RESUMEN
INTRODUCTION: The evaluation of myeloma cells in multiple myeloma (MM) patients has generally been limited to the assessment of bone marrow involvement because of the sensitivity limitations of traditional minimal-residual-disease-detection methods. MATERIALS AND METHODS: We developed a sequencing-based method to identify myeloma cells in bone marrow (BM) and peripheral blood (PB) samples, based on their unique immunoglobulin gene rearrangements, that can detect cancer clones at levels well below 1 in 1 million leukocytes (0.0001%). In this multisite study, we used this sequencing method to determine the fraction of patients with myeloma cells in their PB at diagnosis and posttreatment time points. RESULTS: Using this sequencing approach, we detected myeloma cells in the PB in the vast majority of MM patients (44/46, 96%). We demonstrated a clear correlation (R(2) = 0.57) between myeloma clone levels in paired BM and PB samples, and noted that PB clone levels were approximately 100-fold lower than levels in BM samples. The sequencing assay demonstrated a clear sensitivity advantage in the BM compartment and at least equivalent sensitivity in the PB compared with that of monoclonal-protein results. CONCLUSION: This study highlights the promise of a blood-based, sequencing minimal-residual-disease assay that can be used to measure MM disease burden at different time points and various disease stages.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mieloma Múltiple/sangre , Mieloma Múltiple/genética , Células Neoplásicas Circulantes/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/metabolismo , Médula Ósea/patología , Células Clonales/metabolismo , Células Clonales/patología , Femenino , Citometría de Flujo , Reordenamiento Génico de Linfocito B , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Masculino , Persona de Mediana Edad , Mieloma Múltiple/diagnóstico , Proteínas de Mieloma/metabolismo , Neoplasia Residual/sangre , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Células Neoplásicas Circulantes/patología , Sensibilidad y Especificidad , Recombinación V(D)J/genéticaRESUMEN
BACKGROUND: Development and progression of multiple myeloma is dependent on the bone marrow (BM) microenvironment, and within the BM, a number of factors are secreted, including the Wnt ligands. Bone marrow stromal cells (BMSC) secrete Wnt ligands that activate Wnt signaling in multiple myeloma. The canonical Wnt pathway which is mediated through the transcriptional effector ß-catenin (ß-cat) is commonly de-regulated in many cancers. Cells with active ß-cat-regulated transcription (CRT) are protected against apoptosis; conversely, inhibition of CRT may prevent cell proliferation. MATERIALS AND METHODS: In this study, we tested the efficacy of recently described inhibitors of CRT (iCRTs; oxazole and thiazole) for their selective antagonistic effect on Wnt-ß-cat response in MM cells MM.1, U266, BMSC and primary BMMC obtained from patient samples (n=16). RESULTS: We demonstrated that iCRTs we used, block Wnt/ß-cat reporter activity, down regulate ß-cat expression and inhibit cell proliferation in a dose-dependent manner with an optimal dose closer to 15 µM. Our data further indicate that iCRTs do not influence the expression of the upstream components of the Wnt pathway DKK1 at the optimal dose, suggesting that iCRTs may specifically target ß-cat in MM cells. Additionally, iCRT-treatment of MM cells, co-cultured with BMSC, showed an inhibitory effect on VEGF and cell migration. CONCLUSION: This study provides the first in vitro data evaluation of newly-described iCRTs as potential Wnt-ß-cat/VEGF pathway antagonists in multiple myeloma.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Mieloma Múltiple/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Wnt/antagonistas & inhibidores , beta Catenina/antagonistas & inhibidores , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Oxazoles/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Tiazoles/farmacología , Transfección , Factor A de Crecimiento Endotelial Vascular/biosíntesisRESUMEN
Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exact biological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavily disputed, since data supporting both views have been reported in several independent studies. To clarify this issue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the present study, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis in thymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and after induction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering of apoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptotic processes. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expression was restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolytic cleavage site and is therefore predicted to encode a monomeric protein. The biological function under normal circumstances, however, will need further investigations for clarification. While apoptosis could not modulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted in up-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control of cell activation-mediated rather than apoptosis-induced signals.