TECHNICAL ADVANCE Open Access
Simul aneous de ec ion o lung usions
using a mul iplex RT-PCR nex gene a ion
sequencing-based app oach: a mul i-
ins i u ional esea ch s udy
Cecily P. Vaughn
1
, José Luis Cos a
2,3,4*
, Ha ie E. Feilo e
5
, Rosella Pe a oli
6
, Va un Bagai
6
,
Anna Ma ia Rachiglio
7
, Fede ica Zi o Ma ino
8
, Bas iaan Tops
9
, Hen ie e M. Ku h
10
, Kazuko Sakai
11
,
And ea Ma icini
12
, Roy R. L. Bas ien
1
, Anne Reiman
21
, Delphine Le Co e
14,15
, Alexande Boag
5
, Susan C ocke
5
,
Michel Bihl
16
, As id Hi schmann
17
, Aldo Sca pa
12
, José Ca los Machado
2,3,4
, Hélène Blons
14,15
, O la Sheils
18
,
Kelli B amle
6
, Ma jolijn J. L. Lig enbe g
9,19
, Ian A. C ee
13
, Nicola No manno
20
, Kazu o Nishio
11
and
Pie e Lau en -Puig
14,15
Abs ac
Backg ound: Gene usion e en s esul ing om ch omosomal ea angemen s play an impo an ole in ini ia ion
o lung adenoca cinoma. The ecen associa ion o ou oncogenic d i e genes, ALK,ROS1,RET, and NTRK1, as lung
umo p edic i e bioma ke s has inc eased he need o de elopmen o up- o-da e echnologies o de ec ion o
hese bioma ke s in limi ed amoun s o ma e ial.
Me hods: We desc ibe he e a mul i-ins i u ional s udy using he Ion AmpliSeq™RNA Fusion Lung Cance Resea ch
Panel o in e oga e p e iously cha ac e ized lung umo samples.
Resul s: Rep oducibili y be ween labo a o ies using dilu ed usion-posi i e cell lines was 100%. A coho o lung
clinical esea ch samples om di e en o igins ( issue biopsies, issue esec ions, lymph nodes and pleu al luid
samples) we e used o e alua e he panel. We obse ed 97% conco dance o ALK (28/30 posi i e; 71/70 nega i e
samples), 95% o ROS1 (3/4 posi i e; 19/18 nega i e samples), and 93% o RET (2/1 posi i e; 13/14 nega i e samples)
be ween he AmpliSeq assay and o he me hodologies.
Conclusion: This me hodology enables simul aneous de ec ion o mul iple ALK,ROS1,RET,andNTRK1 gene usion
ansc ip s in a single panel, enhanced by an in eg a ed analysis solu ion. The assay pe o ms well on limi ed amoun s
o inpu RNA (10 ng) and o e s an in eg a ed single assay solu ion o de ec ion o ac ionable usions in lung
adenoca cinoma, wi h po en ial sa ings in bo h cos and u n-a ound- ime compa ed o he combina ion o all
ou assays by o he me hods.
Keywo ds: Gene usions, De ec ion, Bioma ke , Lung cance , Nex -gene a ion sequencing, FFPE
* Co espondence: [email p o ec ed]
2
i3S - Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o,
Rua Al edo Allen 208, 4200-135 Po o, Po ugal
3
IPATIMUP - Ins i u e o Molecula Pa hology and Immunology o he
Uni e si y o Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© The Au ho (s). 2018 Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0
In e na ional License (h p://c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o
he C ea i e Commons license, and indica e i changes we e made. The C ea i e Commons Public Domain Dedica ion wai e
(h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle, unless o he wise s a ed.
Vaughn e al. BMC Cance (2018) 18:828
h ps://doi.o g/10.1186/s12885-018-4736-4
Backg ound
Non-small cell lung ca cinoma (NSCLC) has been
ca ego ized in o se e al dis inc en i ies by molecula
cha ac e iza ion o gene ic al e a ions occu ing du ing
epi helial cell ans o ma ion. These al e a ions lead
mainly o he ac i a ion o oncogenes such as EGFR,
KRAS,NRAS,BRAF o ERBB2, [1–3] which occu
h ough poin mu a ions, small dele ions o inse ions
and, mo e a ely, ampli ica ions. Se e al o he key
d i e s ha e been implica ed in lung cance ca cinogen-
esis h ough o he mechanisms. Indeed, ch omosomal
ea angemen s in ol ing he y osine kinase ecep o
genes ALK,[4]ROS1,[5]RET,[6–8] and NTRK1,[9]
ha e been mo e ecen ly desc ibed, ex ending he epe -
oi e o molecula al e a ions ound in NSCLC. These
usion e en s, in ol ing a a ie y o pa ne genes, esul
in he o ma ion o chime ic usion kinases capable o
oncogenic ans o ma ion and induc ion o oncogene de-
pendency wi hin he neoplas ic cells. The p e alence o
each o hese ch omosomal ea angemen s indi idually
is 1–7% in NSCLC [4,6,10,11], and al oge he can be
iden i ied in app oxima ely 5–9% o NSCLC [7,12,13].
The de elopmen o d ugs ha speci ically a ge
usion p o eins encoded by hese ea angemen s [9,11,
14] has d i en he need o sys ema ic sensi i e assays o
de ec hem. Lung cance usions ha e adi ionally been
de ec ed using FISH, IHC, o RT-PCR. While FISH is
conside ed he gold s anda d, especially o ALK es ing
due o he a ailabili y o an FDA-app o ed ALK FISH
assay, FISH analysis o mul iple a ge s pe sample can
be cos ly. The massi ely pa allel na u e o nex gene -
a ion sequencing (NGS) allows a apid cha ac e iza ion
o poin mu a ions, small inse ions and dele ions. Add-
i ionally, NGS can be used o he de ec ion o ch omo-
some ea angemen s in a la ge se o genes by a ge ed
sequencing o he usion junc ions o by pai ed-end
mapping me hods. In his s udy we alida ed a new
lib a y ki , he Ion AmpliSeq™RNA Fusion Lung Cance
Resea ch Panel, o cha ac e iza ion o he mos equen
ch omosome ea angemen s in lung adenoca cinoma
by NGS. This lib a y ki is based on he high-mul iplex-
ing capabili ies o PCR and ocuses on he iden i ica ion
o 72 di e en ansc ip s. We epo he sensi i i y and
speci ici y o his assay o he de ec ion o gene usions
implica ed in NSCLC.
Me hods
Samples
A o al o 138 clinical esea ch samples p e iously es ed
o ALK,ROS1, and/o RET ea angemen s we e col-
lec ed om 10 pa icipa ing labo a o ies. All clinical
esea ch samples we e s udied in he labo a o y o
o igin. All samples we e om esec ions o biopsies ha
had been o malin- ixed and pa a in-embedded (FFPE),
wi h he excep ion o h ee esh ozen samples (one
esec ion and wo pleu al e usions). These included 128
samples p e iously es ed o ALK ea angemen s by
luo escence in si u hyb idiza ion (FISH). Six y- i e o
hese samples had also been es ed o ALK ea ange-
men s by ano he me hod: immunohis ochemis y
(IHC), e e se ansc ip ion (RT)-PCR, and/o mass
spec ome y (pe o med on he MassARRAY Sys em
om Agena Bioscience, San Diego, CA). Ca ego iza ion
o he ALK- es ed samples as posi i e, nega i e o incon-
clusi e was de e mined by he FISH esul s, as his
me hodology is conside ed he gold s anda d o ALK
es ing. Fo hose samples p e iously es ed by mul iple
me hods, any disc epancies in esul s be ween he me h-
odologies we e no ed. Thi een o he ALK samples had
also been es ed o ROS1 and/o RET ea angemen s.
An addi ional 10 clinical esea ch samples p e iously
es ed o ROS1 and/o RET, bu o which ALK es ing
esul s we e una ailable, we e also included in his s udy.
Ca ego iza ion o he ROS1 and RET samples was based
on he esul s om any a ailable me hod, including
FISH, IHC, RT-PCR and/o mass spec ome y, since
he e is no an es ablished gold-s anda d o de ec ion o
hese ea angemen s.
RNA was ex ac ed om each o he clinical esea ch
samples by he pa icipa ing labo a o ies using hei
espec i e s anda d ex ac ion p ocedu es. Six o he en
labo a o ies used he Reco e All To al Nucleic Acid
Isola ion Ki o FFPE (The mo Fishe Scien i ic,
Wal ham, MA); emaining labs used he Qiagen RNeasy
FFPE Ki (Qiagen, Hilden, Ge many), he Qiagen AllP ep
DNA/RNA FFPE Ki , o he Maxwell LEV RNA FFPE
Pu i ica ion Ki (P omega, Madison, WI). RNA was
quan i ied using he Qubi RNA assay ki s (The mo
Fishe ) a eigh o he labo a o ies; Quan -iT RiboG een
RNA Assay Ki (The mo Fishe ) and he Nanod op 2000
ins umen (The mo Scien i ic) we e also used o
quan i ica ion.
In addi ion o he clinical esea ch samples, a cock ail o
RNA isola ed om he ALK usion-posi i e H2228 (ATCC
CRL-5935), ROS1 usion-posi i e HCC-78 (DSMZ ACC
563), and RET usion-posi i e LC-2/ad (ECACC LC-2/ad)
cell lines was p epa ed by The mo Fishe Scien i ic and
supplied o each o he pa icipa ing labo a o ies. Selec
labo a o ies also p epa ed and es ed RNA isola ed om
FFPE e sions o hese cell lines and RNA isola ed om
he ALK usion-posi i e cell line H3122 (ECACC
NCI-H322) and he NTRK1 usion-posi i e cell line
KM-12.
Ion AmpliSeq RNA usion lung Cance esea ch panel design
P ime s spanning 72 usions (37 ALK,9RET,15ROS1,
and 11 NTRK1) we e designed by a esea ch eam a
The mo Fishe . These p ime s we e designed o span all
Vaughn e al. BMC Cance (2018) 18:828 Page 2 o 8
p e iously desc ibed usions, a he ime o de elopmen ,
o ALK,ROS1,RET, and NTRK1 in lung cance s.
Sou ces used o he cu a ion o known usions included
he COSMIC and NCBI da abases, and e iew o cu en
medical li e a u e. Ta ge ed usion genes a e shown in
Table 1. The mul iplex p ime mix also included p ime s
o he ampli ica ion o i e housekeeping genes: HMBS,
ITGB7,LMNA,MYC, and TBP.
Addi ionally, p ime s designed o ampli y 5′and 3′
egions o ALK,ROS1,RET, and NTRK1 we e included
in he p ime mix. Ampli ica ion o hese egions o
each gene o in e es allowed o he compa ison o
exp ession le els be ween he 3′end o he gene, which
is pa o he esul ing usion, and he non-in ol ed 5′
end o he gene. A lis o all a ge s in he mul iplex
PCR –including a ge ed usions (genes and exons),
exp ession con ol genes, and 3′and 5′ egions –is a ail-
able in Addi ional ile 1: Table S1.
De ec ion o usions
A minimum o 10 ng o o al RNA was e e se an-
sc ibed using he Supe Sc ip VILO cDNA Syn hesis Ki
ollowed by lib a y gene a ion using he Ion AmpliSeq
Lib a y Ki 2.0 and he Ion AmpliSeq RNA Fusion Lung
Cance Resea ch Panel (he ea e , AmpliSeq Fusion
Lung Panel). Ba codes we e u ilized du ing lib a y
gene a ion using he Ion Xp ess Ba code Adap e s.
Lib a ies we e quan i ied using he Qubi DNA assay,
he 2100 BioAnalyze (Agilen Technologies, San a
Cla a, CA) o he Ion Lib a y Quan i a ion Ki , hen
pooled in equimola concen a ions o sequencing.
Eigh o six een lib a ies we e mul iplexed and em-
pla ed using he Ion OneTouch2 Sys em wi h he Ion
PGM Templa e OT2 200 Ki . Lib a ies we e sequenced
using he Ion PGM Sequencing 200 2 ki on an Ion 316
2 o 318 2 chip on he Ion PGM ins umen . (All
eagen s and ins umen a ion abo e a e om The mo
Fishe Scien i ic, wi h he excep ion o he BioAnalyze .)
Typically, eigh samples we e sequenced pe 316 chip
and six een samples pe 318 chip.
A e sequencing, unaligned BAM iles we e ans-
e ed o he Ion Repo e So wa e 4.2 and analyzed
using he AmpliSeq Lung Fusion single sample wo k-
low. This wo k low u ilizes a BED ile comp ised o
chime ic sequences o a ge ed usion ansc ip s along
wi h sequences o he exp ession con ol genes and he
3′and 5′ egions o ALK,ROS1,RET, and NTRK1. The
alignmen consis s o h ee main s eps. In he i s s ep,
he aligne equi es ha he eads align end o end (i.e,
eads ha a e immed, o so clipped, a he ends a e
no allowed). Each ead is hen aligned o he bes
p ima y alignmen and il e ing c i e ia a e applied.
Alignmen s o he usion a ge s a e coun ed only i he
ead o e laps a leas 70% o he expec ed usion inse
wi h high local alignmen sco e. Alignmen s o he
imbalance and con ol a ge s a e coun ed i he ead
o e laps a leas 50%. In he second s ep, all unaligned
eads, and eads ha aligned bu we e il e ed ou , a e
spli in o wo agmen s. These agmen ed eads a e
hen e-aligned o he same e e ence ile. T imming o
he eads is allowed in his s ep and all he alignmen s o
e e y ead (no jus he p ima y alignmen s) a e kep in
he alignmen s iles. This s ep helps eco e mo e coun s
o he a ge s in he e e ence ile and also inds any
non- a ge ed usion iso o ms ha a e no p esen in he
o iginal lis o a ge s. A no el usion iso o m in ol ing
exis ing p ime s is epo ed in he ou pu i he e is
e idence om a leas 100 di e en pai s o agmen s.
Las ly, coun s om s eps one and wo a e agg ega ed
and all he usion a ge s ha ha e coun s highe han
he h eshold a e epo ed as “ usion p esen .”The
algo i hm gene a es a 3′/5′exp ession imbalance me ic
o each o he d i e genes based on he indi idual
coun s o he 5′assay and 3′assay. I is calcula ed by
sub ac ing he coun o 5′ eads om he coun o 3′
eads, and di iding he esul by he sum o coun s o all
con ol a ge s. This me ic can be used o con i m he
de ec ion o a known usion o o p edic a usion in he
sample ha is no co e ed by he iso o ms in he panel.
Resul s
Cell lines
Using he cock ail o RNA om he ALK,ROS1, and
RET usion-posi i e cell lines, each o he en pa icipa -
ing labo a o ies success ully de ec ed all h ee ea ange-
men s using he AmpliSeq Fusion Lung Panel assay (see
Table 2). The usions de ec ed co esponded o he ea -
angemen s p e iously desc ibed o hese cell lines [11,
15–18] (see Table 3). The expec ed ea angemen s we e
also de ec ed om RNA isola ed om he FFPE cell
blocks o he same cell lines and om he ALK-posi i e
H3122 cell line [11] (Tables 2and 3). In he KM12 cell
line [19], p ime s o he speci ic NTRK usion we e no
included in he assay design, bu he ea angemen was
Table 1 Ta ge ed Pa ne s o ALK,RET,ROS1, and NTRK1
ALK RET ROS1 NTRK1
EML4 KIF5B CD74 CEL
KIF5B CCDC6 SDC4 NFASC
KLC1 CUX1 SLC34A2 IRF2BP2
HIP1 EZR TFG
TPR TPM3 SQSTM1
LRIG3 SSBP2
GOPC CD74
DYNC2H1
MPRIP
Vaughn e al. BMC Cance (2018) 18:828 Page 3 o 8
de ec ed by a posi i e 3′/5′imbalance esul o 0.076,
abo e he cu -o o 0.025 (Table 2).
ALK clinical samples
O he 138 clinical esea ch samples es ed, 117 (84.5%)
passed he QC equi emen o a minimum o 20,000
o al eads. One hund ed o hese samples had p e i-
ously been es ed o ALK ea angemen s by FISH wi h
conclusi e esul s; AmpliSeq Fusion Lung Panel esul s
we e 97% conco dan (97/100 samples) wi h FISH
analysis (see Table 4).ALK ea angemen s we e
de ec ed in 28/30 ALK FISH-posi i e samples, wi h a
sensi i i y o 93.3%; exac usions we e iden i ied in 24
o he samples and an addi ional 4 samples showed
e idence o ea angemen using he 3′/5′imbalance
calcula ion. In samples nega i e o ALK ea angemen s
by FISH, 69 o 70 samples we e also nega i e by he
AmpliSeq assay, hus esul ing in a speci ici y o 98.6%.
De ails on all ALK clinical esea ch samples a e shown
in Addi ional ile 1: Table S2.
Close analysis o he disco dan esul s (see
Table 5), e ealed ha wo o he FISH-posi i e ALK
samples wi hou a de ec ed usion showed 10%
ea anged cells (below he usual cu -o o 15%). One
o hese samples showed only weak s aining by IHC,
and subsequen e- es ing by he AmpliSeq Fusion
LungPanelga eaposi i e esul o a usiono
HIP1-ALK wi h 30 eads. Fi e o he emaining se en
disco dan ALK FISH-posi i e samples showed an
a ypical FISH esul o one ed signal, a he han he
spli ed-g een signal; o hese, wo we e IHC nega i e
o ALK and one was posi i e by IHC o iginally, bu
nega i e upon epea es ing. The emaining wo
FISH-posi i e, AmpliSeq usion-nega i e samples
showed ypical FISH esul s wi h 19% and 20% ea -
anged cells, espec i ely, and bo h we e posi i e o
ALK IHC s aining.
One disco dan sample was nega i e by FISH and
posi i e by AmpliSeq. This sample showed an EML4-
ALK usion wi h 6137 usion eads, and he 3′/5′im-
balance in his sample also showed a posi i e esul .
Addi ionally, his sample had p e iously es ed posi-
i e o ALK p o ein exp ession by IHC.
ROS1 and RET clinical samples
Panel esul s o ROS1 and RET we e conco dan in 21/
22 samples (95.5%) o ROS1 and 14/15 (93.3%) o RET
as compa ed o p e ious es ing using a ailable me hods,
Table 2 Con ol Sample Resul s Ac oss Pa icipa ing Labo a o ies
Labo a o y Sample ALK
usion
ROS1
usion
RET
usion
NTRK1
usion
ALK 3′/5′
imbalance
ROS1 3′/5′
imbalance
RET 3′/5′
imbalance
NTRK 3′/5′
imbalance
INSERM Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0044 0.3031 0.0603 n/a
Kinki Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0405 0.4648 0.0434 n/a
ARUP Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0172 0.6141 0.0421 n/a
ARC-Ne Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0086 0.3458 0.0353 n/a
Viollie Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.043 0.3398 0.046 n/a
Radboudumc Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0414 0.2502 0.0856 n/a
CROM Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.012 0.054 0.0813 n/a
IPATIMUP Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0238 0.2877 0.0539 n/a
Wa wick Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0064 0.3 0.055 n/a
Queen’s Cell Line RNA cock ail
a
De ec ed De ec ed De ec ed n/a 0.0123 0.4158 0.0478 n/a
ARUP FFPE cell block H2228 De ec ed n/a n/a n/a 0.0312 n/a n/a n/a
ARUP FFPE cell block HCC78 n/a De ec ed n/a n/a n/a 3.8313 n/a n/a
ARUP FFPE cell block LC-2/ad n/a n/a De ec ed n/a n/a n/a 0.2521 n/a
Kinki F esh cells H3122 De ec ed n/a n/a n/a 0.3165 n/a n/a n/a
ARUP FFPE cell block KM-12 n/a n/a n/a No co e ed n/a n/a n/a 0.076
a
A mix u e o RNA om h ee cell lines (H2228, HCC78, and LC-2/ad)
Table 3 Cell Line Fusions
Cell line Expec ed usion (s) De ec ed usion (s)
H2228 EML4(6a,6b)-ALK(20) EML4(6a,6b)-ALK(20)
HCC78 SLC34A2(4)-ROS1(32,34) SLC34A2(4)-ROS1(32,34,35,36)
LC-2/ad CCDC6(1)-RET(12) CCDC6(1)-RET(12)
H3122 EML4(13)-ALK(20) EML4(13)-ALK(20)
KM-12 TPM3(7)-NTRK1(8) no co e ed
Table 4 Conco dance Be ween FISH and AmpliSeq o De ec ion
o ALK Fusions
FISH + FISH -
AmpliSeq + 28 1
AmpliSeq - 2 69
Vaughn e al. BMC Cance (2018) 18:828 Page 4 o 8
including FISH, IHC, RT-PCR, and mass spec ome y.
The AmpliSeq assay de ec ed he app op ia e usions in
3/4 ROS1-posi i e samples and in 1/1 RET-posi i e
sample. Samples p e iously de e mined o be nega i e
o ROS1 using o he me hodologies (18 samples) we e
all nega i e using he AmpliSeq Fusion Lung Panel. In
samples p e iously de e mined o be nega i e o RET
usions, 13 o 14 we e nega i e by he AmpliSeq panel,
and one showed a posi i e imbalance esul o 0.197, in
he absence o a de ec ed usion iso o m (Addi ional ile
1: Table S3). This sample was subsequen ly es ed using
he RET FISH b eak-apa p obe and also e- un using
he AmpliSeq panel; bo h esul s we e nega i e.
De ec ion and con i ma ion o addi ional usions
Tes ing o he clinical esea ch samples yielded he
de ec ion o ROS1 usions in wo samples which we e
ALK-nega i e by FISH (Samples 63 and 67, Addi ional
ile 1: Table S2). In bo h cases, he CD74-ROS1 usion
was de ec ed and subsequen es ing using a TaqMan
assay wi h p ime s speci ic o he de ec ed usion
con i med he esul s. P io o his s udy, nei he o
hese samples had been es ed o ROS1 ea angemen s.
RET usions we e de ec ed in h ee o he ALK-nega-
i e samples (Samples 48, 55, and 98, Addi ional ile 1:
Table S2). In Sample 98, he KIF5B-RET usion was
de ec ed and subsequen ly con i med by a TaqMan
assay. Samples 48 and 55 showed posi i e RET 3′/5′im-
balance esul s o 0.041 and 0.271, espec i ely. Subse-
quen FISH analysis o Sample 55 showed 10% spli
signals in he issue a ea used o ex ac ion o RNA o
he AmpliSeq Fusion Lung Panel assay. Addi ional
ma e ial o con i ma o y es ing was no a ailable o
Sample 48.
Las ly, a ROS1 usion was de ec ed wi h 83 usion
eads in one o he ALK FISH-posi i e samples (Sample
8, Addi ional ile 1: Table S2). The p esence o wo
usion e en s is unlikely, and subsequen es ing by RT-
PCR did no con i m he p esence o his usion.
Samples wi h low To al eads
Upon ini ial e alua ion o he 138 clinical esea ch sam-
ples, samples below he QC cu -o o 20,000 we e
epea ed wi h ei he 30 PCR cycles o simply e-pooling
p io o bead empla ing. Fi e o en samples success ully
epea ed, and hose samples a e included in he da a
abo e. In he o he i e samples and in samples o
which epea es ing was no possible, easons o ailu e
included insu icien RNA quan i y (< 10 ng), deg aded
RNA, and imp ope pooling o lib a ies.
Discussion
The ad en o he apies a ge ing he usion p o eins
a ising om ALK,ROS1, and RET gene usions makes
he ou ine de ec ion o hese e en s impo an in
pa ien s wi h lung adenoca cinoma. We ha e desc ibed
he e an in e na ional, mul i-ins i u ional s udy using a
mul iplex RT-PCR nex gene a ion sequencing-based
me hod ha enables simul aneous de ec ion o ALK,
RET,ROS1, and NTRK1 gene usion ansc ip s in a
single assay. The simul aneous de ec ion o hese usions
has impo an implica ions o u n-a ound- ime and
cos . Fu he , i can be pe o med wi h e y li le inpu
RNA. This is pa icula ly a ac i e o an assay a ge ed
a lung cance s, as hese samples a e o en biopsies wi h
limi ed a ailable issue. Lung cance usions ha e
adi ionally been de ec ed using FISH, IHC, o RT-PCR.
While FISH is conside ed he gold s anda d, especially
o ALK es ing due o he a ailabili y o an FDA-ap-
p o ed ALK FISH assay, FISH analysis o mul iple a -
ge s pe sample can be cos ly. O en hese analyses a e
done in s ep-wise ashion, which can educe he o e all
cos o pe o ming mul iple FISH assays, bu po en ially
ex end he ime needed o ule ou all ele an gene e-
a angemen s. Immunohis ochemis y s aining o e s a
cheape al e na i e; howe e , his me hodology is sub-
jec i e, some imes making in e p e a ion di icul . [20]
RT-PCR, on he o he hand, can o e p ecise de ec ion
o usions, including iden i ica ion o bo h pa ne genes
and he exons in ol ed. The main limi a ion o ad-
i ional RT-PCR is ha i ypically ocuses on only he
mos common usion e en s and is hus limi ed in de-
ec ing a e exon combina ions. [21]
In con as o FISH o IHC, he de ec ion o ALK,
ROS1,RET, and NTRK1 usions a e combined in a single
assay wi h he AmpliSeq design. F om he 70 clinical
esea ch samples ha p e iously had been de e mined o
be ALK-nega i e by FISH, we de ec ed wo ROS1 usions
and h ee RET usions. Bo h o he ROS1 usions and
wo o RET usions we e con i med o be posi i e by
o hogonal me hods; issue o addi ional es ing was no
a ailable o he hi d RET-posi i e sample. Fu he , he
de ec ion o usions by NGS o e s a imely me hodology
ha can also be designed o accommoda e he
Table 5 Disco dan ALK clinical samples
Sample Labo a o y FISH ( ea anged cells) AmpliSeq esul (n° eads) 3′-5′imbalance IHC esul s
26 Viollie Posi i e (19%) Nega i e (136146) 0,0099 Posi i e
30 CROM Posi i e (20%) Nega i e (166403) 0,0008 2+
107 Queen’s Nega i e EML4(6a)-ALK(20) (1835387) 0,3179 Posi i e
Vaughn e al. BMC Cance (2018) 18:828 Page 5 o 8

simul aneous de ec ion o poin mu a ions and inse -
ions and dele ions in he DNA o ele an genes in a
single assay. Analysis o hese ypes o mu a ions, pa -
icula ly in EGFR and KRAS, is ypically pa o he
wo k-up o lung adenoca cinoma pa ien s. Me hods o
de ec bo h DNA mu a ions and usion e en s in a
imely manne a e pa icula ly impo an in hese
pa ien s due o he agg essi e na u e o he disease.
While combined analysis o DNA and RNA was no
he ocus o his s udy, i is cu en ly being pe o med
by many o he ins i u ions ha pa icipa ed in his
s udy.
The me hodology desc ibed in his pape elies on
RT-PCR o he ini ial ampli ica ion o usion e en s;
howe e , he design o his assay ci cum en s a limi a-
ion o adi ional RT-PCR. The AmpliSeq Fusion Lung
Panel assay includes mul iplexing o p ime s o 72 di -
e en usion combina ions and hus is no limi ed o
only he mos common usions. A second limi a ion o
adi ional RT-PCR is ha one mus ha e p e ious
knowledge o all possible ele an usions. The AmpliSeq
assay add esses his issue in wo ways. Fi s , du ing he
analysis o he sequenced eads, all eads ha a e ini ially
unaligned o he e e ence sequence a e spli in hal and
allowed o e-align. This s ep os e s he de ec ion o
no el usions in ol ing exis ing p ime s. Secondly, he
assay includes a me hod o de ec ion usions in ol ing
unknown pa ne s using he 3′/5′imbalance calcula ion.
This s ep analyzes he exp ession le els o he 3′and 5′
ends o each d i e gene. Fo genes in ol ed in a usion
e en , he 3′end o he gene is now unde di e en
egula o y con ol and shows o e exp ession ela i e o
he 5′end o he gene. Ano he ecen ly desc ibed
me hodology using NanoS ing echnology also exploi s
his phenomenon o 3′o e exp ession. [22] Tha s udy
ound ha e alua ion o he imbalance be ween 3′and
5′exp ession wo ks ela i ely well o ALK and RET,
which a e no mally no exp essed in lung issue, bu ha
his calcula ion was mo e di icul o ROS1 as his gene
is no mally exp essed a high le els. Gi en ha a posi i e
imbalance esul is sugges i e o a usion e en , bu
alone does no iden i y an exac usion, ou sugges ion
o he AmpliSeq assay is o use he imbalance calcula-
ion as a me hod o iden i ying possible usions ha
should be ollowed up wi h o hogonal es ing me hods
i desi ed.
Fu he analysis o disco dan samples wi hin ou
s udy ound ha some samples had ei he low le els o
ea anged cells by FISH o disco dan esul s be ween
FISH and IHC. One o he samples o which FISH
es ing showed 10% ea anged cells, was posi i e o a
HIP1-ALK usion upon epea es ing wi h he AmpliSeq
assay. The epea esul had usion eads alling jus
abo e he cu -o , while he ini ial nega i e esul did
iden i y he same usion bu wi h a numbe o eads
alling below he cu -o , indica ing he sample was likely
app oaching he limi o de ec ion o he assay. Dis-
co dance be ween ALK FISH and o he me hods has
been no ed p e iously [23–25] and b ings up he ques-
ion o a ue “gold s anda d.”Th ee o he ALK
FISH-posi i e samples o which he AmpliSeq assay was
nega i e, we e also nega i e by IHC. Addi ionally, we
ound ha i e o he disco dan samples displayed
single ed signals by FISH. This phenomenon o a single
ed signal ep esen s a likely dele ion o he 5′end o
ALK and is no unusual o his s uc u al a ian ; how-
e e , p e ious s udies ha e also shown a simila disco d-
ance be ween ALK FISH-posi i e esul s displaying a
dele ion o he 5′ALK p obe and IHC [24] o PCR. [26]
The exac na u e o hese usion e en s may be o in e -
es o u u e s udies. We also obse ed disco dan
esul s o one o he ALK FISH-nega i e samples. In
his case, he AmpliSeq assay iden i ied an EML4-ALK
usion wi h a high numbe o eads and he sample was
also posi i e by IHC. While his sample was o icially
classi ied as an AmpliSeq “ alse posi i e,”i likely ep e-
sen s a ue posi i e in which FISH es ing ailed o
de ec he usion.
A ecen s udy using he AmpliSeq me hod o usion
de ec ion epo ed 100% conco dance be ween his and
o he me hodologies. [27] I is unknown, bu p obable,
ha he es ing o his s udy was pe o med a a single
ins i u ion. The di e ence be ween a single o limi ed
ins i u ion s udy and a la ge s udy (in his case, en
ins i u ions) may explain he di e ence in conco dance
esul s be ween he P a s udy [27]and hes udy
desc ibed he e. The in e na ional, mul i-ins i u ional
na u e o his s udy p esen ed many challenges. Sco ing
c i e ia be ween labo a o ies o en a ies e en o
well-es ablished e e ence me hods, e.g., some samples in
his s udy we e deemed FISH-posi i e, ye ell below he
cu -o o 15% used by o he ins i u ions. A lack o
conco dance be ween mul iple ins i u ions o de ec ion
o ALK ea angemen s has been p e iously obse ed, [20,
28] and his phenomenon may ha e con ibu ed o he
lowe conco dance o compa ed me hods in his s udy. A
u he challenge o he mul i-ins i u ional s udy included
a lack o ma e ial o ollow up on disc epan samples, as
he samples we e no only om he pa icipa ing ins i u-
ions bu in some cases we e om addi ional labo a o y
pa ne s. Howe e , we belie e ha he ad an ages o his
mul i-ins i u ional s udy a ou weigh he disad an ages.
Rep oducibili y ac oss di e en labo a o ies using cell line
mix u es was 100%, despi e po en ial di e ences in
labo a o y p ac ices and pe sonnel. Addi ionally, an in e -
na ional, mul i-ins i u ional s udy such as his allows o
he inclusion o mo e a ied samples and mo e ully
explo es he pe o mance o he assay.
Vaughn e al. BMC Cance (2018) 18:828 Page 6 o 8
Conclusion
The RT-PCR NGS assay desc ibed he e o e s many
ad an ages o labo a o y es ing in lung adenoca cin-
oma samples. This me hodology allows de ec ion o
mul iple usions in a single assay and can easily also be
mul iplexed wi h de ec ion o poin mu a ions and small
inse ions and dele ions in genes such as KRAS and
EGFR ha a e also impo an in he wo k up o hese
pa ien s. The single-assay o ma po en ially allows o
as e u n-a ound- ime and lowe cos han doing he
assays sepa a ely. Fu he , he small amoun o inpu
RNA equi ed is e y ad an ageous o hese samples.
Howe e , he AmpliSeq assay p ima ily a ge s known
usions. Inclusion o he 3′/5′imbalance calcula ion aims
o add ess his limi a ion, bu could likely bene i om
u he e inemen o cu -o s alues as mo e da a is
gene a ed by his assay. Las ly, e o s o pe iodically
upda e he p ime pool as addi ional pa ne genes o
ALK,ROS1,RET, and NTRK1 usions a e iden i ied
would aid in he con inuing u ili y o his assay.
Addi ional ile
Addi ional ile 1: Table S1. RT-PCR Ta ge s wi hin he Ion AmpliSeq™
Lung Fusion Resea ch Panel. A lis o all a ge s in he mul iplex PCR s udied
–including a ge ed usions (genes and exons), exp ession con ol genes,
and 3′and 5′ egions. Table S2. ALK Clinical Samples. Table depic ing de ails
on all ALK clinical esea ch samples analyzed in he s udy. Table S3. ROS1
and RET clinical samples. Table depic ing de ails on all ROS1 and RET clinical
esea ch samples analyzed in he s udy. (DOCX 57 kb)
Abb e ia ions
FFPE: Fo malin- ixed pa a in-embedded; FISH: Fluo escence in si u
hyb idiza ion; IHC: Immunohis ochemis y; NGS: Nex gene a ion sequencing;
NSCLC: Non-small cell lung cance ; RT-PCR: Re e se ansc ip ase-polyme ase
chain eac ion
Acknowledgemen s
The au ho s wish o hank Xiao Zhang (Queen’s Uni e si y, Kings on On a io,
Canada), Miguel Sil a (Ipa imup, Po o, Po ugal) and Ana Jus ino (Ipa imup,
Po o, Po ugal).
Funding
This wo k esul ed om p ojec s “Ins i u e o Resea ch and Inno a ion in
Heal h Sciences”(POCI-01-0145-FEDER-007274), “GenomePT”(POCI-01-0145-
FEDER-022184), “Ad ancing cance esea ch: om knowledge o applica ion”
(NORTE-01-0145-FEDER-000029) suppo ed by COMPETE 2020 - Ope a ional
P og amme o Compe i i eness and In e na ionalisa ion (POCI), No e
Po ugal Regional P og amme (NORTE 2020), Lisboa Po ugal Regional
Ope a ional P og amme (Lisboa2020), Alga e Po ugal Regional Ope a ional
P og amme (CRESC Alga e2020), unde he PORTUGAL 2020 Pa ne ship
Ag eemen , h ough he Eu opean Regional De elopmen Fund (ERDF), and
by FCT - Fundação pa a a Ciência e a Tecnologia (PTDC/DTP-PIC/2500/2014).
This wo k was suppo ed by a g an om he Associazione I aliana pe la
Rice ca sul Canc o (AIRC) o N. No manno (G an numbe : IG17135).
A ailabili y o da a and ma e ials
The da ase s used and analyzed du ing he cu en s udy a e a ailable om
he co esponding au ho on a easonable eques .
Au ho ’s con ibu ions
S udy concep and design: IC, PLP, NN, OS, RP, KB, JC, CV, AM. Da a acquisi ion,
analysis and in e p e a ion: IC, AR, PLP, VB, RB, BT, NN, MJ, FM, HF, OS, KB, AS, JC,
CV, MB, KN, KS, AM, AR, SC, DC, HK. D a ing o he manusc ip o e ising i
c i ically o impo an in ellec ual con en : IC, PLP, VB, NN, MJ, HF, AB, OS, AS,
JC, CV, JM, AM, SC. All au ho s ha e gi en inal app o al o he e sion o be
published.
E hics app o al and consen o pa icipa e
Samples we e e ie ed om he a chi es o he collabo a ing ins i u ions
and de-iden i ied. Fo he ollowing ins i u ions, he s udy was in e p e ed as
a se ice imp o emen no equi ing speci ic esea ch e hics commi ee
app o al as s a ed in he EU Clinical T ials Di ec i e (2001/20/EC): Radboud
uni e si y medical cen e , VIOLLIER (assay pe o med by Viollie , samples
mainly om Uni e si y Hospi al Basel and Luze ne Kan onsspi al,
Swi ze land), Applied Resea ch on Cance Cen e (ARC-NET), Uni e si y
Hospi als Co en y and Wa wickshi e (UHCW), Ins i u Na ional de la San e e
de la Reche che Medicale (INSERM), and Cen o di Rice che Oncologiche di
Me cogliano (CROM). Samples om he ollowing ins i u ions we e used
unde app o ed Ins i u ional Re iew Boa d p o ocols: ARUP Labo a o ies
(ARUP Labo a o ies E hics Commi ee app o al 24,487), Queen’s Uni e si y
(Heal h Sciences and A ilia ed Teaching Hospi als Resea ch E hics Boa d ap-
p o al 6,010,968), and Kinki Uni e si y (Ins i u ional Re iew Boa d o Kindai
Uni e si y Facul y o Medicine app o al 22–106). Samples om Ipa imup
we e used in acco dance wi h A icle 19 (“DNA Banks and O he Biological
P oduc s”) o Po uguese Law No. 12/2005 o 26 Janua y (“Pe sonal gene ic
in o ma ion and heal h in o ma ion”).
Compe ing in e es s
The mo Fishe Scien i ic p o ided eagen s o his s udy o pa icipa ing
labo a o ies a a discoun . T a el unds we e pa ially eimbu sed o
conso ium membe s o a end a g oup mee ing. Au ho s R.P., V.B., and K.B.
a e employed by The mo Fishe Scien i ic. NN is a membe o he edi o ial
boa d (Associa e Edi o ) o his jou nal.
Publishe ’sNo e
Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in
published maps and ins i u ional a ilia ions.
Au ho de ails
1
ARUP Ins i u e o Clinical and Expe imen al Pa hology, Sal Lake Ci y, UT,
USA.
2
i3S - Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do
Po o, Rua Al edo Allen 208, 4200-135 Po o, Po ugal.
3
IPATIMUP - Ins i u e
o Molecula Pa hology and Immunology o he Uni e si y o Po o, Rua
Al edo Allen 208, 4200-135 Po o, Po ugal.
4
Medical Facul y o he
Uni e si y o Po o, Po o, Po ugal.
5
Depa men o Pa hology and Molecula
Medicine, Queen’s Uni e si y, Kings on, ON, Canada.
6
The mo Fishe
Scien i ic, Aus in, TX, USA.
7
Labo a o y o Pha macogenomics, Cen o di
Rice che Oncologiche di Me cogliano (CROM)-Is i u o Nazionale Tumo i
“Fondazione G. Pascale”-IRCCS, Naples, I aly.
8
Pa hology Uni , Is i u o
Nazionale Tumo i “Fondazione G. Pascale”-IRCCS, Naples, I aly.
9
Depa men
o Pa hology, Radboud Uni e si y Medical Cen e , Nijmegen, he
Ne he lands.
10
Viollie AG, Depa men o Gene ics/Molecula Biology, Basel,
Swi ze land.
11
Depa men o Genome Biology, Kinki Uni e si y Facul y o
Medicine, Osaka, Japan.
12
ARC-NET: Cen e o Applied Resea ch on Cance ,
Depa men o Pa hology and Diagnos ic, Uni e si y and Hospi al T us o
Ve ona, Ve ona, I aly.
13
Depa men o Pa hology, Uni e si y Hospi als
Co en y and Wa wickshi e, Walsg a e, Co en y, UK.
14
Uni e si y Pa is
Desca es, Pa is, F ance.
15
Biology Depa men , Assis ance Publique-Hôpi aux
de Pa is, Eu opean Geo ges Pompidou Hospi al, Pa is, F ance.
16
Ins i u e o
Pa hology, Uni e si y Hospi al Basel, Basel, Swi ze land.
17
Luze ne
Kan onsspi al, Luze n, Swi ze land.
18
T ini y T ansla ional Medicine Ins i u e
(TTMI), T ini y College Dublin, Dublin, I eland.
19
Depa men o Human
Gene ics, Radboud Uni e si y Medical Cen e , Nijmegen, he Ne he lands.
20
Cell Biology and Bio he apy Uni , Is i u o Nazionale Tumo i “Fondazione G.
Pascale”-IRCCS, Naples, I aly.
21
Cen e o Spo , Exe cise and Li e Sciences,
Co en y Uni e si y, Co en y, UK.
Recei ed: 9 No embe 2017 Accep ed: 9 Augus 2018
Re e ences
1. Cance Genome A las Resea ch N. Comp ehensi e molecula p o iling o
lung adenoca cinoma. Na u e. 2014;511(7511):543–50.
Vaughn e al. BMC Cance (2018) 18:828 Page 7 o 8
2. Ding L, Ge z G, Wheele DA, Ma dis ER, McLellan MD, Cibulskis K, Sougnez
C, G eulich H, Muzny DM, Mo gan MB, e al. Soma ic mu a ions a ec key
pa hways in lung adenoca cinoma. Na u e. 2008;455(7216):1069–75.
3. Sholl LM, Aisne DL, Va ella-Ga cia M, Be y LD, Dias-San aga a D, Wis uba II,
Chen H, Fujimo o J, Kugle K, F anklin WA, e al. Mul i-ins i u ional
oncogenic d i e mu a ion analysis in lung adenoca cinoma: he lung
Cance mu a ion conso ium expe ience. J Tho ac Oncol. 2015;10(5):768–77.
4. Soda M, Choi YL, Enomo o M, Takada S, Yamashi a Y, Ishikawa S, Fujiwa a S,
Wa anabe H, Ku ashina K, Ha anaka H, e al. Iden i ica ion o he
ans o ming EML4-ALK usion gene in non-small-cell lung cance . Na u e.
2007;448(7153):561–6.
5. Riko a K, Guo A, Zeng Q, Possema o A, Yu J, Haack H, Na done J, Lee K,
Ree es C, Li Y, e al. Global su ey o phospho y osine signaling iden i ies
oncogenic kinases in lung cance . Cell. 2007;131(6):1190–203.
6. Kohno T, Ichikawa H, To oki Y, Yasuda K, Hi amo o M, Nammo T, Sakamo o
H, Tsu a K, Fu u a K, Shimada Y, e al. KIF5B-RET usions in lung
adenoca cinoma. Na Med. 2012;18(3):375–7.
7. Takeuchi K, Soda M, Togashi Y, Suzuki R, Saka a S, Ha ano S, Asaka R,
Hamanaka W, Ninomiya H, Ueha a H, e al. RET, ROS1 and ALK usions in
lung cance . Na Med. 2012;18(3):378–81.
8. Li F, Feng Y, Fang R, Fang Z, Xia J, Han X, Liu XY, Chen H, Liu H, Ji H.
Iden i ica ion o RET gene usion by exon a ay analyses in “pan-nega i e”
lung cance om ne e smoke s. Cell Res. 2012;22(5):928–31.
9. Vaishna i A, Capelle i M, Le AT, Kako S, Bu aney M, E can D, Mahale S,
Da ies KD, Aisne DL, Pilling AB, e al. Oncogenic and d ug-sensi i e NTRK1
ea angemen s in lung cance . Na Med. 2013;19(11):1469–72.
10. Uguen A, De B aekelee M. ROS1 usions in cance : a e iew. Fu u e Oncol.
2016;12(16):1911–28.
11. Koi unen JP, Me mel C, Zejnullahu K, Mu phy C, Li shi s E, Holmes AJ, Choi
HG, Kim J, Chiang D, Thomas R, e al. EML4-ALK usion gene and e icacy o
an ALK kinase inhibi o in lung cance . Clin Cance Res. 2008;14(13):4275–83.
12. Sca pino S, Rampioni Vincigue a GL, Di Napoli A, Foche i F, Uccini S, Iacono
D, Ma che i P, Ruco L. High p e alence o ALK+/ROS1+ cases in pulmona y
adenoca cinoma o adoloscen s and young adul s. Lung Cance . 2016;97:95–8.
13. Pan Y, Zhang Y, Li Y, Hu H, Wang L, Li H, Wang R, Ye T, Luo X, Zhang Y, e al.
ALK, ROS1 and RET usions in 1139 lung adenoca cinomas: a comp ehensi e
s udy o common and usion pa e n-speci ic clinicopa hologic, his ologic and
cy ologic ea u es. Lung Cance . 2014;84(2):121–6.
14. Gaino JF, Shaw AT. No el a ge s in non-small cell lung cance : ROS1 and
RET usions. Oncologis . 2013;18(7):865–75.
15. Phelps RM, Johnson BE, Ihde DC, Gazda AF, Ca bone DP, McClin ock PR,
Linnoila RI, Ma hews MJ, Bunn PA J , Ca ney D, e al. NCI-na y medical
oncology b anch cell line da a base. J Cell Biochem Suppl. 1996;24:32–91.
16. Vi mani AK, Fong KM, Kodagoda D, McIn i e D, Hung J, Tonk V, Minna JD,
Gazda AF. Allelo yping demons a es common and dis inc pa e ns o
ch omosomal loss in human lung cance ypes. Genes ch omosomes
cance . 1998;21(4):308–19.
17. Ma suba a D, Kanai Y, Ishikawa S, Oha a S, Yoshimo o T, Saka ani T,
Oguni S, Tamu a T, Ka aoka H, Endo S, e al. Iden i ica ion o CCDC6-
RET usion in he human lung adenoca cinoma cell line, LC-2/ad. J
Tho ac Oncol. 2012;7(12):1872–6.
18. Da ies KD, Le AT, Theodo o MF, Skokan MC, Aisne DL, Be ge EM,
Te acciano LM, Cappuzzo F, Inca bone M, Roncalli M, e al. Iden i ying and
a ge ing ROS1 gene usions in non-small cell lung cance . Clin Cance Res.
2012;18(17):4570–9.
19. A dini E, Boso i R, Bo gia AL, De Pon i C, Somaschini A, Camma o a R,
Amboldi N, Radd izzani L, Milani A, Magnaghi P, e al. The TPM3-NTRK1
ea angemen is a ecu ing e en in colo ec al ca cinoma and is
associa ed wi h umo sensi i i y o TRKA kinase inhibi ion. Mol Oncol.
2014;8(8):1495–507.
20. Tembuyse L, Tack V, Zwaenepoel K, Pauwels P, Mille K, Bubendo L, Ke K,
Schuu ing E, Thunnissen E, Dequeke EM. The ele ance o ex e nal quali y
assessmen o molecula es ing o ALK posi i e non-small cell lung
cance : esul s om wo pilo ounds show oom o op imiza ion. PLoS
One. 2014;9(11):e112159.
21. Wallande ML, Geie sbach KB, T ipp SR, Lay ield LJ. Compa ison o e e se
ansc ip ion-polyme ase chain eac ion, immunohis ochemis y, and
luo escence in si u hyb idiza ion me hodologies o de ec ion o
echinode m mic o ubule-associa ed p o einlike 4-anaplas ic lymphoma
kinase usion-posi i e non-small cell lung ca cinoma: implica ions o
op imal clinical es ing. A ch Pa hol Lab Med. 2012;136(7):796–803.
22. Li a ME, Choi YL, Lim SM, Deng S, Huang D, Ozeck M, Han J, Jeong JY, Shim
HS, Cho BC, e al. A single- ube mul iplexed assay o de ec ing ALK, ROS1,
and RET usions in lung cance . J Mol Diagn. 2014;16(2):229–43.
23. To KF, Tong JH, Yeung KS, Lung RW, Law PP, Chau SL, Kang W, Tong CY,
Chow C, Chan AW, e al. De ec ion o ALK ea angemen by
immunohis ochemis y in lung adenoca cinoma and he iden i ica ion o a
no el EML4-ALK a ian . J Tho ac Oncol. 2013;8(7):883–91.
24. Gao X, Sholl LM, Nishino M, Heng JC, Janne PA, Oxna d GR. Clinical
implica ions o a ian ALK FISH ea angemen pa e ns. J Tho ac Oncol.
2015;10(11):1648–52.
25. Ali SM, Hensing T, Sch ock AB, Allen J, San o d E, Gowen K, Kulka ni A, He J,
Suh JH, Lipson D, e al. Comp ehensi e genomic p o iling iden i ies a subse o
C izo inib- esponsi e ALK- ea anged non-small cell lung Cance no de ec ed
by luo escence in si u hyb idiza ion. Oncologis . 2016;21(6):762–70.
26. Dai Z, Kelly JC, Meloni-Eh ig A, Slo ak ML, Boles D, Ch is acos NC, B yke CR,
Schonbe g SA, O ani-Rosa J, Pan Q, e al. Incidence and pa e ns o ALK
FISH abno mali ies seen in a la ge unselec ed se ies o lung ca cinomas.
Mol Cy ogene . 2012;5(1):44.
27. P a N, S enzinge A, Penzel R, Wa h A, Dienemann H, Schi mache P,
Weiche W, End is V. High- h oughpu diagnos ic p o iling o clinically
ac ionable gene usions in lung cance . Genes ch omosomes cance .
2016;55(1):30–44.
28. Ma che i A, Ba be is M, Papo i M, Rossi G, F anco R, Mala es a S, Bu i a F,
A dizzoni A, C ino L, G idelli C, e al. ALK ea angemen es ing by FISH
analysis in non-small-cell lung cance pa ien s: esul s o he i s i alian
ex e nal quali y assu ance scheme. J Tho ac Oncol. 2014;9(10):1470–6.
Vaughn e al. BMC Cance (2018) 18:828 Page 8 o 8