Supplemental Table S1. The PCR primers for analyzing the mutation hotspots of CIC, PLCG1, and KDR genes.
Primer Pair Primer Sequence
Annealing Temperature
(℃)
Amplico n Size
(bp) CIC exon 5 forward 5'-GTACTTTATCCCTGCCTGTCCTG 71℃ x 10 cycles
66℃ x 25 cycles 333 CIC exon 5 reverse 5'-CACTCACCCACCCCACCAG
CIC exon 15 forward 5'-GTGTCAGATCAACCCAGAGCAGAG
65 499
CIC exon 15 reverse 5'-GAGTAGGACAGAAAAGCAAAGACG
CIC exon 18 forward 5'-CTTAGGTGGAGGGCAGACTGG 71℃ x 10 cycles
66℃ x 25 cycles 268 CIC exon 18 reverse 5'-CTCTCACTCTCTAACCGCCTTCC
CIC exon 19 forward 5'-GGAAGGCGGTTAGAGAGTGAGAG 71℃ x 10 cycles
66℃ x 25 cycles 457 CIC exon 19 reverse 5'-GATAGAAAAACGGTGGGGAGCAC
CIC exon 20 forward 5'-GTCTCGGAGTCTTGGGGTCAC
65 561
CIC exon 20 reverse 5'-CAAACTGGAGAGGACGAAATGG PLCG1 exon 11 forward 5'-CATCTGACCATACCTACCTGCCTCTC
66 201
PLCG1 exon 11 reverse 5'-CACTGGGGAGCAACATCACC PLCG1 exon 18 forward 5'-GCTAATGCGCGTCCCTCGTG
66 161
PLCG1 exon 18 reverse 5'-CTTCACAAGGACAAAACAGTCACAC KDR exon 15 forward 5'-GAAAAACTTCACAGACTACATC
62 258
KDR exon 15 reverse 5'-ATTCCTTTTTACGGCTGC KDR exon 16 forward 5'-GCTTGGGATTTTGCTTTAGTGC
62 287
KDR exon 16 reverse 5'-
GAAGAAACAACTCAAAGAACCCCAG KDR exon 24 forward 5'-CCAGATTATGTCAGAAAAGGAG
62 329
KDR exon 24 reverse 5'-CTTTGGAGTCTGGATGGAA
Supplemental Table S2. Custom bacterial artificial chromosome (BAC) probes used for fluorescence in situ hybridization assay.
BAC clones Cytoband Genes GP-starting GP-ending
RP11-98A7 19q13.2 C-CIC 42509683 42707772
RP11-688M4 19q13.2 C-CIC 42331074 42514687
RP11-343B1 19q13.2 C-CIC 42181928 42355256
RP11-778C1 19q13.2 T-CIC 42835831 43042674
RP11-165I8 19q13.2 T-CIC 43042754 43238787
RP11-766P9 19q13.2 -13.31 T-CIC 43225331 43400179
RP11-1101H9 19q13.11 M-PEPD 33876931 34081543
RP11-440N18 8q24.21 M-MYC 128596756 128777986
RP11-586L9 5q35.3 M-FLT4 179971355 180139031
RP11-71G19 22q11.23 M-SMARCB1 24052413 24200221 GP, genomic position; T, telomeric; C, centromeric; M: intragenic
Supplemental Table S3. The gene expression level of endothelial receptors, angiogenesis, and CIC related fusion signature among 2 CIC-rearranged AS (AS1-2), 3 classical AS (AS25, AS32, AS101), and 3 CIC-rearranged SBRCTs.
Gene AS1 AS2 Median AS
(Median)
SBRCT (Median)
CIC-rearrangement signature genes
ETV1 18.09 46.04 32.06 1.67 64.62
ETV4 429.00 510.87 469.94 4.71 556.44 ETV5 115.62 171.13 143.38 4.47 239.47 CCND1 201.55 106.91 154.23 58.16 116.64 VGF 1106.56 1343.83 1225.19 1.02 2509.42
DIO3 11.63 59.93 35.78 1.02 213.76
WT1 8.60 5.04 6.82 1.00 12.73
Endothelial differentiation
PECAM1 42.07 13.52 27.80 116.53 5.43
EPHA2 47.71 32.75 40.23 28.32 29.68
ANGPT2 6.28 5.99 6.14 7.81 8.38
EDNRB 1.83 2.09 1.96 25.67 1.49
PGF 19.06 83.59 51.33 51.94 11.82
FLI1 10.20 7.37 8.78 18.75 12.36
VWF 4.24 3.91 4.07 14.84 5.32
Angiogenesis
TIE1 35.78 39.55 37.66 232.01 21.11
SNRK 3.54 6.39 4.97 12.72 5.42
TEK 1.69 2.19 1.94 17.71 2.56
KDR 2.82 4.04 3.43 63.97 2.36
FLT4 169.40 100.56 134.98 218.92 47.15
FLT1 3.23 3.76 3.50 2.41 1.90
SNRK 3.54 6.39 4.97 12.72 5.42
THBS1 42.30 45.87 44.09 5.31 11.50
ANTXR1 12.87 6.11 9.49 7.98 6.43
RASA1 6.92 8.20 7.56 5.12 10.15
GLMN 3.62 4.58 4.10 2.10 5.92
KRIT1 4.05 5.83 4.94 3.07 6.37
CCM2 16.71 17.48 17.09 22.98 12.08
ACVRL1 65.62 14.17 39.90 78.05 6.56
ENG 53.18 46.20 49.69 145.12 25.93
PTPRB 1.39 1.77 1.58 6.34 1.57
DLL4 10.09 25.18 17.64 44.64 6.01
*Unit: RPMK, reads per kilobase per million mapped reads.
AS
#
PLCG1 or KDR Mutation
Age Sex Location Etiology Recurrence (mo)
Follow-up (mo)
Final Status
4 PLCG1, p.R707Q 40 F Breast Primary 17 28 DOD
10 PLCG1, p.R707Q 41 F Breast Primary 19 23 DOU
11 PLCG1, p.R707Q 30 F Breast Primary - - -
12 PLCG1, p.S345F 82 F Breast Primary 14 45 DOD 13 PLCG1, p.R707L 69 F Chest wall RT-related 5 35 DOD 14 PLCG1, p.R707Q 65 F Chest wall RT-related 11 14 NED 15 PLCG1, p.R707Q 75 F Chest wall RT-related 22 43 DOD 16 PLCG1, p.R707Q 63 F Chest wall RT-related 0.8 47 NED 17 PLCG1, p.R707Q 65 F Chest wall RT-related 16 98 NED
18 PLCG1, p.S345F 34 M Spleen Primary - 10 DOD
19 PLCG1, p.R707Q 23 F Heart Primary - 24 DOD
20 KDR, p.A1065T 74 F Chest wall RT-related 10 81 DOD
21 KDR, p.T771R 41 F Breast Primary 48 55 DOD
22 KDR, p.T771R 77 F Breast Primary 93 106 DOD
23 KDR, p.T771R 66 F Breast Primary 68 86 NED
24 KDR, p.T771R 23 F Breast Primary - - -
25 KDR, p.D731E 29 F Breast Primary 2 NED
26 KDR, p.T771K 66 F Spine/bone Primary - - - 27 KDR, p.D717V 76 F Breast RT-related 9 29 DOD F, female; M, male; RT, radiotherapy; DOD, dead of disease; DOU, dead of other causes; NED, no evidence of disease
Supplemental Table S4. Summary of clinical findings in PLCG1 or KDR-mutated AS.
Supplemental Table S5. AS survival analysis related to clinicopathologic factors and genetic alterations.
Clinicopathologic Factors Disease-Free Survival
Overall Survival
Median survival (months) 95% CI
p-value Median survival
(months) 95% CI
p-value
Age (years) 0.328
0.244
≤65 (n=53; 63) 16.6
12.2-21.1 57.8
27.2-88.4
>65 (n=43; 45) 14.0
9.0-19.1 43.7
21.5-65.9
Gender 0.630
0.470
Male (n=24; 31) 12.5
0-30.6 39.8
14.6-64.9
Female (n=72; 77) 16.2
13.0-19.4 55.0
33.8-76.3
Location
<0.001
<0.001
Breast/chest (n=52; 52) 16.2
13.1-19.4 58.2
31.0-85.3
Cutaneous (n=19; 19) 12.5
4.1-20.8 54.7
29.3-80.1
Soft tissue (n=17; 20) 18.6
5.9-31.2 137.1*
79.6-194.6
Bone (n=1; 6) 0.37*
- 22.0
7.5-36.5
Viscera (n=7; 11) 12.8*
2.4-23.2 10.3
0-20.8
Etiology 0.004
0.208
Primary (n=53; 63) 19.8
14.4-25.3 57.8
49.6-66.0
Secondary (n=43; 45) 11.8
6.5-17.1 35.9
27.6-44.2
PLCG1/KDR mutation 0.474
0.659
Absent(n=72; 82) 18.6
13.6-23.5 55.6
30.9-80.3
Present (n=14; 16) 16.2
9.9-22.5 45.4
24.0-66.7
MYC amplification 0.083
0.062
Absent (n=55; 65) 19.8
14.5-22.7 57.8
38.0-77.6
Present (n=36; 37) 11.8
6.1-17.5 35.5
25.4-45.6
FLT4 amplification 0.250
0.037
Absent (n=82; 93) 16.4
11.6-21.1 54.7
37.8-71.6
Present (n=6; 6) 7.8
4.3-11.3 22.7
16.0-29.4
CIC alteration 0.040
0.849
Absent (n=73; 84) 18.6
14.7-22.5 57.8
39.5-76.1
Present (n=9; 9) 12.5
2.9-22.0 46.4*
25.6-67.2
*mean survival (months) , CI: confidence interval.
