Our hereby presented methodology is suitable for reliable assessment of the most common unavoidable DNA modifications which arise as a product of fundamental metabolic processes. 8-Oxoguanine, one of the oxidatively modified DNA bases, is a typical biomarker of oxidative stress. A noncanonical base, uracil, may be also present in small quantities in DNA. A set of ten-eleven translocation (TET) proteins are involved in oxidation of 5-methylcytosine to 5-hydroxymethylcytosine which can be further oxidized to 5-formylcytosine and 5-carboxycytosine. 5-Hydroxymethyluracil may be formed in deamination reaction of 5-hydroxymethylcytosine or can be also generated by TET enzymes. All of the aforementioned modifications seem to play some regulatory roles. We applied isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) for direct measurement of the 5-methyl-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, 5-carboxy-2'-deoxycytidine, 5-(hydroxymethyl)-2'-deoxyuridine, 2'-deoxyuridine, and 8-oxo-2'-deoxyguanosine. Analyses of DNA extracted from matched human samples showed that the 5-(hydroxymethyl)-2'-deoxycytidine level was 5-fold lower in colorectal carcinoma tumor in comparison with the normal one from the tumor's margin; also 5-formyl-2'-deoxycytidine and 5-carboxy-2'-deoxycytidine were lower in colorectal carcinoma tissue (ca. 2.5- and 3.5-fold, respectively). No such differences was found for 2'-deoxyuridine and 5-(hydroxymethyl)-2'-deoxyuridine. The presented methodology is suitable for fast, accurate, and complex evaluation of an array of endogenously generated DNA deoxynucleosides modifications. This novel technique could be used for monitoring of cancer and other diseases related to oxidative stress, aberrant metabolism, and environmental exposure. Furthermore, the fully automated two-dimensional separation is extremely useful for analysis of material containing a considerable amount of coeluting interferents with mass-spectrometry-based methods.

Genomic instability is a hallmark of cancer, and it is well-known that in several cancers the karyotype is unstable and rapidly evolving. Molecular cytogenetics has contributed to the description and interpretation of cancer karyotypes, in particular through multicolor FISH approaches which can define even complex chromosome rearrangements. The introduction of genome-wide methods has made available a powerful set of tools with higher resolution than cytogenetics, thus appropriate to comprehend the huge variability of cancer cells. This review focuses on novel findings deriving from the combination of cytogenetic and genomic approaches in cancer research.

Induction pemetrexed platin doublet is the standard of care in locally advanced and metastatic non squamous NSCLC. Maintenance TKI has been successfully explored to sustain benefit achieved after induction therapy especially in EGFR mutation positive NSCLC. The aim of this study is to evaluate outcomes with maintenance TKI in EGFR mutation positive metastatic non squamous NSCLC after induction pemetrexed-platin doublet. The objective is to calculate progression freesurvival rate, overall survival rate and factors affecting outcomes.

Iatrogenic tumorigenesis is a major limitation for the use of human induced pluripotent stem cells (hiPSCs) in hematology. The teratoma risk comes from the persistence of hiPSCs in differentiated cell populations. Our goal was to evaluate the best system to purge residual hiPSCs before graft without compromising hematopoietic repopulation capability. Teratoma risk after systemic injection of hiPSCs expressing the reporter gene luciferase was assessed for the first time. Teratoma formation in immune-deficient mice was tracked by in vivo bioimaging. We observed that systemic injection of hiPSCs produced multisite teratoma as soon as 5 weeks after injection. To eliminate hiPSCs before grafting, we tested the embryonic-specific expression of suicide genes under the control of the pmiR-302/367 promoter. This promoter was highly active in hiPSCs but not in differentiated cells. The gene/prodrug inducible Caspase-9 (iCaspase-9)/AP20187 was more efficient and rapid than thymidine kinase/ganciclovir, fully specific, and without bystander effect. We observed that iCaspase-9-expressing hiPSCs died in a dose-dependent manner with AP20187, without reaching full eradication in vitro. Unexpectedly, nonspecific toxicity of AP20187 on iCaspase-9-negative hiPSCs and on CD34+ cells was evidenced in vitro. This toxic effect strongly impaired CD34+ -derived human hematopoiesis in adoptive transfers. Survivin inhibition is an alternative to the suicide gene approach because hiPSCs fully rely on survivin for survival. Survivin inhibitor YM155 was more efficient than AP20187/iCaspase-9 for killing hiPSCs, without toxicity on CD34+ cells, in vitro and in adoptive transfers. hiPSC purge by survivin inhibitor fully eradicated teratoma formation in immune-deficient mice. This will be useful to improve the safety management for hiPSC-based medicine. Stem Cells Translational Medicine 2017;6:382-393.

Glioblastoma (GBM), an aggressive grade IV astrocytoma, is the most common primary malignant adult brain tumor characterized by extensive invasiveness, heterogeneity, and angiogenesis. Standard treatment options such as radiation and chemotherapy have proven to be only marginally effective in treating GBM because of its invasive nature. Therefore, extensive efforts have been put forth to develop tumor-tropic stem cells as viable therapeutic vehicles with potential to treat even the most invasive tumor cells that are harbored within areas of normal brain. To this end, we discovered a newly described NG2-expressing cell that we isolated from a distinct pericyte subtype found abundantly in cultures derived from peripheral muscle. In this work, we show the translational significance of these peripherally derived neural-like stem cells (NLSC) and their potential to migrate toward tumors and act as therapeutic carriers. We demonstrate that these NLSCs exhibit in vitro and in vivo GBM tropism. Furthermore, NLSCs did not promote angiogenesis or transform into tumor-associated stromal cells, which are concerns raised when using other common stem cells, such as mesenchymal stem cells and induced neural stem cells, as therapeutic carriers. We also demonstrate the potential of NLSCs to express a prototype therapeutic, tumor necrosis factor α-related apoptosis-inducing ligand and kill GBM cells in vitro. These data demonstrate the therapeutic potential of our newly characterized NLSC against GBM. Stem Cells Translational Medicine 2017;6:471-481.

Tumor development and the generation of induced pluripotent stem cells are highly comparable processes with striking similarities. Cellular plasticity is inherent to tumor evolution, rendering cells that acquire a stem cell-like phenotype, for which Sox2 activation has proved instrumental for the plastic acquisition of stemness properties in tumor cells. Understanding the molecular mechanisms underlying both events might uncover novel approaches for the development of anticancer therapeutics and constitute model systems for understanding tumor generation and ensuring the biosafety of cell-based therapies. Stem Cells Translational Medicine 2017;6:335-339.

Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c-Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c-Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum-free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS-like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient-derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c-Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS-like cells by Rb knockdown and c-Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512-526.

Survival and proliferation of Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (CHL), are dependent on constitutive activation of nuclear factor kB (NF-κB). A20, encoded by TNF alpha-induced protein 3 (TNFAIP3), one of the inhibitors of NF-kB, was found to be inactivated by deletions and/or point mutations in CHL.

An important challenge in cancer biology is to understand the complex aspects of the disease. It is increasingly evident that genes are not isolated from each other and the comprehension of how different genes are related to each other could explain biological mechanisms causing diseases. Biological pathways are important tools to reveal gene interaction and reduce the large number of genes to be studied by partitioning it into smaller paths. Furthermore, recent scientific evidence has proven that a combination of pathways, instead than a single element of the pathway or a single pathway, could be responsible for pathological changes in a cell.

More than fifty percent of neuroblastoma (NB) patients with adverse prognosis do not benefit from treatment making the identification of new potential targets mandatory. Hypoxia is a condition of low oxygen tension, occurring in poorly vascularized tissues, which activates specific genes and contributes to the acquisition of the tumor aggressive phenotype. We defined a gene expression signature (NB-hypo), which measures the hypoxic status of the neuroblastoma tumor. We aimed at developing a classifier predicting neuroblastoma patients' outcome based on the assessment of the adverse effects of tumor hypoxia on the progression of the disease.

In the study of complex diseases using genome-wide expression data from clinical samples, a difficult case is the identification and mapping of the gene signatures associated to the stages that occur in the progression of a disease. The stages usually correspond to different subtypes or classes of the disease, and the difficulty to identify them often comes from patient heterogeneity and sample variability that can hide the biomedical relevant changes that characterize each stage, making standard differential analysis inadequate or inefficient.

Detecting somatic mutations in whole exome sequencing data of cancer samples has become a popular approach for profiling cancer development, progression and chemotherapy resistance. Several studies have proposed software packages, filters and parametrizations. However, many research groups reported low concordance among different methods. We aimed to develop a pipeline which detects a wide range of single nucleotide mutations with high validation rates. We combined two standard tools - Genome Analysis Toolkit (GATK) and MuTect - to create the GATK-LODN method. As proof of principle, we applied our pipeline to exome sequencing data of hematological (Acute Myeloid and Acute Lymphoblastic Leukemias) and solid (Gastrointestinal Stromal Tumor and Lung Adenocarcinoma) tumors. We performed experiments on simulated data to test the sensitivity and specificity of our pipeline.

Kinase over-expression and activation as a consequence of gene amplification or gene fusion events is a well-known mechanism of tumorigenesis. The search for novel rearrangements of kinases or other druggable genes may contribute to understanding the biology of cancerogenesis, as well as lead to the identification of new candidate targets for drug discovery. However this requires the ability to query large datasets to identify rare events occurring in very small fractions (1-3 %) of different tumor subtypes. This task is different from what is normally done by conventional tools that are able to find genes differentially expressed between two experimental conditions.

ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian histone H3K4 methyltransferase complexes, including mixed lineage leukemia (MLL) complexes. ASH2L protein levels in primary leukemia patient samples have not yet been defined. We analyzed ASH2L protein expression in 511 primary AML patient samples using reverse phase protein array (RPPA) technology. We discovered that ASH2L expression is significantly increased in a subset of patients carrying fms-related tyrosine kinase 3 (FLT3) mutations. Furthermore, we observed that low levels of ASH2L are associated with increased overall survival. We also compared ASH2L levels to the expression of 230 proteins previously analyzed on this array. ASH2L expression was inversely correlated with 32 proteins, mostly involved in cell adhesion and cell cycle inhibition, while a positive correlation was observed for 50 proteins, many of which promote cell proliferation. Together, these results indicate that a lower level of ASH2L protein is beneficial to AML patients.

Mouse F9 cells differentiate into primitive endoderm (PrE) following the activation of the canonical WNT-β-catenin pathway. The upregulation of Wnt6 and activation of β-catenin-TCF-LEF-dependent transcription is known to accompany differentiation, but the Frizzled (FZD) receptor responsible for transducing the WNT6 signal is not known. Eight of the 10 Fzd genes were found to be expressed in F9 cells, with Fzd7 being the most highly expressed, and chosen for further analysis. To alter steady-state Fzd7 levels and test the effect this has on differentiation, siRNA and overexpression approaches were used to knock-down and ectopically express the Fzd7 message, respectively. siRNA knock-down of Fzd7 resulted in reduced DAB2 levels, and the overexpression activated a TCF-LEF reporter, but neither approach affected differentiation. Our focus turned to how canonical WNT6 signaling was attenuated to allow PrE cells to form parietal endoderm (PE). Dkk1, encoding a WNT antagonist, was examined and results showed that its expression increased in F9 cells treated with retinoic acid (RA) or overexpressing Wnt6. F9 cells overexpressing human DKK1 or treated with DKK1-conditioned medium and then treated with RA failed to differentiate, indicating that a negative feedback loop involving WNT6 and DKK1 attenuates canonical WNT-β-catenin signaling, thereby allowing PE cells to differentiate.

PRPS1 (phosphoribosyl pyrophosphate synthetase 1), which drives the nucleotide biosynthesis pathway, modulates a variety of functions by providing central building blocks and cofactors for cell homeostasis. As tumor cells often display abnormal nucleotide metabolism, dysregulated de-novo nucleotide synthesis has potential impacts in cancers. We now report that PRPS1 is specifically and highly expressed in chemoresistant (CR) cancer cells derived from cisplatin-resistant human breast cancer cell lines SK-BR-3 and MCF-7. The inhibition of PRPS1 activity in CR cells by genetic silencing reduces cell viability and increases apoptosis in vitro, both of which can be further potentiated by cisplatin treatment. Significantly, such down-regulation of PRPS1 in CR cells when administered to nude mice enhanced the survival of those animals, as demonstrated by decreased tumor growth. Knockdown of PRPSI may cause these effects by potently inducing autonomous activation of caspase-3 and inhibiting the proliferation in the engrafted CR tumors. As a result, cisplatin sensitivity in a xenograft model of CR cancer cells can be restored by the down-regulation of PRPS1. Thus, PRPS1 inhibition may afford a therapeutic approach to relapsed patients with breast cancer, resistant to chemotherapy.

E-cadherin and β-catenin are two molecules implicated in cellular adhesion. The reduced expression of β-catenin and E-cadherin is associated with the tumoral epithelial-mesenchymal transition process, a key event in the development of endometrial carcinoma. The aim of our study was to investigate the association between the immunoexpression of β-catenin÷E-cadherin and the tumor differentiation degree, presence of lymph nodes, depth of tumor invasion and pTNM stage of endometrioid endometrial carcinomas in order to enhance the potential aggressiveness of these tumors. Our results revealed significant differences in the expression of β-catenin, when grouping for the tumor stage, invasion in the myometrium and degree of differentiation, as well as significant differences in the expression of E-cadherin for tumor degrees of differentiation. E-cadherin and β-catenin expression was stronger in well-differentiated tumors, superficial myometrium invasion and early tumor stages I or II, thus was associated with better prognostic forms of endometrioid endometrial carcinoma. Our study indicated that decreased of the E-cadherin and β-catenin expression is useful for the assessment of tumor aggressivity.

A tumor represents an abnormal tissue growth that can arise from any ocular structure, such as eyelids, muscles or the optic nerve. At the eyelids, there are two main tumor types: basal cell carcinoma and squamous cell carcinoma. Angiogenesis plays a crucial role in growth, invasion and metastasis processes of any tumor. It is well known the fact that without new vessels formation tumors cannot exceed 1-2 mm diameter. Immunohistochemical analysis has been performed on 43 cases of primary carcinomas of the eyelid, diagnosed between 2010 and 2014 in the Laboratory of Pathological Anatomy of the University Emergency County Hospital of Craiova, Romania. Biological material was represented by surgical resection samples, coming from the Clinic of Ophthalmology the anteriorly named Hospital. Within the immunohistochemical study, we have evaluated epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) expression in a group of 43 cutaneous carcinomas of the eyelid, depending on the type and differentiation grade of the tumor. Of the 43 samples, 23 came from patients with eyelid basal cell carcinoma and 20 came from patients with eyelid squamous cell carcinoma. In our study, EGFR and VEGF immunoexpression was superior for squamous cell carcinomas, compared to basal cell carcinomas, fact that was statistically significant. Regarding squamous cell carcinomas, the immunoexpression of these two markers was superior in moderate÷poor differentiated forms, compared to well differentiated forms, fact that was statistically significant. The markers used in this study were found to be associated with the acquisition of aggression and angiogenic phenotypes by analyzed carcinomas.