Supplementary Information RET, ROS1, and ALK Fusions in ...Supplementary Information RET, ROS1, and ALK Fusions in Lung Cancer Kengo Takeuchi, 1,2 Manabu Soda,3 Yuki Togashi, 1,2 Ritsuro

Supplementary Information

RET, ROS1, and ALK Fusions in Lung Cancer

Kengo Takeuchi,1,2 Manabu Soda,3 Yuki Togashi,1,2 Ritsuro Suzuki,4 Seiji Sakata,1,2

Satoko Hatano,1,2 Reimi Asaka,1,2 Wakako Hamanaka,2 Hironori Ninomiya,2 Hirofumi

Uehara,5 Young Lim Choi,6 Yukitoshi Satoh,5* Sakae Okumura,5 Ken Nakagawa,5

Hiroyuki Mano,3,6 Yuichi Ishikawa.2

1Pathology Project for Molecular Targets, the Cancer Institute, Japanese Foundation for

Cancer Research, Tokyo, Japan; 2Division of Pathology, the Cancer Institute, Japanese

Foundation for Cancer Research, Tokyo, Japan; 3Division of Functional Genomics, Jichi

Medical University, Tochigi, Japan; 4Department of HSCT Data Management and

Biostatistics, Nagoya University Graduate School of Medicine, Nagoya, Japan;

5Department of Thoracic Surgical Oncology, Thoracic Center, the Cancer Institute

Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan; 6Department of

Medical Genomics, Graduate School of Medicine, The University of Tokyo, Tokyo,

Japan

*Present address: Department of Thoracic Surgery, Kitasato University School of

Medicine, Kanagawa 252-0375, Japan.

Nature Medicine doi:10.1038/nm.2658

Takeuchi et al. 1

Method summary Patients and sample preparation All 1,529 patients with lung cancer underwent surgery at the Cancer Institute Hospital between 1995 and 2009. The following specimens were consecutively collected from the pathology archives of the hospital: 1122 adenocarcinomas, 233 squamous cell carcinomas, 38 adenosquamous carcinomas, 34 large-cell carcinomas, 36 large-cell neuroendocrine carcinomas, seven mucoepidermoid carcinomas, four adenoid cystic carcinomas, 12 sarcomatoid carcinomas, and 43 small-cell carcinomas. Previously reported cases of ALK fusion-positive (n = 20) and ALK fusion-negative tumors (n = 304), all of which were screened with multiplex RT-PCR, were included1-5 because specimens of these 324 patients were collected consecutively during the period of tissue collection, and they served as positive and negative controls, respectively. The 1,528 cohort contained 100 adenocarcinomas with EGFR mutation. This study was approved by the institutional review board of the Cancer Institute Hospital. Identification and transforming assays of kinase fusions Our screening system consisted of two steps: (1) histopathology-based screening for gene rearrangement-positive candidates using tissue microarrays, and (2) PCR-based studies for partner gene detection using corresponding snap-frozen materials. In brief, ALK expression was screened by anti-ALK immunohistochemistry by using the iAEP method, and ROS1, KIF5B, and RET rearrangements were screened using FISH. Positive cases were further examined by rapid amplification of cDNA ends (RACE) or inverse RT-PCR assays for the detection of fusion partners. Fusion-positive cases were confirmed by fusion-specific RT-PCR, genomic PCR, and FISH. The transforming ability of novel kinase fusions were examined using mouse 3T3 fibroblasts infected with expression plasmids for each fusion kinase. Mouse BA/F3 cells induced to express EML4-ALK (E13;A20) or KIF5B-RET (K15;R12L and K15;R12S) were used to confirm the transforming ability of the fusions and the growth inhibition of vandetanib (please see Appendix for Methods). Physical and biologic containment procedures for recombinant DNA followed institutional protocols in accordance with the National Institutes of Health Guidelines. Detection of EGFR and KRAS mutations and TTF1 and p63 expression Mutation analyses were performed using a Cycleave PCR core kit (TaKaRa) for EGFR (G719X, exon 19 deletion, T790M, L858R, L861Q) using a direct sequencing assay for KRAS (codons 12 and 13). The expression of TTF1 and p63 was examined by immunohistochemistry. Statistical analysis The smoking index is defined as a product of cigarettes per day and duration in years. Overall survival was calculated from the date of surgery to the date of death. Pairwise comparisons were performed using the χ2 test and Fisher’s exact test for categorical variables and the Mann-Whitney U test for continuous variables. The Kruskal-Wallis test was used to compare multiple groups. P values less than 0.05 obtained in two-sided tests were considered statistically significant. Patient survival data were analyzed by the method of Kaplan and Meier and were compared using the log-rank test. Univariate and multivariate analyses were performed using the Cox proportional hazard regression model, and variables were selected using the stepwise method. Data were analyzed with the statistical software STATA version 11 (STATA Corp).


Takeuchi et al. 2

References 1. Takeuchi, K., et al. Clin Cancer Res 15, 3143-3149 (2009). 2. Takeuchi, K., et al. Clin Cancer Res 14, 6618-6624 (2008). 3. Inamura, K., et al. J Thorac Oncol 3, 13-17 (2008). 4. Soda, M., et al. Nature 448, 561-566 (2007). 5. Choi, Y.L., et al. Cancer Res 68, 4971-4976 (2008).


ab dd

c Sup

plem

enta

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igur

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P im

mun

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toch

emic

al a

naly

sis


ab dd

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plem

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spl

it FI

SH

ass

ay


ab dd

c Sup

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sion

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so

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ur

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su

Moc

k*N

egat

ive

cont

rol f

or L

–T

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ock*

Neg

ativ

e co

ntro

l for

A, C

–I

Mk*

Nti

tlf

BJ

dK

wM

ock*

Neg

ativ

e co

ntro

l for

B, J

and

K*p

MX

s re

trovi

rus

vect

er

Sup

plem

enta

ry F

igur

e 5.

Tra

nsfo

rmin

g as

says

for n

ovel

fusi

ons


Sup

plem

enta

ry F

igur

e L

egen

ds

Sup

plem

enta

ry F

igur

e 1:

Ant

i-A

LK

iAE

P im

mun

ohis

toch

emic

al a

naly

sis

All

car

cino

ma

cell

s st

aine

d fo

r AL

K e

xhib

ited

a c

ytop

lasm

ic s

tain

ing

patt

ern.

Pan

els

a, b

, c, a

nd d

are

fro

m A

LK

#4, #

16, #

27,a

nd #

28, r

espe

ctiv

ely

(sca

le b

ar

yp

gp

py

(10

0 m

,×20

obj

ecti

ve).

Sup

plem

enta

ry F

igur

e 2:

AL

K s

plit

FIS

H a

ssay

5′-

and

3′-A

LK

gen

es a

re s

tain

ed g

reen

and

red

, res

pect

ivel

y. W

ild

type

AL

K a

ppea

rs y

ello

w. T

he 5′ s

igna

ls a

re n

ot d

etec

ted

in p

anel

c, i

ndic

atin

g th

e de

leti

on

of th

e re

gion

. The

refo

re, t

he e

valu

atio

n of

the

AL

K s

plit

sta

tus

is e

asy

in p

anel

c b

ut m

ay b

e di

ffic

ult i

n pa

nels

a, b

, and

d b

ecau

se o

f th

e pr

oxim

ity

of th

e 2

diff

eren

tly

colo

red

sign

als,

whi

ch is

der

ived

fro

m th

e pr

oxim

ity

of E

ML4

and

ALK

(sca

le b

ars,

40 m

). P

anel

s a,

b, c

, and

d a

re f

rom

AL

K#4

, #16

, #27

, and

#2

8, r

espe

ctiv

ely.

Sl

tF

i3

EM

L4

AL

Kf

iF

ISH

Sup

plem

enta

ry F

igur

e 3:

EM

L4-

AL

K f

usio

n F

ISH

ass

ay

EM

L4

and

AL

K a

re s

tain

ed r

ed a

nd g

reen

, res

pect

ivel

y. T

he f

usio

n ge

ne a

ppea

rs y

ello

w (

scal

e ba

rs, 4

0 m

). P

anel

s a,

b, c

, and

d a

re f

rom

pat

ient

s #4

, #16

, #2

7, a

nd #

28, r

espe

ctiv

ely.

Spp

lem

enta

rF

igre

4:M

orph

olog

ofm

cino

scr

ibri

form

carc

inom

aS

uppl

emen

tary

Fig

ure

4: M

orph

olog

y of

muc

inou

s cr

ibri

form

car

cino

ma

Rep

rese

ntat

ive

hist

opat

holo

gy o

f m

ucin

ous

crib

rifo

rm c

arci

nom

a is

sho

wn.

Pan

els

a, b

, c, a

nd d

are

fro

m p

atie

nts

#4, #

16, #

27, a

nd #

28, r

espe

ctiv

ely

(sca

le b

ar

100 m

,×20

obj

ecti

ve).

Sup

plem

enta

ryF

igur

e5:

Tra

nsfo

rmin

gas

says

for

nove

lfus

ions

Sup

plem

enta

ry F

igur

e 5:

Tra

nsfo

rmin

g as

says

for

nov

el f

usio

ns

The

ful

l-le

ngth

cD

NA

s w

ere

isol

ated

fro

m c

orre

spon

ding

spe

cim

ens

wit

h R

T-P

CR

and

wer

e us

ed to

gen

erat

e re

com

bina

nt r

etro

viru

ses

expr

essi

ng e

ach

fusi

on

prot

ein

wit

h a

FL

AG

epi

tope

tag.

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ecti

on o

f 3T

3 ce

lls

wit

h th

e vi

rus

expr

essi

ng K

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(tra

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ipt v

aria

nt 2

)-R

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1, o

r K

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T (

both

the

long

er a

nd s

hort

er is

ofor

ms

of R

ET

) le

d to

the

form

atio

n of

mul

tipl

e tr

ansf

orm

ed

foci

in c

ultu

re a

nd s

ubcu

tane

ous

tum

ors

in a

nud

e m

ouse

tum

orig

enic

ity

assa

y (f

or e

ach

fusi

on, a

tum

or f

orm

ed in

all

4 o

f th

ein

ject

ed m

ice)

, dem

onst

rati

ng th

e po

tent

tran

sfor

min

g ab

ilit

y of

eac

h fu

sion

kin

ase.

Int

eres

ting

ly, f

or L

RIG

3-R

OS

1, o

nly

tran

scri

pt v

aria

nt 2

of

LR

IG3

was

tum

orig

enic

(j a

nd k

). W

e fa

iled

to

obta

in 2

ful

l-le

ngth

cD

NA

s fo

r S

LC

34A

2-R

OS

1 (S

13de

l204

6;R

34 o

f R

OS

#6 in

whi

ch th

e 56

8th

nucl

eoti

de o

f ex

on 1

3 of

SL

C34

A2

fuse

d to

exo

n 34

of

RO

S1)

and

the

long

er is

ofor

m o

f K

IF5B

-RE

T (

K24

;R11

L o

f R

ET

#12)

.


1E+09

1E+10

1E+11

109

1011

1010

a1.00E+08

1.00E+09

BA

/F3:

CD

8(+)

IL-3

109

108

bA

n in

hibi

tor

spec

ific

onl

y to

RE

T is

not

cu

rren

tly

avai

labl

e, b

ut th

ere

are

agen

ts th

at h

ave

acti

vity

aga

inst

sev

eral

kin

ases

, inc

ludi

ng R

ET.

To

1000000

0000000

0000000

CD8(‐)IL‐3

CD8(+)IL‐3

()

108

107

106

105

1.00E+05

1.00E+06

1.00E+07

0 μM

01

M

Vand

etanib

107

106

105

test

whe

ther

suc

h in

hibi

tors

mig

ht b

e ef

fect

ive

for

lung

can

cers

wit

h R

ET

fus

ions

, we

indu

ced

Fla

g-ta

gged

EM

L4-

AL

K (

E13

;A20

) or

KIF

5B-R

ET

(K

15;R

12S

and

K15

;R12

L)

in B

A/F

3 ce

lls,

whi

ch

are

inte

rleu

kin-

3(I

L-3

)-de

pend

entf

orgr

owth

All

100

1000

10000

100000

EA(‐)IL‐3

EA(+)IL

‐3KR

L(‐)IL‐3

KRL(+)IL‐3

KRS(‐)IL‐3

KRS(+)IL‐3

105

104

103

102

1.00E+02

1.00E+03

1.00E+04

0.1 μM

0.5 μM

1 μM

5 μM

10 μM

104

103

102

are

inte

rleu

kin

3 (I

L3)

depe

nden

t for

gro

wth

. All

tr

ansf

ecte

d ce

lls

incl

udin

g th

ose

wit

hout

a k

inas

e fu

sion

(ex

pres

sing

CD

8 on

ly)

prol

ifer

ated

in th

e pr

esen

ce o

f IL

-3, b

ut o

nly

thos

e ex

pres

sing

EM

L4-

AL

K o

r K

15;R

12L

gre

w in

the

abse

nce

of I

L-3

(a)

. T

hifi

dif

tht

dth

i100

day0

day1

day2

day3

day4

day5

day6

day7

1.00E+08

1.00E+09

BA

/F3:

KIF

5B-R

ET(

-)IL

-310

9

108

c1.00E+08

1.00E+09

BA

/F3:

EM

L4-A

LK(-

)IL-3

109

108

d

day0

day1

day2

day3

day4

day5

10T

his

find

ing

furt

her

supp

orte

d th

e on

coge

nic

pote

ntia

l of

KIF

5B-R

ET.

Int

eres

ting

ly, B

A/F

3 ce

lls

expr

essi

ng K

15;R

12S

did

not

gro

w in

the

abse

nce

of

IL-3

alt

houg

h th

e fu

sion

indu

ced

in th

e 3T

3 ce

lls

clea

rly

show

ed o

ncog

enic

abi

lity

. Thi

s su

gges

ts th

at

1.00E+06

1.00E+07

10 107

106

Vand

etanib

1.00E+06

1.00E+07

1.00E08

108

107

106

Vand

etanib

the

C-t

erm

inal

dif

fere

nce

betw

een

RE

T9

and

RE

T51

(t

he w

ild

type

cou

nter

part

s fo

r K

15;R

12S

and

K

15;R

12L

, res

pect

ivel

y) is

cri

tica

l for

the

IL-3

-in

depe

nden

t gro

wth

of

BA

/F3

cell

s bu

t not

for

tr

ansf

orm

ing

3T3

cell

s.IL

-3-d

epen

dent

grow

thof

1.00E+03

1.00E+04

1.00E+05

0 μM

0.1 μM

0.5 μM

1 μM

5 μM

105

104

103

Vand

etanib

1.00E+03

1.00E+04

1.00E+05

0 μM

0.1 μM

0.5 μM

1 μM

5 μM

105

104

103

Vand

etanib

tran

sfor

min

g 3T

3 ce

lls.

IL

3de

pend

ent g

row

th o

f B

A/F

3 ce

lls

was

sli

ghtl

y af

fect

ed b

y ≥5μM

va

ndet

anib

, whi

ch is

a s

mal

l-m

olec

ule

inhi

bito

r of

V

EG

FR

-2 th

at h

as a

ddit

iona

l act

ivit

y ag

ains

t V

EG

FR

-3, E

GF

R, a

nd R

ET

(b)

. In

cont

rast

, va

ndet

anib

noti

ceab

lyin

hibi

ted

the

prol

ifer

atio

nof

1.00E+02 da

y0day1

day2

day3

day4

day5

10 μM

102

1.00E+02 da

y0day1

day2

day3

day4

day510

μM

102

vand

etan

ib n

otic

eabl

y in

hibi

ted

the

prol

ifer

atio

n of

B

A/F

3 ce

lls

expr

essi

ng K

15;R

12L

(c)

but

not

that

of

BA

/F3

cell

s ex

pres

sing

E13

;A20

in th

e ab

senc

e of

IL

-3 (

d). C

ell n

umbe

rs w

ere

dete

rmin

ed a

t the

in

dica

ted

tim

es. D

ata

are

mea

ns p

lus

s.d.

of

valu

es

Sup

plem

enta

ry F

igur

e 6.

RE

T fu

sion

as

a po

tent

ial m

olec

ular

targ

et

from

thre

e se

para

te e

xper

imen

ts.


Criz

otin

ib: 5

00nM

1.00

E+08

1.00

E+09

109

108

BA

/F3

cell

s ex

pres

sing

CD

8 ei

ther

alo

ne o

r to

geth

er w

ith

EM

L4-

AL

K (

E13

;A20

) or

K

IF5B

RE

T(K

15;R

12L

)w

ere

cult

ured

in

1.00

E+06

1.00

E+07

10 107

106

KIF

5B-R

ET

(K

15;R

12L

) w

ere

cult

ured

in

the

abse

nce

(–)

or p

rese

nce

(+)

of I

L-3

(1

ng/m

l). B

A/F

3 ce

lls

expr

essi

ng C

D8

alon

e w

ere

incu

bate

d w

ith

IL-3

and

500

nM

criz

otin

ib, a

nd th

ose

expr

essi

ng K

15;R

12L

13

A20

ld

ih

3b

1.00

E+03

1.00

E+04

1.00

E+05

CD8(+)IL‐3

KRL(‐)IL‐3

EA(‐)IL‐3

105

104

103

or E

13;A

20 w

ere

cult

ured

wit

hout

IL

-3 b

ut

wit

h 50

0 nM

cri

zoti

nib.

Cri

zoti

nib

was

not

ef

fect

ive

to B

A/F

3 ce

lls

expr

essi

ng E

13;A

20

but n

ot to

thos

e ex

pres

sing

K15

;R12

L. C

ell

num

bers

wer

e de

term

ined

at t

he in

dica

ted

1.00

E+02

day0

day1

day2

day3

day4

day5

()

102

tim

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Supplementary Table 1 Patient characteristics

Patient # age sex Mucinouscribriform Sig TTF1 p63 Fusion

partner Fusion pattern Dead Observation(day) Cause of death PY Diameter

(mm) p-stage Other therapy

ALK#1 38 F - - + +w EML4 E13;A20 - 4808 0 30 2A -ALK#2 52 M MCC - + +w EML4 E13;A20 - 4487 1000 23 3A -ALK#3 70 M MCC + + - EML4 E13;A20 + 478 PD 620 22 3B -ALK#4 51 M MCC + + +w EML4 E13;A20 - 3702 4 15 2A CT (post)ALK#5 58 F - - + - EML4 E13;A20 - 2277 0 27 1A -ALK#6 36 M - - + - EML4 E13;A20 - 2271 0 25 2A -ALK#7 56 M - - + - EML4 E13;A20 + 971 PD 1600 35 3A CT (pre+post)ALK#8 59 F MCC + + - EML4 E13;A20 - 764 0 34 3A -ALK#9 43 F - - + - EML4 E13;A20 - 2260 10 12 1A -ALK#10 66 F MCP+ + + - EML4 E13;A20 - 2212 0 33 3B RT (post)ALK#11 70 F MCP+ - + +w EML4 E13;A20 - 2177 0 18 1A -ALK#12 66 F - - + +w EML4 E13;A20 - 1462 0 15 1A -ALK#13 59 F - - + - EML4 E13;A20 - 1056 0 20 1A -ALK#14 46 M MCC + + - EML4 E13;A20 - 918 0 14 2B -ALK#15 59 F MCC - + +w EML4 E13;A20 - 736 0 18 1A -ALK#16 66 F MCC - + - EML4 E20;A20 + 2102 Other cancer 0 28 1A NAALK#17 64 M MCC + + - EML4 E20;A20 - 2711 220 18 3B NAALK#18 43 M MCP+ + + - EML4 E20;A20 + 537 PD 160 23 4 CT+RT (post)ALK#19 56 M MCC - - - EML4 E20;A20 - 1524 700 60 2B -ALK#20 66 M MCC - + +w EML4 E20;A20 - 995 1380 19 2A -ALK#21 52 M MCP+ - + - EML4 E20;A20 - 405 0 12 3B -ALK#22 50 F MCP+ - + - EML4 E20;A20 - 222 6 15 1A -ALK#23 55 M MCC - - - EML4 E6a/b;A20 - 4661 600 18 3B -ALK#24 57 F MCP+ - + +w EML4 E6a/b;A20 - 4019 0 30 1A -ALK#25 62 F MCC - + - EML4 E6a/b;A20 + 528 PD 0 19 2A NAALK#26 47 M MCP+ - + - EML4 E6a/b;A20 - 2675 540 17 3A -ALK#27 73 M MCP+ - + - EML4 E6a/b;A20 - 2292 300 13 1A -ALK#28 61 M MCC - + - EML4 E6a/b;A20 + 1647 PD 0 13 3A -ALK#29 61 F - - + +w EML4 E6a/b;A20 - 1462 435 13 1A -ALK#30 34 F - - + - EML4 E6a/b;A20 - 1461 0 16 1A -ALK#31 75 F MCP+ - + - EML4 E6a/b;A20 - 233 0 43 3B NAALK#32 68 M MCC - + - EML4 E6a/b;A20 - 1280 0 18 3B -ALK#33 47 F - - + +w EML4 E6a/b;A20 - 1140 0 11 3B -ALK#34 76 F MCC - + - EML4 E6a/b;A20 - 547 0 26 2A -ALK#35 44 F - - + +w EML4 E6a/b;A20 - 492 0 12 1A -ALK#36 84 M MCC - + - EML4 E6a/b;A20 - 365 840 14 1A -


ALK#37 50 M MCC - + - EML4 E6a/b;A20 - 124 0 9 1A -ALK#38 52 F MCC - + - EML4 E14;ins11;del49A20 - 2556 0 24 1A -ALK#39 44 M MCP+ - + - EML4 E2a/b;A20 - 2367 0 15 1A -ALK#40 65 F - + + +w EML4 E13;ins69A20 - 599 0 18 1A -ALK#41 26 F MCC - + - EML4 E14;del14A20 - 365 0 32 1B -ALK#42 59 F - - + - KIF5B K17;A20 - 4018 0 22 1A -ALK#43 72 F - - + - KIF5B K24;A20 - 4313 0 24 1A -ALK#44 75 F - - + +w KIF5B K24;A20 - 734 0 15 3A CT (post)ROS#1 50 M - - + - TPM3 T8;R35 - 2562 500 12 1A -ROS#2 51 F - - + - TPM3 T8;R35 - 1352 190 25 2A CT (post)ROS#3 70 F - - + - SDC4 S2;R32 + 405 PD 0 28 1A -ROS#4 60 F - - + - SDC4 S2;R32 - 2745 0 50 1B -ROS#5 77 F - - + - SDC4 S2;R32, S2;R34 - 582 0 36 1B -

ROS#6 69 F - - + - SLC34A2 S13del2046;R32,S13del2046;R34 - 2146 0 35 1B -

ROS#7 57 M MCP+ - + - CD74 C6;R34 - 1835 1480 20 1A -ROS#8 60 M MCC - + - CD74 C6;R34 + 3169 other disease 930 18 3B NAROS#9 79 F MCC - + - CD74 C6;R32, C6;R34 - 2939 0 9 1A -ROS#10 36 M - - + - EZR E10;R34 - 1771 0 19 2A CT (post)ROS#11 43 F - - + - EZR E10;R34 - 2150 0 11 1A -ROS#12 57 M - - + - LRIG3 L16;R35 - 1114 100 26 1A CT (post)ROS#13 55 F - - + - unknown + 1259 PD 0 105 4 -RET#1 59 M - - + - KIF5B K15;R12 + 1159 PD 0 18 2A -RET#2 53 F - - + - KIF5B K15;R12 - 3361 0 19 1A -RET#3 43 F - - + - KIF5B K15;R12 - 2556 0 23 1A -RET#4 53 F - - + - KIF5B K15;R12 - 2927 150 19 1A -RET#5 74 F - - + - KIF5B K15;R12 - 2553 0 45 2B -RET#6 60 F - - + - KIF5B K15;R12 - 1087 0 22 1A -RET#7 60 F - - + - KIF5B K15;R12 - 729 0 22 1A -RET#8 62 M - - + - KIF5B K15;R12 - 570 720 21 3B CT (post)RET#9 62 M - - + - KIF5B K16;R12 - 1034 390 29 3B CT (post)

RET#10 55 M - - + - KIF5B K22;R12 - 211 0 12 1A -RET#11 58 F - - + - KIF5B K23;R12 - 2021 0 11 1A -RET#12 56 F - - + - KIF5B K24;R11 - 3553 0 19 1A -RET#13 68 F - - + - CCDC6 C1;R12 - 1224 0 20 3A CT (post)RET#14 76 F MCC + + - CCDC6 C1;R12 - 582 0 34 1B -

Abbreviations: Sig, signet ring cell; +w, weakly positive; PY, pack years; MCP, mucinous cribriform pattern (+ for cases with more than 5% area of MCP); MCC, mucinouscribriform carcinoma (for cases with more than 25% area of MCP); PD, progressive disease; CT, chemotherapy; RT, radiotherapy; NA, not available; pre, pre-operative;post, post-operative


Supplementary Table 2 Clinicopathological features of kinase fusion-positive lung cancer

Fusion(-) NSCLC P Fusion(+) P Fusion(-) Adeno.N=1447 N=71 N=1045

Age, median (range) 65 (25-86) <0.0001 59 (26-84) <0.0001 64 (30-86)

Sex (M:F) 851:596 0.001 28:43 0.13 509:536 Male% 59% 39% 49%

Pack years (PY) Median (range) 570 (0-5000) <0.0001 0 (0-1600) <0.0001 141 (0-4500)Mean 678 181 482

PY < 400 631 58 591PY ≥ 400 816 13 454

Smoking historyNever smoker 477 <0.001 48 <0.001 453Smoker 970 23 592

Diameter (D) Median (range) 28 (5-150) <0.0001 20 (9-105) 0.0001 26 (5-150)Mean 32.5 23.4 29.4

D ≤30mm 771 58 665D >30mm 624 11 374

Pathological stage 0.001 0.0041A 544 34 4691B 333 5 2132A 62 9 402B 131 3 603A 168 7 1093B 166 11 1194 34 2 31


Supplementary Table 3 Prognostic factors for adenocarcinoma

Hazard ratio (CI) P Hazard ratio (CI) P

Age ≥ 50 years 1.57 (0.97-2.54) 0.07 1.83 (1.13-2.96) 0.01Sex Male 1.94 (1.52-2.47) < 0.001 1.70 (1.33-2.18) < 0.001Pathological stage 2-4 4.15 (3.24-5.31) < 0.001 4.16 (3.24-5.35) < 0.001Pack years ≥ 400 1.81 (1.43-2.29) < 0.001 -ALK Negative 1.82 (0.81-4.10) 0.15 -ROS1 Negative 1.13 (0.36-3.53) 0.83 -RET Negative 3.31 (0.46-23.6) 0.23 -Kinase fusion Negative 1.78 (0.95-3.35) 0.07 2.16 (1.15-4.08) 0.02

Abbreviation: CI, confidence interval

Variables Unfavorable factorsUnivariate Multivariate


Supplementary Table 4. Clinicopathological features of ALK, ROS1 or RET-positive lung cancers

ALK(-) NSCLC P ALK+ P ALK(-) Adeno. ROS1(-) NSCLC P ROS1+ P ROS1(-) Adeno.N =1441 N =44 N =1077 N=1463 N=13 N=1103

Age, median (range) 65 (25-86) 0.0001 59 (26-84) 0.0005 64 (30-86) 65 (25-86) 0.09 57 (36-79) 0.14 64 (26-86)

Sex (M:F) 835:606 0.051 19:25 0.50 521:556 844:619 0.16 5:8 0.48 533:570 Male% 58% 43% 48% 58% 38% 48%

Pack years (PY) Median (range) 520 (0-5000) <0.0001 0 (0-1600) 0.001 118 (0-4500) 500 (0-5000) 0.03 0 (0-1480) 0.18 100 (0-4500)Mean 660 191 474 650 246 465

PY < 400 652 35 617 672 10 639PY ≥ 400 782 9 453 784 3 457

Smoking history <0.001 0.005 0.05 0.23Never smoker 497 29 475 514 8 493Smoker 937 15 595 942 5 603

Diameter (D) Median (range) 28 (5-150) <0.0001 18 (9-60) <0.0001 25 (5-150) 28 (5-150) 0.50 26 (9-105) 0.94 25 (5-150)Mean 32.5 21.3 29.4 32.2 31.9 29.0

D ≤ 30mm 796 38 688 821 8 715D > 30mm 635 6 382 633 4 382

Pathological stage 0.001 0.002 0.19 0.281A 550 20 485 561 6 4971B 334 1 218 329 3 2142A 58 6 43 61 2 472B 129 2 61 131 0 633A 161 6 111 166 0 1173B 164 8 122 171 1 1294 36 1 33 35 1 32


RET(-) NSCLC P RET+ P RET(-) Adeno.N=1512 N=14 N=1106

65 (25-86) 0.06 60 (43-76) 0.13 64 (26-86)

880:632 0.03 4:10 0.14 535:57158% 29% 48%

540 (0-5000) 0.0005 0 (0-720) 0.008 100 (0-4500)661 90 468

680 13 638832 1 468

0.001 0.01517 11 492995 3 614

28 (5-150) 0.02 21 (11-45) 0.10 25 (5-150)32.2 22.4 29.1

822 12 714637 2 385

0.68 0.78573 8 497339 1 21871 1 48

133 1 62175 1 115175 2 12837 0 34


Takeuchi et. al. 1

Appendix for Methods Sample preparation Small pieces were grossly dissected out of the tumors removed from the patients, rapidly frozen in liquid nitrogen, cryosectioned, microscopically examined to ensure adequate tumor tissue content, and stored at -80°C. The remaining tissues were fixed in formalin and routinely subjected to pathological diagnosis.

Screening for kinase fusions Tissue microarrays containing tumor cores (1 mm in diameter) from all patients were cut at 4-μm thickness and subjected to hematoxylin and eosin staining, immunohistochemical analysis, and fluorescein in situ hybridization (FISH). For ALK fusion screening, anti-ALK immunohistochemical analysis was performed. An anti-ALK mouse monoclonal antibody, 5A4, and the iAEP method were used.1,2 The methods for FISH analyses have been previously described.1-4 The 1-tube multiplex RT-PCR assay for all possible fusion variants of both EML4-ALK and KIF5B-ALK transcripts was performed using RNA extracted from snap-frozen materials taken from the same tumors to determine the fusion pattern.1,2 In 1 patient, formalin-fixed paraffin-embedded material was subjected to RT-PCR assays specific to each known fusion variants because frozen material was not available. PCR products of the multiplex RT-PCR-positive tumors were directly sequenced for verification. FISH analysis was performed for the other kinase screenings with bacterial artificial chromosome (BAC) clone-derived DNA probes appropriate for the assay intended. For the split assay of ROS1, RP1-179P9 and RP11-323I17 BAC clones were used. For the fusion FISH assays, the following BAC clones were used: RP1-179P9, RP3-453C12, RP11-1120I24, RP11-507C10, RP11-752I1, RP11-557F20, and RP11-205-M9 for ROS1, SDC4, CD74, EZR, SLC34A2, LRIG3, and TPM3, respectively. For KIF5B and RET, the following BAC clones were used: KIF5B (RP11-460H18 and RP11-167O6), and RET (RP11-124O11, RP11-351D16, and RP11-718J13). See details in the Clone Registry in National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genome/clone/). Hybridized slides were then stained with 4,6-diamino-2-phenylindole and examined with a BX51 fluorescence microscope (Olympus, Tokyo, Japan). For each split assay screening, a patient was considered positive if more than 20% of tumor cells exhibited individual 5′-side signals for KIF5B or individual 3′-side signals for ALK, ROS1, and RET. Isolation of ROS1 fusions To reveal suspected ROS1 partner genes, we used a 5′-RACE method using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instruction with a minor modification; ROS1-5992R (5′-ATTTGCTCATCAGATGTGCCTCCTTCAG-3′) was used as the gene-specific reverse primer. Fusion-specific RT-PCR was performed with the following primers: TPM3-705F (5′-AGAGACCCGTGCTGAGTTTGCTG-3′) and ROS1-5674R (5′-CTGTCACCCCTTCCTTGGCACT-3′) for TPM3-ROS1; SDC4-48F (5′-AGTCGCCGAGTCGATCCGAG-3′) and ROS1-5674R for SDC4-ROS1, SLC34A2-1927F (5′-AAGTGCTGCGAGGACTTGGAGG-3′) and ROS1-5674R for SLC34A2-ROS1; CD74-531F (5′-CTGGAAGGTCTTTGAGAGCTGGAT-3′) and ROS1-5674R for CD74-ROS1, EZR-958F (5′-GATGATGCGCGAGAAGGAGGA-3′) and ROS1-5992R (5′-ATTTGCTCATCAGATGTGCCTCCTTCAG-3′) for EZR-ROS1; and LRIG3-2531F (5′-ACACAGATGAGACCAACTTGC-3′) and ROS1-5676R (5′-CACTGTCACCCCTTCCTTG-3′) for LRIG3-ROS1.


Takeuchi et. al. 2

Isolation of RET fusions To obtain the suspected KIF5B partner genes, we used a 3′-RACE method using SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instruction with a minor modification; KIF5B-exon23F (5′-TGAACAGCTCACTAAAGTGCACAAACAG-3′) was used as a gene-specific forward primer. To detect all possible KIF5B-RET fusion variants, a multiplex RT-PCR was performed using 3 forward primers, KIF5B-272F (5′-AGACACACACAATGGAGGGTA-3′), KIF5B-1241F (5′-CTGATGCTGAAAGAAGAAAGTGT-3′), and KIF5B 2265F (5′-AGCCACAGATCAGGAAAAGA-3′), and a reverse primer, RET-2381R (5′-CAGGCCCCATACAATTTGAT-3′). To determine the RET partner gene in RET split FISH-positive patients, we used an inverse RT-PCR method. Double-stranded cDNA was synthesized from 2 μg of total RNA with 1 pM of the primer RET-2746R (5′-TAACTGGAATCCGACCCTGG-3′) and a cDNA Synthesis System (Roche), and was self-ligated by incubation overnight with T4 DNA ligase (TaKaRa Bio). We subjected the resulting circular cDNA to PCR (35 cycles at 94°C for 15 s, 62°C for 30 s, and 72°C for 1 min) with primers RET-2381R (5′-CAGGCCCCATACAATTTGAT-3′) and RET-2271F (5′-GAAGATGCTGAAAGAGAACGC-3′) in a final volume of 20 μL. We subjected 1 μL of the 1:100 diluted reaction products to a second PCR step (the same settings as above) with primers RET-2208R (5′-TCCAAATTCGCCTTCTCCTA-3′) and RET-2605F (5′-AAGCTCGTTCATCGGGACT-3′) in a final volume of 20 μL. The resulting products were purified by gel extraction and directly sequenced in both directions with primers RET-2605F and RET-2208R. To enable the multiplex RT-PCR system for KIF5B-RET to also detect CCDC6-RET, the forward primer CCDC6-197F (5′-TGCAGCAAGAGAACAAGGTG-3′) was added. Multiplex RT-PCR system for simultaneous detection of EML4-ALK, KIF5B-ALK, KIF5B-RET, and CCDC6-RET To capture EML4-ALK, KIF5B-ALK, KIF5B-RET, and CCDC6-RET fusions in a single RT-PCR, we used the following 7 primers simultaneously in 1 assay: EML4-72F (5′-GTCAGCTCTTGAGTCACGAGTT-3′), Fusion-RT-S (5′-GTGCAGTGTTTAGCATTCTTGGGG-3′), ALK-3078RR (5′-ATCCAGTTCGTCCTGTTCAGAGC- 3′), KIF5B-867F (5′-ATTAGGTGGCAACTGTAGAACC-3′), KIF5B-2265F (5′-AGCCACAGATCAGGAAAAGA-3′), CCDC6-197F, and RET-2381R (5′-CAGGCCCCATACAATTTGAT-3′).

Isolation of full-length cDNA From the cDNA of the corresponding tumor tissues, full-length cDNA for novel fusions were amplified by PCR with PrimeSTAR HS DNA polymerase (TakaraBio, Shiga, Japan) using the following primers: KA-w-cDNA-out-S (5′-GCGCGTTCCCTGCAAGACTGAG-3′) and KA-w-cDNA-out-AS (5′-CCACGGTCTTAGGGATCCCAAGG-3′) for KIF5B-ALK; KIF5B-5′UTR2 (5′-CGGAAGTGAGCATTAGGGTTAACGGC-3′) and RET-3′UTR (5′-CATGTTTCTTCCCCTTGTGAGTCCA-3) or RET-3′UTR2 (5′-GGTTCAGAGCAGACTTTGGTTTTGTTCA-3′) for the shorter isoform of RET for KIF5B-RET; SDRO-w-cDNA-in-S (5′-CCTTCTCCAGTCCGCGGTGCC-3′) and ROSfu-w-cDNA-in-A (5′-ACGCTATTAATCAGACCCATCTCC-3′) for SDC4-ROS1; CD74RO-w-cDNA-S (5′-TCCCAGATGCACAGGAGGAGAA-3′) and ROSfu-w-cDNA-out-A (5′-CTCAACTCTCTATTTCCCAAACAAC-3′) for CD74-ROS1 (C6;R34); CD74RO-w-cDNA-S and ROSfu-cDNA-out-A


Takeuchi et. al. 3

(5′-CTCTCTATTTCCCAAACAACGC-3′) for CD74-ROS1 (C6;R32); EZRO-w-cDNA-in-S (5′-CCAGAAACCGAAAATGCCGAAACC-3′) and ROSfu-w-cDNA-in-A for EZR-ROS1; SLC34-cDNA-in-S (5′-TGACCATGGCTCCCTGGCCTGAAT-3′) and ROSfu-w-cDNA-in-A for SLC34A2-ROS1; LRIG3v1-F48 (5′-CGGTCATTCTCCAAGTTATGGTGG-3′) and ROSfu-w-cDNA-in-A for LRIG3 (transcript variant 1)-ROS1; and LRIG3v2-F208 (5′-CTGGGAGCTTCGGGTAGAGACCTA-3′) and ROSfu-w-cDNA-in-A for LRIG3 (transcript variant 2)-ROS1. For TPM3-ROS1, 3 pieces amplified with the following primer sets were ligated: block 1 (5′-GTGTGAATTCGTGAGCCTACCAACAGCCACTGCT-3′) and (5′-CACGGGTCTCTGCCTCCTTGAGTT-3′), block 2 (5′-CCTGAAGTGTCTGAGTGCTGCTG-3′) and (5′-CTTTCTGGAGCCATCCACCGAAC-3′), and block 3 (5′-CCAGTCTCCAGTTCTCCAGTGT-3′) and (5′-CCCAAACAACGCTATTAATCAGAC-3′). Transformation assay for fusion proteins Analysis of the transforming activity of kinase fusions was performed as described previously.1,3,5 A pMXS-based expression plasmid for each fusion was used to generate recombinant ecotropic retroviruses,6 which were then used individually to infect mouse 3T3 fibroblasts. The formation of transformed foci was evaluated after culturing the cells for 14 days. The same set of 3T3 cells was injected subcutaneously into nu/nu mice (96 males), and tumor formation was examined after 21 days. For mouse BA/F3 cell experiments, cDNAs for KIF5B-RET (K15;R12S and K15;R12L) and EML4-ALK (E13;A20) were inserted into the plasmid pMX-iresCD8 to enable simultaneous expression of mouse CD8 and the protein of interest.7 CD8-positive BA/F3 cells infected with recombinant retroviruses generated from each plasmid were purified using a miniMACS cell separation column and magnetic beads attached with antibodies against CD8 (both from Miltenyi Biotec). The purified cells were then incubated with various concentrations of vandetanib. The animal experiments were approved by the animal ethics committee of Jichi Medical University. 1. Takeuchi, K., et al. Multiplex reverse transcription-PCR screening for EML4-ALK

fusion transcripts. Clin Cancer Res 14, 6618-6624 (2008). 2. Takeuchi, K., et al. KIF5B-ALK, a novel fusion oncokinase identified by an

immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 15, 3143-3149 (2009).

3. Choi, Y.L., et al. Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res 68, 4971-4976 (2008).

4. Sakairi, Y., et al. EML4-ALK fusion gene assessment using metastatic lymph node samples obtained by endobronchial ultrasound-guided transbronchial needle aspiration. Clin Cancer Res 16, 4938-4945 (2010).

5. Soda, M., et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 448, 561-566 (2007).

6. Onishi, M., et al. Applications of retrovirus-mediated expression cloning. Exp Hematol 24, 324-329 (1996).

7. Yamashita, Y., et al. Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase. J Biol Chem 276, 39012-39020 (2001).


Takeuchi et al. 1

Supplementary Appendix

Nucleotide sequences of full-length cDNAs for the identified fusion kinases.

Every cDNA was isolated by RT-PCR from the corresponding tumor specimen.

Nucleotides for the 5’- and 3’-genes in the fusions are shown in blue and red letters,

respectively. Further, noncoding and coding regions for each cDNA are depicted by

lower and upper letters, respectively.


Takeuchi et al. 2

KIF5B-ALK K17;A20 cDNA sequence

In this cDNA, exon 17 of KIF5B is ligated in an in-frame manner to exon 20 of ALK.

Position 3307, polymorphism (rs1670283).

gcgcgttccctgcaagactgagcggcggggagtggctcccggccgccggccccggctgc

gagaaagATGGCGGACCTGGCCGAGTGCAACATCAAAGTGATGTGTCGCTTCAGACCTC

TCAACGAGTCTGAAGTGAACCGCGGCGACAAGTACATCGCCAAGTTTCAGGGAGAAGAC

ACGGTCGTGATCGCGTCCAAGCCTTATGCATTTGATCGGGTGTTCCAGTCAAGCACATC

TCAAGAGCAAGTGTATAATGACTGTGCAAAGAAGATTGTTAAAGATGTACTTGAAGGAT

ATAATGGAACAATATTTGCATATGGACAAACATCCTCTGGGAAGACACACACAATGGAG

GGTAAACTTCATGATCCAGAAGGCATGGGAATTATTCCAAGAATAGTGCAAGATATTTT

TAATTATATTTACTCCATGGATGAAAATTTGGAATTTCATATTAAGGTTTCATATTTTG

AAATATATTTGGATAAGATAAGGGACCTGTTAGATGTTTCAAAGACCAACCTTTCAGTT

CATGAAGACAAAAACCGAGTTCCCTATGTAAAGGGGTGCACAGAGCGTTTTGTATGTAG

TCCAGATGAAGTTATGGATACCATAGATGAAGGAAAATCCAACAGACATGTAGCAGTTA

CAAATATGAATGAACATAGCTCTAGGAGTCACAGTATATTTCTTATTAATGTCAAACAA

GAGAACACACAAACGGAACAAAAGCTGAGTGGAAAACTTTATCTGGTTGATTTAGCTGG

TAGTGAAAAGGTTAGTAAAACTGGAGCTGAAGGTGCTGTGCTGGATGAAGCTAAAAACA

TCAACAAGTCACTTTCTGCTCTTGGAAATGTTATTTCTGCTTTGGCTGAGGGTAGTACA

TATGTTCCATATCGAGATAGTAAAATGACAAGAATCCTTCAAGATTCATTAGGTGGCAA

CTGTAGAACCACTATTGTAATTTGCTGCTCTCCATCATCATACAATGAGTCTGAAACAA

AATCTACACTCTTATTTGGCCAAAGGGCCAAAACAATTAAGAACACAGTTTGTGTCAAT

GTGGAGTTAACTGCAGAACAGTGGAAAAAGAAGTATGAAAAAGAAAAAGAAAAAAATAA

GATCCTGCGGAACACTATTCAGTGGCTTGAAAATGAGCTCAACAGATGGCGTAATGGGG

AGACGGTGCCTATTGATGAACAGTTTGACAAAGAGAAAGCCAACTTGGAAGCTTTCACA

GTGGATAAAGATATTACTCTTACCAATGATAAACCAGCAACCGCAATTGGAGTTATAGG

AAATTTTACTGATGCTGAAAGAAGAAAGTGTGAAGAAGAAATTGCTAAATTATACAAAC

AGCTTGATGACAAGGATGAAGAAATTAACCAGCAAAGTCAACTGGTAGAGAAACTGAAG

ACGCAAATGTTGGATCAGGAGGAGCTTTTGGCATCTACCAGAAGGGATCAAGACAATAT

GCAAGCTGAGCTGAATCGCCTTCAAGCAGAAAATGATGCCTCTAAAGAAGAAGTGAAAG

AAGTTTTACAGGCCCTAGAAGAACTTGCTGTCAATTATGATCAGAAGTCTCAGGAAGTT

GAAGACAAAACTAAGGAATATGAATTGCTTAGTGATGAATTGAATCAGAAATCGGCAAC

TTTAGCGAGTATAGATGCTGAGCTTCAGAAACTTAAGGAAATGACCAACCACCAGAAAA

AACGAGCAGCTGAGATGATGGCATCTTTACTAAAAGACCTTGCAGAAATAGGAATTGCT

GTGGGAAATAATGATGTAAAGCAGCCTGAGGGAACTGGCATGATAGATGAAGAGTTCAC

TGTTGCAAGACTCTACATTAGCAAAATGAAGTCAGAAGTAAAAACCATGGTGAAACGTT


Takeuchi et al. 3

GCAAGCAGTTAGAAAGCACACAAACTGAGAGCAACAAAAAAATGGAAGAAAATGAAAAG

GAGTTAGCAGCATGTCAGCTTCGTATCTCTCAACATGAAGCCAAAATCAAGTCATTGAC

TGAATACCTTCAAAATGTGGAACAAAAGAAAAGACAGTTGGAGGAATCTGTCGATGCCC

TCAGTGAAGAACTAGTCCAGCTTCGAGCACAAGTGTACCGCCGGAAGCACCAGGAGCTG

CAAGCCATGCAGATGGAGCTGCAGAGCCCTGAGTACAAGCTGAGCAAGCTCCGCACCTC

GACCATCATGACCGACTACAACCCCAACTACTGCTTTGCTGGCAAGACCTCCTCCATCA

GTGACCTGAAGGAGGTGCCGCGGAAAAACATCACCCTCATTCGGGGTCTGGGCCATGGC

GCCTTTGGGGAGGTGTATGAAGGCCAGGTGTCCGGAATGCCCAACGACCCAAGCCCCCT

GCAAGTGGCTGTGAAGACGCTGCCTGAAGTGTGCTCTGAACAGGACGAACTGGATTTCC

TCATGGAAGCCCTGATCATCAGCAAATTCAACCACCAGAACATTGTTCGCTGCATTGGG

GTGAGCCTGCAATCCCTGCCCCGGTTCATCCTGCTGGAGCTCATGGCGGGGGGAGACCT

CAAGTCCTTCCTCCGAGAGACCCGCCCTCGCCCGAGCCAGCCCTCCTCCCTGGCCATGC

TGGACCTTCTGCACGTGGCTCGGGACATTGCCTGTGGCTGTCAGTATTTGGAGGAAAAC

CACTTCATCCACCGAGACATTGCTGCCAGAAACTGCCTCTTGACCTGTCCAGGCCCTGG

AAGAGTGGCCAAGATTGGAGACTTCGGGATGGCCCGAGACATCTACAGGGCGAGCTACT

ATAGAAAGGGAGGCTGTGCCATGCTGCCAGTTAAGTGGATGCCCCCAGAGGCCTTCATG

GAAGGAATATTCACTTCTAAAACAGACACATGGTCCTTTGGAGTGCTGCTATGGGAAAT

CTTTTCTCTTGGATATATGCCATACCCCAGCAAAAGCAACCAGGAAGTTCTGGAGTTTG

TCACCAGTGGAGGCCGGATGGACCCACCCAAGAACTGCCCTGGGCCTGTATACCGGATA

ATGACTCAGTGCTGGCAACATCAGCCTGAAGACAGGCCCAACTTTGCCATCATTTTGGA

GAGGATTGAATACTGCACCCAGGACCCGGATGTAATCAACACCGCTTTGCCGATAGAAT

ATGGTCCACTTGTGGAAGAGGAAGAGAAAGTGCCTGTGAGGCCCAAGGACCCTGAGGGG

GTTCCTCCTCTCCTGGTCTCTCAACAGGCAAAACGGGAGGAGGAGCGCAGCCCAGCTGC

CCCACCACCTCTGCCTACCACCTCCTCTGGCAAGGCTGCAAAGAAACCCACAGCTGCAG

AGGTCTCTGTTCGAGTCCCTAGAGGGCCGGCCGTGGAAGGGGGACACGTGAATATGGCA

TTCTCTCAGTCCAACCCTCCTTCGGAGTTGCACAAGGTCCACGGATCCAGAAACAAGCC

CACCAGCTTGTGGAACCCAACGTACGGCTCCTGGTTTACAGAGAAACCCACCAAAAAGA

ATAATCCTATAGCAAAGAAGGAGCCACACGACAGGGGTAACCTGGGGCTGGAGGGAAGC

TGTACTGTCCCACCTAACGTTGCAACTGGGAGACTTCCGGGGGCCTCACTGCTCCTAGA

GCCCTCTTCGCTGACTGCCAATATGAAGGAGGTACCTCTGTTCAGGCTACGTCACTTCC

CTTGTGGGAATGTCAATTACGGCTACCAGCAACAGGGCTTGCCCTTAGAAGCCGCTACT

GCCCCTGGAGCTGGTCATTACGAGGATACCATTCTGAAAAGCAAGAATAGCATGAACCA

GCCTGGGCCCTGAgctcggtcgcacactcacttctcttccttgggatccctaagaccgt

gg


Takeuchi et al. 4

TPM3-ROS1 cDNA sequence

The TPM3 region of this fusion cDNA is composed of exons 1-5 of TPM3 variant 1

(NM_152263) followed by exon 5 of variants 2/3 (NM_153649/NM_001043352) and

exons 7-8 of variant 1, which is then ligated to exon 35 of ROS1.

gtgagcctaccaacagccactgctcATGATGGAGGCCATCAAGAAAAAGATGCAGATGC

TGAAGTTAGACAAGGAGAATGCTCTGGATCGGGCAGAGCAAGCTGAAGCTGAGCAGAAG

CAGGCAGAAGAAAGAAGTAAACAGCTGGAGGATGAGCTGGCAGCCATGCAGAAGAAGCT

GAAAGGGACAGAGGATGAGCTGGACAAGTATTCTGAAGCTTTGAAGGATGCCCAGGAGA

AGCTGGAACTGGCAGAGAAGAAGGCTGCTGATGCTGAGGCTGAGGTGGCCTCCTTGAAC

CGTAGGATCCAGCTGGTTGAAGAAGAGCTGGACCGTGCTCAGGAGCGCCTGGCCACTGC

CCTGCAAAAGCTGGAAGAAGCTGAAAAAGCTGCTGATGAGAGTGAGAGAGGTATGAAGG

TTATTGAAAACCGGGCCTTAAAAGATGAAGAAAAGATGGAACTCCAGGAAATCCAACTC

AAAGAAGCTAAGCACATTGCAGAAGAGGCAGATAGGAAGTATGAAGAGGTGGCTCGTAA

GTTGGTGATCATTGAAGGAGACTTGGAACGCACAGAGGAACGAGCTGAGCTGGCAGAGT

CCCGTTGCCGAGAGATGGATGAGCAGATTAGACTGATGGACCAGAACCTGAAGTGTCTG

AGTGCTGCTGAAGAAAAGTACTCTCAAAAAGAAGATAAATATGAGGAAGAAATCAAGAT

TCTTACTGATAAACTCAAGGAGGCAGAGACCCGTGCTGAGTTTGCTGAGAGATCGGTAG

CCAAGCTGGAAAAGACAATTGATGACCTGGAAGTCTGGCATAGAAGATTAAAGAATCAA

AAAAGTGCCAAGGAAGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCT

GCGAGGTCTGGCAGCCGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTC

CAACCCAAGAGGAGATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGT

CTCTTGCTGGGAAGTGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTT

AGGAGTTGGAAGTGGAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAG

ACCAGGAGAAGATTGAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCC

AACATTCTGAAGCAGCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGA

ACTGATGGAGGGAGGAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTT

ATGGTCCTTTACTCACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGC

TGTGTCTACTTGGAACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCT

TGTTTCCGTGAAAGACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCG

CCAGAGACATCTATAAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTT

CGGTGGATGGCTCCAGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATG

GTCTTTTGGAATTCTGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTC

ATTCCAACCTTGATGTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGA

AATTGTCCTGATGATCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCA

AAGACCTACTTTTCATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAA


Takeuchi et al. 5

ATAGCATTTATAAGTCCAGAGATGAAGCAAACAACAGTGGAGTCATAAATGAAAGCTTT

GAAGGTGAAGATGGCGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTT

AATGGAAACGAAGAACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCC

AAGGTGAAGAAAAGTCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTG

AGGAAAGAAGAGAAGGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGC

TTACTGCCCTTCTGGCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGAT

ATGGAGATGGGTCTGATTAAtagcgttgtttggg


Takeuchi et al. 6

SDC4-ROS1 S2;R32 cDNA sequence

In this cDNA, exon 2 of SDC4 is ligated in an in-frame manner to exon 32 of ROS1.


ccttctccagtccgcggtgccATGGCCCCCGCCCGTCTGTTCGCGCTGCTGCTGCTCTT

CGTAGGCGGAGTCGCCGAGTCGATCCGAGAGACTGAGGTCATCGACCCCCAGGACCTCC

TAGAAGGCCGATACTTCTCCGGAGCCCTACCAGACGATGAGGATGTAGTGGGGCCCGGG

CAGGAATCTGATGACTTTGAGCTGTCTGGCTCTGGAGATCTGGCTGGAGTCCCAAATAA

ACCAGGCATTCCCAAATTACTAGAAGGGAGTAAAAATTCAATACAGTGGGAGAAAGCTG

AAGATAATGGATGTAGAATTACATACTATATCCTTGAGATAAGAAAGAGCACTTCAAAT

AATTTACAGAACCAGAATTTAAGGTGGAAGATGACATTTAATGGATCCTGCAGTAGTGT

TTGCACATGGAAGTCCAAAAACCTGAAAGGAATATTTCAGTTCAGAGTAGTAGCTGCAA

ATAATCTAGGGTTTGGTGAATATAGTGGAATCAGTGAGAATATTATATTAGTTGGAGAT

GATTTTTGGATACCAGAAACAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGT

TGTTACAATCCCACTGACCTTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCA

AGGAAGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTG

GCAGCCGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGA

GGAGATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGG

GAAGTGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGA

AGTGGAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAA

GATTGAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGA

AGCAGCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAG

GGAGGAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTT

ACTCACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACT

TGGAACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTG

AAAGACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACAT

CTATAAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGG

CTCCAGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGA

ATTCTGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCT

TGATGTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTG

ATGATCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACT

TTTCATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTA

TAAGTCCAGAGATGAAGCAAACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAG

ATGGCGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACG

AAGAACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGA

AAAGTCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAG


Takeuchi et al. 7

AGAAGGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCT

TCTGGCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGG

GTCTGATTAAtagcgt


Takeuchi et al. 8




ccttctccagtccgcggtgccATGGCCCCCGCCCGTCTGTTCGCGCTGCTGCTGTTCTT



CAGGAATCTGATGACTTTGAGCTGTCTGGCTCTGGAGATCTGGATGACTTGGAAGACTC

CATGATCGGCCCTGAAGTTGTCCATCCCTTGGTGCCTCTAGATAACCATATCCCTGAGA

GGGCAGGGTCTGGGAGCCAAGTCCCCACCGAACCCAAGAAACTAGAGGAGAATGAGGTT

ATCCCCAAGAGAATCTCACCCGTTGAAGAGAGTGAGGATGTGTCCAACAAGGTGTCAAT

GTCCAGCACTGTGCAGGGCAGCAACATCTTTGAGAGAACGGAGGTCCTGGCAGCTGGAG

TCCCAAATAAACCAGGCATTCCCAAATTACTAGAAGGGAGTAAAAATTCAATACAGTGG

GAGAAAGCTGAAGATAATGGATGTAGAATTACATACTATATCCTTGAGATAAGAAAGAG

CACTTCAAATAATTTACAGAACCAGAATTTAAGGTGGAAGATGACATTTAATGGATCCT

GCAGTAGTGTTTGCACATGGAAGTCCAAAAACCTGAAAGGAATATTTCAGTTCAGAGTA

GTAGCTGCAAATAATCTAGGGTTTGGTGAATATAGTGGAATCAGTGAGAATATTATATT

AGTTGGAGATGATTTTTGGATACCAGAAACAAGTTTCATACTTACTATTATAGTTGGAA

TATTTCTGGTTGTTACAATCCCACTGACCTTTGTCTGGCATAGAAGATTAAAGAATCAA

AAAAGTGCCAAGGAAGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCT

GCGAGGTCTGGCAGCCGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTC

CAACCCAAGAGGAGATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGT

CTCTTGCTGGGAAGTGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTT

AGGAGTTGGAAGTGGAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAG

ACCAGGAGAAGATTGAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCC

AACATTCTGAAGCAGCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGA

ACTGATGGAGGGAGGAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTT

ATGGTCCTTTACTCACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGC

TGTGTCTACTTGGAACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCT

TGTTTCCGTGAAAGACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCG

CCAGAGACATCTATAAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTT

CGGTGGATGGCTCCAGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATG

GTCTTTTGGAATTCTGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTC

ATTCCAACCTTGATGTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGA

AATTGTCCTGATGATCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCA

AAGACCTACTTTTCATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAA


Takeuchi et al. 9

ATAGCATTTATAAGTCCAGAGATGAAGCAAACAACAGTGGAGTCATAAAGGAAAGCTTT

GAAGGTGAAGATGGCGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTT

AATGGAAACGAAGAACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCC

AAGGTGAAGAAAAGTCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTG

AGGAAAGAAGAGAAGGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGC

TTACTGCCCTTCTGGCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGAT

ATGGAGATGGGTCTGATTAAtagcgt


Takeuchi et al. 10




ccttctccagtccgcggtgccATGGCCCCCGCCCGTCTGTTCGCGCTGCTGCTGTTCTT



CAGGAATCTGATGACTTTGAGCTGTCTGGCTCTGGAGATCTGGATGACTTGGAAGACTC

CATGATCGGCCCTGAAGTTGTCCATCCCTTGGTGCCTCTAGATAACCATATCCCTGAGA

GGGCAGGGTCTGGGAGCCAAGTCCCCACCGAACCCAAGAAACTAGAGGAGAATGAGGTT

ATCCCCAAGAGAATCTCACCCGTTGAAGAGAGTGAGGATGTGTCCAACAAGGTGTCAAT

GTCCAGCACTGTGCAGGGCAGCAACATCTTTGAGAGAACGGAGGTCCTGGCAGATGATT

TTTGGATACCAGAAACAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGTTGTT

ACAATCCCACTGACCTTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAGGA

AGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCAG

CCGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGGAG

ATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAAG

TGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAGTG

GAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGATT

GAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAGCA

GCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGGAG

GAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTACTC

ACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTGGA

ACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAAG

ACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCTAT

AAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTCC

AGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAATTC

TGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTGAT

GTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGATGA

TCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTTTC

ATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATAAG

TCCAGAGATGAAGCAAACAACAGTGGAGTCATAAAGGAAAGCTTTGAAGGTGAAGATGG

CGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAAGA

ACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAAAG

TCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGAA

GGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCTG


Takeuchi et al. 11

GCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGTCT

GATTAAtagcgt


Takeuchi et al. 12

SLC34A2-ROS1 cDNA sequence

In this cDNA, nucleotide position 568 of exon 13 of SLC34A2 is ligated in an in-frame

manner to exon 32 of ROS1.

gaccATGGCTCCCTGGCCTGAATTGGGAGATGCCCAGCCCAACCCCGATAAGTACCTCG

AAGGGGCCGCAGGTCAGCAGCCCACTGCCCCTGATAAAAGCAAAGAGACCAACAAAACA

GATAACACTGAGGCACCTGTAACCAAGATTGAACTTCTGCCGTCCTACTCCACGGCTAC

ACTGATAGATGAGCCCACTGAGGTGGATGACCCCTGGAACCTACCCACTCTTCAGGACT

CGGGGATCAAGTGGTCAGAGAGAGACACCAAAGGGAAGATTCTCTGTTTCTTCCAAGGG

ATTGGGAGATTGATTTTACTTCTCGGATTTCTCTACTTTTTCGTGTGCTCCCTGGATAT

TCTTAGTAGCGCCTTCCAGCTGGTTGGAGGAAAAATGGCAGGACAGTTCTTCAGCAACA

GCTCTATTATGTCCAACCCTTTGTTGGGGCTGGTGATCGGGGTGCTGGTGACCGTCTTG

GTGCAGAGCTCCAGCACCTCAACGTCCATCGTTGTCAGCATGGTGTCCTCTTCATTGCT

CACTGTTCGGGCTGCCATCCCCATTATCATGGGGGCCAACATTGGAACGTCAATCACCA

ACACTATTGTTGCGCTCATGCAGGTGGGAGATCGGAGTGAGTTCAGAAGAGCTTTTGCA

GGAGCCACTGTCCATGACTTCTTCAACTGGCTGTCCGTGTTGGTGCTCTTGCCCGTGGA

GGTGGCCACCCATTACCTCGAGATCATAACCCAGCTTATAGTGGAGAGCTTCCACTTCA

AGAATGGAGAAGATGCCCCAGATCTTCTGAAAGTCATCACTAAGCCCTTCACAAAGCTC

ATTGTCCAGCTGGATAAAAAAGTTATCAGCCAAATTGCAATGAACGATGAAAAAGCGAA

AAACAAGAGTCTTGTCAAGATTTGGTGCAAAACTTTTACCAACAAGACCCAGATTAACG

TCACTGTTCCCTCGACTGCTAACTGCACCTCCCCTTCCCTCTGTTGGACGGATGGCATC

CAAAACTGGACCATGAAGAATGTGACCTACAAGGAGAACATCGCCAAATGCCAGCATAT

CTTTGTGAATTTCCACCTCCCGGATCTTGCTGTGGGCACCATCTTGCTCATACTCTCCC

TGCTGGTCCTCTGTGGTTGCCTGATCATGATTGTCAAGATCCTGGGCTCTGTGCTCAAG

GGGCAGGTCGCCACTGTCATCAAGAAGACCATCAACACTGATTTCCCCTTTCCCTTTGC

ATGGTTGACTGGCTACCTGGCCATCCTCGTCGGGGCAGGCATGACCTTCATCGTACAGA

GCAGCTCTGTGTTCACGTCGGCCTTGACCCCCCTGATTGGAATCGGCGTGATAACCATT

GAGAGGGCTTATCCACTCACGCTGGGCTCCAACATCGGCACCACCACCACCGCCATCCT

GGCCGCCTTAGCCAGCCCTGGCAATGCATTGAGGAGTTCACTCCAGATCGCCCTGTGCC

ACTTTTTCTTCAACATCTCCGGCATCTTGCTGTGGTACCCGATCCCGTTCACTCGCCTG

CCCATCCGCATGGCCAAGGGGCTGGGCAACATCTCTGCCAAGTATCGCTGGTTCGCCGT

CTTCTACCTGATCATCTTCTTCTTCCTGATCCCGCTGACGGTGTTTGGCCTCTCGCTGG

CCGGCTGGCGGGTGCTGGTTGGTGTCGGGGTTCCCGTCGTCTTCATCATCATCCTGGTA

CTGTGCCTCCGACTCCTGCAGTCTCGCTGCCCACGCGTCCTGCCGAAGAAACTCCAGAA

CTGGAACTTCCTGCCGCTGTGGATGCGCTCGCTGAAGCCCTGGGATGCCGTCGTCTCCA

AGTTCACCGGCTGCTTCCAGATGCGCTGCTGCTGCTGCTGCCGCGTGTGCTGCCGCGCG


Takeuchi et al. 13

TGCTGCTTGCTGTGTGGCTGCCCCAAGTGCTGCCGCTGCAGCAAGTGCTGCGAGGACTT

GGAGGAGGCGCAGGAGGGGCAGGATGTCCCTGTCAAGGCTCCTGAGACCTTTGATAACA

TAACCATTAGCAGAGAGGCTCAGGCTGGAGTCCCAAATAAACCAGGCATTCCCAAATTA

CTAGAAGGGAGTAAAAATTCAATACAGTGGGAGAAAGCTGAAGATAATGGATGTAGAAT

TACATACTATATCCTTGAGATAAGAAAGAGCACTTCAAATAATTTACAGAACCAGAATT

TAAGGTGGAAGATGACATTTAATGGATCCTGCAGTAGTGTTTGCACATGGAAGTCCAAA

AACCTGAAAGGAATATTTCAGTTCAGAGTAGTAGCTGCAAATAATCTAGGGTTTGGTGA

ATATAGTGGAATCAGTGAGAATATTATATTAGTTGGAGATGATTTTTGGATACCAGAAA

CAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGTTGTTACAATCCCACTGACC

TTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAGGAAGGGGTGACAGTGCT

TATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCAGCCGGAGTAGGCCTGG

CTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGGAGATTGAAAATCTTCCT

GCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAAGTGGAGCCTTTGGAGA

AGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAGTGGAGAAATCAAAGTAG

CAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGATTGAATTCCTGAAGGAG

GCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAGCAGCTTGGAGTTTGTCT

GCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGGAGGAGACCTTCTTACTT

ATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTACTCACCTTGGTTGACCTT

GTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTGGAACGGATGCATTTCAT

TCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAAGACTATACCAGTCCAC

GGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCTATAAAAATGATTACTAT

AGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTCCAGAAAGTTTGATGGA

TGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAATTCTGATTTGGGAGATTT

TAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTGATGTGTTAAACTATGTG

CAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGATGATCTGTGGAATTTAAT

GACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTTTCATAGAATTCAGGACC

AACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATAAGTCCAGAGATGAAGCA

AACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAGATGGCGATGTGATTTGTTT

GAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAAGAACCGAGAAGGGTTAA

ACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAAAGTCTGAGGGTCCTCTA

GGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGAAGGAACCACATGCAGA

CAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCTGGCAAGCCTGAAGGCC

TGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGTCTGATTAAtagcgt


Takeuchi et al. 14

CD74-ROS1 C6;R32 cDNA sequence

In this cDNA, exon 6 of CD74 is ligated in an in-frame manner to exon 32 of ROS1.


tcccagATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATGGA

TGACCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTG

GGGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTG

ACTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGGCCG

GCTGGACAAACTGACAGTCACCTCCCAGAACCTGCAGCTGGAGAACCTGCGCATGAAGC

TTCCCAAGCCTCCCAAGCCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATGCAG

GCGCTGCCCATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAA

CATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACC

CGCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACC

ATAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAG

GCACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAGCTGGAGTCCCAAATAAAC

CAGGCATTCCCAAATTACTAGAAGGGAGTAAAAATTCAATACAGTGGGAGAAAGCTGAA

GATAATGGATGTAGAATTACATACTATATCCTTGAGATAAGAAAGAGCACTTCAAATAA

TTTACAGAACCAGAATTTAAGGTGGAAGATGACATTTAATGGATCCTGCAGTAGTGTTT

GCACATGGAAGTCCAAAAACCTGAAAGGAATATTTCAGTTCAGAGTAGTAGCTGCAAAT

AATCTAGGGTTTGGTGAATATAGTGGAATCAGTGAGAATATTATATTAGTTGGAGATGA

TTTTTGGATACCAGAAACAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGTTG

TTACAATCCCACTGACCTTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAG

GAAGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGC

AGCTGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGG

AGATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGA

AGTGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAG

TGGAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGA

TTGAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAG

CAGCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGG

AGGAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTAC

TCACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTG

GAACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAA

AGACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCT

ATAAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCT

CCAGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAAT

TCTGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTG


Takeuchi et al. 15

ATGTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGAT

GATCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTT

TCATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATA

AGTCCAGAGATGAAGCAAACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAGAT

GGCGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAA

GAACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAA

AGTCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAG

AAGGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTC

TGGCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGT

CTGATTAAtagcgttgtttgggaaatagagag


Takeuchi et al. 16

CD74-ROS1 C6;R34 cDNA sequence

In this cDNA, exon 6 of CD74 is ligated in an in-frame manner to exon 34 of ROS1.

cccagATGCACAGGAGGAGAAGCAGGAGCTGTCGGGAAGATCAGAAGCCAGTCATGGAT

GACCAGCGCGACCTTATCTCCAACAATGAGCAACTGCCCATGCTGGGCCGGCGCCCTGG

GGCCCCGGAGAGCAAGTGCAGCCGCGGAGCCCTGTACACAGGCTTTTCCATCCTGGTGA

CTCTGCTCCTCGCTGGCCAGGCCACCACCGCCTACTTCCTGTACCAGCAGCAGGGCCGG

CTGGACAAACTGACAGTCACCTCCCAGAACCTGCAGCTGGAGAACCTGCGCATGAAGCT

TCCCAAGCCTCCCAAGCCTGTGAGCAAGATGCGCATGGCCACCCCGCTGCTGATGCAGG

CGCTGCCCATGGGAGCCCTGCCCCAGGGGCCCATGCAGAATGCCACCAAGTATGGCAAC

ATGACAGAGGACCATGTGATGCACCTGCTCCAGAATGCTGACCCCCTGAAGGTGTACCC

GCCACTGAAGGGGAGCTTCCCGGAGAACCTGAGACACCTTAAGAACACCATGGAGACCA

TAGACTGGAAGGTCTTTGAGAGCTGGATGCACCATTGGCTCCTGTTTGAAATGAGCAGG

CACTCCTTGGAGCAAAAGCCCACTGACGCTCCACCGAAAGATGATTTTTGGATACCAGA

AACAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGTTGTTACAATCCCACTGA

CCTTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAGGAAGGGGTGACAGTG

CTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCAGCCGGAGTAGGCCT

GGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGGAGATTGAAAATCTTC

CTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAAGTGGAGCCTTTGGA

GAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAGTGGAGAAATCAAAGT

AGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGATTGAATTCCTGAAGG

AGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAGCAGCTTGGAGTTTGT

CTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGGAGGAGACCTTCTTAC

TTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTACTCACCTTGGTTGACC

TTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTGGAACGGATGCATTTC

ATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAAGACTATACCAGTCC

ACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCTATAAAAATGATTACT

ATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTCCAGAAAGTTTGATG

GATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAATTCTGATTTGGGAGAT

TTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTGATGTGTTAAACTATG

TGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGATGATCTGTGGAATTTA

ATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTTTCATAGAATTCAGGA

CCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATAAGTCCAGAGATGAAG

CAAACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAGATGGCGATGTGATTTGT

TTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAAGAACCGAGAAGGGTT

AAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAAAGTCTGAGGGTCCTC


Takeuchi et al. 17

TAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGAAGGAACCACATGCA

GACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCTGGCAAGCCTGAAGG

CCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGTCTGATTAAtagcgtt

gtttgggaaatagagagttgag


Takeuchi et al. 18

EZR-ROS1 E10;R34 cDNA sequence

In this cDNA, exon 10 of EZR is ligated in an in-frame manner to exon 34 of ROS1.


cagaaaccgaaaATGCCGAAACCAATCAATGTCCGAGTTACCACCATGGATGCAGAGCT

GGAGTTTGCAATCCAGCCAAATACAACTGGAAAACAGCTTTTTGATCAGGTGGTAAAGA

CTATCGGCCTCCGGGAAGTGTGGTACTTTGGCCTCCACTATGTGGATAATAAAGGATTT

CCTACCTGGCTGAAGCTGGATAAGAAGGTGTCTGCCCAGGAGGTCAGGAAGGAGAATCC

CCTCCAGTTCAAGTTCCGGGCCAAGTTCTACCCTGAAGATGTGGCTGAGGAGCTCATCC

AGGACATCACCCAGAAACTTTTCTTCCTCCAAGTGAAGGAAGGAATCCTTAGCGATGAG

ATCTACTGCCCCCCTGAGACTGCCGTGCTCTTGGGGTCCTACGCTGTGCAGGCCAAGTT

TGGGGACTACAACAAAGAAGTGCACAAGTCTGGGTACCTCAGCTCTGAGCGGCTGATCC

CTCAAAGAGTGATGGACCAGCACAAACTTACCAGGGACCAGTGGGAGGACCGGATCCAG

GTGTGGCATGCGGAACACCGTGGGATGCTCAAAGATAATGCTATGTTGGAATACCTGAA

GATTGCTCAGGACCTGGAAATGTATGGAATCAACTATTTCGAGATAAAAAACAAGAAAG

GAACAGACCTTTGGCTTGGAGTTGATGCCCTTGGACTGAATATTTATGAGAAAGATGAT

AAGTTAACCCCAAAGATTGGCTTTCCTTGGAGTGAAATCAGGAACATCTCTTTCAATGA

CAAAAAGTTTGTCATTAAACCCATCGACAAGAAGGCACCTGACTTTGTGTTTTATGCCC

CACGTCTGAGAATCAACAAGCGGATCCTGCAGCTCTGCATGGGCAACCATGAGTTGTAT

ATGCGCCGCAGGAAGCCTGACACCATCGAGGTGCAGCAGATGAAGGCCCAGGCCCGGGA

GGAGAAGCATCAGAAGCAGCTGGAGCGGCAACAGCTGGAAACAGAGAAGAAAAGGAGAG

AAACCGTGGAGAGAGAGAAAGAGCAGATGATGCGCGAGAAGGAGGAGTTGATGCTGCGG

CTGCAGGACTATGAGGAGAAGACAAAGAAGGCAGAGAGAGATGATTTTTGGATACCAGA

AACAAGTTTCATACTTACTATTATAGTTGGAATATTTCTGGTTGTTACAATCCCACTGA

CCTTTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAGGAAGGGGTGACAGTG

CTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCAGCTGGAGTAGGCCT

GGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGGAGATTGAAAATCTTC

CTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAAGTGGAGCCTTTGGA

GAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAGTGGAGAAATCAAAGT

AGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGATTGAATTCCTGAAGG

AGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAGCAGCTTGGAGTTTGT

CTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGGAGGAGACCTTCTTAC

TTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTACTCACCTTGGTTGACC

TTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTGGAACGGATGCATTTC

ATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAAGACTATACCAGTCC

ACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCTATAAAAATGATTACT


Takeuchi et al. 19

ATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTCCAGAAAGTTTGATG

GATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAATTCTGATTTGGGAGAT

TTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTGATGTGTTAAACTATG

TGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGATGATCTGTGGAATTTA

ATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTTTCATAGAATTCAGGA

CCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATAAGTCCAGAGATGAAG

CAAACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAGATGGCGATGTGATTTGT

TTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAAGAACCGAGAAGGGTT

AAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAAAGTCTGAGGGTCCTC

TAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGAAGGAACCACATGCA

GACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCTGGCAAGCCTGAAGG

CCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGTCTGATTAAtagcgt


Takeuchi et al. 20

LRIG3 (variant 1)-ROS1 cDNA sequence

In this cDNA, exon 16 of LRIG3 variant 1 (NM_001136051) is ligated in an in-frame

manner to exon 35 of ROS1. Position 2633, polymorphism (rs55687640).

cggtcattctccaagttATGGTGGACGTACTTCTGTTGTTCTCCCTCTGCTTGCTTTTT

CACATTAGCAGACCGGACTTAAGTCACAACAGATTATCTTTCATCAAGGCAAGTTCCAT

GAGCCACCTTCAAAGCCTTCGAGAAGTGAAACTGAACAACAATGAATTGGAGACCATTC

CAAATCTGGGACCAGTCTCGGCAAATATTACACTTCTCTCCTTGGCTGGAAACAGGATT

GTTGAAATACTCCCTGAACATCTGAAAGAGTTTCAGTCCCTTGAAACTTTGGACCTTAG

CAGCAACAATATTTCAGAGCTCCAAACTGCATTTCCAGCCCTACAGCTCAAATATCTGT

ATCTCAACAGCAACCGAGTCACATCAATGGAACCTGGGTATTTTGACAATTTGGCCAAC

ACACTCCTTGTGTTAAAGCTGAACAGGAACCGAATCTCAGCTATCCCACCCAAGATGTT

TAAACTGCCCCAACTGCAACATCTCGAATTGAACCGAAACAAGATTAAAAATGTAGATG

GACTGACATTCCAAGGCCTTGGTGCTCTGAAGTCTCTGAAAATGCAAAGAAATGGAGTA

ACGAAACTTATGGATGGAGCTTTTTGGGGGCTGAGCAACATGGAAATTTTGCAGCTGGA

CCATAACAACCTAACAGAGATTACCAAAGGCTGGCTTTACGGCTTGCTGATGCTGCAGG

AACTTCATCTCAGCCAAAATGCCATCAACAGGATCAGCCCTGATGCCTGGGAGTTCTGC

CAGAAGCTCAGTGAGCTGGACCTAACTTTCAATCACTTATCAAGGTTAGATGATTCAAG

CTTCCTTGGCCTAAGCTTACTAAATACACTGCACATTGGGAACAACAGAGTCAGCTACA

TTGCTGATTGTGCCTTCCGGGGGCTTTCCAGTTTAAAGACTTTGGATCTGAAGAACAAT

GAAATTTCCTGGACTATTGAAGACATGAATGGTGCTTTCTCTGGGCTTGACAAACTGAG

GCGACTGATACTCCAAGGAAATCGGATCCGTTCTATTACTAAAAAAGCCTTCACTGGTT

TGGATGCATTGGAGCATCTAGACCTGAGTGACAACGCAATCATGTCTTTACAAGGCAAT

GCATTTTCACAAATGAAGAAACTGCAACAATTGCATTTAAATACATCAAGCCTTTTGTG

CGATTGCCAGCTAAAATGGCTCCCACAGTGGGTGGCGGAAAACAACTTTCAGAGCTTTG

TAAATGCCAGTTGTGCCCATCCTCAGCTGCTAAAAGGAAGAAGCATTTTTGCTGTTAGC

CCAGATGGCTTTGTGTGTGATGATTTTCCCAAACCCCAGATCACGGTTCAGCCAGAAAC

ACAGTCGGCAATAAAAGGTTCCAATTTGAGTTTCATCTGCTCAGCTGCCAGCAGCAGTG

ATTCCCCAATGACTTTTGCTTGGAAAAAAGACAATGAACTACTGCATGATGCTGAAATG

GAAAATTATGCACACCTCCGGGCCCAAGGTGGCGAGGTGATGGAGTATACCACCATCCT

TCGGCTGCGCGAGGTGGAATTTGCCAGTGAGGGGAAATATCAGTGTGTCATCTCCAATC

ACTTTGGTTCATCCTACTCTGTCAAAGCCAAGCTTACAGTAAATATGCTTCCCTCATTC

ACCAAGACCCCCATGGATCTCACCATCCGAGCTGGGGCCATGGCACGCTTGGAGTGTGC

TGCTGTGGGGCACCCAGCCCCCCAGATAGCCTGGCAGAAGGATGGGGGCACAGACTTCC

CAGCTGCACGGGAGAGACGCATGCATGTGATGCCCGAGGATGACGTGTTCTTTATCGTG

GATGTGAAGATAGAGGACATTGGGGTATACAGCTGCACAGCTCAGAACAGTGCAGGAAG


Takeuchi et al. 21

TATTTCAGCAAATGCAACTCTGACTGTCCTAGAAACACCATCATTTTTGCGGCCACTGT

TGGACCGAACTGTAACCAAGGGAGAAACAGCCGTCCTACAGTGCATTGCTGGAGGAAGC

CCTCCCCCTAAACTGAACTGGACCAAAGATGATAGCCCATTGGTGGTAACCGAGAGGCA

CTTTTTTGCAGCAGGCAATCAGCTTCTGATTATTGTGGACTCAGATGTCAGTGATGCTG

GGAAATACACATGTGAGATGTCTAACACCCTTGGCACTGAGAGAGGAAACGTGCGCCTC

AGTGTGATCCCCACTCCAACCTGCGACTCCCCTCAGATGACAGCCCCATCGTTAGACGA

TGACGGATGGGCCACTGTGGGTGTCGTGATCATAGCCGTGGTTTGCTGTGTGGTGGGCA

CGTCACTCGTGTGGGTGGTCATCATATACCACACAAGGCGGAGGAATGAAGATTGCAGC

ATTACCAACACAGATGAGACCAACTTGCCAGCAGATATTCCTAGTTATTTGTCATCTCA

GGGAACGTTAGCTGACAGGCAGGATGGGTACGTGTCTTCAGAAAGTGGAAGCCACCACC

AGTTTGTCACATCTTCAGGTGCTGGATTTTTCTTACCACAACATGACAGTAGTGTCTGG

CATAGAAGATTAAAGAATCAAAAAAGTGCCAAGGAAGGGGTGACAGTGCTTATAAACGA

AGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCAGCTGGAGTAGGCCTGGCTAATGCCT

GCTATGCAATACATACTCTTCCAACCCAAGAGGAGATTGAAAATCTTCCTGCCTTCCCT

CGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAAGTGGAGCCTTTGGAGAAGTGTATGA

AGGAACAGCAGTGGACATCTTAGGAGTTGGAAGTGGAGAAATCAAAGTAGCAGTGAAGA

CTTTGAAGAAGGGTTCCACAGACCAGGAGAAGATTGAATTCCTGAAGGAGGCACATCTG

ATGAGCAAATTTAATCATCCCAACATTCTGAAGCAGCTTGGAGTTTGTCTGCTGAATGA

ACCCCAATACATTATCCTGGAACTGATGGAGGGAGGAGACCTTCTTACTTATTTGCGTA

AAGCCCGGATGGCAACGTTTTATGGTCCTTTACTCACCTTGGTTGACCTTGTAGACCTG

TGTGTAGATATTTCAAAAGGCTGTGTCTACTTGGAACGGATGCATTTCATTCACAGGGA

TCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAAGACTATACCAGTCCACGGATAGTGA

AGATTGGAGACTTTGGACTCGCCAGAGACATCTATAAAAATGATTACTATAGAAAGAGA

GGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTCCAGAAAGTTTGATGGATGGAATCTT

CACTACTCAATCTGATGTATGGTCTTTTGGAATTCTGATTTGGGAGATTTTAACTCTTG

GTCATCAGCCTTATCCAGCTCATTCCAACCTTGATGTGTTAAACTATGTGCAAACAGGA

GGGAGACTGGAGCCACCAAGAAATTGTCCTGATGATCTGTGGAATTTAATGACCCAGTG

CTGGGCTCAAGAACCCGACCAAAGACCTACTTTTCATAGAATTCAGGACCAACTTCAGT

TATTCAGAAATTTTTTCTTAAATAGCATTTATAAGTCCAGAGATGAAGCAAACAACAGT

GGAGTCATAAATGAAAGCTTTGAAGGTGAAGATGGCGATGTGATTTGTTTGAATTCAGA

TGACATTATGCCAGTTGCTTTAATGGAAACGAAGAACCGAGAAGGGTTAAACTATATGG

TACTTGCTACAGAATGTGGCCAAGGTGAAGAAAAGTCTGAGGGTCCTCTAGGCTCCCAG

GAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGAAGGAACCACATGCAGACAAAGATTT

CTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCTGGCAAGCCTGAAGGCCTGAACTATG

CCTGTCTCACTCACAGTGGATATGGAGATGGGTCTGATTAAtagcgt


Takeuchi et al. 22

LRIG3 (variant 2)-ROS1 cDNA sequence

In this cDNA, exon 16 of LRIG3 variant 2 (NM_153377) is ligated in an in-frame

manner to exon 35 of ROS1. Position 120, polymorphism (rs61754220). Position 2835,

polymorphism (rs55687640).

ctgggagcttcgggtagagacctaggccgctggaccgcgATGAGCGCGCCGAGCCTCCG

TGCGCGCGCCGCGGGGTTGGGGCTGCTGCTGTGCGCGGTGCTGGGGCGCGCTGGCCGGT

CAGACAGCGGCGGTCGCGGGGAACTCGGGCAGCCCTCTGGGGTAGCCGCCGAGCGCCCA

TGCCCCACTACCTGCCGCTGCCTCGGGGACCTGCTGGACTGCAGTCGTAAGCGGCTAGC

GCGTCTTCCCGAGCCACTCCCGTCCTGGGTCGCTCGGCTGGACTTAAGTCACAACAGAT

TATCTTTCATCAAGGCAAGTTCCATGAGCCACCTTCAAAGCCTTCGAGAAGTGAAACTG

AACAACAATGAATTGGAGACCATTCCAAATCTGGGACCAGTCTCGGCAAATATTACACT

TCTCTCCTTGGCTGGAAACAGGATTGTTGAAATACTCCCTGAACATCTGAAAGAGTTTC

AGTCCCTTGAAACTTTGGACCTTAGCAGCAACAATATTTCAGAGCTCCAAACTGCATTT

CCAGCCCTACAGCTCAAATATCTGTATCTCAACAGCAACCGAGTCACATCAATGGAACC

TGGGTATTTTGACAATTTGGCCAACACACTCCTTGTGTTAAAGCTGAACAGGAACCGAA

TCTCAGCTATCCCACCCAAGATGTTTAAACTGCCCCAACTGCAACATCTCGAATTGAAC

CGAAACAAGATTAAAAATGTAGATGGACTGACATTCCAAGGCCTTGGTGCTCTGAAGTC

TCTGAAAATGCAAAGAAATGGAGTAACGAAACTTATGGATGGAGCTTTTTGGGGGCTGA

GCAACATGGAAATTTTGCAGCTGGACCATAACAACCTAACAGAGATTACCAAAGGCTGG

CTTTACGGCTTGCTGATGCTGCAGGAACTTCATCTCAGCCAAAATGCCATCAACAGGAT

CAGCCCTGATGCCTGGGAGTTCTGCCAGAAGCTCAGTGAGCTGGACCTAACTTTCAATC

ACTTATCAAGGTTAGATGATTCAAGCTTCCTTGGCCTAAGCTTACTAAATACACTGCAC

ATTGGGAACAACAGAGTCAGCTACATTGCTGATTGTGCCTTCCGGGGGCTTTCCAGTTT

AAAGACTTTGGATCTGAAGAACAATGAAATTTCCTGGACTATTGAAGACATGAATGGTG

CTTTCTCTGGGCTTGACAAACTGAGGCGACTGATACTCCAAGGAAATCGGATCCGTTCT

ATTACTAAAAAAGCCTTCACTGGTTTGGATGCATTGGAGCATCTAGACCTGAGTGACAA

CGCAATCATGTCTTTACAAGGCAATGCATTTTCACAAATGAAGAAACTGCAACAATTGC

ATTTAAATACATCAAGCCTTTTGTGCGATTGCCAGCTAAAATGGCTCCCACAGTGGGTG

GCGGAAAACAACTTTCAGAGCTTTGTAAATGCCAGTTGTGCCCATCCTCAGCTGCTAAA

AGGAAGAAGCATTTTTGCTGTTAGCCCAGATGGCTTTGTGTGTGATGATTTTCCCAAAC

CCCAGATCACGGTTCAGCCAGAAACACAGTCGGCAATAAAAGGTTCCAATTTGAGTTTC

ATCTGCTCAGCTGCCAGCAGCAGTGATTCCCCAATGACTTTTGCTTGGAAAAAAGACAA

TGAACTACTGCATGATGCTGAAATGGAAAATTATGCACACCTCCGGGCCCAAGGTGGCG

AGGTGATGGAGTATACCACCATCCTTCGGCTGCGCGAGGTGGAATTTGCCAGTGAGGGG

AAATATCAGTGTGTCATCTCCAATCACTTTGGTTCATCCTACTCTGTCAAAGCCAAGCT


Takeuchi et al. 23

TACAGTAAATATGCTTCCCTCATTCACCAAGACCCCCATGGATCTCACCATCCGAGCTG

GGGCCATGGCACGCTTGGAGTGTGCTGCTGTGGGGCACCCAGCCCCCCAGATAGCCTGG

CAGAAGGATGGGGGCACAGACTTCCCAGCTGCACGGGAGAGACGCATGCATGTGATGCC

CGAGGATGACGTGTTCTTTATCGTGGATGTGAAGATAGAGGACATTGGGGTATACAGCT

GCACAGCTCAGAACAGTGCAGGAAGTATTTCAGCAAATGCAACTCTGACTGTCCTAGAA

ACACCATCATTTTTGCGGCCACTGTTGGACCGAACTGTAACCAAGGGAGAAACAGCCGT

CCTACAGTGCATTGCTGGAGGAAGCCCTCCCCCTAAACTGAACTGGACCAAAGATGATA

GCCCATTGGTGGTAACCGAGAGGCACTTTTTTGCAGCAGGCAATCAGCTTCTGATTATT

GTGGACTCAGATGTCAGTGATGCTGGGAAATACACATGTGAGATGTCTAACACCCTTGG

CACTGAGAGAGGAAACGTGCGCCTCAGTGTGATCCCCACTCCAACCTGCGACTCCCCTC

AGATGACAGCCCCATCGTTAGACGATGACGGATGGGCCACTGTGGGTGTCGTGATCATA

GCCGTGGTTTGCTGTGTGGTGGGCACGTCACTCGTGTGGGTGGTCATCATATACCACAC

AAGGCGGAGGAATGAAGATTGCAGCATTACCAACACAGATGAGACCAACTTGCCAGCAG

ATATTCCTAGTTATTTGTCATCTCAGGGAACGTTAGCTGACAGGCAGGATGGGTACGTG

TCTTCAGAAAGTGGAAGCCACCACCAGTTTGTCACATCTTCAGGTGCTGGATTTTTCTT

ACCACAACATGACAGTAGTGTCTGGCATAGAAGATTAAAGAATCAAAAAAGTGCCAAGG

AAGGGGTGACAGTGCTTATAAACGAAGACAAAGAGTTGGCTGAGCTGCGAGGTCTGGCA

GCTGGAGTAGGCCTGGCTAATGCCTGCTATGCAATACATACTCTTCCAACCCAAGAGGA

GATTGAAAATCTTCCTGCCTTCCCTCGGGAAAAACTGACTCTGCGTCTCTTGCTGGGAA

GTGGAGCCTTTGGAGAAGTGTATGAAGGAACAGCAGTGGACATCTTAGGAGTTGGAAGT

GGAGAAATCAAAGTAGCAGTGAAGACTTTGAAGAAGGGTTCCACAGACCAGGAGAAGAT

TGAATTCCTGAAGGAGGCACATCTGATGAGCAAATTTAATCATCCCAACATTCTGAAGC

AGCTTGGAGTTTGTCTGCTGAATGAACCCCAATACATTATCCTGGAACTGATGGAGGGA

GGAGACCTTCTTACTTATTTGCGTAAAGCCCGGATGGCAACGTTTTATGGTCCTTTACT

CACCTTGGTTGACCTTGTAGACCTGTGTGTAGATATTTCAAAAGGCTGTGTCTACTTGG

AACGGATGCATTTCATTCACAGGGATCTGGCAGCTAGAAATTGCCTTGTTTCCGTGAAA

GACTATACCAGTCCACGGATAGTGAAGATTGGAGACTTTGGACTCGCCAGAGACATCTA

TAAAAATGATTACTATAGAAAGAGAGGGGAAGGCCTGCTCCCAGTTCGGTGGATGGCTC

CAGAAAGTTTGATGGATGGAATCTTCACTACTCAATCTGATGTATGGTCTTTTGGAATT

CTGATTTGGGAGATTTTAACTCTTGGTCATCAGCCTTATCCAGCTCATTCCAACCTTGA

TGTGTTAAACTATGTGCAAACAGGAGGGAGACTGGAGCCACCAAGAAATTGTCCTGATG

ATCTGTGGAATTTAATGACCCAGTGCTGGGCTCAAGAACCCGACCAAAGACCTACTTTT

CATAGAATTCAGGACCAACTTCAGTTATTCAGAAATTTTTTCTTAAATAGCATTTATAA

GTCCAGAGATGAAGCAAACAACAGTGGAGTCATAAATGAAAGCTTTGAAGGTGAAGATG

GCGATGTGATTTGTTTGAATTCAGATGACATTATGCCAGTTGCTTTAATGGAAACGAAG

AACCGAGAAGGGTTAAACTATATGGTACTTGCTACAGAATGTGGCCAAGGTGAAGAAAA


Takeuchi et al. 24

GTCTGAGGGTCCTCTAGGCTCCCAGGAATCTGAATCTTGTGGTCTGAGGAAAGAAGAGA

AGGAACCACATGCAGACAAAGATTTCTGCCAAGAAAAACAAGTGGCTTACTGCCCTTCT

GGCAAGCCTGAAGGCCTGAACTATGCCTGTCTCACTCACAGTGGATATGGAGATGGGTC

TGATTAAtagcgt


Takeuchi et al. 25

KIF5B-RET (variant 4) K15;R12 cDNA sequence

In this cDNA, exon 15 of KIF5B is ligated in an in-frame manner to exon 12 of RET

variant 4 (NM_020630). Position 2105, polymorphism (rs1800861). Position 3032, a

C-to-T change in the 5’-untranslated region of RET cDNA.

cggaagtgagcattagggttaacggctgccggcgccggctcttcaagtcccggctcccc

ggccgcctccacccggggaagcgcagcgcggcgcagctgactgctgcctctcacggccc

tcgcgaccacaagccctcaggtccggcgcgttccctgcaagactgagcggcggggagtg

gctcccggccgccggccccggctgcgagaaagATGGCGGACCTGGCCGAGTGCAACATC

AAAGTGATGTGTCGCTTCAGACCTCTCAACGAGTCTGAAGTGAACCGCGGCGACAAGTA

CATCGCCAAGTTTCAGGGAGAAGACACGGTCGTGATCGCGTCCAAGCCTTATGCATTTG

ATCGGGTGTTCCAGTCAAGCACATCTCAAGAGCAAGTGTATAATGACTGTGCAAAGAAG

ATTGTTAAAGATGTACTTGAAGGATATAATGGAACAATATTTGCATATGGACAAACATC

CTCTGGGAAGACACACACAATGGAGGGTAAACTTCATGATCCAGAAGGCATGGGAATTA

TTCCAAGAATAGTGCAAGATATTTTTAATTATATTTACTCCATGGATGAAAATTTGGAA

TTTCATATTAAGGTTTCATATTTTGAAATATATTTGGATAAGATAAGGGACCTGTTAGA

TGTTTCAAAGACCAACCTTTCAGTTCATGAAGACAAAAACCGAGTTCCCTATGTAAAGG

GGTGCACAGAGCGTTTTGTATGTAGTCCAGATGAAGTTATGGATACCATAGATGAAGGA

AAATCCAACAGACATGTAGCAGTTACAAATATGAATGAACATAGCTCTAGGAGTCACAG

TATATTTCTTATTAATGTCAAACAAGAGAACACACAAACGGAACAAAAGCTGAGTGGAA

AACTTTATCTGGTTGATTTAGCTGGTAGTGAAAAGGTTAGTAAAACTGGAGCTGAAGGT

GCTGTGCTGGATGAAGCTAAAAACATCAACAAGTCACTTTCTGCTCTTGGAAATGTTAT

TTCTGCTTTGGCTGAGGGTAGTACATATGTTCCATATCGAGATAGTAAAATGACAAGAA

TCCTTCAAGATTCATTAGGTGGCAACTGTAGAACCACTATTGTAATTTGCTGCTCTCCA

TCATCATACAATGAGTCTGAAACAAAATCTACACTCTTATTTGGCCAAAGGGCCAAAAC

AATTAAGAACACAGTTTGTGTCAATGTGGAGTTAACTGCAGAACAGTGGAAAAAGAAGT

ATGAAAAAGAAAAAGAAAAAAATAAGATCCTGCGGAACACTATTCAGTGGCTTGAAAAT

GAGCTCAACAGATGGCGTAATGGGGAGACGGTGCCTATTGATGAACAGTTTGACAAAGA

GAAAGCCAACTTGGAAGCTTTCACAGTGGATAAAGATATTACTCTTACCAATGATAAAC

CAGCAACCGCAATTGGAGTTATAGGAAATTTTACTGATGCTGAAAGAAGAAAGTGTGAA

GAAGAAATTGCTAAATTATACAAACAGCTTGATGACAAGGATGAAGAAATTAACCAGCA

AAGTCAACTGGTAGAGAAACTGAAGACGCAAATGTTGGATCAGGAGGAGCTTTTGGCAT

CTACCAGAAGGGATCAAGACAATATGCAAGCTGAGCTGAATCGCCTTCAAGCAGAAAAT

GATGCCTCTAAAGAAGAAGTGAAAGAAGTTTTACAGGCCCTAGAAGAACTTGCTGTCAA

TTATGATCAGAAGTCTCAGGAAGTTGAAGACAAAACTAAGGAATATGAATTGCTTAGTG

ATGAATTGAATCAGAAATCGGCAACTTTAGCGAGTATAGATGCTGAGCTTCAGAAACTT


Takeuchi et al. 26

AAGGAAATGACCAACCACCAGAAAAAACGAGCAGCTGAGATGATGGCATCTTTACTAAA

AGACCTTGCAGAAATAGGAATTGCTGTGGGAAATAATGATGTAAAGGAGGATCCAAAGT

GGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAATTTGGA

AAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCACGGTGGC

CGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTGCGAGACCTGCTGTCAGAGT

TCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCCTGCAGC

CAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCGGGGCTT

CCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACT

CCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATTT

GCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCATCGGGA

CTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGGATTTCG

GCTTGTCCCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGTCGGATT

CCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGA

TGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACCCCTATC

CTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATGGAGAGG

CCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCAGGAGCC

GGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGGTTAAGA

GGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTATGACGAC

GGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCCTCGAGC

CCTCCCTTCCACATGGATTGAAAACAAACTCTATGGTAGAATTTCCCATGCATTTACTA

GATTCTAGcaccgctgtcccctttgcactatccttcctctctgtgatgctttttaaaaa

tgtttctggtctgaacaaaaccaaagtctgctctgaacc


Takeuchi et al. 27



variant 2 (NM_020975). Position 2105, polymorphism (rs1800861).


































Takeuchi et al. 28

AGACCTTGCAGAAATAGGAATTGCTGTGGGAAATAATGATGTAAAGGAGGATCCAAAGT

GGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAATTTGGA

AAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCACGGTGGC

CGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTGCGAGACCTGCTGTCAGAGT

TCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCCTGCAGC

CAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCGGGGCTT

CCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACT

CCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATTT

GCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCATCGGGA

CTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGGATTTCG

GCTTGTCCCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGTCGGATT

CCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGA

TGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACCCCTATC

CTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATGGAGAGG

CCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCAGGAGCC

GGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGGTTAAGA

GGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTATGACGAC

GGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCCTCGAGC

CCTCCCTTCCACATGGATTGAAAACAAACTCTATGGCATGTCAGACCCGAACTGGCCTG

GAGAGAGTCCTGTACCACTCACGAGAGCTGATGGCACTAACACTGGGTTTCCAAGATAT

CCAAATGATAGTGTATATGCTAACTGGATGCTTTCACCCTCAGCGGCAAAATTAATGGA

CACGTTTGATAGTTAAcatttctttgtgaaaggtaatggactcacaaggggaagaaaca

tg


Takeuchi et al. 29





































Takeuchi et al. 30

AGACCTTGCAGAAATAGGAATTGCTGTGGGAAATAATGATGTAAAGCAGCCTGAGGGAA

CTGGCATGATAGATGAAGAGTTCACTGTTGCAAGACTCTACATTAGCAAAATGAAGTCA

GAAGTAAAAACCATGGTGAAACGTTGCAAGCAGTTAGAAAGCACACAAACTGAGAGCAA

CAAAAAAATGGAAGAAAATGAAAAGGAGTTAGCAGCATGTCAGCTTCGTATCTCTCAAG

AGGATCCAAAGTGGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAA

GGCGAATTTGGAAAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTA

CACCACGGTGGCCGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTTCGAGACC

TGCTGTCAGAGTTCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTAT

GGGGCCTGCAGCCAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTC

CCTGCGGGGCTTCCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAG

GCAGCCGCAACTCCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGAC

CTCATCTCATTTGCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCT

CGTTCATCGGGACTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGA

TTTCGGATTTCGGCTTGTCCCGAGATGTTTATGAAGAGGATTCGTACGTGAAGAGGAGC

CAGGGTCGGATTCCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACAC

CACGCAAAGTGATGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGG

GAAACCCCTATCCTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCAC

CGGATGGAGAGGCCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTG

GAAGCAGGAGCCGGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGA

TGATGGTTAAGAGGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTG

ATTTATGACGACGGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCC

CCTCCCTCGAGCCCTCCCTTCCACATGGATTGAAAACAAACTCTATGGTAGAATTTCCC

ATGCATTTACTAGATTCTAGcaccgctgtcccctctgcactatccttcctctctgtgat

gctttttaaaaatgtttctggtctgaacaaaaccaaagtctgctctgaacc


Takeuchi et al. 31





































Takeuchi et al. 32




CAAAAAAATGGAAGAAAATGAAAAGGAGTTAGCAGCATGTCAGCTTCGTATCTCTCAAG

AGGATCCAAAGTGGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAA

GGCGAATTTGGAAAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTA

CACCACGGTGGCCGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTTCGAGACC

TGCTGTCAGAGTTCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTAT

GGGGCCTGCAGCCAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTC

CCTGCGGGGCTTCCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAG

GCAGCCGCAACTCCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGAC

CTCATCTCATTTGCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCT

CGTTCATCGGGACTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGA

TTTCGGATTTCGGCTTGTCCCGAGATGTTTATGAAGAGGATTCGTACGTGAAGAGGAGC

CAGGGTCGGATTCCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACAC

CACGCAAAGTGATGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGG

GAAACCCCTATCCTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCAC

CGGATGGAGAGGCCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTG

GAAGCAGGAGCCGGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGA

TGATGGTTAAGAGGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTG

ATTTATGACGACGGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCC

CCTCCCTCGAGCCCTCCCTTCCACATGGATTGAAAACAAACTCTATGGCATGTCAGACC

CGAACTGGCCTGGAGAGAGTCCTGTACCACTCACGAGAGCTGATGGCACTAACACTGGG

TTTCCAAGATATCCAAATGATAGTGTATATGCTAACTGGATGCTTTCACCCTCAGCGGC

AAAATTAATGGACACGTTTGATAGTTAAcatttctttgtgaaaggtaatggactcacaa

ggggaagaaacatg


Takeuchi et al. 33



variant 4 (NM_020630). Position 2819, polymorphism (rs1800861). Position 3746, a

C-to-T change in the 5’-untranslated region of RET cDNA.

































Takeuchi et al. 34





CAAAAAAATGGAAGAAAATGAAAAGGAGTTAGCAGCATGTCAGCTTCGTATCTCTCAAC

ATGAAGCCAAAATCAAGTCATTGACTGAATACCTTCAAAATGTGGAACAAAAGAAAAGA

CAGTTGGAGGAATCTGTCGATGCCCTCAGTGAAGAACTAGTCCAGCTTCGAGCACAAGA

GAAAGTCCATGAAATGGAAAAGGAGCACTTAAATAAGGTTCAGACTGCAAATGAAGTTA

AGCAAGCTGTTGAACAGCAGATCCAGAGCCATAGAGAAACTCATCAAAAACAGATCAGT

AGTTTGAGAGATGAAGTAGAAGCAAAAGCAAAACTTATTACTGATCTTCAAGACCAAAA

CCAGAAAATGATGTTAGAGCAGGAACGTCTAAGAGTAGAACATGAGAAGTTGAAAGCCA

CAGATCAGGAAAAGAGCAGAAAACTACATGAACTTACGGTTATGCAAGATAGACGAGAA

CAAGCAAGACAAGACTTGAAGGGTTTGGAAGAGACAGTGGCAAAAGAACTTCAGACTTT

ACACAACCTGCGCAAACTCTTTGTTCAGGACCTGGCTACAAGAGTTAAAAAGGAGGATC

CAAAGTGGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAA

TTTGGAAAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCAC

GGTGGCCGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTGCGAGACCTGCTGT

CAGAGTTCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCC

TGCAGCCAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCG

GGGCTTCCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCC

GCAACTCCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATC

TCATTTGCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCA

TCGGGACTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGG

ATTTCGGCTTGTCCCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGT

CGGATTCCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCA

AAGTGATGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACC

CCTATCCTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATG

GAGAGGCCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCA

GGAGCCGGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGG

TTAAGAGGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTAT

GACGACGGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCC

TCGAGCCCTCCCTTCCACATGGATTGAAAACAAACTCTATGGTAGAATTTCCCATGCAT

TTACTAGATTCTAGcaccgctgtcccctttgcactatccttcctctctgtgatgctttt

taaaaatgtttctggtctgaacaaaaccaaagtctgctctgaacc


Takeuchi et al. 35





































Takeuchi et al. 36













ACACAACCTGCGCAAACTCTTTGTTCAGGACCTGGCTACAAGAGTTAAAAAGGAGGATC

CAAAGTGGGAATTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAA

TTTGGAAAAGTGGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCAC

GGTGGCCGTGAAGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTGCGAGACCTGCTGT

CAGAGTTCAACGTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCC

TGCAGCCAGGATGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCG

GGGCTTCCTCCGCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCC

GCAACTCCAGCTCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATC

TCATTTGCCTGGCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCA

TCGGGACTTGGCAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGG

ATTTCGGCTTGTCCCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGT

CGGATTCCAGTTAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCA

AAGTGATGTATGGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACC

CCTATCCTGGGATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATG

GAGAGGCCAGACAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCA

GGAGCCGGACAAAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGG

TTAAGAGGAGAGACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTAT

GACGACGGCCTCTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCC

TCGAGCCCTCCCTTCCACATGGATTGAAAACAAACTCTATGGCATGTCAGACCCGAACT

GGCCTGGAGAGAGTCCTGTACCACTCACGAGAGCTGATGGCACTAACACTGGGTTTCCA

AGATATCCAAATGATAGTGTATATGCTAACTGGATGCTTTCACCCTCAGCGGCAAAATT

AATGGACACGTTTGATAGTTAAcatttctttgtgaaaggtaatggactcacaaggggaa

gaaacatg


Takeuchi et al. 37



variant 4 (NM_020630).


































Takeuchi et al. 38













ACACAACCTGCGCAAACTCTTTGTTCAGGACCTGGCTACAAGAGTTAAAAAGAGTGCTG

AGATTGATTCTGATGACACCGGAGGCAGCGCTGCTCAGAAGCAAAAAATCTCCTTTCTT

GAAAATAATCTTGAACAGCTCACTAAAGTGCACAAACAGGAGGATCCAAAGTGGGAATT

CCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAATTTGGAAAAGTGG

TCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCACGGTGGCCGTGAAG

ATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTTCGAGACCTGCTGTCAGAGTTCAACGT

CCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCCTGCAGCCAGGATG

GCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCGGGGCTTCCTCCGC

GAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACTCCAGCTC

CCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATTTGCCTGGC

AGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCATCGGGACTTGGCA

GCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGGATTTCGGCTTGTC

CCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGTCGGATTCCAGTTA

AATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGATGTATGG

TCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACCCCTATCCTGGGAT

TCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATGGAGAGGCCAGACA

ACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCAGGAGCCGGACAAA

AGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGGTTAAGAGGAGAGA

CTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTATGACGACGGCCTCT

CAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCCTCGAGCCCTCCCT

TCCACATGGATTGAAAACAAACTCTATGGTAGAATTTCCCATGCATTTACTAGATTCTA

Gcaccgctgtcccctctgcactatccttcctctctgtgatgctttttaaaaatgtttct

ggtctgaacaaaaccaaagtctgctctgaacc


Takeuchi et al. 39





































Takeuchi et al. 40















GAAAATAATCTTGAACAGCTCACTAAAGTGCACAAACAGGAGGATCCAAAGTGGGAATT

CCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAATTTGGAAAAGTGG

TCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCACGGTGGCCGTGAAG

ATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTTCGAGACCTGCTGTCAGAGTTCAACGT

CCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCCTGCAGCCAGGATG

GCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCGGGGCTTCCTCCGC

GAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACTCCAGCTC

CCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATTTGCCTGGC

AGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCATCGGGACTTGGCA

GCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGGATTTCGGCTTGTC

CCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGTCGGATTCCAGTTA

AATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGATGTATGG

TCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACCCCTATCCTGGGAT

TCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATGGAGAGGCCAGACA

ACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCAGGAGCCGGACAAA

AGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGGTTAAGAGGAGAGA

CTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTATGACGACGGCCTCT

CAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCCTCGAGCCCTCCCT

TCCACATGGATTGAAAACAAACTCTATGGCATGTCAGACCCGAACTGGCCTGGAGAGAG

TCCTGTACCACTCACGAGAGCTGATGGCACTAACACTGGGTTTCCAAGATATCCAAATG

ATAGTGTATATGCTAACTGGATGCTTTCACCCTCAGCGGCAAAATTAATGGACACGTTT

GATAGTTAAcatttctttgtgaaaggtaatggactcacaaggggaagaaacatg


Takeuchi et al. 41





































Takeuchi et al. 42















GAAAATAATCTTGAACAGCTCACTAAAGTGCACAAACAGTTGGTACGTGATAATGCAGA

TCTCCGCTGTGAACTTCCTAAGTTGGAAAAGCGACTTCGAGCTACAGCTGAGAGAGTGA

AAGCTTTGGAATCAGCACTGAAAGAAGCTAAAGAAAATGCATCTCGTGATCGCAAACGC

TATCAGCAAGAAGTAGATCGCATAAAGGAAGCAGTCAGGTCAAAGAATATGGCCAGAAG

AGGGCATTCTGCACAGATTGATCCACTGTGCGACGAGCTGTGCCGCACGGTGATCGCAG

CCGCTGTCCTCTTCTCCTTCATCGTCTCGGTGCTGCTGTCTGCCTTCTGCATCCACTGC

TACCACAAGTTTGCCCACAAGCCACCCATCTCCTCAGCTGAGATGACCTTCCGGAGGCC

CGCCCAGGCCTTCCCGGTCAGCTACTCCTCTTCCGGTGCCCGCCGGCCCTCGCTGGACT

CCATGGAGAACCAGGTCTCCGTGGATGCCTTCAAGATCCTGGAGGATCCAAAGTGGGAA

TTCCCTCGGAAGAACTTGGTTCTTGGAAAAACTCTAGGAGAAGGCGAATTTGGAAAAGT

GGTCAAGGCAACGGCCTTCCATCTGAAAGGCAGAGCAGGGTACACCACGGTGGCCGTGA

AGATGCTGAAAGAGAACGCCTCCCCGAGTGAGCTTCGAGACCTGCTGTCAGAGTTCAAC

GTCCTGAAGCAGGTCAACCACCCACATGTCATCAAATTGTATGGGGCCTGCAGCCAGGA

TGGCCCGCTCCTCCTCATCGTGGAGTACGCCAAATACGGCTCCCTGCGGGGCTTCCTCC

GCGAGAGCCGCAAAGTGGGGCCTGGCTACCTGGGCAGTGGAGGCAGCCGCAACTCCAGC

TCCCTGGACCACCCGGATGAGCGGGCCCTCACCATGGGCGACCTCATCTCATTTGCCTG

GCAGATCTCACAGGGGATGCAGTATCTGGCCGAGATGAAGCTCGTTCATCGGGACTTGG

CAGCCAGAAACATCCTGGTAGCTGAGGGGCGGAAGATGAAGATTTCGGATTTCGGCTTG

TCCCGAGATGTTTATGAAGAGGATTCCTACGTGAAGAGGAGCCAGGGTCGGATTCCAGT

TAAATGGATGGCAATTGAATCCCTTTTTGATCATATCTACACCACGCAAAGTGATGTAT

GGTCTTTTGGTGTCCTGCTGTGGGAGATCGTGACCCTAGGGGGAAACCCCTATCCTGGG

ATTCCTCCTGAGCGGCTCTTCAACCTTCTGAAGACCGGCCACCGGATGGAGAGGCCAGA


Takeuchi et al. 43

CAACTGCAGCGAGGAGATGTACCGCCTGATGCTGCAATGCTGGAAGCAGGAGCCGGACA

AAAGGCCGGTGTTTGCGGACATCAGCAAAGACCTGGAGAAGATGATGGTTAAGAGGAGA

GACTACTTGGACCTTGCGGCGTCCACTCCATCTGACTCCCTGATTTATGACGACGGCCT

CTCAGAGGAGGAGACACCGCTGGTGGACTGTAATAATGCCCCCCTCCCTCGAGCCCTCC

CTTCCACATGGATTGAAAACAAACTCTATGGTAGAATTTCCCATGCATTTACTAGATTC

TAGcaccgctgtcccctctgcactatccttcctctctgtgatgctttttaaaaatgttt

ctggtctgaacaaaaccaaagtctgctctgaacc


Documents

Supplementary Information RET, ROS1, and ALK Fusions in ...Supplementary Information RET, ROS1, and ALK Fusions in Lung Cancer Kengo Takeuchi, 1,2 Manabu Soda,3 Yuki Togashi, 1,2 Ritsuro