Suzanne Vercauteren

Detection and clinical significance of human acute myeloid leukaemia progenitors capable of long-term proliferation in vitro

Acute myeloid leukaemia (AML) blasts within individual patients are heterogeneous in their surface antigen expression and proliferative ability suggesting that, subsequent to transformation, differentiation occurs creating a hierarchy of progenitors in AML. Cells that can produce AML colonies (colony forming units, CFU) in growth factor containing suspension cultures (SC) over 4–8 weeks have a phenotype similar to AML progenitors that engraft non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice, but different from bulk AML blasts, suggesting that these AML SC‐initiating cells (SC‐IC) may be early progenitors. In this study, we evaluated culture conditions that provide for optimal growth of AML progenitors capable of long‐term proliferation. The frequency of CFU, both initially and after 2–4 weeks of SC, varied over four logs between individual patients and was not related to French–American–British subtype. Using limiting dilution, the proliferative potential of individual SC‐IC varied from 1 to 480 CFU. The frequency of CFU from SC after > 4 weeks was prognostic for patient survival, and correlated with NOD/SCID engrafting ability. These results suggest the use of long‐term culture as an assay for AML cells with leukaemia sustaining potential.

JAK1 gain-of-function causes an autosomal dominant immune dysregulatory and hypereosinophilic syndrome

Kate L. Del Bel, MSc, Robert J. Ragotte, BSc, Aabida Saferali, MSc, Susan Lee, RN, Suzanne M. Vercauteren, MD PhD, Sara A. Mostafavi, PhD, Richard A. Schreiber, MD, Julie S. Prendiville, MD, Min S. Phang, MD, Jess Halperin, MD, Nicholas Au, MD, John M. Dean, MD BS, Emily Jewels, RN, Anne K. Junker, MD, Paul C. Rogers, MB ChB MBA, Michael Seear, MB ChB, Margaret L. McKinnon, MD, Stuart E. Turvey, MB BS, DPhil

Copy number alterations at polymorphic loci may be acquired somatically in patients with myelodysplastic syndromes

Loss of genomic integrity is thought to be one of the underlying causes of myelodysplastic syndromes (MDS). However, it is unclear whether changes in copy number at loci that are common sites of copy number polymorphisms play a pathogenic role. Here we show that copy number changes in the MDS clone that occur at polymorphic loci are frequently somatic alterations rather than constitutional variants, and the extent of copy number changes at polymorphic loci is increased in CD34(+) cells of MDS patients compared to age-matched controls. This study suggests a potential pathophysiological role for copy number alterations at polymorphic loci in patients with MDS, and highlights the need for somatic control tissues for each patient studied in high-resolution genome-wide investigations.

Evaluation of two methods to measure hemoglobin concentration among women with genetic hemoglobin disorders in Cambodia: a method-comparison study.

BACKGROUND Genetic hemoglobin (Hb) E variants are common in Cambodia and result in an altered and unstable Hb molecule. We evaluated two methods to measure Hb concentration among individuals with and without Hb variants using a hemoglobinometer (HemoCue) and a hematology analyzer (Sysmex XT-1800i). METHODS We determined the bias and concordance between the methods among 420 Cambodian women (18-45 y). RESULTS Bias and concordance appeared similar between methods among women with no Hb disorders (n=195, bias=2.5, ρc=0.68), women with Hb E variants (n=133, bias=2.5, ρc=0.78), and women with other Hb variants (n=92, bias=2.7, ρc=0.73). The overall bias was 2.6g/l, resulting in a difference in anemia prevalence of 11.5% (41% using HemoCue and 29.5% using Sysmex, p<0.001). Based on visual interpretation of the concordance plots, the HemoCue device appears to underestimate Hb concentrations at lower Hb concentrations and to overestimate Hb concentrations at higher Hb concentrations (in comparison to the Sysmex analyzer). CONCLUSIONS Bias and concordance were similar across groups, suggesting the two methods of Hb measurement were comparable. We caution field staff, researchers and policy makers in the interpretation of data and the impact that bias between methods can have on anemia prevalence rates.

The Homozygous Hemoglobin EE Genotype and Chronic Inflammation Are Associated with High Serum Ferritin and Soluble Transferrin Receptor Concentrations among Women in Rural Cambodia

BACKGROUND Ferritin and soluble transferrin receptor (sTfR) concentrations are commonly used to assess iron deficiency (ID); however, they are influenced by multiple factors. OBJECTIVES We assessed associations between numerous variables and both ferritin and sTfR concentrations in Cambodian women and compared ID prevalence through the use of study-generated correction factors (CFs) for ferritin with those from a published meta-analysis. METHODS Venous blood from 450 women (aged 18-45 y) was assessed for hemoglobin (Hb), ferritin, sTfR, retinol binding protein, folate, vitamin B-12, C-reactive protein, α-1 acid glycoprotein (AGP), and genetic Hb disorders. Linear regression was used to calculate geometric mean ratios (95% CIs) for ferritin and sTfR concentrations. RESULTS The variant Hb EE genotype was associated with 50% (14%, 96%) and 51% (37%, 66%) higher geometric mean ferritin and sTfR concentrations, respectively, than was the normal Hb AA genotype; a 1-g/L increase in AGP was associated with 99% (50%, 162%) and 48% (33%, 64%) higher concentrations in the same variables, respectively. ID prevalence in nonpregnant women (n = 420) was 2% (n = 9) with the use of ferritin <15 μg/L and 18% (n = 79) with the use of sTfR >8.3 mg/L as criteria. ID prevalence with the use of sTfR was higher in women with the Hb EE genotype (n = 17; 55%) than in those with the Hb AA genotype (n = 20; 10%); and in women with the Hb AA genotype and chronic inflammation (n = 10; 18%) than in that group of women without chronic inflammation (n = 10; 7%) (P < 0.05). No differences in ID prevalence were found with the use of ferritin between women with Hb EE and AA genotypes (P = 1.0) or by chronic inflammation status (P = 0.32). There were no differences in mean ferritin concentrations among all 450 women when study-generated CFs were compared with those from the meta-analysis (P = 0.87). CONCLUSIONS Compared with sTfR, ferritin concentrations appear to reflect more accurately true ID in rural Cambodian women. The CFs from a published meta-analysis were appropriate for use in this population with a high prevalence of Hb disorders and inflammation.

Policy recommendations for addressing privacy challenges associated with cell-based research and interventions

BackgroundThe increased use of human biological material for cell-based research and clinical interventions poses risks to the privacy of patients and donors, including the possibility of re-identification of individuals from anonymized cell lines and associated genetic data. These risks will increase as technologies and databases used for re-identification become affordable and more sophisticated. Policies that require ongoing linkage of cell lines to donors’ clinical information for research and regulatory purposes, and existing practices that limit research participants’ ability to control what is done with their genetic data, amplify the privacy concerns.DiscussionTo date, the privacy issues associated with cell-based research and interventions have not received much attention in the academic and policymaking contexts. This paper, arising out of a multi-disciplinary workshop, aims to rectify this by outlining the issues, proposing novel governance strategies and policy recommendations, and identifying areas where further evidence is required to make sound policy decisions. The authors of this paper take the position that existing rules and norms can be reasonably extended to address privacy risks in this context without compromising emerging developments in the research environment, and that exceptions from such rules should be justified using a case-by-case approach. In developing new policies, the broader framework of regulations governing cell-based research and related areas must be taken into account, as well as the views of impacted groups, including scientists, research participants and the general public.SummaryThis paper outlines deliberations at a policy development workshop focusing on privacy challenges associated with cell-based research and interventions. The paper provides an overview of these challenges, followed by a discussion of key themes and recommendations that emerged from discussions at the workshop. The paper concludes that privacy risks associated with cell-based research and interventions should be addressed through evidence-based policy reforms that account for both well-established legal and ethical norms and current knowledge about actual or anticipated harms. The authors also call for research studies that identify and address gaps in understanding of privacy risks.

T cells of patients with myelodysplastic syndrome are frequently derived from the malignant clone.

T cell clonality is a common finding in patients with myelodysplastic syndrome (MDS), but is currently thought to be a reactive phenomenon (van Lom et al, 1995; Epling-Burnette et al, 2007). Recent evidence points to a stem or multipotent progenitor cell as the MDS-initiating cell in some patients, suggesting that the lymphoid lineage may also be involved in the disease. Clonal circulating myeloid and lymphoid precursor dendritic cells have been detected in patients with MDS (Ma et al, 2004) and a high percentage of monosomy 7 in pluripotent stem cells, B cell progenitors and T/Natural Killer (NK) progenitor cells was reported in three of four MDS patients analysed (Miura et al, 2000). In a series of MDS cases that progressed to T cell acute lymphoid leukaemia (T-ALL), the MDS karyotypic aberration was also detected in the T-ALL cells (Disperati et al, 2006). Many genome-wide studies in MDS have used CD3+ cells from the same patient to represent a patient normal control in order to distinguish between constitutional and acquired variants (Starczynowski et al, 2008). The present study investigated whether T cells are derived from the malignant MDS clone using DNA microarrays in 40 MDS patients. CD34+ and CD3+ cells were selected from marrow or peripheral blood by immunomagnetic separation (Stem Cell Technologies, Vancouver, BC, Canada). Genomic DNA was extracted with the AllPrep DNA/RNA Mini Kit (QIAGEN, Valencia, CA, USA). Normal reference DNA was purchased as a pool of either male or female genomic DNA (Novagen, Madison, WI, USA). Details of whole genome array comparative genomic hybridization (aCGH) including DNA extraction, labelling and hybridization as well as image analysis have been described previously (Shah et al, 2006; Coe et al, 2007; Starczynowski et al, 2008). A region was considered altered when a minimum of two overlapping consecutive clones showed the change. The multiplex polymerase chain reaction protocol and primers used for T cell receptor gamma (TRG@) gene analysis followed the standardized BIOMED-2 protocols, followed by analysis on an ABI3730 capillary electrophoresis instrument (van Dongen et al, 2003). Intracellular immunohistochemical staining of marrow/peripheral blood cells with anti-CD3cytoplasmic antibody (Dako North America Inc., Carpenteria, CA, USA) followed by AlexaFluor 594 (Molecular probes Inc., Eugene, OR, USA) preceded the fluorescence in situ hybridization (FISH) procedure with FISH probes (20q or 8 or 11q)(Abbott/Vysis, Downers Grove, IL, USA). aCGH was performed on 40 DNA samples of matched CD34+ stem/progenitor cells and CD3+ T cells from patients with MDS or MDS/myeloproliferative neoplasm (MPN). Fourteen patients had known cytogenetic abnormalities as identified by conventional karyotyping (Table I). Of these 14 patients, two male patients had a deletion of the Y chromosome. In 11 of the remaining 12 MDS patients the cytogenetic abnormalities could be detected in the CD34+ cells using aCGH. In one patient (Patient 33) with trisomy 8 (4/20 metaphases by conventional karyotyping) and deletion 5q23.1-q31.2 (11/20 metaphases by conventional karyotyping), only the deletion 5q was detectable by aCGH, consistent with a detection threshold of 25–30% aberrant cells (Coe et al, 2007). Additionally, aCGH detected deletion of chromosome 20q11.21-13.33 in this patient. Patient 32 showed deletion of chromosome 11q14.1-q23.1 as well as amplification of 11q12.3-13.4. In five patients (Patients 29, 30, 34, 39 and 40) conventional karyotyping failed or was not performed. One of these patients (Patient 39) revealed partial trisomy 9 from q33.3 to q34.3 as well as trisomies 19 and 22 by aCGH in the CD34+ cells. Table I Clinical, chromosomal and TCR rearrangement characteristics of 40 patients with MDS or MDS/MPN analyzed by array CGH for chromosomal changes in T cells.. aCGH analysis of CD3+ T cells demonstrated the same cytogenetic abnormalities in nine of 12 MDS patients with karyotypic abnormalities, excluding the two patients with –Y (Table I and Fig 1). Deletion of chromosome 20q (Patients 4 and 5), isodicentric X chromosome (Patients 14 and 38), trisomy 8 (Patients 25 and 37), 11q abnormalities (Patient 32) and deletion 5q (Patient 33), partial trisomy 9 and trisomies 19 and 22 (Patient 39) were detected in the T cells of these patients. Consistent with the findings in the CD34+ cells, deletion of chromosome 5q23.1-q31.2 but not trisomy 8, was identified in the CD3+ cells of Patient 33. The presence of deletion 20q in the T cells from the marrow of Patient 5 (60% of CD3+ cells), trisomy 8 in the marrow T cells of Patient 25 (15% of CD3+ cells) and deletion of chromosome arm 11q in the peripheral blood T cells of Patient 32 (8% of CD3+ cells) was confirmed by FISH (Fig 1, and data not shown). Fig 1 Large genomic alterations in marrow CD34+ cells are detected in matched T cells of some patients with MDS. Array comparative genomic hybridization (aCGH) (A) and fluorescence in situ hybridization (FISH) (B) show deletions of chromosome 20 (1 and 3) as ... T cell clonality of 14 MDS cases was determined by analysing TRG@ rearrangement (Table I). Six of the 14 patients analysed had karyotypic abnormalities, four of whom had the identical copy number variant identified in both the CD34+ and CD3+ populations by aCGH. These four patients either had clonal (Patients 5, 14 and 37) or oligoclonal (Patient 33) TRG@ rearrangements. In contrast, the two patients without the genetic abnormality in the T cells, showed polyclonal (Patient 17) and oligoclonal (Patient 18) TRG@ rearrangement. Clonal TRG@ rearrangement was detected in only one of the eight patients with a normal karyotype. Here we show that cytogenetic abnormalities, identical to those seen in stem/progenitor cells, are present in the T cells of some MDS patients. We speculate that low numbers of T cells derived from the malignant clone are probably also present in other cases, but that this population may be smaller and thus not detectable by aCGH. Alternatively, the aberrant CD3+ cells may undergo apoptosis in the marrow before entering the circulation, and again may not be detectable in cases in which peripheral blood rather than marrow T cells are examined. This is in agreement with one report, in which a high percentage of monosomy 7 cells was identified in marrow-derived stem cells, B cell progenitors and T/NK progenitor cells but not in peripheral blood B and T cells (Miura et al, 2000). A recent publication has described the presence of TET2 mutations in T cells of a significant number of MDS patients (Smith et al, 2010). We conclude that, in a large proportion of MDS cases, at least a proportion of the T cells are part of the malignant clone, and suggest that CD3+ cells do not represent an appropriate patient normal control for genome-wide studies, but rather a non-haematopoietic cell type should be used.

Primitive AML progenitors from most CD34(+) patients lack CD33 expression but progenitors from many CD34(-) AML patients express CD33.

BACKGROUND AML blast populations are heterogeneous in their phenotype and functional properties, and contain a small subset of cells that regenerate leukemia in immunocompromised mice or produce clonogenic progeny in long-term cultures. This suggests the existence of a hierarchy of AML progenitor cells. CD33 is a myeloid marker absent on normal hematopoietic stem cells but expressed in about 75% of AML patients, and has been used for BM purging strategies and Ab-targeted therapies. These CD33 Ab therapies benefit only a minority of AML patients, suggesting that AML stem cells are heterogeneous in their CD33 expression. METHODS In order to evaluate this question, we determined expression levels of CD34 and CD33 on AML progenitors with long-term in vitro proliferative ability and NOD/SCID engrafting ability. RESULTS The CD34(+) CD33(-) subfraction contained the majority of progenitors detected in vitro and most often engrafted the mice. This proliferation was leukemic from the CD34(+) AML patients, however from the CD34(-) AML patients only normal progenitors were detected in this fraction in some cases. DISCUSSION These data suggest that most leukemic progenitors of CD34(+) patients do not express CD33. In contrast, CD34(-) AML primitive leukemic progenitors may be CD33(+). CD34(-) AML patients could potentially benefit most from CD33-targeted therapies or purging.

Comparison of four immunoassays to measure serum ferritin concentrations and iron deficiency prevalence among non-pregnant Cambodian women and Congolese children

Abstract Background: Global standardization of ferritin assays is lacking, which could have direct implications on the accurate measurement and comparability of ferritin concentration and iron deficiency (ID) prevalence rates in at-risk populations. Methods: We measured serum ferritin concentrations using four immunoassays: the s-ELISA and the AxSYM™ analyzer were compared among 420 non-pregnant Cambodian women; the Centaur® XP analyzer, s-ELISA, and AxSYM™ analyzer were compared among a subset of 100 Cambodian women; and the s-ELISA and the Elecsys® 2010 analyzer were compared among 226 Congolese children aged 6–59 months. Results: Median ferritin concentrations (adjusted for inflammation) ranged between 48 and 91 μg/L among Cambodian women and between 54 and 55 μg/L among Congolese children. ID prevalence ranged from 2% to 10% among Cambodian women and 5% to 7% among Congolese children. Bias between methods varied widely (–9 to 45 μg/L) among women, and was 43 μg/L among children. Bias was lower when ferritin values outside of the s-ELISA measurement range (>250 μg/L) were excluded. Conclusions: The observed differences in ferritin concentrations likely reflect different ferritin isoforms, antibodies, and calibrators used across assays and by different laboratories. However, despite differences in ferritin concentrations, ID prevalence was relatively similar and low across all methods.

Elevated levels of iron in groundwater in Prey Veng province in Cambodia : a possible factor contributing to high iron stores in women

Iron is a natural element found in food, water and soil and is essential for human health. Our aim was to determine the levels of iron and 25 other metals and trace elements in groundwater from 22 households in Prey Veng, Cambodia. Water analyses were conducted using inductively coupled plasma-mass spectrometry and optical emission spectrometry. Compared to the 2011 World Health Organization guidelines for drinking water quality, aluminum, iron and manganese exceeded maximum levels (in 4.5, 72.7 and 40.9% of samples, respectively). Compared to the 2004 Cambodian drinking water quality standards, iron and manganese exceeded maximum levels (in 59.1 and 36.4% of samples, respectively). We found no evidence of arsenic contamination. Guidelines for iron were established primarily for esthetic reasons (e.g. taste), whereas other metals and elements have adverse effects associated with toxicity. Iron in groundwater ranged from 134 to 5,200 μg/L (mean ∼1,422 μg/L). Based on a daily consumption of 3 L groundwater, this equates to ∼0.4-15.6 mg iron (mean ∼4.3 mg/day), which may be contributing to high iron stores and the low prevalence of iron deficiency anemia in Prey Veng women. Elevated levels of manganese in groundwater are a concern and warrant further investigation.