Monika Jasek

Characterization of chromosome arm 20q abnormalities in myeloid malignancies using genome‐wide single nucleotide polymorphism array analysis

Deletion of the long arm of chromosome 20 is a common abnormality associated with myeloid malignancies. We characterized abnormalities of chromosome 20 as defined by metaphase cytogenetics (MC) in patients with myeloid neoplasms to define commonly deleted regions (CDR) and commonly retained regions (CRR) using genome‐wide, high resolution single nucleotide polymorphism array (SNP‐A) analysis. We reviewed the MC results of a cohort of 1,162 patients with myeloid malignancies, including myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasia (MDS/MPN), and acute myeloid leukemia (AML). We further analyzed a subcohort of 532 patients by SNP‐A using the Affymetrix Genome‐Wide Human SNP Array 6.0 and GeneChip Human Mapping 250K Nsp arrays. By MC, 5% (54/1,162) harbored a deletion of 20q; in 30% (16/54), del(20q) was the sole cytogenetic abnormality. By SNP‐A analysis, we identified del(20q) in 23 patients, 3 not detected by MC. In four cases, monosomy 20 with a marker chromosome by MC was proven to be an interstitial deletion of 20q by SNP‐A. We defined 2 CDR and 2 CRR on chromosome arm 20q: CDR1 spanned 2.5 Mb between bands 20q11.23 and 20q12, while CDR2 encompassed 1.8 Mb within 20q13.12. CRR1 spanned 1.9 Mb within 20q11.21 and CRR2 encompassed 2.5 Mb within 20q13.33. In contrast to other chromosomes frequently affected by deletions, no somatic copy neutral loss of heterozygosity (CN‐LOH) was detected. Our data suggest that SNP‐A is useful for the detection of cryptic aberrations of chromosome 20q and allows for a more precise characterization of complex karyotypes. Furthermore, SNP‐A allowed definition of a CDR on 20q.

TP53 Mutations in Myeloid Malignancies are either Homozygous or Hemizygous due to Copy Number-Neutral Loss of Heterozygosity or Deletion of 17p

Our previous studies demonstrated that single nucleotide polymorphism arrays (SNP-A), applied as a karyotyping platform, complement traditional metaphase cytogenetics (MC) and improve the diagnostic yield of cytogenetic studies because SNP-A can more precisely resolve smaller genetic defects and allow for detection of copy number-neutral loss of heterozygosity (CN-LOH), a defect frequently found in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Like balanced translocations, CN-LOH represents a chromosomal abnormality that occurs without a concurrent change in gene copy number (CN). CN-LOH is an increasingly recognized chromosomal mechanism by which homozygous somatic mutations may be acquired during evolution of hematologic malignancies and can pinpoint the location of such gene(s); examples include CEBPA, FLT3, WT1, RUNX1, JAK2, and NF11.

MICA polymorphism identified by whole genome array associated with NKG2D-mediated cytotoxicity in T-cell large granular lymphocyte leukemia

Background Large granular lymphocyte leukemia is a semi-autonomous clonal proliferation of cytotoxic T cells accompanied by immune cytopenias and various autoimmune conditions. Due to the rarity of this disease and its association with autoimmune diseases, a theoretical germline or somatic mutation might have significant penetrance, thus enabling detection, even from samples of suboptimal size, through genome-wide association studies. Design and Methods To investigate a non-mendelian genetic predisposition to large granular lymphocyte leukemia, we used a step-wise method for gene discovery. First, a modified ‘random forests’ technique was used for candidate gene identification: this was followed by traditional allele-specific polymerase chain reaction, sequencing modalities, and mechanistic assays. Results Our analysis found an association with MICA, a non-peptide-presenting, tightly regulated, stress-induced MHC-like molecule and cognate receptor for NKG2D, found abundantly on large granular lymphocyte leukemia cells. Sequencing of germline DNA revealed a higher frequency of MICA*00801/A5.1 in patients with large granular lymphocyte leukemia than in matched controls (64% versus 41%, P<0.001, homozygous 40% versus 15%, P<0.001). Flow cytometry was employed to determine the expression of MICA within hematologic compartments, showing that the signal intensity of MICA was increased in granulocytes from neutropenic patients with large granular lymphocyte leukemia in comparison with that in controls (P=0.033). Furthermore, neutrophil counts were inversely correlated with MICA expression (R2=0.50, P=0.035). Finally, large granular lymphocyte leukemia cells were able to selectively kill MICA+ Ba/F3 lymphocytes transfected with human MICA*019 in a dose-dependent manner compared to naïve cells (P<0.001), an effect mitigated by administration of an anti-NKG2D antibody (P=0.033). Conclusions Our results illustrate that MICA-NKG2D played a role in disease pathogenesis in the majority of patients in our cohort of cases of large granular lymphocyte leukemia and further investigation into this signaling axis may provide potent therapeutic targets.

Molecular lesions in childhood and adult acute megakaryoblastic leukaemia

While acute megakaryoblastic leukaemia (AMKL) occurs in children with (DS‐AMKL) and without (paediatric non‐DS‐AMKL) Down syndrome, it can also affect adults without DS (adult non‐DS‐AMKL). We have analysed these subgroups of patients (11 children with DS‐AMKL, 12 children and four adults with non‐DS‐AMKL) for the presence of molecular lesions, including mutations and chromosomal abnormalities studied by sequencing and single nucleotide polymorphism array‐based karyotyping, respectively. In children, AMKL was associated with trisomy 21 (somatic in non‐DS‐AMKL), while numerical aberrations of chromosome 21 were only rarely associated with adult AMKL. DS‐AMKL was also associated with recurrent somatic gains of 1q (4/11 DS‐AMKL patients). In contrast to trisomy 21 and gains of 1q, other additional chromosomal lesions were evenly distributed between children and adults with AMKL. A mutational screen found GATA1 mutations in 11/12 DS‐AMKL, but mutations were rare in paediatric non‐DS‐AMKL (1/12) and adult AMKL (0/4). JAK3 (1/11), JAK2 (1/11), and TP53 mutations (1/11) were found only in patients with DS‐AMKL. ASXL1, IDH1/2, DNMT3A, RUNX1 and CBL mutations were not found in any of the patient group studied, while NRAS mutation was identified in two patients with paediatric non‐DS‐AMKL.

Graft-versus-host disease: role of inflammation in the development of chromosomal abnormalities of keratinocytes.

Graft-versus-host disease (GVHD) is a major risk factor for secondary malignancy after hematopoietic stem cell transplantation. Squamous cell carcinoma (SCC) of the skin and mucous membranes are especially frequent in this setting where aneuploidy and tetraploidy are associated with aggressive disease. The current study is directed at the mechanism of neoplasia in this setting. Unmanipulated keratinocytes from areas of oral GVHD in 9 patients showed tetraploidy in 10% to 46% of cells when examined by florescein in situ hybridization (FISH). Keratinocytes isolated from biopsy sites of GVHD but not from normal tissue showed even greater numbers of tetraploid cells (mean = 78%, range: 15%-85%; N = 9) after culture. To mimic the inflammatory process in GVHD, allogeneic HLA-mismatched lymphocytes were mixed with normal keratinocytes. After 2 weeks, substantial numbers of aneuploid and tetraploid cells were evident in cultures with lymphocytes and with purified CD8 but not CD4 cells. Telomere length was substantially decreased in the lymphocyte-treated sample. No mutations were present in the p53 gene, although haploinsufficiency for p53 due to the loss of chromosome 17 was common in cells exposed to lymphocytes. These findings suggest that in GVHD, inflammation and repeated cell division correlate with the development of karyotypic abnormalities.