identification of novel alk and ret gene fusions from ...genomic dna sequencing. dna sequencing was...
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Identification of novel ALK and RET gene fusions from colorectal and lung cancer
biopsies
Doron Lipson, Marzia Capelletti, Roman Yelensky, Geoff Otto, Alex Parker, Mirna Jarosz, John A Curran, Sohail Balasubramanian, Troy Bloom, Kristina W Brennan, Amy Donahue, Sean R Downing, Garrett M Frampton, Lazaro Garcia, Frank Juhn, Kathy C Mitchell, Emily White, Jared White, Zac Zwirko, Tamar Peretz, Hovav Nechushtan, Lior Soussan-Gutman, Jhingook Kim, Hidefumi Sasaki, Hyeong Ryul Kim, Seung-il Park, Dalia Ercan, Christine E Sheehan, Jeffrey S Ross, Maureen T Cronin, Pasi A Jänne and Philip J Stephens
Supplementary Methods
Patients and samples. CRCs included 24 male and 16 female patients of mean age 61.8
years (range 56 to 87) with 1 Stage I, 4 Stage II, 14 Stage III and 17 Stage IV cases. NSCLCs
included 18 female and 6 male patients of mean age 66.9 years (range 36 to 82). Patients’
smoking status was unavailable. Stage included: 9 Stage I, 3 Stage II, 1 Stage III and 5 Stage
IV. For all cases CRC/NSCLC diagnosis was confirmed and thyroid cancer ruled out by at
least two pathologists reviewing clinical history, radiologic images, gross appearance and
routine histology.
Genomic DNA sequencing. DNA sequencing was performed for 2574 exons of 145 cancer
genes on indexed, adaptor ligated, hybridization-captured (Agilent SureSelect custom kit)
libraries using DNA isolated from 40 microns of formalin fixed paraffin embedded (FFPE)
tumor. For all specimens ≥25% of the nuclear area was malignant tumor cells so no
micro/macro dissection tissue enrichment was performed. Sequencing on the HiSeq2000
instrument (Illumina) was with 36 bp paired reads to average depth of 229X.
Nature Medicine doi:10.1038/nm.2673
Sequence data analysis. Sequence data from gDNA and cDNA was mapped to the
reference human genome (hg19) using the BWA aligner1 and processed using publicly
available SAMtools2, Picard (http://picard.sourceforge.net) and GATK3. Genomic base
substitutions and indels were detected using custom tools optimized for mutation calling in
heterogeneous tumor samples, based on statistical modeling of sequence quality scores and
local sequence assembly. Variations were filtered using dbSNP and a custom artifact
database, then annotated for known and likely somatic mutations using COSMIC4. Copy
number alterations were detected by comparing targeted genomic DNA sequence coverage
with a process-matched normal control sample. Genomic rearrangements were detected by
clustering chimeric reads mapping to targeted introns. Expression levels were determined
by analyzing cDNA sequence coverage of targeted exons.
Analytical validation. For analytical validation, we obtained results from previous clinical
genotyping for 59/64 cases: 38 CRCs and 2 NSCLCs for KRAS codons 12 and 13 by allele-
specific primer extension (Genzyme Genetics); 3 CRCs for BRAF V600 by pyro-sequencing
(ARUP); 19 NSCLCs for exon 18 through 21 of EGFR by sequencing (Genzyme Genetics).
Mutation calls were completely concordant between methods, giving estimates of 100%
sensitivity (95% CI 74%-100%) and 100% specificity (95% CI 93%-100%) for our assay.
RET protein immunohistochemistry. Tumors from 55 female and 62 male patients of
mean age 65.7 years (range 40 to 86) were tested for RET expression using an anti-RET
mouse monoclonal antibody (Vector Laboratories, clone: 3F8). Antibody specificity was
confirmed using control tissues which showed cytoplasmic immunoreactivity for RET in
normal human intestine and enteric ganglion cells. Twenty-two tumors were RET
Nature Medicine doi:10.1038/nm.2673
immunopositive and 95 were negative.
Additional RET rearrangement screening was performed by qRT-PCR on cDNA from frozen
tumors or RET IHC in FFPE specimens. cDNA sequencing from FFPE tissues confirmed
expression of novel gene fusions. Sequencing matched normal genomic DNA from blood for
the index KIF5B-RET NSCLC patient and two additional cases confirmed the somatic origin
of the rearrangement.
CRC ALK IHC. CRC specimens were sectioned at 5 microns and stained for ALK expression
using the IgG3 Clone CD 246 anti-ALK antibody (CD 246; Dako Corp) with the Ventana
Benchmark automated IHC stainer (Ventana Medical Systems).
NSCLC RT-PCR. Specimens from never or limited (< 10 pack years) former smoking NSCLC
patients without prior chemotherapy or radiation were identified (347 Korean, 58
Japanese and 121 CALGB tumor bank protocol 14202 European). All patients provided
written informed consent and studies were approved by local Institutional Review Boards.
cDNA sequencing. RNA from 10 micron FFPE sections (Roche High Pure Kit) was reverse
transcribed with random hexamers using SuperScript®III First-Strand Synthesis System
(Invitrogen). cDNA made double-stranded using NEBNext® mRNA Second Strand
Synthesis Module (New England Biolabs)5 was used for sequencing library construction,
hybridization selection and sequencing6.
RT-PCR for fusion gene products. Tumors obtained at surgery were snap frozen in liquid
nitrogen, embedded in OCT and sectioned. RNA was prepared using Trizol (Invitrogen) and
purified using RNeasy mini-eluate cleanup kit (Qiagen). cDNA was transcribed with
Nature Medicine doi:10.1038/nm.2673
Quantiscript Reverse Transcriptase (Qiagen). qRT-PCR used primers designed from
genomic sequence data reconstructing fusion events:
F5'-AAATGAGCTCAACAGATGGCGTAA-3'; R5’-AGAACCAAGTTCTTCCGAGGGAAT-3'.
Genotyping was performed using RT-PCR for EGFR, EML4-ALK, KRAS, BRAF, ERBB2, BRAF,
CD74-ROS and KIF5B-RET. All PCR-positive specimens were verified by direct sequencing.
Specific PCR primers are available upon request.
Plasmid Construction, Cell Culture, and Transfection studies. Full length KIF5B-RET
cDNA was constructed by RT-PCR amplifying two overlapping fragments, each containing a
unique EcoRI site. PCR primers used to generate the N-terminal and C-terminal
overlapping fragments are:
F5’-ATACGAAGTTATCAGTCGACCAGCTGACTGCTGCCTCTCAC-3’;
R5’-CAGATACTGCATCCCCTGTGAGAT-3’;
F5’-TAAGGAAATGACCAACCACCAGAA-3’;
R5’-ACGAATGGTCTAGAAAGCTTTTAACTATCAAACGTGTCCATTAATTTTGCCGCTGA-3’
cDNA was inserted into pDNR-Dual vector (BD Biosciences) using SalI/HindIII sites and
recombined into lentiviral expression vector JP1698 as previously described6. Full length
cDNA was confirmed by sequencing. Ba/F3 cell culture, lentivirus production, titrations
and infections were performed as previously described7,8. Polyclonal cell lines were
established by blastocidin selection then cultured without interleukin-3 (IL-3). Cell
proliferation and growth were performed as previously described8. Gefitinib, sorafenib,
sunitinib and vandetinib were prepared in DMSO and stored at -20oC. Protein detection by
immunoblotting was performed according to antibody manufacturer's recommendations.
Anti-RET and anti-phospho-RET (Tyr 905) antibodies were from Cell Signaling Technology.
Nature Medicine doi:10.1038/nm.2673
Anti-tubulin antibody was from Sigma Aldrich.
References
1. Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler
transform. Bioinformatics, 25, 1754-60. [PMID: 194511682. Li, H., et al. The Sequence
Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).
2 Li, H., et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079 (2009).
3. McKenna, A., et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing
next-generation DNA sequencing data. Genome Res 20, 1297-1303 (2010). 4. Forbes, S.A., et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic
Mutations in Cancer. Nucleic Acids Res 39, D945-950 (2011). 5. D'Alessio, J.M. & Gerard, G.F. Second-strand cDNA synthesis with E. coli DNA polymerase I
and RNase H: the fate of information at the mRNA 5' terminus and the effect of E. coli DNA ligase. Nucleic Acids Res 16, 1999-2014 (1988).
6. Levin, J.Z., et al. Targeted next-generation sequencing of a cancer transcriptome enhances
detection of sequence variants and novel fusion transcripts. Genome Biol 10, R115 (2009).
7. Ercan, D., et al. Amplification of EGFR T790M causes resistance to an irreversible EGFR inhibitor. Oncogene. 16, 2346-56. (2010).
8. Sasaki, T., et al. A novel ALK secondary mutation and EGFR signaling cause resistance to
ALK kinase inhibitors. Cancer Res. 18, 6051-60. (2011).
Nature Medicine doi:10.1038/nm.2673
Identification of recurrent KIF5B-RET gene fusions from lung cancer biopsies
Doron Lipson, Marzia Capelletti, Roman Yelensky, Geoff Otto, Alex Parker, Mirna Jarosz, John A Curran, Sohail Balasubramanian, Troy Bloom, Kristina W Brennan, Amy Donahue, Sean R Downing, Garrett M Frampton, Lazaro Garcia, Frank Juhn, Kathy C Mitchell, Emily White, Jared White, Zac Zwirko, Tamar Peretz, Hovav Nechushtan, Lior Soussan-Gutman, Jhingook Kim, Hidefumi Sasaki, Hyeong Ryul Kim, Seung-il Park, Dalia Ercan, Christine E Sheehan, Jeffrey S Ross, Maureen T Cronin, Pasi A Jänne and Philip J Stephens
Gene RefSeq Gene RefSeq Gene RefSeq Gene RefSeq ABL1 NM_007313 PTCH1 NM_000264 HOXA3 NM_030661 RUNX1 NM_001754 AKT1 NM_005163 PTEN NM_000314 HSP90AA1 NM_005348 SMAD2 NM_005901 AKT2 NM_001626 RB1 NM_000321 IDH1 NM_005896 SMAD3 NM_005902 AKT3 NM_181690 RET NM_020630 IDH2 NM_002168 SMAD4 NM_005359 ALK NM_004304 SMO NM_005631 IGF1R NM_000875 SMARCA4 NM_003072 APC NM_000038 STK11 NM_000455 IGF2R NM_000876 SMARCB1 NM_003073 AR NM_000044 TP53 NM_000546 IKBKE NM_014002 SOX10 NM_006941
BRAF NM_004333 ABL2 NM_005158 INHBA NM_002192 SOX2 NM_003106 CCND1 NM_053056 ATM NM_000051 IRS2 NM_003749 SRC NM_005417 CDK4 NM_000075 AURKA NM_003600 JAK3 NM_000215 TET2 NM_017628
CDKN2A NM_000077 AURKB NM_004217 KDR NM_002253 TGFBR2 NM_003242 CEBPA NM_004364 BCL2 NM_000633 MAP2K4 NM_003010 TOP1 NM_003286
CTNNB1 NM_001904 BCL2L1 NM_001191 MCL1 NM_021960 TSC1 NM_000368 EGFR NM_005228 BCL2L2 NM_004050 MDM2 NM_002392 TSC2 NM_000548
ERBB2 NM_004448 BCL6 NM_001706 MDM4 NM_002393 VHL NM_000551 ESR1 NM_000125 BRCA1 NM_007294 MEN1 NM_000244 WT1 NM_000378
FGFR1 NM_015850 BRCA2 NM_000059 MITF NM_198159 ARFRP1 NM_003224 FGFR2 NM_000141 CBL NM_005188 MLH1 NM_000249 BCL2A1 NM_004049 FGFR3 NM_000142 CCNE1 NM_001238 MPL NM_005373 CDH20 NM_031891 FLT3 NM_004119 CDH1 NM_004360 MRE11A NM_005590 CDH5 NM_001795 HRAS NM_005343 CDH2 NM_001792 MSH2 NM_000251 EPHA3 NM_005233 JAK2 NM_004972 CDK6 NM_001259 MSH6 NM_000179 EPHA5 NM_004439 KIT NM_000222 CDK8 NM_001260 MTOR NM_004958 EPHA7 NM_004440
KRAS NM_004985 CHEK1 NM_001274 MYCL1 NM_005376 EPHB1 NM_004441 MAP2K1 NM_002755 CHEK2 NM_007194 MYCN NM_005378 FOXP4 NM_138457 MAP2K2 NM_030662 CRKL NM_005207 NF2 NM_000268 GPR124 NM_032777
MET NM_000245 EPHA6 NM_173655 NKX2-1 NM_003317 GUCY1A2 NM_000855 MLL NM_005933 EPHB4 NM_004444 NTRK1 NM_002529 LRP1B NM_018557 MYC NM_002467 EPHB6 NM_004445 PAX5 NM_016734 LTK NM_002344 NF1 NM_000267 ERBB3 NM_001982 PDGFRB NM_002609 PAK3 NM_002578
NOTCH1 NM_017617 ERBB4 NM_005235 PKHD1 NM_138694 PLCG1 NM_002660 NPM1 NM_002520 FBXW7 NM_018315 PRKDC NM_006904 PTPRD NM_002839 NRAS NM_002524 FGFR4 NM_002011 PTPN11 NM_002834 TBX22 NM_016954
NTRK3 NM_002530 FLT1 NM_002019 RAF1 NM_002880 USP9X NM_001039590 PDGFRA NM_006206 FLT4 NM_182925 RARA NM_000964 PIK3CA NM_006218 GATA1 NM_002049 RICTOR NM_152756 PIK3R1 NM_181523 GNAS NM_016592 RPTOR NM_020761
Supplementary Table 1a. Genes sequenced across entire coding sequence (n=145)
Nature Medicine doi:10.1038/nm.2673
Gene RefSeq Introns sequenced ALK NM_004304 19
BCR NM_004327 8,13,14
BRAF NM_004333 7,8,9,10
EGFR NM_005228 7
ETV1 NM_004956 3,4
ETV4 NM_001986 8
ETV5 NM_004454 6,7
ETV6 NM_001987 5,6
EWSR1 NM_005243 8,9,10,11,12,13
MLL NM_005933 6,7,8,9
RAF1 NM_002880 5,6,7,8,9
RARA NM_000964 2
RET NM_020630 9,10,11
TMPRSS2 NM_005656 1,2
Supplementary Table 1b. Genes sequenced across selected introns (n=14)
Nature Medicine doi:10.1038/nm.2673
Sam
ple n
ame
Cancer
Gender
Tota
l pai
rs
% p
ass f
ilter
% u
niquely
aligned
Error r
ate
Read p
air le
ngth
% ch
imer
as
Mean
inse
rt siz
e
Fold
enric
hment
Mean
exo
n cove
rage
Media
n exo
n cove
rage
100X cove
red e
xons
150X cove
red e
xons
250X cove
red e
xons
On-targ
et % d
uplicatio
n
Est o
n-targ
et lib
rary
size
On-targ
et dist
inct
pai
rs
% u
sable
pai
rs
SM3 CRC Male 27,019,766 94% 94% 0.23% 36, 36 1.11% 176.0 2067.3 161.7 159 81% 55% 10% 87% 2,247,862 2,246,894 8%
SM4 CRC Male 16,509,937 94% 93% 0.22% 36, 36 1.08% 174.2 2396.6 277.6 277 93% 86% 59% 71% 3,650,254 3,517,635 21%
SM6 CRC Male 19,231,329 95% 94% 0.22% 36, 36 0.92% 161.1 2486.5 261.8 251 92% 83% 50% 78% 3,306,316 3,266,131 17%
SM7 CRC Female 16,194,851 95% 90% 0.23% 36, 36 1.73% 159.1 1971.2 215.6 212 89% 77% 33% 72% 2,755,942 2,667,390 16%
SM8E CRC Female 11,945,498 91% 91% 0.28% 36, 36 1.77% 154.1 2199.1 156.1 159 79% 55% 9% 74% 1,960,454 1,916,603 16%
SM9 CRC Female 18,744,036 94% 92% 0.25% 36, 36 2.39% 141.1 2536.0 199.9 201 89% 74% 25% 83% 2,410,330 2,402,503 13%
SM11 CRC Male 16,023,607 95% 94% 0.22% 36, 36 1.21% 173.9 2345.6 215.6 218 88% 76% 36% 76% 2,787,679 2,745,027 17%
SM12 CRC Male 18,079,049 95% 89% 0.24% 36, 36 2.35% 161.8 1715.3 274.8 279 93% 86% 61% 62% 3,828,343 3,481,145 19%
SM13E CRC Female 17,743,604 91% 92% 0.29% 36, 36 1.25% 156.8 2211.9 176.3 184 83% 67% 17% 80% 2,230,888 2,217,154 12%
SM14 CRC Male 15,093,799 95% 94% 0.23% 36, 36 1.61% 154.9 2505.4 215.8 214 87% 74% 36% 77% 2,699,597 2,660,457 18%
SM16 CRC Male 20,624,203 95% 93% 0.22% 36, 36 1.16% 174.4 2330.9 303.7 301 95% 89% 67% 73% 4,096,015 3,984,814 19%
SM18 CRC Female 15,332,978 95% 93% 0.22% 36, 36 1.39% 164.7 2319.5 253.9 256 91% 84% 52% 70% 3,368,662 3,237,097 21%
SM19 CRC Male 10,247,815 92% 88% 0.28% 36, 36 2.71% 135.2 1737.8 117.9 119 62% 29% 1% 71% 1,485,566 1,431,053 14%
SM20 CRC Male 21,069,786 88% 92% 0.30% 36, 36 0.82% 158.8 2151.4 185.3 190 84% 69% 22% 81% 2,409,046 2,396,207 11%
SM21 CRC Male 17,431,311 95% 92% 0.22% 36, 36 2.34% 167.2 2273.0 253.3 250 92% 83% 50% 73% 3,287,666 3,199,956 18%
SM23 CRC Female 19,255,847 90% 89% 0.31% 36, 36 2.80% 139.4 1655.0 91.1 92 41% 7% 0% 87% 1,111,921 1,111,459 6%
SM28 CRC Female 15,487,496 90% 87% 0.41% 36, 36 3.63% 170.6 1586.9 177.2 176 83% 63% 18% 65% 2,448,828 2,283,271 15%
SM29 CRC Female 11,187,953 90% 89% 0.34% 36, 36 2.34% 169.1 1568.7 156.2 155 77% 53% 11% 59% 2,312,007 2,043,114 18%
SM32 CRC Female 18,846,140 95% 92% 0.23% 36, 36 2.68% 185.1 2147.2 275.4 274 92% 86% 59% 71% 3,804,394 3,664,524 19%
SM34 CRC Male 19,892,704 95% 87% 0.29% 36, 36 4.41% 144.2 1918.7 96.8 94 44% 12% 0% 89% 1,183,986 1,183,855 6%
SM36E CRC Male 38,125,948 64% 87% 0.84% 36, 36 2.36% 170.4 1438.7 228.2 233 85% 75% 43% 73% 3,030,432 2,948,401 8%
SM39 CRC Male 21,261,869 94% 93% 0.26% 36, 36 2.77% 171.6 2293.7 141.3 142 77% 44% 3% 87% 1,858,817 1,858,188 9%
SM40 CRC Male 24,280,140 94% 91% 0.25% 36, 36 1.79% 173.6 2054.5 141.5 145 79% 46% 2% 87% 1,926,458 1,925,750 8%
SM41E CRC Female 18,542,548 94% 94% 0.22% 36, 36 2.45% 201.6 2252.6 211.4 207 90% 77% 31% 78% 2,930,814 2,900,347 16%
SM42E CRC Male 18,988,365 95% 95% 0.22% 36, 36 0.74% 184.3 2342.0 152.7 159 81% 56% 4% 85% 2,086,042 2,083,697 11%
SM45 CRC Female 26,249,436 94% 94% 0.23% 36, 36 1.06% 181.1 2329.5 182.9 187 86% 69% 18% 87% 2,490,649 2,489,484 9%
SM54 CRC Female 25,197,937 94% 92% 0.27% 36, 36 3.02% 175.0 2258.3 167.9 167 86% 61% 9% 87% 2,217,645 2,216,705 9%
SM61 CRC Male 39,256,837 63% 89% 0.83% 36, 36 1.98% 168.1 1308.2 229.6 239 82% 73% 46% 70% 3,160,993 3,036,072 8%
SM67 CRC Male 28,245,093 96% 88% 0.31% 36, 36 3.58% 161.3 1379.8 230.3 216 86% 73% 38% 74% 3,068,169 2,997,557 11%
SM73 CRC Male 23,936,537 94% 93% 0.26% 36, 36 3.45% 196.3 2229.3 145.5 144 79% 45% 3% 88% 2,022,561 2,022,050 8%
SM74NE CRC Female 10,955,649 95% 94% 0.21% 36, 36 1.63% 199.9 2213.0 203.4 204 87% 74% 28% 64% 3,016,934 2,786,571 25%
SM75 CRC Female 9,434,682 95% 87% 0.25% 36, 36 4.62% 189.3 1572.2 152.8 147 79% 48% 9% 54% 2,439,252 2,046,162 22%
SM77E CRC Male 7,025,793 94% 91% 0.24% 36, 36 3.43% 181.4 2090.1 108.6 103 52% 18% 2% 68% 1,504,042 1,426,893 20%
SM78E CRC Female 7,055,984 95% 91% 0.23% 36, 36 3.15% 207.6 1942.8 155.3 154 80% 52% 10% 51% 2,653,615 2,149,238 30%
SM81 CRC Male 24,702,715 94% 94% 0.21% 36, 36 1.07% 182.7 2325.8 353.5 344 93% 89% 74% 74% 4,742,318 4,633,928 19%
SM83 CRC Male 19,069,353 94% 95% 0.20% 36, 36 0.84% 187.3 2328.7 287.9 288 91% 84% 62% 73% 3,862,786 3,758,336 20%
SM88 CRC Male 40,923,233 95% 93% 0.22% 36, 36 1.63% 171.2 2273.0 304.7 304 93% 87% 67% 86% 3,972,120 3,969,048 10%
SM98E CRC Male 20,847,010 94% 92% 0.24% 36, 36 1.34% 213.2 1754.8 292.6 293 92% 86% 65% 65% 4,521,756 4,189,272 20%
SM100E CRC Female 18,406,316 95% 93% 0.22% 36, 36 1.47% 186.0 2160.3 274.2 277 91% 85% 60% 71% 3,813,874 3,674,132 20%
SM110E CRC Male 15,076,598 95% 93% 0.21% 36, 36 1.17% 176.1 2065.7 216.5 223 83% 73% 39% 70% 3,018,578 2,900,594 19%
Average 19,588,594 92% 92% 0.28% 36, 36 2.08% 172.5 2068.4 206.2 205.9 83% 66% 31% 75% 2,793,090 2,691,718 15%
Median 18,795,088 94% 92% 0.24% 36, 36 1.78% 172.6 2179.7 207.4 205.5 86% 73% 30% 74% 2,727,770 2,663,924 16%
Supplementary Table 2a. CRC assay sequencing statistics
Sam
ple n
ame
Cancer
Gender
Tota
l pai
rs
% p
ass f
ilter
% u
niquely
aligned
Error r
ate
Read p
air le
ngth
% ch
imer
as
Mean
inse
rt siz
e
Fold
enric
hment
Mean
exo
n cove
rage
Media
n exo
n cove
rage
100X cove
red e
xons
150X cove
red e
xons
250X cove
red e
xons
On-targ
et % d
uplicatio
n
Est o
n-targ
et lib
rary
size
On-targ
et dist
inct
pai
rs
% u
sable
pai
rs
SM44 NSCLC Female 17,364,255 95% 88% 0.26% 36, 36 2.80% 166.6 1539.2 201.4 200 85% 70% 29% 68% 2,671,898 2,535,020 15%
SM46 NSCLC Female 20,948,260 94% 93% 0.24% 36, 36 1.77% 180.5 2281.0 178.9 182 86% 68% 15% 84% 2,426,651 2,421,216 12%
SM48 NSCLC Female 14,499,848 95% 92% 0.23% 36, 36 1.11% 163.4 2013.3 211.8 217 88% 77% 34% 69% 2,905,679 2,769,458 19%
SM49A5E NSCLC Female 19,297,409 95% 85% 0.37% 36, 36 6.16% 140.9 1510.8 173.4 172 83% 61% 15% 74% 2,150,042 2,098,151 11%
SM51 NSCLC Female 15,247,561 95% 92% 0.26% 36, 36 3.21% 140.5 2497.1 140.6 139 73% 43% 5% 85% 1,654,976 1,652,751 11%
SM53 NSCLC Female 15,976,709 95% 94% 0.21% 36, 36 0.77% 175.9 2423.8 285.8 283 92% 86% 61% 69% 3,888,103 3,713,704 23%
SM55 NSCLC Male 22,322,494 95% 87% 0.32% 36, 36 2.60% 144.2 1440.1 186.6 187 85% 67% 22% 75% 2,320,955 2,276,351 10%
SM63 NSCLC Female 13,442,277 95% 94% 0.21% 36, 36 2.09% 173.6 2352.7 238.1 243 88% 80% 47% 69% 3,198,092 3,050,028 23%
SM64E NSCLC Female 17,310,249 95% 93% 0.23% 36, 36 1.21% 185.7 2028.1 293.4 293 93% 87% 64% 65% 4,219,443 3,911,941 23%
SM70E NSCLC Female 15,525,662 95% 94% 0.19% 36, 36 0.95% 201.9 2207.0 314.2 319 92% 87% 70% 61% 4,731,278 4,275,065 28%
SM71E NSCLC Male 16,233,125 95% 91% 0.25% 36, 36 2.95% 153.1 2371.5 188.4 188 86% 68% 22% 80% 2,322,055 2,303,999 14%
SM86 NSCLC Female 37,176,479 94% 92% 0.26% 36, 36 2.12% 156.8 2287.5 179.4 186 89% 71% 11% 91% 2,291,983 2,291,945 6%
SM87 NSCLC Female 20,631,365 94% 93% 0.21% 36, 36 2.70% 197.2 2123.6 330.9 317 93% 88% 70% 68% 4,666,235 4,414,860 21%
SM89 NSCLC Female 20,486,730 95% 93% 0.23% 36, 36 2.05% 172.3 2146.7 192.7 199 85% 72% 25% 82% 2,527,689 2,516,344 12%
SM90 NSCLC Female 19,073,249 94% 94% 0.21% 36, 36 1.13% 215.6 1970.2 287.2 282 92% 86% 61% 68% 4,135,076 3,915,497 21%
SM91A3 NSCLC Female 22,892,049 94% 92% 0.23% 36, 36 1.07% 183.1 1952.5 131.2 136 70% 40% 3% 87% 1,839,751 1,838,841 8%
SM92 NSCLC Male 20,707,344 95% 92% 0.23% 36, 36 1.40% 167.9 2009.9 230.1 229 88% 78% 41% 77% 3,048,336 3,003,074 15%
SM93 NSCLC Unknown 20,162,905 94% 93% 0.24% 36, 36 1.06% 221.9 1912.4 301.7 290 90% 85% 62% 65% 4,663,117 4,348,135 22%
SM96 NSCLC Female 26,403,071 95% 94% 0.21% 36, 36 0.88% 197.2 2258.9 314.6 320 93% 88% 72% 78% 4,288,387 4,237,268 16%
SM107 NSCLC Female 31,404,026 94% 92% 0.25% 36, 36 2.63% 156.6 2393.8 139.9 137 74% 41% 4% 92% 1,695,838 1,695,835 5%
SM109 NSCLC Female 18,778,284 95% 93% 0.21% 36, 36 0.79% 199.4 1944.0 294.6 293 90% 84% 63% 65% 4,408,833 4,102,176 22%
SM112 NSCLC Male 16,541,878 95% 92% 0.25% 36, 36 2.56% 155.8 2216.2 193.1 196 87% 71% 24% 78% 2,456,445 2,428,207 15%
SM113 NSCLC Female 17,328,887 95% 89% 0.26% 36, 36 2.72% 164.4 1550.8 217.3 216 90% 78% 34% 65% 2,962,814 2,764,578 16%
SM114 NSCLC Male 39,093,969 94% 95% 0.62% 49, 49 1.75% 178.8 1735.3 1096.5 1173 95% 92% 88% 47% 17,022,547 13,072,965 33%
Average 20,785,337 95% 92% 0.26% 36, 36 2.02% 174.7 2048.6 263.4 266.5 87% 74% 39% 73% 3,687,343 3,401,559 17%
Median 19,185,329 95% 93% 0.24% 36, 36 1.91% 173.0 2075.8 214.5 216.5 88% 78% 34% 72% 2,934,247 2,767,018 16%
Supplementary Table 2b. NSCLC assay sequencing statistics
Nature Medicine doi:10.1038/nm.2673
Sample
Total
alterations
Substitut
ions INDELs
Copy
number
changes
Rearrang
ement APC TP53 KRAS BRAF FBXW7 CDH1 MYC ATM BCL2L1 BRCA2 ERBB3 GNAS PIK3CA SMAD4 ALK CDK8 LRP1B MSH6 RICTOR SMAD2 STK11
SM77E 3 3
S1346*(37%)
R213*(31%) R213*(72%)
SM18 3 3 D1394fs*21(42%) R248W(43%) S464*(13%)
SM41E 3 2 1 G1357fs*7(25%) G245S(22%) G12A(32%)
SM98E 6 3 3 K1878fs*4(33%)
R273C(32%)
R181C(38%) V600E(33%) S353fs*24(29%) T3033fs*29(44%)
SM75 3 1 2
Q1193fs*14(18%)
E1309fs*12(25%) C242F(38%)SM16 2 1 1 E1309fs*4(38%) R306*(42%)
SM21 4 4
E1353*(31%)
E1374*(31%) R175H(58%) G284R(36%)
SM9 3 2 1
E1544*(27%)
K1250fs*5(26%) V272M(36%)
SM7 3 2 1
G1288*(18%)
S1100fs*26(29%) R248W(55%)SM42E 2 2 K1370*(3%) G12V(3%)SM83 2 2 K534*(29%) C238Y(32%)
SM20 4 3 1
L1488fs*19(36%)
R213*(27%) G12A(21%) E545K(25%)
SM32 3 2 1
P1324fs*91(32%)
Q1406*(9%) K132R(26%)
SM40 3 1 2
P1373fs*42(37%)
E1317fs*4(17%) C176Y(41%)SM4 3 3 Q1294*(53%) G12D(32%) R278*(64%)
SM6 4 3 1
Q1406*(21%)
L1129S(31%) R280T(15%) Amp
SM28 3 3 Q1429*(32%) G12C(27%) E545K(11%)
SM23 4 4 Q1625*(10%) V600E(10%) V104M(16%) R201H(15%)
SM81 3 2 1
Q789*(28%)
L235fs*58(16%) L194F(40%)SM88 3 2 1 R1399fs*9(37%) E258G(32%) R658*(25%)
SM8E 9 3 6
R1450*(15%)
T1556fs*9(16%) R196*(18%) V600E(13%) P126fs*89(5%) K2811fs*46(26%)
C3869fs*1(9%)
T406fs*8(15%)
N854fs*12(26%)
SM45 3 3 R213*(25%) R282W(24%) G12V(19%)
SM100E 3 2 1
R216*(14%)
C1270*(22%) V122fs*26(25%)
SM67 3 2 1
R232*(26%)
Q1131*(30%) AmpSM110E 3 2 1 R564*(18%) F212fs*3(10%) G12V(13%)
SM3 4 3 1
R564*(64%)
E1322*(24%) R282W(88%) Amp
SM54 5 1 4
S1465fs*3(18%)
L519fs*18(24%)
S215_V218>RR(1
5%) V600E(21%) T576fs*29(13%)SM29 3 1 1 1 C135F(46%) Amp Rearranged
SM14 2 1 1 W91fs*32(26%) G12V(38%)SM13E 1 1 T253fs*11(10%)
SM34 1 1 E204fs*43(39%)
SM19 4 2 2 E258A(58%) V600E(18%) S668fs*26(17%) D360fs*24(12%)
SM36E 3 0 2 1 K292fs*54(21%) S1982fs*22(64%) Rearranged
SM12 7 3 4 P250H(11%) V600E(10%)
R74*(11%)
P201fs*14(5%)
P126fs*89(4%) R1875*(12%) P1087fs*5(5%)
SM11 2 2 R248W(30%) G12V(30%)
SM39 3 2 1
R273C(30%)
L114fs*9(21%) A118V(37%)
SM73 2 2 S240R(20%) Q214*(22%)SM74NE 2 2 V173M(10%) G12C(7%)
SM61 1 1 201H(22%)
SM78E 0
Total 125 80 39 4 2 42 34 10 6 5 4 3 2 2 2 2 2 2 2 1 1 1 1 1 1 1
Supplementary Table 3a. Alterations in 40 CRC cases . Mutations with the percent mutant allele frequency (in brackets) are shown.
Sample
Total
alterations
Substitut
ions
Total
INDELs
Copy
number
changes
Gene
fusion KRAS TP53 STK11 LRP1B JAK2 CTNNB1 RET EGFR BRAF CDKN2A MDM2 PIK3CA ATM TSC1 CCNE1 NF1 RB1 APC MLH1 MSH6 CDK4SM109 1 1 G12F(24%)
SM86 4 4 G12C(28%) V617F(4%) I35S(2%) E545K(5%)
SM51 3 3 G12C(11%) E165*(14%) G466A(12%)
SM71E 1 1 G12C(12%)
SM89 2 1 1 G12C(13%) V997fs*1(9%)SM90 2 2 G12V(12%) M237I(5%)
SM96 3 3 G12V(14%) Q2940*(10%) H83Y(27%)
SM44 3 2 1 G12V(27%) S232fs*55(19%) S37Y(10%)
SM70E 1 1 G12V(16%)
SM107 1 1 G12A(21%)SM91A3 7 6 1 C229fs*10(26%) D194Y(18%) K4112*(7%) V617F(10%) Y1635*(8%) E280(23%) L1129S(41%)
SM93 2 2 C242F(55%) E13*(27%)
SM63 1 1 G245S(7%)
SM48 3 2 1 K132*(31%) E3508*(16%)
R124_L130delRDV
ARYL(22%)
SM114 3 1 1 1 R248L(29%) Hom del RearrangedSM53 3 1 1 1 Y163C(50%) L90fs*9(41%) Amp
SM92 6 4 2 V617F(8%) G466V(21%) Amp N345K(32%) V509A(51%) Amp
SM87 2 2 Amp Amp
SM64E 1 1
D770_N771insSV
D(20%)
SM113 1 1 N429fs*7(42%)SM46 0
SM112 0
SM49A5E 0
SM55 0
Total 50 36 7 6 1 10 7 4 3 3 2 1 2 2 2 2 2 2 1 1 1 1 1 1 1 1
Supplementary Table 3b. Alterations in 24 NSCLC cases . Mutations with the percent mutant allele frequency (in brackets) are shown.
Nature Medicine doi:10.1038/nm.2673
Gene No. of Mutated
Samples Potential therapeutic treatment or clinical trial
TP53 32 Presently unknown
APC 27 Presently unknown
KRAS 10 Resistance to cetuximab and panitumumab
BRAF 6 Resistance to cetuximab and panitumumab
FBXW7 5 Potential resistance to tubulins
ATM 2 PARP inhibitors
BCL2L1 2 Presently unknown
BRCA2 2 PARP inhibitors
CDH1 2 Presently unknown
ERBB3 2 Presently unknown
GNAS 2 MEK or ERK inhibitors
PIK3CA 2 PI3 kinase/mTOR inhibitors
SMAD4 2 Prognostic factor
ALK 1 ALK inhibitors e.g. Crizotinib
CDK8 1 CDK inhibitors e.g. Flavopiridol
LRP1B 1 Presently unknown
MYC 1 Presently unknown
MSH6 1 Prognostic factor
RICTOR 1 Presently unknown
SMAD2 1 Presently unknown
STK11 1 Presently unknown
Supplementary Table 4a. CRC alterations that could be linked to a clinical treatment option or clinical
trial of novel targeted therapies.
Nature Medicine doi:10.1038/nm.2673
Gene
No. of Mutated Samples Potential therapeutic treatment or clinical trial
KRAS 10 Resistance to EGFR kinase inhibitors, clinical trials of PI3K and MEK inhibitors
STK11 4 Presently unknown
JAK2 3 JAK2 inhibitors
EGFR 2 Erlotinib or gefitinib
BRAF 2 Vemurafenib and GSK 2118436
CDKN2A 2 CDK inhibitors e.g.PD0332991
RET 2 RET inhibitors e.g. Sorafenib or sunitinib
CTNNB1 2 Presently unknown
MDM2 2 Nutlins
PIK3CA 2 PI3 kinase/mTOR inhibitors
ATM 2 PARP inhibitors
TSC1 1 mTOR inhibitors
CCNE1 1 CDK4 inhibitors e.g PD0332991
NF1 1 Presently unknown
RB1 1 Presently unknown
MLH1 1 Presently unknown
MSH6 1 Presently unknown
CDK4 1 CDK4 inhibitors e.g PD0332991
Supplementary Table 4b. NSCLC alterations that could be linked to a clinical treatment option or clinical
trial of novel targeted therapies.
Nature Medicine doi:10.1038/nm.2673
Characteristic No. of Patients (n = 117) Gender Male 62 Female 55 Histology Adenocarcinoma 83 Squamous cell Carcinoma 26 Carcinoid 8 Smoking Never 5 Limited former 53 Current 34 Unknown 25 Stage I 77 II 16 III 13 IV 8 N/A 3 RET IHC 0 78 1+/2+ 17 3+/4+ 22
No of KIF5B-RET fusions: 1
Frequency in all patients: 1/117: 0.85%
Frequency Adenocarcinoma: 1/89: 1.1%
Supplementary Table 5a. Summary of NSCLC patients analyzed by RET Immunohistochemistry.
Nature Medicine doi:10.1038/nm.2673
Characteristic No. of Patients (n= 121) Gender Male 16 Female 35 N/A 70 Histology Adenocarcinoma 96 Squamous cell Carcinoma 11 Carcinoid 10 Other 4 Smoking Never 25 Limited former or current 26 N/A 70 Mutation EGFR 47 (39%) KRAS 13 (11%) EML4-ALK 4 (3%) BRAF 3 (2.5%) CD74-ROS 3 (2.5%) ERBB2 2 (2%) None 49 (40%)
No of KIF5B-RET fusions: 1
Frequency in all patients: 1/121: 0.8%
Frequency in adenocarcinoma: 1/96: 1.0%
Frequency in WT patients: 1/49: 2.0%
Supplementary Table 5b. Summary of European ancestry NSCLC patients analyzed by RT-PCR.
Nature Medicine doi:10.1038/nm.2673
Characteristic No. of Patients (n = 405) Gender Male 84 Female 321 Histology Adenocarcinoma 382 Squamous cell Carcinoma 17 Carcinoid 2 Other 4 Smoking Never 373 Limited former 32 Mutation EGFR 228 (56%) EML4-ALK 30 (7.4%) KRAS 20 (5.5%) ERBB2 10 (2.5%) BRAF 4 (1%) CD74-ROS 3 (0.5%) None 110 (27%)
Note – 1 patient has concurrent KRAS and BRAF mutations
No of KIF5B-RET fusions: 9
Frequency in all patients: 9/405: 2%
Frequency in all adenocarcinoma: 9/382: 2.4%
Frequency in WT patients: 9/110: 8.2%
Supplementary Table 5c. Summary of Asian NSCLC patients analyzed by RT-PCR.
Nature Medicine doi:10.1038/nm.2673
Supplementary Figure 1. Predicted C2orf44-ALK gene fusion variant sequence.
1 ATGGAGTTGG GAAAAGGAAA ACTACTCAGG ACTGGACTGA ATGCGTTGCA TCAAGCAGTG
61 CATCCGATCC ATGGCCTTGC CTGGACCGAT GGGAATCAAG TTGTCCTAAC TGATTTGCGG
121 CTTCACAGTG GAGAGGTCAA GTTTGGGGAC TCCAAAGTCA TTGGACAGTT TGAATGTGTC
181 TGTGGGTTGT CCTGGGCCCC ACCTGTTGCA GATGATACAC CTGTTCTACT CGCTGTCCAG
241 CATGAGAAGC ATGTCACTGT GTGGCAGCTG TGTCCCAGCC CTATGGAGTC AAGCAAATGG
301 CTGACGTCTC AGACTTGTGA GATTAGAGGA TCACTACCTA TCCTTCCCCA GGGCTGTGTG
361 TGGCACCCAA AATGTGCTAT TCTGACTGTG TTGACTGCTC AGGATGTCTC CATTTTCCCT
421 AATGTTCACT CTGATGATTC CCAGGTAAAG GCAGACATCA ACACCCAGGG CCGCATTCAC
481 TGTGCATGTT GGACCCAGGA TGGCCTGAGG CTGGTGGTGG CAGTAGGCAG CAGCCTGCAT
541 TCTTATATTT GGGACAGCGC TCAGAAGACT CTTCACAGGT GCTCCTCCTG CCTGGTGTTT
601 GATGTGGACA GCCACGTCTG CTCCATCACA GCAACTGTGG ACTCACAGGT TGCTATAGCT
661 ACTGAGCTTC CATTGGATAA GATCTGTGGC TTAAATGCAT CTGAAACCTT TAATATCCCA
721 CCTAACAGTA AAGACATGAC TCCGTATGCT TTACCAGTTA TTGGTGAAGT ACGCTCTATG
781 GATAAAGAGG CAACTGATTC TGAAACAAAT TCTGAAGTAT CAGTTTCTTC TTCCTATTTA
841 GAACCTCTGG ATCTAACTCA CATACATTTC AATCAACATA AGTCTGAGGG TAATTCTCTT
901 ATTTGTCTAA GAAAAAAGGA CTACTTGACA GGAACTGGCC AAGATTCTTC ACATTTGGTC
961 CTTGTGACCT TTAAGAAGGC AGTTACCATG ACGAGAAAAG TCACTATTCC AGGCATTCTG
1021 GTTCCTGATC TGATAGCATT TAATCTTAAA GCCCACGTAG TGGCAGTGGC TTCCAACACT
1081 TGTAATATAA TTTTGATCTA CTCTGTCATT CCATCTTCAG TCCCAAACAT CCAGCAAATT
1141 CGATTAGAGA ACACTGAAAG ACCAAAAGGG ATATGTTTCT TGACAGACCA ACTATTACTA
1201 ATTTTGGTAG GAAAACAAAA ACTCACTGAT ACAACATTTC TTCCTTCTTC AAAGTCTGAT
1261 CAGTATGCCA TTAGCTTGAT TGTTAGAGAA ATAATGTTGG AAGAAGAACC TTCAATAACA
1321 TCAGGTGAAA GCCAGACTAC CTACTCTACT TTCAGTGCTC CGTTAAATAA AGCAAATAGA
1381 AAAAAGTTAA TTGAAAGTCT TTCCCCAGAT TTTTGTCACC AAAACAAAGG GCTGTTGCTG
1441 ACAGTTAATA CCAGTAGTCA GAATGGAAGG CCTGGAAGAA CACTTATTAA AGAAATCCAG
1501 AGTCCTCTGT CTAGTATCTG TGATGGCTCC ATAGCTCTAG ATGCTGAGCC TGTTACCCAG
1561 CCAGCATCGC TGCCCAGACA CAGCAGCACA CCAGACCACA CCAGCACACT GGAGCCTCCT
1621 CGTTTGCCTC AAAGAAAGAA CTTACAAAGT GAAAAGGAAA CTTATCAGCT GTCTAAGGAA
1681 GTGGAAATTT TATCTAGGAA CCTGGTTGAA ATGCAACGGT GTCTTTCTGA ACTTACAAAC
1741 CGTCTGCATA ATGGGAAGAA ATCCTCTTCA GTGTATCCAC TCTCTCAAGA TCTTCCTTAT
1801 GTTCACATCA TTTACCAGAA ACCTTATTAT CTAGGTCCTG TTGTTGAAAA AAGAGCGGTG
1861 CTTCTCTGTG ATGGTAAACT AAGGCTCAGT ACAGTTCAGC AGACTTTTGG CCTTTCTCTC
1921 ATTGAAATGC TACATGATTC CCACTGGATT CTTCTCTCTG CTGACAGTGA GGGCTTTATC
1981 CCGTTAACCT TCACAGCCAC ACAGGAAATA ATCATAAGAG ATGGCAGCCT GTCCAGGCTG
2041 GAGTGCATTG GCACAATCTT GGCTCACTGC AACCTCCAAC TCCCGGGTTC AAACCGTTCA
2101 GAGCTCAGGG GAGGATATGG AGATCCAGGG AGGCTTCCTG TAGGAAGTGG CCTGTGTAGT
Nature Medicine doi:10.1038/nm.2673
2161 GCTTCAAGGG CCAGGCTGCC AGGCCATGTT GCAGCTGACC ACCCACCTGC AGTGTACCGC
2221 CGGAAGCACC AGGAGCTGCA AGCCATGCAG ATGGAGCTGC AGAGCCCTGA GTACAAGCTG
2281 AGCAAGCTCC GCACCTCGAC CATCATGACC GACTACAACC CCAACTACTG CTTTGCTGGC
2341 AAGACCTCCT CCATCAGTGA CCTGAAGGAG GTGCCGCGGA AAAACATCAC CCTCATTCGG
2401 GGTCTGGGCC ATGGCGCCTT TGGGGAGGTG TATGAAGGCC AGGTGTCCGG AATGCCCAAC
2461 GACCCAAGCC CCCTGCAAGT GGCTGTGAAG ACGCTGCCTG AAGTGTGCTC TGAACAGGAC
2521 GAACTGGATT TCCTCATGGA AGCCCTGATC ATCAGCAAAT TCAACCACCA GAACATTGTT
2581 CGCTGCATTG GGGTGAGCCT GCAATCCCTG CCCCGGTTCA TCCTGCTGGA GCTCATGGCG
2641 GGGGGAGACC TCAAGTCCTT CCTCCGAGAG ACCCGCCCTC GCCCGAGCCA GCCCTCCTCC
2701 CTGGCCATGC TGGACCTTCT GCACGTGGCT CGGGACATTG CCTGTGGCTG TCAGTATTTG
2761 GAGGAAAACC ACTTCATCCA CCGAGACATT GCTGCCAGAA ACTGCCTCTT GACCTGTCCA
2821 GGCCCTGGAA GAGTGGCCAA GATTGGAGAC TTCGGGATGG CCCGAGACAT CTACAGGGCG
2881 AGCTACTATA GAAAGGGAGG CTGTGCCATG CTGCCAGTTA AGTGGATGCC CCCAGAGGCC
2941 TTCATGGAAG GAATATTCAC TTCTAAAACA GACACATGGT CCTTTGGAGT GCTGCTATGG
3001 GAAATCTTTT CTCTTGGATA TATGCCATAC CCCAGCAAAA GCAACCAGGA AGTTCTGGAG
3061 TTTGTCACCA GTGGAGGCCG GATGGACCCA CCCAAGAACT GCCCTGGGCC TGTATACCGG
3121 ATAATGACTC AGTGCTGGCA ACATCAGCCT GAAGACAGGC CCAACTTTGC CATCATTTTG
3181 GAGAGGATTG AATACTGCAC CCAGGACCCG GATGTAATCA ACACCGCTTT GCCGATAGAA
3241 TATGGTCCAC TTGTGGAAGA GGAAGAGAAA GTGCCTGTGA GGCCCAAGGA CCCTGAGGGG
3301 GTTCCTCCTC TCCTGGTCTC TCAACAGGCA AAACGGGAGG AGGAGCGCAG CCCAGCTGCC
3361 CCACCACCTC TGCCTACCAC CTCCTCTGGC AAGGCTGCAA AGAAACCCAC AGCTGCAGAG
3421 ATCTCTGTTC GAGTCCCTAG AGGGCCGGCC GTGGAAGGGG GACACGTGAA TATGGCATTC
3481 TCTCAGTCCA ACCCTCCTTC GGAGTTGCAC AAGGTCCACG GATCCAGAAA CAAGCCCACC
3541 AGCTTGTGGA ACCCAACGTA CGGCTCCTGG TTTACAGAGA AACCCACCAA AAAGAATAAT
3601 CCTATAGCAA AGAAGGAGCC ACACGACAGG GGTAACCTGG GGCTGGAGGG AAGCTGTACT
3661 GTCCCACCTA ACGTTGCAAC TGGGAGACTT CCGGGGGCCT CACTGCTCCT AGAGCCCTCT
3721 TCGCTGACTG CCAATATGAA GGAGGTACCT CTGTTCAGGC TACGTCACTT CCCTTGTGGG
3781 AATGTCAATT ACGGCTACCA GCAACAGGGC TTGCCCTTAG AAGCCGCTAC TGCCCCTGGA
3841 GCTGGTCATT ACGAGGATAC CATTCTGAAA AGCAAGAATA GCATGAACCA GCCTGGGCCC
3901 TGA
Nucleotides derived from C2orf44 are shown in blue and nucleotides derived from ALK are
shown in red.
Nature Medicine doi:10.1038/nm.2673
Supplementary Figure 2. Predicted KIF5B-RET gene fusion variant sequence.
a) K15;R12 (Variant 1): KIF5B exon 15 fused to RET exon 12.
1 ATGGCGGACC TGGCCGAGTG CAACATCAAA GTGATGTGTC GCTTCAGACC TCTCAACGAG
61 TCTGAAGTGA ACCGCGGCGA CAAGTACATC GCCAAGTTTC AGGGAGAAGA CACGGTCGTG
121 ATCGCGTCCA AGCCTTATGC ATTTGATCGG GTGTTCCAGT CAAGCACATC TCAAGAGCAA
181 GTGTATAATG ACTGTGCAAA GAAGATTGTT AAAGATGTAC TTGAAGGATA TAATGGAACA
241 ATATTTGCAT ATGGACAAAC ATCCTCTGGG AAGACACACA CAATGGAGGG TAAACTTCAT
301 GATCCAGAAG GCATGGGAAT TATTCCAAGA ATAGTGCAAG ATATTTTTAA TTATATTTAC
361 TCCATGGATG AAAATTTGGA ATTTCATATT AAGGTTTCAT ATTTTGAAAT ATATTTGGAT
421 AAGATAAGGG ACCTGTTAGA TGTTTCAAAG ACCAACCTTT CAGTTCATGA AGACAAAAAC
481 CGAGTTCCCT ATGTAAAGGG GTGCACAGAG CGTTTTGTAT GTAGTCCAGA TGAAGTTATG
541 GATACCATAG ATGAAGGAAA ATCCAACAGA CATGTAGCAG TTACAAATAT GAATGAACAT
601 AGCTCTAGGA GTCACAGTAT ATTTCTTATT AATGTCAAAC AAGAGAACAC ACAAACGGAA
661 CAAAAGCTGA GTGGAAAACT TTATCTGGTT GATTTAGCTG GTAGTGAAAA GGTTAGTAAA
1201 ACTGGAGCTG AAGGTGCTGT GCTGGATGAA GCTAAAAACA TCAACAAGTC ACTTTCTGCT
1261 CTTGGAAATG TTATTTCTGC TTTGGCTGAG GGTAGTACAT ATGTTCCATA TCGAGATAGT
1321 AAAATGACAA GAATCCTTCA AGATTCATTA GGTGGCAACT GTAGAACCAC TATTGTAATT
1381 TGCTGCTCTC CATCATCATA CAATGAGTCT GAAACAAAAT CTACACTCTT ATTTGGCCAA
1441 AGGGCCAAAA CAATTAAGAA CACAGTTTGT GTCAATGTGG AGTTAACTGC AGAACAGTGG
1501 AAAAAGAAGT ATGAAAAAGA AAAAGAAAAA AATAAGATCC TGCGGAACAC TATTCAGTGG
1561 CTTGAAAATG AGCTCAACAG ATGGCGTAAT GGGGAGACGG TGCCTATTGA TGAACAGTTT
1621 GACAAAGAGA AAGCCAACTT GGAAGCTTTC ACAGTGGATA AAGATATTAC TCTTACCAAT
1681 GATAAACCAG CAACCGCAAT TGGAGTTATA GGAAATTTTA CTGATGCTGA AAGAAGAAAG
1741 TGTGAAGAAG AAATTGCTAA ATTATACAAA CAGCTTGATG ACAAGGATGA AGAAATTAAC
1801 CAGCAAAGTC AACTGGTAGA GAAACTGAAG ACGCAAATGT TGGATCAGGA GGAGCTTTTG
1861 GCATCTACCA GAAGGGATCA AGACAATATG CAAGCTGAGC TGAATCGCCT TCAAGCAGAA
1921 AATGATGCCT CTAAAGAAGA AGTGAAAGAA GTTTTACAGG CCCTAGAAGA ACTTGCTGTC
1981 AATTATGATC AGAAGTCTCA GGAAGTTGAA GACAAAACTA AGGAATATGA ATTGCTTAGT
2041 GATGAATTGA ATCAGAAATC GGCAACTTTA GCGAGTATAG ATGCTGAGCT TCAGAAACTT
2101 AAGGAAATGA CCAACCACCA GAAAAAACGA GCAGCTGAGA TGATGGCATC TTTACTAAAA
2161 GACCTTGCAG AAATAGGAAT TGCTGTGGGA AATAATGATG TAAAGGAGGA TCCAAAGTGG
2221 GAATTCCCTC GGAAGAACTT GGTTCTTGGA AAAACTCTAG GAGAAGGCGA ATTTGGAAAA
2281 GTGGTCAAGG CAACGGCCTT CCATCTGAAA GGCAGAGCAG GGTACACCAC GGTGGCCGTG
2341 AAGATGCTGA AAGAGAACGC CTCCCCGAGT GAGCTGCGAG ACCTGCTGTC AGAGTTCAAC
2401 GTCCTGAAGC AGGTCAACCA CCCACATGTC ATCAAATTGT ATGGGGCCTG CAGCCAGGAT
2461 GGCCCGCTCC TCCTCATCGT GGAGTACGCC AAATACGGCT CCCTGCGGGG CTTCCTCCGC
2521 GAGAGCCGCA AAGTGGGGCC TGGCTACCTG GGCAGTGGAG GCAGCCGCAA CTCCAGCTCC
Nature Medicine doi:10.1038/nm.2673
2581 CTGGACCACC CGGATGAGCG GGCCCTCACC ATGGGCGACC TCATCTCATT TGCCTGGCAG
2641 ATCTCACAGG GGATGCAGTA TCTGGCCGAG ATGAAGCTCG TTCATCGGGA CTTGGCAGCC
2701 AGAAACATCC TGGTAGCTGA GGGGCGGAAG ATGAAGATTT CGGATTTCGG CTTGTCCCGA
2761 GATGTTTATG AAGAGGATTC CTACGTGAAG AGGAGCCAGG GTCGGATTCC AGTTAAATGG
2821 ATGGCAATTG AATCCCTTTT TGATCATATC TACACCACGC AAAGTGATGT ATGGTCTTTT
2881 GGTGTCCTGC TGTGGGAGAT CGTGACCCTA GGGGGAAACC CCTATCCTGG GATTCCTCCT
2941 GAGCGGCTCT TCAACCTTCT GAAGACCGGC CACCGGATGG AGAGGCCAGA CAACTGCAGC
3001 GAGGAGATGT ACCGCCTGAT GCTGCAATGC TGGAAGCAGG AGCCGGACAA AAGGCCGGTG
3061 TTTGCGGACA TCAGCAAAGA CCTGGAGAAG ATGATGGTTA AGAGGAGAGA CTACTTGGAC
3121 CTTGCGGCGT CCACTCCATC TGACTCCCTG ATTTATGACG ACGGCCTCTC AGAGGAGGAG
3181 ACACCGCTGG TGGACTGTAA TAATGCCCCC CTCCCTCGAG CCCTCCCTTC CACATGGATT
3241 GAAAACAAAC TCTATGGTAG AATTTCCCAT GCATTTACTA GATTCTAG
b) K16;R12 (Variant 2): KIF5B exon 16 fused to RET exon 12.
1 ATGGCGGACC TGGCCGAGTG CAACATCAAA GTGATGTGTC GCTTCAGACC TCTCAACGAG
61 TCTGAAGTGA ACCGCGGCGA CAAGTACATC GCCAAGTTTC AGGGAGAAGA CACGGTCGTG
121 ATCGCGTCCA AGCCTTATGC ATTTGATCGG GTGTTCCAGT CAAGCACATC TCAAGAGCAA
181 GTGTATAATG ACTGTGCAAA GAAGATTGTT AAAGATGTAC TTGAAGGATA TAATGGAACA
241 ATATTTGCAT ATGGACAAAC ATCCTCTGGG AAGACACACA CAATGGAGGG TAAACTTCAT
301 GATCCAGAAG GCATGGGAAT TATTCCAAGA ATAGTGCAAG ATATTTTTAA TTATATTTAC
361 TCCATGGATG AAAATTTGGA ATTTCATATT AAGGTTTCAT ATTTTGAAAT ATATTTGGAT
421 AAGATAAGGG ACCTGTTAGA TGTTTCAAAG ACCAACCTTT CAGTTCATGA AGACAAAAAC
481 CGAGTTCCCT ATGTAAAGGG GTGCACAGAG CGTTTTGTAT GTAGTCCAGA TGAAGTTATG
541 GATACCATAG ATGAAGGAAA ATCCAACAGA CATGTAGCAG TTACAAATAT GAATGAACAT
601 AGCTCTAGGA GTCACAGTAT ATTTCTTATT AATGTCAAAC AAGAGAACAC ACAAACGGAA
661 CAAAAGCTGA GTGGAAAACT TTATCTGGTT GATTTAGCTG GTAGTGAAAA GGTTAGTAAA
1201 ACTGGAGCTG AAGGTGCTGT GCTGGATGAA GCTAAAAACA TCAACAAGTC ACTTTCTGCT
1261 CTTGGAAATG TTATTTCTGC TTTGGCTGAG GGTAGTACAT ATGTTCCATA TCGAGATAGT
1321 AAAATGACAA GAATCCTTCA AGATTCATTA GGTGGCAACT GTAGAACCAC TATTGTAATT
1381 TGCTGCTCTC CATCATCATA CAATGAGTCT GAAACAAAAT CTACACTCTT ATTTGGCCAA
1441 AGGGCCAAAA CAATTAAGAA CACAGTTTGT GTCAATGTGG AGTTAACTGC AGAACAGTGG
1501 AAAAAGAAGT ATGAAAAAGA AAAAGAAAAA AATAAGATCC TGCGGAACAC TATTCAGTGG
1561 CTTGAAAATG AGCTCAACAG ATGGCGTAAT GGGGAGACGG TGCCTATTGA TGAACAGTTT
1621 GACAAAGAGA AAGCCAACTT GGAAGCTTTC ACAGTGGATA AAGATATTAC TCTTACCAAT
1681 GATAAACCAG CAACCGCAAT TGGAGTTATA GGAAATTTTA CTGATGCTGA AAGAAGAAAG
1741 TGTGAAGAAG AAATTGCTAA ATTATACAAA CAGCTTGATG ACAAGGATGA AGAAATTAAC
1801 CAGCAAAGTC AACTGGTAGA GAAACTGAAG ACGCAAATGT TGGATCAGGA GGAGCTTTTG
1861 GCATCTACCA GAAGGGATCA AGACAATATG CAAGCTGAGC TGAATCGCCT TCAAGCAGAA
Nature Medicine doi:10.1038/nm.2673
1921 AATGATGCCT CTAAAGAAGA AGTGAAAGAA GTTTTACAGG CCCTAGAAGA ACTTGCTGTC
1981 AATTATGATC AGAAGTCTCA GGAAGTTGAA GACAAAACTA AGGAATATGA ATTGCTTAGT
2041 GATGAATTGA ATCAGAAATC GGCAACTTTA GCGAGTATAG ATGCTGAGCT TCAGAAACTT
2101 AAGGAAATGA CCAACCACCA GAAAAAACGA GCAGCTGAGA TGATGGCATC TTTACTAAAA
2161 GACCTTGCAG AAATAGGAAT TGCTGTGGGA AATAATGATG TAAAGCAGCC TGAGGGAACT
2221 GGCATGATAG ATGAAGAGTT CACTGTTGCA AGACTCTACA TTAGCAAAAT GAAGTCAGAA
2281 GTAAAAACCA TGGTGAAACG TTGCAAGCAG TTAGAAAGCA CACAAACTGA GAGCAACAAA
2341 AAAATGGAAG AAAATGAAAA GGAGTTAGCA GCATGTCAGC TTCGTATCTC TCAAGAGGAT
2401 CCAAAGTGGG AATTCCCTCG GAAGAACTTG GTTCTTGGAA AAACTCTAGG AGAAGGCGAA
2461 TTTGGAAAAG TGGTCAAGGC AACGGCCTTC CATCTGAAAG GCAGAGCAGG GTACACCACG
2521 GTGGCCGTGA AGATGCTGAA AGAGAACGCC TCCCCGAGTG AGCTGCGAGA CCTGCTGTCA
2581 GAGTTCAACG TCCTGAAGCA GGTCAACCAC CCACATGTCA TCAAATTGTA TGGGGCCTGC
2641 AGCCAGGATG GCCCGCTCCT CCTCATCGTG GAGTACGCCA AATACGGCTC CCTGCGGGGC
2701 TTCCTCCGCG AGAGCCGCAA AGTGGGGCCT GGCTACCTGG GCAGTGGAGG CAGCCGCAAC
2761 TCCAGCTCCC TGGACCACCC GGATGAGCGG GCCCTCACCA TGGGCGACCT CATCTCATTT
2821 GCCTGGCAGA TCTCACAGGG GATGCAGTAT CTGGCCGAGA TGAAGCTCGT TCATCGGGAC
2881 TTGGCAGCCA GAAACATCCT GGTAGCTGAG GGGCGGAAGA TGAAGATTTC GGATTTCGGC
2941 TTGTCCCGAG ATGTTTATGA AGAGGATTCC TACGTGAAGA GGAGCCAGGG TCGGATTCCA
3001 GTTAAATGGA TGGCAATTGA ATCCCTTTTT GATCATATCT ACACCACGCA AAGTGATGTA
3061 TGGTCTTTTG GTGTCCTGCT GTGGGAGATC GTGACCCTAG GGGGAAACCC CTATCCTGGG
3121 ATTCCTCCTG AGCGGCTCTT CAACCTTCTG AAGACCGGCC ACCGGATGGA GAGGCCAGAC
3181 AACTGCAGCG AGGAGATGTA CCGCCTGATG CTGCAATGCT GGAAGCAGGA GCCGGACAAA
3241 AGGCCGGTGT TTGCGGACAT CAGCAAAGAC CTGGAGAAGA TGATGGTTAA GAGGAGAGAC
3301 TACTTGGACC TTGCGGCGTC CACTCCATCT GACTCCCTGA TTTATGACGA CGGCCTCTCA
3361 GAGGAGGAGA CACCGCTGGT GGACTGTAAT AATGCCCCCC TCCCTCGAGC CCTCCCTTCC
3421 ACATGGATTG AAAACAAACT CTATGGTAGA ATTTCCCATG CATTTACTAG ATTCTAG
c) K22;R12 (Variant 3): KIF5B exon 22 fused to RET exon 12 1 ATGGCGGACC TGGCCGAGTG CAACATCAAA GTGATGTGTC GCTTCAGACC TCTCAACGAG
61 TCTGAAGTGA ACCGCGGCGA CAAGTACATC GCCAAGTTTC AGGGAGAAGA CACGGTCGTG
121 ATCGCGTCCA AGCCTTATGC ATTTGATCGG GTGTTCCAGT CAAGCACATC TCAAGAGCAA
181 GTGTATAATG ACTGTGCAAA GAAGATTGTT AAAGATGTAC TTGAAGGATA TAATGGAACA
241 ATATTTGCAT ATGGACAAAC ATCCTCTGGG AAGACACACA CAATGGAGGG TAAACTTCAT
301 GATCCAGAAG GCATGGGAAT TATTCCAAGA ATAGTGCAAG ATATTTTTAA TTATATTTAC
361 TCCATGGATG AAAATTTGGA ATTTCATATT AAGGTTTCAT ATTTTGAAAT ATATTTGGAT
421 AAGATAAGGG ACCTGTTAGA TGTTTCAAAG ACCAACCTTT CAGTTCATGA AGACAAAAAC
481 CGAGTTCCCT ATGTAAAGGG GTGCACAGAG CGTTTTGTAT GTAGTCCAGA TGAAGTTATG
Nature Medicine doi:10.1038/nm.2673
541 GATACCATAG ATGAAGGAAA ATCCAACAGA CATGTAGCAG TTACAAATAT GAATGAACAT
601 AGCTCTAGGA GTCACAGTAT ATTTCTTATT AATGTCAAAC AAGAGAACAC ACAAACGGAA
661 CAAAAGCTGA GTGGAAAACT TTATCTGGTT GATTTAGCTG GTAGTGAAAA GGTTAGTAAA
1201 ACTGGAGCTG AAGGTGCTGT GCTGGATGAA GCTAAAAACA TCAACAAGTC ACTTTCTGCT
1261 CTTGGAAATG TTATTTCTGC TTTGGCTGAG GGTAGTACAT ATGTTCCATA TCGAGATAGT
1321 AAAATGACAA GAATCCTTCA AGATTCATTA GGTGGCAACT GTAGAACCAC TATTGTAATT
1381 TGCTGCTCTC CATCATCATA CAATGAGTCT GAAACAAAAT CTACACTCTT ATTTGGCCAA
1441 AGGGCCAAAA CAATTAAGAA CACAGTTTGT GTCAATGTGG AGTTAACTGC AGAACAGTGG
1501 AAAAAGAAGT ATGAAAAAGA AAAAGAAAAA AATAAGATCC TGCGGAACAC TATTCAGTGG
1561 CTTGAAAATG AGCTCAACAG ATGGCGTAAT GGGGAGACGG TGCCTATTGA TGAACAGTTT
1621 GACAAAGAGA AAGCCAACTT GGAAGCTTTC ACAGTGGATA AAGATATTAC TCTTACCAAT
1681 GATAAACCAG CAACCGCAAT TGGAGTTATA GGAAATTTTA CTGATGCTGA AAGAAGAAAG
1741 TGTGAAGAAG AAATTGCTAA ATTATACAAA CAGCTTGATG ACAAGGATGA AGAAATTAAC
1801 CAGCAAAGTC AACTGGTAGA GAAACTGAAG ACGCAAATGT TGGATCAGGA GGAGCTTTTG
1861 GCATCTACCA GAAGGGATCA AGACAATATG CAAGCTGAGC TGAATCGCCT TCAAGCAGAA
1921 AATGATGCCT CTAAAGAAGA AGTGAAAGAA GTTTTACAGG CCCTAGAAGA ACTTGCTGTC
1981 AATTATGATC AGAAGTCTCA GGAAGTTGAA GACAAAACTA AGGAATATGA ATTGCTTAGT
2041 GATGAATTGA ATCAGAAATC GGCAACTTTA GCGAGTATAG ATGCTGAGCT TCAGAAACTT
2101 AAGGAAATGA CCAACCACCA GAAAAAACGA GCAGCTGAGA TGATGGCATC TTTACTAAAA
2161 GACCTTGCAG AAATAGGAAT TGCTGTGGGA AATAATGATG TAAAGCAGCC TGAGGGAACT
2221 GGCATGATAG ATGAAGAGTT CACTGTTGCA AGACTCTACA TTAGCAAAAT GAAGTCAGAA
2281 GTAAAAACCA TGGTGAAACG TTGCAAGCAG TTAGAAAGCA CACAAACTGA GAGCAACAAA
2341 AAAATGGAAG AAAATGAAAA GGAGTTAGCA GCATGTCAGC TTCGTATCTC TCAACATGAA
2401 GCCAAAATCA AGTCATTGAC TGAATACCTT CAAAATGTGG AACAAAAGAA AAGACAGTTG
2461 GAGGAATCTG TCGATGCCCT CAGTGAAGAA CTAGTCCAGC TTCGAGCACA AGAGAAAGTC
2521 CATGAAATGG AAAAGGAGCA CTTAAATAAG GTTCAGACTG CAAATGAAGT TAAGCAAGCT
2581 GTTGAACAGC AGATCCAGAG CCATAGAGAA ACTCATCAAA AACAGATCAG TAGTTTGAGA
2641 GATGAAGTAG AAGCAAAAGC AAAACTTATT ACTGATCTTC AAGACCAAAA CCAGAAAATG
2701 ATGTTAGAGC AGGAACGTCT AAGAGTAGAA CATGAGAAGT TGAAAGCCAC AGATCAGGAA
2761 AAGAGCAGAA AACTACATGA ACTTACGGTT ATGCAAGATA GACGAGAACA AGCAAGACAA
2821 GACTTGAAGG GTTTGGAAGA GACAGTGGCA AAAGAACTTC AGACTTTACA CAACCTGCGC
2881 AAACTCTTTG TTCAGGACCT GGCTACAAGA GTTAAAAAGG AGGATCCAAA GTGGGAATTC
2941 CCTCGGAAGA ACTTGGTTCT TGGAAAAACT CTAGGAGAAG GCGAATTTGG AAAAGTGGTC
3001 AAGGCAACGG CCTTCCATCT GAAAGGCAGA GCAGGGTACA CCACGGTGGC CGTGAAGATG
3061 CTGAAAGAGA ACGCCTCCCC GAGTGAGCTG CGAGACCTGC TGTCAGAGTT CAACGTCCTG
3121 AAGCAGGTCA ACCACCCACA TGTCATCAAA TTGTATGGGG CCTGCAGCCA GGATGGCCCG
3181 CTCCTCCTCA TCGTGGAGTA CGCCAAATAC GGCTCCCTGC GGGGCTTCCT CCGCGAGAGC
3241 CGCAAAGTGG GGCCTGGCTA CCTGGGCAGT GGAGGCAGCC GCAACTCCAG CTCCCTGGAC
3301 CACCCGGATG AGCGGGCCCT CACCATGGGC GACCTCATCT CATTTGCCTG GCAGATCTCA
Nature Medicine doi:10.1038/nm.2673
3361 CAGGGGATGC AGTATCTGGC CGAGATGAAG CTCGTTCATC GGGACTTGGC AGCCAGAAAC
3421 ATCCTGGTAG CTGAGGGGCG GAAGATGAAG ATTTCGGATT TCGGCTTGTC CCGAGATGTT
3481 TATGAAGAGG ATTCCTACGT GAAGAGGAGC CAGGGTCGGA TTCCAGTTAA ATGGATGGCA
3541 ATTGAATCCC TTTTTGATCA TATCTACACC ACGCAAAGTG ATGTATGGTC TTTTGGTGTC
3601 CTGCTGTGGG AGATCGTGAC CCTAGGGGGA AACCCCTATC CTGGGATTCC TCCTGAGCGG
3661 CTCTTCAACC TTCTGAAGAC CGGCCACCGG ATGGAGAGGC CAGACAACTG CAGCGAGGAG
3721 ATGTACCGCC TGATGCTGCA ATGCTGGAAG CAGGAGCCGG ACAAAAGGCC GGTGTTTGCG
3781 GACATCAGCA AAGACCTGGA GAAGATGATG GTTAAGAGGA GAGACTACTT GGACCTTGCG
3841 GCGTCCACTC CATCTGACTC CCTGATTTAT GACGACGGCC TCTCAGAGGA GGAGACACCG
3901 CTGGTGGACT GTAATAATGC CCCCCTCCCT CGAGCCCTCC CTTCCACATG GATTGAAAAC
3961 AAACTCTATG GTAGAATTTC CCATGCATTT ACTAGATTCT AG
d) K15;R11 (Variant 4): KIF5B exon 15 fused to part RET exon 11
1 ATGGCGGACC TGGCCGAGTG CAACATCAAA GTGATGTGTC GCTTCAGACC TCTCAACGAG
61 TCTGAAGTGA ACCGCGGCGA CAAGTACATC GCCAAGTTTC AGGGAGAAGA CACGGTCGTG
121 ATCGCGTCCA AGCCTTATGC ATTTGATCGG GTGTTCCAGT CAAGCACATC TCAAGAGCAA
181 GTGTATAATG ACTGTGCAAA GAAGATTGTT AAAGATGTAC TTGAAGGATA TAATGGAACA
241 ATATTTGCAT ATGGACAAAC ATCCTCTGGG AAGACACACA CAATGGAGGG TAAACTTCAT
301 GATCCAGAAG GCATGGGAAT TATTCCAAGA ATAGTGCAAG ATATTTTTAA TTATATTTAC
361 TCCATGGATG AAAATTTGGA ATTTCATATT AAGGTTTCAT ATTTTGAAAT ATATTTGGAT
421 AAGATAAGGG ACCTGTTAGA TGTTTCAAAG ACCAACCTTT CAGTTCATGA AGACAAAAAC
481 CGAGTTCCCT ATGTAAAGGG GTGCACAGAG CGTTTTGTAT GTAGTCCAGA TGAAGTTATG
541 GATACCATAG ATGAAGGAAA ATCCAACAGA CATGTAGCAG TTACAAATAT GAATGAACAT
601 AGCTCTAGGA GTCACAGTAT ATTTCTTATT AATGTCAAAC AAGAGAACAC ACAAACGGAA
661 CAAAAGCTGA GTGGAAAACT TTATCTGGTT GATTTAGCTG GTAGTGAAAA GGTTAGTAAA
1201 ACTGGAGCTG AAGGTGCTGT GCTGGATGAA GCTAAAAACA TCAACAAGTC ACTTTCTGCT
1261 CTTGGAAATG TTATTTCTGC TTTGGCTGAG GGTAGTACAT ATGTTCCATA TCGAGATAGT
1321 AAAATGACAA GAATCCTTCA AGATTCATTA GGTGGCAACT GTAGAACCAC TATTGTAATT
1381 TGCTGCTCTC CATCATCATA CAATGAGTCT GAAACAAAAT CTACACTCTT ATTTGGCCAA
1441 AGGGCCAAAA CAATTAAGAA CACAGTTTGT GTCAATGTGG AGTTAACTGC AGAACAGTGG
1501 AAAAAGAAGT ATGAAAAAGA AAAAGAAAAA AATAAGATCC TGCGGAACAC TATTCAGTGG
1561 CTTGAAAATG AGCTCAACAG ATGGCGTAAT GGGGAGACGG TGCCTATTGA TGAACAGTTT
1621 GACAAAGAGA AAGCCAACTT GGAAGCTTTC ACAGTGGATA AAGATATTAC TCTTACCAAT
1681 GATAAACCAG CAACCGCAAT TGGAGTTATA GGAAATTTTA CTGATGCTGA AAGAAGAAAG
1741 TGTGAAGAAG AAATTGCTAA ATTATACAAA CAGCTTGATG ACAAGGATGA AGAAATTAAC
1801 CAGCAAAGTC AACTGGTAGA GAAACTGAAG ACGCAAATGT TGGATCAGGA GGAGCTTTTG
1861 GCATCTACCA GAAGGGATCA AGACAATATG CAAGCTGAGC TGAATCGCCT TCAAGCAGAA
1921 AATGATGCCT CTAAAGAAGA AGTGAAAGAA GTTTTACAGG CCCTAGAAGA ACTTGCTGTC
Nature Medicine doi:10.1038/nm.2673
1981 AATTATGATC AGAAGTCTCA GGAAGTTGAA GACAAAACTA AGGAATATGA ATTGCTTAGT
2041 GATGAATTGA ATCAGAAATC GGCAACTTTA GCGAGTATAG ATGCTGAGCT TCAGAAACTT
2101 AAGGAAATGA CCAACCACCA GAAAAAACGA GCAGCTGAGA TGATGGCATC TTTACTAAAA
2161 GACCTTGCAG AAATAGGAAT TGCTGTGGGA AATAATGATG TAAAGTTTGC CCACAAGCCA
2221 CCCATCTCCT CAGCTGAGAT GACCTTCCGG AGGCCCGCCC AGGCCTTCCC GGTCAGCTAC
2281 TCCTCTTCCG GTGCCCGCCG GCCCTCGCTG GACTCCATGG AGAACCAGGT CTCCGTGGAT
2341 GCCTTCAAGA TCCTGGAGGA TCCAAAGTGG GAATTCCCTC GGAAGAACTT GGTTCTTGGA
2401 AAAACTCTAG GAGAAGGCGA ATTTGGAAAA GTGGTCAAGG CAACGGCCTT CCATCTGAAA
2461 GGCAGAGCAG GGTACACCAC GGTGGCCGTG AAGATGCTGA AAGAGAACGC CTCCCCGAGT
2521 GAGCTGCGAG ACCTGCTGTC AGAGTTCAAC GTCCTGAAGC AGGTCAACCA CCCACATGTC
2581 ATCAAATTGT ATGGGGCCTG CAGCCAGGAT GGCCCGCTCC TCCTCATCGT GGAGTACGCC
2641 AAATACGGCT CCCTGCGGGG CTTCCTCCGC GAGAGCCGCA AAGTGGGGCC TGGCTACCTG
2701 GGCAGTGGAG GCAGCCGCAA CTCCAGCTCC CTGGACCACC CGGATGAGCG GGCCCTCACC
2761 ATGGGCGACC TCATCTCATT TGCCTGGCAG ATCTCACAGG GGATGCAGTA TCTGGCCGAG
2821 ATGAAGCTCG TTCATCGGGA CTTGGCAGCC AGAAACATCC TGGTAGCTGA GGGGCGGAAG
2881 ATGAAGATTT CGGATTTCGG CTTGTCCCGA GATGTTTATG AAGAGGATTC CTACGTGAAG
2941 AGGAGCCAGG GTCGGATTCC AGTTAAATGG ATGGCAATTG AATCCCTTTT TGATCATATC
3001 TACACCACGC AAAGTGATGT ATGGTCTTTT GGTGTCCTGC TGTGGGAGAT CGTGACCCTA
3061 GGGGGAAACC CCTATCCTGG GATTCCTCCT GAGCGGCTCT TCAACCTTCT GAAGACCGGC
3121 CACCGGATGG AGAGGCCAGA CAACTGCAGC GAGGAGATGT ACCGCCTGAT GCTGCAATGC
3181 TGGAAGCAGG AGCCGGACAA AAGGCCGGTG TTTGCGGACA TCAGCAAAGA CCTGGAGAAG
3241 ATGATGGTTA AGAGGAGAGA CTACTTGGAC CTTGCGGCGT CCACTCCATC TGACTCCCTG
3301 ATTTATGACG ACGGCCTCTC AGAGGAGGAG ACACCGCTGG TGGACTGTAA TAATGCCCCC
3361 CTCCCTCGAG CCCTCCCTTC CACATGGATT GAAAACAAAC TCTATGGTAG AATTTCCCAT
3421 GCATTTACTA GATTCTAG
In all panels, nucleotides derived from KIF5B are shown in blue and nucleotides derived
from RET are shown in red.
Nature Medicine doi:10.1038/nm.2673
a) K15;R12
(Variant 1, 8 cases)
1 638 1040
1 575 977
Kinesin Tyrosine
Kinase
Coiled
coil
KIF5B Exon 15 RET Exon 12
cDNA confirmation
of fusion junction
KIF5B Exon 16 RET Exon 12
T G T A A A G G A G G A T C
C T C T C A A G A G G A T C
b) K16;R12
(Variant 2, 3 cases)
cDNA confirmation
of fusion junction
Kinesin Tyrosine
Kinase
Coiled
Coil
c) K22;R12
(Variant 3, 1 case)
cDNA confirmation
of fusion junction
Kinesin Tyrosine
Kinase
Coiled
Coil
1 852 1254
T A A A A A G G A G G A T C
KIF5B Exon 22 RET Exon 12
d) K15;R11*
(Variant 4, 1 case)
1 575 1027
Kinesin Tyrosine
Kinase
Coiled
Coil
cDNA confirmation
of fusion junction
T G T A A A G T T T G C C C
KIF5B Exon 15 RET Exon 11*
Supplementary Figure 3. KIF5B-RET fusion transcripts
The total length and the position of the fusion breakpoint are shown above each variant protein
and capillary sequence confirmation of the exon junction boundaries derived from cDNA is shown
below. *This case also harbored a KIF5B-RET gene fusion transcript variant 1.
Nature Medicine doi:10.1038/nm.2673