An invasion front gene expression signature for higher-risk patient selection in stage IIA MSS colon cancer.

Stage II colon cancer (CC) encompasses a heterogeneous group of patients with diverse survival experiences: 87% to 58% 5-year relative survival rates for stages IIA and IIC, respectively. While stage IIA patients are usually spared the adjuvant chemotherapy, some of them relapse and may benefit from it; thus, their timely identification is crucial. Current gene expression signatures did not specifically target this group nor did they find their place in clinical practice. Since processes at invasion front have also been linked to tumor progression, we hypothesize that aside from bulk tumor features, focusing on the invasion front may provide additional clues for this stratification. A retrospective matched case-control collection of 39 stage IIA microsatellite-stable (MSS) untreated CCs was analyzed to identify prognostic gene expression-based signatures. The endpoint was defined as relapse within 5 years vs. no relapse for at least 6 years. From the same tumors, three different classifiers (bulk tumor, invasion front, and constrained baseline on bulk tumor) were developed and their performance estimated. The baseline classifier, while the weakest, was validated in two independent data sets. The best performing signature was based on invasion front profiles [area under the receiver operating curve (AUC) = 0.931 (0.815-1.0)] and contained genes associated with KRAS pathway activation, apical junction complex, and heme metabolism. Its combination with bulk tumor classifier further improved the accuracy of the predictions.

Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies.

This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

A Tagging Polymorphism in Fat Mass and Obesity-Associated (FTO) Gene Is Associated with Sepsis Status in Children.

INTRODUCTION: Sepsis is one of the most common causes of death in patients admitted to intensive care units (ICUs). The development of sepsis is significantly influenced by genetic predisposition. In this study, we highlight a potential association between a variant of the fat mass and obesity-associated (FTO) gene and risk of sepsis in children and adolescents. METHODS: We investigated a first-intron tagging FTO polymorphism (rs17817449) by comparing a severe condition (SC) group, comprising 598 paediatric patients (ages 0-19 years) admitted to an ICU with fever, systemic inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, or multiple organ dysfunction syndrome (MODS), with a control group consisting of 616 healthy young adults. RESULTS: We observed a lower prevalence (p < 0.01; OR = 0.59, 95% CI = 0.39-0.87) of the FTO TT genotype in febrile and SIRS patients compared to patients with severe illness. There was a borderline trend towards a lower prevalence of the FTO TT genotype in the control group compared to the SC group (p < 0.09, OR = 0.81, 95% CI = 0.62-1.06). CONCLUSIONS: Our findings suggest that rs17817449, a common FTO polymorphism, may be a predictor of sepsis in paediatric patients, and that higher body weight is protective against this clinical complication.

Identification of Plasmatic MicroRNA-206 as New Predictor of Early Recurrence of Atrial Fibrillation After Catheter Ablation Using Next-generation Sequencing.

BACKGROUND: Catheter ablation (CA) of atrial fibrillation (AF) is indicated in patients with recurrent and symptomatic AF episodes. Despite the strict inclusion/exclusion criteria, AF recurrence after CA remains high. Identification of a novel biomarker that would predict AF recurrence would help to stratify the patients. The aim of the study was to seek novel biomarkers among the plasmatic microRNAs (miRNAs, miRs). METHODS: A prospective monocentric study was conducted. A total of 49 consecutive AF patients indicated for CA were included. Blood sampling was performed prior to CA. RNA was isolated from plasma using commercial kits. In the exploration phase, small RNA sequencing was performed in ten AF patients (five with and five without AF recurrence) using Illumina instrument. In the validation phase, levels of selected miRNAs were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in all participants. RESULTS: Altogether, 22 miRNAs were identified as altered between the groups by next-generation sequencing (using the DESeq2 algorithm). Using qRT-PCR, levels of the five most altered miRNAs (miR-190b/206/326/505-5p/1296-5p) were verified in the whole cohort. Plasma levels of hsa-miR-206 were significantly higher in patients with early (within 6 months) AF recurrence and showed an increase of risk recurrence,2.65 times by every increase in its level by 1 unit in the binary logistic regression. CONCLUSION: We have identified a set of 22 plasmatic miRNAs that differ between the patients with and without AF recurrence after CA and confirmed hsa-miR-206 as a novel miRNA associated with early AF recurrence. Results shall be verified in a larger independent cohort.

Critical appraisal of the piRNA-PIWI axis in cancer and cancer stem cells.

Small noncoding RNAs play an important role in various disease states, including cancer. PIWI proteins, a subfamily of Argonaute proteins, and PIWI-interacting RNAs (piRNAs) were originally described as germline-specific molecules that inhibit the deleterious activity of transposable elements. However, several studies have suggested a role for the piRNA-PIWI axis in somatic cells, including somatic stem cells. Dysregulated expression of piRNAs and PIWI proteins in human tumors implies that, analogously to their roles in undifferentiated cells under physiological conditions, these molecules may be important for cancer stem cells and thus contribute to cancer progression. We provide an overview of piRNA biogenesis and critically review the evidence for the role of piRNA-PIWI axis in cancer stem cells. In addition, we examine the potential of piRNAs and PIWI proteins to become biomarkers in cancer.

PredictONCO: a web tool supporting decision-making in precision oncology by extending the bioinformatics predictions with advanced computing and machine learning.

PredictONCO 1.0 is a unique web server that analyzes effects of mutations on proteins frequently altered in various cancer types. The server can assess the impact of mutations on the protein sequential and structural properties and apply a virtual screening to identify potential inhibitors that could be used as a highly individualized therapeutic approach, possibly based on the drug repurposing. PredictONCO integrates predictive algorithms and state-of-the-art computational tools combined with information from established databases. The user interface was carefully designed for the target specialists in precision oncology, molecular pathology, clinical genetics and clinical sciences. The tool summarizes the effect of the mutation on protein stability and function and currently covers 44 common oncological targets. The binding affinities of Food and Drug Administration/ European Medicines Agency -approved drugs with the wild-type and mutant proteins are calculated to facilitate treatment decisions. The reliability of predictions was confirmed against 108 clinically validated mutations. The server provides a fast and compact output, ideal for the often time-sensitive decision-making process in oncology. Three use cases of missense mutations, (i) K22A in cyclin-dependent kinase 4 identified in melanoma, (ii) E1197K mutation in anaplastic lymphoma kinase 4 identified in lung carcinoma and (iii) V765A mutation in epidermal growth factor receptor in a patient with congenital mismatch repair deficiency highlight how the tool can increase levels of confidence regarding the pathogenicity of the variants and identify the most effective inhibitors. The server is available at https://loschmidt.chemi.muni.cz/predictonco.

Tazemetostat in the therapy of pediatric INI1-negative malignant rhabdoid tumors.

Rhabdoid tumors are aggressive tumors that may arise in the kidney, soft tissue, central nervous system, or other organs. They are defined by SMARCB1 (INI1) or SMARCA4 alterations. Often, very young children are affected, and the prognosis is dismal. Four patients with primary atypical teratoid rhabdoid tumor (AT/RT, a rhabdoid tumor in the central nervous system) were treated by resection and high dose chemotherapy. Tazemetostat was introduced after completion of chemotherapy. Three patients have achieved an event free survival of 32, 34, and 30 months respectively. One progressed and died. His overall survival was 20 months. One patient was treated for a relapsed atypical teratoid rhabdoid tumor. The treatment combined metronomic therapy, radiotherapy, tazemetostat and immunotherapy. This patient died of disease progression, with an overall survival of 37 months. One patient was treated for a rhabdoid tumor of the ovary. Tazemetostat was given as maintenance after resection, chemotherapy, and radiotherapy, concomitantly with immunotherapy. Her event free survival is 44 months. Only approximately 40% of patients with rhabdoid tumors achieve long-term survival. Nearly all relapses occur within two years from diagnosis. The event free survival of four of the six patients in our cohort has exceeded this timepoint. Tazemetostat has been mostly tested as a single agent in the relapsed setting. We present promising results when applied as maintenance or add on in the first line treatment.

Molecular portraits of colorectal cancer morphological regions.

Heterogeneity of colorectal carcinoma (CRC) represents a major hurdle towards personalized medicine. Efforts based on whole tumor profiling demonstrated that the CRC molecular subtypes were associated with specific tumor morphological patterns representing tumor subregions. We hypothesize that whole-tumor molecular descriptors depend on the morphological heterogeneity with significant impact on current molecular predictors. We investigated intra-tumor heterogeneity by morphology-guided transcriptomics to better understand the links between gene expression and tumor morphology represented by six morphological patterns (morphotypes): complex tubular, desmoplastic, mucinous, papillary, serrated, and solid/trabecular. Whole-transcriptome profiling by microarrays of 202 tumor regions (morphotypes, tumor-adjacent normal tissue, supportive stroma, and matched whole tumors) from 111 stage II-IV CRCs identified morphotype-specific gene expression profiles and molecular programs and differences in their cellular buildup. The proportion of cell types (fibroblasts, epithelial and immune cells) and differentiation of epithelial cells were the main drivers of the observed disparities with activation of EMT and TNF-α signaling in contrast to MYC and E2F targets signaling, defining major gradients of changes at molecular level. Several gene expression-based (including single-cell) classifiers, prognostic and predictive signatures were examined to study their behavior across morphotypes. Most exhibited important morphotype-dependent variability within same tumor sections, with regional predictions often contradicting the whole-tumor classification. The results show that morphotype-based tumor sampling allows the detection of molecular features that would otherwise be distilled in whole tumor profile, while maintaining histopathology context for their interpretation. This represents a practical approach at improving the reproducibility of expression profiling and, by consequence, of gene-based classifiers.

Role of the microbiome in pathophysiology of necrotising enterocolitis in preterm neonates.

Although necrotising enterocolitis (NEC) is a serious, life-threatening disease, improved neonatal care is increasing the number of survivors with NEC among extremely preterm neonates. Therapy is nevertheless mostly symptomatic and the mortality rate remains high, especially among neonates requiring surgery. Therefore, it is important to focus on preventing the disease and modifiable risk factors. NEC's pathophysiology is multifaceted, with key factors being immaturity of the immune and barrier protective mechanisms of the premature gut and exaggerated proinflammatory reaction to insults like gut hypoxia, enteral nutrition or microbial dysbiosis. The role of the intestinal microbiome in the pathophysiology of NEC has been a subject of research for many years, but to date no specific pathogen or type of dysbiosis has been connected with NEC development. This review assesses current knowledge as to the role of the intestinal microbiota in the pathophysiology of NEC and the possibilities for positively influencing it.

MiR-4649-5p acts as a tumor-suppressive microRNA in triple negative breast cancer by direct interaction with PIP5K1C, thereby potentiating growth-inhibitory effects of the AKT inhibitor capivasertib.

BACKGROUND: Triple negative breast cancer (TNBC) is a particularly aggressive and difficult-to-treat subtype of breast cancer that requires the development of novel therapeutic strategies. To pave the way for such developments it is essential to characterize new molecular players in TNBC. MicroRNAs (miRNAs) constitute interesting candidates in this regard as they are frequently deregulated in cancer and contribute to numerous aspects of carcinogenesis. METHODS AND RESULTS: Here, we discovered that miR-4649-5p, a miRNA yet uncharacterized in breast cancer, is associated with better overall survival of TNBC patients. Ectopic upregulation of the otherwise very low endogenous expression levels of miR-4646-5p significantly decreased the growth, proliferation, and migration of TNBC cells. By performing whole transcriptome analysis and physical interaction assays, we were able to identify the phosphatidylinositol phosphate kinase PIP5K1C as a direct target of miR-4649-5p. Downregulation or pharmacologic inhibition of PIP5K1C phenocopied the growth-reducing effects of miR-4649-5p. PIP5K1C is known to play an important role in migration and cell adhesion, and we could furthermore confirm its impact on downstream PI3K/AKT signaling. Combinations of miR-4649-5p upregulation and PIP5K1C or AKT inhibition, using the pharmacologic inhibitors UNC3230 and capivasertib, respectively, showed additive growth-reducing effects in TNBC cells. CONCLUSION: In summary, miR-4649-5p exerts broad tumor-suppressive effects in TNBC and shows potential for combined therapeutic approaches targeting the PIP5K1C/PI3K/AKT signaling axis.

CCL2/MCP-1, interleukin-8, and fractalkine/CXC3CL1: Potential biomarkers of epileptogenesis and pharmacoresistance in childhood epilepsy.

OBJECTIVE: The pathophysiological processes leading to epileptogenesis and pharmacoresistance in epilepsy have been the subject of extensive preclinical and clinical research. The main impact on clinical practice is the development of new targeted therapies for epilepsy. We studied the importance of neuroinflammation in the development of epileptogenesis and pharmacoresistance in childhood epilepsy patients. METHODS: A cross-sectional study conducted at two epilepsy centers in the Czech Republic compared 22 pharmacoresistant patients and 4 pharmacodependent patients to 9 controls. We analyzed the ProcartaPlex™ 9-Plex immunoassay panel consisting of interleukin (IL)-6, IL-8, IL-10, IL-18, CXCL10/IP-10, monocyte chemoattractant protein 1 (CCL2/MCP-1), B lymphocyte chemoattractant (BLC), tumor necrosis factor-alpha (TNF-α), and chemokine (C-X3-X motif) ligand 1 (fractalkine/CXC3CL1) to determine their alterations in cerebrospinal fluid (CSF) and blood plasma, concurrently. RESULTS: The analysis of 21 paired CSF and plasma samples in pharmacoresistant patients compared to controls revealed a significant elevation of CCL2/MCP-1 in CSF (p < 0.000512) and plasma (p < 0.00.017). Higher levels of fractalkine/CXC3CL1 were revealed in the plasma of pharmacoresistant patients than in controls (p < 0.0704), and we determined an upward trend in CSF IL-8 levels (p < 0.08). No significant differences in CSF and plasma levels were detected between pharmacodependent patients and controls. CONCLUSION: Elevated CCL2/MCP-1 in CSF and plasma, elevated levels of fractalkine/CXC3CL1 in CSF, and a trend toward elevated IL-8 in the CSF of patients with pharmacoresistant epilepsy indicate these cytokines as potential biomarkers of epileptogenesis and pharmacoresistance. CCL2/MCP-1was detected in blood plasma; this assessment may be easily achieved in clinical practice without the invasiveness of a spinal tap. However, due to the complexity of neuroinflammation in epilepsy, further studies are warranted to confirm our findings.

Targeted treatment of severe vascular malformations harboring PIK3CA and TEK mutations with alpelisib is highly effective with limited toxicity.

This was a prospective cohort study of eighteen patients with large and debilitating vascular malformations with one or more major systemic complications. In all patients, we discovered activating alterations in either TEK or PIK3CA. Based on these findings, targeted treatment using the PI3K inhibitor alpelisib was started with regular check-ups, therapy duration varied from 6 to 31 months. In all patients, marked improvement in quality of life was observed. We observed radiological improvement in fourteen patients (two of them being on combination with either propranolol or sirolimus), stable disease in 2 patients. For 2 patients, an MRI scan was not available as they were shortly on treatment, however, a clinically visible response in size reduction or structure regression, together with pain relief was observed. In patients with elevated D-dimer levels before alpelisib administration, a major improvement was noted, suggesting its biomarker role. We observed overall very good tolerance of the treatment, documenting a single patient with grade 3 hyperglycemia. Patients with size reduction were offered local therapies wherever possible. Our report presents a promising approach for the treatment of VMs harboring different targetable TEK and PIK3CA gene mutations with a low toxicity profile and high efficacy.

Driver and actionable mutations in younger patients with lung cancer - are we searching properly?

AIMS: The authors focused on a group of young lung cancer patients with the aim of better understanding the mechanisms of tumor pathogenesis in these patients and search for potential targetable mutations. METHODS: We collected retrospective data on patients under 40 years diagnosed with lung cancer (NSCLC or small-cell lung cancer) from 2011-2020 at the Department of Respiratory Diseases, University Hospital Brno, Czech Republic. Tumor tissue of these patients was analysed by next-generation sequencing (NGS, a panel of 550 variants in 19 genes). Demographic characteristics, smoking history, histology, molecular-genetic results and clinical stage of the disesase were recorded in all eligible patients from accessible medical databases. RESULTS: Of 17 identified patients in only 8 cases was successful NGS carried out due to lack of sufficient good quality material in the other cases. The most frequently found molecular genetic changes were EGFR, RICTOR and HER2 amplification and MET and FGFR1 amplification. In addition, we found rare pathogenic variants in BRAF and PIK3CA genes. Actionable variants were detected in 75% patients. CONCLUSION: We detected very frequent driver and potentially actionable alterations in young patients with lung cancer. This suggests different mechanisms of carcinogenesis in these patients and indicates that they might benefit more from a specific approach than older lung cancer patients.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Combination of Urinary MiR-501 and MiR-335 With Current Clinical Diagnostic Parameters as Potential Predictive Factors of Prostate Biopsy Outcome.

BACKGROUND: The detection of prostate cancer (PCa) is currently based on prostate-specific antigen (PSA) quantification as an initial screening followed by ultrasound-guided transrectal biopsy. However, the high rate of false-negative biopsies often leads to inappropriate treatment. Therefore, new molecular biomarkers, such as urine microRNAs (miRNAs), are a possible way to redefine PCa diagnostics. PATIENTS AND METHODS: Urine samples of 356 patients undergoing prostate biopsy (256 cases with confirmed prostate cancer, 100 cases with negative prostate biopsy) at the Masaryk Memorial Cancer Institute (Czech Republic) and additional 36 control subjects (healthy controls, benign prostatic hyperplasia - BPH) were divided into the discovery and validation cohorts and analyzed. In the discovery phase, small RNA sequencing was performed using the QIAseq miRNA Library Kit and the NextSeq 500 platform. Identified miRNA candidates were validated by the RT-qPCR method in the independent validation phase. RESULTS: Using the small RNA sequencing method, we identified 12 urine miRNAs significantly dysregulated between PCa patients and controls. Furthermore, independent validation showed the ability of miR-501-3p and the quantitative miR-335:miR-501 ratio to distinguish between PCa patients and patients with negative prostate biopsy. The subsequent combination of the miR-335:miR-501 ratio with PSA and total prostate volume (TPV) using logistic regression exceeded the analytical accuracy of standalone parameters [area under curve (AUC)=0.75, positive predictive value (PPV)=0.85, negative predictive value (NPV)=0.51)] and discriminated patients according to biopsy outcome. CONCLUSION: Combination of miR-335:miR-501 ratio with PSA and total prostate volume was able to identify patients with negative prostate biopsy and could potentially streamline decision making for biopsy indication.

Long non-coding RNAs enable precise diagnosis and prediction of early relapse after nephrectomy in patients with renal cell carcinoma.

PURPOSE: Renal cell carcinoma belongs among the deadliest malignancies despite great progress in therapy and accessibility of primary care. One of the main unmet medical needs remains the possibility of early diagnosis before the tumor dissemination and prediction of early relapse and disease progression after a successful nephrectomy. In our study, we aimed to identify novel diagnostic and prognostic biomarkers using next-generation sequencing on a novel cohort of RCC patients. METHODS: Global expression profiles have been obtained using next-generation sequencing of paired tumor and non-tumor tissue of 48 RCC patients. Twenty candidate lncRNA have been selected for further validation on an independent cohort of paired tumor and non-tumor tissue of 198 RCC patients. RESULTS: Sequencing data analysis showed significant dysregulation of more than 2800 lncRNAs. Out of 20 candidate lncRNAs selected for validation, we confirmed that 14 of them are statistically significantly dysregulated. In order to yield better discriminatory results, we combined several best performing lncRNAs into diagnostic and prognostic models. A diagnostic model consisting of AZGP1P1, CDKN2B-AS1, COL18A1, and RMST achieved AUC 0.9808, sensitivity 95.96%, and specificity 90.4%. The model for prediction of early relapse after nephrectomy consists of COLCA1, RMST, SNHG3, and ZNF667-AS1 and achieved AUC 0.9241 with sensitivity 93.75% and specificity 71.07%. Notably, no combination has outperformed COLCA1 alone. Lastly, a model for stage consists of ZNF667-AS1, PVT1, RMST, LINC00955, and TCL6 and achieves AUC 0.812, sensitivity 85.71%, and specificity 69.41%. CONCLUSION: In our work, we identified several lncRNAs as potential biomarkers and developed models for diagnosis and prognostication in relation to stage and early relapse after nephrectomy.

Disclosing quantitative RT-PCR raw data during manuscript submission: a call for action.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Challenges of in situ hybridization in miRNA analysis of triple-negative breast cancer morphological diversity.

miRNA expression in triple-negative breast cancers (TNBC) has mainly been studied from a methodological viewpoint. However, it has not been considered that miRNA expression profile may be associated with a specific morphological entity inside every tumor. The verification of this hypothesis on a set of 25 TNBCs was the subject of our previous work, where we confirmed specific expression of the studied miRNAs in 82 samples of different morphologies including inflammatory infiltrate, spindle cell, clear cell, and metastases after RNA extraction and purification as well as microchip and biostatistical analysis. In the current work, we demonstrate a low suitability of in situ hybridization method for miRNA detection compared to RT-qPCR, and in detail discuss the biological role of 8 miRNAs with the most significant changes of expression.

Spinal Metastasis in a Patient with Supratentorial Glioblastoma with Primitive Neuronal Component: A Case Report with Clinical and Molecular Evaluation.

Glioblastoma (GBM) is regarded as an aggressive brain tumor that rarely develops extracranial metastases. Despite well-investigated molecular alterations in GBM, there is a limited understanding of these associated with the metastatic potential. We herein present a case report of a 43-year-old woman with frontal GBM with primitive neuronal component who underwent gross total resection followed by chemoradiation. Five months after surgery, the patient was diagnosed with an intraspinal GBM metastasis. Next-generation sequencing analysis of both the primary and metastatic GBM tissues was performed using the Illumina TruSight Tumor 170 assay. The number of single nucleotide variants observed in the metastatic sample was more than two times higher. Mutations in TP53, PTEN, and RB1 found in the primary and metastatic tissue samples indicated the mesenchymal molecular GBM subtype. Among others, there were two inactivating mutations (Arg1026Ile, Trp1831Ter) detected in the NF1 gene, two novel NOTCH3 variants of unknown significance predicted to be damaging (Pro1505Thr, Cys1099Tyr), one novel ARID1A variant of unknown significance (Arg1046Ser), and one gene fusion of unknown significance, EIF2B5-KIF5B, in the metastatic sample. Based on the literature evidence, the alterations of NF1, NOTCH3, and ARID1A could explain, at least in part, the acquired invasiveness and metastatic potential in this particular GBM case.