Supplementary Methods:
PCR detection of fusion gene
To confirm the suspected the BCR-FGFR1 fusion, genomic DNA and total RNA from
the patient and a healthy donor were isolated using AllPrep DNA/RNA/Protein kit
(Qiagen), and reverse-transcription on RNA was performed with SuperScript II Reverse
Transcriptase (Life Technologies). For detection of the BCR-FGFR1 fusion gene from
genomic DNA, forward primer on the intron between BCR exon 4 and exon 5 and
reverse primer on the intron between exon 8 and exon 9 of FGFR1 were used, while the
fusion transcript was detected with forward primer on BCR exon 3 and reverse primer
on FGFR1 exon 9. PCR products verification and breakpoint mapping were performed
with Sanger sequencing. The reciprocal FGFR1-BCR fusion gene from genomic DNA
and cDNA was also detected by PCR with primer pairs flanking FGFR1 upstream of the
breakpoint and BCR downstream of the breakpoint. Detection of BCR and FGFR1 with
primers flanking within the genes was performed as controls. To measure fusion gene
allele burden, nested quantitative real-time PCR on gDNA was performed with Power
SYBR® Green PCR Master Mix (Life Technologies) on 7900HT System (Applied
Biosystems) following manufacturer’s instruction. Fifteen nanograms of genomic DNA
was initially amplified by PCR. This PCR product was then used in a quantitative realtime PCR assay with a second set of nested primers.
All primers are listed in
Supplementary Table 1. The level of RNase P, a target that exists as a single copy
gene per haploid genome was used as internal control for the quantitative real-time
PCR1.
Supplementary Figure 1. Morphologic, flow cytometric, and cytogenetic findings
at presentation. (A) The peripheral blood showed a marked leukocytosis with an
absolute neutrophilia, monocytosis, and 42% blasts with a variety of morphologic forms
including round to highly convoluted nuclei and delicate to deeply basophilic cytoplasm
(60x).
(B&C) The bone marrow was hypercellular with similar composition and
morphology to that noted in the peripheral blood with occasional eosinophils and mast
cells also present (60x). (D) Cytogenetic findings included a t(8;22) with two copies of
the der(22). (E) BCR-ABL1 fusion probe analysis demonstrated an abnormal signal
pattern with four BCR signals (green); arrows indicate one BCR signal localizing to the
der(8)t(8;22).
(F) FGFR1 break-apart probe analysis demonstrates a 5’/3’ signal
separation consistent with an FGFR1 rearrangement.
PCR analysis (not shown)
confirmed the BCR-FGFR1 fusion product. (G-I) Flow cytometric findings demonstrate
three distinct populations of MPO+ myeloblasts, CD19+/CD79a+/CD22+ lymphoblasts,
and cytoplasmic CD3+ T-lymphoblasts. The bright CD117+ events are consistent with
increased mast cells.
Key:
SSC:
side
scatter;
TdT:
terminal
deoxynucleotidyl
transferase;
MPO:
myeloperoxidase.
Supplementary Figure 2. Quantitative nested PCR detection of BCR-FGFR1
fusion. (A) Quantitative nested PCR was performed with primer sets specific for the
patient’s BCR-FGFR1 fusion breakpoint or RNase P.
Threshold cycle (Ct) versus
dilution is shown, demonstrating satisfactory efficiency of the PCR reaction. (B) Allele
burden for each sample was detected in triplicate. Allele burden was normalized to the
average value at diagnosis. Two replicate experiments are shown.
Supplementary Figure 1. Morphologic, flow cytometric, and cytogenetic findings
at presentation.
A
B
C
E
D
F
p11.2
q11.2
der(22) der(22)
8
G
22
der(8)
262144
H
14.4%
I
2.6%
105
15.2%
0.2%
105
SSC-A
Blast
131072
104
cCD3 PE-A
79a PE-A
196608
103
65536
29.2%
102
103
104
105
-101
102
CD45 PerCP-Cy5-5-A
1.2%
3.3%
K
103
51.6%
102
-101
-102
12.9%
103
104
CD19 APC-A
105
10.4%
103
102
102
74.6%
-101
9.9%
102
103
104
105
TdT FITC-A
104
-101
-101
-102
105
105
CD34 PE-A
CD10 PE-A
104
-102
104
MPO FITC-A
105
82.6%
103
L
3.2%
2.1%
105
CD117 PE-A
J
-101
53.8%
-103
-102 -101
103
102
-102
0
104
104
103
102
35.3%
-101
102
103
104
CD22 FITC-A
2.7%
36.3%
105
-101
58.4%
102
103
104
CD15 FITC-A
105
Supplementary Figure 2.
Supplementary Table 1. Primers for detection and quantification of BCR-FGFR1
fusion gene
BCRFGFR1
FGFR1BCR
gDNA
cDNA
gDNA
cDNA
gDNA
BCR
cDNA
gDNA
FGFR1
cDNA
First round
of nested
PCR
RNase P
Second
round of
nested PCR
RNase P
BCRFGFR1
BCRFGFR1
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
5’-CACTTGCTTGCCACAGGTTC-3’
5’-GGGGTGACTCCTTCCCATTC-3’
5’- GCTGGACTTGGAAAAGGGCT-3’
5’- CGGGAAGCTCATACTCAGAGAC-3’
5’-TGTCAAGCTGGAAGAGGTGC-3
5’-ACTGCACCACTGTACTCCAGA-3’
5’-GACGCAGGGGAGTATACGTG-3’
5’-ACTCCGTAGTTGTCCACGAA-3’
5’- GCTGGACTTGGAAAAGGGCT-3’
5’-AGAGCAGAAAACTCAGCCCC-3’
5’- GCTGGACTTGGAAAAGGGCT-3’
5’-ACTCCGTAGTTGTCCACGAA-3’
5’- TGTCAAGCTGGAAGAGGTGC-3’
5’-GGGGTGACTCCTTCCCATTC-3’
5’- GACGCAGGGGAGTATACGTG-3’
5’-CGGGAAGCTCATACTCAGAGAC-3’
5'-TAGGATGTTTTTCGCATCTGGC-3'
5'-GGATACAGATTTGCCACGTCAT-3'
5'-CACTTGCTTGCCACAGGTTC-3'
5'-CGGGAAGCTCATACTCAGAGAC-3'
Forward
5'-CCCACTTTCTTTAGGCTTCAACTT-3'
Reverse
Forward
Reverse
5'-GGCCCTCTTATTTCTAAAGGCTGA-3'
5'-GACCAGCTCAGTAGGGTTGG-3'
5'-GGGGTGACTCCTTCCCATTC-3'
1.
Szantai E, Elek Z, Guttman A, et al: Candidate gene copy number analysis by PCR and
multicapillary electrophoresis. Electrophoresis 30:1098-101, 2009