Bone marrow WT1 levels at diagnosis, post-induction and post-intensification in adult de novo AML.

We retrospectively assessed whether normalized bone marrow WT1 levels could be used for risk stratification in a consecutive series of 584 acute myeloid leukemia (AML) patients. A cutoff value of 5065 copies at diagnosis identified two prognostic groups (overall survival (OS): 44 ± 3 vs 36 ± 3%, P=0.023; leukemia-free survival (LFS): 47 ± 3 vs 36 ± 4%, P=0.038; and cumulative incidence of relapse (CIR): 37 ± 3 vs 47 ± 4%, P=:0.043). Three groups were identified on the basis of WT1 levels post-induction: Group 0 (WT1 between 0 and 17.5 copies, 134 patients, OS: 59 ± 4%, LFS:59 ± 4% and CIR: 26 ± 4%); Group 1 (WT1 between 17.6 and 170.5 copies, 160 patients, OS: 48 ± 5%, LFS:41 ± 4% and CIR: 45 ± 4%); and Group 2 (WT1 >170.5 copies, 71 patients, OS: 23 ± 6%, LFS: 19 ± 7% and CIR: 68 ± 8%) (P<0.001). Post-intensification samples distinguished three groups: patients with WT1 >100 copies (47 patients, 16%); an intermediate group of patients with WT1 between 10 and 100 copies (148 patients, 52%); and a third group with WT1 <10 copies (92 patients, 32%). Outcomes differed significantly in terms of OS (30 ± 7%, 59 ± 4%, 72 ± 5%), LFS (24 ± 7%, 46 ± 4%, 65 ± 5%) and relapse probability (CIR 72 ± 7%, 45 ± 4%, 25 ± 5%), all P<0.001. WT1 levels in bone marrow assayed using the standardized ELN method provide relevant prognostic information in de novo AML.

Adverse impact of IDH1 and IDH2 mutations in primary AML: experience of the Spanish CETLAM group.

The study of genetic lesions in AML cells is helpful to define the prognosis of patients with this disease. This study analyzed the frequency and clinical impact of recently described gene alterations, isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) mutations, in a series of homogeneously treated patients with primary (de novo) AML. Two-hundred and seventy-five patients enrolled in the CETLAM 2003 protocol were analyzed. IDH1 and IDH2 mutations were investigated by well-established melting curve-analysis and direct sequencing (R140 IDH2 mutations). To establish the percentage of the mutated allele a pyrosequencing method was used. Patients were also studied for NPM, FLT3, MLL, CEBPA, TET2 and WT1 mutations. IDH1 or IDH2 mutations were identified in 23.3% AML cases and in 22.5% of those with a normal karyotype. In this latter group, mutations were associated with short overall survival. This adverse effect was even more evident in patients with the NPM or CEBPA mutated/FLT3 wt genotype. In all the cases analyzed, the normal allele was detected, suggesting that both mutations act as dominant oncogenes. No adverse clinical impact was observed in cases with TET2 mutations. IDH1 and IDH2 mutations are common genetic alterations in normal karyotype AML. Favourable genotype NPM or CEBPA mutated/FLT3 wt can be further categorized according to the IDH1 and IDH2 mutational status.

Immunophenotype of acute myeloid leukemia with NPM mutations: prognostic impact of the leukemic compartment size.

NPM mutations are the most common genetic abnormalities found in non-promyelocytic AML. NPM-positive patients usually show a normal karyotype, a peculiar morphologic appearance with frequent monocytic traits and good prognosis in the absence of an associated FLT3 mutation. This report describes the immunophenotypic and genetic characteristics of a consecutive series of NPM-mutated de novo AML patients enroled in the CETLAM trial. Eighty-three patients were included in the study. Complete immunophenotype was obtained using multiparametric flow cytometry. Associated genetic lesions (FLT3, MLL, CEBPA and WT1 mutations) were studied by standardized methods. Real-time PCR was employed to assess the minimal residual status. The most common pattern was CD34-CD15+ and HLA-DR+. Small CD34 populations with immunophenotypic aberrations (CD15 and CD19 coexpression, abnormal SSC) were detected even in CD34 negative samples. Nearly all cases expressed CD33 (strong positivity), CD13 and CD117, and all were CD123+. The stem cell marker CD110 was also positive in most cases. Biologic parameters such as a high percentage of intermediate CD45+ (blast gate) (>75% nucleated cells), CD123+ and FLT3-ITD mutations were associated with a poor outcome. Quantitative PCR positivity had no prognostic impact either after induction or at the end of chemotherapy. Only PCR positivity (greater than 10 copies) detected in patients in haematological remission was associated with an increased relapse rate. Further studies are required to determine whether the degree of leukemic stem cell expansion (CD45+CD123+cells) increases the risk of acquisition of FLT3-ITD and/or provides selective advantages.

Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia.

Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.

Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)].

Most patients with acute myeloid leukemia (AML) and t(8;21) or inv(16) have a good prognosis with current anthracycline- and cytarabine-based protocols. Tandem analysis with flow cytometry (FC) and real-time RT-PCR (RQ-PCR) was applied to 55 patients, 28 harboring a t(8;21) and 27 an inv(16), including one case with a novel CBFbeta/MYH11 transcript. A total of 31% (n=17) of CR patients relapsed: seven with t(8;21) and 10 with inv(16). The mean amount of minimal residual disease (MRD) detected by FC in relapsed and nonrelapsed patients was markedly different: 0.3 vs 0.08% (P=0.002) at the end of treatment. The mean number of fusion transcript copies/ ABL x 10(4) also differed between relapsed and non-relapsed patients: 2385 vs 122 (P=0.001) after induction, 56 vs 7.6 after intensification (P=0.0001) and 75 vs 3.3 (P=0.0001) at the end of chemotherapy. Relapses were more common in patients with FC MRD level >0.1% at the end of treatment than in patients with < or = 0.1%: cumulative incidence of relapse (CIR) was 67 and 21% (P=0.03), respectively. Likewise, using RQ-PCR, a cutoff level of >10 copies at the end of treatment correlated with a high risk of relapse: CIR was 75% for patients with RQ-PCR >10 compared to 21% for patients with RQ-PCR levels < or = 10 (P=0.04). Combined use of FC and RQ-PCR may improve MRD detection, and provide useful clinical information on relapse kinetics in AML patients.

Genetic abnormalities associated with the t(12;21) and their impact in the outcome of 56 patients with B-precursor acute lymphoblastic leukemia.

The ETV6/RUNX1 rearrangement is found in 20-30% of children with B-cell precursor acute lymphoblastic leukemia and is associated with a good outcome. To determine the cytogenetic and molecular abnormalities associated with the ETV6/RUNX1 rearrangement and the influence of this rearrangement in patients' evolution, we analyzed the molecular cytogenetic profiles of 56 children with this rearrangement and B-cell precursor acute lymphoblastic leukemia. Secondary changes detected with conventional cytogenetics and with fluorescence in situ hybridization were found in 71.4% of cases, the most frequent being the loss of the normal ETV6 allele, 12p aberrations, duplication of the fusion gene, and trisomy 21, as in replicating the results of previous studies. In this preliminary series, with a mean follow-up of 69.3 months, secondary abnormalities did not influence patients' outcome. It seems therefore that the prognostic value of the t(12;21) does not vary and that ETV6/RUNX1 rearrangement is an independent indicator of good prognosis.

Clinical utility of bone marrow flow cytometry in B-cell non-Hodgkin lymphomas (B-NHL).

AIM: To determine the efficacy of flow cytometry (FC) in the assessment of bone marrow (BM) in B-cell non-Hodgkin lymphoma (B-NHL). FC is a common practice, but is far from being validated. METHODS AND RESULTS: Morphological analysis and FC immunophenotyping were performed on 421 samples. T-cell lymphomas, Hodgkin's disease, chronic lymphocytic leukaemia and hairy cell leukaemia were not included in the study. Clonality was assessed by the standard kappa/lambda/CD19 test. Aberrant immunophenotypes present in the B-cell subpopulation were also investigated. A double-step procedure was employed in all cases to increase the sensitivity of the FC procedure. Of 380 evaluable samples, 188 corresponded to follicular lymphoma (FL), 58 to diffuse large B-cell lymphoma (DLBCL), 57 to mantle cell lymphoma (MCL), seven to Burkitt's lymphoma and the remaining 70 samples to other low-grade lymphomas. Morphological marrow infiltration was found in 148 cases, and flow immunophenotyping identified 138 cases with BM involvement. A concordance between the two methods was detected in 298 cases (79%). There was a discordance in 82 cases (21%): morphology positive/FC negative in 46 cases and morphology negative/FC positive in 36 (61% of all cases with discordance were from FL). There was no difference in outcome when patients with discordances were compared with patients without discordances. CONCLUSIONS: Most samples showed concordance between morphological and FC results. FC identified BM involvement in the absence of morphological infiltration. Morphology/FC discordance seems to have no influence on the outcome of FL patients.

ETV6/RUNX1 rearrangement in childhood B-precursor acute lymphoblastic leukemia with normal karyotypes or without cytogenetic results.

The ETV6/RUNX1 rearrangement (also known as TEL/AML1) was evaluated in 39 children with B-precursor acute lymphoblastic leukemia (ALL) who had a normal karyotype or lack of mitoses. Forty-one point six percent of patients with normal karyotypes and 66.6% of patients without mitoses presented with the ETV6/RUNX1 rearrangement. In addition to this rearrangement, eight patients showed loss of the normal ETV6 allele; of three patients without mitoses, two showed an extra signal of the RUNX1 gene and the third showed the fusion gene duplicated and loss of the normal ETV6 allele. One patient without the ETV6/RUNX1 rearrangement and without mitoses showed two extra signals of the RUNX1 gene.

MEIS 1 expression is downregulated through promoter hypermethylation in AML1-ETO acute myeloid leukemias.

Retroviral insertional mutagenesis in BXH2 mice commonly induces myeloid leukemias. One of the most frequently involved genes in experimental studies is Meis 1. In contrast to other genes in murine models, Meis 1 has not been affected by recurrent chromosomal translocations or point mutations in human leukemias. We found a constant downregulation of the Meis 1 gene mRNA in AML1-ETO acute myeloid leukemias and in those cases harboring in frame mutations in the bZIP domain of CEBPalpha. The absence of the Meis 1 mRNA was not caused by inactivating point mutations in the coding sequence. Promoter hypermethylation was present in more than half of the cases (9/14), including samples obtained from the widely employed Kasumi-1 cell line. Double treatment with 5-Aza-2'-deoxycytidine and trichostatin A of the Kasumi-1 cell line partially reverses Meis 1 inhibition. HoxA9 levels were also low. In a cell line model (U937 Tet AML1-ETO), AML1-ETO expression was not associated with Meis 1 suppression at 72 h. Nevertheless, Meis 1 repression is dependent on the AML1-ETO transcript levels in treated leukemic patients. Chimeric products that arise from chromosomal translocations may be associated with locus-specific epigenetic inactivation. It remains to be investigated when this methylation process is acquired and which are the basic mechanisms underlying these molecular events in AML1-ETO and CEBPalpha-mutated AML.

Insertion of the CCND1 gene into the IgH locus in a case of leukaemic small cell mantle lymphoma with normal chromosomes 11 and 14.

The t(11;14)(q13;q32) translocation is considered to be the cytogenetic hallmark of mantle cell lymphoma. This report describes a case of leukaemic mantle cell lymphoma in which conventional cytogenetics on stimulated peripheral blood cells showed a 46,XY, t(1;12)(p21;q23)/46,XY karyotype. Fluorescence in situ hybridisation analysis using a dual colour immunoglobulin heavy chain (IgH)/CCND1 probe showed a fusion hybridisation signal on one normal chromosome 14, indicating that an insertion of the CCND1 gene into the 14q32/IgH locus had taken place. Overexpression of the cyclin D1 protein was demonstrated on bone marrow trephine by immunohistochemical staining.

MLL tandem duplication in two cases of acute myelocytic leukemia with unbalanced translocations: der(16)t(11;16)(q23;p13) and der(18)t(11;18)(q22;p11.2).

We describe two cases of acute myelocytic leukemia (AML), classified as M4 and M1 in the French-American-British classification, with unbalanced translocations der(16)t(11;16)(q23;p13) and der(18)t(11;18) (q22;p11.2), respectively. Molecular studies using Southern blot and reverse transcriptase-polymerase chain reaction showed an MLL rearrangement due to an internal duplication of the gene in both cases. Fluorescence in situ hybridization disclosed the presence of an extra copy of the MLL gene on 16p13 and 18p11.2, respectively, as a result of the partial trisomy of chromosome 11q. Our two cases clearly show that tandem duplication of the MLL gene may occur in AML with a partial 11q trisomy. Thus, systematic screening of this molecular defect should be performed in patients with unbalanced translocations involving 11q22 approximately q23-->qter.

Prognostic factors influencing feasibility of cytogenetic and molecular analysis in leukapheresis products in chronic myelogenous leukemia patients.

Conventional cytogenetic (CC) study and molecular analysis were performed in 150 leukapheresis products from 36 patients diagnosed with chronic myelogenous leukemia who were included in an autologous stem cell transplantation program. The aims of the study were to evaluate the effectiveness of these two methods for the detection of residual disease in the harvest and to identify the factors influencing the number of cycling cells present in the apheresis products. Progenitor cell mobilization procedures performed late after diagnosis (>12 months), a short interval between interferon-alpha discontinuation and mobilization (<3.5 months), and an intensive mobilization regimen (idarubicin, cytarabine, and etoposide, ICE protocol) were associated with a low probability of obtaining 25 metaphases, which was achieved in only 41 instances (25% of the samples). In 38 samples, less than ten metaphases were obtained; a peripheral blood leukocyte count <1.0x10(9)/l at mobilization and mononuclear cell counts in the bag <0.5x10(8)/kg significantly increased the probability to obtain less than ten metaphases for CC analysis. Previous interferon-alpha treatment during > or =12 months and low mononuclear cell counts in the bag (<0.5x10(8)/kg) increased the probability of not obtaining mitosis for cytogenetic analysis. Molecular analysis by the polymerase chain reaction (PCR) technique did not give discriminate information in the samples not evaluable by cytogenetics due to the high frequency of PCR-positive results. We conclude that new techniques such as hypermetaphase fluorescence in situ hybridization (FISH), interphase FISH, or quantitative PCR need to be routinely employed in the study of leukapheresis samples of chronic myelogenous leukemia patients for a better assessment of the neoplastic contamination of the infused products.

Interleukin-3 receptor alpha chain (CD123) is widely expressed in hematologic malignancies.

BACKGROUND AND OBJECTIVES: The hematopoietic system is controlled by growth factors (cytokines) which can influence cell survival, proliferation, differentiation and functional activation. The study of cytokine receptor expression by flow cytometry could allow us to differentiate between normal and tumoral cells. DESIGN AND METHODS: We analyzed the expression of the interleukin-3 (IL-3) receptor a chain (CD123) in 22 normal samples and in a wide panel of hematologic malignancies using flow cytometry. We found that CD123 was expressed in the myeloid progenitor subpopulation but in contrast, normal lymphoid progenitors lacked CD123. We analyzed the CD123 expression pattern in 64 patients with acute leukemia, 45 with acute myeloid leukemia (AML) and 19 with acute lymphocytic leukemia (ALL) (13 B-cell lineage ALL and 6 T-cell lineage ALL). RESULTS: All the AML cases except two patients with M7 and all the B-cell lineage ALL patients were CD123 positive. In contrast, all the T-cell lineage ALL cases tested were CD123 negative. We also studied the CD123 expression pattern in 122 patients with a B-cell chronic lymphoproliferative disease (B-CLPD). CD123 was positive in three situations: 1) typical cases of hairy cell leukemia showed a specific, strong CD123 expression, 2) a subgroup of atypical chronic lymphocytic leukemia with a marked CD11c expression was also CD123 positive, and finally 3) transformed B-CLPD showed the phenomenon of transition from CD123 negativity to CD123 positivity simultaneuosly with morphologic changes. INTERPRETATION AND CONCLUSIONS: In summary, our data show high expression of IL-3 receptor a chain in hematologic malignancies. Given the high frequency of CD123 reactivity in blast cells in contrast to in normal precursors, this antigen could be applied to the study of minimal residual disease in acute leukemia. CD123 is expressed with a characteristic pattern in cases of hairy cell leukemia.

Usefulness and reproducibility of cytomorphologic evaluations to differentiate myeloma from monoclonal gammopathies of unknown significance.

We attempted to differentiate monoclonal gammopathies of unknown significance (MGUS) and multiple myeloma (MM) on morphologic grounds and to determine interobserver reproducibility of the differentiation. Cytologists blindly evaluated bone marrow smears from 154 patients with bone marrow plasmacytosis for the proportion of plasma cells with predefined cellular atypias. The single morphologic characteristic that most strongly differentiated MM from MGUS was the presence of nucleoli. The percentage of plasma cells, cytoplasmic contour irregularities, and anisocytosis also predicted a diagnosis of myeloma in multivariate analysis. Six cytologists independently evaluated 68 consecutive cases to determine sensitivity and specificity of these cytomorphologic features. The interobserver coefficient of variation for the plasma cell count was 33%. On consideration of the diagnosis, 36 of 41 MGUS cases and all 24 cases of myeloma were classified correctly. The use of a predesigned score system did not present such a bias, although it did not improve overall efficiency. The plasma cell count is the most predictive characteristic of myeloma from a cytologic viewpoint, but the interobserver variability is high. Interobserver variability is also high in the assessment of morphologic atypia, and atypical traits are not uncommon in plasma cells in MGUS.