Natalie P. H. Chan

Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia

RUNX3/AML2 is a Runt domain transcription factor like RUNX1/AML1 and RUNX2/AML3. Regulated by 2 promoters P1 and P2, RUNX3 is frequently inactivated by P2 methylation in solid tumors. Growing evidence has suggested a role of this transcription factor in hematopoiesis. However, genetic alterations have not been reported in blood cancers. In this study on 73 acute myeloid leukemia (AML) patients (44 children and 29 adults), we first showed that high RUNX3 expression among childhood AML was associated with a shortened event-free survival, and RUNX3 was significantly underexpressed in the prognostically favorable subgroup of AML with the t(8;21) and inv(16) translocations. We further demonstrated that this RUNX3 repression was mediated not by P2 methylation, but RUNX1-ETO and CBFbeta-MYH11, the fusion products of t(8;21) and inv(16), via a novel transcriptional mechanism that acts directly or indirectly in collaboration with RUNX1, on 2 conserved RUNX binding sites in the P1 promoter. In in vitro studies, ectopically expressed RUNX1-ETO and CBFbeta-MYH11 also inhibited endogenous RUNX3 expression. Taken together, RUNX3 was the first transcriptional target found to be commonly repressed by the t(8;21) and inv(16) fusion proteins and might have an important role in core-binding factor AML.

Alterations of RAS signalling in Chinese multiple myeloma patients: absent BRAF and rare RAS mutations, but frequent inactivation of RASSF1A by transcriptional silencing or expression of a non-functional variant transcript

Summary.  The methylation status, mutation and expression of RASSF1A, and mutations of RAS and BRAF were studied in 52 patients with multiple myeloma (MM), one plasma cell leukaemia (PCL) patient and four MM‐derived cell lines. Aberrant methylation of RASSF1A was found in nine of 32 MM patients and in one of four MM cell lines (U266), where the associated loss of transcription was reversible by demethylation treatment. RASSF1A transcription was further investigated on anti‐CD138‐sorted plasma cell‐enriched bone marrow samples from 10 MM, one PCL and three reactive plasmacytosis patients. While the wild‐type RASSF1A transcript was detected in all three reactive plasmacytosis and the PCL samples, we found no detectable wild‐type transcripts in six of 10 MM samples studied. In two MM samples, only the non‐functional variant transcript was detected, whereas the other four showed loss of transcription. In great contrast to western data, RAS mutations were identified in only four of 31 (13%) MM patients. While no RASSF1A or BRAF mutation (V599E) was detected in any of the primary MM studied (n = 21), the latter was found in the U266 cell line. Taken together, these data indicate that alterations of RAS signalling are critical in MM pathogenesis. In our current studies of Chinese MM patients, these alterations involved frequent RASSF1A inactivation (60%) as a result of transcriptional silencing or expression of a non‐functional variant transcript.

Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma

Platelet factor 4 (PF4) is an angiostatic chemokine that suppresses tumor growth and metastasis. We previously revealed frequent transcriptional silencing of PF4 in multiple myeloma, but the functional roles of this chemokine are still unknown. We studied the apoptotic effects of PF4 on myeloma cell lines and primary myeloma in vitro, and investigated the involved signaling pathway. The in vivo effects were also studied using a mouse model. PF4 not only suppressed myeloma-associated angiogenesis, but also inhibited growth and induced apoptosis in myeloma cells. We found that PF4 negatively regulated STAT3 and concordantly inhibited constitutive and interleukin-6-induced phosphorylation of STAT3, and down-regulated the expression of STAT3 target genes (Mcl-1, survivin and VEGF). Overexpression of constitutively activated STAT3 could rescue PF4-induced apoptotic effects. Furthermore, we found that PF4 induced the expression of SOCS3, a STAT3 inhibitor, and gene silencing of SOCS3 abolished its ability to inhibit STAT3 activation, suggesting a critical role of SOCS3 in PF4-induced STAT3 inhibition. Knockdown of LRP1, a putative PF4 receptor, could also abolish PF4-induced apoptosis and STAT3 inhibition. Finally, the tumor growth inhibitory effect of PF4 was confirmed by in vivo mouse models. Immunostaining of rabbit bone xenografts from PF4-treated mice showed induction of apoptosis of myeloma cells and inhibition of angiogenesis, which was associated with suppression of STAT3 activity. Together, our preclinical data indicate that PF4 may be a potential new targeting agent for the treatment of myeloma.

Secreted-frizzled related protein 1 is a transcriptional repression target of the t(8;21) fusion protein in acute myeloid leukemia

Secreted-frizzled related proteins (SFRPs) are modulators of the Wnt signaling pathway that is closely involved in normal and malignant hematopoiesis. Epigenetic deregulation of Wnt modulators leading to aberrant signaling has been reported in adult patients with acute myeloid leukemia (AML), but its occurrence in childhood patients with AML and the role of individual modulators are unclear. In this study, we examined SFRP1, SFRP2, SFRP4, and SFRP5 promoter methylation in 83 patients with AML (59 children and 24 adults) and found preferential SFRP1 methylation and mRNA down-regulation in the prognostically favorable subgroup of AML with t(8;21) translocation. Among the 4 genes, SFRP1 methylation independently predicted prolonged event-free and relapse-free survivals in childhood patients with nonacute promyelocytic leukemia with nonadverse cytogenetics. Mechanistically, we further demonstrated that RUNX1-ETO, the t(8;21) fusion product, specifically bound the SFRP1 promoter and repressed its transcription via a consensus RUNX binding site. In t(8;21)-leukemia cells, SFRP1 selectively inhibited canonical Wnt signaling and cellular proliferation that were associated with concomitant down-regulation of Wnt/β-catenin target genes, including CCND1 and MYC. Taken together, we identified SFRP1 as a transcriptional repression target of the t(8;21) fusion protein and demonstrated a novel mechanism of Wnt activation in a specific subtype of AML.

JAK2 V617F mutation is associated with increased risk of thrombosis in Chinese patients with essential thrombocythaemia

Manuela Mancini Elisa Zuffa Nevena Veljkovic Gianluca Brusa Patrizia Corrado Valentina Corradi Giovanni Martinelli Enza Barbieri Maria Alessandra Santucci Istituto di Ematologia e Oncologia Medica ‘‘Lorenzo e Ariosto Seràgnoli’’, University of Bologna-Medical School, Bologna, Italy, Centre for Multidisciplinary Research, Institute of Nuclear Sciences VINCA, Belgrade, Serbia, Dipartimento Farmaco Clinico Tecnologico, University of Siena, Siena, and Istituto di Radioterapia ‘‘Luigi Galvani’’, University of Bologna-Medical School, Bologna, Italy. E-mail: mancini_manu@yahoo.com

Minimal Residual Disease-Based Risk Stratification in Chinese Childhood Acute Lymphoblastic Leukemia by Flow Cytometry and Plasma DNA Quantitative Polymerase Chain Reaction

Minimal residual disease, or MRD, is an important prognostic indicator in childhood acute lymphoblastic leukemia. In ALL-IC-BFM 2002 study, we employed a standardized method of flow cytometry MRD monitoring for multiple centers internationally using uniformed gating, and determined the relevant MRD-based risk stratification strategies in our local patient cohort. We also evaluated a novel method of PCR MRD quantitation using peripheral blood plasma. For the bone marrow flow MRD study, patients could be stratified into 3 risk groups according to MRD level using a single time-point at day-15 (Model I) (I-A: <0.1%, I-B: 0.1–10%, I-C: >10%), or using two time-points at day-15 and day-33 (Model II) (II-A: day-15<10% and day-33<0.01%, II-B: day-15≥10% or day-33≥0.01% but not both, II-C: day-15≥10% and day-33≥0.01%), which showed significantly superior prediction of relapse (p = .00047 and <0.0001 respectively). Importantly, patients with good outcome (frequency: 56.0%, event-free survival: 90.1%) could be more accurately predicted by Model II. In peripheral blood plasma PCR MRD investigation, patients with day-15-MRD≥10−4 were at a significantly higher risk of relapse (p = 0.0117). By multivariate analysis, MRD results from both methods could independently predict patients’ prognosis, with 20–35-fold increase in risk of relapse for flow MRD I-C and II-C respectively, and 5.8-fold for patients having plasma MRD of ≥10−4. We confirmed that MRD detection by flow cytometry is useful for prognostic evaluation in our Chinese cohort of childhood ALL after treatment. Moreover, peripheral blood plasma DNA MRD can be an alternative where bone marrow specimen is unavailable and as a less invasive method, which allows close monitoring.

A polymorphism in the 3'-untranslated region of the NPM1 gene causes illegitimate regulation by microRNA-337-5p and correlates with adverse outcome in acute myeloid leukemia

Nucleophosmin, encoded by NPM1, is a haploinsufficient suppressor in hematologic malignancies. NPM1 mutations are mostly found in acute myeloid leukemia patients with normal karyotype and associated with favorable prognosis. A polymorphic nucleotide T deletion with unknown significance is present in the NPM1 3′-untranslated region. Here, we showed that the homozygous nucleotide T deletion was associated with adverse outcomes and could independently predict shortened survival in patients with de novo acute myeloid leukemia. Mechanistically, we demonstrated that the nucleotide T deletion created an illegitimate binding NPM1 for miR-337-5p, which was widely expressed in different acute myeloid leukemia subtypes and inhibited NPM1 expression. Accordingly, NPM1 levels were found to be significantly reduced and correlated with miR-337-5p levels in patients carrying a homozygous nucleotide T-deletion genotype. Together, our findings uncover a microRNA-mediated control of NPM1 expression that contributes to disease heterogeneity and suggest additional prognostic values of NPM1 in acute myeloid leukemia.

DNA of Erythroid Origin is Present in Human Plasma and Informs the Types of Anemia

BACKGROUND There is much interest in the tissue of origin of circulating DNA in plasma. Data generated using DNA methylation markers have suggested that hematopoietic cells of white cell lineages are important contributors to the circulating DNA pool. However, it is not known whether cells of the erythroid lineage would also release DNA into the plasma. METHODS Using high-resolution methylation profiles of erythroblasts and other tissue types, 3 genomic loci were found to be hypomethylated in erythroblasts but hypermethylated in other cell types. We developed digital PCR assays for measuring erythroid DNA using the differentially methylated region for each locus. RESULTS Based on the methylation marker in the ferrochelatase gene, erythroid DNA represented a median of 30.1% of the plasma DNA of healthy subjects. In subjects with anemia of different etiologies, quantitative analysis of circulating erythroid DNA could reflect the erythropoietic activity in the bone marrow. For patients with reduced erythropoietic activity, as exemplified by aplastic anemia, the percentage of circulating erythroid DNA was decreased. For patients with increased but ineffective erythropoiesis, as exemplified by β-thalassemia major, the percentage was increased. In addition, the plasma concentration of erythroid DNA was found to correlate with treatment response in aplastic anemia and iron deficiency anemia. Plasma DNA analysis using digital PCR assays targeting the other 2 differentially methylated regions showed similar findings. CONCLUSIONS Erythroid DNA is a hitherto unrecognized major component of the circulating DNA pool and is a noninvasive biomarker for differential diagnosis and monitoring of anemia.

HLA-B67 may be a male-specific HLA marker of susceptibility to relapsed childhood ALL in Hong Kong Chinese and HLA-A33 or HLA-B17 signifies a higher presentation leukocytosis: a retrospective analysis on 53 transplant candidates (1989–2003)

We performed a retrospective analysis on the human leukocyte antigen (HLA) data of 53 consecutive Chinese patients with high-risk childhood acute lymphoblastic leukemia (ALL) diagnosed from 1989 to 2003. A significantly higher frequency of HLA-B67 in the male relapse group of patients [OR, 23.08; 95% CI, 5.31–100.36; p=0.0042; for statistical significance after Bonferroni correction (Bc) pBc<0.0083] was identified after Bonferroni correction. Although not surviving the Bonferroni correction, gender effects on the association were also observed with HLA-A11, HLA-A32, HLA-A33, and HLA-B22, which were however more prevalent in the female patients and particularly those developing relapse. Two patients with HLA-A29 and HLA-B7 revealed significantly shortened survivals, suggestive of their potential prognostic impacts. Notably, for the first time, we found a significant correlation of leukocyte count with HLA types, where HLA-A33 (p=0.006) or HLA-B17 (p<0.001) signifies higher leukocytosis at presentation. Taken together, our findings support the involvement of HLA in Chinese high-risk childhood ALL.

FNDC3B is another novel partner fused to RARA in the t(3;17)(q26;q21) variant of acute promyelocytic leukemia

To the editor: Acute promyelocytic leukemia (APL) is characterized by the promyelocytic leukemia-retinoic acid receptor α ( PML-RARA ) fusion. In rare instances, RARA is fused to other partners, which dictate sensitivity to targeted therapies. Chen et al previously reported in Blood a novel TBLR1