PD L1 expression is associated with advanced non small cell lung cancer

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1 ONCOLOGY LETTERS 12: , 2016 PD L1 expression is ssocited with dvnced non smll cell lung cncer ZHIQUAN CHEN 1 3, JIANDONG MEI 3 5, LUNXU LIU 4,5, GUOCHEN WANG 2, ZUOSHENG LI 2, JINGPU HOU 2, QIUYANG ZHANG 3, ZONGBING YOU 3 nd LIU ZHANG 1,6 1 Deprtment of Surgery, The Fourth Hospitl of Hebei Medicl University, Shijizhung, Hebei ; 2 Deprtment of Thorcic Surgery, Affilited Hospitl of North Chin University of Science nd Technology, Tngshn, Hebei , P.R. Chin; 3 Deprtment of Structurl nd Cellulr Biology, Tulne University, New Orlens, LA 70112, USA; 4 Deprtment of Thorcic Surgery, West Chin Hospitl, Sichun University; 5 Western Chin Collbortive Innovtion Center for Erly Dignosis nd Multidisciplinry Therpy of Lung Cncer, Sichun University, Chengdu, Sichun ; 6 Deprtment of Surgery, Affilited Hospitl of North Chin University of Science nd Technology, Tngshn, Hebei , P.R. Chin Received August 24, 2015; Accepted My 16, 2016 DOI: /ol Abstrct. Lung cncer is the most common cuse of cncer ssocited mortlities worldwide. Novel immunotherpies hve been developed to improve the clinicl outcomes of non smll cell lung cncer (NSCLC). Antibodies ginst progrmmed cell deth protein 1 (PD 1) nd progrmmed cell deth protein 1 lignd 1 (PD L1) hve been tested in clinicl trils, nd nti PD 1 ntibody hs been pproved for the tretment of NSCLC. The im of the present study ws to ssess expression of PD 1, PD L1 nd progrmmed cell deth protein 1 lignd 2 (PD L2) in 48 ptients with NSCLC, using immunohistochemicl stining. The results found tht 35.4% (17/48) of ptients were positive for PD 1 expression, 64.6% (31/48) were positive for PD L1 expression nd 45.8% (22/48) were positive for PD L2 expression. Neither PD 1 nor PD L2 expression ws ssocited with gender, histology, differentition sttus, tumor stge or lymph node metstsis. PD L1 expression ws not ssocited with gender, histology, differentition sttus or lymph node metstsis; however, PD L1 expression ws significntly incresed in stge III NSCLC (85.7% PD L1+) compred with stge I/II NSCLC (55.9% PD L1+) (P=0.049). Correspondence to: Dr Liu Zhng, Deprtment of Surgery, The Fourth Hospitl of Hebei Medicl University, 12 Jinkng Rod, Shijizhung, Hebei , P.R. Chin E mil: zhliu130@sohu.com Dr Zongbing You, Deprtment of Structurl nd Cellulr Biology, Tulne University, 1430 Tulne Avenue, New Orlens, LA 70112, USA E mil: zyou@tulne.edu Key words: lung cncer, progrmmed cell deth protein 1, progrmmed cell deth protein 1 lignd 1, progrmmed cell deth protein 1 lignd 2, immunohistochemistry Introduction Lung cncer is the most common cuse of cncer ssocited mortlities worldwide (1). The Americn Cncer Society estimted tht there would be ~221,200 novel cses nd ~158,040 mortlities cused by lung cncer in the USA in 2015 (1,2). Lung cncer is the second most common mlignncy nd the most common cuse of cncer ssocited mortlity in Americn men nd women (1). In Chin, novel cses nd lung cncer ssocited mortlities were estimted to be 536,407 nd 475,768, respectively, in 2005 (2). Globlly, it hs been estimted tht there were 1,824,700 novel cses nd 1589,900 lung cncer ssocited mortlities in 2012 (3). Currently, surgicl resection remins the stndrd of cre for the mjority of ptients with non metsttic non smll cell lung cncer (NSCLC). Cncer immunotherpy hs recently received ttention (4), since the United Sttes Food nd Drug Administrtion (FDA) pproved Provenge (sipuleucel T) for the tretment of metsttic cstrtion resistnt prostte cncer nd Yervoy (ipilimumb) for the tretment of metsttic melnom (5,6). Inhibitors of the progrmmed cell deth protein 1 (PD 1), n immunosuppressive checkpoint protein, nd the progrmmed cell deth protein 1 lignd 1 (PD L1) nd lignd 2 (PD L2), hve demonstrted positive outcomes in the tretment of cncers, including lung cncer, in clinicl trils (7). A phse I clinicl tril reported objective responses in pproximtely 1/4 to 1/5 of ptients with NSCLC, melnom nd renl cell cncer, who were treted with nti PD 1 ntibodies (8). Another phse I clinicl tril reported objective response rtes of 6 17% nd stbiliztion of disese t rtes of 12 41% t 24 weeks in ptients with dvnced cncers, including NSCLC, melnom nd renl cell cncer, who were treted with nti PD L1 ntibodies (9). Three ptients sustined long term prtil or complete response in 16 months to 3 yers following tretment (10). Subsequent studies showed tht nti PD 1 ntibody (lmbrolizumb)

2 922 CHEN et l: PD-L1 EXPRESSION IN LUNG CANCER produced response rte of ~38% in melnom ptients, with or without prior ipilimumb tretment (11). A combintion of nti PD 1 ntibody (nivolumb) nd ipilimumb produced 53% objective response in the ptients with dvnced melnom (12). A phse III tril showed tht nti PD 1 ntibody (pembrolizumb, lso clled lmbrolizumb or MK 3475) produced significntly better response rte (~33%) compred with ipilimumb (11.9%; P<0.001) in the tretment of dvnced melnom (13). A recent phse I tril showed tht pembrolizumb produced n objective response rte of 19.4% in 495 ptients with NSCLC. The medin durtion of progression free survivl ws 3.7 months nd the medin durtion of overll survivl ws 12.0 months (14). Therefore, on September 4, 2014, the FDA grnted ccelerted pprovl to the nti PD 1 ntibody pembrolizumb (Keytrud ; Merck & Co, Inc., Whitehouse Sttion, NJ, USA) for the tretment of ptients with unresectble or metsttic melnom nd disese progression following ipilimumb nd, if B Rpidly Accelerted Fibrosrcom (BRAF) V600 muttion positive, BRAF inhibitor such s vemurfenib, sorfenib or dbrfenib. The FDA lso pproved nivolumb (Opdivo ; Bristol Myers Squibb Compny, Princeton, NJ, USA) for the tretment of ptients with unresectble or metsttic melnom nd disese progression following ipilimumb nd, if BRAF V600 muttion positive, BRAF inhibitor for the tretment of ptients with metsttic squmous NSCLC with progression during or following pltinum bsed chemotherpy, on December 22, 2014 nd Mrch 4, 2015, respectively. FDA ssigned priority review designtion to pembrolizumb (Keytrud ) s tretment for ptients with dvnced NSCLC nd finl pprovl decision will be mde in the future. Anti PD L1 ntibody (MPDL3280A; Genentech; Roche, South Sn Frncisco, CA, USA) showed responsive rtes of 13 26% in solid tumors, including NSCLC (15). On Februry 2, 2015, the FDA gve MPDL3280A brekthrough therpy designtion for the tretment of PD L1 positive NSCLC tht hs progressed during or following pltinum bsed chemotherpy, s well s trgeted therpy for ptients with epiderml growth fctor receptor (EGFR) positive or nplstic lymphom kinse (ALK) positive tumors. MPDL3280A is currently undergoing phse II nd III trils to obtin FDA pprovl (16). PD 1 ws originlly identified by Ishid et l (17) in serch of genes responsible for progrmmed cell deth. The study cloned gene encoding protein with 288 mino cids, which ws ctivted during progrmmed cell deth; therefore, the protein ws nmed PD 1 (17). Disruption of the PD 1 gene led to development of lupus like rthritis nd glomerulonephritis, indicting tht PD 1 is negtive regultor of immune responses (18,19). Honjo nd Freemn et l (20) collbortively identified PD L1, which is identicl to B7 H1 reported by Dong et l (21). Ltchmn et l (22) further identified second PD 1 lignd PD L2, which is identicl to B7 DC (23). The binding of PD 1 by PD L1 nd PD L2 is now known to inhibit T cell receptor medited lymphocyte prolifertion nd cytokine secretion, thus suppressing immune responses (24). In the tumor microenvironment, the PD 1 PD L1/L2 pthwy is upregulted, resulting in the immune evsion of tumor cells (22,25). Therefore, the ntibodies ginst PD 1, PD L1 nd likely PD L2 my block the immune evsion response nd induce tumor regression. PD 1, negtive costimultory receptor, is primrily expressed on the cellulr surfce of ctivted T cells (26,27). PD L1 is expressed by tumor cells nd tumor infiltrting immune cells, including mcrophges, dendritic cells nd T cells (15). PD L1 nd PD L2 mrnas re expressed in the humn hert, plcent, spleen, lymph nodes nd thymus tissues. In ddition, PD L2 messenger RNA (mrna), but not PD L1 mrna, is expressed in the humn lung, liver, smooth muscle nd pncres tissues (22). In cohort of 824 NSCLC ptients, 50% of tumor cells stined positive for PD L1 in 23.2% of ptients, 1 49% of tumor cells stined positive for PD L1 in 37.6% of ptients nd <1% of tumor cells stined positive for PD L1 in 39.2% of ptients (14). The objective response rte (ORR) to pembrolizumb tretment is positively ssocited with the percentge of tumor cells with membrnous PD L1 stining, for exmple: Ptients tht were <1% PD L1+ exhibited n 8.1% ORR; ptients tht were 1 24% PD L1+ exhibited 12.9% ORR; ptients tht were 25 49% PD L1+ exhibited 19.4% ORR; ptients tht were 50 74% PD L1+ exhibited 29.6% ORR; nd ptients tht were % PD L1+ exhibited 45.4% ORR (14). In contrst, in cohort of 272 squmous NSCLC, the ORRs to nivolumb tretment were similr between PD L1+ nd PD L1 tumors, nmely: Ptients tht were <1% PD L1+ exhibited 17% ORR; ptients tht were 1% PD L1+ exhibited 17% ORR; ptients tht were <5% PD L1+ exhibited 15% ORR; ptients tht were 5% PD L1+ exhibited 21% ORR; ptients tht were <10% PD L1+ exhibited 16% ORR; nd ptients tht were 10% PD L1+ exhibited 19% ORR). This discrepncy my be due to the differences in smple size or ntibodies. However, dditionl studies re required to ssess expression of PD 1, PD L1 nd PD L2 in NSCLC. Although Keytrud nd Opdivo re not yet pproved for use in Chin, their eventul pprovl is possible. Therefore, the objective of this study ws to ssess expression of PD 1, PD L1, nd PD L2 in 48 cses of NSCLC in Chin. We found tht PD L1, but not PD 1 or PD L2 expression ws ssocited with stge III NSCLC. Mterils nd methods Humn lung cncer tissue smples. The present study ws pproved by the Institutionl Review Bord of The Fourth Hospitl of Hebei Medicl University (Shijizhung, Chin). The procedures to obtin humn lung cncer tissue nd follow up informtion were in ccordnce with the Ethicl Principles for Medicl Reserch Involving Humn Subjects, s formulted in the World Medicl Assocition Declrtion of Helsinki (revised 2008). All humn lung cncer tissue smples were obtined from the rchives of formlin fixed, prffin embedded tissue blocks in the Deprtment of Thorcic Surgery t The Fourth Hospitl of Hebei Medicl University (Shijizhung, Chin). The specimens were collected from surgeries performed between April 2010 nd Mrch Written informed consent ws obtined from ll ptients prior to surgery. The ptients were followed up until Mrch 2015, through outptient visits or correspondences to fmily members. In totl, 48 ptients were included in this retrospective study. Tumor stge ws evluted ccording to the Union for Interntionl Cncer Control (UICC) 7th TNM clssifiction system nd histologicl evlution ws bsed on the World Helth Orgniztion criteri (28). The clinicopthologicl chrcteristics of the ptients re summrized in Tble I.

3 ONCOLOGY LETTERS 12: , Tble I. Clinicopthologicl chrcteristics of ptients (n=48). Chrcteristic No. of ptients Age, yers 59.3±7.6 Mle 33 Femle 15 SCC 23 ADC 25 Differentition Well 40 Poor 8 Tumor stge I 17 II 17 III 14 Lymph node metstsis No 30 Yes 18 Dt re presented s men ± stndrd devition. SCC, squmous cell crcinom; ADC, denocrcinom. Tble II. Assocition between PD 1 expression nd clincopthologicl chrcteristics of ptients. PD 1 expression Chrcteristic + P vlue No. of ptients Age, yers 58.7± ± Mle Femle 6 9 SCC ADC 8 17 Differentition Well Poor 1 7 Tumor stge I/II III 4 10 Lymph node metstsis No Yes 5 13 Dt presented s men ± stndrd devition. PD 1, progrmmed cell deth protein 1; SCC, squmous cell crcinom; ADC, denocrcinom. Immunohistochemistry. Tissue sections (4 µm thick) were bked t 60 C for 60 min, deprffinized in xylene nd rehydrted through grded ethnol solutions to wter. Antigens were retrieved by heting the tissue sections in 0.01 M ethylenediminetetrcetic cid buffer t 95 C for 5 min nd then cooling down to room temperture in 20 min. Endogenous peroxidse ctivity ws blocked by 0.3% H 2 O 2 for 5 min. Non specific binding ws blocked with 1.5% norml got or horse serum (VECTASTAIN Elite ABC kit; Vector Lbortories, Burlingme, CA, USA). The sections were incubted with primry ntibodies in humid chmber t 4 C overnight: Rbbit nti humn PD L1 polyclonl ntibodies (ctlog no., b58810; dilution, 1:40; Abcm, Cmbridge, MA, USA), rbbit nti humn PD L2 polyclonl ntibodies (ctlog no., SAB UG; dilution, 1:800; Sigm Aldrich, St. Louis, MO, USA) nd mouse nti humn cluster of differentition (CD)279 (PD 1) purified monoclonl ntibodies (ctlog no., ; dilution, 1:25; ebioscience, Inc., Sn Diego, CA, USA) were used s the primry ntibodies. Subsequent to being wshed 3 times in phosphte buffered sline, the sections were incubted with secondry ntibodies from the VECTA- STAIN Elite ABC kit for 120 min. The color ws developed using 3,3' diminobenzidine (DAB) substrte kit (Vector Lbortories) following the mnufcturer's protocol. The sections were then counterstined with hemtoxylin. Tissue sections tht hd previously stined positively were used s positive control nd tissue sections stined with non immune serum rther thn primry ntibodies served s negtive control. Positive stining showed brown prticles t the cytoplsmic membrne or in the cytoplsm. Under microscope, 5 representtive high power (mgnifiction, x400) fields, contining tumor islet cells nd strom, per tissue section were rndomly selected nd evluted by two investigtors (Dr Zhiqun Chen from Hebei Medicl University, Shijizhung, Chin, nd Dr Jindong Mei from Sichun University, Chengdu, Chin), who were blinded to the clinicopthologicl chrcteristics. An verge of the scores obtined by the two exminers ws used to represent ech cse. A two score system bsed on proportion score nd n intensity score, previously described by Allred et l (29), ws used. The proportion scores were ssigned bsed on the percentge of positive stining: 0, none; 1, <1%; 2, 1 10%; 3, %; 4, %; nd 5, >66.7%. The intensity scores were ssigned bsed on the estimted verge stining intensity of positive stining: 0, none; 1, wek; 2, intermedite; nd 3, strong. The overll Allred scores (29) were the sum of the proportion score nd intensity score of ech cse (rnge, 0 8). Sttisticl nlysis. Sttisticl nlysis ws performed using the Sttisticl Pckge for the Socil Sciences (SPSS) version 16.0 for Windows (SPSS, Chicgo, IL, USA). The results were presented s the men ± stndrd devition (SD) or medin nd rnge for numericl vribles. The comprison of clinicopthologicl chrcteristics between vrious groups ws performed using the χ 2 test. Spermn's rnk correltion coefficient ws clculted to revel the correltion between PD 1, PD L1 nd PD L2 scores. The survivl time of vrious groups ws described using Kpln Meier curves, nd the sttisticl significnce ws nlyzed using the log rnk test. P<0.05 ws considered to indicte sttisticlly significnt difference.

4 924 CHEN et l: PD-L1 EXPRESSION IN LUNG CANCER Tble III. Assocition between PD L2 expression nd clincopthologicl chrcteristics of ptients. PD L2 expression Chrcteristic + P vlue No. of ptients Age, yers 60.6± ± Mle Femle 7 8 SCC ADC Differentition Well Poor 3 5 Tumor stge I/II III 8 6 Lymph node metstsis No Yes 7 11 Dt presented s men ± stndrd devition. PD L2, progrmmed cell deth protein 1 lignd 2; SCC, squmous cell crcinom; ADC, denocrcinom. Tble IV. Assocition between PD L1 expression nd clincopthologicl chrcteristics of ptients. PD L1 expression Chrcteristic + P vlue No. of ptients Age, yers 60.5± ± Mle Femle 8 7 SCC ADC 17 8 Differentition Well Poor 7 1 Tumor stge I/II III 12 2 Lymph node metstsis No Yes 13 5 Dt presented s men ± stndrd devition. PD L1, progrmmed cell deth protein 1 lignd 1; SCC, squmous cell crcinom; ADC, denocrcinom. Results PD 1, PD L1 nd PD L2 re expressed in NSCLC. Immunohistochemicl stining reveled tht PD 1 ws expressed in the immune cells tht were locted mostly in the strom of lung denocrcinoms nd squmous cell crcinoms (Fig. 1). PD L1 nd PD L2 were expressed in the cncer cells of lung denocrcinoms nd squmous cell crcinoms (Fig. 1). PD L1, but not PD 1 or PD L2, is ssocited with stge III lung cncer. To ssess whether the expression of PD 1, PD L1 nd PD L2 is correlted with ny clinicopthologicl chrcteristics of the ptients, ny stining (Allred score rnges 1 8) ws defined s positive (+) nd no stining (Allred score=0) ws defined s negtive ( ). Anlysis reveled tht neither PD 1 nor PD L2 expression ws ssocited with the ptients' gender, tumor histologicl types, tumor differentition, tumor stge or sttus of lymph node metstsis (Tbles II nd III). PD L1 expression ws not ssocited with the ptients' gender, tumor histologicl types, tumor differentition or sttus of lymph node metstsis (Tble IV). However, PD L1 expression ws ssocited with the tumor stge (P=0.049). The positive stining rte ws 55.9% (19/34) in the stge 1/II tumors, wheres it ws 85.7% (12/14) in the stge III tumors (Tble IV). PD 1, PD L1 nd PD L2 expression is independent of ech other in lung cncer. Correltion nlysis found tht the expressions of PD 1, PD L1 nd PD L2 were independent of Tble V. Correltion between PD 1 nd PD L1 or PD L2 expression. PD 1 expression Protein + P vlue PD L PD L PD 1, progrmmed cell deth protein 1; PD L1/2, progrmmed cell deth protein 1 lignd 1/2. ech other. No correltion ws identified between PD 1 nd PD L1 expression, PD 1 nd PD L2 expression or PD L1 nd PD L2 expression (Fig. 2; Tble V). PD 1, PD L1 nd PD L2 expression is not ssocited with the survivl time in lung cncer ptients. Kpln Meier nlysis showed tht PD 1, PD L1 nd PD L2 expression ws not ssocited with the survivl time of ptients with lung cncer (Fig. 3). Incresed levels of PD 1 expression ppered to be inversely

5 ONCOLOGY LETTERS 12: , Figure 1. Representtive photomicrogrphs of immunohistochemicl stining. Arrows indicte the positively stined cells. Originl mgnifiction, x400. PD 1, progrmmed cell deth protein 1; PD L1/2, progrmmed cell deth protein 1 lignd 1/2. Figure 2. Correltion nlysis of PD 1, PD L1 nd PD L2 expression. The expression levels re represented by Allred scores nd ssessed by Spermn's rnk correltion coefficient between ech pir of proteins. PD 1, progrmmed cell deth protein 1; PD L1/2, progrmmed cell deth protein 1 lignd 1/2. Figure 3. Correltion nlysis between PD 1, PD L1 nd PD L2 expression nd the survivl time of the lung cncer ptients. The expression levels re represented by Allred scores nd ssessed by Spermn's rnk correltion coefficient between the expression level nd survivl time. PD 1, progrmmed cell deth protein 1; PD L1/2, progrmmed cell deth protein 1 lignd 1/2. Figure 4. Kpln Meier curves of the lung cncer ptients with positive nd negtive stining. The sttisticl significnce ws nlyzed using the log rnk test. PD 1, progrmmed cell deth protein 1; PD L1/2, progrmmed cell deth protein 1 lignd 1/2; Cum., cumultive.

6 926 CHEN et l: PD-L1 EXPRESSION IN LUNG CANCER ssocited with the survivl time; however, this result ws not sttisticlly significnt (Fig. 3). In ddition, the survivl time of ptients with tumors tht were positively stined for PD 1, PD L1 nd PD L2 expression ws not significntly different from the survivl time of ptients with negtively stined tumors (Fig. 4). Discussion In the present study of cohort of 48 ptients with NSCLC, 35.4% (17/48) of ptients were positive for PD 1 expression, 64.6% (31/48) of ptients were positive for PD L1 expression nd 45.8% (22/48) of ptients were positive for PD L2 expression. Neither PD 1 nor PD L2 expression ws ssocited with gender, histology, differentition sttus, tumor stge or lymph node metstsis. PD L1 expression ws not ssocited with gender, histology, differentition sttus or lymph node metstsis. However, PD L1 expression ws significntly incresed in stge III NSCLC (85.7% PD L1+) compred with stge I/II NSCLC (55.9% PD L1+) (P=0.049). The lck of sttisticlly significnt ssocitions with the mjority of the clinicopthologicl chrcteristics my be due to the smll smple size used in the present study. In cohort of 331 ptients with squmous NSCLC in previous study, neither PD L1 nor PD L2 expression ws ssocited with gender, ge, smoking history, tumor size, tumor stge or lymph node metstsis (30). However, PD L1 expression ws mrginlly ssocited with tumor stge (P=0.059) (30). The present study lso found tht the expressions of PD 1, PD L1 nd PD L2 were independent of ech other, which is consistent with the previous study (30). This independence my suggest tht ny component of the PD 1 PD L1/L2 pthwy my be upregulted to suppress immune responses in the tumor microenvironment. In ddition, the present study indicted tht the expression of PD 1, PD L1 nd PD L2 ws not ssocited with the survivl of the ptient. In met nlysis of 9 studies tht included 1,550 NSCLC ptients, PD L1 expression ws ssocited with differentition sttus, but not with gender, smoking sttus, histology, tumor stge or lymph node sttus (31). These findings suggest tht PD L1 my hve limited use for predicting prognosis. The present study provides essentil informtion regrding the expression of PD 1, PD L1 nd PD L2 in ptients with NSCLC, which my be useful for guiding future tretment with Keytrud nd Opdivo. Given the unstisfctory clinicl outcomes with current therpies, the doption of immunotherpy my help to improve the survivl rte of our ptients. Acknowledgements Dr Zongbing You ws supported prtilly by Ntionl Institutes of Helth (Bethesd, MD, USA; grnt nos. P20GM nd R01CA174714), Deprtment of Defense (Fort Detrick, MD, USA; grnt nos. W81XWH , W81XWH , W81XWH nd W81XWH ), the Developmentl Fund of Tulne Cncer Center, Louisin Cncer Reserch Consortium Fund nd Tulne's Institute of Integrted Engineering for Helth nd Medicine (New Orlens, LA, USA; grnt no. TI2EHM). Dr Lunxu Liu ws prtilly supported by Ntionl Nturl Science Foundtion of Chin (Beijing, Chin; grnt nos. NSFC 81,172,236, 'The mechnism of TAMs ctivtion in lung cncer nd novel immunotherpy' nd NSFC 81,372,505, 'The role of IL 17 in formtion nd progression of primry lung cncer nd the underlying moleculr mechnisms') nd the Key Science nd Technology Progrm of Sichun Province (Chengdu, Chin; grnt no., 2013SZ0005). Dr Jindong Mei ws visiting scholr t Tulne University School of Medicine sponsored by the Chin Scholrship Council (Beijing, Chin; grnt no., 201,406,240,145). References 1. Siegel RL, Miller KD nd Jeml A: Cncer sttistics, CA Cncer J Clin 65: 5 29, Chen W, Zhng S nd Zou X: Evlution on the incidence, mortlity nd tendency of lung cncer in Chin. Thorcic Cncer 1: 35 40, Torre LA, Bry F, Siegel RL, Ferly J, Lortet Tieulent J nd Jeml A: Globl cncer sttistics, CA Cncer J Clin 65: , Ledford H: Cncer tretment: The killer within. Nture 508: 24 26, Kntoff PW, Higno CS, Shore ND, Berger ER, Smll EJ, Penson DF, Redfern CH, Ferrri AC, Dreicer R, Sims RB, et l: Sipuleucel T immunotherpy for cstrtion resistnt prostte cncer. N Engl J Med 363: , Hodi FS, O'Dy SJ, McDermott DF, Weber RW, Sosmn JA, Hnen JB, Gonzlez R, Robert C, Schdendorf D, Hssel JC, et l: Improved survivl with ipilimumb in ptients with metsttic melnom. N Engl J Med 363: , Hrvey RD: Immunologic nd clinicl effects of trgeting of pd 1 in lung cncer. Clin Phrmcol Ther: Toplin SL, Hodi FS, Brhmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Crvjl RD, Sosmn JA, Atkins MB, et l: Sfety, ctivity, nd immune correltes of nti pd 1 ntibody in cncer. N Engl J Med 366: , Brhmer JR, Tykodi SS, Chow LQ, Hwu WJ, Toplin SL, Hwu P, Drke CG, Cmcho LH, Kuh J, Odunsi K, et l: Sfety nd ctivity of nti pd l1 ntibody in ptients with dvnced cncer. N Engl J Med 366: , Lipson EJ, Shrfmn WH, Drke CG, Wollner I, Tube JM, Anders RA, Xu H, Yo S, Pons A, Chen L, et l: Durble cncer regression off tretment nd effective reinduction therpy with n nti pd 1 ntibody. Clin Cncer Res 19: , Hmid O, Robert C, Dud A, Hodi FS, Hwu WJ, Kefford R, Wolchok JD, Hersey P, Joseph RW, Weber JS, et l: Sfety nd tumor responses with lmbrolizumb (nti pd 1) in melnom. N Engl J Med 369: , Wolchok JD, Kluger H, Cllhn MK, Postow MA, Rizvi NA, Lesokhin AM, Segl NH, Ariyn CE, Gordon RA, Reed K, et l: Nivolumb plus ipilimumb in dvnced melnom. N Engl J Med 369: , Robert C, Schchter J, Long GV, Arnce A, Grob JJ, Mortier L, Dud A, Crlino MS, McNeil C, Lotem M, et l: Pembrolizumb versus ipilimumb in dvnced melnom. N Engl J Med 372: , Gron EB, Rizvi NA, Hui R, Leighl N, Blmnoukin AS, Eder JP, Ptnik A, Aggrwl C, Gubens M, Horn L, et l: Pembrolizumb for the tretment of non smll cell lung cncer. N Engl J Med 372: , Herbst RS, Sori JC, Kownetz M, Fine GD, Hmid O, Gordon MS, Sosmn JA, McDermott DF, Powderly JD, Gettinger SN, et l: Predictive correltes of response to the nti pd l1 ntibody mpdl3280 in cncer ptients. Nture 515: , Ch E, Wllin J nd Kownetz M: Pd l1 inhibition with mpdl3280 for solid tumors. Semin Oncol 42: , Ishid Y, Agt Y, Shibhr K nd Honjo T: Induced expression of pd 1, novel member of the immunoglobulin gene superfmily, upon progrmmed cell deth. EMBO J 11: , Nishimur H, Minto N, Nkno T nd Honjo T: Immunologicl studies on pd 1 deficient mice: Impliction of pd 1 s negtive regultor for b cell responses. Int Immunol 10: , Nishimur H, Nose M, Hii H, Minto N nd Honjo T: Development of lupus like utoimmune diseses by disruption of the pd 1 gene encoding n itim motif crrying immunoreceptor. Immunity 11: , 1999.

7 ONCOLOGY LETTERS 12: , Freemn GJ, Long AJ, Iwi Y, Bourque K, Chernov T, Nishimur H, Fitz LJ, Mlenkovich N, Okzki T, Byrne MC, et l: Enggement of the pd 1 immunoinhibitory receptor by novel b7 fmily member leds to negtive regultion of lymphocyte ctivtion. J Exp Med 192: , Dong H, Zhu G, Tmd K nd Chen L: B7 h1, third member of the b7 fmily, co stimultes t cell prolifertion nd interleukin 10 secretion. Nt Med 5: , Ltchmn Y, Wood CR, Chernov T, Chudhry D, Borde M, Chernov I, Iwi Y, Long AJ, Brown JA, Nunes R, et l: Pd l2 is second lignd for pd 1 nd inhibits t cell ctivtion. Nt Immunol 2: , Tseng SY, Otsuji M, Gorski K, Hung X, Slnsky JE, Pi SI, Shlbi A, Shin T, Prdoll DM nd Tsuchiy H: B7 dc, new dendritic cell molecule with potent costimultory properties for t cells. J Exp Med 193: , Okzki T nd Honjo T: Pd 1 nd pd 1 lignds: From discovery to clinicl ppliction. Int Immunol 19: , Dong H, Strome SE, Slomo DR, Tmur H, Hirno F, Flies DB, Roche PC, Lu J, Zhu G, Tmd K, et l: Tumor ssocited b7 h1 promotes t cell poptosis: A potentil mechnism of immune evsion. Nt Med 8: , Keir ME, Butte MJ, Freemn GJ nd Shrpe AH: Pd 1 nd its lignds in tolernce nd immunity. Annu Rev Immunol 26: , Shrpe AH nd Freemn GJ: The b7 cd28 superfmily. Nt Rev Immunol 2: , Trvis WD, Brmbill E, Muller Hermelink HK nd Hrris CC (eds): World Helth Orgniztion Clssifiction of Tumours. Pthology nd Genetics of Tumours of the Lung, Pleur, Thymus nd Hert. Lyon, IARC Press, pp9 11, Allred DC, Clrk GM, Elledge R, Fuqu SA, Brown RW, Chmness GC, Osborne CK nd McGuire WL: Assocition of p53 protein expression with tumor cell prolifertion rte nd clinicl outcome in node negtive brest cncer. J Ntl Cncer Inst 85: , Kim MY, Koh J, Kim S, Go H, Jeon YK nd Chung DH: Clinicopthologicl nlysis of pd l1 nd pd l2 expression in pulmonry squmous cell crcinom: Comprison with tumor infiltrting t cells nd the sttus of oncogenic drivers. Lung Cncer 88: 24 33, Pn ZK, Ye F, Wu X, An HX nd Wu JX: Clinicopthologicl nd prognostic significnce of progrmmed cell deth lignd1 (pd l1) expression in ptients with non smll cell lung cncer: A met nlysis. J Thorc Dis 7: , 2015.