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Ana Ba bosa de Sousa Nogal
P o eomic cha ac e iza ion o lung cance and ch onic
obs uc i e pulmona y disease: a b onchoal eola la age luid
analysis
Tese de Candida u a ao g au de Dou o em
Ciências Biomédicas subme ida ao Ins i u o de
Ciências Biomédicas Abel Salaza da
Uni e sidade do Po o.
O ien ado – Dou o Luis Paz-A es
Ca ego ia – Di ec o do Se iço de Oncologia
Médica e P o esso Ti ula de Medicina.
A iliação – Se iço de Oncologia Médica
(Hospi al Uni e si á io Vi gen del Rocío - HUVR,
Se ilha). Ins i u o de Biomedicina de Se ilha
(HUVR, Conselho Supe io de In es igação
Cien í ica - CSIC, Uni e sidade de Se ilha)
Co-o ien ado – Dou o a Ma ia Dolo es Pas o
Ca ego ia – In es igado a pós-dou o al
A iliação – Ins i u o de Biomedicina de Se ilha
(HUVR, Conselho Supe io de In es igação
Cien í ica - CSIC, Uni e sidade de Se ilha)
Co-o ien ado – Dou o Rui Manuel Medei os
Sil a
Ca ego ia – P o esso associado com
ag egação
A iliação – Ins i u o de Ciências Biomédicas
Abel Salaza da Uni e sidade do Po o. Ins i u o
Po uguês de Oncologia do Po o FG, EPE.
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Depois da empes ade…
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TABLE OF CONTENTS
ACKNOWLEDGMENTS!.......................................................................................................................!IX!
LIST!OF!ABBREVIATIONS!...................................................................................................................!XI!
LIST!OF!PUBLICATIONS!...................................................................................................................!XVII!
ABSTRACT!........................................................................................................................................!XIX!
RESUMO!........................................................................................................................................!XXIII!
INTRODUCTION!..................................................................................................................................!1!
1.1. Lung cance ........................................................................................................... 3
1.1.1. Epidemiology and causes ................................................................................ 3
1.1.2. Classi ica ion and s aging ................................................................................. 5
1.1.3. Diagnosis and ea men .................................................................................. 7
1.2. Ch onic obs uc i e pulmona y disease ................................................................. 9
1.2.1. Epidemiology and causes ................................................................................ 9
1.2.2. Diagnosis, classi ica ion, and ea men ......................................................... 10
1.3. Links be ween LC and COPD .............................................................................. 12
1.4. P o eomics ........................................................................................................... 15
1.4.1. In oduc ion o p o eomics .............................................................................. 15
1.4.2. P o eomics o COPD ...................................................................................... 19
1.4.3. P o eomics o LC ............................................................................................ 19
HYPOTHESIS!AND!OBJECTIVES!.........................................................................................................!23!
3.1. Main objec i e ...................................................................................................... 25
3.2. Speci ic objec i es ................................................................................................ 25
MATERIALS!AND!METHODS!.............................................................................................................!27!
4.1. Pa ien selec ion and sample collec ion ............................................................... 29
4.2. Sample p ocessing ............................................................................................... 29
4.2.1. Sample concen a ion ..................................................................................... 29
4.2.2. P o ein quan i a ion ........................................................................................ 30

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4.2.3. Deple ion o high abundance p o eins ............................................................ 30
4.2.4. Sample cleaning ............................................................................................. 30
4.3. Two-dimensional gel elec opho esis ................................................................... 31
4.3.1. Isoelec ic ocusing ......................................................................................... 31
4.3.2. S ip equilib a ion ............................................................................................ 32
4.3.3. 2D- PAGE ....................................................................................................... 32
4.3.4. Gel s aining and imaging ................................................................................ 33
4.3.5. Gel image analysis ......................................................................................... 34
4.4. Mass spec ome y ............................................................................................... 35
4.4.1. MS sample p epa a ion and analysis ............................................................. 35
4.4.2. P o ein iden i ica ion ....................................................................................... 36
4.5. An ibody a ays .................................................................................................... 37
4.6. Wes e n blo ......................................................................................................... 40
4.7. Enzyme-Linked Immunoso ben Assay ................................................................ 41
4.8. Bioin o ma ics analysis and s a is ical analysis .................................................... 42
4.8.1. 2D-PAGE bioin o ma ics analysis .................................................................. 42
4.8.2. An ibody a ays s a is ical analysis ................................................................. 43
4.8.3. An ibody a ays exp ession da a analysis ...................................................... 43
4.8.4. Diagnos ic es alidi y analysis ...................................................................... 43
RESULTS!...........................................................................................................................................!45!
5.1. Fi s speci ic objec i e: Iden i ica ion o di e en ly exp essed p o eins in BALF
om pa ien s wi h LC, COPD, COPD wi h LC, and wi hou LC o COPD by compa a i e
p o eomic analysis. ........................................................................................................ 47
5.1.1. Selec ion o pa ien s ....................................................................................... 47
5.1.2. P o eomic p o iling .......................................................................................... 48
5.1.3. Selec ion o p o eins and wes e n blo alida ion ........................................... 53
5.1.4. LC and COPD pa hway analysis .................................................................... 54
5.1.5. NF-kB unc ional analysis ............................................................................... 56
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5.2. Second speci ic objec i e: Analysis o he di e en ial exp ession o
in lamma ion ela ed p o eins (cy okines and g ow h ac o s) in LC and COPD
pa ien s. ...................................................................................................................... 58
5.2.1. Pa ien selec ion. ............................................................................................ 58
5.2.2. Exp ession o cy okines and g ow ac o s in BALF om LC and COPD
pa ien s. ...................................................................................................................... 61
5.2.3. Analysis o Il-11 and CCL1 as adenoca cinoma bioma ke s .......................... 64
GENERAL!DISCUSSION!AND!MAIN!CONCLUSIONS!...........................................................................!73!
FUTURE!PERSPECTIVES!....................................................................................................................!87!
REFERENCES!.....................................................................................................................................!91!
ARTICLES!........................................................................................................................................!111!
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ACKNOWLEDGMENTS
Ao D . Luis Paz-A es, meu o ien ado , po me e pe mi ido cump i um sonho de
ju en ude, pela opo unidade de aze pa e de uma no a ealidade, de um no o g upo de
in es igação, pelos b aços abe os com que me ecebeu, pela sua capacidade de
lide ança que inspi a odos a alcança semp e algo melho . Ob igada po oda a sua
ajuda, simpa ia e disponibilidade.
À D a. Ma ia Dolo es Pas o , minha co-o ien ado a, não exis em linhas su icien es pa a
ag adece udo o que ez po mim: mui o mais do que imagina e ei o. Mui o ob igada
pela comp eensão, pela paciência com o meu mau espanhol (de início), pela amizade e
simpa ia ex emas, pelo ca inho nos momen os mais complicados e pela apidez com que
os esol ia, pela sua de e minação e diligência e sob e udo ob igada pelo exemplo, pela
inspi ação e pela on ade de se algum dia algo pa ecido com ela.
Ao D . Rui Medei os, meu co-o ien ado , que me pe mi iu en a no campo da
in es igação quando e a apenas uma “jo em” em 2005, mui o ob igada pelo apoio e
amizade demons ados ao longo des es anos, pelas opo unidades e po as que me
ab iu, pela disponibilidade que demons a semp e que lhe en o no gabine e po qualque
mo i o.
À Fundação pa a a Ciência e Tecnologia, que me concedeu a bolsa de dou o amen o e
me pe mi iu aze , du an e 4 anos, aquilo que mais gos o.
Ao abuloso g upo de “Oncología molecula y nue as e apias”, o meu gigan e
ag adecimen o. Ob igada Sonia, Rocío, Ana, Rica do e demais colegas do HUVR e IBIS
pelo companhei ismo, pela o ça que me de am, pela comp eensão e paciência quando
me explica am as coisas mui o len amen e, pela momen os di e idos, pelas “se illanas
mane as” que me o am ensinando, pelo abalho que desen ol em dia iamen e e do qual
sin o mui o o gulho e saudade. Ob igada sob e udo po me aze em sen i em casa e po
me aze em esquece du an e a semana que es a a longe...
À Ana Coelho, D . A aújo e Raquel Ca a ino, pela o ien ação que me dão nes as
andanças, mesmo sem acha que o es ão a aze . Ob igada pelo apoio, amizade, po
aze em c esce como pessoa e como in es igado a.
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P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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LIST OF PUBLICATIONS
Acco ding o a icle 31 o he Dec ee-Law nº230/2009, he p esen Thesis has al eady
p oduced he ollowing publica ions in pee - e iewed jou nals in addi ion o a book
chap e :
Nogal A, Pas o MD, Molina-Pinelo S, Ca ne o A, Paz-A es L. P o eomic bioma ke s in
lung cance . Clinical & ansla ional oncology: o icial publica ion o he Fede a ion o
Spanish Oncology Socie ies and o he Na ional Cance Ins i u e o Mexico.
2013;15(9):671-82 (M. D. Pas o and A. Nogal con ibu ed equally o he p esen wo k).
Nogal A, Pas o MD, Molina-Pinelo S, Melendez R, Rome o-Rome o B, Mediano MD, e
al. Iden i ica ion o oxida i e s ess ela ed p o eins as bioma ke s o lung cance and
ch onic obs uc i e pulmona y disease in b onchoal eola la age. In e na ional jou nal o
molecula sciences. 2013;14(2):3440-55 (The i s wo au ho s con ibu ed equally o his
wo k).
Nogal A, Pas o MD, Molina-Pinelo S, Melendez R, Salinas A, Gonzalez De la Pena M, e
al. Iden i ica ion o p o eomic signa u es associa ed wi h lung cance and COPD. Jou nal
o p o eomics. 2013;89:227-37 (The i s wo au ho s con ibu ed equally o his wo k).
Ma Dolo es Pas o , Ana Nogal, Sonia Molina-Pinelo, Luis Paz-A es and Amancio
Ca ne o (2013). Oncop o eomic App oaches in Lung Cance Resea ch, Oncogenomics
and Cance P o eomics - No el App oaches in Bioma ke s Disco e y and The apeu ic
Ta ge s in Cance , D . Cesa Lopez (Ed.), ISBN: 978-953-51-1041-5, InTech, DOI:
10.5772/53873. A ailable om: h p://www.in echopen.com/books/oncogenomics-and-
cance -p o eomics-no el-app oaches-in-bioma ke s-disco e y-and- he apeu ic- a ge s-in-
cance /oncop o eomic-app oaches-in-lung-cance - esea ch
I he eby decla e ha I ha e ac i ely pa icipa ed in he ga he ing and s udy o he ma e ial
included in each o he publica ions p esen ed and ha e w i en he manusc ip s in
collabo a ion wi h he o he au ho s.
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ABSTRACT
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ABSTRACT
Cance is a majo wo ldwide public heal h issue and con inues o be a leading
cause o dea h in de eloped coun ies. On he op o incidence and mo ali y a es lies
lung cance (LC). This disease, caused mainly by obacco smoking, has one o he lowes
5-yea su i al a es among all ypes o cance s: only 15%. Despi e he ecen ad ances
in sys emic he apies, such as chemo he apy and a ge ed he apies, p ognosis emains
s ongly linked o ea ly diagnosis. Un o una ely, mos pa ien s a e diagnosed in ad anced
s ages, when cu a i e op ions a e limi ed. Consequen ly, he e is a p essing need o
de ec ion me hods ha may allow he diagnosis o LC ea ly in i s na u al his o y,
especially among high- isk indi iduals such as smoke s, pa icula ly i ch onic obs uc i e
pulmona y disease (COPD) is also p esen . The p ima y isk ac o o COPD is, as o
LC, obacco smoking. Howe e , his in lamma o y disease is an independen isk ac o o
he de elopmen o LC and some pa hogenic p ocesses a e hough o be impo an in he
de elopmen o bo h diseases. A be e unde s anding o he molecula and cellula
mechanisms by which ch onic in lamma o y diseases, such as COPD, lead o he ini ia ion
o LC, may iden i y ea ly diagnos ic bioma ke s and sc eening es s, aluable a ge s, and
mo e e ec i e he apeu ic s a egies.
The sea ch o ea ly de ec ion ma ke s has gained an ally in he las se e al yea s.
P o eomic echnologies ha e begun o unco e he molecula complexi y o lung umou s
by allowing a apid and comple e analysis o he p o eins ha a e exp essed in he con ex
o his disease. The analysis o lung samples om LC pa ien s and high- isk pa ien s by
p o eomic me hodologies migh allow us o gain mo e knowledge on he mechanisms o
bo h diseases and possibly iden i y LC diagnos ic bioma ke s ha could help in he clinical
p ac ice. The majo aim o he p esen wo k was o s udy he p o ein p o ile o
b onchoal eola la age luid (BALF) in pa ien s wi h LC and/o COPD using p o eomic
me hodologies in an a emp o iden i y new LC bioma ke s among high- isk pa ien s.
Pa ien s who had equi ed lexible b onchoscopy o diagnos ic pu poses a he
Hospi al Uni e si a io Vi gen del Rocío (Se ille, Spain) we e ec ui ed o his s udy. BALF
samples we e ea ed and submi ed o di e en p o eomic echnologies such as wo-
dimensional polyac ylamide gel elec opho esis (2D-PAGE), mass spec ome y (MS),
wes e n blo , an ibody a ays, and enzyme-linked immunoso ben assay (ELISA).
The use o a 2D-PAGE MALDI-TOF/TOF MS me hodology allowed he de ec ion
o 40 di e en ially exp essed p o eins be ween he con ol g oup (wi hou LC o COPD)
and he LC, COPD, and LC&COPD g oups. Ingenui y pa hway analysis (IPA) o hese
p o eins e ealed h ee op molecula mechanisms occu ing in bo h LC and COPD:
glycolysis and gluconeogenesis, ee adical sca enging and oxida i e s ess esponse,

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and in lamma ion. The majo connec o o all h ee pa hways was he nuclea ac o
kappa-ligh -chain-enhance o ac i a ed B cells (NF-κB), a key ansc ip ion ac o in he
in lamma o y p ocess and ca cinogenesis. The impo ance o in lamma ion in LC and
COPD was u he e alua ed by applica ion o BALF samples o cy okine and g ow h
ac o an ibody a ays. This analysis e ealed IL-11 and CCL1 as ma ke s o
adenoca cinoma o he lung wi h a eas unde he cu e (AUC) o 93 and 83%,
espec i ely. Sensi i i y and speci ici y alues we e 90 and 88% when e alua ing IL-11
and 80 and 74% when conside ing CCL1.
In conclusion, he p esen wo k has e ealed he p o eomic di e ences and
simila i ies be ween LC and COPD. In addi ion, he p o eomic me hodology used has
allowed he iden i ica ion o wo possible adenoca cinoma bioma ke s ha could be used
o de ec his ype o LC. The applica ion o a simple ELISA assay could be used o
imp o e he diagnosis o lung adenoca cinoma in smoke s, despi e he p esence o
absence o COPD. The use o his assay in ou ine clinical p ac ice, i alida ed in u he
s udies, could in he u u e imp o e he ea ly de ec ion o lung adenoca cinoma, cu en ly
he mos common o m o NSCLC, and hope ully con ibu e o an inc ease o he 5-yea
su i al a e associa ed wi h his disease.
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RESUMO
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RESUMO
O canc o é um p oblema de saúde pública em odo o mundo e con inua a se uma
das p incipais causas de mo e nos países desen ol idos. No opo das axas de
incidência e mo alidade encon a-se o canc o do pulmão. Es a neoplasia, causada
p incipalmen e pelo umo do abaco, em uma das meno es axas de sob e i ência a 5
anos en e odos os ipos de canc o: apenas 15%. Apesa dos ecen es a anços nos
a amen os sis émicos como a quimio e apia e medicamen os di ecionados, o
p ognós ico pe manece o emen e ligado ao diagnós ico p ecoce. In elizmen e, a maio ia
dos pacien es é diagnos icada em es adios a ançados da doença, onde as opções
e apêu icas são limi adas. Consequen emen e, há uma necessidade p emen e de
encon a mé odos de de ecção que pe mi iam o diagnós ico do canc o do pulmão no
início de sua his ó ia na u al, especialmen e en e os indi íduos de al o isco, como
umado es e pessoas com doença pulmona obs u i a c ónica. O p incipal a o de isco
pa a a doença pulmona obs u i a c ónica é, al como pa a o canc o do pulmão, o umo
do abaco. No en an o, es a doença in lama ó ia é um ac o de isco independen e pa a o
desen ol imen o de canc o do pulmão e pensa-se que alguns p ocessos pa ogénicos
possam se impo an es pa a o desen ol imen o de ambas as doenças. Uma melho
comp eensão dos mecanismos molecula es e celula es pelos quais doenças
in lama ó ias c ónicas, como a doença pulmona obs u i a c ónica, le am ao
desen ol imen o de canc o do pulmão, pode iden i ica bioma cado es de diagnós ico
p ecoce, al os e apêu icos, e es a égias de comba e à doença mais e icazes.
A p ocu a de ma cado es de de ecção p ecoce ganhou um o e aliado nos úl imos
anos. O uso de ecnologias p o eómicas pe mi iu começa a descob i a complexidade
molecula dos umo es pulmona es, já que pe mi em uma análise ápida e comple a das
p o eínas que são exp essas no con ex o des a doença. A análise de amos as de
pacien es com canc o de pulmão e pacien es de al o isco a a és de écnicas
p o eómicas pode pe mi i um maio conhecimen o sob e os mecanismos de ambas as
doenças e possi elmen e iden i ica bioma cado es de diagnós ico de canc o do pulmão
que possam auxilia na p á ica clínica.
O p incipal obje i o do p esen e abalho oi es uda o pe il p o eómico de la ado
b onco-al eola em pacien es com canc o do pulmão e/ou doença pulmona obs u i a
c ónica a a és de mé odos p o eómicos, na en a i a de iden i ica no os bioma cado es
diagnós icos de canc o do pulmão em pacien es de al o isco.
Pa a o es udo o am ec u ados pacien es aos quais inham sido e ec uadas
b oncoscopias pa a ins de diagnós ico no Hospi al Uni e si á io Vi gen del Rocío
(Se ilha, Espanha). Amos as de la ado b onco-al eola o am a adas e subme idas a
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cases. Adenoca cinomas a e cu en ly mo e o en diagnosed in women, young males
(<50 yea s old), non-smoke s and Asians (20). This his ological ype is de ined as a
malignan epi helial umou wi h glandula di e en ia ion o mucin p oduc ion and ends o
a ise in he pe iphe y o he lungs and he al eoli (Figu e 3B). The e a e se e al sub ypes
o adenoca cinoma, such as acina , papilla y, b onchioloal eola , solid wi h mucin
p oduc ion, and mixed. Howe e , mos adenoca cinomas a e his ologically he e ogeneous
and consequen ly classi ied as mixed (20-22).
Malignan epi helial umou s ha show ke a inisa ion and/o in e cellula b idges
a e de ined as SCC (23). Also known as epide moid ca cinomas, his g oup o lung
umou s was he mos equen bu now ep esen s app oxima ely 30% o all diagnosis
(16). They a e mo e common in men and smoke s and he majo i y occu as slow g owing
umou s in he b onchial epi helium o he cen al ai ways (Figu e 3C) (21). All NSCLCs
ha lack he cy ological and a chi ec u al cha ac e is ics o he adenoca cinomas o SCCs
a e classi ied as LCCs (24). They ep esen app oxima ely 10% o all lung cance cases
and hei incidence is dec easing mos likely due o imp o ed diagnos ic echniques
(Figu e 3D) (25). Al hough he accu a e classi ica ion o a lung umou is ex emely
impo an s ep in he e alua ion o a pa ien , he co ec de e mina ion o ex en o he
pa ien s’ disease is also necessa y o de e mine he cou se o ea men and p ognosis.
Figu e 3: His ological pa e ns o LC. (A) Small cell lung cance , om Jackman e al. (2005); (B)
adenoca cinoma, om Colby e al. (2004); (C) squamous cell ca cinoma, om Hamma e al.
(2004); (D) la ge cell ca cinoma, om Hamma e al. (2004).

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Lung cance s aging is based on he TNM classi ica ion o malignan umou s
(TNM). This classi ica ion was o iginally p oposed by Moun ain and adop ed by he
Ame ican Join Commi ee on Cance (AJCC) in 1973 and one yea la e by he Union o
In e na ional Cance Con ol (UICC) (26). The TNM sys em is used o he majo i y o
malignan umou s and is based on he ex en o he disease. I s goal is o assis
ea men planning, e alua ion o ea men ou comes, and p ognosis de e mina ion (27).
The le e T s ands o p ima y umou and de e mines he size and deg ee o loco egional
in asion. The N indica es he ex en o egional lymph node in ol emen , and he M
e eals he p esence o absence o in a ho acic o dis an me as asis (27, 28). The la es
edi ion o he TNM classi ica ion o lung cance can be ound on Figu e 4.
Figu e 4: Cha o he la es TNM classi ica ion o lung cance , om Uybico e al. (2010).
1.1.3. Diagnosis and ea men
Lung cance diagnosis includes e alua ion o clinical his o y, se e al ou ine blood
exams such as anaemia and li e enzymes, b onchoscopy o allow he e alua ion o he
ex en o he disease in he acheob onchial ee, and he umou iden i ica ion a e
biopsy (29). In o ma ion on umou size and local in asion a e ob ained by ches
adiog aphy (30). Compu e ized omog aphies (CT) ha e been o g ea help in LC s aging,
gi en ha hey o e 3D in o ma ion o he disease ex en such as medias inal in asion,
ches wall in asion, o he p esence o o he pulmona y nodules (29). Medias inum
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s aging is u he amended by endob onchial ul asound, endoscopic ul asound,
medias inoscopy, and posi on emission omog aphy. The la e is also a sensi i e me hod
o de ec me as a ic sp ead o he umou . Following he classi ica ion and s aging o a
pa ien s’ umou , cou se o ea men is decided, selec ing be ween he a ailable
he apeu ic op ions: su ge y, adio and chemo he apy, used sepa a ely o in di e en
combina ions.
Lung cance is usually asymp oma ic du ing i s de elopmen and his causes he
majo i y o pa ien s o be diagnosed a ad anced s ages o he disease. The accu a e
diagnosis o LC, including classi ica ion and s aging, is essen ial o he co ec ea men
and p ognosis o a gi en pa ien .
Tumou esec ion by su ge y is he mos success ul cu a i e op ion a ailable o
pa ien s wi h ea ly s age disease: s age I, II, and selec ed pa ien s wi h s age IIIA.
None heless, he su i al a e o hese pa ien s a ies g ea ly depending on hei disease
s age (28). S ages IIIB and IV, he g and majo i y o diagnoses, a e conside ed inope able
and o he ea men s, such as adio he apy and chemo he apy, a e applied.
Radio he apy is a aluable he apeu ic op ion o pa ien s who, despi e he
p esence o ea ly disease, a e medically un i o e use o unde go su gical esec ion. I is
also o use as adju an he apy o pa ien s wi h incomple e esec ion o node-posi i e
disease and as pallia i e he apy, p o iding symp oma ic imp o emen (31).
Chemo he apy was ini ially used in pa ien s wi h ad anced me as a ic disease as a
pallia i e measu e. Since hen, he applica ion o d ugs (cispla in, axanes,
an ime aboli es, opoisome ase inhibi o s, among o he s) has been used wi h cu a i e
in en combined wi h su ge y, neoadju an o adju an , and/o adio he apy, in an a emp
o imp o e he su i al o NSCLC pa ien s (32). In addi ion o hese adi ional d ugs,
a ge ed he apy has been imp o ing in he las yea s, ga he ing in o ma ion om
molecula s udies ha iden i ied speci ic al e a ions in g oups o LC pa ien s. The g ea es
achie emen s o his a ge ed he apy ha e been he de elopmen o monoclonal
an ibodies and y osine kinase inhibi o s agains he epide mal g ow h ac o ecep o
(EGFR), such as ce uximab and e lo inib, and he ascula endo helial g ow h ac o
(VEGF), such as be acizumab and so a enib, among o he s (Figu e 5) (33). In addi ion,
no el agen s ha a ge he echinode m mic o ubule-associa ed p o ein-like 4 (EML4) and
he anaplas ic lymphoma kinase (ALK) gene usion, o example c izo inib, he insulin-like
g ow h ac o -1 ecep o (IGF-1R), such as cixu umumab o linsi imib, he mammalian
a ge o apamycin (mTOR), o example de o olimus, and he hepa ocy e g ow h ac o
ecep o (MET), such as i an inib, a e cu en ly being in es iga ed in NSCLC clinical ials
(Figu e 5) (33).
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Al hough a lo o p og ess has been made, he 5-yea su i al a e o lung cance
is s ill app oxima ely 15%, ein o cing he need o diagnos ic ma ke s ha can aid in he
ea ly de ec ion o his umou .
Figu e 5: Mos ele an pa hways in NSCLC and de eloped a ge ed he apies, adap ed om Pal e
al. (2010).
1.2. Ch onic obs uc i e pulmona y disease
1.2.1. Epidemiology and causes
Ch onic obs uc i e pulmona y disease (COPD) is, acco ding o he Global
Ini ia i e o Ch onic Obs uc i e Pulmona y Disease (GOLD), a “p e en able and
ea able disease wi h signi ican ex apulmona y e ec s. The pulmona y componen o
COPD is cha ac e ized by a p og essi e ai low limi a ion associa ed wi h an a ypical
in lamma o y esponse o he lungs o noxious pa icles and gases” (34). The ai low
limi a ion p esen in COPD pa ien s is he esul o damages o he small and la ge
ai ways (ch onic b onchioli is and b onchi is, Figu e 6A) and o he pulmona y
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pa enchyma (emphysema, Figu e 6B) (35). The majo i y o pa ien s p esen signs o bo h
b onchi is and emphysema ins ead o one componen exclusi ely (36).
Figu e 6: His ological pa e ns o b onchi is wi h inc eased numbe o glands and hicke smoo h
muscle bundles (A) and emphysema wi h enla ged ai way spaces (B), om Macnee e al. (2007).
COPD is a leading cause o wo ldwide mo bidi y and mo ali y, apa om being a
majo economic and social bu den. The WHO es ima ed ha COPD was he i h leading
cause o dea h in 2006 and is expec ed o ise o ou h place by 2020 (3). I is clea ly
mo e equen in men and he elde ly, bu incidence is inc easing in women e lec ing
changes in hei exposu e o COPD isk ac o s since he middle o he 21s cen u y
The p ima y isk ac o o COPD is, as o LC, obacco smoking. The WHO
assessed ha 40 o 70% o all COPD mo ali y is ela ed o smoking (37). Age o smoking
ini ia ion, pack-yea s smoked, and cu en smoking s a us, in luence COPD de elopmen ,
p ognosis, and mo ali y (38). E en so, epo s s a e ha 15-50% o smoke s will de elop
some o m o COPD o e ime (36, 37). The e is a na u al decline in lung unc ion wi h
age, bu ac i e smoke s ha e an accele a ed a e o de e io a ion ha can be e e sed o
no mal le els o decline wi h sus ained smoking cessa ion.
1.2.2. Diagnosis, classi ica ion, and ea men
COPD is a mul icomponen and he e ogeneous disease ha a ies wi hin a g oup
o pa ien s wi h espec o lung pa hology, disease p og ession, and como bidi ies. Gi en
ha he clinical p esen a ion is a om uni o m, heal hca e p ac i ione s should conside
he diagnosis o COPD i any pa ien is o e 40 yea s o age, has been exposed o he
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a o emen ioned isk ac o , and p esen s symp oms such as b ea hlessness du ing
exe cise, inc eased e o o b ea h, cough, o excessi e mucus p oduc ion (39, 40). I
hese symp oms a e p esen , physical examina ion and spi ome ic es s a e equi ed o
make he accu a e diagnosis and disease s aging.
Spi ome y is he gold s anda d o he de ec ion o COPD, gi en i s high
ep oducibili y and a ailabili y (38). I is a simple and non-in asi e es ha measu es lung
unc ion: he amoun ( olume) and/o speed ( low) o ai ha can be inhaled and exhaled.
The e alua ed pa ame e s a e: pos -b onchodila o o ced expi a o y olume in 1 second
(FEV1) and he a io be ween FEV1 and o ced i al capaci y (FEV1/FVC). These wo
measu emen s a e used o de ec COPD and assess i s se e i y by compa ison wi h
e e ence alues ha a y wi h age, gende , ace and heigh (34).
The classi ica ion o COPD anges om mild o e y se e e. A no mal spi ome y
implies a FEV1/FVC a io supe io o 70% and a FEV1 supe io o 80%. COPD is
diagnosed when he FEV1/FVC a io is below 70% and i s se e i y a ies om mild o
e y se e e depending on he educ ion o pos -b onchodila o FEV1 compa ed o he
p edic ed in a heal hy popula ion (Table 1) (41). Radiological imaging can be used in he
assessmen o COPD se e i y since i can p o ide an accu a e cha ac e iza ion o
changes in he lung pa enchyma and ai way wall hickness, as well as exclude al e na i e
diagnoses (42). A e ial blood gas measu emen , α1-an i ypsin de iciency sc eening, and
he six minu e walk es a e also use ul in he diagnosis o his disease (34).
Table 1: COPD classi ica ion o pa ien s wi h a FEV1/FVC a io below 70%
Gold s age
Desc ip ion
FEV1 alues
GOLD 1
Mild
FEV1 ≥ 80% p edic ed
GOLD 2
Mode a e
50% ≤ FEV1 < 80% p edic ed
GOLD 3
Se e e
30% ≤ FEV1 < 50% p edic ed
GOLD 4
Ve y se e e
FEV1 < 30% p edic ed
Gi en ha no cu e is cu en ly a ailable and he e a e no he apies han can hal
he lung unc ion decline, he ea men o COPD a ies eno mously ac oss pa ien s. The
main goals o he ea men a e o educe symp oms and imp o e quali y o li e, educe
he equency and se e i y o he exace ba ions, and educe mo ali y (43). One o he i s
s eps in he ea men and managemen o hese pa ien s is smoking cessa ion. Non-
pha macological ea men s include long e m oxygen he apy ( ha can modi y long e m
lung unc ion decline), non-in asi e en ila ion, lung olume educ ion su ge y ( o pa ien s
wi h emphysema), and pulmona y ehabili a ion ha can p o ide elie om dyspnoea,

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inc eases exe cise ole ance, and imp o es quali y o li e (34). Pha macological
ea men s a e pa ien -speci ic and can include he use o an ibio ics, b onchodila o s,
co icos e oids, and mucoly ic agen s, alone o in combina ions (34, 42).
1.3. Links be ween LC and COPD
Lung cance and COPD a e wo o he majo causes o mo ali y in he wo ld. Apa
om being caused by he same majo e iological ac o , obacco smoking, epidemiological
da a demons a es a clea connec ion be ween bo h diseases. The odds a io (OR) alue
o LC inc eases 2.8 old in pa ien s wi h mode a e o se e e COPD (44). Independen ly o
smoking his o y, educed FEV1 is associa ed wi h an inc eased isk o de eloping LC:
e en small changes in his pa ame e , such as 10% di e ences, aise LC isk by 30-60%
(45). Indeed, 50 o 70% o all LC pa ien s ha e spi ome ic e idences o COPD (46). In
addi ion, he p esence o mild emphysema, also independen o ciga e e smoking and
his o y o o he lung diseases, con e s a g ea e isk o de elop LC (47). Since only a
ac ion o long- e m smoke s de elop hese diseases, complex mechanisms and
in e ac ions a e hough o de e mine an indi idual’s isk o de eloping COPD, LC, o bo h.
A a i s glance, i is di icul o en ision any common mechanism be ween bo h
diseases: LC is cha ac e ized by unlimi ed cell p oli e a ion, sus ained angiogenesis and
sel -su iciency in g ow h ac o s, issue in asion, e asion o apop osis, and insensi i i y o
an i-g ow h signals, whe eas COPD has inc eased le els o apop osis, ex acellula ma ix
deg ada ion, limi ed angiogenesis, and ine ec i e issue epai (48, 49). Unde s anding
he sha ed mechanisms be ween hese wo dissimila diseases is c i ical o de eloping
new me hods o ea ly diagnosis and ea men o LC.
Th oughou he yea s speci ic p ocesses ha e been sugges ed o be o impo ance
in he pa hogenesis o bo h COPD and LC (Figu e 7). Reac i e oxygen species (ROS)
caused by obacco smoke a e p oduced in excess in he se ing o COPD and can
ul ima ely p oduce DNA al e a ions ha lead o LC de elopmen . The balance be ween
cellula p oli e a ion and cell dea h is also impo an in he lung gi en i s con ibu ion o he
de elopmen o bo h diseases. Hypoxia and angiogenesis can also a ec he
de elopmen o COPD and LC: ai low limi a ion can lead hypoxia ha may cause
des uc ion o al eola capilla ies and ai way enla gemen , in addi ion o being capable o
p omo ing, h ough angiogenesis, he p og ession and me as asis o lung umou s (46,
50). Howe e , hese p ocesses a e all in luenced by and in e ac wi h a majo mechanism
ac i a ed by obacco smoking ha is in lamma ion.
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Figu e 7: Mechanisms and pa hways po en ially in ol ed in he de elopmen o COPD and LC,
adap ed om Yang e al. (2011).
In lamma ion is one o he body’s na u al p o ec i e mechanisms agains ha m ul
s imuli. I is he esul o complex in e ac ions be ween immune cells and soluble ac o s
ha a ises in issues as a esponse o in ec ions, auma, o oxic subs ances such as he
ones p esen in obacco smoke (51). I s majo ole is o emo e he sou ce o agg ession
and o ini ia e he healing p ocess. Ne e heless, a pe sis en in lamma o y esponse in
he lung can esul in he de elopmen o lung diseases, such as COPD and lung umou s.
COPD was ea ly ecognized as an in lamma o y disease, wi h p o ound
abno mali ies in he in lamma o y pa hways, and ha ai way in lamma ion s a s a an
ea ly s age, many yea s p io o he onse o clinical symp oms. Tobacco smoke induces
in lamma ion in all smoke s, bu hose who de elop COPD ha e a g ea e deg ee o
in lamma ion ha pe sis s e en a e smoking cessa ion (50). Tobacco smoke is a
complex mix u e o housands o chemicals gene a ed by obacco combus ion such as
ca cinogenic oxins, addic i e subs ances, and mo e han 1014 oxidan s and ee adicals
(46). These compounds i s in e ace wi h he lungs a he mucosal su aces lining he
ai ways. The e hey can damage epi helial cells and esiden in lamma o y cells by
inducing pe oxida ion o lipids and o he cell memb ane cons i uen s, ac i a e oxida i e-
sensi i e cellula pa hways, induce DNA damage, and ac i a e epi helial cell in acellula
signalling cascades ha lead o in lamma o y gene ac i a ion (52). The elease o hese
in lamma o y media o s ini ia es he ec ui men and ac i a ion o mo e p o-in lamma o y
cells such as neu ophils, mac ophages, and lymphocy es ha p opaga e he
in lamma o y esponse by eleasing mo e cy okines, o example in e leukin 8 (IL-8) and
umou nec osis ac o α (TNF-α), and ROS, gene a ing a icious cycle o pe sis en
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in lamma ion. In e es ingly, he immune cells and media o s ha d i e ch onic
in lamma ion and he p og ession o COPD a e also known o p omo e a umou iendly
mic oen i onmen (53).
The obse a ion o a ela ionship be ween cance and in lamma ion is no a ecen
one. Almos 2000 yea s ago, Galenus desc ibed he simila i ies be ween in lamed issue
and cance ous issue. Rudolph Vi chow, in he yea o 1863, epo ed he in il a ion o
leukocy es in malignan issues and sugges ed ha cance s a ise in si es o ch onic
in lamma ion (54). Mo e han one hund ed yea s la e D o ak obse ed ha cance and
in lamma ion sha e some de elopmen al mechanisms and ypes o in il a ing immune
cells, apa om classi ying umou s as “wounds ha do no heal” (55).
The ul ima e ecogni ion o in lamma ion as a majo playe in cance de elopmen
came wi h he 2011 upda e a icle on cance hallma ks by Hanahan and Weinbe g whe e
i was classi ied as an enabling cha ac e is ic o umou s. The e idence ga he ed o e he
las yea s showed ha in lamma ion con ibu es o he appea ance o mul iple cance
hallma k capabili ies by supplying impo an molecules o he umou mic oen i onmen .
Those molecules include g ow h ac o s ha sus ain he p oli e a i e signalling, su i al
ac o s ha limi apop osis, p o-angiogenic ac o s, ex acellula ma ix-modi ying enzymes
ha a ou angiogenesis, in asion, and me as asis. Fu he mo e, in lamma ion mani es s
i sel a he ea lies s ages o umou p og ession and is capable o nu u ing insipien
neoplasias in o de eloped cance s. In addi ion, in lamma o y cells can elease a numbe
o chemicals, such as ROS, ha a e ac i ely mu agenic and p omo e malignancy e en
u he (14).
S udies on LC and ch onic in lamma ion ha e e ealed ha he likelihood o
de eloping LC was inc eased in pa ien s wi h highe le els o C- eac i e p o ein (CRP), an
acu e-phase p o ein ha is a ma ke o in lamma ion (56). Also, he isk o de eloping LC
was dec eased among COPD pa ien s who had been ea ed wi h inhaled co icos e oids
(57). S udies on animal models ha e showed ha , a e in ec ion by Haemophilus
in luenza, mice de elop a b onchial in lamma ion simila o COPD and a subsequen
p edisposi ion o lung ca cinogenesis (58). The cons an lung inju y and epai cycle
igge ed by ch onic in lamma ion, such as he one ha occu s in COPD, can enhance cell
p oli e a ion and gene ic damage accumula ion, epi helial o mesenchymal ansi ion
(EMT), and ul ima ely lung ca cinogenesis (46).
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1.4. P o eomics
In he las se e al yea s genomic and p o eomic echnologies ha e begun o
unco e he molecula complexi y o lung umou s by allowing a apid and comple e
analysis o he genes and p o eins ha a e exp essed in he con ex o his disease. The
use o genomic echnologies such as mic oa ays and quan i a i e e e se ansc ip ion
polyme ase chain eac ion (RT-PCR) on LC s udies has allowed he sepa a ion o
adenoca cinoma pa ien s in o new molecula sub-classes wi h di e en ou comes, he
ea ly iden i ica ion o high isk pa ien s, and he de ec ion o molecula signa u es
associa ed wi h me as asis (59).
While genomic echnologies ha e e ealed impo an da a ega ding he biology o
LC, he di ec e alua ion o p o eins o e s in o ma ion ha is no ob ainable h ough he
s udy o DNA and RNA. P o eins a e c ucial ope a o s in he majo i y o biological sys ems
and signalling pa hways. Al hough hey can de e mine he cells’ pheno ype, RNA le els
o en poo ly co ela e wi h p o ein exp ession (60).
1.4.1. In oduc ion o p o eomics
The p o eome is de ined as he en i e collec ion o p o eins ha is p oduced by a
cell o issue in a gi en ime, making i a e y dynamic en i y: he ype o exp essed
p o eins, hei ela i e abundance, and hei subcellula loca ion depend on he
physiological s a e o a cell o issue (61).
P o eomics, he analysis o he p o eome, is pe o med h ough a a ie y o
echnologies ha a e con inuously e ol ing. T adi ional p o ein analysis echnologies
include app oaches such as wes e n blo , immunohis ochemis y (IHC), and enzyme-
linked immunoso ben assay (ELISA). P o ein mic oa ays, which consis o la ge numbe
o an ibodies obo ically immobilized on a slide, ha e a highe h oughpu and allow he
iden i ica ion o se e al p o eins a a ime in he same sample (62). P o ein mic oa ays
a e apid, au oma ed, economical, and highly sensi i e, consuming small quan i ies o
samples and eagen s. Howe e , he p e ious echniques a e a ge d i en, meaning hey
equi e a p e ious knowledge o he p o eins ha a e expec ed o appea in a gi en
sample. In con as o hese echniques, wo me hods do no equi e a p e ious
assump ion o he iden i y o numbe o p o eins in a sample and a e conside ed classical
p o eomic echnologies: wo-dimensional polyac ylamide gel elec opho esis (2D-PAGE)
and mass spec ome y (MS). The use o elec osp ay ioniza ion (ESI) and liquid
ch oma og aphy, second gene a ion p o eomics o sho gun p o eomics, has mo e ecen ly
allowed he iden i ica ion o complex and so a unknown p o eomes.
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The analysis o he p o eomic p o iles o LC and COPD pa ien s migh allow us o
gain mo e knowledge on he mechanisms o bo h diseases, o iden i y possible LC
p o eomic diagnos ic bioma ke s, as well as elucida e why some COPD pa ien s end up
de eloping LC.

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HYPOTHESIS AND OBJECTIVES
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2. HYPOTHESIS
The e a e di e ences in he p o ein exp ession (in lamma ion and/o o he
pa hways) o BALF among pa ien s wi h LC and/o COPD as compa ed o heal hy
indi iduals. These di e en ially exp essed p o eins could help o unde s and he molecula
pa hogens o LC and COPD, and may p o ide use ul bioma ke o diagnosis.
3. OBJECTIVES
3.1. Main objec i e
The p esen wo k ocused on iden i ying new bioma ke s as well as biological
pa hways in ol ed in he de elopmen o LC and/o COPD among high- isk
pa ien s by using p o eomic me hodologies, ocusing pa icula ly on he
in lamma o y pa hway.
3.2. Speci ic objec i es
3.2.1. Iden i ica ion o di e en ly exp essed p o eins in BALF om pa ien s
wi h LC, COPD, COPD wi h LC, and wi hou LC o COPD by
compa a i e p o eomic analysis.
3.2.2. Analysis o he di e en ial exp ession o in lamma ion ela ed p o eins
(cy okines and g ow h ac o s) in LC and COPD pa ien s.
!
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MATERIALS AND METHODS

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4. MATERIALS AND METHODS
4.1. Pa ien selec ion and sample collec ion
F om 2009 o 2011, a o al o 925 pa ien s who had equi ed lexible b onchoscopy o
diagnos ic pu poses a he Hospi al Uni e si a io Vi gen del Rocío (Se ille, Spain), we e
collec ed o his p ospec i e s udy. F om he o iginal i s g oup only 419 pa ien s
espec ed he selec ion c i e ia: 1) pa ien s we e unde pneumologis consul a ion due o
haemop ysis and/o a pulmona y nodule; 2) pa ien s we e eques ed by hei physician o
pe o m a lexible b onchoscopy o diagnos ic pu poses; 3) pa ien s we e smoke s o ex-
smoke s o mo e han 20 pack-yea s; 4) pa ien s had o be olde han 40 yea s o age.
Exclusion c i e ia included: 1) diagnosis o a neoplas ic disease o he han lung cance ; 2)
ac i e pulmona y ube culosis; 3) p e ious lung esec ion; 4) his o y o d ug abuse; 5)
p esence o o he acu e o ch onic in lamma o y diseases.
The s udy p o ocol was conduc ed in ag eemen wi h he Helsinki decla a ion and
app o ed by he Hospi al's E hical Commi ee, and a w i en in o med consen was
ob ained om all pa ien s p io o hei addi ion o he s udy.
Subjec s we e p epa ed wi h a combina ion o opical anaes hesia (20%
benzocaine sp ay o he pha ynx plus 2% opical lidocaine as needed) and conscious
seda ion using midazolam and mepe idine acco ding o ins i u ional guidelines. All BALF
samples we e ob ained by ins illa ion and aspi a ion o 40 o 60 ml aliquo s o 0.9% s e ile
saline in he app op ia e b onchopulmona y segmen . Reco e ed luid was immedia ely
passed h ough a 100 µm s e ile nylon il e (Bec on Dickinson, San Jose, CA) o emo e
mucus and anspo ed on ice o he labo a o y. The o al olume was hen cen i uged o
10 min a 1800xg and 4ºC. The supe na an was aliquo ed in o 2 ml ubes and ozen a –
80ºC un il u he use.
4.2. Sample p ocessing
4.2.1. Sample concen a ion
The amoun o p o eins in BALF samples is e y small, dilu ed in he s e ile saline
used o i s eco e y. In o de o use hese samples o p o eomics, hey equi ed
concen a ion and his was accomplished by he use o a Concen a o Plus acuum
concen a o (Eppendo , Hambu g, Ge many). BALF samples we e hawed on ice wi h a
p o ease inhibi o cock ail ki (Pie ce, The mo Scien i ic, Rock o d, IL, USA). Samples
we e hen aliquo ed in o new ubes and placed on he acuum concen a o . The ini ial
olume o he samples, usually 4-8 ml, was educed o 250-450 µl in 2-6 hou s.
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4.2.2. P o ein quan i a ion
A numbe o colo ime ic me hods we e es ed, such as bicinchoninic acid assay
(BCA om Pie ce, The mo Scien i ic, Rock o d, IL, USA, Rock o d, IL, USA), B ad o d
(Bio-Rad, He cules, CA, USA) and RC-DC (Bio-Rad, He cules, CA, USA). The RC-DC
me hod was selec ed gi en ha i was educing agen s and de e gen compa ible. I is
based on he p inciple o Low y es ima ion whe e p o eins eac wi h alkaline coppe and
subsequen ly educe he olin eagen , leading o colou de elopmen .
P o ein quan i a ion was assessed acco ding o he manu ac u e ’s ins uc ions.
Abso bance was measu ed a 750 nm and he linea equa ion c ea ed by di e en bo ine
se um albumin (BSA) dilu ions allowed he de e mina ion o he p o ein concen a ion in
ou samples. P o ein quan i a ion was assessed be o e and a e sample concen a ion,
deple ion o high abundance p o eins, and sample cleaning p ocedu es, which will be
desc ibed in he ollowing co esponding sec ions.
4.2.3. Deple ion o high abundance p o eins
The p esence o high abundance plasma p o eins obscu es he p esence o low
abundan ones o possible in e es . Re ealing hese impo an p o eins equi ed ha a
deple ion p ocess be applied o he analysis o BALF p o eomes.
Se e al comme cial ki s o deple ion columns we e es ed: P o eomine (Bio-Rad,
He cules, CA, USA), P o eoP ep (Sigma-Ald ich, S . Louis, MO, USA), and SpinT ap (GE
Heal hca e, Waukesha, WI, USA). The la e one showed he bes esul s in albumin and
immunoglobulin G emo al in ou samples.
Deple ion p o ocol ollowed he manu ac u e ’s ins uc ions. The SpinT ap columns
we e in e ed epea edly o esuspend he s o age medium, and hen cen i uged o
emo e i . Binding bu e was added and cen i uged ou o wash he column be o e he
addi ion o he concen a ed BALF sample. The sample was allowed o incuba e o 5
minu es o le he exis en albumin and immunoglobulin G bind o he column. The column
was hen cen i uged and he elua e collec ed. Binding bu e was added wice o he
column and he elua e was again collec ed. The deple ed BALF samples we e cleaned
immedia ely o ozen a -80ºC un il u he use.
4.2.4. Sample cleaning
A e he deple ion s ep BALF samples equi ed he emo al o sal s, hiols,
dena u an s, and o he con aminan s ha could be p esen and in e e e wi h he 2D-
PAGE p o ocol. O all a ailable me hods o clean p o ein samples ( ichlo oace ic acid
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p ecipi a ion, wo-s ep p ecipi a ion, ul a il a ion, and dialysis) and aking in o accoun
hei ad an ages and disad an ages, deple ed samples we e cleaned by he 2-D clean-up
ki (GE Heal hca e, Waukesha, WI, USA) ollowing he manu ac u e ’s di ec ions. A he
end, he p o ein pelle was ehyd a ed wi h a solu ion con aining u ea a 7M, hiou ea a
2M, and 2% CHAPS, sui able o 2D-PAGE (all h ee eagen s om GE Heal hca e,
Waukesha, WI, USA).
4.3. Two-dimensional gel elec opho esis
Two-dimensional gel elec opho esis is a p o ein analysis me hod ha allows he
sepa a ion o p o eins acco ding o wo inna e cha ac e is ics: isoelec ic poin (IP) and
molecula weigh (MW).
In he i s dimension, also desc ibed as isoelec ic ocusing (IEF), p o eins a e
sepa a ed in a gel s ip ha con ains a p ede e mined pH ange, allowing he p o eins o
sepa a e acco ding o hei isoelec ic poin . In he second dimension, he s ip is placed
on a SDS-PAGE gel in which he ocused p o eins mig a e acco ding o hei molecula
weigh . The inal esul is a SDS-PAGE gel whe e, a e s aining, p o ein spo s a e
obse ed.
4.3.1. Isoelec ic ocusing
T ea ed BALF samples (n=60), equally dis ibu ed among ou g oups o pa ien s
(con ols, LC, COPD, and LC&COPD), we e independen ly submi ed o IEF in 7 cm IPG
D yS ips wi h a 3-11 NL pH ange.
A mix u e o 75 µg o p o ein om each sample, DeS eak ehyd a ion solu ion,
and 0.5% o 3-11 NL pH IPG Bu e , in a inal olume o 125 µl, was p epa ed. This
mix u e was hen applied o he s ip holde . The s ip’s co e oil was ca e ully emo ed
om he posi i e end and he s ip placed, gel-side down and posi i e end i s , on o he
s ip holde , dis ibu ing he solu ion e enly unde he s ip. The s ip was hen o e laid
wi h co e luid and he s ip holde s placed on he E an IPGpho II o he IEF p o ocol
(Table 2). All eagen s and ins umen s we e om GE Heal hca e (Waukesha, WI, USA).
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GenePix 4100 A (Molecula De ices, Sunny ale, CA, USA), which was also used o
pe o m da a ex ac ion.

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Table 3: Cy okines and g ow h ac o s e alua ed by he an ibody a ay
Cy okine
symbol
Cy okine
name
G ow h ac o
symbol
G ow h ac o
name
BLC
B-cell lymphoma
AR
And ogen ecep o
Eo axin
Eo axin
BNDF
B ain-de i ed neu o ophic ac o
Eo axin2
(CCL24)
Eo axin 2
bFGF
Basic ib oblas g ow h ac o
G-CSF
G anulocy e colony-s imula ing ac o
BMP4
Bone mo phogene ic p o ein 4
GM-CSF
G anulocy e-mac ophage colony-s imula ing ac o
BMP5
Bone mo phogene ic p o ein 5
I309 (CCL1)
Chemokine (C-C mo i ) ligand 1
BMP7
Bone mo phogene ic p o ein 7
ICAM-1
In e -Cellula Adhesion Molecule 1
B-NGF
Be a ne e g ow h ac o
IFN γ
In e e on-gamma
EGF
Epide mal g ow h ac o
IL-1α
In e leukine-1 alpha
EGFR
Epide mal g ow h ac o ecep o
IL-1β
In e leukine-1 be a
EG-VEGF
Endoc ine gland-de i ed ascula
endo helial g ow h ac o
IL-1Ra
In e leukin-1 ecep o an agonis
FGF4
Fib oblas g ow h ac o 4
IL-2
In e leukine-2
FGF7
Fib oblas g ow h ac o 7
IL-4
In e leukine-4
GDF15
G ow h di e en ia ion ac o 15
IL-5
In e leukine-5
GDNF
Glial cell-de i ed neu o ophic ac o
IL-6
In e leukine-6
GH
G ow h ho mone
IL-6sR
In e leukine-6 soluble ecep o
HB-EGF
Hepa in-binding EGF-like g ow h ac o
IL-7
In e leukine-7
HGF
Hepa ocy e g ow h ac o
IL-8
In e leukine-8
IGFBP1
Insulin-like g ow h ac o -binding p o ein
1
IL-10
In e leukine-10
IGFBP2
Insulin-like g ow h ac o -binding p o ein
2
IL-11
In e leukine-11
IGFBP3
Insulin-like g ow h ac o -binding p o ein
3
IL-12p40
In e leukine-12 p40
IGFBP4
Insulin-like g ow h ac o -binding p o ein
4
IL-12p70
In e leukine-12 p70
IGFBP6
Insulin-like g ow h ac o -binding p o ein
6
IL-13
In e leukine-13
IGF-1
Insulin-like g ow h ac o 1
IL-15
In e leukine-15
Insulin
Insulin
IL-16
In e leukine-16
MCFR
Mac ophage chemo ac ic ac o ecep o
IL-17
In e leukine-17
NGFR
Ne e g ow h ac o ecep o
MCP-1 (CCL2)
Monocy e chemo ac ic p o ein-1/
Chemokine (C-C mo i ) ligand 2
NT-3
Neu o ophin 3
MCSF
Mouse s em cell ac o
NT-4
Neu o ophin 4
MIG (CXCL9)
Monokine induced by IFN-Gamma/
Chemokine (C-X-C mo i ) ligand 9
OPG
Os eop o ege in
MIP-1α
(CCL3)
Mac ophage in lamma o y p o ein 1 alpha/
Chemokine (C-C mo i ) ligand 3
PDGFAA
Pla ele -de i ed g ow h ac o AA
MIP-1β
(CCL4)
Mac ophage in lamma o y p o ein 1 be a/
Chemokine (C-C mo i ) ligand 4
PIGF
Phospha idylinosi ol-glycan biosyn hesis
class F
MIP-1δ
Mac ophage in lamma o y p o ein 1 del a
SCF
S em cell ac o
PDGFBB
Pla ele -de i ed g ow h ac o BB
SCFR
S em cell ac o ecep o
Ran es
(CCL5)
Regula ed upon Ac i a ion, No mal T-cell Exp essed, and
Sec e ed/ Chemokine (C-C mo i ) ligand 5
TGF-α
T ans o ming g ow h ac o alpha
TIMP-1
Tissue inhibi o o me allop o einases 1
TGF-β1
T ans o ming g ow h ac o be a 1
TIMP-2
Tissue inhibi o o me allop o einases 2
TGF-β3
T ans o ming g ow h ac o be a 3
TNF-α
Tumou nec osis ac o alpha
VEGF
Vascula endo helial g ow h ac o
TNF-β
Tumou nec osis ac o be a
VEGFR2
Vascula endo helial g ow h ac o
ecep o 2
TNFRI
Tumou nec osis ac o ecep o 1
VEGFR3
Vascula endo helial g ow h ac o
ecep o 3
TNFRII
Tumou nec osis ac o ecep o 2
VEGFD
Vascula endo helial g ow h ac o D
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4.6. Wes e n blo
Wes e n blo analyses we e pe o med o allow he alida ion o esul s om he
p e ious expe imen s,
The BALF samples we e p epa ed wi h Laemmli bu e 4X ha con ained a T is-
HCl 250 mM solu ion wi h a pH o 6.8, SDS a 4% (w/ ), glyce ol a 60% ( / ), and 2-
me cap oe hanol a 10% ( / ). They we e submi ed o SDS-PAGE sepa a ion a e boiling
o 5 min a 100ºC o dena u e p o eins. Depending on he molecula weigh o he p o ein,
SDS-PAGE sepa a ing gels (1.5 mm) we e made wi h 7.5, 10, and 12.5% ac ylamide as
p e iously desc ibed. Gels we e co e ed wi h wa e and le o polyme ize. As soon as he
gels we e se , a s acke gel wi h 4% ac ylamide was made and placed on op o he
sepa a ing gels wi h a comb o c ea e wells. The elec opho esis cell was illed wi h 1x
T is/Glycine/SDS Bu e unning bu e (T is-Base 25mM, GIycine 192mM, SDS 0.1%
(w/ )) (Bio-Rad, He cules, CA, USA) and he elec opho esis began. The un s a ed a a
low ol age (30-50 V) un il he samples had le he s acke gel. Then, he ol age was
aised o 100-120 V and was kep cons an un il he end o he un.
Gels we e hen ans e ed o poly inylidene luo ide (PVDF) memb anes (Bio-
Rad, He cules, CA, USA) in a ans e cell wi h 1x T is/Glycine ans e bu e (25 mM T is,
192 mM glycine, 0.1% SDS, and 20% me hanol) (Bio-Rad, He cules, CA, USA), a 4ºC o
3-4 hou s. The memb anes we e hen emo ed om he ans e casse es and blocked
o 1 hou in T is-bu e ed saline (TBS) wi h 0.05% Tween-20 (Sigma-Ald ich, S . Louis,
MO, USA) (0.05% TBST) and wi h 5% BSA (Sigma-Ald ich, S . Louis, MO, USA).
A e wa ds, he blo s we e incuba ed o e nigh a 4ºC wi h hei co esponding p ima y
an ibody (in TBST wi h 1% BSA), in he app op ia e p opo ion. The used an ibodies and
hei espec i e a ios can be seen on Table 4. Memb anes we e hen washed o emo e
he excess p ima y an ibodies and he seconda y an ibody, in he sui able a io, was
applied o he memb anes o 1 hou in TBST wi h 1% BSA, a oom empe a u e.
Memb anes we e hen washed o emo e he excess seconda y an ibodies and placed on
wa e .
Memb anes we e e ealed using enhanced chemiluminescence ECL acco ding o
he ins uc ions p o ided by he manu ac u e (GE Heal hca e, Waukesha, WI, USA) and
he memb anes we e exposed in he image analyse Mini LAS-3000 (Fuji ilm, Tokyo,
Japan). The ela i e p o ein le els we e calcula ed by compa ison o he amoun o β-ac in
p o ein (1:1000 Abcam, Camb idge, MA, USA). The expe imen s we e epea ed h ee
imes independen ly. The analysis o he di e en exp ession alues o he 8 p o eins
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ob ained by wes e n blo was pe o med by densi ome y. The densi ome y analysis o
he scanned blo s was done using Image J so wa e (118). Resul s we e no malized wi h
he exp ession o he con ol p o ein (β-ac in).
Table 4: Wes e n blo alida ion an ibodies and used a ios
Pu pose
Name
Ra ios
2D-PAGE alida ion
An i-HSP70, An i-AKR1B10, An i-PRDX1
(Epi omics, Bu lingame, CA, USA), An i-
PKM2, An i-NF-κBp65, An i-pNF-κBp65
(Cell Signaling, Be e ly, MA, USA)
1/1000
An ibody a ay alida ion
An i-IL-11, An i-CCL1 (I309) (Abcam,
Camb idge, MA, USA)
1/200
4.7. Enzyme-Linked Immunoso ben Assay
P o eins p e iously alida ed by wes e n blo we e e alua ed by ELISA in a i s
alida ion coho o 139 pa ien s and la e in a second alida ion coho o 160 pa ien s.
BALF samples we e applied as ins uc ed by he manu ac u e , using sandwich ELISA ki s
(RayBio ech, No c oss, GA, USA).
All eagen s we e placed a oom empe a u e (18 - 25°C) be o e use. S anda ds
we e p epa ed a di e en concen a ions o c ea e a s anda d cu e. One hund ed ml o
each s anda d and samples we e added in o hei espec i e wells. All samples and
s anda ds we e assayed in duplica e and he same pla e was used. The ELISA pla e was
co e ed and le o incuba e o 2.5 hou s a oom empe a u e wi h gen le shaking. The
wells we e washed 4 imes wi h 1x Wash Solu ion by illing each well wi h Wash Bu e
(300 µl). A e he las wash, he pla e was in e ed and blo ed agains clean pape owels
o emo e all excess Wash Solu ion. The 1x p epa ed bio inyla ed an ibody was added o
each well (100 µl) and le o incuba e o 1 hou a oom empe a u e wi h gen le shaking.
The wells we e hen washed 4 imes, as p e iously desc ibed. P epa ed S ep a idin
solu ion (100 µl) was added o each well and incuba ed o 45 minu es a oom
empe a u e wi h gen le shaking. The wells we e hen washed 4 imes, as p e iously
desc ibed. Finally, 100 µl o TMB One-S ep Subs a e Reagen we e placed on each well.
Incuba ion was pe o med o 30 minu es a oom empe a u e in he da k wi h gen le
shaking. The S op Solu ion was hen added o each well and pla es we e ead a 450 nm
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immedia ely on an EMax Mic opla e Reade (Molecula De ices, Minneapolis, Minn.,
USA). The mean abso bance o each se o duplica e s anda ds and samples was
sub ac ed by he a e age ze o s anda d op ical densi y. The s anda d cu e was c ea ed
on an Excel sp ead shee , wi h he s anda d concen a ion on he x-axis and abso bance
on he y-axis. Sample concen a ion was calcula ed by using he equa ion p o ided by
linea end g aph.
A g aphic ep esen a ion o he p o eomic analysis o BALF om LC and COPD
pa ien s by an ibody a ays and ELISA can be ound below on Figu e 13.
Figu e 13: Wo k low o he employed an ibody a ays and ELISA me hodology.
4.8. Bioin o ma ics analysis and s a is ical analysis
4.8.1. 2D-PAGE bioin o ma ics analysis
The p o eins selec ed om he 2D-PAGE expe imen s and subsequen ly iden i ied
by MS we e analysed by he Ingenui y Pa hway Analysis (IPA) web-based applica ion
(www.ingenui y.com, Ingenui y® Sys ems, Redwood Ci y, CA, USA). I uses a da abase
ha con ains up- o-da e in o ma ion on housands o genes and p o eins, o e one million
biological in e ac ions, and one hund ed canonical pa hways. This allows he iden i ica ion
o ela ionships, biological mechanisms, unc ions, and ele an pa hways associa ed wi h
he s udied p o eins. The IPA Co e analysis allows he assessmen o he pa hways and
biological p ocesses mos signi ican ly al e ed in ou da ase , he associa ed diseases and
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diso de s, he mos impo an molecula and cellula unc ions, and he iden i ica ion o he
signalling and me abolic pa hways mos ele an in ou da a se . Only p o eins iden i ied
by he IPA so wa e we e submi ed o co e analysis, ha e ealed he mos impo an
biological unc ions and pa hways associa ed wi h ou p o eins, and also c ea ed
in e ac ion ne wo ks be ween hem.
4.8.2. An ibody a ays s a is ical analysis
All con inuous a iables o pa ien s’ cha ac e is ics we e exp essed as a median o
each a iable (in e qua ile [IQR] ange) and ca ego ical a iables as numbe o cases and
pe cen age. The mean and SD o selec ed p o eins we e calcula ed o e e y pa ien in all
s udy g oups. The di e ences among g oups we e e alua ed by he Mann–Whi ney U
es . Di e ences we e conside ed s a is ically signi ican i hei p alue was ≤0.05
(*p≤0.05, **p<0.01, ***p<0.001).
S a is ical analysis was pe o med using he S a is ical Package o he Social
Sciences so wa e (SPSS 17, Chicago, Illinois, USA).
4.8.3. An ibody a ays exp ession da a analysis
Hie a chical supe ised clus e ing analysis was pe o med using he unc ion
UPGMA (Unweigh Pai G oup Me hod wi h A i hme ic Mean). Be o e s a is ical analysis,
p o ein exp ession le els we e s anda dized, p o ein by p o ein, ac oss all condi ions
using he medians and s anda d de ia ion (SD) alues. The sample condi ions we e
clus e ed using euclidean dis ance me ic es s. The esul s we e isualized and analysed
wi h Babelomics 4.2 (babelomics.bioin o.cip .es) (119). The exp ession le el o each
p o ein, ela i e o i s median exp ession le el ac oss all condi ions, was ep esen ed by a
colou , wi h ed ep esen ing exp ession g ea e han he median, blue ep esen ing
exp ession lesse han he median, and a ious in e media e colou in ensi ies
ep esen ing he magni ude o a iance om he median.
4.8.4. Diagnos ic es alidi y analysis
Recei e ope a ing cha ac e is ics cu es we e cons uc ed o assess sensi i i y,
speci ici y, and espec i e a eas unde he cu e (AUCs) wi h 95% con idence in e al (CI)
o possible LC bioma ke s. We in es iga ed he op imum cu -o alue, which was chosen

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in o de o e lec he bes coope a ion be ween sensi i i y, speci ici y, posi i e p edic i e
alue, nega i e p edic i e alue, and likelihood a io o p edic he diagnosis o lung
adenoca cinoma. To es he diagnos ic accu acy o se e al bioma ke s measu ed
oge he , he chi-squa ed es was used.
!
!
RESULTS
!
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5. RESULTS
5.1. Fi s speci ic objec i e: Iden i ica ion o di e en ly exp essed p o eins in BALF
om pa ien s wi h LC, COPD, COPD wi h LC, and wi hou LC o COPD by
compa a i e p o eomic analysis.
5.1.1. Selec ion o pa ien s
Six y samples om he pa ien s in he es coho we e di ided in o ou g oups:
con ol (wi hou LC o COPD), COPD, LC, and LC&COPD. The p incipal cha ac e is ic
om es coho , we e all male, wi h a median age o 61 (41-80) yea s. The majo i y o
pa ien s we e cu en smoke s, wi h simila pack-yea s 30 (28.6- 37.2). In he COPD
g oup, he majo i y o pa ien s had mode a e and se e e cases o COPD, and in he
LC&COPD g oup, o e hal o pa ien s had mild COPD. The adenoca cinoma his ology
was he p edominan his ology o LC in he LC and LC&COPD g oups (Table 5). O e all
pa ien cha ac e is ics we e well ma ched among he ou s udy g oups.
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The wes e n blo analysis o he selec ed p o eins, oge he wi h he le els o he
e e ence p o ein β-ac in, a e shown in Figu e 17. The samples used o wes e n blo
alida ion we e andomly selec ed.
Ou esul s showed ha he AKR1B10 p o ein had inc eased exp ession only in
he LC g oup. On he o he hand, HSP70 was o e -exp essed by all disease g oups when
compa ed o he con ol g oup. The PRDX1 p o ein showed an inc eased exp ession in
he COPD and LC&COPD g oups compa ed o he LC and con ol g oups. Finally, PKM2
was clea ly o e -exp essed in he BALF o he pa ien s in he LC and LC&COPD g oups
compa ed o hose in he COPD and con ol g oups. The esul s o he wes e n blo
alida ion p ocess con i med hose ob ained by he 2D-PAGE expe imen s.
Figu e 17: Wes e n blo s o AKR1B10, HSP70, PKM2, PRDX1, and β-ac in in he con ol, COPD,
LC, and LC&COPD g oups. Di e ences in exp ession, no malized wi h he β-ac in p o ein, a e
illus a ed by a ba cha on he igh .
5.1.4. LC and COPD pa hway analysis
The connec ions and in e ac ions be ween ou 40 di e en ially exp essed p o eins
and also he biological mechanisms, unc ions, and pa hways in which hey we e possibly
in ol ed in we e e alua ed by he IPA so wa e. In addi ion, he associa ion be ween
ce ain diseases and diso de s and ou da a se we e also analysed by IPA.
Co e analysis was pe o med o include di ec and indi ec connec ions, and
endogenous chemicals, and o conside only molecules and/o associa ions, c ea ing an
in e ac ion ne wo k ha can be seen on Figu e 18. Cellula compa men s as designa ed
by IPA (gene on ology based), indica ed ha he majo i y o iden i ied p o eins we e o
cy oplasma ic o igin.

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Figu e 18: IPA in e ac ion ne wo k conside ing he di e en ially exp essed p o eins be ween he
con ol, LC, COPD, and LC&COPD g oups.
The mapping o ou di e en ially exp essed p o eins on o known molecula
pa hways and biological unc ions e ealed h ee op p ocesses occu ing in he BALF
samples om ou ou g oups o pa ien s: in lamma ion, ee adical sca enging and
oxida i e s ess esponse, and glycolysis and gluconeogenesis. Toge he wi h hese
indings, he ecogni ion o he NF-kB complex as he majo connec ion node be ween
hese h ee mechanisms was also accomplished.
The majo i y o iden i ied p o eins, a o al o 22 p o eins, we e associa ed o
in lamma o y mechanisms (AKR1B10, AKR1C3, ALDOA, ANXA1, ANXA2, ANXA5,
ARHGDIB, CA1, CAT, CFL1, CRP, ENO1, EZR, GSR, HSP70, LCN2, PEBP4, PPIA,
PRDX1, PRDX2, SELENBP1, and TKT), 16 p o eins we e ela ed o ee adical
sca enging and oxida i e s ess (ARHGDIB, CAT, CRP, CTSD, EZR, GSR, GSTA1,
GSTA2, GSTP, IDH1, PPIA, PRDX1, PRDX2, PRDX5, SERPINB1, and TXN), and 8
p o eins we e ound o be connec ed o he glycolysis and gluconeogenesis p ocesses
(ALDH3A1, ALDOA, ENO1, FBP1, IDH1, PEBP4, PKM2, and PYGM), as seen on Figu e
19.
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Figu e 19: Top biological unc ions and pa hways iden i ied by IPA; in lamma ion ela ed p o eins (p
alue: 1,35*10-08–1,42*10-02), ee adical sca enging and oxida i e s ess esponse (p alue:
4,93*10-11–1,27*10-02), and glycolysis and gluconeogenesis ela ed p o eins (p alue: 7,39*10-09–
1,58*10-02).
5.1.5. NF-kB unc ional analysis
NF-κB was he main connec o be ween he di e en ially exp essed p o eins, e en
hough his p o ein was no iden i ied as di e en ially exp essed among he ou g oups o
pa ien s analysed by 2D-PAGE.
Consequen ly, we analysed NF-kB in BALF samples om he con ol, COPD, LC,
and LC&COPD g oups o pa ien s, by measu ing he exp ession le els o he NF-κBp65
p o ein and i s phospho yla ed (ac i a ed) o m, pNF-κBp65, by wes e n blo (Figu e 20).
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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Figu e 20: Wes e n blo s o NF-κBp65, p NF-κBp65, and β-ac in in he con ol, COPD, LC, and
LC&COPD g oups. Di e ences in exp ession, no malized wi h he β-ac in p o ein, a e illus a ed by
a ba cha on he igh .
High exp ession le els o NF-κBp65 we e de ec ed in all g oups, wi h a sligh ly
highe in ensi y in he COPD g oup. The phospho yla ed o m o NF-κBp65 showed highe
exp ession le els only in he disease g oups (COPD, LC, and LC&COPD), wi h he LC
g oup p esen ing he highes in ensi y. These wes e n blo analyses a e compa ible wi h
he ac ha ac i a ion o NF-κBp65 migh be in ol ed in he pa hogenesis o bo h
diseases.
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5.2. Second speci ic objec i e: Analysis o he di e en ial exp ession o
in lamma ion ela ed p o eins (cy okines and g ow h ac o s) in LC and COPD
pa ien s.
5.2.1. Pa ien selec ion.
The an ibody a ay me hodology was applied o samples om 60 pa ien s, whose
cha ac e is ics can be ound on Table 8. All selec ed pa ien s we e male, wi h median
ages a ound 61 (41-80) yea s. The majo i y o pa ien s we e cu en smoke s, wi h simila
pack-yea s smoking exposu e. Mode a e and se e e COPD we e he mos common
s ages in he COPD g oup o pa ien s, and mild COPD was p edominan in he LC&COPD
g oup o pa ien s. The adenoca cinoma his ology was he mos common among he
pa ien s in he LC g oup, con a y o he pa ien s in he LC&COPD g oup, who had been
p edominan ly diagnosed wi h he SCC his ology. All o he cha ac e is ics we e well
ma ched among he s udy g oups.
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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Table 8: Cha ac e is ics o he disco e y coho pa ien s o he an ibody a ays me hodology
Con ols
n=16
COPD
n=15
LC
n=17
LC&COPD
n=12
Gende
Male
100.0% (16)
100.0% (15)
100.0% (17)
100.0% (12)
Female
0.0% (0)
0.0% (0)
0.0% (0)
0.0% (0)
Median age
[ ange]
61.3
[41.0-80.0]
61.5
[45.0-78.0]
60.7
[46.0-69.0]
60.7
[49.0-68.0]
Smoking s a us
Smoke s
68.8% (11)
53.3% (8)
52.9% (9)
83.3% (10)
Ex-smoke s
31.2% (5)
46.7% (7)
47.1% (8)
16.7% (2)
Pack-yea s
[ ange]
38.8
[31.0-53.2]
50.0
[42.0-65.0]
58.2
[41.0-65.7]
52.3
[41.0-63.2]
COPD
Mild
0.0% (0)
20.0% (3)
0.0% (0)
58.3% (7)
Mode a e
0.0% (0)
33.3% (5)
0.0% (0)
25.0% (3)
Se e e
0.0% (0)
26.7% (4)
0.0% (0)
0.0% (0)
Ve y se e e
0.0% (0)
20.0% (3)
0.0% (0)
16.7% (2)
LC His ology
Adenoca cinoma
S ages I-II
0.0% (0)
0.0% (0)
11.8% (2)
0.0% (0)
S ages III-IV
0.0% (0)
0.0% (0)
58.8% (10)
33.3% (4)
SCC
S ages I-II
0.0% (0)
0.0% (0)
5.9% (1)
8.3% (1)
S ages III-IV
0.0% (0)
0.0% (0)
23.5% (4)
58.4% (7)
SCC: squamous cell ca cinoma
To alida e he esul s ob ained by he aining coho , a g oup o 299 pa ien s was
selec ed and wo dis inc alida ion coho s we e c ea ed. The i s alida ion coho was
made up o 139 pa ien s ha we e sepa a ed in o ou dis inc g oups. The pa ien s in his
coho had simila clinical and pa hological cha ac e is ics o hose in he aining coho s
(Table 9).

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Table 9: Cha ac e is ics o he pa ien s in he i s alida ion coho
Con ols
n=20
COPD
n=29
LC
n=40
LC&COPD
n=50
Gende
Male
60.0% (12)
86.2% (25)
82.5% (33)
96.0% (48)
Female
40.0% (8)
13.8% (4)
17.5% (7)
4.0% (2)
Median age
[ ange]
52.3
[42.0-58.0]
65.2
[45.0-78.0]
61.2
[48.0-75.0]
64.3
[48.0-75.0]
Smoking s a us
Smoke s
100.0% (20)
51.7% (15)
50.0% (20)
52.0% (26)
Ex-smoke s
0.0% (0)
48.3% (14)
50.0% (20)
48.0% (24)
Pack-yea s
[ ange]
41.8
[33.0-57.2]
52.8
[41.0-69.2]
55.0
[39.0-73.2]
57.0
[43.0-75.2]
COPD
Mild
0.0% (0)
27.6% (8)
0.0% (0)
26.0% (13)
Mode a e
0.0% (0)
51.7% (15)
0.0% (0)
50.0% (25)
Se e e
0.0% (0)
20.7% (6)
0.0% (0)
24.0% (12)
Ve y se e e
0.0% (0)
0.0% (0)
0.0% (0)
0.0% (0)
LC His ology
Adenoca cinoma
S ages I-II
0.0% (0)
0.0% (0)
10.0% (4)
10.0% (5)
S ages III-IV
0.0% (0)
0.0% (0)
27.5% (11)
36.0% (18)
SCC
S ages I-II
0.0% (0)
0.0% (0)
25.0% (10)
30.0% (15)
S ages III-IV
0.0% (0)
0.0% (0)
37.5% (15)
24.0% (12)
SCC: squamous cell ca cinoma
The cha ac e is ics o he 160 pa ien s in he second alida ion coho can be seen
on Table 10. This coho was signi ican ly di e en om he i s one gi en ha i did no
include a g oup o pa ien s wi h only COPD and ha he LC g oups had pa ien s
diagnosed no only wi h adenoca cinoma and SCC, bu also LCC and SCLC.
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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Table 10: Cha ac e is ics o he pa ien s in he second alida ion coho
Con ols
n=20
LC
n=66
LC&COPD
n=74
Gende
Male
60.0% (12)
83.3% (55)
94.6% (70)
Female
40.0% (8)
16.7% (11)
5.4% (4)
Median age
[ ange]
52.3
[42.0-58.0]
61.2
[48.0-75.0]
64.3
[48.0-75.0]
Smoking s a us
Smoke s
100.0% (20)
50.0% (33)
52.7% (39)
Ex-smoke s
0.0% (0)
50.0% (33)
47.3% (35)
Pack-yea s
[ ange]
33.6
[30.0-51.2]
58.8
[41.0-77.2]
62.0
[43.0-79.2]
LC His ology
Adenoca cinoma
S ages I-II
0.0% (0)
4.5% (3)
2.7% (2)
S ages III-IV
0.0% (0)
18.2% (12)
13.6% (10)
SCC
S ages I-II
0.0% (0)
6.1% (4)
8.1% (6)
S ages III-IV
0.0% (0)
12.1% (8)
5.4% (4)
LCC
S ages I-II
0.0% (0)
4.5% (3)
8.1% (6)
S ages III-IV
0.0% (0)
7.6% (5)
10.8% (8)
SCLC
Limi ed s age
0.0% (0)
19.7% (13)
21.6% (16)
Ex ensi e s age
0.0% (0)
27.3% (18)
29.7% (22)
SCC: squamous cell ca cinoma; LCC: la ge cell ca cinoma; SCLC: small cell lung cance
5.2.2. Exp ession o cy okines and g ow ac o s in BALF om LC and COPD pa ien s.
The ini ial p o ec i e esponse, elici ed by he damaging complex mix u e o
chemicals p esen in obacco smoke, can gene a e he elease and ec ui men o mo e
p o-in lamma o y immune cells and cy okines and c ea e a ch onic in lamma ion scena io
and a umou iendly mic oen i onmen . So, we aimed a analysing which in lamma o y
p o eins had al e ed exp ession in ou ou g oups o pa ien s. Fo his pu pose we used
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an ibody a ays ha measu ed he le els o 80 cy okines and g ow h ac o s implica ed in
he in lamma o y p ocess.
A o al o 15 cy okines and g ow h ac o a ays we e ca ied ou and he da a
analysis was pe o med by measu ing he di e en exp ession le els be ween he 80
in lamma o y p o eins and he posi i e con ols p esen in he a ays. P o ein exp ession
le els we e hen s anda dized, p o ein by p o ein, ac oss all condi ions by using he
medians and SD alues. Resul s we e isualized and analysed wi h Babelomics 4.2
(babeIomics.bioin o.cip .es). We c ea ed a hie a chical supe ised clus e ing analysis o
he p o ein a ays using he UPGMA unc ion. The gene a ed dendog ams o he
cy okines and g ow h ac o s exp ession le els and he g oups o pa ien s, and hea map
a e ep esen ed below in Figu e 21.
The o iginal ou g oups o pa ien s (con ol, COPD, LC, and LC&COPD) we e sub-
di ided in o six g oups aking in o conside a ion he his ological sub- ypes o NSCLC;
con ol, COPD, adenoca cinoma (ADC), SCC, ADC&COPD, and SCC&COPD. Analysis
e ealed ha 20% o he e alua ed p o eins in he cy okine and g ow h ac o a ays we e
o e -exp essed in he disease g oups compa ed o he con ol g oup. These 16 p o eins
we e MIP-1β, MIG, IGFBP1, EGF, VEGF, TNFRI and TNFRII, IL-6S , GDF15, IL-1Ra,
MCP-1, Eo axin2, PDGFAA, IGFBP2, IL-11, and CCL1.
A i s g oup o p o eins (MIP-1β, MIG, IGFBP2, and EGF) was highly exp essed
ac oss all disease g oups, compa ed o he con ol g oup. A second g oup o p o eins
(VEGF, TNFRI and TNFRII, IL-6S , GDF15, IL-1Ra, MCP-1, Eo axin2, and PDGFAA) also
had a highe exp ession in he disease g oups, bu no as high as he p e ious g oup o
p o eins. In e es ingly, he e we e clea di e ences in p o ein exp ession be ween he
adenoca cinoma his ological sub ype and he o he g oups. In ac , he exp ession o IL-11
and CCL1 was inc eased di e en ially in adenoca cinoma.
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Figu e 21: Hea map o he di e ences in p o ein exp ession ac oss he disease g oups, compa ed
o he con ol g oup, ob ained om he in lamma o y p o ein a ays; p o ein exp ession le els
ela i e o i s median exp ession le el ac oss all condi ions, a e ep esen ed by a colou , wi h ed
ep esen ing exp ession g ea e han he median, blue ep esen ing exp ession lesse han he
median, and a ious in e media e colou in ensi ies ep esen ing he magni ude o a iance om
he median.
S a is ical analysis o he cy okine and g ow h ac o a ays, seen on Figu e 22,
e ealed signi ican s a is ical di e ences (p≤0.05) among g oups o he majo i y o
p o eins, despi e he exis ing dispe sion be ween hem due o pa ien a iabili y.
Among he 16 in lamma o y p o eins ha had s a is ically signi ican di e en
exp ession le els when compa ed be ween he pa ien s o he disease g oups and he
con ol g oup pa ien s, IL-11 and CCL1 we e obse ed o ha e a simila beha iou . These
wo p o eins we e only o e -exp essed in he BALF samples om pa ien s wi h ADC and
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Figu e 28: IL-11 and CCL1 exp ession le els measu ed by ELISA in he second alida ion coho .
S a is ically signi ican exp ession di e ences (p<0.0001), ob ained by he Mann-Whi ney U es ,
we e ound be ween he ADC g oups and all emaining g oups.
The ROC cu e analysis o he second alida ion coho can be seen on Figu e 29.
This analysis e ealed ha he op imum diagnos ic cu -o alue o IL-11 was 29.5 pg/ml
(AUC – 0.95; 95%CI: 0.92 - 0.98). The AUC associa ed wi h his cu -o indica ed ha a
pa ien wi h lung adenoca cinoma had highe le els o IL-11 han 95% o con ols. The
es ima ed cu -o alue o CCL1 was 24.25 pg/ml (AUC – 0.91; 95%CI: 0.87 - 0.96),
which showed ha a lung adenoca cinoma pa ien would ha e highe le els o CCL1 han
91% o pa ien s in he con ol g oup.

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Figu e 29: Diagnos ic ou comes o IL-11 and CCL1 le els in BALF in he diagnosis o
adenoca cinoma ( e sus all g oups) in he second alida ion coho .
In o de o u he alida e he esul s om coho alida ion i s , he cu -o alues
om he i s coho we e applied o he da a om he second alida ion coho . P edic i e
alues and likelihood a ios o IL-11 and CCL1 in he second alida ion coho can be
ound on Table 12.
Posi i e le els o IL-11, CCL1, IL-11 and CCL1, and IL-11 and/o CCL1 we e
de ec ed on app oxima ely 91%, 92%, 71%, and 92% o pa ien s wi h lung
adenoca cinoma (Figu e 30). These pe cen ages we e simila o hose ob ained in he i s
alida ion coho . Likewise, speci ici y and PPV inc eased when bo h ma ke s we e
analysed oge he compa ed o measu ing each one in sepa a e (96.3% and 84.1%
espec i ely). Inc eased alues o sensi i i y (92.3%) and NPV (98.1%) we e obse ed
when he e alua ion o only one ma ke was equi ed o posi i ely p edic he
adenoca cinoma ou come, simila o wha occu ed wi h he i s alida ion coho .
Speci ici y (84%) and PPV (62.5%) also inc eased in his case, con a y o wha occu ed
in he i s alida ion coho .
1-
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Table 12: Analysis o IL-11 and CCL1 le els as diagnos ic ma ke s o adenoca cinoma in BALF o
pa ien s om he second alida ion coho , wi h cu -o alues om he i s alida ion coho
Adenoca cinoma
s all pa ien s
IL-11
CCL1
IL-11
and CCL1
IL-11
and/o CCL1
AUC (95%CI)
0.95
(0.92-0.98)
0.91
(0.87-0.96)
-
-
Sensi i i y
(95%CI)
90.6%
(79.7-95.9%)
91.7%
(80.4-96.7%)
71.2%
(57.7-81.7%)
92.3%
(82.6-98.1%)
Speci ici y
(95%CI)
83%
(80.8-87.7%)
77.5%
(71.0-82.9%)
96.3%
(92.5-98.2%)
84%
(78-88.5%)
PPV
(95%CI)
60.8%
(49.7-70.8%)
51.2%
(40.8-61.4%)
84.1%
(70.6-92.4%)
62.5%
(51.5-72.3%)
NPV
(95%CI)
96.8%
(92.7-98.6%)
97.3%
(93.3-99%)
92.3%
(87.7-95.3%)
98.1%
(94.6-99.4%)
Posi i e LR
5.32
(3.81-7.41)
4.08
(3.09-5.04)
19.1
(9.0-41.13)
5.88
(4.21-8.22)
Nega i e LR
0.11
(0.05-0.26)
0.11
(0.04-0.28)
0.3
(0.19-0.46)
0.07
(0.02-0.20)
AUC= a ea unde cu e. PPV= posi i e p edic i e alue. NPV= nega i e p edic i e alue. LR= likelihood a io. The
diagnos ic cu -o alues o IL-11 and CCL1 we e 42 pg/ml and 39.5 pg/ml espec i ely.
Figu e 30: P opo ion o adenoca cinoma pa ien s om he second alida ion coho wi h posi i e
le els o IL-11, CCL1, IL-11 and CCL1, and IL-11 o CCL1.
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GENERAL DISCUSSION
AND MAIN CONCLUSIONS
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6. GENERAL DISCUSSION AND MAIN CONCLUSIONS
Lung cance is one o he mos commonly diagnosed malignancies and kills mo e
pa ien s han any ype o cance in he wo ld. App oxima ely 90% o all hese LC ypes a e
a ibu able o one majo ac o : obacco smoking.
The diagnosis o LC is a c ucial s ep gi en i s in luence in he p ognosis o a
pa ien . The use o ches adiog aphies, b onchoscopies, among o he es s, is common
when sea ching o LC. A e wa ds, a ailable he apeu ic op ions include su ge y, adio
and chemo he apy, used sepa a ely o in di e en combina ions. Howe e , LC is usually
asymp oma ic du ing i s de elopmen and his causes he majo i y o pa ien s o be
diagnosed a ad anced s ages o he disease, limi ing he apeu ic op ions and p ognosis.
When a pa ien is diagnosed a an ea ly s age, he 5-yea su i al a e lies be ween 43-
73%. Wi h a s age II diagnosis, 25-45% o people will li e o a leas 5 yea s. When
conside ing mo e ad anced s ages, he ou comes change d ama ically: only 7-24% o
pa ien s diagnosed wi h s age III will li e mo e han 5 yea s a e hei ini ial diagnosis;
s age IV, cha ac e ized by he p esence o me as asis, has a 5-yea su i al a e be ween
2 and 13% (120).
Radiological sc eenings ha e been used o many yea s o he ea ly de ec ion o
LC. S ill, da a om se e al sc eening ials has e ealed ha pe o ming annual ches
adiog aphies in smoke s, o me smoke s, and non-smoke s is no e ec i e in educing
LC mo ali y and canno be ecommended o clinical p ac ice (121). Mo e ecen ly, low
dose CT scanning was associa ed wi h a signi ican educ ion in LC mo ali y (20%), in
one la ge s udy o high- isk indi iduals. Howe e , mo e da a a e needed on he cos
e ec i eness o sc eening ha akes in o accoun he equency o sc eening and bo h he
bene i s and ha ms (such as alse posi i es and o e diagnosis) be o e la ge-scale
sc eening p og ammes a e c ea ed (122).
In addi ion o adiological me hods, he cy ological analysis o ce ain samples is
also help ul o he diagnosis o LC. Howe e , pulmona y cy ology has a iable sensi i i y
and speci ici y alues, and alse posi i e diagnoses a e somewha common, gi ing ha
he cy ological e alua ion o he lungs has some limi a ions (123). The use o
b onchoscopy o localize lesions and ea ly LC p esen s some challenges: SCC is mos ly
de ec ed in he cen al a eas o he lungs, whe eas adenoca cinomas, he mos common
ype o LC in ecen yea s, a e mos ly ound in he pe iphe al ai ways o he lungs,
un eachable o he majo i y o b onchoscopes. In conclusion, he diagnosis o LC in ol es
a combina ion o he adiological and his ological e alua ion o a symp oma ic pa ien o a
suspicious lesion, equi ing he cy ological examina ion spu um o in b onchoscopy

P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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ob ained samples, such as biopsies and BALF (124). These es s a e some imes
inconclusi e, making i necessa y o pe o m in asi e su gical p ocedu es in pa ien s ha
may o may no ha e he disease.
The abo emen ioned classical me hods o diagnosing LC a e cu en ly e ol ing o
less in asi e, molecula o ien ed app oaches o he de ec ion o his disease. The use o
au o luo escence b onchoscopy, lung issue molecula ma ke s, blood based LC ma ke s,
ai way based bioma ke s, among o he s, a e cu en ly being e alua ed o he ea ly
de ec ion o LC. Al hough some ecen s udies ha e shown ha hese molecula
bioma ke s can ha e a posi i e impac in he imp o emen o LC diagnosis, ex ensi e
alida ion o he mos p omising molecules ha a ise om hese s udies is c ucial be o e
hey can be used o sc een indi iduals a high isk o de eloping LC (125-127).
Apa om being he majo cause o LC de elopmen , ciga e e smoke is also
esponsible o ano he common illness ha a ec s he espi a o y sys em, which is
COPD. Fo a long ime i was hough ha he link be ween bo h diseases was hei
ae iological ac o . None heless, he obse a ion ha COPD pa ien s had high LC mo ali y
a es challenged ha concep . Despi e he inc eased a en ion gi en o he s udy o he
commonali ies be ween bo h diseases, ei he by clinical s udies and esea ch, he ue
na u e o he link ha connec s bo h diseases, emains elusi e. Howe e , as discussed
below, he e a e key sha ed mechanisms ha may ep esen links be ween he wo
diseases, and po en ially diagnos ic and he apeu ic a ge s.
Some common gene ic al e a ions play a ole in he de elopmen o COPD and
LC, o example, he α1 an i ypsin p o ein inhibi s he neu ophil elas ase ha deg ades
elas in and has an i apop o ic capabili ies. The α1 an i ypsin de iciency causes he
de elopmen o emphysema and inc eases he isk o de eloping LC by 70% (128). O he
genes ha ha e been associa ed wi h LC and COPD include hose ha code p o einases,
de oxi ying enzymes, nico ine ecep o s, and in lamma o y p o eins (129). The enzymes
ha con ibu e o he me abolism o he compounds ound in obacco smoke can
occasionally ans o m hese molecules in o mo e ha m ul subs ances, such as
ca cinogens and ROS. The ac ion o hese me aboli es in he lung epi helial cells can
p omo e he de elopmen o bo h LC and COPD.
Ano he seemingly impo an mechanism connec ing bo h diseases is
in lamma ion. Mac ophage and neu ophil in il a ion is a common occu ence in all
smoke s, bu hose who ha e COPD de elop a mo e ma ked in lamma o y esponse ha
co ela es wi h disease se e i y (130). In addi ion, he cells ha con ibu e o he
de elopmen o , o example, emphysema exis bo h in he ai ways and al eola ai spaces
and can he e o e con ibu e o he de elopmen o p oximal and dis al LC (129). Al hough
bo h diseases ha e ch onic in lamma ion as a ea u e, he na u e o he mic oen i onmen
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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in COPD and LC is di e en and con ibu es o he dis inc beha iou s ha cha ac e ize
bo h diseases. The COPD mic oen i onmen is mo e cy o oxic, geno oxic, and has mo e
ma ix deg ading capabili ies han he LC mic oen i onmen , which is mo e angiogenic
and g ow h p omo ing (129).
The e a e s ill many unanswe ed ques ions conce ning LC and COPD and he
links be ween bo h diseases. In addi ion, a p essing need exis s o disco e new
molecula bioma ke s ha can aid in he ea ly de ec ion o LC. This la e a ea o esea ch
has been g an ed a majo ally wi h he de elopmen and he imp o emen o ce ain
analy ical echnologies, such as genomics and p o eomics. Bo h echnologies we e
designed o pe o m he apid and comple e analysis o he genes and p o eins ha a e
exp essed in a gi en cell o issue in a gi en ime. Despi e he be e unde s anding o he
gene ic al e a ions p esen in LC gi en by genomic app oaches, such as mu a ions and
polymo phism in key genes, he impo ance o he e alua ion o p o eins canno be
o e looked.
P o eomics complemen s he genomic-based app oaches by p o iding addi ional
in o ma ion ha canno be ob ained by he s udy o genes alone. One o he mos
impo an ad an ages o he p o eomic app oach in cance esea ch is based on he ac
ha he e is gene ally a poo co ela ion be ween he ansc ip ional le els o many genes
and he ela i e abundance o he co esponding p o ein. Fu he mo e, due o di e en ial
splicing, one gene can encode se e al p o ein a ian s wi h dis inc p ope ies (131). In
addi ion o ela i e abundance, he ac i i y o a gi en p o ein is no a di ec consequence
o i s gene ic exp ession, bu he esul o pos - ansla ional modi ica ions such as
phospho yla ion, glycosyla ion, me hyla ion, ubiqui ina ion, clea age, and ace yla ion
(132). Despi e he many ad an ages when compa ed o o he app oaches, he use o
p o eomic echnologies has o be applied ca e ully in o de o p o ide ele an biological
esul s and o be success ully ansla ed in o he clinical p ac ice. Challenges associa ed
wi h p o eomics include he he e ogenei y o cells ha exis in he lungs, which can be
malignan o no mal, he b oad dynamic ange o p o ein abundances in p oximal body
luids and se um ha di icul he iden i ica ion o bioma ke s in hose ypes o samples,
and inally he need o ex ensi e alida ion o he molecula changes in independen
coho s o pa ien s using di e en echniques (131).
P o eomic me hodologies can be applied o he disco e y o new LC bioma ke s as
well as aid wi h he p ognosis, p edic ion and moni o ing o a pa ien esponse o a gi en
he apeu ic in e en ion. Such ma ke s o disease can be eleased di ec ly om cance
cells o can be a ibu able o he hos ’s esponse agains he malignancy i sel . The
de ec ion o hese candida e bioma ke s can be pe o med in a a ie y o body luids
collec ed by a non-in asi e me hod. Bioma ke s ob ained by hese me hods a e usually
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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mo e sensi i e, mo e speci ic, and mo e easily de ec ed han in asi e bioma ke s
(ob ained by p ocedu es such as su ge y). In addi ion, hey a e associa ed wi h less
anxie y and discom o o he pa ien s. Samples such as blood, u ine, se um, and sali a
a e majo sou ces o non-in asi e bioma ke s and commonly used in cance esea ch.
Al hough mo e in asi e han he p e ious samples, BALF is a g ea sou ce o possible
bioma ke s o lung diseases gi en ha i is hough o e lec mos ai h ully he p o ein and
cell composi ion o he pulmona y ai ways among he non-in asi e echniques (133). The
p esen wo k aimed a e alua ing his pa icula ype o sample by di e en p o eomic
echniques wi h he aim o iden i ying p o eins ha could be used as bioma ke s o he
diagnosis o LC, some hing ha o he bes o ou knowledge has no been done be o e.
The i s p o eomic echnique used in his s udy was 2D-PAGE. This ool has been
he d i ing o ce behind he de elopmen o p o eomics and p o ein analysis h oughou
he yea s. This echnique has been ex ensi ely es ed and compa ed be ween di e en
labo a o ies h oughou he yea s makes i one o he mos obus p o eomic me hods.
Ou esul s showed ha he p o ein composi ion o BALF was di e en among he
ou g oups o s udied pa ien s, especially when we compa ed he con ol g oup o he
disease g oups. A o al o 40 we e di e en ially exp essed among he ou g oups o
pa ien s and he exp ession changes we e supe io o 2 old (p<0.05). The dis ibu ion o
hese di e en ially exp essed p o eins allowed us o de e mine he speci ic p o eomic
p o iles o he a ious g oups o pa ien s.
Se en een p o eins we e o e -exp essed in all he pa ien s om he disease
g oups (LC, COPD, and LC&COPD) compa ed o hose in he con ol g oup. These
p o eins we e AMY1A, AMY2A, ANXA1, ANXA2, ANXA5, ARHGDIB, CA1, CRP, C3,
ENO1, GSR, HSP70, IDH1, PEBP4, SERPINB1, TPPP3, and TXN. The exp ession o
HSP70, which in he p esen s udy had an a e age o e -exp ession o 3.6 old ac oss he
h ee disease g oups, was also ound signi ican ly ele a ed in se um samples om COPD
pa ien s, when compa ed o con ols (134). I has also been linked o he numbe o
ciga e es smoked daily in a s udy o lung adenoca cinoma in smoke s (135). The use o
p o eomic app oaches has e ealed he associa ion be ween he o e -exp ession o
HSP70 and he di e en ia ion le el and/o agg essi eness o se e al ypes o cance ,
such as gas ic adenoca cinomas, hepa oca cinomas, and oesophageal cance (136-
138). Fu he mo e, a ious oncop o eomic s udies ha e co ela ed he ele a ed le els o
HSP70 wi h he apeu ic esis ance (139-142).
Ano he impo an p o ein in his g oup is CA1. This p o ein is a ma ke o cellula
hypoxia, which is a na u al pheno ype o solid umou s. Se e al s udies ha e in es iga ed
he po en ial o ca bonic anhyd ases as cance diagnos ic, p ognos ic, and he apeu ic
ma ke s (143). Recen ly, he o e -exp ession o CA1 was epo ed in ca cinoma cell lines
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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om mul iple o gans, such as lung, b eas , ce ix, bladde , oesophagus, colo ec al,
kidney, and head and neck (144). PEBP4 which plays a ole in he inhibi ion o he
mi ogen-ac i a ed p o ein kinase (MAPK) signalling pa hway bu is also in ol ed in he
inhibi ion o he c-Jun N- e minal kinases (JNK) pa hway ha p omo es he ac i a ion o
p o ein kinase B (AKT), was ecen ly associa ed wi h an inc eased in asion and
me as asis in NSCLC and colo ec al umou s (145-147).
The IDH1 enzyme is essen ial o cell me abolism and ene gy p oduc ion, gi en i s
ole in he K ebs cycle. In he p esen s udy, he IDH1 p o ein had an a e age o e -
exp ession o 2.3 old in he disease g oups. The oncogenic po en ial o his enzyme has
been s udied in acu e myeloid leukaemia (148). An also ecen epo has obse ed he
o e -exp ession o IDH1 in mo e han 70% o NSCLC umou s. I was also co ela ed wi h
lowe 5-yea su i al a es and conside ed an independen un a ou able p ognos ic
ma ke o o e all su i al o NSCLC pa ien s in a mul i a ia e analysis (149). In addi ion,
IDH1 plasma le els we e conside ed a bioma ke o lung adenoca cinoma wi h ela i ely
high sensi i i y and speci ici y (150).
The nex g oup o di e en ially exp essed p o eins appea ed in he LC and
LC&COPD g oups o pa ien s. In he LC g oup o pa ien s he AKR1B10, ALDOA, CTSD,
EZR, FBP1, and TKT p o eins we e o e -exp essed when compa ed o he con ol g oup
and he SELENBP1 p o ein was unde -exp essed in his g oup o pa ien s compa ed o
hose in he con ol g oup. A numbe o s udies ha e associa ed he p esence o CTSD,
Ez in, and SELENBP1 wi h inc eased cance g ow h, in asion, and me as asis in a ious
ypes o umou s (151-153). Fu he mo e, h ee di e en s udies ha e highligh ed he
impo ance and po en ial ole o hese p o eins as p ognos ic bioma ke s o lung umou s
(154-156). Ano he o e -exp essed p o ein in ou esul s was he AKR1B10 p o ein. In
o he s udies, his o e -exp ession was associa ed in mos cases wi h smoking,
sugges ing a possible in ol emen o his enzyme in obacco-induced LC (157-159). In
ac , AKR1B10 has a high ca aly ic e iciency o he educ ion o e inoids, and e inoic
acid de iciency has been linked o ai way epi helial squamous me aplasia and epi helial-
o-mesenchymal ansi ion (160). In addi ion, AKR1B10 has also been ecen ly in ol ed in
he esis ance o di e en chemo he apeu ic d ugs, such as cispla in (161).
Apa om hese di e en ially exp essed p o eins, he LC g oup o pa ien s
oge he wi h he LC&COPD g oup o pa ien s, had an o e -exp ession o he ollowing
p o eins when compa ed o he con ol g oup; ALDH3A1, AKR1C3, PYGM, PKM2, and
PPIA. The ALDH3A1 p o ein is a phase II d ug-me abolizing enzyme ha is highly
exp essed in he lung, s omach, ke a inocy es, and co nea. Cy osolic ALDH3A1, which is
induced by polycyclic a oma ic hyd oca bons o chlo ina ed compounds (p esen in
obacco smoke), is hough o play an impo an ole in al eola pneumocy e physiology,
P o eomic!cha ac e iza ion!o !lung!cance !and!ch onic!obs uc i e!pulmona y!disease:!a!b onchoal eola !la age! luid!analysis!
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encou aging. The p esen wo k, h ough he applica ion o a 2D-PAGE MALDI-TOF/TOF
me hodology, iden i ied dis inc p o eomic p o iles cha ac e is ic o LC, COPD and
LC&COPD, which we e la e alida ed. The bioin o ma ics analysis o he 40 di e en ially
exp essed p o eins iden i ied in BALF sugges ed ha LC and COPD sha ed some
pa hogenic pa hways such as in lamma ion, ee adical sca enging and oxida i e s ess
esponse, and glycolysis and gluconeogenesis. In addi ion, IPA analysis exposed a majo
connec o o hese pa hogenic mechanisms: he NF-kB ansc ip ion ac o . The u he
explo a ion o he in lamma o y p ocess in ou pa ien s e ealed ha he de e mina ion o
IL-11 and CCL1 le els in BALF samples by a simple ELISA assay, could be used o
imp o e he diagnosis o lung adenoca cinoma in smoke s, ega dless he p esence o
absence o COPD. The applica ion o his assay o ou ine clinical p ac ice could in he
u u e imp o e he ea ly de ec ion o lung adenoca cinoma, he cu en ly mos common
o m o NSCLC, and possibly con ibu e o an inc ease in he 5-yea su i al a e
associa ed wi h his disease.

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FUTURE PERSPECTIVES
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Ana!Ba bosa!de!Sousa!Nogal! ! 89!
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7. FUTURE PERSPECTIVES
The sea ch o molecula bioma ke s in easily accessible samples is o i al
in e es in LC esea ch. The diagnosis o LC is cu en ly commonly ob ained in ad anced
s ages o he disease and is un o una ely associa ed wi h poo su i al and a lack o
he apeu ic op ions.
Ou esul s e ealed he exis ence o di e en p o eomic p o iles be ween pa ien s
wi h LC and COPD. The p o eins included in hese signa u es dese e u he alida ion
and in es iga ion as po en ial bioma ke s o ea ly diagnosis and p ognosis, and possibly
as he apeu ic a ge s. The explo a ion o he in lamma o y p ocess in he samples om
ou pa ien s e ealed he po en ial o wo p o eins as LC bioma ke s. Highe le els o IL-
11 and CCL1 in BALF om smoke s we e associa ed wi h he p esence o
adenoca cinoma o he lung.
Fu u e s udies aiming a alida ing he indings in he p esen wo k should include
a la ge sample size, bo h in LC cases and con ols. Fu he alida ion o he diagnos ic
pe o mance o IL-11 and CCL1 in mo e accessible luids such as plasma would be
desi able and mo e p ac ical o e en ual sc eening p og ams o ou ine diagnosis. In
addi ion, he e alua ion o IL-11 and CCL1 as p ognos ic and he apeu ic ma ke s should
also be add essed in u u e in es iga ions. Fu he mo e, he pe o mance o he IL-11 and
CCL1 p o eins on he di e en ial diagnosis o pleu al e usions (be ween lung
adenoca cinoma, meso helioma, adenoca cinoma o o he o igins, and non umo al)
would also be use ul. The associa ion o hese wo p o eins wi h he de elopmen o o he
ypes o in lamma ion- ela ed cance s should also be o in e es .
Finally, he unc ional ole o IL-11 and CCL1 in he ca cinogenic p ocess should
also be assessed. S udies aiming a e alua ing he capabili ies o hese wo p o eins in
di e en cell lines and animal models could shed some ligh on hei ac ion in he lung
ca cinogenic p ocess and also unco e new possible a ge ed he apies.
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REFERENCES

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!
!
ARTICLES
PRDX2, and ecep o o ac i a ed p o ein kinase 1 [60].
Fi e p o eins we e ound o be unde -exp essed in he same
samples: c ea ine kinase B (CKB), SCCA1 as down- egu-
la ed [58], ca hepsin D p ep o ein (CTSD), e i in hea y
chain (FTH1), and ANXA3 (unde -exp essed) [59]. The
iden i ied annexins in hese p o eomic s udies ha e a
po en ial ole in cellula signal ansduc ion, exocy osis,
in lamma ion, coagula ion, cellula g ow h and di e en i-
a ion. Using issue and se um om SCC and heal hy
indi iduals, Yang and co-wo ke s de ec ed an inc ease in
au oan ibodies agains TIM and SOD2 ha could aid in he
diagnosis o his ype o lung cance [61]. Finally, by
compa ing se um om SCC pa ien s and con ols, i e
o e -exp essed (HP, APO-A4, complemen componen
C3c, SAA, and Ras- ela ed p o ein 7B—RAB7B) and i e
unde -exp essed (AHSG, hemopexin p ecu so —HPX,
p oapolipop o ein, an i h ombin III—SERPINC1, and
CLU) p o eins we e iden i ied and we e able o dis inguish
SCC pa ien s [62].
A summa y o he mos ecu en ly iden i ied diagnos ic
bioma ke s on lung cance issue samples and in luid
samples can be ound on Tables 1and 2, espec i ely.
P ognos ic
When he exp ession le els o a p o ein co ela e wi h he
na u al his o y o he disease hey a e conside ed o ha e
p ognos ic alue. The s udy o p ognos ic bioma ke s in
lung cance has been made by co ela ing he exp ession o
a molecule o he pa ien su i al. An al e na i e app oach
is o compa e g oups o pa ien s wi h di e en clinical
s ages o disease, based on he assump ion ha a mo e
ad anced umou is mo e agg essi e and may exp ess
p o eins ha d i e he me as a ic p ocess. The published
p o eomic s udies ha ha e ocused on inding p ognos ic
bioma ke s o lung cance ha e only made use o samples
om NSCLC pa ien s as a whole o in sepa a e as adeno-
ca cinoma o SCC pa ien s.
The g and majo i y o s udies using samples om
NSCLC pa ien s we e pe o med using issue. The p o eins
small ubiqui in- ela ed modi ie -2 p o ein (SUMO-2),
TMSB4, and ubiqui in we e iden i ied in a 15 MS peak
p o ile ha dis inguished be ween pa ien s wi h esec ed
NSCLC who had poo o good p ognosis [63]. Using a
simila app oach, 25 p o eins (no all iden i ied, bu
including TMSB4, TMSB10, ibosomal p o ein L39 and
S30, S100A6, and his one H2A.2) we e associa ed wi h
su i al among NSCLC pa ien s and appea ed o dis in-
guish hose wi h poo p ognosis om hose wi h good
p ognosis [46]. In a di e en s udy, he le els o TMSB4,
TMSB10, and calmodulin we e also associa ed wi h pa ien
su i al. In addi ion, low le els o CFL1 we e associa ed
wi h be e ou come o pa ien s wi h nega i e lymph nodes
and high le els o CFL1 wi h be e ou come o hose wi h
posi i e lymph nodes [64]. TMSB4 is a egula o o ac in
polyme isa ion whose o e -exp ession seems o s imula e
lung umou me as asis [64]. Imp o ed su i al o NSCLC
was addi ionally associa ed wi h he le els o S100A6 [65].
The S100 p o eins a e in ol ed in he egula ion o a
numbe o cellula p ocesses such as cell cycle p og ession,
di e en ia ion, and also in lamma ion.
P o eomic s udies o adenoca cinoma issue samples
iden i ied CFL1, an ac in-modula ing p o ein ha is
inc eased in some in asi e cance s, and PKM2, a key
glycoly ic enzyme du ing umo igenesis, as associa ed
wi h poo p ognosis in adenoca cinoma pa ien s [54]. The
compa ison be ween adenoca cinoma and adjacen lung
issue e ealed ha speci ic iso o ms o CK7, 8, 18, and
19 we e associa ed wi h pa ien su i al [66]. Cy oskel-
e al eo ganiza ion is a cen al p ocess egula ing cell
mig a ion and me as asis, and CKs, a amily o cy o-
skele al in e media e ilamen s, ha e been sugges ed o
play a ole in ca cinogenesis, by p omo ing cellula
a chi ec u e eo ganisa ion du ing umou de elopmen
and p og ession. Ano he adenoca cinoma s udy associ-
a ed he exp ession o phosphoglyce a e kinase 1 (PGK1),
HSP70, CK19, PGAM1, and G p o ein-coupled ecep o 4
(GRK4) wi h poo su i al [67]. Se e al annexins
(ANXA1, ANXA2, and ANXA3) we e associa ed wi h
ad anced clinical s age, by p esen ing highe exp ession
le els in lymph node me as a ic issue [68,69]. The
de egula ion o annexins has been epo ed in nume ous
cance s and is hough o in luence he pa e ns o cellula
beha iou , such as cell p oli e a ion, mo ili y, in asi e-
ness and signalling pa hways. Finally, Maeda and col-
leagues e ealed ha he absence o exp ession o LC–
MS/MS iden i ied myosin IIA and imen in, wo p o eins
associa ed wi h he cy oskele on and cell mo ili y, co e-
la ed wi h good p ognosis in adenoca cinoma pa ien s
who did no ecei e pos -ope a i e adju an chemo he -
apy, indica ing ha hese pa ien s do no equi e such
ea men [70].
Conside ing SCC p o eomic s udies sea ching o
p ognos ic bioma ke s, he p e iously iden i ied IDH1
p o ein was associa ed wi h poo o e all su i al [60]. The
e alua ion o mic o-dissec ed p ima y SCC and ma ched
lymph node me as a ic issues e ealed he unde -exp es-
sion o SNF in lymph node me as a ic umou e sus p i-
ma y SCC [71]. Yao and co-wo ke s compa ed issue om
SCC wi h and wi hou lymph node me as asis. The
ANXA2, HSP27, and CK17 p o eins we e o e -exp essed
in me as a ic SCC, and SNF was unde -exp essed in hese
issues [72]. A summa y o hese po en ial p ognos ic
bioma ke s can be ound on Table 3.
676 Clin T ansl Oncol (2013) 15:671–682
123

Table 1 Candida e p o eomic diagnos ic bioma ke s o lung cance iden i ied in issue samples
Diagnos ic bioma ke Lung cance ype P o eomic echnique
S100
A6 SCC MALDI–TOF–MS [38]
NSCLC MALDI–MS [46]
A8 SCC iTRAQ, LC-Q-MS/MS [58]
A9 SCC iTRAQ, LC-Q-MS/MS [58]
SCCA1 (squamous cell ca cinoma an igen 1) SCC iTRAQ, LC-Q-MS/MS [58]
Cys a in A Lung cance MALDI–TOF–MS [24]
Tubulin (a,b) SCLC 2D-PAGE; 2D-PAGE, MALDI–TOF–MS [35,36]
UCH-L1 (ubiqui in ca boxy- e minal hyd olase
L1)
Lung cance 2D-PAGE, MALDI–TOF–MS [26]
SCLC 2D-PAGE, MALDI–TOF–MS [36]
Adenoca cinoma 2D-PAGE, MALDI–MS [51]
Pe oxi edoxin
PRDX4 NSCLC,
Adenoca cinoma
1D-LC–ESI–MS/MS [44]; 2D-PAGE, MALDI–MS [51]
PRDX1 NSCLC 2D-PAGE [43]
PRDX2 SCC 2D-DIGE, MALDI–TOF–TOF [60]
PRDX3 NSCLC 2D-PAGE [43]
PRDX6 SCC 2D-PAGE, MALDI–TOF–MS [61]
TIM ( iose-phospha e isome ase) Adenoca cinoma 2D-PAGE, MALDI–MS [51]
SCC 2D-PAGE, MALDI–TOF–MS [57,61]
MIF (mac ophage mig a ion inhibi o y ac o ) Lung cance MALDI–TOF–MS [22]
NSCLC MALDI–TOF–MS; IMAC, LC–MS/MS [24,45]
CyP-A (Cyclophilin A) Lung cance MALDI–TOF–MS [22]
Adenoca cinoma 2D-DIGE, LC–MS/MS [52]
TAGLN (T ansgelin) NSCLC 2D-PAGE, MALDI–TOF–MS [42]
Adenoca cinoma 2D-DIGE, LC–MS/MS [52]
CA (Ca bonic anhyd ase) SCLC 2D-PAGE, MALDI–TOF–MS [36]
NSCLC 2D-PAGE, MALDI–TOF–MS [42]
ENO1 (Alpha enolase) NSCLC 2D-PAGE, MALDI–TOF–MS [42]
SCC 2D-PAGE, MALDI–TOF–MS [57,61]
14-3-3
gLung cance 1D-PAGE, nanoESI–MS/MS [23]
(SNF) Adenoca cinoma 2D-PAGE, MALDI–TOF–MS, Q-TOF–MS/MS [53]
SCC 2D-DIGE, MALDI–TOF–TOF [60]
SOD2 (Supe oxide dismu ase 2) Adenoca cinoma 2D-PAGE, MALDI–TOF–MS, Q-TOF–MS/MS [53]
SCC 2D-DIGE, MALDI–TOF–TOF; 2D-PAGE, MALDI–TOF–MS
[60,61]
Hea shock p o ein
HSP73 SCLC 2D-PAGE [35]
HSP90 SCLC 2D-PAGE [35]
HSP20-like NSCLC 2D-PAGE, MALDI–TOF–MS [42]
HSP70 SCC 2D-PAGE, MALDI–TOF–MS [61]
HSP60 SCC 2D-PAGE, MALDI–TOF–MS [59,61]
HSP27 SCC iTRAQ, LC-Q-MS/MS [58]
Annexin
ANXA1 Adenoca cinoma 2D-PAGE, MALDI–TOF–MS, Q-TOF–MS/MS [53]
ANXA2 SCC 2D-PAGE, MALDI–TOF–MS [61]
ANXA3 SCC 2D-PAGE, MALDI–TOF–MS [59]
ANXA5 SCC 2D-PAGE, MALDI–TOF–MS [59]
Clin T ansl Oncol (2013) 15:671–682 677
123
Table 1 con inued
Diagnos ic bioma ke Lung cance ype P o eomic echnique
ANXA6 SCC 2D-PAGE, MALDI–TOF–MS [59]
ACTG1 (Gamma ac in) SCLC 2D-PAGE, MALDI–TOF–MS [36]
SCC 2D-PAGE, MALDI–TOF–MS [59]
Thymosin
b4 Lung cance MALDI–TOF–MS [24]
NSCLC MALDI–MS [46]
b10 NSCLC MALDI–MS [46]
CFL1 (Co ilin-1) Adenoca cinoma 2D-PAGE, ESI-Q-TOF–MS/MS [54]
NSCLC non-small cell lung cance , SCLC small cell lung cance , SCC squamous cell ca cinoma
Table 2 Candida e p o eomic diagnos ic bioma ke s o lung cance iden i ied in luid samples
Diagnos ic bioma ke Sample ype Lung cance
ype
P o eomic echnique
CLU (Clus e in) U ine Adenoca cinoma 1D-PAGE, HPLC–MS/MS [20]
Se um SCC 2D-DIGE, MALDI–TOF–MS [62]
Gelsolin U ine Adenoca cinoma 1D-PAGE, HPLC–MS/MS [20]
Se um NSCLC 2D-DIGE, MALDI–TOF–MS [49]
Pleu al e usion,
plasma
Adenoca cinoma iTRAQ, LC–MS/MS [56]
LRG1 (leucine- ich alpha-2-
glycop o ein)
U ine Adenoca cinoma 1D-PAGE, HPLC–MS/MS [20]
Se um Lung cance 2D-DIGE, LC–MS/MS [27]
U ine NSCLC 1D-PAGE, nanoHPLC-MS/MS [50]
SAA (Se um amyloid A) Se um Lung cance SELDI–TOF–MS; MALDI–MS [30,32]
NSCLC MALDI–TOF–MS [48]
Adenoca cinoma 2D-PAGE, MALDI–TOF–PMF [55]
SCC 2D-DIGE, MALDI–TOF–MS [62]
Pleu al e usion,
se um
Lung cance 1D-PAGE, LC–ESI–MS/MS [31]
HP (hap oglobin) Se um Lung cance 2D-DIGE, LC–MS/MS; 2D-DIGE, MALDI–TOF–MS
[27,29]
SCLC SDS–PAGE, MALDI–TOF–MS [39]
NSCLC 2D-DIGE, MALDI–TOF/TOF [47]
Adenoca cinoma 2D-PAGE, MALDI–TOF–PMF [55]
SCC 2D-DIGE, MALDI–TOF–MS [62]
Plasma, se um Lung cance 1D-PAGE, LC–ESI–MS/MS [28]
Sali a Lung cance 2D-DIGE, MALDI–TOF–MS, LC–MS/MS [21]
TTR (T ans hy e in) Se um Lung cance SELDI–TOF–MS [33]
Adenoca cinoma 2D-PAGE, MALDI–TOF–PMF [55]
Complemen componen
C4 Se um Lung cance 2D-DIGE, LC–MS/MS [27]
C3, C3c Lung cance ,
SCC
2D-DIGE, LC–MS/MS [27]; 2D-DIGE, MALDI–TOF–
MS [62]
A1AT (a-1-an i ypsin) Se um Lung cance 2D-DIGE, MALDI–TOF–MS [29]
Pleu al e usion,
plasma
Adenoca cinoma iTRAQ, LC–MS/MS [56]
678 Clin T ansl Oncol (2013) 15:671–682
123
Table 2 con inued
Diagnos ic bioma ke Sample ype Lung cance
ype
P o eomic echnique
Apolipop o ein
A1 Se um Adenoca cinoma 2D-PAGE, MALDI–TOF–PMF [55]
A4 SCC 2D-DIGE, MALDI–TOF–MS [62]
CK8 (Cy oke a in 8) Pleu al e usion,
plasma
Adenoca cinoma iTRAQ, LC–MS/MS [56]
AHSG (alpha-2-HSglycop o ein) Pleu al e usion Lung cance 1D-PAGE, LC–ESI–MS/MS [34]
Se um SCC 2D-DIGE, MALDI–TOF–MS [62]
S100A8 and S100A9 Pleu al e usion,
se um
NSCLC 2D-DIGE, MALDI–TOF–MS [49]
SCCA1 (Squamous cell ca cinoma
an igen 1)
Se um Lung cance 2D-DIGE, MALDI–TOF–MS [29]
Cys a in C3 Pleu al e usion Lung cance 1D-PAGE, LC–ESI–MS/MS [34]
Pleu al e usion,
plasma
Adenoca cinoma iTRAQ, LC–MS/MS [56]
NSCLC non-small cell lung cance , SCLC small cell lung cance , SCC squamous cell ca cinoma
Table 3 Candida e p o eomic p ognos ic bioma ke s o lung cance
P ognos ic bioma ke Sample ype Lung cance
ype
P o eomic echnique
S100A6 Tissue NSCLC MALDI–MS [46]
Tissue, plasma, pleu al
e usion
NSCLC SELDI–TOF–MS [65]
Thymosin
b4 Tissue NSCLC MALDI–MS; MALDI–TOF–MS [63,64]
b10 NSCLC MALDI–MS; MALDI–TOF–MS [46,64]
CFL1 (Co ilin-1) Tissue NSCLC MALDI–TOF–MS [64]
Adenoca cinoma 2D-PAGE, ESI-Q-TOF–MS/MS [54]
IDH1 (isoci a e
dehyd ogenase 1)
Tissue SCC 2D-DIGE, MALDI–TOF–TOF [60]
Cy oke a in
CK7, CK8, CK18 Tissue Adenoca cinoma 2D-PAGE, MALDI–TOF–MS [66]
CK19 Adenoca cinoma 2D-PAGE, MALDI–TOF–MS; 2D-PAGE [66,67]
SCC 2D-DIGE, MALDI–TOF–PMF [72]
Hea shock p o ein
HSP70 Tissue Adenoca cinoma 2D-PAGE, MALDI–MS [51]
HSP27 SCC 2D-DIGE, MALDI–TOF–PMF [72]
Annexin
ANXA1 Tissue Adenoca cinoma 2D-DIGE, MALDI–TOF–PMF [69]
ANXA2 Adenoca cinoma 2D-DIGE, MALDI–TOF–PMF [69]
SCC 2D-DIGE, MALDI–TOF–PMF [72]
ANXA3 Adenoca cinoma 2D-DIGE, MALDI–TOF–MS; 2D-DIGE, MALDI–TOF–
PMF [68,69]
Myosin IIA Tissue Adenoca cinoma LC–MS/MS [70]
Vimen in
SNF (14-3-3 ) Tissue SCC 2D-PAGE, MALDI–TOF–MS; 2D-DIGE, MALDI–TOF–
PMF [71,72]
NSCLC non-small cell lung cance , SCC squamous cell ca cinoma
Clin T ansl Oncol (2013) 15:671–682 679
123
P edic i e
A good p edic i e bioma ke can an icipa e he e icacy o
a speci ic ea men . I aims a indi idualising he apies in
lung cance and s udies ha e been based on s udying
clinical samples om esponding and non- esponding
pa ien s and hen alida ing esul s on selec ed coho s.
P o eomic s udies de eloped wi h his pu pose ha e
ocused on he esponse o EGFR inhibi o s.
Okano and co-wo ke s analysed lung adenoca cinoma
issue om pa ien s who showed a di e en esponse o
ge i inib ea men by 2D-DIGE–LC–MS/MS. High le els
o a y acid-binding p o ein hea (H-FABP) we e asso-
cia ed wi h pa ial and comple e esponses. This p o ein
pa icipa es in in acellula lipid anspo , s o age, and
me abolism, in addi ion o ha ing a possible ole in cance
biology [73]. In addi ion o his s udy, a se um MALDI–
MS s udy conduc ed by Taguchi and co-wo ke s in NSCLC
pa ien s ea ed wi h ge i inib and e lo inib e ealed an
8-peak p o ile p edic i e o ou come [74]. This 8-peak
signa u e was comme cially launched as a comme cial
p oduc (Ve is a
Ò
, Biodesix, B oom ield, CO, USA) and
has been used o disc imina e NSCLC pa ien s ea ed wi h
combina ions o e lo inib, ge i inib, be acizumab, o ce-
uximab wi h good and poo p ognosis in a numbe o
s udies [75–82]. The SAA1 p o ein was ecen ly iden i ied
as pa o his p o eomic signa u e [82]. A summa y o he
abo emen ioned p edic i e bioma ke s can be ound on
Table 4.
Conclusions
The de ec ion o new bioma ke s is o o emos impo ance
in lung cance esea ch. The as majo i y o lung cance s
a e s ill diagnosed a ad anced s ages o he disease,
educing in some cases he a ailable he apeu ic op ions o
only pallia i e ca e, esul ing in one o he lowes 5-yea
su i al a es among cance s.
The p o eomic ield is expanding and he de elopmen
o high- h oughpu echnologies has allowed he iden i i-
ca ion o se e al p o ein p o iles wi h diagnos ic, p og-
nos ic, and p edic i e alue o lung cance . None heless,
i is necessa y o ake in o accoun se e al key poin s
o de o conside his p o eins as bioma ke s. Fi s , an
ex ensi e clinical alida ion wi h s ic s a is ical c i e ia
is equi ed o e alua e hese p o iles, o educe he
occu ence o alse posi i e esul s. Second, la ge coho s
o ca e ully selec ed pa ien s o de e mine he ac ual
use ulness o he bioma ke s a e necessa y. Thi d, i
would be in e es ing o s udy he ep oducibili y o he
ob ained p o ein p o iles and his equi es a alida ion in
di e en biological samples and di e en labo a o ies o
p o e hei sensi i i y. Finally, i is necessa y o add ess
he p oblems o he e ogenei y o he lung cance pa ien s.
S udies conside ing he TNM classi ica ion, umou g ade,
and demog aphic cha ac e is ics o he popula ion would
be equi ed.
In summa y, p o eomics echnologies a e one o he
mos powe ul ools o expand he epe oi e o known
bioma ke s o lung cance ea ly diagnosis, p ognosis, and
p edic ion o esponse o he apy. Mo eo e , many o he
iden i ied candida e bioma ke s could se e as a ge s o
mo e a ionale and e ec i e he apeu ic s a egies due o
hei ole in umo igenesis and cance p og ession.
Con lic o in e es The au ho s decla e o ha e no con lic o
in e es .
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In . J. Mol. Sci. 2013, 14, 3440-3455; doi:10.3390/ijms14023440
In e na ional Jou nal o
Molecula Sciences
ISSN 1422-0067
www.mdpi.com/jou nal/ijms
A icle
Iden i ica ion o Oxida i e S ess Rela ed P o eins as
Bioma ke s o Lung Cance and Ch onic Obs uc i e
Pulmona y Disease in B onchoal eola La age
Ma ia Dolo es Pas o 1,†, Ana Nogal 1,†, Sonia Molina-Pinelo 1, Rica do Meléndez 1,
Bea iz Rome o-Rome o 2, Ma ia Dolo es Mediano 1, Jose L. López-Campos 1,2,
Rocío Ga cía-Ca bone o 1,3, Ampa o Sanchez-Gas aldo 1,3, Amancio Ca ne o 1,4 and
Luis Paz-A es 1,3,*
1 Biomedicine Ins i u e o Se ille, Se ille 41013, Spain;
E-Mails: ma ilopas o @gmail.com (M.D.P.); [email p o ec ed] (A.N.);
[email p o ec ed] (S.M.-P.); ica domelendezcaden[email p o ec ed] (R.M.);
mm [email protected] (M.D.M.); josel.lopezcam[email p o ec ed] (J.L.L.-C.);
[email p o ec ed] (R.G.-C.); klim a77@ho mail.com (A.S.-G.); aca [email p o ec ed] (A.C.)
2 Respi a o y Diseases Medical and Su gical Uni o he Vi gen del Rocío Uni e si y Hospi al,
Se ille 41013, Spain; E-Mail: [email p o ec ed]
3 Oncology Uni o he Vi gen del Rocío Uni e si y Hospi al, Se ille 41013, Spain
4 Supe io Counsil o Scien i ic Resea ch, Se ille 41013, Spain
† The au ho s con ibu ed equally o his wo k.
* Au ho o whom co espondence should be add essed;
E-Mail: luis.paza es.sspa@jun adeandalucia.es; Tel.: +34-955-013-414; Fax: +34-955-013-292.
Recei ed: 24 Decembe 2012; in e ised o m: 23 Janua y 2013 / Accep ed: 31 Janua y 2013 /
Published: 6 Feb ua y 2013
Abs ac : Lung cance (LC) and ch onic obs uc i e pulmona y disease (COPD)
commonly coexis in smoke s, and he p esence o COPD inc eases he isk o de eloping
LC. Ciga e e smoke causes oxida i e s ess and an in lamma o y esponse in lung cells,
which in u n may be in ol ed in COPD and lung cance de elopmen . The aim o his
s udy was o iden i y di e en ial p o eomic p o iles ela ed o oxida i e s ess esponse ha
we e po en ially in ol ed in hese wo pa hological en i ies. P o ein con en was assessed
in he b onchoal eola la age (BAL) o 60 pa ien s classi ied in ou g oups: COPD,
COPD and LC, LC, and con ol (nei he COPD no LC). P o eins we e sepa a ed in o spo s
by wo dimensional polyac ylamide gel elec opho esis (2D-PAGE) and examined by
OPEN ACCESS
In . J. Mol. Sci. 2013, 14 3441
ma ix-assis ed lase deso p ion/ioniza ion ime o ligh mass spec ome y
(MALDI-TOF/TOF). A o al o 16 oxida i e s ess egula o y p o eins we e di e en ially
exp essed in BAL samples om LC and/o COPD pa ien s as compa ed wi h he con ol
g oup. A dis inc p o eomic eac i e oxygen species (ROS) p o ein signa u e eme ged ha
cha ac e ized lung cance and COPD. In conclusion, ou indings highligh he ole o he
oxida i e s ess esponse p o eins in he pa hogenic pa hways o bo h diseases, and p o ide
new candida e bioma ke s and p edic i e ools o LC and COPD diagnosis.
Keywo ds: b onchoal eola la age; lung cance ; sc eening; bioma ke ; in lamma ion;
p o eomics; ROS; oxida i e s ess
1. In oduc ion
Ciga e e smoking has been ecognized as he mos impo an causa i e ac o o COPD and i is
associa ed wi h mo e han 90% o lung cance cases [1]. Lung cance accoun s o 12% o all cance
diagnoses wo ldwide, making i he la ges cause o cance -associa ed dea h wo ldwide, accoun ing o
mo e han one million casual ies pe yea wo ldwide. COPD is also a majo independen isk ac o o
lung ca cinoma, among long- e m smoke s. In ac , he p esence o COPD inc eases he isk o lung
cance up o 4.5- old. Indeed, 50%–70% o pa ien s diagnosed wi h lung cance ha e spi ome ic
e idence o COPD [2]. Ciga e e smoke (CS) con ains o e 1014 ee adicals pe pu ha include
eac i e oxygen species (ROS) [3]. Inhaled oxidan s om smoke gene a e cellula damage by di ec ly
a ge ing p o eins, lipids, and nucleic acids, and deple e he le el o an ioxidan s in he lung, he eby
o e whelming he oxidan /an ioxidan balance o he lung, leading o inc eased oxida i e s ess [4].
ROS can lead o he ac i a ion o a ious cell signaling componen s. Examples include he
ex acellula signal egula ed kinases (ERKs), c-jun N- e minal kinases (JNKs), p38 MAPKs, PKC,
PI3K/Ak , and g ow h ac o y osine kinases ecep o s pa hways, all o which lead o inc eased
in lamma o y gene ansc ip ion. Indeed, oxida i e s ess in he lungs has been implica ed in COPD
se e i y and lung ca cinogenesis [5]. This p ocess is one o he mechanisms p oposed in he common
pa hogenesis o lung cance and COPD, along wi h in lamma ion, epi helial-mesenchymal ansi ion
(EMT), al e ed DNA epai , and cellula p oli e a ion [6]. P o eins a e impo an molecula signpos s
o oxida i e damage. Di e en p o eomic app oaches ha e been de eloped and used o he de ec ion
and iden i ica ion o ROS ela ed p o eins [7]. In he las ew yea s, combined p o eomics, mass
spec ome y (MS), and a ini y chemis y-based me hodologies ha e con ibu ed in a signi ican way
o p o ide a be e unde s anding o p o ein oxida i e modi ica ions occu ing in a ious biological
specimens unde di e en physiological and pa hological condi ions. B onchoal eola la age (BAL) is
he clinical bio luid sampling o he soluble p o eins con en s o he ai way lumen. A compa ison
be ween se um and BAL p o eomes e eals ha a ce ain numbe o p o eins a e p esen a a highe
le el in BAL han in plasma, sugges ing ha hey a e speci ically p oduced in he ai ways. These
p o eins a e, he e o e, po en ial candida es o becoming lung-speci ic bioma ke s [8]. 2D-PAGE is
conside ed one o he bes echniques o sepa a ion o complex mix u es o soluble p o eins [9].
Se e al s udies o BAL p o ein p o iles ob ained by 2D-PAGE analysis aimed a e ealing he
In . J. Mol. Sci. 2013, 14 3442
di e ences be ween smoke s and ne e smoke s [10,11] as well as s udies di ec ed o de e mine he
isk o de eloping COPD [12–14]. Howe e , o he bes o ou knowledge, he e a e no 2D-PAGE
s udies in lung cance using BAL. The 2D-PAGE s udies o LC ha e been pe o med mainly in plasma
and issue. These analyses ha e ocused on a be e unde s anding o he molecula basis o cance
pa hogenesis [15–17], as well as on he iden i ica ion o new diagnos ic, p ognos ic, and p edic i e
ma ke s o lung cance [18,19]. In his ega d, analyzing he p o ein composi ion o BAL media ed by
a high- h oughpu echnology, gi en i s icini y o umo cells and en ichmen in umo -de i ed
p o eins, would be insigh ul. In his s udy, we ha e in es iga ed he changes occu ing in he
p o eome o BAL samples om lung cance and/o COPD pa ien s, and ound a se o edox egula i e
p o eins di e en ially exp essed in each disease.
2. Resul s
2.1. P o eome P o iles o Compa ison o LC and/o COPD
The expe imen s we e pe o med in BAL samples ex ac ed om a coho o 60 pa ien s di ided
in o ou g oups (con ol g oup and LC and/o COPD g oups) whose cha ac e is ics a e desc ibed in
Table 1. The spo s ha showed signi ican inc emen o educ ion o hei exp ession compa ed o
con ol g oup (nei he COPD no LC) we e iden i ied by MALDI-TOF/TOF-MS. The MS/MS da a
we e acqui ed and compa ed o he Swiss-P o da abase using MASCOT so wa e. Candida e p o eins
we e selec ed om each spo , aking in o conside a ion se e al a iables such as isoelec ic poin ,
molecula mass, ma ched pep ides, and sequence co e age (Figu e 1). A o al o 123 p o ein spo s
we e success ully iden i ied. O hese, 40 p o eins spo s had consis en signi ican di e ences (>2- old,
p < 0.05) be ween lung cance and/o COPD g oups and he con ol g oup. Among hem, a majo
g oup o 16 p o eins we e oxida i e s ess egula o y p o eins (Table 2). The spo s co esponding o
his g oup o ROS egula o y p o eins a e ma ked on he ep esen a i e gel 2D image in Figu e 1. The
es o iden i ied p o eins we e dis ibu ed in o he a iable g oups such as in lamma ion, glycolysis
and gluconeogenesis (Da a no show).
Table 1. Pa ien s cha ac e is ics.
Con ols n = 15 COPD n = 15 LC n = 15 LC&COPD n = 15
Gende
Male 100.0% (15) 100.0% (15) 100.0% (15) 100.0% (15)
Female 0.0% (0) 0.0% (0) 0.0% (0) 0.0% (0)
A e age age ( ange) 61.3 (41–80) 61.5 (45–78) 60.7 (46–69) 60.7 (49–68)
Smoking s a us
Smoke s 73.3% (11) 53.3% (8) 53.3% (8) 80.0% (12)
Ex-smoke s 26.7% (4) 46.7% (7) 46.7% (7) 20.0% (3)
Packs-yea 21.82 32.20 35.21 30.78
COPD
Mild - 20.0% (3) - 53.3% (8)
Mode a e - 33.3% (5) - 26.7% (4)
Se e e - 26.7% (4) - -
Ve y se e e - 20.0% (3) - 20.0% (3)
His ology
Adenoca cinoma - - 73.3% (11) 66.7% (10)
Squamous cell ca cinoma - - 26.7% (4) 33.3% (5)
Abb e ia ions: COPD: ch onic obs uc i e pulmona y disease; LC: lung cance .