Mutational Analysis of Uterine Cervical Cancer That Survived Multiple Rounds of Radiotherapy

学位記番号：医博甲175

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer.

Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in >2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R2 = 0.72, p < 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9-3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22-1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors

Incorporating Genetic Biomarkers into Predictive Models of Normal Tissue Toxicity.

There is considerable variation in the level of toxicity patients experience for a given dose of radiotherapy, which is associated with differences in underlying individual normal tissue radiosensitivity. A number of syndromes have a large effect on clinical radiosensitivity, but these are rare. Among non-syndromic patients, variation is less extreme, but equivalent to a ±20% variation in dose. Thus, if individual normal tissue radiosensitivity could be measured, it should be possible to optimise schedules for individual patients. Early investigations of in vitro cellular radiosensitivity supported a link with tissue response, but individual studies were equivocal. A lymphocyte apoptosis assay has potential, and is currently under prospective validation. The investigation of underlying genetic variation also has potential. Although early candidate gene studies were inconclusive, more recent genome-wide association studies are revealing definite associations between genotype and toxicity and highlighting the potential for future genetic testing. Genetic testing and individualised dose prescriptions could reduce toxicity in radiosensitive patients, and permit isotoxic dose escalation to increase local control in radioresistant individuals. The approach could improve outcomes for half the patients requiring radical radiotherapy. As a number of patient- and treatment-related factors also affect the risk of toxicity for a given dose, genetic testing data will need to be incorporated into models that combine patient, treatment and genetic data.NGB is supported by the NIHR Cambridge Biomedical Research Centre.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.clon.2015.06.01

Predictive biomarkers in radioresistant rectal cancer:A systematic review

Background and aims: The treatment of locally advanced rectal cancer often consists of neoadjuvant chemoradiotherapy followed by surgery. However, approximately 15% of patients show no response to this neoadjuvant chemoradiotherapy. This systematic review aimed to identify biomarkers of innate radioresistant rectal cancer. Method: Through a systematic literature search, 125 papers were included and analyzed using ROBINS-I, a Cochrane risk of bias tool for non-randomized studies of interventions. Both statistically significant and nonsignificant biomarkers were identified. Biomarkers mentioned more than once in the results or biomarkers with a low or moderate risk of bias were included as the final results. Results: Thirteen unique biomarkers, three genetic signatures, one specific pathway, and two combinations of two or four biomarkers were identified. In particular, the connection between HMGCS2, COASY, and PI3K-pathway seems promising. Future scientific research should focus on further validating these genetic resistance markers

Radiation Response Biomarkers for Individualised Cancer Treatments

Personalised medicine is the next step in healthcare, especially when applied to genetically diverse diseases such as cancers. Naturally, a host of methods need to evolve alongside this, in order to allow the practice and implementation of individual treatment regimens. One of the major tasks for the development of personalised treatment of cancer is the identification and validation of a comprehensive, robust, and reliable panel of biomarkers that guide the clinicians to provide the best treatment to patients. This is indeed important with regards to radiotherapy; not only do biomarkers allow for the assessment of treatability, tumour response, and the radiosensitivity of healthy tissue of the treated patient. Furthermore, biomarkers should allow for the evaluation of the risks of developing adverse late effects as a result of radiotherapy such as second cancers and non-cancer effects, for example cardiovascular injury and cataract formation. Knowledge of all of these factors would allow for the development of a tailored radiation therapy regime. This Special Issue of the Journal of Personalised Medicine covers the topic of Radiation Response Biomarkers in the context of individualised cancer treatments, and offers an insight into some of the further evolution of radiation response biomarkers, their usefulness in guiding clinicians, and their application in radiation therapy

Predictive Biomarkers of Cellular Radiosensitivity for Clinical Radiotherapy Treatment

Radiotherapy is prescribed to more than 50% of cancer patients during their treatment schedule. Due to intrinsic factors, individual variation in response exists, which will result in side effects or toxicity in a number of patients. Therefore, development of an assay or biomarker for the prediction and assessment of radiosensitivity among cancer patients undergoing radiotherapy would be beneficial. Such an assay would limit toxicities and facilitate dose-escalation for those patients who require it. Assays for predicting intrinsic cellular radiosensitivity remains as the established G2 chromosomal radiosensitivity and most promising, γH2AX foci assay. They can be applied to blood samples from donors and are sensitive enough to detect individual radiosensitivity. Therefore, both assays were applied to normal control cohorts compared to ‘radiosensitive’ cells to assess their efficacy as potential predictive assays for the clinic. Different low doses and energies of Linac radiation was applied to cells to assess their impact on patient intrinsic radiosensitivity and the most radiosensitive dose was confirmed at 0.5Gy (6MV photon beam) Linac radiation in cells. In addition to this, intrinsic radiosensitivity which could be measured at initial diagnosis and treatment planning stages for patients was investigated. The assays were applied to patients sampled at various time-points throughout a course of their radiotherapy treatment. The time-points included pre-treatment, post hormone treatment, last day of radiotherapy and the 2 and 8 month follow ups. Both assays were capable of depicting a dose response and differences between treatment visits. The DNA damage based assays indicated that cell cycle regulation through the DNA damage response (DDR) activated by radiation was central to the underlying mechanistic response. Therefore, the molecular mechanisms of radiosensitivity were studied with an emphasis of genes related to the cell cycle and DDR. Furthermore, genetic targets that emerged from this work could potentially be biomarkers of radiosensitivity that could also be incorporated into the clinic. Following from the emergence of cell cycle and DDR genes, potential biomarkers for predicting radiosensitivity was analysed in a collaboration with Public Health England. This was done using the most sensitive genes which were found from bio dosimetry microarray studies carried out by the group (P21, PCNA, SESN1 and FDXR). Again this work was done on blood from healthy controls, prostate cancer patients and radiosensitive cells from Ataxia Telangiectasia donors. The genes in combination were able to depict a clear difference in the cohorts analysed in which expression was collectively highest in the healthy controls, less expression was observed in the Prostate cancer cohort and the least expression was observed in the radiosensitive cells from Ataxia Telangiectasia donors. Finally, investigation of the miRNA composition of exosomes in healthy cells and cells from Ataxia telangiectasia donors was done to identify novel biomarkers for radiosensitivity prediction, in a collaboration with Trinity College Dublin. A subgroup of the let-7 family of miRNA’s was among the top 88 expressed miRNA’s in this chapter. Additionally, most miRNA’s were not as highly expressed in radiosensitive cells compared to normal healthy cells. This work forms the basis for future work on prostate cancer patient samples

Chromosomal radiosensitivity and instability in triple negative and/or young breast cancer and Fanconi Anaemia patients in South Africa

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, and the Faculty of Medicine and Health Sciences, Ghent University, Belgium in fulfilment of the requirements for the joint degree of Doctor of Philosophy / Doctor in Health Sciences Johannesburg, 2018.Introduction: Breast cancer is the leading cancer in women in South Africa (SA). Triple negative breast cancer (TNBC) is clinically characterised by the lack of expression of estrogen, progesterone and HER2/NEU receptors. These breast cancers occur frequently in young African women and are associated with aggressive disease progression, poor prognosis and BRCA1 mutations. TN patients with operable tumours may undergo surgery under general anaesthetics. Treatment of TNBC poses a clinical challenge as these tumours are unresponsive to hormonal or HER2 targeted therapy. Defects in BRCA1 and other DNA repair genes contribute to chromosomal instability and radiosensitivity and cause irregularities in the cell cycle checkpoints in the S/G2 phase. Studies have shown the overlap of breast cancer susceptibility genes and Fanconi Anaemia (FA) genes. FA is an autosomal recessive disorder defined by cellular hypersensitivity to DNA cross-linking agents such as mitomycin C (MMC) and defects in DNA repair genes. FA patients are known to be radiosensitive and have defects with DNA repair. These patients are at high risk to develop leukaemia and solid tumours that may require radiotherapy. Diagnosis of FA patients often includes detecting chromosomal aberrations induced by a cross-linking agent. Molecular tests are also conducted to identify mutations in FA genes. It has previously been shown that FA patients undergoing radiotherapy display increased clinical radiosensitivity. Evidence suggests that FA patients are chromosomally radiosensitive to ionising radiation (IR). Chromosomal radiosensitivity can be evaluated using the cytokinesis-block micronucleus (CBMN) assay in different phases of the cell cycle. Micronuclei (MNi) serve as biomarkers for radiation-induced DNA damage repair and defects in DNA repair mechanisms can be reflected in chromosomal radiosensitivity. A number of factors could influence the MNi yield such as storage time and temperature, and cytotoxic agents such as anaesthetics. As radiotherapy is considered a principle treatment in the management of TNBC, it is important to investigate in vitro chromosomal radiosensitivity of South African TN breast cancer patients. Chromosomal instability and radiosensitivity of FA patients has previously not been investigated in SA. The overall aim of this study was to investigate chromosomal instability and radiosensitivity of lymphocytes in South African breast cancer patients, FA patients and parents compared to healthy individuals using the G0 and S/G2 CBMN assay. The effect of age, ethnicity and mutations in breast cancer susceptibility genes was also investigated. Furthermore, storage time and effect of anaesthetics on MNi yield was investigated. Methods: For the G0 MN assay, heparinised blood in culture medium was irradiated at 0Gy (Baseline), 2 and 4 Gy followed by the immediate stimulation of lymphocytes using phytohaemagglutinin (PHA). Cytochalasin B was added 23 hours later to inhibit cell division. The S/G2 MN assay is a modified version of the G0 MN assay. In this assay, the cultures are first stimulated with PHA and irradiated 72 hours post stimulation. Eight hours post irradiation cells were fixed. The Mitomycin C (MMC) MN assay is similar to the G0 MN assay except the DNA damage is induced using MMC. Results: Chromosomal instability is significantly elevated in TNBC, young and older breast cancer patients. Radiation-induced MN values in the G0 MN assay are significantly enhanced in a total unselected group of breast cancer patients compared to healthy individuals. However, when subdividing the breast cancer patients in a TNBC group, the enhanced radiation-induced MNi are not observed. We cannot demonstrate a correlation between the age of the patients and chromosomal radiosensitivity but an effect of ethnicity is noted in our breast cancer population. In the S/G2 MN assay, TNBC patients continued to exhibit a decreased chromosomal radiosensitivity. We also demonstrated that increased storage time can influence MNi yields in patients and controls; anaesthetics influenced spontaneous MNi yields. The FA patients in our study demonstrate higher MNi when compared to parents and controls indicating chromosomal instability and chromosomal radiosensitivity in the G0 as well as in the S/G2 phase of the cell cycle. This is not seen in the FA heterozygotes. With the MMC assay, the detection of significantly higher MN is noted in as well the FA patients as well as the FA carriers. Conclusions: Chromosomal instability and radiosensitivity of breast cancer and FA patients are notably higher when compared to healthy individuals. The association of BRCA mutations in TN and young patients highlight the importance of radiosensitivity information in the understudied SA population. FA carriers can be at risk for breast cancer with mutations associated with breast cancer susceptibility genes. As a functional assay, the MMC MN assay will be useful in the identification of FA carriers who may be at risk of breast cancer. Data on radiosensitivity of patients with defects in DNA repair genes could provide important information for radiotherapy management of cancer.LG201