Ann Lab Med.  2016 Mar;36(2):101-110. 10.3343/alm.2016.36.2.101.

Clinicopathological Implications of Mitochondrial Genome Alterations in Pediatric Acute Myeloid Leukemia

• Kang MG
• Kim YN
• Lee JH
• Szardenings M
• Baek HJ
• Kook H
• Kim HR
• Shin MG

• KMID: 2373508
• DOI: http://doi.org/10.3343/alm.2016.36.2.101

Abstract

BACKGROUND
To the best of our knowledge, the association between pediatric AML and mitochondrial aberrations has not been studied. We investigated various mitochondrial aberrations in pediatric AML and evaluated their impact on clinical outcomes.
METHODS
Sequencing, mitochondrial DNA (mtDNA) copy number determination, mtDNA 4,977-bp large deletion assessments, and gene scan analyses were performed on the bone marrow mononuclear cells of 55 pediatric AML patients and on the peripheral blood mononuclear cells of 55 normal controls. Changes in the mitochondrial mass, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were also examined.
RESULTS
mtDNA copy numbers were about two-fold higher in pediatric AML cells than in controls (P<0.0001). Furthermore, a close relationship was found between mtDNA copy number tertiles and the risk of pediatric AML. Intracellular ROS levels, mitochondrial mass, and mitochondrial membrane potentials were all elevated in pediatric AML. The frequency of the mtDNA 4,977-bp large deletion was significantly higher (P< 0.01) in pediatric AML cells, and pediatric AML patients harboring high amount of mtDNA 4,977-bp deletions showed shorter overall survival and event-free survival rates, albeit without statistical significance.
CONCLUSIONS
The present findings demonstrate an association between mitochondrial genome alterations and the risk of pediatric AML.

Keyword

Pediatric; Acute myeloid leukemia; Clinical outcomes; mtDNA; Copy number; 4,977-bp deletion

MeSH Terms

Bone Marrow Cells/metabolism
Case-Control Studies
Child
Cohort Studies
DNA, Mitochondrial/chemistry/genetics/metabolism
Female
Flow Cytometry
Gene Deletion
Gene Dosage
*Genome, Mitochondrial
Humans
Leukemia, Myeloid, Acute/genetics/mortality/*pathology
Male
Membrane Potential, Mitochondrial
Minisatellite Repeats/genetics
Odds Ratio
Reactive Oxygen Species/metabolism
Sequence Analysis, DNA
Survival Rate
DNA, Mitochondrial
Reactive Oxygen Species

Figure

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  Fig. 1 AML cell-specific mtDNA sequence alteration. The mtDNA mutations were only identified in AML cells (CD34+ and CD33+ cells) and not in corresponding non-AML cells (CD2+ and CD3+ cells) from the same pediatric AML patient. (A, B) Sequence chromatogram of mtDNA hypervariable (HV) region 1 from non-AML cells and AML cells of the same patients showed the existence of an AML cell-specific mtDNA substitution mutation. (C, D) Electropherogram identified a length heteroplasmic mutation in the 303 poly C minisatellite in HV2 (nucleotide 303-315) in non-AML cells and in corresponding AML cells from the same patient.Abbreviation: mtDNA, mitochondrial DNA.

• 

  Fig. 2 Increased mtDNA copy number and mtDNA 4,977-bp large deletion in pediatric AML. (A) mtDNA copy number was significantly higher in patients with pediatric AML than in controls. (B) An increased amount of mtDNA 4,977-bp large deletion was observed in pediatric AML patients than in controls.Abbreviation: mtDNA, mitochondrial DNA.

• 

  Fig. 3 Increased intracellular reactive oxygen species (ROS) in pediatric AML cells. (A, B) DCFH-DA and flow cytometry were used to detect changes in intracellular ROS levels. The level of intracellular ROS was significantly increased in pediatric AML cells (4.96±1.96 mean fluorescence intensity [MFI]) than in control peripheral blood cells (2.65±0.41 MFI).Abbreviation: FITC, fluorescein isothiocyanate.

• 

  Fig. 4 Increased mitochondrial mass and membrane potential in pediatric AML cells. MitoTracker Green FM and MitoTracker Red probe were used to determine mitochondrial mass and mitochondrial membrane potential, respectively. (A, B) There was no significant difference of mitochondrial mass between patients (red colored histogram in A and black colored bar in B) and controls. (C, D) Mitochondrial membrane potentials were significantly elevated in pediatric AML cells (red colored histogram in C and black colored bar in D) than in control cells.Abbreviations: PE, phycoerythrin; MFI, mean fluorescence intensity.

• 

  Fig. 5 Impact of mtDNA 4,977-bp large deletion on survival. Overall survival (A) and event-free survival (B) tended to be shorter in pediatric AML patients (n=13) harboring high amount (≥0.00002) of the mtDNA 4,977-bp deletion than those (n=32) with low amount (<0.00002) of mtDNA 4,977-bp deletion. However, there were no significant differences between them.Abbreviation: mtDNA, mitochondrial DNA.

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