Proteomic characterization of lung cancer and chronic obstructive pulmonary disease: a bronchoalveolar lavage fluid analysis

! ! 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. ! ! ! ! Depois da empes ade… ! ! 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! Ana!Ba bosa!de!Sousa!Nogal! V! 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 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! Ana!Ba bosa!de!Sousa!Nogal! VI! 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 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! Ana!Ba bosa!de!Sousa!Nogal! VII! 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! ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal! IX! 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. ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal! XVII! 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. ! ! ! ! ! ! ABSTRACT ! ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal! XXI! 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, 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! ! Ana!Ba bosa!de!Sousa!Nogal! XXII! 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. ! ! ! RESUMO ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal! XXV! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 6! ! 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). 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 7! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 8! ! 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). 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 9! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 10! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 11! ! 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, 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 12! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 13! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 14! ! 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). 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 15! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 22! ! 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. ! ! HYPOTHESIS AND OBJECTIVES ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 25! ! 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. ! ! ! ! MATERIALS AND METHODS ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 29! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 30! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 31! ! 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). 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 38! ! GenePix 4100 A (Molecula De ices, Sunny ale, CA, USA), which was also used o pe o m da a ex ac ion. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 39! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 40! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 41! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 42! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 43! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 44! ! 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 ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 47! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 54! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 55! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 56! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 57! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 58! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 59! ! 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). 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 60! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 61! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 62! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 63! ! 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 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 70! ! 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. 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 71! ! 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- 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! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 72! ! 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. ! ! GENERAL DISCUSSION AND MAIN CONCLUSIONS ! ! ! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal! ! 75! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 76! ! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 77! ! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 78! ! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 79! ! 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! ! ! Ana!Ba bosa!de!Sousa!Nogal!! ! 86! ! 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. ! ! FUTURE PERSPECTIVES ! ! 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! ! Ana!Ba bosa!de!Sousa!Nogal! ! 89! ! 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. 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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 . Re e ences 1. Mal ezzi M, Be uccio P, Le i F, La Vecchia C, Neg i E. Eu opean cance mo ali y p edic ions o he yea 2012. Ann Oncol. 2012;23(4):1044–52. 2. Albe g AJ, Same JM. Epidemiology o lung cance . Ches . 2003;123(1 Sup- pl):21S-49S. 3. Albe g AJ, Fo d JG, Same JM. Epidemiology o lung cance : ACCP e idence- based clinical p ac ice guidelines (2nd edn). Ches . 2007;132(3 Suppl):29S– 55S. 4. Agullo-O uno MT, Lopez-Rios F, Paz-A es L. Lung cance genomic signa- u es. J Tho ac Oncol. 2010;5(10):1673–91. 5. Meye son M, F anklin WA, Kelley MJ. Molecula classi ica ion and molecula gene ics o human lung cance s. Semin Oncol. 2004;1(Suppl 1):4–19. 6. He bs RS, Heymach JV, Lippman SM. Lung cance . N Engl J Med. 2008;359(13):1367–80. 7. 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SAA1 is o e - exp essed in plasma o non small cell lung cance pa ien s wi h poo ou come a e ea men wi h epide mal g ow h ac o ecep o y osine-kinase inhibi o s. J P o eomics. 2012. 682 Clin T ansl Oncol (2013) 15:671–682 123 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 .