RESUMEN
Juvenile xanthogranuloma (JXG) is a histiocytic neoplasm that usually presents in the skin. Rarely, extracutaneous localizations occur; the genetic drivers of this clinical variant of JXG remain incompletely characterized. We present detailed clinicopathologic and molecular data of 16 children with extracutaneous JXG and 5 adults with xanthogranulomas confined to the central nervous system (CNS) or soft tissue. Tissue samples were obtained through the Dutch Nationwide Pathology Databank and analyzed with an innovative sequencing technique capable of detecting both small genomic variants and gene rearrangements. Targetable kinase alterations were detected in 16/16 children and 1/5 adults. Alterations included CLTC::SYK fusions in 6 children and CSF1R mutations in 7 others - all below 2 years old with soft tissue tumors. One child had a CSF1R mutation and MRC1::PDGFRB fusion. Most were treated surgically, although spontaneous regression occurred in 1/6 with CLTC::SYK and 2/7 with CSF1R mutations - underscoring that treatment is not always necessary. Tumors with CLTC::SYK fusions generally lacked Touton giant cells, but exhibited many other histologic features of JXG and concordant methylation profiles. Using multispectral immunofluorescence, phosphorylated-SYK expression was localized to CD163+ histiocytes; tumors with CLTC::SYK fusions also demonstrated mTOR activation, Cyclin D1 expression, and variable phosphorylated-ERK expression. BRAFV600E was detected in 1 child and 1 adult with CNS xanthogranulomas; both responded to BRAF inhibition. Finally, a TPM3::NTRK1 fusion or MAP2K1 deletion were detected in 2 children with systemic JXG who experienced spontaneous disease regression. This study advances the molecular understanding of histiocytic neoplasms and may guide diagnostics and clinical management.
RESUMEN
Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.
Asunto(s)
Distrofina , Edición Génica , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Edición Génica/métodos , Humanos , Células Musculares/metabolismo , Distrofia Muscular de Duchenne/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
During gametogenesis in mammals, meiosis ensures the production of haploid gametes. The timing and length of meiosis to produce female and male gametes differ considerably. In contrast to males, meiotic prophase I in females initiates during development. Hence, the knowledge regarding progression through meiotic prophase I is mainly focused on human male spermatogenesis and female oocyte maturation during adulthood. Therefore, it remains unclear how the different stages of meiotic prophase I between human oogenesis and spermatogenesis compare. Analysis of single-cell transcriptomics data from human fetal germ cells (FGC) allowed us to identify the molecular signatures of female meiotic prophase I stages leptotene, zygotene, pachytene and diplotene. We have compared those between male and female germ cells in similar stages of meiotic prophase I and revealed conserved and specific features between sexes. We identified not only key players involved in the process of meiosis, but also highlighted the molecular components that could be responsible for changes in cellular morphology that occur during this developmental period, when the female FGC acquire their typical (sex-specific) oocyte shape as well as sex-differences in the regulation of DNA methylation. Analysis of X-linked expression between sexes during meiotic prophase I suggested a transient X-linked enrichment during female pachytene, that contrasts with the meiotic sex chromosome inactivation in males. Our study of the events that take place during meiotic prophase I provide a better understanding not only of female meiosis during development, but also highlights biomarkers that can be used to study infertility and offers insights in germline sex dimorphism in humans.
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Cromosomas Humanos X , Células Germinativas , Profase Meiótica I , Factores Sexuales , Transcripción Genética , Citoesqueleto/metabolismo , Metilación de ADN , Femenino , Expresión Génica , Genitales Femeninos/patología , Humanos , Masculino , Oocitos/metabolismoRESUMEN
Tenosynovial giant cell tumors (TSGCTs) are rare tumors arising in tendons or the synoviae of joints and bursae. The localized type is benign while the diffuse type shows expansive growth leading to greater morbidity and is therefore considered locally aggressive. Typical recurrent chromosomal aberrations are found in the majority of TSCGT and the CSF1 gene is frequently involved. In this article, we describe a newly identified gene fusion mediated by an inversion in a case of diffuse TSGCT. Multicolor-fluorescence in situ hybridization (FISH) molecular karyotyping identified a pericentric inversion of chromosome 1 in 7 out of 17 analyzed cells 46,XX,inv(1)(p13.3q24.3) [7]/46,XX [10], and with interphase FISH the involvement the CSF1 locus was detected. After performing transcriptome sequencing analysis for fusion detection, only one out of five fusion gene algorithms detected a fusion involving the CSF1 gene product. The resulting chimera fuses a sequence from a human endogenous retrovirus (HERV) gene to CSF1 Exon 6 on chromosome 1, abrogating the regulatory element of the 3' untranslated region of the CSF1 gene. This new translocation involving Exon 6 of the CSF1 gene fused to 1q24.1, supports the hypothesis that a mutated CSF1 protein is likely to play a vital role in the pathogenesis of TSGCT. The role of the HERV partner identified as a translocation partner, however, remains unclear. Our data add to the complexity of involved translocation partners in TSGCT and point to the potential difficulty of identifying fusion partners in tumor diagnostics using transcriptome sequencing when HERV or other repeat elements are involved.
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Retrovirus Endógenos , Tumor de Células Gigantes de las Vainas Tendinosas , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Retrovirus Endógenos/metabolismo , Hibridación Fluorescente in Situ , Tumor de Células Gigantes de las Vainas Tendinosas/genética , Tumor de Células Gigantes de las Vainas Tendinosas/metabolismo , Translocación GenéticaRESUMEN
Psammomatoid ossifying fibroma (PsOF), also known as juvenile PsOF, is a benign fibro-osseous neoplasm predominantly affecting the extragnathic bones, particularly the frontal and ethmoid bones, with a preference for adolescents and young adults. The clinical and morphologic features of PsOF may overlap with those of other fibro-osseous lesions, and additional molecular markers would help increase diagnostic accuracy. Because identical chromosomal breakpoints at bands Xq26 and 2q33 have been described in 3 cases of PsOF located in the orbita, we aimed to identify the exact genes involved in these chromosomal breakpoints and determine their frequency in PsOF using transcriptome sequencing and fluorescence in situ hybridization (FISH). We performed whole RNA transcriptome sequencing on frozen tissue in 2 PsOF index cases and identified a fusion transcript involving SATB2, located on chromosome 2q33.1, and AL513487.1, located on chromosome Xq26, in one of the cases. The fusion was validated using reverse transcription (RT)-PCR and SATB2 FISH. The fusion lead to a truncated protein product losing most of the functional domains. Subsequently, we analyzed an additional 24 juvenile PsOFs, 8 juvenile trabecular ossifying fibromas (JTOFs), and 11 cemento-ossifying fibromas (COFs) for SATB2 using FISH and found evidence of SATB2 gene rearrangements in 58% (7 of 12) of the evaluable PsOF cases but not in any of the evaluable JTOF (n = 7) and COF (n = 7) cases. A combination of SATB2 immunofluorescence and a 2-color SATB2 FISH in our index case revealed that most tumor cells harboring the rearrangement lacked SATB2 expression. Using immunohistochemistry, 65% of PsOF, 100% of JTOF, and 100% of COF cases showed moderate or strong staining for SATB2. In these cases, we observed a mosaic pattern of expression with >25% of the spindle cells in between the bone matrix, with osteoblasts and osteocytes being positive for SATB2. Interestingly, 35% (8 of 23) of PsOFs, in contrast to JTOFs and COFs, showed SATB2 expression in <5% of cells. To our knowledge, this is the first report that shows the involvement of SATB2 in the development of a neoplastic lesion. In this study, we have showed that SATB2 rearrangement is a recurrent molecular alteration that appears to be highly specific for PsOF. Our findings support that PsOF is not only morphologically and clinically but also genetically distinct from JTOF and COF.
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Neoplasias Óseas , Fibroma Osificante , Proteínas de Unión a la Región de Fijación a la Matriz , Humanos , Fibroma Osificante/genética , Hibridación Fluorescente in Situ , Neoplasias Óseas/genética , Inmunohistoquímica , Reordenamiento Génico , Factores de Transcripción/genética , Proteínas de Unión a la Región de Fijación a la Matriz/genéticaRESUMEN
AIMS: Simple Bone Cysts (SBCs) predominantly occur in long bones and 59% harbour NFATC2 rearrangements. Jaw SBC is rare and was previously referred to as traumatic bone cyst. It can rarely occur in association with cemento-osseous dysplasia (COD). To determine whether jaw SBCs represent the same entity as SBC of the long bones, or if they have a different molecular signature, we collected 48 jaw SBC cases of 47 patients to assess NFATC2 rearrangement. METHODS AND RESULTS: Out of the 48 cases, 36 could be used for fluorescence in-situ hybridization (FISH), of which nine (two of which associated with COD) were successful using an NFATC2 split probe. The remaining cases failed to show adequate FISH signals. All nine cases lacked NFATC2 rearrangement and five of these showed no detectable gene fusions using Archer FusionPlex. CONCLUSION: In our study, NFATC2 rearrangement is absent in solitary jaw SBC (n = 7) and COD-associated SBC (n = 2). Our findings suggest that SBC presenting in the jaw is molecularly different from SBC in long bones. Future molecular studies may confirm the absence of clonal molecular aberrations in SBC of the jaw which would support a non-neoplastic, reactive origin.
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Quistes Óseos , Factores de Transcripción NFATC , Tumores Odontogénicos , Humanos , Quistes Óseos/genética , Tumores Odontogénicos/genética , Factores de Transcripción NFATC/genéticaRESUMEN
Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20-23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic.
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Neoplasias Óseas , Células Madre Mesenquimatosas , Osteosarcoma , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteosarcoma/tratamiento farmacológico , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Genome editing typically involves recombination between donor nucleic acids and acceptor genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, nucleases yield off-target mutations and, most pervasively, unpredictable target allele disruptions. Remarkably, to date, the untoward phenotypic consequences of disrupting allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness as a result of DSBs at allelic and non-allelic target sites and report that simultaneous single-stranded DNA break formation at donor and acceptor DNA by CRISPR-Cas9 nickases (in trans paired nicking) mostly overcomes such disruptive genotype-phenotype associations. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments into essential and multiple-copy genomic sites. As shown herein by genotyping assays and high-throughput genome-wide sequencing of DNA translocations, this is achieved while circumventing most allelic and non-allelic mutations and chromosomal rearrangements characteristic of nuclease-dependent procedures. Our work demonstrates that in trans paired nicking retains target protein dosages in gene-edited cell populations and expands gene editing to chromosomal tracts previously not possible to modify seamlessly due to their recurrence in the genome or essentiality for cell function.
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Sistemas CRISPR-Cas/genética , ADN/genética , Desoxirribonucleasa I/química , Edición Génica/métodos , Secuencia de Bases , ADN/química , Roturas del ADN de Doble Cadena , Roturas del ADN de Cadena Simple , Desoxirribonucleasa I/genética , Endonucleasas/química , Marcación de Gen/métodos , Genoma/genética , Humanos , Mutación/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genéticaRESUMEN
The self-limited nature of nodular fasciitis (NF) is well-known but its precise mechanism has not yet been clarified. We observed that "young" NF (preoperative duration <1 month) consistently contains a higher percentage (~80%) of USP6 break-apart FISH signals than "old" NF (preoperative duration >3 months) (~20%). Thus, we hypothesized that our original observation may reflect a connection with the self-limited nature of NF. Seventeen cases with reliable data concerning the onset were selected, thus approximating the lifetime of each tumor. Besides the USP6 interphase FISH examination, we also checked the most common MYH9-USP6 fusion using RT-PCR. Because of the known pathways of the tumorigenesis of NF, the mRNA level of USP6, TRAIL, IFN-beta, JAK1, STAT1, STAT3, JUN, and CDKN2A was measured using qRT-PCR. Regarding proteins, USP6, p16, p27, TRAIL, and IFN-beta were examined using immunohistochemistry. Targeted gene panel next-generation sequencing (NGS) of three cases was additionally performed. We found a strong negative correlation (p = 0.000) between the lifetime and percentage of USP6 break-apart signals and a strong positive relationship (p = 0.000) between USP6 break-apart signals and mitotic counts. Results of immunostainings, along with qRT-PCR results, favored the previously-suggested USP6-induced negative feedback mechanism through activation of TRAIL and IFN-beta, likely resulting in apoptosis and senescence of tumor cells harboring USP6 fusions. Targeted-NGS resulted in the detection of several variants, but no additional recurrent changes in the pathogenesis of these tumors. We revealed on a cellular level the USP6-induced negative feedback mechanism. In conclusion, we emphasize that in "old" NF, the percentage of USP6 break-apart FISH signals can be as low as 14-27% which can be very important from a differential diagnostic point of view. We emphasize that a careful examination and interpretation of the NGS data is needed before clinical decision-making on treatment.
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Biomarcadores de Tumor/genética , Fascitis/genética , Fusión Génica , Reordenamiento Génico , Neoplasias de Tejido Conjuntivo/genética , Neoplasias de los Tejidos Blandos/genética , Ubiquitina Tiolesterasa/genética , Adolescente , Adulto , Biomarcadores de Tumor/análisis , Niño , Preescolar , Fascitis/metabolismo , Fascitis/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Miofibroblastos/química , Miofibroblastos/patología , Neoplasias de Tejido Conjuntivo/química , Neoplasias de Tejido Conjuntivo/patología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de los Tejidos Blandos/química , Neoplasias de los Tejidos Blandos/patología , Factores de Tiempo , Adulto JovenRESUMEN
Acute lymphoblastic leukemia is the most common pediatric cancer characterized by a heterogeneous genomic landscape with copy number aberrations occurring at various stages of pathogenesis, disease progression, and treatment resistance. In this study, disease-relevant copy number aberrations were profiled in bone marrow samples of 91 children with B- or T-cell precursor acute lymphoblastic leukemia using digital multiplex ligation-dependent probe amplification (digitalMLPATM). Whole chromosome gains and losses, subchromosomal copy number aberrations, as well as unbalanced alterations conferring intrachromosomal gene fusions were simultaneously identified with results available within 36 hours. Aberrations were observed in 96% of diagnostic patient samples, and increased numbers of copy number aberrations were detected at the time of relapse as compared with diagnosis. Comparative scrutiny of 24 matching diagnostic and relapse samples from 11 patients revealed three different patterns of clonal relationships with (i) one patient displaying identical copy number aberration profiles at diagnosis and relapse, (ii) six patients showing clonal evolution with all lesions detected at diagnosis being present at relapse, and (iii) four patients displaying conserved as well as lost or gained copy number aberrations at the time of relapse, suggestive of the presence of a common ancestral cell compartment giving rise to clinically manifest leukemia at different time points during the disease course. A newly introduced risk classifier combining cytogenetic data with digitalMLPATM-based copy number aberration profiles allowed for the determination of four genetic subgroups of B-cell precursor acute lymphoblastic leukemia with distinct event-free survival rates. DigitalMLPATM provides fast, robust, and highly optimized copy number aberration profiling for the genomic characterization of acute lymphoblastic leukemia samples, facilitates the decipherment of the clonal origin of relapse and provides highly relevant information for clinical prognosis assessment.
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Perfilación de la Expresión Génica/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Adolescente , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa Multiplex/métodosRESUMEN
Based on morphology it is often challenging to distinguish between the many different soft tissue sarcoma subtypes. Moreover, outcome of disease is highly variable even between patients with the same disease. Machine learning on transcriptome sequencing data could be a valuable new tool to understand differences between and within entities. Here we used machine learning analysis to identify novel diagnostic and prognostic markers and therapeutic targets for soft tissue sarcomas. Gene expression data was used from the Cancer Genome Atlas, the Genotype-Tissue Expression project and the French Sarcoma Group. We identified three groups of tumors that overlap in their molecular profiles as seen with unsupervised t-Distributed Stochastic Neighbor Embedding clustering and a deep neural network. The three groups corresponded to subtypes that are morphologically overlapping. Using a random forest algorithm, we identified novel diagnostic markers for soft tissue sarcoma that distinguished between synovial sarcoma and MPNST, and that we validated using qRT-PCR in an independent series. Next, we identified prognostic genes that are strong predictors of disease outcome when used in a k-nearest neighbor algorithm. The prognostic genes were further validated in expression data from the French Sarcoma Group. One of these, HMMR, was validated in an independent series of leiomyosarcomas using immunohistochemistry on tissue micro array as a prognostic gene for disease-free interval. Furthermore, reconstruction of regulatory networks combined with data from the Connectivity Map showed, amongst others, that HDAC inhibitors could be a potential effective therapy for multiple soft tissue sarcoma subtypes. A viability assay with two HDAC inhibitors confirmed that both leiomyosarcoma and synovial sarcoma are sensitive to HDAC inhibition. In this study we identified novel diagnostic markers, prognostic markers and therapeutic leads from multiple soft tissue sarcoma gene expression datasets. Thus, machine learning algorithms are powerful new tools to improve our understanding of rare tumor entities.
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Biomarcadores de Tumor/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Aprendizaje Automático , Sarcoma/genética , Biomarcadores de Tumor/análisis , Bases de Datos Genéticas , Descubrimiento de Drogas , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Pronóstico , Sarcoma/diagnóstico , Sarcoma/mortalidad , Sarcoma/terapia , Transcriptoma/genéticaRESUMEN
AIMS: Localised- and diffuse-type tenosynovial giant cell tumours (TGCT) are regarded as different clinical and radiological TGCT types. However, genetically and histopathologically they seem indistinguishable. We aimed to correlate CSF1 expression and CSF1 rearrangement with the biological behaviour of different TGCT-types with clinical outcome (recurrence). METHODS AND RESULTS: Along a continuum of extremes, therapy-naive knee TGCT patients with >3-year follow-up, mean age 43 (range = 6-71) years and 56% females were selected. Nine localised (two recurrences), 16 diffuse-type (nine recurrences) and four synovitis as control were included. Rearrangement of the CSF1 locus was evaluated with split-apart fluorescence in-situ hybridisation (FISH) probes. Regions were selected to score after identifying CSF1-expressing regions, using mRNA ISH with the help of digital correlative microscopy. CSF1 rearrangement was considered positive in samples containing >2 split signals/100 nuclei. Irrespective of TGCT-subtype, all cases showed CSF1 expression and in 76% CSF1 rearrangement was detected. Quantification of CSF1-expressing cells was not informative, due to the extensive intratumour heterogeneity. Of the four synovitis cases, two also showed CSF1 expression without CSF1 rearrangement. No correlation between CSF1 expression or rearrangement with clinical subtype and local recurrence was detected. Both localised and diffuse TGCT cases showed a scattered distribution in the tissue of CSF1-expressing cells. CONCLUSION: In diagnosing TGCT, CSF1 mRNA-ISH, in combination with CSF1 split-apart FISH using digital correlative microscopy, is an auxiliary diagnostic tool to identify rarely occurring neoplastic cells. This combined approach allowed us to detect CSF1 rearrangement in 76% of the TGCT cases. Neither CSF1 expression nor presence of CSF1 rearrangement could be associated with the difference in biological behaviour of TGCT.
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Tumor de Células Gigantes de las Vainas Tendinosas/metabolismo , Rodilla/patología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Neoplasias de los Tejidos Blandos/metabolismo , Adolescente , Adulto , Anciano , Niño , Femenino , Reordenamiento Génico , Tumor de Células Gigantes de las Vainas Tendinosas/genética , Tumor de Células Gigantes de las Vainas Tendinosas/patología , Humanos , Factor Estimulante de Colonias de Macrófagos/genética , Masculino , Persona de Mediana Edad , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/patología , Membrana Sinovial/patología , Adulto JovenRESUMEN
Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). Causative genetic alterations in MF are unknown. The low recurrence of pathogenic small-scale mutations (ie, nucleotide substitutions, indels) in the disease, calls for the study of additional aspects of MF genetics. Here, we investigated structural genomic alterations in tumor-stage MF by integrating whole-genome sequencing and RNA-sequencing. Multiple genes with roles in cell physiology (n = 113) and metabolism (n = 92) were found to be impacted by genomic rearrangements, including 47 genes currently implicated in cancer. Fusion transcripts involving genes of interest such as DOT1L, KDM6A, LIFR, TP53, and TP63 were also observed. Additionally, we identified recurrent deletions of genes involved in cell cycle control, chromatin regulation, the JAK-STAT pathway, and the PI-3-K pathway. Remarkably, many of these deletions result from genomic rearrangements. Deletion of tumor suppressors HNRNPK and SOCS1 were the most frequent genetic alterations in MF after deletion of CDKN2A. Notably, SOCS1 deletion could be detected in early-stage MF. In agreement with the observed genomic alterations, transcriptome analysis revealed up-regulation of the cell cycle, JAK-STAT, PI-3-K and developmental pathways. Our results position inactivation of HNRNPK and SOCS1 as potential driver events in MF development.
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Eliminación de Gen , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Sistema de Señalización de MAP Quinasas , Micosis Fungoide/genética , Neoplasias Cutáneas/genética , Proteína 1 Supresora de la Señalización de Citocinas/genética , Aneuploidia , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Fusión Génica , Reordenamiento Génico , Humanos , Quinasas Janus/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/genética , Micosis Fungoide/enzimología , Polimorfismo de Nucleótido Simple , ARN Neoplásico , Análisis de Secuencia de ARN , Neoplasias Cutáneas/enzimología , Secuenciación Completa del GenomaRESUMEN
Epithelioid hemangioma is a locally aggressive vascular neoplasm, found in bones and soft tissue, whose cause is currently unknown, but may involve oncogene activation. FOS is one of the earliest viral oncogenes to be characterized, and normal cellular FOS forms part of the activator protein 1 (AP-1) transcription factor complex, which plays a pivotal role in cell growth, differentiation, and survival as well as the DNA damage response. Despite this, a causal link between aberrant FOS function and naturally occurring tumors has not yet been established. Here, we describe a thorough molecular and biochemical analysis of a mutant FOS protein we identified in these vascular tumors. The mutant protein lacks a highly conserved helix consisting of the C-terminal four amino acids of FOS, which we show is indispensable for fast, ubiquitin-independent FOS degradation via the 20S proteasome. Our work reveals that FOS stimulates endothelial sprouting and that perturbation of normal FOS degradation could account for the abnormal vessel growth typical of epithelioid hemangioma. To the best of our knowledge, this is the first functional characterization of mutant FOS proteins found in tumors.
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Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/fisiología , Neoplasias Vasculares/genética , Inductores de la Angiogénesis , Carcinogénesis/genética , Carcinogénesis/metabolismo , Diferenciación Celular , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes fos/genética , Hemangioma/genética , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Elementos Reguladores de la Transcripción/genética , Neoplasias Vasculares/metabolismoRESUMEN
The availability of a receptor for theranostic pretargeting approaches was assessed by use of a new click-chemistry-based deactivatable fluorescence-quenching concept. The efficacy was evaluated in a cell-based model system featuring both membranous (available) and internalized (unavailable) receptor fractions of the clinically relevant receptor chemokine receptorâ 4 (CXCR4). Proof of concept was achieved with a deactivatable tracer consisting of a CXCR4-specific peptide functionalized with a Cy5 dye bearing a chemoselective azide handle (N3 -Cy5-AcTZ14011). Treatment with a Cy7 quencher dye (Cy7-DBCO) resulted in optically silent Cy7-[click]-Cy5-AcTZ14011. In situ, a >90 % FRET-based reduction of the signal intensity of N3 -Cy5-AcTZ14011 [KD =(222.4±25.2)â nm] was seen within minutes after quencher addition. In cells, discrimination between the membranous and the internalized receptor fraction could be achieved through quantitative assessment of quenching/internalization kinetics. Similar evaluation of an activatable tracer variant based on the same targeting moiety (Cy5-S-S-Cy3-AcTZ14011) was unsuccessful in vitro. As such, using the described deactivatable approach to screen membrane receptors and their applicability in receptor-(pre-)targeted theranostics can become straightforward.
RESUMEN
Metaplastic breast carcinoma (MBC) is a rare histological breast cancer subtype characterized by mesenchymal elements and poor clinical outcome. A large fraction of MBCs harbor defects in breast cancer 1 (BRCA1). As BRCA1 deficiency sensitizes tumors to DNA cross-linking agents and poly(ADP-ribose) polymerase (PARP) inhibitors, we sought to investigate the response of BRCA1-deficient MBCs to the PARP inhibitor olaparib. To this end, we established a genetically engineered mouse model (GEMM) for BRCA1-deficient MBC by introducing the MET proto-oncogene into a BRCA1-associated breast cancer model, using our novel female GEMM ES cell (ESC) pipeline. In contrast to carcinomas, BRCA1-deficient mouse carcinosarcomas resembling MBC show intrinsic resistance to olaparib caused by increased P-glycoprotein (Pgp) drug efflux transporter expression. Indeed, resistance could be circumvented by using another PARP inhibitor, AZD2461, which is a poor Pgp substrate. These preclinical findings suggest that patients with BRCA1-associated MBC may show poor response to olaparib and illustrate the value of GEMM-ESC models of human cancer for evaluation of novel therapeutics.
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Proteína BRCA1/deficiencia , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Proteína BRCA1/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Carcinosarcoma/tratamiento farmacológico , Carcinosarcoma/genética , Carcinosarcoma/metabolismo , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Metaplasia , Ratones Endogámicos C57BL , Ratones Noqueados , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proto-Oncogenes Mas , Análisis de SupervivenciaRESUMEN
Genetic abnormalities associated with the development, progression and treatment resistance of hematological malignancies are extensively characterized. Rapid, reliable and cost-efficient techniques are needed to screen the clinically relevant aberrations in routine diagnostics. Multiplex ligation-dependent probe amplification is an efficient tool to analyze genomic copy number aberrations at 55-60 different genomic loci. The method allows the profiling of prognostic and predictive markers; thus, it can efficiently be combined with karyotyping and fluorescence in situ hybridization, the most commonly used diagnostic techniques to detect cytogenetic lesions. Furthermore, the method can interrogate methylation status and unravel point mutations at specific sites, providing results in 24 hours. Here, we describe the technical background of multiplex ligation-dependent probe amplification, summarize its advantages and limitations as well as discuss its role in oncohematological diagnostics and research. Finally, future outlook is provided, with emphasis on recent technological advances related to next-generation sequencing. Orv Hetil. 2018; 159(15): 583-592.
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Variaciones en el Número de Copia de ADN/genética , Neoplasias Hematológicas/genética , Cariotipificación/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Aberraciones Cromosómicas , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodos , PronósticoRESUMEN
BACKGROUND: Ewing sarcoma is an aggressive, highly metastatic primary bone and soft tissue tumor most frequently occurring in the bone of young adolescents. Patients, especially those diagnosed with a metastatic disease, have a poor overall survival. Chemokine receptor CXCR4 has a key pro-tumorigenic role in the tumor microenvironment of Ewing sarcoma and has been suggested to be involved in the increased metastatic propensity. Earlier studies on CXCR4 protein expression in Ewing sarcoma yielded contradictory results when compared to CXCR4 RNA expression studies. Previously, we demonstrated that CXCR4 expression could be detected in vivo using the fluorescently tagged CXCR4-specific peptide MSAP-Ac-TZ14011. Therefore, we studied the membranous CXCR4 expression in Ewing sarcoma cell lines using MSAP-Ac-TZ14011. METHODS: The CXCR4 membrane expression levels were studied in EWS cell lines by flow cytometry using the hybrid peptide MSAP-Ac-TZ14011 and were correlated to CXCR4 RNA expression levels. The measurements were compared to levels detected using the CXCR4 antibody ab2074 under various cell preparation conditions. In addition, the staining patterns were analyzed by confocal fluorescence microscopy over time. RESULTS: The hybrid peptide MSAP-Ac-TZ14011 levels showed a strong and better correlation of CXCR4 membrane expression with the CXCR4 RNA expression levels than observed with the anti-CXCR4 antibody ab2074. With the hybrid peptide MSAP-Ac-TZ14011 using live cell confocal microscopy CXCR4 membrane staining and internalization was detected and the signal intensity correlated well with CXCR4 mRNA expression levels. CONCLUSIONS: The fluorescently labeled CXCR4 targeting peptide-based method provides a reliable alternative to antibody staining to study the CXCR4 membrane expression in live cells using either flow cytometry or live cell fluorescence microscopy. The fluorescently tagged CXCR4 targeting peptide could enable in vivo detection of CXCR4 expression in Ewing sarcoma which may help to stratify cases for anti-CXCR4 therapy.
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Biomarcadores de Tumor/análisis , Neoplasias Óseas , Receptores CXCR4/análisis , Sarcoma de Ewing , Línea Celular Tumoral , Colorantes Fluorescentes , Humanos , Imagen Óptica , PéptidosRESUMEN
In vitro and in vivo studies attribute potent immune regulatory properties to the vitamin D receptor (VDR). Yet, it is unclear to what extend these observations translate to the clinical context of (vascular) inflammation. This clinical study evaluates the potential of a VDR agonist to quench vascular inflammation. Patients scheduled for open abdominal aneurysm repair received paricalcitol 1 µg daily during 2-4 weeks before repair. Results were compared with matched controls. Evaluation in a parallel group showed that AAA patients are vitamin D insufficient (median plasma vitamin D: 43 (30-62 (IQR)) nmol/l). Aneurysm wall samples were collected during surgery, and the inflammatory footprint was studied. The brief paricalcitol intervention resulted in a selective 73% reduction in CD4+ T-helper cell content (P<0.024) and a parallel 35% reduction in T-cell (CD3+) content (P<0.032). On the mRNA level, paricalcitol reduced expression of T-cell-associated cytokines IL-2, 4, and 10 (P<0.019). No effect was found on other inflammatory mediators. On the protease level, selective effects were found for cathepsin K (P<0.036) and L (P<0.005). Collectively, these effects converge at the level of calcineurin activity. An effect of the VDR agonist on calcineurin activity was confirmed in a mixed lymphocyte reaction. In conclusion, brief course of the VDR agonist paricalcitol has profound effects on local inflammation via reduced T-cell activation. The anti-inflammatory potential of VDR activation in vitamin D insufficient patients is highly selective and appears to be mediated by an effect on calcineurin-mediated responses.
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Aneurisma de la Aorta Abdominal/tratamiento farmacológico , Aneurisma de la Aorta Abdominal/metabolismo , Calcineurina/metabolismo , Ergocalciferoles/farmacología , Mediadores de Inflamación/metabolismo , Receptores de Calcitriol/agonistas , Anciano , Antiinflamatorios/farmacología , Aneurisma de la Aorta Abdominal/inmunología , Citocinas/genética , Citocinas/metabolismo , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Masculino , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Mesenchymal chondrosarcomas are rare and highly aggressive sarcomas occurring in bone and soft tissue, with poor overall survival. Bcl-2 expression was previously shown to be upregulated in mesenchymal chondrosarcomas. We here report on a newly derived mesenchymal chondrosarcoma cell line, MCS170, in which we investigated treatment with the BH3 mimetic ABT-737 alone or in combination with conventional chemotherapy as a possible new therapeutic strategy. The presence of the characteristic HEY1-NCOA2 fusion was confirmed in the MCS170 cell line using FISH, RT-PCR, and sequencing. The MCS170 cell line was treated with ABT-737 alone or in combination with doxorubicin or cisplatin. Cell viability and proliferation was determined using WST-1 viability assays and the xCELLigence system. Expression of Bcl-2 family members was studied using immunohistochemistry. Apoptosis was determined using the caspase-glo 3/7 assay and western blot for PARP cleavage. The MCS170 cell line was sensitive to doxorubicin treatment with an IC50 of 0.09 µM after 72 h, but more resistant to cisplatin treatment with an IC50 of 4.5 µM after 72 h. Cells showed little sensitivity toward ABT-737 with an IC50 of 1.8 µM after 72 h. Combination treatments demonstrated ABT-737 synergism with cisplatin as well as doxorubicin as shown by induction of apoptosis and reduction in cell proliferation. Restoration of the apoptotic machinery by inhibition of Bcl-2 family members sensitizes MCS170 mesenchymal chondrosarcoma cells to conventional chemotherapy. This indicates that combining the inhibition of Bcl-2 family members with conventional chemotherapy can be a possible therapeutic strategy for patients with mesenchymal chondrosarcoma.