RESUMEN
ABSTRACT: Amyloidogenic serum free light chains (sFLCs) drive disease progression in AL amyloidosis. Matrix-assisted laser desorption/ionization time of flight mass spectrometry-based FLC assay (FLC-MS) has greater sensitivity than conventional sFLC assays allowing for the detection of serological residual disease. We report the utility of FLC-MS in a large series of patients with AL amyloidosis assessing the impact of FLC-MS negativity after treatment on overall survival (OS) and organ response rates. Serum samples were analyzed using FLC-MS at diagnosis and at 6 and 12 months after treatment. The impact of FLC-MS negativity over standard hematologic responses on survival and organ response was assessed. A total of 487 patients were included; 290 (59%) and 349 (71.5%) had cardiac and renal involvement, respectively. There was 100% concordance between the light chain (LC) fibril type and LC isotype identified by FLC-MS. At 6 and 12 months, 81 (16.6%) and 101 (20.7%) were FLC-MS negative. Of those achieving a conventional hematologic complete response (CR) at 6 and 12 months, 45 (27.7%) and 64 (39%) were FLC-MS negative. At 12 months, median OS for CR + FLC-MS negative was not reached vs 108 months in CR + FLC-MS positive (P = .024). At 12 months, 70% of patients with FLC-MS negativity (vs 50% FLC-MS positive) achieved a cardiac response (P = .015). In a multivariate analysis, FLC-MS negativity at 12 months was an independent predictor of better outcomes. FLC-MS can detect persistent monoclonal light chains in a significant proportion of patients in a conventional hematologic CR. FLC-MS assessment promises to be a new standard for response assessment in AL amyloidosis.
Asunto(s)
Amiloidosis , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas , Humanos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Amiloidosis/diagnóstico , Cadenas Ligeras de Inmunoglobulina , Respuesta Patológica Completa , Progresión de la EnfermedadRESUMEN
Smoldering multiple myeloma (SMM) is an asymptomatic precursor to multiple myeloma. Here we define the epidemiological characteristics of SMM in the general population in Iceland. The iStopMM study (ClinicalTrials.gov ID: NCT03327597 ) is a nationwide screening study for multiple myeloma precursors where all residents in Iceland 40 years or older were invited to participate. SMM was defined as 10-60% bone marrow plasma cells and/or monoclonal (M) protein concentration ≥3 g dl-1, in the absence of myeloma-defining events. Of the 80,759 who gave informed consent to participate, 75,422 (93%) were screened. The prevalence of SMM in the total population was 0.53% (95% confidence interval (CI) = 0.49-0.57%) in individuals 40 years or older. In men and women, the prevalence of SMM was 0.67% (95% CI = 0.62-0.73%) and 0.39% (95% CI = 0.35-0.43%), respectively; it increased with age in both sexes. For the 193 individuals with SMM, median age was 70 years (range 44-92 years) and 60% were males. The mean M protein concentration of individuals with SMM was 0.62 g dl-1 (range 0.01-3.5 g dl-1) and 73% had 11-20% bone marrow plasma cell infiltration. The high prevalence of SMM has implications for future treatment policies in multiple myeloma as the evidence supporting treatment initiation at the SMM stage is emerging.
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Mieloma Múltiple , Mieloma Múltiple Quiescente , Humanos , Femenino , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Mieloma Múltiple/terapia , Prevalencia , Factores de Riesgo , Progresión de la EnfermedadRESUMEN
Mass spectrometry (MS) is a promising tool for monitoring monoclonal protein in plasma cell dyscrasias. We included 480 transplant-eligible newly-diagnosed multiple myeloma (MM) patients from the GMMG-MM5 trial (EudraCT No. 2010-019173-16) and performed a retrospective MS analysis at baseline (480 patients) and at the pre-defined, consecutive time points after induction (444 patients), prior to maintenance (305 patients) and after one year of maintenance (227 patients). We found that MS negativity was significantly associated with improved progression-free survival (PFS) even in patients with complete response (CR) at all investigated follow-up time points. The prognostic impact was independent of established risk factors, such as the revised International Staging System. Combining MS and baseline cytogenetics improved the prediction of outcome: MS-positive patients with high-risk cytogenetics had a dismal PFS of 1.9 years (95% confidence interval [CI]: 1.6-2.3 years) from the start of maintenance. Testing the value of sequential MS prior to and after one year of maintenance, patients converting from MS positivity to negativity had an excellent PFS (median not reached) while patients converting from MS negativity to positivity progressed early (median 0.6 years, 95% CI: 0.3-not reached). Among patients with sustained MS positivity, the baseline high-risk cytogenetic status had a significant impact and defined a group with poor PFS. Combining minimal residual disease (MRD) in the bone marrow and MS allowed the identification of double negative patients with a favorable PFS (median 3.33 years, 95% CI: 3.08-not reached) and no overall survival events. Our study provides strong evidence that MS is superior to conventional response monitoring, highlighting the potential of MS to become a new standard. Our data indicate that MS should be performed sequentially and combined with baseline disease features and MRD to improve its clinical value.Clinical Trials Register: EudraCT No. 2010-019173-16.
Asunto(s)
Mieloma Múltiple , Humanos , Médula Ósea , Mieloma Múltiple/diagnóstico , Mieloma Múltiple/tratamiento farmacológico , Neoplasia Residual/diagnóstico , Pronóstico , Estudios Retrospectivos , Resultado del Tratamiento , Espectrometría de MasasRESUMEN
Salvage of endogenous immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) is implicated in many clinical areas, including therapeutic monoclonal antibody kinetics, patient monitoring in IgG multiple myeloma, and antibody-mediated transplant rejection. There is a clear clinical need for a fully parameterized model of FcRn-mediated recycling of endogenous IgG to allow for predictive modeling, with the potential for optimizing therapeutic regimens for better patient outcomes. In this paper we study a mechanism-based model incorporating nonlinear FcRn-IgG binding kinetics. The aim of this study is to determine whether parameter values can be estimated using the limited in vivo human data, available in the literature, from studies of the kinetics of radiolabeled IgG in humans. We derive mathematical descriptions of the experimental observations-timecourse data and fractional catabolic rate (FCR) data-based on the underlying physiological model. Structural identifiability analyses are performed to determine which, if any, of the parameters are unique with respect to the observations. Structurally identifiable parameters are then estimated from the data. It is found that parameter values estimated from timecourse data are not robust, suggesting that the model complexity is not supported by the available data. Based upon the structural identifiability analyses, a new expression for the FCR is derived. This expression is fitted to the FCR data to estimate unknown parameter values. Using these parameter estimates, the plasma IgG response is simulated under clinical conditions. Finally a suggestion is made for a reduced-order model based upon the newly derived expression for the FCR. The reduced-order model is used to predict the plasma IgG response, which is compared with the original four-compartment model, showing good agreement. This paper shows how techniques for compartmental model analysis-structural identifiability analysis, linearization, and reparameterization-can be used to ensure robust parameter identification.
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Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Modelos Biológicos , Receptores Fc/inmunología , HumanosRESUMEN
Cardiac involvement and presenting dFLC (difference between involved and uninvolved free light chains) are independent predictors of outcome in systemic AL amyloidosis. These markers have limited prognostic utility in patients surviving the initial months following diagnosis. Here we assessed immunoparesis, as determined by novel heavy+light chain (HLC) immunoassays, as a prognostic marker for survival in AL amyloidosis. HLC measurements identified immunoparesis of at least one immunoglobulin (Ig) isotype in 145 (85%) patients; and severe immunoparesis (≥2 Ig isotypes suppressed by >50% below normal levels) in 29 (17%) patients. Median overall survival (OS) on intention to treat (ITT) analysis was 26·2 months. In the ITT cohort, dFLC >180 mg/l was associated with shorter OS (P = 0·05); whereas HLC immunoparesis was not prognostic. On a landmark analysis of 127 patients alive at 6 months, presenting dFLC was not prognostic for OS (P = 0·33) and severe HLC immunoparesis trended towards poorer survival (20·2 vs. 42·8 months; P = 0·09). In the subset of patients with cardiac involvement, severe HLC immunoparesis conferred very poor outcome (median OS 8·8 vs. 29·9 months, P = 0·007). In conclusion, severe HLC immunoparesis is an independent marker of long-term poor prognosis in AL patients with cardiac involvement. The pathophysiological significance of this observation needs further study.
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Cardiomiopatías/etiología , Cadenas Pesadas de Inmunoglobulina/sangre , Cadenas Ligeras de Inmunoglobulina/sangre , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/complicaciones , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Cardiomiopatías/inmunología , Femenino , Humanos , Inmunoensayo/métodos , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/diagnóstico , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/mortalidad , Masculino , Persona de Mediana Edad , Paraproteinemias , Paresia/inmunología , Pronóstico , Tasa de SupervivenciaRESUMEN
Heavy light chain (HLC) assays allow precise measurement of the monoclonal and of the noninvolved polyclonal immunoglobulins of the same isotype as the M-protein (e.g., monoclonal IgAκ and polyclonal IgAλ in case of an IgAκ myeloma), which was not possible before. The noninvolved polyclonal immunoglobulin is termed 'HLC-matched pair'. We investigated the impact of the suppression of the HLC-matched pair on outcome in 203 patients with multiple myeloma, a phenomenon that likely reflects the host's attempt to control the myeloma clone. Severe (>50%) HLC-matched pair suppression was identified in 54.5% of the 156 newly diagnosed patients and was associated with significantly shorter survival (45.4 vs. 71.9 months, P = 0.019). This correlation was statistically significant in IgG patients (46.4 vs. 105.1 months, P = 0.017), but not in patients with IgA myelomas (32.9 vs. 54.1 months, P = 0.498). At best response, HLC-matched pair suppression improved only in patients with ≥VGPR, indicating partial or complete humoral immune reconstitution during remission in those with excellent response. Severe HLC-matched pair suppression retained its prognostic impact also during follow-up after first response. In the 47 pretreated patients with relapsed/refractory disease, a similar correlation between severe HLC suppression and survival was noted (22.8 vs. not reached, P = 0.028). Suppression of the polyclonal immunoglobulins of the other isotypes than the myeloma protein correlated neither with HLC-matched pair suppression, nor with outcome. Multivariate analysis identified severe HLC-matched pair suppression as independent risk factor for shorter survival, highlighting the impact of isotype specific immune dysregulation on outcome in multiple myeloma.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cadenas Pesadas de Inmunoglobulina/sangre , Cadenas Ligeras de Inmunoglobulina/sangre , Inmunosupresores/uso terapéutico , Mieloma Múltiple/inmunología , Mieloma Múltiple/mortalidad , Trasplante de Células Madre , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Terapia Combinada , Femenino , Humanos , Isotipos de Inmunoglobulinas/sangre , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Inmunosupresores/administración & dosificación , Masculino , Persona de Mediana Edad , Mieloma Múltiple/sangre , Mieloma Múltiple/terapia , Análisis Multivariante , Proteínas de Mieloma/análisis , Pronóstico , Análisis de SupervivenciaRESUMEN
Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.
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Inmunoensayo/métodos , Inmunoglobulina A/sangre , Humanos , Cadenas alfa de Inmunoglobulina/sangre , Cadenas kappa de Inmunoglobulina/sangre , Cadenas lambda de Inmunoglobulina/sangre , Paraproteinemias/diagnóstico , Paraproteinemias/inmunología , Paraproteínas/análisis , RecurrenciaRESUMEN
BACKGROUND: In previous studies, we showed that gene activated matrices (GAMs) containing nonviral vectors successfully deliver genes to neurons after optic nerve and spinal cord injury. In the present study, we evaluated whether adenoviral vectors delivered within a GAM increase the efficiency of local gene delivery to injured CNS neurons. Lyophilized GAMs containing collagen and adenoviral vectors were assessed in vitro and in vivo. METHODS: We evaluated viral vector stability, release kinetics and efficiency of transduction for this GAM formulation in vitro using the quantitative polymerase chain reaction (qPCR), flow cytometry and fluorescence microscopy. Using PCR, reverse transcriptase-PCR and confocal microscopy, we assessed viral DNA retrograde axonal transport, green fluorescent protein (GFP) expression in retinal ganglion cells (RGCs) after GAM implantation into the wound of the rat transected optic nerve. RESULTS: qPCR analyses demonstrated that 100% of viral particles were retained within the collagen after lyophilization. In vitro studies demonstrated that 60% of the particles within the GAM were infective and not released from the collagen matrix when placed in water. By 24 h, GFP expression was detected within cells that have invaded the GAM. In vivo studies demonstrated that adenoviral particles were retrogradely transported in axons from the GAM implanted at the lesion site to the RGC in the retina where the corresponding mRNA was expressed. Analysis of the efficiency of cell transduction indicated that 69% of RGC express GFP. CONCLUSIONS: These studies demonstrate that lyophilized GAMs containing adenoviral particles within collagen are stable, retain a significant proportion of their infectivity and successfully and efficiently deliver genes to neurons after central nervous system injury.
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Adenoviridae/genética , Axones/metabolismo , Materiales Biocompatibles/metabolismo , Sistema Nervioso Central/patología , Terapia Genética , Activación Transcripcional , Transgenes/genética , Animales , Axones/patología , Línea Celular , Supervivencia Celular , ADN Viral/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Nervio Óptico/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patologíaRESUMEN
Murine haematopoietic stem cells (HSCs) are contained in the Kit+Sca1+Lin(-) (KSL) population of bone marrow and are able to repopulate lethally irradiated mice. Myeloproliferative disorders (MPDs) are thought to be clonogenic diseases arising at the level of the HSC. Here, we show that mice expressing low levels of the transcription factor c-Myb, as the result of genetic knockdown, develop a transplantable myeloproliferative phenotype that closely resembles the human disease essential thrombocythaemia (ET). Unlike wild-type cells, the KSL population in c-myb knockdown bone marrow cannot repopulate irradiated mice and does not transfer the disease. Instead, cells positive for Kit and expressing low to medium levels of CD11b acquire self-renewing stem cell properties and are responsible for the perpetuation of the myeloproliferative phenotype.
Asunto(s)
Células Madre Hematopoyéticas/fisiología , Proteínas Proto-Oncogénicas c-myb/fisiología , Trombocitemia Esencial/patología , Animales , Técnicas de Silenciamiento del Gen , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-myb/genéticaRESUMEN
BACKGROUND: Glycoprotein VI (GPVI) is a physiologic receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerization of GPVI has been proposed as being necessary for the interaction with collagen, although direct evidence of dimerization has not been reported in cell lines or platelets. OBJECTIVES: To investigate oligomerization of GPVI in transfected cell lines and in platelets under non-stimulated conditions. METHODS AND RESULTS: By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extracellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerization of GPVI under these conditions. A chemical crosslinker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the Fc receptor (FcR) gamma-chain. CONCLUSIONS: The present results directly demonstrate GPVI-FcR gamma-chain oligomerization at the surface of the platelet, and thereby add to the growing evidence that oligomerization of GPVI may be a prerequisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.
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Biopolímeros/metabolismo , Plaquetas/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Secuencia de Bases , Línea Celular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , HumanosRESUMEN
B-Myb is an essential transcription factor involved in control of the cell cycle and the regulation of tissue-specific gene expression in a wide range of cell types. Loss of both alleles results in early embryonic lethality at E4.5-6.5. To address the function of B-Myb in later stages of embryogenesis and in specific adult tissues, a floxed B-myb allele (B-mybF) was generated. Cre-mediated deletion in vivo was demonstrated by breeding with a transgenic GATA-Cre mouse line. An intermediate allele produced in the creation of the floxed allele, in which the PGK-neo(R) cassette is present in intron 3 (B-myb(loxneo)), was deduced to be a weak hypomorph based on the later embryonic death of homozygotes compared to B-myb(-/-) embryos. To demonstrate the efficiency and possible consequences of B-myb inactivation, we performed conditional deletion in cultured MEFs and observed decreased growth that correlated with aberrant nuclear DNA replication.
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Proteínas de Ciclo Celular/genética , Recombinación Genética , Factores de Transcripción/genética , Alelos , Animales , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Células Cultivadas , Cruzamientos Genéticos , Replicación del ADN , Genotipo , Homocigoto , Intrones , Ratones , Ratones Noqueados , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVES: GPIIb integrin expression has been found on platelets and megakaryocytes, and more recently on immature hematopoietic progenitors. We set out to investigate expression of GPIIb in other hematopoietic cell lineages and, having detected it on mast cells, aimed to determine what possible role it might perform. METHODS: We have made use of cultured human and murine bone marrow mast cells (BMMC) in order to characterize the expression of GPIIb. Further, BMMC cultures from wild type and GPIIb deficient (gpIIb-/-) mice were used for comparison of the adhesive properties mediated by this receptor. Finally, peritoneal mast cells were analyzed from both wild type and (gpIIb-/-) mice. RESULTS: We demonstrate expression of GPIIb on cultured BMMC. Using cells derived from mice homozygous for a null allele of gbIIb we show that the absence of GPIIb has no effect on mast cells with respect to a number of measures of cell growth and differentiation. However, loss of GPIIb on BMMC results in an increase in surface expression of aV integrin, the alternative partner of GPIIIa. CONCLUSION: The results in this study demonstrate that GPIIb is expressed in human and murine mast cells. A function for GPIIb on mast cells is suggested by the altered adhesion of gbIIb-/- BMMC to fibronectin- and vitronectin-coated surfaces. Moreover, comparison of mast cells from the peritoneal cavity of wild type and gbIIb-/- mice indicates that GPIIb could influence the in vivo differentiation or homing of tissue mast cells.
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Mastocitos/citología , Glicoproteína IIb de Membrana Plaquetaria/genética , Glicoproteína IIb de Membrana Plaquetaria/fisiología , Animales , Células de la Médula Ósea/citología , Adhesión Celular , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Fibronectinas/metabolismo , Humanos , Integrina alfaV/análisis , Integrina alfaV/genética , Mastocitos/química , Ratones , Ratones Noqueados , Glicoproteína IIb de Membrana Plaquetaria/análisis , Vitronectina/metabolismoRESUMEN
There is extensive evidence that FcR gamma-chain couples to the collagen receptor glycoprotein VI (GPVI) and becomes phosphorylated on tyrosines upon receptor cross-linking. However, it is not established whether this receptor complex is sufficient to initiate the signalling cascade. We transfected GPVI and the FcR gamma-chain into the human erythroleukaemia cell line K562, which lacks detectable expression of GPVI and the FcR gamma-chain. The results show that GPVI is unable to signal when expressed alone, despite its surface expression, upon stimulation with the snake C-type lectin, convulxin. Coexpression of the FcR gamma-chain confers signalling properties on the receptor. Furthermore, cotransfection of the FcR gamma-chain and two mutant versions of GPVI shows that the transmembrane arginine and cytoplasmic tail of GPVI are necessary for association with the FcR gamma-chain. These results demonstrate that reconstitution of the GPVI-FcR gamma-chain complex in cells expressing the necessary signalling network is sufficient to initiate signalling events in response to convulxin and collagen-related peptide.