M. J. Moro

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.

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/sIgλ/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 sIgκ/sIgλ/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 sIgκ/sIgλ/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.

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).

Immunophenotypic heterogeneity of multiple myeloma: influence on the biology and clinical course of the disease

In 112 untreated myeloma patients we have analysed the immunophenotype of plasma cells both by immunofluorescence (IF) and immunocytochemistry (APAAP). Both techniques yielded similar results pointing to aH important degree of heterogeneity in antigenic expression not only between different patients but also within the same patient. The expression of CD38 and Han‐PCl antigens (Ags) was almost constant (> 90% positive cases), while CD9 was detected in 66% of the cases. On the other hand, less than one third of patients were positive for CD 10, CD20 and HLA‐DR and generally with a weak expression (< 30% positive plasma cells). In occasional cases plasma cells were weakly positive for the myelomonocytic markers CD13 (9%), CD15 (25%) and CD14 (6%). The possibility that this heterogeneity might be the result of different stages of differentiation of the neoplastic clone is suggested both by the positive correlation in the expression of some of these antigens (CD10, CD9, CD20, HLA‐DR) and by the relationship between CD10 and myeloid antigens with immature plasma cell morphology. Finally, the cALLA antigen does not seem to be of significant value in predicting survival. Moreover, none of the other markers explored showed a clear influence in the course of the disease, although the tendency towards a lower survival found for the CD20+ cases as well as the association of the expression of some antigens and advanced clinical stage, may warrant further studies in a larger series of patients.

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.

Prognostic implications of DNA aneuploidy in 156 untreated multiple myeloma patients

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 02M levels, higher proliferative activity within the residual normal haemopoietic cells, increased expression of CD 5 6 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).

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 β2‐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 β2-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 β2-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).

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 β2M, 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.

Lymphoid subsets in acute myeloid leukemias: Increased number of cells with NK phenotype and normal T-cell distribution

SummaryNatural killer (NK) and T subsets were analyzed with appropriate dual labeling by flow cytometry in peripheral blood (PB) (66 cases) and bone marrow (BM) (55 cases) from patients with de novo AML in order to determine: (a) their distribution at diagnosis, (b) the correlation between PB and BM in NK subpopulations, (c) their relationship with the clinical and hematological disease characteristics, and (d) the changes occurring upon achieving complete remission (CR). NK cells defined by the expression of CD56 in the absence of CD3 were significantly increased at diagnosis and their levels in PB correlated with those of BM. By contrast, NK subsets defined by CD16 expression (CD16+ CD2+ and CD16+ CD2− NK-cell subsets) as well as T lymphocytes with NK activity (CD56+ CD3+), although increased in PB, displayed normal levels in BM. An additional observation of interest was the expansion of an immature NK population lacking CD16 Ag expression (CD56+CD16−). AML cases were divided into two groups according to the absolute number of NK cells in PB; patients with the highest levels showed an increased proportion of blast cells in PB (p=0.01), monocytic subtypes (p=0.03), and expression of CD11b, CD14, and CD4 antigens (p=0.05). Infections at diagnosis were not related to the level of NK cells. In 19 patients who achieved complete remission the number of CD56+CD3− cells tended to be reduced to within the normal range. Other T-cell populations, including the CD4 naive and memory cells, were also explored, their distribution being normal in the PB of AML patients. By contrast, the cytotoxic subset CD8+/CD57+was significantly increased (p< 0.001). These data point to the existence of marked alterations of NK cells in AML patients, possibly reflecting a host-tumor immunological interaction.