Development of a QPCR-Block Assay to Quantify Platinum Drug Adducts In Vitro

Abstract

Cisplatin, oxaliplatin and carboplatin are chemotherapy drugs that are used in the treatment of cancer. Treatment response to the platinum chemotherapy can vary from high, such as with testicular and ovarian cancer, to quite low, such as with lung cancer. Treatment using the platinum chemotherapy drugs can become less effective over time as the patient’s cancer acquires resistance mechanisms, such as improved efflux, DNA repair mechanisms and inactivation of the platinum chemotherapy, preventing the effective treatment of the cancer. Patients can also have intrinsic resistance to platinum chemotherapy, rendering the therapy ineffective from onset. This thesis aimed to investigate possible predictors of patient outcomes to platinum chemotherapy. Firstly, a systematic review was conducted to assess any associations between DNA repair genes and patient outcomes to cisplatin, oxaliplatin, or carboplatin in prospective clinical studies. The systematic review identified no single candidate genes that could confidently predict outcomes in patients treated with platinum drugs. This finding indicates the need for alternative methods of identifying whether patients will respond to platinum chemotherapy. Secondly, a published QPCR-block phenotypic assay was adapted for the assessment of platinum DNA adducts. A novel primer set targeting a 3.3 kb amplicon in the human TP53 gene was designed and optimised. These primers were then assessed for their ability to detect platinum drug DNA adducts in vitro and compared to a 1.6 kb primer set previously optimised for assessment of other DNA damaging drugs. Whilst the 3.3 kb primer set could successfully amplify purified genomic DNA, they failed to replicate DNA in lysate from cultured cells treated with oxaliplatin or carboplatin. However, the published assay, using the 1.6 kb primer set, could both replicate the DNA in cell lysate and detect platinum drug DNA adducts. The results reported in this thesis indicate the need for a paradigm shift away from single candidate gene studies and presents a phenotypic assay that can be further developed to assess DNA damage in cultured cells, and with further work, patient samples.