset of patients with advanced lung adenocarcinoma. Immunotherapies such as the Programmed Death 1 (PD-1)/Programmed Death Ligand 1 (PD-L1) inhibitors are also improving progression-free survival in non-small cell lung cancer (NSCLC) patients irrespective of the subtype.

PD-L1/PD1 blockade boosts T cell activation and killing of tumour cells. Scoring of PD-L1 immunohistochemical staining is the only biomarker used to identify patients that are more likely to benefit from immunotherapies as first line (>50 % PD-L1 tumour cell staining) or second line treatment (>1% PD-L1 tumour cell staining). Whilst immunotherapy has improved survival rates in NSCLC patients with elevated PD-L1 expression, this response rate does not exceed 25-30%. Hence alternative therapeutic targets are needed to further improve survival rates.  The chronic inflammatory environment has been previously identified as enabling tumour development. Patients with the chronic obstructive pulmonary disease (COPD) are six times more likely to develop lung cancer, yet mechanisms underlying this increased risk have not been fully elucidated. It is known that chronic smokers and people with COPD are more likely to develop Squamous cell carcinoma (SCC).  I hypothesised that the inflammatory microenvironment is distinct between the major NSCLC subtypes, which is driven by smoking history and COPD. This has important implications as strategies that target the immune system will be influenced by the function and frequency of immune cells surrounding the tumour.

The role of innate immune cell types including macrophages and neutrophils were investigate using retrospectively-collected NSCLC biopsy specimens from resection surgery patients including adenocarcinoma (n=48) and squamous cell carcinoma (SCC, n=40). In particular, increased neutrophilic inflammation, observed in both COPD and lung cancer, is a major source of the matrix degrading enzyme MMP-9 that contributes to tumour invasion and metastasis. Using RTqPCR profiling of NSCLC tumour biopsies and brushings, I simultaneously profiled expression levels of 24 gene transcripts using low density TaqManTM arrays from a single small tumour biospecimen. I observed that MMP-9 expression was highly increased in NSCLC biopsies and its natural inhibitors TIMP2 and TIMP3 were significantly decreased. With MMP-9 transcription consistently elevated in early stage malignant NSCLC tumours, we demonstrate for the first time the diagnostic potential of the MMP-9:TIMP3 transcript ratio to discriminate malignant and benign EBUS specimens.

Next, I developed a novel digital droplet PCR (ddPCR) assay to evaluate the MMP9:TIMP3 ratio as ddPCR is a more sensitive and reliable PCR platform that is also suitable for multiplexing targets. This allowed for the addition of PD-L1 quantification by ddPCR in the same biospecimen, which revealed high concordance between PD-L1 transcription and PD-L1 tumour cell staining, determined by ddPCR and immunohistochemistry, respectively. Simultaneous assessment of MMP-9, TIMP3 and PD-L1 transcripts may have the potential to improve the molecular diagnosis of NSCLC by reducing the time and cost associated with identifying suitable candidates for PD-1/PD-L1 inhibitors. Based on our data, we proposed an alternative workflow for the preservation of a single-pass EBUS bronchoscopy specimen for molecular analysis. This specimen is not only suitable for multiplexing by ddPCR but is also highly suitable for mutational analysis, as we detected clinically relevant mutations by performing targeted sequencing on the same single cytology specimen. Integration of multiplex ddPCR also outperforms RTqPCR in that it requires less starting material to identify malignant tumour biopsies with elevated PD-L1 expression and can be used to develop new thresholds or cut-offs for diagnosing lung cancer and identify suitable candidates for targeted therapies including immunotherapy. Further investigation is warranted in a larger cohort to establish appropriate thresholds and to determine whether this approach can be applied to all bronchoscopy-directed specimens.

Consistent with recent studies, we observed that neutrophils dominate the immune landscape in primary NSCLC tumours, especially in SCC. In this study, gene expression analysis of a select panel of chemotactic factors suggests that alternative pathways may be involved in driving neutrophilic inflammation in each NSCLC subtype. The prognostic potential of the neutrophil-to-macrophage (NMR) ratio in patients with early stage SCC was identified. This data demonstrated that a high NMR was prognostic of increased survival, to indicate that neutrophils may be protective in early stage SCC, but not adenocarcinoma. A limitation of this study is that factors responsible for driving increased neutrophilic infiltration in SCC was not yet established. Therefore, further studies are needed to evaluate the role of alternative neutrophilic chemotactic factors, including matrikines, in SCC.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Respiratory Diseases
Keyword(s) lung cancer
EBUS Bronchoscopy
molecular profiling
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Created: Wed, 02 Oct 2019, 13:44:54 EST by Keely Chapman
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