2018RakhitCPhD.pdf (6.52 MB)
Circulating DNA for the in vivo detection and monitoring of lung cancer
thesisposted on 2018-06-21, 09:25 authored by Callum Paul Rakhit
Lung cancer is the most lethal cancer globally, in part because it is often diagnosed at an incurable stage. Furthermore, limited options exist for effective patient treatment stratification within the disease. Circulating cell free (cf) DNA provides an accessible non-invasive source of patient tumour DNA, potentially useful for the detection of early stage lesions, or for identifying biomarkers predictive of response to treatment in more advanced disease. The molecular chaperone Heat Shock Protein 90 (HSP90) is a promising therapeutic target in cancer, though predictive biomarkers remain elusive. Using cfDNA it was possible to identify genetic alterations predictive of patient survival in GALAXY-1, a clinical trial designed to assess the efficacy of the HSP90 inhibitor ganetespib. It was also found that low cfDNA concentration was prognostic of improved survival, and a high somatic tumour burden predicted improved survival under the treatment arm containing ganetespib. Furthermore, using FFPE samples it was also possible to identify somatic copy number alterations (SCNAs) predictive of survival, and a positive correlation between the number of SCNAs found and worsened survival was observed. Experimental workflows established that peptide nucleic acid (PNA) clamps can improve the sensitivity of mutation detection in next generation sequencing (NGS) workflows using cfDNA. It was also demonstrated that NGS data that is routinely discarded, such as information on synonymous mutations that alter tRNA pools, can give potentially useful information on somatic alterations in a patient’s cancer. Reduced cfDNA integrity was found to be prognostic of worsened survival in the GALAXY-1 patients. To explore the hypothesis that analysis of cfDNA could allow for the identification of early stage preneoplastic lung cancer the genetically engineered KRAS+/LSL-G12D mouse model was utilised. cfDNA levels were found to correlate with tumour burden, with tumour bearing mice having significantly greater levels of cfDNA versus mice without premalignant lesions, and versus earlier samples taken from the same mice before the development of a tumour burden. It was also shown that the recombined KRAS allele could be detected in circulation. Additionally, CCR1 inhibition was demonstrated to have potential therapeutic benefit in the KRASLSL-G12D model, by causing significant alterations in the recruitment of hematopoietic cells to KRAS mutant murine lungs.
Date of award2018-05-04
Author affiliationMRC Toxicology Unit
Awarding institutionUniversity of Leicester