Journal of Hematology, ISSN 1927-1212 print, 1927-1220 online, Open Access
Article copyright, the authors; Journal compilation copyright, J Hematol and Elmer Press Inc
Journal website http://www.thejh.org

Original Article

Volume 3, Number 2, June 2014, pages 34-42

Study of Correlation Between SFRP-1 and SFRP-2 Hypermethylation With Relapse, Complete Remission, Genetic Mutations of FLT3-ITD and NPM1 and Immunophenotypes of Leukemic Cells in Patients With De Novo Acute Myeloblastic Leukemia

Figures

Figure 1.
Figure 1. MSP analysis of SFRP-1 in AML patients and normal control. PC: positive control; NC: negative control; NC: normal control; P: patient; M: methylated; U: unmethylated. dH2O served as a blank control.
Figure 2.
Figure 2. MSP analysis of SFRP-2 in AML patients and normal control. PC: positive control; NC: negative control; NC: normal control; P: patient; M: methylated; U: unmethylated. dH2O served as a blank control.
Figure 3.
Figure 3. Mutation analysis by fragment analysis to assess NPM1 and FLT3/ITD mutations. (A) The peaks in blue and red show the results from amplification of the unmutated NPM1 and FLT3 alleles, respectively. (B) The peaks in blue and red on the right of the corresponding wild type alleles represent the mutated NPM1 and FLT3-ITD-positive alleles, respectively.

Tables

Table 1. SFRP-1 and SFRP-2 Gene Primers Sequences, Annealing Temperature and Product Size for MSP Assays
 
PrimerSequenceAnnealing temperatureProduct size (bp)
M: methylated; U: unmethylated; F: forward; R: reverse.
SFRP-1 MFTGTAGTTTTCGGAGTTAGTGTCGCGC62126
SFRP-1 MRCCTACGATCGAAAACGACGCGAACG
SFRP-1 UFGTTTTGTAGTTTTTGGAGTTAGTGTTGTGT54135
SFRP-1 URCTCAACCTACAATCAAAAACAACACAAACA
SFRP-2 MFGGGTCGGAGTTTTTCGGAGTTGCGC62138
SFRP-2 MRCCGCTCTCTTCGCTAAATACGACTCG
SFRP-2 URTTTTGGGTTGGAGTTTTTTGGAGTTGTGT64145
SFRP-2 URAACCCACTCTCTTCACTAAATACAACTCA

 

Table 2. Primer Sequences for Detection of Mutations in Nucleophosmin 1 (NPM1) Exon 12 and FLT3-ITD Exon 14
 
PrimerSequenceProduct size (bp)
NPM-FFAM-AGTTAACTCTCTGGTGGTAGAATGAAA236
NPM-RAGGACAGCCAGATATCAACTGTTAC
FLT3-FROX-GCAATTTAGGTATGAAAGCCAGC329
FLT3-RCTTTCAGCATTTTGACGGCAACC

 

Table 3. Correlations Between Hypermethylation of SFRP-1 and -2 Genes and Laboratory and Clinical Symptoms of AML Patients
 
CharacteristicsSFRP-1SFRP-2
MUPMUP
AML: acute myeloblastic leukemia; Hb: hemoglobin; WBC: white blood cell; FAB: French-American-British; NPM1: nucleophosmin 1; M: methylated; U: unmethylated.
Number of patients, %13 (30.2)30 (69.7)9 (20.9)34 (79.1)
Age (yeas), median (range)45.4 (23 - 60)39.6 (15 - 60)0.31946 (24 - 70)57 (15 - 72)0.692
Sex, %0.6510.692
  Male1021625
  Female3939
WBC count, 109/L, median15.731.70.24266.114.40.182
Platelet count, 109/L, median105.295.60.630891180.408
Hb level, g/dL, median8.99.90.1908.99.90.096
FAB type, n (%)
  M02 (15.3)00.0282 (22.2)00.004
  M12 (15.3)5 (16.6)0.91907 (20.5)0.646
  M24 (30.7)8 (26.6)0.7893 (33.3)9 (26.4)0.223
  M42 (15.3)8 (26.6)0.4332 (22.2)8 (23.5)0.936
  M52 (15.3)4 (13.3)0.8632 (22.2)8 (23.5)0.936
  M602 (6.66)0.35202 (5.8)0.468
  Unclassified1 (7.6)3 (10)0.81604 (11.7)0.291
Genetic mutation, n (%)
  FLT3-ITD2 (15.3)6 (20)0.9993 (33.3)5(14.7)0.332
  NPM12 (15.3)4 (13.3)0.9992 (22.2)4 (11.7)0.589
Outcome, n (%)
  Complete remission9 (69.2)19 (63.3)0.7175 (55.5)25 (73.5)0.308
  Failure3 (23)7 (23.3)0.9682 (22.2)8 (23.5)0.142
    Death1 (7.6)1 (3.3)0.5441 (11.1)1 (2.9)0.312
    Relapse2 (15.3)4 (13.3)0.8631 (11.1)5 (14.7)0.788