Angiogenesis and Ovarian Cancer: What Potential Do Different Subtypes of Circulating Endothelial Cells Have for Clinical Application?

Ovarian cancer (OC) remains the most fatal disease of gynaecologic malignant tumours. The neovasculature in the tumour microenvironment principally comprises endothelial cells. Haematogenous cancer metastases are significantly impacted by tumour neovascularisation, which predominantly depends on the tumour-derived endothelial vasculogenesis. There is an urgent need for biomarkers for the diagnosis, prognosis and prediction of drug response. Endothelial cells play a key role in angiogenesis and other forms of tumour vascularisation. Subtypes of circulating endothelial cells may provide interesting non-invasive biomarkers of advanced OC that might have the potential to be included in clinical analysis for patients' stratification and therapeutic management. In this review, we summarise the reported studies on circulating endothelial subtypes in OC, detailing their isolation methods as well as their potential diagnostic, prognostic, predictive and therapeutic utility for clinical application. We highlight key biomarkers for the identification of circulating endothelial cell subtypes and their targets for therapies and critically point out future challenges.

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.

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.

Expression of RAS and RAB interactor 1 (RIN1) in head and neck tumors at selected hospital in Ghana.

BACKGROUND: Head and neck tumors (HNT) are tumors of the paranasal sinuses, the salivary glands and the upper aerodigestive tract. RIN1 is a Ras effector protein regulating epithelial cell properties and has been implicated in a number of cancers. METHOD: The aim of this study was to investigate the expression of RIN1 in head and neck tumors. RIN1 expression was assessed using quantitative real-time PCR (qRT-PCR) and immunohistochemical staining on archival head and neck tissue samples between 2014 and 2020. RESULTS: RIN1 expression was low in tissue samples as compared with the normal head and neck tissues. High and low RIN1 levels were compared with ages ≤40, >40 in the head and neck tumors of p-value 0.02. There was a significant difference with p-values of 0.001 when poor and well-moderate malignant tumors were compared. CONCLUSION: Our data suggests that RIN1may play an important role in head and neck tumor progression and that its expression may provide baseline data to facilitate identification of new molecular targeted therapeutics.

Therapeutic Benefit of Vernonia amygdalina in the Treatment of Diabetes and Its Associated Complications in Preclinical Studies.

Diabetes mellitus (DM), a complex heterogeneous metabolic disorder characterized by a defect in the function of insulin, is on the rapid rise globally. Sustained hyperglycemia which is a major sign of DM is linked to the generation of reactive oxygen species which promotes adverse complications of the disorder. Traditional herbal treatment of DM is a common practice in Africa and other tropical parts of the world. Vernonia amygdalina (VA), one of the highly researched species in the Asteraceae family, has proven to possess potent antidiabetic properties. Several phytochemicals identified in multiple extracts from VA are purported to be responsible for the antidiabetic potential of the plant. In this review, we discuss the therapeutic potential of VA in diabetes and its associated complications. We appraise the current evidence and further suggest potential areas that could be effectively exploited in future VA research on diabetes.

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.

Molecular Analysis of HBV in Histologically Confirmed Hepatocellular Carcinoma in a Tertiary Hospital in Ghana.

Background: Hepatocellular carcinoma (HCC) is a leading cause of death in Africa. Viral hepatitis B is a leading cause of hepatocellular cancer in Ghana and most African countries except Egypt where hepatitis C virus is more prevalent. This study aims at reviewing the histopathological patterns of HCC and its association with hepatitis B virus in our environment. Methodology: Demographics and histological diagnosis were retrieved from the surgical daybook and archival FFPE tissue samples with histopathologically confirmed HCC were used for this study. Sections (10µm) were taken from the tissues and digested to obtain DNA lysates. The DNA lysates were used in polymerase chain reaction (PCR) to determine the prevalence of HBV in the biopsies. Result: Of the 24 confirmed cases of HCC seen in the 5-year period, there were 17 males and 7 females with M:F ratio of 2.4:1. The mean age of our patients was 39.92 ± 1.98 years with age range 13-85 years. 50% of the cases were moderately differentiated while 25% each were well and poorly differentiated. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Out of the 24 archival HCC biopsies screened, HBV DNA PCR amplification was achieved in 11 (45.83%) after the restriction fragment length polymorphism PCR reaction. Eight of the 11 cases were found in the male and 3 in females. Of the 11 (45.83%) samples that were positive for HBV DNA, 3 were above 40 years and 8 were 40 years and younger. Conclusion: The overall prevalence of HBV DNA in our study was 45.83% and a greater proportion seen in ≤ 40 years. This suggests that most of our patients are infected with HBV early in life in our environment.

Evaluation of PD-L1 expression on circulating tumour cells in small-cell lung cancer.

Background: Antibodies against the programmed death-1 (PD-1) receptor and its ligand (PD-L1) have been recently approved for small-cell lung cancer (SCLC) treatment. Circulating tumour cells (CTCs) have emerged as an appealing liquid biopsy candidate that could enhance treatment decision-making in systemic therapy for SCLC patients. Several current technologies enrich CTCs using specific surface epitopes, size, rigidity, or dielectric properties. However, they are hampered by the heterogeneity of the enriched cells from blood samples. Methods: We evaluated two CTC enrichment systems: EpCAM conjugated to magnetic beads and a microfluidic device (Parsortix, Angle plc). PD-L1 expression was evaluated on the isolated CTCs. Twenty-three blood samples were collected from 21 patients with SCLC. PD-L1 expression was determined on CTCs through immunofluorescent staining. Results: CTCs were found in 14/23 (60.9%) of the samples, with 11/23 (47.8%) through EpCAM-coated magnetic beads (range, 4-1,611 CTCs/8 mL; median =5) and 11/20 (55.0%) using the Parsortix system (range, 1-165 CTCs/8 mL; median =4). Notably, a total of 17 EpCAM-negative CTCs were isolated using the Parsortix system. PD-L1 expression was detected on 268 of the 3,501 (7.7%) CTCs isolated with EpCAM-coated beads and in 33/366 (9.0%) of the CTCs isolated with the Parsortix system. No vimentin expression was observed in any of the detected CTCs. Conclusions: Overall, we identified a population of EpCAM-negative SCLC CTCs and showed that PD-L1 expression can be assessed on CTCs from SCLC patients. Comparison to tumour and treatment outcomes is needed to validate the potential of CTCs as an alternative sample for the assessment of PD-L1 expression in SCLC.

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.

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.

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 < 0.05) higher in EthOL than EthYL, while saponins were significantly higher (p < 0.05) in EthYL than EthOL. The antioxidant ability and total antioxidant capacity were significantly (p < 0.05) higher in EthYL than EthOL. However, this was significantly (p < 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.

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.

Immunotherapy for acute myeloid leukemia (AML): a potent alternative therapy.

The standard therapy of AML for many years has been chemotherapy with or without stem transplantation. However, there has not been any tangible improvement in this treatment beyond induction through chemotherapy and consolidation with allogeneic stem cell transplantation or chemotherapy. Residual AML cells which later cause relapse mostly persist even after rigorous standard therapy. It is imperative therefore to find an alternative therapy that can take care of the residual AML cells. With a better understanding of how the immune system works to destroy tumor cells and inhibit their growth, another therapeutic option immunotherapy has emerged to address the difficulties associated with the standard therapy. Identification of leukemia-associated antigens (LAA) and the fact that T and NK cells can be activated to exert cytotoxicity on AML cells have further introduced diverse immunotherapeutic development strategies. This review discusses the merits of current immunotherapeutic strategies such as the use of antibodies, adoptive T cells and alloreactive NK cell, and vaccination as against the standard therapy of AML.

Corrigendum to "Antidiabetic Effect of Young and Old Ethanolic Leaf Extracts of Vernonia amygdalina: A Comparative Study".

[This corrects the article DOI: 10.1155/2016/8252741.].

Antidiabetic Effect of Young and Old Ethanolic Leaf Extracts of Vernonia amygdalina: A Comparative Study.

The young leaves of Vernonia amygdalina are often utilized as vegetable and for medicinal purpose compared to the old leaves. This study was designed to evaluate and compare the antidiabetic effects between ethanolic leaf extracts of old and young V. amygdalina on streptozotocin (STZ) induced diabetic rat for four weeks. Preliminary screening of both young and old ethanolic extracts revealed the presence of the same phytochemicals except flavonoids which was only present in the old V. amygdalina. Difference in antioxidant power between the young and old leaf extracts was statistically significant (p < 0.05). Both leaf extracts produced a significant (p < 0.05) antihyperglycaemic effect. Also results from treated rats revealed increasing effect in some haematological parameters. Similarly, the higher dose (300 mg/kg) of both extracts significantly (p < 0.05) reduced serum ALT, AST, and ALP levels as compared to the diabetic control rats. Results also showed significant (p < 0.05) decrease in LDL-C and VLDL-C in the extract-treated rats with a corresponding increase in HDL-C, as compared to the diabetic control rats. Moreover histopathological analysis revealed ameliorative effect of pathological insults induced by the STZ in the pancreas, liver, and spleen, most significantly the regeneration of the beta cells of the islets of Langerhans in treated rats.