deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
h ps://doi.o g/10.1186/s13046‑022‑02379‑1
RESEARCH
Ex acellula esicle PD‑L1 dynamics p edic
du able esponse oimmune‑checkpoin
inhibi o s andsu i al inpa ien s
wi hnon‑small cell lung cance
Diego de Miguel‑Pe ez1,2, Alessand o Russo2,3, Osca A ie a4, Mu a Ak5,6, Feliciano Ba on4,
Mu hukuma Gunaseka an2, P iyada shini Mamindla6, Luis La a‑Mejia4, Ch is ine B. Pe e son7, Mehme E. E 5,6,
Vishal Peddagangi eddy5, F ancesco Buemi3, B andon Coope 2, Paolo Manca8, Rena G. Lapidus2,
Ru‑Ching Hsia2, And es F. Ca dona9, Aung Naing10, Sunjay Kaushal2, F ed R. Hi sch1, Philip C. Mack1,
Ma ia Jose Se ano11, Vincenzo Adamo3, Ri ka R. Colen5,6 and Ch is ian Rol o1,2*
Abs ac
Backg ound: Immune‑checkpoin inhibi o s (ICIs) changed he he apeu ic landscape o pa ien s wi h lung cance .
Howe e , only a subse o hem de i ed clinical bene i and e idenced he need o iden i y eliable p edic i e bio‑
ma ke s. Liquid biopsy is he non‑in asi e and epea able analysis o biological ma e ial in body luids and a p omis‑
ing ool o cance bioma ke s disco e y. In pa icula , he e is g owing e idence ha ex acellula esicles (EVs) play
an impo an ole in umo p og ession and in umo ‑immune in e ac ions. Thus, we e alua ed whe he ex acellula
esicle PD‑L1 exp ession could be used as a bioma ke o p edic ion o du able ea men esponse and su i al in
pa ien s wi h non‑small cell lung cance (NSCLC) unde going ea men wi h ICIs.
Me hods: Dynamic changes in EV PD‑L1 we e analyzed in plasma samples collec ed be o e and a 9 ± 1 weeks du ‑
ing ea men in a e ospec i e and a p ospec i e independen coho s o 33 and 39 pa ien s, espec i ely.
Resul s: As a esul , an inc ease in EV PD‑L1 was obse ed in non‑ esponde s in compa ison o esponde s and was
an independen bioma ke o sho e p og ession‑ ee su i al and o e all su i al. To he con a y, issue PD‑L1
exp ession, he commonly used bioma ke , was no p edic i e nei he o du able esponse no su i al.
Conclusion: These indings indica e ha EV PD‑L1 dynamics could be used o s a i y pa ien s wi h ad anced NSCLC
who would expe ience du able bene i om ICIs.
Keywo ds: Ex acellula esicles, PD‑L1, Bioma ke s, Immuno he apy, NSCLC
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Backg ound
Immune checkpoin inhibi o s (ICIs) ha e e olu ion-
ized he ea men o se e al malignancies, including
non-small cell lung cance (NSCLC). P og ammed cell
dea h p o ein ligand-1 (PD-L1) exp ession in cance cells
is one o he inhibi o y mechanisms in ol ed in umo
immune e asion by PD-1 binding and subsequen T cell
impai men [1]. Se e al ials ha e epo ed imp essi e
Open Access
*Co espondence: ch is ian. ol [email p o ec ed]
1 Cen e o Tho acic Oncology, Tisch Cance Ins i u e, Icahn School
o Medicine a Moun Sinai, New Yo k, NY, USA
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Page 2 o 14
deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
ac i i y o an i-PD-(L)1 monoclonal an ibodies alone
o in combina ion wi h chemo he apy o o he immu-
no he apeu ic d ugs such as an i-CTLA-4 in pa ien s
wi hNSCLC [2–4]. Consequen ly, he FDA app o ed he
i s - o second-line use o d ugs such as pemb olizumab
(an i-PD-1), a ezolizumab (an i-PD-L1), ni olumab (an i-
PD-1), o ecen ly cemiplimab (an i-PD-1) [5] in se e al
umo s including lung cance .
Signi ican ad ances ha e been made in he sea ch
o he ideal p edic i e bioma ke , including he ecen
accep ance o umo mu a ional bu den (TMB) and
mic osa elli e ins abili y-high (MSI-H) as p edic i e bio-
ma ke s in he umo -agnos ic use o pemb olizumab
[6]. Howe e , PD-L1 de ec ion by immunohis ochem-
is y (IHC) is he FDA-app o ed and mos commonly
used p edic i e bioma ke in hese pa ien s. Ne e he-
less, many pa ien s exp essing high PD-L1 did no ben-
e i om he ea men and a conside able pe cen age
o hose wi h low/nega i e PD-L1 exp ession did, which
migh be caused by i s high a iabili y [7, 8]. Along wi h
in e - umo a iabili y, pa ien s wi hNSCLC show sub-
s an ial in a- umo al he e ogenei y and changes in
PD-L1 exp ession can occu a e i s -line ea men s,
hinde ing he accu a e classi ica ion o PD-L1 s a us [9].
The e o e, aside om he lack o “ eal- ime” in o ma ion,
a single issue biopsy may no be able o ecapi ula e he
exac s a us o he umo mic oen i onmen a he ime
o ea men ha , in some cases, can be mon hs o e en
yea s a e umo collec ion. Thus, he e is a huge need
o iden i y eliable p edic i e bioma ke s o an i-PD(L)1
agen s ha can e lec he s a us o he umo mic oen i-
onmen in eal- ime.
PD-L1 p o ein exp ession can also be ound in ex acel-
lula esicles (EVs) [10]. These esicles a e double‐mem-
b ane s uc u es o 20 – 2000nm in ol ed in in e cellula
communica ion and ound in body luids such as blood
[11]. EVs a e in ol ed in he c oss- alk wi hin he umo
mic oen i onmen and play a ole in he inhibi ion o he
an i- umo immune esponse and me as asis, in pa icu-
la by PD-L1 p esen a ion [12–15] So a , only ew s udies
ha e e alua ed he po en ial o PD-L1 exp ession inEVs
as a p edic ing bioma ke in pa ien s wi hlung cance
unde going ICIs and hey showed con lic ing esul s ha
wa an u he in es iga ion [16, 17].
On he o he hand, adiomics is a apidly g owing
ield in imaging, which can con e a pa ien ’s imaging
scans in o mineable quan i a i e da a o be e unde -
s and he umo he e ogenei y and mic oen i onmen
[18]. In pa icula , adiomics may p edic immuno he -
apy esponse and ou come in mul iple cance s, includ-
ing NSCLC and o he ad anced solid umo s [18–21].
Al hough combining independen p edic i e ma ke s has
been ecommended o imp o e accu acy o ea men
esponse p edic ion, o he bes o ou knowledge, only
one s udy has a emp ed o combine adiomics and liquid
biopsy da a o p edic he esponse o immuno he apy in
pa ien s wi hNSCLC [16]. Conside ing he limi a ions o
ha s udy ega ding a e y small coho , using non-con-
as compu ed omog aphy (CT), and mos likely model
o e i ing, we belie e ha u he clinical e alua ion o
hese combined ma ke s is needed.
The e is compelling e idence ha he an i- umo
immune esponse is a complex p ocess egula ed by he
in e ac ion be ween he umo , he immune sys em, and
mul iple hos ac o s in which EVs play a c i ical ole.
Thus, we aimed o iden i y and alida e he p edic i e
ole o EV PD-L1 dynamics in pa ien s wi had anced/
me as a ic NSCLC ea ed wi h ICIs compa ed o he
s anda d-o -ca e issue PD-L1. Addi ionally, we aimed o
c ea e and e alua e he pe o mance o a mul ipa ame ic
p edic i e model wi h he inclusion o adiomics analysis
in ou ini ial coho .
Ma e ials andme hods
S udy design andpa ien s
We conduc ed he e ospec i e analysis o blood sam-
ples and CT scan images om pa ien s wi had anced/
me as a ic NSCLC ea ed wi h an i-PD-1 an ibodies a
he Medical Oncology Uni o A.O. Papa do o Messina,
I aly, be ween May 2018 and No embe 2019 wi h ol-
low-up un il Augus 2021 (T aining coho A). Then,
we p ospec i ely analyzed blood samples ompa ien s
wi h ad anced/me as a ic NSCLC en olled in he phase
2 PROLUNG clinical ial [22], unde going Pemb oli-
zumab + Doce axel o Doce axel alone a he Na ional
Cance Ins i u e, Mexico, wi h ollow-up un il Augus
2021 (Valida ion coho B) (Fig.1). All pa ien s p o ided
w i en in o med consen and he s udy was app o ed by
each ins i u ional e iew boa d. Inclusion c i e ia consid-
e ed pa ien s olde han 18yea s old a he ime o diag-
nosis, s age IIIB o IV acco ding o he 8 h edi ion o he
Ame ican Join Commi ee on Cance TNM manual [23],
and immune-naï e pa ien s ea ed wi h an i-PD(L)-1
(ni olumab, pemb olizumab) inhibi o s in he i s , sec-
ond, o hi d line.
His ological PD-L1 exp ession was assessed by immu-
nohis ochemis y using SP263, 22C3, o 28–8 clones as
pe clinical p ac ice in he mos ecen o malin- ixed
pa a in-embedded issue block om he p ima y is-
sue o me as asis and classi ied acco ding o he umo
p opo ion sco e (TPS). Tumo assessmen du ing ea -
men was e alua ed by CT scan a e 9 ± 1weeks and
a 21 ± 3weeks o ea men acco ding o he Response
E alua ion C i e ia in Solid Tumo s (RECIST) e sion
1.1 [24]. Addi ionally, an independen ins i u ion e iew
o he CT scan om he disco e y coho was conduc ed
Page 3 o 14
deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
using RECIST 1.1 and Immune- ela ed Response E alu-
a ion C i e ia In Solid Tumo s (i RECIST) [25] o he
adiomics analysis. Du able esponde s included hose
pa ien s demons a ing comple e esponse (CR), pa ial
esponse (PR), o s able disease (SD) a he 21 ± 3 week
e alua ion while non- esponde s included hose wi h
p og essi e disease (PD). P og ession- ee su i al (PFS)
was de ined as he ime elapsed be ween he s a o he
ea men o he i s adiological o clinical p og ession
and o e all su i al (OS) as he ime om he s a o he
he apy ill exi us.
Blood samples
Th ee mL o pe iphe al blood we e collec ed in o EDTA
Vacu aine ® ubes a baseline (T1) and a he i s
esponse e alua ion (T2). Blood samples we e cen i-
uged a 2,000 × g o 15min and plasma was isola ed and
ozen a -80°C.
Ex acellula esicle isola ion
Ex acellula esicles we e isola ed acco ding o s and-
a d p o ocols om ou g oup [26, 27]. B ie ly, plasma
aliquo s we e hawed and each500µl we e dilu ed in o
1mL o 1X PBS and cen i uged a 3,000 × g o 20min a
4°C and la e a 10,000 × g o 30min a 4°C o emo e
cell deb is. Supe na an s we e eco e ed, dilu ed wi h 1X
PBS, and cen i uged a 100,000 × g o 70min a 4°C
in o 6.5mL, Open-Top Thickwall Polyca bona e Tubes
(Beckman Coul e ) in an Op ima MAX Ul acen i uge
(Beckman Coul e ). Finally, EVs pelle s we e esuspended
in 1X PBS o lysed wi h 1X RIPA lysis bu e (Cell Signal-
ing) and sonica ed o 2min o u he analysis.
Ex acellula esicle PD‑L1 cha ac e iza ion
Following he las ecommenda ions o he In e na ional
Socie y o Ex acellula Vesicles (ISEV) [28], EVs we e
cha ac e ized by nanopa icle acking analysis (NTA),
ansmission elec on mic oscopy (TEM), and wes e n
blo ollowing ou s anda dized me hodology [27]. In
addi ion, immunogold TEM cha ac e iza ion o PD-L1
exp ession in EVs was pe o med. EV PD-L1 exp ession
was e alua ed by immunoblo and EV PD-L1 dynam-
ics (ΔEV PD-L1) we e calcula ed as he exp ession o
PD-L1 no malized agains CD9 in he second ime poin
di ided by he same alue in he pai ed baseline sample
[(PD-L1/CD9) T2 / (PD-L1/CD9) T1]. A ull desc ip ion
o he EV cha ac e iza ion me hodology can be ound in
he Supplemen a y Me hods. Inc ease in EV PD-L1 was
de ined as pa ien s wi h ΔEV PD-L1 > 1 and dec ease in
hose wi h ΔEV PD-L1 < 1. Pa ien s wi h lowe olume o
quali y o a ailable plasma we e excluded om he s udy
(Coho B: 2 pa ien s in he Pemb olizumab + Doce axel
and 4 in he Doce axel g oup).
Radiomics imaging analysis
Radiomics analysis o a ge and non- a ge lesions was
execu ed acco ding o ou es ablished me hodology
[21]. B ie ly, all lesions we e segmen ed using 3D Slice
4.10.1 module (Slice 4.10.1: Summa y, Highligh s and
Changelog—Announcemen s / Release No es—3D Slice
Communi y) by a di e en colo label. Addi ional ol-
umes o in e es (VOI) o he no mal pec o alis majo
muscle we e segmen ed o wi hin-phase no maliza ion.
Ten in ensi y-le el his og am ea u es and 195Gy le el
co-occu ence ma ix (GLCM) ea u es we e ex ac ed
[29, 30]. We calcula ed he a e age, ange, and angula
a iance o each ea u e o di e en angles, esul ing
in 39 o a ion-in a ian ex u e ea u es calcula ed o
each VOI o i e g ay le els. Addi ionally, we compu ed
195 olume-dependen second-o de ea u es by di id-
ing each GLCM ea u e by he olume o he segmen ed
lesions; he e o e, a o al o 400 adiomics ea u es we e
Fig. 1 S udy design: G aphical scheme o pa ien acc ual, ollow‑up, and bioma ke analysis [c ea ed wi h Bio ende .com]
Page 4 o 14
deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
acqui ed. Radiomics analysis was pe o med using ou
in-house pipeline in Ma lab ( e sion 2017b; Ma hWo ks
Inc) and Phy on P og amming Language ( e sion Phy on
3.7).
Bioin o ma ics ands a is ical analysis
S a is ical analysis and g aphs we e done using SPSS
[SPSS S a is ics o Windows, Ve sion 22.0 (IBM Co p.,
A monk NY, US), G aphPad P ism Ve sion 8.4 (G aph-
Pad So wa e Inc., San Diego CA, US) and R so wa e
( e sion 3.4.0, R Founda ion o S a is ical Compu ing,
Vienna, Aus ia). Non-pa ame ic es e alua ed di e -
ences be ween a iables. Uni a ia e p edic i e models
we e gene a ed wi h logis ic eg ession using glmne
unc ion om he glmne package in R so wa e. Reg es-
sion analysis was pe o med wi h he leas absolu e
sh inkage and selec ion ope a o (LASSO) ea u e selec-
ion me hod o ind he mos ele an adiomics ea u es
associa ed wi h he esponse [31]. Selec ed ea u es we e
en e ed in o eX eme G adien Boos ing (XGBoos )
o build a classi ica ion model o p edic ing o umo
esponse o immuno he apy [32]. Finally, lea e-one-ou
c oss- alida ion (LOOCV) was applied o assess he
obus ness o ou models. Fea u e selec ion, model build-
ing, and ecei e ope a ing cha ac e is ics (ROC) analy-
ses we e implemen ed using he R packages XGBoos
( e sion 0.6.4.1), ml ( e sion 2.11), and pROC ( e sion
1.9.1). The a ea unde he cu e (AUC) was calcula ed
o each ROC cu e and sensi i i y and speci ici y alues
we e shown o he op imal cu -poin alue om each
cu e, esul ed by selec ing he alue p o iding highe
o e all sensi i i y & speci ici y (Youden’s index). Su i al
analyses we e pe o med by Kaplan–Meie (log‐ ank
es ) and Cox P opo ional‐Haza ds Reg ession wi h
backwa d s epwise selec ion o he mul i a ia e model.
Two- ailed p alues < 0.05 we e conside ed s a is ically
signi ican .
Resul s
This s udy en olled wo independen coho s o pa ien s
wi had anced/me as a ic NSCLC. The aining coho
(A) included 33 pa ien s unde going ICIs wi h a median
ollow-up o 12.4mon hs ( ange 2.5 – 33.1). The ali-
da ion coho (B) en olled 39 pa ien s wi h median ol-
low-up o 13.1mon hs ( ange 3.5 – 56.5) om which 24
ecei ed Pemb olizumab + Doce axel and 15 Doce axel
alone. Pa ien s cha ac e is ics a e summa ized in Supple-
men a y Table S1.
EV PD‑L1 cha ac e iza ion
Concen a ion, mo phology, size, and speci ic ma k-
e s in plasma EVs we e analyzed o p o e hei na u e
and pu i y. Fi s , he NTA showed ha plasma EVs had
a concen a ion o 2.15 × 108 pa icles/ mL and a mean
diame e o 99.4nm (Fig.2A). Second, he immunogold
TEM cha ac e iza ion depic ed EVs o simila size wi h
posi i e PD-L1 memb anous exp ession (Fig.2B). Thi d,
he wes e n-blo e ealed exp ession o PD-L1 and he
EV ma ke s CD9 and Flo illin-1 in EVs while absence o
he non-EV ma ke GM130, commonly used as con ol
Fig. 2 EVs cha ac e iza ion: (A) Nanopa icle acking analysis (NTA) o EVs isola ed om ad anced NSCLC plasma samples showing a concen a ion
o 2.15 × 108 pa icles/mL wi h a mode diame e o 68.4 nm. (B) The immunogold ansmission elec on mic oscopy (TEM) depic ed EVs o simila
size wi h exp ession o PD‑L1 in he memb ane. (C) Wes e n blo (WB) images e ealed exp ession o PD‑L1, Flo illin‑1, and CD9 in he plasma EVs
and lung cance cul u e EVs, while low exp ession o GM130
Page 5 o 14
deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
o non-EV con amina ion (Fig.2C). Then, PD-L1 and
CD9 exp ession we e analyzed in pai ed samples om
each pa ien , calcula ing he ΔEV PD-L1 (Supplemen a y
Fig. S1).
EV PD‑L1 dynamics a e abe e p edic i e bioma ke
o du able esponse oICIs han issue PD‑L1
We analyzed he associa ions be ween he EV PD-L1
dynamics and he di e en clinical cha ac e is ics,
including age, gende , line o ea men , his ology, is-
sue PD-L1 (TPS), e c. in ou h ee subg oups o pa ien s
wi h NSCLC, obse ing no s a is ical associa ion wi h
any o hem (Supplemen a y Table S2). Then, we exam-
ined he po en ial ole o his dynamic bioma ke as
a p edic o o du able ICIs esponse compa ed o he
s anda d-o -ca e bioma ke , issue PD-L1 (Rep esen a-
i e images om esponde s and non- esponde s and
he issue and EV PD-L1 cha ac e iza ion a e shown
in Fig.3A-C). We ound an inc ease in EV PD-L1 du -
ing ea men in non- esponde s in compa ison wi h
dec easing le els in esponde s in ou coho A o ICIs
pa ien s (p = 0.017) (Fig.3D). Simila ly, in he Pemb oli-
zumab + Doce axel g oup, non- esponde s showed a
end owa ds inc eased EV PD-L1 in compa ison o
esponde s (p = 0.050) (Fig.3E) while no di e ences we e
obse ed in he Doce axel ea ed pa ien s (Fig.3F). No
di e ences in hese dynamics we e ound be ween he
di e en ea men s (Supplemen a y Fig. S2). Mo eo-
e , no associa ion was ound be ween he issue PD-L1
exp ession and he du able esponse in any g oup. Thus,
ΔEV PD-L1 ou pe o med issue PD-L1 as a p edic i e
ac o o iden i ying pa ien s wi h non-du able clinical
bene i s om ICIs. ΔEV PD-L1 showed an a ea-unde -
he-cu e (AUC) o 77.3% in coho A (Fig. 3G) and
75% in he Pemb olizumab + Doce axel g oup (Fig.3H),
while he issue PD-L1 showed only an AUC o 62.7%
and 64.1%, espec i ely. Poo p edic i e alues we e
obse ed o bo h bioma ke s in he Doce axel g oup
(Fig. 3I). When conside ing he ea ly esponse e alu-
a ed a he i s CT scan, simila bu no s a is ically sig-
ni ican di e ences we e obse ed in Coho A. Howe e ,
as only one pa ien showed ea ly PD in he Pemb oli-
zumab + Doce axel g oup, no signi ican di e ences we e
ound (Supplemen a y Fig. S3).
Fu he mo e, when he du able esponse was ana-
lyzed in all 57 pa ien s unde going ea men wi h ICIs,
he dynamics o EV PD-L1 showed di e ences be ween
pa ien s wi h PR, SD, and PD (p = 0.009) since i was pos-
i i ely co ela ed wi h lesion size (p = 0.040) (Supplemen-
a y Fig. S4). Indeed, pa ien s wi h inc eased EV PD-L1
showed an inc ease in lesion size (p = 0.036), bu no asso-
cia ion was ound be ween he issue PD-L1 TPS and
umo size (p = 0.330) o pa ien s’ esponse (p = 0.561)
(Fig. 4A). Mo eo e , inc eased EV PD-L1 iden i ied
non- esponde s wi h 73% sensi i i y and 61% speci ic-
i y (p = 0.009) (Fig.4B). On he o he hand, high issue
PD-L1 was no associa ed wi h a du able esponse ei he
when conside ing pa ien s wi h TPS ≥ 50% (p = 0.192)
o wi h TPS ≥ 1% (p = 0.370) (Fig.4B). Addi ional sub-
g ouped analysis o he p edic i e pe o mance o EV
PD-L1 ac oss di e en ypes and lines o he apy o TPS
g oups a e shown in Supplemen a y Fig. S5.
EV PD‑L1 dynamics a e ap edic i e bioma ke o su i al
Du ing he ollow-up o hese coho s, 28 (84.8%)
pa ien s p og essed om Coho A, 22 (91.7%) pa ien s
unde going Pemb olizumab + Doce axel, and 15 (100%)
o hose who unde wen Doce axel in coho B (Sup-
plemen a y Table S1). Fi s , we analyzed he p edic i e
alue o PFS o he dynamics o EV PD-L1 in coho
A. Pa ien s wi h EV PD-L1 dec ease end o expe ience
longe PFS han hose wi h inc easing le els (Haza d
a io (HR) = 0.36; p = 0.097) (Fig.5A). This was alida ed
in pa ien s unde going Pemb olizumab + Doce axel,
whe e hose wi h EV PD-L1 dec ease showed longe PFS
(HR = 0.18; p = 0.020) (Fig. 5B). To he con a y, no di -
e ences in PFS we e obse ed in he Doce axel g oup
(HR = 13.3; p = 0.784) (Fig.5C). The mul i a ia e Cox’s
eg ession analysis o he o al 57 pa ien s ecei ing ICIs
e ealed ha ΔEV PD-L1 was an independen p edic i e
bioma ke o PFS, wi h dec eased le els associa ed wi h
longe PFS (HR) = 0.45; p = 0.008) while issue PD-L1
exp ession was no (Supplemen a y Table S3) (Fig.5G).
Rega ding he mo ali y o hese pa ien s, 19 (57.6%)
pa ien s died in coho A. In coho B, 18 (75%) pa ien s
died in he Pemb olizumab + Doce axel g oup and 12
(80%) in he Doce axel g oup (Supplemen a y Table S1).
As p e iously desc ibed o he PFS, we obse ed ha
pa ien s wi h dec easedEV PD-L1 p esen ed longe OS
in coho A (HR = 0.22; p = 0.031) (Fig.5D). As a ali-
da ion, i was also associa ed wi h longe OS in pa ien s
unde going Pemb olizumab + Doce axel (HR = 0.23;
p = 0.039) (Fig.5E) and no di e ences we e obse ed in
he Doce axel g oup (HR = 1.63; p = 0.202) (Fig.5F). The
mul i a ia e Cox’s eg ession analysis o all 57 pa ien s
unde going ICIs demons a ed ha hedec ease in EV
PD-L1 was an independen p edic i e bioma ke o
longe OS (HR = 0.35; p = 0.004); howe e , he issue
PD-L1 was no (Supplemen a y Table S4) (Fig.5G).
Radiomic ea u es complemen EV PD‑L1 o  hep edic ion
o  he esponse
Addi ionally, we pe o med an explo a o y analysis o
adiomics da a ob ained om baseline CT scans om
27 pa ien s om ou aining coho (A). Among hese
pa ien s, 11 (40.7%) we e classi ied as du able esponde s

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Fig. 3 EV PD‑L1 dynamics ou pe o med issue PD‑L1 as a p edic o o ICIs esponse: (A) Rep esen a i e axial sec ion compu ed omog aphy
(CT) images om a esponde and a non‑ esponde a baseline and du ing ICIs ea men . (B) Examples o immunohis ochemis y mic og aphs
o posi i e and nega i e issue PD‑L1 s aining (scale ba s 5 µm) and (C) EV PD‑L1 blo s om a esponde wi h dec easing EV PD‑L1 (0.29) and
a non‑ esponde showing an inc ease (1.55). (D) ICIs coho A (n = 33), non‑ esponde s (NR) showed inc eased EV PD‑L1 du ing ea men in
compa ison o esponde s (p = 0.017) (Mann–Whi ney U es ). (E) In he alida ion coho , non‑ esponde s unde going Pemb olizumab + Doce axel
(n = 24) showed a end owa ds inc eased EV PD‑L1 in compa ison o esponde s (p = 0.050) while hose ea ed wi h Doce axel alone (n = 15)
showed no di e ences (p = 0.794) (F) (Mann–Whi ney U es ). (G) As obse ed in he ROC cu e, EV PD‑L1 dynamics was a be e p edic o han
issue PD‑L1 TPS wi h an AUC = 74.4% s. 62.6% o he issue (bina y logis ic eg ession). (H) This was also obse ed in he alida ion coho o
pa ien s ea ed wi h ICIs wi h AUC = 75% o he EVs s. 64.1% o he issue. (I) In compa ison, simila AUCs we e obse ed in he Doce axel ea ed
g oup wi h 54.5% and 59.1%, espec i ely (bina y logis ic eg ession)
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deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
by RECIST and 16 (59.3%) by i RECIST, while 15
(55.6%) pa ien s we e conside ed ea ly esponde s by
RECIST and 18 (66.7%) by i RECIST, in he i s CT
scan. Figu e6A depic s he adiomics pipeline o ea-
u e ex ac ion and model selec ion, whe e he mos el-
e an ea u es o p edic RECIST du able esponse we e
selec ed and combined, esul ing in a model o 6 LASSO
ea u es (Supplemen a y Table S5). We compa ed he
p edic i e alue o his signa u e wi h he issue and EV
PD-L1. We obse ed ha o RECIST esponse, he com-
bina ion o ΔEV PD-L1 and he adiomics signa u e was
he bes model, able o iden i y non- esponde s wi h an
81.5% accu acy. A he same ime, o du able i RECIST,
indi idual ΔEV PD-L1 was he bes p edic i e model wi h
74.1% accu acy (Fig.6B & C). Simila ly, when p edic ing
ea ly esponse, he combina ion wi h adiomics also
imp o ed he p edic i e accu acy o he dynamics o EV
PD-L1 o ea ly RECIST esponse bu no o i RECIST
esponse (Supplemen a y Fig. S6). Fu he mo e, hese
six ea u es we e used o p edic su i al, which showed
ha only low TL_FLV7 was associa ed wi h wo se PFS
(HR = 5.52, p = 0.019) (Supplemen a y Fig. S7 & S8).
Discussion
Ad anced s ages o NSCLC a e cha ac e ized wi h a
supp essed immune sys em wi h educed coun s o
CD8 + T-cells, esponsible o he immune esponse
agains umo s [33]. Consequen ly, ICIs ha e become
one o he mos p omising he apeu ic op ions, e o-
lu ionizing he he apeu ic landscape o hese pa ien s.
Fig. 4 Changes in lesion size o du able esponse co ela ed wi h EV PD‑L1 dynamics in pa ien s unde going ICIs. (A) As obse ed in he co ela ion
ma ix, la ge inc eases in he umo lesion we e obse ed in pa ien s wi h inc eased EV PD‑L1 (p = 0.036) (Mann–Whi ney U es ) bu we e
independen o he le els o issue PD‑L1 (p = 0.330) (K uskal–Wallis es ). No associa ion was ound be ween he issue PD‑L1 TPS and he umo
esponse (p = 0.561) (Chi‑squa e es ). (B) Inc ease in EV PD‑L1 iden i ied non‑ esponde s (p = 0.009), howe e , nei he high issue PD‑L1 TPS > 50%
(p = 0.192) o TPS > 1% (p = 0.370) we e associa ed wi h du able esponse (Chi‑squa e es s)
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deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
Ne e heless, he e icacy o hese ea men s can s ill
be p ima ily imp o ed wi h he adequa e use o eliable
p edic i e bioma ke s ha could s a i y which pa ien s
would bene i om hem and a oid unnecessa y ad e se
e en s o hose who would no de i e bene i . Nowadays,
issue PD-L1 is he s anda d-o -ca e o pa ien s a i ica-
ion, howe e , i ails a p edic ing he e icacy o ICIs due
o se e al echnical and biological issues associa ed wi h
Fig. 5 EV PD‑L1 inc ease as a p edic i e bioma ke o PFS and OS. (A) Pa ien s wi h an inc easing EV PD‑L1 (blue) showed a end o sho e PFS
(p = 0.097) in he ICIs coho and demons a ed sho e PFS in he Pemb olizumab + Doce axel ea ed g oup (p = 0.020). S ill, no associa ion wi h
PFS was obse ed in he Doce axel g oup (p = 0.784) (C). (D) Longe OS was depic ed in pa ien s wi h EV PD‑L1 inc ease (blue) in he ICIs coho
(p = 0.031) and he Pemb olizumab + Doce axel g oup (p = 0.038) (E) while no in he Doce axel con ol g oup (p = 0.202) (F) (log‐ ank es s).
Numbe o pa ien s a isk o he e en is shown e e y 6 mon hs and he pe cen age o ee o e en (p og ession o dea h) pa ien s is shown a
12 and 24 mon hs. (G) In he 57 pa ien s unde going ICIs, an EV PD‑L1 inc ease was obse ed in hose wi h sho e PFS and OS while issue PD‑L1
was no ( issue PD‑L1 TPS, da k ed = > 50%, ed = 1–49%, pink < 1%, whi e = unknown; a ow = ongoing ea men ; black & whi e squa es ba = OS
a e ea men discon inua ion; x = exi us (dea h); o ange ci cles = p og essi e disease; illed da k blue ec angles = EV PD‑L1 inc ease
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deMiguel‑Pe eze al. J Exp Clin Cance Res (2022) 41:186
PD-L1 IHC [34]. Mo eo e , he high complexi y o he
immune landscape o NSCLC sugges ha many ma k-
e s migh be in ol ed in he esponse [35] and hence
a e needed o i s p edic ion. In his scena io, liquid
biopsy holds p omise as he eal- ime cha ac e iza ion o
umo s h ough he s udy o molecules ound in human
body luids, able o ack lung umo s e olu ion o e ime
[11]. Tissue and blood TMB o dynamics o c DNA ha e
been p oposed as po en ial bioma ke s. Howe e , hey
a e s ill no widely used in clinical p ac ice due o he
lack o me hod s anda diza ion and unce ain p edic i e
alue. Indeed, ecen analyses o pi o al s udies, includ-
ing KEYNOTE-189 [36], KEYNOTE-021 [37], o he
ecen esul s o he BFAST coho C s udy [38], aised
se e al conce ns on he alidi y o hese be o ehand
p omising bioma ke s, equi ing he e alua ion o o he
al e na i e ci cula ing ma ke s o e icacy. The e o e, we
examined he p edic i e ole o plasma EV PD-L1 exp es-
sion in a e ospec i e coho o pa ien s wi had anced/
me as a ic NSCLC unde going ea men wi h ICIs and
alida ed i in a p ospec i e analysis o a sub-coho om
he phase 2 PROLUNG clinical ial [22]. Fu he mo e, in
an explo a o y analysis, we included adiomics da a in a
mul ipa ame ic p edic i e model in combina ion wi h
he EV PD-L1.
Fi s , we demons a ed ha PD-L1 can be exp essed in
EVs in plasma om pa ien s wi h ad ancedNSCLC, con-
i ming p e ious indings obse ed in o he solid umo s
[39]. Then, we analyzed he dynamics o EV PD-L1 le els
om baseline o 8weeks o an i-PD-1 ea men in hese
pa ien s. We obse ed ha hese dynamic changes we e
associa ed wi h du able esponses o he ea men , since
non- esponde s showed inc eased le els in compa ison
o a dec ease in esponde s, e en co ela ed wi h he
change in lesion size. This concu s wi h p e ious s ud-
ies on EV PD-L1 in pa ien s and mice models ecei -
ing ICIs in which heinc ease in EV PD-L1 co ela ed
wi h poo esponse, disease ac i i y, o lesion size [15,
39, 40]. Simila ly, a smalle s udy analyzed he dynam-
ics o PD-L1 exp ession bu ocused only on exosomes
om 44 pa ien s wi hmelanoma unde going pemb oli-
zumab [13]. Con a y o ou esul s, his s udy desc ibed
inc eased le els o exosomal PD-L1 in bo h esponde s
and non- esponde s by i RECIST, obse ing a highe
inc ease du ing he 6 h week o ea men in esponde s
bu equal le els a he 9 h week. The idea o inc eased le -
els being a p edic o o be e esponse con adic s hei
esul s and hose om o he s udies which demons a ed
ha exosomal PD-L1 inhibi ed CD8 + T-cell unc ion
and acili a ed umo g ow h in in i o and in i o mod-
els [13, 15, 41]. This disc epancy could be caused by hei
use o he bes esponse by i RECIST as classi ica ion o
he clinical esponse, which would include as espond-
e s, pa ien s wi h new me as asis o oligop og ession, o
e en hose who sho ly esponded and hen p og essed.
In addi ion, he di e ence in imepoin s o second blood
Fig. 6 Combina ion o adiomics and EV PD‑L1 dynamics o p edic ing du able esponse: (A) Cha ac e is ic pipeline o adiomic analysis including
CT scan image segmen a ion, ea u e ex ac ion, and ea u e and model selec ion by machine lea ning. (B) The in oduc ion o he 6‑ ea u es
adiomic signa u e in o he ΔEV PD‑L1 p edic i e model o RECIST imp o ed i s pe o mance as obse ed in he conside able inc ease o sensi i i y
and speci ici y, wi h an accu acy o 81.5%. (C) On he con a y, he bes model o p edic ion o i RECIST only included he ΔEV PD‑L1 wi h an
accu acy o 74.1% (bina y logis ic eg ession)