Current status of in vitro models for rare gynaecological cancer research.

Gynaecological cancers originate within the female reproductive system and are classified according to the site in the reproductive system where they arise. However, over 50 % of these malignancies are categorized as rare, encompassing 30 distinct histological subtypes, which complicates their diagnosis and treatment. The focus of this review is to give an overview of established in vitro models for the investigation of rare gynaecological cancers, as well as an overview of available online databases that contain detailed descriptions of cell line characteristics. Cell lines represent the main models for the research of carcinogenesis, drug resistance, pharmacodynamics and novel therapy treatment options. Nowadays, classic 2D cell models are increasingly being replaced with 3D cell models, such as spheroids, organoids, and tumoroids because they provide a more accurate representation of numerous tumour characteristics, and their response to therapy differs from the response of adherent cell lines. It is crucial to use the correct cell line model, as rare tumour types can show characteristics that differ from the most common tumour types and can therefore respond unexpectedly to classic treatment. Additionally, some cell lines have been misclassified or misidentified, which could lead to false results. Even though rare gynaecological cancers are rare, this review will demonstrate that there are available options for investigation of such cancers in vitro on biologically relevant models.

HIGH-DOSE INSULIN EUGLYCEMIC THERAPY.

Calcium channel blockers and beta-blockers toxicity/poisoning are one of the most common causes of poisoning. More importantly, they are among the deadliest types of poisoning caused by cardiac drugs that emergency physicians can encounter. Common toxidrome caused by these medications includes the following symptoms: hypotension, bradycardia, hypoglycemia/hyperglycemia, hypothermia, arrhythmia, and seizures. Treatment is usually complex, It consists of administration of various medications, such as crystalloids, intravenous calcium, glucagon, vasopressors/inotropes, and especially high-dose insulin euglycemic therapy. In this paper, we will review the mechanism for this type of treatment, propose a potential protocol for its application and address possible adverse effects. High-dose insulin euglycemic therapy should be an integral part of the treatment protocol for calcium channel blockers and beta-blockers toxicity.

cfDNA methylation in liquid biopsies as potential testicular seminoma biomarker.

Background: Seminoma is a testicular tumor type, routinely diagnosed after orchidectomy. As cfDNA represents a source of minimally invasive seminoma patient management, this study aimed to investigate whether cfDNA methylation of six genes from liquid biopsies, have potential as novel seminoma biomarkers. Materials & methods: cfDNA methylation from liquid biopsies was assessed by pyrosequencing and compared with healthy volunteers' samples. Results: Detailed analysis revealed specific CpGs as possible seminoma biomarkers, but receiver operating characteristic curve analysis showed modest diagnostic performance. In an analysis of panels of statistically significant CpGs, two DNA methylation panels emerged as potential seminoma screening panels, one in blood CpG8/CpG9/CpG10 (KITLG) and the other in seminal plasma CpG1(MAGEC2)/CpG1(OCT3/4). Conclusion: The presented data promote the development of liquid biopsy epigenetic biomarkers in the screening of seminoma patients.

Seminoma belongs to testicular cancer, which represents a common malignancy among men of reproductive age. Diagnosis of seminoma is a multistep process that also includes checking tumor biomarkers from blood. However, these biomarkers are not specific for seminoma and to conclude a definite diagnosis of seminoma immunohistochemical analysis is needed, which requires the removal of a whole or partial testicle. Therefore, there is a need for novel, noninvasive biomarkers. cfDNA is the most extensively investigated source of minimally invasive tumor markers. Therefore, this study investigated cfDNA methylation of six genes as potential noninvasive biomarkers for the management of seminoma patients. By examining CpG sites of selected genes by pyrosequencing, the authors detected significant differences. However, receiver operating characteristic curve analysis showed modest results. Therefore, the authors tested possible panels of significantly different CpGs and detected two possible DNA methylation panels for seminoma screening. These findings suggest the further investigation of possible epigenetic biomarkers for seminoma patient management from liquid biopsies.

CNV Hotspots in Testicular Seminoma Tissue and Seminal Plasma.

Seminoma (SE) is the most frequent type of testicular tumour, affecting predominantly young men. Early detection and diagnosis of SE could significantly improve life quality and reproductive health after diagnosis and treatment. Copy number variation (CNV) has already been associated with various cancers as well as SE. In this study, we selected four genes (MAGEC2, NANOG, RASSF1A, and KITLG) for CNV analysis in genomic DNA (gDNA), which are located on chromosomes susceptible to gains, and whose aberrant expression was already detected in SE. Furthermore, CNV was analysed in cell-free DNA (cfDNA) from seminal plasma. Analysis was performed by droplet digital polymerase chain reaction (ddPCR) on gDNA from SE and nonmalignant testicular tissue. Seminal plasma cfDNA from SE patients before and after surgery was analysed, as well as from healthy volunteers. The CNV hotspot in gDNA from SE tissue was detected for the first time in all analysed genes, and for two genes, NANOG and KITLG it was reflected in cfDNA from seminal plasma. Although clinical value is yet to be determined, presented data emphasize a potential use of CNV as a potential SE biomarker from a liquid biopsy.

Epigenetically inactivated RASSF1A as a tumor biomarker.

RASSF1A, one of the eight isoforms of the RASSF1 gene, is a tumor suppressor gene that influences tumor initiation and development. In cancer, RASSF1A is frequently inactivated by mutations, loss of heterozygosity, and, most commonly, by promoter hypermethylation. Epigenetic inactivation of RASSF1A was detected in various cancer types and led to significant interest; current research on RASSF1A promoter methylation focuses on its roles as an epigenetic tumor biomarker. Typically, researchers analyzed genomic DNA (gDNA) to measure the amount of RASSF1A promoter methylation. Cell-free DNA (cfDNA) from liquid biopsies is a recent development showing promise as an early cancer diagnostic tool using biomarkers, such as RASSF1A. This review discusses the evidence on aberrantly methylated RASSF1A in gDNA and cfDNA from different cancer types and its utility for early cancer diagnosis, prognosis, and surveillance. We compared methylation frequencies of RASSF1A in gDNA and cfDNA in various cancer types. The weaknesses and strengths of these analyses are discussed. In conclusion, although the importance of RASSSF1A methylation to cancer has been established is included in several diagnostic panels, its diagnostic utility is still experimental.

In Search of TGCT Biomarkers: A Comprehensive In Silico and Histopathological Analysis.

Testicular germ cell tumors (TGCTs) are ever more affecting the young male population. Germ cell neoplasia in situ (GCNIS) is the origin of TGCTs, namely, seminomas (SE) and a heterogeneous group of nonseminomas (NS) comprising embryonal carcinoma, teratoma, yolk sac tumor, and choriocarcinoma. Response to the treatment and prognosis, especially of NS, depend on precise diagnosis with a necessity for discovery of new biomarkers. We aimed to perform comprehensive in silico analysis at the DNA, RNA, and protein levels of six prospective (HOXA9, MGMT, CFC1, PRSS21, RASSF1A, and MAGEC2) and six known TGCT biomarkers (OCT4, SOX17, SOX2, SALL4, NANOG, and KIT) and assess its congruence with histopathological analysis in all forms of TGCTs. Cancer Hallmarks Analytics Tool, the Search Tool for the Retrieval of Interacting Genes/Proteins database, and UALCAN, an interactive web resource for analyzing cancer OMICS data, were used. In 108 TGCT and 48 tumor-free testicular samples, the immunoreactivity score (IRS) was calculated. SE showed higher frequency in DNA alteration, while DNA methylation was significantly higher for all prospective biomarkers in NS. In GCNIS, we assessed the clinical positivity of RASSF1 and PRSS21 in 52% and 62% of samples, respectively, in contrast to low or nil positivity in healthy seminiferous tubules, TGTCs as a group, SE, NS, or all NS components. Although present in approximately 80% of healthy seminiferous tubules (HT) and GCNIS, HOXA9 was diagnostically positive in 64% of TGCTs, while it was positive in 82% of NS versus 29% of SE. Results at the DNA, mRNA, and protein levels on putative and already known biomarkers were included in the suggested panels that may prove to be important for better diagnostics of various forms of TGCTs.