Immunophenotypic characterization of plasma cells from monoclonal gammopathy of undetermined significance patients. Implications for the differential diagnosis between MGUS and multiple myeloma

Es el artículo seminal para distinguir entre célula plasmática normal y patológica en las gamm-patías monoclonales.[EN]Although the immunophenotype of plasma cells (PCs) from multiple myeloma (MM) patients has been extensively explored, information on the phenotypic characteristics of PCs in monoclonal gammopathy of undetermined significance (MGUS) patients is scanty and frequently controversial. Thus, the question of whether or not PCs are phenotypically different in the two disorders and whether this criteria could be useful for the differential diagnosis between MGUS and MM remains to be explored. In the present study, the immunophenotypic profile of bone marrow PCs (BMPCs) from a group of 76 MGUS patients has been analyzed by flow cytometry and compared with that of BMPCs present in both MM patients (n = 65) and control subjects (n = 10). For that purpose, a large panel of monoclonal antibodies against PC-related antigens was used together with a sensitive methodology in which a minimum of 10(3) PCs were studied. In all MGUS cases studied, two clearly defined and distinct PC subpopulations could be identified. One PC subpopulation, population A (33 +/- 31% of total PCs), constantly displayed a high CD38 expression with low forward light scatter (FSC)/side light scatter (SSC) and was positive for CD19 and negative for CD56 (only a small proportion of these PCs were weakly positive for CD56). The other PC subpopulation, population B (67 +/- 31% of total PCs), showed the opposite pattern; the antigen CD56 was strongly positive and CD19 was constantly negative, and it showed a lower CD38 expression and higher FSC/SSC values than population A. Clonality studies (cytoplasmic light chain restriction, DNA content studies, and polymerase chain reaction assessment) confirmed the clonal nature of PCs from population B and the polyclonal origin of PCs from population A. Moreover, the polyclonal PCs from MGUS displayed a phenotypic profile identical to that found in PCs from healthy individuals. By contrast, clonal PCs from all MGUS patients displayed a similar antigenic profile to myelomatous PCs, with clear phenotypic differences with respect to normal PCs: lower intensity of CD38 expression and a variable reactivity for markers that were not expressed in normal PCs, such as CD28, CD117, and sIg. Although the presence of residual polyclonal PCs was a constant finding in MGUS patients, it was a rare event in MM and, when present (only 22% of MM cases), its frequency was significantly lower than that observed in MGUS (0.25% versus 32.9%, respectively; P < 0.0001). Only 1.5% of patients with MM had more than 3% of normal PCs, whereas 98% of patients with MGUS had more than 3%. Moreover, as shown by multivariate analysis, the number of residual polyclonal PCs was the most powerful single parameter for the discrimination between MGUS and MM patients at diagnosis, even when only stage I MM cases were considered.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Zalypsis has in vitro activity in acute myeloid blasts and leukemic progenitor cells through the induction of a DNA damage response.

Background: Although the majority of patients with acute myeloid leukemia initially respond to conventional chemotherapy, relapse is still the leading cause of death, probably because of the presence of leukemic stem cells that are insensitive to current therapies. We investigated the antileukemic activity and mechanism of action of zalypsis, a novel alkaloid of marine origin. Design and methods: The activity of zalypsis was studied in four acute myeloid leukemia cell lines and in freshly isolated blasts taken from patients with acute myeloid leukemia before they started therapy. Zalypsis-induced apoptosis of both malignant and normal cells was measured using flow cytometry techniques. Gene expression profiling and western blot studies were performed to assess the mechanism of action of the alkaloid. Results: Zalypsis showed a very potent antileukemic activity in all the cell lines tested and potentiated the effect of conventional antileukemic drugs such as cytarabine, fludarabine and daunorubicin. Interestingly, zalypsis showed remarkable ex vivo potency, including activity against the most immature blast cells (CD34(+) CD38(-) Lin(-)) which include leukemic stem cells. Zalypsis-induced apoptosis was the result of an important deregulation of genes involved in the recognition of double-strand DNA breaks, such as Fanconi anemia genes and BRCA1, but also genes implicated in the repair of double-strand DNA breaks, such as RAD51 and RAD54. These gene findings were confirmed by an increase in several proteins involved in the pathway (pCHK1, pCHK2 and pH2AX). Conclusions: The potent and selective antileukemic effect of zalypsis on DNA damage response mechanisms observed in acute myeloid leukemia cell lines and in patients' samples provides the rationale for the investigation of this compound in clinical trials.Funding: this work was supported by a grant from the Ministry of Science and Innovation of Spain (BFU2006-01813/BMC and RD06/0020/ 0041).The CIC receives support from the European Community through the regional development funding program (FEDER).This work was also supported by the ‘AcciónTransversal del Cáncer’ project, through an agreement between the Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Science and Innovation, and the Cancer Research Foundation of Salamanca University. Our group also receives support from the Junta de Castilla y Léon through ‘Ayudas destinadas a financiar programas de actividad investigadora a realizar por grupos de investigación de excelencia de Castilla y León’

New criteria to identify risk of progression in monoclonal gammopathy of uncertain significance and smoldering multiple myeloma based on multiparameter flow cytometry analysis of bone marrow plasma cells

[EN] Monoclonal gammopathy of uncertain significance (MGUS) and smoldering multiple myeloma (SMM) are plasma cell disorders with a risk of progression of approximately 1% and 10% per year, respectively. We have previously shown that the proportion of bone marrow (BM) aberrant plasma cells (aPCs) within the BMPC compartment (aPC/BMPC) as assessed by flow cytometry (FC) contributes to differential diagnosis between MGUS and multiple myloma (MM). The goal of the present study was to investigate this parameter as a marker for risk of progression in MGUS (n = 407) and SMM (n = 93). Patients with a marked predominance of aPCs/BMPC (> or = 95%) at diagnosis displayed a significantly higher risk of progression both in MGUS and SMM (P or = 95%) as the most important independent variable, together with DNA aneuploidy and immunoparesis, for MGUS and SMM, respectively. Using these independent variables, we have identified 3 risk categories in MGUS (PFS at 5 years of 2%, 10%, and 46%, respectively; P< .001) and SMM patients (PFS at 5 years of 4%, 46%, and 72%, respectively; P < .001). Our results show that multiparameter FC evaluation of BMPC at diagnosis is a valuable tool that could help to individualize the follow-up strategy for MGUS and SMM patients

Minimal residual disease in adolescent (older than 14 years) and adult acute lymphoblastic leukemias: early immunophenotypic evaluation has high clinical value

Se trata de un trabajo que demostró por primera vez que la citometría de flujo es una excelen-te metodología para analizar la enfermedad residual mínima en la leucemia aguda linfoblástica. Hasta entonces sólo se había usado la PCR alelo-específica y a partir de ahí se dio pie a su uso en muchos ensayos, algo que ha ocurrió de forma sistemática en nuestro país desde la publicación este trabajo.[EN]Investigation of minimal residual disease (MRD) in acute leukemias by immunophenotyping and/or molecular techniques is proving to be increasingly valuable for disease monitoring. In acute lymphoblastic leukemia (ALL), most MRD studies have focused on children, whereas in contrast, information on the value of MRD on adult ALL is scanty, and almost exclusively restricted to polymerase chain reaction (PCR) studies. Early response to therapy is one of the most important prognostic factors in acute leukemia, which prompted us to investigate whether or not early immunophenotypic assessment of MRD could also be a valuable tool for predicting relapse in adult patients with ALL. For that purpose we have analyzed the level of MRD during the initial phase of treatment (induction phase) by multiparameter flow cytometry in a series of 102 adolescent (older than 14 years) and adult patients with ALL. Immunophenotypic evaluation of the bone marrow (BM) at day +35 showed that patients with low MRD levels (< 0.05% leukemia-associated phenotype [LAP+] cells) had a significantly longer relapse-free survival (RFS) than patients with high MRD levels, and this prognostic influence was retained when only those patients in morphologic complete remission (mCR) at day +35 were considered (median RFS: 42 months vs 16 months; P =.001). Moreover, immunophenotyping helped to identify a small subset of patients (n = 12) with negative or low MRD levels (< 0.03% LAP+ cells) by day +14, with an excellent prognosis (projected RFS of 90% at 5 years). The contrary is true of patients who achieved late mCR (after day +35), since immunophenotypic investigation of MRD showed that, in spite of the mCR, none of the cases with more than 0.1% LAP+ cells would be relapse-free after 2 years. Multivariate analysis showed that the immunologic evaluation of MRD at day +35 was the most relevant independent prognostic parameter for adult patients with ALL, and together with age, white blood cell (WBC) count at diagnosis, and presence of the Philadelphia (Ph) chromosome, represented the most informative combination of variables for predicting relapse-free survival.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique

Este trabajo generó la validación de una técnica para estudiar la fase de síntesis de las células plasmáticas en mieloma múltiple y gammapatías monoclonales de significado incierto. Su desarrollo permitió luego aplicar la técnica a una serie de pacientes con mieloma múltiple y determinar su valor pronóstico. Fue la base de la tesis del Dr. García-Sanz permitiendo iniciar una carrera y una línea de investigación del Servicio de hematología que actualmente ha permitido a dicho servicio ser la referencia nacional en esta enfermedad, incluyendo tanto el desarrollo del programa de trasplante, los ensayos clínicos y la línea de citometría de flujo del servicio.[EN]In the present paper a CD38/propidium iodide double staining technique is described which separately assesses the cell cycle distribution of myelomatous plasma cells from that of the residual normal hemopoietic cells. For this purpose, bone marrow (BM) cells from a group of 42 untreated multiple myeloma patients were analyzed. Of these, 23 cases were aneuploid (55%) and 19 diploid (45%). The use of the CD38/propidium iodide double staining method allowed a clear separation between CD38 strong positive cells from the remaining bone marrow populations, cell sorting experiments confirming that plasma cells were almost exclusively contained in the former fraction where they represented 97 +/- 2% of the total cells sorted. In all cases, the S-phase in plasma cells and in the remaining normal hemopoietic bone marrow cells was assessed, being higher in normal hemopoietic cells (8.0 +/- 6.3%) than in plasma cells (3.3 +/- 2.6%, P 0.10); this work therefore shows that the assessment of the total proliferative rate of bone marrow samples does not reflect either the proliferation of normal cells or that of neoplastic plasma cells but will depend on the proliferative rate and the percentage of each population within the sample, which can be assessed by the technique described here.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc