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Background/Aim: Brain metastases (BMs) are the most frequent intracranial tumors in adults and one of the greatest challenges for modern oncology. Most are derived from lung, breast, renal cell, and colorectal carcinomas and melanomas. Up to 14% of patients are diagnosed with BMs of unknown primary, which are commonly characterized by an early and aggressive metastatic spread. It is important to discover novel biomarkers for early identification of BM origin, allowing better management of patients with this disease. Our study focused on microRNAs (miRNAs), which are very stable in frozen native and FFPE tissues and have been shown to be sensitive and specific diagnostic biomarkers of cancer. We aimed to identify miRNAs with significantly different expression in the five most frequent groups of BMs and develop a diagnostic classifier capable of sensitive and specific classification of BMs. Materials and Methods: Total RNA enriched for miRNAs was isolated using the mirVana miRNA Isolation Kit from 71 fresh-frozen histopathologically confirmed BM tissues originating in 5 cancer types. Sequencing libraries were prepared using the QIAseq miRNA Library Kit and sequenced on the NextSeq 500 platform. MiRNA expression was further validated by RT-qPCR. Results: Differential analysis identified 373 miRNAs with significantly different expression between 5 BM groups (p

B-cell receptor (BCR) signaling and T-cell interactions play a pivotal role in chronic lymphocytic leukemia (CLL) pathogenesis and disease aggressiveness. CLL cells can use microRNAs (miRNAs) and their targets to modulate microenvironmental interactions in the lymph node niches. To identify miRNA expression changes in the CLL microenvironment, we performed complex profiling of short noncoding RNAs in this context by comparing CXCR4/CD5 intraclonal cell subpopulations (CXCR4dimCD5bright vs CXCR4brightCD5dim cells). This identified dozens of differentially expressed miRNAs, including several that have previously been shown to modulate BCR signaling (miR-155, miR-150, and miR-22) but also other candidates for a role in microenvironmental interactions. Notably, all 3 miR-29 family members (miR-29a, miR-29b, miR-29c) were consistently down-modulated in the immune niches, and lower miR-29(a/b/c) levels associated with an increased relative responsiveness of CLL cells to BCR ligation and significantly shorter overall survival of CLL patients. We identified tumor necrosis factor receptor–associated factor 4 (TRAF4) as a novel direct target of miR-29s and revealed that higher TRAF4 levels increase CLL responsiveness to CD40 activation and downstream nuclear factor-κB (NF-κB) signaling. In CLL, BCR represses miR-29 expression via MYC, allowing for concurrent TRAF4 upregulation and stronger CD40–NF-κB signaling. This regulatory loop is disrupted by BCR inhibitors (bruton tyrosine kinase [BTK] inhibitor ibrutinib or phosphatidylinositol 3-kinase [PI3K] inhibitor idelalisib). In summary, we showed for the first time that a miRNA-dependent mechanism acts to activate CD40 signaling/T-cell interactions in a CLL microenvironment and described a novel miR-29–TRAF4–CD40 signaling axis modulated by BCR activity.

Introduction: Glioblastoma (GBM) is the most frequent primary astrocytoma characterized by an aggressive and diffuse growth and a poor prognosis. The median survival of patients with GBM is only 12 to 16 months from diagnosis despite conventional therapy. It is, therefore, important to identify new therapeutic targets, which would allow a more effective treatment. In our present study, we focus on long non-coding RNAs (lncRNAs), the regulators of gene expression in both physiological conditions and GBM pathology, and their potential as targets for future therapy. Material and Methods: Our study included 219 GBM patients and 29 intractable epilepsy patients as controls who signed an informed consent prior to treatment. Previously, cDNA libraries were prepared from 77 rRNA-depleted RNA samples and sequenced, and the expression of 11 significantly dysregulated lncRNAs was validated in 188 specimens by RT-qPCR. Results were newly validated in an independent TCGA-GBM cohort. Moreover, LINC00634 expression was upregulated in U251 and T98G cells. Cells transfected with an empty vector served as negative controls (NCs). The effect of upregulated LINC00634 expression on viability, migration, and clonogenicity was studied in vitro, and, in the case of U251-derived cell line, in vivo in immunodeficient mice with the focus on GBM growth and diffuse character. Results: Out of the 538 significantly dysregulated lncRNAs (P < 0.001) identified by transcriptome sequencing, a panel of 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM was validated previously by qPCR in another cohort (P < 0.0001 for all lncRNAs) and newly in an independent TCGA-GBM dataset. The upregulated LINC00634 expression in U251 and T98G cells was confirmed by qPCR to be approximately 11,000-fold and 600-fold, respectively, compared to the expression in NCs. Although no significant effect on viability, migration and clonogenicity was observed in vivo, a less diffuse growth pattern was observed in U251-derived GBMs with upregulated expression of LINC00634 in vivo compared with NCs. Conclusion: A significant dysregulation of lncRNAs was previously observed in GBM compared to non-tumor controls using both transcriptome sequencing and RT-qPCR and was newly confirmed also by analyzing the TCGA-GBM dataset. We also showed possible effect of LINC00634 upregulation on the growth pattern of U251-derived GBM tumors in vivo. Our study shows that lncRNAs are dysregulated in GBM and could, therefore, serve as promising diagnostic biomarkers in GBM as well as potential therapeutic targets. The research was supported by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102, funded by the European Union - Next Generation EU). Citation Format: Marek Vecera, Lenka Radova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Dagmar Al Tukmachi, Tana Machackova, Petra Pokorna, Jiri Sana, Ondrej Slaby. Long non-coding RNAs are dysregulated in glioblastoma and LINC00634 may affect the diffuse growth of U251-derived tumors in vivo. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3765.

Brain metastases (BMs) comprise a heterogeneous group of the most frequent intracranial tumors in adults, most originating in lung, breast, renal cell, and colorectal carcinomas and melanomas. Despite the recent improvements in imaging methodology resulting in earlier BM identification and advancements in treatment strategies, BMs are still a significant cause of patient morbidity. Furthermore, BMs frequency increases due to more prolonged survival of cancer patients and population aging. Since the most widely used prognostic scoring systems for BMs require prior knowledge of the primary origin and up to 14% of BMs are classified as BMs of unknown primary, there is an urgent unmet need for accurate biomarkers for identification of BM origin. MiRNAs are non-coding RNAs with an approximate length of 22 nucleotides, functioning as post-transcriptional regulators of gene expression. Dysregulated miRNA expression profile has been observed in many pathological processes, including the complex and not fully understood metastatic cascade. These molecules are very stable and present not only in tissues but also in human body fluids, including blood plasma and cerebrospinal fluid (CSF). Based on these facts, both tissue and circulating miRNAs are extensively studied as potential diagnostic biomarkers. Specific miRNA signatures of BMs were obtained using high-throughput miRNA profiling (Illumina small RNA sequencing) on 3 types of samples (metastatic tissue, blood plasma, CSF) from a cohort of 30 patients with BMs originating in the 5 tumor types – lung, breast, renal cell and colorectal carcinomas and melanomas (6 patients per group, 87 samples in total, only 3 CSF samples from RCC patients available). We identified significantly differentially expressed miRNAs in BM tissues with the ability to differentiate between primary origins. Tissue miRNAs could identify BMs originating from breast, colorectal and renal cell carcinomas and melanomas with high specificity and sensitivity. Interestingly, the heterogeneity of lung carcinomas was also characteristic for the corresponding BMs, making it challenging to distinguish accurately from other BMs. Even though the tissue-specific miRNA signature was the most precise, our results suggest a significant diagnostic potential of circulating miRNAs from CSF for BM patients. Therefore, these short and stable molecules could potentially help identify the origin of BMs of unknown primary. The research is supported by project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Dagmar Al Tukmachi, Michaela Ruckova, Marek Vecera, Tana Machackova, Petra Pokorna, Marketa Hermanova, Michal Hendrych, Leos Kren, Ivana Roskova, Vaclav Vybihal, Hana Valekova, Radim Jancalek, Jiri Sana, Martin Smrcka, Ondrej Slaby. Tumor tissue and cerebrospinal fluid microRNA profiles enable the classification of brain metastasis accordingly to their origin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3758.

Carney triad is a multitumor syndrome affecting almost exclusively young women in a nonfamilial setting, which manifests by multifocal gastric gastrointestinal stromal tumors, paragangliomas, and pulmonary chondroma. The Carney triad-associated tumors are characterized by a deficiency of the mitochondrial succinate dehydrogenase enzymatic complex. Recently, it has been observed that the deficiency results from epigenetic silencing of the SDHC gene by its promoter hypermethylation. To elucidate anatomic distribution of SDHC promoter methylation in Carney triad patients and thus to shed some light on the possible natural development of this epigenetic change, both neoplastic and available non-neoplastic tissues of 3 patients with Carney triad were tested for hypermethylation at the SDHC promoter site. SDHC promoter hypermethylation was proven in all tumors studied. Lack of SDHC epigenetic silencing in the non-neoplastic lymphoid and duodenal tissue (ie, tissues not involved in the development of Carney triad-associated tumors) together with the finding of SDHC promoter hypermethylation in the non-neoplastic gastric wall favors the hypothesis of postzygotic somatic mosaicism as the biological background of Carney triad; it also offers an explanation of the multifocality of gastrointestinal stromal tumors of the stomach occurring in this scenario as well. However, the precise mechanism responsible for the peculiar organ-specific distribution of Carney triad-associated tumors is still unknown.

Background/Aim: Glioblastoma (GBM) is one of the deadliest human cancers responding very poorly to therapy. Although the central nervous system has been traditionally considered an immunologically privileged site with an enhanced immune response, GBM appears to benefit from this immunosuppressive milieu. Immunomodulatory molecules play an important role in immune tumor-host interactions. Non-classical human leukocyte antigens (HLA) class Ib molecules HLA-E, HLA-F, and HLA-G have been previously described to be involved in protecting semi-allogeneic fetal allografts from the maternal immune response and in transplant tolerance as well as tumoral immune escape. Unfortunately, their role in GBM remains poorly understood. Our study, therefore, aimed to characterize the relationship between the expression of these molecules in GBM on the transcriptional level and clinico-pathological and molecular features of GBM as well as the effect of ionizing radiation. Materials and Methods: We performed the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in 69 GBM tissue samples and 21 non-tumor brain tissue samples (controls) by reverse transcription polymerase chain reaction. Furthermore, two primary GBM cell cultures had been irradiated to identify the effect of ionizing radiation on the expression of non-classical HLA molecules. Results: Analyses revealed that both HLA-E and HLA-F are significantly up-regulated in GBM samples. Subsequent survival analysis showed a significant association between low expression of HLA-E and shorter survival of GBM patients. The dysregulated expression of both molecules was also observed between patients with methylated and unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Finally, we showed that ionizing radiation increased HLA-E expression level in GBM cells in vitro. Conclusion: HLA-E and HLA-F play an important role in GBM biology and could be used as diagnostic biomarkers, and in the case of HLA-E also as a prognostic biomarker.

Many growth factors have been studied as additives accelerating lumbar fusion rates in different animal models. However, their low hydrolytic and thermal stability both in vitro and in vivo limits their workability and use. In the proposed work, a stabilized vasculogenic and prohealing fibroblast growth factor-2 (FGF2-STAB®) exhibiting a functional half-life in vitro at 37 °C more than 20 days was applied for lumbar fusion in combination with a bioresorbable scaffold on porcine models. An experimental animal study was designed to investigate the intervertebral fusion efficiency and safety of a bioresorbable ceramic/biopolymer hybrid implant enriched with FGF2-STAB® in comparison with a tricortical bone autograft used as a gold standard. Twenty-four experimental pigs underwent L2/3 discectomy with implantation of either the tricortical iliac crest bone autograft or the bioresorbable hybrid implant (BHI) followed by lateral intervertebral fixation. The quality of spinal fusion was assessed by micro-computed tomography (micro-CT), biomechanical testing, and histological examination at both 8 and 16 weeks after the surgery. While 8 weeks after implantation, micro-CT analysis demonstrated similar fusion quality in both groups, in contrast, spines with BHI involving inorganic hydroxyapatite and tricalcium phosphate along with organic collagen, oxidized cellulose, and FGF2- STAB® showed a significant increase in a fusion quality in comparison to the autograft group 16 weeks post-surgery (p = 0.023). Biomechanical testing revealed significantly higher stiffness of spines treated with the bioresorbable hybrid implant group compared to the autograft group (p &lt, 0.05). Whilst histomorphological evaluation showed significant progression of new bone formation in the BHI group besides non-union and fibrocartilage tissue formed in the autograft group. Significant osteoinductive effects of BHI based on bioceramics, collagen, oxidized cellulose, and FGF2-STAB® could improve outcomes in spinal fusion surgery and bone tissue regeneration.

Recirculation of chronic lymphocytic leukemia (CLL) cells between the peripheral blood and lymphoid niches plays a critical role in disease pathophysiology, and inhibiting this process is one of the major mechanisms of action for B-cell receptor (BCR) inhibitors such as ibrutinib and idelalisib. Migration is a complex process guided by chemokine receptors and integrins. However, it remains largely unknown how CLL cells integrate multiple migratory signals while balancing survival in the peripheral blood and the decision to return to immune niches. Our study provided evidence that CXCR4/CD5 intraclonal subpopulations can be used to study the regulation of migration of CLL cells. We performed RNA profiling of CXCR4dimCD5bright vs CXCR4brightCD5dim CLL cells and identified differential expression of dozens of molecules with a putative function in cell migration. GRB2–associated binding protein 1 (GAB1) positively regulated CLL cell homing capacity of CXCR4brightCD5dim cells. Gradual GAB1 accumulation in CLL cells outside immune niches was mediated by FoxO1-induced transcriptional GAB1 activation. Upregulation of GAB1 also played an important role in maintaining basal phosphatidylinositol 3-kinase (PI3K) activity and the “tonic” AKT phosphorylation required to sustain the survival of resting CLL B cells. This finding is important during ibrutinib therapy, because CLL cells induce the FoxO1-GAB1-pAKT axis, which represents an adaptation mechanism to the inability to home to immune niches. We have demonstrated that GAB1 can be targeted therapeutically by novel GAB1 inhibitors, alone or in combination with BTK inhibition. GAB1 inhibitors induce CLL cell apoptosis, impair cell migration, inhibit tonic or BCR-induced AKT phosphorylation, and block compensatory AKT activity during ibrutinib therapy.

Follicular lymphoma (FL) is a common indolent B-cell malignancy with a variable clinical course. An unfavorable event in its course is histological transformation to a high-grade lymphoma, typically diffuse large B-cell lymphoma. Recent studies show that genetic aberrations of MYC or its overexpression are associated with FL transformation (tFL). However, the precise molecular mechanisms underlying tFL are unclear. Here we performed the first profiling of expression of microRNAs (miRNAs) in paired samples of FL and tFL and identified 5 miRNAs as being differentially expressed. We focused on one of these miRNAs, namely miR-150, which was uniformly downmodulated in all examined tFLs (∼3.5-fold), and observed that high levels of MYC are responsible for repressing miR-150 in tFL by binding in its upstream region. This MYC-mediated repression of miR-150 in B cells is not dependent on LIN28A/B proteins, which influence the maturation of miR-150 precursor (pri-miR-150) in myeloid cells. We also demonstrated that low miR-150 levels in tFL lead to upregulation of its target, namely FOXP1 protein, which is a known positive regulator of cell survival, as well as B-cell receptor and NF-κB signaling in malignant B cells. We revealed that low levels of miR-150 and high levels of its target, FOXP1, are associated with shorter overall survival in FL and suggest that miR-150 could serve as a good biomarker measurable in formalin-fixed paraffin-embedded tissue. Overall, our study demonstrates the role of the MYC/miR-150/FOXP1 axis in malignant B cells as a determinant of FL aggressiveness and its high-grade transformation.

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through ATM or TP53 defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of ATM and TP53 mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated ATM and TP53 alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated TP53 gene was associated with the significantly reduced PFS and OS and the same output was observed when ATM and TP53 defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.

Background: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. Methods: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. Results: We describe a strong correlation between WES-based and panel-based TMBs, however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients, however, this significance decreases with the increasing cut-off values. Conclusions: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines.

Glioblastoma stem-like cells (GSCs) are critical for the aggressiveness and progression of glioblastoma (GBM) and contribute to its resistance to adjuvant treatment. MicroRNAs (miRNAs) are small, non-coding RNAs controlling gene expression at the post-transcriptional level, which are known to be important regulators of the stem-like features. Moreover, miRNAs have been previously proved to be promising diagnostic biomarkers in several cancers including GBM. Using global expression analysis of miRNAs in 10 paired in-vitro as well as in-vivo characterized primary GSC and non-stem glioblastoma cultures, we identified a miRNA signature associated with the stem-like phenotype in GBM. 51 most deregulated miRNAs classified the cell cultures into GSC and non-stem cell clusters and identified a subgroup of GSC cultures with more pronounced stem-cell characteristics. The importance of the identified miRNA signature was further supported by demonstrating that a Risk Score based on the expression of seven miRNAs overexpressed in GSC predicted overall survival in GBM patients in the TCGA dataset independently of the IDH1 status. In summary, we identified miRNAs differentially expressed in GSCs and described their association with GBM patient survival. We propose that these miRNAs participate on GSC features and could represent helpful prognostic markers and potential therapeutic targets in GBM.

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain malignancy of astrocytic origin. The prognosis remains very poor with the median overall survival (OS) being between 12 and 16 months from diagnosis despite early use of conventional medical therapy. Identifying new therapeutic targets, as well as prognostic and predictive biomarkers for accurate stratification of patients is therefore of utmost importance. Long non-coding RNAs (lncRNAs) are regulators of gene expression having critical impact on both physiological processes and the molecular pathology of GBM, indicating their potential as biomarkers and therapeutic targets. Material and Methods: Our study included 219 GBM patients and 29 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. RNA (RIN > 8) from 77 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were done with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was performed using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 8,414 lncRNAs and their sequential variants with non-zero RPKM at least in one sample were statistically evaluated. The alignment and target counts were performed with CLC genomic workbench. Selected significantly dysregulated lncRNAs between GBM and non-tumor controls were analyzed in a larger cohort of 188 specimens by qRT-PCR and the expression data normalized to PPIA was then evaluated by Mann-Whitney U test. Results: Statistical analysis revealed 538 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The expression of top 10 downregulated lncRNAs (SNAI3-AS1, LINC00882, RFPL1S, MIR137HG, TTLL7-IT1, PWAR6, LINC00634, LINC00632, DGCR5, LINC00982; logFC ≤ -2; P < 0.001) and 1 upregulated lncRNA (BTN2A3P; logFC ≥ 2; P < 0.001) in GBM and non-tumor controls was successfully validated by qRT-PCR (P < 0.0001). Moreover, the statistical analysis revealed 22 lncRNAs significantly dysregulated between patients with OS less than 12 months and those with OS equal or more than 12 months (P < 0.01). Conclusion: We observed significant dysregulation of lncRNAs in GBM tissues compared to non-tumor controls based on the results of both RNASeq and qRT-PCR. We also found 22 lncRNAs to be dysregulated in relation to overall survival. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic grant nr. NV18-03-00398, grant of Czech Grant Agency nr. 17-17636S, and by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601). Citation Format: Marek Vecera, Jan Oppelt, Lenka Radova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Jiri Sana, Alena Kopkova, Julia Kovacova, Ondrej Slaby. Dysregulated expression of lncRNAs in glioblastoma multiforme and their association with overall survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3575.

Current progress in the field of next-generation transcriptome sequencing have contributed significantly to the study of various malignancies including glioblastoma multiforme (GBM). Differential sequencing of transcriptomes of patients and non-tumor controls has a potential to reveal novel transcripts with significant role in GBM. One such candidate group of molecules are long non-coding RNAs (lncRNAs) which have been proved to be involved in processes such as carcinogenesis, epigenetic modifications and resistance to various therapeutic approaches. To maximize the value of transcriptome sequencing, a proper protocol for library preparation from tissue-derived RNA needs to be found which would produce high quality transcriptome sequencing data and increase the number of detected lncRNAs. It is important to mention that success of library preparation is determined by the quality of input RNA, which is in case of real-life tissue specimens very often altered in comparison to high quality RNA commonly used by manufacturers for development of library preparation chemistry. In the present study, we used GBM and non-tumor brain tissue specimens and compared three different commercial library preparation kits, namely NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), SENSE Total RNA-Seq Library Prep Kit (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries generated using SENSE kit were characterized by the most normal distribution of normalized average GC content, the least amount of over-represented sequences and the percentage of ribosomal RNA reads (0.3-1.5%) and highest numbers of uniquely mapped reads and reads aligning to coding regions. However, NEBNext kit performed better having relatively low duplication rates, even transcript coverage and the highest number of hits in Ensembl database for every biotype of our interest including lncRNAs. Our results indicate that out of three approaches the NEBNext library preparation kit was most suitable for the study of lncRNAs via transcriptome sequencing. This was further confirmed by highly consistent data reached in an independent validation on an expanded cohort.

In rare disorders, there are often no standard therapy recommendations. Patients with refractory disease may require novel experimental approaches. Applied as second- up to fourth-line treatment, lenalidomide (10–25 mg perorally on days 1–21 in a 28-day cycle) was used in our cohort of four adult patients with aggressive, multisystem and relapsing diseases. Complete and long-lasting remissions (more than 1 year, no maintenance therapy) were achieved in patients with Langerhans cell histiocytosis (11 cycles, combination with dexamethasone and etoposide, consolidated by allogeneic blood stem cell transplant) and plasma-cell Castleman disease (15 cycles, monotherapy). Mixed response with complete disappearance of brain infiltrates was reached in Erdheim-Chester disease (6 cycles, monotherapy) and gastrointestinal bleeding was well controlled in multiple angiomatosis (9 cycles, combination with thalidomide). For disease activity evaluation each patient underwent fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography scan imaging, which was complemented by clinical and laboratory investigations.

Glioblastoma is the most common and the most aggressive type of brain cancer. Aberrations of the RTK/RAS/PI3K-, p53-, and RB cell signaling pathways were recognized as a core requirement for pathogenesis of glioblastoma. The p53 tumor suppressor functions as a transcription factor transactivating expression of its target genes in response to various stress stimuli. We determined the p53 status in 36 samples of glioblastoma by functional analyses FASAY and split assay. Seventeen p53 mutations were detected and further analyzed by cDNA and gDNA sequencing in 17 patients (47.2 %). Fifteen (88.2 %) of the mutations were missense mutations causing amino acid substitutions, seven of them exhibited temperature-sensitivity. Two mutations were determined as short deletions, one of them causing formation of premature termination codon in position 247. Fluorescent in situ hybridization revealed the loss of the p53-specific 17p13.3 locus in four of 33 analyzed samples (12 %). In 12 out of 30 samples (40 %), the p53 protein accumulation was shown by immunoblotting. There was high (80 %) concordance between the presence of the clonal p53 mutation and the p53 protein accumulation.

Research has exposed cancer to be a heterogeneous disease with a high degree of inter-tumoral and intra-tumoral variability. Individual tumors have unique profiles, and these molecular signatures make the use of traditional histology-based treatments problematic. The conventional diagnostic categories, while necessary for care, thwart the use of molecular information for treatment as molecular characteristics cross tissue types. This is compounded by the struggle to keep abreast the scientific advances made in all fields of science, and by the enormous challenge to organize, cross-reference, and apply molecular data for patient benefit. In order to supplement the site-specific, histology-driven diagnosis with genomic, proteomic and metabolomics information, a paradigm shift in diagnosis and treatment of patients is required. While most physicians are open and keen to use the emerging data for therapy, even those versed in molecular therapeutics are overwhelmed with the amount of available data. It is not surprising that even though The Human Genome Project was completed thirteen years ago, our patients have not benefited from the information. Physicians cannot, and should not be asked to process the gigabytes of genomic and proteomic information on their own in order to provide patients with safe therapies. The following consensus summary identifies the needed for practice changes, proposes potential solutions to the present crisis of informational overload, suggests ways of providing physicians with the tools necessary for interpreting patient specific molecular profiles, and facilitates the implementation of quantitative precision medicine. It also provides two case studies where this approach has been used.

BACKGROUND: Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner. METHODS: We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival. FINDINGS: We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084). INTERPRETATION: The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection. FUNDING: Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Introduction. This study describes the results achieved using a combination of allogeneic mesenchymal stem cells (MSCs) with chondrocytes (CHC) and a new scaffold consisting of type-I collagen and chitosan nanofibers in the prevention of partial growth plate arrest after iatrogenic injury in pigs. Material and methods. The miniature pig was selected as an experimental model to compare the results in the left femoral bones (MSCs and CHC in scaffold transplantation into the iatrogenic partial distal growth plate defect) and right femoral bones (scaffold alone transplantation). The experimental group consisted of 10 animals. Bone marrow from os ilium as the source of MSCs was used. A porous cylinder consisting of 0.5% by weight type-I collagen and 30% by weight chitosan, was the optimal choice. The length of the bone and angular deformity of distal femur after the healing period was measured and the quality and structure of the newly formed cartilage was histologically examined. Results. Transplantation of the composite scaffold in combination with MSCs and chondrocytes led to the prevention of growth disorder and angular deformity in the distal epiphysis of the left femur. Compared to the right (control) femur, tissue similar to hyaline cartilage with signs of columnar organization typical of the growth plate occurred in most cases. Conclusions. The promising results of this study reveal the new and effective means for the prevention of bone bridge formation after growth plate injury.

Although the role of the non-classical human leukocyte antigen E (HLA-E) was originally thought to be limited to the development of a maternal tolerance to a semiallogeneic fetal graft, it is now known that HLA-E exerts multiple immunoregulatory functions. The significance of the presence of HLA-E in neoplastic cells and/or background microenvironment cells in classical Hodgkin lymphoma (cHL) is not well characterized. In our study, we evaluated the presence of HLA-E in both neoplastic and background cells in 40 cases of cHL. We found HLA-E in both neoplastic and background cells. There was a positive statistical correlation between the expression of HLA-E in neoplastic cells and the clinical stage of the disease, which indicates an immune-tolerogenic property of this multiple-purpose molecule. The presence of HLA-E in background cells seems to be prognostically neutral but its significance still needs to be determined. The mechanisms regulating the immunopathology of cHL neoplastic cells with respect to the presence of these molecule deserve further studies.

Down-regulation of miR-150 and up-regulation of its target FOXP1 is associated with transformation of follicular lymphoma.Down-regulation of miR-150 and up-regulation of its target FOXP1 is associated with transformation of follicular lymphoma.

Glioblastoma multiforme (GBM) is the most frequently occurring primary malignant brain tumor; patients with GBM often have a very poor prognosis and differing responses to treatment. Therefore, it is very important to find new biomarkers that can predict clinical outcomes and help in treatment decisions. MicroRNAs are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and play a key role in the pathogenesis of GBM. In a group of 38 patients with primary GBM, we analyzed the expression of eight microRNAs (miR-21, miR-128a, miR-181c, miR-195, miR-196a, miR-196b, miR-221, and miR-222). In addition, we examined the methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) promoter by high-resolution melting analysis, as this has been shown to be a predictive marker in GBM. MGMT methylation status correlated with progression-free survival (P = 0.0201; log-rank test) as well as with overall survival (P = 0.0054; log-rank test). MiR-195 (P = 0.0124; log-rank test) and miR-196b (P = 0.0492; log-rank test) positively correlated with overall survival. Evaluation of miR-181c in combination with miR-21 predicted time to progression within 6 months of diagnosis with 92% sensitivity and 81% specificity (P < 0.0001). Our data confirmed that the methylation status of MGMT but also miR-21, miR-181c, miR-195, and miR-196b to be associated with survival of GBM patients. Above all, we suggest that the combination of miR-181c and miR-21 could be a very sensitive and specific test to identify patients at high risk of early progression after surgery.

Caudal regression syndrome is a rare congenital condition characterized by varying degrees of developmental failure ranging from a partial sacral agenesis to the absence of lumbosarcal spine, hypoplasia, or fusion of the lower extremities and visceral anomalies. This is the third case of only one of the twins involved by this syndrome described in the literature and the second case of the selective involvement in dizygotic twins. Selective involvement of only one twin suggests that factors other than hyperglycemia and 7q deletions may be involved in the pathogenesis.

The goal of this pilot study was to create an experimental model of myocardial infarction (for subsequent evaluation of the effectiveness of an alternative way of stem cell application – intracoronary cell infusion in the management of acute myocardial infarction). Four experimental animals, female pigs weighing between 30 and 40 kg, were used in the initial phase of this study to create an experimental model of acute myocardial infarction. An experimental myocardial infarction was performed via occlusion of the interventricular arm of the left coronary artery for 90 min. The hearts were examined 1 h, 3 days, 5 days, and 7 days after the procedure. Macroscopically, red infarction characteristic of reperfusion was found. Microscopically, the healing process with granulation tissue production/collagen deposition was remarkably accelerated compared to literature data. Repair processes in reperfused experimental myocardial infarction and/or reperfused autopsy specimens should not be evaluated on the basis of literature data only. Large collections of extracellular calcium were present. This phenomenon is not well described in the literature and probably has the potential for significantly interfering with the repair process. The histopathology of reperfused acute myoardial infarction deserves to be studied in further investigations.

Introduction: Glioblastoma (GBM) is the most frequent primary brain tumor of astrocytic origin. The prognosis is unfavourable with the median overall survival (OS) being between 12 and 15 months from diagnosis. Identification of new therapeutic targets, as well as new prognostic and predictive biomarkers for more accurate stratification of patients presents significant unmet medical needs. Long non-coding RNAs (lncRNAs) are regulators of gene expression playing important roles in the molecular pathology of GBM, indicating their potential as biomarkers and therapeutics targets. Material and Methods: Our study included 80 GBM patients treated with Stupp protocol and 16 patients with non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient. IDH1 mutations and MGMT methylation status were evaluated in all GBMs. RNA (RIN > 8) from 96 specimens was used for next-generation RNA sequencing (RNAseq). rRNA depletion and cDNA library preparation were performed with RiboCop rRNA Depletion Kit V1.2 (Lexogen) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB), respectively. RNAseq was done using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). 24,087 protein-coding and 8,414 non-coding RNAs and their sequential variants with non-zero RPKM at least in one sample underwent statistical evaluation. CLC genomic workbench was used for the alignment and target counts. Results: Statistical analysis revealed 84 (P < 0.001) dysregulated lncRNAs in GBMs compared to non-tumor brain tissue samples. The results also showed 485 dysregulated protein-coding RNAs with P < 0.001 and 24 protein-coding RNAs with P < 0.000001. 35 lncRNAs showed significant dysregulation when lncRNA profiles of GBM tissues with methylated MGMT promoter (≥ 25% methylation) were compared to those with unmethylated promoter (P < 0.01). When lncRNA patterns of GBM samples with mutated IDH1 were compared to those with wild-type IDH1, 60 lncRNAs were found to be significantly dysregulated (P < 0.001). Correlating lncRNA expression patterns with OS uncovered 6 lncRNA signature which enabled identifying patients with significantly worse prognosis (OS < 6 months). Conclusion: We described significant dysregulation of lncRNAs and protein-coding RNAs in GBM tissue compared to non-tumor brain tissue and specific lncRNA patterns linked to MGMT methylation and IDH1 mutation status. We also identified 6 lncRNA signature allowing sensitive prognostic stratification of GBM patients. Our study indicates that lncRNAs could serve as promising diagnostic and prognostic biomarkers in GBM. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Citation Format: Marek Vecera, Romana Butova, Radim Lipina, Stefan Reguli, Martin Smrcka, Radim Jancalek, Michal Filip, Marketa Hermanova, Leos Kren, Pavol Mojak, Jaroslav Juracek, Tana Machackova, Natalia A. Gablo, Jiri Sana, Ondrej Slaby. Clinicopathological subgroups of glioblastoma patients are characterized by specific lncRNA expression patterns [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2459.

The possible role of immune-modulatory nonclassical molecules HLA-G and HLA-E in an anti-tumoral response and development of glioblastoma is not well characterized. In this study, we evaluated an expression of HLA-G and HLA-E by activated tumor infiltrating microglia/macrophages. We found production of HLA-G and HLA-E by tumor infiltrating activated microglia/macrophages in a majority of glioblastomas. We speculate that the expression of these molecules by activated microglia/CNS macrophages plays a role in the anti-tumoral immunity in the development of glioblastoma. Mechanisms of microglia–glioblastoma cell interactions with respect to the expression of these molecules deserves further study.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Despite radical surgery and radiotherapy supported by chemotherapy, the disease still remains incurable with an extremely low median survival rate of 12-15 months from the time of initial diagnosis. The main cause of treatment failure is considered to be the presence of cells that are resistant to the treatment. MicroRNAs (miRNAs) as regulators of gene expression are involved in the tumor pathogenesis, including GBM. MiR-338 is a brain-specific miRNA which has been described to target pathways involved in proliferation and differentiation. In our study, miR-338-3p and miR-338-5p were differentially expressed in GBM tissue in comparison to non-tumor brain tissue. Overexpression of miR-338-3p with miRNA mimic did not show any changes in proliferation rates in GBM cell lines (A172, T98G, U87MG). On the other hand, pre-miR-338-5p notably decreased proliferation and caused cell cycle arrest. Since radiation is currently the main treatment modality in GBM, we combined overexpression of pre-miR-338-5p with radiation, which led to significantly decreased cell proliferation, increased cell cycle arrest, and apoptosis in comparison to irradiation-only cells. To better elucidate the mechanism of action, we performed gene expression profiling analysis that revealed targets of miR-338-5p being Ndfip1, Rheb, and ppp2R5a. These genes have been described to be involved in DNA damage response, proliferation, and cell cycle regulation. To our knowledge, this is the first study to describe the role of miR-338-5p in GBM and its potential to improve the sensitivity of GBM to radiation.

The transcription factor c-Myb is required for modulation of progenitor cells in several tissues, including skeletal muscle and its upregulation is observed in many human malignancies. Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors with features of developing skeletal muscle. Several miRNAs are downregulated in RMS, including miR-150, a negative regulator of c-Myb expression. Using the C2C12 myoblast cell line, a cellular model of skeletal muscle differentiation, we showed that miR-150 controls c-Myb expression mainly at the level of translation. We hypothesized that a similar mechanism of c-Myb regulation operates in RMS tumors. We examined expression of c-Myb by immunohistochemistry and revealed c-Myb positivity in alveolar and embryonal tumors, the two most common subgroups of RMS. Furthermore, we showed direct correlation between c-Myb production and myogenin expression. Interestingly, high myogenin levels indicate poor prognosis in RMS patients. c-Myb could, therefore, contribute to the tumor phenotype by executing its inhibitory role in skeletal muscle differentiation. We also showed that c-Myb protein is abundant in migratory C2C12 myoblasts and its ectopic expression potentiates cell motility. In summary, our results implicate that metastatic properties of some RMS subtypes might be linked to c-Myb function.

Background: Children with high-grade glioma still have a poor prognosis despite the use of multimodal therapy including surgery, radiotherapy, and chemotherapy. New therapeutic strategies and methods evaluating such therapies are needed. Observation: Here we describe a child with anaplastic oligodendroglioma of the spinal cord who was unable to tolerate standard chemoradiotherapy and who had still-vital residual tumour during therapy. A good response was obtained with low-dose metronomic treatment containing vinblastine. The treatment was guided according to gradual response assessed using various positron-emission tomography tracers. Conclusions: Metronomic treatment guided by positron-emission tomography could be a reasonable option in some high-risk pediatric tumours.

Background Chylothorax is an uncommon medical condition. To the best of our knowledge, there have been no detailed English-language reports dealing with its cytopatholoogic findings and diagnostic pitfalls. Cases A 12-year-old boy, hemodialysis dependent, with congenital nephrotic syndrome due to focal segmental glomerular sclerosis and it failed renal transplant, developed shortness of breath. Physical and radiologic examinations revealed a left pleural effusion. A 7-year-old boy developed shortness of breath, with a subsequent finding of a left pleural effusion. Multiple osteolytic skeletal lesions were found in this patient. Both patients underwent thoracocentesis. Cytologically, both fluids contained many relatively uniform, large lymphoid cells with high nuclear/cytoplasmic (N/C) ratio, condensed chromatin and occasional nucleoli, resembling blasts. Some nuclei were convoluted. Mitotic figures were present. Foamy macrophages were present ill both cases. The differential diagnosis of these populations of cells included a lymphoroliferative disorder. However, the 'mature T-lymphocytic nature of the cells was confirmed by immunobistochemistry performed on cell block preparations, confirming the clinical impression of chylothorax in both cases. The first patient had chylothorax as a result of trauma due to therapeutic interventions (subclavian vein cannulation), in the second patient the chylothorax was a part of Gorbam-Stout syndrome. Conclusion The large T-lymphocytes that are the major cellular component of chylothorax may arouse suspicion of a lymphoproliferative disorder. Attention to the clinical history and immunophenotyping confirm the benign nature of the pleural space fluid. Also, abundant foamy macrophages can be considered a low-power clue to this diagnosis.

Inhibitors of apoptosis, including bcl-2 and survivin (a novel gene encoding a unique apoptosis inhibitor), regulate cell proliferation by promoting cell survival. Although survivin has been detected in several human cancers, its prognostic significance and relationship to bcl-2 are not well characterized in lung cancer. Tissue sections from 102 non-small cell lung carcinomas (NSCLC) were immunostained using antibodies against survivin and bcl-2. Staining results were correlated with prognostic variables. Immunoreactivity for survivin and bcl-2 was observed in 53% and 21% of NSCLCs, respectively. Fifty-two percent of the 50 squamous cell carcinomas and 54% of the 52 adenocarcinomas expressed survivin. Survivin positivity correlated with tumor stage in squamous cell carcinoma. On univariate analysis, survivin expression correlated with decreased patient survival in NSCLC and in the subset of squamous cell carcinomas, but not in adenocarcinomas. On multivariate analysis, survivin was an independent predictor, along with distant metastasis and large tumor size. Eighteen percent of squamous cell carcinomas and 24% of adenocarcinomas expressed bcl-2. On univariate analysis, bcl-2 expression correlated with increased patient survival in NSCLC and in the subset of squamous cell carcinomas. An inverse correlation between the expression of survivin and bcl-2 was noted. Survivin immunoreactivity is an independent predictor of shortened survival in NSCLC, while bcl-2 protein expression correlated with prolonged patient survival. These findings indicate an inverse relationship between survivin and bcl-2 expression and suggest that these two inhibitors of apoptosis function through different pathways in the regulation of tumorigenesis in NSCLC.

The multistep process of carcinogenesis implies the accumulation of multiple molecular defects. Alteration of tumor suppressor and metastasis suppressor genes are the important steps. Increasing experimental evidence indicates that decreased expression of tumor-metastasis/suppressor genes and gene products are involved in the progression of a variety of human malignancies. In the present study, we have extended this analysis to non-small cell lung carcinomas (NSCLC). The expression and prognostic significance of the tumor suppressor gene PTEN and metastasis suppressor genes nm23-H1 and KAI-1 was evaluated in NSCLCs. Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded tissues from 53 bronchogenic adenocarcinomas and 51 squamous cell carcinomas using monoclonal antibodies against PTEN, nm23H-1, and KAI-1 proteins. Immunohistochemical results were correlated with tumor stage, grade, lymph nodes positivity, metastasis, and patient survival. Significant co-expression of PTEN, nm23-H1 and KAI-1 was observed in NSCLC (P

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and are frequently aberrantly expressed in cancer. We aimed to understand their role in the transformation of indolent follicular lymphoma (FL) into an aggressive diffuse large B cell lymphoma. This happens in ~3% of cases per year during the course of the disease, and is associated with median survival of only 2 years. The NGS revealed number of aberrations associated with transformed FL (tFL), including frequent high-level activity of MYC (amplifications, translocations, and mutations) or loss of DNA damage regulators (p53, CDKN2A/B). Firstly, we performed a miRNA profiling (TaqMan miRNA Arrays) in paired FL and tFL samples (N=8 pairs). This revealed a relatively small group of 5 miRNAs that are consistently differentially expressed in tFL (P1.5). Since the most frequently acquired aberration in tFL is the high-level activity of MYC we performed a correlation analysis of MYC levels and expression of these miRNAs in additional samples of FL, tFL, and CLL samples with/without MYC duplication (N=40 FL/tFL, N=39 CLL). This revealed that at least one of these miRNAs is significantly down-modulated (P Citation Format: Katerina Musilova, Gabriela Pavlasova, Vaclav Seda, Eva Vojackova, Katerina Cerna, Veronika Svobodova, Robert Pytlik, Vit Prochazka, Zuzana Prouzova, Sarka Pospisilova, Lenka Zlamalikova, Heidi Mocikova, Lenka Kruzova, Marie Jarosova, Andrew Evans, Clive Zent, Leos Kren, Marek Trneny, Jiri Mayer, Andrea Janikova, Marek Mraz. Differential expression of microRNAs in transformation of follicular lymphoma to diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1479. doi:10.1158/1538-7445.AM2017-1479

Introduction: Glioblastoma multiforme (GBM) is the most frequent primary brain tumor of astrocytic origin characterized by very poor prognosis. Despite conventional therapeutic protocol the prognosis of GBM patients is very poor with median of overall survival ranging between 12 and 15 months from diagnosis. Therefore, many financial charges and lot of effort is spent in research of new therapeutic approaches that could prolong the survival of GBM patients. Long non-coding RNAs (lncRNAs) are a relatively new class of noncoding gene regulators playing critical roles in tumor biology, including GBM. From this perspective, lncRNAs seem to be promising therapeutic targets in GBM patients. Material and Methods: We performed next-generation sequencing analysis of fresh-frozen histopatologically confirmed 45 GBM tissues and 5 non-tumor brain tissues obtained from non-dominant anterior temporal cortexes resected during surgery for intractable epilepsy. Informed consent approved by the local Ethical Commission was obtained from each patient before the treatment. rRNA depletion and cDNA library preparation were performed with GeneRead rRNA Depletion Kit (Qiagen) and NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific), respectively. Sequencing was held using NextSeq 500 High Output Kit and NextSeq 500 instrument (both Illumina). Statistical analysis evaluated 24 087 protein-coding and 8 414 non-coding RNAs and their sequential variants with non-zerou RPKM (Reads Per Kilobase of transcript per Million mapped reads) at least in one sample. We used CLC genomic workbench for the alignment and target counts. Targeted regulation of ZFAS1 level have been carried out by the transient transfection of specific siRNA in GBM stable cell lines (A172, T98G, U87MG, U251). Viability and migration were analyzed in vitro using MTT and scratch wound healing assay, respectively. Results: Statistical analysis has revealed 274 (P < 0.01) deregulated lncRNAs in GBMs in comparison with non-tumor brain samples. Moreover, the results have showed also 489 deregulated proten-coding RNAs with P value less than 0.001 and 26 protein-coding RNAs with P value less than 0.000001. For subsequent in vitro functional analyses was chosen one of the most upregulated lncRNAs in GBM samples ZFAS1. Targeted downregulation of this molecule led to the significant reduction of viability in all examined GBM cell lines. Decreasing of proliferation potential was observed only in A172 a U251 cell lines. Conclusion: We have demonstrated a deregulation of many lncRNAs and protein-coding RNAs in GBM tissue in comparison with non-tumor brain tissue. Moreover, ZFAS1, one of the most upregulated lncRNAs in GBM tissue, is involved in regulation of viability and migration of GBM cell lines in vitro. This work was supported by Ministry of Health of the Czech Republic, grant nr. 15-33158A, 15-34553A, 15-31627A, 15-34678A, 16-31314A, 16-31765A and by grant of Czech Grant Agency nr. 16-18257S. Note: This abstract was not presented at the meeting. Citation Format: Jiri Sana, Marek Vecera, Romana Butova, Jaroslav Juracek, Tana Machackova, Parwez Ahmad, Natalia Anna Gablo, Kamila Souckova, Leos Kren, Radim Lipina, Martin Smrcka, Ondrej Slaby. ZFAS1 is upregulated in GBM tissue and affects viability and migration of GBM cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3445. doi:10.1158/1538-7445.AM2017-3445

Background: Glioblastoma multiforme (GBM) is the most frequently occurring primary malignant brain tumor with very poor prognosis, varying therapeutic response. Therefore, it is very important to find new biomarkers which can predict a clinical outcomes and help in treatment decisions. MicroRNAs (miRNAs) are small, non-coding RNAs that function as post-transcriptional regulators of gene expression and play key role in pathogenesis of wide range of cancer, including GBM. Aim of our study is to identify miRNA signature associated with the more aggressive phenotype of GBMs, which will enable sensitive prediction of clinical outcome in GBM patients under standard therapeutic protocols. Methods: We determined the expression profile of 667 miRNAs in tumor tissues of 58 patients with primary GBM with completed concomitant chemoradiotherapy with temozolomide according to standard Stupp protocol and 12 non-malignant brain tissues obtained from patients with drug-resistant anteromedial temporal lobe epilepsy. Methylation status of MGMT promoter (methylation-specific HRM), isocitrate dehydrogenase (IDH1) status (immunohistochemistry) and co-deletion of 1p/19q (FISH) was also evaluated. Results: We have confirmed frequencies of commonly used prognostic biomarkers in GBM patients (MGMT, IDH1, 1p/19q deletion), and observed significant correlation of MGMT methylation status, response to chemoradiotherapy with temozolomide and survival of GBM patients. We have identified specific signature of miRNAs diferentially expressed between GBM tissue and non-tumoral brain tissue from epilepsy surgeries (28 miRNAs, p

Purpose Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. Patients and Methods Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. Results Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. Conclusion Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

We and others have shown that deregulation of microRNAs (miRNAs) is associated with the biology of B cell malignancies, including regulation of B cell proliferation and survival (Musilova & Mraz, Leukemia, 2015). We focused on studying miRNAs that associate with the aggressiveness of FL and its transformation to diffuse large B cell lymphoma (DLBCL). First, we analyzed the expression of 380 miRNAs (TaqMan Arrays, ABI) in 8 paired primary samples of FL that subsequently transformed to DLBCL. We identified statistically significant changes (P1.8) in the expression of 5 miRNAs. The most significant change was the down-regulation of miR-150 (~5 fold, P=0.01). Similarly, we observed significantly reduced miR-150 levels in an independent cohort of non-paired samples of FL before vs. after transformation to DLBCL, and miR-150 was significantly less expressed in de novo DLBCL in comparison with FL. MicroRNA miR-150 is of particular interest as we have shown that its expression determines BCR signaling propensity in chronic lymphocytic leukemia (CLL) B cells, and low levels associated with worse survival (Mraz et al., Blood, 2014). Therefore, we analyzed miR-150 expression in a cohort of 89 FL samples. We noticed that miR-150 expression was lower in samples from patients with a FLIPI score ≥3 (P=0.03), and with high Ki67 positivity (>20%; P=0.003). Moreover, FL patients with low miR-150 levels ( To determine the potential reason for variable miR-150 levels in FL B cells, we tested the effect of microenvironmental interactions on its expression. In this experiment, a short term (48hrs) co-culture of B cell lymphoma cells with stromal cells (HS-5) led to down-regulation of miR-150 levels (P CONCLUSION: Low miR-150 levels associate with a shorter overall survival in FL. This could be used as a reasonable prognostic marker since high miRNA stability allows reliable analyses of miR-150 levels from formalin-fixed, paraffin-embedded (FFPE) samples. Interactions with stromal cells and/or soluble microenvironmental factors down-modulate miR-150 levels in B cells, which support their BCR signalling potential. We are further investigating to what extent the miR-150 down-regulation is causally connected with the aggressiveness and/or transformation of FL. This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; the research grant GACR (16-13334Y); the Ministry of Health of the Czech Republic, grant nr. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein; Masaryk university as part of the project "New approaches in research, diagnostics and therapy of hematological malignancies III", number MUNI/A/1028/2015 with the support of the Specific University Research Grant, as provided by the Ministry of Education, Youth and Sports of the Czech Republic in the year 2016; the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16); the Ministry of Education, Youth and Sports of the Czech Republic, grant nr. LD15144 (COST CZ); the research grant TACR (TEO2000058/2014-2019); and EHA Research Fellowship award granted by the European Hematology Association. G.P. is a city of Ostrava scholarship holder. Disclosures No relevant conflicts of interest to declare.