RESUMEN
The increased risk of Richter transformation (RT) in patients with chronic lymphocytic leukaemia (CLL) due to Epstein-Barr virus (EBV) reactivation during immunosuppressive therapy with fludarabine other targeted agents remains controversial. Among 31 RT cases classified as diffuse large B-cell lymphoma (DLBCL), seven (23%) showed EBV expression. In contrast to EBV- tumours, EBV+ DLBCLs derived predominantly from IGVH-hypermutated CLL, and they also showed CLL-unrelated IGVH sequences more frequently. Intriguingly, despite having different cellular origins, clonally related and unrelated EBV+ DLBCLs shared a previous history of immunosuppressive chemo-immunotherapy, a non-germinal centre DLBCL phenotype, EBV latency programme type II or III, and very short survival. These data suggested that EBV reactivation during therapy-related immunosuppression can transform either CLL cells or non-tumoural B lymphocytes into EBV+ DLBCL. To investigate this hypothesis, xenogeneic transplantation of blood cells from 31 patients with CLL and monoclonal B-cell lymphocytosis (MBL) was performed in Rag2-/- IL2γc-/- mice. Remarkably, the recipients' impaired immunosurveillance favoured the spontaneous outgrowth of EBV+ B-cell clones from 95% of CLL and 64% of MBL patients samples, but not from healthy donors. Eventually, these cells generated monoclonal tumours (mostly CLL-unrelated but also CLL-related), recapitulating the principal features of EBV+ DLBCL in patients. Accordingly, clonally related and unrelated EBV+ DLBCL xenografts showed indistinguishable cellular, virological and molecular features, and synergistically responded to combined inhibition of EBV replication with ganciclovir and B-cell receptor signalling with ibrutinib in vivo. Our study underscores the risk of RT driven by EBV in CLL patients receiving immunosuppressive therapies, and provides the scientific rationale for testing ganciclovir and ibrutinib in EBV+ DLBCL. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Herpesvirus Humano 4/efectos de los fármacos , Inmunosupresores/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Adulto , Anciano , Linfocitos B/efectos de los fármacos , Linfocitos B/patología , Transformación Celular Neoplásica/patología , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/patología , Femenino , Herpesvirus Humano 4/genética , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Masculino , Persona de Mediana EdadRESUMEN
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.
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Metilación de ADN/genética , Epigénesis Genética , MicroARNs/biosíntesis , Mieloma Múltiple/genética , Linfocitos B/patología , Línea Celular Tumoral , Islas de CpG , Elementos E-Box , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Masculino , MicroARNs/genética , Mieloma Múltiple/patología , Regiones Promotoras GenéticasRESUMEN
The B-cell compartment in which multiple myeloma stem cells reside remains unclear. We investigated the potential presence of mature, surface-membrane immunoglobulin-positive B lymphocytes clonally related to the tumor bone marrow plasma cells among different subsets of peripheral blood B cells from ten patients (7 with multiple myeloma and 3 with monoclonal gammopathies of undetermined significance). The presence of clonotypic immunoglobulin heavy chain gene rearrangements was determined in multiple highly-purified fractions of peripheral blood B-lymphocytes including surface-membrane IgM(+) CD27(-) naïve B-lymphocytes, plus surface-membrane IgG(+), IgA(+) and IgM(+) memory CD27(+) B cells, and normal circulating plasma cells, in addition to (mono)clonal plasma cells, by a highly-specific and sensitive allele-specific oligonucleotide polymerase chain reaction directed to the CDR3 sequence of the rearranged IGH gene of tumor plasma cells from individual patients. Our results showed systematic absence of clonotypic rearrangements in all the different B-cell subsets analyzed, including M-component isotype-matched memory B-lymphocytes, at frequencies <0.03 cells/µL (range: 0.0003-0.08 cells/µL); the only exception were the myeloma plasma cells detected and purified from the peripheral blood of four of the seven myeloma patients. These results indicate that circulating B cells from patients with multiple myeloma and monoclonal gammopathies of undetermined significance are usually devoid of clonotypic B cells while the presence of immunophenotypically aberrant myeloma plasma cells in peripheral blood of myeloma patients is a relatively frequent finding.
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Linfocitos B/metabolismo , Células Clonales/metabolismo , Gammopatía Monoclonal de Relevancia Indeterminada/metabolismo , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/patología , Linfocitos B/patología , Humanos , Inmunofenotipificación , Recuento de Linfocitos , Células Plasmáticas/metabolismo , Células Plasmáticas/patología , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
The gene expression profiles (GEPs) of 96 selected genes were analysed by real-time quantitative polymerase chain reaction (qPCR) with a TaqMan low-density array card in isolated tumour plasma cells (PCs) from 157 newly diagnosed multiple myeloma (MM) patients. This qPCR-based GEP correctly classified cases following the Translocation-cyclin D classification. Classic prognostic parameters and qPCR-based GEP predicted MM patient outcome and, although multivariate analyses revealed that cytogenetic risk (standard vs. high risk) was the variable that most strongly predicted prognosis, GEP added significant information for risk stratification. Considering only the standard risk cytogenetic patients, multivariate analyses revealed that high ß2-microglobulin, low CDKN1A and high SLC19A1 gene expression levels independently predicted a short time-to-progression (TTP), while high International Staging System stage, low CDKN2B and high TBRG4 gene expression predicted poor overall survival (OS). A gene expression risk score enabled the division of standard risk patients into two groups with different TTPs (83% vs. 38% at 3 years, P < 0·0001) and OS rates (88% vs. 61% at 5 years; P = 0·003). This study demonstrates that quantitative PCR is a robust, accurate and feasible technique for implementing in the daily routine as a surrogate for GEP-arrays.
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Perfilación de la Expresión Génica , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/genética , Aberraciones Cromosómicas , Progresión de la Enfermedad , Humanos , Análisis por Micromatrices , Mieloma Múltiple/mortalidad , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Factores de RiesgoRESUMEN
MicroRNA have been demonstrated to be deregulated in multiple myeloma. We have previously reported that miR-214 is down-regulated in multiple myeloma compared to in normal plasma cells. The functional role of miR-214 in myeloma pathogenesis was explored by transfecting myeloma cell lines with synthetic microRNA followed by gene expression profiling. Putative miR-214 targets were validated by luciferase reporter assay. Ectopic expression of miR-214 reduced cell growth and induced apoptosis of myeloma cells. In order to identify the potential direct target genes of miR-214 which could be involved in the biological pathways regulated by this microRNA, gene expression profiling of the H929 myeloma cell line transfected with precursor miR-214 was carried out. Functional analysis revealed significant enrichment for DNA replication, cell cycle phase and DNA binding. miR-214 directly down-regulated the expression of PSMD10, which encodes the oncoprotein gankyrin, and ASF1B, a histone chaperone required for DNA replication, by binding to their 3'-untranslated regions. In addition, gankyrin inhibition induced an increase of P53 mRNA levels and subsequent up-regulation of CDKN1A (p21Waf1/Cip1) and BAX transcripts, which are direct transcriptional targets of p53. In conclusion, MiR-214 functions as a tumor suppressor in myeloma by positive regulation of p53 and inhibition of DNA replication.
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Proliferación Celular , Replicación del ADN , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteína p53 Supresora de Tumor/genética , Regiones no Traducidas 5'/genética , Apoptosis/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Metilación de ADN , Perfilación de la Expresión Génica , Humanos , Immunoblotting , MicroARNs/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
We have evaluated the use of CD138+ positively selected bone marrow samples to identify a molecular target for minimal residual disease assessment by polymerase chain reaction (PCR) in 25 untreated patients with multiple myeloma. A fraction of each sample was used for CD138+ selection, and the rest served as a reference control. VDJH, DJH, and Kde gene rearrangements were tested for amplification according to the BIOMED-2 Concerted Action. PCR products were directly sequenced in an automated ABI 3130 DNA sequencer using Big-Dye terminators. Within the CD138+ selected group, VDJH rearrangements were detected in all cases (100 %), DJH in 16 (64 %), and Kde in 18 (72 %) cases; whereas in the control samples, VDJH, DJH, and Kde rearrangements were detected in 19 (76 %), 11 (44 %), and 12 (48 %) cases, respectively. After sequencing, 24 (96 %) cases within the CD138+ group had a PCR target for MRD detection compared with 15 (60 %) cases in the control group. We conclude that the use of CD138+ positively selected bone marrow samples increases the applicability of minimal residual disease studies by PCR in patients with multiple myeloma.
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Examen de la Médula Ósea/métodos , Médula Ósea/patología , Separación Celular/métodos , Reordenamiento Génico de Linfocito B , Mieloma Múltiple/patología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sindecano-1/análisis , Células Clonales/patología , ADN de Neoplasias/genética , Humanos , Proteínas de Mieloma/genética , Neoplasia Residual/diagnóstico , Recombinación V(D)JRESUMEN
UNLABELLED: Background Recent findings suggest that a specific deletion of Dicer1 in mesenchymal stromal cell-derived osteoprogenitors triggers several features of myelodysplastic syndrome in a murine model. Our aim was to analyze DICER1 and DROSHA gene and protein expression in mesenchymal stromal cells (the osteoblastic progenitors) obtained from bone marrow of myelodysplastic syndrome patients, in addition to microRNA expression profile and other target genes such as SBDS, a DICER1-related gene that promotes bone marrow dysfunction and myelodysplasia when repressed in a murine model. DESIGN AND METHODS: Mesenchymal stromal cells from 33 bone marrow samples were evaluated. DICER, DROSHA and SBDS gene expression levels were assessed by real-time PCR and protein expression by Western blot. MicroRNA expresion profile was analyzed by commercial low-density arrays and some of these results were confirmed by individual real-time PCR. RESULTS: Mesenchymal stromal cells from myelodysplastic syndrome patients showed lower DICER1 (0.65±0.08 vs. 1.91±0.57; P=0.011) and DROSHA (0.62±0.06 vs. 1.38±0.29; P=0.009) gene expression levels, two relevant endonucleases associated to microRNA biogenesis, in comparison to normal myelodysplastic syndrome. These findings were confirmed at protein levels by Western blot. Strikingly, no differences were observed between paired mononuclear cells from myelodysplastic syndrome and controls. In addition, mesenchymal stromal cells from myelodysplastic syndrome patients showed significant lower SBDS (0.63±0.06 vs. 1.15±0.28; P=0.021) gene expression levels than mesenchymal stromal cells from healthy controls. Furthermore, mesenchymal stromal cells from myelodysplastic syndrome patients showed an underlying microRNA repression compared to healthy controls. Real-time PCR approach confirmed that mir-155, miR-181a and miR-222 were down-expressed in mesenchymal stromal cells from myelodysplastic syndrome patients. Conclusions This is the first description of an impaired microRNA biogenesis in human mesenchymal stromal cells from myelodysplastic syndrome patients, where DICER1 and DROSHA gene and protein downregulation correlated to a gene and microRNA abnormal expression profile, validating the animal model results previously described.
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ARN Helicasas DEAD-box/genética , Regulación Neoplásica de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Síndromes Mielodisplásicos/genética , Proteínas/genética , Ribonucleasa III/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) assessment by PCR in multiple myeloma (MM) has several shortcomings, including the lack of a suitable target. Kappa deleting element (KDE) rearrangements occur in virtually all Ig-lambda B-cell malignancies and in 1/3 of Ig-kappa are not affected by somatic hypermutation and, as in ALL, could be used as PCR targets. METHODS: We have first investigated the incidence, gene segment usage, and CDR3 composition of IGK-KDE rearrangements in 96 untreated myeloma patients. Second, we tested 16 KDE gene rearrangements as molecular targets for MRD assessment by RQ-PCR using a germline reverse primer and a germline Taqman probe in combination with allele-specific oligonucleotides (ASO) as forward primers. RESULTS: Monoclonal KDE rearrangements were amplified in 45% (43/96) of cases, monoallelic in 2/3 of them (29 cases), and biallellic in the remaining 14 cases. Overall, 88% of cases were successfully sequenced, KDE being equally frequently rearranged with VK and with intron-Recombination signal sequence (RSS). Median numbers of inserted and deleted nucleotides in the junctional region were one and five, respectively. CONCLUSIONS: Using KDE rearrangements as additional PCR target for MRD assessment in MM improves the applicability of these studies in 9% of cases overall and in 20% of lambda cases. Its use in the latter subset could represent a significant advance.
Asunto(s)
Cadenas kappa de Inmunoglobulina/genética , Mieloma Múltiple/patología , Neoplasia Residual/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuencia de Bases , Cartilla de ADN , Humanos , Intrones , Mieloma Múltiple/genética , Mutación , Neoplasia Residual/genéticaRESUMEN
We have evaluated 9 new molecular markers (ERG, EVI1, MLL-PTD, MN1, PRAME, RHAMM, and WT1 gene-expression levels plus FLT3 and NPM1 mutations) in 121 de novo cytogenetically normal acute myeloblastic leukemias. In the multivariate analysis, high ERG or EVI1 and low PRAME expressions were associated with a shorter relapse-free survival (RFS) and overall survival (OS). A 0 to 3 score was given by assigning a value of 0 to favorable parameters (low ERG, low EVI1, and high PRAME) and 1 to adverse parameters. This model distinguished 4 subsets of patients with different OS (2-year OS of 79%, 65%, 46%, and 27%; P = .001) and RFS (2-year RFS of 92%, 65%, 49%, and 43%; P = .005). Furthermore, this score identified patients with different OS (P = .001) and RFS (P = .013), even within the FLT3/NPM1 intermediate-risk/high-risk subgroups. Here we propose a new molecular score for cytogenetically normal acute myeloblastic leukemias, which could improve patient risk-stratification.
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Biomarcadores de Tumor/genética , Leucemia Mieloide Aguda/genética , Modelos Genéticos , Adulto , Anciano , Antígenos de Neoplasias/genética , Análisis Citogenético , Proteínas de Unión al ADN/genética , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Marcadores Genéticos , Humanos , Leucemia Mieloide Aguda/mortalidad , Proteína del Locus del Complejo MDS1 y EV11 , Masculino , Persona de Mediana Edad , Mutación , Nucleofosmina , Pronóstico , Proto-Oncogenes/genética , Factores de Riesgo , España/epidemiología , Tasa de Supervivencia , Transactivadores/genética , Factores de Transcripción/genética , Regulador Transcripcional ERGRESUMEN
Dicer and Drosha are key enzymes in the miRNA-processing pathway which is altered in many human cancers. We analyzed Dicer and Drosha expression levels by quantitative PCR in 151 patients with monoclonal gammopathies: 102 symptomatic myeloma patients, 23 smoldering myelomas and 26 monoclonal gammopathy of undetermined significance. We found that Dicer expression values were significantly higher in monoclonal gammopathy of undetermined significance than in smoldering myelomas and symptomatic myeloma (mean ± SD, 0.84 ± 0.36 vs. 0.60 ± 0.23 and 0.62 ± 0.51; P<0.01). Moreover, the median progression-free survival was significantly longer in symptomatic myeloma patients with high expression of Dicer (not reached vs. 23.6 months; P=0.02). By contrast, no differences in the expression of Drosha among these groups of patients were observed. Our data suggest that Dicer expression may play an important role in the progression and prognosis of monoclonal gammopathies.
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ARN Helicasas DEAD-box/genética , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Mieloma Múltiple/genética , Ribonucleasa III/genética , Anciano , Biomarcadores/análisis , ARN Helicasas DEAD-box/metabolismo , Supervivencia sin Enfermedad , Expresión Génica , Trasplante de Células Madre Hematopoyéticas , Humanos , Melfalán/administración & dosificación , Melfalán/uso terapéutico , MicroARNs/análisis , Persona de Mediana Edad , Gammopatía Monoclonal de Relevancia Indeterminada/mortalidad , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Gammopatía Monoclonal de Relevancia Indeterminada/terapia , Mieloma Múltiple/mortalidad , Mieloma Múltiple/patología , Mieloma Múltiple/terapia , Pronóstico , Ribonucleasa III/metabolismo , España , Regulación hacia ArribaRESUMEN
We have explored the potential role of genetics in the development of osteonecrosis of the jaw (ONJ) in multiple myeloma (MM) patients under bisphosphonate therapy. A genome-wide association study was performed using 500 568 single nucleotide polymorphisms (SNPs) in 2 series of homogeneously treated MM patients, one with ONJ (22 MM cases) and another without ONJ (65 matched MM controls). Four SNPs (rs1934951, rs1934980, rs1341162, and rs17110453) mapped within the cytochrome P450-2C gene (CYP2C8) showed a different distribution between cases and controls with statistically significant differences (P = 1.07 x 10(-6), P = 4.231 x 10(-6), P = 6.22 x 10(-6), and P = 2.15 x 10(-6), respectively). SNP rs1934951 was significantly associated with a higher risk of ONJ development even after Bonferroni correction (P corrected value = .02). Genotyping results displayed an overrepresentation of the T allele in cases compared with controls (48% vs 12%). Thus, individuals homozygous for the T allele had an increased likelihood of developing ONJ (odds ratio 12.75, 95% confidence interval 3.7-43.5).
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Hidrocarburo de Aril Hidroxilasas/genética , Difosfonatos/efectos adversos , Genoma Humano/genética , Enfermedades Maxilomandibulares/genética , Mieloma Múltiple/complicaciones , Osteonecrosis/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Citocromo P-450 CYP2C8 , Difosfonatos/uso terapéutico , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Enfermedades Maxilomandibulares/inducido químicamente , Enfermedades Maxilomandibulares/complicaciones , Enfermedades Maxilomandibulares/enzimología , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/enzimología , Mieloma Múltiple/genética , Osteonecrosis/inducido químicamente , Osteonecrosis/complicaciones , Osteonecrosis/enzimologíaRESUMEN
We have analyzed brain and acute leukemia, cytoplasmic (BAALC) gene expression and other genetic markers (ERG, EVI1, MN1, PRAME, WT1, FLT3, and NPM1 mutations) in 127 intermediate-risk acute myeloid leukemia (AML) patients: 98 cytogenetically normal and 29 with intermediate-risk cytogenetic alterations. High versus low BAALC expressers showed a higher refractoriness to induction treatment (31% vs 10%; p = .005), lower complete remission rate after salvage therapy (82% vs 97%; p = .010), and lower 3-year overall (23% vs 58%, p < .001) and relapse-free survival (26% vs 52%, p = .006). Similar results were found when cytogenetic subgroups were analyzed separately. Multivariate models confirmed the unfavorable prognosis of this marker. In conclusion, BAALC is a relevant prognostic marker in intermediate-risk AML.
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Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/biosíntesis , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biosíntesis , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Nucleofosmina , Valor Predictivo de las Pruebas , Factores de Riesgo , Tasa de Supervivencia/tendencias , Resultado del Tratamiento , Adulto JovenRESUMEN
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).
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Péptidos y Proteínas de Señalización Intracelular/fisiología , Mieloma Múltiple/patología , Células Plasmáticas/patología , Linfocitos B , Desdiferenciación Celular , Diferenciación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/análisis , Pronóstico , Células Tumorales CultivadasRESUMEN
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.
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Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Técnicas de Diagnóstico Molecular , Mieloma Múltiple/genética , Anciano , Femenino , Frecuencia de los Genes , Genes Relacionados con las Neoplasias , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , MutaciónRESUMEN
INTRODUCTION: Molecular alterations leading progression of asymptomatic CLL-like high-count monoclonal B lymphocytosis (hiMBL) to chronic lymphocytic leukemia (CLL) remain poorly understood. Recently, genome-wide association studies have found 6p21.3, where the human leukocyte antigen (HLA) system is coded, to be a susceptibility risk region for CLL. Previous studies have produced discrepant results regarding the association between HLA and CLL development and outcome, but no studies have been performed on hiMBL. AIMS: We evaluated the role of HLA class I (-A, -B and -C) and class II (-DRB1 and -DQB1) in hiMBL/CLL susceptibility, hiMBL progression to CLL, and treatment requirement in a large series of 263 patients diagnosed in our center with hiMBL (n = 156) or Binet A CLL (n = 107). RESULTS: No consistent association between HLA specificities and hiMBL or CLL susceptibility was found. With a median follow-up of 7.7 years, 48/156 hiMBLs (33%) evolved to asymptomatic CLLs, while 16 hiMBLs (10%) and 44 CLLs (41%) required treatment. No HLA specificities were found to be significantly associated with hiMBL progression or treatment in the whole cohort. However, within antigen-experienced immunoglobulin heavy-chain (IGHV)-mutated hiMBLs, which represents the highest proportion of hiMBL cases (81%), the presence of HLA-DQB1*03 showed a trend to a higher risk of progression to CLL (60% vs. 26%, P = 0.062). Moreover, HLA-DQB1*02 specificity was associated with a lesser requirement for 15-year treatment (10% vs. 36%, P = 0.012). CONCLUSION: In conclusion, our results suggest a role for HLA in IGHV-mutated hiMBL prognosis, and are consistent with the growing evidence of the influence of 6p21 on predisposition to CLL. Larger non-biased series are required to enable definitive conclusions to be drawn.
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Genes de las Cadenas Pesadas de las Inmunoglobulinas , Antígenos de Histocompatibilidad Clase I/genética , Linfocitosis/genética , Mutación , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/patología , Cromosomas Humanos Par 6/genética , Femenino , Humanos , Recuento de Linfocitos , Linfocitosis/sangre , Masculino , Persona de Mediana Edad , PronósticoAsunto(s)
Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Antineoplásicos/uso terapéutico , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/prevención & control , Trasplante de Células Madre/efectos adversos , Acondicionamiento Pretrasplante/efectos adversos , Trasplante Homólogo/efectos adversos , Adulto , Enfermedad Crónica , Femenino , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Humanos , Inmunosupresores/uso terapéutico , Linfoma Folicular/patología , Recurrencia , Inducción de Remisión , RituximabRESUMEN
PURPOSE: Dysregulation of one of the three D-cyclin genes has been observed in virtually all multiple myeloma tumors. The mechanisms by which CCND2 is upregulated in a set of multiple myeloma are not completely deciphered. We investigated the role of post-transcriptional regulation through the interaction between miRNAs and their binding sites at 3'UTR in CCND2 overexpression in multiple myeloma. EXPERIMENTAL DESIGN: Eleven myeloma cell lines and 45 primary myeloma samples were included in the study. Interactions between miRNAs deregulated in multiple myeloma and mRNA targets were analyzed by 3'UTR-luciferase plasmid assay. The presence of CCND2 mRNA isoforms different in length was explored using qRT-PCR, Northern blot, mRNA FISH, and 3' rapid amplification of cDNA ends (RACE)-PCR. RESULTS: We detected the presence of short CCND2 mRNA, both in the multiple myeloma cell lines and primary cells. The results obtained by 3'RACE experiments revealed that changes in CCND2 3'UTR length are explained by alternative polyadenylation. The luciferase assays using plasmids harboring the truncated CCND2 mRNA strongly confirmed the loss of miRNA sites in the shorter CCND2 mRNA isoform. Those multiple myelomas with greater abundance of the shorter 3'UTR isoform were associated with significant higher level of total CCND2 mRNA expression. Furthermore, functional analysis showed significant CCND2 mRNA shortening after CCND1 silencing and an increased relative expression of longer isoform after CCND1 and CCND3 overexpression, suggesting that cyclin D1 and D3 could regulate CCND2 levels through modifications in polyadenylation-cleavage reaction. CONCLUSIONS: Overall, these results highlight the impact of CCND2 3'UTR shortening on miRNA-dependent regulation of CCND2 in multiple myeloma.
Asunto(s)
Ciclina D2/genética , Regulación Neoplásica de la Expresión Génica , Mieloma Múltiple/genética , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 3' , Empalme Alternativo , Línea Celular Tumoral , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclina D2/metabolismo , Ciclina D3/genética , Ciclina D3/metabolismo , Metilación de ADN , Humanos , MicroARNs/genética , Mieloma Múltiple/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Translocación Genética , Regulación hacia ArribaRESUMEN
BACKGROUND AND OBJECTIVES: Analysis of IgH rearrangements in B-cell malignancies has provided clinical researchers with a wide range of information during the last few years. However, only a few studies have contributed to the characterization of these features in multiple myeloma (MM), and they have been focused on the analysis of the expressed IgH allele only. Comparison between the expressed and the non-functional IgH alleles allows further characterizion of the selection processes to which pre-myeloma cells are submitted. DESIGN AND METHODS: We analyzed a cohort of 84 untreated MM patients in order to characterize their functional VDJH and non-functional DJH rearrangements. The pattern of mutations and gene segment usage for both types of rearrangements was analyzed by polymerase chain reaction and sequencing. RESULTS: VH3 and VH1 family members were over- and under-represented, respectively. VH3-30 and VH3-15 segments were the most frequently used, whereas VH4-34 was found only in non-functional or heavily mutated VDJH rearrangements. DH2 and DH3 family members were over-represented in both VDJH and DJH repertoires, while the DH1 family was under-represented only in the productive VDJH rearrangements. Finally, DH3-22 and DH2-21 gene segments were found to be over-represented in the functional repertoire while segments commonly used by less mature B-cell malignancies, such as DH6-19 or DH3-3, were under-represented. INTERPRETATION AND CONCLUSIONS: Data reported here help to identify the clonogenic MM cell as a post-germinal center B cell that has undergone selection processes during the germinal center reaction.
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Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas/genética , Mieloma Múltiple/genética , Diversidad de Anticuerpos , Linfocitos B , Salud de la Familia , Reordenamiento Génico , Humanos , Región de Unión de la Inmunoglobulina , Región Variable de Inmunoglobulina , Mieloma Múltiple/inmunología , Ácidos Nucleicos Heterodúplex/química , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Mieloma Múltiple/fisiopatología , Sindecano-1/genética , Sindecano-1/fisiología , Animales , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Doxorrubicina/farmacología , Xenoinjertos , Humanos , Inmunofenotipificación , Melfalán/farmacología , Ratones SCID , Mieloma Múltiple/inmunología , Células Madre Neoplásicas , Fenotipo , Células Plasmáticas/inmunología , Células Precursoras de Linfocitos B , Pirazinas/farmacología , Sindecano-1/biosíntesisRESUMEN
Most patients with pancreatic ductal adenocarcinoma (PDAC) die within 6 months of diagnosis. However, 20% to 25% patients undergoing total tumor resection remain alive and disease-free 5 years after diagnostic surgery. Few studies on tumor markers have predicted patient prognosis and/or survival. We evaluated the effect of tumor cytogenetic copy number changes detected by interphase fluorescence in situ hybridization on overall survival (OS) of 55 PDAC patients. The prognostic value of copy number changes showing an effect on OS was validated in an external cohort of 44 surgically resected PDAC patients by comparative genomic hybridization arrays, and the genes coded in altered chromosomes with prognostic value were identified by high-density single-nucleotide polymorphism arrays in 20 cases. Copy number changes of chromosomes 4 and 9q34 with gains of 8q24 were independently associated with shorter OS. On the basis of these three chromosomal alterations, a score is proposed that identifies patients with significantly different (P < 0.001) 5-year OS rates: 60% ± 20%, 16% ± 8%, and 0% ± 0%, respectively. Our results show an association between tumor cytogenetics and OS of PDAC patients and provide the basis for further prognostic stratification of patients undergoing complete tumor resection. Further studies to identify specific genes coded in these chromosomes and their functional consequences are necessary to understand the clinical effect of these changes.