Patients participating in the study.

<p>MDS/MPD-U: myeloproliferative disorder/myelodysplastic syndrome overlap, unclassifiable; RARSt: refractory anemia with ringed sideroblasts and thrombocytosis; PV: polycythemia vera; IMF: idiopathic myelofibrosis,</p

Cryptic chromosomal abnormalities identified in patients with MDS/MPD-U and secondary AML with and without the JAK2 V617F mutation.

<p>NOTE: copy number lesions identified by MC were confirmed by SNP-A. Any significant changes in the size of regions initially reported by MC are noted in the right-hand column along with other additional lesions found by SNP-A.</p><p>Abbreviations: N/A: no aspirate obtained; NAL: no additional lesions found; *SNP-A data obtained using granulocyte DNA.</p

List of commonly deleted regions in MDS/MPD-U patients.

<p>List of commonly deleted regions in MDS/MPD-U patients.</p

SNP karyograms confirm loss of heterozygosity in patients homozygous for JAK2 V617F.

<p>SNP-A based karyotypic analysis on chromosome 9 for (A) a patient heterozygous for the JAK2 V617F mutation and (B,C) two patients homozygous for the JAK2 V617F mutation. (A,B and C, left portion) Signal intensity and SNP karyograms for each corresponding patient; the blue line represents the average fluorescent signal intensity of each SNP and oscillates around the diploid marker line; green tics represent heterozygous calls for each individual SNP. Areas of UPD are associated with the absence of heterozygous calls and are highlighted by blue and pink bars. Extraneous calls in regions of UPD occur as a result of contamination by non-clonal cells. UPD was confirmed by microsatellite analysis (data not shown). (C) In addition to chromosome 9, patient #36 also exhibited a segmental deletion in chromosome 12 as indicated by decreases in the copy number and frequency of heterozygous calls. (A,B, and C, right portion) Corresponding ARMS-PCR analysis of the JAK2 V617F mutation in each patient confirms heterozygous (A) and homozygous (B,C) mutational status (gel images are cropped and enhanced).</p

SNP-A can be used to identify lesions acquired during AML evolution.

<p>SNP-A karyograms demonstrate that before transformation (A), patient #38 showed only UPD9p at initial diagnosis as a sole abnormality (consistent with a homozygous JAK2 V617F mutation) along with normal chromosomes 4 and 19. However, after transformation to AML (B), repeated SNP-A analysis showed the presence of a V617F- leukemic clone with a normal chromosome 9 and newly-acquired micro-deletions on both chromosomes 4 and 19.</p

UPD found in MDS/MPD-U patients both positive and negative for the JAK2 V617F mutation.

<p>UPD found in MDS/MPD-U patients both positive and negative for the JAK2 V617F mutation.</p

Profile of genetic mutations and aberrant methylation.

<p>(A) Mutation status of RAS pathway genes and secondary genes (<i>SETBP1</i> and <i>JAK3</i>) identified as gene targets. Aberrant methylation scores (AMS) in a cohort of 92 patients with juvenile myelomonocytic leukemia are summarized. A rhombus denotes a patient with Noonan syndrome-associated myeloproliferative disorder. (B) Mutations in <i>SETBP1</i> and <i>JAK3</i> were associated with a higher AMS. The mean AMS of patients with <i>SETBP1</i> and/or <i>JAK3</i> mutations was higher than that of patients without secondary mutations (p = 0.03).</p

Summary of DNA methylation in candidate genes.

<p>(A) The dot plot represents the frequencies of methylated CpG sites for each candidate gene in the 92 patients with juvenile myelomonocytic leukemia. Aberrant hypermethylation was defined as >3 standard deviations above the mean methylation level of the healthy control population. The threshold values of each gene are shown as red broken lines. (B) Kaplan–Meier plots of the patient groups, defined by aberrant methylation of the indicated genes, are shown for <i>BMP4</i>, <i>CALCA</i>, <i>CDKN2A</i>, <i>CDKN2B</i>, <i>H19</i>, and <i>RARB</i>.</p

Hypermethylation status and clinical outcome in patients with juvenile myelomonocytic leukemia (JMML).

<p>(A) Kaplan–Meier curves represent the probability of transplantation-free survival (TFS) in the 92 patients with JMML. TFS was defined as the probability of being alive and transplantation free. Both death and transplantation were considered events. The probability of 5-year TFS in the aberrant methylation score (AMS) 0 cohort (solid line) was significantly higher than that in the AMS 1–2 (long dashed line) and AMS 3–4 cohorts (dashed line), p < 0.001. (B) Kaplan–Meier curves represent the probability of overall survival (OS) in the 92 patients with JMML. Death was considered an event. The probability of OS in both the AMS 0 (solid line) and 1–2 cohorts (long dashed line) was significantly higher than that in the AMS 3–4 cohort (dashed line), p < 0.001.</p

Patient characteristics.

<p>Patient characteristics.</p