Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation.
Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients).
Analysis of pooled, published data established three DNA methylation subgroups as a de facto standard. Unfavorable prognostic parameters (PTPN11 mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS.
This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.

T-cell prolymphocytic leukemia (T-PLL) is a rare, mature T-cell neoplasm with a heterogeneous clinical course. With the advent of novel treatment options that will potentially change the management of patients with T-PLL, it has become necessary to produce consensus guidelines for the design and conduct of clinical trials. The T-PLL International Study group (TPLL-ISG) set out to define standardized criteria for diagnosis, treatment indication, and evaluation of response. These criteria will facilitate comparison of results from clinical trials in T-PLL, and will thus support clinical decision making, as well as the approval of new therapeutics by healthcare authorities.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important curative therapy for patients with leukemia. However, relapse remains the leading cause of death after transplantation. In recent years, substantial progress has been made by Chinese physicians in the field of establishment of novel transplant modality, patient selection, minimal residual disease (MRD) monitoring, and immunological therapies, such as modified donor lymphocyte infusion (DLI) and chimeric antigen receptor T (CART) cells, as well as MRD-directed intervention for relapse. Most of these unique systems are distinct from those in the Western world. In this consensus, we reviewed the efficacy of post-HSCT relapse management practice from available Chinese studies on behalf of the HSCT workgroup of the Chinese Society of Hematology, Chinese Medical Association, and compared these studies withthe consensus or guidelines outside China. We summarized the consensus on routine practices of post-HSCT relapse management in China and focused on the recommendations of MRD monitoring, risk stratification directed strategies, and modified DLI system. This consensus will likely contribute to the standardization of post-HSCT relapse management in China and become an inspiration for further international cooperation to refine global practices.

PML/RARalpha is of crucial importance in acute promyelocytic leukemia (APL) both pathologically and therapeutically. Using a genome-wide approach, we identified in vivo PML/RARalpha binding sites in a PML/RARalpha-inducible cell model. Of the 2979 targeted regions, >62% contained canonical PU.1 motifs and >84% of these PU.1 motifs coexisted with one or more RARE half (RAREh) sites in nearby regions. Promoters with such PU.1-RAREh binding sites were transactivated by PU.1. PU.1-mediated transactivation was repressed by PML/RARalpha and restored by the addition of all-trans retinoic acid (ATRA). Genes containing such promoters were significantly represented by genes transcriptionally suppressed in APL and/or reactivated upon treatment with ATRA. Thus, selective targeting of PU.1-regulated genes by PML/RARalpha is a critical mechanism for the pathogenesis of APL.

Human beta-globin disorders are relatively common genetic diseases cause by mutations in the beta-globin gene. Increasing the expression of the gamma-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the gamma-globin gene. The human gamma-globin genes (both Agamma and Ggamma-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing gamma-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the gamma-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, alpha2gamma2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous gamma-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous gamma-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the gamma-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the gamma-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and beta-thalassemia.

Interferon consensus sequence binding protein (ICSBP) was first identified as a transcription factor of the interferon (IFN) regulatory factor family (IRF) which regulates expression of IFN-dependent genes by binding to DNA at specific sites, IFN-stimulated responsive elements. Analysis of ICSBP-deficient mice showed hematologic alterations similar to chronic myelogenous leukemia (CML) in humans and suggested a novel role for ICSBP in regulating proliferation and differentiation of hematopoietic progenitor cells. Here we show that ICSBP-mRNA expression is impaired in human myeloid leukemias: 27 of 34 CML patients (79%) and 21 of 32 patients with acute myeloid leukemia (AML) (66%) showed very low or absent transcript numbers of ICSBP. In contrast, only 2 of 33 normal volunteers (6%) showed low transcription of ICSBP (P < . 0001 both for CML and AML values). The lack of expression was not associated with lack of lymphatic cells, which normally have been shown to express ICSBP at the highest level. More detailed analysis showed an absence of ICSBP-mRNA also in sorted B cells derived from CML patients. To analyze whether ICSBP may be induced in leukemic cells, ex vivo experiments using a known inducer of ICSBP, IFN-gamma, were performed. Ex vivo treatment of primary CML cells using IFN-gamma resulted in induction of ICSBP transcripts. Furthermore, samples of CML patients during IFN-alpha treatment were analyzed. In 11 of 12 CML patients ICSBP-mRNA was inducible upon in vivo treatment with IFN-alpha, but decreased with progression of CML. Stable transfection of K-562 cell line with ICSBP led to no difference in bcr-abl expression in vitro, although two patients showed an inverse correlation between bcr-abl and ICSBP in vivo. These data suggest that lack of ICSBP may have an important role also in human myeloid leukemogenesis.