Academic literature on the topic 'Circulating tumour cell clusters'

Circulating tumour cells (CTCs) are the metastatic precursors to distant disease in head and neck cancers (HNCs). Whilst the prognostic and predictive value of single CTCs have been well documented, the role of CTC clusters, which potentially have a higher metastatic capacity are limited. In this study, the authors used a novel straight microfluidic chip to focus and capture CTCs. The chip offers high cell recoveries with clinically relevant numbers (10–500 cells/mL) without the need for further purification. Single CTCs were identified in 10/21 patient samples (range 2–24 CTCs/mL), CTC clusters in 9/21 patient samples (range 1–6 CTC clusters/mL) and circulating tumour microemboli (CTM) in 2/21 samples. This study demonstrated that CTC clusters contain EGFR amplified single CTCs within the cluster volume. This novel microfluidic chip demonstrates the efficient sorting and preservation of single CTCs, CTC clusters and CTMs. The authors intend to expand this study to a larger cohort to determine the clinical implication of the CTC subsets in HNC.

AbstractCirculating tumour cell (CTC) clusters have been proposed to be major players in the metastatic spread of breast cancer, particularly during advanced disease stages. Yet, it is unclear whether or not they manifest in early breast cancer, as their occurrence in patients with metastasis-free primary disease has not been thoroughly evaluated. In this study, exploiting nanostructured titanium oxide-coated slides for shear-free CTC identification, we detect clustered CTCs in the curative setting of multiple patients with early breast cancer prior to surgical treatment, highlighting their presence already at early disease stages. These results spotlight an important aspect of metastasis biology and the possibility to intervene with anti-cluster therapeutics already during the early manifestation of breast cancer.

Multicellular aggregates of circulating tumor cells (CTC clusters) are potent initiators of distant organ metastasis. However, it is currently assumed that CTC clusters are too large to pass through narrow vessels to reach these organs. Here, we present evidence that challenges this assumption through the use of microfluidic devices designed to mimic human capillary constrictions and CTC clusters obtained from patient and cancer cell origins. Over 90% of clusters containing up to 20 cells successfully traversed 5- to 10-μm constrictions even in whole blood. Clusters rapidly and reversibly reorganized into single-file chain-like geometries that substantially reduced their hydrodynamic resistances. Xenotransplantation of human CTC clusters into zebrafish showed similar reorganization and transit through capillary-sized vessels in vivo. Preliminary experiments demonstrated that clusters could be disrupted during transit using drugs that affected cellular interaction energies. These findings suggest that CTC clusters may contribute a greater role to tumor dissemination than previously believed and may point to strategies for combating CTC cluster-initiated metastasis.

Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

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.

AbstractEven after a decade of use, CAR-T cell therapy for non-Hodgkin lymphoma (NHL) is still evolving, and disease control is now the main concern in the majority of experienced centres. Indeed, despite highly appealing objective response (OR) rates in refractory patients, the long-term overall survival (OS) of this population has only slightly improved. Pivotal studies have suggested that 2-year OS rates do not surpass 30%, even though results improve when complete response (CR) is achieved within the first 3 months after treatment (Wang et al. 2020; Schuster et al. 2019; Neelapu et al. 2017). Although achieving this exceptionally high level of OR is praiseworthy, similar improvements have not been made regarding OS, and current OS probabilities are not satisfactory. Of course, there are multiple reasons for this; a substantial proportion of patients either do not achieve an initial response or experience progression very soon after treatment, with poor OS (Chow et al. 2019). Both populations present with disease burden or aggressive cancer prior to CAR-T cell therapy, possibly having been referred too late in the course of treatment or waited too long before CAR-T cells were processed for them. Both of these issues have potential solutions, such as more widely publicizing the efficacy of CAR-T cells, which may increase referrals at an earlier stage, and developing methods, which are already being heavily investigated, for shortening the manufacturing process (Rafiq et al. 2020). In the latter case, the use of allogeneic lymphocytes could allow for already prepared cells to be readily used when needed and would most likely be the most efficient strategy as long as the risk of graft-versus host disease is offset (Graham and Jozwik 2018). Thus, achieving CR is a crucial step in increasing OS, as patients with partial response (PR) or stable disease (SD) present with lower OS, while currently, recurrence appears to be rare when CR is maintained for more than 6 months (Komanduri 2021). However, the disease will likely recur in more than half of patients in the months following treatment, possibly due to issues such as the poor persistence of CAR-T cells (which may not be as crucial as once thought for acute lymphoblastic leukaemia (Komanduri 2021)) or the loss of target antigen expression (which has been regularly documented (Rafiq et al. 2020)). Both of these mechanisms could potentially be used to develop methods that reduce recurrence after CAR-T cell therapy. In fact, the most popular approaches currently being investigated are attempting to either use two CAR-T cell types that each target different antigens or to create CAR-T cell constructs that target either multiple antigens or an antigen other than CD19 (Shah et al. 2020). The concomitant infusion of CAR-T cells with targeted therapies is also being explored in other B-cell malignancies and appears to both increase the CR rate and decrease recurrence (Gauthier et al. 2020). When recurrence does occur, patient OS is rather dismal, and the best remaining option would most likely be inclusion in a clinical trial. If this option is not available, salvage therapy may be attempted, although cytotoxic treatments are extremely limited given that most diseases have been refractory to numerous lines of treatment prior to immunotherapy. A few case reports and studies with a small patient population receiving anti-PD-1 antibodies, ibrutinib, or ImiDs have been reported with largely anecdotal supporting evidence (Byrne et al. 2019). However, even in the case of a new objective response (OR), the subsequent risk of recurrence is substantial and may invite further consolidation with allogeneic haematopoietic stem cell transplantation (Byrne et al. 2019), which has already been performed in patients treated for acute lymphoblastic leukaemia (Hay et al. 2019). However, the efficacy of this strategy remains to be validated in NHL patients in clinical trials. Further supporting evidence, although limited, has recently been reported concerning an additional treatment with CAR-T cells inducing an OR. Of the 21 NHL patients included in the study, the OR rate after the second infusion was 52% (CR, n = 4; PR, n = 7), with some durable responses inviting further investigations (Gauthier et al. 2021). Overall, with such poor outcomes after recurrence, current efforts are also focused on predicting the patients most likely to experience disease progression and that are potential candidates for preemptive consolidation therapy, although there is no doubt that patients who do not achieve a rapid CR should be the first candidates. Additionally, immune monitoring should encompass not only CAR-T cell survival but also the detection of circulating tumour DNA (Komanduri 2021) because this could aid in detecting subclinical recurrence and in deciding whether consolidation or maintenance therapy should be administered. However, currently, all these approaches are highly speculative and require further clinical study.

Short fragments of cell-free DNA are released into the plasma when cells die. In patients with cancer some of this circulating DNA is released by tumour cells; in pregnant women some is derived from the fetus; and increased amounts are found in many pathological conditions associated with cell death. In each of these circumstances, analysis of cell-free DNA can provide useful clinical information (e.g. detection or monitoring of cancer, determination of mutation status of a fetus). With further improvement in analytical technologies and developments of new markers, it is likely that the application of circulating cell-free DNA and cell-free RNA species in molecular diagnostics will increase in the future.

This chapter discusses haematological emergencies, including blood transfusion reactions, sickle-cell crisis, bleeding disorders, abnormal coagulation, circulating inhibitors of coagulation, abnormal platelets, anticoagulant therapy, bleeding with fibrinolytic therapy, bleeding in liver disease, bleeding in uraemia, massive transfusion/cardiopulmonary bypass, haemophilia and related disorders, combined thrombotic and haemorrhagic disorders, disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP) and haemolytic uraemic syndrome (HUS), microangiopathic haemolytic anaemia, heparin-associated thrombocytopenia, acute leukaemias, early complications of bone marrow transplantation (BMT), the febrile neutropenic patient, infections in the transplant patient, cytomegalovirus (CMV) infections in transplant patients, hyperviscosity syndrome, tumour lysis syndrome, hypercalcaemia of malignancy, superior vena cava obstruction, and massive mediastinal mass.

Pituitary adenomas are discovered in up to 25% of unselected autopsies, however, clinically apparent tumours are considerably less common. The pituitary gland is composed of differentiated cell types: somatotrophs, lactotrophs, corticotrophs, thyrotrophs, and gonadotrophs. Tumours may arise from any of these cell types and their secretory products depend on the cell of origin. The functional classification of pituitary tumorus is based on identification of cell gene products by immunostaining or mRNA detection, as well as measurement of circulating tumour and target organ hormone levels. Oversecretion of adrenocorticotropic hormone (ACTH) results in cortisol excess with Cushing’s disease. Growth hormone overproduction leads to acromegaly with typical acral overgrowth and metabolic abnormalities. Prolactin hypersecretion results in hypogonadism and galactorrhoea. Rarely, thyroid-stimulating hormone (TSH) hypersecretion leads to goitre and thyrotoxicosis, and gonadotropin excess results in gonadal dysfunction (1). Mixed tumours cosecreting growth hormone with prolactin, TSH, or ACTH may also arise from single cells. Clinically nonfunctional tumours are those that do not efficiently secrete their gene products, and most commonly they are derived from gonadotroph cells. Pituitary tumours are further defined radiographically as microadenomas (<1 cm in diameter) or macroadenomas (>1 cm in diameter). However, this classification does not reflect whether the pituitary tumour is amenable to total resection and limits assessment of invasive progression during serial imaging. Therefore, it is useful to apply the classification proposed by Hardy in 1973 and modified by Wilson in 1990 (Table 2.3.2.1), whereby pituitary tumours are classified into one of five grades and one of six stages, providing important preoperative information. Pituitary tumours cause morbidity by both abnormal hormone secretion as well as compression of regional structures. As a considerable proportion of patients do not achieve optimal therapeutic control of mass effects and/or hormone hypersecretion despite advances in therapeutic approaches, understanding pathogenesis and pituitary tumour growth patterns in individual patients will enable identification of subcellular treatment targets, ultimately decreasing tumour-related morbidity and mortality. Determinants of initiation and progression of pituitary adenomas are not fully understood. This chapter describes a spectrum of mechanisms implicated in pituitary tumorigenesis, including the role of pituitary plasticity, imbalances in cell cycle regulation, transcription factors, signalling pathways, and angiogenesis (Fig. 2.3.2.1). Molecular events related to tumorigenesis in human pituitary adenoma subtypes are summarized in Table 2.3.2.2. The causal role for selected genetic imbalances leading to development of pituitary tumours has been confirmed in several transgenic mouse models (Table 2.3.2.3).

Usual diagnostic methods of leptomeningeal metastases (LM) in CerebroSpinal fluid (CSF), lack both specificity and sensitivity. The Veridex CellSearch® technique quantifying circulating tumour cells (CTCs) in blood was adapted to detect Tumour Cells (CSFTCs) in CSF from cancer patients with LM. CSF samples from 60 patients with established or suspected breast cancer or lung cancer LM and/or melanoma were evaluated. 5 mL CSF samples were collected on CellSave® preservative and analyzed within 3 days after CSF sampling. Gold Standard cytological analysis on 1 to 10 mL CSF samples from patients with established LM allowed sometimes the detection but usually not the quantification of TCs. In established LM, EpCAM+/cytokeratin+ or CD146+/HMW-MAA+ nucleated (DAPI+) cells were observed and enumerated with precision from one to up to 10 000 cells/mL. Their morphology on digital images galleries could be discriminant between breast and lung cancer. This methodology, established on a limited volume of CSF compared to the Gold Standard and allowing delayed processing, is of great interest in the diagnosis and follow-up of cancer patients with LM. The reliability of the method also opens new fields of investigation for other biological fluids and to precise the stem cell potential of metastatic cells in CSF.

Anaemia is very common, affecting over one- third of the world’s population and can be defined as a reduction in the haemoglobin content of red blood cells (RBC). The normal range varies slightly according to the population being tested, but typically in the UK anaemia in males can be diagnosed if the haemoglobin falls to below 135 g/ L and in females below 115 g/ L. In addition to a reduction in the haemoglobin concentration there is usually an as­sociated reduction in the number of circulating red cells and a low haematocrit. Anaemia is not a diagnosis, it is an abnormality that has an underlying cause and, therefore, a determination of that cause must be made before effective treatment can begin. The production of red cells is termed ‘haematopoiesis’ and occurs in the bone marrow (liver and spleen in foetal life). The bones involved in production change as we age from almost all bones in neonates to long bones, pelvis, and thoracic cage when we reach our 4th decade. As with all blood cells, production of RBCs begins with a pluripotent stem cell that is capable of forming many progenitor cells, including those of the erythroid (red cell) lineage (Figure 12.1). It is estimated that a single pluripotent stem cell, fol­lowing 18– 20 successful divisions, is able to produce 10 million mature erythrocytes. For this process to occur a number of growth factors (GF) are required, which act in synergy and enable the process of haematopoiesis to follow a stepwise maturation process, ending in the release of mature erythrocytes into the blood stream. Examples of such factors include the interleukins (IL), i.e. IL- 1, IL- 3, IL- 4, IL- 5, and IL- 6. Growth factors also act on the bone marrow stromal cells, enabling the correct environment for cell maturation and development. Tumour necrosis factor (TNF) and IL- 1 are particularly important stromal acting growth factors and can stimulate the stromal cells to produce many of the IL factors described above. The GF erythropoietin (EPO) is required for successful red cell maturation. Many of the growth factors work by binding to cell sur­face receptors.