Prognostic value of numerical chromosome aberrations in multiple myeloma: A FISH analysis of 15 different chromosomes

Este trabajo proporcionó la primera idea de que la deleción del Cr13 tiene valor pronóstico pe-yorativo en MM. Proporcionó las primeras pistas del valor de la FISH en MM, que sigue siendo la herramienta fundamental para evaluar el pronóstico en estos pacientes en todo el mundo[EN]Recent observations indicate that chromosome aberrations are important prognostic factors in patients with multiple myeloma (MM) treated with high-dose chemotherapy. Nevertheless, the inherent problems of conventional cytogenetics have hampered the systematic evaluation of this parameter in series of patients treated with conventional chemotherapy. Fluorescence in situ hybridization (FISH) analysis is an attractive alternative for evaluation of numerical chromosomal changes. In the present study, we analyze the relationship between aneuploidies of 15 different chromosomes assessed by FISH and prognosis in a series of 63 patients with MM treated with conventional chemotherapy. After a median follow-up of 61 months (range, 6 to 109), 49% of patients are still alive with a median survival of 33 months. The overall incidence of numerical chromosome abnormalities was 70%. This incidence significantly increased when seven or more chromosomes were analyzed (53 patients), reaching 81%. Trisomies of chromosomes 6, 9, and 17 were associated with prolonged survival (P = .033, P = .035, and P = .026, respectively); by contrast, overall survival (OS) was lower in cases with monosomy 13 (as assessed by deletion of Rb gene, P = .0012). From the clinical point of view, loss of Rb gene was associated with a poor performance status; low hemoglobin levels; high creatinine, C-reactive protein, and lactic dehydrogenase serum levels; high percentage of bone marrow plasma cells (BMPC); extensive bone lytic lesions; and advanced clinical stage. Other chromosome abnormalities such as trisomy of chromosome 9 and 17 were associated with good prognostic features including high hemoglobin levels, early clinical stage, beta2microglobulin less than 6 micro/mL, and low percentage of BMPC. A multivariate analysis for OS showed that S-phase PC greater than 3% (P = .010) and beta2microglobulin serum levels greater than 6 micro/mL (P = .024), together with monosomy of chromosome 13 (P = .031) and nontrisomy of chromosome 6 (P = .048) was the best combination of independent parameters for predicting survival in patients with MM. According to these results, chromosomal analysis is of great use in patients with MM at diagnosis to have a correct prognostic evaluation for clinical decision making.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

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

A new staging system for multiple myeloma based on the number of S-phase plasma cells

Fue el que primer artículo que dio origen a la exitosa línea de investigación en mieloma del grupo de Salamanca, y por añadidura, del Grupo español de mieloma. Aplicó una nueva metodología diseñada por el grupo de Salamanca para evaluar el pronóstico de los pacientes y lo aplicó con éxito a una serie de 156 enfermos con esta patología. Este trabajo permitió desarrollar una patente de la USAL que se comercializó con la empresa Cytognos (Cycloscope™ MM) para estudiar el ADN de células plasmáticas en Gammapatías Monoclonales. https://www.cytognos.com/products/cyt-cs-138-38[EN]In the present study, we analyzed the cell cycle distribution of bone marrow (BM) cells in 120 untreated multiple myeloma patients using a DNA/CD38 double-staining technique at flow cytometry in which plasma cells (PCs) can be clearly discriminated from residual BM cells based on their CD38 expression. This approach allows us to determine the proliferative activity of both PCs and residual normal BM cells. The percentage of S-phase cells in the myelomatous population was found to be significantly lower than that of the residual normal BM cells (P 3%) showed a significantly (P < .05) increased incidence of anemia and hypercalcemia; higher values of beta 2-microglobulin (beta 2M), urea, and creatinine; and higher numbers of peripheral blood natural killer cells, as well as a poor prognosis as assessed both by response duration and overall survival. With respect to the residual BM normal fraction, a low proliferative activity was significantly (P < .05) associated with the presence of anemia and neutropenia together with increased numbers of BM PCs, a higher incidence of Bence Jones myelomas, and DNA diploidy. Multivariate analysis showed that the number of S-phase PCs was the most important independent prognostic factor, allowing us to discriminate two subgroups of patients with different prognoses, even within the same clinical stage. Moreover, the S-phase PCs, together with beta 2M, age, and performance status, represent the best combination of disease characteristics for stratifying patients according to prognosis and allow the establishment of a simple and powerful staging system for multiple myeloma patients. In addition, this classification can be used for planning treatment in patients who are candidates for transplantation.Castellano-Leonés Group of Monoclonal Gammopathies University Hospital of Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Serum lactate dehydrogenase level as a prognostic factor in Hodgkin's disease

Este trabajo permitió consolidar la relevancia de la LDH como factor pronóstico en el linfoma de Hodgkin, algo utilizado después para predecir el pronóstico de estos pacientes en nuestro servicio durante muchos años. Además, sirvió para iniciar la carrera del autor 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 trasplante Haploidéntico y el diseño de los ensayos clínicos más relevantes sobre esta patología en España[EN]The efficacy of currently available treatments for Hodgkin's disease (HD) has led to a substantial modification in the prognosis of this disease; nevertheless there is still a group of patients that cannot be cured with conventional treatments and who will be candidates for alternative therapy. In the present work we analysed the prognostic influence of the most relevant clinico-biological characteristics of HD in a consecutive series of 137 patients diagnosed and treated in a single institution. Univariate analyses identified six variables with significant prognostic influence, both on achieving complete remission (CR) and overall survival (OS); LDH > 320 U ml-1, age > 45 years, stages IIB, III and IV, extranodal involvement, alkaline phosphatase > 190 UI dl and ESR > 40 mm h. In addition, Hb or = 2) affected a lower survival. In the multivariate analysis only LDH, age and the clinical stage retained a significant prognostic influence for achieving CR, while the two first factors above, together with performance status were the variables with independent prognostic value with respect to OS. Moreover, only LDH > 320 U ml-1 had prognostic influence in the probability of relapse and disease free survival (DFS), both in the univariate and multivariate analyses. According to the three independent factors obtained in the multivariate analysis for CR (LDH, age and stage) a predictive model was established that allows the stratification of patients into two prognostic groups: one with poor prognosis that includes patients with the three adverse prognostic factors, or two if one of them was elevated LDH, and the other with good prognosis that includes the remaining patients. This model was also able to separate two independent groups of patients with respect to OS and to DFS. In conclusion, the present study shows that LDH is one of the most important prognostic factors in HD.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Primary plasma cell leukemia: clinical, immunophenotypic, DNA ploidy, and cytogenetic characteristics

Ha sido y sigue siendo una de las principales referencias en leucemia de células plasmáticas, con múltiples citaciones, incluyendo libros de texto en medicina interna tan prestigiosos como el Harrison.[EN]We report on a series of 26 patients diagnosed with primary (de novo) plasma cell (PC) leukemia (PCL) in whom we analyzed the clinicobiologic characteristics of the disease together with the immunophenotype, DNA cell content, proliferative index, and numeric chromosomal aberrations of the neoplastic PC, and compared them with 664 multiple myeloma (MM) patients at diagnosis. The median age, sex ratio, and bone lesion extension were similar, but PCL cases displayed a higher prevalence of clinical stage III, extramedullary involvement, and Bence Jones cases, with fewer IgA cases than for MM patients. In addition, according to several prognostic indicators (beta2-microglobulin serum level, proportion of S-phase PCs, proteinuria, calcium serum level, lactate dehydrogenase [LDH] and renal function), the incidence of adverse prognostic factors was significantly higher in PCL versus MM. Immunophenotypic expression was similar for CD38, CD138, CD2, CD3, CD16, CD10, CD13, and CD15, but PCL differed from MM in the expression of CD56, CD9 HLA-DR, CD117, and CD20 antigens. Twenty-two PCL cases were diploid and one was hypodiploid, while most MM cases (57%) showed DNA hyperdiploidy. With the fluorescent in situ hydridization (FISH) technique, 12 of 13 PCL cases displayed the numeric aberrations, -13 (86%), +/-1 (57%), +18 (43%), and -X in women (25%), but they lacked several numeric aberrations usually found in MM such as +3, +6, +9, +11, and +15. PCL cases had a lower overall response to therapy than MM cases (38% v 63%, P =.01332). Among PCL patients, a trend for a worse response was observed in cases treated with melphalan and prednisone (MP) versus polychemotherapy. Overall survival was significantly worse in PCL versus MM patients (8 v 36 months, P <.0001), but it was significantly better in PCL patients treated with polychemotherapy versus MP (18 v 3 months, P =.0137). By contrast, MM patients did not show significant differences in overall survival according to the treatment used, MP or polychemotherapy. Ten variables seemed to predict survival in PCL patients, but only the beta2-microglobulin level and S-phase PCs retained an independent value in multivariate analysis. In summary, our study illustrates that PCs from PCL display singular phenotypic, DNA cell content, and cytogenetic characteristics that lead to a different disease evolution versus MM.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

Methylation is an inactivating mechanism of the p16 gene in multiple myeloma associated with high plasma cell proliferation and short survival

[EN]In order to gain further insights into the role of the p16 gene in cell cycle regulation and the prognostic implications of its inactivation, we investigated the methylation status of the p16 gene in 98 untreated patients using a polymerase chain reaction assay based on the inability of some restriction enzymes to digest methylated sequences. Forty-one patients showed a p16 methylated gene (42%). The percentage of S-phase plasma cells (PC) in these patients was almost three times higher than in those with an unmethylated p16 gene (4Æ16% ± 3Æ37% vs 1Æ5% ± 1Æ41%, P < 0Æ001). The presence of p16 methylation also correlated with both elevated b2-microglobulin serum levels and high C-reactive protein values. Patients with a p16 methylated gene had shorter overall and progression-free survival than those patients without p16 methylation. However, this feature did not retain independent prognostic influence on multivariate analysis, probably due to its association with the S-phase PC, which had more potent statistical significance in the Cox model. These findings showed methylation of the p16 gene was a frequent event in MM patients at diagnosis, and was associated with an increased proliferative rate of plasma cells and a poor prognosis, indicating an important role for p16 gene in the cell cycle regulation of multiple myeloma tumour cells, and thus in the clinical outcome of the disease

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

Prognostic implications of DNA aneuploidy in 156 untreated multiple myeloma patients

Fue el primer artículo que, en contra de lo esperado, incidió en el buen pronóstico de los pacientes con mieloma hiperdiploide, hecho confirmado posteriormente. Fue uno de los primeros artículos que consolidó los primeros pasos de la exitosa línea de investigación en mieloma del grupo de Salamanca, y por añadidura, del Grupo español de mieloma.[EN]In this study the incidence of DNA aneuploidy in a large series of untreated multiple myeloma (MM) patients was assessed in order to determine its clinical and prognostic significance. A total of 156 MM patients were included in the study. DNA measurements were performed in all cases at diagnosis using two different flow cytometry methods: (1) propidium iodide (PI) staining on isolated nuclei, and (2) CD38/PI double staining on whole cells. The DNA ploidy status was correlated with the most relevant clinical and haematological disease characteristics. From the 156 cases analysed, 91 (58%) were aneuploid (56% hyperdiploid and 2% hypodiploid). The correlation between the two techniques on the detection of DNA aneuploidy was excellent, although CD38/PI double staining would be preferable in cases with < 5% of DNA aneuploid plasma cells (PC). Upon comparing the clinical and haematological disease characteristics of hyperdiploid versus diploid cases, the former group was characterized by a lower age, reduced incidence of anaemia, lower beta 2M levels, higher proliferative activity within the residual normal haemopoietic cells, increased expression of CD56 antigen in PC, and higher proportion of PB CD4+ T cells. In contrast, diploid cases had a higher expression of the CD10, CD20 and CD15 antigens and greater numbers of PB CD56+CD3- NK cells (P < 0.05). Circulating PC were identified in six cases, all being diploid. Overall survival was significantly longer in hyperdiploid compared to diploid MM (P = 0.02). These results show that over 50% of MM patients are aneuploid, almost all of them being hyperdiploid. This characteristic is associated with better prognosis.Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

The oral combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex) is effective in relapsed/refractory multiple myeloma

Fue el trabajo que ha permitido tratar con éxito a muchos pacientes con mieloma múltiple resis-tente. Ha sido citado múltiples veces y es una referencia esencial en el tratamiento con asocia-ciones de inmunomoduladores y alquilantes Fue utilizado por el grupo inglés como referencia para el ensayo MRC IX, que incluyó casi 2000 pacientes.[EN]We evaluate the efficacy of the oral combination of thalidomide, cyclophosphamide and dexamethasone (ThaCyDex) in 71 refractory/relapsed multiple myeloma patients, including a prognostic analysis to predict both response and survival. Patients received thalidomide at escalating doses (200-800 mg/day), daily cyclophosphamide (50 mg/day) and pulsed dexamethasone (40 mg/day, 4 days every 3 weeks). On an intention-to-treat basis and using the EBMT response criteria, 2% patients reached complete response (CR), 55% partial response (PR) and 26% minor response (MR) yielding a total response (CR+PR+MR) rate of 83% after 3 months of therapy. After 6 months of therapy, responses were maintained including a 10% CR. The 2-year progression free and overall survival were 57 and 66%, respectively. A favorable response was associated with beta2 microglobulin 80 x 10(9)/l and nonrefractory disease. Regarding survival, low beta2 microglobulin (< or =4 mg/dl), age (< or =65 years) and absence of extramedullary myelomatous lesion were associated with a longer survival. Major adverse effects included constipation (24%), somnolence (18%), fatigue (17%) and infection (13%). Only 7% of patients developed a thrombo-embolic event. ThaCyDex is an oral regimen that induces a high response rate and long remissions, particularly in relapsing patients with beta2 microglobulin < or =4 mg/dl and < or =65 years.Grupo Español de Mieloma (GEM/PETHEMA) Hospital Universitario de Salamanca Universidad de SalamancaHospital Universitario de Salamanc

MYD88 L265P is a marker highly characteristic of, but not restricted to, Waldenström's macroglobulinemia

Evaluamos la mutación MYD88 L265P en la macroglobulinemia de Waldenström (WM) y los tras-tornos linfoproliferativos de células B mediante reacción en cadena de la polimerasa específica (PCR) (sensibilidad ∼10(-3)). No se observó mutación en donantes normales, mientras que estuvo presente en 101/117 (86%) pacientes con WM, 27/31 (87%) gammapatías monoclonales IgM de significado incierto (GMSI), 3/14 (21%) linfomas esplénicos de la zona marginal y 9/48 (19%) lin-fomas difusos de células B grandes (DLBCL) del centro no germinal (GC). La mutación estuvo ausente en los 28 DLBCL GC, 13 DLBCL no subclasificados, 35 leucemias de células pilosas, 39 leucemias linfocíticas crónicas (16 con componente M), 25 IgA o IgG-GMSI, 24 mieloma múltiple (3 con un isotipo IgM), 6 amiloidosis, 9 linfomas linfoplasmocíticos y 1 neuropatía relacionada con IgM. Entre las WM y las IgM-GMSI, la mutación MYD88 L265P se asoció con algunas diferencias en las características clínicas y biológicas, aunque por lo general menores; Los casos de MYD88 de tipo salvaje tuvieron menor componente M (1,77 vs. 2,72 g/dl, p=0,022), más linfocitosis (24 vs. 5%, p=0,006), mayor nivel de lactato deshidrogenasa (371 vs. 265 UI/L, p=0,002), inmunofe-notipo atípico (CD23-CD27+ +FMC7++), menos hipermutación somática del gen de la cadena pesada de inmunoglobulina variable (IGHV) (57 vs. 97%, p=0,012) y menor selección del gen IGHV3-23 (9 vs. 27%, P=0,014). Estas pequeñas diferencias no condujeron a diferencias en el tiempo transcurrido hasta el primer tratamiento, la respuesta al tratamiento o la supervivencia libre de progresión o global. Ramón García-Sanz fue el INVESTIGADOR SENIOR de este trabajo, responsable de la idea ini-cial, obtención de la financiación, diseño del estudio y de los experimentos, análisis estadístico, revisión bibliográfica definitiva y redacción definitiva del artículo. La Licenciada Jiménez preparó las muestras, llevó a cabo los experimentos de PCR y secuenciación, recogió los datos y llevó a cabo su análisis para ver la asociación con la clínica. Fue responsable del primer borrador. La Dra. Sebastián aportó la casuística en LNH difuso de célula grande. La licenciada Balanzategui ayudo de forma directa en los estudios moleculares. El resto de los autores participó en el trabajo aportando casuística, experimentos paralelos, datos, y revisión del texto, en especial el Dr. San Miguel y el Dr. González, que revisaron el texto en profundidad. El trabajo ha sido citado en 49 ocasiones en otras revistas internacionales y es un trabajo conti-nuamente citado en foros internacionales. Fue y sigue siendo uno de los trabajos más relevantes en detección de mutaciones en Macroglobulinemia de Waldenström, en especial porque es el que mayor casuística aporta y el que definió definitivamente que la mutación MYD88 está presente no sólo en MW sino también en gammapatía monoclonal de significado incierto.[EN]We evaluated the MYD88 L265P mutation in Waldenström's macroglobulinemia (WM) and B-cell lymphoproliferative disorders by specific polymerase chain reaction (PCR) (sensitivity ∼10(-3)). No mutation was seen in normal donors, while it was present in 101/117 (86%) WM patients, 27/31 (87%) IgM monoclonal gammapathies of uncertain significance (MGUS), 3/14 (21%) splenic marginal zone lymphomas and 9/48 (19%) non-germinal center (GC) diffuse large B-cell lymphomas (DLBCLs). The mutation was absent in all 28 GC-DLBCLs, 13 DLBCLs not subclassified, 35 hairy cell leukemias, 39 chronic lymphocytic leukemias (16 with M-component), 25 IgA or IgG-MGUS, 24 multiple myeloma (3 with an IgM isotype), 6 amyloidosis, 9 lymphoplasmacytic lymphomas and 1 IgM-related neuropathy. Among WM and IgM-MGUS, MYD88 L265P mutation was associated with some differences in clinical and biological characteristics, although usually minor; wild-type MYD88 cases had smaller M-component (1.77 vs 2.72 g/dl, P=0.022), more lymphocytosis (24 vs 5%, P=0.006), higher lactate dehydrogenase level (371 vs 265 UI/L, P=0.002), atypical immunophenotype (CD23-CD27+ +FMC7+ +), less Immunoglobulin Heavy Chain Variable gene (IGHV) somatic hypermutation (57 vs 97%, P=0.012) and less IGHV3-23 gene selection (9 vs 27%, P=0.014). These small differences did not lead to different time to first therapy, response to treatment or progression-free or overall survival.University Hosptial of SalamancaHospital Universitario de Salamanc