6q deletion in Waldenström macroglobulinemia is associated with features of adverse prognosis.

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenström macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of beta2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q- among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series.

Clonal plasma cells from monoclonal gammopathy of undetermined significance, multiple myeloma and plasma cell leukemia show different expression profiles of molecules involved in the interaction with the immunological bone marrow microenvironment.

The immunological bone marrow (BM) microenvironment plays a major role in controlling growth and survival of clonal plasma cells (PC); this might translate into different patterns of expression of molecules involved in immune responses on PC from different types of monoclonal gammopathies (MG). We have studied the expression of a group of nine such molecules on both BMPC and the plasma of 61 newly diagnosed MG patients (30 MG of undetermined significance (MGUS), 27 multiple myeloma (MM) and four plasma cell leukemia (PCL)) and five normal individuals. Clonal PC from all MG displayed significantly increased levels of CD56, CD86 and CD126, and decreased amounts of CD38 (P<0.001). Additionally, HLA-I and beta2-microglobulin were abnormally highly expressed in MGUS, while CD40 expression was decreased in MM and PCL (P<0.05). Interestingly, a progressive increase in the soluble levels of beta2-microglobulin was found from MGUS to MM and PCL patients (P=0.03). In contrast, all groups showed similar surface and soluble amounts of CD126, CD130 and CD95, except for increased soluble levels of CD95 observed in PCL. Overall, those phenotypic differences are consistent with increased antigen presentation and costimulatory capacities in MGUS, which progressively deteriorate in malignant MG (MM and PCL).

Interaction between clonal plasma cells and the immune system in plasma cell dyscrasias.

The term "monoclonal gammopathy" (MG) includes a group of clonal plasma cell disorders, which show heterogeneous clinical behavior. While multiple myeloma (MM) and plasma cell leukemia (PCL) are incurable malignant diseases, most patients with MG of undetermined significance (MGUS) show an indolent/benign clinical course. Evidence has accumulated which supports the role of the bone marrow microenvironment in MG. Accordingly, the survival, drug-resistance and proliferation of MM cells have been shown to be largely dependent on a supportive microenvironment. Among the different environment-associated parameters, those related to the status/activity of the immune system are particularly relevant. This review focuses on the different ways clonal plasma cells (PC) interact with the immune system in different models of MG, to characterize crucial events in the development and progression of MG. These advances may support the design of novel therapeutic approaches in patients with MG.

Immunophenotypic analysis of Waldenstrom's macroglobulinemia.

Immunophenotyping has become an essential tool for diagnosis of hematological malignancies. By contrast, for diagnosis of Waldenstrom's macroglobulinemia (WM) immunophenotyping is used only occasionally. From 150 patients with a IgM monoclonal gammopathy we have selected 60 cases with (1) morphological lymphoplasmocytoid bone marrow (BM) infiltration (>20%); (2) IgM paraprotein (>10g/L); and (3) absence of features of other lymphoma types. Immunophenotypic analysis was based on the use of the triple or quadruple monoclonal antibody (MoAb) combinations. To increase the sensitivity of the analysis of antigen expression, selected CD19(+)CD20(+) B cells were targeted. We have also explored the antigenic characteristics of both the plasma cell (PC) and mast cell (MC) compartments present in the BM from 15 WM patients. Clonal WM lymphocytes were characterized by the constant expression of pan-B markers (CD19, CD20, CD22, CD24) together with sIg, predominantly kappa (5:1, kappa:lambda ratio). A high proportion of cases (75%) were positive for FMC7 and CD25, but in contrast to hairy cell leukemia (HCL), these lymphocytes were always negative for CD103 and CD11c. CD10 antigen was also absent in all WM patients and less than one fifth of patients were positive for CD5 and CD23, while CD27, CD45RA, and BCL-2 were present in most malignant cells. In two cases, the coexistence of two different clones of B lymphocytes was identified, and in eight additional cases, intraclonal phenotypic heterogeneity was observed. As far as PCs are concerned, in most patients (85%) the number of PCs was within the normal range (median, 0.36%). The antigenic profile of these PCs differed from that observed in normal and myelomatous PC (CD38(++)CD19(++/-)CD56(-)CD45(++)CD20(+)). In three cases, PCs showed aberrant expression for CD5, CD22, or FMC7. Finally, the number of mast cells was significantly higher (0.058 +/- 0.13) as compared to normal BM (0.019 +/- 0.02) (P <.01), although they were immunophenotypically normal (CD117(+)CD2(-)CD25(-)).

Incidence of phenotypic aberrations in a series of 467 patients with B chronic lymphoproliferative disorders: basis for the design of specific four-color stainings to be used for minimal residual disease investigation.

Multiparameter immunophenotypic analysis of neoplastic cells has proven to be of great help for the investigation of minimal residual disease in acute leukemias; however, its utility has not been systematically explored in B cell chronic lymphoproliferative disorders. The aim of the present study was to investigate the incidence of phenotypic aberrations in a series of 467 consecutive leukemic B cell chronic lymphoproliferative disorders through the comparison of the phenotypic characteristics of tumor vs normal peripheral blood (n = 10) and bone marrow (n = 10) B cells, in order to explore the applicability of this strategy for minimal residual disease monitoring. An additional goal of our study was to evaluate the sensitivity of multiparameter flow cytometry for the detection of minimal residual disease in leukemic B cell chronic lymphoproliferative disorders through dilutional experiments (n = 19). From the patients analyzed 382 corresponded to B cell chronic lymphocytic leukemia/small lymphocytic lymphoma (353 typical and 29 atypical); five to prolymphocytic leukemia; 13 to hairy cell leukemias; 12 to lymphoplasmacytic lymphomas; 14 to splenic marginal zone lymphomas; 22 were follicular lymphomas; and 19 mantle cell lymphomas. The following triple stainings were systematically applied to both normal and leukemic samples: FMC7/CD5/CD19, CD22/CD23/CD19, CD103/CD25/CD19, CD10/CD11c/CD19 and sIg/sIg(lambda)/CD19. Overall, 98% of the leukemic B cell chronic lymphoproliferative disorders cases displayed aberrant phenotypes at diagnosis with no significant differences being found between cases analyzed in peripheral blood vs bone marrow samples. The most common types of aberrant criteria detected included asynchronous antigen expression (92%) and antigen over-expression (54%); abnormally light scatter characteristics were found in 17% of the cases. Most of the cases studied (90%) displayed four or more phenotypic aberrations. Once patients were divided according to the different diagnostic subgroups, the overall incidence of aberrant phenotypes ranged from 79 to 80% among atypical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia to 97% of follicular lymphoma and 100% of typical B cell chronic lymphocytic leukemia/small lymphocytic lymphoma, hairy cell leukemia, lymphoplasmacytic lymphomas, splenic marginal zone lymphomas and mantle cell lymphomas. Based on the aberrant phenotypes detected unique four-color stainings could be built for the specific identification of aberrant phenotypes. These include CD22/CD23/CD19/CD5 and sIg(kappa)/sIg(lambda)/CD19/CD5 for lymphocytic leukemia/small lymphocytic lymphoma and prolymphocytic leukemia, CD103/CD25 or CD22/CD19/CD11c for hairy cell leukemia, FMC7/CD22/CD19/CD103 and sIg(kappa)/sIg(lambda)/CD22/CD19 for splenic marginal zone lymphomas, CD22/CD23/CD19/CD10 for follicular lymphomas and CD10/CD22/CD19/CD5 for mantle cell lymphomas. Serial dilutional experiments showed that the sensitivity level of immunophenotyping ranges between 10(-4) and 10(-5). In summary, the present study shows that immunophenotypic analysis allows the identification of aberrant phenotypes in 98% of leukemic B cell chronic lymphoproliferative disorders and these phenotypes can be used for minimal residual disease monitoring with a sensitivity limit of 10(-4)-10(-5).

Continuous oral cytarabine ocfosfate with interferon-alpha-2b for patients with newly diagnosed chronic myeloid leukaemia: a pilot study.

Recombinant(R) interferon alpha (r-IFN-alpha) has been shown to be an effective drug for chronic myeloid leukaemia (CML). However, higher response rates can be achieved using cytarabine along with r-IFN-alpha. YNK01 is a derivative of cytosine arabinoside for oral administration. So far, the only published experience with continuous YNK01 was in advanced CML (10 cases). We have performed a pilot study to evaluate the efficacy and toxicity of the combined therapy r-IFN-alpha and daily oral YNK01 in patients with newly diagnosed Ph+ CML. Ten previously untreated patients were included in the study. Among those patients evaluable for cytogenetic response, 87% (seven out of eight) reached a major cytogenetic response with four reaching complete cytogenetic response (50%). The most significant side-effects were gastrointestinal. Macrocytic anaemia was observed in three patients. In conclusion, continuous oral administration of YNK01 in combination with IFN-alpha is safe and can result in high-cytogenetic response rates.

p16/INK4a gene inactivation by hypermethylation is associated with aggressive variants of monoclonal gammopathies.

INTRODUCTION: A model of a stepwise malignant transformation has been proposed for the pathogenesis of monoclonal gammopathies. In this model, cell cycle regulators play a central role as a source of genetic events; particularly, p16/INK4a gene acts as a tumoral suppressor gene and, recently, inactivation of this gene through a methylation mechanism, has been observed in multiple myeloma patients. Under the diagnosis of monoclonal gammopathies there is a broad spectrum of disorders with very different outcomes, ranging from indolent courses, such as those of monoclonal gammopathy of undetermined significance, Waldeströn macroglobulinemia and smoldering multiple myeloma, to aggressive diseases such as symptomatic MM and primary plasma cell leukemia. To the best of our knowledge, the activity of p16 gene has not been evaluated and compared in these different subtypes of monoclonal gammopathies. MATERIALS AND METHODS: The methylation status of the p16 gene was analysed in a group of 159 patients with monoclonal gammopathies (40 monoclonal gammopathy of uncertain significance, eight Waldenström Macroglobulinemia, eight smoldering multiple myeloma, 98 symptomatic multiple myeloma and five primary plasma cell leukemia) using three different assays (restriction enzymes and PCR or S-B and modification by sodium bisulphite). RESULTS: Forty-one of 98 MM patients (41.8%) as well as four of the five (80%) primary PCL patients showed methylation of the p16 gene, while none of the patients with monoclonal gammopathy of undetermined significance, Waldenström Macroglobulinemia or smoldering multiple myeloma displayed a methylation status. CONCLUSION: These findings suggest that the methylation of the p16 gene could be a relevant oncogenic event in the monoclonal gammopathies evolution being associated with the most aggressive forms.

Increased conventional chemotherapy does not improve survival in multiple myeloma: long-term results of two PETHEMA trials including 914 patients.

BACKGROUND: Melphalan and prednisone (MP) has been the standard treatment for multiple myeloma (MM) for the last 30 years. Combination chemotherapy at conventional doses has not shown a significant prolongation of survival when compared to MP. There are few data comparing conventional chemotherapy at standard doses with conventional treatment at higher doses. We present the long-term outcome of 914 patients from two randomized trials comparing three different dose intensity regimens. METHODS: From 1 January, 1985 to 31 December, 1989, 487 patients were randomized between MP (melphalan 9 mg/m(2) p.o. and prednisone 60 mg/m(2) days 1-4) and alternating VCMP (vincristine 1 mg i.v. on day 1, cyclophosphamide 500 mg/m(2) i.v. on day 1, melphalan 6 mg/m(2) p.o. on days 1-4, and prednisone 60 mg/m(2) on days 1-4) and VBAP (vincristine 1 mg i.v. on day 1, BCNU and doxorubicin 30 mg/m(2) i.v. each on day 1, and prednisone 60 mg/m(2) on days 1-4). From 1 January, 1990 to 31 May, 1994, 427 patients were randomized between VCMP/VBAP at the above detailed doses (VCMP/VBAP 'SD') and the same regimen increasing the doses of cyclophosphamide and doxorubicin from 500 to 1200 mg/m(2) and from 30 to 50 mg/m(2), respectively (VCMP/VBAP 'HD'). RESULTS: Increasing dose intensity produced a significantly higher partial response rate (31% vs 45% vs 51% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P < 0.01). However, a significantly early death rate was observed in the HD arm (7.7, 7.5 and 12.1% for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = 0.05). Median duration of response (20 vs 18 vs 19 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) and median survival (25 vs 31 vs 29 months for MP, VCMP/VBAP 'SD', and VCMP/VBAP 'HD', respectively; P = NS) were similar in the three groups. MP produced a higher degree of thrombocytopenia than combination chemotherapy at standard (P = 0.002) or high dose (P = 0.01), this leading to a significantly higher dose reduction in the MP arm (P < 0.001 and P = 0.003 for VCMP/VBAP 'SD' and VCMP/VBAP 'HD', respectively). CONCLUSION: In these trials the response rate significantly correlated with the regimen intensity. However, no significant differences in response duration and survival were found. This highlights the limited role of conventional chemotherapy in MM and the need for further trials, aimed at determining the impact of new treatment approaches such as high-dose therapy/autotransplantation.

[Effect of mitomycin C on corneal endothelium cells. In vitro study]. / Efecto de la mitomicina C sobre el endotelio corneal. Estudio in vitro.

PURPOSE: Mitomycin C is an antibiotic with a demonstrated antiproliferative capacity as an inhibitor of fibroblastic cells proliferation. Its use has been extended in glaucoma surgery. In the present study, we evaluated mitomycin C effect on cell culture monolayer of rabbit corneal endothelium. METHODS: The source of corneal endothelium for cell culture was New Zealand albino rabbit eyes. Desegregation of cells was carried out with mechanic and enzymatic dissociation from corneal endothelium and Descemet membrane. Culture medium was EMEM. Three treatment groups of plates were exposed to three different concentrations of mitomycin C 2x10(-3) mg/ml, 2x10(-2) mg/ml and 2x10(-1) mg/ml. Control and witness plate groups were also established. The morphometric study was performed through quantitative analysis with a video system connected to the light microscope. RESULTS: Different morphological changes related with cell size, cytoplasm and dyeing were seen at the morphological study and several degenerative signs were established indicating cellular death and a very decrease of the cellular population. In the groups treated with minimal dose (2x10(-3) mg/ml) and 3 days evolution time, cellular population was 434 cels/mm(2), 7 evolution days group cell density was 300.97cels/mm(2), and at 14 days it was 201.88 cels/mm(2). The percentage of survival in all the groups of treated cells was under 50%. CONCLUSIONS: Mitomycin C in concentrations and exposure time as used in this study has a potent lethal effect on this cellular type that compromises to a greater or smaller extent their function and integrity.

High-sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma.

Sensitive techniques for monitoring minimal residual disease (MRD) in multiple myeloma (MM) are needed to evaluate the effectiveness of new intensive treatment strategies. The aim of the present study was to explore the applicability and sensitivity of flow cytometry immunophenotyping and DNA ploidy studies for the investigation of residual myelomatous plasma cells (PC) in MM patients. Bone marrow (BM) samples from 61 untreated MM patients were immunophenotypically analysed with a panel of 21 monoclonal antibodies, using a high-sensitive method based on a two-step acquisition procedure through a SSC/CD38 -CD138+ 'live-gate'. Overall, in 87% of MM cases, PC displayed an aberrant phenotype at diagnosis. The most important aberrant criteria were: antigen over-expression of CD56 (62%), CD28 (16%) and CD33 (6%) and asynchronous expression of CD117 (28%), sIg (21%) and CD20 (10%). DNA aneuploidy was found in 62% of cases. The simultaneous use of these two techniques allowed the detection of aberrant/aneuploid PC in 95% of the cases. Based on dilutional experiments, the detection limit of both techniques ranged from 10(-4) to 10(-5). In 29 stem cells harvests and 19 BM samples obtained 3 months after autologous transplantation, we have investigated the presence of residual myelomatous PC; they were detected in 44% of the stem cell collections and in 61% of the BM samples obtained after transplant. The percentage of pathological PC did not significantly change during the days of harvest. In summary, the present study shows that the combined use of immunophenotyping and DNA ploidy studies is a suitable approach for MRD investigation in MM patients based on their applicability (95% of cases) and sensitivity (up to 10(-5)).

An abnormal CD34+ myeloid/CD34+ lymphoid ratio at the end of chemotherapy predicts relapse in patients with acute myeloid leukemia.

During conventional follow-up of patients with acute myeloid leukemia (AML), the emergence of cytopenias is considered to be a sign of impending relapse, and it represents an example of how leukemic hematopoiesis affects normal hemopoietic differentiation. In the present study, we have explored the possible value of the analysis of the distribution of CD34+ myeloid and CD34+ lymphoid progenitor cells in follow-up complete remission bone marrow samples from de novo AML patients as a prognostic parameter for predicting relapse. A total of 213 bone marrow samples from 36 AML patients in morphological complete remission, obtained at the end of induction, consolidation, and intensification therapy and every six months thereafter were analyzed. The normal CD34+ myeloid/CD34+ lymphoid ratio ranged between 2.4 and 8.9. In contrast, in most AML cases an abnormally high ratio (> or =10) was observed at the end of induction and consolidation therapy: 96% and 75% of cases, respectively. On the other hand, at the end of intensification, 70% of the patients displayed a normal CD34+ ratio. Patients with a myeloid/lymphoid CD34+ ratio higher than 10 at the end of intensification showed a significantly lower overall survival (median survival of 19 months versus median not reached, P = 0.05), as well as a lower disease-free survival (median of 7 months versus 30 months, P = 0.0001). Regarding sequential studies, 67% of the relapses were preceded by the re-appearance of an abnormal CD34 ratio, whereas relapse was not predicted in four patient with leukemia classified as M3 undergoing maintenance therapy. From the remaining 18 patients who are still in continuous complete remission, all except 3 cases (17%) displayed a normal CD34 myeloid/lymphoid ratio. In summary, the present study shows that the persistence at the end of chemotherapy of an abnormally high (> or =10) ratio between CD34+ myeloid and CD34+ lymphoid progenitors in the bone marrow of AML patients is associated with high risk of relapse and a shorter overall survival.

Immunophenotypic and DNA content characteristics of plasma cells in multiple myeloma and monoclonal gammopathy of undetermined significance.

In the present paper we review the immunophenotypic characteristics of plasma cells (PC) and the PC DNA contents from multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), and its value for the differential diagnosis between both entities. The strong reactivity for CD38 and the positivity for CD138 are the two best markers for identifying PC. Myelomatous PC display an heterogeneous phenotype consistent with the fact that the neoplastic clone is able to undergo a certain degree of differentiation. In addition, PC from MM patients usually lack surface expression of B-cell associated antigens and frequently display reactivity for markers which are not restricted to the B-cell lineage. In MGUS patients, two clearly defined and distinct PC subpopulations can be identified. One of these PC subpopulations shows phenotypic characteristics identical to those of normal PC, including a very strong reactivity for the CD38 antigen, intermediate/low light scatter characteristics and positivity for CD19, in the absence of CD56, and corresponds to the residual normal bone marrow PC. The second PC subpopulation shows an immunophenotype similar to that of myelomatous PC, characterized by a slightly lower reactivity for CD38 and strong CD56 expression, on the absence of positivity for CD19, these PC corresponding to the clonal counterpart. Using a simultaneous staining for PC and DNA, around 60% of MM and 73% of MGUS patients display DNA aneuploidy, the majority of them being hyperdiploid. However, in contrast to MM patients, in MGUS patients two clearly different PC subsets can be discriminated in most cases (73%): a diploid and an aneuploid (hyperdiploid) subset, corresponding to normal and clonal PC, respectively. Upon comparing hyperdiploid with diploid patients in MM, the former display a better prognosis, in line with the higher incidence of DNA hyperdiploidy in MGUS. A clear correlation between the percentage of S-phase PC and several prognosis features of MM has been found. In spite of these findings, no significant differences in the percentage of pathological S-phase PC are detected between MM and MGUS patients. Regarding the differential diagnosis between MGUS and MM, multivariate analysis shows that the ratio between the number of clonal and normal residual PC is the best single parameter.

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

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.

Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia.

PURPOSE: The identification of immunophenotypic aberrancies through multiparametric flow cytometry makes the differentiation between normal and leukemic cells relatively simple and quick, and is therefore an attractive method for the investigation of minimal residual disease (MRD). In this report, we have analyzed the impact on relapse and relapse-free survival (RFS) of detecting immunophenotypical aberrant cells in acute lymphoblastic leukemia (ALL) patients in cytomorphologic complete remission (CR). MATERIALS AND METHODS: Two hundred eleven bone marrow (BM) samples from 53 consecutive ALL (37 precursor B-ALL and 16 T-ALL) patients were analyzed. The only selection criteria were to have at least one aberrant immunophenotypic feature at diagosis and to have achieved cytomorphologic CR after induction therapy. For MRD detection, all follow-up samples were analyzed with triple labelings using a two-step acquisition procedure, in which 106 cells were screened for the possible persistence of residual leukemic cells with the same phenotypic aberrancy as that identified diagnosis. RESULTS: Patients who displayed a gradual increase in MRD levels showed a higher relapse rate (90% v22%; P < .00001) and shorter median RFS (12 months v not reached; P < .0001) than those with stable or decreasing MRD levels. This adverse prognostic influence also was observed when children and adults, as well as B-ALL and T-ALL patients, were analyzed separately. An MRD level > or = or greater than 10(-3) discriminated two risk groups of ALL patients with significantly different relapse rates and RFS at all treatment phases (end of induction, consolidation, maintenance, and out of treatment). CONCLUSION: Multiparametric flow cytometry of MRD in ALL patients is a valuable tool for relapse prediction and for the identification of a cohort of patients with very poor prognosis.

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

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.

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

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.

Functional expression of MDR-1 in acute myeloid leukemia: correlation with the clinical-biological, immunophenotypical, and prognostic disease characteristics.

Up till now, clinical data on the possible prognostic influence of multidrug resistance (MDR) in hematological malignancies have been inconsistent, probably due to technical pitfalls. Moreover, in most studies qualitative information on the presence/absence of MDR-1 expression has been used instead of quantitative results. In addition, results usually refer to the total BM population and not specifically to blast cells. In the present study we analyzed the expression of MDR-1 in a series of 50 newly diagnosed de novo AML using a double-staining technique: (a) monoclonal antibodies for the specific identification of blast cells and (b) the rhodamine-123 efflux assay, which allows a quantitative and calibrated measurement of MDR-1 function. Expression of MDR-1 was correlated with clinical, biological, and immunophenotypical disease characteristics. All patients were uniformly treated according to the AML 87/91 protocols of the Spanish Pethema Cooperative Group; the median age was 51 +/- 19 years and the FAB distribution was as follows: 2 M0, 9 M1, 9 M2, 12 M3, 11 M4, 5 M5, and 2 M6 cases. Upon grouping the 50 AML patients analyzed according to the level of rh123 elimination, it was observed that those cases with > or = 30% decrease in rh123 fluorescence displayed higher WBC counts (9 +/- 12 vs 37 +/- 73 x 10(9)/l, p = 0.02) and platelet numbers (94 +/- 11 vs 35 +/- 25 x 10(9)/l, p = 0.02), together with a higher incidence of extramedullary involvement (35% vs 24%, p = 0.02). Half of the patients (47%) displaying a low rh123 elimination (< 30%) showed M3 morphology, while among the 33 patients with a higher rate of rh123 elimination (> or = 30%), only four (12%) corresponded to the M3 morphological subtype (p = 0.0006). From the immunophenotypic point of view, a low rate of rh123 elimination was associated with a lower expression of HLADR antigen (p = 0.003) and a higher expression of CD117 (p = 0.01). Regarding the possible prognostic influence of MDR1 expression, we found that a high rate of rh123 elimination (> 30%) was associated with a tendency towards poor disease outcome, illustrated by both a lower complete remission rate with the first cycle of chemotherapy (36% vs 56%) and a lower median disease-free survival (22 months vs median DFS not reached), although differences did not reach statistical significance (p = 0.1 in both comparisons). This data shows that although MDR-1 can be a relevant parameter in the evaluation of AML patients, larger series of patients using appropriate techniques for specifically analyzing the MDR of blast cells will be necessary in order to establish the final clinical value of this parameter.

Treatment of multiple myeloma in elderly people: long-term results in 178 patients.

The purposes of the present study have been to analyse the presenting features, response to therapy and survival of myeloma patients aged 70 years or more, in comparison to younger patients. From January 1985 to December 1989, 487 patients with multiple myeloma (MM) were randomized to receive melphalan and prednisone (MP) versus alternating cycles of vincristine, cyclophosphamide, melphalan, and prednisone (VCMP) and vincristine, BCNU, adriamycin, and prednisone (VBAP). The subset of 178 patients who were 70 or more years is the subject of this study, whereas the 309 patients younger than 70 years were used as a control group. The presenting features and response to chemotherapy of older patients were no different to those of the younger population. However, the survival of elderly patients was significantly shorter (median 23.4 vs. 33.5 months, p < 0.001). The overall response rate to MP in older patients was 50% (28% objective plus 22% partial response) compared with 61% (44% objective plus 17% partial response) to combination chemotherapy (p = not significant). Myelosuppression was moderate in both arms, although MP produced a higher degree of thrombocytopenia. There were no significant differences in survival between patients given MP versus VCMP/ VBAP (median, 20 vs. 27 months, p = 0.2). Response to treatment was associated with a significantly longer survival. Older patients with symptomatic myeloma tolerate chemotherapy and should be offered treatment.

Survival of multiple myeloma patients who are potential candidates for early high-dose therapy intensification/ autotransplantation and who were conventionally treated.

PURPOSE: To analyze the outcome of patients with multiple myeloma (MM) who were potential candidates for early high-dose therapy (HDT) intensification followed by autotransplantation from a series treated with conventional chemotherapy. PATIENTS AND METHODS: From January 1985 through December 1989, 487 patients with symptomatic MM were entered onto a randomized study to compare melphalan and prednisone (MP) versus vincristine, cyclophosphamide, melphalan, and prednisone (VCMP) /vincristine, carmustine (BCNU), doxorubicin, and prednisone (VBAP). The sub-group of 77 patients who could have been candidates for early intensification with HDT followed by stem-cell support (ie, < 65 years of age, stage II or III disease, performance status < 3, and objective or partial response to initial chemotherapy) are the subjects of this report. RESULTS: Seventy-seven of 487 patients could have been candidates for early intensification. The median age was 56 years (range, 27 to 64). At diagnosis, 12% had abnormal renal function, 16% hypercalcemia, and 42% serum beta 2-microglobulin level > or = 6 mg/L; 62% had stage III disease at diagnosis. Thirty-six patients were initially treated with MP and 41 with VCMP/VBAP. The median response duration to initial chemotherapy was 22 months, and the actuarial probability of being in continued first response at 5 years was 14%. After a median follow-up time of 58 months, 59 patients have died, one was lost to follow-up evaluation, and 17 are still alive 69 to 119 months after initial chemotherapy. The median survival time from initiation of treatment was 60 months and from the time when autotransplantation would be considered, 52 months. The only independent prognostic parameter for survival was renal function at diagnosis. CONCLUSION: The median survival time of patients with MM who are less than 65 years of age and who respond to initial chemotherapy is 5 years. This survival duration is similar to that reported in selected series of patients given early HDT and stresses the importance of ongoing randomized trials to determine the role of HDT in the treatment of younger myeloma patients.

Analysis of natural killer-associated antigens in peripheral blood and bone marrow of multiple myeloma patients and prognostic implications.

The aim of this study was to analyse the expression of NK-associated antigens in both peripheral blood and bone marrow lymphocytes from a large series of newly diagnosed multiple myeloma patients. 112 patients with untreated multiple myeloma (MM) were included in the study. 36 sex- and age-matched healthy volunteers were used as controls for peripheral blood (PB) studies and 14 for the bone marrow (BM) studies. Simultaneous stainings with the CD3/CD56, CD2/CD16 and CD8/CD57 monoclonal antibodies were systematically performed in PB and CD3/CD56 and CD2/CD16 in BM in order to analyse their relationship with the clinical and biological characteristics of the disease and survival. The expression of NK-associated antigens (CD56, CD16 and CD57) assessed within the lymphoid gate, was significantly increased (P < 0.001) in the PB of MM patients both in relative and absolute numbers. In the BM a significant increase in the percentage of CD56+ lymphocytes (P < 0.001) was also observed; in contrast, the proportion of CD16+ cells did not differ significantly from that of normal BM samples. The number of CD56+CD3- lymphocytes increased significantly within high-risk patients (869 +/- 671) as compared to intermediate (388 +/- 212) and low-risk patients (274 +/- 199) (P = 0.04). Moreover, patients with high values of CD56+CD3- lymphocytes showed a statistically significant association with several adverse prognostic factors including anaemia, hypoalbuminaemia, renal failure, high beta 2M, DNA diploidy and high S-phase plasma cells. In addition, patients with higher absolute numbers of PB CD56+CD3-lymphocytes displayed a poorer prognosis, whereas patients with higher values of CD57+CD8- cells had a better outcome.