Sandy Sung

Identification of miR-145 and miR-146a as mediators of the 5q– syndrome phenotype

5q– syndrome is a subtype of myelodysplastic syndrome characterized by severe anemia and variable neutropenia but normal or high platelet counts with dysplastic megakaryocytes. We examined expression of microRNAs (miRNAs) encoded on chromosome 5q as a possible cause of haploinsufficiency. We show that deletion of chromosome 5q correlates with loss of two miRNAs that are abundant in hematopoietic stem/progenitor cells (HSPCs), miR-145 and miR-146a, and we identify Toll–interleukin-1 receptor domain–containing adaptor protein (TIRAP) and tumor necrosis factor receptor–associated factor-6 (TRAF6) as respective targets of these miRNAs. TIRAP is known to lie upstream of TRAF6 in innate immune signaling. Knockdown of miR-145 and miR-146a together or enforced expression of TRAF6 in mouse HSPCs resulted in thrombocytosis, mild neutropenia and megakaryocytic dysplasia. A subset of mice transplanted with TRAF6-expressing marrow progressed either to marrow failure or acute myeloid leukemia. Thus, inappropriate activation of innate immune signals in HSPCs phenocopies several clinical features of 5q– syndrome.

High-resolution whole genome tiling path array CGH analysis of CD34+ cells from patients with low-risk myelodysplastic syndromes reveals cryptic copy number alterations and predicts overall and leukemia-free survival

Myelodysplastic syndromes (MDSs) pose an important diagnostic and treatment challenge because of the genetic heterogeneity and poorly understood biology of the disease. To investigate initiating genomic alterations and the potential prognostic significance of cryptic genomic changes in low-risk MDS, we performed whole genome tiling path array comparative genomic hybridization (aCGH) on CD34(+) cells from 44 patients with an International Prognostic Scoring System score less than or equal to 1.0. Clonal copy number differences were detected in cells from 36 of 44 patients. In contrast, cells from only 16 of the 44 patients displayed karyotypic abnormalities. Although most patients had normal karyotype, aCGH identified 21 recurring copy number alterations. Examples of frequent cryptic alterations included gains at 11q24.2-qter, 17q11.2, and 17q12 and losses at 2q33.1-q33.2, 5q13.1-q13.2, and 10q21.3. Maintenance of genomic integrity defined as less than 3 Mb total disruption of the genome correlated with better overall survival (P = .002) and was less frequently associated with transformation to acute myeloid leukemia (P = .033). This study suggests a potential role for the use of aCGH in the clinical workup of MDS patients.

Correlation of clinical response and response duration with miR-145 induction by lenalidomide in CD34+ cells from patients with del(5q) myelodysplastic syndrome

We examined whether lenalidomide exposure up-regulates miRNAs and mRNAs, previously shown to play a role in the disease phenotype of del(5q) myelodysplastic syndrome, in pre-treatment CD34+ marrow cells. We hypothesized that increased expression would predict for clinical response. Changes in miR-143, miR-145, miR-146a, miR-146b, miR-378, miR-584, SPARC and RPS14 were examined in del(5q) (n=10) and non-del(5q) (n=18) myelodysplastic syndrome patient samples. Significantly increased expression of miR-143 (1.8-fold and 1.5-fold in del(5q) and non-del(5q), respectively), and miR-145 (1.9-fold and 1.6-fold in del(5q) and non-del(5q), respectively) was observed. In the del(5q) myelodysplastic syndrome cohort, transfusion independence correlated with a 1.3-fold or more increase in miR-145 expression and response over 12 months correlated with a 1.5-fold or more increase. Knockdown of miR-143 and miR-145 in cord blood CD34+ cells resulted in increased erythroid progenitor activity. Lenalidomide selectively abrogated progenitor activity in cells depleted of miR-143 and miR-145 supporting a key role for miR-143/145 in the sensitivity to lenalidomide of del(5q) myelodysplastic syndrome patients.

Heterogeneity of breast cancer stem cells as evidenced with Notch-dependent and Notch-independent populations

Studies have suggested the potential importance of Notch signaling to the cancer stem cell population in some tumors, but it is not known whether all cells in the cancer stem cell fraction require Notch activity. To address this issue, we blocked Notch activity in MCF‐7 cells by expressing a dominant‐negative MAML‐GFP (dnMAML) construct, which inhibits signaling through all Notch receptors, and quantified the effect on tumor‐initiating activity. Inhibition of Notch signaling reduced primary tumor sphere formation and side population. Functional quantification of tumor‐initiating cell numbers in vivo showed a significant decrease, but not a complete abrogation, of these cells in dnMAML‐expressing cells. Interestingly, when assessed in secondary assays in vitro or in vivo, there was no difference in tumor‐initiating activity between the dnMAML‐expressing cells and control cells. The fact that a subpopulation of dnMAML‐expressing cells was capable of forming primary and secondary tumors indicates that there are Notch‐independent tumor‐initiating cells in the breast cancer cell line MCF‐7. Our findings thus provide direct evidence for a heterogeneous cancer stem cell pool, which will require combination therapies against multiple oncogenic pathways to eliminate the tumor‐initiating cell population.

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.

Array comparative genomic hybridization of peripheral blood granulocytes of patients with myelodysplastic syndrome detects karyotypic abnormalities.

The diagnosis of myelodysplastic syndromes (MDSs) relies largely on morphologic and karyotypic abnormalities, present in about 50% of patients with MDS. Array-based genomic platforms have identified copy number alterations in 50% to 70% of bone marrow samples of patients with MDS with a normal karyotype, suggesting a diagnostic role for these platforms. We investigated whether blood granulocytes harbor the same copy number alterations as the marrow of affected patients. Of 11 patients, 4 had cytogenetic abnormalities shown by conventional karyotyping involving chromosomes 5, 8, 11, 20, and X, and these changes were seen in the granulocytes of all 4 patients by using array comparative genomic hybridization (aCGH). Cryptic alterations were identified at a significantly higher level in marrow CD34+ cells compared with granulocytes (P < .0001). These data suggest that aCGH analysis of circulating granulocytes may be useful in detecting gross karyotypic alterations in patients with MDS when marrow examination has failed or not been done.

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.