Real-world data on diffuse large B-cell lymphoma in 2010-2019: usability of large data sets of Finnish hospital data lakes.

Background: Real-world data on diffuse large B-cell lymphoma (DLBCL) has remained incomplete. In Finland, health record data originally recorded in different hospital data record systems are collectively available via data lake technology, enabling efficient extraction and analysis of large data sets. The usability of Finnish data lake data in the assessment of DLBCL was evaluated. Methods: Adult DLBCL patients diagnosed between 2010 and 2019, home municipality in the Hospital District of Southwest Finland and data available in respective data lake were included. Results: The algorithmic determination of treatment lines and respective survival was successful. Patient characterization was feasible, albeit partly incomplete because of limited data content/availability and coverage. Stage, International Prognostic Index and cell of origin were available for 63.0, 68.3 and 28.4% of patients, respectively. Genetic aberrations were not structurally available or feasible to extract without a manual chart review. Conclusion: Finnish data lakes represent an efficient way to analyze large DLBCL data sets. The current study provides a tool for developing recording practices in routine care.

Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses.

Neuronal ceroid lipofuscinoses (NCLs) are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5) disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs) harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X), were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

Deregulation of biometal homeostasis: the missing link for neuronal ceroid lipofuscinoses?

Neuronal ceroid lipofuscinoses (NCLs), a group of genetically distinct fatal neurodegenerative disorders with no treatment or cure, are clinically characterised by progressive motor and visual decline leading to premature death. While the underlying pathological mechanisms are yet to be precisely determined, the diseases share several common features including inflammation, lysosomal lipofuscin deposits and lipid abnormalities. An important hallmark of most common neurodegenerative disorders including Alzheimer's, Parkinson's and motor neuron diseases is deregulation of biologically active metal homeostasis. Metals such as zinc, copper and iron are critical enzyme cofactors and are important for synaptic transmission in the brain, but can mediate oxidative neurotoxicity when homeostatic regulatory mechanisms fail. We previously demonstrated biometal accumulation and altered biometal transporter expression in 3 animal models of CLN6 NCL disease. In this study we investigated the hypothesis that altered biometal homeostasis may be a feature of NCLs in general using 3 additional animal models of CLN1, CLN3 and CLN5 disease. We demonstrated significant accumulation of the biometals zinc, copper, manganese, iron and cobalt in these mice. Patterns of biometal accumulation in each model preceded significant neurodegeneration, and paralleled the relative severity of disease previously described for each model. Additionally, we observed deregulation of transcripts encoding the anti-oxidant protein, metallothionein (Mt), indicative of disruptions to biometal homeostasis. These results demonstrate that altered biometal homeostasis is a key feature of at least 4 genetically distinct forms of NCL disease.

Exacerbated neuronal ceroid lipofuscinosis phenotype in Cln1/5 double-knockout mice.

Both CLN1 and CLN5 deficiencies lead to severe neurodegenerative diseases of childhood, known as neuronal ceroid lipofuscinoses (NCLs). The broadly similar phenotypes of NCL mouse models, and the potential for interactions between NCL proteins, raise the possibility of shared or convergent disease mechanisms. To begin addressing these issues, we have developed a new mouse model lacking both Cln1 and Cln5 genes. These double-knockout (Cln1/5 dko) mice were fertile, showing a slight decrease in expected Mendelian breeding ratios, as well as impaired embryoid body formation by induced pluripotent stem cells derived from Cln1/5 dko fibroblasts. Typical disease manifestations of the NCLs, i.e. seizures and motor dysfunction, were detected at the age of 3 months, earlier than in either single knockout mouse. Pathological analyses revealed a similar exacerbation and earlier onset of disease in Cln1/5 dko mice, which exhibited a pronounced accumulation of autofluorescent storage material. Cortical demyelination and more pronounced glial activation in cortical and thalamic regions was followed by cortical neuron loss. Alterations in lipid metabolism in Cln1/5 dko showed a specific increase in plasma phospholipid transfer protein (PLTP) activity. Finally, gene expression profiling of Cln1/5 dko cortex revealed defects in myelination and immune response pathways, with a prominent downregulation of α-synuclein in Cln1/5 dko mouse brains. The simultaneous loss of both Cln1 and Cln5 genes might enhance the typical pathological phenotypes of these mice by disrupting or downregulating shared or convergent pathogenic pathways, which could potentially include interactions of CLN1 and CLN5.

Cln5-deficiency in mice leads to microglial activation, defective myelination and changes in lipid metabolism.

CLN5 disease, late infantile variant phenotype neuronal ceroid lipofuscinosis, is a severe neurodegenerative disease caused by mutations in the CLN5 gene, which encodes a lysosomal protein of unknown function. Cln5-deficiency in mice leads to loss of thalamocortical neurons, and glial activation, but the underlying mechanisms are poorly understood. We have now studied the gene expression of Cln5 in the mouse brain and show that it increases gradually with age and differs between neurons and glia, with the highest expression in microglia. In Cln5(-/-) mice, we documented early and significant microglial activation that was already evident at 3 months of age. Loss of Cln5 also leads to defective myelination in vitro and in the developing mouse brain. This was accompanied by early alterations in serum lipid profiles, dysfunctional cellular metabolism and lipid transport in Cln5(-/-) mice. Taken together, these data provide significant new information about events associated with Cln5-deficiency, revealing altered myelination and disturbances in lipid metabolism, together with an early neuroimmune response.

Amplification and overexpression of KIT, PDGFRA, and VEGFR2 in medulloblastomas and primitive neuroectodermal tumors.

Medulloblastomas (MB) and primitive neuroectodermal tumors (PNET) are the most common malignant brain tumors in children. These two tumor types are histologically similar, but have different genetic backgrounds and clinical outcomes. Other brain tumors, such as gliomas, frequently have coamplification and overexpression of receptor tyrosine kinases KIT, platelet-derived growth factor receptor alpha (PDGFRA), and vascular endothelial growth factor receptor 2 (VEGFR2). We investigated protein expression and gene copy numbers of KIT, PDGFRA, and VEGFR2 in 41 MB and 11 PNET samples by immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH). KIT and PDGFRA expression was detected in both MBs and PNETs, whereas VEGFR2 expression was weak in these tumors. KIT, PDGFRA, and VEGFR2 amplifications were all present in 4% of MBs/PNETs, and KIT amplification was associated with concurrent PDGFRA and VEGFR2 amplifications (P

Mutation and copy number analysis of LNX1 and Numbl in nervous system tumors.

Alterations at chromosome locus 4q12 are frequently found in gliomas; this locus contains the receptor tyrosine kinase--encoding genes KIT, PDGFRA, and KDR (alias VEGFR2). Notable among the genes at this locus is LNX1, the ligand of Numb protein X. LNX1 encodes a PDZ domain containing protein, which interacts with the cell fate determinant Numbl, a Numb homolog-like gene involved in the maintenance of neural progenitor cells during embryonic neurogenesis. We performed a mutation analysis for LNX1 and Numbl genes. In addition, gene copy numbers of LNX1, Numbl, and KIT in human nervous system tumors were analyzed by chromogenic in situ hybridization. Tissue samples from 90 patients were screened for LNX1 and Numbl mutations, and tissue sections from 56 samples were analyzed for gene amplification status. Our analysis revealed missense mutations in LNX1 exons 3 and 5 and a single-nucleotide polymorphism in Numbl exon 6. In addition, polyglutamine repeat polymorphism was found in Numbl exon 10. Chromogenic in situ hybridization showed gene amplification of LNX1 in 10%, Numbl in 5%, and KIT in 6% of nervous system tumors. Both gene sequence alterations and amplifications of LNX1 and Numbl are present in a subset of human gliomas, and the role of these genes in neurogenesis suggests that they may contribute to development of glial tumors.

KIT overexpression induces proliferation in astrocytes in an imatinib-responsive manner and associates with proliferation index in gliomas.

Activating gene mutations, gene amplifications and overexpressed proteins may be useful as targets for novel therapies. Alterations at chromosome locus 4q12 are associated with gliomas and the region harbors the receptor tyrosine kinase gene KIT, which is frequently amplified in gliomas, and also overexpressed in a subset of gliomas. KIT and its ligand stem cell factor are widely expressed in embryonic and adult mouse brain, and they play a role in many signal transduction pathways involved in cellular proliferation, differentiation and cancer cell metastasis. However, the function of KIT in gliomagenesis or disease progression remains unresolved as well as its role in neural and brain tumor development. In this study, we utilized lentivirus-mediated gene transfer to deliver the KIT gene into mouse astrocytes. The growth properties of KIT overexpressing cells were analyzed using several in vitro functional assays. The effect of receptor tyrosine kinase inhibitor imatinib on astrocyte growth was also investigated. Our results indicate that overexpression of KIT in mouse astrocytes promotes cell proliferation, and the increased proliferation is partly inhibited by imatinib treatment. Furthermore, KIT overexpression induces phenotypic changes in the cells suggesting that KIT may play a role in astrocyte growth regulation.

Copy number alterations of the polycomb gene BMI1 in gliomas.

Gliomas are heterogeneous tumours that grow in an uninhibited fashion, and these brain tumour cells share numerous characteristics with neural stem cells. The BMI1 gene encodes a component of the polycomb protein complex regulating epigenetically gene activity via histone modification. It functions for instance during the development of the central nervous system and maturation of neural cells. BMI-1 protein expression is deregulated in several forms of cancer and gene amplification has been identified in mantle cell lymphomas. Since BMI1 is located at chromosome 10p, a region implicated frequently in brain tumourigenesis, we investigated the genetic status and the corresponding expression patterns of BMI1 in a series of 100 low- and high-grade primary and recurrent gliomas. Chromogenic in situ hybridisation (CISH) with probes directed against BMI1 at 10p13 and the centromere of chromosome 10 was used in the analyses. Of all gliomas, 59% demonstrated aberrant copy numbers of BMI1. Deletions of the BMI1 locus were found in most types of tumours, and in a univariate survival analysis these cases had poor prognosis. Increased copy numbers of the BMI1 locus (3-5 copies) were found in all histological types, especially in high-grade astrocytomas. No difference in prognosis between cases with normal copy numbers and cases with increased copy numbers could be observed. This data suggests that BMI1 gene is aberrant at the chromosomal level in a subset of gliomas, and possibly contributes to brain tumour pathogenesis.

Characterization and biological role of the O-polysaccharide gene cluster of Yersinia enterocolitica serotype O:9.

Yersinia enterocolitica serotype O:9 is a gram-negative enteropathogen that infects animals and humans. The role of lipopolysaccharide (LPS) in Y. enterocolitica O:9 pathogenesis, however, remains unclear. The O:9 LPS consists of lipid A to which is linked the inner core oligosaccharide, serving as an attachment site for both the outer core (OC) hexasaccharide and the O-polysaccharide (OPS; a homopolymer of N-formylperosamine). In this work, we cloned the OPS gene cluster of O:9 and identified 12 genes organized into four operons upstream of the gnd gene. Ten genes were predicted to encode glycosyltransferases, the ATP-binding cassette polysaccharide translocators, or enzymes required for the biosynthesis of GDP-N-formylperosamine. The two remaining genes within the OPS gene cluster, galF and galU, were not ascribed a clear function in OPS biosynthesis; however, the latter gene appeared to be essential for O:9. The biological functions of O:9 OPS and OC were studied using isogenic mutants lacking one or both of these LPS parts. We showed that OPS and OC confer resistance to human complement and polymyxin B; the OPS effect on polymyxin B resistance could be observed only in the absence of OC.

Construction and biological activity of a full-length molecular clone of human Torque teno virus (TTV) genotype 6.

Torque teno virus (TTV) is a non-enveloped human virus with a circular negative-sense (approximately 3800 nucleotides) ssDNA genome. TTV resembles in genome organization the chicken anemia virus, the animal pathogen of the Circoviridae family, and is currently classified as a member of a new, floating genus, Anellovirus. Molecular and cell biological research on TTV has been restricted by the lack of permissive cell lines and functional, replication-competent plasmid clones. In order to examine the key biological activities (i.e. RNA transcription and DNA replication) of this still poorly characterized ssDNA virus, we cloned the full-length genome of TTV genotype 6 and transfected it into cells of several types. TTV mRNA transcription was detected by RT-PCR in all the cell types: KU812Ep6, Cos-1, 293, 293T, Chang liver, Huh7 and UT7/Epo-S1. Replicating TTV DNA was detected in the latter five cell types by a DpnI-based restriction enzyme method coupled with Southern analysis, a novel approach to assess TTV DNA replication. The replicating full-length clone, the cell lines found to support TTV replication, and the methods presented here will facilitate the elucidation of the molecular biology and the life cycle of this recently identified human virus.

Amplification of KIT, PDGFRA, VEGFR2, and EGFR in gliomas.

Receptor tyrosine kinase aberrations are implicated in the genesis of gliomas. We investigated expression and amplification of KIT, PDGFRA, VEGFR2, and EGFR in 87 gliomas consisting of astrocytomas, anaplastic astrocytomas, oligodendrogliomas, or oligoastrocytomas in tumor samples collected at the time of the diagnosis and in samples of the same tumors at tumor recurrence. Gene amplifications were investigated using either chromogenic in situ hybridization or fluorescence in situ hybridization, and protein expression using immunohistochemistry. In samples collected at glioma diagnosis, KIT and PDGFRA amplifications were more frequent in anaplastic astrocytomas than in astrocytomas, oligodendrogliomas, and oligoastrocytomas [28% versus 5% (P = 0.012) and 33% versus 2% (P = 0.0008), respectively]. VEGFR2 amplifications occurred in 6% to 17% of the gliomas at diagnosis, and EGFR amplifications in 0% to 12%. Amplified KIT was more frequently present in recurrent gliomas than in newly diagnosed gliomas (P = 0.0066). KIT amplification was associated with KIT protein expression and with presence of PDGFRA and EGFR amplifications both at the time of the first glioma diagnosis and at tumor recurrence, and with VEGFR2 amplification at tumor recurrence. Three (4%) primary gliomas and 10 (14%) recurrent gliomas that were evaluable for coamplification of KIT, PDGFRA, and VEGFR2 showed amplification of at least two of these genes; the amplicon contained amplified KIT in all 13 cases. In conclusion, besides glioblastoma, amplified KIT, PDGFRA, and VEGFR may also occur in lower-grade gliomas and in their recurrent tumors. It is currently not known whether specific tyrosine kinase inhibitors are effective in the treatment of such gliomas.

Molecular genetic analysis of the REST/NRSF gene in nervous system tumors.

The gene for RE1-silencing transcription factor (REST) alias neuron-restrictive silencer factor NRSF, acts as a transcriptional repressor in the neuronal differentiation pathways in non-neuronal cells, and plays an important role in neuronal development. Inactivating mutations or overexpression of REST have previously been reported in various types of cancer, but no data is available for the role of REST alterations in gliomas. REST gene was screened for mutations in 161 nervous system tumors consisting of astrocytomas, glioblastomas, oligodendrogliomas, oligoastrocytomas, medulloblastomas, meningiomas and schwannomas. REST exons 1-3 were analyzed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. The gene copy numbers of REST were investigated by chromogenic (CISH) and fluorescence in situ hybridization (FISH) techniques. Non-synonymous SNPs (P797L, P815S) were found in eight different brain tumor samples. No truncating or activating novel mutations of REST were discovered. Since REST is located at 4q12, a chromosome region implicated in brain tumorigenesis, we conducted gene copy number analyses in medulloblastomas and gliomas. The majority of gliomas (67%) demonstrated low-level amplifications of REST, and only one oligodendroglioma showed high-level amplification of the gene. In medulloblastomas, 38% of samples were determined as aneuploidic, no high-level amplifications were found. Our data suggests that REST is neither activated nor inactivated via mutations in gliomas, while high-level amplification may rarely occur.