Liquid biopsy in ovarian cancer using circulating tumor DNA and cells: Ready for prime time?

Liquid biopsies hold the potential to inform cancer patient prognosis and to guide treatment decisions at the time when direct tumor biopsy may be impractical due to its invasive nature, inaccessibility and associated complications. Specifically, circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have shown promising results as companion diagnostic biomarkers for screening, prognostication and/or patient surveillance in many cancer types. In ovarian cancer (OC), CTC and ctDNA analysis allow comprehensive molecular profiling of the primary, metastatic and recurrent tumors. These biomarkers also correlate with overall tumor burden and thus, they provide minimally-invasive means for patient monitoring during clinical course to ascertain therapy response and timely treatment modification in the context of disease relapse. Here, we review recent reports of the potential clinical value of CTC and ctDNA in OC, expatiating on their use in diagnosis and prognosis. We critically appraise the current evidence, and discuss the issues that still need to be addressed before liquid biopsies can be implemented in routine clinical practice for OC management

Detection of Human Papillomavirus Genotypes and Epstein-Barr Virus in Nasopharyngeal Carcinomas at the Korle-Bu Teaching Hospital, Ghana

Nasopharyngeal carcinomas (NPC) are endemic in Far East Asia and commonly harbour Epstein-Barr virus (EBV) which is known to serve as a key oncogenic promoter. Human papillomavirus (HPV) is known to contribute to the pathogenesis of NPC. However, in Ghana these two viruses have not been linked to NPC prevalence. This study was designed to determine the HPV genotypes and EBV involved in NPC tissue biopsies. A retrospective study design involving 72 formalin-fixed paraffin-embedded tissue (FFPET) samples of NPC from 2006 to 2012 were retrieved from the Department of Pathology, University of Ghana School of Biomedical and Allied Health Sciences. Sections were taken for histological analysis and for DNA lysate preparation. The DNA lysates were subjected to polymerase chain reaction (PCR) analysis to determine the presence of HPV genotypes and EBV. HPV specific primers were used to type for fourteen HPV genotypes (HPV-16, 18, 6/11, 31, 33, 35, 44, 42, 43, 45, 56, 52, 58, and 59). Out of the 72 NPC biopsies analyzed by PCR, EBV DNA was present in 18 (25%) cases and HPV DNA in 14 (19.23%). High risk HPV (HR-HPV) genotypes 18 and 31 were associated with the NPC. There were 3 (4.2%) cases of coinfection by both viruses. The EBV DNA present in the undifferentiated variant of the NPC and the histopathology of the NPC in Ghana is similar to the type described in endemic areas

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

Effect of xylopic acid on alloxan-induced diabetic neuropathy in rats

Background: Neuropathic pain is a very disturbing condition commonly found in diabetic patients. This study investigated xylopic acid (XA), the major constituent of Xylopia aethiopica in diabetic neuropathy as well as established possible toxicity of the compound on some selected tissues.Methods: Diabetes was induced in six groups of male rats with 120 mg/kg alloxan monohydrate. Diabetes was confirmed as a blood glucose level >15 mmol/dl. Neuropathic pain was confirmed on day three post-diabetes induction and treatment with 10 mg/kg, 30 mg/kg or 100 mg/kg xylopic acid, 10 mg/kg glibenclamide, 10 mg/kg morphine, and 10 ml/kg normal saline were initiated and continued for the next 15 days. The effects of the treatments on cold allodynia (cold water at 4°C) and thermal hyperalgesia (hot water at 55 ± 1°C) were evaluated within the duration of treatments. Histology of the liver and kidney, as well as haematological, serum biochemical, and semen analyses, were done after the fifteenth day of the experiment.Results: Xylopic acid produced significant anti-hyperglycaemic and analgesic effects in the cold allodynia and thermal hyperalgesia tests. Sperm motility, viability and count were significantly restored at 10 mg/kg XA as compared higher doses and negative control. The outcome of haematological analysis revealed a protective effect of XA although histological damage liver and kidney due to alloxan treatment was observable.Conclusions: Xylopic acid ameliorates diabetic neuropathy in rats and does not exert detrimental effects at low doses

Chemical profile and in vivo toxicity evaluation of unripe Citrus aurantifolia essential oil

Citrus aurantifolia (Christm.) Swingle (syn. C. MEDICA var. ACIDA Brandis) (family: Rutaceae) essential oil is one of the cheapest oils found in local markets. Although, it is generally accepted as non-toxic to vital organs and cells, majority of people are cynical about it usage. Herein, the present study reports the chemical composition and in vivo oral toxicity study of unripe C. aurantifolia essential oil found in Ghana. The toxicity of C. aurantifolia essential oil extract was investigated via oral administration using two methods: The acute toxicity single dose study (SDS) and the repeated dose method. The oil exhibited no acute toxicity but in the sub-chronic studies, the effects was dose and time-dependent. Chemical profile investigation of the oil showed 9 constituent of phytochemicals (Germacrene isomers (61.2%), Pineen (14%), Linalool dimmer (2.9%), Bornane (11%), Citral (2.9%), Anethole (1.5%), Anisole (1.1%), Safrole (0.3%) and Demitol (0.6%)). Histopathological studies revealed conditions such as necrosis, edema and inflammatory reaction in the liver, spleen and kidneys. Marginal upsurge of biochemical parameters above normal and elevated levels of lymphocytes (35.20–46.40 g/dL) demonstrated mild toxicity among the 100 mg/kg and 500 mg/kg dose groups at the sub-chronic stage. Low levels of hemoglobin (13.60 to 12.70 g/dL), MCV (34.20–24.0 fL), MCH (40.20–36.40 g/dL) along with high levels of liver enzymes confirmed the mild toxicity of the oil at sub-chronic stage. These results demonstrate that, despite consideration of lime essential oil as safe, it can have mild hematotoxic, nephrotoxic and hepatotoxic effects

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

Anti-inflammatory, anti-nociceptive and antipyretic activity of young and old leaves of Vernonia amygdalina

Background Both young and old leaves of Vernonia amygdalina (VA) are traditionally used to treat inflammation, pain and fever. However, the efficacy of young and old leaves for treating these ailments have not been compared till date. Aim To ascertain the effect of young and old leaves of VA in managing inflammation, pain and fever. Methods Both quantitative and qualitative phytochemical screening of ethanol extracts of young (EthYL) and old (EthOL) leaves of VA were performed. The anti-inflammatory activity of orally administered EthYL and EthOL (50–200 mg/kg) and Diclofenac (10 mg/kg) were evaluated in carrageenan-induced inflammation model in rats. Antipyretic activity of EthYL, EthOL and Aspirin (25 mg/kg) were assessed in the Baker’s yeast-induced pyrexia model. Anti-allodynic effect of both extracts were evaluated by inserting inflamed paws of rats in cold water. Antinociceptive property of the extracts were assessed using tail withdrawal and formalin-induced nociception test. Histopathological examination of the paws was performed, in addition to formalin test to understand the possible mechanism of action of the extracts. Negative control rats received 2 ml/kg normal saline in all tests. Results The amount of flavonoids, alkaloids, tannins, and phenolics were significantly (p \u3c 0.05) higher in EthOL than EthYL, while saponins were significantly higher (p \u3c 0.05) in EthYL than EthOL. The antioxidant ability and total antioxidant capacity were significantly (p \u3c 0.05) higher in EthYL than EthOL. However, this was significantly (p \u3c 0.05) lower than the anti-oxidant activity of Ascorbic acid. A dose-dependent increase in anti-inflammatory, antipyretic and antinociceptive properties were observed in both EthYL and EthOL, similar to the standard drugs. Mast cell degranulation accompanied by vasodilatation and high leukocytosis were observed in the negative control, but were markedly low in extract treated groups. Both extracts mediated their analgesic effect through opioidergic and nitric oxide pathways with EthYL additionally implicating the muscarinic cholinergic system. Conclusion Although both EthYL and EthOL alleviate inflammation, pyrexia and nociception, EthYL of VA was found to be more potent than EthOL

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

HER-2 Protein Overexpression in Patients with Gastric and Oesophageal Adenocarcinoma at a Tertiary Care Facility in Ghana

The prognosis of gastric and oesophageal adenocarcinoma remains generally poor. However, mounting evidence suggests a positive role of human epidermal growth factor receptor-2 (HER-2) expression in the prognosis of patients with these cancers. In this work, the patterns of HER-2 protein expression were determined in patients with gastric or oesophageal adenocarcinoma. Retrospectively, we reviewed records of gastric and oesophageal biopsies received from 2008 to 2012 and their corresponding archived formalin-fixed paraffin-embedded tissue blocks selected for immunohistochemical analysis. The prevalence of gastric and oesophageal adenocarcinomas and their association with HER-2 protein overexpression were evaluated. Gastric adenocarcinoma made up 18.79% of the gastric biopsies reviewed, and majority of these cancers occurred in males. Regarding the tumour type, HER-2 overexpression was common in the intestinal subtype compared to the diffuse type. Although squamous cell carcinoma was observed to be the commonest (31%) tumour type in the oesophagus compared to adenocarcinoma (8.79%), HER-2 was overexpressed in 42.9% of oesophageal adenocarcinomas, like gastric adenocarcinoma (41.4%). There is a high prevalence of gastric and oesophageal adenocarcinoma, with significant overexpression of HER-2 in these tumours, a window of hope for the management of patients with these cancers

Development and evaluation of methodologies for analysis of CTC and ctDNA in patients with ovarian carcinoma

About 70% of patients with ovarian cancer (OC) are diagnosed at an advanced stage (III-IV), with associated poor prognosis, even after chemotherapeutic interventions (mostly platinum taxane-based), leading to poor survival rates. The current biomarkers available in the clinic are not enough for efficient prognostication and surveillance of OC. Hence, more accurate and reliable biomarkers of patient disease status are urgently required. Currently, histopathological evaluation of tumour biopsies is the gold standard in clinical practice with the purpose of evaluating specific biomarkers to predict therapeutic response. However, this is invasive, and associated complications may occur. Liquid biopsy is a minimally invasive test and has the advantage of allowing real-time monitoring of treatment response and comprehensive serial/repetitive phenotypic and genotypic profiling of the primary, metastatic and recurrent tumours. Circulating tumour cells (CTC) and circulating tumour DNA (ctDNA) represent a new generation of biomarkers that can be used for predicting therapy responsiveness and longitudinal monitoring of OC patients undergoing therapy. This thesis describes a series of investigations in OC which include, a methodological study to improve phenotypic characterization of detected CTCs, genomic validation of putative CTCs, and the evaluation of the clinical validity of ctDNA as biomarker of response to neoadjuvant chemotherapy (NACT). This thesis consists of six chapters: a general introduction to OC (Chapter 1); a thorough review of the literature regarding CTCs and ctDNA in OC (Chapter 2); three results chapters (Chapters 3 - 5); and a final chapter that comprises of a general discussion of the main findings, the limitations of the study and future directions (Chapter 6). The first chapter (Chapter 1) of the thesis includes a review of the literature, which consist of the characterisation and clinical landscape of OC, while briefly introducing the topic of liquid biopsy. This is then followed by a comprehensive review (Chapter 2) that highlights the progress in the field of liquid biopsy for OC from 2011 to 2019, focusing specifically on CTCs and ctDNA as potential biomarkers for the management of the disease. Based on the limitations identified in this review (in Chapter 2), a multi-marker antibody staining protocol was developed to target epithelial (cytokeratin (CK) and EpCAM), mesenchymal (vimentin), and OC specific (PAX8) markers for detection of CTCs (Chapter 3). CD45/16 and CD31 were used for the exclusion of white blood and vascular endothelial cells, respectively. PD-L1 was used for CTC characterisation. Optimisation of the CTC markers were done using OC cell lines, SKOV-3 and OVCA432. CTCs were enriched using the ParsortixTM (Angle plc.) system, and further detected using the multifluorescent markers from 16 OC patients. Patients were found to have heterogeneous CTCs, with 113/157 (72%) cells positive for CK/EpCAM (epithelial marker), 58/157 (37%) positive for vimentin (mesenchymal marker), and 17/157 (11%) positive for both (hybrid). Positivity for PD-L1 was significantly associated with the hybrid phenotype when compared with the epithelial (p = 0.0009) and mesenchymal (p = 0.007) expressing CTCs. In Chapter 4, we performed copy number alteration (CNA) profiling on putative CTCs that were previously identified in Chapter 3. CNA were detected in both CK/EpCAM and vimentin positive CTCs. However, a proportion of cells expressing CK/EpCAM, PD-L1 and CD31 were found not to carry CNAs. Further characterisation of CTCs in combination with clinical outcomes is needed to provide insight into the role that epithelial to mesenchymal plasticity and vasculogenic mimicry plays in OC and its relationship with PD-L1 expression. In addition, ctDNA was analysed in 58 patients with high grade serous ovarian cancer (HGSOC) enrolled in a neoadjuvant chemotherapy (NACT) plus immune checkpoint inhibitor clinical trial (iPRIME, ACTRN12618000109202) (Chapter 5). Cell free DNA (cfDNA) was sequenced using the Oncomine™ Breast cfDNA Research Assay V2, and selected mutations were validated using droplet digital PCR (ddPCR). For ctDNA, the frequencies of genes affected by non-synonymous somatic mutations included TP53 (67%), KRAS (11%), PIK3CA (2%) and SF3B1 (2%). A total of 41 gene variants were identified in either pre- or post-NACT samples; 36 (87.8%) of them had variants in TP53 alone, and 3 (7.4%) in KRAS and 1 (2.4%) each for PIK3CA and SF3B1. Undetectable levels of ctDNA post, but not prior to NACT was associated with chemotherapy response score 3 (CRS3) (p = 0.04) when compared with CRS1/2. Comparison of mutant copies per mL of plasma quantified by NGS and ddPCR demonstrated a strong correlation (Pearson’s r = 0.929; p \u3c 0.0001) between the two platforms. Longitudinal monitoring of ctDNA specific mutant gene variants using ddPCR, showed a predictive lead time, and anticipates disease progression earlier (7-12 weeks) compared to monitoring patients by analysis of serum levels of CA-125. These results suggest that ctDNA could be used as a biomarker to determine patients’ clinical outcomes and serve as a potential endpoint for clinical trials. Finally, Chapter 6 provides a general discussion of the studies covered in this thesis, highlighting potential contributions to the growing field of liquid biopsy and its application for the clinical management of OC patients. The limitations inherent to both CTCs and ctDNA analysis, and suggestions for improvements before its successful implementation in routine clinical practice, are comprehensively discussed