Optimisation of the isolation and identification of circulating melanoma cells

Although melanoma is largely curable when detected in its earliest stages, it can metastasise to other tissues, drastically reducing survival rates. The most recent therapies used to treat metastatic melanoma are effective long-term in only 11 to 33% of patients. Our ability to monitor treatment failure is limited. New prognostic markers are urgently required to allow monitoring of treatment response and disease progression. Circulating tumour cells (CTCs) are released into the bloodstream by the tumours within a patient, this being a key step in melanoma spread. Since CTCs can be detected in the blood of metastatic melanoma patients, these cells can be used as a “liquid biopsy”, providing critical insight into each person’s melanoma biology. Melanoma CTCs have been described as very heterogeneous, hindering their isolation via commonly used CTC capturing methods. To address this, microfluidic devices have been developed to isolate viable CTCs from blood, independently of their marker expression. This study aimed to determine the effectiveness of two different microfluidic devices (Slanted and A5) in recovering melanoma cell lines, and their potential use in the isolation of CTCs from the blood of metastatic melanoma patients. It also aimed to study additional cancer or melanoma specific markers to be used in immunostaining protocols for detection of CTCs after microfluidic enrichment. The optimal isolation procedure was identified as two rounds of enrichment with the Slanted spiral device, after which we obtained a 3-log depletion of white blood cells and a recovery of over 60% when cells from two melanoma cell lines were spiked into blood samples from healthy volunteers. In addition, we optimised the detection of CTCs using four melanoma markers (gp100, Melan-A, s100 and MCSP) combined in a multimarker immunocytochemistry staining protocol. The optimised enrichment and detection procedures were validated in a cohort of ten metastatic melanoma patients. Results showed that 40% of the patients had one or more CTCs in their blood (1-4 CTCs/8 mL of blood). Furthermore, three additional markers (Vimentin, RANK, and ABCB5) were trialled so as to increase detection of highly heterogeneous melanoma CTCs in samples that have been processed through the Slanted microfluidic device. The improved enrichment and detection of CTCs in the blood of melanoma patients using the methods developed as part of this study will facilitate the molecular, genomic and functional characterisation of melanoma CTCs. This will ultimately improve our understanding of the biology of melanoma CTCs and their role in metastatic spread and treatment response

Evaluation of PD-L1 expression in various formalin-fixed paraffin embedded tumour tissue samples using SP263, SP142 and QR1 antibody clones

Background & objectives: Cancer cells can avoid immune destruction through the inhibitory ligand PD-L1. PD-1 is a surface cell receptor, part of the immunoglobulin family. Its ligand PD-L1 is expressed by tumour cells and stromal tumour infltrating lymphocytes (TIL). Methods: Forty-four cancer cases were included in this study (24 triple-negative breast cancers (TNBC), 10 non-small cell lung cancer (NSCLC) and 10 malignant melanoma cases). Three clones of monoclonal primary antibodies were compared: QR1 (Quartett), SP 142 and SP263 (Ventana). For visualization, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform for immunohistochemical staining Ventana BenchMark GX. Results: Comparing the sensitivity of two different clones on same tissue samples from TNBC, we found that the QR1 clone gave higher percentage of positive cells than clone SP142, but there was no statistically significant difference. Comparing the sensitivity of two different clones on same tissue samples from malignant melanoma, the SP263 clone gave higher percentage of positive cells than the QR1 clone, but again the difference was not statistically significant. Comparing the sensitivity of two different clones on same tissue samples from NSCLC, we found higher percentage of positive cells using the QR1 clone in comparison with the SP142 clone, but once again, the difference was not statistically significant. Conclusion: The three different antibody clones from two manufacturers Ventana and Quartett, gave comparable results with no statistically significant difference in staining intensity/ percentage of positive tumour and/or immune cells. Therefore, different PD-L1 clones from different manufacturers can potentially be used to evaluate the PD- L1 status in different tumour tissues. Due to the serious implications of the PD-L1 analysis in further treatment decisions for cancer patients, every antibody clone, staining protocol and evaluation process should be carefully and meticulously validated

Neuronal differentiation markers in basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common skin cancer in humans. The demonstration of genetic and protein alterations has, so far, had limited correlation with either biological behaviour or histological classification of these tumours. It was observed that Glil-overexpressing keratinocytes express elevated levels of genes known to be associated with neuronal development, including p-tubulin III, GAP-43, Arc and neurofilament. It was proposed that these genes may similarly be overexpressed in BCCs and that different levels of expression may be present in different BCC subtypes Immunohistochemistry of BCCs demonstrated that neuronal differentiation marker proteins are expressed in BCCs, but that this expression is significantly reduced in tumours that behave aggressively. Elevated neuronal differentiation marker gene expression was shown in BCCs. Again, expression was more prominent in tumour types that behave indolently. Results were obtained for tumour samples processed by laser capture microdissection, needle microdissection and homogenised tissue. Expression of neuronal differentiation marker genes in Gli-overexpressing keratinocytes was examined by semi-quantitiative PCR. Neuronal differentiation marker expression was associated with GUI and GH2 over-expression in some cases {lcub}P-tubulin III and Arc). GUI and GH2 also promoted the expression of each other in a positive-feedback loop. Expression of these markers was examined in archival tumours for which the clinical outcome was known in terms of recurrence. In completely excised tumours P-tubulin III was significantly reduced in tumours that went on to subsequently recur. Other markers were not expressed in significantly different amounts. In summary, I have shown that expression of markers associated with neuronal development is a feature of Basal Cell carcinoma, and that the expression of these markers correlates strongly with the tumour histological subtype but only weakly with tumour recurrence. More work will be required to discover further alterations in BCC molecular biology that impact significantly on tumour behaviour

Proteomic Characterization of Ovarian and Breast Cancer Microenvironments for Improved Diagnostics and Therapeutic Targeting

Cancers exist within complex microenvironments formed by heterogeneous cell types. This diversity creates significant challenges for detection, diagnosis and treatment. Mass spectrometry-based proteomics is a powerful approach capable of characterizing complex biological systems which are characteristic of cancer biology. In this thesis, proteomics was utilized to answer several questions related to ovarian cancer diagnosis and detection, and the effects of NODAL, an embryonic morphogen, on the breast cancer secretome and stromal cell recruitment. First, I compared multiple sample preparation techniques and found high-pH/low-pH fractionation to yield the greatest proteome coverage over commonly used approaches. Second, I compared the proteomes from two ovarian cancer subtypes (high-grade serous and endometrioid) for which histological discrimination remains difficult in a proportion of cases. I documented several unknown proteins, including KIAA1324, which were validated and confirmed to improve the differential diagnosis of endometrial ovarian cancer. Third, I extensively characterized extracellular vesicle proteomes from biological fluids (conditioned media, plasma and ascites) to catalogue potential biomarkers associated with malignant ovarian cancer. I detected many factors associated with advanced stage, high-grade serous ovarian cancer including CFHR4, MUC1, APCS and PZP that may be useful for early detection. Last, I characterized the global effects of the Transforming Growth Factor-β superfamily member NODAL on the breast cancer secretome and stromal cell recruitment in vitro. I found a previously unknown role for NODAL in modulating pro-inflammatory factors, including CXCL1 and IL6 that were correlated with multipotent stromal cell recruitment. In summary, this work represents a significant contribution to the histological assessment and detection of ovarian cancer and our understanding of the malignant properties of NODAL within the breast cancer microenvironment

Molecular magnetic resonance imaging for tumour targeting

During my postgraduate training in Radiology at the University of Genoa, Italy, I developed research projects on the application of molecular magnetic resonance imaging for tumour targeting. Molecular imaging (MI) aims to provide “pictures of what is happening inside the body at molecular and cellular level”. Magnetic resonance imaging (MRI) has been applied to many aspects of MI. Although it offers better temporal and spatial resolution than other methodologies, it is less sensitive for molecular or cellular activities, and therefore there is a need to develop more efficient contrast agents. The publications included in this PhD thesis demonstrate the successful application of two classes of MRI contrast agents: ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) and manganese (Mn2+). Novel USPIO-antibody-conjugated probes for investigating lymphoma tumours were applied, and the potential of labelling natural killer cells by SPIO was demonstrated, offering a great opportunity for in vivo investigation of these lymphocytes that play an essential role in cell-based immune defence. The development of a birdcage prototype coil for a clinical 3T MR scanner with a commercial scientific collaboration was carried out. Research projects for investigating tumour calcium metabolism and risk of bone metastases were developed preclinically (by using Mn2+ in human preclinical cancer animal models) and clinically (by using in vivo proton magnetic resonance spectroscopy to investigate human breast cancer). An in vivo Manganese-enhanced-MRI (MEMRI) technique to visualise brown adipose tissue, its physiopathology, and its role in breast or prostate cancer progression, has been included as well. The last research projects carried out as the conclusion of this PhD were focused on: 1) the development of a novel USPIO-MR imaging approach to monitor chronic lymphocytic leukaemia (CLL), induced by interfering with both miR-15 and miR-16 expression; and 2) evaluating response to a potential treatment with the use of miRNA mimics or inhibitors

An analysis of the proliferative response of oral keratinocytes during the re-epithelialization of a chemically induced wound

The wound healing response requires the interaction of a series of events, culminating in the restoration of tissue integrity. In the present study, the proliferative phase of this process was examined with particular reference to re-epithelialization of the wound defect and the manner in which epithelial keratinocytes respond. PC-lO (Dako), a mouse monoclonal antibody against proliferating cell nuclear antigen, was employed as a marker of cell division. This 36kDa auxilliary protein 2 for DNA polymerase-delta is essential for DNA replication at S-phase and has been employed with varying success as a marker of cell proliferation. The majority of work utilizing PCNA to evaluate proliferative activity has been performed on histopathological tissue. Studies examining PCNA in non-neoplastic tissue, and in particular, oral epithelium, are limited. The proliferative status of tongue for instance, one of the more readily accessible regions of the oral cavity, has been determined using analagous techniques, however there is a paucity of information regarding the application of PCNA. In fact, research by Warnakulasuriya and Johnson (1993) investigating cell proliferation in hamster tongue appears to be the only equivalent study. Consequently, in the present investigation the performance of PCNA was examined in both normal and wounded rat tongue. An animal model, adapted from unpublished work by Shaw and Young (1983), was established and involved the subepithelial injection of 35ul of the sclerosant sodium tetradecyl sulphate (STS) into the right ventrolateral aspect of the tongue of anaesthetised animals using a Hamilton microsyringe. The resulting wound was assessed at two, four, seven and fourteen days subsequent to the STS injection. The excised tongues were microwave irradiated in Kryofix (Merck) and submitted for immunostaining with PC-lO (Dako). Detection of the incorporated antibody was performed using an indirect PAP technique. Stained sections were quantified using a "chromatic" image analysis system to determine the proliferative activity in the healing epithelium . A PCNA labelling index, expressed as the number of PCNA-stained cells per 100 epithelial basal cells, was calculated for the dorsal, ventral and total epithelial surface. Each experimental wound was mapped to indicate the spatial distribution of proliferating cells, as determined by PCNA with regard to the wound margin. The results of this study showed that epithelial regeneration of the STS induced wound began within forty eight hours and confluence was achieved fourteen days following the injection. PCNA labelling indices for the dorsal, ventral and total basal epithelial surfaces of normal rat tongue, expressed as the mean :- SD, were 23: 6.3, 21 t 16.7 and 22 i 9.1 respectively. These findings reflect the more complex proliferative pattern that occurs in dorsal epithelium to maintain the undulating rete ridge pattern. Comparison of the PCNA indices for the four experimental groups with those obtained for normal rat tongue demonstrated that maximal epithelial activity occurred at seven days post-wounding in the dorsal surface and four days post-wounding in the ventral surface. Quantitative analysis of the PCNA labelling combined with microscopic evaluation also enabled a general sequence of healing to be established, based on proliferative activity. From these data, a number of events known to occur during the reparative process could be identified, namely the lag phase, a period of peak proliferative activity and an interval of maximal migratory activity. lmmunohistochemical detection of PCNA also revealed that following wounding, oral keratinocytes undergo cell division in a regular temporal distribution rather than in random order. The findings of this investigation suggest that the STS animal model is reproducible and results in a slow healing wound that allows various aspects of the reparative process to be considered. The STS model, when used in conjunction with the methods employed for the quantitative assessment of PCNA, may have utility in wound healing studies. In particular, this procedure would be of benefit in evaluating the contribution of proliferation to the reparative process