Methodological aspects of minimal residual disease assessment by flow cytometry in acute lymphoblastic leukemia: A French multicenter study.

BACKGROUND: Minimal residual disease (MRD) assessment provides a powerful prognostic factor for therapeutic stratification in acute lymphoblastic leukemia (ALL). Multiparameter flow cytometry (MFC) has the potential for a rapid and sensitive identification of high risk patients. Our group has previously published that MRD levels analyzed by clone specific Ig/TcR-QPCR and MFC were concordant at a sensitivity of 10(-4) . Here we report the MFC methodological aspects from this multi-center experience. METHODS: MRD was assessed by MFC in 1030 follow-up samples from 265 pediatric and adult patients with de novo ALL treated in the FRALLE, EORTC, or GRALL clinical trials. MRD assessment as applied by the eight participating MFC laboratories is described in detail regarding cell preparation, leukemia-associated immunophenotype (LAIP) markers and data analysis. Samples were obtained from bone marrow (BM) and peripheral blood (PB). Immunostaining was performed after erythrocyte lysis or Ficoll enrichment. RESULTS: This study confirms the applicability of MFC-based MRD assessment in 97% of patients with ALL at the 10(-4) cut-off. MRD values after Ficoll enrichment and erythrocyte lysis were found comparable. Higher MRD values were obtained in BM than in PB, especially for B-lineage ALL. CONCLUSIONS: Measurement of MRD by MFC at the 10(-4) cut-off is applicable within a few hours for almost all patients and using a comparable analytical strategy allows for multicenter collaborative studies. The method can be introduced in a strategy aimed at defining the risk of failure of patients with childhood or adult ALL.

Mutual benefits of B-ALL and HLDA/HCDM HLDA 9th Barcelona 2010.

The B-cell panel of the ninth HLDA was applied in a multicentre fashion to cryopreserved cells from 46 patients with acute lymphoblastic leukemia. The reagents were aliquoted and shipped to volunteer participants from the French Groupe d'Etude Immunologique des Leucémies (GEIL). All samples were tested in flow cytometry, and the results collected as of the strength of labeling of the leukemic clone as negative, weak or strong. Among the 64 antibodies tested, the strongest and most frequent staining was observed for CD305 (LAIR), CD229 (Ly9), CD200 (OX-2) and, to a lesser extent, CD361 (EVI2b). Details of the observations, and information about the molecules tested are provided in the manuscript as well as a summary table.

Adverse prognostic significance of CD20 expression in adults with Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia.

The prognostic significance of CD20 expression in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been mostly studied in children and yielded conflicting results. In 143 adults with Philadelphia chromosome-negative BCP-ALL treated in the multicentric GRAALL 2003 trial, CD20 positivity over 20% was observed in 32% of patients. While not influencing complete remission achievement, CD20 expression was associated with a higher cumulative incidence of relapse (CIR) at 42 months (P=0.04), independently of the ALL high-risk subset (P=0.025). Notably, the negative impact of CD20 expression on CIR was only observed in patients with a white blood cell count (WBC) over 30x10(9)/L (P=0.006), while not in those with a lower WBC. In the former subgroup, this impact translated into lower event-free survival (15% vs. 59% at 42 months, P=0.003). CD20 expression thus appears to be associated with a worse outcome, which reinforces the interest of evaluating rituximab combined to chemotherapy in CD20-positive adult BCP-ALL. ClinicalTrials.gov ID, NCT00222027.

Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia.

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4(+) CD56(+) lineage(neg) CD45RA(+)/RO(neg) CD11c(neg) CD116(low) CD123(+) CD34(neg) CD36(+) HLA-DR(+). Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC - lymphoid or myeloid - acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4(+) CD56(+/-) MPO(neg) cCD3(neg) cCD79a(neg) CD11c(neg) profile and then the CD123(high), BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL.

A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5.

We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3' rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre-B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

Age-related phenotypic and oncogenic differences in T-cell acute lymphoblastic leukemias may reflect thymic atrophy.

Postnatal thymic involution occurs progressively throughout the first 3 decades of life. It predominantly affects T-cell receptor (TCR) alphabeta-lineage precursors, with a consequent proportional increase in multipotent thymic precursors. We show that T-acute lymphoblastic leukemias (T-ALLs) demonstrate a similar shift with age from predominantly TCR expressing to an immature (IM0/delta/gamma) stage of maturation arrest. Half demonstrate HOX11, HOX11L2, SIL-TAL1, or CALM-AF10 deregulation, with each being associated with a specific, age-independent stage of maturation arrest. HOX11 and SIL-TAL represent alphabeta-lineage oncogenes, whereas HOX11L2 expression identifies an intermediate alphabeta/gammadelta-lineage stage of maturation arrest. In keeping with preferential alphabeta-lineage involution, the incidence of SIL-TAL1 and HOX11L2 deregulation decreased with age. In contrast, HOX11 deregulation became more frequent, suggesting longer latency. TAL1/LMO1 deregulation is more frequent in alphabeta-lineage T-ALL, when it is predominantly due to SIL-TAL1 rearrangements in children but to currently unknown mechanisms in adolescents and adults. LMO2 was more frequently coexpressed with LYL1, predominantly in IM0/delta/gamma adult cases, than with TAL1. These age-related changes in phenotype and oncogenic pathways probably reflect progressive changes in the thymic population at risk of malignant transformation.

Chronic myeloid leukemia associated with B-cell chronic lymphocytic leukemia: evidence of two separate clones as shown by combined cell-sorting and fluorescence in situ hybridisation.

The simultaneous occurrence of Philadelphia positive chronic myeloid leukemia (Ph+ CML) and B-cell chronic lymphocytic leukemia (B-CLL) is a rare event which raises the possibility that the two malignant clones derive from a common, or distinct, malignant stem cells. In this study, we used combined CD19-based cell-sorting and fluorescence in situ hybridisation (FISH) to investigate whether or not the BCR-ABL fusion gene was present in the malignant B-cells of a patient who presented a Ph+ CML/B-CLL association. The CD19+ cells lacked the BCR-ABL rearrangement whereas all CD19-cells exhibited the fusion gene. This result demonstrates that B-cell transformation occurred in a Ph-B-cell subset.

Clinical value of the quantitative expression of the three epitopes of CD34 in 300 cases of acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: The various epitopes of the CD34 molecule have been classified according to their different sensitivities to enzymatic cleavage by neuraminidase, chymopapain and a glycoprotease from Pasteurella haemolytica. Although monoclonal antibodies have been developed that specifically identify these epitopes, few studies have evaluated the distribution and quantitative expression of such epitopes on leukemic blasts. DESIGN AND METHODS: We report here a prospective multicenter study in which we examined and quantified the expression of the 3 classes of CD34 on fresh leukemic blast cells from 300 cases of acute myeloid leukemia (AML). The binding of monoclonal antibodies was studied by flow cytometry, allowing evaluation of blast cell positivity as well as their mean fluorescence intensity. These quantitative data were made comparable between centers by means of a calibration curve established with the same reagents in all laboratories. RESULTS: Quantitative expression of class I epitope was significantly higher than that of class II and class III epitopes (p<0.0001). The three classes were more frequently expressed in M0 and M1 and less in M3 and M5. The highest levels of CD34 expression were observed in M2, M0 and M1 and the lowest in M3, M5 and BAL for class II and III. CD34 expression was lower for all classes in cases with a normal karyotype, compared to in cases with structural or numerical abnormalities. INTERPRETATION AND CONCLUSIONS: In cases with a t(9;22) the expression of class I was significantly higher than that of class II and III and the opposite was observed in AML with t(15;17). Moreover, as a whole, a high intensity of class III CD34 appeared to be a marker of good prognosis.

Clinical and biologic features of CD4(+)CD56(+) malignancies.

CD4(+)CD56(+) malignancies are rare hematologic neoplasms, which were recently shown to correspond to the so-called type 2 dendritic cell (DC2) or plasmacytoid dendritic cells. This study presents the biologic and clinical features of a series of 23 such cases, selected on the minimal immunophenotypic criteria defining the DC2 leukemic counterpart, that is, coexpression of CD4 and CD56 in the absence of B, T, and myeloid lineage markers. Clinical presentation typically corresponded to cutaneous nodules associated with lymphadenopathy or spleen enlargement or both. Cytopenia was frequent. Circulating malignant cells were often detected. Massive bone marrow infiltration was seen in 20 of 23 (87%) patients. Most tumor cells exhibited nuclei with a lacy chromatin, a blastic aspect, large cytoplasm-containing vacuoles or microvacuoles beside the plasma membrane, and cytoplasmic expansions resembling pseudopodia. Other immunophenotypic characteristics included both negative (CD16, CD57, CD116, and CD117) and positive (CD36, CD38, CD45 at low levels, CD45RA, CD68, CD123, and HLA DR) markers. The prognosis was rapidly fatal in the absence of chemotherapy. Complete remission was obtained in 18 of 23 (78%) patients after polychemotherapy. Most patients had a relapse in less than 2 years, mainly in the bone marrow, skin, or central nervous system. Considering these clinical and biologic features, the conclusion is made that CD4(+)CD56(+) malignancies constitute a genuine homogeneous entity. Furthermore, some therapeutic options were clearly identified. Finally, relationships between the pure cutaneous indolent form of the disease and acute leukemia as well as with the lymphoid/myeloid origin of the CD4(+)CD56(+) malignant cell are discussed.