Dissertations / Theses on the topic 'Circulating tumour cell clusters'

Metastasis in head and neck cancer patients is responsible for over 50% of deaths. There are currently no tools to identify patients at risk of developing metastasis. Circulating tumour cells (CTC) represent a transient cancer cell population in the blood. In this study, the researcher has developed CTC isolation methodologies and used novel culture formulations to expand patient derived CTCs for therapy testing. Furthermore, the researcher identified biomarkers present on CTCs which could select patients for immunotherapies, a current unmet need. This work sets the foundation for a personalized medicine approach to treating head and neck cancer patients.

There is growing concern about the relevance of epithelial mesenchymal plasticity (EMP) status of primary tumours in influencing their metastatic potential. Circulating tumour cells (CTCs) provide a window into the metastatic process, and molecular characterisation of CTCs could lead to better understanding of the mechanisms involved in the metastatic cascade. This thesis is an investigation of molecular characteristics of EMP in tumours and CTCs using patient-derived xenograft models and patient blood samples. The CTC heterogeneity observed emphasises the complexity in CTC isolation and classification and supports the increasingly recognised importance of the epithelial-mesenchymal hybrid state in cancer progression and metastasis.

Circulating tumour cells (CTCs) have the potential to transform the management of patients with non-small cell lung cancer (NSCLC). The applications of CTCs can identify clinically actionable targets to predict treatment response and to better understand metastasis. CTCs isolated using microfluidics can be used as prognostic indicators of NSCLC as well as characterizing for markers of immunotherapy (PD-L1), molecular targets (ALK, EGFR). Short term cultures were successfully expanded in 9/70 NSCLC patients and cultured for up to 3 months. Optimization of this novel CTC culture model provides opportunity to identify new therapeutics for NSCLC patients in a precision medicine approach.

Lung cancer is among the most prevalent forms of cancer and remains the leading cause of cancer-associated deaths globally. Traditionally, lung cancers are classified as either non-small cell lung cancer (NSCLC) (85%) or small cell lung cancer (SCLC) (15%). About 60% of all cases are diagnosed at an advanced stage, at which the 5-year survival is only 4%. Anti-programmed cell death-1 and its ligand 1 (anti-PD-1/PD-L1) therapies have significantly improved the outcomes for lung cancer patients in recent years. However, prognosis and understanding of an individual patient’s lung cancer are often limited by tumour accessibility. Tissue biopsies are invasive, costly, and technically challenging procedures, posing risks to the patient. Circulating tumour cells (CTCs) are very attractive tumour surrogates that could serve as “liquid biopsy” with the advantage to be a low–to–null invasive and real-time approach compared to conventional tissue biopsies. Increasing evidence suggests that CTCs counts can serve as a prognostic biomarker for lung cancers. Notably, phenotypic, and molecular characterisation of CTCs may offer important clinical information for guiding personalised medicine. The studies in this dissertation assessed the potential of CTCs to provide information that could aid the management of lung cancer patients. We carried out a series of investigations covering a systematic review and meta-analysis of programmed cell death ligand-1 (PD-L1) expression on tumour samples and CTCs, a methodological study to improve phenotypic characterisation of CTCs for PD-L1 expression and its application in the clinical settings, and a study using singlecell genomics to uncover novel subpopulations of CTCs. The first chapter of the thesis includes an introduction to lung cancer and a thorough review of the literature on immunotherapy in lung cancer as well as CTCs. Chapter 2 describes a comprehensive review and meta-analysis of PD-L1 expression on tumour cells in SCLC from 27 studies enrolling a total of 27,292 patients. Our results revealed that the prevalence of PD-L1 expression in SCLC tumour cells was heterogeneous across studies. This heterogeneity was significantly moderated by factors such as cut-off values used for scoring PD-L1 staining by immunohistochemistry, and assessment of PD-L1 staining patterns as membranous and/or cytoplasmic. Following these findings, Chapter 3 covers a study carried out to address the feasibility to quantify PD-L1 expression on CTCs in SCLC patients. We develop an EpCAM targeting magnetic bead-based CTC isolation method as a surrogate for the CellSearch method, as this is the gold standard for CTC enumeration and the most used SCLC CTC isolation platform in the clinical setting. Using our immunomagnetic isolation technique, we compared detection rates of CTCs to those isolated using the microfluidic CTC enrichment device - Parsortix system, which separates cells by size exclusion. Detected CTCs were used to assess PD-L1 expression. We identified a subpopulation of EpCAM-negative SCLC CTCs, indicating that epitope-independent methods can detect additional CTCs missed by EpCAM basedcapture. The study also demonstrated that PD-L1 expression can be quantified on CTCs detected in SCLC patients. In parallel, we questioned whether blood is the alternative for PD-L1 expression in NSCLC patients based on several published studies that have assessed PD-L1 expression on CTCs in NSCLC patients. The review in Chapter 4 indicates that the analysis of PD-L1 on CTCs is feasible and PD-L1 expression could be detected before and after first-line therapy. However, there was limited evidence of whether PD-L1 expression on CTCs could predict response to anti-PD-1/PDL1 treatment. Chapter 5 describes a study in NSCLC patients to improve the detection of relevant CTC phenotypes and interrogate them for PD-L1 expression. We simultaneously identified circulating cells with epithelial origin and cells with mesenchymal features in patients with NSCLC by combining the Parsortix system with a modified sequential fluorescent quenching and restaining protocol. Nevertheless, none of the detected circulating cells expressed PD-L1 protein. Furthermore, a subset of mesenchymal-featured cells was confirmed as cancer cells via whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) which revealed copy number alterations (CNAs) in several genomic regions. Lastly, the general discussion underscores how specific CTCs enrichment techniques are required for lung cancers according to their phenotypic characteristics. The results question the potential of CTCs for evaluating PD-L1 expression and the need for systematic clinical validation. Finally, the prospect of CTC genomic analysis is highlighted as it provides an opportunity to timely recognise patients harbouring deleterious alteration and new treatment targets. We conclude by proposing future directions building upon the findings presented in this thesis.

There is a need for informative biomarkers in localised urological cancers. At present, no method can accurately distinguish between indolent and aggressive prostate cancers, and men often require repeated biopsies. Patients with muscle invasive bladder cancer undergo neo-adjuvant chemotherapy (NAC) to improve survival. However many do not respond to NAC, delaying definitive treatment. Cell-free mutant DNA (mutDNA) analysis represents an opportunity for non-invasive monitoring of cancer through tumour genome analysis. MutDNA derived from plasma can monitor tumour burden. There is emerging evidence that mutDNA can identify mutations from multiple clones and is abundant in adjacent body fluids. This work explores the utility of plasma and urinary mutDNA in localised prostate and bladder cancers. This thesis describes the optimisation of urinary mutDNA analysis by assessing urinary DNA processing and extraction methods using healthy volunteer and bladder cancer patient urine samples. Primer panels were designed and validated to target frequently mutated regions in prostate and bladder cancers, as well as for analysis of patient-specific mutations. Sequencing-based methods and dPCR were employed to analyse clinical samples including plasma and urine, to detect and quantify mutDNA. Molecular and clinical data were integrated to explore potential areas of application of mutDNA analysis. For bladder cancer, mutDNA was analysed from liquid-biopsy samples including plasma, cell pellets from urine and urine supernatant from multiple time-points of 17 MIBC patients undergoing NAC. I showed that mutDNA was more frequently detected and was present at higher AFs in urine compared to plasma samples. Of potential clinical relevance, I showed that the presence of mutDNA after starting NAC was associated with disease recurrence. This original contribution to knowledge could offer patients an opportunity to expedite surgical resection in a timely manner, if corroborated in large-scale trials. For prostate cancer, a TP53 specific panel was applied to men with metastatic disease, to demonstrate that clones containing TP53 mutations, which are dominant in at the metastatic stage were present in historical prostatectomy samples taken when then patient was believed to have localised disease only. Furthermore, I showed that these TP53 mutations could be detected at the localised stage of disease. To investigate the ability of mutDNA detection private clonal mutations I developed a method for higher sensitivity analysis (MRD-Seq). This was applied to a clinical cohort of 2 men with multi-focal localised prostate cancer to demonstrate the though the overall levels of mutDNA is low, private clonal mutations may be detectable. Taken together, these original contributions to knowledge could allow for less invasive surveillance of men with low risk prostate cancer and warrants further investigation. In this thesis, I used a range of molecular methods were applied to small cohorts of clinical samples from patients with urological malignancies, in an exploratory analysis. The molecular data was analysed in conjunction with clinical information to draw hypotheses on the biology and natural history of these cancer, and to suggest possible utility of mutDNA analysis in their clinical management. Some of the findings suggest areas of potential utility, which merit further validation or investigation in larger cohorts or clinical studies.

This thesis describes the development of integrated microfluidic technology for single cell proteomic analysis, focusing on circulating tumour cells (CTCs). While single cell proteomic analysis has wide applicability across biology and medicine, CTCs form an ideal first application. Circulating tumour cells are intimately involved in metastasis, the step in cancer overwhelmingly responsible for death, yet have proved hard to study. Single cell microfluidic technology is ideal first because the quantity of material available is inherently at the level of a few cells and second because cell to cell variation is of great interest. Chapter 1 is an introduction to the field. In chapter 2 a microfluidic sandwich assay for quantification of protein at the single cell level is described. In chapter 3 the isolation of CTCs in a microfluidic device is described. This relies on taking the output of the CellSearch® system and inputing it to a microfluidic device. While CTCs were identified, the result showed that a more systematic approach is required for counting and integration with the single cell assay previously described. Chapters 4 and 5 describe development of technology suitable for counting and isolation of CTCs integrated into a microfluidic device with single cell proteomic analysis, although the work done here makes use of fluorescently labelled beads and model cell lines rather than CTCs from patient samples. Chapter 4 describes microfluidic cytometry that can be used to count and identify a labelled population of cells, such as stained CTCs. Chapter 5 describes the prelimary development of a sorting system suitable for isolation of CTCs integrated with the cytometer.

Cutaneous melanoma accounts for 90% of all skin cancer deaths (Balch et al., 2010) and is responsible for 3.6% of deaths from cancer in Australia (Australian Institute of Health and Welfare, 2016). Whilst early detection and successful surgical removal of primary melanomas have improved survival rates (DeSantis et al., 2014), approximately 30% of these patients will have disease recurrence at some point in their lives (Soong et al., 1992; Soong et al., 1998). This is despite being considered disease free following treatment, which may have included surgical removal of the primary and/or its metastasis/es, radiation and/or systemic therapy. Whilst the risk of melanoma recurrence may correlate to some extent with the stage of the primary melanoma in terms of its size and thickness and whether it has metastasised (Shaw et al., 1987; Soong et al., 1992; Soong et al., 1998), recurrences occur even after thin melanomas (associated with low-risk for recurrence) that have been completely excised (Dalal et al., 2007; Jones et al., 2013; Leiter et al., 2012; Meier et al., 2002; Salama et al., 2013; Soong et al., 1998). Melanoma may recur at any point in time, even 10 or more years after a primary melanoma has been excised (Crowley et al., 1990; Dong et al., 2000; Hohnheiser et al., 2011; Kalady et al., 2003; Tsao et al., 1997). Recurrences may present in the same or in areas adjacent to the primary melanoma, however the majority of recurrences appear in lymph nodes or other organs, at which point the disease is among the most aggressive and treatment-resistant of all human cancers (Kenessey et al., 2012; Luke et al., 2017; Mocellin et al., 2013; Sanmamed et al., 2015; Ti'mar et al., 2013). In the metastatic setting, resective surgery of solitary metastases is associated with the most favourable outcome (Chua et al., 2010; Petersen et al., 2007; Sanki et al., 2009; Wasif et al., 2011), however systemic therapy options are dramatically improving survival of patients with unresectable metastases (Garbe et al., 2016). Overall, the greatest treatment efficacy is associated with a low disease burden at time of therapy (Hodi et al., 2010; Luke et al., 2017; McArthur et al., 2016; Sosman et al., 2011) and therefore early detection of melanoma recurrence is critical for improved survival. To date, there are no reliable early markers of melanoma recurrence. Radiological imaging techniques and sentinel lymph node (SLN) biopsies (SLNB) are currently the methods employed to stage primary melanomas and detect metastases. Positron emission tomography (PET) with a labelled glucose analogue fluorine 18 fluorodeoxyglucose (18F-FDG) combined with computed tomography (CT) scans (FDG-PET/CT), are used routinely to determine disease burden. These have limited sensitivity however for the detection of early stage melanoma micro-metastases (Meyers et al., 2009; Pfannenberg et al., 2015), thus cannot provide timely clinical evidence of disease recurrence (Belhocine et al., 2002; Hindié et al., 2011; Krug et al., 2008). Fluorine 18 fluorodeoxyglucose Positron Emission Tomography combined with Computed Tomography (FDGPET/ CT) may be used routinely for monitoring of melanoma patients at high risk of disease recurrence, but it is expensive (Gellén et al., 2015) and subjects patients to excessive radiation exposure (Rueth et al., 2015). Whilst routine SLNBs offer a survival advantage in monitoring recurrence in patients with >1.0mm thick melanomas (Faries et al., 2017; Morton et al., 2014), they are relatively invasive for routine monitoring (Agnese et al., 2003; Lens et al., 2002). Early stage melanoma patients who are considered disease free and are not at high risk for a recurrence, are not routinely assessed by SLNB, or PET/CT or LNB, but rather by physical examinations (Australian Cancer Network Melanoma Guidelines Revision Working Party, 2008). Thus, an additional monitoring regime that can be performed regularly and in conjunction with physical examinations could lead to timely interventions resulting in improved treatment options that will positively impact on the patient’s quality of life and survival. The detection and analysis of mutant specific circulating tumour DNA (ctDNA) is an emerging tool for detection of residual disease and for prognosis and monitoring of different cancers (Bettegowda et al., 2014; Dawson et al., 2013; Gray et al., 2015; Spindler et al., 2012). There is however, limited use of ctDNA for monitoring of residual disease and recurrence in clinically disease free patients v (Oshiro et al., 2015; Tie et al., 2016) and to date, this has not been assessed in melanoma. In melanoma, mainly V-raf murine sarcoma viral oncogene homolog B1 (BRAF) and to some extent, neuroblastoma RAS viral oncogene (NRAS) mutant ctDNA are utilised to monitor patients during therapy in the research setting (Ascierto et al., 2013a; Girotti et al., 2015; Gray et al., 2015; Sanmamed et al., 2015; Santiago-Walker et al., 2015). Notably, telomerase reverse transcriptase (TERT) promoter mutations are present in 50-70% of melanomas and confer a significantly poorer prognosis if found concurrently with BRAF or NRAS mutations relative to the occurrence of each mutation alone. Thus, the ability to monitor patients at all disease stages for the presence of BRAF, NRAS as well as TERT mutant ctDNA, would be advantageous even in BRAF and NRAS wild-type patients. The overall aim of this thesis was to further develop existing tools that could regularly, inexpensively and non-invasively monitor melanoma patients for melanoma recurrence. Firstly, we focused on increasing the number of patients that could be monitored through ctDNA analysis. To do this we developed a new and innovative ddPCR TERT mutation assay and investigated its sensitivity alongside current assays in detecting mutations in melanoma tissue containing a small fraction of tumour cells. The significance of ctDNA for patient monitoring relative to current methods of clinical monitoring was then investigated in relation to melanoma recurrence. Finally, we conducted a retrospective analysis of ctDNA levels relative to metabolic tumour burden (MTB) derived from FDG-PET/CT to determine the lower limit of disease burden detectable by ctDNA using ddPCR. In the first study of this thesis, a novel droplet digital PCR (ddPCR) assay for the concurrent detection of C228T and C250T TERT promoter mutations was designed and developed to display a lower limit of detection (LOD) of 0.17%. The assay was validated using 22 matched plasma and vi tumour samples and showed a 68% concordance rate, with a sensitivity of 53% (95% CI, 27%- 79%) and a specificity of 100% (95% CI, 59%-100%). Plasma samples from 56 metastatic melanoma patients and 56 healthy controls were tested for TERT promoter mutations confirming a specificity of 100% (95% CI, 94%-100%). Importantly, we not only detected TERT mutant specific ctDNA in 4 BRAF mutant cases, but this assay allowed ctDNA quantification in 11 BRAF wild-type cases, which allows for an increased number of patients to be monitored using ctDNA. To monitor patients for recurrence using ctDNA, the mutational profile must first be determined from a patient’s tumour. However, this may be difficult to obtain from tumours that have limited and/or low tumour cellularity and high heterogeneity, particularly when sourced from SLNB and fine needle aspiration biopsies of metastatic sites. Consequently, only limited, low-quality DNA may be isolated for use on different mutation detection platforms, each with varying analytical sensitivities. Limited previous studies focused predominantly on assessment of the BRAF V600 mutation (as the only actionable mutation), and, notably, in tumour samples with more than 50% cellularity. Given the prevalence of TERT promoter mutations which, together with BRAF and NRAS mutations provide prognostic significance, the ability to assess the presence of such mutations in patient tumours, at high sensitivity, would dramatically improve assessment of mutations. In the second study presented here, we evaluated the sensitivity of detection of BRAF, NRAS and TERT promoter mutations in 40 melanoma tissues, using ddPCR relative to Sanger sequencing and pyrosequencing. Tumour cellularity in our samples ranged from 5-50% (n=28) and 50-90% (n=12). Overall, ddPCR was the most sensitive, detecting one of the tested hotspot mutations in a total of 77.5% (31 of 40) of cases, including in 12.5% and 23% of samples deemed as wild-type by pyrosequencing and Sanger sequencing, respectively. The ddPCR sensitivity was particularly apparent among samples with less than 50% tumour cellularity. Therefore, implementation of ddPCR based assays could facilitate mutation detection of early stage tumours and support research aimed at using ctDNA to improve early detection of residual disease and disease recurrence or progression. In the third paper presented here, we assessed the sensitivity of ctDNA to detect disease recurrence. A cohort of 139 patients diagnosed with AJCC stages 0-III in the preceding 10 years were enrolled in the study between January 2015 and February 2017. A blood sample was collected at enrolment and on average 11 months thereafter. Patients were followed up for disease progression for a median time of 50.2 months. From the remaining cohort, three patients developed metastatic disease. The median follow-up from diagnosis of the primary tumour to stage IV disease was 34.4 months. The remaining patients had no clinical evidence of disease recurrence at last follow-up or at death from other causes. We analysed the primary tumour of 37 patients for mutations in BRAF, NRAS and TERT, and identified mutations in 30 patients (three patients with recurrence and 27 patients without recurrence). Using our proven, highly sensitive ddPCR tests we analysed BRAF, NRAS and TERT promoter mutated ctDNA in all available blood samples. Three serial plasma samples were available for each of the three patients who had recurred. CtDNA was detected at the time of radiological or biopsy confirmation of metastases in all three patients. Moreover, ctDNA was detectable in earlier plasma samples from one of the three patients; in this one patient, ctDNA was detected four months prior to clinical detection of gastric and ileum metastases by gastroscopy and biopsy. We detected no mutant specific ctDNA at any time point in the patients without recurrence. Whilst this data is limited because of the limited number of patients and the limited rates of recurrence in early disease stages (2.15%), it provides proof of concept that ctDNA may be a valuable tool to monitor early disease recurrence. Additionally, our assessments were limited by our knowledge of the level of sensitivity of the ctDNA analyses. There was therefore, a robust need to understand the correlation between ctDNA levels and the patient’s tumour burden as assessed by metabolic activity using PET. Given that the metabolic activities of tumours are measured routinely during clinical disease monitoring by assessment of FDG uptake using PET/CT (Larson et al., 1999), we hypothesised that if ctDNA levels correlate with metabolic tumour burden (MTB) derived from FDG-PET/CT scans in melanoma patients, we could determine the limit of detection (LOD) of ctDNA to signify disease recurrence which would indicate the limitations of ctDNA as a biomarker to identify low disease burden. Thus, the indications of ctDNA in the clinical setting will be more clearly identified OR, the need to improve the sensitivity of ctDNA is therefore apparent. Consequently, in the fourth paper of this thesis, we conducted a retrospective analysis of the ctDNA levels in 32 stage IV melanoma patients with active disease prior to systemic therapy. Corresponding FDG-PET/CT scans were examined and the MTB was determined from metabolic tumour volume (MTV) and tumour lesion glycolysis (TLG) (Larson et al., 1999; Winther-Larsen et al., 2017). Within this cohort of patients, ctDNA was detected in 72% of cases with the number of mutated copies per mL of plasma ranging from 1.6 to 52,440. A significant correlation between the MTB and allele frequency was found (P Overall, ctDNA tests were developed to monitor TERT promoter mutations in cell free DNA (cfDNA) in addition to those currently available for BRAF and NRAS therefore maximising the number of patients whose disease status can be monitored using ctDNA. We also demonstrated that ddPCR is a highly sensitive method for detection of BRAF, NRAS and TERT promoter mutations in tumour tissue. Using these tests, we identified a strong correlation between the level of ctDNA and metabolic tumour burden, suggesting, for the first time in melanoma, that ctDNA reflects melanoma disease burden. We also detected ctDNA in early stage melanoma patients that suffered disease recurrence. Prospective studies are now warranted to serially assess the amount of ctDNA after resective surgery to determine if the presence of ctDNA can detect residual disease, and whether ix rising levels of ctDNA in the blood can detect disease recurrence earlier than current clinical methods. This will ultimately provide a sensitive method with which to monitor patients, to ensure timely, earlier interventions thereby improving melanoma survival rates.

Circulating tumour cells (CTCs) are cancer cells shed from a primary tumour site into the bloodstream, where they have the potential to invade other tissues in the body, and thus become the seed of metastases. CTCs have great potential to monitor disease progression and guide cancer treatment, but a key technical challenge for their isolation and characterization is their extreme rarity in blood. CTCs are commonly enriched using immunoaffinity, which while being highly selective, may fail to capture cells that have weak antigen expression. The biophysical properties of CTCs offer a compelling alternative to immunoenrichment. CTCs are much larger in size than erythrocytes, but are similar to leukocytes. Owing to their epithelial origin however, CTCs are likely to be more rigid than leukocytes which allows for deformability based methods to separate these cells. Previously, our group has demonstrated the continuous flow microfluidic ratchet device for deformability based separation of CTCs. Here, an improved version of the device has been developed to be compatible with pre-enrichment methods, allowing for a dramatic increase in throughput. While similar in principle to the previous version, this work specifically improves the design of the sample infusion area to increase the points of contact between the sorting matrix and sample inlet, in order to prevent the accumulation of cell debris. Using this new design, epoxy resin devices and supporting instrumentation were developed to provide a pathway towards scale-up production and automation. These combined improvements allow biology laboratory technicians to enrich CTCs without significant training. The improved device is capable of capturing > 80% CTCs from whole blood at a throughput of 1 mL/hr, which when combined with a red blood cell lysis pre-enrichment step, increases to 8 mL/hr. Finally, devices were used to enrich CTCs from patients with metastatic castration-resistant prostate cancer. CTCs were found in 3 out of 11 patients, with an average count of 78. The enriched cells were further processed to perform single cell genomic sequencing where CTCs were found to contain driver mutations including those commonly associated with prostate cancer.
Applied Science, Faculty of
Mechanical Engineering, Department of
Graduate

Background: Uveal melanoma (UM) is an extremely aggressive disease with approximately 50% of patients developing incurable metastatic disease. Therefore, accurate prognosis of a patient is necessary for closer follow up and the earlier implementation of systemic adjuvant therapies in those most likely to develop metastatic disease. Fortunately, UM can be classified into two distinct molecular classes based on clinically validated gene expression profiling, chromosomal aberrations and specific driver mutations, which accurately predict the metastatic propensity of the primary tumour. However, genetic testing currently requires biopsy of the eye which can lead to serious complications including permanent blindness. Therefore, an alternative source of primary tumour genetic material is needed to avoid these complications. Aims: We proposed that circulating tumour cells (CTCs) are a viable source of tumour genetic material in which patient prognosis could be analysed. Firstly, we aimed to increase the sensitivity of an immunomagnetic enrichment protocol to capture CTCs. Secondly, we aimed to evaluate whole genome amplification methods for accurate single cells analysis to determine the genomic profile of UM cells. The combination of both aims would allow the use of UM CTCs for determining disease prognosis from an easily accessible blood sample. Methodologies: Aim 1 - To refine and evaluate methods for multi-marker immunomagnetic capture of UM CTCs. A tissue microarray (TMA) was created from 1mm cores taken from archived primary UM tissue. Normal tissue and cutaneous melanoma were added as controls. The TMA was stained by immunohistochemistry (IHC) for melanoma, melanocyte, and stem cell markers. Stained tissue was assessed to determine intensity and coverage of staining. In addition to primary UM tissue, five UM cell lines were assessed for the same markers using flow cytometry and immunocytochemistry. Given their high level of staining of UM, 5HT2B, ABCB5, surface gp100 (BETEB), MCAM, and MCSP were coated to immunomagnetic beads and used to determine the retrieval rate of UM cell lines cells spiked into peripheral blood mononuclear cells at a known quantity. CTCs could be detected by immunofluorescent staining of MART1, gp100, and S100β. Aim 2 - Aim 2: To develop methodologies for the detection of genetic markers of metastatic propensity using single UM cells. Single UM cell line cells plus respective bulk genomic DNA whole genome amplified and bulk genomic DNA were amplified using PicoPlex and Repli-G WGA kits to determine each kits’ respective viability of detecting CNVs using low-pass (0.01-0.1x) whole genome sequencing (WGS) on the IonPGM platform. Peripheral blood mononuclear cells (PBMCs) were used as negative controls. In addition, we tested if these methods allowed accurate CNV data after fixation, permeabilisation, and immunostaining. After ensuring cell processing had no significant effects on genomic profile of single cells, blood samples from patients were processed to isolate CTCs from PBMCs. Isolated CTCs were then whole genome amplified using PicoPlex and shallow sequenced using the IonPGM system. Results: We validated several melanoma, melanocyte, and stem cell markers which have been previously shown to be expressed in cancer, cutaneous melanoma, or UM. We found that 5HT2B, and ABCB5, surface gp100 (BETEB), MCAM, and MCSP were highly expressed in primary UM tissue or UM cell lines and were able to immunomagnetically capture UM cell line cells. Concurrently, we validated the use of shallow (0.01x-0.1x depth) whole genome sequencing of single UM cells amplified using the PicoPlex WGA Kit and found that PicoPlex offered a robust method of amplifying single cells that have undergone immunomagnetic isolation, fixation, staining, and capture whilst retaining the original genetic profile of the parent cell line. Upon testing this in a patient, we found a gain of chromosome 8 which is an early event in UM tumourigenesis; aneuploidy of chromosome 8 is a genetic feature that may, with the aid of future studies, delineate patient metastatic risk.

Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths in the UK, and patient mortality is strongly correlated to the cancer stage at diagnosis. Circulating cell-free DNA (cfDNA) are short DNA fragments released into the blood from dying cells, and the use of cfDNA as a predictive and diagnostic biomarker for CRC patients has gained traction recently. The work undertaken in the thesis aimed to assess whether cfDNA could be a suitable biomarker for detection and monitoring of early colorectal lesions in preclinical and clinical settings. Custom qPCR and ddPCR assays were designed and implemented for quantitative cfDNA analysis and for detection of tumour-derived mutations in plasma samples. Preclinical studies investigating the cfDNA dynamics during early tumourigenesis were conducted with the Lgr5-EGFP-IRES-creERT2+/0;Apcfl/fl mouse model of CRC. In parallel, plasma cfDNA was also assessed in an adenoma patient cohort (n=76) against a ‘polyp-free’ control group (n=37). Analysis of mice failed to show a correlation between total cfDNA levels and the adenoma development in three separate preclinical studies, whereas detection of a surrogate tumour-derived mutation in animal plasma samples showed sensitivities between 16 and 25%. Similarly, total plasma cfDNA concentrations between the adenoma patient and control group showed no significant difference (p=0.1012). Targeted detection of BRAF and KRAS mutations in the plasma was achievable, but hampered by low sensitivities (0-25%). Multi-regional targeted next-generation sequencing (NGS) was performed on selected patient cases. The data unmasked extensively and previously unappreciated intra-tumour heterogeneity in colorectal adenomas. Nonetheless, identification and plasma targeting of tumour truncal mutations did not improve the detection rate. In conclusion, these results suggested that further methodological optimisation is necessary to achieve improved diagnostic sensitivity with plasma cfDNA liquid biopsy for the early detection of colorectal lesions.

Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs.
Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.

碩士
國立清華大學
工程與系統科學系
99
The presence and number of CTCs in blood has recently been demonstrated to provide significant prognostic information for patients with metastatic breast cancer. While finding as few as five CTCs in about 1 mL of blood is clinically significant, however, detection of CTCs is currently difficult and time consuming. CTC enrichment is performed by either gradient centrifugation of CTC based on their buoyant density or magnetic separation of epithelial CTC, both of which are laborious procedures with variable efficiency, and these processes may take hours, if not days. Igor Sokolov et al. found that the villous structures of the CTC cell membrane surface is longer than normal cells, which are close to the characteristics length of nano-pillar structure and may be useful for CTC capture. Our work provides a chip based device consisting of nanowires to help on the retention of CTC cells from normal cells. This in situ cell capturing device provides simple control, cheap and high throughput way to enrich CTCs by the higher interaction forces generated between the longer villous nanostructure of CTCs and the nanostructures with designated structure length. While normal blood cells, with much shorter villous on surface, will not have significant retention on the the nanowire. With different incubated time, nanowire chip reach the cancer cell capture rate with 22% and WBCs capture rate with 1.53% at 4 hours incubation；With different length of nanowire, nanowire chip reach the cancer cell capture rate with 1.53% and WBCs capture rate with 26.4% at 14.8 um length .

Colorectal cancer (CRC) is the third most common cancer worldwide; it is responsible for nearly 10% of all newly diagnosed cancers and is the second most cause of cancer related death in Europe. Biomarkers for therapy guidance, targeted therapy and survival prognosis are still limited. As CRC is a heterogeneous disease, different parts of the tumor might have varying molecular characteristics which may change during therapy or disease progression. Through solid biopsies and screenings, these local or temporal differences are impossible to monitor. To facilitate detection of these possible temporal changes, a regularly and non-invasively accessible biomarker is required for disease monitoring. Circulating tumor cells (CTCs) might represent such a biomarker as they have been shown to be fluid surrogates of the solid tumor. EpCAM positive CTCs have shown to be prognostic in CRC for survival, but their full potential has not yet been evaluated further. By using the High Definition Single Cell Analysis (HD-SCA) workflow, we were able to analyze the entire spectrum of CTCs and categorize them as the regular CTCs (HD-CTC), CTCs with a smaller nuclear area (CTC-Small), CTCs with low expression of epithelial marker cytokeratin (CTC-LowCK) and CTCs undergoing apoptosis and therefore releasing cell free DNA...