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BACKGROUND & AIMS: Constitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype-phenotype correlations using novel MSI markers selected for instability in blood. METHODS: Three CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls. RESULTS: Thirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%-100.0%) and specificity (95% CI 97.9%-100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor. CONCLUSIONS: We present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk.
Assuntos
Neoplasias Encefálicas , Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Colorretais , Síndromes Neoplásicas Hereditárias , Humanos , Neoplasias Colorretais Hereditárias sem Polipose/genética , Instabilidade de Microssatélites , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Encefálicas/diagnóstico , Genótipo , Reparo de Erro de Pareamento de DNA/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genéticaRESUMO
Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.
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Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA , Estudos de Associação Genética , Predisposição Genética para Doença , Leucócitos/metabolismo , Instabilidade de Microssatélites , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/genética , Alelos , Estudos de Associação Genética/métodos , Mutação em Linhagem Germinativa , Humanos , Repetições de MicrossatélitesAssuntos
Síndrome Linfoproliferativa Autoimune , Proteínas Sanguíneas , Síndrome da Plaqueta Cinza , Proteínas do Tecido Nervoso , Receptores de Superfície Celular , Síndrome Linfoproliferativa Autoimune/diagnóstico , Síndrome Linfoproliferativa Autoimune/genética , Síndrome Linfoproliferativa Autoimune/metabolismo , Síndrome Linfoproliferativa Autoimune/patologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Pré-Escolar , Proteína Ligante Fas/genética , Proteína Ligante Fas/metabolismo , Feminino , Síndrome da Plaqueta Cinza/diagnóstico , Síndrome da Plaqueta Cinza/genética , Síndrome da Plaqueta Cinza/metabolismo , Síndrome da Plaqueta Cinza/patologia , Humanos , Lactente , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMO
This article presents the international dataset of cases in which the association of Langerhans cell Histiocytosis (LCH) with other malignancies (AM) was documented occurring at any age before, concurrently or after LCH. These data are mostly derived from previously published manuscripts or from completed case report forms (CRFs) by Histiocyte Society (HS) members or colleagues. In particular, for each case of LCH-AM, the database reports all the available data about clinical and biologic characteristics of the two tumors, as well about treatment and status at follow-up. The AM were categorized as: i) leukemias [acute lymphoblastic or myeloid leukemia (ALL and AML, respectively), other leukemias] and myeloproliferative disorders; ii) lymphomas [Hodgkin lymphoma (HL) and non-Hodgkin lymphomas (NHL)] and iii) solid tumors. A total of 270 LCH-AM cases were documented, of which 116 (43%) occurred among children. After stratification by age at LCH diagnosis, using 18 years as cut-off between children and adults, we here provide details on the clinical characteristics in terms of LCH system involvement and affected organs, as well on the temporal relationship between the LCH and AM diagnoses, including details on the AM malignancy types. In 19 cases the LCH and the corresponding AM occurred in a different age group. The data set is available for future studies in view of new insights of the genetic or environmental determinants of LCH and/or of treatment related subsequent neoplasms.
RESUMO
PURPOSE: The frequency of Langerhans cell histiocytosis (LCH) and associated malignancies (AM) is greater than statistically expected. Here, we analyze LCH-AM co-occurrence in both children and adults. METHODS: Between 1991 and 2015, data were collected by regular questionnaires to members of the Histiocyte Society and searches in PubMed and Abstract Books. Patients were grouped by age at LCH diagnosis (≤ and >18 years), and types and timing of AM occurrence were plotted with respect to the LCH diagnosis. For the statistical analysis, only the first AM were considered. RESULTS: A total of 285 LCH-AM in 270 patients were identified, 116 (43%) ≤ 18 years, and 154 (57%) >18 years. In childhood LCH-AM pairs, leukemias and myeloproliferative disorders (n = 58; 50.0%) prevailed over solid tumors (n = 43; 37.1%) and lymphoma (n = 15; 12.9%). In adults, solid tumors were reported in 61 patients (39.6%), lymphoma, and leukemias and myeloproliferative disorders in 56 (36.4%) and 37 (24.0%) patients, respectively. In most children, AM followed LCH (n = 69, 59.5%), whereas in adults, LCH and AM occurred concurrently in 69 patients (44.8%). In children, T-lineage acute lymphoblastic leukemia (ALL) and promyelocytic acute myeloid leukemia (AML) and retinoblastoma were over-represented and thyroid carcinoma in adults. CONCLUSIONS: The largest collection of data on LCH-AM to date clearly indicates inherent relationships between specific types of AM and LCH, which may be due to therapy effects, clonal evolution, and germ-line predisposition, respectively. Prospective thorough genetic analysis is warranted and will hopefully shed light on the association of LCH and second neoplasms.
Assuntos
Histiocitose de Células de Langerhans , Leucemia Mieloide Aguda , Linfoma , Adulto , Criança , Histiocitose de Células de Langerhans/epidemiologia , Histiocitose de Células de Langerhans/genética , Histiocitose de Células de Langerhans/patologia , Humanos , Estudos Prospectivos , Estudos RetrospectivosRESUMO
PURPOSE: The alkylating agent treosulfan exerts a high cytotoxic activity against various malignant cells. Due to limited non-hematological toxicity, treosulfan might be a promising compound in myeloablative therapy for hematopoietic transplantation in children. Since in vitro data regarding the activity of treosulfan against childhood leukemic cells are limited, we compared the effect of treosulfan and busulfan against pediatric leukemic and non-malignant cells. EXPERIMENTAL DESIGN: Both agents were tested alone and in combination with fludarabine by means of the MTT and/or a five color-flow cytometric assay. Moreover, the induction of apoptosis by treosulfan was investigated via regulation of the proteinase caspase 3. RESULTS: Treosulfan was more active against leukemic cells of 20 children as well as against 3 leukemia-derived cell lines than busulfan, with increasing IC50 values from initial diagnosis to relapse. Overall purified stem cells were most sensitive, followed by CD56+CD3- NK and CD3+ T cells. The combination of treosulfan with fludarabine resulted in a synergistic effect against leukemic cells. In malignant cells, treosulfan induced rapid cell apoptosis measured by the activation of the centrally proteinase caspase 3. CONCLUSION: Our results indicate that treosulfan has activity against pediatric leukemic cells, myeloablative potential and immunosuppressive properties suitable for conditioning regimen in childhood malignancies.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Bussulfano/análogos & derivados , Leucemia/tratamento farmacológico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bussulfano/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Criança , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Leucemia/patologia , Células-Tronco/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Sais de Tetrazólio , Tiazóis , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Selective BRAF inhibitors such as vemurafenib have become a treatment option in patients with Langerhans cell Histiocytosis (LCH). To date, only 14 patients receiving vemurafenib for LCH have been reported. Although vemurafenib can stabilize the clinical condition of these patients, it does not seem to cure the patients, and it is unknown, when and how to stop vemurafenib treatment. We present a girl with severe multisystem LCH who responded only to vemurafenib. After 8 months of treatment, vemurafenib was tapered and replaced by prednisone and vinblastine, a strategy which has not been described to date. Despite chemotherapy, early relapse occurred, but remission was achieved by re-institution of vemurafenib. Further investigation needs to address the optimal duration of vemurafenib therapy in LCH and whether and which chemotherapeutic regimen may prevent disease relapse after cessation of vemurafenib.
RESUMO
For the evaluation of novel therapies, and for initial in vitro testing of potential in vivo graft-versus-tumour-effects (GvT), cytotoxicity of effector cells against target tumour cells needs to be determined in a reliable fashion. Recently Zimmermann et al. [Zimmermann, S.Y., Esser, R., Rohrbach, E., Klingebiel, T., Koehl, U., 2005. A novel four-colour flow cytometric assay to determine natural killer cell or T-cell-mediated cellular cytotoxicity against leukaemia cells in peripheral or bone marrow specimens containing greater than 20% of normal cells. J. Immunol. Methods. 296(1-2), 63-76] introduced a single platform, no-wash flow cytometric assay to quantify natural killer (NK) cell cytotoxicity against leukaemia cells. Here we have optimised this method introducing a novel five-colour flow cytometric assay for the evaluation of NK cell activity against adherent tumour cells, in particular neuroblastoma cells (NB cells). Beside an enhanced cytotoxic activity corresponding to increasing effector/target (E:T) ratios, we could demonstrate an increasing cytotoxicity in a time-dependent manner over a time period of 8 h. The usefulness of this novel method was also confirmed with human tumour cells lines of various other origin including breast and ovarian carcinoma and Wilms tumour cells freshly isolated from a patient after surgery. In addition to flow cytometric analysis, we monitored NK-cell-mediated induction of target cell apoptosis via the caspase cascade in attacked NB cells by fluorescence microscopy after immunofluorescence staining of activated Caspase-3 (Casp-3) in combination with detection of CD45(+) and CD9(+) for discrimination between NK and NB cells. In summary, this novel flow cytometric cytotoxicity assay enables efficient quantification of the phenotype of both, effector and adherent target tumour cells, and therefore represents a useful tool for research on immunotherapies that rely on cytotoxic effector cells.
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Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Antígenos CD/análise , Apoptose/imunologia , Caspase 3/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/imunologia , Células Cultivadas , Humanos , Células K562 , Células Matadoras Naturais/citologia , Microscopia de Fluorescência , Neoplasias/imunologia , Neoplasias/patologia , Neuroblastoma/imunologia , Neuroblastoma/patologiaRESUMO
During recent years, attempts have been made to complement more classical concepts of medical teaching by introducing card and board games on medical topics. These teaching tools cover every age and education group, and many different medical topics. In this article we have reviewed all card and board games for medical education purpose listed in NCBI PubMed database and Internet game databases (n = 29). It summarizes games that might be useful to medical teaching staff. To categorize these games, a new schema for medical games categorization, based on the game mechanism and theories on experiential learning circles, is proposed and discussed. Additionally we have a view on card and board games with medical topics for entertainment (n = 22).
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Educação Médica/métodos , Teoria dos Jogos , Materiais de Ensino , Educação Médica/tendências , Humanos , Jogos e Brinquedos/psicologia , Psicologia Educacional , Estudantes de Medicina/psicologia , Ensino/métodosRESUMO
INTRODUCTION: Bacteria are a significant cause of infectious complications, in particular in immunocompromised patients. There is an increasing understanding that Natural Killer (NK) cells not only exhibit direct activity against bacteria, but also exert indirect antibacterial activity through interaction with other immune cells via cytokines and interferons. Areas covered: This review seeks to give a global overview of in vitro and in vivo data how NK cells interact with bacteria. In this regard, the review describes how NK cells directly damage and kill bacteria by soluble factors such as perforin, the impact of NK cells on other arms of the immune system, as well as how bacteria may inhibit NK cell activities. Expert commentary: A better characterization of the antibacterial effects of NK cells is urgently needed. With a better understanding of the interaction of NK cells and bacteria, NK cells may become a promising tool to prevent or to combat bacterial infections, e.g. by adoptively transferring NK cells to immunocompromised patients.
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Bactérias/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade , Células Matadoras Naturais/imunologia , Animais , Infecções Bacterianas/metabolismo , Comunicação Celular , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Imunomodulação , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismoRESUMO
To be able to determine the cytotoxic activity of NK cells or T cells against leukaemic cells in patient samples containing >20% of normal peripheral blood cells, we have developed a four-colour flow cytometric cytotoxicity assay. The assay is based on differential immunostaining of both leukaemic cells and effector cells in combination with propidium iodide (PI). The cytometer is set for measuring the fluorescence of the monoclonal antibody (mAb) bound fluorochromes, with moderate overcompensation of the third and fourth fluorescence signals. PI-positive events were excluded from analysis by their characteristic uncompensated signal on these two detectors. Thus, all four fluorescence ranges can be used for detection of mAb-derived signals and this allows discrimination between various populations contained in effector and target cell samples. The cytotoxic activity in our assay is calculated by the absolute loss of vital leukaemic cells. For this purpose, fluorescent beads are included as an internal standard. When calculating the effector concentrations after coculture, characteristic changes can be seen which yield additional information about the presence of cytotoxic activity and the active effector cell subset. With this assay, we present a versatile tool that combines minimum cell manipulation before coculture with maximum information from a sample. The assay is suitable for the analysis of complex samples with regard to different cell subsets, their decrease or increase, and conjugate formation.
Assuntos
Testes Imunológicos de Citotoxicidade/métodos , Citometria de Fluxo/métodos , Células Matadoras Naturais/imunologia , Leucemia/imunologia , Linfócitos T Citotóxicos/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Células Sanguíneas/imunologia , Células da Medula Óssea/imunologia , Antígenos CD4/análise , Antígenos CD4/imunologia , Antígenos CD8/análise , Antígenos CD8/imunologia , Cor , Corantes Fluorescentes , Humanos , Células Matadoras Naturais/química , Leucemia/patologia , Propídio/química , Linfócitos T Citotóxicos/químicaRESUMO
As a result of improved experimental methodologies and a better understanding of the immune system, there is increasing insight into the antifungal activity of natural killer (NK) cells. Murine and human NK cells are able to damage fungi of different genera and species in vitro, and they exert both direct and indirect antifungal activity through cytotoxic molecules such as perforin and through cytokines and interferons, respectively. On the other hand, recent data suggest that fungi exhibit immunosuppressive effects on NK cells. Whereas clear in vivo data are lacking in humans, the importance of NK cells in the host response against fungi has been demonstrated in animal models. Further knowledge of the interaction of NK cells with fungi might help to better understand the pathogenesis of invasive fungal infections and to improve treatment strategies.
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Fungos/imunologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/microbiologia , Micoses/imunologia , Animais , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Humanos , Evasão da Resposta Imune , Tolerância Imunológica , CamundongosRESUMO
Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.
RESUMO
To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56CD3 donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16 and CD16 subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR) NK cells were overgrown by KIR proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2-stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.