Molecular genetic techniques for gains and losses of genomic material in a case of acute myeloid leukemia.

A 67-year-old male presented with a four-week history of weakness. The complete blood count showed: hemoglobin: 5.1 g/dL, leukocytes 182.55 × 103/μL (23% blasts and 34% monocytes) and platelets: 31 × 109/L. A bone marrow aspirate showed 50.4% of myeloid myeloperoxidase (MPO)-blast cells and 42.4% of dysplastic granulocytic-monocytic series, with alpha-naphthyl acetate esterase positivity in 30% of total nucleated cells. Flow cytometry identified two distinct aberrant blasts (CD4-CD7-CD11c-CD13-CD34-CD117-HLA-DR-cMPO+) and myeloid/monocytic (CD14-CD33-CD35-HLA-DR-CD11b+) populations. Karyotyping showed monosomy 7 and additional material in the long arm of chromosome 2 (Figure 1A). Acute myeloid leukemia (AML)-M4 (FAB classification) or AML with myelodysplasia-related changes (WHO 2008 classification) was diagnosed. Patient died two months after without response to therapy.

Figure 1

(A) Karyotype (G-band): 45,XY,add(2)(q35),-7[20]. (B) FISH (Del (7q) Deletion Probe, ref: RU-LPH 025; Cytocell, Cambridge, UK): Deletion of RELN gene (chromosome 7) in 96% of the analyzed nuclei. The absence of a green and a red signal may indicate the monosomy of the chromosome.

Apart from karyotyping, other molecular genetic techniques can detect gains and losses of genomic material.1, 2, 3 In this case, the additional material in chromosome 2 was elucidated and chromosome 7 monosomy was confirmed using fluorescence in situ hybridization, multiplex ligation-dependent probe amplification and single nucleotide polymorphism-array methodologies (Figure 1, Figure 2).

Figure 2

(A) MLPA with SALSA MLPA probemix P144-A2 (above) and P145-A2 (below) kits (MRC-Holland, Amsterdam, The Netherlands). Dosage quotient of the patients’ probes in relation to a control group. Probes positioned below the 0.65 limit indicate deletion; probes positioned above the 1.35 limit indicate duplication. Probes in red indicate monosomy 7 (deletion of all probes of chromosome 7) and probes in black are normal for the other chromosomes studied in these kits. (B) SNP-A (CytoScanHD Array; Affymetrix, Santa Clara, USA): Diagrams generated by ChAS (Affymetrix) for chromosome 2 (above) and chromosome 7 (below). In “Copy number state”, line in 2.00 indicates a normal copy number (diploid), line in 1.00 indicates a deletion and line in 3.00 indicates a duplication/gain. Lines with intermediate values between 1.00 and 2.00; 2.00 and 3.00 indicate mosaicism. In “Allele Difference”, three lines indicate a normal genotype (AA, AB and BB alleles), two lines indicate deletion (A and B alleles) or loss of heterozygosity (AA and BB alleles) and four lines indicate duplication (AAA, AAB, ABB and BBB alleles). SNP-A revealed the result arr[hg19] 2p25.3p16.2(12770_54276429)x3[0.8],2q35q37.3 (219576639_242783384)x1,(7)x1 where there was a partial duplication of the short arm of chromosome 2 (A and C), presenting clonal mosaic data of the duplicated region in 80% of the cells, and partial deletion of the long arm of chromosome 2 (B and D). This result can explain add (2) (q35) present in the karyotype, indicating the origin of the additional material. In addition, the monosomy of chromosome 7 was confirmed.

Conflicts of interest

The authors declare no conflicts of interest.