PMC4779576_01
Microscopic picture of peripheral blood smear of the AML patient from the time of admission to the local pediatric department showing a a myeloblast (arrow) and a lymphocyte. b A promyelocyte (arrow) and a vacuolated monocyte. Magnification 100x
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Fig1
Blood tests revealed anemia (hemoglobin 8.2 g/dL), thrombocytopenia (64 x 109/L), normal white blood cell count (11.9 x 109/L) with monocytosis (1.5 x 109/L), and immature myeloid cells were seen in the blood smear (Fig. 1). 
Bivariate dot plots of the flow cytometric analysis of the bone marrow aspirate. The color codes for the different cell subsets are as follows: green for granulocytes, blue for monocytes, purple for CD34 positive precursors, yellow for lymphocytes, and dark blue for erythroid precursors. Dot plot
a and b show normal light scatter signals and normal expression of CD45 of all cell populations. No increase of CD34/CD117 positive cells was demonstrated as can be seen in dot plot
c. Dot plot 
d shows partial loss of CD13 expression on monocytes as well as on granulocytes and shift to immaturity of the granulocytes of which the majority were negative for CD11b. The aberrant expression of CD56 by granulocytes and monocytes is demonstrated in dot plot
e. Immature granulocytes were positive for CD4 as shown in dot plot
f
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Fig2
Further, flow cytometry analysis (Fig. 2) confirmed the normal distribution of CD34+ or CD117+ precursor cells but revealed two distinct myeloid populations comprised of 68 % CD15 bright +/HLA-DR- granulocytic cells and 15 % CD64 bright +/HLA-DR- monocytic cells.
Coronal water-sensitive, fat-suppressed MRI-sequences of the mandible (a), whole-body (b), pelvis, and femurs of the AML patient (c, d) showing extensive infarcts in several bones (arrows). In the mandible and iliac wings, there was marked, spiculated cortical reaction and new bone formation (arrowheads) with destruction of the underlying skeleton (asterisk). Subtracted post contrast T1 fat suppressed images of the pelvis and femurs showed low-signal avascular areas with a peripheral rim of enhancement caused by the multiple infarcts (arrows). There was soft tissue edema in the lower extremities. The kidneys were slightly enlarged
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Fig3
Whole body magnetic resonance imaging (WBMRI) showed marked spiculated periosteal reaction and new bone formation in the mandible, iliac bones, and bilaterally in the distal femur (Fig. 3a-d). There was soft tissue involvement of the lower extremities with edema in the muscles and subcutaneous tissue and fluid along the facial planes (Fig. 3a-d). 
Cytogenetic, FISH, and PCR analyses of the AML patient. a G-banded karyotype showing trisomy 6, der(8), der(11), and der(19) of the t(8;19;11)(q24;p13;q23) together with the corresponding normal chromosome homologs; breakpoint positions are indicated by arrows. b FISH using an MLL breakapart probe showed rearrangement of MLL. The 3 -end part of the MLL gene (red probe) has moved to the q arm of the der(8), while the 5 -end part of the gene (green probe) remains on 11q23 of the der(11). c The initial RT-PCR amplifications for the detection of a possible MLL-ELL fusion transcript. Lane 1, nested PCR with the forward primers located in exon 7 of MLL and reverse primers located in exon 4 of ELL (MLL-3947F1/ELL-415R) failed to amplify any cDNA fragments. Lane 2, amplification of a cDNA fragment of the ABL1 gene using the primers ABL1-91F1 and AsBL1-404R1 suggested that the synthesized cDNA was of good quality. d RT-PCR using a new reverse primer located in exon 8 of ELL (primer ELL-1044R1) and a forward primer located in exon 7 of MLL (primer MLL-3878F) amplified a cDNA fragment. M, 1 Kb DNA ladder. e Partial sequence chromatogram of the amplified fragment using the primers MLL-3878F and ELL-1044R1 showing the junction of the MLL-ELL chimeric transcript
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Fig4
FISH analyses of bone marrow cells using the Cytocell multiprobe ALL panel (Cytocell, http://www.cytocell.co.uk/) showed a split signal of the MLL locus in 146 out of 201 investigated interphase nuclei (data not shown). FISH analysis of metaphase spreads using the MLL breakpoint probe (Cytocell, http://www.cytocell.co.uk/) showed that the red signal (distal) had moved not to chromosome 19 but to the q arm of chromosome 8 (Fig. 4b). Thus, the modified karyotype after G-banding analysis and FISH was 47,XY, +6,t(8;19;11)(q24;p13;q23) (Fig. 4a, b). The initial RT-PCR with the primer set MLL-3878F/ELL-498R1 as well as nested PCR with the primers MLL-3947F1/ELL-415R (the sequences of the primers are listed in Table 1) failed to amplify any cDNA fragments (Fig. 4c). At the same time, use of the primer set ABL1-91F/ABL1-404R1 led to amplification of an ABL1 cDNA fragment suggesting that the synthesized cDNA was of good quality (Fig. 4c).  PCR with the MLL-3878F and ELL-1044R1 primer combination (Table 1) amplified a fragment from the patient's bone marrow cDNA (Fig. 4d). Sanger sequencing of the amplified product showed that it was a chimeric MLL-ELL cDNA fragment in which exon 9 of MLL was fused to exon 6 of ELL, i.e., the same MLL-ELL fusion transcript found by RNA-sequencing (Fig. 4e; Table 2).