Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Microsatellite instability assessment is instrumental for Predictive, Preventive and Personalised Medicine: status quo and outlook.

A form of genomic alteration called microsatellite instability (MSI) occurs in a class of tandem repeats (TRs) called microsatellites (MSs) or short tandem repeats (STRs) due to the failure of a post-replicative DNA mismatch repair (MMR) system. Traditionally, the strategies for determining MSI events have been low-throughput procedures that typically require assessment of tumours as well as healthy samples. On the other hand, recent large-scale pan-tumour studies have consistently highlighted the potential of massively parallel sequencing (MPS) on the MSI scale. As a result of recent innovations, minimally invasive methods show a high potential to be integrated into the clinical routine and delivery of adapted medical care to all patients. Along with advances in sequencing technologies and their ever-increasing cost-effectiveness, they may bring about a new era of Predictive, Preventive and Personalised Medicine (3PM). In this paper, we offered a comprehensive analysis of high-throughput strategies and computational tools for the calling and assessment of MSI events, including whole-genome, whole-exome and targeted sequencing approaches. We also discussed in detail the detection of MSI status by current MPS blood-based methods and we hypothesised how they may contribute to the shift from conventional medicine to predictive diagnosis, targeted prevention and personalised medical services. Increasing the efficacy of patient stratification based on MSI status is crucial for tailored decision-making. Contextually, this paper highlights drawbacks both at the technical level and those embedded deeper in cellular/molecular processes and future applications in routine clinical testing.

Extracellular vesicles derived from dental mesenchymal stem/stromal cells with gemcitabine as a cargo have an inhibitory effect on the growth of pancreatic carcinoma cell lines in vitro.

Extracellular vesicles (EVs) are nowadays a target of interest in cancer therapy as a successful drug delivering tool. Based on their many beneficial biocompatible properties are designed to transport nucleic acids, proteins, various nanomaterials or chemotherapeutics. Extracellular vesicles derived from mesenchymal stem/stromal cells (MSCs) possess their tumor-homing abilities. This inspired us to engineer the MSC's EVs to be packed with chemotherapeutic agents and deliver it as a Trojan horse directly into tumor cells. In our study, human dental pulp MSCs (DP-MSCs) were cultivated with gemcitabine (GCB), which led to its absorption by the cells and subsequent secretion of the drug out into conditioned media in EVs. Concentrated conditioned media containing small EVs (potentially exosomes) significantly inhibited the cell growth of pancreatic carcinoma cell lines in vitro. DP-MSCs were simultaneously engineered to express a suicide gene fused yeast cytosinedeaminase:uracilphosphoribosyltransferase (yCD::UPRT). The product of the suicide gene converts non-toxic prodrug 5-fluorocytosine (5-FC) to highly cytotoxic chemotherapeutic drug 5-fluorouracil (5-FU) in the recipient cancer cells. Conversion of 5-FC to 5-FU had an additional effect on cancer cell's growth inhibition. Our results showed a therapeutic potential for DP-MSC-EVs to be designed for successful delivering of chemotherapeutic drugs, together with prodrug suicide gene therapy system.

Induction of AML Preleukemic Fusion Genes in HSPCs and DNA Damage Response in Preleukemic Fusion Gene Positive Samples.

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by several research groups and estimated to be 1-5% in umbilical cord blood (UCB) of healthy newborns, PFGs that are relevant to acute myeloid leukemia (AML) were poorly investigated. Therefore, this study is focused on the estimation of the incidence of the most frequent AML PFGs in newborns. For the first time, this study considered the inducibility of AML PFGs in different subsets of UCB HSPCs by low-dose γ-rays and also compared endogenous DNA damage, apoptosis, and reactive oxygen species (ROS) level between UCB samples containing or lacking AML PFGs. We found that: (i) the incidence of AML PFGs in UCB was 3.19% for RUNX1-RUNX1T1, 3.19% for PML-RARα, and 1.17% for KMT2A-MLLT3, (ii) 50 cGy of γ-rays did not induce RUNX1-RUNX1T1, PML-RARα, or KMT2A-MLLT3 PFGs in different subsets of sorted and expanded HSPCs, and (iii) the AML PFG+ samples accumulated the same level of endogenous DNA damage, as measured by the γH2AX/53BP1 focus formation, and also the same ROS level, and apoptosis as compared to PFG- controls. Our study provides critical insights into the prevalence of AML PFGs in UCB of newborns, without the evidence of a specific HSPC population more susceptible for PFG formation after irradiation to low-dose γ-rays or increased amount of ROS, apoptosis and DNA damage.

Partial hydatidiform moles with unclear histopathological profile - importance of genetic diagnostics.

OBJECTIVE: The aim of the study was the genetic characterization of a set of cases with an unclear morphological profile of the placental tissue suspected of a partial hydatidiform mole. PATIENTS AND METHODS: This work presents the results of a genetic analysis of a group of 10 patients with various clinical manifestations of reproductive loss, where a partial hydatidiform mole was suspected on the basis of a histopathological examination. The composition of the genome of the products of conception was determined by short tandem repeats (STR) genotyping using a commercial kit;Devyser Compact v3 (Devyser). RESULTS AND CONCLUSIONS: Out of 10 analyzed cases, five had diandric monogynic triploid genome, characteristic for a partial mole. Aneuploidies of chromosomes 13, 18, 21, X and Y were excluded in four cases and Pataus syndrome was dia-gnosed in one case. In the case of an unclear histopathological profile, consultative DNA analysis (ideally STR genotyping) can significantly help the pathologist in the differential dia-gnosis of a partial mole. The histopathological profile of a partial hydatidiform mole may be in some cases incomplete and unclear, especially in the early weeks of gestation, which can lead to false negativity of the examination. On the other hand, other pathologies, for example aneuploides or digynic triploidy, may produce a histopathological profile similar to a partial mole, which leads to false positivity. Accurate dia-gnosis of a partial hydatidiform mole using molecular genetic methods contributes to the determination of adequate dispensary care for patients.

Preleukemic fusion genes typical for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a highly heterogeneous subtype of leukemia, accounting for 25 % of childhood leukemias. By the presence of genetic mutations in hematopoietic/ progenitor stem cells, the bone marrow produces a large number of abnormal undifferentiated leukocytes (blasts), which significantly impairs the proper differentiation of cells. AML is induced by two interventions. Chromosomal translocation during hematopoiesis of intrauterine development is the first intervention. This creates preleukemic fusion genes (PFG), which can later be transformed by a second intervention (point genetic mutation - deletion, insertion ) into a functional malignant clone. Characteristic AML fusion genes include AML1-ETO, PML-RARA or MLL-AF9, which in turn produce hybrid proteins with altered function. Several studies suggest that these PFGs are considered an important prognostic tool in disease assessment. While the incidence of PFG characteristic of acute lymphoblastic leukemia (ALL) has been relatively well studied by several research groups and has been estimated at 1 to 5% in the umbilical cord blood of healthy neonates, PFG relevant to AML are still not sufficiently clarified.