ONLINE SUPPLEMENTARY MATERIAL FOR:
Synergistic interaction of ABCB1 and ABCG2 polymorphisms predicts the prevalence of toxic encephalopathy during
anticancer chemotherapy
DJ Erdélyi, E Kámory, B Csókay, H Andrikovics, A Tordai, C Kiss, Á Félné-Semsei, I Janszky, A Zalka, G Fekete, A Falus, GT Kovács, C
Szalai
Hospitals participating in this study:
1st and 2nd Dept. Pediatrics, Semmelweis University, Budapest; Heim Pál Children Hospital, Budapest; Madarász street Children Hospital,
Budapest; Markusovszky Hospital, Szombathely; Dept. Pediatrics, University of Pécs, Pécs; Dept. Pediatrics, University of Debrecen, Debrecen;
Dept. of Pediatrics, University of Szeged, Szeged; Borsod County Hospital, Miskolc, all in Hungary. Patients were treated according to the same
chemotherapy protocols at all institutes (Table S1).
Table S1
Flowchart of the intensive chemotherapy phase of the ALL BFM 90 and 95 protocols, low risk and medium risk arms.
Induction
Prednisone 60mg/m2 orally for 36 days (including tapering at start and end)
Vincristine 4 x 1,5 mg/m2 i.v. bolus
Daunorubicin 2-4 x 30 mg/m2 / 3 hours infusion i.v.a
L-asparaginase 8 x 10.000 U/m2 / 1 hour i.v. infusion
Methotrexate 3-5 x 6-12 mg intrathecally (dose depending on age)b
Intensification
Cyclophosphamide 1-2 x 1 g/m2 / 1 hour infusion c
Cytosine arabinoside 8-16 x 75mg/m2 i.v. bolus c
Mercaptopurine 60 mg/m2 orally for 14-28 days c
Methotrexate 2 x 6-12 mg intrathecally (dose depending on age)
Consolidation
Methotrexate 4 x 5 g/m2/24 hours infusion
Mercaptopurine 25 mg/m2 orally for 56 days
Methotrexate 4 x 6-12 mg intrathecally (dose depending on age)
Reinduction
Dexamethasone 10mg/m2 orally for 30 days (including tapering)
Vincristine 4 x 1,5 mg/m2 i.v. bolus
Doxorubicin 4 x 30 mg/m2 / 3 hours infusion i.v.
L-asparaginase 4 x 10.000 U/m2 / 1 hours i.v.
Methotrexate 0-4 x 6-12 mg intrathecally (dose depending on age)b
Reintensification
Cyclophosphamide 1 x 1 g/m2 / 1 hour infusion
Cytosine arabinoside 8 x 75mg/ m2 i.v. bolus
Thioguanine 60 mg/m2 orally for 14 days
Methotrexate 2 x 6-12 mg intrathecally (dose depending on age)
The intensive chemotherapy phase was followed by an oral maintenance chemotherapy period completed 2 or 3 years after the start of intensive
therapy.
a
Patients assigned to the medium risk arm were administered 8 doses of anthracyclines, while 6 doses were given at the low risk arm. In the
latter group, two daunorubicin doses were omitted.
b
Number of doses depending on central nervous system involvement of ALL at diagnosis.
c
Patients received a shorter protocol in the period of 1995-1999 according to the lower limits of dose number ranges indicated. In the altered
protocol in use afterwards, the upper limits of the ranges were applied.
Abbreviations: ALL: acute lymphoblastic leukemia; BFM: Berlin-Münster-Frankfurt workgroup; i.v.: intravenous
Method for testing ABCB1 polymorphisms
ABCB1 3435C/T, 2677G/T,A and 1236C/T genotypes were determined by single base extension method. This method and the primer design
were based on the work of Gwee et al.,1 however numerous minor changes were made. Three fragments including exon 12, 21 and 26 were
amplified in a single multiplex PCR reaction. The sequences of home made primers are shown in Table S2. We used 20 ng of genomic DNA per
sample in a total volume of 20 μl containing 5 mM MgCl2 (Applied Biosystems), 200 μM of each dNTP (Promega), 0.15, 0.075 and 0.3 pmol/μl
of the primers, and 1.5 unit of AmpliTaq Gold polymerase (Applied Biosystems) in the PCR buffer (Applied Biosystems). The PCR reaction
began with an initial denaturation step at 95 ºC for 10 min, followed by 40 cycles of 95 ºC for 30 s, 58 ºC for 30 s and 72 ºC for 60 s, a final
extension at 72 ºC for 5 min was also performed. Electrophoresis was carried out by an Agilent lab-on-chip instrument.
Unincorporated dNTPs and primers were degraded by the addition of 1 unit of shrimp alkaline phosphatase (SAP, USB) and 0.5 unit of
exonuclease I (USB) to 3 μl of PCR product in a final volume of 5 μl. The mixture was incubated at 37 ºC for 1 hour, and then the enzymes were
inactivated at 75 ºC for 15 min. The treated PCR products were subjected to a multiplex minisequencing reaction to interrogate the three
simultaneous SNP loci. The reaction contained 1 μl of the treated PCR product 0.4, 0.1 and 0.2 pmol/μl of the minisequencing primers, 2.5 μl of
SNaPshot Multiplex Ready Reaction Mix (Applied Biosystems) in a total volume of 5 μl. The reaction contained 25 cycles of 96 ºC for 10 s, 54
ºC for 5 s and 60 ºC for 30 s. The unincorporated fluorescent ddNTPs were degraded by the addition of 1 unit of SAP at 37 ºC for 1 hour, and
then the enzyme was inactivated at 75 ºC for 15 min. 0.7 μl of the digested product was added to 9 μl of HiDi formamide and 0.5 μl of
GeneScan-120 LIZ size standard (Applied Biosystems). The electrophoresis was performed for 17 min with ABI 310 Genetic Analyzer (Applied
Biosystems). The runs were analyzed with Genotyper software (Applied Biosystems) and home made template. The relative position of each
peak indicated the SNP locus, and the peak color indicated the specific genotype.
Genotype findings were verified for the three ABCB1 polymorphisms on 2 samples for each genotype by single PCR followed by bidirectional
sequencing using the BigDye v3.1 kit on an ABI310 sequencer.
Table S2 Sequences of oligonucleotides used for genotyping ABCB1 SNPs
Forward primer
Reverse primer
Minisequencing primer
1236C/T
tctcaccatcccctctgt
tctttgtcacttatccagc
ct(c)12gactctgcatcttcaggttcag1
2677G/T,A
tagggagtaacaaaataacac
tgcaggctataggttccagg
(tgac)6ttagtttgactcaccttcccag
3435C/T
cttacattaggcagtgac
ctcacagtaacttggcag
(tgac)12ctcctttgctgccctcac
1
Different from that of Gwee et al., changes made due to hairpin formation
Method for testing ABCG2 polymorphisms
ABCG2 34G/A and 421C/A genotyping was performed using the LightCycler (Roche Diagnostics) allelic discrimination system. Amplification
primers and hybridization probes were designed by the LightCycler Probe Design software (Roche Diagnostics). Table S3 shows the
oligonucleotide sequences. Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, USA). PCR was performed by rapid
cycling in glass capillaries in a reaction volume of 10 l with 50 ng genomic DNA, 5 µl 2x PCR Master Mix (Promega), supplemented by 0.7 U
Taq DNA polymerase (Finnzyme, Espoo, Finnland), and 1.5 mM MgCl2, and 2.5 pmol of labelled oligonucleotides (sensor and anchor). An
asymmetric PCR was used for amplification of the two loci with 5:1.5 pmol forward:reverse amplification oligonuclicleotides and 2:20 F:R
amounts for 34G>A and 421C>A SNP-s respectively.2 Cycling conditions were the following: initial denaturation at 94C for 2 min, followed by
70 cycles of denaturation 94C for 0 s, annealing at 50C for 10 s, and extension at 72C for 15 s, with a ramping rate of 20C/s. After
amplification, melting curve analysis was performed by cooling the samples to 38C, then gradually heating them to 85C with a ramp rate of
0.1C/s. The decline of fluorescence was continuously monitored. Melting curves were converted to melting peaks with wild type and variant
alleles showing distinct melting points.
Table S3
Sequences of oligonucleotides used for genotyping ABCG2 SNPs
Designation
Function
Sequence
ABCG2-34-F
amplification primer
5’atgtattgtcacctagtgtttgc
ABCG2-34-R
amplification primer
5’agctccttcagtaaatgcc
ABCG2-34-ANC
hybridization probe
5’gcggggaagccattggtgt/36-FAM/
ABCG2-34-SENS
hybridization probe
5’/Bodipy650/ccttgtgacactgggat/3Phos/
ABCG2-421-F
amplification primer
5’tatgtatactaaacagtcatggtcttaga
ABCG2-421-R
amplification primer
5’tggagtctgccactttatccagacct
ABCG2-421-ANC
hybridization probe
5’gatgatgttgtgatgggcactctgacggtgaga/36-FAM/
ABCG2-421-SENS
hybridization probe
5’/Bodipy650/aaacttacagttctcagcagctcttcgg/3Phos/
Nucleotides corresponding to the localization of the investigated sequence variants in the sensor hybridization probes are typed with underline
bold face characters.
Table S4.
CSF methotrexate concentrations (µmol/l) in genotype groups
Genotype groups
N
Median
Q25
Q75
Minimum
Maximum
ABCB1 3435TT
45 (14)
1.95
1.47
3.35
0.76
70.8
ABCB1 3435CC+CT
79 (26)
2.34
1.72
4.82
0.78
81.8
ABCB1 2xTTT
42 (13)
1.94
1.45
3.07
0.76
70.8
ABCB1 non-2xTTT
82 (27)
2.34
1.71
4.83
0.78
81.8
ABCG2 421CC
107 (34)
2.22
1.50
4.59
0.76
81.8
17 (6)
2.33
2.02
2.64
1.12
44.7
ABCG2 421AA+AC
N: number of CSF methotrexate concentration data available (number of patients the data are originated from); Q25: 25% quantile; Q75: 75%
quantile; 2xTTT: 3435T-2677T-1236T haplotype homozygote.
REFERENCES
1. Gwee PC, Tang K, Chua JM, Lee EJ, Chong SS, Lee CG. Simultaneous genotyping of seven single-nucleotide polymorphisms in the
MDR1 gene by single-tube multiplex minisequencing. Clin Chem 2003; 49: 672-676.
2. Szilvasi A, Andrikovics H, Kalmar L, Bors A, Tordai A. Asymmetric PCR increases efficiency of melting peak analysis on the
LightCycler. Clin Biochem 2005; 38: 727-730.