Ricardo Morilla

Levels of expression of CD19 and CD20 in chronic B cell leukaemias

AIMS: To investigate whether the antigen levels of the B cell lineage markers CD19 and CD20 can distinguish between normal and neoplastic B cells or characterise distinct expression patterns among the chronic B cell leukaemias. METHODS: Peripheral blood cells from 70 patients with B cell disorders and 17 healthy donors were analysed by quantitative flow cytometry. Direct immunofluorescence staining was performed with phycoerythrin conjugated CD19 and CD20 monoclonal antibodies. Standard microbeads with different capacities to bind mouse immunoglobulins were used to convert the mean fluorescence intensity (MFI) values into number of antigen molecules/cell, expressed as antibody binding capacity (ABC). RESULTS: CD19 and CD20 ABC values in leukaemic B cells differed from those of normal blood B lymphocytes. The results identified distinct profiles of CD19 and CD20 expression in the various types of B cell leukaemias. In all leukaemias studied except hairy cell leukaemia (HCL), CD19 expression was significantly lower than the mean (SD) value in normal B cells (22 (7) x 10(3) molecules/cell), as follows: chronic lymphocytic leukaemia (CLL), 13 (7) x 10(3); B prolymphocytic leukaemia (B-PLL), 16 (9) x 10(3); splenic lymphoma with villous lymphocytes (SLVL), 15 (11) x 10(3); mantle cell lymphoma (MCL), 10 (7) x 10(3). In HCL there was strong CD19 expression (38 (16) x 10(3)). In contrast, the level of expression of membrane CD20 was higher than the mean (SD) value in normal B cells (94 (16) x 10(3) molecules/cell) in MCL (123 (51) x 10(3)); B-PLL (129 (47) x 10(3)); SLVL (167 (72) x 10(3)); and HCL (312 (110) x 10(3)); while it was significantly lower (65 (11) x 10(3)) in CLL compared with normal B cells and the other B cell leukaemias. CONCLUSIONS: Quantitative determination of CD19 and CD20 may provide useful diagnostic information for the study of B lymphoproliferative disorders.

Levels of expression of CD52 in normal and leukemic B and T cells: Correlation with in vivo therapeutic responses to Campath-1H

The CD52 antigen is expressed on most normal and neoplastic lymphoid cells. The reshaped humanized IgG1 anti-CD52 monoclonal antibody (Campath-1H) has been used in the treatment of hemopoietic and non-hemopoietic diseases for its ability to induce lymphocyte depletion both in vitro and in vivo. Good activity has been shown in patients with chronic T and B cell leukemias, in particular T-prolymphocytic leukemia (T-PLL). However, the response to treatment is not uniform and this variability may depend on differences in the level of antigen expression on the leukemic cells. To test this hypothesis, we used quantitative flow cytometry to investigate the intensity of the expression of CD52 in 45 cases of lymphoid leukemia, 24 with B-cell chronic lymphocytic leukemia (CLL), 21 with T-PLL and 12 normal controls. Normal T lymphocytes expressed higher CD52 antigen than B lymphocytes (p < 0.005) and the antigen was also significantly higher in T-PLL compared to CLL (p < 0.001). Moreover, the differences in CD52 expression were somewhat higher in Campath-1H treated patients who responded than in non responders. Although other factors may play a role in the response to Campath-1H in vivo, the quantitative estimation of CD52 expression may provide a rationale for the greater response in T-PLL and help select those patients with a higher probability of responding to this therapy.

Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission.

Quantitative flow cytometry can distinguish between normal and leukaemic B-cell precursors

Summary. The immunological detection of minimal residual disease in B‐lineage acute lymphoblastic leukaemia (ALL) has been hampered by the fact that the leukaemic cells represent the malignant counterparts of normal haemo‐poietic precursors expressing terminal deoxynucleotidyl transferase (TdT), CD10 and CD19. We have used quantitative double‐labelling flow cytometry with standard fluorescent beads to convert the mean fluorescence to the number of antigen molecules per cell. The number of TdT, CD10 and CD19 molecules per cell was determined in normal B‐cell precursors from 22 healthy donors and eight regenerating marrows from patients with various malignancies and in 20 cases of B‐lineage ALL. In normal bone marrow we characterized two different B‐cell populations: TdT+/CD10+/ CD19+ and TdT/CD10+/CD19+. We demonstrated a major difference in the level of expression of TdT, CD 10 and CD 19 between normal bone marrow and B‐lineage ALL blasts. Normal TdT+ precursors have significantly higher number of TdT (>100×l03) and lower number of CD10 (<50×l03) and CD19 (<10×103) molecules per cell than B‐lineage ALL blasts (<100, >50, >10×l03 molecules per cell respectively); these differences were statistically highly significant. Furthermore, regenerating marrows had a significantly higher percentage of B‐cell precursors than healthy donors. This increase was at the expense of the TdT−/ CD10+/CD19+ population which, in the context of B‐lineage ALL, could be wrongly interpreted as evidence of relapse if TdT is not included in the analysis. Therefore the quantitative analysis of TdT combined with CD10 and CD19 may allow a clear distinction between normal precursors and minimal residual leukaemia in B‐lineage ALL and avoid the pitfall of misinterpreting regenerating B‐cells as evidence of relapse.

Differential expression of CD3 and CD7 in T-cell malignancies: a quantitative study by flow cytometry.

Most T‐cell antigens are expressed on normal and neoplastic T lymphocytes and for this reason it is not easy to distinguish between the immunophenotype of normal and malignant T cells. We have addressed this problem by comparing the levels of expression of CD3 and CD7 on T lymphocytes from 18 healthy donors with those of 61 cases of T‐cell leukaemia using quantitative flow cytometry with a method that converts fluorescence intensity into number of antigen molecules per cell. Normal T lymphocytes expressed 124 ± 25 CD3 and 20 ± 3 × 103 CD7 molecules per cell. The mean CD3 values were significantly lower in all types of T‐cell leukaemia than in normal T cells ( P < 0.05), with the exception of Sezary syndrome. The lowest CD3 values were found in T‐lymphoblastic leukaemia (T‐ALL), 30 ± 21 × 103, and adult T‐cell leukaemia/lymphoma (ATLL), 38 ± 31 × 103, followed by T‐prolymphocytic leukaemia (T‐PLL), 92 ± 47 × 103, and granular lymphocyte leukaemia (GLL), 95 ± 21 × 103. In contrast, the number of CD7 molecules was significantly higher in T‐ALL, 35 ± 7 × 103 ( P < 0.01), and T‐PLL, 29 ± 12 × 103, than the normal controls ( P < 0.01), whereas ATLL and GLL showed a low CD7 expression, 13 ± 3 and 12 ± 3 × 103, respectively. Our results show that the quantitative analysis of CD3 and CD7 and their combined evaluation may enable a distinction between normal and leukaemic T cells and could facilitate the monitoring of minimal residual disease. This study has also defined the T prolymphocyte as a cell of intermediate maturity between thymic derived and peripheral T lymphocytes.

ζ‐Chain associated protein 70 and CD38 combined predict the time to first treatment in patients with chronic lymphocytic leukemia

ζ‐Chain associated protein (ZAP)‐70 has been proposed as a surrogate marker for immunoglobulin heavy‐chain variable region (IgVH) mutation in chronic lymphocytic leukemia (CLL), but it is still not clear whether it is an independent prognostic factor.

The role of ultrastructural cytochemistry and monoclonal antibodies in clarifying the nature of undifferentiated cells in acute leukaemia

Summary. The nature of the cells in 21 cases of acute leukaemia with blasts which were undifferentiated by light microscopy criteria was investigated by immunophenotyping, ultrastructural cytochemistry and DNA analysis. Two groups of cases were recognized. Fourteen cases were negative with B and T lymphoid markers and expressed one or two myeloid antigens detected by the monoclonal antibodies (McAb) MCS2 (CD13) and MY9 (CD33). Peroxidase activity was demonstrated at ultrastructural level by the method of Roels on unfixed cells in eight out of 10 cases; rearrangement of the immunoglobulin (Ig) genes was demonstrated in one of the three cases investigated. These cases are proliferations of early, MO, myeloblasts which can only be recognized by immunological and ultrastructural cytochemical methods. The remaining seven cases revealed a complex phenotype with expression of myeloid and lymphoid antigens. Peroxidase activity was detected in blasts from two cases with rearrangement of the Ig‐heavy chain gene: in one of them the T cell receptor β and γ chain genes were also found in rearranged configuration. This group comprises cases of biphenotypic and mixed acute leukaemia which probably involve multipotent stem cells. This study demonstrates that the expression of myeloid antigens on blast cells parallels closely the presence of peroxidase activity and that lymphoid markers correlate with gene rearrangements at DNA level. Our findings are reassuring with respect to the specificity of the antimyeloid McAb for the diagnosis of cases which are unclassifiable by conventional methods.

Adhesion receptors on peripheral blood leukemic B cells. A comparative study on B cell chronic lymphocytic leukemia and related lymphoma/leukemias

The expression of a series of adhesion receptors: L-selectins (CD62L): Leu-8, several integrins (LFA-1: CD11a/CD18, VLA-4: CD49d/CD29 and VLA-5: CD49e/CD29), ICAM-1(CD54) and the ‘homing receptor’ (CD44) were investigated by a dual color flow cytometry in 56 cases of B cell disorders namely, 39 chronic lymphocytic leukemias (CLL), four hairy cell leukemia (HCL), seven splenic lymphoma with villous lymphocytes (SLVL) and six other non-Hodgkin’s lymphoma (NHL). The functional activity of L-selectins was assessed with L-selectin ligand analogs (polyphosphomonester core polysaccharide: PPME and fucoidin). Leukemic B cells were identified with phycoerythrin-conjugated monoclonal antibodies (McAbs) anti-CD19, anti-kappa/lambda investigated simultaneously for the expression of adhesion receptors estimated with fluorescein-isothiocyanate (FITC) conjugated McAbs. The percentage of leukemic cells expressing L-selectins (Leu-8) was high in CLL (52% of positive cases) and integrin expression (LFA-1, VLA-4, 5) was low (19 and 33%, respectively), while a reverse pattern, low Leu-8 (17%), and a high VLA-4 (77%), was observed in non-CLL cases. The expression of LFA-1 α-chain was variable in non-CLL cases, and the LFA-1 heterodimer was expressed on most clonal B cell in NHLs (92%). LFA-1 α-chain was detected on cells from only one HCL case, while β2 integrin was regularly expressed on hairy cells. VLA-5 integrin was found on a relatively small number (26%) of mature B cell leukemias. A remarkable finding was the detection of ICAM-1 in all CLL cases albeit the number of positive cells was significantly lower (P < 0.05) compared to non-CLL cases. CD44 was expressed on a high number of neoplastic cells in all the investigated categories. There was no correlation between the expression of the adhesion molecules and clinical and laboratory parameters except for CD18 which was expressed on a significantly (P < 0.05) higher number of leukemic cells in CLL with more advanced stages. This study demonstrates that even closely related B cell leukemia/lymphomas have a certain well defined and strictly variable adhesion profile which is characteristic of the disease entity and therefore, the adhesion profile may offer additional information useful for differential diagnosis and study of disease pathogenesis.

Detection of minimal residual disease in B-lineage acute lymphoblastic leukaemia by quantitative flow cytometry

The clinical significance of detecting minimal residual disease (MRD) in B‐lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B‐cell precursor ALL were followed during and after completion of treatment (median follow‐up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5–15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease‐free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P < 0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B‐lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with ‘true’ remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT‐negative B‐cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B‐lineage ALL and should be tested in prospective clinical trials.

The role of an anti‐myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry

Summary. The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti‐MPO and immunocytochemistry (alkaline phosphatase anti‐alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti‐MPO was positive (> 3% blasts) in all but one of the 90 AML positive by LM cytochemistry. Of 13 AML cases negative by MPO cytochemistry, six showed 3.10% blasts reactive with anti‐MPO and were also positive with antibodies to CD13 arid/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti‐MPO in AML was 92%. Anti‐MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also GD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO cytochemistry and anti‐MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti‐MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.