Advances in Intraoperative Flow Cytometry.

Flow cytometry is the gold-standard laser-based technique to measure and analyze fluorescence levels of immunostaining and DNA content in individual cells. It provides a valuable tool to assess cells in the G0/G1, S, and G2/M phases, and those with polyploidy, which holds prognostic significance. Frozen section analysis is the standard intraoperative assessment for tumor margin evaluation and tumor resection. Here, we present flow cytometry as a promising technique for intraoperative tumor analysis in different pathologies, including brain tumors, leptomeningeal dissemination, breast cancer, head and neck cancer, pancreatic tumor, and hepatic cancer. Flow cytometry is a valuable tool that can provide substantial information on tumor analysis and, consequently, maximize cancer treatment and expedite patients' survival.

Enhancer DNA methylation in acute myeloid leukemia and myelodysplastic syndromes.

DNA methylation (CpG methylation) exerts an important role in normal differentiation and proliferation of hematopoietic stem cells and their differentiated progeny, while it has also the ability to regulate myeloid versus lymphoid fate. Mutations of the epigenetic machinery are observed in hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) resulting in hyper- or hypo-methylation affecting several different pathways. Enhancers are cis-regulatory elements which promote transcription activation and are characterized by histone marks including H3K27ac and H3K4me1/2. These gene subunits are target gene expression 'fine-tuners', are differentially used during the hematopoietic differentiation, and, in contrast to promoters, are not shared by the different hematopoietic cell types. Although the interaction between gene promoters and DNA methylation has extensively been studied, much less is known about the interplay between enhancers and DNA methylation. In hematopoiesis, DNA methylation at enhancers has the potential to discriminate between fetal and adult erythropoiesis, and also is a regulatory mechanism in granulopoiesis through repression of neutrophil-specific enhancers in progenitor cells during maturation. The interplay between DNA methylation at enhancers is disrupted in AML and MDS and mainly hyper-methylation at enhancers raising early during myeloid lineage commitment is acquired during malignant transformation. Interactions between mutated epigenetic drivers and other oncogenic mutations also affect enhancers' activity with final result, myeloid differentiation block. In this review, we have assembled recent data regarding DNA methylation and enhancers' activity in normal and mainly myeloid malignancies.

Platelet activation after endovascular repair of abdominal aortic aneurysm.

AIM: To investigate the effect of endovascular aneurysm repair (EVAR) on platelet (PLT) function and reveal possible associated factors. METHODS: Fifty consecutive patients were included. PLT count and activation (CD62P-CD36), white blood cell (WBC) count, and high sensitivity C-reactive protein (hs-CRP) were measured preoperatively, at the first and third postoperative day. RESULTS: EVAR elicited a significant reduction in PLT count from baseline to first day after EVAR (p < 0.001), while no significant difference was noted between the first and third day. Furthermore, CD62P expression was markedly elevated at the first day after EVAR (median % positive PLTs from 13.7 at baseline to 22.1, p = 0.05), but returned to baseline levels by the third day. Maximum abdominal aortic aneurysm diameter was the only factor that significantly affected the CD62P values (p = 0.005). Postoperative CD36 values were significantly correlated with total aneurysm volume (p = 0.05) and were higher in endografts made from polyester (p = 0.01). There were no correlation between PLT activation and hs-CRP, WBC, maximum temperature, and 30-day morbidity. CONCLUSION: EVAR has elucidated a significant reduction in PLT count and increase in PLT activation at the immediate postoperative period. The type of the endograft material and the aneurysm maximum diameter and volume appear to play an important role in PLT activation.

Polycomb group proteins and MYC: the cancer connection.

Polycomb group proteins (PcGs) are transcriptional repressors involved in physiological processes whereas PcG deregulation might result in oncogenesis. MYC oncogene is able to regulate gene transcription, proliferation, apoptosis, and malignant transformation. MYC deregulation might result in tumorigenesis with tumor maintenance properties in both solid and blood cancers. Although the interaction of PcG and MYC in cancer was described years ago, new findings are reported every day to explain the exact mechanisms and results of such interactions. In this review, we summarize recent data on the PcG and MYC interactions in cancer, and the putative involvement of microRNAs in the equation.

DLK1-DIO3 imprinted cluster in induced pluripotency: landscape in the mist.

DLK1-DIO3 represents an imprinted cluster which genes are involved in physiological cell biology as early as the stem cell level and in the pathogenesis of several diseases. Transcription factor-mediated induced pluripotent cells (iPSCs) are considered an unlimited source of patient-specific hematopoietic stem cells for clinical application in patient-tailored regenerative medicine. However, to date there is no marker established able to distinguish embryonic stem cell-equivalent iPSCs or safe human iPSCs. Recent findings suggest that the DLK1-DIO3 locus possesses the potential to represent such a marker but there are also contradictory data. This review aims to report the current data on the topic describing both sides of the coin.

The Role of Fast Cell Cycle Analysis in Pediatric Brain Tumors.

Cell cycle analysis by flow cytometry has not been adequately studied in pediatric brain tumors. We investigated the value of a modified rapid (within 6 min) cell cycle analysis protocol for the characterization of malignancy of pediatric brain tumors and for the differentiation of neoplastic from nonneoplastic tissue for possible intraoperative application. We retrospectively studied brain tumor specimens from patients treated at our institute over a 5-year period. All tumor samples were histopathologically verified before flow-cytometric analysis. The histopathological examination of permanent tissue sections was the gold standard. There were 68 brain tumor cases. All tumors had significantly lower G0/G1 and significantly higher S phase and mitosis fractions than normal brain tissue. Furthermore low-grade tumors could be differentiated from high-grade tumors. DNA aneuploidy was detected in 35 tumors. A correlation between S phase fraction and Ki-67 index was found in medulloblastomas and anaplastic ependymomas. Rapid cell cycle analysis by flow cytometry is a promising method for the identification of neoplastic tissue intraoperatively. Low-grade tumors could be differentiated from high-grade tumors. Thus, cell cycle analysis can be a valuable adjunct to the histopathological evaluation of pediatric brain tumors, whereas its intraoperative application warrants further investigation.

The microRNAs within the DLK1-DIO3 genomic region: involvement in disease pathogenesis.

The mammalian genome is transcribed in a developmentally regulated manner, generating RNA strands ranging from long to short non-coding RNA (ncRNAs). NcRNAs generated by intergenic sequences and protein-coding loci, represent up to 98 % of the human transcriptome. Non-coding transcripts comprise short ncRNAs such as microRNAs, piwi-interacting RNAs, small nucleolar RNAs and long intergenic RNAs, most of which exercise a strictly controlled negative regulation of expression of protein-coding genes. In humans, the DLK1-DIO3 genomic region, located on human chromosome 14 (14q32) contains the paternally expressed imprinted genes DLK1, RTL1, and DIO3 and the maternally expressed imprinted genes MEG3 (Gtl2), MEG8 (RIAN), and antisense RTL1 (asRTL1). This region hosts, in addition to two long intergenic RNAs, the MEG3 and MEG8, one of the largest microRNA clusters in the genome, with 53 miRNAs in the forward strand and one (mir-1247) in the reverse strand. Many of these miRNAs are differentially expressed in several pathologic processes and various cancers. A better understanding of the pathophysiologic importance of the DLK1-DIO3 domain-containing microRNA cluster may contribute to innovative therapeutic strategies in a range of diseases. Here we present an in-depth review of this vital genomic region, and examine the role the microRNAs of this region may play in controlling tissue homeostasis and in the pathogenesis of some human diseases, mostly cancer, when aberrantly expressed. The potential clinical implications of this data are also discussed.

Non-coding RNAs and EZH2 interactions in cancer: long and short tales from the transcriptome.

A large amount of data indicates that non-coding RNAs represent more than the "dark matter" of the genome. Both microRNAs and long non-coding RNAs are involved in several fundamental biologic processes, and their deregulation may lead in oncogenesis. Interacting with the Polycomb-repressive complex 2 subunit EZH2, they could affect the expression of protein-coding genes and form feedback networks and autoregulatory loops. They can also form networks with upstream and downstream important factors, in which EZH2 represent the stabilizing factor of the pathway. As such non-coding RNAs affect the epigenetic modifications leading to malignant transformation.

MicroRNAs mark in the MLL-rearranged leukemia.

MicroRNAs are short noncoding RNAs, known regulators of several signaling pathways cell differentiation and proliferation, development, and apoptosis, which are deregulated in acute leukemia. Mixed lineage leukemia (MLL) gene encodes a protein with histone methyltransferase activity, which is essential for the fine tuning of hematopoietic stem cell development and differentiation through the regulation of HOXA and MEIS1. MLL gene rearrangements characterize both acute myeloid and acute lymphoblastic leukemia associated with poor outcomes. MicroRNAs and MLL rearrangements are in tight association regulating each other expression, affecting cell cycle regulators, and composing complex networks with factors involved in leukemogenesis such as MYC and FLT3. MLL fusion genes are also capable of recruiting DNA methyltransferases at microRNAs promoters controlling their expression through epigenetic changes. Direct drug targeting of MLL has been difficult to achieve, and in this context, microRNA expression modulation represents an attractive approach.

Differences in the Profile of Circulating Immune Cell Subsets in Males with Type 2 Cardiorenal Syndrome versus CKD Patients without Established Cardiovascular Disease.

Maladaptive activation of the immune system plays a key role in the pathogenesis of chronic kidney disease (CKD). Our aim was to investigate differences in circulating immune cells between type 2 cardiorenal syndrome (CRS-2) patients and CKD patients without cardiovascular disease (CVD). CRS-2 patients were prospectively followed up, with the primary endpoint being all-cause and cardiovascular mortality. METHOD: A total of 39 stable males with CRS-2 and 24 male CKD patients matched for eGFR (CKD-EPI) were enrolled. A selected panel of immune cell subsets was measured by flow cytometry. RESULTS: Compared to CKD patients, CRS-2 patients displayed higher levels of proinflammatory CD14++CD16+ monocytes (p = 0.04) and T regulatory cells (Tregs) (p = 0.03), lower lymphocytes (p = 0.04), and lower natural killer cells (p = 0.001). Decreased lymphocytes, T-lymphocytes, CD4+ T-cells, CD8+ T-cells, Tregs, and increased CD14++CD16+ monocytes were associated with mortality at a median follow-up of 30 months (p < 0.05 for all). In a multivariate model including all six immune cell subsets, only CD4+ T-lymphocytes remained independent predictors of mortality (OR 0.66; 95% CI 0.50-0.87; p = 0.004). CONCLUSION: Patients with CRS-2 exhibit alterations in immune cell profile compared to CKD patients of similar kidney function but without CVD. In the CRS-2 cohort, CD4+ T-lymphocytes independently predicted fatal cardiovascular events.

Correlation of Lymphocyte Subpopulations, Clinical Features and Inflammatory Markers during Severe COVID-19 Onset.

BACKGROUND: Dysregulation of the immune response in the course of COVID-19 has been implicated in critical outcomes. Lymphopenia is evident in severe cases and has been associated with worse outcomes since the early phases of the pandemic. In addition, cytokine storm has been associated with excessive lung injury and concomitant respiratory failure. However, it has also been hypothesized that specific lymphocyte subpopulations (CD4 and CD8 T cells, B cells, and NK cells) may serve as prognostic markers for disease severity. The aim of this study was to investigate possible associations of lymphocyte subpopulations alterations with markers of disease severity and outcomes in patients hospitalized with COVID-19. MATERIALS/METHODS: A total of 42 adult hospitalized patients were included in this study, from June to July 2021. Flow-cytometry was used to calculate specific lymphocyte subpopulations on day 1 (admission) and on day 5 of hospitalization (CD45, CD3, CD3CD8, CD3CD4, CD3CD4CD8, CD19, CD16CD56, CD34RA, CD45RO). Markers of disease severity and outcomes included: burden of disease on CT (% of affected lung parenchyma injury), C-reactive protein and interleukin-6 levels. PO2/FiO2 ratio and differences in lymphocytes subsets between two timepoints were also calculated. Logistic and linear regressions were used for the analyses. All analyses were performed using Stata (version 13.1; Stata Corp, College Station, TX, USA). RESULTS: Higher levels of CD16CD56 cells (Natural Killer cells) were associated with higher risk of lung injury (>50% of lung parenchyma). An increase in CD3CD4 and CD4RO cell count difference between day 5 and day 1 resulted in a decrease of CRP difference between these timepoints. On the other hand, CD45RARO difference was associated with an increase in the difference of CRP levels between the two timepoints. No other significant differences were found in the rest of the lymphocyte subpopulations. CONCLUSIONS: Despite a low patient number, this study showed that alterations in lymphocyte subpopulations are associated with COVID-19 severity markers. It was observed that an increase in lymphocytes (CD4 and transiently CD45RARO) resulted in lower CRP levels, perhaps leading to COVID-19 recovery and immune response homeostasis. However, these findings need further evaluation in larger scale trials.

Intraoperative Flow Cytometry for the Evaluation of Meningioma Grade.

Meningiomas are the most frequent central nervous system tumors in adults. The majority of these tumors are benign. Nevertheless, the intraoperative identification of meningioma grade is important for modifying surgical strategy in order to reduce postoperative complications. Here, we set out to investigate the role of intraoperative flow cytometry for the differentiation of low-grade (grade 1) from high-grade (grade 2-3) meningiomas. The study included 59 patients. Intraoperative flow cytometry analysis was performed using the 'Ioannina Protocol' which evaluates the G0/G1 phase, S-phase, mitosis and tumor index (S + mitosis phase fraction) of a tumor sample. The results are available within 5 min of sample receipt. There were 41 grade 1, 15 grade 2 and 3 grade 3 meningiomas. High-grade meningiomas had significantly higher S-phase fraction, mitosis fraction and tumor index compared to low-grade meningiomas. High-grade meningiomas had significantly lower G0/G1 phase fraction compared to low-grade meningiomas. Thirty-eight tumors were diploids and twenty-one were aneuploids. No significant difference was found between ploidy status and meningioma grade. ROC analysis indicated 11.4% of tumor index as the optimal cutoff value thresholding the discrimination between low- and high-grade meningiomas with 90.2% sensitivity and 72.2% specificity. In conclusion, intraoperative flow cytometry permits the detection of high-grade meningiomas within 5 min. Thus, surgeons may modify tumor removal strategy.

Assessment of Gliomas' Grade of Malignancy and Extent of Resection Using Intraoperative Flow Cytometry.

BACKGROUND: Intraoperative Flow Cytometry (iFC) is a novel technique for the assessment of the grade of malignancy and the diagnosis of tumor type and resection margins during solid tumor surgery. Herein, we set out to analyze the role of iFC in the grading of gliomas and the evaluation of resection margins. MATERIAL AND METHODS: iFC uses a fast cell cycle analysis protocol (Ioannina Protocol) that permits the analysis of tissue samples within 5-6 min. Cell cycle analysis evaluated the G0/G1 phase, S-phase, mitosis, and tumor index (S + mitosis phase fraction) and ploidy status. In the current study, we evaluated tumor samples and samples from the peripheral borders from patients with gliomas who underwent surgery over an 8-year period. RESULTS: Eighty-one patients were included in the study. There were sixty-eight glioblastoma cases, five anaplastic astrocytomas, two anaplastic oligodendrogliomas, one pilocytic astrocytoma, three oligodendrogliomas and two diffuse astrocytomas. High-grade gliomas had a significantly higher tumor index than low grade gliomas (median value 22 vs. 7.5, respectively, p = 0.002). Using ROC curve analysis, a cut-off value of 17% in the tumor index could differentiate low- from high-grade gliomas with a 61.4% sensitivity and 100% specificity. All low-grade gliomas were diploid. From the high-grade gliomas, 22 tumors were aneuploid. In glioblastomas, aneuploid tumors had a significantly higher tumor index (p = 0.0018). Twenty-three samples from glioma margins were evaluated. iFC verified the presence of malignant tissue in every case, using histology as the gold standard. CONCLUSION: iFC constitutes a promising intraoperative technique for glioma grading and resection margin assessment. Comparative studies with additional intraoperative adjuncts are necessary.

DLK1-MEG3 imprinted domain microRNAs in cancer biology.

A rapidly growing body of evidence highlights the involvement of DLK1-MEG3 imprinted domain in cell biology and cancer pathogenesis. The imprinted domain contains protein-coding genes, long non-coding RNAs, and various small non-coding RNAs. The imprinted microRNAs located here interact with important transcription factors, modulate fundamental signaling cascades, form molecular signatures with diagnostic and prognostic potential, and could differentiate chemoresistant from chemosensitive disease. Moreover, as they can be detected in patients' serum, are easy to obtain, and can be used as adjuvant diagnostic biomarkers with the potential of monitoring disease progression and response to treatment.

Deregulated microRNAs in multiple myeloma.

MicroRNAs are short noncoding RNAS involved in gene expression regulation under physiological and pathological situations. They bind to mRNA of target genes and are potential regulators of gene expression at a post-transcription level through the RNA interference pathway. They are estimated to represent 1% to 2% of the known eukaryotic genome, and it has been demonstrated that they are involved in the pathogenesis of neurodegenerative diseases, cancer, metabolism disorders, and heart disease. MicroRNAs are known to act as tumor suppressors or oncogenes in cancer biology. The authors describe the current knowledge on microRNA involvement in regulatory pathways that characterize multiple myeloma pathogenesis gained from in vitro and in vivo studies. These small molecules interact with important factors such as p53, SOCS1, IGF-1, IGF-1R, vascular endothelial growth factor, NF-κB, and others. As such, microRNAs represent an attractive therapeutic target in the context of multiple myeloma interfering with the myeloma regulatory networks. Further studies are needed to better understand their role in myelomagenesis and their therapeutic potential.