The expression level of BAALC -associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (⩾60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P =0.0025), shorter leukemia-free survival (P =0.026) and higher cumulative incidence of relapse (P =0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P =0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML

The lincRNA HOTAIRM1, located in the HOXA genomic region, is expressed in acute myeloid leukemia, impacts prognosis in patients in the intermediate-risk cytogenetic category, and is associated with a distinctive microRNA signature

Altres ajuts: SDCSD from School of Medicine, University of BarcelonaLong non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown.We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients.The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6;9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival (OR:2.04;P = 0.001), shorter leukemia-free survival (OR:2.56; P < 0.001) and a higher cumulative incidence of relapse (OR:1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33-microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004).Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signatur

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by diff

2001; 12

acute leukemia. CD123 is expressed with a characteristic pattern in cases of hairy cell leukemia. ©2001, Ferrata Storti Foundation Key words: acute leukemia, B-cell lymphoproliferative disorders, CD123 expression, IL-3 receptor α chain, hairy cell leukemia, minimal residual disease. © F e r r a t a S t o r t i F o u n d a t i o n er signal-regulatory proteins such as CD90, AC133 and CD117. Antigen expression is probably a gradually increasing or decreasing process reflecting the fluent maturation progress and at the end of this process differentiated cells display distinct patterns of antigen expression. 11 Acute myeloid leukemias (AMLs) are considered to be clonal disorders involving early hematopoietic progenitor cells. Differences and similarities in phenotype, genotype and biology are described for leukemic cells and normal hematologic progenitors. One potential difference between normal and leukemic cells lies in their response to hematopoietic growth factors. Design and Methods Patients Bone marrow (BM) n=12 , peripheral blood (PB) n=7 and non-neoplastic lymph nodes (LN) n=3, from healthy donors were used as controls. Sixtyfour BM samples from patients with acute leukemia (AL) were analyzed for the expression of the IL-3α chain receptor (IL-3αR). The patients were categorized as follows: 45 with acute myeloid leukemia (AML) and 19 with acute lymphoblastic leukemia (ALL), 13 with B-cell lineage ALL and 6 with T-cell lineage ALL. The diagnosis of AL was based on standard morphologic and immunophenotypic criteria. Flow cytometry analysis Sample preparation. The number of cells was quantified by microscopy and adjusted to 2×10 6 in each tube. The immunophenotypic analysis was performed on lysed whole BM samples with direct conjugated monoclonal antibodies (MoAbs). Antigen expression was analyzed using triple combinations of the following MoAbs conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinine chlorophyll protein (PerCp) or phycoerythrin-cyanine 5 (PE/Cy 5) fluorochrome tandem. The MoAbs used in the study were: CD22 (4KB128 FITC), glycophorin A (JC 159 PE), CD41 (5B 12 PE), IgM (rabbit anti-human, PE), CD79a (HM57 PE) and TDT (HT-6 FITC) from DAKO, Glostrup, Denmark; CD15 (MMA-FITC), CD34 (8G12-FITC, PE), HLA-Dr (L243 PetCp), CD10 (W8E7 FITC), CD 20 (L27 PE), CD2 (S5.2 FITC), CD33 (67.6 PE), CD7 (4H9 FITC), CD45 (2D1 PerCp), CD13 (L138 PE), CD14 (M0P9 FITC), CD3 (SK7 PerCp), CD4 (Leu 3 FITC), CD5 (Leu 1 FITC), CD8 (Leu 2 PE) purchased from Becton Dickinson, San José, CA, USA (BDIS); CD19 (SJ25-C1 PE/Cy 5) and MPO (H-43-5 FITC) from Caltag Laboratories, Burlingame, CA, USA; CD 123 (9F5 PE), CD10 (HI10a, Cy-Chrome) from Pharmingen, San Diego, CA, USA; CD36 (FAG-52 FITC) from Immunotech, Marseille, France. In B-CLPD samples a different panel of monoclonal antibodies was used to study the complete immunophenotype of each clonal sample: FMC7-FITC (Harlan SeraLab, Sussex), CD103-FITC (Immunoquality, Groningen), CD-10 PE-Cy5, CD123 (9F5 PE), (Pharmingen, San Diego, CA, USA), CD19-RPe/Cy5, CD22-FITC and CD79b-FITC (Dako), CD5-PE, CD23-PE, CD25-PE, CD20-PE, CD10-FITC, CD11c-PE, CD10-FITC, CD11c-PE (Becton Dickinson, San José, CA, USA). The clonality study of B-lymphocytes was undertaken using a triple reagent consisting of a combination of κ-FITC, λ-PE and CD19 PE/Cy5 in a single tube (K/L, Simultest® purchased from Becton Dickinson, San José, CA, USA and CD19-PE/Cy5 from Caltag, San Francisco, USA). Direct immunofluorescence was performed by incubating 2×10 6 cells with the specific MoAb for 15 minutes in the dark at room temperature. An isotype-matched negative control (BDIS) was used in all cases to assess background fluorescence intensity. Cells were lysed (FACS Lysis solution, BDIS) for 3 to 5 minutes and centrifuged at 250 g for 5 min. The cells were washed twice with phosphate buffered saline (PBS) before being resuspended in PBS and examined. Measurements were performed on a FACScan flow cytometer (BDIS). For data acquisition the LYSIS-II (BD) software program (BDIS) was used. At least 10,000 events/tube were measured. The PAINT-A-GATE PRO software program (BDIS) was employed for further data analysis. Thresholds for positivity were based on isotype negative controls. Analytical gates were set on desired viable cells based on forward light scatter and side light scatter. The positivity threshold was 20% for all markers except for cytoplasmic or intranuclear antigens for which a 10% threshold was used. The normal precursor cells were analyzed using a two-step acquisition procedure. In the first step, acquisition of 10,000 cells was performed and information stored for all these events. In the second step, a minimum of 300,000 cells was measured, information being stored only for the precursor cells, which were acquired employing a preestablished CD34 live gate. The level of fluorescence in cells which expressed CD123 was measured in arbitrary units as the mean fluorescence intensity (MFI). Results Normal expression of the IL-3 αR in bone marrow, peripheral blood and lymph nodes In control BM from healthy volunteers, CD123 was expressed in 0.27% (0.1%-0.6%) of the total nucleated cells. In the precursor cell compartment, the percentage of CD123 expression was 53%, ranging between 29% and 78% of the CD34 + cells. We studied the expression of this interleukin receptor in myeloid and lymphoid progenitor subpopulations. In the myeloid compartment (CD34 + CD33 + CD19 -) CD123 was positive in 63% (38%-100%) of cells with a dim-moderate intensity (MFI= 33 ± 21). In contrast, CD123 was negative in normal lymphoid progenitors (CD34 + CD33 -CD19 + CD10 + ) We also analyzed CD123 expression in PB. In our samples, cells with the strongest CD123 expression corresponded to dendritic cells, monocytes were positive for CD123 but showed a very faint reactivity and there was a minor population of B-cells expressing CD123. Granulocytes appeared to be CD123 negative. We tested the CD123 expression in 3 normal lymph nodes. In all the cases the CD19 + cells showed dim CD123 expression (MFI= 16± 12) and T-cells were CD123 negative. IL-3 αR expression in AML CD123 was expressed in 42 out of 45 AML samples assayed (93%). Regarding the FAB classification, CD123 expression was detected in all subtypes except in patients with a megakaryoblastic phenotype (n=2) The IL-3 αR pattern was similar in all the AML cases. CD123 was expressed in the totality of the blast cell population with a homogeneous pattern. The intensity of the expression was moderate (MFI= 69±40) ( CD34 expression was analyzed in the blast cells of AML patients. We found no differences in CD123 expression between CD34 + and CD34 -cases. Specific molecular lesions in AML were studied in order to detect differences in IL-3 αR. Six patients were PML/RARα positive, two patients had MLL rearrangements, and two patients were CBFβ/MYH11 positive. No differences in the CD123 pattern expression were found in patients with these molecular lesions. © F e r r a t a S t o r t i F o u n d a t i o n IL-3 αR expression in ALL In ALL samples, IL-3 αR was restricted to the Bcell lineage. Molecular lesions were studied in order to compare the CD123 expression in different biological subgroups. Three patients were bcr/abl positive and one patient had c-myc rearrangements. No differences in CD123 expression were found in these patients harboring specific molecular lesions. CD123 was expressed in all B-cell ALL samples (n=13) and this high expression was in striking contrast to lack of expression of CD123 on normal lymphoid precursors. The intensity of the expression in ALL was higher than in AML samples (MFI= 119±84) IL-3 αR expression in B-cell chronic lymphoproliferative disorders We analyzed IL-3 αR expression in 122 samples of BM, PB or lymph nodes from patients with B-CLPD. In the CLL group (n=77), 7 patients showed CD123 reactivity MFI= 41±42) ( Interestingly, 3 out of 4 patients with aggressive B-cell CLPD (two patients with transformed NHL from mantle and follicular lymphoma and one patient with Burkitt&apos;s lymphoma) expressed CD123 with a moderate intensity. The results in the hairy leukemia group were striking. In 6 out of 7 patients studied CD123 was positive with strong intensity, (MFI=120±288) ( © F e r r a t a S t o r t i F o u n d a t i o n Discussion Interleukin-3 is a regulatory glycoprotein known to support the survival, proliferation, and development of progenitor cells from multiple hematopoietic lineages. We investigated IL-3 αR expression in bone marrow and in peripheral blood from normal samples in order to establish normality patterns. In our study, normal lymphoid precursors did not show CD123 reactivity and only a proportion of myeloid progenitors expressed CD123 with a moderate intensity. These findings are in agreement with results from previous authors 13 who found a small CD123 expression in the normal CD34 + population while CD123 was negative in the more primitive CD34 + /CD38 -compartment. In contrast with the small expression of receptor in normal hematopoietic precursors, we found IL-3 αR expression in the majority of myeloid and B-cell leukemic cells. This feature may be useful in MRD studies. 14 Our data show that the combined use of CD123/CD34 with B-cell associated antigens such as CD19/CD10 may be a great help for identifying residual leukemic cells in B-cell leukemia cases. In addition, the CD123 + /CD34 + /CD33 + is a minor phenotype in normal BM. The detection of cells with this phenotype in a very superior number to the standard range could suggest the presence of myeloid leukemic cells. The wide expression of this marker in the majority of AML and B-cell lineage ALL cases analyzed and the low CD123 expression in normal hematopoietic stem cells suggest that IL-3 αR could be a suitable target for immunotherapeutic intervention. © F e r r a t a S t o r t i F o u n d a t i o n expressed in the majority of aggressive or transformed B-CLPD analyzed. We observed the acquisition of CD123 simultaneously with blast transformation in one patient with a mantle-cell lymphoma ( In conclusion, we have shown that CD123 is a good marker of myeloid and B-cell lymphoid leukemic cells and this feature could be used in MRD analysis. CD123 is expressed with a characteristic pattern in HCL cases and this antigen could be important in the differential diagnosis of B-CLPD. Nevertheless, further analysis is necessary to understand better the biological function of the IL-3 αR and the mechanisms by which this receptor transduces possible aberrant regulatory signals in malignant cells. If the biological role of the CD123 is confirmed, this antigen could be used as a target in the selective destruction of malignant cells in the majority of myeloid and B-cell leukemia patients. © F e r r a t a S t o r t i F o u n d a t i o n Contributions and Acknowledgments LM was primarily responsible for the article, was responsible for the analysis and interpretation of data and drafted the article. JFN was responsible for its conception and revised it critically for impo

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real time quantitative PCR

Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/?l. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f)

GENETIC LESIONS ASSOCIATED WITH BLASTIC TRANSFORMATION OF POLYCYTHEMIA VERA AND ESSENTIAL THROMBOCYTHEMIA

Polycythemia vera (PV) and essential thrombocythemia (ET) are chronic myeloproliferative disorders that may progress to acute leukemia in a subset of patients. This study aimed at investigating the genetic lesions associated with the blastic transformation of PV and ET. A panel of PV and ET cases at different stages of disease was analyzed for the presence of genetic alterations of TP53, NRAS, KRAS, and MDM2 by a combination of mutational analysis and Southern blot hybridization. The occurrence of microsatellite instability (MSI) was also tasted in selected cases. Samples of PV and ET analyzed in chronic phase disease were consistently devoid of all genetic lesions tested, suggesting that alterations of TP53, NRAS, KRAS, and MDM2 do not contribute significantly to development of chronic phase PV and ET. Conversely, mutations of TP53 were detected in 7/15 (46.6%) blastic phase cases, including 3/5 PV and 4/10 ET. In blastic phase patients for whom the corresponding chronic phase DNA was also available, it could be documented that the genetic lesion had arisen at the time of blastic transformation. In addition to TP53 mutations, cases of blastic phase PV and ET occasionally harbored mutations of NRAS (one case of blastic phase ET) or displayed MSI (one case of blastic phase PV). These data indicate that inactivation of TP53 is a relatively frequent event associated with the blastic transformation of PV and ET and may be responsible for the tumor progression of these disorders