Philadelphia-Negative Classical Myeloproliferative Neoplasms: Critical Concepts and Management Recommendations From European LeukemiaNet

We present a review of critical concepts and produce recommendations on the management of Philadelphia-negative classical myeloproliferative neoplasms, including monitoring, response definition, first-and second-line therapy, and therapy for special issues. Key questions were selected according the criterion of clinical relevance. Statements were produced using a Delphi process, and two consensus conferences involving a panel of 21 experts appointed by the European LeukemiaNet (ELN) were convened. Patients with polycythemia vera (PV) and essential thrombocythemia (ET) should be defined as high risk if age is greater than 60 years or there is a history of previous thrombosis. Risk stratification in primary myelofibrosis (PMF) should start with the International Prognostic Scoring System (IPSS) for newly diagnosed patients and dynamic IPSS for patients being seen during their disease course, with the addition of cytogenetics evaluation and transfusion status. High-risk patients with PV should be managed with phlebotomy, low-dose aspirin, and cytoreduction, with either hydroxyurea or interferon at any age. High-risk patients with ET should be managed with cytoreduction, using hydroxyurea at any age. Monitoring response in PV and ET should use the ELN clinicohematologic criteria. Corticosteroids, androgens, erythropoiesis-stimulating agents, and immunomodulators are recommended to treat anemia of PMF, whereas hydroxyurea is the first-line treatment of PMF-associated splenomegaly. Indications for splenectomy include symptomatic portal hypertension, drug-refractory painful splenomegaly, and frequent RBC transfusions. The risk of allogeneic stem-cell transplantation-related complications is justified in transplantation-eligible patients whose median survival time is expected to be less than 5 years.

RNAi screen identifies Jarid1b as a major regulator of mouse HSC activity

Histone methylation is a dynamic and reversible process proposed to directly impact on stem cell fate. The Jumonji (JmjC) domain-containing family of demethylases comprises 27 members which can demethylate mono-, di- and tri-methylated lysine residues of histone (or non-histone) targets. To evaluate their role in regulation of hematopoietic stem cell (HSC) behaviour we performed a RNAi-based screen and found that demethylases JARID1B (H3K4) and JHDM1F (H3K9) play opposing roles in regulation of HSC activity. Decrease in Jarid1b levels correlated with an in vitro expansion of HSC with preserved long term in vivo lympho-myeloid differentiation potential. Jarid1b knockdown was associated with an increase in expression levels of 5’ Hoxa cluster genes and CxCl5 , and reduced levels of Pu.1, Egr1 and Cav1. shRNA against Jhdmlf, in contrast, impaired hematopoietic reconstitution of bone marrow cells. Together, our studies identified Jarid1b as a negative, and Jhdmlf as a positive regulator of HSC activity.

Changes of 5-hydroxymethylcytosine distribution during myeloid and lymphoid differentiation of CD34+ cells

BACKGROUND: Hematopoietic stem cell renewal and differentiation are regulated through epigenetic processes. The conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5hmC) by ten-eleven-translocation enzymes provides new insights into the epigenetic regulation of gene expression during development. Here, we studied the potential gene regulatory role of 5hmC during human hematopoiesis.

RESULTS: We used reduced representation of 5-hydroxymethylcytosine profiling (RRHP) to characterize 5hmC distribution in CD34+ cells, CD4+ T cells, CD19+ B cells, CD14+ monocytes and granulocytes. In all analyzed blood cell types, the presence of 5hmC at gene bodies correlates positively with gene expression, and highest 5hmC levels are found around transcription start sites of highly expressed genes. In CD34+ cells, 5hmC primes for the expression of genes regulating myeloid and lymphoid lineage commitment. Throughout blood cell differentiation, intragenic 5hmC is maintained at genes that are highly expressed and required for acquisition of the mature blood cell phenotype. Moreover, in CD34+ cells, the presence of 5hmC at enhancers associates with increased binding of RUNX1 and FLI1, transcription factors essential for hematopoiesis.

CONCLUSIONS: Our study provides a comprehensive genome-wide overview of 5hmC distribution in human hematopoietic cells and new insights into the epigenetic regulation of gene expression during human hematopoiesis.

ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia.

Recent evidence suggests that - in addition to 17p deletion - TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4-9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.

The Clinical Impact and Molecular Biology of del(17p) in Multiple Myeloma Treated with Conventional or Thalidomide-Based Therapy

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P <0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group. (C) 2011 Wiley-Liss, Inc.

Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL.

Early detection of leukaemic relapse after bone marrow transplantation using the polymerase chain reaction

We report a case of acute lymphoblastic leukaemia relapsing after allogeneic bone marrow transplantation in which the polymerase chain reaction (PCR) was used to assess chimeric status. This technique demonstrated the progressive reappearance of host cells prior to clinical relapse. The relapse was of host cell origin as shown by the presence of female (recipient) metaphases containing an abnormal chromosomal marker (iso 9q) which had also been present at initial diagnosis. The emergence of host cells in this case, detected only by PCR techniques but not by cytogenetic methods, appeared to herald overt relapse. PCR analysis provides a sensitive tool for detecting a progressive rise in host cell numbers which may predict clinical relapse.

Cytohesin Binder and Regulator (Cybr) is Not Essential for T- and Dendritic-Cell Activation and Differentiation

Cybr (also known as Cytip, CASP, and PSCDBP) is an interleukin-12-induced gene expressed exclusively in hematopoietic cells and tissues that associates with Arf guanine nucleotide exchange factors known as cytohesins. Cybr levels are dynamically regulated during T-cell development in the thymus and upon activation of peripheral T cells. In addition, Cybr is induced in activated dendritic cells and has been reported to regulate dendritic cell (DC)-T-cell adhesion. Here we report the generation and characterization of Cybr-deficient mice. Despite the selective expression in hematopoietic cells, there was no intrinsic defect in T- or B-cell development or function in Cybr-deficient mice. The adoptive transfer of Cybr-deficient DCs showed that they migrated efficiently and stimulated proliferation and cytokine production by T cells in vivo. However, competitive stem cell repopulation experiments showed a defect in the abilities of Cybr-deficient T cells to develop in the presence of wild-type precursors. These data suggest that Cybr is not absolutely required for hematopoietic cell development or function, but stem cells lacking Cybr are at a developmental disadvantage compared to wild-type cells. Collectively, these data demonstrate that despite its selective expression in hematopoietic cells, the role of Cybr is limited or largely redundant. Previous in vitro studies using overexpression or short interfering RNA inhibition of the levels of Cybr protein appear to have overestimated its immunological role.

Isolation of retinal progenitor and stem cells from the porcine eye

Purpose: Retinal progenitor cells (RPCs) and retinal stem cells (RSCs) from rodents and humans have been isolated and characterized in vitro. Transplantation experiments have confirmed their potential as tools for cell replacement in retinal degenerative diseases. The pig represents an ideal pre-clinical animal model to study the impact of transplantation because of the similarity of its eye to the human eye. However, little is known about porcine RPCs and RSCs. We aimed to identify and characterize in vitro RPCs and RSCs from porcine ocular tissues. Methods: Cells from different subregions of embryonic, postnatal and adult porcine eyes were grown in suspension sphere culture in serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Growth curves and BrdU incorporation assays were performed to establish the proliferative capacity of isolated porcine retina-derived RPCs and ciliary epithelium (CE)-derived RSCs. Self-renewal potential was investigated by subsphere formation assays. Changes in gene expression were assayed by reverse transcription polymerase chain reaction (RT-PCR) at different passages in culture. Finally, differentiation was induced by addition of serum to the cultures and expression of markers for retinal cell types was detected by immunohistochemical staining with specific antibodies. Results: Dissociated cells from embryonic retina and CE at different postnatal ages generated primary nestin- and Pax6-immunoreactive neurosphere colonies in vitro in numbers that decreased with age. Embryonic and postnatal retina-derived RPCs and young CE-derived RSCs displayed self-renewal capacity, generating secondary neurosphere colonies. However, their self-renewal and proliferation capacity gradually decreased and they became more committed to differentiated states with subsequent passages. The expansion capacity of RPCs and RSCs was higher when they were maintained in monolayer culture. Porcine RPCs and RSCs could be induced to differentiate in vitro to express markers of retinal neurons and glia. Conclusions: Porcine retina and CE contain RPCs and RSCs which are undifferentiated, self-renewing and multipotent and which show characteristics similar to their human counterparts. Therefore, the pig could be a useful source of cells to further investigate the cell biology of RPCs and RSCs and it could be used as a non-primate large animal model for pre-clinical studies on stem cell-based approaches to regenerative medicine in the retina.

CCN3-A key regulator of the hematopoietic compartment

CCN3, a founding member of the CCN family of growth regulators, was linked with hematology in 2003(1) when it was detected in human serum. CCN3 is expressed and secreted by hematopoietic progenitor cells in normal bone marrow. CCN3 acts through the core stem cell signalling pathways including Notch and Bone Morphogenic Protein, connecting CCN3 with the modulation of self-renewal and maturation of a number of cell lineages including hematopoietic, osteogenic and chondrogenic. CCN3 expression is disrupted in Chronic Myeloid Leukemia as a consequence of the BCR-ABL oncogene and allows the leukemic clone to evade growth regulation. In contrast, naive cord blood progenitors undergo enhanced clonal expansion in response to CCN3. Altered CCN3 expression is associated with numerous solid tumors including glioblastoma, melanoma. adrenocortical tumours, prostate cancer and bone malignancies including osteosarcoma. Mature CCN3 protein has five distinct modules and truncated protein variants with altered function are found in many cancers. Regulation by CCN3 is therefore cell type and isoform specific. CCN3 has emerged as a key player in stem cell regulation, hematopoiesis and a crucial component within the bone marrow microenvironment. (c) 2008 Elsevier Ltd. All rights reserved.

Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients

A clinical trial using human embryonic stem cell (hESC) therapy for an inherited retinal degenerative disease is about to commence. The Advanced Cell Technology (ACT) trial will treat patients with Stargardt's macular dystrophy using transplanted retinal pigment epithelium derived from hESCs. Currently, no effective treatment is available for Stargardt's disease so a stem cell-based therapy that can slow progression of this blinding condition could represent a significant breakthrough. While there are some hurdles to clear, the ACT trial is a fine example of translational research that could eventually pave the way for a range of stem cell therapies for the retina and other tissues.

Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-alpha is rescued by HDAC inhibitor trichostatin A

Inflammation and TNF-alpha signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-alpha receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-alpha stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-alpha stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.

Limbal stem cells of the corneal epithelium

Stem cells have certain unique characteristics, which include longevity, high capacity of self-renewal with a long cell cycle time and a short S-phase duration, increased potential for error-free proliferation, and poor differentiation. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. Stem cells for the cornea reside at the corneoscleral limbus. The limbal palisades of Vogt and the interpalisade rete ridges are believed to be repositories of stem cells. The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. Limbal stem cells also act as a 'barrier' to conjunctival epithelial cells and normally prevent them from migrating on to the corneal surface. Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell deficiency with resulting abnormalities in the corneal surface. Such deficiency of limbal stem cells leads to 'conjunctivalization' of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. This results in ocular discomfort and reduced vision. Partial stem cell deficiency can be managed by removing the abnormal epithelium and allowing the denuded cornea, especially the visual axis, to resurface with cells derived from the remaining intact limbal epithelium. In total stem cell deficiency, autologous limbus from the opposite normal eye or homologous limbus from living related or cadaveric donors can be transplanted on to the affected eye. With the latter option, systemic immunosuppression is required. Amniotic membrane transplantation is a useful adjunct to the above procedures in some instances. Copyright (C) 2000 Elsevier Science Inc.

Granulocyte-macrophage precursor cell and colony-stimulating factor responses of mice infected with Salmonella typhimurium

The response of granulocyte-macrophage progenitor cells (in vitro colony-forming cells) and of colony-stimulating (CS) factor in serum were studied in mice infected intraperitoneally with 10(3) viable Salmonella typhimurium. Increases in the number of colony-forming cells in marrow and spleen and increases in the serum level of CS factor occurred during the infection. There was no evidence to suggest that progressive infection was associated with failure of macrophage production. Medium rich in CS factor increased the bactericidal activity of macrophages in vitro and it was suggested that CS factor could be involved in macrophage activation.