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1.
Nucleic Acids Res ; 49(21): 12007-12016, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34230973

RESUMO

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.


Assuntos
Sistemas CRISPR-Cas , DNA/metabolismo , Neuroblastoma/genética , Linhagem Celular Tumoral , Aberrações Cromossômicas , Deleção Cromossômica , Humanos
2.
Haematologica ; 107(3): 655-667, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33567812

RESUMO

In myelodysplastic syndromes (MDS) the immune system is involved in pathogenesis as well as in disease progression. Dendritic cells (DC) are key players of the immune system by serving as regulators of immune responses. Their function has been scarcely studied in MDS and most of the reported studies didn't investigate naturally occurring DC subsets. Therefore, we here examined the frequency and function of DC subsets and slan+ non-classical monocytes in various MDS risk groups. Frequencies of DC as well as of slan+ monocytes were decreased in MDS bone marrow compared to normal bone marrow samples. Transcriptional profiling revealed down-regulation of transcripts related to pro-inflammatory pathways in MDS-derived cells as compared to normal bone marrow. Additionally, their capacity to induce T-cell proliferation was impaired. Multidimensional mass cytometry showed that whereas healthy donor-derived slan+ monocytes supported Th1/Th17/Treg differentiation/expansion their MDS-derived counterparts also mediated substantial Th2 expansion. Our findings point to a role for an impaired ability of DC subsets to adequately respond to cellular stress and DNA damage in the immune escape and progression of MDS. As such, it paves the way toward potential novel immunotherapeutic interventions.


Assuntos
Monócitos , Síndromes Mielodisplásicas , Medula Óssea/patologia , Células Dendríticas , Humanos , Ativação Linfocitária , Síndromes Mielodisplásicas/patologia
3.
Haematologica ; 106(1): 87-97, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31974198

RESUMO

Complex karyotype (CK) identified by chromosome-banding analysis (CBA) has shown prognostic value in chronic lymphocytic leukemia (CLL). Genomic arrays offer high-resolution genome-wide detection of copy-number alterations (CNAs) and could therefore be well equipped to detect the presence of a CK. Current knowledge on genomic arrays in CLL is based on outcomes of single center studies, in which different cutoffs for CNA calling were used. To further determine the clinical utility of genomic arrays for CNA assessment in CLL diagnostics, we retrospectively analyzed 2293 arrays from 13 diagnostic laboratories according to established standards. CNAs were found outside regions captured by CLL FISH probes in 34% of patients, and several of them including gains of 8q, deletions of 9p and 18p (p<0.01) were linked to poor outcome after correction for multiple testing. Patients (n=972) could be divided in three distinct prognostic subgroups based on the number of CNAs. Only high genomic complexity (high-GC), defined as ≥5 CNAs emerged as an independent adverse prognosticator on multivariable analysis for time to first treatment (Hazard ratio: 2.15, 95% CI: 1.36-3.41; p=0.001) and overall survival (Hazard ratio: 2.54, 95% CI: 1.54-4.17; p<0.001; n=528). Lowering the size cutoff to 1 Mb in 647 patients did not significantly improve risk assessment. Genomic arrays detected more chromosomal abnormalities and performed at least as well in terms of risk stratification compared to simultaneous chromosome banding analysis as determined in 122 patients. Our findings highlight genomic array as an accurate tool for CLL risk stratification.


Assuntos
Leucemia Linfocítica Crônica de Células B , Aberrações Cromossômicas , Genoma Humano , Genômica , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Linfocítica Crônica de Células B/genética , Estudos Retrospectivos
4.
Haematologica ; 105(4): 961-971, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31273091

RESUMO

The bone marrow of patients with low-risk myelodysplastic syndromes (MDS) is often an inflammatory environment and associated with an active cellular immune response. An active immune response generally contributes to antitumor responses and may prevent disease progression. However, chronic immune stimulation can also induce cell stress, DNA damage and contribute to the pathogenesis of MDS. The protective mechanisms against excessive immune activation are therefore an important aspect of the pathophysiology of MDS and characterizing them may help us to better understand the fine balance between protective and destabilizing inflammation in lower-risk disease. In this study we investigated the role of thrombomodulin (CD141/BDCA-3) expression, a molecule with anti-inflammatory properties, on monocytes in the bone marrow and peripheral blood of MDS patients in different risk groups. Patient-derived classical monocytes showed high expression levels of thrombomodulin, whereas monocytes from healthy donors hardly expressed any thrombomodulin. The presence of thrombomodulin on monocytes from MDS patients correlated with lower-risk disease groups and better overall and leukemia-free survival. Using multidimensional mass cytometry, in an in-vitro setting, we showed that thrombomodulin-positive monocytes could polarize naïve T cells toward cell clusters which are closer to T helper type 2 and T regulatory cell phenotypes and less likely to contribute to effective immune surveillance. In conclusion, the expression of thrombomodulin on classical monocytes is a favorable and early prognostic marker in patients with low-risk MDS and may represent a new mechanism in the protection against disproportionate immune activation.


Assuntos
Monócitos , Síndromes Mielodisplásicas , Medula Óssea , Progressão da Doença , Humanos , Trombomodulina/genética
5.
Blood ; 127(4): 458-63, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26582376

RESUMO

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Tretinoína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/análise , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Camundongos SCID , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Células Mieloides/patologia , Fatores de Transcrição/análise , Células Tumorais Cultivadas , Regulação para Cima
8.
Am J Med Genet A ; 161A(2): 244-53, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23303641

RESUMO

In a screening project of patients with (complex) craniosynostosis using genomic arrays, we identified two patients with craniosynostosis and microcephaly with a deletion in the 2p15p16.1 chromosomal region. This region has been associated with a new microdeletion syndrome, for which patients have various features in common, including microcephaly and intellectual disability. Deletions were identified using Affymetrix 250K SNP array and further characterized by fluorescence in situ hybridization (FISH) analysis and qPCR. The deletions in our two patients overlapped within the 2p15p16.1 microdeletion syndrome area and were 6.8 and 6.9 Mb in size, respectively. FISH and qPCR confirmed the presence of only one copy in this region. Finemapping of the breakpoints indicated precise borders in our patients and were further finemapped in two other previously reported patients. Clinical features of patients with deletions in the 2p15p16.1 region vary. Including data from our patients, now eight out of nine reported patients have microcephaly, one of the major features, and all had intellectual disability. The current reported two patients add different forms of craniosynostosis to the clinical spectrum of this recently recognized microdeletion syndrome.


Assuntos
Anormalidades Múltiplas/genética , Cromossomos Humanos Par 2 , Craniossinostoses/genética , Microcefalia/genética , Cariótipo Anormal , Anormalidades Múltiplas/diagnóstico , Adolescente , Adulto , Transtorno do Deficit de Atenção com Hiperatividade/genética , Criança , Pré-Escolar , Deleção Cromossômica , Craniossinostoses/diagnóstico , Deficiências do Desenvolvimento/genética , Feminino , Dedos/anormalidades , Estudos de Associação Genética , Humanos , Lactente , Masculino , Microcefalia/diagnóstico , Técnicas de Diagnóstico Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Síndrome
9.
Case Rep Oncol ; 16(1): 1443-1450, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38028572

RESUMO

Introduction: Acute promyelocytic leukemia (APL) is characterized by the PML::RARa gene fusion and treatment consists of all-trans retinoic acid (ATRA). Rarely, genetic APL variants have been described which are insensitive to ATRA treatment and are therefore associated with a worse prognosis. Rapid identification of the APL variant is essential to start the correct treatment. Case Presentation: Here, we present a case of a 66-year-old male patient with weight loss and arthralgia. Laboratory results showed an anemia and mild leukocytosis with predominantly monocytes. Bone marrow investigation unexpectedly revealed a t(11;17)(q23;q21). This raised suspicion of an ATRA-resistant APL. By demonstrating the ZBTB16::RARa gene fusion, the diagnosis was confirmed. Conclusion: This case study emphasizes the importance of integrated diagnostics and provides guidance to recognize the ZBTB16::RARa APL, which is the most prevalent ATRA-resistant APL. Furthermore, an overview of other genetic APL variants is presented and how to treat these uncommon diseases in clinical practice.

10.
Dev Med Child Neurol ; 53(5): 417-21, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21410694

RESUMO

AIM: To evaluate survival, clinical, and genetic characteristics of all patients with classic or type 1 lissencephaly born between 1972 and 1990 in the Netherlands, who at the time were enrolled in an observational study. METHOD: We re-evaluated 24 patients (11 males, 13 females) for long-term follow-up and survival information. RESULTS: Mean length of follow-up was 14 years (SD 9 y 8 mo). Eleven patients were alive at follow-up. All patients showed severe intellectual disability, intractable epilepsy, and complete dependency on care. Life expectancy was related to the severity of the lissencephaly on neuroimaging. Molecular analysis of the LIS1 gene was not possible at the time of the original study and was now requested by eight parents. This revealed a pathogenic nonsense mutation or deletion in seven patients. INTERPRETATION: Our study provides information about the long-term course of lissencephaly and the relationship between lissencephaly severity and prognosis. It also shows that renewed attention to genetic counselling remains valued by families of patients with a severe congenital neurological disease.


Assuntos
Encéfalo/anormalidades , Encéfalo/patologia , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/mortalidade , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/patologia , Adulto , Lissencefalias Clássicas e Heterotopias Subcorticais em Banda/diagnóstico por imagem , Feminino , Humanos , Estudos Longitudinais , Masculino , Análise de Sobrevida , Tomografia Computadorizada por Raios X/métodos , Adulto Jovem
11.
Elife ; 102021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33459596

RESUMO

Germline mutations in the Folliculin (FLCN) tumor suppressor gene cause Birt-Hogg-Dubé (BHD) syndrome, a rare autosomal dominant disorder predisposing carriers to kidney tumors. FLCN is a conserved, essential gene linked to diverse cellular processes but the mechanism by which FLCN prevents kidney cancer remains unknown. Here, we show that disrupting FLCN in human renal tubular epithelial cells (RPTEC/TERT1) activates TFE3, upregulating expression of its E-box targets, including RRAGD and GPNMB, without modifying mTORC1 activity. Surprisingly, the absence of FLCN or its binding partners FNIP1/FNIP2 induces interferon response genes independently of interferon. Mechanistically, FLCN loss promotes STAT2 recruitment to chromatin and slows cellular proliferation. Our integrated analysis identifies STAT1/2 signaling as a novel target of FLCN in renal cells and BHD tumors. STAT1/2 activation appears to counterbalance TFE3-directed hyper-proliferation and may influence immune responses. These findings shed light on unique roles of FLCN in human renal tumorigenesis and pinpoint candidate prognostic biomarkers.


Assuntos
Proteínas de Transporte/genética , Células Epiteliais/metabolismo , Rim/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Proteínas de Transporte/metabolismo , Mutação em Linhagem Germinativa , Humanos , Interferons/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/metabolismo
12.
Am J Med Genet A ; 152A(9): 2221-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20683990

RESUMO

Supernumerary marker chromosomes (SMC) originating from chromosome 15 are the most common SMCs. They encompass clinically irrelevant SMC(15)s containing only heterochromatin and 15p material, and clinically relevant SMC(15)s that consist of both eu- and heterochromatic 15q material. On the basis of size, the clinically relevant SMC(15)s can be subdivided into type A, "large" asymmetric and type B, "small" symmetric SMC(15)s. Type B SMC(15)s contain the triplicated 15pter to BP3 (located at 26.5 Mb) region, while type A SMC(15)s consist of 15pter --> BP4(28.5 Mb)::BP5(30.5 Mb) --> 15pter. In this study, the clinical and molecular features of 18 patients with A and B SMC(15)s and two patients with a partial trisomy 15q were reviewed. Questionnaires (including Child Behavior Check Lists) were used to assess behavior and developmental features in more detail. The marker size and parental origin were determined by multiplex ligation-dependent probe amplification (MLPA). Based on the MLPA results, the majority of patients (14/18) had type A SMC(15)s. The phenotype observed did not show significant differences between types A and B SMC(15)s. A high prevalence of autistic-like behavior, attention problems, aggressive behavior, anxiety, and sleeping problems was reported. Motor and speech development varied extensively among the patients. An association was found between positive seizure history and degree of intellectual disability.


Assuntos
Cromossomos Humanos Par 15/genética , Deficiência Intelectual/genética , Transtornos Mentais/genética , Adolescente , Criança , Pré-Escolar , Feminino , Marcadores Genéticos , Humanos , Masculino , Fenótipo , Convulsões/genética , Inquéritos e Questionários
13.
Am J Med Genet A ; 152A(6): 1488-97, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20503325

RESUMO

The combination of megalencephaly, perisylvian polymicrogyria, polydactyly and hydrocephalus (MPPH) is a rare syndrome of unknown cause. We observed two first cousins affected by an MPPH-like phenotype with a submicroscopic chromosome 5q35 deletion as a result of an unbalanced der(5)t(5;20)(q35.2;q13.3) translocation, including the NSD1 Sotos syndrome locus. We describe the phenotype and the deletion breakpoints of the two MPPH-like patients and compare these with five unrelated MPPH and Sotos patients harboring a 5q35 microdeletion. Mapping of the breakpoints in the two cousins was performed by MLPA, FISH, high density SNP-arrays and Q-PCR for the 5q35 deletion and 20q13 duplication. The 5q35 deletion area of the two cousins almost completely overlaps with earlier described patients with an atypical Sotos microdeletion, except for the DRD1 gene. The five unrelated MPPH patients neither showed submicroscopic chromosomal aberrations nor DRD1 mutations. We reviewed the brain MRI of 10 Sotos patients and did not detect polymicrogyria in any of them. In our two cousins, the MPPH-like phenotype is probably caused by the contribution of genes on both chromosome 5q35 and 20q13. Some patients with MPPH may harbor a submicroscopic chromosomal aberration and therefore high-resolution array analysis should be part of the diagnostic workup.


Assuntos
Cromossomos Humanos Par 5/genética , Hidrocefalia/genética , Malformações do Desenvolvimento Cortical/genética , Polidactilia/genética , Translocação Genética , Encéfalo/anormalidades , Pré-Escolar , Mapeamento Cromossômico , Feminino , Humanos , Hidrocefalia/diagnóstico , Hibridização in Situ Fluorescente , Cariotipagem , Angiografia por Ressonância Magnética , Masculino , Malformações do Desenvolvimento Cortical/diagnóstico , Linhagem , Polidactilia/diagnóstico , Deleção de Sequência , Síndrome
14.
Chromosome Res ; 17(6): 737-44, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19672683

RESUMO

Apparently balanced chromosomal inversions may lead to disruption of developmentally important genes at the breakpoints of the inversion, causing congenital malformations. Characterization of such inversions may therefore lead to new insights in human development. Here, we report on a de novo inversion of chromosome 7 (p15.2q36.3) in a patient with postaxial polysyndactyly. The breakpoints do not disrupt likely candidate genes for the limb phenotype observed in the patient. However, on the p-arm the breakpoint separates the HOXA cluster from a gene desert containing several conserved noncoding elements, suggesting that a disruption of a cis-regulatory circuit of the HOXA cluster could be the underlying cause of the phenotype in this patient.


Assuntos
Cromossomos Humanos Par 7/genética , Cromossomos Humanos Y/genética , Deformidades Congênitas do Pé/genética , Deformidades Congênitas da Mão/genética , Proteínas de Homeodomínio/genética , Polidactilia/genética , Sequência de Bases , Southern Blotting , Quebra Cromossômica , Inversão Cromossômica , Deformidades Congênitas do Pé/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Deformidades Congênitas da Mão/patologia , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Polidactilia/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
Am J Med Genet A ; 149A(7): 1468-75, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19449434

RESUMO

We present a family with multiple cytogenetic abnormalities, identified through a girl with several dysmorphic features and cardiac problems, suspected for Jacobsen syndrome. Cytogenetic analysis showed a 46,XX,del(11)(qter) karyotype, which was confirmed by fluorescence in situ hybridization (FISH). Cytogenetic investigation of the parents showed a chromosome aberration in both: the father had a t(11;12)(p13;q22) translocation and the mother was carrier of an ins(4;11)(p14;q24q25). FISH analysis with an 11q-subtelomeric probe from the second-generation telomere clone set and BACs from 11q24-q25 suggested a complex maternal rearrangement. However, subsequent array analysis showed a single interstitial deletion in the proband, derived from the maternal insertion. The aberrant karyotypes in both parents implicated an increased risk of unbalanced fetal chromosome composition, thus high risk for a child with multiple congenital abnormalities. Therefore, during the next pregnancy, the couple opted for prenatal diagnosis by means of amniocentesis. An interphase FISH strategy for uncultured amniotic fluid cells predicted two possible unbalanced fetal chromosome constitutions. Karyotyping of cultured amniotic cells confirmed one of the predicted unbalanced cytogenetic options, demonstrating the value of a fast interphase strategy for parents who both are carriers of a chromosomal abnormality. In addition, we present an overview of patients with Jacobsen syndrome and an interstitial 11q deletion reported thus far in literature.


Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 4 , Mães , Translocação Genética , Pré-Escolar , Família , Feminino , Humanos , Recém-Nascido , Padrões de Herança , Síndrome da Deleção Distal 11q de Jacobsen/diagnóstico , Síndrome da Deleção Distal 11q de Jacobsen/genética , Masculino
16.
Eur J Med Genet ; 50(2): 112-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17161033

RESUMO

We describe a unique family with two children having a delay in psychomotor development. In both children we identified an interstitial duplication dup(2)(q34q33) using multiple, complementary molecular cytogenetic techniques. Comparative genomic hybridisation (CGH) and array-CGH were used to determine the size and the location of the duplicated region, the orientation of the duplicated region was identified with fluorescence in situ hybridisation (FISH). Both parents demonstrated a normal karyotype and normal CGH and array-CGH-profiles. However, FISH on peripheral blood cells from the mother showed the inv dup(2) in 9% of metaphases and 19% of interphase nuclei. To our knowledge this is the first report of a mosaic carrier of duplication in the long arm of chromosome 2. The finding of chromosomal mosaicism of at least 19% in the mother increases the recurrence risk. The exact characterisation of the inv dup(2) with FISH probes enabled us to offer a reliable prenatal FISH test. Comparison of the clinical features of the two children with those of previously described cases supports the hypothesis that the characteristic facial phenotype is linked to the distal part of the 2q33-q37 region. This report illustrates that in case of two sibs with an identical structural chromosomal abnormality the possibility of parental chromosomal mosaicism must be thoroughly investigated.


Assuntos
Inversão Cromossômica , Cromossomos Humanos Par 2 , Análise Citogenética/métodos , Duplicação Gênica , Amostra da Vilosidade Coriônica , Mapeamento Cromossômico , Coloração Cromossômica , DNA/genética , DNA/isolamento & purificação , Feminino , Heterozigoto , Humanos , Cariotipagem , Mosaicismo , Hibridização de Ácido Nucleico , Linhagem , Irmãos
17.
Clin Cancer Res ; 23(24): 7498-7511, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29025767

RESUMO

Purpose: Daratumumab treatment results in a marked reduction of CD38 expression on multiple myeloma cells. The aim of this study was to investigate the clinical implications and the underlying mechanisms of daratumumab-mediated CD38 reduction.Experimental Design: We evaluated the effect of daratumumab alone or in combination with lenalidomide-dexamethasone, on CD38 levels of multiple myeloma cells and nontumor immune cells in the GEN501 study (daratumumab monotherapy) and the GEN503 study (daratumumab combined with lenalidomide-dexamethasone). In vitro assays were also performed.Results: In both trials, daratumumab reduced CD38 expression on multiple myeloma cells within hours after starting the first infusion, regardless of depth and duration of the response. In addition, CD38 expression on nontumor immune cells, including natural killer cells, T cells, B cells, and monocytes, was also reduced irrespective of alterations in their absolute numbers during therapy. In-depth analyses revealed that CD38 levels of multiple myeloma cells were only reduced in the presence of complement or effector cells, suggesting that the rapid elimination of CD38high multiple myeloma cells can contribute to CD38 reduction. In addition, we discovered that daratumumab-CD38 complexes and accompanying cell membrane were actively transferred from multiple myeloma cells to monocytes and granulocytes. This process of trogocytosis was also associated with reduced surface levels of some other membrane proteins, including CD49d, CD56, and CD138.Conclusions: Daratumumab rapidly reduced CD38 expression levels, at least in part, through trogocytosis. Importantly, all these effects also occurred in patients with deep and durable responses, thus excluding CD38 reduction alone as a mechanism of daratumumab resistance.The trials were registered at www.clinicaltrials.gov as NCT00574288 (GEN501) and NCT1615029 (GEN503). Clin Cancer Res; 23(24); 7498-511. ©2017 AACR.


Assuntos
ADP-Ribosil Ciclase 1/genética , Anticorpos Monoclonais/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , ADP-Ribosil Ciclase 1/imunologia , Idoso , Anticorpos Monoclonais/efeitos adversos , Linfócitos B/imunologia , Linhagem Celular Tumoral , Dexametasona/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Humanos , Células Matadoras Naturais/imunologia , Lenalidomida , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Linfócitos T , Talidomida/administração & dosagem , Talidomida/efeitos adversos
18.
Eur J Cancer ; 54: 49-56, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26720403

RESUMO

Mandatory for the diagnosis of myelodysplastic syndromes (MDS) is the presence of dysplasia in >10% of cells within one or more cell lineages or presence of >15% ring sideroblasts or presence of MDS-associated cytogenetic (CG) abnormalities. Discrimination between neo-plastic and non-neoplastic causes of cytopenias can be challenging when dysplastic features by cytomorphology (CM) are minimal and CG abnormalities are absent or non-discriminating from other myeloid neoplastic disorders. This study evaluated a standard diagnostic approach in 379 patients with unexplained cytopenias and highlights the additional value of flow cytometry (FC) in patients with indeterminate CM and CG. CM reached no clear-cut diagnosis in 44% of the patients. Here, CG was able to identify two additional patients with MDS; other CG results did not reveal abnormalities or were not contributory. Based on the FC results, patients without a diagnosis by CM and CG were categorized 'no MDS-related features' (65%), 'limited number of MDS-related changes' (24%), and 'consistent with MDS' (11%). Patients were followed over time in an attempt to establish or confirm a diagnosis (median follow-up 391 d, range 20-1764). The specificity (true negative) of MDS-FC analysis calculated after follow-up was 95%. FC can aid as a valuable tool to exclude MDS when CM and additional CG are not conclusive in patients with cytopenia.


Assuntos
Citometria de Fluxo/métodos , Imunofenotipagem/métodos , Leucopenia/diagnóstico , Síndromes Mielodisplásicas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/análise , Biomarcadores/análise , Exame de Medula Óssea , Diagnóstico Diferencial , Feminino , Humanos , Antígenos Comuns de Leucócito/análise , Leucopenia/etiologia , Leucopenia/genética , Leucopenia/imunologia , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/complicações , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/imunologia , Células Progenitoras Mieloides/imunologia , Fenótipo , Células Precursoras de Linfócitos B/imunologia , Valor Preditivo dos Testes , Prognóstico , Fatores de Risco , Fatores de Tempo , Adulto Jovem
19.
Oncotarget ; 7(46): 74779-74796, 2016 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-27542283

RESUMO

PSMB5 mutations and upregulation of the ß5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.


Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos , Exocitose , Leucemia/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Resistencia a Medicamentos Antineoplásicos/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Leucemia/genética , Leucemia/mortalidade , Leucemia/terapia , MicroRNAs/genética , Substrato Quinase C Rico em Alanina Miristoilada/genética , Prognóstico , Ubiquitina/metabolismo , Ubiquitinação , Regulação para Cima
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