Genetic markers in circulating tumour cells as a measure of the metastatic propensity of uveal melanoma

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

Incidence and mortality of conjunctival melanoma in Australia (1982 to 2014)

Purpose: The purpose of this study was to estimate the incidence and mortality of conjunctival melanoma in Australia from 1982 to 2014. Methods: De-identified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1982 to 2014. Conjunctival melanoma cases were extracted, and the incidence and mortality were analyzed. Incidence rates were age-standardized against the 2001 Australian Standard Population. Mortality was assessed using log-rank and Cox regression. Results: From 1982 to 2014, there were 299 cases of conjunctival melanoma. The age-standardized incidence rate was 0.48 (95% confidence interval [CI] = 0.41 to 0.54) per million per year. Women (0.52, 95% CI = 0.42 to 0.62) had a higher incidence than men (0.42, 95% CI = 0.33 to 0.51). The incidence of conjunctival melanoma increased in men (+1.46%) and significantly women (+1.41%, P = 0.023) over the study period. The mean 5-, 10-, and 15-year disease-specific survival were 90%, 82%, and 80%, respectively, during the 33-year interval. Comparisons of survival among age, sex, and state revealed no significant differences when tested using log-rank or Cox regression. Conclusions: In conclusion, we found an increase in the rate of conjunctival melanoma diagnoses in Australia from 1982 to 2014. Over the same period, disease survival remained unchanged at a mean of 90%

Incidence and mortality of uveal melanoma in Australia (1982–2014)

Aims: We aimed to estimate the incidence and mortality of uveal melanoma (UM) in Australia from 1982 to 2014. Methods: Deidentified unit data for all cases of ocular melanoma were extracted from the Australian Cancer Database from 1 January 1982 to 31 December 2014. UM cases were extracted and trends in incidence and disease-specific mortality were calculated. Incidence rates were age-standardised against the 2001 Australian Standard Population. Mortality was assessed using Cox regression. Results: From 1982 to 2014, there were 5087 cases of ocular melanoma in Australia, of which 4617 were classified as UM. The average age-standardised incidence rate of UM was 7.6 (95% CI 7.3 to 7.9) per million. There was an increase (p=0.0502) in the incidence of UM from 1982 to 1993 with an annual percent change (APC) of +2.5%, followed by a significant decrease in the incidence of UM from 1993 to 2014 (APC −1.2%). The average 5-year survival from 1982 to 2011 did not significantly change from an average of 81%, with an average APC (AAPC) of +0.1%. A multivariate Cox regression revealed that residence in Western Australia (p=0.001) or Tasmania (p=0.05), age ≥60 years (p \u3c 0.001) and histological classification as mixed (p \u3c 0.001) or epithelioid cells (p \u3c 0.001) were significantly associated with reduced survival. Conclusion: In conclusion, we found that the incidence of UM peaked in the 1990s. Although treatment for primary UM has improved in the last 30 years, overall survival did not change significantly in the last 30 years

Locally performed postoperative circulating tumour DNA testing performed during routine clinical care to predict recurrence of colorectal cancer

Background: Identifying patients at high risk for colorectal cancer recurrence is essential for improving prognosis. In the postoperative period, circulating tumour DNA (ctDNA) has been demonstrated as a significant prognostic indicator of recurrence. These results have been obtained under the strict rigours of clinical trials, but not validated in a real-world setting using in-house testing. We report the outcomes of locally performed postoperative ctDNA testing conducted during routine clinical care and the association with the recurrence of colorectal cancer. Methods: We recruited 36 consecutive patients with newly diagnosed colorectal cancer between 2018 and 2020. Postoperative plasma samples were collected at the first outpatient review following resection. Tumour-informed ctDNA analysis was performed using droplet digital polymerase chain reaction or targeted next-generation sequencing. Results: At the time of surgery, there were 24 patients (66.7%) with localized cancer, nine (25%) with nodal spread, and three (8.3%) with metastatic disease. The median time from surgery to plasma sample donation was 22 days (IQR 20–28 days). At least one somatic mutation was identified in primary tumour tissue for 28 (77.8%) patients. Postoperative ctDNA was detected in five patients (13.9%). The median duration of follow-up was 32.0 months (IQR 27.2–38.1 months). Two patients (5.56%) developed metastatic recurrence. However, neither had detectable postoperative ctDNA. There were no instances of loco-regional recurrence. Conclusion: Analysis of postoperative ctDNA testing can be performed locally, however this study did not reproduce the adverse association between detectable postoperative ctDNA and the development of colorectal cancer recurrence seen in clinical trials

Powering single-cell genomics to unravel circulating tumour cell subpopulations in non-small cell lung cancer patients

Background Circulating tumour cells (CTCs) are attractive “liquid biopsy” candidates that could provide insights into the different phenotypes of tumours present within a patient. The epithelial-to-mesenchymal transition (EMT) of CTCs is considered a critical step in tumour metastasis; however, it may confound traditional epithelial feature-based CTC isolation and detection. We applied single-cell copy number alteration (CNA) analysis for the identification of genomic alterations to confirm the neoplastic nature of circulating cells with only mesenchymal phenotypes. Methods We isolated CTCs from blood samples collected from 46 NSCLC patients using the Parsortix system. Enriched cells were subjected to immunofluorescent staining for CTC identification using a multi-marker panel comprising both epithelial and mesenchymal markers. A subset of isolated CTCs was subjected to whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for the analysis of copy number alterations (CNAs). Results CTCs were detected in 16/46 (34.8%) patients, inclusive of CK+/EpCAM+ CTCs (3/46, 6.5%) and Vim+ CTCs (13/46, 28.3%). Clusters of Vim+ cells were detected in 8 samples, which constitutes 50% of the total number of NSCLC patients with CTCs. No patients had detectable hybrid CK+/EpCAM+/Vim+ cells. All of the tested CK+/EpCAM+ CTCs and 7/8 Vim+ CTCs or CTC clusters carried CNAs confirming their neoplastic nature. Notably, the Vim+ cluster with no CNAs was characterised by spindle morphology and, therefore, defined as normal mesenchymal circulating cells. Conclusion Our results revealed that CK-negative, vimentin-expressing cells represent a large proportion of CTCs detected in NSCLC patients, which are likely missed by standard epithelial-marker-dependent CTC categorisation

Genetic analysis of heterogeneous subsets of circulating tumour cells from high grade serous ovarian carcinoma patients

Circulating tumour cells (CTCs) are heterogenous and contain genetic information from the tumour of origin. They bear specific intra- and extra-cellular protein markers aiding in their detection. However, since these markers may be shared with other rare cells in the blood, only genetic testing can confirm their malignancy. Herein, we analyse different CTC subsets using single cell whole genome DNA sequencing to validate their malignant origin. We randomly selected putative CTCs identified by immunostaining that were isolated from 4 patients with high grade serous ovarian cancer (HGSOC) and one with benign cystadenoma. We specifically targeted CTCs positive for epithelial (CK/EpCAMpos), mesenchymal (vimentinpos), and pseudoendothelial (CK/EpCAMpos plus CD31pos) markers. We isolated these cells and performed whole genome amplification (WGA) and low-pass whole-genome sequencing (LP-WGS) for analysis of copy number alterations (CNA). Of the CK/EpCAMpos cells analysed from the HGSOC patients, 2 of 3 cells showed diverse chromosomal CNAs. However, the 4 pseudoendothelial cells (CK/EpCAMpos plus CD31pos) observed in the HGSOC cases did not carry any CNA. Lastly, two of the clusters of vimentin positive cells sequenced from those found in the benign cystadenoma case had CNA. Despite the low number of cells analysed, our results underscore the importance of genetic analysis of putative CTCs to confirm their neoplastic origin. In particular, it highlights the presence of a population of CK/EpCAMpos cells that are not tumour cells in patients with HGSOC, which otherwise would be counted as CTCs

INTRODUCING AUTHENTIC RESEARCH EXPERIENCE AT THE UNDERGRADUATE LEVEL

One of the challenges facing new graduates is that they don’t necessarily know what lies out there for them once they have finished their degrees. Not all students are aware of the jobs they are qualified for, or the post-graduate opportunities that may be available to them. In an attempt to engage with undergraduate students and give them a glimpse into life after graduation, we ran a pilot Cancer Research Summer Project (CRSP). In this pilot program we took four high-achieving second-year biomedical science students through a three-week research project, which exposed them to a real-life laboratory experience, and also provided them with additional skills training in areas such as scientific journal article writing and database mining. Students were given a fully immersive laboratory experience, receiving the type of instruction and supervision they could expect in either post-graduate study or out in the workforce, with some autonomy, and successes and failures driven by their own hands. Students reported that the CRSP provided a fantastic opportunity and made them aware of a new world of possibilities after graduation. At the end of the project the students were provided with a supporting letter to include in their curriculum vitae, showing evidence of the industry-relevant training they undertook. Work experience is often required of new employees, especially in the field of biomedical research. This project had the additional benefit of providing students with vitally important first-hand experience. It also provided a vehicle to demonstrate to the students that the skills and theories they learn in their undergraduate courses can be directly transferred to the workplace or in their further studies. With the continuation of this project into the future, the hope is that students can gain a broader understanding of the opportunities available to them and that more students will be encouraged to take up post-graduate study

Is tissue still the issue? The promise of liquid biopsy in uveal melanoma

Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients\u27 uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years

The SLUGGS survey: probing the supermassive blackhole connection with bulges and haloes using red and blue globular cluster systems

Understanding whether the bulge or the halo provides the primary link to the growth of supermassive black holes has strong implications for galaxy evolution and supermassive black hole formation itself. In this paper, we approach this issue by investigating extragalactic globular cluster (GC) systems, which can be used to probe the physics of both the bulge and the halo of the host galaxy. We study the relation between the supermassive black hole masses (MBH) and the GC system velocity dispersions (σGC) using an updated and improved sample of 21 galaxies. We exploit the dichotomy of GC system colours, to test if the blue and red GCs correlate differently with black hole mass. This may be expected if they trace the potentially different formation history of the halo and of the bulge of the host galaxy, respectively. We find that MBH correlates with the total GC system velocity dispersion, although not as strongly as claimed by recent work of Sadoun & Colin. We also examine the MBH-σGC relation for barred/barless and core/non-core galaxies, finding no significant difference, and for the first time we quantify the impact of radial gradients in the GC system velocity dispersion profile on the MBH-σGC relation. We additionally predict MBH in 13 galaxies, including dwarf elliptical galaxies and the cD galaxy NGC 3311. We conclude that our current results cannot discriminate between the bulge/halo scenarios. Although there is a hint that the red GC velocity dispersion might correlate better with MBH than the blue GC velocity dispersion, the number statistics are still too low to be certain

Multi-marker immunofluorescent staining and pd-l1 detection on circulating tumour cells from ovarian cancer patients

Detection of ovarian cancer (OC) circulating tumour cells (CTCs) is primarily based on targeting epithelial markers, thus failing to detect mesenchymal tumour cells. More importantly, the immune checkpoint inhibitor marker PD-L1 has not been demonstrated on CTCs from OC patients. An antibody staining protocol was developed and tested using SKOV-3 and OVCA432 OC cell lines. We targeted epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC-specific (PAX8) markers for detection of CTCs, and CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. CTCs were enriched using the Parsortix™ system from 16 OC patients. Results revealed the presence of CTCs in 10 (63%) cases. CTCs were heterogeneous, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) for both (hybrid). PAX8 was only found in 11/157 (7%) CTCs. In addition, 62/157 (39%) CTCs were positive for PD-L1. Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.007) and mesenchymal (p = 0.0009) expressing CTCs. Characterisation of CTC phenotypes in relation to clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity plays in OC and its relationship with PD-L1