Plasma samples of APL patients
An elderly Japanese man aged 85 years and a Japanese girl aged 4 years were diagnosed as having APL [9, 10]. They were treated with ATO (Trisenox®; Nippon Shinyaku, Kyoto, Japan) at Shimane University Hospital after giving informed consent. ATO was intravenously administered at a dose of 0.15 mg/kg/day for 6 weeks for the induction therapy. Two courses of consolidation therapy (0.15 mg/kg/day, 3 days a week for 5 weeks) were administered with a 6-week interval after a 4-week withdrawal period. Plasma samples from the older patient were collected during the two consolidation therapies while those of the pediatric patient were collected during the induction therapy. Plasma control specimens were obtained healthy Japanese volunteers (n = 76; mean age ± standard deviation, 36.8 ± 10.9 years). Written informed consent was obtained from the elderly patient, from the parents of the pediatric patient, and from the healthy volunteers. This study was approved by the Human Ethics Committee of Shimane University School of Medicine.
Extraction of cfDNA
Plasma cfDNA was extracted from 1 mL plasma by using the Maxwell® RSC ccfDNA Plasma Kit (Promega Corp., Madison, WI) with the Maxwell® RSC Instrument (Promega Corp.) in accordance with our previous studies [6, 7]. The cfDNA (2 µL) was plated on Thermo Scientific µDrop™ plates and concentrations were determined with a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA) by measuring spectrophotometric absorbances at 260 nm, 280 nm, and 320 nm.
Mutation analysis
Using cfDNA from APL patients as shown in Table 1 and in accordance with previous studies [17–21], mutations in FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), GATA-binding protein 2 gene (GATA2), Janus-associated kinase 2 gene (JAK2), Wilms’ Tumor 1 (WT1), Kirsten-ras (KRAS), and neuroblastoma-ras (NRAS) were assayed using the allele-specific primer-polymerase chain reaction (PCR) method, amplification-refractory mutation system (ARMS)-PCR, and PCR-restriction fragment length polymorphism (RFLP). cfDNA was amplified in a 20-µL reaction mixture by using a thermal cycler (T100 Thermal Cycler; Bio-Rad Laboratories, Inc., Hercules, CA). The reaction mixture contained forward and reverse primers and Green Master Mix (GoTaq®; Promega Corp.).
Table 1. FLT3-ITD mutation, GATA2 rs2713604, JAK2 rs10974944, WT1 rs16754, KRAS, and NRAS genotypes in APL patients
Gene mutation
|
Method
|
Restriction enzyme
|
Fragment size
|
Genotype
|
Elderly patient
|
Pediatric patient
|
FLT3-ITD mutation
|
ASP-PCR [16]
|
-
|
Mutation+: more than 329 bp
|
FLT3-ITD−
|
Mutation−: 329 bp
|
GATA2 rs2713604 (G > A)
|
PCR-RFLP [17]
|
Ava I
|
G allele: 227 bp
|
AG
|
A allele: 157 and 70 bp
|
JAK2 rs10974944 (C > G)
|
PCR-RFLP [18]
|
Mbo I
|
C allele: 176, 37, 23 and 7 bp
|
CG
|
CC
|
G allele: 213, 23 and 7 bp
|
WT1 rs16754 (A > G)
|
ARMS-PCR [19]
|
-
|
A allele: 240, 604 bp
|
GG
|
AA
|
G allele: 400, 604 bp
|
KRAS (codon 12 mutation)
|
PCR-RFLP [20]
|
Mva I
|
WT allele: 113, 29, 15 bp
|
Heterozygous mutation
|
Mutant allele: 142, 15 bp
|
NRAS (codon 12 mutation)
|
WT allele: 41, 23, 19 bp
|
Homozygous mutation
|
Mutant allele: 60, 23 bp
|
FLT3-ITD: FMS-like tyrosine kinase 3-internal tandem duplication
GATA2: GATA-binding protein 2
JAK2: Janus-associated kinase 2 gene
WT1: Wilms' Tumor 1
KRAS: Kirsten-ras
NRAS: Neuroblastoma-ras
ASP-PCR: allele-specific primer-polymerase chain reaction
PCR-RFLP: polymerase chain reaction-restriction fragment length polymorphism
ARMS-PCR: amplification-refractory mutation system-polymerase chain reaction
Microchip electrophoresis
Plasma cfDNA was electrophoresed using an MCE-202 MultiNA automated microchip-based electrophoresis system equipped with MultiNA Viewer software (Shimadzu Corp., Kyoto, Japan) in accordance with our previous studies [6, 7]. cfDNA samples were run with separation buffer reagents and DNA marker reagent (DNA-1000 kit; Shimadzu), and a 100-bp DNA ladder (Takara Bio, Inc., Shiga, Japan) was used as a marker. Amplified cfDNA samples for gene mutations were run with the separation buffer reagents and DNA marker reagent (DNA-500 kit; Shimadzu), and 25 bp DNA Step Ladder (Promega Corp.) was used as a marker.
Total arsenic analysis
The total arsenic concentration in plasma was determined by high-performance liquid chromatography (LC10A Series, Shimadzu) coupled with inductively coupled plasma mass spectrometry (HP-4500; Hewlett-Packard, Avondale, PA) in accordance with our previous reports [11, 12]. Plasma was filtered and analyzed without dilution. The concentrations of total arsenic in plasma are expressed as µg/mL.
Statistical analysis
The differences in cfDNA concentrations between the control group and APL patients were analyzed with Dunnett’s test. In addition, the correlation between arsenic and the cfDNA concentration was analyzed with Spearman’s rank correlation test. These analyses were performed by using Bell Curve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan).