Ales Vicha | Charles University, Prague (original) (raw)

Papers by Ales Vicha

Child's Nervous System

Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology... more Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology varying from indolent low-grade gliomas to aggressive diffuse intrinsic pontine glioma (DIPG). Making the correct tumor diagnosis in the pontine location is thus critical. Here, we report a case study of a 14-month-old patient initially diagnosed as histone H3 wild-type DIPG. Due to the low age of the patient, the MRI appearance of DIPG, and anaplastic astrocytoma histology, intensive chemotherapy based on the HIT-SKK protocol with vinblastine maintenance chemotherapy was administered. Rapid clinical improvement and radiological regression of the tumor were observed with nearly complete remission with durable effect and excellent clinical condition more than 6.5 years after diagnosis. Based on this unexpected therapeutic outcome, genome-wide DNA methylation array was employed and the sample was classified into the methylation class “Low-grade glioma, MYB(L1) altered.” Additionally, RT-P...

Pathology Research and Practice, Jun 1, 2023

Neoplasma, 2019

Paragangliomas and pheochromocytomas are rare, mostly benign neuroendocrine tumors, which are emb... more Paragangliomas and pheochromocytomas are rare, mostly benign neuroendocrine tumors, which are embryologically derived from neural crest cells of the autonomic nervous system. Paragangliomas are essentially the extra-adrenal counterparts of pheochromocytomas. As such, this family of tumors can be subdivided into head and neck paragangliomas, pheochromocytomas, and thoracic and abdominal extra-adrenal paragangliomas. Ten out of fifteen genes that contribute to the development of paragangliomas are more susceptible to the development of head and neck paragangliomas when mutated. Gene expression profiling revealed that pheochromocytomas and paragangliomas could be classified into two main clusters (C1 and C2) based on transcriptomes. These groups were defined according to their mutational status and as such strongly associated with specific tumorigenic pathways. The influence of the main genetic drivers on the somatic molecular phenotype was shown by DNA methylation and miRNA profiling. Certain subunits of succinate dehydrogenase (SDHx), von Hippel-Lindau (VHL) and transmembrane protein 127 (TMEM127) still have the highest impact on development of head and neck paragangliomas. The link between RAS proteins and the formation of pheochromocytomas and paragangliomas is clear due to the effect of receptor tyrosine-protein kinase (RET) and neurofibromatosis type 1 (NF1) in RAS signaling and recent discovery of the role of HRAS. The functions of MYC-associated factor X (MAX) and prolyl hydroxylase 2 (PHD2) mutations in the contribution to the pathogenesis of paragangliomas still remain unclear. Ongoing studies give us an insight into the incidence of germline and somatic mutations, thus offering guidelines for early detection. Furthermore, these also show the risk of mistakenly assuming sporadic cases in the absence of definitive family history in head and neck paragangliomas.

PubMed, 2022

The current progress and increasing knowledge about the genetic causes of cancer opens up new pos... more The current progress and increasing knowledge about the genetic causes of cancer opens up new possibilities for its treatment. However, it is necessary to combine the results obtained using classical pathological methods with sensitive, multiplex molecular pathological methods. The method that meets the required criteria is MLPA based on multiplex PCR reaction. This method detects both changes in gene copy number and DNA methylation and, last but not least, point mutations. The MLPA reaction is applicable to even highly fragmented DNA. At the same time, it is a robust method that can be performed on standard thermocyclers, the fluorescent tip label requires automatic sequencers. Up to 50 genetic markers can be tested in one reaction, a number that allows a diagnostic and prognostic conclusion. All these features lead to the routine use of MLPA analysis not only in diagnosis but also in cancer research. The present article aims to summarize the different types of MLPA reactions, its benefits, but also the potential pitfalls.

PubMed, 2022

The new WHO classification of CNS tumors is largely based on molecular diagnostic. Without molecu... more The new WHO classification of CNS tumors is largely based on molecular diagnostic. Without molecular methods some entities can no longer be diagnosed. We are trying to show a rational approach to the CNS tumors diagnostics, which is based on conventional molecular methods such as RT-PCR, Sanger sequencing, MLPA, extended by the next generation sequencing (NGS) and methylation SNP array.

1 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Pra... more 1 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 2 Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol in Prague, Czech Republic 3 Department of Oncology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 4 Institute of Pathology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 5 Institute of Endocrinology, Prague, Czech Republic 6 Department of Nephrology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 7 Department of Paediatrics of the 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol in Prague, Czech Republic *Coressponding Author’s E-mail: musil.z@seznam.cz Tel.: +420-224968164; Fax.: +420224918666 Renal anomalies are quite a common medical condition thought to be genetically influenced. The subject of this work was to conduct molecular genetic analysis of two human renal agenesis causing candidate genes encoding the RET receptor thyrosin kinase and GDNF neutrophic factor. The mutational analysis of twenty RET exons and three GDNF exons in twenty patients diagnosed with unilateral renal agenesis was carried out. Furthermore, copy number changes in both genes were investigated. The aim of this work was to identify potential mutations of RET/GDNF genes and thus to prove their association with renal agenesis. In this group of patients, no known pathogenic mutations were discovered, only three known single nucleotide polymorphisms of the RET gene were detected. Polymorphism rs1800860 (GCG-GCA, Ala432) was detected in 11/20 patients, two of them in a homozygous state. Polymorphism rs1800861 (CTT-CTG, Leu769) was identified in 6/20 patients, where one patient was a homozygote for the minor allele G. Polymorphism rs1800863 (TCC-TCG, Ser904) was detected in 5/20 patients, always in heterozygous combinations. All these polymorphisms are common ones with the minor allele frequency in population higher than 10%. Results of this study did not confirm an increased incidence of RET and GDNF mutations in patients diagnosed with renal agenesis and thus the association of these genes with renal agenesis cannot be proved. Nevertheless, with regard to a limited size of the patient group, the association between RET/GDNF signal complex and renal agenesis cannot be excluded. Submitted paper is a pilot study of patients with CAKUT in the Czech Republic, we intend to continue collecting samples and examine more genes relating to these anomalies.

Pediatric Blood & Cancer, Sep 12, 2020

To the Editor: Lipoblastomas are rare, benign adipose tissue tumors that occur primarily in infan... more To the Editor: Lipoblastomas are rare, benign adipose tissue tumors that occur primarily in infants and children below 3 years of age. Although lipoblastomas are usually localized and well-circumscribed tumors, part of them can have diffuse infiltrative growth patterns known as lipoblastomatosis. Lipoblastomatosis has a higher risk of recurrence than ordinary lipoblastoma. Histologically, lipoblastomas are composed of an admixture of mature adipocytes and lipoblasts at various stages of development.1 Lipoblastomas are characterized by chromosome rearrangement of chromosomearm8p,which occurs in about 70%of cases. Rearrangements concern the pleomorphic adenoma gene 1 (PLAG1).2,3 The 8q12 rearrangements in lipoblastoma are associated with promoter swapping, in which the PLAG1 promoter is replaced by an active promoter from the other gene, for exampleHAS2 (hyaluronic acid synthase 2) or COL1A2 (collagen 1 α 2),4,5 COL3A1 (collagen 3 α 1), or in single case RAB2A (Ras-related protein)4 or BOC (BOC cell adhesion associated, oncogene regulated).6 The promoter swapping mechanism results in overexpression of a normal PLAG1 protein. In contrast, PLAG1 rearrangement is uncommon in other types of adipose tumors, including lipoma and liposarcoma. We examined whether our lipomatous tumors had one of the two most common fusion genes, HAS2-PLAG1 and COL1A2-PLAG1 (Table S1). Due to RNA degradation and impossibility to amplify longer PCR products from FFPE material, next-generation sequencing using Sarcoma FusionPlex panel (Archer) was performed to identify molecular alteration of PLAG1 gene in Case 1. We identified a novel, yet undescribed ZEB2-PLAG1 fusion gene. Exon 1 of ZEB2 fused to exon 2 (Figure 1) ofPLAG1 and exon1ofZEB2 to exon 3ofPLAG1 (Supplemental Figure S1). The two fusion transcripts are the result fromalternative splicing. HAS2-PLAG1 fusion gene was found in one lipoma and two lipoblastomas. In all three cases, we identified two fusion transcripts (HAS2 ex1-PLAG1 ex3 and HAS2 ex1-PLAG1 ex2) resulting from alternative splicing. COL1A2-PLAG1 fusion was detected in one lipoblastoma. In one case, we identified fusion gene RAB2A-PLAG1 (Supplemental Figure S2) using primers described by Yoshida et al.4 In this case, we found hyper rearrangement in the chromosome 8 (Supplemental Figure S3) using single nucleotide polymorphismmicroarray. We describe four different PLAG1 gene fusion partners in five patients with lipoblastoma and in one lipoma. PLAG1 is an oncogene that was first observed in pleomorphic adenoma of salivary glands.4,5 This gene encodes a zinc finger protein. Its oncogenicity is partly because of activation of the insulin-like growth factor 2 (IGF2) mitogenic pathway.4,7 The entire PLAG1 coding sequence, which begins in the exon 4, is placed under the transcriptional control of an active promoter region of its fusion partner. The PLAG1 rearrangement results in promoter swapping, in which PLAG1 promoter is replaced by promoter region from another gene as: HAS2 at 8q24.1, COL1A2 at 7q22, COL3A1 at 2q31 or RAB2A at 8q12.1. We found a novel fusion gene ZEB2-PLAG1. ZEB2 (Zinc Finger E-Box Binding Homeobox 2) in 2q22.3 is the gene encoding protein, which is a member of the Zfh1 family of two-handed zinc finger/homeodomain proteins. It is located in the nucleus and functions as a DNA-binding transcriptional repressor that interacts with activated SMADs, the transducers of TGF-beta signaling, and interacts with the nucleosome remodeling and histone deacetylation complex.8 Sequence analysis of ZEB2-PLAG1 fusion gene showed that the exon 1 of ZEB2 fused to the exon 2 or 3 of PLAG1. We are of the opinion that placing all functional domains of PLAG1 under control of ZEB2 promoter represents the oncogenic mechanism for the ZEB2-PLAG1 fusion gene, similar to other PLAG1 fusion partners. PLAG1 overexpression in lipoblastoma with PLAG1 rearrangement is postulated to be result of a promoter swapping event.5,6 From our quantitative expression analysis (Supplemental Figure S4), it is apparent that high expression of the PLAG1 gene is a common feature of all PLAG1 fusion rearrangements.

Pediatric Blood & Cancer, Apr 3, 2023

Oncotarget, Dec 16, 2017

from horse kidney and characterized by Margoshes and Vallee [1]. All vertebrates examined contain... more from horse kidney and characterized by Margoshes and Vallee [1]. All vertebrates examined contain at least two or more distinct MT isoforms designated MT-1 through MT-4. MT-3 was originally dubbed neuronal growth-inhibitory factor (GIF) due to its neuroinhibitory activity [2]. Besides,

Cancer genetics and cytogenetics, Nov 1, 2002

Ewing family of tumors is a group of highly aggressive neoplasias that occur most commonly in the... more Ewing family of tumors is a group of highly aggressive neoplasias that occur most commonly in the first two decades of life. These tumors are most frequently localized in bones, less frequently in soft tissues. They usually appear as undifferentiated small round-cell tumors. With current treatment regiments, 5-year disease-free survival rates exceed 60% in patients with a localized disease. Patients

Neuro-oncology, Dec 1, 2020

induced significant cell death in all 4 cell lines in vitro. Temozolomide, difluoromethyornithine... more induced significant cell death in all 4 cell lines in vitro. Temozolomide, difluoromethyornithine and chloroquine (CQ) were then tested together with pegArg-I in U87 in vitro. We found that only CQ showed additive effect with pegArg-I against glioma in vitro. Such additive cytotoxic effect may be associated with enhanced autophagy and necrosis as shown in transmission electron microscopy and autophagy markers' expression by Western blotting. PegArg-I prolonged the survival of glioma mice, suggesting its possible anti-glioma efficacy. However, CQ+pegArg-I didn't show further significant anti-cancer efficacy in vivo. CONCLUSION: PegArg-I may be useful in slowing the progression of glioma, but additional drug candidate which works synergistically with pegArg-I remains to be explored.

Acta Neuropathologica, Aug 5, 2021

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur ... more Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2-and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017

Fishbein et al. show that neuroendocrine tumors pheochromocytomas and paragangliomas have a low g... more Fishbein et al. show that neuroendocrine tumors pheochromocytomas and paragangliomas have a low genome alteration rate but diverse driver alterations, which coalesce into four molecular subtypes. The Wnt-altered subtype, driven by MAML3 fusions and CSDE1 somatic mutations, correlates with poor clinical outcome.

Klinická onkologie, May 15, 2018

Rezistence nadorových buněk k cytostatikům je způsobena řadou mechanismů, ktere se casto kombinuj... more Rezistence nadorových buněk k cytostatikům je způsobena řadou mechanismů, ktere se casto kombinuji. Bylo popsano, že vysoka koncentrace thiolových skupin v cytoplazmě važe platinove alkylacni derivaty a chemorezistence je způsobena přenosem platiny z cytostatika na MT, ktere je inaktivuji. Protože jsme v nasich předchozich studiich prokazali zvýseni hladin MT v rezistentnich neuroblastomových /NB/ liniich, ale ne v senzitivnich liniich po inkubaci s platinovými cytostatiky zabývali jsme se významem MT-3 pro buňky NB. Metoda: Buňky NB linie SiMa transfekovane vektorem obsahujicim lidský MT-3 a GFP nebo pouze GFP (kontrola). Expresni microarray (Custom Array, Bothell, WA, USA), exprese MT-3 a nejvice exprimovaných genů validovana RT PCR. Citlivost k CDDP: MTT test, vysetřeni klonogenicity, průkaz stěpeni caspasy 3 blotem a volne kyslikove radikaly /ROS/ fluorescencni mikroskopii. Hladiny mRNA MT-3 ve 23 vzorcich vysoce rizikových NB, buňkach normalni lidske kůry nadlediny byly vysetřeny RT PCR.. Výsledky: Expresni microarray prokazala downregulaci 3 a overexpresi 19 genů u MT-3 transfekovaných NB buněk. Pomoci genove ontologie bylo zjistěno, že overexprimovane geny řidi onkogeny indukovanou senescenci ( CDKN2B a ANAPC5 ), a zvýseně exprimovane byly i geny glutathion S-transferasy M3, caspasy 4 a DNAJB6 (chaperon neuronalnich proteinů). Prokazali jsme sniženou senzitivitu MT-3 transfekovaných buněk k CDDP (24h IC 50 7,48± 0,97 a 19,81± 1,2 µg/ml), vyssi pocet kolonii po inkubaci s CDDP, snižene stěpeni caspasy 3 po inkubaci s CDDP a nižsi ROS po jejich indukci CDDP. Buňky vysoce rizikových NB exprimovaly MT-3 významně vice než nenadorove buňky nadledviny, ale nepodařilo se prokazat jednoznacný vztah k průběhu onemocněni. Zavěr: Prokazali jsme vztah mezi MT-3 a geny onkogeny indukovane senescence a některými dalsimi geny významnými pro osud buňky ( glutathion S-transferasa M3, caspasa 4 a DNAJB6 ) a významný podil MT-3 na rezistenci k CDDP. Vysoke hladiny MT-3 u vysoce rizikoveho NB by mohly být jednou z přicin castých recidiv u tohoto nadoru.

Neoplasma, 2012

Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: s... more Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133-ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs.

Československá patologie, 2022

Acta Neuropathologica Communications

Gliomas are the most common central nervous tumors in children and adolescents. However, spinal c... more Gliomas are the most common central nervous tumors in children and adolescents. However, spinal cord low-grade gliomas (sLGGs) are rare, with scarce information on tumor genomics and epigenomics. To define the molecular landscape of sLGGs, we integrated clinical data, histology, and multi-level genetic and epigenetic analyses on a consecutive cohort of 26 pediatric patients. Driver molecular alteration was found in 92% of patients (24/26). A novel variant of KIAA1549:BRAF fusion (ex10:ex9) was identified using RNA-seq in four cases. Importantly, only one-third of oncogenic drivers could be revealed using standard diagnostic methods, and two-thirds of pediatric patients with sLGGs required extensive molecular examination. The majority (23/24) of detected alterations were potentially druggable targets. Four patients in our cohort received targeted therapy with MEK or NTRK inhibitors. Three of those exhibited clinical improvement (two with trametinib, one with larotrectinib), and two p...

Neuro-Oncology

INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological ... more INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological behaviour with dismal prognosis of long-term survival 10-15%. Current molecular-biological diagnostic approaches allow for more precise characterization and determination of new unique subgroups of HGG. Our aim was to identify novel and rare HGG subgroups within our institution cohort. PATIENTS AND METHODS: Our reference centre patients′ cohort consisted of 97 clinically annotated patients with HGG diagnosed between 2000 and 2021. Sanger sequencing was used for screening of the most common HGG-related oncogenic drivers; furthermore we employed whole genome methylation array (Illumina Infinium MethylationEPIC BeadChip) and for selected samples RNA sequencing and expression profiling. RESULTS: Based on H3 status and previous radiotherapy we separated our HGG cases into the RIG, H3mut and H3wt groups. In contrast to H3mut(n=35) and RIG(n=11) that were uniformly fatal, H3wt group contained a...

Child's Nervous System

Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology... more Pontine gliomas represent difficult to treat entity due to the location and heterogeneous biology varying from indolent low-grade gliomas to aggressive diffuse intrinsic pontine glioma (DIPG). Making the correct tumor diagnosis in the pontine location is thus critical. Here, we report a case study of a 14-month-old patient initially diagnosed as histone H3 wild-type DIPG. Due to the low age of the patient, the MRI appearance of DIPG, and anaplastic astrocytoma histology, intensive chemotherapy based on the HIT-SKK protocol with vinblastine maintenance chemotherapy was administered. Rapid clinical improvement and radiological regression of the tumor were observed with nearly complete remission with durable effect and excellent clinical condition more than 6.5 years after diagnosis. Based on this unexpected therapeutic outcome, genome-wide DNA methylation array was employed and the sample was classified into the methylation class “Low-grade glioma, MYB(L1) altered.” Additionally, RT-P...

Pathology Research and Practice, Jun 1, 2023

Neoplasma, 2019

Paragangliomas and pheochromocytomas are rare, mostly benign neuroendocrine tumors, which are emb... more Paragangliomas and pheochromocytomas are rare, mostly benign neuroendocrine tumors, which are embryologically derived from neural crest cells of the autonomic nervous system. Paragangliomas are essentially the extra-adrenal counterparts of pheochromocytomas. As such, this family of tumors can be subdivided into head and neck paragangliomas, pheochromocytomas, and thoracic and abdominal extra-adrenal paragangliomas. Ten out of fifteen genes that contribute to the development of paragangliomas are more susceptible to the development of head and neck paragangliomas when mutated. Gene expression profiling revealed that pheochromocytomas and paragangliomas could be classified into two main clusters (C1 and C2) based on transcriptomes. These groups were defined according to their mutational status and as such strongly associated with specific tumorigenic pathways. The influence of the main genetic drivers on the somatic molecular phenotype was shown by DNA methylation and miRNA profiling. Certain subunits of succinate dehydrogenase (SDHx), von Hippel-Lindau (VHL) and transmembrane protein 127 (TMEM127) still have the highest impact on development of head and neck paragangliomas. The link between RAS proteins and the formation of pheochromocytomas and paragangliomas is clear due to the effect of receptor tyrosine-protein kinase (RET) and neurofibromatosis type 1 (NF1) in RAS signaling and recent discovery of the role of HRAS. The functions of MYC-associated factor X (MAX) and prolyl hydroxylase 2 (PHD2) mutations in the contribution to the pathogenesis of paragangliomas still remain unclear. Ongoing studies give us an insight into the incidence of germline and somatic mutations, thus offering guidelines for early detection. Furthermore, these also show the risk of mistakenly assuming sporadic cases in the absence of definitive family history in head and neck paragangliomas.

PubMed, 2022

The current progress and increasing knowledge about the genetic causes of cancer opens up new pos... more The current progress and increasing knowledge about the genetic causes of cancer opens up new possibilities for its treatment. However, it is necessary to combine the results obtained using classical pathological methods with sensitive, multiplex molecular pathological methods. The method that meets the required criteria is MLPA based on multiplex PCR reaction. This method detects both changes in gene copy number and DNA methylation and, last but not least, point mutations. The MLPA reaction is applicable to even highly fragmented DNA. At the same time, it is a robust method that can be performed on standard thermocyclers, the fluorescent tip label requires automatic sequencers. Up to 50 genetic markers can be tested in one reaction, a number that allows a diagnostic and prognostic conclusion. All these features lead to the routine use of MLPA analysis not only in diagnosis but also in cancer research. The present article aims to summarize the different types of MLPA reactions, its benefits, but also the potential pitfalls.

PubMed, 2022

The new WHO classification of CNS tumors is largely based on molecular diagnostic. Without molecu... more The new WHO classification of CNS tumors is largely based on molecular diagnostic. Without molecular methods some entities can no longer be diagnosed. We are trying to show a rational approach to the CNS tumors diagnostics, which is based on conventional molecular methods such as RT-PCR, Sanger sequencing, MLPA, extended by the next generation sequencing (NGS) and methylation SNP array.

1 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Pra... more 1 Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 2 Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol in Prague, Czech Republic 3 Department of Oncology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 4 Institute of Pathology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 5 Institute of Endocrinology, Prague, Czech Republic 6 Department of Nephrology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic 7 Department of Paediatrics of the 2nd Faculty of Medicine, Charles University in Prague and University Hospital Motol in Prague, Czech Republic *Coressponding Author’s E-mail: musil.z@seznam.cz Tel.: +420-224968164; Fax.: +420224918666 Renal anomalies are quite a common medical condition thought to be genetically influenced. The subject of this work was to conduct molecular genetic analysis of two human renal agenesis causing candidate genes encoding the RET receptor thyrosin kinase and GDNF neutrophic factor. The mutational analysis of twenty RET exons and three GDNF exons in twenty patients diagnosed with unilateral renal agenesis was carried out. Furthermore, copy number changes in both genes were investigated. The aim of this work was to identify potential mutations of RET/GDNF genes and thus to prove their association with renal agenesis. In this group of patients, no known pathogenic mutations were discovered, only three known single nucleotide polymorphisms of the RET gene were detected. Polymorphism rs1800860 (GCG-GCA, Ala432) was detected in 11/20 patients, two of them in a homozygous state. Polymorphism rs1800861 (CTT-CTG, Leu769) was identified in 6/20 patients, where one patient was a homozygote for the minor allele G. Polymorphism rs1800863 (TCC-TCG, Ser904) was detected in 5/20 patients, always in heterozygous combinations. All these polymorphisms are common ones with the minor allele frequency in population higher than 10%. Results of this study did not confirm an increased incidence of RET and GDNF mutations in patients diagnosed with renal agenesis and thus the association of these genes with renal agenesis cannot be proved. Nevertheless, with regard to a limited size of the patient group, the association between RET/GDNF signal complex and renal agenesis cannot be excluded. Submitted paper is a pilot study of patients with CAKUT in the Czech Republic, we intend to continue collecting samples and examine more genes relating to these anomalies.

Pediatric Blood & Cancer, Sep 12, 2020

To the Editor: Lipoblastomas are rare, benign adipose tissue tumors that occur primarily in infan... more To the Editor: Lipoblastomas are rare, benign adipose tissue tumors that occur primarily in infants and children below 3 years of age. Although lipoblastomas are usually localized and well-circumscribed tumors, part of them can have diffuse infiltrative growth patterns known as lipoblastomatosis. Lipoblastomatosis has a higher risk of recurrence than ordinary lipoblastoma. Histologically, lipoblastomas are composed of an admixture of mature adipocytes and lipoblasts at various stages of development.1 Lipoblastomas are characterized by chromosome rearrangement of chromosomearm8p,which occurs in about 70%of cases. Rearrangements concern the pleomorphic adenoma gene 1 (PLAG1).2,3 The 8q12 rearrangements in lipoblastoma are associated with promoter swapping, in which the PLAG1 promoter is replaced by an active promoter from the other gene, for exampleHAS2 (hyaluronic acid synthase 2) or COL1A2 (collagen 1 α 2),4,5 COL3A1 (collagen 3 α 1), or in single case RAB2A (Ras-related protein)4 or BOC (BOC cell adhesion associated, oncogene regulated).6 The promoter swapping mechanism results in overexpression of a normal PLAG1 protein. In contrast, PLAG1 rearrangement is uncommon in other types of adipose tumors, including lipoma and liposarcoma. We examined whether our lipomatous tumors had one of the two most common fusion genes, HAS2-PLAG1 and COL1A2-PLAG1 (Table S1). Due to RNA degradation and impossibility to amplify longer PCR products from FFPE material, next-generation sequencing using Sarcoma FusionPlex panel (Archer) was performed to identify molecular alteration of PLAG1 gene in Case 1. We identified a novel, yet undescribed ZEB2-PLAG1 fusion gene. Exon 1 of ZEB2 fused to exon 2 (Figure 1) ofPLAG1 and exon1ofZEB2 to exon 3ofPLAG1 (Supplemental Figure S1). The two fusion transcripts are the result fromalternative splicing. HAS2-PLAG1 fusion gene was found in one lipoma and two lipoblastomas. In all three cases, we identified two fusion transcripts (HAS2 ex1-PLAG1 ex3 and HAS2 ex1-PLAG1 ex2) resulting from alternative splicing. COL1A2-PLAG1 fusion was detected in one lipoblastoma. In one case, we identified fusion gene RAB2A-PLAG1 (Supplemental Figure S2) using primers described by Yoshida et al.4 In this case, we found hyper rearrangement in the chromosome 8 (Supplemental Figure S3) using single nucleotide polymorphismmicroarray. We describe four different PLAG1 gene fusion partners in five patients with lipoblastoma and in one lipoma. PLAG1 is an oncogene that was first observed in pleomorphic adenoma of salivary glands.4,5 This gene encodes a zinc finger protein. Its oncogenicity is partly because of activation of the insulin-like growth factor 2 (IGF2) mitogenic pathway.4,7 The entire PLAG1 coding sequence, which begins in the exon 4, is placed under the transcriptional control of an active promoter region of its fusion partner. The PLAG1 rearrangement results in promoter swapping, in which PLAG1 promoter is replaced by promoter region from another gene as: HAS2 at 8q24.1, COL1A2 at 7q22, COL3A1 at 2q31 or RAB2A at 8q12.1. We found a novel fusion gene ZEB2-PLAG1. ZEB2 (Zinc Finger E-Box Binding Homeobox 2) in 2q22.3 is the gene encoding protein, which is a member of the Zfh1 family of two-handed zinc finger/homeodomain proteins. It is located in the nucleus and functions as a DNA-binding transcriptional repressor that interacts with activated SMADs, the transducers of TGF-beta signaling, and interacts with the nucleosome remodeling and histone deacetylation complex.8 Sequence analysis of ZEB2-PLAG1 fusion gene showed that the exon 1 of ZEB2 fused to the exon 2 or 3 of PLAG1. We are of the opinion that placing all functional domains of PLAG1 under control of ZEB2 promoter represents the oncogenic mechanism for the ZEB2-PLAG1 fusion gene, similar to other PLAG1 fusion partners. PLAG1 overexpression in lipoblastoma with PLAG1 rearrangement is postulated to be result of a promoter swapping event.5,6 From our quantitative expression analysis (Supplemental Figure S4), it is apparent that high expression of the PLAG1 gene is a common feature of all PLAG1 fusion rearrangements.

Pediatric Blood & Cancer, Apr 3, 2023

Oncotarget, Dec 16, 2017

from horse kidney and characterized by Margoshes and Vallee [1]. All vertebrates examined contain... more from horse kidney and characterized by Margoshes and Vallee [1]. All vertebrates examined contain at least two or more distinct MT isoforms designated MT-1 through MT-4. MT-3 was originally dubbed neuronal growth-inhibitory factor (GIF) due to its neuroinhibitory activity [2]. Besides,

Cancer genetics and cytogenetics, Nov 1, 2002

Ewing family of tumors is a group of highly aggressive neoplasias that occur most commonly in the... more Ewing family of tumors is a group of highly aggressive neoplasias that occur most commonly in the first two decades of life. These tumors are most frequently localized in bones, less frequently in soft tissues. They usually appear as undifferentiated small round-cell tumors. With current treatment regiments, 5-year disease-free survival rates exceed 60% in patients with a localized disease. Patients

Neuro-oncology, Dec 1, 2020

induced significant cell death in all 4 cell lines in vitro. Temozolomide, difluoromethyornithine... more induced significant cell death in all 4 cell lines in vitro. Temozolomide, difluoromethyornithine and chloroquine (CQ) were then tested together with pegArg-I in U87 in vitro. We found that only CQ showed additive effect with pegArg-I against glioma in vitro. Such additive cytotoxic effect may be associated with enhanced autophagy and necrosis as shown in transmission electron microscopy and autophagy markers' expression by Western blotting. PegArg-I prolonged the survival of glioma mice, suggesting its possible anti-glioma efficacy. However, CQ+pegArg-I didn't show further significant anti-cancer efficacy in vivo. CONCLUSION: PegArg-I may be useful in slowing the progression of glioma, but additional drug candidate which works synergistically with pegArg-I remains to be explored.

Acta Neuropathologica, Aug 5, 2021

Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur ... more Ependymomas encompass a heterogeneous group of central nervous system (CNS) neoplasms that occur along the entire neuroaxis. In recent years, extensive (epi-)genomic profiling efforts have identified several molecular groups of ependymoma that are characterized by distinct molecular alterations and/or patterns. Based on unsupervised visualization of a large cohort of genome-wide DNA methylation data, we identified a highly distinct group of pediatric-type tumors (n = 40) forming a cluster separate from all established CNS tumor types, of which a high proportion were histopathologically diagnosed as ependymoma. RNA sequencing revealed recurrent fusions involving the pleomorphic adenoma gene-like 1 (PLAGL1) gene in 19 of 20 of the samples analyzed, with the most common fusion being EWSR1:PLAGL1 (n = 13). Five tumors showed a PLAGL1:FOXO1 fusion and one a PLAGL1:EP300 fusion. High transcript levels of PLAGL1 were noted in these tumors, with concurrent overexpression of the imprinted genes H19 and IGF2, which are regulated by PLAGL1. Histopathological review of cases with sufficient material (n = 16) demonstrated a broad morphological spectrum of tumors with predominant ependymoma-like features. Immunohistochemically, tumors were GFAP positive and OLIG2-and SOX10 negative. In 3/16 of the cases, a dot-like positivity for EMA was detected. All tumors in our series were located in the supratentorial compartment. Median age of the patients at the time of diagnosis was 6.2 years. Median progression-free survival was 35 months (for 11 patients with data available). In summary, our findings suggest the existence of a novel group of supratentorial neuroepithelial tumors that are characterized by recurrent PLAGL1 fusions and enriched for pediatric patients.

Carolina Digital Repository (University of North Carolina at Chapel Hill), 2017

Fishbein et al. show that neuroendocrine tumors pheochromocytomas and paragangliomas have a low g... more Fishbein et al. show that neuroendocrine tumors pheochromocytomas and paragangliomas have a low genome alteration rate but diverse driver alterations, which coalesce into four molecular subtypes. The Wnt-altered subtype, driven by MAML3 fusions and CSDE1 somatic mutations, correlates with poor clinical outcome.

Klinická onkologie, May 15, 2018

Rezistence nadorových buněk k cytostatikům je způsobena řadou mechanismů, ktere se casto kombinuj... more Rezistence nadorových buněk k cytostatikům je způsobena řadou mechanismů, ktere se casto kombinuji. Bylo popsano, že vysoka koncentrace thiolových skupin v cytoplazmě važe platinove alkylacni derivaty a chemorezistence je způsobena přenosem platiny z cytostatika na MT, ktere je inaktivuji. Protože jsme v nasich předchozich studiich prokazali zvýseni hladin MT v rezistentnich neuroblastomových /NB/ liniich, ale ne v senzitivnich liniich po inkubaci s platinovými cytostatiky zabývali jsme se významem MT-3 pro buňky NB. Metoda: Buňky NB linie SiMa transfekovane vektorem obsahujicim lidský MT-3 a GFP nebo pouze GFP (kontrola). Expresni microarray (Custom Array, Bothell, WA, USA), exprese MT-3 a nejvice exprimovaných genů validovana RT PCR. Citlivost k CDDP: MTT test, vysetřeni klonogenicity, průkaz stěpeni caspasy 3 blotem a volne kyslikove radikaly /ROS/ fluorescencni mikroskopii. Hladiny mRNA MT-3 ve 23 vzorcich vysoce rizikových NB, buňkach normalni lidske kůry nadlediny byly vysetřeny RT PCR.. Výsledky: Expresni microarray prokazala downregulaci 3 a overexpresi 19 genů u MT-3 transfekovaných NB buněk. Pomoci genove ontologie bylo zjistěno, že overexprimovane geny řidi onkogeny indukovanou senescenci ( CDKN2B a ANAPC5 ), a zvýseně exprimovane byly i geny glutathion S-transferasy M3, caspasy 4 a DNAJB6 (chaperon neuronalnich proteinů). Prokazali jsme sniženou senzitivitu MT-3 transfekovaných buněk k CDDP (24h IC 50 7,48± 0,97 a 19,81± 1,2 µg/ml), vyssi pocet kolonii po inkubaci s CDDP, snižene stěpeni caspasy 3 po inkubaci s CDDP a nižsi ROS po jejich indukci CDDP. Buňky vysoce rizikových NB exprimovaly MT-3 významně vice než nenadorove buňky nadledviny, ale nepodařilo se prokazat jednoznacný vztah k průběhu onemocněni. Zavěr: Prokazali jsme vztah mezi MT-3 a geny onkogeny indukovane senescence a některými dalsimi geny významnými pro osud buňky ( glutathion S-transferasa M3, caspasa 4 a DNAJB6 ) a významný podil MT-3 na rezistenci k CDDP. Vysoke hladiny MT-3 u vysoce rizikoveho NB by mohly být jednou z přicin castých recidiv u tohoto nadoru.

Neoplasma, 2012

Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: s... more Cancer stem cells (CSCs) form a small proportion of tumor cells that have stem cell properties: self-renewal capacity, the ability to develop into different lineages and proliferative potential. The interest in CSCs emerged from their expected role in initiation, progression and recurrence of many tumors. They are generally resistant to conventional chemotherapy and radiotherapy. There are two hypotheses about their origin: The first assumes that CSCs may arise from normal stem cells, and the second supposes that differentiated cells acquire the properties of CSCs. Both hypotheses are not mutually exclusive, as it is possible that CSCs have a diverse origin in different tumors. CD133+ cells (CD133 is marker of CSC in some tumors) isolated from NBL, osteosarcoma and Ewing sarcoma cell lines are resistant to cisplatin, carboplatin, etoposide and doxorubicin than the CD133-ones. Being resistant to chemotherapy, there were many attempts to target CSCs epigenetically including the use of histone deacetylase inhibitors. The diverse influence of valproic acid (histone deacetylase inhibitor) on normal and cancer stem cells was proved in different experiments. We have found an increase percentage of CD133+ NBL cells after their incubation with VPA in a dose that does not induce apoptosis. Further researches on CSCs and clinical application for their detection are necessary: (i) to define the CSC function in carcinogenesis, cancer development and their role in metastasis; (ii) to find a specific marker for CSCs in different tumors; (iii) to explain the role of different pathways that determine their behavior and (iv) to explain mechanisms of chemoresistance of CSCs.

Československá patologie, 2022

Acta Neuropathologica Communications

Gliomas are the most common central nervous tumors in children and adolescents. However, spinal c... more Gliomas are the most common central nervous tumors in children and adolescents. However, spinal cord low-grade gliomas (sLGGs) are rare, with scarce information on tumor genomics and epigenomics. To define the molecular landscape of sLGGs, we integrated clinical data, histology, and multi-level genetic and epigenetic analyses on a consecutive cohort of 26 pediatric patients. Driver molecular alteration was found in 92% of patients (24/26). A novel variant of KIAA1549:BRAF fusion (ex10:ex9) was identified using RNA-seq in four cases. Importantly, only one-third of oncogenic drivers could be revealed using standard diagnostic methods, and two-thirds of pediatric patients with sLGGs required extensive molecular examination. The majority (23/24) of detected alterations were potentially druggable targets. Four patients in our cohort received targeted therapy with MEK or NTRK inhibitors. Three of those exhibited clinical improvement (two with trametinib, one with larotrectinib), and two p...

Neuro-Oncology

INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological ... more INTRODUCTION: Paediatric high-grade gliomas (HGG) are characterised by the aggressive biological behaviour with dismal prognosis of long-term survival 10-15%. Current molecular-biological diagnostic approaches allow for more precise characterization and determination of new unique subgroups of HGG. Our aim was to identify novel and rare HGG subgroups within our institution cohort. PATIENTS AND METHODS: Our reference centre patients′ cohort consisted of 97 clinically annotated patients with HGG diagnosed between 2000 and 2021. Sanger sequencing was used for screening of the most common HGG-related oncogenic drivers; furthermore we employed whole genome methylation array (Illumina Infinium MethylationEPIC BeadChip) and for selected samples RNA sequencing and expression profiling. RESULTS: Based on H3 status and previous radiotherapy we separated our HGG cases into the RIG, H3mut and H3wt groups. In contrast to H3mut(n=35) and RIG(n=11) that were uniformly fatal, H3wt group contained a...