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1.
J Mol Diagn ; 22(1): 60-71, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31605801

RESUMO

Acute myeloid leukemias (AMLs) are currently genomically characterized by karyotype, fluorescence in situ hybridization (FISH), real-time quantitative PCR, and DNA sequencing. Next-generation sequencing offers the promise of detecting all genomic lesions in a single run. However, technical limitations have hampered the detection of chromosomal rearrangements, so most studies are limited to somatic mutation assessment or require the use of RNA-based strategies. To overcome these limitations, we designed a targeted-DNA capture next-generation sequencing approach associated with easy-to-perform public bioinformatic tools for one-step identification of translocations, inversions, and somatic mutations in AML. Thirty well-characterized newly diagnosed myeloid leukemia patients (27 AML and 3 chronic myeloid leukemia) were tested with the panel. Twenty-three of 24 known rearrangements, as well as one novel fusion gene that could not be detected by karyotype/fluorescence in situ hybridization/real-time quantitative PCR, were detected. This strategy also identified all chromosomal breakpoints as potential targets for future high-sensitive minimal residual disease studies. In addition, mutation analysis revealed the presence of missense protein-coding alterations in at least 1 of the 32 genes evaluated in 21 of 30 patients (70%). This strategy may represent a time- and cost-effective diagnostic method for molecular characterization in AML.

3.
Blood Cancer J ; 9(12): 90, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31748515

RESUMO

Primary plasma cell leukemia (pPCL) is a highly aggressive plasma cell dyscrasia characterised by short remissions and very poor survival. Although the 17p deletion is associated with poor outcome and extramedullary disease in MM, its presence does not confer the degree of aggressiveness observed in pPCL. The comprehensive exploration of isoform expression and RNA splicing events may provide novel information about biological differences between the two diseases. Transcriptomic studies were carried out in nine newly diagnosed pPCL and ten MM samples, all of which harbored the 17p deletion. Unsupervised cluster analysis clearly distinguished pPCL from MM samples. In total 3584 genes and 20033 isoforms were found to be deregulated between pPCL and MM. There were 2727 significantly deregulated isoforms of non-differentially expressed genes. Strangely enough, significant differences were observed in the expression of spliceosomal machinery components between pPCL and MM, in respect of the gene, isoform and the alternative splicing events expression. In summary, transcriptome analysis revealed significant differences in the relative abundance of isoforms between pPCL and MM, even when they both had the 17p deletion. The mRNA processing pathway including RNA splicing machinery emerged as one of the most remarkable mechanisms underlying the biological differences between the two entities.

4.
Stem Cells ; 37(10): 1357-1368, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31184411

RESUMO

Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC-EV. MSC-EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC-EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)-STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho-STAT5 were confirmed by WES Simple in CD34+ cells with MSC-EV. In addition, these cells displayed a higher colony-forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC-EV was significantly increased in the injected femurs. In summary, the incorporation of MSC-EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. Stem Cells 2019;37:1357-1368.

5.
Noncoding RNA ; 5(1)2019 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654527

RESUMO

Intensive research has been undertaken during the last decade to identify the implication of microRNAs (miRNAs) in the pathogenesis of multiple myeloma (MM). The expression profiling of miRNAs in MM has provided relevant information, demonstrating different patterns of miRNA expression depending on the genetic abnormalities of MM and a key role of some miRNAs regulating critical genes associated with MM pathogenesis. However, the underlying causes of abnormal expression of miRNAs in myeloma cells remain mainly elusive. The final expression of the mature miRNAs is subject to multiple regulation mechanisms, such as copy number alterations, CpG methylation or transcription factors, together with impairment in miRNA biogenesis and differences in availability of the mRNA target sequence. In this review, we summarize the available knowledge about the factors involved in the regulation of miRNA expression and functionality in MM.

6.
Infect Immun ; 86(12)2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30275011

RESUMO

High-risk hematological malignancies are a privileged setting for infection by opportunistic microbes, with invasive mycosis being one of the most serious complications. Recently, genetic background has emerged as an unanticipated risk factor. For this reason, polymorphisms for genes encoding archetypal receptors involved in the opsonic and nonopsonic clearance of microbes, pentraxin-3 (PTX3) and Dectin-1, respectively, were studied and correlated with the risk of infection. Fungal, bacterial, and viral infections were registered for a group of 198 patients with high-risk hematological malignancies. Polymorphisms for the pentraxin-3 gene (PTX3) showed a significant association with the risk of fungal infection by Candida spp. and, especially, by Aspergillus spp. This link remained even for patients undergoing antifungal prophylaxis, thus demonstrating the clinical relevance of PTX3 in the defense against fungi. CLEC7A polymorphisms did not show any definite correlation with the risk of invasive mycosis, nor did they influence the expression of Dectin-1 isoforms generated by alternative splicing. The PTX3 mRNA expression level was significantly lower in samples from healthy volunteers who showed these polymorphisms, although no differences were observed in the extents of induction elicited by bacterial lipopolysaccharide and heat-killed Candida albicans, thus suggesting that the expression of PTX3 at the start of infection may influence the clinical outcome. PTX3 mRNA expression can be a good biomarker to establish proper antifungal prophylaxis in immunodepressed patients.


Assuntos
Proteína C-Reativa/genética , Neoplasias Hematológicas/complicações , Lectinas Tipo C/genética , Infecções Oportunistas/imunologia , Fagocitose , Componente Amiloide P Sérico/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antifúngicos/uso terapêutico , Aspergilose/imunologia , Candidíase/imunologia , Criança , Pré-Escolar , Feminino , Neoplasias Hematológicas/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/microbiologia , Infecções Oportunistas/virologia , Polimorfismo Genético , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
7.
J Clin Gastroenterol ; 52(2): e11-e17, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28059940

RESUMO

BACKGROUND AND AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer; most patients die during the first 6 months after diagnosis. With a 5% 5-year survival rate, is the fourth leading cause of cancer death in developed countries. In this regard, several clinical, histopathologic and biological characteristics of the disease favoring long-term survival after pancreaticoduodenectomy have been reported to be significant prognostic factors. Despite the availability of this information, there is no consensus about the different prognostic factors reported in the literature, probably due to variations in patient selection, methods, and sample size studied. The aim of this study was to identify the clinical and pathologic features associated to prognosis of the disease after pancreaticoduodenectomy. MATERIALS AND METHODS: The clinical and pathologic data from 78 patients who underwent a potentially curative resection for PDAC at our institution between 2003 and 2014 were analyzed retrospectively. RESULTS: Overall, high-grade PDAC cases showed larger tumor size (P=0.009) and a higher frequency of deaths in association with a nonsignificantly shortened patient overall survival (median of 12.5 vs. 21.7 mo; P=0.065) as compared with low-grade PDAC patients. High histologic grade (P=0.013), preoperative drainage on the main bile duct (P=0.014) and absence of adjuvant therapy (P=0.035) were associated with a significantly poorer outcome. Overall survival multivariate analysis showed histologic grade (P=0.019) and bile duct preoperative drainage (P=0.016) as the sole independent variables predicting an adverse outcome. CONCLUSIONS: Our results indicate that histologic tumor grade and preoperative biliary drainage are the only significant independent prognostic factors in PDAC patients after pancreatectomy.


Assuntos
Carcinoma Ductal Pancreático/patologia , Drenagem/métodos , Neoplasias Pancreáticas/patologia , Pancreaticoduodenectomia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/cirurgia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Neoplasias Pancreáticas/cirurgia , Cuidados Pré-Operatórios/métodos , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Resultado do Tratamento
8.
Biol Blood Marrow Transplant ; 24(3): 443-451, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29155314

RESUMO

Bone marrow mesenchymal stromal cells (MSCs) are precursors of adipocytes and osteoblasts and key regulators of hematopoiesis. Irradiation is widely used in conditioning regimens. Although MSCs are radio-resistant, the effects of low-dose irradiation on their behavior have not been extensively explored. Our aim was to evaluate the effect of 2.5 Gy on MSCs. Cells from 25 healthy donors were either irradiated or not (the latter were used as controls). Cells were characterized following International Society for Cellular Therapy criteria, including in vitro differentiation assays. Apoptosis was evaluated by annexin V/7-amino-actinomycin staining. Gene expression profiling and reverse transcriptase (RT)-PCR of relevant genes was also performed. Finally, long-term bone marrow cultures were performed to test the hematopoietic-supporting ability. Our results showed that immunophenotypic characterization and viability of irradiated cells was comparable with that of control cells. Gene expression profiling showed 50 genes differentially expressed. By RT-PCR, SDF-1 and ANGPT were overexpressed, whereas COL1A1 was downregulated in irradiated cells (P = .015, P = .007, and P = .031, respectively). Interestingly, differentiation of irradiated cells was skewed toward osteogenesis, whereas adipogenesis was impaired. Higher expression of genes involved in osteogenesis as SPP1 (P = .039) and lower of genes involved in adipogenesis, CEBPA and PPARG (P = .003 and P = .019), together with an increase in the mineralization capacity (Alizarin Red) was observed in irradiated cells. After differentiation, adipocyte counts were decreased in irradiated cells at days 7, 14, and 21 (P = .018 P = .046, and P = .018, respectively). Also, colony-forming unit granulocyte macrophage number in long-term bone marrow cultures was significantly higher in irradiated cells after 4 and 5 weeks (P = .046 and P = .007). In summary, the irradiation of MSCs with 2.5 Gy improves their hematopoietic-supporting ability by increasing osteogenic differentiation and decreasing adipogenesis.


Assuntos
Adipogenia/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Raios gama , Hematopoese/efeitos da radiação , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos da radiação , Adulto , Idoso , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/patologia , Pessoa de Meia-Idade
9.
Haematologica ; 102(12): 2113-2124, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28860344

RESUMO

Kinesin spindle protein inhibition is known to be an effective therapeutic approach in several malignancies. Filanesib (ARRY-520), an inhibitor of this protein, has demonstrated activity in heavily pre-treated multiple myeloma patients. The aim of the work herein was to investigate the activity of filanesib in combination with pomalidomide plus dexamethasone backbone, and the mechanisms underlying the potential synergistic effect. The ability of filanesib to enhance the activity of pomalidomide plus dexamethasone was studied in several in vitro and in vivo models. Mechanisms of this synergistic combination were dissected by gene expression profiling, immunostaining, cell cycle and short interfering ribonucleic acid studies. Filanesib showed in vitro, ex vivo, and in vivo synergy with pomalidomide plus dexamethasone treatment. Importantly, the in vivo synergy observed in this combination was more evident in large, highly proliferative tumors, and was shown to be mediated by the impairment of mitosis transcriptional control, an increase in monopolar spindles, cell cycle arrest and the induction of apoptosis in cells in proliferative phases. In addition, the triple combination increased the activation of the proapoptotic protein BAX, which has previously been associated with sensitivity to filanesib, and could potentially be used as a predictive biomarker of response to this combination. Our results provide preclinical evidence for the potential benefit of the combination of filanesib with pomalidomide and dexamethasone, and supported the initiation of a recently activated trial being conducted by the Spanish Myeloma group which is investigating this combination in relapsed myeloma patients.


Assuntos
Dexametasona/uso terapêutico , Cinesina/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Talidomida/análogos & derivados , Tiadiazóis/uso terapêutico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Humanos , Camundongos , Talidomida/uso terapêutico , Resultado do Tratamento
10.
J Hematol Oncol ; 10(1): 92, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28420429

RESUMO

BACKGROUND: The B cell maturation process involves multiple steps, which are controlled by relevant pathways and transcription factors. The understanding of the final stages of plasma cell (PC) differentiation could provide new insights for therapeutic strategies in multiple myeloma (MM). Here, we explore the role of DEPTOR, an mTOR inhibitor, in the terminal differentiation of myeloma cells, and its potential impact on patient survival. METHODS: The expression level of DEPTOR in MM cell lines and B cell populations was measured by real-time RT-PCR, and/or Western blot analysis. DEPTOR protein level in MM patients was quantified by capillary electrophoresis immunoassay. RNA interference was used to downregulate DEPTOR in MM cell lines. RESULTS: DEPTOR knockdown in H929 and MM1S cell lines induced dedifferentiation of myeloma cells, as demonstrated by the upregulation of PAX5 and BCL6, the downregulation of IRF4, and a clear reduction in cell size and endoplasmic reticulum mass. This effect seemed to be independent of mTOR signaling, since mTOR substrates were not affected by DEPTOR knockdown. Additionally, the potential for DEPTOR to be deregulated in MM by particular miRNAs was investigated. The ectopic expression of miR-135b and miR-642a in myeloma cell lines substantially diminished DEPTOR protein levels, and caused dedifferentiation of myeloma cells. Interestingly, the level of expression of DEPTOR protein in myeloma patients was highly variable, the highest levels being associated with longer progression-free survival. CONCLUSIONS: Our results demonstrate for the first time that DEPTOR expression is required to maintain myeloma cell differentiation and that high level of its expression are associated with better outcome. Primary samples used in this study correspond to patients entered into GEM2010 trial (registered at www.clinicaltrials.gov as #NCT01237249, 4 November 2010).


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mieloma Múltiplo/patologia , Plasmócitos/patologia , Linfócitos B , Desdiferenciação Celular , Diferenciação Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Prognóstico , Células Tumorais Cultivadas
11.
Clin Cancer Res ; 23(1): 225-238, 2017 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-27440267

RESUMO

PURPOSE: PIM kinases are a family of serine/threonine kinases recently proposed as therapeutic targets in oncology. In the present work, we have investigated the effects of the novel pan-PIM kinase inhibitor, PIM447, on myeloma cells and myeloma-associated bone disease using different preclinical models. EXPERIMENTAL DESIGN: In vitro/ex vivo cytotoxicity of PIM447 was evaluated on myeloma cell lines and patient samples. Synergistic combinations with standard treatments were analyzed with Calcusyn Software. PIM447 effects on bone cells were assessed on osteogenic and osteoclastogenic cultures. The mechanisms of PIM447 were explored by immunoblotting, qPCR, and immunofluorescence. A murine model of disseminated multiple myeloma was employed for in vivo studies. RESULTS: PIM447 is cytotoxic for myeloma cells due to cell-cycle disruption and induction of apoptosis mediated by a decrease in phospho-Bad (Ser112) and c-Myc levels and the inhibition of mTORC1 pathway. Importantly, PIM447 demonstrates a very strong synergy with different standard treatments such as bortezomib + dexamethasone (combination index, CI = 0.002), lenalidomide + dexamethasone (CI = 0.065), and pomalidomide + dexamethasone (CI = 0.077). PIM447 also inhibits in vitro osteoclast formation and resorption, downregulates key molecules involved in these processes, and partially disrupts the F-actin ring, while increasing osteoblast activity and mineralization. Finally, PIM447 significantly reduced the tumor burden and prevented tumor-associated bone loss in a disseminated murine model of human myeloma. CONCLUSIONS: Our results demonstrate dual antitumoral and bone-protective effects of PIM447. This fact, together with the very strong synergy exhibited with standard-of-care treatments, supports the future clinical development of this drug in multiple myeloma. Clin Cancer Res; 23(1); 225-38. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Substâncias Protetoras/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/genética , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Expressão Gênica , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/genética , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Padrão de Cuidado , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Mol Diagn ; 19(1): 99-106, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27863261

RESUMO

Identification and characterization of genetic alterations are essential for diagnosis of multiple myeloma and may guide therapeutic decisions. Currently, genomic analysis of myeloma to cover the diverse range of alterations with prognostic impact requires fluorescence in situ hybridization (FISH), single nucleotide polymorphism arrays, and sequencing techniques, which are costly and labor intensive and require large numbers of plasma cells. To overcome these limitations, we designed a targeted-capture next-generation sequencing approach for one-step identification of IGH translocations, V(D)J clonal rearrangements, the IgH isotype, and somatic mutations to rapidly identify risk groups and specific targetable molecular lesions. Forty-eight newly diagnosed myeloma patients were tested with the panel, which included IGH and six genes that are recurrently mutated in myeloma: NRAS, KRAS, HRAS, TP53, MYC, and BRAF. We identified 14 of 17 IGH translocations previously detected by FISH and three confirmed translocations not detected by FISH, with the additional advantage of breakpoint identification, which can be used as a target for evaluating minimal residual disease. IgH subclass and V(D)J rearrangements were identified in 77% and 65% of patients, respectively. Mutation analysis revealed the presence of missense protein-coding alterations in at least one of the evaluating genes in 16 of 48 patients (33%). This method may represent a time- and cost-effective diagnostic method for the molecular characterization of multiple myeloma.


Assuntos
Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular , Mieloma Múltiplo/genética , Idoso , Feminino , Frequência do Gene , Genes Neoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/diagnóstico , Mutação
13.
Oncotarget ; 7(49): 80664-80679, 2016 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-27811368

RESUMO

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.


Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Metilação de DNA , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Mieloma Múltiplo/genética , Integração de Sistemas , Linhagem Celular Tumoral , Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Estudos de Associação Genética , Predisposição Genética para Doença , Glicosiltransferases/genética , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Mieloma Múltiplo/patologia , Mieloma Múltiplo/terapia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores Purinérgicos P2Y/genética , Recidiva , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética
14.
Am J Pathol ; 186(8): 2171-2182, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27301357

RESUMO

IL-8 promotes cancer cell growth, survival, angiogenesis, and metastasis in several tumors. Herein, we investigated the sources of IL-8 production in multiple myeloma (MM) and its potential roles in MM pathogenesis. We found that bone marrow cells from patients with MM secreted higher amounts of IL-8 than healthy donors. IL-8 production was detected in cultures of CD138(+) plasma cells and CD138(-) cells isolated from bone marrows of MM patients, and in three of seven human myeloma cell lines (HMCLs) analyzed. Interactions between MM and stromal cells increased IL-8 secretion by stromal cells through cell-cell adhesion and soluble factors. Interestingly, IL8 expression also increased in HMCLs, stromal cells, and osteoclasts after treatment with the antimyeloma drugs melphalan and bortezomib. In fact, the effect of bortezomib on IL-8 production was higher than that exerted by stromal-MM cell interactions. Addition of exogenous IL-8 did not affect growth of HMCLs, although it protected cells from death induced by serum starvation through a caspase-independent mechanism. Furthermore, IL-8 induced by stromal-MM cell interactions strongly contributed to osteoclast formation in vitro, because osteoclastogenesis was markedly reduced by IL-8-specific neutralizing antibodies. In conclusion, our results implicate IL-8 in myeloma bone disease and point to the potential utility of an anti-IL-8 therapy to prevent unwanted effects of IL-8 up-regulation on survival, angiogenesis, and osteolysis in MM.


Assuntos
Interleucina-8/biossíntese , Mieloma Múltiplo/patologia , Osteogênese/fisiologia , Separação Celular , Sobrevivência Celular , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Humanos , Mieloma Múltiplo/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Reação em Cadeia da Polimerase , Células Estromais/metabolismo , Regulação para Cima
15.
Blood ; 127(24): 3035-9, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27069257

RESUMO

Immunoglobulin light-chain amyloidosis (AL) and multiple myeloma (MM) are 2 distinct monoclonal gammopathies that involve the same cellular compartment: clonal plasma cells (PCs). Despite the fact that knowledge about MM PC biology has significantly increased in the last decade, the same does not apply for AL. Here, we used an integrative phenotypic, molecular, and genomic approach to study clonal PCs from 24 newly diagnosed patients with AL. Through principal-component-analysis, we demonstrated highly overlapping phenotypic profiles between AL and both monoclonal gammopathy of undetermined significance and MM PCs. However, in contrast to MM, highly purified fluorescence-activated cell-sorted clonal PCs from AL (n = 9) showed almost normal transcriptome, with only 38 deregulated genes vs normal PCs; these included a few tumor-suppressor (CDH1, RCAN) and proapoptotic (GLIPR1, FAS) genes. Notwithstanding, clonal PCs in AL (n = 11) were genomically unstable, with a median of 9 copy number alterations (CNAs) per case, many of such CNAs being similar to those found in MM. Whole-exome sequencing (WES) performed in 5 AL patients revealed a median of 15 nonrecurrent mutations per case. Altogether, our results show that in the absence of a unifying mutation by WES, clonal PCs in AL display phenotypic and CNA profiles similar to MM, but their transcriptome is remarkably similar to that of normal PCs.


Assuntos
Amiloidose/genética , Cadeias Leves de Imunoglobulina/genética , Paraproteinemias/genética , Plasmócitos/metabolismo , Transcriptoma , Amiloidose/metabolismo , Amiloidose/patologia , Células Clonais/metabolismo , Células Clonais/patologia , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Análise em Microsséries , Paraproteinemias/metabolismo , Paraproteinemias/patologia , Fenótipo , Plasmócitos/patologia
16.
Blood ; 127(15): 1896-906, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-26755711

RESUMO

Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered atwww.clinicaltrials.gov as #NCT01237249.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/genética , Idoso , Bortezomib/administração & dosagem , Moléculas de Adesão Celular/metabolismo , Dexametasona/administração & dosagem , Progressão da Doença , Regulação para Baixo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imunofenotipagem , Integrinas/metabolismo , Lenalidomida , Masculino , Melfalan/administração & dosagem , Modelos Genéticos , Mieloma Múltiplo/patologia , Neoplasia Residual/patologia , Fenótipo , Plasmócitos/patologia , Prednisona/administração & dosagem , Prognóstico , Talidomida/administração & dosagem , Talidomida/análogos & derivados
17.
Blood ; 127(9): 1151-62, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26668134

RESUMO

There is significant interest in immunotherapy for the treatment of high-risk smoldering multiple myeloma (SMM), but no available data on the immune status of this particular disease stage. Such information is important to understand the interplay between immunosurveillance and disease transformation, but also to define whether patients with high-risk SMM might benefit from immunotherapy. Here, we have characterized T lymphocytes (including CD4, CD8, T-cell receptor γδ, and regulatory T cells), natural killer (NK) cells, and dendritic cells from 31 high-risk SMM patients included in the treatment arm of the QUIREDEX trial, and with longitudinal peripheral blood samples at baseline and after 3 and 9 cycles of lenalidomide plus low-dose dexamethasone (LenDex). High-risk SMM patients showed at baseline decreased expression of activation-(CD25/CD28/CD54), type 1 T helper-(CD195/interferon-γ/tumor necrosis factor-α/interleukin-2), and proliferation-related markers (CD119/CD120b) as compared with age-matched healthy individuals. However, LenDex was able to restore the normal expression levels for those markers and induced a marked shift in T-lymphocyte and NK-cell phenotype. Accordingly, high-risk SMM patients treated with LenDex showed higher numbers of functionally active T lymphocytes. Together, our results indicate that high-risk SMM patients have an impaired immune system that could be reactivated by the immunomodulatory effects of lenalidomide, even when combined with low-dose dexamethasone, and support the value of therapeutic immunomodulation to delay the progression to multiple myeloma. The QUIREDEX trial was registered to www.clinicaltrials.gov as #NCT00480363.


Assuntos
Dexametasona/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/imunologia , Talidomida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Demografia , Dexametasona/farmacologia , Feminino , Humanos , Imunofenotipagem , Quimioterapia de Indução , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Lenalidomida , Estudos Longitudinais , Quimioterapia de Manutenção , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Talidomida/farmacologia , Talidomida/uso terapêutico
18.
Blood ; 125(15): 2370-80, 2015 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-25655603

RESUMO

Although information about the molecular pathogenesis of Waldenström macroglobulinemia (WM) has significantly advanced, the precise cell of origin and the mechanisms behind WM transformation from immunoglobulin-M (IgM) monoclonal gammopathy of undetermined significance (MGUS) remain undetermined. Here, we undertook an integrative phenotypic, molecular, and genomic approach to study clonal B cells from newly diagnosed patients with IgM MGUS (n = 22), smoldering (n = 16), and symptomatic WM (n = 11). Through principal component analysis of multidimensional flow cytometry data, we demonstrated highly overlapping phenotypic profiles for clonal B cells from IgM MGUS, smoldering, and symptomatic WM patients. Similarly, virtually no genes were significantly deregulated between fluorescence-activated cell sorter-sorted clonal B cells from the 3 disease groups. Interestingly, the transcriptome of the Waldenström B-cell clone was highly different than that of normal CD25(-)CD22(+) B cells, whereas significantly less genes were differentially expressed and specific WM pathways normalized once the transcriptome of the Waldenström B-cell clone was compared with its normal phenotypic (CD25(+)CD22(+low)) B-cell counterpart. The frequency of specific copy number abnormalities [+4, del(6q23.3-6q25.3), +12, and +18q11-18q23] progressively increased from IgM MGUS and smoldering WM vs symptomatic WM (18% vs 20% and 73%, respectively; P = .008), suggesting a multistep transformation of clonal B cells that, albeit benign (ie, IgM MGUS and smoldering WM), already harbor the phenotypic and molecular signatures of the malignant Waldenström clone.


Assuntos
Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Gamopatia Monoclonal de Significância Indeterminada/genética , Macroglobulinemia de Waldenstrom/genética , Linfócitos B/metabolismo , Transformação Celular Neoplásica/patologia , Células Clonais , Citometria de Fluxo , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Imunoglobulina M/análise , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fenótipo , Macroglobulinemia de Waldenstrom/patologia
19.
Biochim Biophys Acta ; 1849(3): 353-66, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25497370

RESUMO

CONTEXT: MiR-155 plays a critical role in the development of B-cell malignancies. Previous studies have shown a deregulation of miR-155 in specific cytogenetic subtypes of multiple myeloma (MM). However, the mechanisms that regulate miR-155 expression in MM are not fully understood. OBJECTIVE: In the present study, we explored the regulation of miRNA-155 in MM by DNA methylation mechanisms and the impact of miR-155 expression in survival of MM patients. METHOD: Primary samples were obtained from 95 patients with newly diagnosed myeloma. Methylation was analyzed by Methylation Specific PCR, sequencing of bisulfite treated DNA and luciferase assay. RESULTS: qRT-PCR analysis revealed that miR-155 was differentially expressed in MM and its upregulation was associated with longer survival. DNA methylation of CpG island present in the first exon of miR-155 host gene was associated with its low expression in MM cell lines and patient samples. Our results showed for the first time that in vitro methylation of part of the promoter and first exon abrogated the miR-155 expression. We further showed that miR-155 expression in MM cell lines was increased by demethylating 5-aza-dC treatment and decreased by RNA-directed DNA methylation. Additionally, we found that LPS "immunological challenge" was insufficient to induce miR-155 expression in MM cell lines with methylated DNA around transcription start site (TSS). CONCLUSION: This study provides evidence that DNA methylation contributes to miR-155 expression in myeloma cells. Interestingly, the survival data showed an association between miR-155 expression and outcome of MM.


Assuntos
Metilação de DNA/genética , Epigênese Genética , MicroRNAs/biossíntese , Mieloma Múltiplo/genética , Linfócitos B/patologia , Linhagem Celular Tumoral , Ilhas de CpG , Elementos E-Box , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , Mieloma Múltiplo/patologia , Regiões Promotoras Genéticas
20.
PLoS One ; 9(3): e92378, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24658332

RESUMO

Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations--CD138++ (95-99%) and CD138low (1-5%)--in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells.


Assuntos
Mieloma Múltiplo/fisiopatologia , Sindecana-1/genética , Sindecana-1/fisiologia , Animais , Ácidos Borônicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Doxorrubicina/farmacologia , Xenoenxertos , Humanos , Imunofenotipagem , Melfalan/farmacologia , Camundongos SCID , Mieloma Múltiplo/imunologia , Células-Tronco Neoplásicas , Fenótipo , Plasmócitos/imunologia , Células Precursoras de Linfócitos B , Pirazinas/farmacologia , Sindecana-1/biossíntese
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