Major impact of an early bone marrow checkpoint (day 21) for minimal residual disease in flow cytometry in childhood acute lymphoblastic leukemia.

The early persistence of minimal residual disease (MRD) is considered a poor prognostic factor indicative of chemoresistance in acute lymphoblastic leukemia. In French children, chemosensitivity is assessed at day 21 post-induction by cytomorphology. Here, it was investigated whether a more precise evaluation could be obtained at this time point with multiparameter flow cytometry (MFC). This study enrolled 123 children with de novo acute lymphoblastic leukemia. MRD0 was investigated at day 21 in MFC with a combination of antibodies based on the immunophenotype of diagnosis. It was also evaluated at day 35 by immunoglobulin/T-cell receptor quantitative real-time polymerase chain reaction (MRD1). Three risk groups could be delineated based on MRD0. Patients with MFC/MRD0 levels >10-2 (n = 25) were considered high risk, those with levels between 10-2 and 10-4 (n = 46) intermediate risk, and those <10-4 (n = 50) low risk. Overall survival (p = 0.048) and event-free survival (EFS, p = 0.00017) were significantly different between these three groups. EFS of the 14 corticoresistant patients strongly depended on their MRD0 level (p = 0.004). Similarly, both EFS (p = 0.0004) and overall survival (p = 0.02) were significantly different in the 109 chemosensitive patients, according to MRD0 levels. MRD0 and MRD1 levels, compared with 112 patients, were consistent (-/- or +/+) in 57.2% of the cases. Both MRD0+/MRD1+ and MRD0+/MRD1- patients had a significantly worse EFS (p = 0.0001) than those with undetectable MRD at both MRD0 and MRD1. This study confirms the usefulness and superiority of an early point of MRD detection by MFC. In addition, MRD0 in MFC identifies a subgroup of patients with poorer prognosis (MRD0+/MRD1-). Copyright © 2015 John Wiley & Sons, Ltd.

CD304 is preferentially expressed on a subset of B-lineage acute lymphoblastic leukemia and represents a novel marker for minimal residual disease detection by flow cytometry.

Minimal residual disease (MRD) has emerged as a major prognostic factor for monitoring patients with B-lineage acute lymphoblastic leukemia (B-ALL). The quantification of MRD by flow cytometry (FC) is based on the identification of a leukemia-associated phenotype (LAP). Because phenotypic switch is common during treatment, multiple LAPs must be available and used for MRD detection over time. We evaluated the potential usefulness of CD304 as a new marker for monitoring MRD. CD304 was expressed in 48% of B-ALL (24/50) with discriminative fluorescence intensity compared with CD304-negative normal B-cell precursors (n = 15). The sensitivity of CD304-based MRD detection reached 10(-4), as with some of established LAPs. The stability of CD304 expression evaluated during therapy and at relapse confirms the usefulness of this marker for MRD quantification. Finally, CD304 was repeatedly expressed in patients with TEL-AML1 gene rearrangement, which warrants further investigation on its potential relevance as a prognosis marker or therapeutic target.

Clinical features and course of refractory anemia with ring sideroblasts associated with marked thrombocytosis.

BACKGROUND: Refractory anemia with ring sideroblasts associated with marked thrombocytosis was proposed as a provisional entity in the 2001 World Health Organization classification of myeloid neoplasms and also in the 2008 version, but its existence as a single entity is contested. We wish to define the clinical features of this rare myelodysplastic/myeloproliferative neoplasm and to compare its clinical outcome with that of refractory anemia with ring sideroblasts and essential thrombocythemia. DESIGN AND METHODS: We conducted a collaborative retrospective study across Europe. Our database included 200 patients diagnosed with refractory anemia with ring sideroblasts and marked thrombocytosis. For each of these patients, each patient diagnosed with refractory anemia with ring sideroblasts was matched for age and sex. At the same time, a cohort of 454 patients with essential thrombocythemia was used to compare outcomes of the two diseases. RESULTS: In patients with refractory anemia with ring sideroblasts and marked thrombocytosis, depending on the Janus Kinase 2 V617F mutational status (positive or negative) or platelet threshold (over or below 600 × 10(9)/L), no difference in survival was noted. However, these patients had shorter overall survival and leukemia-free survival with a lower risk of thrombotic complications than did patients with essential thrombocythemia (P<0.001) but better survival (P<0.001) and a higher risk of thrombosis (P=0.039) than patients with refractory anemia with ring sideroblasts. CONCLUSIONS: The clinical course of refractory anemia with ring sideroblasts and marked thrombocytosis is better than that of refractory anemia with ring sideroblasts and worse than that of essential thrombocythemia. The higher risk of thrombotic events in this disorder suggests that anti-platelet therapy might be considered in this subset of patients. From a clinical point of view, it appears to be important to consider refractory anemia with ring sideroblasts and marked thrombocytosis as a distinct entity.

Morphology, cytogenetics, and survival in myelodysplasia with del(20q) or ider(20q): a multicenter study.

Isochromosome of the long arm of chromosome 20 with interstitial loss of material [ider(20q)] is a rare cytogenetic abnormality reported in myelodysplastic syndrome (MDS), with neither specific morphological pattern nor clear prognostic significance. The aim of this retrospective multicentric study is to compare the peripheral blood and bone marrow morphology of MDS patients with ider(20q) (n = 13) and del(20q) (n = 21) and controls (n = 47) in order to investigate whether the ider(20q) harbors specific morphological features. The secondary objective is to compare the outcome of patients from both groups. This study performed on the largest cohort of MDS patients with ider(20q) is the first that identifies specific morphological features (hypogranulated and vacuolized neutrophils and neutrophil erythrophagocytosis) allowing the identification of this cytogenetic abnormality with high sensitivity (70%) and specificity (85.7%). Suspected ider(20q) by morphology should therefore support targeted FISH tests in case of non informative karyotype. This combined approach will allow a better estimation of the prevalence of this underdiagnozed entity. The overall survival and progression-free survival did not statistically differ in both groups. However, hypogranulated and vacuolized neutrophils were significantly associated with survival.

Childhood near-tetraploid acute lymphoblastic leukemia: an EGIL study on 36 cases.

OBJECTIVES: Patients with near-tetraploid (karyotype: 81 - 103 chromosomes) acute lymphoblastic leukemia (NT-ALL) constitute about 1% of childhood ALL and data reported on them are limited and controversial. The aim of the study was to enlarge the knowledge on these rarely occurring ALL. METHODS: The members of the European Group for Immunophenotyping of Leukemias (EGIL) searched retrospectively their databases for NT-ALL patients. RESULTS: We collected data of 36 European children from seven European countries with NT-ALL diagnosed since 1992. All patients reached complete remission (CR) after induction chemotherapy. Their blasts were negative for peroxidase and BCR-ABL1. Ten children were diagnosed as T-cell ALL (T-ALL) EGIL categories (T-I n=2, T-II n=2, T-III n=3, T-IV n=3) and four displayed various structural chromosomal abnormalities. Eight of 10 T-ALL remained in 1st CR; one died in CR from sepsis and one is alive in 2nd CR. Median survival was 88 (7-213) months. B-cell precursor (BCP) ALL was diagnosed in 26 children. Thirteen were positive for ETV6-RUNX1 and are alive in 1st CR for 32-147 months. Ten children were ETV6-RUNX1 negative and remained in 1st CR for 16-163 months. One girl with hypodiploid and NT metaphases and ETV6-RUNX1-negative BCP-ALL and one of two boys with NT-BCP-ALL not examined for ETV6-RUNX1 died of infection after stem cell transplantation in 2nd/3rd CR. Secondary myelodysplastic syndrome developed in two patients with NT-BCP-ALL. CONCLUSIONS: Our data demonstrate immunophenotypic, cytogenetic, and molecular heterogeneity of NT-ALL and favorable prognosis of most NT-ALL across different immunophenotypic and/or genetic ALL subtypes.

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.

Combination of chemotherapy and gemtuzumab ozogamicin in adult Philadelphia positive acute lymphoblastic leukemia patient harboring CD33 expression.

Gemtuzumab Ozogamicin (GO) is a humanized anti-CD33 antibody conjugated with a cytotoxic antitumor antibiotic, calicheamicin-g1. It was developed at the end of the nineties as 90% of the leukemic blast population of patients with acute myeloid leukaemia (AML) express the CD33 surface antigen (Dinndorf et al. [1] Blood 1986;67:1048-53). GO is currently approved in monotherapy for the treatment of CD33+ AML patients in first relapse, showing a 26% overall response rate and a median disease-free-survival of 5.2 months for responders (Larson et al. [2] Cancer 2005;104:1442-52). CD33 antigen expression is also observed at diagnosis (in 15% of cases) (Pui et al. [3] J Clin Oncol 1998;16:3768-73) or at relapse (Guglielmi et al. [4] Leukemia 1997; 11:1501-7) of acute lymphoblastic leukaemia (ALL), representing a potential cellular target for ALL patients. Case series have already demonstrated the efficacy of GO in children with relapsed CD33+ ALL with documentation of complete remission (CR) (Balduzzi et al. [5] Leukemia 2003;17:2247-8; Cotter et al. [6] Br J Haematol 2003;122:686-91; Zwaan et al. [7] Leukemia 2003;17:468-70). In the other hand, there is no report at our knowledge of the use of GO in the setting of adult CD33+ ALL patient. Here we report the case of a 30-year-old man with a refractory CD33+ ALL who received a salvage regimen combining chemotherapy + GO and achieved a transient CR.

The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement.

BACKGROUND AND OBJECTIVES: Diffuse large B-cell lymphomas (DLBCL) are common lymphomas that have been classified into three subgroups on the basis of their patterns of gene expression. The aim of this study was to characterize the clinical, biological, immunophenotypic and cytogenetic features of DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement. DESIGN AND METHODS: Sixteen cases of DLBCL with the dual translocation were identified between 1998 and January 2006. The clinical features of these cases were examined and morphological, immunohistochemical, flow cytometric and cytogenetic analyses were performed. RESULTS: All patients had aggressive features: B symptoms (81%), ECOG performance status >2 (81%), elevated lactate dehydrogenase (100%), stage IV disease (100%) with at least one extra-nodal localization (bone marrow, blood and central nervous system involvement in 93%, 50% and 50%, respectively) and age-adjusted IPI score of 3 in 81%. Despite intensive chemotherapy regimens (including allogeneic transplants), all patients died of disease progression. Progression-free and overall survival was 4 and 5 months, respectively. Immunophenotyping analysis (CD20, CD10, Bcl-6, Mum-1, Bcl-2 CD138, MIB1, CD19, CD5, CD38 and sIg) was performed and showed DLBCL with a germinal center (GC) profile. Ki-67 staining ranged from 70 to 90%. All cases assessed by cytogenetics analysis [conventional cytogenetic and/or fluorescence in situ hybridization (FISH)] had a complex karyotype. In one case, we identified a 8q24/c-MYC translocation variant never reported in DLBCL before: t(8;9)(q24;p13) and t(14;18)(q32;q21). The BCL-6 rearrangement was investigated by FISH and found to rearranged in four cases. INTERPRETATION AND CONCLUSIONS: In conclusion, DLBCL with concurrent t(14;18) and 8q24/c-MYC rearrangement is a subgroup of GC-DLBCLwith poor outcome. It is worth searching for the coexistence of dual translocations in Bcl-2-positive DLBCL with unusual aggressive presentation.

Administration of mylotarg 4 days after beginning of a chemotherapy including intermediate-dose aracytin and mitoxantrone (MIDAM regimen) produces a high rate of complete hematologic remission in patients with CD33+ primary resistant or relapsed acute myeloid leukemia.

We have used the dose of 9 mg/m(2) of mylotarg 4 days after the beginning of a chemotherapy including intermediate-dose aracytin and mitoxantrone (MIDAM) in 17 patients with refractory (n=4) or relapsed (n=13) AML. Thirteen patients (76%) achieved CR (n=12) or partial CR (n=1). All four refractory patients and all four patients with poor risk cytogenetic achieved CR or CRp. Although the dose of mylotarg given in combination with chemotherapy was not reduced, the toxicity profile was acceptable (1VOD/17 patients). The MIDAM protocol appears to be highly effective especially in patients with poor risk cytogenetic and/or refractory disease.

Four-color flow cytometry bypasses limitations of IG/TCR polymerase chain reaction for minimal residual disease detection in certain subsets of children with acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: Competitive immunoglobulin/T-cell receptor polymerase-chain reaction (PCR) analysis with fluorescent detection is a rapid, cheap and reproducible method for quantifying minimal residual disease (MRD), which is well adapted to the recognition of high-risk childhood acute lymphoblastic leukemia (ALL). We aimed at defining whether flow cytometry (FC) techniques can bypass limitations of PCR for MRD determination. DESIGN AND METHODS: We analyzed 140 remission samples from 91 patients using both competitive PCR amplification of antigen-receptor genes and four-color FC identification of leukemia immunophenotype. These methods were chosen with the aim of detecting at least 0.1% blasts. RESULTS: MRD was measured using both PCR and FC methods in 123 samples and the two methods provided concordant results in 119 of them (97%). Moreover, three out of the four discordant results appeared minor since MRD was detectable by both methods, but at different levels. In 12 of 13 samples from nine patients, mainly infants with early CD10- and/or t(4;11) B-cell ALL and children with immature T-cell ALL, MRD could be determined using FC whereas PCR failed. Conversely, FC methods were unfeasible due to inappropriate leukemia immunophenotype in three additional children (including two with T-cell ALL) for whom PCR successfully provided MRD results. INTERPRETATION AND CONCLUSIONS: The MRD results provided by FC techniques were highly concordant with those of competitive PCR. Moreover, the applicability of FC appeared higher in certain ALL subsets, although the appropriateness of this technique in terms of outcome prediction remains to be demonstrated.

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.

14q32 Translocations discriminate IgM multiple myeloma from Waldenstrom's macroglobulinemia.

Even though the diagnosis of Waldenstrom's macroglobulinemia WM is usually clear, the differential diagnosis with IgM multiple myeloma (MM) might be possible. IgM MM is usually characterized by the accumulation of small mature plasma cells within the bone marrow, and the detection of a monoclonal IgM in the serum. However, in contrast with classical MM, IgM MM is rarely associated with these patients' extensive osteolytic lesions. We analyzed eight cases of IgM MM. None presented with extensive bone lesions. All cases were characterized by the presence of small mature plasma cells within the bone marrow. Molecular cytogenetic analysis revealed a t(11;14) in seven of the eight cases. In contrast, a similar analysis in 17 WM cases failed to detect any t(11;14) cases. We performed further fluorescence in situ hybridization (FISH) experiments, focused on the 14q32 region, and especially on the IgH gene. In contrast to MM (in which illegitimate IgH rearrangements are common), we did not detect any abnormality in the WM cases. In conclusion, even though the cells of origin in WM and MM are mature heavily mutated cells, they differ by the IgH gene rearrangements. Especially in IgM MM, the search for t(11;14) might be useful in difficult cases to discriminate with WM.

Acute megakaryoblastic leukaemia: a national clinical and biological study of 53 adult and childhood cases by the Groupe Français d'Hématologie Cellulaire (GFHC).

Since the WHO classification of haematological malignancies recommended the description of global entities, we performed a national M7-AML study to correlate morphological, immunological and cytogenetic features, and to find new clinically relevant M7 entities. This study is based on accurate morphological and immunological study to select pure megakaryoblastic proliferations and to eliminate megakaryocytic participation in haemopathies. We collected 53 cases: 23 adults and 30 children. We confirm the wide heterogeneity of adult M7. In adults, the cytogenetic abnormalities are frequently those of secondary leukaemia while a few patients have a previous history and morphological features of dyshaematopoiesis; their outcome is very poor. Among children, besides the well-known Down syndrome M7, we in particular, studied ten t(1;22) M7 and one OTT-MAL transcript positive case with normal karyotype presenting specific features. We were already aware of their younger age, female and tumoral presentation, but we also found a lower percentage of bone marrow blasts, sometimes without any megakaryoblastic bone marrow involvement, but always, with a dysmegakaryocytopoiesis associated with micromegakaryocytes. They are generally good responders to intensive AML chemotherapy with very long disease-free survivals (DFS). Accordingly, OTT-MAL transcript study, in infant M7 with normal karyotype, is recommended and we feel that this entity should be added to the WHO AML classification.

Normal levels of peripheral CD19(+) CD5(+) CLL-like cells: toward a defined threshold for CLL follow-up -- a GEIL-GOELAMS study.

BACKGROUND: The development of flow cytometry as a useful tool for the detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) is potentially hampered by the fact that a normal subset of B-cells with a similar immunophenotype is present in the peripheral blood. This subset of CLL-like cells is not well defined in terms of frequency. METHODS: Here, we performed a multicenter study with a panel of four-color antibody combinations possibly useful for the detection of MRD in CLL, to establish the levels of normal CLL-like cells in 49 healthy controls. ROC curves established the upper level of such cells at 4 × 10(-4) . The two best combinations were further applied to 419 samples from 117 treated CLL patients. RESULTS: The combinations CD19/CD5/CD43/CD79b and CD19/CD5/CD81/CD22 appeared very robust and well correlated to enumerate normal CLL-like cells in a lysis no-wash approach. In follow-up samples from CLL patients, they disclosed only 9.8% of the samples within the normal range. In more than 90% of the cases, it was thus possible to report confidently on the absence or presence of MRD in these patients. CONCLUSIONS: This manuscript reports on the frequency of CD19(+) CD5(+) B-cells in normal peripheral blood and confirms the combinations recommended by the European research initiative on CLL as being performing to assess remaining CLL cells above a threshold of 4 × 10(-4) white blood cells.

Evaluation of a telemedicine system for the transmission of morpho/immunological data aiming at the inclusion of patients in a therapeutic trial.

Due to their high levels of achievement and efficiency, image digitalization and teletransmission tools are more and more frequently used. Applied to cellular haematology, these tools often contribute to diagnosis confrontation, sometimes within the framework of therapeutic trials. We present one of the first approaches of the use of telehaematology for the inclusion of patients in the GOELAMS chronic lymphocytic leukaemia 98 trial. The advantages were (1) the creation of a unique, protected, stable data bank that could be remotely consulted, (2) the use of digitized pictures which made expertise on identical documents possible, (3) the facility of computer exchanges between experts, in terms of reception as well as replying time delays. We were able to set out new standards of image sampling for CLL, solve the semantic divergences, and point out interobserver variability as regards morphology. The limiting factors were the important need for expert investment, but they more importantly concerned the first line morphologists who should benefit from adequate tools, in terms of computer equipment as well as members of staff, so as to apprehend this second reading system as a quality control procedure.

A phase II trial of high-dose imatinib mesylate for relapsed or refractory c-kit positive and Bcr-Abl negative acute myeloid leukaemia: the AFR-15 trial.

This was a phase II investigation of high-dose imatinib in 15 adult patients with relapsed or refractory c-kit positive and Bcr-Abl negative acute myeloid leukaemia (AML). Imatinib 600 mg daily was administered for 1 month followed by dose escalation to 800 mg daily for a maximum of 2 months. No clinical responses were reported with early death due to disease progression reported in 7 patients. While no activity was seen in this phase II trial, the findings of the study do not rule out efficacy in subsets of AML with imatinib-sensitive Kit mutations.